hist.library.paho.orghist.library.paho.org/English/ACHR/ACMR11_RP.pdf · FOREWORD The Pan American...

Researchin Progress

1972

A Summary

PAN AMERICAN HEALTH ORGANIZATIONPan American Sanitary Bureau - Regional Office of the

WORLD HEALTH ORGANIZATION

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RESEARCH IN PROGRESS1972

Ref: RD 11/5

PAN AMERICAN HEALTH ORGANIZATIONPan American Sanitary Bureau, Regional Office of the

WORLD HEALTH ORGANIZATIONWashington, D.C.

June 1972

FOREWORD

The Pan American Health Organization's research program turned10 years old in 1971. Its accomplishments have been numerous andimportant. Governments have become more conscious of the significance,

relevance, and necessity of biomedical research to the health of theirpopulations. They have invested progressively greater sums in the

training of investigators, the strengthening of laboratories, thefinancing of important studies, and the application of research to

current health problems. A research program encompassing a variety

of subjects and providing assistance to practically all Latin Americancountries has been firmly established. The Organization's own research

centers, and others that have benefited from PAHO's support and guidance

have won international recognition. A Pan American communicationsnetwork for the health sciences, centered at the PAHO Regional Library

of Medicine in Sao Paulo, is being developed. Scientific meetings andconferences have been held that presented invaluable opportunities forexchanges among workers in specific fields. One hundred and fifty

reports and 25 scientific monographs have been published.

Notwithstanding the results achieved, there are still imbalances

to be corrected. Thus, in the years ahead, the Organization will focus

its research efforts on new priority areas, including the administration

and delivery of health services as a system, Chagas' disease, mycotic

infections, tuberculosis, venereal diseases, heart disease and cancer,

environmental sciences, and human reproduction and development.

The present volume, issued by the Department of Research Develop-

ment and Coordination, summarizes the information available on 133

projects currently sponsored by PAHO, and provides a panoramic view of

the breadth of our research involvement.

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CONTENTS

Page

NUTRITION AND FOOD SCIENCE

Protein-Calorie Malnutrition: Effects, Mechanisms ofAdaptation, Pathophysiology, and Treatment 1

Hair-Root Morphology and Early Detection of Protein-Calorie Malnutrition 12

Evaluation of the Effects of Nutrition Education onNutritional Status 14

The Effects of Malnutrition on Physical Growth andMental Development 16

Adaptive Metabolic Changes in Children DuringNutritional Stress 25

Forces of Infection Acting on the Central AmericanPopulation 27

Colonization of the Intestine by Microorganisms 30

Association Between Nutrition and Fertility Variables 33

Standardizing Methodology and Computer Analysis forAssessing Nutritional Status 35

Assessment of Tests of Behavior from Studies of

Nutrition in the Western Hemisphere 37

Hematologic Investigations in the Central AmericanPopulation 39

Protein Requirements of Children of Preschool Age 42

Feeding the Preschool Child 44

Milk Intolerance 46

Effects of Oral Contraceptives on Lactation 48

Workshop to Prepare Guidelines for a Food Enrichmentand Fortification Nutrition Program 50

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Studies of the Protein Quality of Foods: Conventionaland Protein-Rich Formulas, and Effects of Amino Acidand Protein Supplementation on Staple Cereals 52

Development of Unconventional Protein Sources forHuman or Animal Nutriment 56

Improvement of the Nutritive Quality of Cereal-Based Diets 59

Processing of Beans to Produce an Instant Bean Flour 62

Corn Fortification: A Field Demonstration Model 64

Control of Hypovitaminosis A (Fortification of Sugarwith Retinol Palmitate) 66

Utilization of Coffee Byproducts 69

National Food and Nutrition Survey of Guyana 72

Food Consumption Survey of Trinidad and Tobago 74

Effect of Early Undernutrition on Central NervousSystem Development 76

ENDEMIC GOITER

Goiter and Goitrogenic Factors in Indians ofWestern Argentina 80

Endemic Goiter and the Pathophysiology of ThyroidDisease in Chile 82

Pathophysiology of Endemic Goiter in the Cauca Valleyof Colombia 85

Pathophysiology of Endemic Goiter and Cretinism inEcuador and their Prevention with Iodinated Oil 88

Pathophysiology, Prevention, and Treatment of EndemicGoiter in Andean Peru 94

Biochemical Constituents of Endemic Goiter GlandsBefore and One Year After Iodized Oil Injection 96

Endemic Goiter and Cretinism in Bolivia 98

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OCCUPATIONAL HEALTH

Chronic Manganese Poisoning: A Metabolic Disorder 100

GENETICS

Multidisciplinary Studies on Primitive Populationsin Latin America 105

MATERNAL AND CHILD HEALTH

Fetal Growth and Cord Immunoglobulins in CentralAmerican Populations 111

Intensive Care of the Mother, Fetus, and Newborn 113

Fetal Cephalic Deformation and Umbilical Cord

Compression During Labor 116

VACCINE TRIALS

Trials with Measles Vaccine in a Virgin Population 120

Pilot Typhus Vaccination Project in Bolivia 122

IMMUNOLOGY

Mechanism of Anaphylaxis Induced with Soluble orInsoluble Antigens 126

Study of Homocytotropic Antibodies in the Guinea Pig 128

MALARIA ERADICATION

Economic Impact of Malaria 130

Research on the Epidemiology of Malaria Eradicationin Problem Areas 132

Field Trial of a Long-Acting Antimalarial Drug 135

Sporozoite-Induced Immunity in Mammalian Malaria 139

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AEDES AEGYPTI ERADICATION

Field Test for Insecticide Effectiveness

MULTIDISCIPLINARY STUDIES

Surveillance and Research on Infectious DiseasesAlong the Transamazon Highway

MYCOLOGY

Field Trial of Spherulin, a New Diagnostic Reagent inCoccidioidomycosis

ARTHROPOD-BORNE VIRUSES

Studies on Arthropod-Borne Viruses

Birds in the Ecology of North American EncephalitisViruses

CANCER ETIOLOGY

Herpesviruses of South American Monkeys and MalignantLymphomas

FOOT-AND-MOUTH DISEASE AND VESICULAR STOMATITIS

Identification of Types and Subtypes of Foot-and-Mouth Disease Virus

Identification of Types and Subtypes of VesicularStomatitis Virus

Determination of Foot-and-Mouth Disease and VesicularStomatitis Antibodies in the Serum of Cattle,Horses, and Sheep

Latent Infections in Foot-and-Mouth Disease

Studies of Inactivated Vaccines Against Foot-and-Mouth Disease

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142

147

150

153

155

158

163

165

167

144

Improvements of the Efficacy of Inactivated Foot-and-Mouth Disease Vaccines 169

Development of Modified Live Virus Vaccines AgainstFoot-and-Mouth Disease 171

Laboratory Control of Foot-and-Mouth Disease Vaccines 174

ZOONOSES

The Specificity of Purified Protein Derivatives (PPD)of Mammalian Tuberculin Prepared from Mycobacteriumtuberculosis and M. bovis 176

The Specificity of Tuberculin PPD Prepared fromHeated and Unheated Culture Filtrates, with orwithout High-Speed Centrifugation 179

Survey of Bovine Tuberculosis in Argentina 182

Standardization of the Agglutination Test in Brucellosis 185

Serologic Response of Calves to Different VaccinationSchedules with Brucella abortus Strain 19 Vaccine 187

Studies on the Serologic Diagnosis or Caprine Brucellosisin Vaccinated and Unvaccinated Goats 190

Experimental Infection of Goats with Brucella melitensis 192

Production of Brucella melitensis Rev. 1 Vaccine ina Fermentation Tank 194

Vaccines Against Caprine Brucellosis 196

Typing of Brucella Strains Isolated from Man andAnimals in Latin America 198

Development of a Rapid Diagnostic Test for Ram Epididymitis 200

Pathogenesis of Ram Epididymitis Caused by Brucella ovis:Experimental Studies in Laboratory Animals 203

Experimental Infection of Male Goats with Brucella ovis 205

Brucella melitensis Rev. 1 and B. abortus 45/20 Vaccines:Pattern of Immunoglobulin Production FollowingVaccination and Challenge 207

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Evaluation of Vaccines Against Swine Brucellosis 209

Epidemiologic Studies on Hydatid Disease in the Americas 211

Immunologic Studies of Ovine Hydatidosis 213

The Immunoglobulin Response to Human Hydatid Infection 215

Evaluation of Immunodiagnostic Tests for EpidemiologicScreening for Hydatid Disease 217

IgG and Albumin Levels in Hydatid Cyst Fluid 220

Studies on Echinococcosis in Argentine Sylvatic Animals 222

A Study of Host-Parasite Relationships FollowingPrimary Oral Infection of Laboratory Animals withEchinococcus granulosus 224

Studies on Antihelminthics Active Against Echinococcusgranulosus in Dogs 226

Measurement of Ion Flux Across Hydatid Cyst Membranes 228

Effects of Dimethyl Sulfoxide (DMSO) on Hydatid CystPermeability 230

Studies of the Susceptibility of Echinococcus granulosusEggs to Selected Chemical and Physical Agents 232

Improvement of Laboratory Methods for Rabies Diagnosis 235

Evaluation of the Corneal Test as an AntemortemLaboratory Method for Rabies Diagnosis 237

Ecologic and Virologic Studies of Bat Rabies 239

Immunogenicity and Safety Tests in Cattle with ERA-StrainRabies Virus Propagated in BHK Cells 241

Transference of Rabies Neutralizing Antibodies ThroughColostrum and Milk from Vaccinated Cows to theirOffspring 243

Immune Response of Man Following Different Schedules ofAdministration of Suckling Mouse Brain (SMB) RabiesVaccine 245

Neurologic Complications in Man Associated with RabiesVaccine of Suckling Mouse Brain Origin 247

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Serologic Evaluation of Different Short Schedules forHuman Rabies Preexposure Vaccination 250

Survey of Rabies Vaccine Production in Latin America 252

Potency of Rabies Vaccine Used in the Americas 253

Laboratory Investigations on the EncephalitogenicActivity of Antirabies Vaccines 255

Sensitivity of Tissue Culture Systems Supplemented withDEAE-Dextran for Isolation of Street Rabies Virus 257

Antibiotic Resistance Patterns and Resistance TransferFactor of Enterobacteriaceae Isolated from Animals,Foods of Animal Origin, and Food Handlers 259

Detection of Enteropathogenic Escherichia coli inSamples of Human and Animal Origin 261

Resistance Transfer Factor (RTF) in EnterobacteriaceaeIsolated from Pigs Receiving Prophylactic Doses ofAntibiotics 263

Contamination of Horse Meat with Salmonellae: AnEcologic Study 265

Sanitary Control of Mussels (Mytilus platensis)Sold in the Greater Buenos Aires Area 267

HEALTH STATISTICS

Inter-American Investigation of Mortality in Childhood 269

Study on Multiple Causes of Death 273

Survey of Smoking Patterns in Latin American Cities 275

ECOLOGY

Ecologic Implications of Insect Control: Agriculture,Public Health, and Economic Development 278

ENVIRONMENTAL SCIENCES AND ENGINEERING

Water Distribution 281

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Computer Design of Water Distribution Networks 283

Basic Data for the Design of Water Supply andSewerage Systems 284

Filtering Properties of Natural Sands 286

Model Four-Filter Water Treatment Plant for theCity of El Imperial 288

Evaluation of the Influence of Filtration Rate onWater Quality at the Las Vizcachas Water TreatmentPlant in Santiago 290

Increase in Filtration Rates at the Lo Castillo Plant,its Influence on Water Quality, and the Possibilityof Extending the Rate of Increase to ConventionalPlants 292

Usefulness and Behavior Patterns of'Dynamic andUp-Flow Slow Filters 294

Sedimentation Tanks 296

Stabilization Ponds 298

Stabilization Ponds in Argentina 300

Performance of Facultative Stabilization Ponds inTreating Milk Industry Wastes 302

Orthokinetic Flocculation 304

Biologic Treatment of Toxic Wastes from Manioc Flour Mills 305

Dust Pollution in Working Areas 307

OPERATIONS RESEARCH

An Evaluation Method to Measure the Effectiveness ofPan American Health Organization Programs 309

A Systems Study of the Kingston, Jamaica, Blood Bank 311

HEALTH PLANNING

The Link Model 313

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Health Financing Submodel 315

Methodologic Model 317

Health Typologies 319

National System for Maintenance and Engineering ofHealth Care Facilities 321

BIOMEDICAL COMMUNICATION

PAHO Regional Library of Medicine: A Center of BiomedicalCommunication for Latin America 324

REFERENCE CENTER ACTIVITIES AND RESEARCH TRAINING

PAHO/WHO Immunology Research and Training Centers 328

Research Training Program in Virology, Ornithology,Ecology, and Tropical Medicine 333

Research Training in Clinical Medicine 336

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PROTEIN-CALORIE MALNUTRITION: EFFECTS, MECHANISMS OFADAPTATION, PATHOPHYSIOLOGY, AND TREATMENT

PROBLEM

Although much is known about the effects of protein and caloriedeficiency, the pathophysiology of these nutritional deficiencies andthe mechanisms by which man adapts to them still remain obscure. Thereis a definite need of better understanding of: (1) the effects ofvarious degrees of protein and calorie deficiency; (2) the reversibilityof the changes; (3) the mechanisms by which man is able to survive whensubjected to calorie and protein restrictions; and (4) a better under-standing of the changes expected during rehabilitation.

Among the various areas of protein-calorie deficiency that needinvestigation, we devoted special attention during 1971 to the followingin children during malnourishment and recovery, which constitute sub-projects: (1) quantitative evaluation of protein and calorie deficiencyand of the rates of recovery attained with various dietary regimens;(2) the relationship between protein deficiency and energy metabolism;(3) regulation of erythropoiesis; (4) gastrointestinal function; and(5) mechanisms by which growth is depressed in caloric deficiency.

Subproject I: Means of measuring the degree of deficiency incalories and protein in humans are needed, because only with quantitativeinformation about the degree of depletion of these nutrients can theeffects of these deficiencies be evaluated. Also, by determining therelative depletion of these nutrients, preventive measures and treatmentguides can be devised. Our previous studies have shown that creatinineexcretion can be used to measure the degree of protein deficiency andthe changes that occur during protein repletion, when related to theexpected creatinine excretion of normal children of the same height(creatinine height index or CHI). We have also shown that there is noimpairment in creatinine excretion by the kidney, but we have no infor-mation on the rate of creatinine synthesis in muscle, which is greatlydependent on the activity of creatine phosphokinase. The activity ofthis enzyme in the muscle of malnourished children and during proteinrepletion was investigated by needle muscle biopsies. Total musclecreatine and protein, as well as the CHI were measured simultaneously.The specific objective of this subproject was to further validate theuse of creatinine excretion as representative of muscle and lean bodymass during malnutrition and recovery.

Subprojects II and III: Previous studies have demonstrated thatcreatinine excretion is able to measure the degree of protein depletionand repletion. Considering that muscle is almost the exclusive sourceof creatinine as well as the major protein reserve, it was importantto determine whether creatinine excretion reflects only the depletion of

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protein or whether it reflects total body protein depletion, includingvisceral mass. Knowing that more than 80 per cent of basal oxygenconsumption (Basal VO 2 ) is the consequence of visceral oxygen consumption,simultaneous measurements of basal VO and creatinine excretion have been

made. The specific objective of this phase of the study was to correlatea measure of visceral function (and visceral mass) with the one we haveused to measure body protein depletion and repletion.

Previous findings have also shown that in the process of protein-calorie repletion in malnourished children, caloric intake must be muchhigher than established requirements. This has been interpreted as arelative inefficiency of caloric utilization in protein-calorie mal-nutrition. However, no information exists on the total caloric expenditureof children during malnourishment and recovery, nor its relation withbasal caloric expenditure. Consequently, we have measured total caloricexpenditure in children with various degrees of protein and caloriedeficiencies and also during their recovery by insensible weight losses,time motion studies, and changes in basal VO2 and in body composition.Through these measurements we are calculating efficiency of caloricutilization and routes of caloric loss in malnourished and recoveringchildren.

Investigations into the relation between total circulating hemoglobin,total body protein, basal V02, and total caloric expenditure have conti-nued. In previous studies we showed that the child with uncomplicatedmalnourishment does not have a true anemia and that his total circulatinghemoglobin is normal for the amount of lean body mass and active tissuemass, both on hospital admission and, in general, during recovery.Furthermore, the erythropoeitic responses to iron, folate, vitamin B1 2,and vitamin E are absent when there is no increase in lean body and activetissue mass.

During 1970 and 1971, we have conducted studies to determine if theerythropoietic responses in malnourished children can be altered bychanging the rate of recovery of lean body mass, basal V02, and totalcaloric expenditure.

Subproject IV: Diarrhea is an almost constant finding in thehistory of severely malnourished children. It is also one of the majorcauses of death, not only among severely malnourished populations butalso in the general population of developing countries. An equallyfrequent finding in severely malnourished children is a variable degreeof intestinal malabsorption. The pathophysiology of these gastro-intestinal alterations is poorly understood and in consequence, theirprevention and treatment are still quite empirical. We think that thestudy of severely malnourished children can yield important insightsinto the etiology of nonspecific diarrhea and malabsorption. Studiesstarted earlier of this matter were continued in 1971. They dealt withmorphologic changes in the small intestine, alterations in fat micellari-zation at the duodenal level, and the presence of high concentrationsof unconjugated bile acids in duodenal aspirates, possibly in relation

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to altered intestinal microflora. Specifically, the objectives ofthese studies are:

(1) to provide an understanding of the etiology of non-specific diarrhea and intestinal morphologic changescommonly observed in underdeveloped areas and particularlyin malnourished children.

(2) to determine if altered intestinal motility is a probablefactor in altering the intestinal milieu and consequently,through bacterial overgrowth, induces changes in bileacid composition of the intestinal contents of malnourishedchildren, and

(3) to investigate the role of altered bile acids in causingfat malabsorption and intestinal morphologic changes.The interactions of protein deficiency and altered micro-flora with all of these factors are also specific objectives.

Subproject V: Although retardation of growth is a universal findingin calorically deprived children, very little is known about the retar-dation mechanisms. This is particularly true when one considers thatcaloric requirements for growth are but a minimal part of the totalcaloric requirement of children.

On the other hand, reduced caloric intake in adult and childrenresults in the adaptation mechanism of reduced physical activity. At thesame time, physical activity is able to induce hormonal responses andchanges in body composition in adolescents and adults. Our hypothesishas been that inactivity produced by decreased caloric intake is primarilyresponsible for growth retardation. Our specific aim has been to investi-gate the effects of inactivity in producing growth retardation in theface of adequate nutrient intake and of restricted intake of completediets. These investigations have been performed in rats.

METHOD

The methods used for each subproject have been the following:

Subproject I: Percutaneous needle biopsy specimens of quadricepsmuscle have been obtained from children during malnourishment and periodi-cally during recovery. Muscle creatine phosphokinase, total muscleprotein, and total muscle creatinine have been determined. CHI has beenmeasured simultaneously. The activity of creatine phosphokinase has beenrelated to creatinine excretion and to the other measurements obtainedfrom analyses of the muscle biopsies specimens.

Subprojects II and III: Creatinine excretion, basal VO2, and in-sensible weight measurements loss, time motion studies, and various

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anthropometric measurements have been performed periodically in childrenduring malnourishment and recovery who have been given experimentaldiets providing different amounts of protein (2, 3, and 4 g of protein/kg),caloric intake remaining essentially constant at 120 and 150 calories/kg/day. For the evaluation of hematologic response in these same children,reticulocyte response and total circulating hemoglobin have been measuredon hospital admission, two weeks after therapeutic diet, and when thechild was fully recovered. It has been possible to show that differentrates of recovery in the parameters investigated can be obtained byvarying the protein intake.

Subproject IV: Severely malnourished children are studied uponadmission to the Clinical Center and periodically thereafter until theyare fully recovered. The presence or absence of diarrhea on admissionand through recovering is closely monitored. Intestinal morphology andfunction are studied by means of peroral biopsies and specific absorptiontests. Gastrointestinal motility is studied by measuring the time lapsebetween the oral administration of a marker and its appearance in thefeces. Micelle formation from a fat emulsion instilled into the duodenum,bile salt content in the duodenal fluid, and specific conjugated andunconjugated bile salts are measured quantitatively. Simultaneously,studies of gastrointestinal flora are done on samples obtained from thestomachÍ duodenum, jejunum, and feces. Fourteen children have beenstudied longitudinally with this protocol and several cross-sectionalstudies have been carried out in children at different stages of recovery.

Morphologic studies have been conducted of infants born dead andof children who have died during the neonatal period and first months oflife from causes other than malnutrition. To investigate the effects ofaltered bile salts in the gastrointestinal tract in producing morphologicalterations and nonspecific diarrhea, studies in an animal model (hamster)fed various amounts of free deoxycholic acid have been performed.

The recovery of children studied has been closely monitored usingthe techniques described for other studies.

Subproject V: Male weanling rats have been divided into twogroups: one kept free in metabolic cages and receiving additional periodsof measured exercise on a treadmill, and the other group kept in activity-restricting metabolic cages that we designed. Both groups of rats havebeen fed either at maximal food intake or at various levels of restrictedintake. Nitrogen and potassium balances have been checked at weeklyintervals, and the animals were measured both for height and weight.After five weeks the animals were sacrificed and their body compositiondetermined.

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RESULTS TO DATE

Subproject I: The activity of creatine phosphokinase was lowin the severely malnourished children and rose progressively as theyrecovered. The creatinine phosphokinase activity correlated significantlywith total muscle creatine, total muscle protein, and creatinine heightindex. The ratio of creatine phosphokinase per unit of muscle proteinremained essentially constant throughout recovery. Total muscle creatinealso maintained an essentially constant ratio with muscle protein. Theseresults indicate that creatine and creatinine metabolism in muscle isunimpaired and substantiate the use of creatinine excretion as an indi-cator of muscle mass and most probably of total body protein in childrenduring malnourishment and recovery.

Subproject II: A close correlation was found between creatinineexcretion and basal VO in children during malnourishment and recovery.However, the slope of the linear regression between creatinine excretionand basal oxygen consumption in absolute units was different in protein-malnourished children with different degrees of caloric depletion. Thesame applied to the correlation between basal oxygen consumption and bodysurface area, and when both creatinine excretion and surface area wereconsidered. In contrast, when the CHI was correlated with the relativebasal VO2 expected of a normal child of the same height as well as whenbasal VO , in absolute units, was correlated with surface area times theCHI relative creatinine excretion, the relationship was constant through-out recovery and independent of the degree of caloric depletion.

It has also been found that total caloric expenditure in protein-malnourished children with severe caloric depletion was higher than inthose with smaller caloric deficits during recovery. This was true whenexpressed per unit of surface area, per unit of creatinine excretion, orper unit of basal oxygen consumption. Based on these findings and insequential estimations of body composition, the efficiency of caloricutilization is decreased in children with protein deficiency and severecaloric depletion in relation to those with lesser degrees of caloricdepletion. The main routes of caloric losses that account for thedifferences observed were conduction, convection, and radiation. How-ever, the calories retained were used with the same efficiency forgrowth in both groups of children. The efficiency of caloric utilizationin this regard was found to be around 0.7, which is similar to publisheddata about growing animals.

Subproject III: Rates of recovery of lean body mass and activetissue mass were different in the children receiving 2, 3, and 4 gramsof protein/kg/day. The greater the protein intake, the faster the rateof recovery. The initial hematologic recovery rates also appeared tobe slightly faster in the children whose body composition was recoveringfaster. During later therapy the children receiving greater amounts ofprotein also showed greater increases in total circulating hemoglobin.There was still a disparity between hematologic and lean body mass

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recovery rates in children receiving 4 grams of protein/kg/day, whichresulted in a temporary "anemia."

Subproject IV: Fat micellarization in malnourished childrenis decreased and is accompanied by fat malabsorption. The capacity tomicellarize fat increases with recovery but this increase is hamperedby diarrhea. Furthermore, severely malnourished children with andwithout diarrhea have similarly decreased fat micellarization, butfat micellarization in fully recovered children with diarrhea decreasesto levels close to those observed in malnourished children. In fullyrecovered children without diarrhea, fat micellarization is the sameas in normal children.

The duodenal contents of bile salts in malnourished and recoveredchildren indicate that the malnourished child has decreased total bilesalt concentration with relative increases in unconjugated bile saltsin relation to conjugated bile salts. With protein recovery, conjugatedbile salt concentration increases dramatically and unconjugated bilesalts increase slightly. This results in a higher concentration oftotal bile salts and in a normalization of the ratio of unconjugated toconjugated bile salts. In diarrhea. this ratio tends to increase due toboth an increase in unconjugated bile salts and a decrease in conjugatedbile salts. From statistical analysis it has been found that elevatedunconjugated bile salt concentration impairs micellarization, whileconjugated bile salts improve it. When fat micellarization is plottedagainst the ratio of unconjugated to conjugated bile salts, it becomesevident that fat micellarization increases exponentially as the ratiodecreases. This occurs in severely malnourished, recovering, or fullyrecovered children with or without diarrhea.

Results from bacteriologic studies of the microflora of thesechildren are reported by the Division of Microbiology at INCAP.

Newborns who die from nonnutritional disease have an intestinalmorphology identical to that described in developed areas. Two monthslater and in a few cases even earlier, however, the intestinal morphologyof the children resembles that observed in malnourished and recoveredchildren as well as in normal controls who live in an unsanitary envi-ronment.

Results from feeding hamsters with 0.25 per cent free dehydrocholicacid indicate that changes in the intestinal mucosa closely resemblethose observed in children and adults of developing areas and are alreadypresent after four weeks of feeding. Such animals, furthermore, growless than controls.

Subproject V: Rats restricted in activity and fed an optimal dietas well as those restricted to amounts equalling 70, 60, and 40 per centof the same diet, grow less in length and weight compared to rats leftfree and subjected to treadmill exercise. Whole body analysis after fiveweeks of experimentation showed that restricted rats have less nitrogen

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and potassium than active ones. Potassium retention is also decreasedin the restricted animals but nitrogen retention appeared to be thesame for most groups of animals. This indicates that the restrictedanimals are insensibly loosing extra amounts of nitrogen. Excretionof catecholamines is not increased in the exercise-restricted groups.

SIGNIFICANCE

The significance of the research carried out in this project ismultiple. Most important is an understanding of the basic mechanismsand effects of protein and calorie deficiency, which should result infundamental knowledge necessary for rational treatment and preventivemeasures. We are already conducting investigations of therapeuticmeasures derived from these studies which we hope to extend in the nearfuture. It is important to mention that the areas so far investigated,severe protein-calorie malnutrition in children older than eight monthsdoes not appear to leave permanent impairment. This may not be thecase with somatic and psychologic development, which are probablyaffected permanently. In this last area, we have conducted studies incooperation with the Division of Human Growth at INCAP.

PUBLICATIONS

1. Alleyne, G.A.O., F.E. Viteri, and J. Alvarado. Indices ofbody composition in infantile malnutrition. Total bodypotassium and urinary creatinine. Am J Clin Nutr 23:875-78,1970.

2. Alvarado, J., B.L. Nichols, C.F. Hazlewood, J. Barnes, andF. Viteri. Concentraciones de potasio muscular en ladesnutrición protéico calórica: respuesta a la repleciónde proteina. In Memorias XII Cong Internac Pediatria,México, D.F., Impresiones Modernas, S.A., 1968, vol. III,p. 151.

3. Alvarado, J., B.L. Nichols, C.F. Hazlewood, H. Mansylla,and F. Viteri. Desnutrición proteínico calórica: El usode la biopsia muscular percutánea en la valoración de larecuperación nutricional. Rev Col Med 21:100-10, 1970.

4. Alvarado, J., and F.E. Viteri. Erythropoietic changesduring recovery of protein calorie malnutrition and itsrelation to lean body mass. In Abs Internat Cong Hematol,Munich, August 1970, Munich. J.F. Lehmanns Verlag, 1970,vol. XIII, p. 300.

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5. Alvarado, J., F. Viteri, and M. Béhar. Tratamientohospitalario en la desnutrición proteínico calórica severa.Rev Col Med 21:231-45, 1971.

6. Alvarado, J., F. Viteri, D. Luthringer, and H. Mansylla.Relaci¿n entre factores eritropoyéticos y depleci¿n protéicocal¿rica. In Memorias XII Cong Pediatría, Mexico, D.F.,Impresiones Modernas, S.A., 1968, vol. III, p. 175-76.

7. Arroyave, G., A. Lechtig, F. Viteri, and L.J. Mata. Influenciade la ingesta proteínica sobre las inmunoglobulinas séricasen pre-escolares. In Resúmenes X Cong Internac Microbiol,México, D.F., Editorial Muñoz, S.A., 1970, vol. VIII. p. 72.

8. Beghin, I.D., and F. Viteri. Nutritional rehabilitationcenters: An evaluation of their performance. Backgroundpaper for the Protein Advisory Group Meeting, Geneva, December1971.

9. Burk, R.F. Jr., W.N. Pearson, R.P. Wood II and F. Viteri.Blood selenium levels and red blood cell 75Se-uptake inkwashiorkor. Am J Clin Nutr 20:365, 1967.

10. Burk, R.F., Jr., W.N. Pearson, R.P. Wood II, and F. Viteri.Blood-selenium levels and in vitro red blood cell uptakeof 7 5Se in kwashiorkor. Am J Clin Nutr 20:723-33, 1967.

11. Contreras, C., 0. Pineda, F. Viteri, and G. Arroyave.Effect of protein-calorie malnutrition on muscle creatine,phosphokinase activity. Fed Proc 30:231, 1971.

12. Contreras, C., and F.E. Viteri. Sialic acid content ofred blood cells in children during protein-calorie mal-nourishment recovery, and from normal children and adults.Blood, in press.

13. De Le¿n, R., and F. Viteri. Problemas de salud en Guatemala.Bol Of San Pan, in press.

14. Lechtig, A., G. Arroyave, F. Viteri, and L.J. Mata. Inmuno-globulinas séricas en la desnutrición proteínico calóricaen niños pre-escolares. Arch Lat Nutr 20:321-32, 1970.

15. Lechtig, A., G. Arroyave, F. Viteri, and L.J. Mata.Influencia de la ingesta de proteinicas sobre la concen-traci¿n de inmunoglobulinas séricas en ninos pre-escolares.Arch Lat Nutr, 20:333-43, 1970.

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16. Nichols, B.L., C.F. Hazlewood, J. Alvarado, and F. Viteri.Relationship between tissue glycogen and potassium concen-trations in malnourished children. Fed Proc 28:807, 1969.

17. Schneider, R., J. Alvarado, and F. Viteri. Histopathologicalstudy of the intestinal mucosa in malnutrition. A preliminaryreport. In Pediatrics in Latin America. Report Josiah Macy Jr.Foundation Conf, Santiago, Chile, March 1969, p. 34-41.

18. Schneider, R.E., and R. Chang. A pediatric tube and capsulefor suction biopsy of the small intestinal mucosa designedfor direct nasogastric intubation. Gut 12:399-402, 1971.

19. Schneider, R.E., and F.E. Viteri. Some morphologic andfunctional alterations of the gastrointestinal tract in protein-calorie malnourished children. Workshop on Malabsorption andNutrition, National Academy of Sciences, Washington, April 1971.Am J Clin Nutr, in press.

20. Schneider, R.E., and F.E. Viteri. Técnica de intubaciónintestinal usando un tubo de cuatro luces para el estudio dedistintos eventos gastro intestinales en niños. Rev Col Med,in press.

21. Viteri, F.E., J.M. Flores, and M. Béhar. Intestinal absorptionin protein-calorie malnutrition. Abs VII Internat Cong Nutr,Hamburg, 1966, vol. VIII, p. 4 6.

22. Viteri, F., G. Arroyave, and M. Béhar. Estimation of proteindepletion in malnourished children by a creatinine height index.VII Internat Cong Nutr, Hamburg, 1966, vol. VIII, p. 46-47.

23. Viteri, F., J. Alvarado, D.G. Luthringer, and R.P. Wood II.Adaptation of the erythropoietic system to protein caloriedeficiency. Fed Proc 27:485, 1968.

24. Viteri, F.E., J. Alvarado, D.G. Luthringer, and R.P. Wood II.Hematological changes in protein calorie malnutrition. InR.S. Harris, I.G. Wool, J.A. Loraine and K.V. Thimann (eds.)Vitamins and Hormones (Internat Symp on Vitamin-RelatedAnemias, Skytop, Pennsylvania, May 1968), New York, AcademicPress, Inc., 1968, 26:573-615.

25. Viteri, F., J. Alvarado, and M. Béhar. Evaluaci¿n cuantitativade la desnutrición protéico calórica. In Memorias XII CongInternac Pediatria, México, D.F., Impresiones Modernas, S.A.,1968, vol. III, p. 148.

9

26. Viteri, F.E., and J. Alvarado. Cuantificaci¿n del estadode nutrición protéico calórico por medio de indicadoresde composición corporal. XIV Reuni¿n Soc Arg Invest ClinValdivia, Chile, December 1969.

27. Viteri, F.E., and J. Alvarado. Body composition and oxygenconsumption in protein-calorie malnourished children andduring recovery. Am J Clin Nutr 23:675-76, 1970.

28. Viteri, F.E., and J. Alvarado. The creatinine height index:Its use in the estimation of the degree of protein depletionand repletion in protein-calorie malnourished children.Pediatrics 46:696-706, 1970.

29. Viteri, F.E., M. Béhar, and J. Alvarado. El problema de ladesnutrición proteinico cal¿rica en el Istmo Centroamericano;introducción e epidemiologia. Rev Col Med, 21:137-47, 1970.

30. Viteri, F.E. Composición corporal y requerimientos cal¿ricosy proteínicos en relaci¿n a la edad. Rev Col Med, 21:148-73,1970.

31. Viteri, F.E., and J. Alvarado. Aspectos fisiopatol¿gicos yrespuestas adaptativas en la desnutrición calórica y en ladesnutrición proteinica. Rev Col Med, 21:175-230, 1970.

32. Viteri, F.E. Efecto de dos grados extremos de actividadfisica en el crecimiento de ratas con restricción cal¿rica.In Resúmenes II Reunión Cient SLAN and II Cong Soc ChilNutrici6n, Bromatologia y Toxicologia, Viña del Mar, Chile,December 1970, p. 42. Also published in Arch Lat Nutr21: 274-75, 1971.

33. Viteri, F. Effect of activity restriction and exercise ongrowth of calorically restricted rats. Fed Proc 30:645,1971.

34. Viteri, F.E., J. Alvarado, J.R. Aguilar, J.T. Rodriguez, andE. Cifuentes. Basal oxygen consumption, body surface areaand creatinine excretion. Studies in protein-calorie mal-nourished children and during recovery. Pediatrics, in press.

35. Viteri, F.E., and 0. Pineda. Effects of starvation on theindividual; effects on body composition and body function;psychological effects. In Proc Symp on Famine Dealing withNutrition and Relief Operations in Times of Disaster,Saltsjobaden, Sweden, August 1970, in press.

10

36. Viteri, F.E., L.J. Mata, and M. Béhar. Métodos de evaluacióndel estado nutricional proteinico calórico en pre-escolaresde condiciones socioeconómicas diferentes. Repercusiónnutricional del sarampión en niños crónicamente subalimentados.Arch Lat Nutr, in press.

37. Viteri, F. Infecci6n y Nutrici6n. To be published in apediatrics textbook edited by J. Meneghello, Santiago, Chile.

38. Viteri, F.E., J. Alvarado, D.G. Luthringer, and R.P. Wood II.Three articles on hematologic changes in protein-caloriemalnutrition and recovery from it are in the final stage ofpreparation.

OTHER DATA

Grantee: Dr. Fernando E. Viteri, Institute of Nutrition ofCentral America and Panama (INCAP), Guatemala City,Guatemala, C.A.

Funded by: National Institutes of Health/U.S. Public HealthService and others.

Timetable: 1968-1973

11

HAIR-ROOT MORPHOLOGY AND EARLY DETECTIONOF PROTEIN-CALORIE MALNUTRITION

PROBLEM

Simple, practical methods, independent of exact age assessment, areneeded for the early detection of protein-calorie malnutrition (PCM). Astudy was needed to show whether the changes in hair-root morphology inprotein deficiency, which occur before serum albumin levels decline, canbe correlated well enough with various degrees of PCM to make them ofvalue in nutrition surveys and community health and nutrition programs.

METHOD

The weight-for-age status of 72 young children, including 15 controlswith normal weight and 12 patients with severe clinical PCM, was comparedwith the degree of their deviation from normal hair-root morphology.Children were weighed nude on beam balances, their ages were verified frombirth certificates, and the grade of their nutrition was determined accord-ing to their deficit in weight for age (Boston Standards). Approximately300 hairs from the occipital area were rapidly epilated, a process quickerand no more painful than a venipuncture. (The only laboratory equipmentneeded is a low-power microscope.)

RESULTS TO DATE

It seems possible to screen a population at three levels in the PCMsequence: hair-root morphology may be used as an index of early malnutrition(reduction of normal bulb diameter), severe PCM (increase in bulb atrophy),and chronicity (anagen-telogen shift).

SIGNIFICANCE

A contribution toward solution of the problem above has been made.

PUBLICATIONS

1. Bradfield, R.B., and E.F. Jelliffe. Early assessment ofmalnutrition. Nature (Lond) 225:283-84, 1970.

12

OTHER DATA

Grantee:

Funded by:

Timetable:

Caribbean Food and Nutrition Institute,Kingston, Jamaica

Research Corporation of New York

1969-1972

13

EVALUATION OF THE EFFECTS OF NUTRITION EDUCATIONON NUTRITIONAL STATUS

PROBLEM

Nutrition education has always been a part of programs to combatmalnutrition. Its specific contribution to the betterment of nutritionalstatus has been difficult to assess, however, because it has usually beenintegrated with other inputs such as food aid or specially recruitedpersonnel.

Not only is it important to try to define the role of nutritioneducation per se, but it is equally if not more important to make modelsof nutrition programs that will be easily replicable in other regionswhere childhood malnutrition occurs.

Consequently, the need exists for devising a program in which thedominant input is nutrition education provided as economically as possible,and having as its main contributors people indigenous to the region.

METHOD

A rural area, the Lambs River district of the parish of Westmoreland,Western Jamaica, was selected as the study area, and a similar area,Darliston, as a control.

Socioeconomic and relevant anthropomeric data were collected fromboth areas during the first six months of the project. Such data haveestablished the basic similarities between the two areas.

A staged program of nutrition education will now be developed inthe study area, principally involving schools, the health service,agricultural extension services, community development services, voluntaryorganizations, and volunteer groups. Meanwhile, no similar activitiesare planned for the control area.

During the project we intend to use child anthropometry, nutritionknowledge and practices, especially in relation to home food productionand use, and morbidity trends, as objective parameters of change. Inaddition, there will of necessity be some aspects that only allow sub-jective assessment.

RESULTS TO DATE

None to date.

14

SIGNIFICANCE

None has been clearly evident to date. We can only observe thateven among the most rural peoples there is scope for the development ofself-help activities of this type, even though the process may take timeand perseverance.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Caribbean Food and Nutrition Institute,Kingston, Jamaica

Funded by:

Timetable:

Freedom From Hunger Committee, United Kingdom

1971-1974

15

THE EFFECTS OF MALNUTRITION ON PHYSICAL GROWTHAND MENTAL DEVELOPMENT

PROBLEM

Human growth in general is considered as an increase in size andas a change in function. Although hereditary factors determine thecharacteristics of growth to a large extent, growth is affected in oneor more ways by disease and/or nutrition, both environmental factors.

The purposes of this study are: (1) to determine the modificationsin physical growth and mental development that occur in preschool chil-dren as a result of varying degrees of nutritional deficiencies; (2) todetermine at what age adequate nutritional supplements promote favorablechanges in physical growth and mental development; and (3) to explorewhich cultural and socioeconomic factors affect physical growth andmental development.

METHOD

Two groups of preschool children, including newborn infants enter-ing each group during that period, will be compared in physical growthand mental development on a longitudinal basis over six years. Bothgroups were drawn from communities previously matched for the mainvariables. The experimental group in one community is being given anutritionally adequate supplement that provides 130 per cent of theallowances recommended by INCAP. The control group in the other commu-nity is subsisting on its usual (deficient) dietary regimen.

Food supplementation is principally directed toward subjectsunder three years of age, pregnant women, and lactating mothers in theexperimental communities.

The subjects are assessed periodically at predetermined agelevels, by anthropometric measurements, biochemical determinations,dietary surveys, demographic and socio-cultural studies, and neurologicand psychologic examinations.

RESULTS TO DATE

Food supplementation produces a significant improvement inphysical growth among preschool children and growth rates for thetreated group are now similar to North American norms. Moreover,providing a food supplement to pregnant women produces a significant

16

increase in newborn birth weight. Food supplementation was also foundto reduce the incidence of diarrhea among preschool children.

The socioeconomic status of the family of the preschool childunder study is a powerful predictor of both attained physical sizeand psychologic test performance. Preliminary findings suggestthat nutritional status, as estimated by attained size, contributesimportantly to variance in psychological test performance, above andbeyond the contribution of socio-cultural factors. Further, nutri-tional status has been found to heavily influence performance on testsof perceptual processes, tests of memory somewhat less, and tests oflanguage development least.

SIGNIFICANCE

The majority of preschool children in developing countries,which encompass over 66 per cent of the world population, are nowmalnourished to varying degrees. Growth and development rates, whichare fastest in preschool children are seriously impaired by theprotein and calorie deficiencies as well as the infectious diseasesprevalent in such children.

This study is yielding valuable data on the effects of environ-mental conditions on human growth and development potentials, and therelationship between the age of onset and severity of undernourishmentand permanent sequelae.

If it is proved that under-nourishment during the first threeyears of life has lasting detrimental effects on human intellectualdevelopment, immediate measures to prevent malnutrition during thoseyears should receive highest priority in public health programs.

PUBLICATIONS

1. Adinolfi, A.A., and R.E. Klein. The value orientation ofGuatemalan subsistence farmers: measurement and implications.XIII Interam Cong Psych, Panama City, Panama, December 1971.

2. Arroyave, G., A. Lechtig, F. Viteri, and J.L. Mata. In-fluencia de la ingesta protéica sobre las inmunoglobulinasséricas en niños preescolares. Resúmenes, X Cong InternacMicrobiologia, Mexico, D.F., Editorial Muñoz, S.A., 1970,

p. 72.

3. Bekker Mendez, L. La prueba perceptual Johns Hopkins comotécnica para explorar la inteligencia según estudio realizadoen una comunidad rural de Guatemala. Unpublished licentiate

17

dissertation, Universidad de San Carlos de Guatemala,Guatemala, 1970.

4. Erdmenger, O.M. Relaci¿n entre maduración osea ydesarrollo mental en un grupo de sujetos preescolares deuna comunidad rural de Guatemala. Unpublished licentiatedissertation, Universidad de San Carlos de Guatemala,Guatemala, 1970.

5. Erdmenger, J.J., L.G. Elias, N. de Souza, J.B. Salomon,R. Bressani, G. Arroyave, and J.P. Habicht. Estudio enratas del efecto de la suplementaci6n proteinica de unadieta tipica de una comunidad rural de Guatemala. ArchLatinoamer Nutr, in press.

6. González Donis, N.V. Estudio exploratorio de prácticas decrianza de preescolares por madres de tres comunidadesrurales de Guatemala. Unpublished licentiate dissertation,Universidad Rafael Landivar, Guatemala, 1970.

7. Habicht, J.P., A. Lechtig, G. Guzman, and E. de Leon.Effect of high protein supplement on growth of ruralGuatemalan children. Resúmenes de temas libres II ReuniónCient Soc Latinoamer Nutrición y II Cong Soc Chilena Nutri-ción, Bromatologia y Toxicologia con la adhesi6n de la SocChilena Tecnologia de Alimentos, Viña del Mar, Chile,December 1970, p. 22.

8. Habicht, J.P., J.A. Schwedes, and G. Arroyave. Biochemicalindices of nutrition reflecting ingestion of a high proteinsupplement in rural Guatemalan children. II Reunión CientSoc Latinoam Nutrici6n. Viña del Mar, Chile, December 1970.

9. Habicht, J.P. Standardization procedures for quantitativeepidemiologic field methods. Incap Publication DE-418, 1971.

10. Habicht, J.P., G. Guzmán, and J.M. Reyna-Barrios. Out-patient curative medical care provided by a paramedical staff:needs, practicability and quality control. Incap PublicationDE-506, 1971.

11. Habicht, J.P., J.M. Reyna-Barrios, G. Guzman, and J.E. Gordon.Health services in field study of malnutrition: professionalcontent, operational expediency, ethical considerations.Incap Publication DE-449, 1971.

18

12. Klein, R.E., O. Gilbert, C.A. Canosa, and R. de Leon.Performance of malnourished in comparison with adequatelynourished children on selected cognitive tasks (Guatemala).Symp Annual Meeting Am Ass Advancement Science, Boston,Mass., December 1969.

13. Klein, R.E. Some considerations in the measurement of theeffects of food supplementation on intellectual developmentand social adequacy. In N. Scrimshaw and J. Gordon (eds.),Proceedings of the International Conference on Amino AcidFortification of Protein Foods, Boston (Mass), MIT Press,in press.

14. Klein, R.E., and A.A. Adinolfi. Measurement of the behavioralcorrelates of malnutrition. In J. Prescott, D.B. Coursin, andM.S. Read (eds.), Malnutrition and Brain Function: Neurophysio-logical Methods of Assessment, Washington, Government PrintingOffice, in press.

15. Klein, R.E., H.E. Freeman, and R. Millet. Indigenous conceptionsof intelligence: their relationship with standard psychologicaltests. Incap Publication DE-652, 1971.

16. Klein, R.E., J.P. Habicht, and C. Yarbrough. Some methodologicalproblems in field studies of nutrition and intelligence. InD. Kallen (ed.), Proc Conf Assessment of Test of Behavior fromStudies of Nutrition in the Western Hemisphere, Washington,Government Printing Office, in press.

17. Klein, R.E., J.P. Habicht, and C. Yarbrough. Effect ofprotein-calorie malnutrition on mental development. AdvancesPediat, in press.

18. Klein, R.E., J.P. Habicht, and C. Yarbrough. Malnutritionand mental development: four cautionary notes in data analysis.III. Proc West Hem Nutr Cong, Miami, Symposia Specialists, inpress.

19. Klein, R.E., B.M. Lester, M.J. Sellers, and C. Yarbrough.Psychological test performance of rural Guatemalan children.XIII Interam Cong Psychology, Panama City, Panama, December

1971.

20. Klein, R.E., and C. Yarbrough. Some considerations in theinterpretation of psychological data as they relate to theeffects of malnutrition. Incap Publication I-553, 1971.

19

21. Klein, R.E., C. Yarbrough, J.P. Habicht, and B.M. Lester.Cross-cultural evaluation of human intelligence. Proc SympLipids and the Developing Brain. London, Ciba Foundation,in press.

22. Lechtig, A., G. Arroyave, F. Viteri, and L.J. Mata. Inmuno-globulinas séricas en niños preescolares. Arch LatinoamNutr 20:321-32, 1970.

23. Lechtig, A., G. Arroyave, F. Viteri, and L.J. Mata. Influenciade la ingesta de proteinas sobre la concentración de inmuno-globulinas séricas en niños preescolares. Arch Latinoam Nutr

20:333-43, 1970.

24. Lechtig, A., G. Arroyave, F. Viteri, and L.J. Mata. Inmuno-globulinas séricas en la desnutrición proteinico-calóricasevera. Resúmenes de temas libres II Reunión Cient SocLatinoam Nutrición y II Cong Soc Chilena Nutrici¿n, Bromato-logia y Toxicologia con la adhesion de la Soc ChilenaTecnología de Alimentos, Viña del Mar, Chile, December 1970.p. 29-30.

25. Lechtig, A., E. de Leon, and M. Flores. Factores que influen-cian el crecimiento fetal en poblaciones rurales de Guatemala.I. Aspectos dietéticos. Resúmenes de temas libres, II ReuniónCient Soc Latinoam Nutrici¿n y II Cong Soc Chilena Nutrición,Bromatologia y Toxicologia con la adhesi6n de la Soc ChilenaTecnologia de Alimentos, Viña del Mar, Chile, December 1970,

P. 5.

26. Lechtig, A., E. de Leon, and J.P. Habicht. Factores queinfluencian el crecimiento fetal en poblaciones rurales deGuatemala. II. Suplementaci¿n alimenticia. Resúmenes detemas libres II Reunión Cient Soc Latinoam Nutrición y IICong Soc Chilena Nutricion, Bromatologia y Toxicologia conla adhesi¿n de la Soc Chilena Tecnología de Alimentos, Viñadel Mar, Chile, December 1970, p. 19-20.

27. Lechtig, A., E.M. Giron, and J.P. Habicht. Factores queinfluencian el crecimiento fetal en poblaciones rurales deGuatemala. III. Características maternas. Resúmenes detemas libres, II Reuni6n Cient Soc Latinoam Nutrición y IICong Soc Chilena Nutrici¿n, Bromatología y Toxicologia conla adhesion de la Soc Chilena Tecnología de Alimentos, Viñadel Mar, Chile, December 1970, p. 20.

28. Lechtig, A., G. Guzmán, E. de Leon, and E.M. Gir6n. Factoresque influencian el crecimiento fetal en poblaciones rurales

20

de Guatemala. IV. Morbilidad prenatal. Resúmenes de temaslibres, II Reunión Cient Soc Nutrición y II Cong Soc ChilenaNutrición, Bromatologia y Toxicología con la adhesion de laSoc Chilena Tecnologia de Alimentos, Viña del Mar, Chile,December 1970, p. 20-21.

29. Lechtig, A., and L.J. Mata. Immunoglobulin levels in cordblood of Guatemalan village children. Fed Proc 29:578, 1970.

30. Lechtig, A., and L.J. Mata. Influencia del saneamientoambiental sobre inmunoglobulinas séricas en recién nacidos.Resúmenes, X Congreso Internacional del Microbiología.Mexico, D.F., Editorial Muñoz, S.A., 1970, p. 72.

31. Lechtig, A., L.J. Mata, and G. Arroyave. Evaluación de latécnica de inmunodifusión radial para la determinación deinmunoglobulinas y una fracción del complemento hemolíticoen el suero. Rev Latinoam Microbiol 12:131-36, 1970.

32. Mata, L., J.J. Urrutia, B. Garcia, and A. Lechtig. Matro-ambiente y antropometría del recién nacido en una comunidadrural de bajo nivel socio-económico. Resúmenes de temas libres,II Reunión Cient Soc Latinoam Nutrición y II Cong Soc ChilenaNutrición, Bromatologla y Toxicología con la adhesión de laSoc Chilena Tecnología de Alimentos. Viña del Mar, Chile,December 1970, p. 5.

33. Lechtig, A., G. Arroyave, J.P. Habicht, and M. Behar. Nutriciónmaterna y crecimiento fetal. (Revisión). Arch Latinoam Nutr,in press.

34. Lechtig, A., G. Arroyave, J.P. Habicht, F. Viteri, L.J. Mata,and M. Behar. Metabolismo, nutrición, crecimiento y desarrollo.(Letter to the Editor). Arch Latinoam Nutr 21:87-88, 1971.

35. Lechtig, A., J.P. Habicht, E. de Leon, G. Guzmán, and M. Flores.Influencia de la nutrición materna sobre el crecimiento fetalen poblaciones rurales de Guatemala. I. Aspectos dietéticos.Arch Latinoam Nutr, in press.

36. Lechtig, A., J.P. Habicht, E. de Leon, and G. Guzmán. In-fluencia de la nutrición materna sobre el crecimiento fetalen poblaciones rurales de Guatemala. II. Suplementaciónalimentaria. Arch Latinoam Nutr, in press.

21

37. Lechtig, A., J.P. Habicht, and G. Guzmán. Fetal growth inrural populations of Guatemala. Proc III West Hem Nutr Cong,Bal Harbour, Fla., August 1971.

38. Lechtig, A., J.P. Habicht, G. Guzmán, and M. Behar. Nutritionof the mother and fetal growth. Latinoam Nutr, in press.

39. Lechtig, A., J.P. Habicht, G. Guzmán, and E. de Leon. In-fluencia de la morbilidad materna sobre el crecimiento fetal.Memorias, I Cong Nac y III Centroam Microbiologia, Guatemala,July 1971, in press.

40. Lechtig, A., J.P. Habicht, G. Guzmán, and E. de Leon. Morbilidadmaterna y crecimiento fetal en poblaciones rurales de Guatemala.Arch Latinoam Nutr, in press.

41. Lechtig, A., J.P. Habicht, G. Guzmán, and E.M. Gir¿n. Influenciade las caracteristicas maternas sobre el crecimiento fetal enpoblaciones rurales de Guatemala. Arch Latinoam Nutr, in press.

42. Lechtig, A., K. Ishizaka, G. Arroyave, R. Maselli, and L.J. Mata.Inmunoglobulinas E (IgE) en la desnutrición proteinico-cal¿ricasevera. Arch Latinoam Nutr 21:197-99, 1971.

43. Lechtig, A., and L.J. Mata. Cord IgM levels in Latin Americanneonates. (Letter to the Editor). J Pediat 78:909-10, 1971.

44. Lechtig, A., and L.J. Mata. Levels of C3 in newborns andmothers from different ecosystems. Rev Latinoam Microbiol, inpress.

45. Lechtig, A., and L.J. Mata. Levels of C'3 in Peruvian newbornsand mothers from different socioeconomic groups. Fed Proc

30:300, 1971.

46. Lechtig, A., and L.J. Mata. Levels of IgG, IgA, and IgM incord blood of Latin American newborns from different ecosystems,Rev Latinoam Microbiol, in press.

47. Lechtig, A., and L.J. Mata. Relaci¿n entre los componentesséricos de la respuesta inmune en reci¿n nacidos y lascondiciones de saneamiento ambiental. I. Inmunoglobulinas M.Memorias, I Cong Nac y III Centroam Microbiolog;ia, Guatemala,July 1971, in press.

48. Lechtig, A., and L.J. Mata. Relación entre los componentessericos de la respuesta inmune en recién nacidos y lascondiciones de saneamiento ambiental. II. Factor C3 delcomplemento hemolitico. Memorias, I Cong Nac y III CentroamMicrobiologia, Guatemala, July 1971, in press.

49. Lechtig, A., and L.J. Mata. Relaci¿n entre higiene ambientaly componentes séricos de la respuesta inmune. V Cong LatinoamMicrobiologia, Punta del Este, Uruguay, December 1971.

50. Lechtig, A., J.J. Ovalle, and L.J. Mata. Niveles de IgG, IgA,IgM, y C3 en niños indígenas de Guatemala durante los primeros6 meses de edad. Rev Latinoam Microbiol, in press.

51. Mata, L.J., A. Cáceres, A. Lechtig, and J.J. Urrutia. Studiesof infection and immune aspects in malnourished village children.Simp Nutr e Infección, Geneva, June 1971.

52. Mata, L.J., J.J. Urrutia, and A. Lechtig. Infection and nutri-tion of children of a low socioeconomic rural community.Amer J Clin Nutr 24:249-59, 1971.

53. Mejia Pivaral, V. Condiciones generales, economía y organizaciónsocial de cuatro comunidades Ladinas rurales de Guatemala.Guatemala Indigena, in press.

54. Reyna-Barrios, J.M., J.P. Habicht, and G. Guzmán. Medidas paraampliar la cobertura y mejorar la calidad de consulta externaen areas rurales de Guatemala, empleando auxiliares de enfer-mería. XXI Cong Med, Guatemala, December 1970.

55. Sellers, M.J., R.E. Klein, J. Kagan, and C. Minton. Develop-mental determinants of attention: A cross-cultural replication.Develop Psychol, in press.

56. Yarbrough, C. An inequality for the combination of probabilities.Incap Publication DE-591, 1971.

57. Yarbrough, C., and J.P. Habicht. Comparison of bone ageestimators. Annual Meeting Am Ass Phy Anthropologists, Boston,April 1971.

58. Yarbrough, C., and J.P. Habicht. Telling biological time:bone age of the hand and wrist. Incap Publication DE-600,1971.

23

59. Yarbrough, C., J.P. Habicht, and R.E. Klein. Combinationof probabilities: the case of 2x2 tables. Incap PublicationDE-590, 1971.

60. Yarbrough, C., J.P. Habicht, R.E. Klein, and S. Sellers.Confounding factors in malnutrition research. XIII InternatCong Psychology, Panama City, Panama, December 1971. IncapPublication DE-518.

61. Klein, R.E., H.E. Freeman, J. Kagan, C. Yarbrough, andJ.P. Habicht. Is fat smart? Incap Publication DE-650, 1972.

62. Klein, R.E., J.P. Habicht, C. Yarbrough, S. Sellers, andM.J. Sellers. Empirical findings with methodological impli-cations in the study of malnutrition and mental development.Seminar on Malnutrition in Early Life and Subsequent Develop-ment of Functions, Kingston, Jamaica, January 10-14, 1972.

OTHER DATA

Grantee: Dr. Robert E. Klein, Division of Human Development,Institute of Nutrition of Central America andPanama (INCAP), Guatemala City, Guatemala

Funded by: National Institute of Child Health and HumanDevelopment


24

ADAPTIVE METABOLIC CHANGES IN CHILDRENDURING NUTRITIONAL STRESS

PROBLEM

Systematic biochemical studies of malnutrition are needed. Inves-tigations of this kind call for the use of a tissue with a high turnoverrate so as to reflect alterations produced in fundamental organs such asthe liver. At the INCAP laboratories, leukocytes have been shown to besuitable for this purpose.

The current research has the following aims: (1) to investigatelongitudinally the adaptive biochemical alterations produced by protein-calorie malnutrition in polymorphonuclear leukocytes obtained fromexperimentally produced exudates, as well as the patterns of alterationswith similar cells derived from blood; (2) to study the relationshipbetween enzyme changes induced in experimental animals in liver and thosein leukocytes, and to compare this information with data obtained frommalnourished children; (3) to develop practical evaluation proceduresfor a more accurate characterization of the nature and severity ofprotein-calorie malnutrition; and (4) to study the phagocytic ability ofleukocytes derived from normal and malnourished children, and to evaluatethe effect of infection on this function.

METHOD

The study was carried out in two parts: (1) observations of mal-nourished children with severe protein-calorie malnutrition on hospitaladmission and after complete recovery; (2) the effect of a temporaryrestriction of dietary calories or protein on recovered children.Isolation and purification of blood polymorphonuclear neutrophiles (PMN)is achieved by differential sedimentation on high molecular weightdextran, followed by sedimentation through centrifugation. Contaminatingerythrocytes are removed by hypoosmotic shock. The cells obtained inthis manner are viable and show a 90-95 per cent homogeneity. In theintact cell, oxygen uptake is measured polarographically. For theseexperiments, a heat-killed culture of E. coli K-12 has been used forphagocytosis.

RESULTS TO DATE

The results showed that protein-calorie malnutrition leads to areduced capacity for phagocytic stimulation as measured by oxygen uptake.Infection, regardless of the nutritional state of the child, is re-flected by a rate of "resting" oxygen uptake that is clearly higher than

25

in uninfected children. In nutritionally recovered children, short-termdietary restriction of calories or protein resulted in reduced oxygenuptake by the PMN. This alteration is rapidly reversed when the childreceives a complete diet. Preliminary results also indicate that thePMN of severely malnourished children not only phagocytize less, butalso have a reduced bactericidal action in that they kill the engulfedbacteria at a slower rate.

SIGNIFICANCE

The higher resting oxygen uptake of infected children is evidenteven before clinical symptoms become obvious. This could be of importancein determining the presence of infection, particularly in severely mal-nourished children in whom clinical characteristics of infection arecommonly masked. Furthermore, the results emphasize the fact that thepolymorphonuclear leukocytes of malnourished children have a doublefunctional defect, reduced phagocytic activity and decreased capacity tokill bacteria. This finding may elucidate the relationship betweennutrition and resistance to infection.

PUBLICATIONS

1. Viteri, F., and 0. Pineda. Nutrition and relief operationsin times of disaster. In G. Blix, Y. Hofvander, and B.Vahlquist (eds.), IX Symposia of the Swedish NutritionFoundation, Uppsala, Almqvist and Wiksells, 1971, p. 64.

2. Pineda, O. Effect ofmetabolic capacity ofActa Cient Venez, 122

malnutrition and infections on thethe human polymorphonuclear leukocyte.(Suppl. 2): R 51, 1971.

3. Pineda, O., F. Viteri, and J.E. Braham.adaptation to low protein-calorie diets.

1971.

Leukocyte enzymeFed Proc 30:231,

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Oscar Pineda, Division of Physiological Chemistry,Institute of Nutritipn of Central America and Panama,Guatemala City, Guatemala

National Institute of Arthritis and Metabolic Diseases,U.S. Public Health Service

1969-1971

26

FORCES OF INFECTION ACTING ON THECENTRAL AMERICAN POPULATION

PROBLEM

Infectious dieseases interact with malnutrition in a synergicmanner. Knowledge of the forces of infection that act on the CentralAmerican population is important in health planning, particularly becausethey are of great significance as to morbidity and mortality and theirinteraction with malnutrition.

The purpose of the present research is to analyze, on a large scale,

a serum bank representing the various ethnic and socioeconomic groups ofthe six Central American countries for antibodies to several infectious

agents that are of known public health significance.

METHOD

A large bank with approximately 17,000 serum specimens was assembledin 1965-1967. The bank is split into two identical collections, onelocated at INCAP and another at the Middle American Research Unit (MARU)in Panama.

Very small amounts of serum are analyzed to determine the prevalenceand titer of antibodies to syphilis, Brucella, Salmonella, Shigella,enteroviruses, adenoviruses, the rubella agent, arboviruses, vesicularstomatitis virus, Toxoplasma, Trypanosoma cruzi, and malaria. Microtechniques are used to process the several thousand sera.

RESULTS TO DATE

A profile of antibodies to the various agents investigated has beenobtained for the various populations of Central America. Data have been

analyzed by country, age, sex, locality, occupation, and other parameters.

The study of epidemics, and even of their anticipation, has beenpossible through analyses of the serum bank.

For understanding of the findings obtained see the attached listof references.

27

SIGNIFICANCE

This is the first survey attempting to describe the characteristicsof a representative sample of the population of Central America bystandard and accurate techniques of serologic analyses.

The information obtained will be helpful in unveiling public healthproblems, in recognizing the relative importance of the various infections,and in directing certain public health measures as vaccination.

PUBLICATIONS

1. Gangarosa, E.J., D.R. Perera, L.J. Mata, C. Mendizabal,G. Morris, G. Guzmán, and L.B. Reller. Epidemic Shigabacillus dysentery in Central America. II. Epidemiologicstudies in 1969. J Infect Dis 122:182-90, 1970.

2. Mata, L.J., E.J. Gangarosa, A. Cáceres, D.R. Perera, andM.L. Mejicanos. Epidemic Shiga bacillus dysentery inCentral America. I. Etiologic investigations in Guatemala.J Infect Dis 122:170-80, 1970.

3. Mata, L.J., A. Cáceres, and M.F. Torres. Epidemic Shigadysentery in Central America. Lancet I:600-01, 1971.

4. Mata, L.J., and R. Wyatt. The uniqueness of human milk.II. Breast-feeding and host resistance to infection.Am J Clin Nutr 24:976, 1971.

5. Mata, L.J., E.J. Gangarosa, A. Cáceres, D.R. Perera, andM.L. Mejicanos. Epidemia de disenteria Shiga en Centroamérica.I. Investigaciones etiol¿gicas en Guatemala, 1969. Bol Of

San Pan 71:93-107, 1971.

6. Mendizabal, C.A., L.J. Mata, E.J. Gangarosa, and G. Guzmán.Epidemic Shiga dysentery in Central America. III. Magnitudeof the outbreak and mortality in Guatemala in 1969. Am JTrop Med Hyg, in press.

7. Mata, L.J., A. Cáceres, R. Fernández, M.F. Torres, M. Cordon,and R. Rosales. Avances sobre el conocimiento de la disenteriaen Guatemala. Rev Latinoam Microbiol, in press.

28

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Leonardo J. Mata, Division of Microbiology,Institute of Nutrition of Central America and Panama,Guatemala City, Guatemala

U.S. Army Medical Research and Development Command

1967-1972

29

COLONIZATION OF THE INTESTINE BY MICROORGANISMS

PROBLEM

The object of this study is to investigate patterns of intestinalcolonization and infection by entero- and adenoviruses, pathogenicenteric bacteria, bacterial groups in the indigenous intestinal micro-flora, and intestinal parasites in children under three years of age,and to correlate such colonizations and infections with the hosts'health status through careful, repeated clinical and laboratory investi-gation.

The study is conducted in a semi-isolated Mayan village in theGuatemalan highlands where children live in primitive and adverseenvironmental conditions.

All Indian children in the cohort were recruited at birth andhave been studied since 1964 through frequent pediatric, anthropometricdietary, and microbiologic examinations, in which every attempt hasbeen made to minimize environmental and social changes.

METHOD

Cohorts of children in a typical Mayan Indian village have beenstudied from birth to seven years of age. Fecal specimens were collectedat weekly intervals to determine the presence of parasites, bacteria,and enteroviruses. Weekly investigation of the diet was made. Closesurveillance of the population was carried out to determine onset,characteristics, and outcome of disease. Growth and development wereevaluated by anthropometric measurements and hand X-rays.

RESULTS TO DATE

A high incidence of fetal growth retardation was noted in thestudy. The frequency of intrauterine infection appeared to be signi-ficantly greater than in industrial societies. Infant mortalitycorrelated with fetal malnutrition. A high proportion of infants hadelevated IgM levels. Growth retardation occurred in most infantsby the first year of life and correlated with poor dietary intake andconsiderable infection.

30

SIGNIFICANCE

Close longitudinal observation of the health status, diseases,and growth and development of poor Indian children from birth to threeyears of age is unique, and has already contributed significantly tothe understanding of interrelated malnutrition and infection in sucha population.

The knowledge to be gained from this study will be fundamental tothe understanding of the etiology of diarrheal disease, by far the mostfrequent cause of death in small children in underdeveloped areas. Thisinformation is needed as a basis for establishing adequate preventivemeasures in areas of poverty, poor sanitation, and bad nutrition.

PUBLICATIONS

1. Wyatt, R.G., and L.J. Mata. Bacteria in colostrum and milkof Guatemalan Indian women. J Trop Ped 15:159-62, 1969.

2. Mata, L.J., and J.J. Urrutia. Intestinal colonization ofbreast-fed children in a rural area of low socioeconomiclevel. Ann N Y Acad Sci 176:93-109, 1971.

3. Mata, L.J., J.J. Urrutia, and A. Lechtig. Infection andnutrition of children of a low socioeconomic rural community.

Am J Clin Nutr 24:249-59, 1971.

4. Melvin, D.M., and L.J. Mata. Intestinal parasites in aMayan-Indian village in Guatemala. Rev Latinoam Microbiol

13:15-19, 1971.

5. Sweeny, E.A., J. Cabrera, J.J. Urrutia, and L.J. Mata.Factors associated with linear hypoplasia of human deciduousincisors. J Dent Res 48:1275-79, 1969.

6. Mata, L.J., J.J. Urrutia, C. Albertazzi, O. Pellecer, andE. Arellano. Influence of recurrent infections on nutritionand growth of children in Guatemala. Am J Clin Nutr, inpress.

7. Mata, L.J., A. Caceres, and J.J. Urrutia. Studies of infectionand immune aspects in malnourished children. In preparation.

8. Mata, L.J., and R.G. Wyatt. Host resistance to infection.Am J Clin Nutr 24:976-86, 1971.

31

9. Lechtig, A., and L.J. Mata. Cord IgM levels in Latin Americanneonates. (Letter to the Editor). J Pediat 78:909-10, 1971.

10. Lechtig, A., and L.J. Mata. Levels of C'3 in newborns andmothers from different ecosystems. Rev Latinoam Microbiol,in press.

11. Lechtig, A., and L.J. Mata. Levels of C'3 in Peruvian newbornsand mothers from different ecosystem groups. Fed Proc 30:300,1971.

12. Mata, L.J., J.J. Urrutia, A. Caceres, M. Guzmán. The biologicenvironment in a Guatemalan rural community. Proc III WestHem Nutr Cong, Miami Beach, Fla., August 30-September 2, 1971.

OTHER DATA

Grantee: Dr. Leonardo J. Mata, Division of Microbiology,Institute of Nutrition of Central America and Panama,Guatemala City, Guatemala

Funded by: National Institute of Allergy and Infectious Diseases/National Institutes of Health


32

ASSOCIATION BETWEEN NUTRITION AND FERTILITY VARIABLES

PROBLEM

The problem of too rapid population growth has received increasingattention in recent years. Population growth in developing countrieshas been identified as one of the leading reasons for their disappoint-ing economic growth and inability to provide basic services to alltheir citizens. Yet little has been done to show the economic, social,and health effects of high birth rates and large family size on theindividual family. This study will help show the effects of fertilityon family health as manifested by nutritional status.

METHOD

1. Association between fertility parameters and nutritionalstatus. In these analyses, unless noted otherwise, a fertility para-meter will be correlated with a selected nutritional parameter.Nutritional status of children under five years of age will be measuredin all six Central American countries. As in the preceding analyses,these are intended to show only associations, although causal relation-ships are hypothesized.

2. Association between maternal fertility parameters andoccurrence of infant and child deaths.

3. Association of fertility variables of women and the socio-cultural level of their households.

RESULTS TO DATE

A detailed plan of operations was prepared and an agreement signedby the collaborating groups.

The project is to be a joint one between the Institute of Nutri-tion of Central America and Panama, and the Institute for the Study ofHuman Reproduction of Columbia University. In general, preparationof the data for punching will take place at Columbia; preparation ofthe data cards will take place at INCAP; and analysis and interpretationwill be done at Columbia.

33

SIGNIFICANCE

The development of a collaborative research project between PAHO/INCAP and Columbia University on maternal nutrition status and fertilityvariables will assist in describing:

(1) The associations between various fertility patterns andthe nutritional parameters in a sample of rural CentralAmerican women and their children.

(2) Supplementary: To describe within the population studiedthe association of fertility variables of women andoccurrence of prenatal, infant, and child deaths.(Mortality, especially in children one to four years old,may be considered the ultimate effect of malnutrition.)

(3) Supplementary: To describe on an individual level, theassociation between fertility variables of women and thesocio-cultural levels of their households.

(4) Supplementary: To provide demographic descriptions of thesample of women in each of the Central American countriesstudied by the Institute of Nutrition of Central Americaand Panama.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Samuel M. Wishik, Division for Program Development& Evaluation, International Institute for the Study ofHuman Reproduction, Columbia University, New York,New York, and Dr. Miguel Guzmán, Statistics Division,Institute of Nutrition of Central America and Panama,Guatemala City, Guatemala

Funded by: Pan American Health Organization/Agency for InternationalDevelopment

1972 - continuingTimetable:

STANDARDIZING METHODOLOGY AND COMPUTER ANALYSISFOR ASSESSING NUTRITIONAL STATUS

PROBLEM

Nutritional deficiency diseases are still considered major publichealth problem. They are responsible for the high infant-mortalityrate and the high morbidity rate in preschool children. Knowledge ofthe nature, magnitude, cause, and potential remedial and preventivemeasures is still fragmentary.

The purpose is to define and provide for the PAHO countries astandardized system for conducting nutrition surveys; for recording andanalyzing the data in a uniform manner; providing a mechanism for acontinuing data bank and retrieval system; reducing and eliminatingduplication of computer survey data formats, codes, edits, and programsfor analysis. To identify and provide a mechanism to correlate nutri-tional health data with other sociologic, economic, environmental, anddisease factors indicative of high-risk groups.

METHOD

A working congress of 10 specialists, who received assistance fromthe Argentina Health Departments Data Processing Center, drafted preli-minary chapters on: (1) basic standardized and homogeneous specializeddata to be collected; (2) an outline of flexible indexing facilities anddata structure with linkage to other data information systems; (3) summa-rization of accepted guides for interpretation of anthropometric,biochemical, clinical, and dietary data; and (4) development of stan-dardized survey data collection forms adapted to the simplest to mostcomplex assessment procedures.

RESULTS TO DATE

Preliminary drafts of chapters drawn up by the working group arebeing circulated for additions and editing. A preliminary test of thedata survey collection format is scheduled to be tested in a small fieldstudy in Salta and Corrientes, Argentina, in the summer of 1972.

SIGNIFICANCE

The project will assist in developing a data retrieval system forsurveillance and evaluation of nutritional status throughout the Region,

35

for use within countries, and for comparison of problems and theirsolutions in the various countries. It will provide a continuous up-dated source of information on nutrition and health status.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Arnold E. Schaefer, Dr. Joginder G. Chopra,Health Services Department, Pan American HealthOrganization, Washington, D.C., and Dr. FranciscoMardoñes, Pan American Health Organization,Buenos Aires, Argentina

Funded by:

Timetable:

Pan American Health Organization

1971

36

ASSESSMENT OF TESTS OF BEHAVIOR FROM STUDIESOF NUTRITION IN THE WESTERN HEMISPHERE

PROBLEM

Over the last decade it has grown increasingly evident that anunderstanding of the relationship between malnutrition and intellectualdevelopment requires the clarification of at least three issues. First,we must identify behavior tests that are significant indicators ofcognitive development and that are applicable across a broad range ofcultures. Second, since it is doubtful that a physiologic stress indevelopment affects all aspects of cognitive growth equally, it isnecessary to identify measurements that are especially useful in esti-mating particular aspects of developmental competence and their relationto nutritional stresses. Third, since malnutrition is almost alwaysassociated with cultural disadvantage and with the general stress ofenvironmental and familial inadequacies that may in themselves affectcognitive growth, an assessment of the particular contribution thatmalnutrition is making to disturbances in cognitive development requiresan appropriate technique for separating nutritional from socio-culturalinfluences as well as a consideration of the interaction between the twosets of variables.

METHOD

Under the joint auspices of the U.S. National Institute of ChildHealth and Human Development and the Pan American Health Organization,a conference on the Assessment of Tests of Behavior from Studies ofNutrition in the Western Hemisphere was held in Puerto Rico from October20 to 23, 1970. Conference participants represented the disciplines ofpediatrics, nutrition, psychology, sociology, anthropology, and publichealth, and included relatively equal numbers of specialists from NorthAmerica and Latin America.

RESULTS TO DATE

The conference was divided into six sessions, the first five ofwhich were focused upon particular aspects of the problem. The finalsession summarized the meeting from an anthropological and biomedicalpoint of view. General group discussions dealt with the issues raisedand with the possible usefulness of the conference for improving thedesign of studies of malnutrition and cognitive growth. It was recognizedthat the conference was the opening stage of a cross-disciplinary dialoguethat needs to be continued.

37

SIGNIFICANCE

In public health practice we have made increasing use of physicalgrowth and development as an indicator of nutritional status and generalbiologic performance. Measurements of physical growth and developmentenable us to detect problems, define their causes, and apply remedialmeasures. To this end we have identified simple measurements to use ona wide scale and promoted their use by health services at all levels.

As we are now aware of the effect of malnutrition on mental develop-ment, we must obviously seek similar measurements in this field. Whilecomplex tests have been developed for use in specific studies and envi-ronments, we must not forget the need for simple tests that can be usedby local services under varying social and economic conditions. Onlywhen we have done so will it be possible to obtain reliable data on awide basis.

PUBLICATIONS

A report will be published in both Spanish and English during 1972.

OTHER DATA

Grantee: Dr. Roberto Rueda Williamson and Dr. JoginderG. Chopra, Nutrition Section, Health ServicesDepartment, Pan American Health Organization,Washington, D.C.

Funded by: Pan American Health Organization/U.S. NationalInstitute of Child Health and Human Development

Timetable: 1970 - continuing

38

HEMATOLOGIC INVESTIGATIONS IN THECENTRAL AMERICAN POPULATION

PROBLEM

Studies carried out during the Central American survey havedisclosed a high prevalence of subnormal hematologic values and ofserum biochemical indices that indicate iron and folate deficiency.From a highly selected population whose biochemical values reflectednormal iron, folate, and vitamin B1 2 nutrition, hematologic norms havebeen obtained for Central America and Panama. Furthermore, studiesin the general population and in pregnant women at term have indicateda negative correlation between the degree of iron deficiency and serumfolate levels.

During 1971 the prevalence of anemia, based on this norm, wasestablished. Therapeutic trials in selected populations given iron

or iron plus folate have also been conducted because it seemed importantto verify the hematologic norms and to explore the possibility that iron

administration alone could induce folate deficiency.

Tests to measure the physical fitness of subjects with different

degrees of anemia have been conducted before and at various intervalsduring iron and folate administration.

METHOD

The prevalence of anemia has been established by statistical

analysis of hemoglobin distribution curves in different age and sex

groups at altitudes between O and 750 m, 750 and 1,500 m, and 1,500 and2,800 m above sea level. Correlations between hemoglobin content and

biochemical indices of iron and folate nutrition are being completed.

The influence of hookworm infection on anemia has also been

investigated, using the quantitative estimation of hookworm load obtainedduring the Central American survey.

Therapeutic tests with iron and folate have been conducted in a

lowland population of Guatemala. The subjects were hookworm-infected

and had a high prevalence of anemia, iron deficiency, and low serum

folate levels. The population was divided into three matched samples,

and each subjected to the following treatments: Group I received an

iron-free tablet daily; Group II received 100 mg of iron as ferrous

sulfate per day in a similar tablet; Group III received 100 mg of iron

as ferrous sulfate, plus 2 mg of folate, per day in a similar tablet.

39

Blood samples from these three groups were obtained before andtwo, four, and six months after the start of treatment. Completehematologic values were obtained from these samples and serum iron,total iron binding capacity, serum folates, and red cell folates weredetermined. Physical fitness as measured by the Harvard Step Test wasdetermined in men from 18 to 44 years of age in the three groups.

RESULTS TO DATE

Based on previously established norms, the prevalence of anemia inthe Central American population is very high, averaging 19.7 per centfor the altitude group between sea level and 750 m, 12.6 per cent forthe group between 750 and 1,500 m, and 11.7 per cent for the group bet-ween 1,500 and 2,800 m. Particularly vulnerable groups are childrenbetween one and four years of age and adolescents of both sexes between13 and 20 years of age. The prevalence of anemia in men and women isapproximately the same (around 14 per cent), with a slightly greaterprevalence among the male population. The prevalence of anemia inpregnant women increases from the second trimester to term, reachingvalues of 22 per cent. Lactating women have a prevalence slightly higherthan that of nonpregnant women between 21 and 49 years of age.

Hookworm infection of even a moderate degree (egg counts between1,000 and 2,000/g of feces) aggravates the basic nutritional anemia pro-blem by raising its prevalence from 12 to 47 per cent in infected indivi-duals in the lowlands. The effects of hookworm infection decrease withaltitude and are more evident in the men than in women. The reasons forthese facts are being investigated.

In the therapeutic trials, the hematologic status of the group thatreceived no extra iron or folate did not significantly change over sixmonths. By contrast, persons receiving iron alone or iron plus folateachieved hematologic values within the established norms. This occurredindependently of hookworm load. The population receiving iron plus folatedisclosed a more normal distribution of values than persons receivingiron alone. A segment of the population receiving iron alone for sixmonths had lower hemoglobin concentrations than those receiving iron plusfolate.

It seemed therefore, that folate could be a limiting factor inerythropoiesis in a segment of the population. Furthermore, serum folatelevels and red cell folate levels did decrease in the group receivingonly iron when compared to the basal folate levels. This decrease infolates was also accompanied by a greater incidence of macrocytosis.

As to physical fitness, we have been able to demonstrate that ashemoglobin concentration increases so does physical fitness, significantdifferences being obtained between groups differing by 3.5 g of hemo-globin. Persons who attained normal hematologic values had normal

40

physical fitness. The effect of hematologic improvement was clearlyevident after only two months of iron therapy.

SIGNIFICANCE

The high prevalence of anemia in Central America and Panama pointsup a serious nutritional problem, which is aggravated by hookworm in-fection. It appears that in the Central American population folatedeficiency is a factor in nutritional anemia in certain groups. Further-more, folate deficiency can be aggravated by the administration of ironalone. The public health implications of these findings are beinginvestigated.

Finally, the validity of the hematologic norms established on thebasis of biochemical criteria has been confirmed by therapeutic trials.

PUBLICATIONS

1. Viteri, F.E., V. de Tuna, and M.A. Guzmán. Establishmentof normal haematological values in the Central Americanpopulation. Br J Haemat, in press.

2. Viteri, F.E., and M.A. Guzmán. Haematological status ofthe Central American population; prevalence of individualswith haemoglobin levels below "normal." Br J Haemat, inpress.

3. Viteri, F.E., and E. Cifuentes. Physical fitness in anemicGuatemalan agricultural laborers before and after irontherapy. J Appl Phys, in press.

4. Viteri, F.E., M.A. Guzmán, and L.J. Mata. Anemias nutri-cionales en Centro América; influencia de infección conuncinaria. Arch Latinoam Nutrición, in press.

OTHER DATA

Grantee: Dr. Fernando E. Viteri, Institute of Nutritionof Central America and Panama (INCAP), GuatemalaCity, Guatemala

Funded by: World Health Organization and other sources


PROTEIN REQUIREMENTS OF CHILDREN OF PRESCHOOL AGE

PROBLEM

Previous studies in our laboratories have shown that an intake of1.25 g/kg/day of egg protein or milk protein is sufficient to meet theneeds of children 2-3 years of chronologic age (1-2 years height/age).The present report covers further work related to protein requirementsof preschool children in which protein from corn and beans has beentested to determine the extent to which this mixture can satisfy children'sprotein requirements.

METHOD

Four normal children who had completely recovered from malnutritionwere given diets that provided all essential nutrients and calories inrequired amounts. The level of protein (corn protein 76%, bean protein24%) was varied from 1.00 to 2.25 grams of protein/kg/day. The levelswere 1.00, 1.25, 1.50, 1.75, 2.00, and 2.25, each level being maintainedfor two weeks. The children were followed clinically, anthropometrically,and biochemically. The following measurements were taken: body weight,body height, creatinine/height index, nitrogen balance, serum proteinelectrophoresis, blood urea, serum amino acids, and urinary urea.

RESULTS TO DATE

The data are still being analyzed but some preliminary results canbe reported. Measurement of the excretion of urea nitrogen, serumalbumin, and nitrogen balance indicate that children need about 1.40 g/kg/day of combined corn/bean protein to support normal growth and maintainbiochemical normality. The ratio of the requirement for egg protein(1.25) and the suggested requirements for this mixed vegetable protein(1.40) give a relative value of 89 per cent utilization. The childrentolerated the corn/bean diet very well at all levels.

SIGNIFICANCE

The results indicated that it is quite feasible to feed childrenof early preschool age diets based exclusively on mixtures of cerealand leguminous seed proteins if the caloric requirement is adequatelymet. In this research, the mixture (87 per cent corn, 13 per cent beans)was based on the customary dietary patterns of Guatemalan rural popula-tions. The mixture contains 9.6 per cent protein and its price is about

42

,i

one-half cent per pound. A child weighing 10 kg would need to consume10 x 1.40 - 14 g of protein at a cost of 1.6 cents per day.

The implications of these considerations are of great importance,not only biologically but also economically, since they indicate thatthe solution to the protein-calorie malnutrition problem could be basedon the rational utilization of local vegetable protein resources inadequate amounts. They also indicate that the main problem in under-developed areas may not be one of protein deficiency per se but rathera deficit in total amount of food or, in other words, caloric deficiency.

PUBLICATIONS

1. Arroyave G. Protein requirements of preschool children.I Asian Cong Nutrition, Hyderabad, India, January 28-February 2, 1971.

2. Lechtig, A., G. Arroyave, F. Viteri, and L. Mata.Influencia de la ingesta de proteinas sobre la concentraciónde inmunoglobulinas séricas en niños pre-escolares. ArchLatinoam Nutr 20:333, 1970.

OTHER DATA

Grantee: Dr. Guillermo Arroyave, Division of PhysiologicalChemistry, Institute of Nutrition of CentralAmerica and Panama, Guatemala City, Guatemala

Funded by: World Health Organization

Timetable: 1968 - open

43

FEEDING THE PRESCHOOL CHILD

PROBLEM

Protein-calorie malnutrition in the preschool child is beyond anypractical doubt one of the most important health problems in the develop-ing world and its solution one of the most difficult and complex. Thetragic consequences of poor feeding practices are too well known torecapitulate here. Feeding programs for preschool children are inoperation in several parts of the world and are being expanded to covermore regions and more children. It is therefore urgent that we pointup one method to utilize most effectively and economically scarce re-sources in personnel, money, and material.

METHOD

A Protein Advisory Group ad hoc working-group meeting was convenedto consider and make specific recommendations on the different aspectsof the problem, taking into account the biologic, socioeconomic, andcultural factors.

RESULTS TO DATE

Guidelines for planning, implementing, and evaluating programs forthe preschool child were prepared for wide circulation.

SIGNIFICANCE

Malnutrition is clearly a gigantic problem affecting millions ofchildren. The recommendations from this meeting should serve as toolsto refine and point up methods for obtaining reliable and comparativedata. They should also be useful in planning and implementing nutri-tional rehabilitation programs.

PUBLICATIONS

A report will be published during 1972.

44

OTHER DATA

Dr. Roberto Rueda Williamson and Dr. JoginderG. Chopra, Secretariat, Protein Advisory Group,Pan American Health Organization, Washington, D.C.

Funded by:

Timetable:

PAHO/Protein Advisory Group

1971 - continuing

45

Grantee:

MILK INTOLERANCE

PROBLEM

Ingestion of milk is known to be associated with untoward reactionsin some people. Such reactions occur mostly in the gastrointestinalsystem and lead to abdominal discomfort, cramps, and diarrhea. Milkintolerance may result from sensitivity to either milk protein or thelactose present in milk. Symptoms of milk allergy generally becomeapparent in very early infancy, and dramatic improvement occurs when milkis withheld. Cases of milk allergy have been reported from differentparts of the world but they have been isolated and so do not constitutea public health problem. There appear to be no racial differences inmilk allergy incidence.

On the other hand, there have been several reports in recent yearsabout lactose intolerance and deficiency of the intestinal enzyme lactase.Infantile milk intolerance due to congenital lactase deficiency is a wellrecognized entity. Lactose intolerance has also been reported to occurin various gastrointestinal disorders. But what concerns nutritionistsmost is the phenomenon of lactose intolerance in apparently healthysubjects. This disorder has been reported to be common among Africansand Asians. In contrast, its incidence is believed to be low in whiteAmericans and Australians. Lactose intolerance among Africans and Asianshas been attributed to an inherited lactase deficiency.

In view of these observations, the question has been raised whetherit is desirable to use milk as a source of supplementary protein forchildren in some of the developing countries of the world.

METHOD

An ad hoc working group was convened to review the availableliterature on the following subjects and make recommendations:

(1) Prevalence of milk intolerance: a critical reviewof published literature about the problem in(a) North America and Europe, (b) Africa and theMiddle East, (c) Asia and Oceania, and (d) LatinAmerica.

(2) Types of milk intolerance: distinguishing features;a review of diagnostic tests and their merits;screening tests for field application and experienceof their use; development of practical guidelinesfor future studies.

46

(3) Pathogenesis of milk (lactose) intolerance: currentconcepts; inconsistency between milk and lactoseintolerance, and between clinical symptoms andlaboratory tests; possibility of adaptation andreestablishment of tolerance.

(4) Practical implications of the use of milk and milkproducts in developing countries.

(5) Prevention of intolerance: value of low lactosepreparations, vegetable milk, etc.

RESULTS TO DATE

The ad hoc working group reviewed available information about milkintolerance and drew up practical proposals for further study to improveour knowledge of its significance.

SIGNIFICANCE

Most of the reports on lactose intolerance pertain to adult subjectsand not much information exists about its incidence in children. Sincepreschool children are the major beneficiaries of many nutrition programsthat utilize skim milk as a supplementary food, we obviously and urgentlyneed information about their tolerance to lactose. We must also deter-mine whether lactose intolerance necessarily implies milk intolerance.

PUBLICATIONS

A report will be published during 1972.

OTHER DATA

Grantee: Dr. Roberto Rueda Williamson and Dr. JoginderG. Chopra, Secretariat, Protein Advisory Group,Pan American Health Organization, Washington, D.C.

Funded by: PAHO/Protein Advisory Group


47

EFFECTS OF ORAL CONTRACEPTIVES ON LACTATION

PROBLEM

Oral contraceptives usually contain greater or lesser amounts ofestrogen. Lactation itself, even if successful, is only a partialprotection against future pregnancy, and certainly cannot be relied on.But estrogen is used (in much larger doses, of course) to suppresslactation What effects, if any, do oral estrogen-containing contra-ceptives have on lactation?

METHOD

A supply of questionnaires was sent to La Leche League Internationalof the United States and to the Nursing Mothers Association of Australia.Notice was given of the intended research in the newsletters of thesetwo organizations, and responding mothers who had breast-fed their childrenand taken oral contraceptives during lactation received a questionnaireand mailed it back direct to the Caribbean Food and Nutrition Institute.

RESULTS TO DATE

Both the dosage of estrogen and the postnatal time at which mothersstart taking estrogen-containing oral contraceptives are related to thedegree of interference with lactation.

SIGNIFICANCE

This inquiry seems to have produced prima facie evidence that thereis less interference with lactation (a) if low-estrogen tablets are taken,and (b) if these drugs are not started or restarted until three monthsafter the baby's birth.

PUBLICATIONS

1. Jelliffe, D.B., and E.F. Jelliffe. Lactation, conception, andnutrition of the nursing couple. J Pediatr, in press.

48

OTHER DATA

Grantee:

Funded by:

Timetable:


Rockefeller Foundation

1971

49

WORKSHOP TO PREPARE GUIDELINES FOR A FOOD ENRICHMENTAND FORTIFICATION NUTRITION PROGRAM

PROBLEM

Nutritional deficiency diseases have long attracted attention as apublic health problem in Latin America and the Caribbean. The PAHOTechnical Advisory Committee meetings in 1968 and 1970 expressed theopinion that such diseases continue to have a major priority for actionthrough national health services.

Following the identification and delineation of nutritional pro-blems in a country or region, there are a number of possible interventionactivities that may be implemented individually or in combination.

Food fortification or enrichment--the addition of nutrients tofoods--is but one of several approaches. Although this interventionmeasure is singled out for special attention in the meeting reported, itis recognized that it may not be the method of choice in a particularsituation and that even when it is selected for implementation, itsmaximum benefit will only be achieved when it is part of a coordinatedprogram involving several complementary-synergistic approaches.

METHOD

A technical group was convened to prepare guidelines that could bemade available to countries to assist them in planning, implementing,and evaluating fortification programs designed for the maintenance andimprovement of adequate standards on a national, regional, or local basis,or for special groups within a population. The present workshop wasdirected to concern itself with programs that might be implemented inLatin America and the Caribbean.

RESULTS TO DATE

The group drew up guidelines for the planning and development offood fortification programs.

SIGNIFICANCE

The guidelines were made available to countries to assist them inplanning, implementing, and evaluating fortification programs designedfor the maintenance and improvement of adequate nutritional standards on

a national, regional, or local basis, or for special groups within apopulation.

PUBLICATIONS

1. Food fortification in Latin America and the Caribbean, inpress.

OTEER DATA

Grantee: Dr. Roberto Rueda Williamson and Dr. Joginder G. Chopra,Nutrition Section, Health Services Department, PanAmerican Health Organization, Washington, D.C.

Funded by:

Timetable:


1971 - continuing

51

STUDIES OF THE PROTEIN QUALITY OF FOODS: CONVENTIONAL ANDPROTEIN-RICH FORMULAS, AND EFFECTS OF AMINO ACID AND

PROTEIN SUPPLEMENTATION ON STAPLE CEREALS

PROBLEM

Protein deficiency is one of the major nutritional problems ofpreschool children in developing countries, where staple cereals andother vegetables constitute the most important source of proteins. Thisis partly due to the generally low concentration of protein in cerealsand to the generally unbalanced amino acid composition of such proteins.

Several approaches can be conceived in trying to correct thissituation: (1) develop protein-rich formulas based on staple cereals towhich vegetable protein concentrates or small amounts of animal proteincan be added, by which both protein concentration and amino acid com-plementation of the staple cereal can be accomplished; (2) add specificlimiting amino acids to staple cereal proteins; (3) develop geneticallyimproved cereals which have both greater protein concentration and betteramino acid composition; and (4) extend animal protein sources that containa high concentration of essential amino acids through dilution of essen-tial nitrogen by the addition of varying amounts of unessential nitrogen.

These four approaches have been tested in preschool children whohave recovered fully from severe protein-calorie malnutrition.

The specific aims of this study have been to determine the tolerance,digestion, and nitrogen retention of preschool children of experimentaldiets in each of the above-mentioned categories.

METHOD

Preschool children, who by clinical and biochemical criteria haverecovered fully from protein-calorie malnutrition, have been fed experi-mental diets containing specific proteins as their only nitrogen sources.The following diets have been tested: (1) eleven protein-rich formulasbased on corn, wheat, and rice, to which leguminous seed, oil seed,andmilk protein have been added in various proportions; (2) fish proteinconcentrates; (3) Opaque 2 corn; (4) Black bean (Phaseolus vulgaris)protein and three black bean protein fractions obtained by differentialsolubility; (5) corn plus soy bean plus lysine; (6) milk to which variousproportions of unessential nitrogen have been added, with or withoutsmall amounts of methionine (limiting amino acid in milk); and (7) mixturesof corn plus bean plus milk.

52

The experimental design has consisted of two nitrogen balancesequences:

(1) An index phase, in which progressively decreasing amountsof the same protein source are fed sequentially over 10to 13 days. Nitrogen balance is measured at each proteinlevel after four days of adaptation on each level.

(2) An experimental phase, in which nitrogen balance has beenmeasured at an isonitrogenous level, changing the nitrogensource from a reference protein (usually milk) to the testproteins. The nitrogen intake level is close to the milknitrogen intake required to maintain a positive nitrogenbalance compatible with normal growth. All the childrenare fed adequate amounts of calories, minerals, and vitamins.

RESULTS TO DATE

(1) Protein-rich formulas: In general, all the proteins testedhave yielded satisfactory results, the nitrogen balance index in eachcase being slightly inferior to that of milk or egg protein.

(2) Fish protein concentrate: This protein resulted in a nitrogenbalance index equal or even slightly superior to those of milk and eggprotein.

(3) Opaque 2 corn: Nitrogen retention of this variety was clearlysuperior to common corn, approaching that of milk, except for greatervariability of results. Corn plus soy bean protein plus lysine resultedin better and more homogeneous results.

(4) Black bean protein: Digestibility was generally poor (40 to60 per cent that of milk). Among its fractions, water soluble proteinhad better absorption (80 per cent that of milk). Nitrogen retentionwas poor (30 to 50 per cent that of milk). The best results as toretained/absorbed nitrogen were obtained with the residual proteinfraction (insoluble in H O and 0.02 M NaOH), but the nitrogen absorptionof this fraction was often lower than the others.

(5) Milk plus nonessential nitrogen with or without methionine:Nitrogen retention decreased progressively as nonessential nitrogenaddition increased above 10 per cent of total milk nitrogen. Methionineaddition improved nitrogen retention only when the nonessential nitrogenadded amounted to more than 25 per cent of the total milk nitrogen.

(6) Corn plus bean plus milk mixtures: corn plus bean proteinmixtures were retained at levels close to 80 per cent those of milk whenfed at a total nitrogen intake that allowed adequate growth on milk.

53

Addition of nitrogen from milk, milk plus 10 and 25 per cent nonessentialnitrogen, nonessential nitrogen only, or nitrogen from the same corn andbean mixture resulted in similar increases in nitrogen retention.

SIGNIFICANCE

Protein-rich formulas, when carefully designed, can be of value asprotein sources. Fish protein concentrate prepared under careful super-vision is an excellent protein source. Opaque 2 corn protein and aminoacid-supplemented corn can be used to improve the protein nutrition ofchildren. On the other hand, addition of nonessential nitrogen to milkresults in decrements in nitrogen retention, and therefore has no practicalvalue. Corn and bean mixtures appear to constitute adequate proteinsources for preschool children. On the contrary, black bean protein alonedoes not, because of both poor absorption and low nitrogen retention. Itappears that nitrogen absorption from black beans is the main limitingfactor in their utilization.

PUBLICATIONS

1. Béhar, M., F. Viteri, and R. Bressani. Protein quality offish protein concentrate. Fed Proc 30:297, 1971.

2. Bressani, R., J. Alvarado, and F. Viteri. Evaluaci¿n enniños de la calidad de la proteína del maiz Opaco-2.Arch Lat Nutr 19:129-40, 1969.

3. Bressani, R., and F. Viteri. Metabolic studies in humansubjects. In Proc Third Internal Cong Food and TechnologySOS/70, Washington, August 1970.

4. Viteri, F.E., and J. Alvarado. Evaluaci¿n de la calidad deproteínas en humanos con énfasis en metodología. Libro deRecursos Proteinicos en la América Latina 1970, p. 53-77.

5. Viteri, F., and R. Bressani. The quality of new sources ofprotein and their suitability for weanlings and youngchildren. Bull Wld Hlth Org, in press.

6. Viteri, F.E., and R. Bressani. Evaluation of protein qualityof fish protein concentrate. I. Animal testing and nitrogenbalance index in children. Am J Clin Nutr, in press.

7. Viteri, F.E. Evaluation of protein quality of fish proteinconcentrate. II. Effectiveness in achieving recovery in

severely protein calorie malnourished children. Am J ClinNutr, in press.

8. Viteri, F.E., V. de Tuna, and J. Alvarado. Interrelation-ships of iron and folate nutrition. Br J Haemat, in press.

OTHER DATA

Grantee:

Funded by:

Timetable:

Drs. Ricardo Bressani, Moisés Béhar, and FernandoE. Viteri, Institute of Nutrition of Central Americaand Panama (INCAP), Guatemala City, Guatemala

National Institutes of Health/U.S. Public HealthService; World Health Organization; and Bureau ofCommercial Fisheries, U.S. Department of the Interior

1968-1972

55

DEVELOPMENT OF UNCONVENTIONAL PROTEIN SOURCESFOR HUMAN OR ANIMAL NUTRIMENT

PROBLEM

A marked scarcity of proteins to satisfy the biologic needs of thepopulation still exists in Central America and areas with similar ecologicconditions. Even though in many countries there have been increases inthe production of foods of animal origin, particularly beef, the increaseshave been assigned for economic reasons to exportation and the domesticavailability and consumption of such products by large sectors of thepopulation has been really diminishing. Even the availability and con-sumption of traditional vegetable protein sources such as beans has alsobeen decreasing. Because of this situation and independently of theefforts being made to correct these problems, exploring the possibilityof using available but not commonly used protein sources is imperative.INCAP's previous studies along these lines led to the development ofvegetable mixtures such as Incaparina. Ways to better use cotton seedare still being studied, while other sources of vegetable protein arebeing explored at the same time.

METHOD

Seeds from the rubber tree (Hevea brasiliensis), the calabash(Crescentia alata), and the annatto (Bixa orellana) have been studied.The first two seeds contain significant quantities of oil. For thatreason the nature and characteristics of their oil and proteins have beenstudied since only the rational use of both products would economicallyjustify the industrial exploitation of these seeds. The annatto seedis really a byproduct of the extraction of the dye bixina, which is widelyused industrially. If the seed as a protein source as well as bixinacould both be used, the economic exploitation of the annatto would bestimulated and at the same time another protein source for animal or humannutriment would exist.

RESULTS TO DATE

We have found that rubber seed oil is toxic and for that reason can-not be used for either animal or human nutriment, although it has semi-siccative properties that make it of interest to some industries, e.g.,painting. The seed's protein is of medium nutritive value, with a PER of1.9 compared to casein's 2.7. Calabash seed oil is fit for human con-sumption and the seed's protein has a moderate nutritive value similarto that of the rubber seed, which could be improved either through com-

56

binations with other proteins or with amino acid supplementation. Therethus exists a possibility of using this seed, which is widely availablein Central America, that will continue to be explored. The annatto seed'sprotein content is relatively low at about 15 per cent. Nevertheless itcontains appreciable quantities of carbohydrates and inasmuch as it isa bixina industry byproduct it could suitably be used to feed animals,thus replacing cereals that are now diverted from human needs. We willcontinue to explore this possibility.

SIGNIFICANCE

These studies are oriented toward a more rational utilization ofCentral America's natural resources in solving one of the area's mostserious nutritional problems, the scarcity of proteins for human as wellas animal nutriment. The results of this investigation may be of importancenot only in public health and nutrition but also in the Central Americancountries' economic development, inasmuch as certain protein sources--especially soya for animal feed--are now being imported.

PUBLICATIONS

1. Bressani, R. Efecto de la substitución del nitrógeno de laproteína de leche por nitrógeno de urea en terneros norumiantes. III Reuni¿n Soc Latinoam Producci¿n Animal,Bogotá, April 1971.

2. Bressani, R. Fuentes vegetales ricas en proteína paraconsumo humano en América Latina. II Cong Nac AsocTécnicos de Alimentos de México. Tecnologia de Alimentos(México) 6:29, 1971.

3. Bressani, R. Application of food science and technologyto the exploitation of new protein sources. III West HemNutr Cong, Bal Harbour, Fla., August-September 1971.

4. De la Fuente, G. Composici¿n química y valor proteínicode la torta de palma africana y corozo. II Reuni¿n CentroamTecnologia de Aceites, Grasas y Proteína, Guatemala, October1971.

5. Elias, L.G. Evaluaci¿n nutricional del aceite y de la tortade la semilla de hule. II Reuni¿n Centroam Tecnología deAceites, Grasas y Proteína, Guatemala, October 1971.

6. Gómez Brenes, R. Evaluación nutricional del aceite y dela torta de la semilla de jícaro o morro (Crescentia alata).

57

II Reunión Centroam Tecnologia de Aceites, Grasas y Proteina,Guatemala, October 1971.

7. Jarquin, R., J.E. Braham, J.M. González, and R. Bressani.Evaluación del valor nutritivo de subproductos del camarónen la alimentación de pollos. Turrialba, in press.

OTHER DATA

Grantee: Dr. Ricardo Bressani, Institute of Nutrition ofCentral America and Panama, Guatemala City, Guatemala

Funded by: Institute of Nutrition of Central America and Panama/National Institutes of Health


58

IMPROVEMENT OF THE NUTRITIVE QUALITYOF CEREAL-BASED DIETS

PROBLEM

Cereals constitute the principal sources of calories and provideabout two-thirds of the dietary proteins of the population of CentralAmerica. We recognize that this situation will long continue, particularlyin tropical countries, because cereals have a relatively high calorie yieldper unit of cultivated area, especially in view of the advances that haverecently been made in improving their yield.

The problem with diets of a high cereal content is that their proteinconcentration is generally low and their protein quality inadequate. Thisis particularly serious for small children. INCAP has identified in pre-vious studies the limiting amino acids of the proteins in cereals commonlyconsumed in Central America, and has demonstrated that these deficienciescan be corrected through the addition of either synthetic amino acids orproteins that provide the amino acids in which the cereals are deficient.Our studies now underway are oriented to exploring ways in which thesefindings can be put into practice within the framework of today's cerealprocessing and consumption habits in Central America.

METHOD

We have tried to determine the most effective formulas to improvethe protein quality of different cereals through the addition of proteinconcentrates with or without synthetic acids, and we are studying waysto put this type of enrichment into practice either in centralizedindustrial flour processing or under the conditions of cereal processingand consumption that obtain in rural communities.

RESULTS TO DATE

A corn formula has been developed that contains a protein concentrateof soya plus lysine, minerals, and vitamins. The addition of this con-centrate to corn flours results in improvement not only of proteinconcentration and quality but also of the general nutritive value of theproduct. Economic studies indicate that the supplementation is perfectlyfeasible and does not change the price of corn flours. We are nownegotiating with companies in Guatemala producing corn flours to use thisenrichment formula. Studies are also being started to determine thefeasibility of enriching corn with this formula in the small mills thatoperate in Guatemala's rural communities.

59

We have determined the economically most suitable way to enrichrice through synthetic amino acids and using synthetic rice grains. Thefeasibility of putting these findings into practice is still limitedbecause the cost of enrichment would be 10 per cent of the grain's cost,an increase difficult to absorb without changing the price of the grain.Work along these lines will be continued.

SIGNIFICANCE

As previously noted, cereals constitute a very important part ofthe diet of large sectors of the Central American population. It wouldbe very difficult for economic reasons and even in certain cases becauseof dietary habits to improve the diet through the addition of otherproducts. But if cereals could be improved without changing their retailprice or taste characteristics, a significant nutritional advance wouldbe achieved.

PUBLICATIONS

1. Bressani, R. La calidad proteinica del maíz Opaco-2 comoingrediente de dietas rurales de Guatemala. XVII Reuni¿n PCCMCA,Panamá, March 1971.

2. Bressani, R. El valor nutricional del arroz en comparación conel de otros cereales en la dieta humana de la América Latina.Seminario sobre políticas arroceras de la América Latina, CIAT,Cali, October 1971.

3. Bressani, R., and L.G. Ellas. La calidad proteínica del maízOpaco-2 como ingrediente de dietas rurales de Guatemala.Arch Lat Nutr, in press.

4. Ellas, L.G. Mejoramiento tecnológico de la calidad proteínicadel maíz. II Cong Nac Asoc Technicos de Alimentos de México,March 1971.

5. Ellas, L.G., and R. Bressani. Improvement of the protein qualityof corn-bean diets by the use of fortified corn or Opaque-2 corn.III West Hem Nutr Cong, Bal Harbour, Fla., August-September 1971.

6. Elias, L.G., and R. Bressani. Valor proteínico de los sub-productos de la industria del trigo. II. Complementación ysuplementación del granillo de trigo con concentrados proteicos.Arch Lat Nutr, in press.

60

7. G6mez Brenes, R. Valor proteínico del arroz. XVII ReuniónPCCMCA, Panamá, March 1971.

OTHER DATA


Funded by:

Timetable:

Institute of Nutrition of Central America andPanama/National Institutes of Health

1968-1972

PROCESSING OF BEANS TO PRODUCE AN INSTANT BEAN FLOUR

PROBLEM

The common bean (Phaseolus vulgaris) has been the traditionalprotein supplement in cereal diets based on corn or rice that are con-sumed by the majority of the Central American population. Because ofchanges in agricultural economics, however, this product is becoming evermore expensive and its consumption is therefore declining. Moreover,small rural producers have problems storing this grain. As a consequence,great fluctuations are observed in bean prices, from the relatively lowones of the harvest period to the periods of much scarcity and high pricesbefore the following harvest.

We have considered the possibility of processing beans to obtaina flour that once mixed with water would yield a product similar to thepaste that is the most common form in which the populations of Guatemalaand other areas consume this grain. This processing would also allowthe improvement of the beans' nutritive value through the addition ofmethionine, the limiting amino acid of beans and a product that alreadyexists in synthetic form at a very low price.

METHOD

We have investigated what would be the most favorable cookingprocess as to economy and the effect of processing on the nutritive valueof beans as well as ingredients causing adverse physiologic effects thatthey are known to contain. Different drying methods are also beingstudied to obtain a flour with desirable qualities of taste at a reason-able processing cost.

RESULTS TO DATE

We have determined that a soaking period of eight hours reducessteam-pressure cooking to 10 minutes, allows better retention of thebeans' nutritive value, and destroys their undesirable substances. Withthe product thus cooked, flours have been prepared through drying at lowtemperature. We have studied the taste characteristics of these flours.which appear to be favorable, and their storability in different typesof packaging and in different atmospheric conditions. Satisfactoryresults have been achieved with storage periods no longer than five monthsup to now. We believe this to be insufficient and are investigating waysto overcome the alteration the flours undergo which is chiefly due tothe rancidification of their fats. These studies will be continued.

62

SIGNIFICANCE

We hope through these studies to help resolve an economic andindustrial problem that is of great significance in animal nutritionin areas such as Central America where beans constitute a very importantsource of dietary protein in human nutriment.

PUBLICATIONS

1. Bressani, R., and L.G. Elias. Legume Foods, New York,Academic Press, in press.

2. Del Busto, J.A. Estabilidad química y nutricional duranteel almacenamiento de harinas precocidas de frijol. II ReuniónCentroam Tecnologia de Aceites, Grasas y Proteína, Guatemala,October 1971.

3. Ellas, L.G. Posibilidades en el mejoramiento proteínicodel frijol y su contribución a elevar el nivel nutricionalde la dieta Centro Americana. XVII Reuni¿n PCCMCA, Panamá,March 1971.

OTHER DATA

Grantee: Dr. Ricardo Bressani, Institute of Nutrition ofCentral America and Panama, (INCAP), GuatemalaCity, Guatemala

Funded by: Institute of Nutrition of Central America andPanama


63

CORN FORTIFICATION: A FIELD DEMONSTRATION MODEL

PROBLEM

The shortage and poor quality of food consumed in many developingareas will not be rapidly overcome by orthodox methods. Other measuresmust therefore be investigated, particularly those with a theoreticalpotential significance. Metabolic ward experiments in animals and humanshave shown that the addition of small amounts of the limiting amino acidsto foods such as corn results in a remarkable improvement in their quality.Such fortification has not been investigated extensively under fieldconditions however.

The purpose of the present research is to conduct a fortified cornfield trial in a whole rural community in which a significant amount ofinformation on growth, nutrition, and infection has been collected duringthe past eight years. Fortification will make the biologic value of corncomparable to that of meat. It is expected that if this measure iseffective, an improvement in fetal growth, postnatal growth, infant mor-tality and general health will be observed in the community within twoyears.

METHOD

The methods to be followed will be basically those used in thestudy of intestinal infection and colonization by microorganisms. Thesupplement consists of soya flour and synthetic lysine added to cornduring the milling process. Corn represents 70 per cent or more of theprotein and calorie intake of villagers. The supplementation will bemonitored by frequent chemical analyses of tortillas. Fetal growth willbe measured by determining time of gestation and birth weight. Morbiditywill be determined by periodic clinical examinations. Postnatal growthwill be measured anthropometrically.

RESULTS TO DATE

The study has just begun and results are not yet available. Thesupplement has been accepted by the community because it is similar tocorn in color, flavor, and texture.

64

SIGNIFICANCE

If corn fortification works at the community level, its introductionin a whole region or even nationally might dramatically change the nutri-tional status and growth pattern of the population. The supplement itselfdoes not cost significantly more than corn.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Leonardo J. Mata, Division of Microbiology,Institute of Nutrition of Central America and Panama,Guatemala City, Guatemala

Funded by:

Timetable:

U.S. Agency for International Development (USAID)

1971-1976

65

CONTROL OF HYPOVITAMINOSIS A (FORTIFICATION OFSUGAR WITH RETINOL PALMITATE)

PROBLEM

The importance of adequate vitamin A nutrition is without question,especially when it is realized that there are millions of people in vastareas of the developing world who are suffering from moderate to severevitamin A deficiency.

The Central American countries and Panama are no exception. Theseverity of this nutritional situation was revealed during the CentralAmerican nutrition surveys which demonstrated that about 1,500,000children from O to 15 years of age have low or deficient serum retinollevels that could be improved by higher intakes of vitamin A. Studies ofdietary adequacy also revealed that around 70 per cent of the familiessurveyed were consuming less than 50 per cent of the recommended dietaryallowance for this essential nutrient. Clinical studies have documentedthe existence of numerous cases of blindness due to keratomalacia,especially among children who are also suffering from protein-caloriemalnutrition.

In view of these considerations a project was initiated to fortifya suitable dietary vehicle with vitamin A and test it to see if thisneeded nutrient could be included in the diet of the vast majority of thepopulation.

METHOD

After careful consideration of the characteristics that a suitablevehicle should have, table sugar was selected. Sugar is almost universallyconsumed by the population and its consumption per capita varies littlefrom person to person and from day to day. A water-dispersable preparationof retinol palmitate (250 SD of Hoffmann-LaRoche) containing 250,000 I.U.per gram was selected as the fortifying material. At the concentrationneeded it does not affect the organoleptic characteristics of the sugar,as tested in different types of taste and acceptability trials.

Cost considerations also indicate that the project is economicallyfeasible for nationwide applications. Fortification would probably costone-tenth of a cent per pound of sugar. Another estimate indicates thatit would cost slightly over three cents to provide sufficient vitamin Afor a whole year to each member of the population.

66

RESULTS TO DATE

A pilot project was completed at El Salto, S.A., one of Guatemala'slargest sugar refineries, where equipment for the addition of retinolpalmitate to sugar was tested on a large industrial scale. Stabilitytrials have been conducted that indicate the mixture has adequate keepingqualities. The potency of the fortified sugar is maintained when it isboiled for 15 minutes with coffee, Incaparina, or similar beverages.Trials in animals and humans have demonstrated its biologic effectiveness.

The project has been proposed to the Governments of Guatemala andEl Salvador. It seems that no major problems will be met in the nationalimplementation of this public health measure. Based on experience fromthe salt iodization program in Central America, the two following stepsare now being taken: (1) study of the financial aspects of the process,with the participation of the sugar manufacturing sector, the Governments,and INCAP; and (2) elaboration of a proposed law to regulate the process.

SIGNIFICANCE

Food sources of preformed vitamin A (the most effective form) arescarce and very expensive, and consequently the majority of the populationdoes not consume them in sufficient amounts. Green leaves and yellowvegetables, the carotene or provitamin A-containing foods, are moreavailable and less expensive, but in actual practice their consumption isalso slight, perhaps largely for cultural reasons. Furthermore, carotenesyield much less vitamin A than retinol.

To alter the present pattern of vitamin A production and consumptionthrough socioeconomic, agricultural, and educational development--thelogical permanent solution--would take many years, but the hypovitaminosisA problem is so severe that emergency measures are needed. The incorpora-tion of an appropriate amount of retinol in the diet of Central Americanpopulations using a convenient vehicle is indicated. The fortificationmethod has been proved effective in many instances with other nutrients.Iodization of salt is perhaps the best example; in Guatemala it reducedthe prevalence of endemic goiter from 38 to 5 per cent in nine years.

PUBLICATIONS

1. Arroyave, G. Control of hypovitaminosis A (Fortification ofsugar with retinol palmitate). Progress Report No. 1,Institute of Nutrition of Central America and Panama, 1970.

2. Arroyave, G. Control de hipovitaminosis A (Fortificaciónde azúcar con palmitato de retinol). Informe de ProgresoNo. 2, Instituto de Nutrici6n de Centro América y Panamá, 1971.

67

3. Arroyave, G. Distribution of vitamin A to population groups.III. West Hem Nutr Cong, Bal Harbour, Fla., August 30 -September 2, 1971.

4. Arroyave, G., 0.G. Retana, and J.R. Aguilar. Control de ladeficiencia de vitamina A en Guatemala. XXII Cong Nac Med,Guatemala, November 1971.

5. Brenes-Brenes, E., G. Arroyave, and C. Tejada. Fortificaciónde azúcar con vitamina A y su impacto sobre la nutrici¿n delniño lactante. XXII Cong Nac Med, Guatemala, November 1971.

OTHER DATA

Grantee: Dr. Guillermo Arroyave, Division of PhysiologicalChemistry, Institute of Nutrition of Central Americaand Panama, Guatemala City, Guatemala

Funded by: Hoffmann-LaRoche and Co., Basle, Switzerland


68

UTILIZATION OF COFFEE BYPRODUCTS

PROBLEM

In processing coffee two subproducts are obtained, pulp and skins.Dry pulp has a chemical composition similar to that of cereals, as aresult of which the possibility of using it for animal feed has beenconsidered. The skins, on the other hand, are a product with a highcellulose content that could also be used as a bulk material for feedingcellulose-using ruminants.

Previous studies at INCAP and other laboratories have neverthessshown that coffee pulp contains products that are toxic or adverselyaffect animals physiologically. The purpose of this investigation is todetermine if there are processes easily applied at field level thatwould allow the elimination or neutralization of coffee pulp's toxicproducts so that it could be used in feeding animals, and also to deter-mine to what degree coffee skins could be used as ruminant food.

METHOD

Two possible ways to use coffee pulp as animal feed have beenconsidered, the first that of processing it so as to destroy harmfulingredients and the second that of extracting its proteins so they wouldbe nontoxic. The effect of ensilage on the pulp has been studied totest the first possibility, and protein extraction through the use ofsuitable enzymes and inorganic solvents is being studied to test thesecond possibility.

RESULTS TO DATE

Unfortunately, it has been demonstrated that the ensilage processincreases rather than reduces the adverse effect. Ruminants fed dietscontaining 30 per cent fermented pulp (ensilage) grow less than thosefed diets of 30 per cent dehydrated pulp. Animals fed 30 per centensilaged pulp also have higher fatty acid blood levels than controlsor those fed 30 per cent dehydrated pulp. This effect is similar to thatobserved with insulin, epinephrine, or caffeine.

The use of enzymes and inorganic solvents to extract proteins fromcoffee pulp has allowed the extraction of about 70 per cent of the pro-tein. Chemical analysis of the protein indicates that in essentialamino acids it is comparable to protein of good biologic value, whichmeans that the process would be of great use if a practical extractionmethod is achieved.

69

It has been demonstrated that coffee bean skins can form up to30 per cent of the bulk material in the diet of ruminants. Pelletizationof the feed improves the utilization of the skins still more. Contactshave already been established with the animal feed industry so that itmay consider applying this finding.

SIGNIFICANCE

If methods can be established for the suitable use of coffee sub-products in animal feed, great economic advantages would accrue forcoffee-producing countries that have enormous quantities of the subproductsand, at the same time, such use would spur the development of the live-stock industry and thus result in more efficient and cheaper production offood from that source.

PUBLICATIONS

1. Bressani, R. Pulpa de café: composición quimica. III ReuniónAsoc Latinoam Producción Animal, Bogotá, April 1971.

2. Bressani, R. Utilizaci¿n de pulpa de cafe: valor nutritivoen monogástricos. III Reuni¿n Asoc Latinoam ProducciónAnimal, Bogotá, April 1971.

3. Bressani, R., E. Estrada, and R. Jarquin. Pulpa y pergaminode café. I. Composición quimica y contenido de aminoácidosde la pulpa de café. Turrialba, in press.

4. Bressani, R., L. Urrutia, E. Estrada, and R. Jarquin.El efecto de la pulpa de café deshidratada en la dieta deratas y pollos. Arch Lat Nutr, in press.

5. Jarquin, R. Utilización de pulpa de café: alimentación derumiantes. III Reuni¿n Asoc Latinoam Producci¿n Animal,Bogotá, April 1971.

6. Jarquin, R., J.M. Gonzáles, J.E. Braham, and R. Bressani.Pulpa y pergamino de café. II. Utilizaci¿n de pulpa de caféen rumiantes. Turrialba, in press.

OTHER DATA


70

Funded by:

Timetable:

Institute of Nutrition of Central Americaand Panama (INCAP), Guatemala City, Guatemala

1970-1974

71

NATIONAL FOOD AND NUTRITION SURVEY OF GUYANA

PROBLEM

Before this survey was made some qualitative data were available

about the food and nutrition situation in the Republic of Guyana, but

there were so many gaps in the information that no basis existed forformulating a National Food and Nutrition Policy.

METHOD

The survey, conducted by the Government of Guyana, with technical

assistance from the Caribbean Food and Nutrition Institute (CFNI), PAHO/

WHO, and FAO, took place from March through June 1971. Under the super-vision of technical staff from the Government and CFNI, 70 locally

assigned survey workers were trained. A stratified random sample ofcensus districts was taken from urban and rural coastal areas. Subsequentcomparison between characteristics of the sample population and those of

the general population as recorded in the 1970 census demonstrated that

the sample was representative of the whole. Over 5,000 persons were

examined clinically and anthropometrically, and food consumption and

sociocultural and socioeconomic studies were carried out on just under

1,000 families. Biochemical, hematologic, and parasitologic investiga-

tions were made in subsamples as were investigations into food productionfor home use or sale and individual food consumption. A less complete

survey was also made of Amerindians in the hinterland. CFNI processed

and tabulated the resulting data.

RESULTS TO DATE

Results were not available as of December 31, 1971, but were ready

to present to the Government of Guyana at an interdisciplinary seminar in

March 1972.

SIGNIFICANCE

Many of the recommendations that will probably form the basis of

Guyana's National Food and Nutrition Policy were made at the March 1972

meeting. These will be refined further at a similar meeting later in

1972.

72

PUBLICATIONS

A report on the National Food and Nutrition Survey of Guyana isin preparation and will probably be published in 1973.

OTHER DATA

Grantee: Caribbean Food and Nutrition Institute,Kingston, Jamaica

Funded by:

Timetable:

Rockefeller Foundation and the Government ofGuyana

1971-1972

FOOD CONSUMPTION SURVEY OF TRINIDAD AND TOBAGO

PROBLEM

The formulation of a National Food and Nutrition Policy forTrinidad and Tobago was hampered by the lack of any food consumptiondata from later than the ICNND survey of August 1961. The Government

of Trinidad and Tobago therefore requested the assistance of theCaribbean Food and Nutrition Institute (CFNI) in carrying out a new

survey.

METHOD

Approximately 1 per cent of all households in Trinidad and Tobago

were chosen by systematic random sampling. Of 1,122 households selected,42 could not be traced, and 30 would not cooperate. The remaining 1,050households were each surveyed by Inventory or Food Accounts techniques

for five consecutive days. The survey was carried out from February toMay 1970 by 15 supervisors and 25 field assistants, all trained at CFNI

and under the overall direction of CFNI's Trinidad Centre.

RESULTS TO DATE

The average household size was 5.5 persons. The mean annual percapita income ranged from T.T.$1,276 (T.T.$2 = U.S.$1) in householdswith one or two members, of which T.T.$485 was spent on food (38 percent of income), to T.T.$396, of which T.T.$230 was spent on food(58 per cent). For the whole sample of 1,050 households, averageintake over requirements was over 100 per cent for vitamin C (333 percent), vitamin A (retinol) (198 per cent), calcium (154 per cent),thiamine (146 per cent), iron (127 per cent), protein (127 per cent),calories (119 per cent), niacin (111 per cent), and riboflavin (108 percent). Because of uneven distribution of income, 39.1 per cent of thehouseholds did not meet their calorie requirements, 30.6 per cent didnot meet their protein requirements, 33.5 per cent did not meet theiriron requirements, and 29.2 per cent did not meet their vitamin Arequirements. Cereals and grains accounted for 39.5 per cent of calorieintake and 34.7 per cent of protein intake. Animal proteins accountedfor 44.1 per cent of protein intake.

74

SIGNIFICANCE

Food patterns, food costs, and failure to meet nutrient require-ments have been identified by household size, income group, geographicareas, and ethnic origin. The Cabinet of Trinidad and Tobago hasdirected that the National Nutrition Council and CFNI organize a seminarin October 1972 to produce recommendations based on this survey's results.

PUBLICATIONS

The Government Printers, Trinidad and Tobago, are to print 1,800copies of the final report.

OTHER DATA

Grantee:

Funded by:

Timetable:


Research Corporation of New York and theGovernment of Trinidad

1970-1972

75

EFFECT OF EARLY UNDERNUTRITION ON CENTRALNERVOUS SYSTEM DEVELOPMENT

PROBLEM

The objectives of this study are to investigate the effects ofearly prenatal and postnatal undernutrition on the cellular growthpatterns and behavioral development of young rats and children. All theanimal work was carried out in the Division of Growth and Developmentof the Department of Pediatrics, Cornell University Medical College,and more recently at the Institute of Human Nutrition, Columbia UniversityCollege of Physicians and Surgeons. All the human data were collected atthe hospital Roberto del Rio, Santiago, Chile.

METHOD AND RESULTS TO DATE

A. Animal Studies. As a result of these investigations, thefollowing conclusions may be drawn:

1. Postnatal malnutrition produced either by feeding the nursingdam a low protein diet or by increasing the number of suckling pups perdam results in a reduced rate of cell division and a curtailment of DNAsynthesis. It also results in an increase in the rate of RNA synthesisaccompanied by an even greater increase in the rate of RNA degradation,which result in a net decrease in cellular RNA content and a reductionin the net rate of protein synthesis.

2. Activity of DNA polymerase reflects the rate of cell divisionduring normal growth of whole brain or of cerebellum.

3. Neonatal malnutrition reduces the activity of this enzyme inboth brain and liver.

4. In liver, hypophysectomy also reduces enzyme activity andgrowth hormone restores this activity.

5. Activity of alkaline RNase (pH 7.8) increases per cell normallyduring normal growth but at a rate similar to the increase in totalcellular RNA, thus keeping a constant ratio between enzyme activity andthe amount of its substrate per cell.

6. Neonatal malnutrition elevates the activity of this enzyme inwhole brain and in various brain regions.

7. Ligating the uterine artery causes an elevation of RNaseactivity in placenta within 24 hours and in fetal brain within 48 hours.

76

These changes disappear in fetal brain by 72 hours but remain elevatedin placenta even at 96 hours.

8. Maternal protein restriction results in a reduced DNA polymeraseactivity in placenta and in an elevated placental RNase activity.

We have concluded from these animal studies that malnutrition affectscellular growth of the brain by increasing RNA turnover, which reducesthe capacity for protein synthesis and the rate of DNA synthesis. Theformer is reflected by an elevation in activity of alkaline RNase and thelatter by a decrease in activity of DNA polymerase.

In addition to the studies outlined, investigations have recentlybeen undertaken to explore the influence of the interrelationship ofearly malnutrition and environmental deprivation or enrichment on thebiochemical development of the brain and the subsequent behavioral changesof the animal. Preliminary data would indicate that there is a synergisticaction between environmental deprivation and early malnutrition, both intheir biochemical and behavioral consequences. Studies in this area arebeing continued.

B. Human Studies. During the past few years we have been able toshow that:

1. Cell division normally ceases in human whole brain and cerebrum,cerebellum, and brain stem between 12 and 18 months of age.

2. Children who died of severe marasmus had a reduced number ofcells in the brain.

3. In addition, such children had a reduced brain total lipidcontent, cholesterol content, and phospholipid content.

4. Placentas of mothers who were severely malnourished had reducedDNA, RNA, and protein content and elevated alkaline RNase activity.

5. Children surviving marasmus during their first six months oflife showed perceptual and cognitive difficulties, even after as manyas five years of subsequent rehabilitation.

We have concluded from these studies that early undernutritionproduces effects on the human brain similar to those demonstrated in therat.

C. Biochemical Markers of Severe Undernutrition

Studies have been under way during the past 18 months to see ifthe enzymatic changes in tissues, induced by malnutrition, are reflectedin body fluids. Specifically, alkaline RNase has been studied. Data

77

indicate that the activity of this enzyme in serum normally increasesbetween birth and two months of age. Undernutrition markedly elevatesthis activity, and nutritional rehabilitation rapidly returns activityto normal. Enzyme activity is also present in urine and amnioticfluid, and changes with nutritional status are being explored. We havenot yet examined the activity of this enzyme under moderate or mildundernutrition, but we plan to do this shortly in the hope of developinga possible screening test for adequacy of nutritional status.

SIGNIFICANCE

These studies were undertaken to better understand the sequence ofnormal cellular growth and the consequences of early undernutrition onthat growth. In addition, the precise time at which undernutritionproduces its severest and most prolonged effects is being sought. Finally,practical methods for screening procedures to quantitate nutritionalstatus are under investigation. All this work is thus designed to answerthe questions: What does severe undernutrition do to the brain? Howdoes this come about? How can we better diagnose undernutrition, andhow can we best monitor the effects of therapy? Finally, we would hopethat this added knowledge, coupled with social and economic realities,will move governments to develop more rational nutrition policies.

PUBLICATIONS

1. Brasel, J.A., et al. DNA polymerase activity in rat brainduring ontogeny. Dev Biol 23:424-32, 1970.

2. Rosso, P., et al. Changes in brain weight, cholesterol,phospholipid and DNA content in marasmic children. Am JClin Nutr 23:1275-79, 1970.

3. Velasco, E., et al. Cellular growth of placenta and fetusduring prenatal undernutrition. In P. White and N. Selvey(eds.), Proceedings of III Western Hemisphere NutritionCongress, 1971, Mt. Kisco, N.Y., Futura Publishing Co., 1972,PP. 336-40.

4. Winick, M. Nutrition growth and mental development: biologicalcorrelations. Am J Dis Child 120:416-18, 1970.

5. Winick, M., et al. Cellular growth of cerebrum, cerebellum,and brain stem in normal and marasmic children. Exp Neurol26:393-400, 1970.

78

6. Winick, M. and P. Rosso. Malnutrition and cellular growthin the brain In J. Masek, K. Osancova and D. P. Cuthbertson(eds.), Proceedings of the Eighth International Congress ofNutrition, Prague, 1969. Amsterdam, Excerpta Medica, 1970,

PP. 531-38.

7. Winick, M., et al.control mechanismsXIII InternationalVienna, Verlag derPP. 53-58.

The effect of undernutrition on certainof cell division. In Proceedings of theCongress of Pediatrics, Vienna, 1971.Wiener Medizinischen Akadamie, 1971,

8. Winick, M., et al. Malnutrition and cellular growth in thebrain: Existence of critical periods. In Proceedings of theCiba and Nestle Foundations' Symposium on Lipids and Develop-ing Brain, 1971. Amsterdam, Associated Scientific Publishers,in press.

OTHER DATA

Grantee: Dr. Myron Winick, Cornell University Medical College,New York, New York

Funded by:

Timetable:

National Institutes of Health

1969-1972

79

GOITER AND GOITROGENIC FACTORS IN INDIANSOF WESTERN ARGENTINA

PROBLEM

Goiter is endemic in certain isolated Indian tribes of WesternArgentina. There is some indication that both genetic and dietary factorsmay contribute to the disease. The present investigation is concerned withdefining its epidemiology and its causes. Attention will also be given tothe use of iodinated oil as a preventive measure for thyroid disease inthese population groups.

METHOD

Surveys of the population, especially among the Mapuche Indian groupsof Chiquillihuin and Aucapán, have defined the extent of the goiter ende-mia. The subjects have been divided into three groups: one receivesiodinated oil by mouth, a second by injection, and a third serves as con-trol. Goitrogenic activity of the pine nut, a common dietary item, isbeing studied in laboratory animals.

RESULTS TO DATE

Surveys to date indicate a sharp decrease in the size of goiters inthe groups of subjects who received iodinated oil, either injected ororally, but an increase in the detectable nodular goiter rate. Only onecase of jodbasedow was found among the 200 patients treated.

SIGNIFICANCE

These investigations should lead to a definition of the cause orcauses of endemic goiter in the Indian isolates of Western Argentina andprovide some information regarding the feasibility and safety of iodinatedoil as well as its effectiveness under the conditions in question.

PUBLICATIONS

1. Degrossi, O.J., N. Altschuler, H. Forcher, A.A. Zaninovich,O.M. Mutchinick, and C.L. Enriori. Characteristics ofendemic goiter in a Mapuche Indian tribe in Chiquillihuin,El Malleo, Province of Neuquén, Argentine Republic.

80

I. General aspects and some functional and genetic studies.In J. B. Stanbury (ed.), Endemic Goiter, Washington, D.C.,Pan American Health Organization, 1969, p. 149. (PAHOScientific Publ. No. 193)

2. Degrossi, O.J., T. Watanabe, N. Altschuler, V. Pecorini,and C. Santillán. Characteristics of endemic goiter in aMapuche Indian tribe in Chiquillihuin, El Malleo, Provinceof Neuquén, Argentine Republic. II. Iodine kinetic studies.In J. B. Stanbury (ed.), Endemic Goiter, Washington, D.C.,Pan American Health Organization, 1969, p. 159. (PAHOScientific Publ. No. 193)

OTHER DATA

Grantee: Dr. Noah Altschuler and Dr. Osvaldo Degrossi,Facultad de Medicina, Comisi6n Nacional de EnergiaAt6mica, Buenos Aires, Argentina

Funded by: Argentine Atomic Energy Commission


81

ENDEMIC GOITER AND THE PATHOPHYSIOLOGYOF THYROID DISEASE IN CHILE

PROBLEM

We showed through metabolic studies of endemic goiter in the geneticisolate of Pedregoso, an Indian reservation in Chile, a possible ineffi-ciency of the compensatory mechanisms expressed by a considerable "iodineleak" and through radiochromatographic studies considerable amounts oftriiodothyronine in the serum of nine of 24 subjects. We then decided tostudy specifically the role of triiodothyronine in another endemic goiterarea and in iodine-deficient rats.

METHOD

A number of field studies have been under way since 1969 in Peumo,an endemic goiter area two hours from Santiago. We have performed iodinekinetic studies and made serum determinations of PB1, T4, T3, and 24-hour urinary iodine excretion in 64 subjects. Six subjects were studiedbefore and after six months of iodine treatment.

To assess the T3 role of T3 in a laboratory model, the secretionrates of T3 and T4 were measured simultaneously in chronically iodine-de£icient rats.

RESULTS TO DATE

The average T3 serum concentration in seven normal subjects ofSantiago, with an urinary iodide excretion averaging 39.8 + 11.7 pg/24 hwas 0.29 + 0.04 pg/%. In 79 patients from Peumo the T3 serum concentrationwas 0.39 + 0.02 Pg% and they have an average urinary iodide excretion of41.9t4.1 Pg/24 h. There is no statistically significant differencebetween T3 concentration in Santiago and Peumo. The T4 disposal rate,calculated in six subjects whose T3 peripheral metabolism we have studiedsimultaneously, is 40.1 Pg/day,which is half the Robbins and Rall figureof 80.5 Wg of thyroxine per day. In these same patients the T3 degra-dation rate is 62.2 PE/day, which is slightly less than double the figureobtained by the calculations we made, based on Oddie and Fischer andBellabarba's 33 pg/day. The six subjects treated with iodine changedtheir T4 degradation rate from 49.2 to 102.9 pg/day and their T3 degra-dation rate from 91.7 to 46.6 pg/day. The T3 serum concentrationdropped from 0.52 pg% to 0.29 ug%, and the T4 serum concentration rosefrom 5.3 to 10.9 Pg%.

82

Chronic iodine deficiency in rats decreased the T4 disposal rate(equal to secretion rates in steady state) but did not modify T3disposal rate. As clearance rates did not change in this experiment, theflux of both hormones became a function only of the plasma concentration.

On the basis of a decrease found in fecal excretion of T4, inhigher proportion than could be expected by the fall in plasma concen-tration, it is postulated that the liver could modify the fecal loss ofT4. Although the total clearance did not vary with iodine deficiency,the hepatic clearance would be lower, thus permitting a constant supplyof T4 to the tissues in spite of a diminished secretion rate.

SIGNIFICANCE

The activity of T3 in endemic goiter is probably an importantmechanism by which individuals adapt themselves to iodine deficiency.This research has helped us clarify the pathophysiology of endemic goiter.

PUBLICATIONS

1. Pineda, G., E. Silva, L. Penoni, J. Hidalgo, and C. Stevenson.Cambios cualitativos de la secreción tiroidea inducidos porla carencia de yodo. Actas, III Cong Bolivariano Endocrinol,Caracas, October 1971.

2. Pineda, G., L. Madariaga, and C. Jara. Determinaci¿n cuanti-tativa de la triiodotironina en el suero humano. Rev MedChile, in press.

3. Silva, E., and C. Stevenson. Efecto combinado del frio y lacarencia de yodo en la distribución de aminoácidos yodadosen el tiroides de la rata. Rev Med Chile 98:776, 1970.

4. Silva, E. Disposal rates of thyroxine and triiodothyronine iniodine deficient rats. Endocrinology, in press.

5. Silva E., G. Pineda, and C. Stevenson. Plasma inorganic iodine(Pll); thyroid clearance and goiter in a goitrous endemia.Acta Endocrinol Panam, in press.

6. Silva, E. Análisis te¿rico de los cambios cualitativos de lasecreción tiroidea en la carencia de yodo. Rev Med Chile,in press.

83

7. Stevenson, C., G. Pineda, and E. Silva. Role of triiodo-thyronine in endemic goiter. Proc, VII Panam Cong Endocrinol,1970, p. 142

8. Stevenson, C. Investigaci¿n en bocio endémico. Rev Med Chile98:787, 1970.

9. Stevenson, C. Bocio endémico. In J. Meneghello (ed.),Pediatria, Buenos Aires, Intermedica, in press.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Carlos Stevenson, Hospital Salvador, Escuelade Medicina, Universidad de Chile, Santiago, Chile

Partially supported by The Chilean NationalCouncil for Scientific and Technological Research

1969-1973

84

PATHOPHYSIOLOGY OF ENDEMIC GOITERIN THE CAUCA VALLEY OF COLOMBIA

PROBLEM

The aim of this project is to define more precisely the epidemiologyof endemic goiter in the Cauca Valley of Colombia, to explore the possibleetiologic factors, and to study the pathophysiology of the thyroid glandin patients from this area.

METHOD

Straightforward surveys are conducted, and an attempt is made tocorrelate the data collected with such factors as diet and water--itshardness, source, and bacteriology. Samples of water are analyzed by gasliquid chromatography and mass spectroscopy.

RESULTS TO DATE

Well water known to be goitrogenic in experimental animals has beendistilled under low pressure and found to render a goitrogenic fractionactive in an in vitro system using slices of calf thyroid. The activefraction appears to be a low molecular weight compound that containssulfur. The material is heterogeneous. Attempts are now being made toidentify the chemical nature of the active fraction. These studies arebeing done in cooperation with the chemistry department of the MassachusettsInstitute of Technology.

In an epidemiologic study of T supplementation of goitrous childrenin the Cauca Valley, no effect was olserved over 10 weeks of treatment.

Epidemiologic studies in two villages of the Cauca Valley haveindicated that a change in the water supply has been responsible for arise in the incidence of goiter. The goiter rate has also been shown toincrease in association with progressively lower economic status. Goiterhas not been correlated with TBG, TBPA, TT4, TSH, RBC-T3, or thyroidantibodies.

SIGNIFICANCE

The evidence to date indicates that goiter in the Cauca Valley of

Colombia has a different origin than simple iodine deficiency. The

significance of the project rests in the ability to define the nature of

the epidemiologic factor.

85

PUBLICATIONS

1. Gaitán, E., H.W. Wahner, and P. Correa. Studies on endemicgoiter in the Valley of Rio Cauca, Colombia, S.A. ExcerptaMedica Internat Cong Series 20:33, 1965.

2. Gaitán, E., H.W. Wahner, and P. Correa. Studies on theendemic goiter of the Cauca River Valley, Colombia, S.A. InRevue Européene d'Endocrinologie, Oxford, Pergamon Press,1967, vol. 3, p. 291.

3. Gaitán, E., D.P. Island, and G.W. Liddle. Identification ofa naturally occurring goitrogen in water. Trans Ass AmerPhysicians 82:141, 1969.

4. Gaitán, E., and H.W. Wahner. Studies on the pathogenesis ofendemic goiter in the Caugca Valley, Colombia, South America.In J.B. Stanbury (ed.), Endemic Goiter, Washington, D.C., PanAmerican Health Organization, 1969, p. 267 (PAHO ScientificPubl. No. 193)

5. Gaitán, E., H.W. Wahner, P. Correa, R. Bernal, W. Jubiz,J.E. Gaitán, and G. Llanos. Endemic goiter in the Cauca Valley.I. Results and limitations of twelve years of iodine prophylaxis.J Clin Endocrinol 28:1730, 1968.

6. Gaitán, E., H.W. Wahner, C. Cuello, P. Correa, W. Jubiz, andJ.E. Gaitán. Endemic goiter in the Cauca Valley. II. Studiesof thyroid pathophysiology. J Clin Endocrinol 29:675, 1969.

7. Gaitan, E. Water-borne goitrogens and their role in theetiology of endemic goiter. In G.W. Bourne (ed.), WorldReview of Nutrition and Dietetics, Basel, S. Karger, 1972,vol. 14.

8. Gaitán, E., R. MacLennan, D.P. Island, and G.W. Liddle.Identification of water-borne goitrogens in the Cauca Valleyof Colombia. In D.P. Hemphill (ed.), Trace Substances inEnvironmental Health, Proc, V Annual Conf, Columbia, Missouri,University of Missouri, 1972.

9. MacLennan. R., E. Gaitán, and M.C. Miller. Observer variationin grading and measuring the thyroid in epidemiological surveys.In J.B. Stanbury (ed.), Endemic Goiter, Washington, D.C., PanAmerican Health Organization, 1969, p. 67. (PAHO ScientificPubl. No. 193)

86

10. Wahner, H.W., E. Gaitán, and P. Correa. EndemischerKropf in Cali, Kolumbien, Sudamerika, MunchenerMedizinischewochenschrift 107:1513, 1965.

11. Wahner, H.W., E. Gaitán, and P. Correa. Studies of iodinemetabolism in endemic nodular goiter. J Clin Endocrinol26:279, 1966.

12. Wahner, H.W., C. Cuello, P. Correa, L.F. Uribe, and E. Gaitan.Thyroid carcinoma in an endemic goiter area, Cali, Colombia.Am J Med 40:58, 1966.

13. Wahner, H.W., and E. Gaitán. Funci¿n tiroidea en adolescentesque habitan la región de bocio endémico del valle del Cauca,Colombia, Sudamérica. Rev Soc Colomb Endocrinol 6:69, 1969.

14. Wahner, H.W., and E. Gaitán. Thyroid function in adolescentsfrom the goiter endemic of the Cauca valley, Colombia, SouthAmerica. In J.B. Stanbury (ed.), Endemic Goiter, Washington,D.C., Pan American Health Organization, 1969, p. 291 (PAHOScientific Publ. No. 193)

15. Wahner, H.W., E. Gaitán, and R. MacLennan. Proteinas fijadorasde tiroxina (globulina y prealbúmina) en nifos de la regiónde bocio endémico del valle del Cauca. Rev Soc Colomb Endocrinol

7:85, 1970.

16. Wahner, H.W., and E. Gaitan. Metabolismo periférico de laL-tiroxina en sujetos de la zona endémica de bocio del valledel Cauca. Rev Soc Colomb Endocrinol 8:5, 1970.

17. Wahner, H.W., W.E. Mayberry, E. Gaitán, and J.E. Gaitán.Endemic goiter in the Cauca Valley. III. Role of serum TSHin goitrogenesis. J Clin Endocrinol 32:491, 1971.

OTHER DATA

Grantee: Dr. Eduardo Gaitán, Facultad de Medicina, Universidaddel Valle, Cali, Colombia

Funded by: U.S. Public Health Service


87

PATHOPHYSIOLOGY OF ENDEMIC GOITER AND CRETINISMIN ECUADOR AND THEIR PREVENTION WITH IODINATED OIL

PROBLEM

The purpose of our work is to study the incidence of goiter andrelated disturbances such as cretinism, short stature, mental retardation,mutism, and deafness with respect to genetic and environmental factors,with major emphasis on pathophysiologic changes in endemic cretinism. Aprophylaxis program has been started to test the effects of iodinated oilon endemic goiter, and in particular on the cretinism, mental deficiency,mutism, or deafness that often coexist with it.

METHOD

A complete health inventory of several villages in rural Ecuadorhas been made. Data on many aspects of the health of the people in thesecommunities and on their family background have been tabulated. Fromthese communities a number of classical cretins have been brought to Quitofor study of their bone development, cardiopulmonary function, iodinemetabolism, sensory perception, and psychologic attainments.

Two of the studied villages were chosen for the prophylactic program.Just before iodized oil administration in these two villages, nutritional,epidemiologic and anthropometric surveys were made. X-ray bone age deter-minations also were made in groups of selected preadolescents in each ofthe villages. All the persons living in one of the villages were injectedwith ethiodol and iodinated oil; the other village remained as the con-trol.

Careful follow-up of all newborns born in the iodized populationand in the untreated population was conducted chronologically. Thestudy was longitudinal in that each child's neuromotor development,dentition, and somatic growth were studied during several periods of time.Every six months before injection in both communities surveys on goiterincidence and prevalence were conducted.

RESULTS TO DATE

The results are summarized in several papers that have derived fromthis study. In general, there has been a sharp reduction in the incidenceof goiter in the injected population, and virtually no goiter in injectedchildren or in children born to injected mothers during the five yearssince the program began in the spring of 1966. Three cases of thyro-toxicosis have developed in the injected group and have been treated.

88

To date, seven cases of major global neuromotor developmentalretardation have appeared among the children born in the untreatedvillage. Severely deficient infants were treated with thyroid but itwould seem that any defects that developed during fetal life cannotbe reversed by thyroid medication. In addition, in the untreatedpopulation there was a significantly higher percentage of childrenwho presented partial developmental retardation, especially in motorand social-longuistic functions. Furthermore, the mental capabilitiesof those children who received iodine treatment early in fetal life weresignificantly higher than those of children born to untreated mothers.

SIGNIFICANCE

This study should establish once and for all the efficacy ofiodine in preventing major global neuromotor developmental retardation(endemic cretinism). It should also demonstrate the specific role ofchronic iodine deficiency in other conditions associated with endemicgoiter such as short stature, mental retardation, and defects relatedto hearing and speech.

PUBLICATIONS

1. Andrade, V.H., G. Acosta, A. Bustamante, and R. Fierro-Benítez.La cirugía tiroidea en el hospital "Eugenio Espejo" (la cirugíatiroidea en la regi¿n andina). Actas, V Cong Med Nac, Quito,1961.

2. Arcos, L., and R. Fierro-Benítez. Bocio intratorácico. RevEcuat Enf Torax 2:67-72, 1961.

3. Chiriboga, C., and R. Fierro-Benítez. Técnicas cromatográficaspara el estudio de algunas afecciones tiroideas. Rev Ecuat MedCienc Biol 1:56-60, 1963.

4. Dodge, P.R., I. Ramirez, and R. Fierro-Benitez. Neurologicalaspects of endemic cretinism. In J.B. Stanbury (ed.), EndemicGoiter, Washington, D.C., Pan American Health Organization,1969, p. 373. (PAHO Scientific Publication 193)

5. Estrella, E., R. Fierro-Benitez, I. Ramirez, A. G¿mez, C. Hermida,C. Jaramillo, J. Urresta, and J. Súarez. Estudio longitudinaldel desarrollo neuro-motor en niños campesinos andinos de ceroa cuatro años de edad. II Cong Nac Neuropsiquiatria Infantil,Madrid, 1971, p. 29.

89

6. Fierro-Benitez, R. El bocio endemico en la región andina.Rev Asoc Esc Politec Nac (Quito) 1:21-34, 1961.

7. Fierro-Benitez, R. Estudio fisiopatol¿gico de la tireopatlasmediante el yodo-131. Actas, V Cong Med Nac, Quito, 1961.

8. Fierro-Benitez, R. La funci¿n tiroidea en el bocio endémico dela Provincia de Pichincha. Quito, Casa Cultura Ecuatoriana, 1961.

9. Fierro-Benitez, R. Main adaptive mechanisms to chronic iodinedeficiency. Proc, VII Pan Amer Pediat Cong, Quito, 1963.

10. Fierro-Benitez, R. La profilaxis del bocio enémico; conoci-mientos actuales. Actas, Jornadas Anuales Asoc Española Endocr,Zaragoza, October 1969.

11. Fierro-Benítez, R. La dentici¿n hasta los cuatro años de edaddel niño campesino serrano. I Cong Odontológico Regional Andino,Quito, 1970, p. 31.

12. Fierro-Benítez, R. Yodo y desarrollo esquelético y dentario.III Cong Bolivariano Endocrinologia, Caracas, 1971, p. 22.

13. Fierro-Benítez, R. Iodide deficiency and thyroid function in"neurological" cretinism. In J.B. Stanbury (ed.), EndemicCretinism, Kroc Foundation, 1972, in press.

14. Fierro-Benitez, R., and L.H. Correa. Endemic goiter in theEcuadorean Pichinca Province. Proc, V Pan Amer Cong Endocr,Lima, 1961.

15. Fierro-Benitez, R., L. DeGroot, and F. Guerrero. Endemic goiterin the Ecuadorean Andean region. Rev Iber Endocr, in press.

16. Fierro-Benitez, R., E. Estrella, I. Ramirez, and A. Gomez.Correlaci6n entre la captación tiroidea de I-131, la excresiónurinaria de yodo y la producción hormonal observada durantecuatro años luego de la administración intramuscular de aceiteyodado, en un programa de profilaxis del bocio endémico.VIII Reuni¿n Luso-Española Endocrinología, Porto, 1970, p. 22.

17. Fierro-Benitez, R., E. Estrella, I. Ramirez, A. Gomez, C. Jara-millo, J. Urresta, and C. Diaz. Profilaxis del bocio endémico,y defectos asociados con la endemia, por medio de la adminis-traci¿n intramuscular de aceite yodado. Simposios, II CongresoBolivariano Endocrinologia, Lima, 1969, p. 45.

90

18. Fierro-Benltez, R., E. Estrella, I. Ramirez, A. G¿mez,C. Jaramillo, and J. Urresta. Resultados de la correcciónde la carencia crónica de yodo por medio de la administra-ción intramuscular de aceite yodado. VIII Reunión Luso-Española Endocrinología, Porto, 1970, p. 21.

19. Fierro-Benitez, R., and I. Ramirez. Utilida le la prueba dela captación de triyodotironina marcada con I por parte deresina en el diagnóstico de Yod-Basedow producido por laadministración intramuscular de aceite yodado. Proc, SimposioMétodos Radioisot¿picos Aplicados "in vitro" en Medicina ClinicaInvestigaci¿n, Vienna, September 1969.

20. Fierro-Benítez, R., I. Ramirez, E. Estrella, A. Gómez, C. Hermida,C. Jaramillo, J. Urresta, and J. Súarez. Prevención del creti-nismo y otros defectos asociados al bocio endémico medianteaceite yodado. Rev Ecuator Med Cienc Biol 8:99-115, 1970.

21. Fierro-Benitez, R., I. Ramirez, E. Estrella, A. G¿mez, C. Hermida,C. Jaramillo, J. Urresta, J. Súarez, and F. Moncayo. Results ofoil prophylaxis in Ecuador. In J.B. Stanbury (ed.), EndemicCretinism, Kroc Foundation, in press.

22. Fierro-Benítez, R., I. Ramirez, E. Estrella, C. Jaramillo, C.Diaz, and J. Urresta. Iodized oil in the prevention of endemicgoiter and associated defects in the Andean region of Ecuador.I. Program design, effects on goiter prevalence, thyroid function,and iodine excretion. In J.B. Stanbury (ed.), Endemic Goiter,Washington, D.C., Pan American Health Organization, 1969, p. 306.(PAHO Scientific Publication No. 193)

23. Fierro-Benítez, R., I. Ramirez, E. Estrella, A. Querido, andJ. B. Stanbury. The effect of goiter prophylaxis with iodizedoil on the prevention of endemic cretinism. VI Internat ThyroidConf, Vienna, 1970, p. 7.

24. Fierro-Benítez, R., I. Ramirez, and J. Garces. Osteocondrodis-plasias de las extremidades inferiores en el cretinismo endémico.VIII Cong Latinoam Ortopedia y Traumatologia, Quito, 1971, p. 63.

25. Fierro-Benítez, R., I. Ramírez, and J. Garcés. The clinicalpattern of cretinism as seen in highland Ecuador. In J.B.Stanbury (ed.), Endemic Cretinism, Kroc Foundation, in press.

26. Fierro-Benítez, R., and J.B. Stanbury. The effectiveness ofiodized oil in goiter prevention in rural Ecuador. Proc, IIWestern Hemisphere Nutrition Congress, Chicago, American MedicalAssociation, 1969, p. 58.

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27. Fierro-Benitez, R., J.B. Stanbury, A. Querido, L.J. DeGroot,R. Alban, and J. Córdova. Endemic cretinism in the Andeanregion of Ecuador. J Clin Endocr 30:228-36, 1970.

28. Harrison, J., R. Fierro-Benitez. I. Ramirez, S. Refetoff, andJ. B. Stanbury. Immunoreactive growth hormone in endemic cretinsin Ecuador. Lancet 936-40. 1968.

29. Harrison, M.R., R. Fierro-Benítez, I. Ramirez, S. Refetoff, andJ. B. Stanbury. Growth hormone in relation to endemic cretinismand dwarfism. In J.B. Stanbury (ed.), Endemic Goiter, Washington,D.C., Pan American Health Organization, 1969, p. 381. (PAHOScientific Publication No. 193)

30. Hermida, C., R. Fierro-Benitez, I. Ramirez. E. Estrella, A. Gómez,C. Jaramillo, J. Urresta and J. Súarez. Diagn¿stico precoz ytratamiento con tiroides del cretinismo endémico. III CongBolivariano Endocrinologia, Caracas, 1971, p. 45.

31. Israel, H., R. Fierro-Benítez, and J. Garcés. Skeletal anddental development in the endemic goiter and cretinism areas ofEcuador.

32. Jaramillo, C., R. Fierro-Benltez, I. Ramirez, E. Estrella,A. Gómez, C. Hermida, J. Urresta, and J. Súarez. Yodo ydesarrollo del lenguaje. III Cong Bolivariano Endocrinologia,Caracas, 1971, p. 46.

33. Kevany, J., R. Fierro-Benltez, E.A. Pretell, and J.B. Stanbury.Prophylaxis and treatment of endemic goiter with iodized oilin rural Ecuador and Peru. Amer J Clin Nutr 22:1597-1607, 1969.

34. Mouriz, J., R. Fierro-Benltez, and J.B. Stanbury. Iodine contentand ratio of S19 to S27 thyroglobulin in endemic goiter fromEcuador. Z Ges Exp Med 152:1-7, 1970.

35. Ramirez, I., R. Fierro-Benitez, E. Estrella, C. Jaramillo,C Díaz, and J. Urresta. Iodized oil in the prevention ofendemic goiter and associated defects in the Andean region ofEcuador. II. Effects on neuromotor development and somaticgrowth in children before two years. In J.B. Stanbury (ed.),Endemic Goiter, Washington, D.C., Pan American Health Organiza-tion, 1969, p. 341. (PAHO Scientific Publication No. 193)

36. Ramírez, I., R. Fierro-Benítez, F. Trowbridge J. Suarez.E. Estrella, A. G¿mez, C. Hermida, C. Jaramillo, and J. Urresta.

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Efecto de la corrección de la carencia cr¿nica de yodosobre la inteligencia. III Cong Bolivariano Endocrinologia,Caracas, 1971, p. 47.

37. Stanbury, J.B., R. Fierro-Benitez, E. Estrella, P.S. Milutinovic,M.U. Tellez, and S. Refetoff. Endemic goiter with hypothyroi-dism in three generations. J Clin Endocr 29:1596, 1969.

38. Vickery, A.L., R. Fierro-Benitez, and B. Kakulas. Skeletalmuscle morphology in endemic cretinism. Amer J Pathol, in press.

OTHER DATA

Grantee: Dr. Rodrigo Fierro-Benitez, National PolytechnicSchool, Quito, Ecuador

Funded by:

Timetable:


1969-1974

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PATHOPHYSIOLOGY, PREVENTION, AND TREATMENT OF ENDEMICGOITER IN ANDEAN PERU

PROBLEM

This project is designed to explore the metabolism of iodine insubjects with endemic goiter, the relationships existing between fetus

and mother during gestation, and the effects of prophylactic administra-tion of iodized oil on gland structure and function.

METHOD

Three villages in Andean Peru near Tarma are being studied. With

suitable controls, members of the population have been given iodized oil.Careful follow-up of these subjects constitutes the source of information.Particular emphasis is placed on pregnant women, who are followed care-fully until delivery. Blood samples taken during pregnancy and deliverylater provide information regarding fetal-maternal relationships. Fre-quent surveys give information on the effects of the iodized oil on goitersize and function.

RESULTS TO DATE

There is no question regarding the efficacy of iodized oil in

preventing the appearance of goiter in the young and in causing regressionof established goiters. One patient who developed thyrotoxicosis follow-ing administration of iodized oil has been studied in some detail. Nonew cretins have appeared among the population receiving the iodized oil.It has been shown that absence of the normal rise in plasma thyroidhormone levels may be a feature of iodide deficiency and this may contri-bute to the appearance of cretinism.

SIGNIFICANCE

Results of this study add to accumulating information that iodizedoil is an effective, safe, and acceptable means of preventing and treating

endemic goiter. The frequency of side effects is acceptably low.

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PUBLICATIONS

1. Pretell, E.A., F. Moncloa, R. Salinas, A. Kawano, R. Guerra-Garcia, L. Gutiérrez, L. Beteta, J. Pretell, and M. Wan.Prophylaxis and treatment of endemic goiter in Peru withiodized oil. J Clin Endocrinol 29:1586, 1969.

2. Pretell, E.A., M. Wan, and P. Palacios. Fetal thyroidfunction in endemic goiter. Sixth Int Thyroid Conf, Vienna,1970.

3. Pretell, E.A., and J.B. Stanbury. Effect of chronic iodinedeficiency on maternal and fetal thyroid hormone synthesis.In B.S. Hetzel and P.O.D. Pharoah (eds.), Endemic Cretinism,Monograph Series No. 2, Surrey Beatty, N.S.W., 1971, p. 117.

4. Pretell, E.A. Fetal-maternal relationships in iodized oilprophylaxis program. Proceedings of a meeting on endemiccretinism. The Kroc Foundation for Medical Research,Santa Ynez, California, January 1972. Plenum Press, inpress.

OTHER DATA

Grantee: Dr. Eduardo Pretell, Departamento de Endocrinologia,Instituto de Investigaciones de la Altura, Lima,Peru

Funded by: U.S. Public Health Service and the Government ofPeru


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BIOCHEMICAL CONSTITUENTS OF ENDEMIC GOITERGLANDS BEFORE AND ONE YEAR AFTER

IODIZED OIL INJECTION

PROBLEM

We have reported elsewhere that endemic goiters have a larger amountof particulate iodoprotein as well as a more active protein synthesis,as judged by higher RNA/DNA ratios. Furthermore, there is chromatographicevidence that these glands have less thyroglobulin and more light iodo-proteins compared with normal specimens. The purpose of this researchis to define whether these biochemical abnormalities could be reversedafter a constant supply of iodine as given in an injection of iodized oil.

METHOD

Endemic goiter specimens are obtained by biopsy from Vatients withlarge, multinodular goiters and prelabeled with 12SI and 13 I. A detailedstudy is made of iodoproteins in each specimen. Total RNA and DNA-phosphorus are also measured. Ten months to one year after an injectionof iodized oil another specimen from the same gland is studied and theresults compared with those obtained initially. The microscopic appear-ance of both specimens is also carefully compared.

RESULTS TO DATE

At the present time only four patients have completed the study.Results to date indicate a striking reversal of the abnormalities foundin the pretreatment glands as well as definite structural changes in themicroscopic appearance of the glands subjected to a constant supply ofiodine.

SIGNIFICANCE

This investigation promises to give further support to the generalopinion that iodized oil is useful in treating endemic goiter as well asthat many of the biochemical abnormalities found in an endemic hyper-plastic gland are mainly due to lack of iodine. This may lead to anadaptative process with glandular hyperplasia and biochemical changesthat are reversed to normal with a constant supply of iodine.

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PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Geraldo A. Medeiros-Neto, Hospital dasClinicas, University of Sao Paulo MedicalSchool

Research fund of the Hospital das Clinicas

1971-1972

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ENDEMIC GOITER AND CRETINISM IN BOLIVIA

PROBLEM

Endemic goiter is a major public health problem in Bolivia. Theentire country is iodine-deficient and the goiter incidence has beenestimated at 40 to 50 per cent among the general population. In manyisolated communities virtually everyone has a goiter. There is con-siderable variation in the incidence of goiter and cretinism amongdifferent ethnic groups.

This investigation has the following objectives: (1) to ascertainthe true incidence of goiter in various parts of Bolivia; (2) to elucidatethe causes of endemic goiter and cretinism; (3) to clarify the role ofgenetic and ethnic factors.

METHOD

Surveys have been conducted among 10,420 adults and children inthe Department of Chuquisaca. The incidence of goiter and cretinism, andurinary iodine levels are being determined. We are carrying out specialstudies of thyroid function, including iodine uptake and clearance,perchlorate discharge, plasma inorganic iodine, and absolute iodine uptake.

RESULTS TO DATE

The incidence of goiter ranged from 25 to 88 per cent in the villagesstudied. The radioiodine uptake ranged from 54 per cent in normals to65 per cent in goitrous subjects. Guarani Indians showed a higher incidence(81 per cent) than Quechua Indians (53 per cent). In preliminary surveysthe incidence of cretinism among Guaranis appeared much lower (1 per cent)than among Quechuas (5 to 10 per cent), despite the higher incidence ofgoiter. In one Quechua village 12 per cent of the entire population werecretins.

Studies in progress include further surveys and more intensiveclinical and laboratory evaluation to clarify these differences in inci-dence of goiter and cretinism among Quechuas and Guaranís.

SIGNIFICANCE

This study will give detailed information on the prevalence andseverity of goiter and cretinism in a large area of Bolivia. Factors thatmay influence the development of goiter and cretinism will be assessed.

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These will include genetic, dietary, and cultural features in additionto iodine deficiency. From this knowledge, a realistic approach toprevention of endemic goiter and cretinism in Bolivia can be planned.

PUBLICATIONS

1. Pardo, Antonio, et al. Bocio endemico en Ayango. Arch BolivMed 19:43-50, 1968.

2. Pardo, Antonio, et al. Resultados de lucha contra el bocioendemica: programa villa serrano. Arch Boliv Med 19:51-58,1968.

3. Pardo, Antonio, et al. Incidencia del bocio endemico en elDepte. de Chuquisaca. V Congreso Medio-Quirurgica Boliviano-Peruano, July 1970.

OTHER DATA

Grantee: Dr. Antonio Pardo S. University of San Fransiscoand Santa Barbara University Hospital, Sucre,Bolivia

Funded by:

Timetable:

Universidad de San Fransisco

1968 - continuing

99

CHRONIC MANGANESE POISONING: A METABOLIC DISORDER

PROBLEM

It is planned to elucidate the mechanisms whereby chronic industrialinhalation of manganese ores induces a schizophrenia-like syndrome,followed by either Parkinsonism or a Wilson's disease-like syndrome.

A close linkage to the brain metabolism of Dopa-Dopamine was found.Therefore the relationships of manganese to the metabolism of brain aro-matic biogenic amines is being investigated.

METHOD

Turnover of manganese in normal subjects and manganese-poisonedindividuals was measured, as was the absorption of radio manganese afterinhalation and ingestion. Simultaneous determinations of intestinalabsorption of radio iron in both normal subjects and patients with chronicmanganese poisoning were also made.

Patients were transported a distance of approximately 400 milesfrom the mining district to the Catholic University Hospital in Santiago,Chile, where they were hospitalized. In addition to an extensive routinechemical and laboratory work-up, detailed neurologic and psychiatricevaluations were conducted on these patients. Attention was given toneutron activation analysis for natural manganese in body fluids, and cellsamples were sent from Chile to the Brookhaven National Laboratory in theUnited States.

Levodopa was administered orally to manganic patients for periods upto 4 years. Healthy miners have received levodopa for periods of 30 days.Several biochemical maneuvers have yielded insight as to the mode of actionof levodopa; dopa decarboxylation inhibitors, nicotinamide, and fusaricacid have been tested. Furthermore in vivo decarboxylation rates of aminoacids have been measured, and finally evaluation of blood brain barrierto manganese in relation to the susceptibility to manganese poisoning issubject to experimental work.

RESULTS TO DATE

The preliminary results of this project with Chilean miners havebeen reported in the literature. Long-term treatment of chronic manganesepoisoning with levodopa has demonstrated for periods of up to four years,a satisfactory control of hypokinetic and rigid patients. There is alsosatisfactory control of dystonic manganic patients with, however, frequent

100

losses of therapeutic effect due to insomnia, physical or psychical

stress, and intercurrent infections. The chief difference from thetreatment in Parkinson's disease has been the absence of induction ofinvoluntary movements. Stability of treatment of dystonic patientshas been enhanced by the adjunct use of levodopa and a dopamine betahydroxylase inhibitor, fusaric acid.

No significant side-effects have been observed in these patients.In vivo measurements of decarboxylation rates of amino acids suggest a

different metabolic fate of levo tyrosine in normals than in manganicand parkinsonian patients. Susceptibility is studied as a function of

parameters influencing the blood brain barrier. An increased uptakeof manganese has been shown in the brains of anemic rats, while it wasdemonstrated previously that anemia also increases the intestinal

absorption of this metal.

SIGNIFICANCE

The present findings viewed together with data on animals suggestthe following conclusions: (1) normal working miners must be livingwith a significant tissue burden of manganese; (2) since patients with

chronic manganese poisoning lose their manganese burdens after removalfrom the mines but do not lose the symptoms and signs of chronicmanganese poisoning, chelation therapy is not indicated in these cases;(3) the relationship between chronic manganese poisoning and the metabo-lism of aromatic biogenic amines is established. An important differencein the treatment of Parkinsonian patients with levodopa has emerged,namely, the absence of induction of involuntary movements in manganicpatients. This complication is an important and frequent side-effectin Parkinsonians. It is expected that further investigations alongthese lines will provide useful information; (4) factors of susceptibilityto chronic manganese poisoning appear to be determined by the following

sequence: (a) inhalation of ore dust; (b) swallowing of the inhaledmaterial; and (c) intestinal absorption of manganese as a linear function

of absorption of iron.

PUBLICATIONS

1. Bertinchamps, A.J., S.T. Miller, and G.C. Cotzias. Inter-dependence of routes excreting manganese. Amer J Physiol211:217-24, 1966.

2. Borg, D.C., and G.C. Cotzias. Incorporation of manganese intoerythrocytes as evidence for a manganese porphyrin in man.Nature 182:1677-78, 1958.

101

3. Britton, A.A., and G.C. Cotzias. Dependence of manganeseturnover upon intake. Amer J Physiol 211:203-06, 1966.

4. Cotzias, G.C. Manganese. In C. L. Comer and F. Bronner (eds.),Mineral Metabolism: an Advanced Treatise, v. 2b. New York,Academic Press, 1962, p. 403.

5. Cotzias, G.C., and A.C. Foradori. Trade metal metabolism.In E.E. Bittar (ed.), The Metabolical Basis of Medicine.New York, Academic Press, v. 1, 1968, p. 105-21.

6. Cotzias, G.C., K. Horicuhi, S. Fuenzalida, and I. Mena. ChronicManganese Poisoning; clearance of tissue manganese concentra-tions with persistence of the neurological picture. Neurology18:376-82, 1968.

7. Cotzias, G.C., S.T. Miller, and J. Edwards. Neutron activationanalysis: the stability of manganese concentration in humanblood and plasma. J Lab Clin Med 67:836-49, 1966.

8. Cotzias, G.C., and P.S. Papavasiliou. Therapeutic studies ofparkinsonian patients: long-term effects of DL and L-Dopa.Reprinted Progress in Neuro-Genetics, v. I, p. 357-65.(Excerpta Medica International Congress Series No. 175, 1967).

9. Cotzias, G.C., P.S. Papavasiliou, R. Gellene, and R.B. Aronson.Parkinsonism and Dopa. Trans Ass Amer Physicians 81:171-83, 1968.

10. Cotzias, G.C., P.S. Papavasiliou, E.A. Hughes, Z. Tang, andD.C. Borg. Slow turnover of manganese in active rheumatoidarthritis accelerated by prednisone. J Clin Invest 47:992-1001,1968.

11. Mena, I., J. Court, S. Fuenzalida, P.S. Papavasiliou, G.C.Cotzias. Chronic manganism, a metabolic disorder of the extra-pyramidal system, treatment with L-Dopa. In Proceedings 16thInternational Congress on Occupational Health. Tokyo, 1969,p. 799-801.

12. Cotzias, G.C., M.H. Van Woert, and L.M. Schiffer. Aromaticamino acids and modification of Parkinsonism. New Eng J Med276-374-79, 1967.

13. Foradori, A.C., A.J. Bertinchamps, J.B. Guliben, and G.C. Cotzias.The discrimination between magnesium and manganese by serumproteins. J Gen Physiol 50:2255-66, 1967.

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14. Fuenzalida, S., and I. Mena. Intoxicación crónica pormanganeso y sus relaciones con las enfermedades del sistemaextrapiramidal. Rev Med Chile 38:667, 1967.

15. Hughes, E.R., S.T. Miller, and G.C. Cotzias. Tissue concen-trations of manganese and adrenal function. Amer J Physiol211:207-10, 1966.

16. Mena, I., S. Fuenzalida, K. Horiuchi, and G.C. Cotzias.Estudios metabólicos en manganismo crónico. Rev Med Chile95:510-17, 1968.

17. Mena, I., O. Marin, S. Fuenzalida, and G.C. Cotzias. Chronicmanganese poisoning; clinical picture and manganese turnover.Neurology 17:128-36, 1967.

18. Mena, I., A. Vasquez, K. Horiuchi, and G.C. Cotzias. Anemias,metales y enfermedades del sistema extrapiramidal. Rev MedChile 96:717-23, 1968.

19. Mena, I., K. Horiuchi, K. Burke, and G.C. Cotzias. Chronicmanganese poisoning: individual susceptibility and absorptionof iron. Neurology 19:(10) 1000-1006, 1969.

20. Papavasiliou, P.S., S.T. Miller, and G.C. Cotzias. Role ofthe liver in regulating the distribution and excretion ofmanganese. Amer J Physiol 211:211-16, 1966.

21. Fuenzalida, S., and I. Mena. Intoxicación crónica por manganesoy sus relaciones con las enfermedades del sistema extrapira-midal. Rev Med Chile 95:(11) 667, 1967.

22. Mena, I., J. Court, S. Fuenzalida, P.S. Papavasiliou, andG.C. Cotzias. Modification of chronic manganese poisoning:treatment with L-Dopa or 5-OH Tryptophane. New Eng J Med282:5-10, 1970.

23. Cotzias, G.C., P.S. Papavasiliou, C. Fehling, B. Kaufman,and I. Mena. Similarities between neurologic effects of L-Dopaand of apomorphine. New Eng J Med 282:31-33, 1970.

24. Mena, I., J. Court, S. Fuenzalida, P.S. Papavasiliou, andG.C. Cotzias. Tratamiento del manganismo con L-Dopa. RevMed Chile, in press.

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25. Mena, I., A. Vasquez, K. Horiuchi, and G.C. Cotzias.Absorcion intestinal de manganeso: factor de susceptibilidadde manganismo. Rev Biologia Med Nuc 1:27-33, 1969.

26. Mena, I. Perspectivas de la L-Dopa en la enfermedad deParkinson. Primero Simposio Sudamericano sobre el estadoactual del tratamiento en la enfermedad de Parkinson yParkinsonismo. Actas. Buenos Aires, 1970, p. 54-59.

27. Court, J., J.C. Kase, E. Palacios, and I. Mena. Tratamientodel Parkinsonismo con L-Dopa. Rev Med Chile 99:399-401, 1971.

28. Orrego, H., O. Fernandez, and I. Mena. Effect of anti-ch 3inergic drugs on intestinal absorption of Ferrous sulphateFe- . Amer J Digest Dis, in press.

OTHER DATA

Grantee: Dr. Humberto Torloni (Project Coordinator), PanAmerican Health Organization, Washington, D.C.Dr. George Cotzias, Brookhaven National Laboratory,Upton, New York, and Dr. Ismael Mena, PontificaUniversidad Catolica de Chile, Santiago, Chile,(Principal Investigators)

Funded by: National Institutes of Health/U.S. Public HealthService


MULTIDISCIPLINARY STUDIES ON PRIMITIVE POPULATIONSIN LATIN AMERICA

PROBLEM

The primary purpose of these studies is to ascertain the populationstructure of primitive man as exemplified by the American Indian, and todefine the selective pressures and the milieu within which that structurefunctioned. Secondary objectives are (1) to collect data on gene fre-quencies which, properly programmed for large-capacity computers, willprovide new insights into the taxonomic relationships of various Indiangroups, and (2) to study changing patterns of disease, especially thosewith a genetic component, as the Indian makes the transition from the stoneage to the atomic age.

METHOD

A multidisciplinary team, always preceded by an anthropologistthoroughly familiar with the group under study, obtains genealogies;performs medical, dental, and anthropometic examinations; and collectsblood, saliva, urine, and stool specimens. Each specimen of blood, saliva,and urine is typed in the laboratory with reference to a variety of genetic-marker systems, and studies are made of the antibodies, biochemical con-stituents, and parasites involved.

RESULTS TO DATE

Results of studies on the Xavante Indians of the Brazilian MatoGrosso and the Makiritare Indians of Venezuela have been published inextenso. Partial reports of findings on Yanomama Indians of Brazil andVenezuela have been published and further reports are in press. Briefly,the genetic structure of these tribal populations appears to differ inmany important ways from that of civilized populations. The consequencesof these differences are being explored by computerized populationsimulation. Furthermore, the possible genetic implications of the differ-ent patterns of morbidity and mortality in primitive populations are beingexplored.

SIGNIFICANCE

Primitive man is rapidly disappearing. These studies are designedto provide additional insights into the circumstances under which humanevolution occurred. They may help to explain some of the biomedical aspectsof modern man, who in the genetic sense, has only very recently left theprimitive state.

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PUBLICATIONS

1. Arends, T., G. Brewer, N. Chagnon, M. Gallango, H. Gershowitz,M. Layrisse, J. Neel, D. Shreffler, R. Tashian, and L. Weitkamp.Intra-tribal genetic differentiation among the Yanomama Indiansof southern Venezuela. Proc Nat Acad Sci 57:1252-1259, 1967.

2. Arends, T., L.R. Weitkamp, M.L. Gallango, J.V. Neel, and J. Schultz.Gene frequencies and microdifferentiation among the MakiritareIndians. 2. Seven serum protein systems. Amer J Hum Genet 22:526-

532, 1970.

3. Bergold, G.H., C.L. Sequera, A. Saturno, and J.V. Neel. Encuestaserologica para los grupos arbovirus entre Indios Venezolanos(Yanomamos, Maquiritare, Bare). Acta Cient Venezolana In press.

4. Bloom, A.D., J.V. Neel, K.D. Choi, S. Iida, and N.A. Chagnon.Chromosome aberrations among the Yanomama Indians. Proc NatAcad Sci 66:920-927, 1970.

5. Chagnon, N.A., J.V. Neel, L.R. Weitkamp, H. Gershowitz, andM. Ayres. The influence of cultural factors on the demographyand pattern of gene flow from the Makiritare to the YanomamaIndians. Amer J Phys Anthropol 32:339-349, 1970

6. Chagnon, N.A. The feast. Natural History 77:34-41, 1968.

7. Chagnon, N.A., P. LeQuesne, and J.M. Cook. Yanomama hallucinogens:anthropological, botanical, and chemical findings. CurrentAnthropol 12:72-74, 1971.

8. Eveland, W.C., W.C. Oliver, and J.V. Neel. Characteristics ofEscherichia Coli serotypes in the Yanomama, a primitive Indiantribe of South America. Infection and Immunity 4:753-756, 1971.

9. Fitch, W.M., and J.V. Neel. The phylogenic relationships ofsome Indian tribes of Central and South America. Amer J HumGenet 21:384-397, 1969.

10. Gershowitz, H., P.C. Junqueira, F.M. Salzano, and J.V. Neel.Further studies on the Xavante Indians. 3. Blood groups andABH-Lea secretor types in the Simoes Lopes and Sao MarcosXavantes. Amer J Hum Genet 19:502-513, 1967.

106

11. Gershowitz, H., M. Layrisse, Z. Layrisse, J. Neel, C. Brewer,N. Chagnon, and M. Ayres. Gene frequencies and the micro-differentiation among the Makiritare Indians. 1. Eleven bloodgroups systems and the ABH-Le secretor traits: a note on Rhgene frequency determinations. Amer J Hum Genet 22:515-525, 1970.

12. Gerahowitz, H., M. Layrisse, Z. Layrisse, J. Neel, N. Chagnon,and M. Ayres. The genetic structure of a tribal population, theYanomama Indians. 2. Eleven blood group systems and the ABH-Lesecretor traits. Ann Hum Genet In press.

13. Layrisse, Z., M. Layrisse, and H. Gershowitz. Blood typingstudies in American Indians: misclassification of Rz phenotypes.Amer J Phys Anthropol 32:465-470, 1970.

14. Neel, J.V., F.M. Salzano, P.C. Junqueira, F. Keiter, and D. Maybury-Lewis. Studies on the Xavante Indians of the Brazilian Mato Grosso.Amer J Hum Genet 16:52-140, 1964.

15. Neel, J.V., and F.M. Salzano. A prospectus for genetic atudieson the American Indians. In The Biology of Human Adaptability.P.T. Baker and J.S. Weiner (eds.), Oxford, Clarendon Press, 1966.

16. Neel, J.V., and F.M. Salzano. Further studies on the XavanteIndians. 10. Some hypotheses-generalizations resulting fromthese studies. Amer J Hum Genet 19:554-574, 1967.

17. Neel, J.V., and N.A. Chagnon. The demography of two tribes ofprimitive relatively unacculturated American Indians. Proc NatAcad Sci 59:680-689, 1968.

18. Neel, J.V., W.M. Mikkelsen, D.L. Rucknagel, E.D. Weinstein,R.A. Goyer, and S.H. Abadie. Further studies of the XavanteIndians. 8. Some observations on blood, urine, and stoolspecimens. Amer J Trop Med Hyg 17:474-485, 1968.

19. Neel, J.V., A.H.P. Andrade, G.E. Brown, W.E. Eveland, J. Goobar,W.A. Sodeman, Jr., G.H. Stollerman, E.D. Weinstein, and A.H.Wheeler. Further studies of the Xavante Indians. 4. Immunologicstatus with respect to various diseases and organisms. Amer JTrop Med Hyg 17:486-498, 1968.

20. Neel, J.V. The American Indian in the International BiologicalProgram. In Biomedical Challenges Presented by the AmericanIndian. Proceedings. Washington, D.C., 25 June 1968. (PanAmerican Health Organization Scientific Publication No. 165,September 1968) p. 47-54.

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21. Neel, J.V., and R.H. Ward. Village and tribal genetic distancesamong American Indians, and the possible implications for humanevolution. Proc Nat Acad Sci 65:323-330, 1970.

22. Neel, J.V., W.R. Centerwall, N.A. Chagnon, and H.L. Casey.Notes on the effect of measles vaccine in a virgin-soilpopulation of South American Indians. Amer J Epidemiol 91:418-429, 1970.

23. Neel, J.V. Lessons from a primitive people. Science 170:815-822,1970.

24. Neel, J.V. Genetic aspects of the ecology of disease in theAmerican Indian. In The Ongoing Evolution of Latin AmericanPopulations. F.A. Salzano (ed.), Springfield, C.C. Thomas,1971, p. 561-590.

25. Neel, J.V. The genetic structure of a tribal population, theYanomama Indians. 1. Introduction. Ann Hum Genet In press.

26. Neel, J.V., T. Arends, C. Brewer, N. Chagnon, H. Gershowitz,M. Layrisse, Z. Layrisse, J. MacCluer, E. Migliazza, W. Oliver,F. Salzano, R. Spielman, R. Ward, and L. Weitkamp. Studies onthe Yanomama Indians. Fourth International Congress of HumanGenetics. Proceedings. In press.

27. Niswander, J.D. Further studies on the Xavante Indians.7. The oral status of the Xavantes of Sim5es Lopes. Amer J HumGenet 19:543-553, 1967.

28. Niswander, J.D., F. Keiter, and J.V. Neel. Further studies onthe Xavante Indians. 2. Some anthropometric, dermatoglyphic,and nonquantitative morphological traits of the Xavantes ofSimoes Lopes. Amer J Hum Genet 19:490-501, 1967.

29. Post, R.H., J.V. Neel, and W.J. Schull. Tabulations of phenotypeand gene frequencies for 11 different genetic systems studiedin the American Indian. In Biomedical Challenges Presented bythe American Indian. Proceedings. Washington, D.C., 25 June 1968.(Pan American Health Organization Scientific Publication No. 165,September 1968) p. 141-185.

30. Salzano, F.M., J.V. Neel, and D. Maybury-Lewis. Further studieson the Xavante Indians. 1. Demographic data on two additionalvillages: genetic structure of the tribe. Amer J Hum Genet19:463-489, 1967.

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31. Salzano, F.M., and J.V. Neel. Fertility patterns and geneticstructure of Xavante Indian populations. Ciencia e Cultura19:64-66, 1967.

32. Salzano, F.M., J.V. Neel, L.R. Weitkamp, and J.P. Woodall.Serum proteins, hemoglobins and erythrocyte enzymes ofBrazilian Cayapo Indians. Human Biol In press.

33. Salzano, F.M., R. Moreno, M. Palatnik, and H. Gershowitz.Demography and H-Lea salivary secretion of the Maca Indiansof Paraguay. Amer J Phys Anthropol 33:383-388, 1970.

34. Shreffler, D.C., and A.G. Steinberg. Further studies on theXavante Indians. 4. Serum protein groups and the SC1 traitof saliva in the Simoes Lopes and Sao Marcos Xavantes. AmerJ Hum Genet 19:514-523, 1967.

35. Tashian, R.E., G.J. Brewer, H. Lehmann, D.A. Davies, andD.L. Rucknagel. Further studies on the Xavante Indians.5. Genetic variability in some serum and erythrocyte enzymes,hemoglobin, and the urinary excretion of Y-aminoisobutyricacid. Amer J Hum Genet 19:524-531, 1967.

36. Ward, R.H., and J.V. Neel. Gene frequencies and micro-differentiation among the Makiritare Indians. 4. A comparisonof a genetic network with ethnohistory and migration matrices;

a new index of genetic isolation. Amer J Hum Genet 22:538-561,

1970.

37. Ward, R.H. The genetic structure of a tribal population, theYanomama Indians. 5. Comparison of a series of genetic networks.Ann Hum Genet In press.

38. Weinstein, E.D., J.V. Neel, and F.M. Salzano. Further studieson the Xavante Indians. 6. The physical status of the Xavantesof Simoes Lopes. Amer J Hum Genet 19:532-542, 1967.

39. Weitkamp, L.R., and J.V. Neel. Gene frequencies and micro-differentiation among the Makiritare Indians. 3. Nineerythrocyte enzyme systems. Amer J Hum Genet 22:533-537, 1970.

40. Weitkamp, L.R., and N.A. Chagnon. Albumin Máku: A new variantof human serum albumin. Nature 217:759-760, 1968.

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41. Weitkamp, L.R., T. Arends, M.L. Gallango, J.V. Neel,J. Schultz, and D.C. Shreffler. The genetic structure ofa tribal population, the Yanomama Indians. 3. Seven serumprotein systems. Ann Hum Genet In press.

42. Weitkamp, L.R., and J.V. Neel. The genetic structure of atribal population, the Yanomama Indians. 4. Eleven erythrocyteenzymes and summary of protein variants. Ann Hum Genet Inpress.

OTHER DATA

Grantee: Dr. James V. Neel, Department of Human Genetics,The University of Michigan Medical School,Ann Arbor, Michigan

Funded by: U.S. Atomic Energy Commission and NationalScience Foundation


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FETAL GROWTH AND CORD IMMUNOGLOBULINSIN CENTRAL AMERICAN POPULATIONS

PROBLEM

As many as 40 per cent of village children in some areas ofGuatemala experience fetal growth retardation and half of the newbornsshow elevated IgM, a sign of fetal antigenic stimulation. These factshave dramatic implications for survival and postnatal growth. Ingeneral, however, there is very little information about the frequencyof fetal growth retardation in the various countries of Central America.

The purpose of this investigation is to stimulate workers in thevarious Central American countries to collect data on birth weight andcord immunoglobulins in rural areas where most of the population lives.

METHOD

At least two rural communities will be chosen for study in eachCentral American country. All births in each community will be accuratelyrecorded and birth weights determined over one year. Families will bevisited periodically to determine survival of children. Cord blood willbe collected for investigation of immunoglobulins.

RESULTS TO DATE

The study is in the preparatory phase.

SIGNIFICANCE

If results obtained in Guatemala are confirmed in other CentralAmerican countries, more emphasis should be given to prenatal care sincestudies in Guatemala have shown that infant mortality is heavily in-fluenced by fetal growth retardation.

PUBLICATIONS

None thus far.

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OTHER DATA

Dr. Leonardo J. Mata, Division of Microbiology,Institute of Nutrition of Central America andPanama, Guatemala City, Guatemala

Funded by:

Timetable:

World Health Organization

1971-1972

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Grantee:

INTENSIVE CARE OF THE MOTHER, FETUS, AND NEWBORN

PROBLEM

The specific aims of this project are to apply and evaluatediagnostic methods for the early detection of latent or actual fetaldistress and to establish the possible causes of such disturbances,and to develop diagnostic and therapeutic procedures that may be usefulin improving fetal status.

Fetal distress may either be chronic or acute. Chronic distur-bances produced by insufficient metabolic exchanges between mother andfetus last throughout pregnancy and cause intrauterine fetal growthretardation. Acute disturbances appear during labor as a consequenceof exaggerated impairment in fetomaternal exchanges caused by uterinecontractions (acute intrapartum fetal distress).

METHOD

High-risk pregnant women (those with severe diabetes, toxemia,arterial hypertension, Rh isoimmunization) are chosen for study. Thefollowing variables are continuously recorded electronically: (1) pres-sure of amniotic fluid (intrauterine pressure for measuring theintensity and frequency of uterine contractions), (2) maternal arterialpressure, heart rate, and ECG, and (3) fetal ECG, heart rate, and EEG.

During labor, periodic microsampling is made of maternal arterialblood and fetal cutaneous (scalp) capillary blood using the Salingmethod. The following variables are measured in both maternal andfetal blood: Hb saturation, PO2, base deficit, and creatinins and uricacid concentration.

At the moment of birth similar measurements are repeated in theblood of the umbilical artery and vein; after birth they are periodicallyrepeated in blood samples obtained from the aorta. In addition, heartrate, ECG, arterial pressure, respiratory movements, temperature, andEEG are continuously recorded in the newborn during the first hours oflife.

The development of the child is followed with emphasis on neuro-logic and psychologic functions.

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RESULTS TO DATE

1. In previous papers it was reported that type 2 dips are a signof fetal distress. These dips are transient decelerations of fetalheart rate caused by uterine contractions. The time between the peakof the contraction and the lower fetal heart rate recorded for thecorresponding dip is, on the average, 40 seconds. But if type 2 dipsare not symmetric, the bottom of the dip is not the most accurate pointfor studying this kind of fetal response. So a new method that includesmany points of the cardiotachometer record has been developed in whichthe measurements are automatically made by a computer.

2. In a series of 56 cases, fetal blood pH was correlated withApgar scores, and a positive correlation between both variables wasfound (=- 0.74) (p < 60.001). From among 17 fetuses with acidosis, 15newborns were found to be depressed (Apgar score 5 or less). In theremaining two, fetal acidosis was due to a maternal acidosis. Thirty-three of 39 fetuses without acidosis were vigorous (Apgar score 8 to10) at delivery. The remaining six were depressed at delivery, buttheir mothers had received large amounts of analgesia, a sufficientexplanation for neonatal depression in the absence of acidosis.

3. Based on previously published data, a new method for evaluatingthe fetal situation in utero is being developed. Para-amino hyppurate(PAH) was administered to six normal pregnant women in an intravenousinfusion lasting 30 minutes. Periodic samples of amniotic fluid wereobtained, and the concentration of PAH in each sample was measured.The PAH concentration in the amniotic fluid increased progressivelyuntil 150 minutes after infusion was discontinued. With these data,a linear correlation coefficient and its corresponding 95 per centconfidence belt were calculated. In all cases newborns were normal atdelivery and continued to be normal during the neonatal period. Thesame experimental design was followed in eight high-risk pregnant patients.Four patients had depressed newborns, two of whom needed intensive careduring the first five days of life; the other two died within the firstday. Each linear correlation coefficient for these four patients wassignificantly lower than that of the "normal" group. The remaining fourpatients' newborns were normal at delivery and throughout the first 28days. In these patients, correlation coefficients were not differentfrom those of the "normal" group.

As of December 1971, 40 Latin American physicians had receivedresearch training under this program.

SIGNIFICANCE

Disturbances in fetal homeostasis have great medical and socialsignificance. Chronic disturbances throughout pregnancy (fetal mal-

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nutrition) impair fetal growth, and the number of cells in the brain andother organs may not reach the normal number dictated by human geneticpotential. Such infants will never reach full development.

Acute disturbances in fetal homeostasis during labor may interferewith cell function and lead to irreversible structural damage and per-manent postnatal sequelae. A substantial proportion of cases of infantswith neurologic deficits and mental retardation are thought to be aresult of damage to the fetal or newborn brain through alterations inthe composition of the blood caused by hypoxemia, hypercapnia, acidemia,and hypoglycemia.

PUBLICATIONS

1. Caldeyro-Barcia, R., R. Bustos, J.L. Diaz Rosselo, C. MéndezBauer, J. Poseiro, S.V., Pose, and C. Casacuberta. Fisio-patología del sufrimiento fetal: equilibrio ácido-base delfeto. V Jornada Med Bienal Hospital de Gineco-Obstetricia,No. 1 (IMSS), May 1972, pp. 31-50.

2. Fernández, A., A.L. Passo, G. Sabini, R. Belitzky, O. Althabe,and H. Difilippo. Estudio en el liquido amniótico de lasconcentraciones de manitol y para-amino-hipurato de sodioadministrados a la mujer grávida. Rev Argent Ginecol Obstet2:353-62, 1971.

3. Myers, G.G., R.D. Petersen, A.J. Kapohl, and R. Caldeyro-Barcia.New method for measuring lag-time between human uterine con-traction and the effect on fetal heart rate. Am J Obstet Gynecol112:39-45, 1972.

OTHER DATA

Grantee: Dr. Roberto Caldeyro-Barcia, Research Unit, LatinAmerican Center of Perinatology and Human DevelopmentMontevideo, Uruguay

Funded by: Ford Foundation


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FETAL CEPHALIC DEFORMATION AND UMBILICAL CORDCOMPRESSION DURING LABOR

PROBLEM

This research project is examining ways to prevent transient andpermanent brain damage to the fetus by studying a widely used obstetricalprocedure--the artificial rupture of the membranes during labor.

Uterine contractions may compress the umbilical cord, causingocclusion of the umbilical vessels. The fetal head may also suffer com-pression with consequent cephalic deformation, cranial hypertension,and cerebral ischemia. Amniotic fluid tends to minimize these effects.When the membranes are ruptured, the amount of amniotic fluid is greatlyreduced and the forewaters disappear, which increases the possibilityof both fetal head and cord compression.

METHOD

Normal pregnant women with intact membranes who are undergoingspontaneous labor are selected for study. Artificial rupture of themembranes is performed at 5 cm of cervical dilatation in certain ran-domly chosen women. In the others membranes remain intact from 5 cmto full cervical dilatation.

Changes in amniotic fluid pressure caused by uterine contractions(intrauterine pressure for measuring the intensity and the frequencyof the contractions) and fetal heart rate are continuously recordedelectronically.

At the moment of birth, samples from the mother's vein and theumbilical artery and vein are obtained to measure the followingvariables: Hb saturation, pO , pCO2, pH, and base deficit. The newbornstate is evaluated by performing: (1) first-minute, fifth-minute, andtenth-minute Apgar scoring, (2) a physical and neurologic examinationand EEG within the first 48 hours, and (3) measurements of the cranialdiameter, a clinical evaluation of the molding of the head, and adetermination of the presence or absence of caput seccedaneum, cephalo-hematoma, or both. Several cross-correlations are made between thevariables measured, which are also correlated with clinical signs inthe mother, the fetus, and the newborn.

The child's development is followed with particular emphasison neurologic and psychologic functions.

116

RESULTS TO DATE

It has already been demonstrated that rupture of the membranesfavors the production of type 1 dips (1,2). Dips are decelerationsof fetal heart rate caused by uterine contractions, and are characterizedby the lag time between the peak of the contraction and the lowest fetalheart rate value reached during the deceleration caused by that con-traction. This lag time for type 1 dips is less than 18 seconds; inother words, the bottom of the deceleration usually coincides with thepeak of the contraction.

It has also been shown that after rupture of the membranes, mostuterine contractions provoke consistent EEG changes in the fetus--namely,the appearance of irregular high-amplitude slow waves of 100 to 200microvolts at intervals of 0.5 to 1 second--suggesting that some degreeof cerebral ischemia could be present due to fetal head compressioncaused by uterine contractions (3).

In a group of 19 laboring women with intact membranes from 5 cmto full dilatation, the incidence of type 1 dips (6 per cent) was muchless than that observed in a control group of 26 patients with rupturedmembranes during the same labor period (33 per cent).

If only those cases in which a nuchal cord is absent are considered,the group with intact membranes (12 patients) has also a lower incidenceof type 1 dips (5 per cent) compared with the group with ruptured mem-branes (28 per cent) (13 patients). As in the absence of a nuchal cord,type 1 dips are mainly the result of cephalic compression. These resultsindicate that the integrity of the membranes during labor until deliveryprotects the fetal head from the compression caused by uterine con-tractions. This thinking is supported by the magnitude of the newborn'shead deformation, which seems to be less in labors with intact membranesthan in those with ruptured membranes.

If we consider those cases that have a nuchal cord, the incidenceof type 1 dips is also lower in those in which membranes were intact

(11 per cent) (7 patients) than in the group with ruptured membranes(40 per cent) (13 patients) since when a nuchal cord is present, type 1dips are fundamentally due to the occlusion of umbilical vessels.These results indicate that the integrity of the membranes protectsthe cord from uterine compression. From the results already stated,it is obvious that the presence of a cord around the neck causes ahigher incidence of type 1 dips in instances of both intact and ruptured

membranes.

When considering the degree of engagement of the fetal head, the

results are the following: fetal head above zero station: the incidence

of type 1 dips with intact membranes is 4 per cent and with rupturedmembranes 11 per cent; fetal head below zero station: the incidence

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of type 1 dips with intact membranes is 26 per cent and with rupturedmembranes 59 per cent. These results show that the incidence of type 1dips is higher when the fetal head is engaged in the pelvis, a findingvalid for both intact or ruptured membranes, and that the integrityof the membranes protects fetal head or umbilical cord against the com-pression caused by uterine contractions, regardless the degree ofengagement of the fetal head.

SIGNIFICANCE

Artificial rupture of the membranes is an obstetrical procedurethat obstetricians in many institutions routinely perform during labor.The procedure is based on the notion, not satisfactorily proved, thatrupture of the membranes shortens labor. The procedure is also performedin various countries before labor to induce it.

The possible danger to fetal brain resulting from ischemia andcephalic deformation caused by uterine contractions and favored by theearly rupture of the membranes may have great practical implications.

PUBLICATIONS

1. Althabe, O., G. Aramburu, R. Schwarcz, and R. Caldeyro-Barcia.Influencia de la rotura de las membranas sobre la compresiónde la cabeza fetal durante el parto. Rev Argent Ginecol Obstet2:251-60, 1971.

2. Caldeyro-Barcia, R., R. Schwarcz, and 0. Althabe. Effectsof rupture of the membranes on fetal heart rate pattern.Int J Gynecol Obstet, in press.

3. Garcia-Austt, E. Effects of uterine contractions on the EEGof the human fetus during labor. In Perinatal Factors AffectingHuman Development, Washington, D.C., PAHO Scientific Publi-cation No. 185, 1969, pp. 127-36.

4'. Schwarcz, R., G. Strada-Saenz, O. Althabe, J. Fernández Funes,and R. Caldeyro-Barcia. Presión ejercida por las contraccionesuterinas sobre la cabeza del feto humano durante el parto.Rev Argent Ginecol Obstet 2:128-42, 1971.

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OTHER DATA

Dr. Roberto Caldeyro-Barcia, Research Unit,Latin American Center of Perinatology and HumanDevelopment, Montevideo, Uruguay

Funded by:

Timetable:

Ford Foundation

1971-1973

Grantee:

119

TRIALS WITH MEASLES VACCINEIN A VIRGIN POPULATION

PROBLEM

To immunize threatened American Indian populations against measleswith live vaccine, using this as a model system to investigate factorsaffecting the susceptibility of these populations to measles virus. Atthe same time, to collect serologic and other data on the history of awide variety of infectious diseases and thus to construct a broad pictureof disease in isolated populations.

METHOD

A population is divided into two comparable groups and live measlesvaccine given to one group. Daily temperature records are kept on thewhole population. At the end of two weeks the second group is vaccinatedand the recordings continued for a total of four weeks. Each group thenacts as control for the other on intercurrent illnesses. Blood specimensare collected at the beginning and end of the study. These are testedfor measles immune response and for the presence of antibody to about 40other viral and other infectious agents. Demographic data are collectedand medical examinations made on each individual.

RESULTS TO DATE

Febrile responses to Schwarz further attenuated vaccine in twotribes were at the same level as responses to Edmonston vaccine in threeCaucasian populations studied in a WHO study. Antibody patterns toother infections fall into three categories: (a) Highly endemic withantibody in all age groups e.g., Herpes simplex, (b) Moderately endemicin diseases where the main reservoir was in some animal population, e.g.,yellow fever, (c) Absent except where recently introduced from outside,e.g., measles, rubella, mumps, influenza.

SIGNIFICANCE

The hyper-responsiveness of the Indian populations is not sufficientto account for the very high measles mortality occasionally observed,but it is a reasonable explanation of the significant mortality observedin epidemics where nursing support has been available. This difference,coupled with the lack of antibody, suggests that measles and the otherdiseases in Class (c) are not endogenous in small populations and mayhave played little part in the evolution of mankind.

120

PUBLICATIONS

1. Black, F.L., W. Hierholzer, J.P. Woodall, and F. Pinheiro.Intensified reactions to measles vaccine in unexposedpopulations of American Indians. J Infect Dis 124: 306-317,1971. A second publication on serology has been delayed toallow incorporation of new data acquired with sera from asecond Kayopo tribe.

OTHER DATA

Grantee: Dr. Francis L. Black, Yale University School ofMedicine, New Haven, Connecticut

Funded by: Pan American Health Organization

Timetable: Original field work completed March, 1970. Laboratorytests continuing as new serological tests and newserum collections become available.

121

PILOT TYPHUS VACCINATION PROJECT IN BOLIVIA

PROBLEM

Disease consistent with louse-borne typhus has been known in themountainous regions of Mexico and Central and South America since colonialtimes. Fiebre altiplanico, an important cause of illness in Bolivia, wasdifferentiated from typhoid fever about 1913, recognized as a probableform of typhus about 1920, and shown to be indistinguishable from classicalor European louse-borne typhus in the early 1940's. Although its reportedincidence has declined in the last two decades in both Bolivia and otherAndean countries, presumably as a result of health education and louse-control measures, it still occurs in the form of occasional, sharp,localized outbreaks with appreciable mortality and probably also in theform of appreciable numbers of unrecognized infections. Bolivian healthauthorities consider it an important health problem.

An agreement was drawn up between the Government of Bolivia and thePan American Health Organization to initiate a program to determine byserosurvey technique the relative importance of louse-borne typhus as acause of febrile disease, to conduct a small vaccine trial, and to determinelouse susceptibility to insecticides.

As a result of a series of consultations, it was agreed to evaluatethe live attenuated E-strain typhus vaccine, to be obtained through thedepartment of microbiology of the University of Maryland School of Medicinefor use in the altiplano. After a preliminary serosurvey in 1968, whichindicated that louse-borne typhus infection was indeed common, that it wasendemic, and that young children constituted a significant fraction of thesusceptible population, a specific plan of action was developed. Its mainobjectives are: (a) to study the epidemiology of louse-borne typhus inBolivia, (b) to assess the possible use, acceptability, and effect of thelive attenuated E-strain typhus vaccine in relatively small, selected andrepresentative populations in the Bolivian typhus, i.e., in the endemicsituation, and (c) to use the information so obtained to make recommendationsfor typhus control specifically applicable to Bolivia and possibly adjacentsimilar areas.

METHOD

The plan for the initial phase of the program, which is still inprogress, was as follows. Three localities in different parts of Bolivia,with a total population of about 8,000 and representing different geo-graphic, climatic, ethnic, and social groups all having a typhus problem,were chosen for the project. The areas were mapped, the houses noted, a

122

census was taken, and the people were told about the proposed project.Before vaccination, 1,500 serum specimens representing 17 per cent ofthe study population were collected on a random basis from people of allages by personnel of the Instituto Nacional de Enfermedades Transmisibles(INET) for analysis at the University of Maryland laboratories in Baltimore.To develop a typhus diagnostic capability in La Paz and permit continuedcollaborative effort in the project, a Bolivian professional from theInstituto Nacional de Laboratorios de Salud in La Paz was given a PAHO/WHOfellowship for training at the department of microbiology laboratories ofthe University of Maryland School of Medicine in Baltimore.

E-strain vaccine lot CRD-2 in a dose containing 105'5 EID wasadministered in the deltoid region by foot-operated jet gun at a0number ofpredesignated vaccination sites, where the vaccinees were registered andissued numbered vaccination cards. Approximately 4,800 people over oneyear old (school age children predominated) were vaccinated.

Observations on the occurrence of adverse reactions to the vaccinewere made by Bolivian Health Personnel to see how the Bolivian populationwould accept the vaccine. A serum was obtained six weeks after vaccinationfrom a representative portion of the vaccinated population, and provisionswere made to gather one-year-postvaccination serum samples to determineantibody conversion, persistence of typhus antibodies, and typhus incidence.

RESULTS TO DATE

The prevaccination serosurvey with typhus group (soluble) antigenin the complement-fixation (CF) test revealed an age-specific distributionof typhus antibodies consistent with major typhus endemicity. There wasevidence of infection in the very young and a high incidence of CF anti-bodies (over 60 per cent) in older adults. Both sexes were similarlyaffected in all age groups. This typhus antibody distribution is compatiblewith the endemic situation in which louse-borne primary typhus infectionstend to occur in the younger persons. In the young the disease tends tobe milder than in adults, may not be recognized as typhus, and may be in-apparent. It is thus likely that primary typhus infections occur at amuch higher rate (100- to 1,000-fold) than actually reported.

Analysis of the six-week-postvaccination serum samples from thevaccinated populations revealed a dramatic shift of the CF antibodiestoward higher titers as compared to the prevaccination state. One-yearsamples should determine antibody longevity.

The population's reactions to the vaccine as judged by INET personnelwere mild and did not require treatment. The Director of INET thought itwas acceptable for use in the Bolivian population. Only early reactionsthat occur in the first few days after vaccination were recognized, however.No late reactions, which usually occur between nine and 14 days after

123

vaccination, were observed. Lack of late reactions is unusual and mayperhaps be partly attributed to Bolivia's high altitude. This requiresfurther study.

No typhus fever has been recognized in the vaccinated populationin the first year of observation. A sharp typhus outbreak occurred bet-ween November 1971 and February 1972 in an unvaccinated population nearone of the vaccination sites.

Observations of the vaccinated population are continuing, and plansare being worked out for additional studies.

SIGNIFICANCE

The incidence and age distribution of typhus antibodies in, andcollection of other epidemiologic information about the Bolivian popula-tion, in which typhus is endemic, have great bearing on both the selectionof population for vaccination and the effective method and distributionof typhus control measures. Elimination of the susceptible populationsegment through E-strain vaccination would prevent (a) sickness and deathsdue to primary typhus, and (b) the creation of new reservoirs, even in theabsence of complete louse control--a formidable logistic problem.

Information derived from detailed epidemiologic studies would furtherour understanding of typhus as it occurs in Bolivia and in similar SouthAmerican areas in Chile, Ecuador, and Peru.

We are planning a second small pilot control vaccination project inwhich E-strain and control (nontyphus) vaccines will be administered toa population in a single area. Detailed observations will be made forthree to four weeks of the extent and occurrence of both early and latepostvaccination reactions in these high-altitude populations. If thevaccine is judged to be acceptable, recommendations will be made forregional control of louse-borne epidemic typhus.

PUBLICATIONS

1. Montoya, J.A. Report to PAHO on a visit to Bolivia.

2. Wisseman, C.L., Jr. Report to PAHO on Field Trip to Bolivia,July 8-20, 1968.

3. Wisseman, C.L., Jr. Report to PAHO on Field Trip to Bolivia,September 5-12, 1970.

124

OTHER DATA

Grantee: Ministry of Health of Bolivia, La Paz

Funded by:

Timetable:

Pan American Health Organization/World HealthOrganization, Washington, D.C.; University ofMaryland, U.S.A.; and U.S. Army Medical Researchand Development Command

1968 - continuing

125

MECHANISM OF ANAPHYLAXIS INDUCED WITHSOLUBLE OR INSOLUBLE ANTIGENS

PROBLEM

In the guinea pig, the reaction of antigen with antibody fixedto target cells provokes the liberation of histamine which plays amajor role in the acute anaphylactic reaction in this species.Histamine is released mainly from mast cells. Since these cells arelocated extravascularly it is assumed, when soluble antigens are used,that antigen diffuses out of the vessels and gets in contact withsensitized mast cells nearby. It is known that particulate antigens,such as red blood cells, can elicit anaphylaxis in spite of theirinability to cross ,the vascular endothelium. This investigation wasundertaken to find out how particulate antigen induces anaphylaxis.

METHOD

Antibodies to fixed chicken red cells (FCRC), fixed rat red cell(FRRC), or trichloroacetic acid-precipitated ovalbumin (EaTCA) wereobtained by immunizing guinea pigs with these antigens using aluminiumhydroxide gel as adjuvant. The animals were boostered one week laterand bled one month after the first injection. The antibody contentwas estimated by passive cutaneous anaphylaxis (PCA). Systemic ana-phylaxis in actively sensitized animals or cutaneous anaphylaxis inpassively sensitized animals were induced with either fresh untreatedantigen or with fixed or precipitated antigen.

RESULTS TO DATE

Challenge of sensitized guinea pigs with either fresh RRC orfresh CRC always elicited fatal anaphylaxis, whereas challenge withred cells elicited only very mild signs of anaphylaxis with no death.In agreement with these results, animals immunized with EaTCA andchallenged with EaTCA showed no anaphylactic shock whereas those chal-lenged with untreated ovalbumin presented typical anaphylaxis and dieda few minutes after antigen injection. Guinea pigs passively sensitizedwith anti-FCRC or anti-FRRC presented positive PCA reactions when chal-lenged with fresh chicken red cells or fresh rat red cells but no PCAreactions when challenged with FRRC or FCRC. Positive PCA reactionswere also obtained when the animals were challenged with the clearsupernatant obtained after centrifuging the fresh cell suspension.

126

SIGNIFICANCE

This investigation aimed to clarify the mechanism through whichantigen causes cell damage in anaphylaxis.

PUBLICATIONS

1. Albuquerque, L.R., A. Perini, and I. Mota. Failure ofinsoluble antigens to elicit anaphylactic reactions.Inter Arch Allergy, in press.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ivan Mota, L.R. Albuquerque, and Adenir Perini(Principal Investigators), PAHO/WHO ImmunologyResearch and Training Center, Butantan Institute,Sao Paulo, Brazil

Pan American Health Organization/World HealthOrganization

1970-1971

127

STUDY OF HOMOCYTOTROPIC ANTIBODIESIN THE GUINEA PIG

PROBLEM

At least two types of homocytotropic antibodies have been describedin different species. One is an antibody that is heat stable and usuallyunable to persist in the passively sensitized skin for more than 48 hoursand the other is an early skin-sensitizing and heat-labile antibodysimilar in its property to human reaginic antibody. In the guinea pig theonly homocytotropic antibody so far firmly established is a heat stable7S '1 antibody. Thus, it was decided to look for homocytotropic antibodiesin this species differing from 7S' 1 antibody.

METHOD

Different antigens and different adjuvants were injected in guineapigs and their early antibody response investigated by the use of passivecutaneous anaphylaxis (PCA) to find out whether antibodies differing from1l have been produced.

RESULTS TO DATE

The results showed that guinea pigs are capable of producing twoantibodies besides the already known 1. One of these is an antibodysimilar in its properties to the reaginic antibody previously found inother species. The guinea pig reaginic antibody is heat-labile, mer-captoethanol-susceptible and persists in the passively sensitized skinfor at least 20 days. The second antibody is heat-stable but mercap-toethanol-labile and persists in the passively sensitized skin for morethan 72 hours but less than one week. The two antibodies were able tosensitize guinea pig mast cells and cause degranulation of these cellson reaction with antigen. Antihistamine produced complete inhibitionof PCA reactions induced with these two antibodies but were much lessefficient against PCA reactions induced with 11 antibody.

SIGNIFICANCE

The research provides information on the types of antibodiesinvolved in anaphylaxis of the guinea pig.

128

PUBLICATIONS

1. Mota, I., and A. Perini. A heat labile mercaptoethanolsusceptible homocytotropic antibody in the guinea pig.Life Sciences 9:923-30, 1970.

2. Perini, A., and I. Mota. Heterogeneity of guinea pighomocytotropic antibodies. Immunology. In press.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ivan Mota and Dr. Adenir Perini, principalinvestigators. PAHO/WHO Immunology Research andTraining Center, Butantan Institute, Sao Paulo,Brazil


1970-1971

129

ECONOMIC IMPACT OF MALARIA

PROBLEM

For over a decade interest has been growing in the application ofcost-benefit analysis to malaria control. Two main difficulties havebeen encountered in most efforts to study the problem as a whole: first,the near impossibility of disentangling the economic effects of thereduction or elimination of malaria from those of other changes takingplace at the same time, and second, the lack of a system for collectingcomplete economic information in malaria-ridden rural areas of developingcountries.

METHOD

To avoid the difficulties of evaluation mentioned above, PAHO/WHOchose a demonstration area where a prospective study of several individualrural families could be carried out to measure the effects on theireconomy of the disappearance of malaria.

Eight study areas, each consisting of about 40 families, were chosenin Paraguay and each family was visited every two weeks between September1968 and June 1970. The malaria eradication campaign started in October1968.

Following a census and collection of other basic information, datawere periodically obtained on the health of the family members; anypopulation movement; and farm work, including number of hours or daysworked, operations performed, the field and crop in which the work wasdone, and whether it was carried out by adult or child family members,by paid help, or by neighbors. Other relevant information was alsocollected.

RESULTS TO DATE

A preliminary comparison of families affected by malaria on 14farms and those free from the disease on 24 farms indicates that:

The families with malaria worked fewer days on their farms than thehealthy ones and used nearly three times as much outside help especiallyduring the final period of the tobacco harvest.

In families with malaria, children accounted for approximately17 per cent of all work days, as compared with approximately 4 per centin healthy families.

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The families with malaria reduced planting in 1970 in comparisonwith 1969 more than healthy families, the yield obtained being muchreduced as indicated by the total tobacco harvested and the kilogramsobtained per plant sown and harvested. Also, the quality of the sickfamilies' crop was lower, as indicated by the average price realizedper kilogram.

SIGNIFICANCE

These studies will provide basic information on malaria's impacton economic development and may serve as a methodologic model for futureinvestigations in other areas.

PUBLICATIONS

1. Conly, G.I Interam

The Impact of Malaria on Economic Development.Symp Malaria Res, San Salvador, November 1971.

OTHER DATA

Dr. Gladys N. Conly, Malaria Eradication Program,Pan American Health Organization, Asunci¿n,Paraguay

Funded by:

Timetable:


1968-1972

131

Grantee:

RESEARCH ON THE EPIDEMIOLOGY OF MALARIA ERADICATIONIN PROBLEM AREAS

PROBLEM

Although physiologic resistance to DDT and dieldrin is the mostwidely recognized problem obstructing progress in malaria eradicationin the Americas, several other problems related to vector behavior,parasite strains, and human and vector ecology have been found and mightbe major contributors to the slow progress of some eradication programs.

The aim of the project is to evaluate the possibility of interruptingmalaria transmission in a problem area with the most promising attack mea-sures, to study in depth the various epidemiologic factors that could havebrought about the results observed, and to search for the existence offoci of persistent transmission and study its epidemiology.

The project is a continuation, with a broader scope, of the workof the insecticide testing team that has carried out investigations ofvarious insecticides in Central America since 1959.

METHOD

We considered on the basis of experience from malaria eradicationprojects and the insecticide testing team's work in Central America thatthe fumigant insecticide Propoxur (OMS-33), which is sprayed on selectedsurfaces in only part of household interiors on a short cycle, mightinterrupt malaria transmission in a test area.

An area of about 30,000 inhabitants and 500 km2 was chosen on thecoastal plain of El Salvador (La Paz Department) for study. Its altitudewas less than 100 meters, and its annual rainfall was 1,745 mm.

Sixty grams of insecticide were sprayed on the average house of 25meters in perimeter, normally as two swaths on the inner side of the roofand the angle between the roof and the wall, on a 35-day cycle. Surfacesknown to be alkaline or absorbent, i.e., whitewash, plaster, tiles,asbestos, mud, etc., were avoided, and instead good surfaces, i.e., wood,thatch (except rice thatch), etc., were sprayed.

The effects of the attack measures and the epidemiology of malariain the area were studied by comparing the trial area with two neighboringareas.

132

RESULTS TO DATE

Entomologic and epidemiologic evaluation of the results of thetrial indicate that transmission might have been interrupted in most ofthe area. Epidemiologic investigations are being continued to disentanglethe effects of possible focal transmission from those of the intensemigratory movements of population in maintaining relatively high prevalencerates.

The project has made very important contributions to the knowledgeof malaria epidemiology in the following fields:

Human Ecology: determining the frequency and timing of houserenovations and changes in sprayable surfaces and types and distributionof building materials; studying the migration of temporary laborers andthe daily habits of the population in relation to their indoor or outdoorpermanence;

Mode insecticide action: duration of effect, influence of meteoro-logical conditions, and methodology of bioassay tests;

Vector behavior: determining vectors' cycles and timing of contactwith man and of permanence inside houses;

Vector resistance to insecticides and their variations in relationto ecologic conditions and insecticide pressure, as well as the importanceof cross-resistance to various insecticides;

Applicability of serologic diagnosis of malaria to the evaluationof malaria transmission in the experimental area, by longitudinal studyof a group of high expected vulnerability.

The present trial was begun in 1970 and should be completed in 1973.

SIGNIFICANCE

All these studies are fundamental for the finding of a methodologyof attack against malaria refractory to conventional attack measures.

Improvements in the methodology of evaluation and supervision willbe of assistance to antimalarial campaigns in general.

PUBLICATIONS

1. Lassen, K., S.Y. Liu, C. Lizarzaburu, and R. Rios. Theevaluation of Propoxur (OMS-33) in Central America, I InteramSymp Malaria Res, San Salvador, November 1971.

133

2. Liu, S.Y., and K. Lassen. Notes on a case of OMS-33 tolerancein Anopheles albimanus in El Salvador, Central America,III Cong Centroam y I Nac Microbiologia, Guatemala, July 1971.

OTHER DATA

Grantee: Dr. Knud Lassen, Malaria Eradication Program,Pan American Health Organization, San Salvador,El Salvador

Funded by: Pan American Health Organization/World HealthOrganization


134

FIELD TRIAL OF A LONG-ACTING ANTIMALARIAL DRUG

PROBLEM

In mass drug-distribution campaigns, the population soon becomes

tired of taking tablets every two weeks, especially when they cause some

discomfort, as in the case of chloroquine. People begin to refuse treat-ment, absent themselves, or pretend to swallow the drug but spit it out.

The purpose of this project is to determine the feasibility ofusing subprofessional medicators to administer intramuscular injections

of long-acting cycloguanil pamoate (CI-5Cl or Camolar) in a large-scaleoperation, the acceptability of this medication to a population requiringmass drug distribution, and its therapeutic effectiveness. In addition,methods and norms for operation need to be established, and recordingsystems, comparative personnel requirements, costs, and the like need tobe studied.

The drug had been given in 15 previous field trials in other parts

of the world, and revised dosages that were demonstrated to be safe and

effective had been worked out.

The Government of Guatemala agreed to furnish a physician and asubprofessional staff. Fewer subprofessional staff members are neededfor an injectable antimalarial drug program such as this than for

projects in which the drug is given by mouth.

METHOD

Personnel to administer the drug--nurses or nurse's aides--weregiven training in intramuscular injections, including special instructionin the handling and administration of Camolar, which is a thick suspension.Seven men and eight women were selected to perform house-to-house injec-

tions, working in pairs whenever possible.

Discardable 6-ml plastic syringes and discardable sterile needles,(1-1/2", 18-gauge) were supplied. To reduce local reactipns, 20-gauge

needles were substituted, and they caused much less apprehension andtrauma.

Attempts were made to induce people to come to injection centers,but at best not more than 50 per cent did so. Those who did not come

were sometimes more difficult to treat in house-to-house clean-up visits,

owing to local reactions to the medication among some of their relatives.

L35

After the first week, all injections were made in visits to houses,with revisits in the late afternoon or next morning to meet absentees;a second sweep of the area was made after the first coverage.

Two districts with populations of about 6,000 each were selected.Each district was covered in about six weeks by 15 medicators. Drugsensitivity tests were performed before the areas were selected, andpreliminary treatment with proguanil was tried in 26 heavily infectedpersons. All of them cleared up eventually, though some Plasmodium vivaxcases lIngered for six days or more.

Malaria surveys were conducted at the time of the first round ofinjections and again at four and 5 1/2 months. In addition. continuousefforts were devoted to passive case detection and to the search forcases of known contact.

All moderate and severe reactions were examined by physicians, andseveral treatments were tried.

RESULTS TO DATA

Four rounds of injections were completed in the trial area inFebruary 1969. Follow-up parasitologic and epidemiologic studies werecompleted by the end of June 1969. The appearance of positive casesless than four months after successful inoculation suggested the possi-bility of a decreased susceptibility of the parasites to the drug. Aneffort carried out between April and August to test this susceptibilityproduced only a very small number of completed tests, and they wereconsidered inconclusive as to the presence or absence of drug tolerance.

Although coverage in each round was always of the order of 74 to81 per cent, a longitudinal study of a sample group showed that thenumber of persons receiving repeated treatments was lower than expected.The age and sex distribution of the subjects indicated high avoidanceof therapy by some groups, especially men. The movement of populationin and out of the area and the psychologic effect of local reactionswere considered to be contributory causes. All efforts to improvecoverage--health education; improvement of injection technique; treatmentof local reactions with analgesics, antihistamines, and corticosteroids;and even police persuasion--failed to produce significant improvements.

There was no evidence of generalized sensitivity, although morethan half the subjects had some local feeling of pain on walking ortenderness to pressure. Most cases were mild and transient, but 2 percent or more were severe enough to prevent the person from working forone or more days. Reactions were of two types: early, which appeared ina few hours or a day or two and were usually mild and short-lived, anddelayed, which appeared after eight to 30 days or more of latency, lasted

136

a longer time, showed more induration, and caused more pain and inter-ference with activity. Fortunately, the latter were quite rare.

The final evaluation of the trial allowed the following conclusions:

(1) It was possible to give the drug as a mass treatment iIthe form of a semestrial injection to people in smallrural area of Guatemala.

(2) The average coverage per administrator was 10.5 injectedpersons per day.

(3) Registered coverage was between 75 and 80 per cent of thepopulation, with variations in age-sex groups, men beingthe least cooperative group.

(4) Participation of previously treated persons was lowerthan the average for the total population.

(5) Local reactions to the injections were considered themain reason for diminished participation.

(6) Intense migratory movements in the coastal areas ofGuatemala greatly interfered with the maintenance ofhigh proportions of protected population due to the longintervals between injections.

(7) Epidemiologic observations did not show significantdifferences in the evolution of malaria incidencebetween the trial area and those neighboring it andsubject to oral mass drug administration.

SIGNIFICANCE

The trial resulted in a good understanding of the problems involvedin field application of a repository drug and provided bases for deter-mining the limited indications for use of this drug in malaria eradicationprograms.

PUBLICATIONS

1. Lassen, K. Field study of cycloguanil pamoate in Guatemala.I Interam Symp Malaria Res, San Salvador, November 1971.

2. S.N.E.M., Guatemala. Informe preliminar sobre cuatro ciclosde cobertura con Camolar. National Malaria EradicationProgram Directors' Meeting, October 1969, Santo Domingo.

137

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. L. J. Uribe, Malaria Eradication Program,Guatemala City, Guatemala


1967-1969

138

SPOROZOITE-INDUCED IMMUNITY IN MAMMALIAN MALARIA

PROBLEM

Sporozoites of rodent malaria have been shown to be highlyimmunogenic and to induce, when irradiated, a considerable degree ofprotective immunity in mice. These immune animals are protectedexclusively against the sporozoite stage, the immune mechanism beingtotally ineffective against the blood stages of the same parasite strain.A considerable degree of cross-protection was observed to occur withsporozoites of the other rodent malarial species, Plasmodium vinckei andP. chabaudi, in animals immunized with sporozoites of P. berghei.

In view of these results and the lack of information about sporo-zoite-induced immunity in other mammalian malarias, studies on theimmunogenicity of sporozoites of simian malaria were undertaken.

The objectives of the present research project are (1) to determinethe feasibility of the use and the efficiency of an immunization procedureusing irradiated sporozoites of simian malaria, (2) to establish a methodto obtain purified parasite material, and (3) to obtain preliminary dataon the immunogenicity of sporozoites of human malaria.

METHOD

To establish a suitable experimental simian malaria model forimmunologic studies, preliminary experiments are being conducted insquirrel monkeys with the New World simian malarias P. brasilianum andP. simium and in Rhesus monkeys with P. cynomolgi bastianelli. Sporo-zoites of these different malarial species have been obtained fromlaboratory-bred Anopheles stephensi, and serologically negative animalsare being immunized with X-irradiated sporozoites obtained from dissectedsalivary glands. The results of these immunizations will be evaluated bydetermination of both antisporozoite antibody formation and protectiveimmunity against sporozoite challenge.

The purification of sporozoites will be attempted by gradientcentrifugation based on the satisfactory results obtained by investigatorsusing avian malaria sporozoites.

RESULTS TO DATE

The results thus far obtained indicate that animals immunized withsporozoites of P. brasilianum develop antisporozoite antibodies afterrepeated boosters. Positive reactions were also observed in animals

139

whose P. brasilianum infections were acquired in nature and thereforesporozoite-induced. The sera of laboratory-born squirrel monkeys gaveconsistently negative results.

Anti-P. brasilianum sera did not react with sporozoites of P. cynomolgior P. berghei. Clear cross-reactions with sporozoites of P. simium have sofar not been observed.

Our present data on human malaria sporozoites indicate that theybecome attenuated and noninfective at a radiation dose similar to that usedwith simian and rodent malaria sporozoites.

We are just starting to attempt the purification of mammalian malariasporozoites. Initial data indicate that sporozoites of P. berghei remaininfective after being submitted to a bovine serum albumin gradient.

SIGNIFICANCE

Intensification of immunologic research in malaria during recent yearsand additional knowledge obtained mainly through the use of the rodentmalaria system have undoubtedly brought the ultimate goal of vaccinationagainst human malaria much closer to realization.

These and similar experiments to produce and characterize the develop-ment of sporozoite-induced immunity in simian malaria are a crucial testof an approach that potentially could be applied to vaccination of humans.

PUBLICATIONS

1. Nussenzweig, R.S., J. Vanderberg, H. Most, and C. Orton.Protective immunity produced by the injection of X-irradiatedsporozoites of P. berghei. Nature 216:160-62, 1967.

2. Nussenzweig, R.S., J. Vanderberg, and H. Most. Protectiveimmunity produced by the injection of X-irradiated sporozoitesof P. berghei. IV. Dose response, specificity, and humoralimmunity. Milit Med 134:1176-82, 1969.

3. Nussenzweig, R.S., J. Vanderberg, H. Most, and C. OrtonImmunity in simian malaria induced by irradiated sporozoites.J. Parasit 56:252, 1970.

4. Nussenzweig, R.S., J. Vanderberg, G.L. Spitalny, C.I.O. Rivera,C. Orton, and H. Most. Sporozoite-induced immunity in mammalianmalaria: a review. Am J-Trop Med Hyg, in press.

140

5. Spitalny, G.L., and R.S. Nussenzweig. Properties of anti-sporozoite malarial antibodies. Fed Proc 30:303, 1971

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ruth S. Nussenzweig, New York UniversityMedical School, New York, New York

Pan American Health Organization, World HealthOrganization, National Institutes of Health,U.S. Public Health Service, U.S. Army Researchand Development Command

1971 - continuing

FIELD TEST FOR INSECTICIDE EFFECTIVENESS

PROBLEM

In the Caribbean area, there are three main factors blocking theeradication of Aedes aegypti. They are: 1) the resistance of the speciesto DDT and other chlorinated hydrocarbons; 2) the division of the areainto small political units; and 3) the insufficiency of resources toadequately solve public health problems. The presence of resistance isthe key factor. The other problems cannot be solved until an effectiveinsecticide is found.

The purpose of the research program of the Jamaica InsecticideTesting Unit is to: 1) Determine the presence and geographic distributionof DDT resistance; 2) Find candidate insecticides that are effectiveagainst Aedes aegypti by laboratory tests; 3) Field test the candidateinsecticides. The successful candidate insecticides can then be consideredfor use in operational programs.

METHOD

The presence of DDT resistance is determined by testing field-caughtstrains from various localities, using the WHO test kit. Candidateinsecticides were tested in the laboratory using the same proceduresinitially. Since 1956, the results of the WHO Insecticide Testing Schemewere used for picking candidate insecticides for field testing.

The field tests consist of treating villages or urban areas of 200to 300 houses and observing the amount of reduction in the Aedes aegyptiinfestation. The usual eradication procedures are used.

RESULTS TO DATE:

Most areas with Aedes aegypti in the Caribbean, South America, andCentral America have been tested. Among the countries tested, the onlyknown susceptible strains are found in El Salvador and Costa Rica.

To date, 5 candidate insecticides have been field tested against DDT-resistant Aedes aegypti. They are fenthion, Abate, Dursban, bromophos,and Baygon. Fenthion, Abate, and Dursban have proven to be successfulusing the perifocal method. Dursban was significantly superior. It wasfound that a two-month cycle was required for perifocal treatments withthese insecticides. Further tests are in progress for Abate, malathion,Gardona, fenthion, and Dursban.

142

SIGNIFICANCE

The research program has found three insecticides that are effectiveagainst DDT-resistant Aedes aegypti. With these insecticides and thosethat will be tested in the future, the means to eradicate Aedes aegyptiare known.

Further research will determine if modifications in technique willaid in the successful operation of eradication programs.

PUBLICATIONS

1. Zwick, R.W. Susceptibility of two DDT-resistant Aedes aegyptistrains to DDT and deutro-DDT as larvicides in laboratory tests.Mosq News 24(2):202-06, 1964.

2. Zwick, R.W. Evaluation of Aedes aegypti larvicides in variousbreeding containers. Mosq News 24(2):206-11, 1964.

3. Kerr, J. Austin, Solon de Camargo, and Z.H. Abedi. Eradicationof Aedes aegypti in Latin America. Mosq News 24(3):276-82,196-

4. Abedi, Z.H., and R.A. Aarons.as Aedes aegypti larvicides.196M.

Evaluation of four insecticidesWest Indies Med J 13(4):217-28.

5. Tinker, M.E. Effectiveness of Abate, fenthion, and bromophosagainst Aedes aegypti in Village tests. In press.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Milton E. Tinker. Jamaica Insecticide TestingUnit, Pan American Health Organization, Kingston,Jamaica

Pan American Health Organization and the JamaicaMinistry of Health

1962 - continuing

143

SURVEILLANCE AND RESEARCH ON INFECTIOUS DISEASESALONG THE TRANSAMAZON HIGHWAY

PROBLEM

The opening of the Transamazon Highway presents an opportunityto carry out research on the incidence of infectious diseases in a vastarea of virgin forest. The highway construction force and settlerswill work in a variety of ecologic environments, and will almost cer-tainly come into contact with certain zoonotic agents such as arbovirusesand Leishmania. In addition, about 80 per cent of the workers will comefrom other parts of the country, which creates the possibility thatother infectious diseases, e.g., schistosomiasis, and their vectors maybe introduced into areas of the Amazon valley where they have been pre-viously unknown. The main purposes of this research are to studyinfectious diseases enzootic in the highway areas that may be of im-portance to man as well as those that are extraneous to the area butwhich can be introduced by man.

METHOD

A field team is collecting clinical material from people andtrapping animals and arthropods in the highway area.

Virology. Sera from humans, wild mammals, and birds are testedfor Amazon arbovirus groups by hemagglutination-inhibition test. Neu-tralization tests in mice and tissue culture are made in some cases.Attempts are made to isolate virus in mice and tissue culture usingblood from sick persons and wild animals. Hematophagous arthropodsare identified, grouped into species-pools of approximately 50 indi-viduals, and inoculated into mice. Clinical specimens from sick personsand their contacts are also collected for serologic analysis and attemptsto isolate other viruses.

Parasitology. The population is being examined for cases ofleishmaniasis, and Leishmania is isolated by inoculation of biopsiedmaterial into hamsters. Wild animals are also examined for Leishmaniaby culturing techniques and hamster inoculation, which also screenTrypanosoma cruzi infections. Sandflies are trapped, using humansand other animals as bait, and samples dissected to pinpoint localvectors of Leishmania. Blood films are examined in suspected casesof malaria, and fecal samples for helminths and protozoa. Searchesare continuing for foci of snails that may act as intermediaries forSchistosoma.

144

Bacteriology. Fecal samples from humans are brought to Belémin Stuart's medium and cultured in EMB, SS agar, bismuth sulfite agar,and tetrathionate broth (Kauffmann). After biochemical identificationand the maximal serologic analysis possible, strains are sent to anInternational Reference Center. Material from wild animals is takenfrom small and large intestine and examined by the methods just mentioned.Sera of animals are examined for agglutinins against Leptospira by micro-serum-agglutination, and urine cultures are made in Fletcher's mediumfor isolation of Leptospira.

RESULTS TO DATE

Eight strains of arboviruses were isolated from wild birds, in-cluding four strains of SLE, two of Turlock, and one each of Murutucuand An 141106 (as yet unnamed) viruses. High antibody rates to SLE,Turlock, and WEE viruses were also found in birds at some localities.Antibodies to Groups A, B, C, Guamá, Bunyamwera, Guaroa, Simbu,phlebotomus fever, Piry, and Tacaiuma viruses were found in humans. Theimmunity rate was higher in people who had been resident in the studyareas (Marabá and Altamira) for more than six months.

On the parasitologic side, Leishmania was isolated from the skinof three specimens of spiny rat (Proechimys guyannensis) and two specimensof the rodent Oryzomys capito. Two further strains of Leishmania wereisolated from two Institute field workers who contracted the diseaseduring their work in these areas. A number of hemogregarine and piro-plasm infections were noted in marsupials (Didelphis marsupialis). Largenumbers of phlebotomine sandflies were trapped, using human and animalbait, in attempts to pinpoint local vectors of leishmaniasis. Somenew species were encountered.

Examination of stool specimens from people working in the areas

revealed two Schistosoma mansoni infections. Bacteriologic examinationof fecal samples from the captured animals resulted in a single isolationof Salmonella Group C2 from one opossum (D. marsupialis) among 107 animals

examined by culture of small and large intestine. Two isolations ofSalmonella Group B and Group C2 were made after culture of fecal specimensfrom 231 people living in the areas of study. Serologic examination ofsera for antibodies against Leptospira spp. gave significant titers infour of 35 animals studied (Proechimys, Didelphis). Attempts to isolateLeptospira spp. by the inoculation of animal urine specimens in Fletcher'smedium have so far given negative results.

SIGNIFICANCE

These studies should give information on the following points:(1) diseases and their causative agents introduced by the highway

145

construction force and colonists coming from other parts of Brazil,and (2) local diseases and infectious agents of the Amazon area thatmay affect the immigrants.

These studies will comprise epidemiologic investigations ofyellow fever and other arboviruses, enteroviruses, rabies, entero-bacterias (with particular attention to salmonellosis and shigellosis),leptospirosis, malaria, leishmaniasis, Chagas' disease, helminthinfestation (especially schistosomiasis), and febre negra. Investiga-tion will also be made of reservoir hosts among wild animals and ofthe role of certain arthropod vectors.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. M. Martins da Silva (Project Coordinator),Pan American Health Organization, Washington, D.C.,and Dr. Miguel Cordeiro de Azevedo, InstitutoEvandro Chagas, Fundagao Instituto Oswaldo Cruz,Belém, Pará, Brazil

Funded by: Pan American Health Organization and the U.S.Army Medical Research and Development Command


146

FIELD TRIAL OF SPHERULIN, A NEW DIAGNOSTIC REAGENTIN COCCIDIOIDOMYCOSIS

PROBLEM

Coccidioidomycosis is a pulmonary fungal disease that is widespreadthroughout the Americas. An estimated total of 55,000 new symptomaticinfections occur yearly. But diagnosis is very difficult and the actualnumber of symptomatic cases may exceed the figure above many times. Innumerous instances the course and pathologic features of coccidioidomycosisparallel tuberculosis, and many documented cases of the former have beeninitially misdiagnosed as the latter.

A most important means for detecting a subject with Coccidioidesimmitis is the indurated delayed cutaneous response that develops whencoccidioidin is administered intradermally. Coccidioidin, which isderived from the mycelial or saprophytic phase of Coccidioides, plays aprime role also in the diagnosis and even in the prognosis of coccidioidaldisease. We showed previously that spherulin, a derivative of the spheruleor parasitic growth phase, was a superior skin-test reagent in mice andguinea pigs. Its efficacy in man was unknown.

Spherules ordinarily occur in coccidioidal lesions but they can beproduced in a chemically defined medium. Spherulin was prepared from suchspherules. In this project, it was tested in residents of Mexico inregions endemic and nonendemic for coccidioidomycosis.

Spherulin was compared directly with coccidioidin in the same subjects.Histoplasmin was also administered for purposes of cross-reactivity con-trol. Two attributes of the reagents relating to their efficacy in manwere studied in detail: sensitivity, or the capacity of one of the reagentsto elicit reactions either missed or equivocally detected by the other;and specificity, or the property of the reagents of not reacting in un-related infections. The findings showed that spherulin was the reagent ofchoice.

METHOD

Spherulin, 2.8 vg per dose, and coccidioidin (lot 64D4; 1:100), 26 Pgper dose, were administered intracutaneously to five groups of people inMexico. Histoplasmin was administered concurrently in the dose the manu-facturer recommended.

The groups included cavalry troops in Esperanza; Yaqui tribesmen inPotam; and prenursing students in Obregon, all in the state of Sonora,

147

which is highly endemic for coccidioidomycosis. As a negative control,selected residents of Mexico City who had never been in a region endemicfor coccidioidomycosis were tested. Finally, a small group of personswith culture-proved coccidioidomycosis was studied.

RESULTS TO DATE

In the endemic regions, spherulin (2.8 Vg) detected numerous re-actors missed by coccidioidin (25 ig). Thus 61.1 per cent of 113 cavalrytroops showed a positive reaction to spherulin compared to 37.2 per centwho reacted to coccidioidin. In 83 Yaqui tribesmen, the positive ratewas 63.8 per cent with spherulin compared to 44.5 per cent with coccidioidin.And, among 47 prenursing students, the rate was 74.4 per cent positive withspherulin compared to 46.8 per cent positive with coccidioidin.

In the negative control group, living in the nonendemic Mexico Cityregion, none of 90 residents reacted either to spherulin or to coccidioidin.

No evidence of cross-reactivity between spherulin and histoplasminreactors was found, nor was evidence that dermatophytoses, tuberculosis,chromomycosis, actinomycosis, Hansen's disease, scabies, impetigo, or acneproduced a positive reaction with spherulin. Spherulin appeared to behighly specific. In cases where subjects reacted to both spherulin andcoccidioidin, the reaction was usually stronger to spherulin. Additionally,some persons responded equivocally to coccidioidin but were frankly posi-tive to spherulin.

In Mexico City, three culture-proved disseminated cases of coccidioi-domycosis with pulmonary, bone, and cutaneous involvement were studied fortheir reactions to coccidioidin and spherulin. All had been previouslydetermined to be nonreactive to coccidioidin. This nonreactivity tococcidioidin was confirmed again in the present study. However, in onecase, in which the patient had shown striking improvement, the skinsensitivity reaction became very strongly positive to spherulin (55 mm x40 mm induration) even though it remained negative to coccidioidin.

SIGNIFICANCE

This research has disclosed a new, more sensitive reagent fordetecting human experience with Coccidioides immitis. The enhanced sen-sitivity permitted the detection of such experience in cases whereresponse to coccidioidin was frankly negative or equivocal. Thus, con-sidering the role of the skin test in identifying respiratory disease,spherulin offers an improved diagnostic tool and a more precise reagentfor epidemiologic studies. The value of spheruliú in prognosis remainsas yet unknown, although in one case of disseminated coccidioidomycosisthat showed dramatic improvement, spherulin detected cellular reactivitywhere coccidioidin failed to do so.

PUBLICATIONS

Manuscript in preparation.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Hillel B. Levine, University of California,Oakland, California


1972 - continuing

149

STUDIES ON ARTHROPOD-BORNE VIRUSES

PROBLEM

Arboviruses, including Venezuelan encephalitis and St. Louisencephalitis viruses and others such as dengue, have caused extensiveepidemics in the Western Hemisphere in recent years.

The present investigations in Central America have the followingpurposes: (1) to study the ecology, geographic distribution, and importanceto man and domestic animals of Venezuelan encephalitis virus and severalother recently discovered arboviruses; (2) to determine the factors in-volved in virus movement from one area to another; and (3) to identify thefactors that may affect the virulence of geographic isolates of Venezuelanencephalitis virus.

METHOD

The program involves the collection of sera for detection of anti-bodies and the gathering of specimens likely to yield arboviruses--such asmosquitoes, birds, and mammals in Central America. Sentinel animals suchas Syrian hamsters are exposed in coarse wire-mesh cages hung under pro-tective roofs in suitable habitats for the detection of virus transmissionby flying arthropods. Virus isolations are carried out in suckling miceand in various cell culture systems. Antibody tests are performed usingneutralization, hemagglutination inhibition, and complement fixationtechniques.

RESULTS TO DATE

The first major epizootic-epidemic of Venezuelan encephalitis virusin Central America (caused by a South American subtype new to the region)was studied in Guatemala in July and August and in November 1969 incooperation with the University of San Carlos School of Medicine (Guatemala)and other groups of investigations. From materials previously collected inthe northwestern and the southern Pacific lowlands of Mexico (and in 1969from northeastern Mexico), it was demonstrated that those areas were highlysusceptible to invasion by Venezuelan encephalitis virus. In July 1970,in cooperation with personnel from the Mexican National Institute ofVirology, the first isolations of the epidemic subtype was made in interiorChiapas, close to the Guatemalan-Mexican border during the early days ofthe 1970 epizootic. From July to October 1970, the virus spread widelythrough the southern and Pacific coastal regions of Mexico, causing 5,000equine cases (Mexican Department of Agriculture figures) and human disease.

150

Deaths of children were also reported. In April of 1971, in cooperationwith the Mexican and the U.S. Departments of Agriculture, equine deathsin northern Veracruz state near the coast of the Gulf of Mexico wereinvestigated. The agent was isolated from horses and a febrile veteri-narian and identified as Venezuelan encephalitis virus. The virussubsequently spread throughout much of central and northern Mexico and oninto Texas.

During field seasons of 1970 and 1971, Venezuelan encephalitis viruscontinued to be active in endemic foci on the Pacific lowlands of Guatemala.Studies are in progress to determine if the epizootic subtype has developedenzootic cycles in that area. Cattle and pigs have been experimentallyinoculated with epidemic strains of Venezuelan encephalitis virus to deter-mine their possible roles as amplifying hosts.

SIGNIFICANCE

The research provides information on arboviruses, especially Vene-zuelan encephalitis virus, in Central America, and it evaluates the role ofbirds and domestic animals in the ecology of arboviruses. Complete under-standing of the natural cycles of arboviruses will make way for intelligentapproaches to the prevention and control of epidemics caused by these agents.

PUBLICATIONS

1. Scherer, W.F., and B.A. Pancake. Cross-protection among virusesof the Venezuelan equine encephalitis complex in hamsters.Amer J Epid 91:225-29, 1970.

2. Scherer, W.F., R.W. Dickerman and J.V. Ordoñez. Discovery andgeographic distribution of Venezuelan encephalitis virus inGuatemala, Honduras, and British Honduras, 1965-1968, and itspossible movement to Central America and Mexico. Am J Trop

Med and Hyg 19:703-11, 1970.

3. Ordoñez, J.V., W.F. Scherer, and R.W. Dickerman. Isolation ofeastern encephalitis virus in Guatemala from sentinel hamstersexposed in 1968. Bol Ofic Sanit Panam 70:371-75, 19T71.

4. Dickerman, R.W., and W.F. Scherer. Serologic survey for anti-bodies to VE virus in western and lnorth-central Mexico. BolOfic Sanit Panam 70:550-56, 1971.

151

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. William F. Scherer, Cornell UniversityMedical College, New York, New York.

United States Army Medical Research and DevelopmentCommand

1961 - continuing

152

BIRDS IN THE ECOLOGY OF NORTH AMERICANENCEPHALITIS VIRUSES

PROBLEM

The effectiveness of wild birds in the natural history and distribu-tion of arboviruses is related to the rapidity of their population turnover,their ability to move quickly over long distances, the regularity of theirmigrations, and their temporal association and mixing with other animalsand with human populations. However, there is still a lack of criticalinformation concerning life histories of many avian species. Likewise,there is a need for biologically sound experimental infection of birds witharboviruses. The purpose of this research program is to evaluate theinvolvement of birds in the natural cycles of arboviruses.

METHOD

Field research in July and August, 1970 and 1971, was carried out onthe Pacific lowlands of Guatemala, at a site where Venezuelan encephalitisvirus demonstrated to be endemic prior to the 1969 Central Americanepizootic. Experimental studies have been continued in the aviary todetermine reactions of birds of different species and ages to arboviruses;to determine the minimal dosage needed to infect a susceptible avian host;to analyze immunoglobulin production following infection. Laboratorystudies of field materials have continued.

RESULTS TO DATE

Preliminary serological surveys of avian sera from Mexico andGuatemala indicate birds were infected with western, eastern, St. Louis,and California encephalitis viruses as well as others of as yet un-evaluated importance in public health. In experimental inoculation studies,younger herons (5-10 days of age) have been found to develop higher levelsof viremia than do slightly older birds (15-30 days of age). Variationsin levels of viremia among 3 species of herons have not been constant. Verylow levels of Venezuelan encephalitis virus are required to infect youngBlack-crowned Night Herons and to stimulate levels of viremia similar tothose stimulated by far higher levels of inoculum. IgM (19S) immuno-globulins appear in some common Egrets by day 7, post-inoculation, andIgG (7S) appear by day 14. The 19S antibody persists to about day 12-14after which only IgG is detected. Both types of immunoglobulin react inHI and NT antibody tests.

153

SIGNIFICANCE

Virus isolation and serological studies of resident and migratorybirds from Mexico indicate little involvement in natural cycles ofVenezuelan encephalitis virus, but rather extensive involvement in cyclesof St. Louis encephalitis virus. The moderate to high viremia levelsfollowing inoculation with mosquito-sized doses of Venezuelan encephalitisvirus developed by nestling herons 5-10 days of age are more than adequateto infect even some nonvector species of mosquitoes, and emphasize thepotential for age colonies of young of this group to act as foci ofamplification.

PUBLICATIONS

1. Dickerman, R.W., and W.F. Scherer. Serologic survey for antibodiesto VE virus in western and northcentral Mexico. Bol Ofic Sanit

Panamer 70:550-553, 1971.

2. Dickerman, R.W., M.L. Zarate, W.F. Scherer, andVenezuelan encephalitis virus along the centralgulf coast of Mexico as of July-September 1969.Panamer 71:143-150, 1971.

J. de Mucha Macias.and northernBol Ofic Sanit

3. Dickerman, R.W., and L.C. Juárez. Nesting studies of the Boat-billed Heron Cochlearius cochlearius at San Blas, Nayarit,Mexico. Ardea 59:1-16, 1971.

4. Dickerman, R.W., W.F. Scherer, andstudies of Venezuelan encephalitisMexico. 1. Introduction and study

Hyg 20:730-739, 1971.

A. Diaz-Nájera. Ecologicviruses in southeasternsites. Am J Trop Med

Note:

See also publications of Research Training Program in Virology,Ornithology, Ecology, and Tropical Medicine, page 334

OTHER DATA

Grantee: Dr. Robert W. Dickerman, Cornell University MedicalCollege, New York, New York

Funded by:

Timetable:

National Institutes of Health/U.S. Public Health Service

1972-1977

154

HERPESVIRUSES OF SOUTH AMERICAN MONKEYSAND MALIGNANT LYMPHOMAS

PROBLEM

Viruses are known to induce neoplastic processes in several animalspecies. However, viruses so far have not been proven to be oncogenicin man, although enough indirect evidence has been accumulated to indicatethat this is possible.

The first evidence that a nonhuman primate derived virus: Herpes-virus saimiri can induce malignant lymphoma and leukemia in other non-human primates was obtained by Melendez et al. in 1967 (Herpesvirussaimiri. II. Experimentally induced malignant lymphoma in primates, byL.V. Melendez, R.D. Hunt, M.D. Daniel, F.G. Garcia and C.E.O. Fraser.Lab Anim Care 19:378-386, 1969). This finding has proved and providedone of the best animal models to study oncogenic processes in nonhumanprimates.

The occurrence of lymphosarcoma in two individuals who were caringfor spider monkeys (Ateles geofroyii) in Guatemala, led us to searchfor Herpesvirus saimiri in these animals, its transmission to man, andits likely etiological role in the development of this malignant disease.

METHOD

1. Tissue specimens (human lymph node biopsies and spidermonkey kidney renal cortex) were collected in Guatemala and in Southboro,Massachusetts, to prepare in vitro cell cultures for isolation ofH. saimiri.

2. Sera was collected from man and spider monkeys to test forantibodies to H. saimiri.

3. Viral isolation and serum neutralization tests and fluorescentantibody tests were conducted in cell lines susceptible to H. saimiriwhich were developed at the Southboro laboratories.

RESULTS TO DATE

1. A human lymph node cell line was obtained from one of thepatients affected with lymphosarcoma.

2. Two new herpesviruses were found in the spider monkey species.One of these viruses was obtained from cultures prepared in Guatemala

155

and is known as Guatemala isolate AT-46. The other was obtained fromsimilar monkey kidney cultures prepared in Southboro. This new agentis known as Herpesvirus ateles.

3. Evidence that the human patients were infected with H. saimiriwas not obtained nor was this agent isolated from any of the human ormonkey cultures.

4. Marmoset monkeys (Saguinus oedipus) inoculated with H. atelesdeveloped malignant lymphoma and leukemia, thus a second lymphoma virusof monkeys was found.

SIGNIFICANCE

These studies have indicated for the first time the following:

(1) The spider monkey species (Ateles geofroyii) is thereservoir host for two new herpesviruses: Guatemalaisolate AT-46 and Herpesvirus ateles.

(2) Herpesvirus ateles is able to induce a neoplastic diseasein cotton-top marmoset monkeys: leukemic and aleukemicmalignant lymphoma developed in these animals afterinoculation with this agent.

(3) The existence of a second lymphoma virus of monkeys(LVM-2) was discovered.

(4) South American monkeys are excellent animal models to studyfor oncogenic diseases of man: malignant lymphoma andleukemia.

PUBLICATIONS

1. Melendez, L.V., H. Castellanos, H.H. Barahona, M.D. Daniel,R.D. Hunt, C.E.O. Fraser, F.G. Garcia, and N.W. King. Twonew herpesviruses from spider monkeys (Ateles geofroyii).Presented at the Symposium on Viruses of South AmericanMonkeys. Pan American Health Organization, Mexico City,Mexico, August 11-13, 1971.

2. Daniel, M.D., L.V. Melendez, and H.H. Barahona. Plaquecharacterization of viruses from South American nonhumanprimates. Presented at the Symposium on Viruses of SouthAmerican Monkeys, Pan American Health Organization, MexicoCity, Mexico, August 11-13, 1971.

156

3. Hunt, R.D., and L.V. Melendez. Clinical, epidemiological,and pathological features of cytocidal and oncogenicherpesviruses in South American monkeys. Presented at theSymposium on Viruses of South American Monkeys, Pan AmericanHealth Organization, Mexico City, Mexico, August 11-13, 1971.

4. King, N.W., M.D. Daniel, H.H. Barahona, and L.V. Melendez.Viruses from South American monkeys: ultrastructural studies.Presented at the Symposium on Viruses of South American Monkeys,Pan American Health Organization, Mexico City, Mexico, August

11-13, 1971.

5. Melendez, L.V., R.D. Hunt, F.G. Garcia, M.D. Daniel, C.E.O.Fraser, H.H. Barahona, and N.W. King. Herpesvirus ateles,a new lymphoma virus of monkeys. Presented at the InternationalSymposium on Comparative Leukemia Research, Padua, Italy,September 13-17, 1971.

OTHER DATA

Grantee: Dr. Luis V. Melendez, Harvard Medical School,New England Regional Primate Research Center,Southboro, Massachusetts

Funded by: National Institute of Health/U.S. Public HealthService, and Pan American Health Organization


157

IDENTIFICATION OF TYPES AND SUBTYPESOF FOOT-AND-MOUTH DISEASE VIRUS

PROBLEM

The plurality of strains of foot-and-mouth disease virus calls forthe continuous typing of specimens taken from sick animals. The spreadof the virus in the vast stock-raising zones of South America, whereecologic conditions vary and the cattle population is of different breeds

and susceptibilities, at times forces the virus strains to undergo trans-formations to subsist.

The identification of strains whose serologic and immunologicbehavior may differ considerably from the prototypes used for vaccine

production is of great importance to the expanded vaccination programsof various countries.

METHOD

The following methods are used for foot-and-mouth disease virus

typing, 50 per cent complement fixation (serologic method); serumprotection of suckling mice, and cross-immunity in guinea pigs andcattle (immunologic methods); passages in guinea pigs, suckling mice,and/or cattle; induction of homologous sera in guinea pigs.

RESULTS TO DATE

A total of 11,000 specimens have been typed and subtyped. These

specimens were received from 17 countries or were available at the PanAmerican Foot-and-Mouth Disease Center.

To date, the following new subtypes of foot-and-mouth disease virushave been identified:

Subtypes of type O Vallée

08 Brazil/60

Subtypes of type A Vallée

A1 7 Brazil/60

418 Venezuela/62

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A13 Brazil/58

A16 Brazil/59

Aig Argentina/63A19

A29 Peru/69

A24 Brazil/55 A30 Uruguay/68

A25 Argentina/59 A31 Colombia/69

A26 Argentina/66 A32 Venezuela/70

A27 Colombia/67

Subtypes of type C Waldmann

C3 Brazil/55

C4 Argentina/66

C5 Argentina/69

These subtypes were recognized by the World Reference Laboratoryat the Animal Virus Research Institute, Pirbright, England, which hasassigned the designations above in the FMDV classification.

SIGNIFICANCE

Results of this investigation point to the importance of continuousscreening of field viruses, as well as the control of the strains usedfor production and control of vaccines. Many subtypes differ from othersto such an extent that it is impossible to reach a correct classificationif specific sera are not used in the complement fixation test. Theserologic difference corresponds to immunologic difference. Therefore,if a new subtype appears in the course of a vaccination campaign andis not recognized as such, and if appropriate measures are not takenimmediately, it may seriously endanger the success of the program.

The Center is collaborating with affected countries and is supplyingthem with subtype-specific sera for subtype identification, and for theproduction and control of vaccines.

PUBLICATIONS

1. Alonso, A.F., K.E. Federer, I. Gomes, and A. Vieira.Comparación inmunológica de dos subtipos del virus aftosotipo C Waldmann. Boletin del CPFA 4:1-10, 1971.

159

2. Federer, K.E., J. Saile, and M.N. Honigman. Identification

d'un nouveau sous-type A de virus aphteux. Bull Off Int Epizoot

57:1171-90, 1962.

3. Federer, K.E., J. Saile, and I. Gomes. Identificación de un

nuevo subtipo "O" del virus de la fiebre aftosa en Sud-América. Bull Off Int Epizoot 61:1563-78, 1964.

4. Federer, K.E., A.F. Alonso, L. Pustiglione Netto, and A.A. Pinto.

Développement d'un nouveau sous-type du virus de la fievre

aphteuse par passage en séries sur bovins partiellement immuns.

Symposium on foot-and-mouth disease virus variants, Lyon, 1967.

5. Lucam, F., G. Dannacher, J.P. Thomas, N. Magallanes, E. Giambruno,J. Marin, K.E. Federer, and A.F. Alonso. Etude immunologique

des sous-types du virus aphteux: Principe-methode-application.Bull Int Off Epizoot 75:1-20, 1971.

6. Sutmuller, P., P.A. de Mello, M.N. Honigman, and K.E. Federer.

Infectivity for cattle and pigs of three strains of foot-and-

mouth disease virus isolated from carrier cattle. Am J Vet Res

28:101-05, 1967.

OTHER DATA

Grantee: Pan American Foot-and-Mouth Disease Center,Rio de Janeiro, Brazil

Funded by:

Timetable:


1957 - continuing

160

IDENTIFICATION OF TYPES AND SUBTYPESOF VESICULAR STOMATITIS VIRUS

PROBLEM

In areas in which both foot-and-mouth disease and vesicularstomatitis viruses are present, differential diagnosis is of greatimportance in deciding on the veterinary control measures to be adopted.The verification of vesicular stomatitis foci in countries where thedisease is not endemic, such as Brazil and Argentina, has provided anopportunity to demonstrate for the first time the existence of vesicularstomatitis virus subtypes.

METHOD

The following methods are used for vesicular stomatitis virustyping, 50 per cent complement fixation and overnight complement fixation(serologic methods); serum neutralization and serum protection in adultmice (immunologic methods); passages in guinea pigs, in adult and sucklingmice, and in tissue cultures; preparation of homologous sera in guineapigs; and plaque-count titrations under various agar overlays.

RESULTS TO DATE

As a result of the serologic research at the Center, the two differ-ent vesicular stomatitis virus (VSV) strains identified in samples fromArgentina and Brazil have been classified as new subtypes of the Indianaserotype. The classification Indiana I now refers to the classicalstrains, while Indiana II is used for the Argentine and Indiana III forthe Brazilian virus.

The difference between these strains is so marked that it isadvisable to use them as subtype-specific sera in screening samples forthe identification of vesicular stomatitis.

When various VSV strains were plated under agar containing dextransulfate or DEAE-dextran, it was found that the New Jersey, Indiana I,and Indiana II strains were producing plaques whose diameter was not toodifferent from that of plaques produced under standard conditions. Thediameter of the plaques produced by Indiana III strain, however, wasdrastically reduced in the presence of dextran sulfate and increasedupon incorporation of DEAE-dextran in the agar overlay.

161

SIGNIFICANCE

The rapid diagnosis of vesicular stomatitis samples receivedfrom countries free of foot-and-mouth disease is of paramount importanceand allows institution of suitable veterinary control measures.

In the case of Indiana subtypes I, II, and III, it has been found

that some of the field viruses are so subtype-specific that they do notreact at all with standard sera. For this reason, the Center suppliesnational diagnostic laboratories with a sufficient amount of subtype-specific sera to enable them to do their own typing.

PUBLICATIONS

1. Federer, K.E., R. Burrows, and J.B. Brooksby. Vesicular

stomatitis virus: relationship between strains of Indianaserotype. Bull Off Int Epizoot 65:879-80, 1966.

2. Federer, K.E., R. Burrows, and J.B. Brooksby. Vesicularstomatitis virus: the relationship between some strainsof the Indiana serotype. Res Vet Sci 8:103-17, 1967.

3. Gomes, I., and A. Vieira. Effect of polyionic compoundson the plating efficiency of some strains of vesicularstomatitis virus. Arch Ges Virusforsch 34:223-31, 1971.

OTHER DATA




162

DETERMINATION OF FOOT-AND-MOUTH DISEASE AND VESICULARSTOMATITIS ANTIBODIES IN THE SERUM OF CATTLE,

HORSES, AND SHEEP

PROBLEM

The Marucci method provides clear data for the detection of anti-bodies to foot-and-mouth disease (FMD) in cattle, sheep, and swine, evenin convalescent animals. Moreover, this method is appropriated forantibody diagnosis in cattle and horses recovering from vesicular stoma-titis.

We investigate CF inhibition and the single and double diffusionin agar gel for the antibody diagnosis for foot-and-mouth disease andvesicular stomatitis in vaccinated or convalescent cattle, sheep, swine,and horses, and to establish the correlation with seroprotection andseroneutralization results.

METHOD

The following techniques are used: CF 50 Ser cent hemolysis with

incubation at 370 C and combined incubations at 4 C and 37°C; inhibitionof FC 50 per cent hemolysis; single and double diffusion in agar gel;and seroprotection and seroneutralization.

RESULTS TO DATE

Through FC 50 per cent hemolysis, combining incubations at 40

and 37 C, it is possible to detect antibodies in cattle, sheep, and swinerecovering from foot-and-mouth disease. This technique is highly sen-sitive and specific for antibody detection in cattle and horses recovering

from vesicular stomatitis, allowing it to be substituted for seroneutrali-zation, A diagnosis can be obtained in a few hours.

The inhibition of FC 50 per cent hemolysis is highly sensitive and

specific for detection of antibody in cattle, sheep, swine, and guineapigs that have been vaccinated or are recovering from foot-and-mouthdisease.

The single diffusion in agar gel is more sensitive than the double

in the diagnosis of antibodies against foot-and-mouth disease and vesicularstomatitis.

Both techniques indicate the antigenic composition of foot-and-mouth disease viral suspensions.

163

SIGNIFICANCE

When it is impossible to collect infectious material for diagnosis,it may be possible to perform a fast diagnosis in serum samples, usingCF', CF' inhibition, and diffusion in agar gel techniques.

PUBLICATIONS

1. Federer, K.E., R. Burrows, and J.B. Brooksby. Vesicularstomatitis virus: the relationship between some strains ofthe Indiana serotype. Res Vet Sci 8:103-17, 1967.

2. Gomes, I., and A. Vieira. Effect of polyionic compounds onthe plating efficiency of some strains of vesicular stomatitisvirus. Arch Ges Virusforsch 34:223-31, 1971.

OTHER DATA


Funded by:

Timetable:


1957 - continuing

164

LATENT INFECTIONS IN FOOT-AND-MOUTH DISEASE

PROBLEM

Natural foot-and-mouth disease (FMD) virus infections in cattle andother ruminant species result in postconvalescent viral persistence inthe pharyngeal region of many animals. Detecting these persistent orlatent infections in 50 per cent of the infected animals five or sixmonths afrer the acute clinical phase of the disease is not unusual. Theduration of the infections varies considerably among individuals, andthe longest reported duration is two years.

The purpose of these studies is to (a) develop more sensitivemethods of detection of latent infections, (b) identify variables thatinfluence the duration of latent infections, (c) establish the epide-miologic significance of the infections, and (d) impart through trainingand technical assistance the knowledge gained in (a), (b), and (c).

METHOD

The majority of specimens are obtained using the apparatus describedby Van Bekkum, which permits the collection of a mucosaliva mixture fromthe esophagus, pharynx, larynx, and mouth. These specimens are extractedwith an organic solvent to remove debris and extraneous proteins, and toreactivate virus neutralized by antibody.

Virus isolation is carried out in suckling mice and various cellcultures. Identification of isolates is performed by complement fixation.

RESULTS TO DATE

1. Cattle vaccinated with modified live-virus vaccines becomechronically infected with the vaccine virus and serve as a source ofinfection for unvaccináted contact animals.

2. In a limited number of cattle, mixed chronic infections havebeen found in which two strains of virus can be isolated from a singleindividual.

3. Preliminary results indicate that viral adsorbents can bepractically employed to concentrate virus in the clinical specimens.

4. Attempts to isolate virus from lymphatic tissues of chronicallyinfected animals have failed.

165

5. A method of increasing the sensitivity of certain cell culturesto aftosa virus has been developed and is currently being evaluated inlatent infection detection.

SIGNIFICANCE

The study of FMD latent infections by many research workers through-out the world has not yet clearly established the epidemiologic importanceof persisting infections. In controlled laboratory experimen;ts, chronicallyinfected animals have not been shown to transmit virulent FMD virus tocontact susceptible animals. It therefore is of prime importance to betterunderstand the role of this natural virus reservoir as a possible initiatorof new outbreaks. It must be remembered that the environmental selectivepressures imposed upon the virus during chronic residence pose a potentialthreat to control of the disease with vaccines. If calculated approachesto prevention and control of FMD are to be made, therefore, it will benecessary to better understand the latent infections and their influenceon the virus and the disease.

PUBLICATIONS

1. Augé de Mello, P., M.N. Honigman, M.V. Fernandes, and I. Gomes.Further information on the survival of modified Foot-and-Mouth Disease Virus in Cattle. Bull Off Int Epizoot 73:489-505,1970.

OTHER DATA




166

STUDIES OF INACTIVATED VACCINES AGAINSTFOOT-AND-MOUTH DISEASE

PROBLEM

In countries where foot-and-mouth disease (FMD) is enzootic,control is based on systematic vaccination of susceptible hosts. Thevaccines most commonly used in South America are inactivated bi- ortrivalent preparations. These vaccines confer immunity in cattlefor only relatively short periods of time and are a weak antigen forswine. The improvement in quality of inactivated FMDV vaccines istherefore desirable.

METHOD

Studies are being carried out to find better methods of virusinactivation and new adjuvants. First-order inactivants are comparedwith conventional formalin and the adjuvant effect of oil-Arlacel iscompared with aluminum hydroxide. In addition, hyperpotentiation ofthe FMDV with polycationic-nucleic acid complex or cationic polymers isanalyzed.

Young rabbits are commonly used as source of antigen in Brazil.The Center has closely followed field vaccination campaigns to obtainmore information about the effectiveness of rabbit-antigen vaccine,and has assisted the Ministry of Agriculture in solving variousproblems related to the purification and concentration of the antigen.

Susceptible animals are used in these experiments, and the levelof antibody as well as protection against challenge are assessed.

RESULTS TO DATE

FMDV vaccines inactivated with acetylethyleneimine and containingan oil-Arlacel adjuvant are considerably more efficient in cattle thanstandard formalin-aluminum hydroxide vaccines, especially in durationof immunity. In swine, vaccines adjuvanted with aluminum hydroxide arenot effective in eliciting immune response but those emulsified in oilare. The potentiating effect of the nucleic acid polymers is mostevident when applied in oil emulsion.

Some preliminary data were obtained with antigen produced inyoung rabbits, but they are not sufficient for a determination of theamount of antigen necessary to induce immunity in cattle. The durationof immunity with this type of vaccine is another point that is not yetknown.

167

SIGNIFICANCE

The FMD vaccine preparations currently available have a shortimmunity and are expensive. It is hoped that the present research willlead to improvement of these products through the use of more efficientand less expensive culture media as well as better methods of inactivation.We also hope to increase the potency and time span of vaccine-inducedimmunity. These are important factors in the development of adequatemethods of cattle protection.

PUBLICATIONS

1. Callis, J.J., P.D. McKercher, J.H. Graves, and M.V. Fernandes.Foot-and-mouth disease virus--Immunization. Proc, World VetCong, Mexico City, August 1971.

2. Maes, R., and M.V. Fernandes. Viability of foot-and-mouthdisease virus in oil emulsions. Proc Soc Exp Biol Med132:447-49, 1969.

3. Maes, R., P. Augé de Mello. and I. Gomes. Hyperpotentiationof antibodies against FMDV in swine with polycationic-nucleicacid complex. Proc Second Internat Cong Virology, Budapest,1971.

OTHER DATA




168

IMPROVEMENTS OF THE EFFICACY OF INACTIVATEDFOOT-AND-MOUTH DISEASE VACCINES

PROBLEM

Many vaccines are available that will induce protection againstfoot-and-mouth disease (FMD) in adult cattle and older calves if thevaccines are properly prepared and properly used. However, theduration of immunity and the required frequency of revaccination withmany of these vaccines now leave something to be desired. In addition,there is currently no available method to effectively vaccinate youngcalves in any consistent manner.

METHOD

To enhance the immunization powers of inactivated vaccines, threedifferent approaches are now being tried in cattle. The first approachis to increase the actual amount of antigenic mass in the vaccines.The second method is to increase the effective antigenicity of thevaccine by aggregation of the virus particles in preparing the vaccine.The third means of increasing the efficacy of vaccines is to improve theaction of the adjuvants used, both by trying new vaccination methodsand by using other adjuvants. Some of our work includes methods usedin other parts of the world that have not been tried in South America.

To develop an acceptable vaccination procedure for young calves,work is first being done to determine if very young calves can respondto FMD vaccination by any means. Attempts are also being made todevelop a practical means of inducing an immune response in calves evenin the presence of maternal antibodies or maternal nonspecific inhibitors,both of which are known to interfere with conventional means of vaccination.This work includes efforts to determine the nature of nonspecific inhi-bitors found in the serums of susceptible cattle.

RESULTS TO DATE

Preliminary work in a limited number of animals has demonstratedthat inactivated vaccines can be prepared that will protect cattle forseveral months following one primary vaccination. Further work with thistype of vaccine is in progress.

169

SIGNIFICANCE

Efforts are being made to improve the efficacy of vaccines usedin older cattle and to develop a means of effectively vaccinatingyoung calves against FMD virus.

PUBLICATIONS

None thus far.

OTHER DATA


Funded by:

Timetable:


1971 - continuing

170

DEVELOPMENT OF MODIFIED LIVE VIRUS VACCINESAGAINST FOOT-AND-MOUTH DISEASE

PROBLEM

The immunization of cattle susceptible to foot-and-mouth diseasehas been repeatedly shown to be possible through the use of modifiedlive-virus vaccines. Virus multiplication in the organism generallyproduces a solid and durable immunity which greatly improves thepossibility of foot-and-mouth disease control in the South Americancountries.

METHOD

The three types of foot-and-mouth disease virus existing in SouthAmerica (O Vallée, A Vallée, and C Waldmann) are adapted to chickenembryonated eggs by intravenous inoculation at 14 days, alternating inadaptation and modification with passages in suckling mice. tissuecultures, and one-day-old chicks, and are finally cloned. Initially theincubation temperature is approximately 34°C, but it is lowered to 28°Cfor studies of cold mutants and markers using tissue cultures. Themodification is accelerated using limiting dilutions and cloning to obtaingenetically more stable strains. Samples are studied in susceptibleanimals from the standpoint of safety, pathogenicity, and immunogenicpower.

The guinea pig was used in the first experiments. We inoculatedit by intramuscular, subcutaneous, or intradermoplantar routes to observethe animal's infectivity, virus tropism (cardiotropism), and immuneresponse. We used serum antibody studies and generalized foot lesions.

Viremia, contagiousness, pathogenicity (postvaccinal reactions),and immunity are studied in cattle by intradermolingual, intramuscular,subcutaneous, oral, and intranasal inoculation and by contact.

Subcutaneous, intramuscular, or intraplantar inoculation is alsoused in swine to study the same aspects.

Vaccines are prepared using the hearts and livers of chickenembryos, suckling mice, and tissue cultures. The materials are titrated,treated with chloroform, and suspended in 50 per cent buffered glycerin,and then centrifuged. The supernatant liquid is used as immunizingmaterial. The vaccine is used in monovalent, bivalent, and trivalentform, according to the requirements of the region or country. The vacci-nation dose is between 107-0and 10. 0 LD50/SM per dose of each virustype.

171

RESULTS TO DATE

The vaccines produced with modified live virus show a widerantigenic spectrum in comparison to inactivated vaccines. This is thecase in experiments performed at the laboratory level in cattle usingthe modified A24 Cruzeiro strain, which antigenically covers theheterologous subtypes A18, Alg, A24, A27, and A32 and the A-ValléeChile, Ecuador, Colombia, and Venezuela strains.

This vaccine shows good immunity in primovaccinated and re-vaccinated young and adult animals, in which immunity was more solid atthe end of one year.

Postvaccinal reactions diminished markedly in studies of clonesin highly sensitive animals and by the selection of the most convenientclone, both from the point of view of pathogenicity and antigenicity.

The strains so far studied at the laboratory and field levelhave not caused disease in contact animals. The role of the oil adjuvantin the immunity was studied in laboratory animals and cattle, and wasfound to be a good agent for obtaining a better immunity. The study ofsuch substances as Polythylene glicol and Furane 113 was started to con-centrate and purify the virus to be used in the modified live virusvaccines.

Vaccines prepared with 50 per cent buffered glycerin and thenlyophilized proved to be- very stable even at high temperatures.

SIGNIFICANCE

Vaccination with modified live virus may be a tool in the controlof foot-and-mouth disease, mainly in those regions where vaccination atshort intervals is difficult and farm management, high environmentaltemperature, and remote locations are an obstacle to conservation of thevaccine.

The vaccine can be lyophilized which permits conservation and easytransportation. The immunity obtained is very durable, especially inrevaccination, and covers many different virus subtypes. The vaccineis very economical and easy to prepare.

PUBLICATIONS

1. Bernal López, C., R.G. Cunha, M.N. Honigman, and I. Gomes.Estudio sobre la modificación de una cepa de virus de fiebreaftosa (tipo "A" Vallée) y su utilización como vacuna. Proc VCong Panamer Med Vet Zootech, Caracas, 1966.

172

2. Cunha, R.G., I. Torturella, C. Bernal L6pez, and M.N. Honigman.

Quelques aspects de l'application du vaccin anti-aphteux avirus vivant atténué. Bull Off Int Epizoot 57:733-47, 1962.

3. Cunha, R.G., C. Bernal López, M.N. Honigman, and I. Gomes.

Vaccination bivalente au moyen de deux souches modifiees devirus aphteux. Bull Off Int Epizoot 61:801-18, 1964.

4. Cunha, R.G., C. Bernal López, M.N. Honigman, and I. Gomes.Studies on a rabbit-adapted foot-and-mouth disease virus of

type C Waldmann. I. Adaptation and immunogenicity. Canad

Comp Med Vet Sci, December 1964.

5. Maes, R.F., P. Augé de Mello, I. Gomes, C. Bernal López,

and K. Costa Freitas. Hyperpotentiation of antigenicity of

foot-and-mouth disease virus in swine with polycationic-nucleic acid complexes. In preparation.

OTHER DATA


Funded by:

Timetable:


1957 - continuing

173

LABORATORY CONTROL OF FOOT-AND-MOUTH DISEASE VACCINES

PROBLEM

It is absolutely necessary to use the best tools available incampaigns against foot-and-mouth disease (FMD). To guarantee anoptimal outcome, effective control of the vaccine applied in the fieldis necessary. It is thought that partially immune animals may be oneof the main causes for the development of subtypes.

Foot-and-mouth disease vaccine quality control is performed throughdirect methods, but it can also be done through indirect methods ifthe results are similar or comparable with the direct methods.

We intend to correlate the direct and indirect methods, analyzingthe quality of different preparation systems of inactivated and modifiedlive-virus vaccines.

METHOD

The following techniques are used:

Direct methods for cattle vaccines: modified K-index analyzinglocal and general immunity.

Direct methods for swine vaccines: inoculation of 40,000 LD 5SM,and new techniques to be investigated.

Indirect methods for cattle and swine vaccines: seroprotection andseroneutralization indices. and guinea pig (I/C) index.

RESULTS TO DATE

Mouth (primary) lesions in cattle appear before hoof (secondary)lesions. Local immunity is less solid than general immunity, but never-theless there is a correlation between these two. Both the seroprotectionand the seroneutralization tests maintain a correlation with the directmethods, that of the seroprotection being closer.

The guinea pig (I/C) index allows for FMD vaccine control and thusthe dispensing of cattle for that purpose. With the same vaccines pre-pared for cattle it is possible to obtain immunity in sheep for one year,since oil adjuvants give a better response than aluminum hydroxide.

174

For swine immunization, special vaccines should be prepared sinceoil-adjuvant vaccines give acceptable results.

SIGNIFICANCE

The wider use of indirect methods in foot-and-mouth diseasevaccine control will allow the control of vaccine production for all ofSouth America.

PUBLICATIONS

1. Alonso Fernández, A., and M.V. Fernandes. Experimentalinoculation of sheep with foot-and-mouth disease virus.Bull Off Int Epizoot 73:507-20, 1970.

2. Alonso Fernández, A., K.E. Federer, I. Gomes, and A. Vieira.Comparación inmunológica y serológica de dos subtipos delvirus aftoso tipo C Waldmann. Bol Centro Panam Fiebre AftosaNo. 4, 1971.

3. Fernandes, M.V., and I. Gomes. Convalescent immunity inswine experimentally infected with foot-and-mouth diseasevirus. In preparation.

OTHER DATA


Funded by:

Timetable:


1969 - continuing

175

THE SPECIFICITY OF PURIFIED PROTEIN DERIVATIVES (PPD)OF MAMMALIAN TUBERCULIN PREPARED FROM MYCOBACTERIUM

TUBERCULOSIS AND M. BOVIS

PROBLEM

In most countries where tuberculin testing in cattle is carried out,a mammalian tuberculin prepared from strains of Mycobacterium tuberculosisis used. The original reason for using human strains of tubercle bacillifor the production of mammlian tuberculin for animals was that the orga-nisms grew on liquid culture media more readily than bovine strains, andthe yield of tuberculoprotein was higher. With improved production tech-niques, these practical disadvantages of bovine strains can now be largelyovercome.

Theoretically, it seems reasonable to suppose that the homologoustuberculin would be preferable to the heterologous for the diagnosis ofbovine tuberculosis. In a pilot trial comparing some different techniquesof tuberculin-testing cattle in one area of Argentina, Weybridge bovine PPDgave better discrimination between tuberculous and nontuberculous cattlethan Weybridge human PPD. Moreover, the countries of the European EconomicCommunity have adopted a batch of bovine PPD, produced in Rotterdam from theWeybridge strain AN5, as their reference preparation of mammalian tuberculinPPD for veterinary use. Further investigations are therefore required tocompare the biologic activity of human and bovine PPD, both in guinea pigsfor purposes of processing control, and in naturally sensitized cattle inthe field.

METHOD

Three tuberculins have been used in these comparisons: (1) Weybridgestandard mammalian tuberculin, PPD 278 (prepared from M. tuberculosis strainsC, DT, and PN); (2) Weybridge bovine PPD, Batch B1/66 (prepared from M. bovisstrain AN5); and (3) Rotterdam bovine PPD, Batch B 60-12 (prepared from M.bovis strain AN5). All three preparations were adjusted to a concentrationof 2 mg of PPD per ml.

To assess the potency and specificity of the three tuberculins, theywere compared for biologic activity both in homologously and in heterolo-gously sensitized guinea pigs. Assays were carried out in four groups ofnine guinea pigs, each group sensitized 30 days previously with a differentspecies of Mycobacterium, as follows: (1) M. tuberculosis, strains C, DT,and PN (mixed); (2) M. bovis, strain AN5; (3) M. avium, strain D4; (4) M.paratuberculosis (syn. M. johnei), strain 316F.

Three fivefold dilutions in isotonic phosphate buffered saline of eachPPD were injected intradermally in randomly allocated sites, four on each side

176

of every guinea pig, using a balanced incomplete block design. Tests wereread by measuring the diameter of reactions 24 hours after injection.

Further comparisons will be carried out in naturally sensitized cattlein the field, followed by postmortem examinations at a slaughterhouse.

RESULTS TO DATE

Potencies (expressed as percentages) of each of the preparations ofbovine PPD relative to the human PPD are given in the following table:

Assays in guinea pigs: potencies (%)relative to Weybridge human PPD (95% limits in parentheses)

Sensitization Weybridge bovine PPD Rotterdam bovine PPD

M. tuberculosis 47.1 (38.5 - 57.6) 59.0 (48.4 - 71.8)

M. bovis 137 (107 - 175) 149 (117 - 192)

M. avium 36.2 (27.0 - 48.5) 61.2 (46.3 - 80.8)

M. paratuberculosis 54.1 (41.1 - 71.2) 73.6 (56.2 - 96.4)

At equal doses by weight, both of the preparations of bovine PPD were

significantly more potent than the human PPD in the bovine-sensitized groupsof guinea pigs. In the other sensitized groups, including the human sensiti-

zation, however, both bovine PPDs were significantly less potent than thehuman PPD, indicating that they have greater specificity than the human PPDfor an M. bovis sensitization.

SIGNIFICANCE

These results demonstrate marked differences in the specific potency ofhuman and bovine PPDs under experimental conditions. The specificity of atuberculin preparation differs with the animal species and the type of hetero-logous sensitization, and may therefore vary with the type of test and the

environmental factors involved. The ultimate trial in the assessment of atuberculin is that carried out in the field in animals with sensitizationsnaturally acquired and in the particular environment in which the test is to

be used.

177

The diagnostic value of bovine tuberculin PPD in both animals and mandeserves further study under field conditions.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ian W. Lesslie, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina

Pan American Health Organization/World HealthOrganization, United Nations Development Program,and the Government of Argentina

1970-1972

178

THE SPECIFICITY OF TUBERCULIN PPD PREPARED FROM HEATEDAND UNHEATED CULTURE FILTRATES, WITH OR WITHOUT

HIGH-SPEED CENTRIFUGATION

PROBLEM

Tuberculin is traditionally heated during preparation. In the case

of old tuberculin (OT), this is implicit in the method of production. Withpurified protein derivative (PPD) of tuberculin, heating has the advantageof helping to extract the tuberculoprotein from the bacillary bodies, thusincreasing the yield, and also of simplifying the technique, particularlyin large-scale tuberculin production, by making the material safe to handle.

The classical methods of preparation of tuberculin involving heatingshow an extraordinary resistance of protein material to heat. Nevertheless,because heating is known to denature proteins readily, investigations arerequired to compare the biologic activity of tuberculin PPD prepared froman unheated culture filtrate with that of OT and of a PPD prepared from thesame culture filtrate heated.

In the preparation of tuberculin PPD it is usual to subject the con-centrated bulk solutions to high-speed centrifugation (45,000 r.p.m.) toremove high-molecular-weight material which is considered to be of lowbiologic potency. It is important to determine the actual value of thisprocedure, which requires the installation of expensive equipment, to beable to advise on the essential requirements for the installation andequipping of laboratories for tuberculin production in the differentcountries of the Americas.

The aims of the present investigations are:

(1) To prepare a PPD of mammalian tuberculin from an unheated culturefiltrate and to compare its specificity in allergic skin tests in guineapigs and cattle with that of a standard mammalian tuberculin PPD preparedfrom heated culture filtrates.

(2) To determine the effect on biologic activity of high-speedcentrifugation used in the preparation of tuberculin PPD.

METHOD

Five preparations are being used in these comparisons:

(1) Standard mammalian tuberculin PPD (Weybridge PPD 278), preparedfrom heated culture filtrates of human strains C, DT, and PN, and subjectedto high-speed centrifugation.

179

(2) Unheated mammalian tuberculin PPD subjected to high-speedcentrifugation.

(3) Unheated mammalian tuberculin PPD, without high-speed centri-fugation. Twelve 12-week cultures (four penicillin flasks each of strainsC, DT, and PN) were used for the preparation of the two batches of unheatedPPD.

The spent liquid medium was drawn off from beneath the pellicle intoa 10-liter bottle. It was filtered through 2 x 30 cm sterilizing filtersheets (Ford SGS 22) into another sterile lO0-liter bottle using slightnegative pressure. Protein was precipitated from the unheated culturefiltrate with trichloracetic acid, washed, and reconstituted in M/30 phos-phate buffered saline at a concentration of approximately 15 mg/ml. Oneportion of this concentrated solution was subjected to high-speed centri-fugation in a Sharples batch bowl (Preparation 2); the other portion wasnot centrifuged (Preparation 3).

Rapid (colorimetric) nitrogen estimation was carried out on eachpreparation. These were diluted to approximately 4 mg of PPD per ml andfiltered through EK/O 14 cm sterilizing pad. Content of PPD was estimatedby the Kjeldahl method and each preparation diluted to a concentration of2 mg/ml.

Before making up into concentrated PPD solution for Preparations 2and 3, the washed protein precipitate from unheated culture filtrates wasexamined microscopically for acid-fast bacilli. Cultural and biologictests for tubercle bacilli were also carried out on this material.

(4) Heated mammalian tuberculin PPD, without high-speed centrifugation.

This material was prepared from heated culture filtrate from the samesowing as used for Preparations 2 and 3. It was treated by the standardmethod, as above, except that high-speed centrifugation was not carriedout. It was diluted to a concentration of 2 mg of PPD per ml.

Preparations 2, 3, and 4 were tested for sterility. Samples wereexamined microscopically for acid-fast bacilli, and tested for toxicityand allergenicity in guinea pigs.

(5) Old Tuberculin (Weybridge, 1950). Heat-concentrated, synthetic-medium tuberculin was prepared from human type strains C, DT, and PN, andadjusted to international standard strength (100,000 International Unitsper ml).

To assess the specificity of different tuberculins, it is essentialthat they should be compared for biologic activity first in homologouslysensitized and then in heterologously sensitized subjects.

Assays were carried out in groups of 15 guinea pigs sensitized 30days previously with different mycobacteria. Three fivefold dilutions in

18o

isotonic phosphate buffered saline of each of the five tuberculinswere injected intradermally in randomly allocated sites, four on eachside of every guinea pig, using a balanced incomplete block design.Tests were read by measuring the diameter of reactions 24 hours afterinjection. Further tests will be carried out in naturally sensitizedcattle in the field, followed by postmortem examinations at a slaughter-house.

RESULTS TO DATE

None of the five tuberculin preparations was allergenic in guineapigs.

The OT was more than twice as potent as any of the PPD preparations

in the homologously sensitized guinea pigs, but was considerably more

potent again in all heterologous sensitizations.

Although at equal doses by weight the unheated PPD was equivalent in

potency to the heated PPD in the homologous sensitization, it was consider-

ably less potent in all heterologously sensitized groups of guinea pigs.

The elimination of the process of high-speed centrifugation in the

preparation of tuberculin PPD did not significantly affect biologic activity.

SIGNIFICANCE

These results of intradermal tests in guinea pigs demonstrate that

tuberculin PPD is more specific in biologic activity than OT. The expensiveprocess of high-speed centrifugation is not essential for the preparationof an efficient tuberculin PPD. Heating the culture filtrate did not affect

the potency of the PPD but reduced its specificity. The diagnostic valueof unheated tuberculin deserves further study.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. Ian W. Lesslie, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina

Funded by: Pan American Health Organization/World HealthOrganization, United Nations Development Program,and the Government of Argentina


181

SURVEY OF BOVINE TUBERCULOSIS IN ARGENTINA

PROBLEM

Tuberculosis in cattle in Argentina represents a problem of considerableeconomic and public health importance whose extent is unknown. At presentthe country has an estimated cattle population of approximately 50 million,of which well over 40 million are concentrated in one large pampa areacomprising the provinces of Buenos Aires, Córdoba, Santa Fé, and Entre Ríos.

Many estimates of the prevalence of bovine tuberculosis have beenmade, all of which have to be considered in the light of the circumstancesin which the results were obtained. Figures are available from slaughter-houses concerning the extent to which cattle are found tuberculous atslaughter. The results of such investigations vary greatly as can beexpected since they are derived from a highly biased population of animalsoffered for slaughter. Results of studies on tuberculin testing sufferfrom the same limitations, and are usually based on casual tests lackingthe necessary background information. Consequently, there is a wide rangeof estimates of the size of the problem.

Most of the available figures refer to the vast pampa area mentionedearlier where the majority of animals are concentrated. Although no doubtthe most important part of the country from this point of view, for anevaluationcof the problem the other provinces of the country cannot beneglected, especially because of the heavy and largely uncontrolled move-ment of animals over distances of several hundred kilometers all over thecountry.

The movement is due to various factors. Cattle breeding is practicedin defined areas from which the animals are moved to areas suitable formeat production. Other animals are transported to the milk-producing areas.Since slaughterhouses are concentrated in certain areas of the country,another type of movement takes place; each concentration of slaughterhousesusually has its own supply area from which it draws its animals.

The complexity of the situation increases since other animal species

are also involved. Of these, pigs are the most important, showing a highpercentage of tuberculous lesions at slaughter. In many instances theslaughterhouse findings show rates of infection three to four times thosein cattle.

Bovine-type tuberculosis also occurs in human beings in Argentina,but the available figures have not been obtained systematically and theactual importance of this problem is not known.

The problem of tuberculosis in cattle needs to be evaluated by asurvey conducted in a limited time, to provide the essential information

182

on which a plan for the control of bovine tuberculosis in Argentina maybe based.

The aim of the survey is to determine the extent, level, and typeof tuberculin sensitivity present in cattle in the different areas ofArgentina. This would make it possible to assess the prevalence oftuberculosis in cattle related to sex, age, breed, size of herd, type ofhusbandry, and geographic and ecologic factors, and to assess the economicand public health importance of the disease in the country.

METHOD

Approximately 11,000 cattle in 18 different areas throughout Argentinawere tested intradermally with avian and mammalian tuberculins.

Due to many factors--for example, the large animal population, and

the size and great geographic variations of the country--the survey could

only be of a limited nature. The open type of cattle farming makes col-

lection of the test animals difficult so that the number of animalsincluded in the sample was the minimum necessary for statistical evaluation.

Argentina can be divided into a number of distinct regions accordingto climate. There are also areas typical for milk production, breeding,or beef production, and some districts where all three types of cattlefarming are practiced. Since a simple countrywide, random sampling couldnot be applied, a selective method was employed. Within the different

areas typical for milk production, breeding and beef production, a randomsample of a number of districts was taken. In those selected, a number of

farms were allocated, again at random, and from each of these a sample of

animals was taken.

Studies in other animal species including man will be undertaken at

a later stage to determine if there is any correlation between the geographic

patterns of tuberculin sensitivity in different species.

One intradermal injection (0.1 ml) of each PPD of avian and mammalian

tuberculin was simultaneously given on one side of the neck of each animal.

All reactions were measuVed by increase in skin-fold thickness (mm). The

thickness of the skin fold at the site of the injection, before and 72 hours

after injection, was measured with calipers to the nearest millimeter.

Avian tuberculin was used, not only because it might detect infection

with Mycobacterium avium but also because it has close antigenic links with

some other microorganisms that have been considered responsible for para-

specific tuberculin reactions in animals and man.

183

RESULTS TO DATE

Of a total of 11,662 cattle tested, 497 (4.3 per cent) werepositive, with reactions greater to mammalian than to avian tuberculin.The mean prevalence of positive tuberculin reactors varied accordingto the type of exploitation, from 2.5 per cent in breeding cattle to8.5 per cent in dairy cattle. Of the 388 farms included in the survey,147 (38 per cent) had positive reactors.

A total of 308 cattle (2.6 per cent) had paraspecific sensitivityto mammalian tuberculin, as indicated by greater reactions to the aviantuberculin.

SIGNIFICANCE

The results are being analyzed and, after full assessment, theyshould provide the factual basis for future planning of the nationalprogram of bovine tuberculosis eradication in Argentina.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ian W. Lesslie, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1970-1972

184

STANDARDIZATION OF THE AGGLUTINATION TEST IN BRUCELLOSIS

PROBLEM

The agglutination test in brucellosis is most often carried outusing an antigen prepared from Brucella abortus. This antigen is usuallystandardized against either a battery of anti-B. abortus sera or the singleInternational Standard anti-B. abortus serum. The use of the latter permitscalculation of the number of International Units of antibody, if any, con-tained in the serum under test. The validity of these two standardizationmethods has been demonstrated for B. abortus and B. suis antigens and forB. abortus and B. suis antisera, but evidence regarding B. melitensis anti-gen and antisera is lacking. This question is of importance in both humanand veterinary medicine.

METHOD

One group of rabbits was infected experimentally with B. abortus,another with B. melitensis, and a third with B. suis. The animals were bledat intervals and the sera were tested in duplicate against B. abortus, B.melitensis, and B. suis antigens standardized (1) to an arbitrarily chosensensitivity against 20 bovine sera, and (2) against the InternationalStandard anti-B. abortus serum. In the first series the agglutination testswere carried out according to the method of the U.S. Department of Agricul-ture with different quantities of undiluted serum, and in the second seriesdoubling dilutions of sera were tested.

The methods described above are also being applied to sera from goatsinfected with B. melitensis; sera from cattle, some infected with B. abortus,others with B. melitensis; and sera from swine, some infected with B. suis,others with B. melitensis.

RESULTS TO DATE

Agglutinating antigens prepared from strains of B. abortus, B. meli-tensis, and B. suis were standardized to equal sensitivity by use of theInternational Standard anti-B. abortus serum, and against a collection of20 bovine (anti-abortus) sera according to the method recommended by the U.S.Department of Agriculture. In subsequent tests against sera obtained froma variety of animal species infected with different biotypes of B. abortus,B. suis, and B. melitensis, it was evident that the sensitivity of theabortus and suis antigens remained the same but that the melitensis antigendiffered from the other two in sensitivity except in tests with the sera ofcattle infected with B. abortus. Two methods for standardizing B. melitensisantigens are proposed for investigation.

185

SIGNIFICANCE

The results indicate that methods of standardization are adequate foragglutinating antigens prepared from B. abortus and B. suis but not forantigens prepared from B. melitensis.

Antigens prepared from B. abortus or B. suis are less sensitive whenused on sera of animals infected with B. melitensis than they are withanti-B. abortus or anti-B. suis sera.

PUBLICATIONS

1. Alton, G.G. Standardization of agglutinating antigens for thediagnosis of brucellosis. Res Vet Sci 12:330-37, 1971.

OTHER DATA

Grantee

Funded by:

Timetable:

Dr. Godfrey G. Alton, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1969-1971

186

SEROLOGIC RESPONSE OF CALVES TO DIFFERENT VACCINATION SCHEDULES

WITH BRUCELLA ABORTUS STRAIN 19 VACCINE

PROBLEM

At the present time, B. abortus strain 19 vaccine is used universallyin the prevention of bovine brucellosis. The main disadvantage in its in-discriminate use is that vaccinated animals develop agglutinin titers thatinterfere with the diagnosis of Brucella infection. Vaccination of calvesbetween four and eight months of age reduces the number of cattle showingpositive agglutination reactions at first pregnancy. These reactions arereduced even further when the strain 19 vaccine is administered at fourmonths. More recently it has been demonstrated that a good immune responsecan be expected in calves vaccinated at three months of age. King and Frank(1961), after conducting experiments with young calves, concluded that vac-cination at three months of age produces as good an immunity as at the ages

of six or nine months. Nagy and Hignett (1967) have shown that newborncalves infected with a virulent strain produce only slight agglutinationreactions, but when they are reinfected at seven months the antibodiesdetected disappear faster than in animals infected for the first time atthat age.

The purpose of this research is to study the effect of vaccinationon eight- to 15-day-old calves, as determined serologically, and also theeffect of revaccination at three or six months on the antibody titers incattle previously vaccinated at eight to 15 days of age.

METHOD

A total of 200 heifer calves eight to 15 days of age were selected

from negative herds.

The B. abortus strain 19 vaccine with an approximately 12 x 109viable cell count per ml was administered subcutaneously in doses of 5 ml.A controlled commercial preparation was used.

If tests were not immediately performed, serum samples were storedat -30 C. The plate and tube agglutination tests are being carried outin the conventional manner, starting with dilutions of 1:25. B. abortus1119-3 antigen is used, according to the International Standard. Serumtiters of 1:25, 1:50, 1:100, etc., indicate 25, 50, and 100 IU/ml, res-pectively. The mercaptoethanol tube test and the complement-fixationtest are also being used.

Two groups of heifer calves from eight to 15 days of age werevaccinated with a dose of B. abortus strain 19. One group was re-

187

vaccinated at three months and the other at six months of age. Thecorresponding control groups were vaccinated at the same ages (threeand six months).

Bleedings are being conducted according to the plan designed.

RESULTS TO DATE

Calves vaccinated at eight to 15 days of age with B. abortus strain19 vaccine showed little or no antibody response. Routine serologictests (agglutination and complement fixation) detected only low antibodytiters or were negative.

Nevertheless, this early vaccination had a remarkable anamnesticeffect on future vaccination, and especially on IgG antibody production.When animals previously vaccinated at such an early age were revaccinatedat either three or six months of age, higher titers of IgG were producedthan was the case with calves vaccinated for the first time at three orsix months.

Revaccination at the specified ages also reduced the persistenceof antibodies.

SIGNIFICANCE

The fact that vaccination of calves first at eight to 15 days ofage and again at three to six months reduces the persistence of post-vaccinal antibodies will aid in the diagnosis of brucellosis in vaccinatedanimals. It is also possible that such revaccination, because of thegreater quantity of IgG produced, will give a more solid immunity thanthe standard method of vaccination.

PUBLICATIONS

1. Garcia Carrillo, C., and B. Szyfres. Efecto de la vacunacióntemprana y de la revacunación con Brucella abortus, cepa 19sobre la respuesta serologica. Rev Med Vet, 51:3-15, 1970.

OTHER DATA

Grantee: Dr. Casimiro Garcia Carrillo, Pan AmericanZoonoses Center, Ramos Mejia, Buenos AiresProvince, Argentina

188

Funded by:

Timetable:

Pan American Health Organization/World HealthOrganization, United Nations Development Program,and the Government .of Argentina

1967-1971

189

STUDIES ON THE SEROLOGIC DIAGNOSIS OF CAPRINE BRUCELLOSISIN VACCINATED AND UNVACCINATED GOATS

PROBLEM

Present methods of serologic diagnosis of caprine brucellosis areunsatisfactory. The tube or plate agglutination test is often used forthe diagnosis of caprine brucellosis, but precise information as to itsefficiency is lacking. Rev. 1 vaccination, even when confined to young

animals, is known to interfere with the agglutination test and is uselessfor testing vaccinated goats.

In the past few years many serologic tests have been developed, butan exhaustive evaluation of their sensitivity and specificity is pending.

METHOD

Different groups of goats at a known stage of infection were used

as experimental animals. Artificially infected animals, vaccinated withlive and inactivated vaccines, and animals from brucellosis-free areaswere included in this study.

The sensitivity and specificity of different tests were evaluated onbacteriologically positive animals and on uninfected animals.

RESULTS TO DATE

Six serologic tests were employed with serum samples obtained atintervals during 13 1/2 months after vaccination with Brucella melitensisRev. 1 vaccine and B. abortus 45/20 adjuvant vaccine.

The normal dose of Rev. 1 gave rise to prolonged serologic reactionto most of the tests. The reduced dose, previously shown to produceimmunity similar to that of the normal dose, caused a transient antibodyresponse and most tests were negative approximately two months aftervaccination.

SIGNIFICANCE

Immunization systems not interfering with diagnostic methods are veryimportant in control programs.

190

Serologic techniques that differentiate the reponse due tovaccination (residual vaccination antibodies) from antibodies due toinfection are also of great value.

PUBLICATIONS

1. Jones, L.M., et al. Brucella melitensis Rev. 1 and Brucellaabortus 45/20 vaccines in goats: serological studies. Inpreparation.

OTHER DATA

Grantee: Dr. Casimiro Garcia Carrillo, Pan American ZoonosesCenter, Ramos Mejia, Buenos Aires Province, Argentina

Funded by:

Timetable:


1971-1973

191

EXPERIMENTAL INFECTION OF GOATS WITH BRUCELLA MELITENSIS

PROBLEM

Information on the development of immunoglobulins in animals infectedwith microorganisms of the genus Brucella is scant. Though studies onthe production of immunoglobulins in small numbers of infected bovines havebeen carried out, information on the normal evolution of postinfectionantibodies in goats is scarce. The influence of antibiotic treatment ontheir serologic response is still controversial.

METHOD

Two groups of young goats were infected with B. melitensis H38 bythe conjunctival route. At the time agglutinins at levels of 50 I.U. orhigher appeared in their sera, one of these groups was treated withacromycin and streptomycin. Blood samples were taken from treated anduntreated goats first at short intervals and then monthly. The sera weresubjected to routine serologic tests and were fractionated in columns toobserve the evolution of the components.

RESULTS TO DATE

Goats infected with Brucella and then treated with antibiotics werefound to lose their IgG agglutinins before the IgM agglutinins. Treatedanimals were found to be negative to all tests three to four months afterinfection, although Brucella organisms were recovered from the tissuesof one goat necropsied six months after infection.

SIGNIFICANCE

The study of immunoglobulins in the course of Brucella infectionand the influence of antibiotic treatment on the antibodies duringinfection is relevant to the better understanding and interpretation ofdiagnostic techniques.

PUBLICATIONS

None thus far.

192

OTHER DATA

Dr. Casimiro Garcia Carrillo, Pan American ZoonosesCenter, Ramos Mejia, Buenos Aires Province, Argentina

Funded by:

Timetable:


1971-1973

193

Grantee:

PRODUCTION OF BRUCELLA MELITENSIS REV. 1 VACCINEIN A FERMENTATION TANK

PROBLEM

B. melitensis Rev. 1 vaccine is a known immunogen for the prevention

of ovine and caprine brucellosis. It is assumed that this vaccine canalso be of value in the immunization of other animals.

At present procedures used for the industrial production of thisvaccine are performed on solid media in Roux bottles containing differentculture media. The yield is low and dissociation is observed, however.

METHOD

The minimum nutritive requirements of B. melitensis Rev. 1 strain were

studied in Erlenmeyer flasks on a multiple agitator. Fermentation assayswere carried out in different conditions of agitation, air flow, and quantityof anti-foam using several media known to be best for growth support ofBrucella.

RESULTS TO DATE

Different factors influencing the preservation of the Rev. 1 strainin its smooth phase, during growth in liquid media have been studied andit has been found that this strain attains a high level of growth in amedium containing peptone M, dextrose, yeast extract, and buffer salts.

Approximately 200,000 doses of vaccine (2 x 109 cells/dose) areobtained using 11 liters of culture medium in a 14-liter fermentation tank.

SIGNIFICANCE

Adaptation of B. melitensis Revy. 1 strain to culture in liquid mediumin a fermentation tank and determination of the optimal growth conditionswould help to solve the problem of commercial vaccine preparation of higheryield and less cost.

PUBLICATIONS

None thus far.

194

OTHER DATA

Dr. Casimiro Garcia Carrillo, Pan American ZoonosesCenter, Ramos Mejia, Buenos Aires Province, Argentina

Funded by:

Timetable:

Pan American Health Organization/World HealthOrganization, United Nations Development Program,and the Government of Argentina.

1971-1972

195

Grantee:

VACCINES AGAINST CAPRINE BRUCELLOSIS

PROBLEM

Brucella melitensis infection in goats is believed to be the source ofmost of the human brucellosis in Latin America. Experience in other conti-nents has shown that the living attenuated Rev. 1 vaccine gives effective,life-long immunity in sheep and goats. Because of the tendency of Rev. 1to produce abortion and to be excreted occasionally in the milk of adults,its use in full doses is restricted to young goats that have not been bredyet. There is need for a method to vaccinate adult animals, and two possibleprocedures appear to merit further investigation: reduced doses of Rev. 1,and the nonagglutinogenic killed adjuvant 45/20 B. abortus vaccine.

METHOD

A group of young goats is vaccinated with the normal dose of Rev. 1vaccine, while one group of adult, pregnant goats receives a reduced dose ofRev. 1, which has been shown to be incapable of causing abortion, and anothergroup of pregnant adults is given Duphavac, the killed B. abortus vaccine.These animals are to be exposed to natural B. melitensis infection by "donor"goats at six months to one year after vaccination, while they are pregnant.Observations on the occurrence of abortion and the excretion of B. melitensisat and after parturition will be made, and the animals will be slaughteredfor autopsy examination.

RESULTS TO DATE

The attenuated live B. melitensis Rev. 1 vaccine in normal and reduceddose provided similar protection. Animals given the normal dose of Rev. 1were compared with those given the reduced dose and no significant differencewas found, whereas animals given the B. abortus 45/20 vaccine were signifi-

cantly more infected after challenge.

SIGNIFICANCE

The discovery of an effective vaccine against B. melitensis infectionwill contribute significantly to the control of human brucellosis in LatinAmerica.

196

PUBLICATIONS

1. Alton, G.G., et al. Brucella melitensis Rev. 1 and Brucellaabortus 45/20 vaccines in goats: immunity experiment.J Am Vet Res, in press.

OTHER DATA

Grantee: Dr. Casimiro Garcia Carrillo, Pan American ZoonosesCenter, Ramos Mejía, Buenos Aires Province, Argentina

Funded by:

Timetable:


1969-1971

197

TYPING OF BRUCELLA STRAINS ISOLATED FROM MAN AND ANIMALSIN LATIN AMERICA

PROBLEM

Few laboratories in Latin America have specialized personnel andadequate means for typing Brucella strains. As a result, many strainshave been classified on the basis of only one or two biochemical charac-teristics or epidemiologic circumstances alone. In Brazil, for example,it is claimed that Brucella melitensis has been isolated from humanpatients, but B. melitensis in animals has not been recognized and isdisclaimed (Pacheco and Thiago de Mello, 1955). A group of strains receivedby the Center from human patients in Córdoba, Argentina, an enzootic goatbrucellosis area, had been previously labeled as B. melitensis, and severalof them turned out to be B. suis. The classification was originally mademerely on the grounds that one of the patients was a shepherd and thatanother habitually consumed goat cheese.

The biotypes existing in Latin America, and their relative frequency,are not known.

METHOD

Brucella strains isolated from both humans and animals are collectedfrom different laboratories throughout Latin America. They are subculturedand studied for dissociation, and colonies are selected for typing.

Typing is performed by the conventional biochemical methods and bymonospecific sera, as recommended by the WHO/FAO Expert Committee onBrucellosis. Urease activity is also studied by the method of Brauer.

All the cultures are studied for phagelysis using 1 RTD and 10,000RTD of Tb Brucella phage.

All cultures that show characteristics different from biotype 1 ofeach species are studied by the oxidative metabolic method.

RESULTS TO DATE

Of a total of 623 strains, 191 were identified as B. abortus, 289 wereB. melitensis, and 89 B. suis, with the remainder still under investigation.The most prevalent biotype was biotype 1 with the following percentages:82.2 per cent, 97.6 per cent, and 100 per cent for B. abortus, B. melitensis,and B. suis, respectively.

198

B. abortus was isolated in the 10 countries that cooperated inthis study. Besides biotype 1, biotype 2 was found in Brazil, Colombia,and Venezuela, and biotype 4 in Argentina, Chile, Cuba, and Ecuador.

The B. melitensis strains belonged to collections from Argentina,Mexico, and Peru where caprine brucellosis is prevalent. Strains ofB. melitensis isolated in Chile before the successful eradication programcarried out in Caj6n del Maipo were typed, and besides biotype 1, biotype2 was also found. The Mexican strains were predominantly biotype 1 withtwo exceptions that were biotype 3.

All B. suis strains belonged to biotype 1. B. ovis strains wereobtained from Argentina, Chile, and Peru. This infection is also recog-nized in southern Brazil and Uruguay.

SIGNIFICANCE

The present research provides new information on the Brucella speciesand their biotypes prevailing in Latin America. These data will be usefulin epidemiologic studies and in the control of the infection.

PUBLICATIONS

1. Garcia Carrillo, C., et al. Tipificación de brucelas aisladasdel hombre y los animales en América Latina. Rev LatinoamMicrobiol, in press.

OTHER DATA

Grantee: Dr. Casimiro Garcia Carrillo, Pan American ZoonosesCenter, Ramos Mejia, Buenos Aires Province, Argentina



199

DEVELOPMENT OF A RAPID DIAGNOSTIC TEST FOR RAM EPIDIDYMITIS

PROBLEM

The only serologic methods now available for the diagnosis of ramepididymitis are the complement-fixation and the hemagglutination testswhich can be performed at just a few laboratories in Latin America. Theanticomplementary effect of antigens is a source of diagnostic problems,and cross-reacting antigens among Brucella species also affect the speci-ficity of the diagnosis. A rapid, easy, and inexpensive test for Brucellaovis that could be carried out in the field by personnel with minimaltraining would be highly useful.

METHOD

Soluble and sonicated antigens from Brucella ovis were used to evaluatethe agar-gel diffusion precipitation microslide test and the complement-fixation test in the diagnosis of ram epididymitis. The B. ovis antigenswere made by treating washed cells by sonication or by extracting the solubleantigen with hot saline.

Sephadex G-200 elution profiles and immunoelectrophoresis analysiswere performed to characterize the specificity and immunologic and electro-phoretic properties of these antigens and those of B. melitensis. Serafrom rams infected with B. ovis were fractionated to determine the patternof immunoglobulin response involved in each diagnostic test.

The gel-diffusion technique is being evaluated on selected farms forthe eradication of ovine brucellosis.

RESULTS TO DATE

A simple gel-diffusion test for the diagnosis of ram epididymitiswas found to correlate very well with results obtained with the complement-fixation test. When results were obtained with the agar-gel diffusionmethod and the complement-fixation test on 1,126 infected and uninfectedsheep sera, the agreement between the two techniques was 96 per cent.

The soluble B. ovis antigen obtained by ultrasonic treatment of washedcells was used in both tests. In view of the extensive cross-reactionsthat exist between other Brucella species, the specificity of the sonicatedB. ovis antigen for the diagnosis of ram epididymitis was investigated.Sonicated and saline-extracted antigens of B. ovis, B. melitensis, B. abortus,and B. canis were compared in gel-diffusion and complement-fixation tests.

200

It was shown that the sonicated antigens cross-reacted with sera fromanimals infected or immunized with other Brucella species. The saline-extracted type of antigens were more specific for the surface antigens,i.e., rough or smooth phase.

The saline-extracted antigens of B. ovis and B. melitensis were botheluted as a single peak in the void volume by Sephadex G-200 column chroma-tography, in gel-diffusion had staining characteristics of lipoproteins,but in immunoelectrophoresis showed distinct mobility patterns. Serologicactivity for both gel-diffusion and complement-fixation tests were demon-strated in the IgG-containing fraction of sera from rams infected withB. ovis.

SIGNIFICANCE

The serologic diagnosis of ram epididymitis is now limited to thecomplement-fixation and hemagglutination tests, both of which require well-equipped laboratories and trained personnel. The development of a rapiddiagnostic test and improved antigenic preparations will be of great valueas field tools for epidemiologic and control purposes.

The saline-extracted antigen of B. ovis was as sensitive for thediagnosis of ram epididymitis as the sonicated antigen in both the gel-diffusion and complement-fixation tests and did not show cross-reactionswith sera from smooth Brucella infections. The saline extract of B. ovishad the additional advantages of being non-anticomplementary, easier toprepare, and more applicable in countries where both B. ovis and B.melitensis are endemic in Sheep.

The involvement of IgG immunoglobulins in both serologic tests mayaccount in part for the close agreement between the results obtained withboth tests in the diagnosis of ovine brucellosis.

PUBLICATIONS

1. Myers, D.M., and A. Siniuk. Preliminary report on the developmentof a rapid diffusion-in-gel method for the diagnosis of ramepididymitis. Appl Microbiol 19:335-37, 1970.

2. Myers, D.M., et al. Studies of antigens for complement-fixationand gel-diffusion tests in the diagnosis of infections causedby B. ovis and other Brucella. Appl Microbiol, in press.

201

OTHER DATA

Grantee:

Funded by:

Timetable:

Mr. Donald M. Myers, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1972

202

PATHOGENESIS OF RAM EPIDIDYMITIS CAUSED BY BRUCELLA OVIS:EXPERIMENTAL STUDIES IN LABORATORY ANIMALS

PROBLEM

Ram epididymitis is an infection of sheep caused by Brucella ovis.During the course of the disease microorganisms may be isolated from liver,kidney, spleen, lymph nodes, epididymis, testicle, and semen. Clinically,the infection is predominantly characterized by its primary epididymallocalization and secondary testicular involvement (epididymitis and orchitis).

The object of this project is to study the pathologic and immunologicfactors responsible for the selective localization of the microorganismsin the male genital tract.

METHOD

The study consists of three stages:

1. Experimental inoculation of several laboratory animal species witha B. ovis isolate from ram semen to determine the species most susceptibleto the infection that may be a useful experimental model of the naturaldisease.

2. The host species indicated above will be studied in terms of thelesions produced and the localization of the microorganism and its antigens.These studies will be performed using light, fluorescent, and electronmicroscopy.

3. Immunopathologic studies of the experimental infection in thelaboratory model of the disease will be performed.

RESULTS TO DATE

The first stage has been terminated. The following laboratory animalspecies were inoculated: mouse, gerbil, Lewis and Wistar rats, guinea pig,hamster, and Flander's rabbit. An animal from each species was necropsiedevery 30 days until day 150 postinoculation. The complement fixation andagar-gel diffusion tests were performed with the sera. Spleen, liver, andtesticles were cultured to isolate the organism. Histopathologic studieson epididymis, testicle, seminal vesicle, prostrate, bladder, kidney, spleen,lymph node, pancreas, heart, thymus, and lung were carried out. Serologicdata and the frequency of epididymal and testicular lesions revealed thatthe gerbil is the species most susceptible to B. ovis.

203

SIGNIFICANCE

Knowledge of the pathogenesis of experimental B. ovis epididymitismay be of importance in the eventual development of disease preventionin rams. Data on the damage to other organs by this organism is equallyimportant. This information may also be of value in understanding theimmunopathologic aspects of similar diseases caused in man and animals byother etiologic agents.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. A. Cuba-Caparo, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1971-1972

204

EXPERIMENTAL INFECTION OF MALE GOATS WITH BRUCELLA OVIS

PROBIEM

Goats and sheep are very susceptible to Brucella melitensis, butthere is no information on the natural occurrence of B. ovis infectionin goats. It is assumed that goats are not susceptible to this infection.The fact that goats are not raised in the principal areas where B. ovisinfection is prevalent should be taken into account in explaining thisphenomenon.

METHOD

A group of 18 male goats serologically negative to B. ovis wereartificially infected with a B. ovis strain recently isolated froma case of ram epididymitis. A group of lambs infected at the same timewas maintained as controls. One group of animals of both species wasinoculated by the conjunctival and another group by the preputial route.Semen and blood samples were taken periodically to verify infection.

RESULTS TO DATE

One month after exposure to B. ovis the appearance of antibodieswas observed in the sera of nine of l`-male goats, while 11 lambswere serologically positive. Three months later all male goats werenegative while 10 out of 11 lambs remained positive.

SIGNIFICANCE

For the control of the infection in goat- and sheep-raising areasit is important to assess the susceptibility of male goats to B. ovis.

In this study a comparison is also being made of the techniquescurrently available for the diagnosis of ram epididymitis.

PUBLICATIONS

None thus far.

205

OTHER DATA

Dr. Casimiro Garcia Carrillo, Pan American ZoonosesCenter, Ramos Mej{a, Buenos Aires Province, Argentina

Funded by:

Timetable:

Pan American Health Organization/World HealthOrganization, United Nations Development Program,

and the Government of Argentina

1971-1972

206

Grantee:

BRUCELLA MELITENSIS REV. 1 AND B. ABORTUS 45/20 VACCINES:PATTERN OF IMMUNOGLOBULIN PRODUCTIONFOLLOWING VACCINATION AND CHALLENGE

PROBLEM

Although the pattern of immunoglobulin production has been determinedin sera from cattle, sheep, and pigs vaccinated with Brucella abortusstrain 19 and in cattle infected with virulent B. abortus, this informationis not available for goats infected with B. melitensis or vaccinated againstthat organism. This is of interest in determining whether the sequence ofimmunoglobulin production following vaccination differs from that followinginfection.

METHOD

Sera were collected at intervals following vaccination and infectionof goats with B. melitensis Rev. 1 and a virulent B. melitensis strain,respectively. The selected sera were fractionated using columns ofSephadex G-200 and by anion exchange chromatography. Antibody activityin the fractions was determined by the complement-fixation (CF) test,the tube-agglutination (STT) test, the mercaptoethanol (ME) test, thecard test, and the antiglobulin (AG) test. The serum fractions wereexamined by immunoelectrophoretic analysis.

RESULTS TO DATE

Positive reactions to the mercaptoethanol, antiglobulin, andcomplement-fixation tests were restricted to the IgG serum fractions.Reactions to the card and agglutination tests appeared in either or bothIgM and IgG serum fractions.

Although both IgM and IgG agglutinins were present 12 to 47 daysafter vaccination, at four months only ME-sensitive IgM agglutinins werepresent in most goats, while sera from two animals had activity in theIgG fraction. Unvaccinated goats had antibody activity in both the IgMand IgG fraction until necropsy 47 days after conjunctival inoculationof virulent organisms.

The eluates of the DEAE cellulose serum fractionations wereanalyzed by immunoelectrophoresis and revealed the presence of both IgG1and IgG2.

207

SIGNIFICANCE

There are no other immunoglobulin studies in goats vaccinatedagainst or infected with B. melitensis. The persistence of IgG agglutininsin two of 30 goats vaccinated with Rev. 1 stands in contrast to the reporteddisappearance of this activity in 20 calves vaccinated with strain 19.Exclusively IgM agglutinins may occur both after vaccination of goats withRev. 1 and after infection. These observations indicate that a greaterproblem exists in differentiating vaccination from infection in caprinethan in bovine brucellosis.

Evaluation of the method of purification of goat serum IgGl andIgG2 using DEAE cellulose revealed that it is not as effective as inthe separation of these immunoglobulin subclasses in sheep serum. Thismay be attributed to the greater difference in electrophoretic mobilitybetween the two sheep IgG subclasses.

PUBLICATIONS

1. Varela-Diaz, V.M., et al. Brucella melitensis Rev. 1 andBrucella abortus 45/20 vaccines in goats: Pattern of immuno-globulin production following vaccination and challenge.Am J Vet Res, in press.

OTHER DATA

Grantee: Dr. V.M. Varela-Diaz, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


Timetable: 1971

208

EVALUATION OF VACCINES AGAINST SWINE BRUCELLOSIS

PROBLEM

The INTA vaccine against swine brucellosis was developed in Argentina

some years ago, but it has not found acceptance outside the country whereit originated. A definitive evaluation of this vaccine is required and,in addition, a comparison against the most promising of the other methodsthat have been put forward experimentally for immunization against swinebrucellosis would be desirable.

METHOD

A virulent Argentine strain of Brucella suis has been isolated andlyophilized for use as a challenge strain. After a preliminary trial toestablish the suitability of the strain, the next step is to determine theID50 of the strain in susceptible swine. It has been suggested that theRev. 1 B. melitensis vaccine might be suitable for vaccinating pigs againstB. suis infection, and a preliminary trial of its infectiveness for swinehas been planned. The major evaluation experiment is to take the form ofan ID5 0 determination in swine vaccinated by the INTA vaccine, in otherswine vaccinated by the vaccine chosen for comparison, and also in ul-vaccinated control animals.

RESULTS TO DATE

The 50 per cent infecting dose (ID50) or the minimum infecting dose(MID) of B. suis 644 (3b) for sows of different breeds in Argentina, to beused in the evaluation of the immunizing effect of anti-Brucella vaccines,was determined.

Twenty-six sows inoculated by the conjunctival and intramuscularroutes with 105, 107, and 109 doses were serologically positive and B. suiswas isolated from tissues.

No difference in severity of infection or frequency in organs wasobserved in relation to the doses or inoculation routes.

Agglutination tests showed some degree of reaction in all 26 sowsartificially infected; titers ranged from 1/25 (25 I.U.) to 1/200 (200 I.U.),with a predominance of low titers. These tests appear to be of low sensi-tivity in the identification of reactors if titers higher than 50 I.U. areconsidered as positives.

209

The 50 per cent hemolysis complement-fixation test andtest showed some degree of reaction in all cases except one.toethanol and the card test were of low sensitivity and eachonly 10 of the 26 infected animals. The least sensitive waswhich detected only six infected animals.

the antiglobulinThe mercap-

of them detectedthe rivanol test

SIGNIFICANCE

The result of the ID5 0 experiment in pigs casts doubt on the uniformityof susceptibility of the animals used. It is considered that real progressin this work can only be made if and when a source of experimental animalscan be found that is guaranteed to be brucellosis-free. The doubtful sus-ceptibility of the swine used also puts in question the validity of theresults obtained with Rev. 1.

PUBLICATIONS

1. Cedro, V.C.F., et al. Brucelosis experimental en porcinos.1. Terminación de la dosis infectante. Rev Invest AgropecINTA (Argentina), 8:91-98, 1971.

2. Garcia Carrillo, C., et al. Brucelosis experimental en porcinos.2. Evaluación de técnicas serológicas en cerdas con infecciónreciente de Brucella suis. Rev Invest Agropec INTA (Argentina),8:99-107, 1971.

OTHER DATA

Grantee: Dr. Casimiro Garcia Carrillo, Pan American ZoonosesCenter, Ramos Mejía, Buenos Aires Province, Argentina

Funded by:

Timetable:


1969 - continuing

210

EPIDEMIOLOGIC STUDIES ON HYDATID DISEASE IN THE AMERICAS

PROBLEM

The lack of available data on the incidence, prevalence, and economicand public health significance of hydatid disease in the Americas is abarrier to the effective evaluation of control measures. Investigative

efforts need to be initiated in each of the severely affected countries ofLatin America, and prophylactic steps must be taken to prevent introductionof the disease into those areas now free of infection.

METHOD

Present data about the prevalence and distribution of hydatid infectionin man and lower animals are collated for the countries of the Americas.This information is regularly updated as new studies are performed.

A series of visits is made to countries about which no information isavailable or about which additional information is required to performnecessary surveys. In some cases retrospective hospital surveys or diag-nostic surveys of dogs or livestock are carried out in collaboration withnational authorities.

Advice and material assistance is being provided for the preparationof research projects and pilot control programs.

RESULTS TO DATE

An account and discussion of the actual state of hydatidosis in theAmericas has been published, based on information in scientific publicationsand government reports. It was pointed out that the major problem areasinclude Argentina, Chile, southern Brazil, the mountainous portion of Peru,and Uruguay, where, aside from the magnitude of the problem in humans, largeeconomic losses are suffered because of the existence of the disease inlivestock.

A survey of infection in humans, dogs, and livestock in Uruguay forthe period 1962-1971 was completed in collaboration with the UruguayanNational Hydatid Control Commission. It was learned that on a nationwidebasis, human prevalence was higher in Uruguay than in any other countryfrom which information has been reported.

Visits to Peru and Bolivia have revealed new information about thedisease, and for the latter country verified the existence of the infectionin man, dogs, and livestock in the altiplano.

211

Assistance and advice were given in Argentina and Uruguay aboutthe progress of pilot control programs, and a report was made to theGovernment of Peru concerning the implementation of control measuresin the Central Sierra.

SIGNIFICANCE

A realistic assessment of the present situation is essential foreffective future evaluation of control efforts against hydatidosis.The research under way should provide much of the data needed.

PUBLICATIONS

1. Purriel, P., and P.M. Schantz. The epidemiology of hydatiddisease in Uruguay: infection in humans, livestock, and dogs,1962-1971. In preparation.

2. Schantz, P.M. Algunas observaciones sobre hidatidosis enBolivia. In preparation.

3. Williams, J.F., et al. An account of the current incidenceand distribution of hydatidosis, with special reference tothe Americas. Am J Trop Med Hyg 20:224-36, 1971.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Peter M. Schantz, Pan American Zoonoses Center,Ramos Mejla, Buenos Aires Province, Argentina

Pan American Health Organization/World HealthOrganization, United Nations Development Program,and the Government of Argentina.

1969-1973

212

IMMUNOLOGIC STUDIES OF OVINE HYDATIDOSIS

PROBLEM

There are no methods currently available for the diagnosis of ovinehydatidosis in vivo. An immunologic approach appears to be most promisingand of greatest potential use in studies of the infection's prevalence inlarge animal populations. Sheep have been shown to respond immunologicallyto infection with Echinococcus granulosus, but the specificity of theserologic reactions studied is extremely low. The spectrum of immunologicresponses in sheep has not been investigated.

METHOD

Using sheep naturally and experimentally infected with E. granulosus,the diagnostic value of an intradermal test and several serologic techniquesis evaluated.

The intradermal test is evaluated at different skin sites and usingdifferent methods of interpretation. Serologic techniques employed includeindirect hemagglutination, latex agglutination, and immunoelectrophoresis.Immunodiagnostic test results are associated with the stage and degree ofinfection as determined at autopsy. Identification of certain other para-sites is made to establish the possibility of cross-reactions.

An attempt is made to determine the class or classes of immunoglobulinsparticipating in these reactions, including the homocytotropic antibodydetectable by PCA reactions.

RESULTS TO DATE

Immunodiagnostic test results have been found to be quite variableamong animals grazed in Azul, Argentina. Detectable immunologic responsesare found in some but not all infected animals, and many positive resultsare found in animals with no apparent cestode infection as well as thosewith the closely related cestode Taenia hydatigena.

Homocytotropic antibody specific to hydatid antigens was detectableby PCA reactions in sheep, although positive animals were not alwaysinfected with E. granulosus.

213

SIGNIFICANCE

Knowledge of the significance of immunologic responses in sheep,the sequential appearance of such responses, and the role of allergicmechanisms in the host-parasite relationship should be gained fromthis research. Results obtained from herds living in an environmentwith a high risk of natural exposure will be of doubtful value, however.

Studies of the anaphylactic response of sheep to hydatid fluidantigens may have special relevance, since anaphylatic responses are acommon feature of human hydatidosis. In addition, the possibility existsof developing a rapid field test, based on immediate skin reactions, forhydatidosis in sheep.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Peter M. Schantz, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1969-1973

214

THE IMMUNOGLOBULIN RESPONSE TO HUMAN HYDATID INFECTION

PROBLEM

Known variations in sensitivity among the different immunodiagnostictests for human hydatid disease require the simultaneous application oftwo or more of these techniques to increase the specificity of the diag-nosis. These differences have been attributed to the choice of antigensand techniques, and to the site of localization and morphologic conditionof the cysts in the various groups of patients in the different studies.Variations in the behavior of different immunoglobulins in differentserologic tests may be equally pertinent. Knowledge of the role of thesefactors in hydatid serologic tests is necessary for their standardizationand for the improvement of their sensitivity and specificity.

METHOD

Preoperative sera from 27 hydatidosis patients, of known clinicalhistory and surgical determination of site and condition of their cysts,were fractionated by column chromatography. The immunoglobulin fractionswere then examined for antibody activity by the immunoelectrophoresistest using the following antigens: whole sheep hydatid fluid, theEchinococcus granulosus-specific antigen fraction 5, and the purifiedlipoprotein antigens (arcs A and B) as well as by the hemagglutinationtest with glutaraldehyde-treated cells and the latex agglutination testsdeveloped at this Center.

RESULTS TO DATE

Activity in the IgM and IgG fractions was detected using thehemagglutination and latex agglutination tests. Antibody activity tothe E. granulosus-specific antigen and to the purified lipoproteinantigens was restricted to the IgG fraction of serum. No relationshipwas observed between the immunoglobulins involved in the serologic res-ponse to hydatid disease and the site or morphologic state of the patients'cysts.

SIGNIFICANCE

These observations have shown that the antigens studied, the local-ization and morphologic state of the cysts, and the method of couplingantigens to cells do not affect the distribution of the immunoglobulinclasses involved in immunodiagnostic tests for hydatid disease. We are

215

not aware of other studies associating immunoglobulin classes and theimmunoelectrophoresis test. The present study also revealed that theantibody response to the known E. granulosus-specific antigens residedin the IgG fraction of serum, and preliminary evidence indicates thatthe antigenic fractions responsible for the formation of arc 5 and arc Aare similar. This suggests the feasibility and underlines the need tostandardize the immunoelectrophoresis test for the diagnosis of hydatiddisease.

PUBLICATIONS

1. Varela-Diaz, V.M.disease in humans.Hidatidosis, V CongUruguay, December 1

The immunoglobulin response to hydatidMesa Redonda sobre la Inmunologia de laLatinoam Microbiol, Punta del Este,

.971.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. V.M. Varela-Diaz, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina

Pan American Health Organization/World HealthOrganization, United Nations Development Program,Government of Argentina, and the Laboratorio deInmunologia Parasitaria, Ministerio de SaludPública, Uruguay

1971-1972

216

EVALUATION OF IMMUNODIAGNOSTIC TESTS FOR EPIDEMIOLOGICSCREENING FOR HYDATID DISEASE

PROBLEM

The most commonly used index of human hydatid infection is the annual

incidence of surgical cases. This is at best only a crude estimate of

hydatid infection rates because of the long period in which the patient may

remain asymptomatic (in some cases this may be his lifetime). The value

of this rate as an index for comparison with other countries or regions is

reduced because reporting rates and the availability of medical facilities

vary widely between countries and regions. The results of mass miniature

radiographic surveys provide useful prevalence information but only detect

intrathoracic cysts. Furthermore, this type of survey is expensive and is

being discarded in many areas as a traditional tuberculosis control measure.

Immunodiagnostic tests have not yet been evaluated as epidemiologic

screening tools for hydatid disease, although they would appear to offer

potential advantage because of their relatively low cost and the fact that

indices thus obtained would not be affected by administrative and political

factors.

METHOD

The general modus operandi of this study is to screen population

samples in different geographic localities with the intradermal test for

hydatid disease using an antigen prepared and standardized at the Pan

American Zoonoses Center (where possible this screening is done in con-

junction with mobile chest X-ray units, thereby giving thoracic radiographs

of each person tested). Serum samples are obtained from all positive

reactors and from an age- and sex-matched control group of negative reactors,

and these sera are studied in the indirect hemagglutination, latex aggluti-nation, and immunoelectrophoresis serologic tests for hydatid disease.Serologically positive persons are examined further by clinical methods

(e.g., liver scanning, cholangiography, etc.) to determine, as far as possi-

ble, the presence or absence of liver cysts.

These studies are carried out on people of all ages and both sexes

to determine age or sex differences in susceptibility. They are also

performed in populations with different prevalences of other parasitic

infections to determine the possibility that the other infections may cause

cross-reactions to hydatid antigens.

217

RESULTS TO DATE

A rural population in southern Rio Negro Province, Argentina, wasstudied in the manner described above. Of 1,699 persons screened withthe intradermal test, there were 141 (8.3 per cent) positive reactors.Blood samples were obtained from 55 positive reactors and an equal numberof matched negative reactors. Positive serologic reactions were observedin 13 (23 per cent) of the positive intradermal-test reactors but in onlyone of the negative reactors. Thoracic radiographs revealed six presumptivehydatid cyst infections, all of which were in intradermal-test positivepersons.

A similar study was carried out in the department of Flores, Uruguay,of nearly 5,000 persons. The rate of positive reactions among this popula-tion was 2.4 per cent. Blood samples were obtained from 170 persons whoincluded positive reactors and a negative control group. Positive serologicreactions were obtained from 20 per cent of the positive intradermal reactorsbut only 2 per cent of the negative reactors. The results of clinicalfollow-up and chest radiography on this population have not yet been obtained.

A group of approximately 200 persons from Bolivia and Peru have beensimilarly screened and a high rate of skin-test positivity has been observed--approximately 30 per cent. Serologic testing of the positive reactors hasyielded entirely negative results to date, indicating that a high rate offalse-positive reactions is occurring in this population, possibly due tothe occurrence of cross-reacting cestode infections such as cysticercosis.

SIGNIFICANCE

Immunodiagnostic tests, if properly standardized and evaluated, area potentially useful and economical tool for obtaining human prevalence datain different endemic populations and would allow the obtention of importantepidemiologic information.

PUBLICATIONS

1. Schantz, P.M., et al. An epidemiological study of hydatid diseasein southern Rio Negro Province, Argentina. In preparation.

2. Schantz, P.M., and P. Purriel. A seroepidemiological study ofhydatid disease in the department of Flores, Uruguay. Inpreparation.

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OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Peter M. Schantz, Pan American Zoonoses Center,Ramos Mejía, Buenos Aires Province, Argentina


1970-1973

219

IgG AND ALBUMIN LEVELS IN HYDATID CYST FLUID

PROBLEM

Besides antigens of parasite origin, sheep hydatid cyst fluid containshost serum components that are thought to affect the sensitivity of theserologic tests for this disease. The accuracy of previous attempts todetermine the IgG and albumin levels in cyst fluid seemed questionablesince parasite antigens are known to have electrophoretic mobilities similarto those of host antigens. Furthermore, whether these host antigens wereactually synthesized by the host or by the parasite was not establishedand therefore was the subject of controvery.

METHOD

IgG and albumin levels in sheep hydatid fluid were determined byradial immunodiffusion studies using antisera prepared with previouslypurified sheep IgG and albumin.

Hydatid cysts were obtained from sheep at slaughter. Fluid fromindividual lung and liver cysts and pools of fluid from each organ sourcewere examined.

Hydatid fluid from human, sheep, gerbil, and mouse cysts was examinedby Ouchterlony tests against the four host species-specific antisera todetermine their host specificity.

RESULTS TO DATE

Sheep hydatid cyst fluid was found to contain between 1.3 and 13.0 pgIgM/ml and albumin at concentrations ranging from 3 to 34 ng/ml. Thealbumin/IgG ratios of the cyst fluids examined were similar to those ofblood, indicating that these proteins had penetrated through the parasitemembranes. The immunoglobulins in the hydatid fluid of cysts from dif-ferent host species showed the species-specific antigenic determinantsof the host from which they were obtained.

SIGNIFICANCE

The levels of IgG and albumin in sheep hydatid fluid have beendetermined without interference by antigens of parasitic origin in themethodology. The possibility that these proteins are synthesized by

220

the parasite has been discarded. Since these host antigens seem tointerfere in serodiagnostic tests, this knowledge will be of value infurther attempts to improve their specificity.

The host specificity of the IgG molecules and the data on the albumin/globulin ratios suggest that immunoglobulins are able to penetrate intohydatid cysts. This information strengthens the feasibility of attemptsto eliminate or sterilize hydatid cysts by immunologic means and is thesubject of continuing study in our laboratory.

PUBLICATIONS

1. Varela-Diaz, V.M., and E.A. Coltorti. Preliminary studies of hostantigens and hydatid cysts. V Conf Int Inmunidad contra Pará-sitos Animales, Mexico, August 1971.

2. Coltorti, E.A., and V.M. Varela-Diaz. IgG levels and hostspecificity in hydatid cyst fluid. J Parasitol, in press.

OTHER DATA

Grantee: Dr. V.M. Varela-Diaz, Pan American Zoonoses Center,Ramos Mejla, Buenos Aires Province, Argentina


Timetable: 1971

221

STUDIES ON ECHINOCOCCOSIS IN ARGENTINE SYLVATIC ANIMALS

PROBLEM

Three species of fox from Argentina had previously been reportedinfected with adult Echinococcus spp. The role of these animals in thedissemination and transmission of E. granulosus to man and domesticanimals was never clarified and the species identification of the parasitesremained controversial.

Because of the potential effect of these factors on the control ofE. granulosus infection, a study was initiated to determine the prevalenceand species identification of Echinococcus spp. in sylvatic definitiveand intermediate hosts as well as the signifiance of these findings.

METHOD

A wide variety of sylvatic animals, both potential definitive andintermediate hosts, are captured and examined by necropsy in search ofadult or larval Echinococcus parasites. Recovered parasites are used forstudies of experimental transmission to domestic and laboratory animalspecies or stained for taxonomic studies. These parasites are comparedmorphologically with E. granulosus recovered from experimental infectionsof dogs and Argentine sheep.

The susceptibility of sylvatic animals to larval and adult E.granulosus is determined by experimental infection of animals maintainedin captivity with protoscolices or eggs of E. granulosus.

RESULTS TO DATE

A survey carried out in the Argentine province of Neuquen revealedadult-stage infection in 30 per cent of 25 South American red foxes(Dusicyon culpaeus) and larval infection in four of 71 European hares.No other infections were observed among four other carnivorous speciesand six other potential intermediate-host species. This was the firstreport of naturally-acquired Echinococcus infection in Lepus europaeusand the first association in South America of infection in foxes withlarval infection in sylvatic animals.

Taxonomic and biologic study of the parasites recovered from foxesand hares revealed no specific differences from the Argentine strain ofE. granulosus.

222

Experimental infection of Dusicyon culpaeus with protoscolices ofovine origin revealed that species susceptible to E. granulosus with thedevelopment of many gravid worms 80 days after infection.

SIGNIFICANCE

The role of sylvatic animals in the epidemiology of E. granulosushas potential implications for the possible control of this infection. Iffoxes, for example, may serve as a source of infection to domestic inter-mediate hosts, it is clear that control measures that successfully interruptthe cycle of transmission between dogs and sheep will not necessarily achievecomplete control of the disease.

On the other hand, some or all species of domestic animals locallypresent, as well as man, may be resistant to the parasite from foxes, andthe sylvatic cycle may thus have little bearing on a control program. Itis important, therefore, to determine these factors in any area where con-trol programs are being contemplated.

PUBLICATIONS

1. Schantz, P.M., et al. Echinococcus infection in South Americanred fox (Dusicyon culpaeus) and the European hare (Lepus europaeus)in the Province of Neuquen, Argentina. Ann Trop Med Parasitol,in press.

OTHER DATA

Grantee: Dr. Peter M. Schantz, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina



223

A STUDY OF HOST-PARASITE RELATIONSHIPS FOLLOWING PRIMARYORAL INFECTION OF LABORATORY ANIMALS

WITH ECHINOCOCCUS GRANULOSUS

PROBLEM

Common livestock species, the natural intermediate hosts of E.

granulosus, are not usually practical for experimental studies because of

their large size and maintenance cost, and the slow development of the

parasite in these hosts. A major advance in the development of laboratory

animal models of this disease was the finding that white mice and some

other animals were susceptible to the intraperitoneal inoculation of pro-

toscolices. In this way the parasite could be maintained by serial passage

of protoscolices without allowing the completion of the parasite cycle to

the infective adult stage, thus obviating the concomitant dangers to

technicians and animal caretakers. Although this method permits the pro-

longed laboratory maintenance of the parasite, it is not suitable for all

studies because it does not allow the full natural development of the

parasite in the intermediate host from egg to larval cyst. Furthermore,

some workers had experienced difficulties in avoiding the incidental

production of peritonitis in inoculated animals. It had been previously

shown in the Center's laboratories that white mice and jirds (Meriones

unguiculatus) were susceptible to the intraperitoneal inoculation of eggs

with the involvement of a high proportion of the embryos. This is useful

but again presents the problem of peritoneal bacterial infection incidental

to inoculation and does not permit the parasite to follow its normal in-

fective route, i.e., by penetration of the intestinal mucosa. It was

believed that primary oral infection of white mice and jirds, if successful,

would provide an excellent laboratory animal system while avoiding the

inconveniences mentioned above.

METHOD

The oral infectivity of E. granulosus eggs to white mice and jirds

is assayed, evaluating the effect of egg dosage and host age, sex, and

genetic strain.

Experimental animals are sacrificed at periodic intervals to determine

cyst growth rates and the host's immunologic and histopathologic responses.

RESULTS TO DATE

Both white mice and jirds were determined to be highly susceptible

to the oral administration of 1,000 eggs of E. granulosus. In jirds the

224

majority of cysts were localized in the lungs, while in white mice mostcysts were located in the liver.

In jirds a direct relationship was observed between the size ofthe egg dose and resulting cyst burdens, but no effect of host sex wasnoted.

In mice a lineal relationship was observed between cyst diameter andduration of infection up to 300 days after infection.

SIGNIFICANCE

Primary oral infection in white mice and jirds could prove veryuseful for immunologic and chemotherapeutic studies of larval E. granulosusin laboratories prepared to work with infective eggs.

Information about the host's serologic response and histopathologicreaction is providing new information useful for the interpretation ofimmunodiagnostic tests in other species.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Peter M. Schantz, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1970-1973

225

STUDIES ON ANTIHELMINTHICS ACTIVE AGAINSTECHINOCOCCUS GRANULOSUS IN DOGS

PROBLEM

An effective chemotherapeutic agent against the intestinal phase ofEchinococcus granulosus would find immediate application in control programsin many countries. The drugs tried so far have all had drawbacks: low orunsatisfactory level of activity (arecoline), prohibitive cost (Yomesan),or toxicity (hexachlorophene).

Bunamidine hydrochloride has shown promise in the treatment of canine

echinococcosis, but definitive experiments on its effectiveness compared to

that of arecoline under Argentine conditions have not been carried out.Other drugs are worthy of attention, although the lack of basic knowledge

on the host-parasite relationship in canine infection renders drug trials

both expensive and difficult to interpret.

It has also been consistently observed that bunamidine is less effective

against immature than mature Echinococcus in the dog. The reasons for this

are not clear, but one hypothesis suggests that there is less contact bet-

ween parasite and drug in the immature form due to their smaller size and

the protective effect of intestinal mucus.

METHOD

New drugs having known taenicidal activity are being screened in

preliminary trials involving small groups of dogs. Those worthy of further

investigation are examined in large-scale trials, and their activity is

critically compared with that of arecoline hydrobromide.

Some of the factors influencing the evolution of infection with E.

granulosus in dogs are being examined, including immune responses, back-

ground level of infection in the natural population, and histopathologicresponses of the intestine during infection. Studies will also be done

on the standardization of methods for determining infective doses.

To increase contact between bunamidine and the immature Echinococcus,

various measures are contemplated, including the use of organic solvents,

antiperistaltics, and topical anesthetics.

RESULTS TO DATE

Preliminary analysis of results in 60 dogs comparing bunamidine alone

with bunamidine given with the organic solvent dimethyl sulfoxide seem to

226

indicate no specific advantage to the combination. Further analysis of

the data is required before a definite conclusion can be drawn.

SIGNIFICANCE

The major drawback to the use of bunamidine is its lack of effective-ness against immature stages of Echinococcus. Until a better drug is found,ways to increase its effectiveness against these stages should be investigated.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. William S. Kammerer, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina

Funded by:

Timetable:


1960-1973

227

MEASUREMENT OF ION FLUX ACROSS HYDATID CYST MEMBRANES

PROBLEM

An understanding of the regulatory mechanisms and the permeabilitycharacteristics of hydatid cysts is crucial to the development of a rationalchemotherapy for hydatid disease. To this end, a study of the flux ofvarious ions, nutrients, and drugs across hydatid cyst membranes has beendeveloped. We plan to characterize both active and passive transport pro-perties of the hydatid cyst and examine ways in which these properties canbe altered.

METHOD

Whole hydatid cysts from experimentally infected mice are immersedin physiologic solutions to which have been added various radioactivelylabeled elements. After varying periods of time, cyst fluid is removedand flux rates are determined by measuring changes in radioactivity, takinginto consideration surface area, volume, and time.

Whole membranes are also mounted between two chambers and, by changesin electrochemical gradients across the membrane, flux rates for certainelements may be calculated. Various compounds, known to effect membranepermeability and function in other systems will then be examined in likefashion.

RESULTS TO DATE

Initial results using radioactive sodium have suggested an activetransport mechanism for this element. Further work characterizing sodiumand water transport is in progress.

SIGNIFICANCE

If the characteristics and mechanisms of control of permeability inhydatid cysts can be adequately defined, the possibilities of deliberatelyaltering them so as to allow entrance of potentially useful drugs intothe cyst will develop.

PUBLICATIONS

None thus far.

228

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. William S. Kammerer, Dr. Marcelino Cerejido,M. Pérez-Esandi, Lic. Chem., Pan American ZoonosesCenter, Ramos Mejia, Buenos Aires Province,Argentina, and CIMAE

Pan American Health Organization/World HealthOrganization, United Nations Development Program,the Government of Argentina, and Centro de Investi-gaciones Médicas "Albert Einstein"

1972-1973

229

EFFECTS OF DIMETHYL SULFOXIDE (DMSO)ON HYDATID CYST PERMEABILITY

PROBLEM

It has long been reasoned that one of the major problems in hydatidchemotherapy is the penetration of effective drugs into hydatid cysts invivo. Studies performed on isolated protoscolices serve well for topicalchemosterilization at surgery but are inapplicable to problems of chemo-therapy. It is necessary, therefore, to find ways of measuring and alteringdrug penetration into intact hydatid cysts. The organic solvent, DMSO,because of its remarkable ability to facilitate the passage of various com-pounds across intact human skin and its effect on collagen tissue, waschosen as an agent that may be capable of altering hydatid cyst permeabilityto potentially effective drugs.

METHOD

Hydatid cysts from experimentally infected laboratory animals areincubated in physiologic solutions containing various drugs and visualmarkers with and without DMSO. Fluid from cysts incubated with and withoutDMSO is removed after varying intervals, and concentrations of the drugsor markers are measured spectrophotometrically and spectrofluorometrically.Once an effective technique has been perfected, these studies will be triedin vivo.

RESULTS TO DATE

Experiments using colored markers have revealed that DMSO does alterthe permeability of the hydatid cyst in vitro to certain of these compounds.Results using various drugs await the location of suitable spectrofluoro-metric equipment for their measurement.

SIGNIFICANCE

The characterization of drug permeability is a necessary prerequisitein the design of rational chemotherapy. It is postulated that results ofthis study will lead to a better understanding of approaches to a chemo-therapy for hydatid disease.

PUBLICATIONS

None thus far.

230

OTHER DATA

Dr. William S. Kammerer, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina

Funded by:

Timetable:


1972-1973

231

Grantee:

STUDIES OF THE SUSCEPTIBILITY OF ECHINOCOCCUS GRANULOSUSEGGS TO SELECTED CHEMICAL AND PHYSICAL AGENTS

PROBLEM

Although the microanatomy of the eggs and embryos of taeniid tape-worms has been thoroughly documented, the physiologic characteristics of

these organisms are not well defined. Establishment of the conditionsrequired for continued survival and infectivity of E. granulosus eggs,in both the field and the laboratory, could be of immediate importance to

control and investigational programs. The susceptibility of eggs to ex-

tremes of temperature, desiccation, ionizing radiation, chemical compounds,

and various natural microclimates still requires precise determination.

DIETHOD

Eggs of E. granulosus are obtained from gravid proglottids of the

adult tapeworm recovered from dogs. Samples of eggs are then subjected

to the following conditions: extremes of temperature, ultraviolet and

X-irradiation, taenicidal drugs and other chemicals that might haveovacidal properties, and exposure to a series of controlled, artificialmicroenvironments.

The infectivity of treated eggs and those maintained under controlconditions is then determined by oral or intraperitoneal inoculation into

white mice or jirds.

RESULTS TO DATE

The influence of extremes of temperature on the infectivity of

E. granulosus eggs has been measured. Eggs can survive -300C for 24

hours without suffering any damage, and a small proportion can survive

at -50C with significant reduction of infectivity. None survives at-70°C. After five minutes at -55 C there is a significant reduction ofviability, and after five minutes at 600C animals no longer becomeinfected.

The effects of X-irradiation have been evaluated. It was foundthat 20 and 30 Kr had a marked inhibitory effect on both the number ofanimals becoming infected and the number of cysts formed. Furthermore,it was observed that eggs stored for 60 days were less susceptible to theeffect of X-irradiation than those stored for only 15 days before expo-sure. These investigations were continued and the X-ray level wasextended to include dosages of 35, 40, 50, and 60 Kr. The full range of

232

dosages was applied to groups of eggs at different times of storage(8, 16, 32, 64, and 128 days). At all the latter X-ray dosage levelsand egg storage times the development of embryos was markedly inhibitedand viable cysts were detected in only a few animals inoculated witheggs irradiated at 35 Kr.

It was determined that the taenicidal drug bunamidine hydrochloridehad a marked ovacidal effect in aqueous and dimethyl sulfoxide solution.

SIGNIFICANCE

There now exists no known chemical compound that, at practical con-centration, effectively destroys E. granulosus eggs and thus permits thesterilization of laboratory surfaces and instruments. The results ofthese studies are expected to yield information that will remedy thisdeficiency.

The finding of the ovacidal properties of bunamidine hydrochloridemay have important consequences in regard to the value of this drug inthe treatment of infected dogs.

The findings of the effects of X-irradiation upon embryos maypermit the development of effective immunization procedures using eggsattenuated by X-irradiation.

PUBLICATIONS

1. Colli, C.W., and J.F. Williams. Influence of temperatureon the infectivity of eggs of Echinococcus granulosusin laboratory rodents. J Parasitol, in press.

2. Williams, J.F., and C.W. Colli. Influence of ionizingirradiation on infectivity of eggs of Echinococcusgranulosus in laboratory rodents. J Parasitol, in press.

3. Williams, J.F., and C.W. Colli. Influence of bunamidinehydrochloride on the infectivity of eggs of Echinococcusgranulosus. In preparation.

OTHER DATA

Grantee: Dr. Peter M. Schantz, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires, Argentina

233

Funded by:

Timetable:


1969-1973

234

IMPROVEMENT OF LABORATORY METHODS FOR RABIES DIAGNOSIS

PROBLEM

Laboratory methods for rabies diagnosis are not completely efficient.The lack of sensitivity of Sellers' stain and the time required for the

performance of the immunofluorescence (IF) and mouse inoculation testsjustify studies to correct some of these drawbacks.

In spite of the fact that several fixatives, times of fixation, and

time and temperature of incubation have been reported for IF, the standardtechnique calls for air drying of the impressions for 30 minutes, fixationin acetone for four hours and incubation at 370°C for 30 minutes--a total

of approximately 5 1/2 hours for the whole test. Other antigens do notrequire fixation for the IF test and it is known that the initial combinationbetween antigen and antibody takes only a few seconds.

METHOD

Impressions obtained from brain samples received for rabies diagnosiswere fixed in acetone at different intervals while some were kept unfixed.All the impressions were stained in accordance with the standard IF techniquefor rabies and the results evaluated.

In the second phase of this study, impressions were fixed with themethod that appeared fastest in phase 1. These impressions were stainedat different temperatures and times. These results were also evaluated bycomparison with those obtained from the classical rabies IF test.

RESULTS TO DATE

Two hundred and four samples, 105 positive and 99 negative, werestudied in phase 1. The results showed that unfixed impressions were asgood as fixed ones for rabies IF.

One hundred fourteen brain samples, 56 positive and 58 negative, werestudied in phase 2. The results showed that the IF test could be incubatedat room temperature for 10 minutes with no influence on the results.

SIGNIFICANCE

The improvement of rabies diagnostic tests would enable the laboratoryto report the results more quickly and accurately to the physician consideringrabies treatment of bitten persons.

235

PUBLICATIONS

1. Larghi, O.P., and E. Jiménez Ch. Métodos para acelerar eldiagnóstico de la rabia. II Jornadas Arg Microbiol, November1970.

2. Larghi, O.P., and E. Jiménez Ch. Methods for accelerating thefluorescent-antibody test for rabies diagnosis. App Microbiol21:611-13, 1971.

3. Larghi, O.P., and E. Jiménez Ch. Método para acelerar la técnicade inmunofluorescencia para el diagnóstico de la rabia. Bol OficSanit Panamer 71:36-40, 1971.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. O.P. Larghi, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1970-1973

236

EVALUATION OF THE CORNEAL TEST AS AN ANTEMORTEMLABORATORY METHOD FOR RABIES DIAGNOSIS

PROBLEM

Until recently, most of the laboratory methods available for rabiesdiagnosis had to be performed after the patient (man or other animal) haddied. Serum neutralization and indirect immunofluorescence were the onlytests that could be done while the patient was alive--and then only ifhe had not been vaccinated.

The corneal test (CT) described by Schneider could be potentiallyvery useful for diagnosis of the disease before necropsy. This author hasreported that 41 out of 43 experimentally infected mice were positive bythe CT either before or during the symptomatic period.

The purpose of this project is to determine the sensitivity andspecificity of the corneal test as an antemortem rabies diagnostic method.

METHOD

Corneal impressions are made by touching a microscope slide to theslightly protruding eyeball. Animal heads sent to the Center for rabiesdiagnosis and animals under observation at a local dog pound, as well ashuman cases at a local hospital, are used as experimental subjects. Theimpressions are stained by immunofluorescence.

The sensitivity and specificity of the CT will be evaluated againstthe results obtained by immunofluorescence and mouse inoculation of thecorresponding brains in cases where they are available.

RESULTS TO DATE

A total of 338 corneal samples from subjects of different species(man, dogs, cats, mice, cattle, etc.) have been studied. According topreliminary results, the sensitivity of the CT is 41.5 per cent and thespecificity is 94.3 per cent.

SIGNIFICANCE

When the sensitivity and the specificity of the corneal test aredetermined in naturally infected animals, the project should provide veryimportant information on the usefulness of this technique for the ante-mortem diagnosis of rabies.

237

PUBLICATIONS

1. Larghi, O.P., et al. Evaluaci¿n del método de impresi¿n decórnea como técnica de diagnóstico de la rabia. II JornadasArg Microbiol, November 1970.

OTHER DATA

Grantee: Dr. O.P. Larghi, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina



238

ECOLOGIC AND VIROLOGIC STUDIES OF BAT RABIES

PROBLEM

Rabies transmitted by vampire bats has been reported in all the LatinAmerican countries except Chile and Ecuador. It causes extensive economiclosses. Additional information on the biology of vampire bats is needed inorder to develop effective control measures.

METHOD

Vampire bat colonies are being studied in the provinces of Misionesand Salta in northern Argentina. Each colony is visited at intervals of nineto 12 weeks. Vampire bats are captured with nylon "mist" nets; examinedto record size, weight, and reproductive condition; and marked with anumbered metal wing band. A saliva sample is then collected for virusstudies, and the animal is released at the point of capture. At the end ofthe study period, serum samples will be obtained from a proportion of thebats in each colony for rabies antibody assays.

RESULTS TO DATE

A total of 1,259 vampire bats (Desmodus rotundus) have been examinedand marked with numbered wing bands in northern Argentina. These bats haveprovided 1,623 saliva samples and, in addition, 184 saliva samples have beencollected from representatives of 16 species of insectivorous, frugivorous,and piscivorous bats. Isolations from these saliva samples showed the pre-sence of virus in three samples from D. rotundus and two samples from non-hematophagous bats. Field observations indicated that virtual exterminationof vampire bats over large geographic areas is economically feasible incertain parts of northern Argentina. A study is presently under way todetermine the influence of vampire bat roost destruction on the prevalenceof cattle rabies in a province of Argentina.

SIGNIFICANCE

A method for the control of paralytic rabies transmitted by vampire

bats is greatly needed. The present study provides the basic biologic datanecessary for the development of bat control programs.

239

PUBLICATIONS

1. Delpietro, H., et al. Estudios para determinar la tasa de ataquede rabia en murciélagos. Bol Ofic Sanit Panamer, in press.

2. Fornes, A., and H. Delpietro. Sobre Pygoderma bilabiatum (Wagner)en la Repíblica Argentina (Chiroptera, Phyllostomidae, Steno-dermatinae). Physis 29: 141-44, 1969.

3. Fuenzalida, E., and O.P. Larghi. Caracteristicas de una cepa devirus rábico aislada del cerebro de un Desmodus rotundus. BolOfic Sanit Panamer 73:128-37, 1972.

OTHER DATA

Grantee:

Funded by:

Timetable:

Members of the staff, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1968-1971

240

IMMUNOGENICITY AND SAFETY TESTS IN CATTLE WITH ERA-STRAINRABIES VIRUS PROPAGATED IN BHK CELLS

PROBLEM

Current production of antirabies vaccines is not adequate for thecontrol of rabies in cattle in Latin America.

Tissue culture vaccines, which can be produced in large quantities,may offer the solution to this problem.

The commercial ERA-strain vaccine has been demonstrated to be veryeffective in experimental trials and also in extensive field vaccinationof cattle. However, conclusive knowledge about the immunogenic capacityand safety of the ERA-BHK strain in cattle has not yet been obtained.

METHOD

Twenty-four yearling cattle, divided into six groups of four, areinoculgted intramuscularly with a ml dose of each of six dilutions (lOto 10- ) of ERA-BHK virus. A blood sample is taken from each calf at thetime of inoculation and 30, 60, and 90 days thereafter. The two highestdilutions of vaccine that produce detectable serum-neutralizing antibodieswill be tested for safety in young susceptible cattle by intracerebralinoculation of a 0.5 ml dose of both dilutions.

RESULTS TO DATE

None of the animals inoculated with the different dilutions developed

an abnormal temperature or clinical signs of disease during a 90-day obser-vation period.

-4Animals inoculated with dilutions of up to 10 geveloped serum-

neutralizing antibodies. Those receiving 10-5 and 10- dilutions did nothave detectable antibodies. All but one of the animals inoculated witha lO- dilution survived after challenge, and only two of the five con-trols died of rabies. A pronounced increase in antibody level was evident10 ays after challenge in all animals except those inoculated with al0 dilution and in the surviving controls.

Two of three calves inoculated intracerebrally with 0.5 ml of the10 dilution, plus one of the three calves inoculated with the 10- 4

dilution, died of rabies. Rabies virus was not present in the saliva,however. The titer of the virus contained in a pool of the brains from

241

these three animals was 10 5 when inoculated intracerebrally in micein a dose of 0.03 ml. Mice inoculated intraperitoneally with two 0.5 mldoses, spaced a week apart, at dilutions at 10-0.7 to 10-6 -7 of the poolmentioned above survived without developing any symptoms of rabies. Miceinoculated intramuscularly with two doses of 0.10 ml of the same pooland at the same dilution also survived without developing rabies symptoms.Rabies was induced in hamsters by inoculating this pool of virus in a doseof 0.03 ml at a 10-1 dilution by the intracerebral route, whereas 0.10 mlof the same dilution inoculated intramuscularly failed to produce thedisease.

A serum neutralizing antibody response followed the inoculation of12 bovines with virus ERA-BHK (WIRAB) at a 10-3 dilution. No neutralizingantibodies were detected in most of the bovines a year postinoculation,and six of these died from rabies when challenged at time, together withseven of eight controls.

A comparative study in bovines of the WIRAB vaccine and the ERAcommercial vaccine is currently in progress.

SIGNIFICANCE

The research is expected to provide information on the immunogeniccapacity and safety of strain ERA-BHK against rabies in cattle.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Dr. E. Fuenzalida, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina



242

TRANSFERENCE OF RABIES NEUTRALIZING ANTIBODIESTHROUGH COLOSTRUM AND MILK FROM VACCINATED COWS

TO THEIR OFFSPRING

PROBLEM

It is necessary to determine the period during which rabies neutral-izing antibodies are present in the milk of vaccinated cows and to establishthe duration and level of such antibodies transferred to the offspring.

The main purpose of the study is to assess whether the transferredantibodies interfere with vaccination.

METHOD

The following samples were taken: (a) blood samples from the dam andher offspring before the ingestion of colostrum by the calf; (b) bloodsamples from the offspring at seven-day intervals after birth during thefirst month and every 15 days thereafter until definite disappearance ofantibodies; and (c) colostrum and milk samples once a week during the firstmonth after birth and every 15 days thereafter until complete disappearanceof antibodies.

A booster of the corresponding vaccine was administered to the motherswhen antibodies disappeared in the offspring. The transfer phenomenon isbeing studied in females immunized with both live and inactivated vaccines.Antirabies antibodies are being studied in sera from dams and their off-spring by the serum neutralization test in mice. This study is beingcarried out qualitatively by screening in all cases and quantitativelyin those cases where persistence is longer. Antibody levels in milk arebeing determined by the "lactoneutralization" test, qualitatively in allsamples and quantitatively in those cases where antibody persistence islonger.

RESULTS TO DATE

All cows with detectable neutralizing antibodies in their sera werefound to contain detectable antibodies in their colostrum and also in theirmilk. The duration of these antibodies in the milk appears to be relatedto the duration of antibodies in their sera.

The sera of offspring bled on the first day after birth were foundto be negative before ingestion of colostrum; antibodies appeared duringthe succeeding days.

243

The persistence of neutralizing antibodies in the offspring serawas longer when the dam had a high antibody titer.

Booster with vaccine produced an important increase of antibodytiters in the sera and milk of the mothers. The same increase was notdetected in the calf sera.

These results confirm that antibodies are transferred throughcolostrum but not by milk.

SIGNIFICANCE

It is hoped that this study when completed may indicate at what agecalves should be vaccinated against rabies.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. E. Fuenzalida, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1967 - continuing

244

IMMUNE RESPONSE OF MAN FOLLOWING DIFFERENT SCHEDULES OFADMINISTRATION OF SUCKLING MOUSE BRAIN (SMB) RABIES VACCINE

PROBLEM

At the Pasteur Institute in Avellaneda, Argentina, as in other partsof the Province of Buenos Aires where rabies prophylaxis is performed, thepostexposure rabies vaccine schedules have been modified.

Over the last five years, some thousands of persons have receivedthe vaccine following a schedule of either three, seven, or 14 doses,always followed by two boosters given 10 and 20 days after the last dose.

To date no failures in any of these schedules have been reported.Nevertheless, there is no experimental information demonstrating thesuperiority of any of these modified schemes over the classical schedule.

METHOD

Sera from 150 persons subjected to one of three rabies prophylaxisschemes were studied using the serum neutralization technique.

Blood samples were drawn before the patients received the first doseand again 10, 21, 90, and 180 days afterwards. The laboratory receivedeach sample coded in such a way that only the order in which they weretaken was known.

All the patients were inoculated with the same lot of SMB vaccineat a tissue concentration of 0.5 per cent.

RESULTS TO DATE

Serum samples were examined from 44 people who had received a three-dose scheme, 42 who had received a seven-dose scheme, and 32 who had re-ceived 14 doses. All had received their scheduled booster doses.

Comparison of the immune responses demonstrated that the three-dosescheme was inferior to the seven- and 14-dose schemes.

There was no statistically significant difference between theresponses of individuals on the seven- and 14-dose schedules when theserum was tested at 90 days. By 180 days, however, the 14-dose reci-pients showed significantly higher levels of immunity than the subjectson the seven-dose scheme.

245

A more consistent response was noted in individuals less than 25years of age.

SIGNIFICANCE

This study should provide important elements for deciding on thedesirability of reducing the number of SMB vaccine doses administered.Final determination of the optimum schedule would depend on furtherstudies using vaccines of higher potency.

PUBLICATIONS

1. Held, J.R., et al. Comparaci¿n de la respuesta a diferentesesquemas de inmunización humana con vacuna antirrábica decerebro de rat¿n lactante. In preparation.

OTHER DATA

Grantee:

Funded by:

Timetable:



1969

246

NEUROLOGIC COMPLICATIONS IN MAN ASSOCIATED WITH RABIESVACCINE OF SUCKLING MOUSE BRAIN ORIGIN

PROBLEM

In 1967, 18 cases of the Guillain-Barré syndrome associated withrabies vaccine of suckling mouse brain (SMB) origin occurred in Venezuela.This was a startling development, because until that time no vaccinationaccidents had been reported among more than 80,000 persons who had re-ceived this preparation.

METHOD

An investigation is being made of the methods used to prepare vaccinein the laboratories incriminated in this outbreak. The vaccine lots fromwhich the accidents have occurred are being examined for their purity,potency, and degree of attenuation, and also for the presence of murineviruses and encephalitogenic impurities.

Serologic tests are being performed on both healthy and ill indi-viduals who have received the vaccine and on others who have not beenvaccinated. One group is in Argentina and the other is in Venezuela. Adetailed study of the murine poliomyelitis virus GD-VII is also beingmade concurrently.

A rabies surveillance program has been established and a neuropatholo-gist has been contracted to review the material available from threecountries.

RESULTS TO DATE

Inadequate methods of vaccine preparation and control have beenencountered in several countries. Some lots have been found to be contami-nated with bacteria, and one contained viable (fixed) virus six monthsafter its preparation. Potency has been satisfactory in all the lotsexamined. Efforts to reproduce experimental allergic encephalitisin laboratory animals using the Freund and Levin techniques have not metwith success.

In some of the ill individuals tested, antibodies have been foundagainst GD-VII antigens as well as against rabies. However, these GD-VIIantibodies have also been detected in apparently healthy, vaccinatedindividuals and in some unvaccinated individuals. In the laboratory,the GD-VII virus is inactivated by the level of ultraviolet irradiationused to prepare the SMB rabies vaccine.

247

Prospective and retrospective investigation has identified 12additional cases in six countries--Argentina, Brazil, Colombia, Mexico,Peru, and Uruguay.

Neuropathologic examination of available materials and clinicaldata suggests an allergic peripheral neuritis--a condition most likelycause by myelin in the cranial nerves.

SIGNIFICANCE

Investigation carried out so far suggests that the murine virusGD-VII is not the etiologic agent responsible for the vaccine accidents.Certain characteristics of the neurologic syndrome associated with someof the vaccine lots--such as focal distribution--suggest that impropervaccine production may be at least partly responsible for the accidents.Moreover, since the complications occur toward the end of the second weekof administration, different vaccination schedules may be called for.

The clarification of this postvaccinal allergic neuritis would bea great aid to understanding other demyelinization processes in man, suchas multiple sclerosis.

PUBLICATIONS

1. Held, J.R., and A. L¿pez A. Complicaciones neurológicasposteriores a la administración de vacuna antirrábica decerebro de rat¿n lactante. Bol Ofic Sanit Panamer71:50-59, 1971.

2. López A., H., and J.R. Held. Guillain-Barré syndromeassociated with immunization against rabies: epidemiologicalaspects. In Immunological disorders of the nervous system.Res Pub A R N M D 49:178-86, 1971.

3. Varela, D., et al. Estudios preliminares sobre la inducciónde encefalitis alérgica experimental usando tejido de origenmurino. VIII Cong Nac Alergia Inmunol, Salta, Argentina,August 1971.

OTHER DATA

Grantee: Members of the staff, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina

248

Funded by:

Timetable:


1968-1972

249

SEROLOGIC EVALUATION OF DIFFERENT SHORT SCHEDULESFOR HUMAN RABIES PREEXPOSURE VACCINATION

PROBLEM

During recent years, importance has been given to rabies prophylaxisbefore exposure. This is justified because the number of persons exposedto infection during their work (veterinarians, ecologists, physiologists,pharmacologists, medical students, dog catchers, and others) is becomingconsiderably larger.

Several experiments have been made using reduced schedules in orderto determine whether it is possible to obtain a rapid and good neutralizingantibody response with a few doses of vaccine, and also to evaluate theantibody response of such vaccinated persons when they received a newinjection of vaccine a year or more after the primary immunization.

METHOD

Persons working with rabies virus or with experimental animals oranimals exposed to natural infection have been the subjects of this study.Suckling mouse brain (SMB) vaccine is being used according to the follow-ing schedules:

The first group receives a single dose of vaccine and abooster injection 25 days later.

The second group receives two doses of vaccine, spaced oneday apart, and a booster 25 days after the second dose.

The third group receives three doses of vaccine, spaced oneday apart, and booster 25 days after the third dose.

The fourth group receives five doses of vaccine, spaced oneday apart, and a booster 25 days after the fifth dose.

Determination of the level and persistence of the antibodies producedwill indicate the most convenient schedule. The effect of a booster oneyear after vaccination on the antibody level will also be determined.

RESULTS TO DATE

Fifteen persons vaccinated with the two, three, and five doses showeda clear neutralizing antibody response after 21 days.

250

On the basis of this preliminary result, the three-dose scheme wasconsidered the most convenient. To date, 52 persons have been submittedto this schedule, and 50 of them have developed neutralizing antibodieswithin 21 days after the first injection.

A total of 17 out of 23 persons vaccinated with the three doses haddetectable neutralizing antibodies a year or more later. At that time,all 23 were given a half-dose booster, and all of them, including thosein whom antibodies had not been detectable prior to administration of thebooster, developed a high level of antibodies within eight days.

SIGNIFICANCE

The three-dose scheme of vaccination with SMB vaccine appears satis-factory for preexposure immunization of man.

These results and the responses to the booster dose indicate thatthis procedure is satisfactory for long-term maintenance of specificprotection against rabies.

PUBLICATIONS

1. Fuenzalida, E. Human preexposure rabies immunization withsuckling mouse brain vaccine. WHO Bulletin, in press.

OTHER DATA

Grantee:

Funded by:

Timetable:



1966-1970

251

SURVEY OF RABIES VACCINE PRODUCTION IN LATIN AMERICA

PROBLEM

Information on antirabies vaccine production capacity in Latin America

is necessary for the programming of rabies control campaigns.

METHOD

Questionnaires were submitted to the laboratories in Latin America

through PAHO representatives to determine the extent of human, canine, and

bovine vaccine production.

RESULTS TO DATE

Questionnaires were received from 35 production laboratories in

Argentina, Bolivia, Brazil, Chile, Colombia, Cuba, Ecuador, Guatemala,

Mexico, Paraguay, Peru, and Venezuela.

SIGNIFICANCE

It was possible from the information obtained to analyze the rabies

control situation as related to the availability of antirabies vaccines.

Latin America needs to increase the present production of canine antirabies

vaccine four times to control the disease in urban areas.

PUBLICATIONS

1. Fuenzalida, E. Vacunas y rabia en Latinoamérica. V Cong Latinoam

Microbiol, in press.

OTHER DATA

Grantee: Dr. Eduardo Fuenzalida, Pan American Zoonoses Center,

Ramos Mejia, Buenos Aires Province, Argentina

Funded by:

Timetable:

Pan American Health Organization/World Health Organization,United Nations Development Program, and the Government of

Argentina.

The survey is repeated every two years.

252

POTENCY OF RABIES VACCINES USED IN THE AMERICAS

PROBLTEM

The purpose of this study is to determine the potency of rabiesvaccines used in the Americas.

METHOD

Vaccine lots were received under refrigeration from differentAmerican countries over a period of approximately three years. Somewere inactivated vaccines of nervous-tissue origin and others weremodified live-virus-type vaccines.

The inactivated vaccines of nervous-tissue origin were examinedby the Habel and NIH tests in mice, and the modified live-virus-typevaccines were examined by the Koprowski test in guinea pigs.

RESULTS TO DATE

Twenty-nine of 33 inactivated suckling mouse brain vaccines passedthe corresponding potency test but four of five other inactivated typesof vaccine failed to pass it.

Only two of 10 modified live-virus vaccines failed to meet thepotency requirements.

It has not been possible to expand this study because many labora-tories are carrying out their own potency tests for rabies vaccines.

SIGNIFICANCE

It is well known that rabies control programs, both canine andbovine, as well as human prophylaxis, depend on the capacity of labora-tories to produce vaccines of high antigenic quality in volumes that areadequate to meet the requirements of the Region.

PUBLICATIONS

1. Fuenzalida, E. Resultados de las pruebas realizadas en vacunasantirrábicas. Zoonos Bull, Buenos Aires, 8:16-17, 1966.

253

2. Fuenzalida, E. Vacunas y rabia en latinoamérica. V CongLatinoam Microbiol, Montevideo, Uruguay, December 1971.

OTHER DATA

Grantee:

Funded by:

Timetable:



1966-1972

254

LABORATORY INVESTIGATIONS ON THE ENCEPHALITOGENIC ACTIVITYOF ANTIRABIES VACCINES

PROBLEM

The feasibility of inducing experimental allergic encephalitis (EAE)in laboratory animals with myelin and nervous tissue of murine origin isof interest in the investigation of postvaccinal neuroparalytic accidentsin antirabies postexposure immunization with suckling mouse brain (SMB)vaccine. The likelihood of a common underlying immunopathogenetic mechanismfor the natural and experimental diseases requires laboratory evidence. Theontogeny of encephalitogenic activity in the suckling mouse is not known.The possibility of detecting and removing myelin contaminants from vaccinepreparations requires investigation.

METHOD

The feasibility of producing EAE is being studied using guinea pigs

and rabbits, mouse brain and peripheral nerve homogenates, and purifiedmyelin from the two tissues. Central nervous system and peripheral nervedissections are being performed on all experimental animals for subsequenthistopathologic analysis. Skin tests are performed with the differentencephalitogenic preparations and clinical data obtained from all experimentalanimals.

Different centrifugation schemes were evaluated for their ability toremove myelin from SMB homogenates. The presence of myelin was assessed inall these studies and in brain preparations from nine-day-old mice byelectronmicroscopy and by tests for encephalitogenicity and histopathologiclesions in guinea pigs.

RESULTS TO DATE

It was possible using mouse myelin to produce EAE in guinea pigs andrabbits ranging in severity from clinically severe to mild and inapparent.Animals were found to exhibit a positive delayed hypersensitivity reactiona week before the onset of symptoms. The results underlined the dependenceof the encephalitogenicity of a preparation on the concentration and stateof the sensitizing dose; the relationship between skin test reactivityand the onset of symptoms of the disease; the role of the nonmyelin com-ponents in the skin reaction, and the role of the type of adjuvant used ineliciting the disease.

255

In contrast, a neuroparalytic involvement of the extremities wasobserved in similar studies with guinea pigs inoculated with peripheralnerve myelin.

SIGNIFICANCE

Success in producing EAE in laboratory animals opens up the possi-bility of devising a method to test SMB vaccines for encephalitogenicactivity. From knowledge of the upper and lower limits of myelin con-centration beyond which EAE is not produced, it may be possible todetermine the concentration of total brain homogenate needed for encephali-togenicity. This information may be of value in attempting to establishthe optimal concentration of antirabies vaccine needed for such testing.

The histologic studies in progress will contribute to determiningthe extent to which the experimental disease is related to the situationin humans. If these studies indicate that either CNS or peripheral nervemyelin of mice is indeed implicated in postvaccinal accidents, the resultsof the studies of the elimination of this substance from the vaccineswill find a practical application. Furthermore, if myelin is found innine-day-old suckling mouse brain, the technique for its elimination maybe incorporated routinely in the preparation of SMB vaccines.

PUBLICATIONS

1. Varela-Diaz, V.M., et al. Estudios preliminares sobre lainducción de encefalitis alérgica experimental usando tejidode origen murino. VIII Cong Nac Alergia Inmunol, Salta,Argentina, August-September 1971.

OTHER DATA

Grantee: Dr. V.M. Varela-Diaz, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina



256

SENSITIVITY OF TISSUE CULTURE SYSTEMS SUPPLEMENTED WITHDEAE-DEXTRAN FOR ISOLATION OF STREET RABIES VIRUS

PROBLEM

It is very difficult to detect rabies antigen in saliva samples withthe fluorescent antibody (FA) technique, and consequently the mouse inocu-lation test has to be performed for this type of specimen. However, pro-cessing large numbers of samples by the latter method is costly and time-consuming.

Street and fixed rabies virus strains have been propagated in anumber of different tissue culture systems, but low efficiency of cellinfection and slow development of the virus within the cells have impairedthe use of these systems for virus isolation.

The purpose of the present study is to determine whether the additionof DEAE-dextran to tissue culture systems increases their sensitivity forstreet rabies virus isolation.

METHOD

Saliva samples taken from both rabies-positive and rabies-negativeanimals are inoculated into mice by the intracerebral route and also intotissue cultures with and without DEAE-dextran. The presence of rabiesvirus in the mouse brains and in the tissue cultures is determined by theFA technique.

A total of 100 samples will be examined, and the results will bestatistically analyzed.

RESULTS TO DATE

Ninety samples have been processed so far. These samples include onedog brain and 89 saliva samples from dogs, cats, bats, monkeys, rats, andrabbits.

Tissue culture supplemented with DEAE-dextran has yielded 28 positivesamples, while only 15 of the same samples were positive by mouse inoculation.

All the positive samples were obtained from animals with positivelaboratory diagnosis for rabies. Of the 62 saliva samples that were negativein tissue culture, 20 were obtained from animals with a positive laboratorydiagnosis and the rest from negative animals. Of the 75 saliva samplesthat were negative by mouse inoculation, 33 were obtained from rabies posi-tive animals and 42 from negative animals.

257

The tissue cultures became positive from one to seven days afterinoculation, whereas the mice developed rabies symptoms from 12 to 21days after inoculation.

SIGNIFICANCE

If tissue culture systems supplemented by DEAE-dextran are demonstratedto be at least as sensitive as mouse inoculation for rabies virus isolation,they will provide a less expensive and more rapid method for rabies diagnosis.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. O.P. Larghi, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1969-1971

258

ANTIBIOTIC RESISTANCE PATTERNS AND RESISTANCE TRANSFER FACTOROF ENTEROBACTERIACEAE ISOLATED FROM ANIMALS,FOODS OF ANIMAL ORIGIN, AND FOOD HANDLERS

PROBLEM

The increasing number of antibiotic-resistant Enterobacteriaceaecapable of transfering such resistance to other microorganisms, which hasbeen evidenced in recent years in isolates from animals and foods of

animal origin, seems to be due to the indiscriminate use of antibioticsfor nontherapeutic purposes. The impact of this phenomenon on publichealth is obvious, since among other reasons it makes the treatment ofhuman diseases caused by these organisms more difficult.

The purpose of this project was to study the antibiotic resistance

patterns and the presence of resistance transfer factors (RTF) in Entero-bacteriaceae isolated from horses and cattle destined for human consumptionthat had not received any antibiotic either in their feeding or for pro-phylatic purposes. Enterobacteriaceae (Escherichia coli and Salmonellae)isolated from the meat of these animals and from meat handlers were alsostudied.

This project is part of a cooperative study with the U.S. Food andDrug Administration.

METHOD

E. coli and Salmonella were isolated in fecal samples from horses,cattle, and meat handlers, as well as in swabs taken from the carcassesof the animals. The sensitivity tests to antibiotics (ampicillin, tetra-cycline, chloramphenicol, streptomycin, kanamycin, and nalidixic acid)were performed by the Bauer and Kirby technique. Multiresistant strains(resistant to two or more drugs) were studied for the presence of resistancetransfer factors using the method described by Schroeder et al.

RESULTS TO DATE

Enterobacteriaceae (E. coli and Salmonella) isolated from feces of40 horses, 40 cattle, and 160 meat handlers and in surface swabs of 40horse and 40 cattle carcasses were studied. In total, 385 E. coli strainsof food animal origin and 380 of human origin were isolated, togetherwith eight Salmonella strains of animal origin and 27 of human origin.

None of the Salmonella strains proved to be multiresistant to theantibiotics assayed.

259

Thirty nine of the E. coli strains showed multiresistance and allof them had been isolated from human beings; 26 of these multiresistantstrains were able to transfer their resistance.

SIGNIFICANCE

The results obtained demonstrate the absence of antibiotic-resistantorganisms in the animals and their carcasses that were studied. Suchresults were to be expected as the drugs had not been administered to themfor either prophylactic or nutritional purposes. The percentage of re-sistant and RTF-positive isolates of human origin is not surprising sincethe use and abuse of antibiotics is more frequent in man.

PUBLICATIONS

1. Michanié, Silvia, Dora Dobosch, and Fernando Quevedo.Transferable drug resistance among E. coli and Salmonellaisolated from horses and horse meat handlers. AntimicrobAgents Chemother, in press.

2. Michanié, Silvia, Dora Dobosch, and Fernando Quevedo.Transferable drug resistance among E. coli isolated fromcattle and meat handlers. In preparation.

OTHER DATA

Grantee: Dr. Fernando Quevedo, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina



260

DETECTION OF ENTEROPATHOGENIC ESCHERICHIA COLIIN SAMPLES OF HUMAN AND ANIMAL ORIGIN

PROBLEM

It seems that enteropathogenic Escherichia coli (EEC), which causesgastroenteritis in children, often with serious consequences, can bespread by foods of animal origin that may be contaminated by human carriers,water, and also probably by animal feces.

Serologic studies are being carried out on E. coli strains isolatedfrom different sources to estimate their incidence in the mentioned habitats,and to contribute to knowledge of the epidemiology of gastroenteritis inArgentine children.

METHOD

E. coli strains isolated from human and animal feces and from foodsof animal origin are sowed on MacConkey agar medium. The strains are thenbiochemically controlled and studied with the corresponding sera.

RESULTS TO DATE

Of 1,100 E. coli strains isolated up to now, five have been serologi-cally studied. Three of the strains isolated from meat handlers and twoof those from horse feces were found to be EEC, 0111 and 0125 respectively.

SIGNIFICANCE

Although to date only a small number of strains have been serologicallystudied, the presence of EEC in food handlers and especially in the fecesof horses destined for human consumption has increased the interest and im-portance of the work.

PUBLICATIONS

None thus far.

261

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Fernando Quevedo, Pan American Zoonoses Center,Ramos Mejia, Buenos Aires Province, Argentina


1971-1972

262

RESISTANCE TRANSFER FACTOR (RTF) IN ENTEROBACTERIACEAEISOLATED FROM PIGS RECEIVING PROPHYLACTIC

DOSES OF ANTIBIOTICS

PROBLEM

The importance of antibiotic-resistant Enterobacteriaceae and theirability to transfer this resistance, increased by the nontherapeutic useof antibiotics, has been pointed out in our research with horses andcattle. The aim of this study is to compare the incidence rates of resis-tant and RTF-positive organisms in two groups of animals, one of themhaving received prophylatic doses of antibiotics.

METHOD

Using the methods currently employed in our laboratory, Escherichiacoli and Salmonella were isolated from feces of two groups of 50 pigs;animals in one of the groups had received prophylactic doses of antibioticsfor the previous five days.

Sensitivity to antibiotics was determined by the Bauer and Kirbymethod. The transference of resistance was tested with the technique des-cribed by Schroeder et al.

RESULTS TO DATE

Two hundred seventy-two strains of E. coli were isolated frompigs of two groups.

Salmonella isolations were obtained from 45 of the 50 control pigsand from 30 pigs receiving antibiotics. Serotypes identified up to noware: Give, Newport, Montevideo, Derby, and Tennessee. The final sero-typing of several strains is in progress.

Results of the sensitivity tests are not yet available.

SIGNIFICANCE

No conclusions can be inferred from results now available.

PUBLICATIONS

None thus far.

263

OTHER DATA

Grantee:

Funded by:

Timetable:



1971-1972

264

CONTAMINATION OF HORSE MEAT WITH SALMONELLAE:AN ECOLOGIC STUDY

PROBLEM

Control laboratories in countries importing Argentine horse meatreport finding a relatively high percentage of Salmonellae in the meat.Several papers and communications confirming this observation have alsobeen published. To adequately choose those points of the processing linewhere the application of preventive measures would be more effective,it is necessary to determine the relationships between the sources, thecarriers, and the mechanisms of transmission and growth of Salmonellae.It is also necessary to identify the most frequent serotypes to learntheir distribution, behavior, and the way in which they reach the finalproduct. Those are the aims of the present study, which is being carriedout in close collaboration with a horse slaughterhouse and a horse meatexport plant.

METHOD

Using the techniques for the detection of Salmonella routinelyemployed at our laboratory, the presence of the organism has been inves-tigated in fecal specimens from meat handlers, horses, and birds capturedin the horse yards, in samples of fresh and frozen horse meat, and insamples taken from surfaces and tools and processing water.

RESULTS TO DATE

Feces from more than 100 horses, 206 sparrows, and 80 meat handlershave been studied. In addition, 400 samples of frozen meat, 100 of freshmeat, 100 samples taken from surfaces and tools, and some samples ofwater have been also examined. Salmonella serotypes found to date (Good,Montevideo, Vaertan, Derby, Anatum, Hato, Meleagridis, Haelsingborg,Bredeney, Oranienburg, Riggil, Typhimurium, Otmarshen, and Raus) and theirfrequent isolation have lead to interesting conclusions.

SIGNIFICANCE

Those points in the processing line requiring greater attention werepinpointed and advice has been given to veterinary inspectors and slaughter-house managers, thus resulting in a decrease in the incidence of contami-nation of Argentine meat.

265

PUBLICATIONS

1. Quevedo, Fernando, et al. Contamination of horse meat withSalmonellae: an ecological study. (a) Horses as carriers ofSalmonellae; (b) Salmonellae carriers among slaugtherhouse workers(c) Presence of Salmonellae on surfaces and tools from samplesfrom a horse slaughterhouse; (d) Salmonellae in fresh and frozenhorse meat.

2. Quevedo, Fernando, et al. Salmonellae in sparrows captured inhorse yards

3. Quevedo, Fernando, et al. Sources of contamination of horse meatwith Salmonellae.

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Fernando Quevedo, Pan American Zoonoses Center,Ramos Mejía, Buenos Aires Province, Argentina


1969-1971

266

SANITARY CONTROL OF MUSSELS (MYTILUS PLATENSIS)SOLD IN THE GREATER BUENOS AIRES AREA

PROBLEM

Mussels are the shell-fish most eaten in Argentina. Inasmuch as theyhave been frequently incriminated as vehicles of various diseases whensanitary control on their fishing, handling, and sale, is not exercised,we believed it suitable to carry out a study on the sanitary quality ofmussels sold in the Greater Buenos Aires area.

METHOD

Using methods currently employed in our laboratories and those recom-mended by the American Public Health Association, batches of 20 musselswere examined for: (a) MPN of fecal coliforms, (b) MPN of fecal streptococci,and (c) presence of Salmonella.

RESULTS TO DATE

Of 100 pools (2,000 mussels), only 30 per cent had to be consideredunsatisfactory according to USHI and the English standards. The unsatis-factory samples were among those examined in spring and summer. On theother hand, those gathered in autumn and winter were satisfactory in allcases. Salmonella organisms were not isolated from any batch.

SIGNIFICANCE

Comparing these results with those obtained with samples taken at theirpoint of fishing, we suspect that contamination occurs on the way to or atthe market. Therefore, the improvement of sanitary conditions during thehandling and sale of mussels will result in a product of a higher hygienicquality. In view of the slight contamination found, the consumption of thisproduct may thereby be promoted.

PUBLICATIONS

1. Dinelli, J.A., and Fernando Quevedo. Control higiénico demejillones que se expenden en el Gran Buenos Aires. Rev AsocBioquim Arg, in press.

267

OTHER DATA

Grantee:

Funded by:

Timetable:



1970-1971

268

INTER-AMERICAN INVESTIGATION OF MORTALITY IN CHILDHOOD

PROBLEM

The overall aim of the Inter-American Investigation of Mortalityin Childhood is to develop death rates as accurate and comparable aspossible in selected communities, taking into account the nutritional,sociological, and environmental factors responsible for excessive mor-tality. The data provided by the study will be used to compare under-lying and associated causes of death and to investigate relationshipswith infectious diseases, nutritional deficiency states, and sociologicalconditions.

METHOD

The deaths of approximately 35,000 infants and children under fiveyears of age in 13 widely separated areas of Latin America are beinginvestigated through interviews in the home, at the hospital or clinic,and with the physician to obtain as complete a record as possible of thefatal illness, including results of laboratory and other examinations andautopsy. To permit a full evaluation of the factors resulting in thedeath of these infants and young children, tabulations on the pregnancyhistory, the status of the infant at birth, breast feeding, growth anddevelopment, and social and environmental conditions have been included.

In order to compare the data on the children who died with similardata for the general population, approximately 20,000 living childrenunder five years of age are being studied through household surveys inwhich probability sampling techniques have been used.

RESULTS TO DATE:

Some of the highlights of the Report for the First Year of theInvestigation of the 13 Latin American projects and of the exploratoryanalyses in five working drafts are below:

a. First, to establish quality in the projects for theinclusion of information regarding all deaths under 5 years of age,a serious situation was discovered in hospital and registrationprocedures. In several areas significant proportions of deathsparticularly of those occurring in the first few days of life and ofinfants of low birth weight were not registered.

b. Excessive mortality in childhood has been revealed - oftenin excess of the official statistics. The situation in rural areas

269

is very serlous with rates as much as twice those in the cities. Thesefindings indicate the need for action in cities and especially in ruralareas.

c. The role of infectious diseases as underlying causes of deathhas been clarified, with diarrheal disease and measles the principalcauses. The synergistic association of infectious diseases and nutri-tional deficiencies indicate that the susceptibility of the host aswell as the specific agent must be considered in effective programs ofcontrol of infectious diseases. From 40 to 76 per cent of the deathsfrom infectious diseases had nutritional deficiency as an associated cause.

d. Only through the study of multiple causes is measurementpossible of the magnitude of health problems in infancy and childhood,such as nutritional deficiency and prematurity as well as the infectiousdiseases, certain perinatal causes, etc., and the interrelationshipsof these causes.

e. Nutritional deficiency constitutes the most serious healthproblem in infancy and childhood in Latin America. Death rates are veryhigh in infancy and the situation is even more serious in the ruralareas. Preliminary analyses of foods provided to deceased childrenindicate probable deficiencies in foods with proteins. Thus, the dataserve to show the need for extended and improved nutrition programs.

f. Excessive reproductive wastage was found in several areasfrom the study of outcome of previous products of pregnancy. In allareas infant mortality among previous products was higher than in theInvestigation. Mortality was greater among infants of higher birthorders and older mothers. Reduction of the birth rate as in the Provinceof Santiago with introduction of family planning practices results in ashift from births of higher birth orders to those of low birth ordersand thus a lower death rate.

g. Limited breast feeding was found in several projectsespecially in those in Brazil and San Salvador. Since higher propor-tions of the deaths were due to diarrhea in infants with weaningstarted, further investigation of these relationships is advisablewhich in turn should lead to improvement in feeding of infants.

h. Socioeconomic factors such as education and occupation arebeing analyzed and the interrelationships studied. The educationallevel of the mother appears to be the best single index of socio-economic status as it measures the effect of several factors such asincome, occupation, nutrition and environment, in addition to education.Thus, the socioeconomic aspects of these problems deserve considerationin planning health programs.

i. Piped water supplies were available to less than 20 per centof the homes of deceased children in two cities and three rural areaswith excessive mortality under 5 years of age. Prevention of spread

270

of disease requires provision of water to the homes.

The Provisional Report of the First Year of the Investigation wasreleased in English and Spanish in 1971, which serves as the basis foractions while the final report is being completed.

SIGNIFICANCE

The Investigation is pointing out important health problems suchas the excessive mortality in nearly all the areas, the serious defi-ciencies in the systems of health statistics and the important role ofthe various social, biological, and environmental factors which are relatedto health problems.

The importance of this research program is being recognized widelyin the Continent for orientation of actions in maternal and child health.In December 1971, the Ministers of Health of the Andean Region stated theneed for strengthening community centered research in their countries inorder to measure and uncover the real magnitude of basic health problemsand also to find the best methods for the efficient use of resources forsolution of the problems disclosed. They pointed out the value ofjoining their efforts for coordinated research such as has been done inthe Inter-American Investigation of Mortality in Childhood.

The complexity of causes and factors determining the type and sizeof health problems as well as their interrelationships which are beingclarified in the Investigation indicate the need for a multisectorialand multidisciplinary approach in research leading to actions and inimplementation of solutions to health problems in infancy and childhood.For this reason it is recognized that the health agencies, both nationaland international, and the universities should exercise joint responsi-bility in promotion and conduct of coordinated epidemiological researchof the collaborative type, and for the development of human resources forsuch activities.

PUBLICATIONS

1. Puffer, R.R. Initial phases of the Inter-American Investigationof Mortality in Childhood. Bol Ofic Sanit Panamer 65:114-126,1968; reviews in 66:341-343, 1969, and English edition, 65:35-46,1968.

2. Puffer, R.R., and C.V. Serrano. Inter-American Investigationof Childhood Mortality. Washington, D.C. Technical FrontSection of War on Hunger. U.S. Agency for InternationalDevelopment, v. 3, No. 11, November-December 1969.

271

3. Puffer, R.R., C.V. Serrano, and A. Dillon. The Inter-American Investigation of Mortality in Childhood.Assignment Children. United Nations Children's Fund, No. 15,July-September 1971.

4. Provisional Report, First Year of Investigation, Inter-American Investigation of Mortality in Childhood. PanAmerican Health Organization, Washington, D.C., September 1971.(In Spanish also).

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. Ruth R. Puffer, Pan American Health Organization,Washington D.C.

U.S. Agency for International Development

Thirteen field projects (24 + 3 months each)May-September 1968 to December 1971

Central office

Processing and analysis October 1968 -MarchReport first six months JuneProvisional report first year SeptemberReport for two years March(followed by additional papers)

1973197019711973

2 72

STUDY ON MULTIPLE CAUSES OF DEATH

PROBLEM

A study of the questionnaires completed in connection with the Inter-American Investigation of Mortality for Bristol and San Francisco wasconducted to evaluate the completeness and accuracy of the reporting ofunderlying and associated causes on death certificates and to determine thefrequency of combinations of causes in terms of age, sex, and residence.

METHOD

Data from questionnaires corresponding to 3,506 deceased personsbetween 35 and 74 years of age who died from natural causes and for whomautopsy records and clinical information were available from two citieswere used for the study. One of the medical referees designated thecauses to be coded, and tabulations were prepared by computer.

RESULTS TO DATE

An exploratory analysis of multiple causes was completed in 1969,and the resulting report was transmitted to the National Center of HealthStatistics, which was the source of funding for the present project.

The consideration of multiple causes of death in contrast to onlythe underlying cause in the analysis of mortality was shown to be usefulin understanding the factors contributing to death.

The World Health Organization convened a small working group toadvise on rules for multiple-cause coding and tabulation. A paper fromthe Inter-American Investigation of Mortality in adults served as areference document. Also, some of the material from this research wasused in a document for the WHO Study Group on the Classification ofDiseases.

SIGNIFICANCE

This research indicates the value of the additional informationavailable on autopsy and clinical records not only for correct designationof underlying causes of death but also for knowledge of the associatedcauses and the frequency of causes contributing to death. Methods need tobe developed to facilitate the combining of data from autopsy and clinicalrecords with those on death certificates in order to determine the under-

273

lying and associated causes. Attention should also be given to thesetting of international standards for comparable tabulations andanalyses.

The findings from this study are valuable to the staff of theInter-American Investigation of Mortality in Childhood, in whichmultiple causes are being coded and analyzed. In order to facilitatemultiple-cause coding and analysis, it is expected that changes willbe incorporated into the Ninth Revision of the International Classifica-tion of Diseases in 1975.

PUBLICATIONS

1. Puffer, R.R. Estudios de Causas Múltiples de Defunción.Bol Ofic Sanit Panamer 69:93-114, 1970.

OTHER DATA

Grantee: Dr. Ruth R. Puffer, Pan American Health Organization,Washington, D.C.

Funded by:

Timetable:

National Center for Health Statistics/U.S. PublicHealth Service

1967-1969

274

SURVEY OF SMOKING PATTERNS IN LATIN AMERICAN CITIES

PROBLEM

Background Information

According to a PAHO investigation in the early sixties, 20 per centof all deaths occurring in men 35 to 64 years of age in 10 Latin Americancities were due to smoking-related diseases. Considerable variation wasnoted among the cities in the mortality from specific forms of cancer,and one of the most striking findings was the very high death rate frommalignant neoplasms of the lung in La Plata, Argentina. Even though thereis some knowledge about tobacco consumption on a national scale in a fewLatin American countries, there is still no detailed information availableon the smoking habits of the Region's urban populations in general.

Research Objectives

1. To estimate the extent of smoking in the populations of eight

large urban centers of Latin America (Bogotá, Caracas, Guatemala City,Lima, La Plata, Mexico City, Santiago, and Sao Paulo), and to investigate

some of the more significant social, cultural, and demographic character-istics of current and former smokers and lifelong nonsmokers.

2. To determine the beliefs and attitude in these communities

about smoking and its effects on health.

3. To correlate smoking patterns with certain types of morbidity,such as respiratory symptoms, disability, and days lost from work orschool, and with mortality rates from smoking-related diseases.

METHOD

A probabilistic 1,600-person sample of the population 15-74 yearsold in each of the cities will be interviewed about their present and

past smoking behavior, their attitudes toward smoking, the reasons thatmotivated their acceptance or rejection of the habit, and a number ofsociocultural variables. The frequency of smoking will be examinedaccording to sex, age, race, education, socioeconomic level, and other

significant variables. The intensity of the habit will be correlatedwith the local patterns of mortality, respiratory symptoms, disability,and absenteeism from work or school.

As far as possible, use will be made of the questions in the surveyinstrument employed by the USPHS National Clearinghouse for Smoking and

275

Health in studies carried out in the fall of 1964, spring of 1966, andfall of 1970. This approach will facilitate comparison of Latin Americanand United States data.

RESULTS TO DATE

Local coinvestigators were selected in each of the cities. In ameeting at PAHO, they agreed on the content and format of the question-naire as well as on procedural norms for the conduct of the field work,including the selection, training, and supervision of interviewers andother local staff. The sampling methodology worked out by a consultantwas discussed at the aforementioned meeting, and the consultant thenvisited the study cities, evaluated locally available demographic infor-mation, and collaborated with each coinvestigator in the initial stagesof the sample selection.

Field work has ended in seven of the eight cities. It is expectedthat the survey will be completed in the last city in July 1972. Dataprocessing has already begun at PAHO Headquarters under the direction ofthe main investigator, who has also closely supervised all stages of thefield work.

SIGNIFICANCE

It is expected that the investigation will:

1. Establish baselines for the evaluation of smoking controlprograms.

2. Provide information to member countries that will help themformulate policies regarding tobacco smoking, and plan and conduct anti-smoking programs.

3. Generate interest and promote active support for the fightagainst cigarette smoking within the health agencies and voluntaryorganizations of the Region's countries.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Daniel J. Joly, M.D., M.P.H., Dr.P.H., RegionalAdviser in Cancer Control, PAHO/WHO

276

Funded by: Pan American Health Organization, United NationsDevelopment Program, American Cancer Society, Inc.,Sociedad Anticancerosa de Venezuela, FundaciónPeruana del Cáncer, and Associagao Paulista deCombate ao Cancer

Planning and design:Field work:Data processing and analysis:Report preparation and publication:

March 1971June 1972November 1972March 1973

277

Timetable:

ECOLOGIC IMPLICATIONS OF INSECT CONTROL: AGRICULTURE,PUBLIC HEALTH, AND ECONOMIC DEVELOPMENT

PROBLEM

Synthetic organic insecticides are being widely used in many lessdeveloped countries for both agricultural and public health purposes.In Central America, they have been used in agriculture in record quan-tities, particularly on cotton in the Pacific coast. Synthetic organicinsecticides (principally DDT and sometimes dieldrin) have also formedthe basis for malaria control.

The purpose of the present investigation in Central America is(a) to study present pest and vector control techniques that depend onintensive use of insecticides, and those techniques' ecologic consequencesfor agriculture, public health, and economic development in the countriesinvolved, and (b) to develop a model interdisciplinary strategy for astudy of complex environmental problems in less developed countriesinduced by the introduction and diffusion of modern technologic innovations.

METHOD

Observations are being made by an interdisciplinary team of scientistsof insecticidal and agricultural techniques, exposure pattern to insecti-cides, and various aspects of the ecosystem. Data are being collected onthe extent of insecticide use among various segments of the population.Questionnaires, interviews, and participant observation techniques are usedto measure differential pesticide diffusion rates as an innovation, versuscountermeasures as adaptive strategies.

A study of the insect communities is under way using various samplingdevices to estimate insect populations on cotton, corn, and weeds. Pestsas well as beneficial species (entomophagous, parasitic, and some patho-genic organisms) are being collected to determine the extent of naturalbiologic control, both under prevailing (treated) and control (untreated)conditions. Samples of human and animal tissue (fat, brain, whole animals,etc.) and products (milk, urine) as well as foods are being collected foranalysis using gas chromatographic techniques to determine residue contentof persistent insecticides.

RESULTS TO DATE

Further investigation has shown the presence and effectiveness ofmany biologic control agents for agricultural pests. The heavy use ofinsecticides on the Pacific coastal plains seems to have influenced the

278

ecologic characteristics of the entire area so that natural pest enemiesare rare in the rainy season.

Review of data from malaria eradication campaigns has shown thatin several countries the programs have met severe obstacles, bothtechnical and administrative. While agricultural spraying seems to beresponsible for the development of high resistance among Anopheles vectors,the eradication programs themselves cannot be totally exempted from thecause.

Analysis of samples of human milk from the three communities of thePacific coast studied shows unprecedented concentrations of DDT and otherpersistent insecticides in milk. Amounts vary from 12 to 436 timeshigher in this part of the country than the WHO/FAO maximum AcceptableDaily Intake (ADI) for neonates. Fish samples analyzed also contained upto 70 ppm of DDT, which is unusually high.

Sociocultural data collected show a high degree of pesticide useby small farmers of the coast. A preliminary analysis of the causes ofhigh accumulation of pesticides in milk implicates the malaria eradicationprograms as the main contributors, but further studies are needed beforea more definitive statement can be made.

SIGNIFICANCE

The total impact of pesticidal chemicals has never been fully inves-tigated in any one region. It is therefore becoming increasingly crucialto examine and analyze the biologic consequences of present pest-controlstrategy, which consists largely of a short-term economic view of pestsas target species. There is usually no consideration of the role of pestspecies within the total complex of biologic systems that constitute thebasis not only for all agriculture, but also for health.

Findings of large quantities of pesticides in mothers' milk arehighly significant. Guatemalan milk at this time has more DDT and severalother insecticides than any other study has revealed in the past. Thepossible implications of such facts for human health, particularly forinfant morbidity and perhaps mortality, as well as for neonatal growthand development, merit serious concern and further investigation. Theyalso demonstrate the urgent need for alternative strategies of pest andvector control using environmental management techniques.

Although the project is designed as basic research, it is expectedthat it will indicate changes that could improve present conditions. Itis also intended that the study will produce a model for interdisciplinarystudies of environmental problems in other less developed regions of theworld.

279

PUBLICATIONS

1. Farvar, M., et al. Ecological aspects of insect control.CBNS Notes, T717-2, 1971.

2. Farvar, M. Ecological implications of insect control:Agriculture, public health and development. Ninth Meeting,Advisory Committee on Medical Research, Pan American HealthOrganization, Washington, D.C., June 1970.

3. Farvar, M. Interactions between agricultural and public healthaspects of insect control through insecticides in CentralAmerica. Symposium on Vector Control and the Recrudescence ofVector-borne Diseases, Pan American Health Organization,Washington, D.C., June 1971.

4. Farvar, M. Interrelations of pesticides in agriculture andpublic health. In R. Van den Bosch, D. Dahlsten, M.T. Farvar,and K.P. Shea (eds.), Report of the Pesticide ReevaluationTask Force, New York, Scientists Institute for Public Informa-tion, in press.

OTHER DATA

Grantee: Dr. M. Taghi Farvar, Center for the Biology ofNatural Systems, Washington University, St. Louis,Missouri

Funded by: National Institutes of Health (USA); National AudubonSociety (USA); Ministerio de Agricultura (Guatemala);and Washington University (St. Louis, USA)


280

WATER DISTRIBUTION

PROBLEM

Water demand, and its seasonal, daily, and special variations, arenot well understood in Latin America. Per capita rates are generallyadopted from textbooks or arrived at on the basis of personal judgment.Once these rates are adopted for a given project, they have a decisiveinfluence on the cost of the entire water supply system. The School ofEngineering of the University of Chihuahua, in cooperation with theCentral and Municipal Boards for Water and Sanitation, has undertaken toconduct the present research on water demand.

METHOD

Water users in Chihuahua will be divided into categories accordingto housing standards. Sample households will be selected in each category,and their water consumption will be registered. Consumption will also bemeasured in public buildings (hospitals, schools, office buildings, etc.)and industrial establishments. The data will be processed and tabulatedby the computer center at the University of Chihuahua.

RESULTS TO DATE

An advance report has been prepared showing preliminary data andestimates. The Secretariat of Water Resources in Mexico, D.F., is nowprocessing all data from the project's first stage (eight months ofcontinuous observations). The second stage began in 1971 and willincrease the observation period to one year.

SIGNIFICANCE

Considerable savings in water costs can be expected by the adoptionof more realistic per capita consumption rates at the time installationsare designed.

PUBLICATIONS

1. Informe preliminar sobre el proyecto de investigación dedemanda de agua en la red de distribución de la ciudad deChihuahua, Facultad de Ingeniera, Universidad de Chihuahua,1971.

281

OTHER DATA

Grantee:

Funded by:

Timetable:

Ing. José Lozano Franco, School of Engineering,University of Chihuahua, Chihuahua, Mexico

Pan American Health Organization and the Universityof Chihuahua, Mexico

Revised: 1970-1972

282

COMPUTER DESIGN OF WATER DISTRIBUTION NETWORKS

PROBLEM

Manual hydraulic computations for the design of water distributionnetworks are lengthy and tedious, and delay the completion of watersupply projects. The use of computers would greatly speed the requiredcomputations.

METHOD

We are formulating specially designed computer programs including:(1) one using the Mannes-Lueger method for economical design of waternetworks; (2) another for cost analysis of sewerage networks; and(3) a third for direct economic analyses of sewerage network operation.

RESULTS TO DATE

No significant results yet. The project, an extension of a projectto obtain basic data on the design of water supply and sewerage systems,started in 1971.

SIGNIFICANCE

The project will enable us to examine and analyze a great numberof variables in water distribution design with accuracy, economy, andspeed.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Ing. Juan Pablo Schifini, School of Physical andMathematical Sciences, University of Chile, Santiago

University of Chile/Pan American Health Organization

1971-1972

283

BASIC DATA FOR THE DESIGN OF WATER SUPPLYAND SEWERAGE SYSTEMS

PROBLEM

Very few countries in Latin America base their design standardsand specifications for water supply systems on national experience andaccumulated statistical data. In general, Latin American countries havetended to rely on considerations and data in textbooks that are usuallybased on practices followed in the United States. The objective of thisresearch project is to evaluate and revise the basic design standardsfor water supply and sewerage systems in Chile.

METHOD

Our investigation is based on the following factors: per capitawater consumption, including averages and variations in the averages;waste water quality, BOD, settleable solids, and per capita contribution;and population growth trends.

RESULTS TO DATE

The first stage of research has been completed. Reports on populationgrowth and the design of water distribution networks and sewerage systemshave been written.

SIGNIFICANCE

Results of the research will help establish design regulations forwater supply and sewerage systems in the light of national conditions andexperiences.

PUBLICATIONS

1. Chechilnitzky, A. Estudio de métodos de proyección depoblaciones con miras a su aplicación en proyectos deingenierla sanitaria. Santiago, Facultad de CienciasFisicas y Matemáticas, Universidad de Chile, 1968.

284

2. Coddou P., R. Aplicación de computadores al cálculo deredes de alcantarillado de aguas servidas. Secci¿n deIngenieria Sanitaria, Facultad de Ciencias Fisicas yMatemáticas, Universidad de Chile, Santiago, 1970.

3. Gundelach L., J.M. Aplicaci¿n del método de Newton-Raphsonal calculo de redes de distribución de agua potable. Secciónde Ingeniería Sanitaria, Facultad de Ciencias Físicas yMatemáticas, Universidad de Chile, Santiago, 1970.

OTHER DATA

Grantee: Ing. Isaac Faiguenbaum, Section of SanitaryEngineering, School of Mathematical and PhysicalSciences, University of Chile, Santiago.



285

FILTERING PROPERTIES OF NATURAL SANDS

PROBLEM

Natural sands have been extensively employed as filtering media inwater treatment plants. Their use requires special grading proceduresthat are costly and time-consuming. In some cases sand for filtrationpurposes has had to be imported.

METHOD

Research is being conducted in three stages: (1) design andconstruction of a pilot filtration plant to use water conditioned in thelaboratory; (2) trials of the plant's effectiveness in treating waterused in several communities of the savannah of Bogotá; and (3) trialswith water from other parts of Colombia. Studies will be made of charac-teristics of the sand used, such as organic material content, granulometry,effective size, and uniformity coefficient, as well as of thickness ofthe filtration bed, loss of head, velocity of filtration, efficiency ofbacteria removal, and rate of filtration. The first stage of the researchhas been completed.

RESULTS TO DATE

The following conclusions have been reached from the first stage ofthe research: (1) the length of the filtration run was limited primarilyby hydraulic head loss; (2) turbidity and color removal were 100 percent when coagulation and rapid filtration were used; (3) water forfilter washing was 11.9 per cent of treated water; and (4) the longevityof slow filters was about 20 days.

SIGNIFICANCE

The following benefits may result from this study: reduced costsin the selection and grading of sand, improved and more economicaloperation of plants, and substantial savings on the importation of sand.

PUBLICATIONS

1. Estudio de las propiedades filtrantes de una arena natural,Facultad de Ingenieria, Universidad Nacional de Colombia, Bogotá,February 1971.

286

OTHER DATA

Grantee:

Funded by:

Timetable:

Ing. Jairo Romero, School of Engineering,National University, Bogotá, Colombia


1969-1970

287

MODEL FOUR-FILTER WATER TREATMENT PLANTFOR THE CITY OF EL IMPERIAL

PROBLEM

One important water treatment plant problem is the complex operationand maintenance because many control panels, valves, and machines are used.This has an impact on the cost of a plant's construction, operation, andmaintenance.

The purpose of this project is to explore the possibilities ofreducing to a minimum the use of mechanical equipment, relying almostentirely on hydraulic conditions.

METHOD

The project consists of the building of a pilot treatment plant witha battery of four filters. Variables will be studied and conclusions willbe applied to the design of the plant for the city of El Imperial of 8,000people in the Department of Lima. The pilot plant is to be built at theLaboratory of Hydraulics of the National University of Engineering.

RESULTS TO DATE

The model plant is being built. The project was approved in November1971.

SIGNIFICANCE

With simplified design, savings in the cost of building and operatinga water treatment plant may be substantial.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Ing. Harry D. Dawson, Department of EnvironmentalSanitation, National University of Engineering,Lima, in cooperation with CEPIS.

288

Funded by:

Timetable:


1971-1972

289

EVALUATION OF THE INFLUENCE OF FILTRATION RATE ON WATER QUALITYAT THE LAS VIZCACHAS WATER TREATMENT PLANT IN SANTIAGO

PROBLEM

Water treatment plants are designed to meet water demands for"design periods" of usually 10, 20, or 30 years. Inability to expandplants' treatment capacity after the design period ends, usually becauseof financial difficulties, is a common problem. To improve the rateof flow through the plant without impairing the quality of the watermight be a good temporary solution. The present project was undertakento study the elasticity of the rapid filters at the Las Vizcachas watertreatment plant in Santiago, Chile, and to determine whether they couldincrease filtration rates.

METHOD

A new experimental filter having a surface of 0.806 m has beenbuilt. Different dosages of coagulants were compared with varying ratesof flow through the filters. Correlation was established between theseparameters and different sizes of sand and the uniformity coefficientin the sand media used for the filtration bed.

RESULTS TO DATE

The collection of data has been completed and the final report

is in preparation.

SIGNIFICANCE

If the research project shows that a greater load can be appliedto the Las Vizcachas plant, the capacity of both the old and the newinstallations will have been greatly expanded.

PUBLICATIONS

A final report will be ready by March 1972.

290

OTHER DATA

Ing. Isaac Faiguenbaum, School of Physical andMathematical Sciences, University of Chile,Santiago

Funded by:

Timetable:


Revised: 1969-1971

291

Grantee:

INCREASE IN FILTRATION RATES AT THE LO CASTILLO PLANT,ITS INFLUENCE ON WATER QUALITY, AND THE POSSIBILITY OFEXTENDING THE RATE OF INCREASE TO CONVENTIONAL PLANTS

PROBLEM

Water treatment plants are designed to meet water demands for"design periods" of 10, 20, 30, or more years. Inability to expandplants' treatment capacity after the design period ends, usually becauseof financial difficulties, is a common problem. To improve the rate offlow through the plant without impairing the quality of water might bea good temporary solution. The present project was undertaken to studythe elasticity of the rapid filters at the Lo Castillo water treatmentplant in Santiago, Chile, and to determine whether they could increasefiltration rates.

METHOD

An experimental filter with a surface of 0.806 m was built.Different dosages of coagulant were compared with varying rates of flowthrough the filters. Correlation was established between these para-meters and different finenesses of sand and the uniformity coefficientin the sand media used for the filtration bed.

RESULTS TO DATE

The project has ended. Some of our conclusions are: (1) averageresidual turbidities measured against different rates of filtration anddifferent kinds of sand do not change significantly; (2) for sand ofthe same characteristics, bacteria removal efficiency seems to be lowerwhen the rate of filtration is increased; (3) bacteria removal efficiencynotably diminishes when coarse sand grains are used; (4) the researchproject's size A sand is most recommended since it has an effective sizeof 0.65 mm and permits a filtration rate of 250 liters per square meterper minute.

SIGNIFICANCE

Higher rates of filtration result in an increase in the load capacityof water treatment plants and thus, in effect, in extending the design period.

292

PUBLICATIONS

1. Unda, F., et al. Filtros experimentales con alta tasa defiltración. Escuela de Salubridad, Universidad de Chile,y Escuela de Ingenieria, Universidad Católica, Santiago, 1970.

OTHER DATA

Grantee: Ing. Francisco Unda, School of Public Health,University of Chile, Santiago, in cooperation withthe Catholic University of Chile.

Funded by:

Timetable:


Revised: 1968-1970

293

USEFULNESS AND BEHAVIOR PATTERNS OFDYNAMIC AND UP-FLOW SLOW FILTERS

PROBLEM

The project will test the behavior and efficiency of dynamic andup-flow slow filters now in operation in water treatment plants.

METHOD

To achieve a more simplified filter design, the plan of studycontemplates:

a) Field research:

1. Selection of three filters2. Studies of filtering media3. Hydraulic studies4. Efficiency of turbidity removal5. Observation of hydraulic head-loss

b) Laboratory research:

1. Design and construction of pilot plants(dynamic and up-flow slow filters)

2. Selection of filtering media3. Observations of performance of pilot plants

c) Processing and interpretation of data gathered

d) Conclusions

RESULTS TO DATE

No results yet. The project was approved in November 1971.

SIGNIFICANCE

To achieve design of filters and, by implication, of settlingtanks, using simplified equipment and thus considerable savings in thecost of water treatment plants, both in their building and in boththeir operation and maintenance.

294

OTHER DATA

Grantee:

Funded by:

Timetable:

Prof. Mario Carcedo, Sanitary Engineering Center,National University of Rosario, Argentina

Pan American Health Organization and the NationalUniversity of Rosario, Argentina

1971-1972

295

SEDIMENTATION TANKS

PROBLEM

No definite conclusions have been reached concerning the advantagesand disadvantages of vertical-flow settling tanks as opposed to horizontal-flow installations. A number of factors affect the performance and effi-ciency of each type.

At the Vizcachas water treatment plant in Santiago, Chile, are circularsettling tanks, originally of horizontal-flow design, that have been modifiedto function through combined vertical and horizontal flow. The purpose ofthe present research is to gain a better understanding of this particulartype of tank and to compare the performance of horizontal- and vertical-flowinstallations.

METHOD

A compilation of existing literature on the subject is being prepared.Two different types of pilot settling tanks were designed and built, andthe efficiency of both units was measured. They were made to work underdifferent conditions, but under the same hydraulic head and with water ofthe same quality.

RESULTS TO DATE

As programmed, pilot plants and scale-model tanks were designed andbuilt. Conditioning of the settling tanks at the Vizcachas plant hasbeen completed.

SIGNIFICANCE

If conclusive results are derived from the research on combinedvertical- and horizontal-flow installations, they can be incorporated inthe design of future circular settling tanks.

PUBLICATIONS

1. Estudios sobre sedimentadores: Informe de avance. SecciónIngenieria Sanitaria, Departamento de Obras Civiles, Facultadde Ciencias Fisicas y Matemáticas, Universidad de Chile,Santiago, December 1970.

296

OTHER DATA

Ing. Juan Pablo Schifini, Faculty of Physical andMathematical Sciences, University of Chile,Santiago.

Funded by:

Timetable:


1970-1972

297

Grantee:

STABILIZATION PONDS

PROBLEM

Stabilization ponds, because of their relatively low cost, arebeing increasingly recognized as one of the most suitable approaches inthe developing countries to sewage treatment. There has been no experiencein Chile with this process. The purpose of the present research is tostudy the efficiency of stabilization ponds under the climatic conditionsprevailing in the city of Melipilla, Chile.

METHOD

A pilot stabilization pond is being built by the Ministry of PublicWorks in the city of Melipilla. At the same time, the existing literatureon stabilization ponds is being compiled and reviewed. Meteorologic dataare being collected. Different loads and rates of flow will be appliedto the lagoon, and BOD, DO, total solids, etc., will be measured to testits efficiency.

RESULTS TO DATE

The project is in its very early stages and no results are available.

SIGNIFICANCE

The performance of the stabilization pond will be compared with thatof a conventional sewage treatment plant already under construction. Theresults will provide realistic information about the feasibility of thisprocess in Chile.

PUBLICATIONS

None thus far.

OTHER DATA

Grantee: Ing. Guillermo Ruiz, School of Physical andMathematical Sciences, University of Chile

298

Funded by:

Timetable:


1970-1973

299

STABILIZATION PONDS IN ARGENTINA

PROBLEM

The stabilization pond is a sewage treatment method that isinexpensive, efficient, and simple to operate and maintain. It can beused successfully to treat wastes from slaughter houses, a matter ofgreat importance in Argentina.

The effects of particular geographic conditions--seasonal variationsin temperature, hours of sunshine, altitude, etc.--on the efficiency oflagoons need to be investigated.

METHOD

Two stabilization ponds were built, one in 1968 and one in 1969,and put into operation. In the first stage of the study, the efficiencyof the lagoons was examined, flow rate control was established, andlaboratory analysis was performed. Rainfall, temperature, evaporation,and sunlight hours were recorded.

In the second stage of the project, the effluent of the facultativelagoon has been studied and influence of algae on BOD has been determined.Performance of the two lagoons under different loading conditions hasbeen recorded.

RESULTS TO DATE

Two stabilization lagoons, one anaerobic and the other facultative,are in full operation. Data have been collected and the final report isin preparation.

SIGNIFICANCE

It is expected that this research will provide information on theefficiency of natural treatment of sewage through the stabilization pondprocess in the area of Buenos Aires.

300

PUBLICATIONS

1. Investigaci¿n sobre lagunas de estabilización; informe parcial

sobre trabajos preliminares. Instituto de Ingenieria Sanitaria,

Universidad Nacional de Buenos Aires, Buenos Aires, 1969.

OTHER DATA

Grantee: Ing. Carlos Carrique, Instituto de IngenierlaSanitaria, Universidad de Buenos Aires

Funded by:

Timetable:


Second stage: 1970-1971

301

PERFORMANCE OF FACULTATIVE STABILIZATION PONDSIN TREATING MILK INDUSTRY WASTES

PROBLEM

Water pollution due to milk industry wastes is spreading in theProvince of Santa Fé, Argentina. The project aims to study the per-formance of the stabilization pond water treatment process when appliedto milk industry wastes.

METHOD

Research will consist of several activities: (1) study of thetechnical literature on stabilization ponds; (2) determination ofphysical, chemical, and biologic characteristics of the waste effluentsfrom selected dairy plants; (3) analysis of the performance of full-sized stabilization ponds; (4) analysis of the performance of laboratory-scale stabilization ponds; (5) study and interpretation of the datagathered; and (6) formulation of conclusions.

RESULTS TO DATE

No results yet. The project was approved at the end of October,1971.

SIGNIFICANCE

We hope to determine the design parameters of stabilization pondswhen used to treat wastes from the dairy industry.

PUBLICATIONS

None thus far.

OTHER DATA

Prof. Mario Carcedo, Center of Sanitary Engineering,National University of Rosario, Argentina

302

Grantee:

Funded by:

Timetable:

Pan American Health Organization and NationalUniversity of Rosario, Argentina

1971-1972

303

ORTHOKINETIC FLOCCULATION

PROBLEM

Orthokinetic flocculation is a hydraulic process that has not beenthoroughly studied. Only scant information is available about thistechnique. A systematic study of the subject would seem very desirable.

METHOD

Water with artificially controlled turbidity will be circulatedthrough a hydraulic flocculator. Rates of flow, velocities, amount ofturbidity, coagulant dosage, and other factors will be varied, and anumber of combinations will be studied.

RESULTS TO DATE

No significant results have been registered yet.

SIGNIFICANCE

It is expected that the results of this project will improve theefficiency of hydraulic flocculators of helicoidal flow through newdesign approaches. These units could be used to serve populations withlimited economic resources.

PUBLICATIONS

None thus far.

OTHER DATA

Ing. Pedro MartinezNational AutonomousD.F., Mexico

Pereda, School of Engineering,University of Mexico, Mexico,

Funded by:

Timetable:


Revised: 1970-1972

304

Grantee:

BIOLOGIC TREATMENT OF TOXIC WASTES FROM MANIOC FLOUR MILLS

PROBLEM

Wastes from the processing of manioc flour have been found to containhydrocyanic acid. The possible biologic flocculation of these wastes wasstudied a few years ago at the University of Sao Paulo. The present re-search, a continuation of that study, is mainly concerned with assessingthe effect of hydrocyanic acid on waste treatment processes.

METHOD

Studies were carried out on the effect of prolonged aeration. Thestabilizing capacity of flocculation was measured using the Warburg micro-respirometric unit. Anaerobic digestion was also investigated. Potentialredox was used to evaluate the oxidizing capacity of microorganisms.

RESULTS TO DATE

In the treatment plant, prolonged aeration helps biologic flocformation, even when concentration of cyanates is 500 mg/l. All cyanatesin water are eliminated in less than 24 hours in the presence of the pre-formed biologic flocs. When cyanates are present, only fungi and bacteriadevelop. Ciliates are adversely affected.

SIGNIFICANCE

Our research may shed light on the impact of the hydrocyanic acidin manioc flour processing on existing waste treatment procedures. On thebasis of the information acquired, we hope to develop an improved methodfor controlling this peculiar and possibly dangerous substance.

PUBLICATIONS

1. Murgel Branco, S. Influencia dos cianetos na estabilizagaobiologica aerobia de residuos de industrializacao de mandioca.Faculdade de Saúde Pública, Universidade de Sao Paulo, 1971.

30o5

OTHER DATA

Dr. Samuel Murgel Branco, Faculdade de Higiene eSaúde Pública, Universidade de Sao Paulo, Sao Paulo,Brazil

Funded by:

Timetable:


1969-1970

306

Grantee:

DUST POLLUTION IN WORKING AREAS

PROBLEM

Dust in working areas is an important occupational health problem.Bloomfield points out that the number of workers exposed to dust particlesin the United States is greater than that exposed to other occupationalrisks. Preliminary surveys in Buenos Aires, C¿rdoba, Mendoza, andTucumán showed that a similar situation exists in Argentina.

METHOD

First stage: A methodology to be followed was established and acomprehensive report prepared on the state of the art, including charac-teristics of aerosols, mechanism of aerosol absorption and deposit onthe respiratory system, and statistical methods used to determine theheterogenic dispersion of aerosols in the air.

Second stage: The methodology established in stage 1 was tested inthe actual working environment of selected plants.

Third stage: Laboratory apparatus and techniques were developed tomeasure the efficiency of dust-treatment equipment and exhaust ducts.

RESULTS TO DATE

A report is in its final stages of preparation.

SIGNIFICANCE

The study's results will help establish regulations for dust controlin the working environment.

PUBLICATIONS

None thus far.

307

OTHER DATA

Grantee:

Funded by:

Timetable:

Ing. Julio C. Durán, Instituto de IngenieriaSanitaria, Universidad Nacional de Buenos Aires


First stage: January - December 1971

308

AN EVALUATION METHOD TO MEASURE THE EFFECTIVENESSOF PAN AMERICAN HEALTH ORGANIZATION PROGRAMS

PROBLEM

A mathematical model that forecasts the vital statistics, lifeexpectancy, and longitudinal population characteristics resulting fromvarious input options is desired to form the basis of cost-effective-ness studies of PAHO activities.

METHOD

A Markovian approach with absorbing barriers that yields mortalityrates by age and disease group, fertility rates by age, and the agestructure of the population over time has been developed. In addition,"steady state" mortality rates, by disease and age grouping, and totalfertility rates are exhibited. The inputs are age- and disease-group-specific mortalities, and age-specific fertilities.

RESULTS TO DATE

The model has been programmed at Johns Hopkins University andapplied in an expository fashion to data obtained in Costa Rica. Theresults of three "simulated" experiments have been obtained. Theyillustrate the impact of changes of a single age- and disease-specificmortality alteration that might ensue from the application of a programto control infectious and parasitic diseases in infancy. In addition,the impact of changes from the baseline fertility structure of Costa Ricais investigated both separately and in conjunction with the mortalitychange mentioned above.

This model is currently being adapted to planning the allocationof human resources in health services and to epidemiologic studies.

SIGNIFICANCE

The model that has been developed is a first step in the study ofthe time-projected impact of PAHO programs.

Program changes are reflected in alterations of age- and disease-group-specific mortalities and/or age-specific fertilities.

309

The computer program calculates such things as life expectancyas a function of one's age, life tables (in the ordinary sense, as wellas in the extended sense of yielding the percentage of a cohort thatwill die in each disease group for each age interval), vital statistics,and population characteristics over time.

The impact of any fertility- and/or disease-specific change forany age group may be examined over time to forecast the total impactof a program. Thus, for example, if one age-specific disease groupmortality rate is drastically reduced, one may see whether this has realimpact on vital statistics or only a nominal impact due to the effectof other disease groups that still affect the cohort and might gain fromthe change.

PUBLICATIONS

1. A birth-life-death model for planning and evaluation ofhealth services programs. Health Services Research 6:120-43,1971.

OTHER DATA

Grantee: Mr. Jorge Ortiz, Pan American Health Organization,Washington, D.C., in collaboration with Dr. RodgerD. Parker, Johns Hopkins University School ofHygiene and Public Health, Baltimore, Maryland



310

A SYSTEMS STUDY OF THE KINGSTON, JAMAICA, BLOOD BANK

PROBLEM

The main purpose of this study is to demonstrate to blood bankadministrators how data records can be processed in different ways togive statistical assistance in decision-making.

METHOD

Daily descriptive tables of collection, use, waste, inventory, andreturn of blood units to the blood bank are drawn up from data in a logbook that lists receipt and chronologic disposition of each unit ofblood. A systems depiction of the blood bank and its users in the formof flow charts is given. Control charts of use, waste, and inventory,useful for noting changes in the blood flow system when they first occur,are provided.

RESULTS TO DATE

The computer programs for this study have been developed and runto produce a report. They are now available for use with other data toproduce periodic reports automatically. Such reports, following theformat of the Jamaican study, would contain general tables involvingnumbers of donors versus blood type and donor characteristics, numberof blood units used versus type and location, number of blood unitswasted versus type and location, etc.; daily tallies of blood dispositionby type; blood flow charts; histograms involving blood units collected,issued, used, wasted due to old age, returned, and in inventory. Inaddition, percentage of IVD findings among various strata of donors,percentage of blood issued by age of blood and location of issue, per-centage of blood used by age, and percentage of blood returned areexamined.

Quality control charts exhibiting trends in blood collected versusdonor type, blood issued by type, blood returned by type, blood used andwasted per type, etc., are given, with emphasis on using these chartsto note changes in the system.

SIGNIFICANCE

The daily descriptive tables provide the administrator with a meansof being aware of the patterns of blood use throughout the system. The

311

systems depiction and control-chart approach are useful as a frameworkin which to monitor changes in the system and relate actions involvingthe operation of the blood bank to their impact on user hospitals.

PUBLICATION

A report has been completed and submitted to the Jamaican Government.

OTHER DATA

Grantee: Mr. Jorge Ortiz, Pan American Health Organization,Washington, D.C., in collaboration with Dr. RodgerD. Parker, Johns Hopkins University School ofHygiene and Public Health, Baltimore, Maryland

Funded by:

Timetable:


1969-1971

312

THE LINK MODEL

PROBLEM

Health planning involves decisions and actions that lead to desiredchanges in the structure and functions of health systems. Such decisionsand actions have very strong political components, both within the healthsector itself and in the economic and social systems of which the healthsector is a part. The planning process will be able to make its mostfeasible proposals when a better knowledge of the structure and operationof the health system, as well as of the internal and external constraintson it, is available.

This project is aimed at developing a theoretical model to representthe totality of the health system and its interconnections with othersocial systems; to analyze the interrelations of its components; to testthe outcomes of alternative policy decisions in health; to link healthplanning with planning for general development, and, finally, to providethe conceptual framework and a heuristic construct for the study ofsubsystems through model-building in the health field.

METHOD

Numerical experimentation methods are used. They are believed tobe the most apt for handling the type of information required for themodels.

RESULTS TO DATE

The theoretical formulation of the link model in very aggregativeterms was developed by the Planning Center. A reduced version for thepurpose of demonstration and testing the feasibility of its implementationwas designed and carried out with data from one country. Several experi-ments were performed at the Computer Center of the Central University ofCaracas.

A global model of this type was constructed in Argentina by theStudy of Health and Medical Education modeling group. It was notimplemented.

Additions and incorporations to improve the workability of the modelare under way.

313

SIGNIFICANCE

1. Recognition is given to the political character of the actof planning, which is aimed deliberately to introduce changes in ahealth system. Value judgments of decision-makers are thus not ruledout; rather, they are introduced into the model.

2. The methods of numerical experimentation are introduced inhealth planning for the first time to study this type of problem.They are substituted for the mechanical procedures that have provedinadequate in dealing with complex and ill-defined situations, such asthat of social systems and in particular the health system.

3. The testing of the feasibility of plans by experimentingwith the outcomes of alternative policy decisions seems to be of theutmost importance.

OTHER DATA

Grantee: Pan American Health Planning Center, Santiago, Chile

Funded by: PAHO and the United Nations Development Program


314

HEALTH FINANCING SUBMODEL

PROBLEM

Financing health services adequately is becoming very complex. Theincreasing costs of existing services due to their striving for bettercare, and the expansion of health facilities to cope with the greaterdemands of a growing population and to meet new health needs result in acritical situation when a government is confronted with decisions as tohow to allocate funds to different sectors of development.

Even though real financing is a function of monetary financing, itis only infrequently that a health plan includes a search for devicesadequate to increase or optimize the financial capacity of the healthsector. It is in this financial area that the social changes expectedto stem from health plans have to be precisely stated and their feasi-bility, coherence, and efficacy spelled out.

This project will study all the financial aspects of health, con-sidering them as a subsystem of the health system. It will try todetermine the financial feasibility of health plans and the probableeffects of policy decisions on allocation of funds to health care pro-grams. The project is being developed at the request of the healthauthorities of the province of Mendoza, Argentina, and will be basedon information from this province.

METHOD

A numerical experimentation model derived from the link modelis used and applied to a concrete situation. Computer services areperformed at the Health Computing Center of Buenos Aires, Argentina.

RESULTS TO DATE

The model has been constructed and implemented. The results oftesting alternative policies in health are now being computerized.

SIGNIFICANCE

The most important problems in financing health are brought tolight and the link model is enriched. Decisions on allocation of fundsand for the search of new financial sources are based on actual knowledgeof the situation.

315

PUBLICATIONS

None thus far.

OTHER DATA

Grantee:

Funded by:

Timetable:

Pan American Health Planning Center/Study of Healthand Manpower (Argentina)

PAHO, the Government of Argentina, and United NationsDevelopment Program

1971-1973

316

METHODOLOGIC MODEL

PROBLEM

The PAHO/CENDES methodology follows a sequence of steps for theprogramming of health activities, but it has been somewhat cumbersomebecause of the lengthy computations involved. The procedure includesa great many variables and numerical indicators whose discriminatingpower, sensitivity, critical values, and other characteristics are notwell known.

The project aims at computerizing a model that simulates the wholeprocedure in order to study and experiment with the intervening variablesand relations.

METHOD

A model of the process of PAHO/CENDES methodology was formalizedand programmed for computation. The model was implemented with realdata from a certain area, and after several experiments have been pro-grammed the outputs will be analyzed.

RESULTS TO DATE

With the exception of a few subroutines, the output of the computeris almost completed. Analysis is under way.

SIGNIFICANCE

The project is significant mostly from a methodologic viewpoint.It will ascertain the precision, sensitivity, and discriminative powerof certain parameters, and will indicate the reliability with which theycould be used in programming. Certain indications will then be obtainedfrom the information systems that collect the data from which they areobtained.

PUBLICATIONS

None thus far.

317

OTHER DATA

Grantee:

Funded by:

Timetable:

Pan American Health Planning Center, Santiago, Chile

PAHO and the United Nations Development Program

1969-1971

318

HEALTH TYPOLOGIES

PROBLEM

It is convenient at times to divide the Region's countries intogroups of similar developmental levels and characteristics. It oftenhappens that specialists in a given subject classify countries by usingindicators that are relevant for their subject only. Decisions thusadopted about the countries in question might result in an optimalallocation of resources in the specialists' area of concern with aprobable loss of efficiency in all or most other areas.

When no particular subject is in mind and all traits of developmentare considered, it is not easy to arrive at a taxonomy since all countriesare at the same developmental level or all are different. Neither ofthese extremes is of any use in efficiently and practically differentiatingcountries under study.

The development of typologies of countries seems to be a worthwhileenterprise, and this project is a first step toward that goal.

METHOD

A total of 30 indicators of health, education, economy, environment,consumption, and population were chosen for 24 countries of the Region.Most of the indicators were obtained from the four-year projections ofPAHO member countries. They were homogenized and combined so as to con-stitute a vector for each country. The distances between vectors werefound and their variability analyzed. Iteration was used to find thequasi-optimum groupings of countries that constitute the typologies ofdevelopment.

A similar procedure was used with an enlarged number of healthindicators, from which health typologies were constructed. The relationbetween the general and special typologies was studied.

RESULTS TO DATE

The typologies of health and development have been constructed andare ready for use.

319

SIGNIFICANCE

From the practical point of view, the typologies may be used as aguide in differentiating groups of countries with similar characteristicsthat are to be dealt with in a like way. This project is also a demonstra-tion of how regularly collected data may be put to good and different usesthrough research.

PUBLICATIONS

The final report of the project is being edited for publication.

OTHER DATA

Grantee: Pan American Health Planning Center, Santiago, Chile

Funded by:

Timetable:

PAHO and the United Nations Development Program

1970-1971

320

NATIONAL SYSTEM FOR MAINTENANCE AND ENGINEERINGOF HEALTH CARE FACILITIES

PROBLEM

Immediate Objectives

Reduce the backlog of essential maintenance and the accumulateddeficit of current investment in maintenance and repairs of health carefacilities. Increase the normal useful life of facilities and equipment.Establish a national system for hospital and health care facilitiesmaintenance and engineering.

Long Range Objectives

Development of human resources for maintenance and engineering.Assignment of economic resources and financing. Development of anational maintenance system, emphasizing regional programs. Managementand administrative improvement in the utilization of the existing andfuture maintenance resources.

METHOD

The establishment of national maintenance and engineering servicesat four levels of operation--central, regional, subregional, and local--byclearly identifying the functional relationship of the various levels ofthe system. Organizational structures will be developed and introducedat each level within the framework of the administrative reform. Themanagement component will be developed and introduced at all levels alongwith the education and training component, special studies, and technicalassistance.

RESULTS TO DATE

A UNDP mission made an on-site evaluation which indicated that theobjective of the plan or operation for the center for hospital maintenanceand engineering were being carried out effectively. All necessary tech-nical manuals have been written, and programs of instruction have beendeveloped and utilized in the education and training of personnel. Inter-ministerial technical committees have been established, through whichtechnical recommendations and modifications can be incorporated during thecourse of design and construction. A comprehensive program for the operation,maintenance, and inspection of equipment through technical training courses

321

in electrical systems, hospital maintenance and engineering, refrigerationand air-conditioning, boiler operation and maintenance, emergency powerplants, environmental sanitation, sterilization equipment, and laundrysupervision were conducted.

SIGNIFICANCE

The special studies carried out as a result of the total programindicate that effective cost savings can be made without jeopardizingmedical care procedures and that special studies through operationsresearch are required in order to maintain the health facility at theoptimum.

PUBLICATIONS

Listed below are technical manuals published by the Center forHospital Maintenance and Engineering, Caracas, Venezuela:

1. Maintenance and Operation of Sterilization Equipment

2. Cleaning Methods and Procedures

3. X-Ray Equipment and Its Maintenance

4. Maintenance of Steam Plants and Distribution Lines

5. Maintenance of Furnishings in Buildings, Installationsand Equipment

6. Maintenance and Operation of Incinerators

7. Maintenance of Fire Prevention Equipment

8. Maintenance of Water Tightness

9. Operation and Maintenance of Refrigeration and Air-Conditioning Equipment

10. Operation and Maintenance of Kitchen Equipment

11. Maintenance of Exterior Areas

12. Water Treatment for Installation and Equipment of Hospitals

13. Hospital Environmental Conditioning

322

14. Organization Study and Project for the Maintenance ofEmergency Electric Plants

15. Maintenance and Operation of Laundry Equipment

16. Training Program for Hospital Maintenance Personnel

17. Training in the Maintenance and Operation of Steam GeneratingPlants

18. Standards of Acceptance of Medical Assistance Centers

OTHER DATA

Grantee:

Funded by:

Timetable:

Center for Hospital Maintenance and Engineering,Caracas, Venezuela

Pan American Health Organization, World HealthOrganization, United Nations Development Program,and the Government of Venezuela

1972-1974

323

PAHO REGIONAL LIBRARY OF MEDICINE: A CENTER OFBIOMEDICAL COMMUNICATION FOR LATIN AMERICA

PROBLEM

A survey of library resources in Latin America conducted by the PanAmerican Health Organization in 1965 revealed serious deficiencies in thesupply of information to the biomedical community and an urgent desireon the part of most of the institutions concerned to resolve the problem.

METHOD

The Regional Library of Medicine (RLM) was established in 1967 tocontribute to the strengthening and improvement of the teaching, research,and practice of medicine and allied professions in Latin America. Itcarries out regular functions designed to accomplish the following aims:

To provide workers in the health sciences with increased access toa more comprehensive collection of publications in these sciences, and,by so doing, to make Latin America self-sufficient in library resourcesand services in the biomedical fields;

To use modern methods of communication, including MEDLARS tapes, tomake these materials more accessible;

To build up a pilot audiovisual medical unit;

To serve as a demonstration and training center to foster andencourage the development of medical libraries in Latin America;

To encourage cooperative library programs among medical librariesin Latin America; and

To build up its own facilities through the development of journalsand monograph collections, as wellas selected secondary referencematerials.

RESULTS TO DATE

A library building provided by the Escola Paulista de Medicina hasbeen adapted for RLM's use, and equipment is being obtained as needed.Since 1971 the building has been expanded from 1,950 m to 3,340 m tosatisfy RIM's growth in services.

324

The systematic acquisition and maintenance of bibliographicmaterials, serial publications, monographs, and documents is being

continued. A collection of periodicals from 1960 onward of 4,288biomedical journal titles--l,4 81 of which are obtained by subscription,235 by exchange, and 675 by grants from the editors--is well on itsway to being completed.

The staff is being organized so as to make the best use of special

techniques for international library service. Its role in the improve-ment of local services is also being developed.

A Scientific Advisory Committee to the Library has held four annualmeetings so far.

In January 1969, interlibrary loan services to all parts of Brazilwere begun, and based on experience in operations in that country RLM

extended its services to other Latin American countries in 1970. Up to1971 a total of 65,287 interlibrary loan requests from 150 librarieswere processed and services were begun to Latin American countries,

especially Argentina, Peru, and Venezuela. RLM was able to satisfy 62per cent of the requests from its own resources. An additional 16 per

cent were answered through other libraries in Sao Paulo participating in

the program, and the remaining 22 per cent were forwarded to the U. S.

National Library of Medicine in Bethesda, Maryland, for processing. Up

to 1971, 661 reference requests for special bibliographies were alsoreceived: 567 of these were attended to by the Regional Library itself;

94 were sent to NLM in Bethesda for bibliographic retrieval by theMEDLARS computer-based system.

Seven subcenters have been developed in Brazil--Fortaleza, Recife,

Salvador, Brasilia, Rio de Janeiro, Belo Horizonte, and Porto Alegre--

and in addition, two specialized subsystems for dentistry and veterinary

medicine were established. Once these libraries' resources are pooled

and operations throughout Brazil are fully established, RLM will cooperate

with national centers elsewhere in Latin America. Gift and exchange

activities were conducted with 641 libraries in Brazil, and 1,355 similarinstitutions in other Latin American countries prior to 1971.

In 1970 a Selected Dissemination Information program was begun tospread biomedical and scientific information to selected groups of re-

search workers in hospital administration, medical education, nuclearmedicine, preventive and social medicine, and parasitology and tropical

medicine. At the present time, 4,183 articles have been delivered.

Advanced courses have been offered to medical librarians from

throughout Latin America. In 1971, 19 librarians received PAHO fellow-

ships to attend the courses.

325

SIGNIFICANCE

By providing specialists in the health sciences with increasedaccess to scientific literature, the Regional Library of Medicine is con-tributing to the acceleration and improvement of biomedical education,research, and practice in Latin America.

Satisfactory, prompt service and improved bibliographic resourcesare generating an increase in requests for scientific literature. Bio-medical librarians throughout Brazil have responded with increasinginterest by producing collective catalogs and improving cooperativeinterlibrary loan service among local medical libraries.

Cooperative efforts have been made to improve scientific informationin most of the Latin American countries. An inter-American network ofbiomedical communications is being planned and studies are being made ofthe existing facilities and needs.

Several projects are being presented to international agenciesinvolved in aiding research, with the idea of giving Latin American accessto a wider and more complete collection of medical information that isbasic for creative research work, and of avoiding repetition of research.

PUBLICATIONS

1. Information Bulletin: 3 volumes

2. List of Joint Duplicates for Exchange: 10 issues

3. Catalogue of Recent Acquisitions: 4 issues

4. Recent Information on RLM: 3 issues

5. The Library for Librarians: 1 issue

6. Suggestions for the Establishment of Library Services

7. Regulations for Interlibrary Loans

8. Minimum Requirements for the Establishment of a MedicalSchool Library (also in Spanish): 3 editions

9. Bibliographic Reference Lists: 3 issues

10. RLM (Information Brochure)

11. RLM (Information Brochure - in Spanish)

326

OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. M. Martins da Silva (Project Coordinator), PanAmerican Health Organization, Washington, D.C., andDr. Amador Neghme, Director, PAHO Regional Libraryof Medicine, Sao Paulo, Brazil

The Government of Brazil, the U.S. National Libraryof Medicine, The Commonwealth Fund, the Pan AmericanHealth Organization, and the Escola Paulista deMedicina

1967 - continuing

327

PAHO/WHO IMMUNOLOGY RESEARCH AND TRAINING CENTERS

PROBLEM

Immunology is a rapidly developing science. There can be no doubtthat postdoctoral workers competent in basic immunology and in modernimmunologic techniques have become indispensable to any major healthcenter. Rapid developments are in progress in transplantation, immuno-pathology, and knowledge of the structure and function of immunoglobulins,cellular differentiation, and genetics. It is clear that immunology nowdeserves attention as a central part of biology and medicine, and thatthe subject is no longer restricted mainly to serologic techniques andimmunization procedures. Despite the broadening scope of activities inthis field, however, the phenomenon of immunity to infectious diseases ispoorly understood and vaccination remains an empirical procedure. Per-tinent knowledge will probably be gained by a closer study of the immunologyof parasitic diseases. For a program in immunology to develop harmoniously,it should keep abreast of research developments throughout the world andcooperate closely with all other institutions engaged in such work.

METHOD

Two PAHO/WHO Immunology Research and Training Centers have been setup for the Americas: one in Sao Paulo, Brazil, at the Butantan Institute,and the other in Mexico City, which uses the laboratory facilities andpermanent staff of seven cooperating institutions.

The PAHO/WHO Immunology Research and Training Center in Sao Paulo,now in its sixth year of operation, holds an annual course in advancedimmunology for students from Brazil and other South American countries.The course lasts four months and is given entirely in English. In additionto providing a formal introduction to basic immunology, it is a means ofdiscovering research talent in a few students, who are then offered oppor-tunities to remain at the Center for a year or two to conduct their owninvestigations. The Center also helps local institutions with immunologyinterests.

The PAHO/WHO Immunology Research and Training Center in Mexico City,in cooperation with its affiliated institutions, offers short coursesand seminars and runs a three-year academic course leading to a Ph.D.degree in immunology. These courses are open to students from all ofLatin America. The Center also conducts research in basic immunology andin the immunology of diseases prevalent in Mexico.

328

RESULTS TO DATE

Visiting lecturers and examiners from the United States and Europehave participated in the Sáo Paulo Center's teaching and research. Pastresearch included work on the responses of patients with Brazilianpemphigus foliaceus to treatment with immunosuppressive drugs, the bio-logic significance of the heterogeneity of immunoglobulins to determinewhich are active in protecting against infectious agents and whichcontribute to tissue damage by causing hypersensitivity reactions, andthe mechanism of the hemorrhagic reaction produced in the skin of normallaboratory animals by the sera of Brazilian pemphigus foliaceus patients.Current research includes work on the mechanism of production of homocyto-tropic antibodies in the guinea pig, the effect of splenectomy and thymectomyon reaginic antibody production, the effect of Bacillus pertussis vaccineon cell-mediated immunity, factors present in B. pertussis cells responsiblefor their adjuvant effect, and the complement-inactivating substances pre-sent in the venom of the Brazilian snake Bothrops jararaca.

The work in each of the Mexico City Center's participating laboratorieshas been enriched by the ideas, advice, and know-how other workers haveoffered and final results are discussed in joint, weekly seminars. The Centerhas brought in visiting professors and investigators who have deliveredlectures or short courses and also acted as temporary advisers in differentlaboratories. Some local workers have had opportunities to make some shortvisits to laboratories abroad.

The graduate program have resulted in two candidates obtaining theirdoctoral degrees. Two candidates will present their final dissertations atthe end of the present year, and eight others are in less advanced stagesof training. Two fellows from South America have received stays of oneyear or longer, and three candidates from Mexico have been working tempo-rarily in laboratories abroad to acquaint themselves with methodology notnow fully developed in Mexico.

The Center has organized two courses of two weeks each on theimmunochemistry of bacterial polysaccharides and general immunology.

Research is being carried out in the following areas: studies of thecommon antigen (Kunin) of E. coli in relation to infant diarrheas; therelationship between malnutrition and immunity; antigenicity to rabbits ofIgG in various states of aggregation, and its implications in immunologictolerance and rheumatoid arthritis; complement components in rabbits inrelation to immune responses, including rejection of transplants; immuno-chemical properties of polysaccharides from M. tuberculosis, M. leprae,N. brasiliensis, N. asteroides, and K. rinoscleromatis; systemic lupus;antienzyme antibodies; laboratory models of anaphylaxis; erythrocytegroups and other genetic markers in normal and atopic populations; cell-mediated immunity in lepromatous and tuberculoid leprosy; anti-DNAantibodies in collagen diseases; experimental induction of antinuclear

329

antibodies by different pharmacologic agents; collagen antigenicity;delayed hypersensitivity with special reference to cytotoxic effectors;and production of antibodies to guinea pig macrophages.

SIGNIFICANCE

Experienced scientists agree that applied research will flourish onlyif it is constantly nourished by ideas, techniques, and findings derivedfrom basic research. It is therefore shortsighted for any region of theworld not to devote a reasonable share of its available resources to thesupport of basic research, particularly in immunology, in which there isoften only a short step between basic findings and their application intherapy and prophylaxis. A laboratory for basic research can be maintainedat relatively small expense and can prove a tremendous asset as a continuingsource of inspiration and intellectual support of those who are applyingimmunology in public health.

The establishment of centers for research and training in immunologyin Mexico City, Sao Paulo, Ibadan (Nigeria), Singapore, and Lausanne byPAHO/WHO is one of the ways in which the Organization is helping to createthe trained scientific manpower essential for promoting regional and localstudies in countries where tropical diseases exist.

PUBLICATIONS

Sao Paulo

1. Albuquerque, L.R., et al. Failure of insoluble antigensto elicit anaphylactic reactions. Inter Arch Allergy, inpress.

2. Buetner, E.H., et al. Immunofluorescent studies of auto-antibodies to intercellular areas of epithelia in Brazilianpemphigus foliaceus. Proc Soc Exp Biol 127:81-86, 1968.

3. del Guercio, P., et al. Opsonic, cytophilic, and agglutinatingactivity of guinea pig y2 and YM anti-Salmonella antibodies.Immunology 16:361-71, 1969.

4. Mota, I., and A. Perini. A heat labile mercaptoethanolsusceptible hemocytotropic antibody in the guinea pig.Life Sciences 9:923-30, 1970.

5. Perini, A., and I. Mota. Heterogeneity of guinea pig homo-cytotropic antibodies. Immunology, in press.

330

6. Prigenzi, L.S., et al. Observations on the responses of patientswith BPF (Brazilian pemphigus foliaceus) to treatment withazathioprine (Imuran) alone or in combination with corticosteroids.II Int Symp Immunodermatol, Warsaw, 1969. Submitted for publicationin Int Arch Allerg.

7. Siqueira, M., et al. The inhibitory effect of large doses ofhistamine on the passive Arthus' reaction. Int Arch Allerg31:117-22, 1967.

8. Uriel, J., et al. Immunoelectrophoretic characterization ofenzymes in the venom of Bothrops jararaca. Submitted for publi-cation in Science.

Mexico City (1971 only)

1. Jaroslow, B.N., and L. Ortiz-Ortiz. Hydroxyurea and cell cyclekinetics of cultured antibody forming cells. Cell Immunol2:164, 1971.

2. Kumate, J., et al. Content of common antigen of Escherichia coliand diarrhea of newborns and infants in a Mexican preindustrialcommunity. Ann NY Acad Sci 176:360-69, 1971.

3. Kumate, J., et al. Neutrophil-mediated immunity in severe humanmalnutrition. Proc III West Hem Nutr Cong, Miami, 1971.

4. Mejia Laguna, J.E., et al. Prolonged survival of kidney xenograftsin leukopenic rabbits. Immunology 21:679-82, 1971.

5. Montfort, I., and R. Pérez Tamayo. Two antigenically differenttypes of macrophages. Proc Soc Exp Biol Med 138:204, 1971.

6. Ok6n, L.M. The use of sensitized bacteria for passive agglutination.Rev Lat Am Microbiol 13:181, 1971.

7. Ortiz-Ortiz, L., et al. Delayed hypersensitivity to ribosomalprotein from BCG. J Immunol 107:1022, 1971.

8. Ortiz-Ortiz, L., et al. Tuberculin activity of somatic fractionsobtained from Mycobacterium. Rev Lat Am Microbiol 13:35, 1971.

9. Rojas, O., et al. Algunos hallazgos serol¿gicos en pacientescon rinoescleroma. Rev Lat Am Microbiol 13:59, 1971.

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OTHER DATA

Dr. Otto G. Bier, PAHO/WHO Immunology Researchand Training Center, Instituto Butantan, Sao Paulo,Brazil, and Dr. Jesús A. Kumate (Project Coordinatorin Mexico), Hospital Infantil de México, México, D.F.,Mexico

Funded by: Pan American Health Organization/World HealthOrganization

Timetable: Sao Paulo:Mexico City:

1966 - continuing1969 - continuing

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Grantee:

RESEARCH TRAINING PROGRAM IN VIROLOGY, ORNITHOLOGY,ECOLOGY, AND TROPICAL MEDICINE

PROBLEM

There is a widely acknowledged shortage of trained investigators inmany fields of medical research, especially investigators competent toplan and carry out programs that include both field and laboratory research.Hence, the purpose of this training program is to develop research investi-gators in virology who are at home not only at the laboratory bench butalso in the field. It is hoped that the predoctoral and postdoctoraltrainees, in addition to receiving their primary training in virology. willalso develop a keen appreciation of entomology, ornithology, mammalogy,and other areas of zoology.

METHOD

Extensive training in virology is given at the Department of Micro-biology, Cornell University Medical College. The curricula for predoctoraland postdoctoral trainees is rounded out with courses in other departmentsat Cornell and, in some cases, courses offered at other graduate institutionsin the New York area or in the field of biology on the campus at Ithaca,New York. Field training has been given through field research and train-ing courses in virology, entomology, vertebrate zoology, and ecology, andthrough graduate student participation in field research programs in Mexico,Guatemala, British Honduras, and Honduras. The students at Cornell maychoose to orient their thesis research around this field work or they mayfollow the materials collected during field periods by participating inweekly seminars at which current research progress is reported.

RESULTS TO DATE

During the field season of 1970-1971 seven trainees from CornellUniversity Medical College participated in field research and training

programs in Guatemala. These included three predoctoral and pre-DVM andtwo postdoctoral fellows from the USA and one pre-Master's fellowshipfrom El Salvador. One of the postdoctoral fellows received initial train-ing in field techniques in preparation for a six-week field research

period in Peru (October-November 1971) under PAHO sponsorship. TheGuatemala programs were carried out in cooperation with the University ofSan Carlos School of Medicine (Guatemala). Six of its medical studentsparticipated in the 1970 field program.

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SIGNIFICANCE

The present program is adding to the corps of qualified investi-gators in the field of arthropod-borne virus research. Although newknowledge is already forthcoming through the research activities ofthe trainees (discovery of Venezuelan encephalitis and other arbovirusespotentially important to man and domestic animals in Guatemala, Mexico,British Honduras, and Honduras), the long-term productivity of thetrainees, once they become independent investigators and educators, willprovide the really significant rewards of the program.

PUBLICATIONS

1. Dickerman, R.W., W.F. Scherer, and A. Diaz-Najera. Ecologicstudies of Venezuelan encephalitis viruses in southeasternMexico. 1. Introduction and study sites. Am J Trop Med Hyg

20:730-39, 1971.

2. Dickerman, R.W., M.L. Zárate, W.F. Scherer, and J. de MuchaMacias. Venezuelan encephalitis virus along the central andnorthern gulf coast of Mexico as of July-September 1969.Bol Ofic Sanit Panam 71:143-51, 1971.

3. Schaffer, P.A., and W.F. Scherer. Stability of virulence andplaque size of Venezuelan encephalitis virus with passage inmosquitoes (Aedes aegypti). Am J Epidemiol 93:68-74, 1971.

4. Scherer, W.F., R.W. Dickerman, C. Campillo-Sainz, M.L. Zárate,and E. Gonzáles. 5. Infection of domestic animals other thanequines. Am J Trop Med Hyg 20:989-93, 1971.

5. Wong-Chia, C., and W.F. Scherer. Aislamiento del virus dela encefalitis venezolana de un murciélago .frugivoro(Artibeus turpis) en México. Bol Ofic Sanit Panam 70:339-43,1971.

6. Zárate, M.L., and W.F. Scherer. A comparative study ofvirulences, plaque morphologies and antigenic characteristicsof Venezuelan encephalitis virus strains. Am J Epidemiol89:489-502, 1969.

7. Zárate, M.L., W.F. Scherer, and R.W. Dickerman. Un casoprobable de encefalitis equina venezolana ocurrido enJaltipan, Veracruz, México, Salud Pub Mex 13:97-99, 1971.

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OTHER DATA

Grantee:

Funded by:

Timetable:

Dr. William F. Scherer, Cornell UniversityMedical Center, New York, New York

National Institutes of Health/U.S. PublicHealth Service

1972-1977

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RESEARCH TRAINING IN CLINICAL MEDICINE

PROBLEM

A young physician from the Caribbean area, Middle America, or SouthAmerica who wants to pursue a career in clinical research generally hastwo possibilities open to him: he may obtain a fellowship to go abroad,usually to the United States, or he may get an appointment, almost invariablypart-time, in his own medical school and seek a grant, most often from anoutside source, to cover his research expenses and the cost of equipment.

Neither of these options is entirely satisfactory. The young medicalgraduate who goes straight into a part-time academic post seldom has ascientific background adequate to ensure success in a research career.Training in highly developed countries is valuable, but it has disadvantages:the subject of study may be quite unrelated to the problems and conditionsthe investigator is likely to encounter on his return home, and the researchfacilities and general approach in his country may be so different as tomake his readaptation very difficult. The frustration that ensues may causehim to emigrate.

A survey in 1968 of approximately 100 institutes and clinical depart-ments in nine countries of Latin America and the Caribbean area identifiedseveral where excellent research is being carried out in various fields.A program for advanced training in clinical research was therefore developedby PAHO to take advantage of the educational potential of these institutions.

METHOD

The program is open to citizens of any country in the Caribbean area,Middle America, or South America to undertake special studies in any otherof these countries.

Candidates must be actively engaged in research in medicine or anallied acience, and must be reasonably fluent in the language of the countryin which they will study or in another language that can be used in theirwork in that country.

Applications are appraised for scientific merit by a Technical ReviewPanel of eight members, appointed on a rotating basis, who currently re-present the fields of internal medicine, pediatrics, orthopedics andrehabilitation, tropical medicine, endocrinology, hematology, immunologyand transplantation, and surgery.

Grantees are generally selected from applicants with less than fiveyears' postdoctoral experience in medicine. Awards are usually made forone year, but may be extended to two years.

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RESULTS TO DATE

Nineteen candidates from 11 countries have so far formally appliedto the program. Four applications were outside the scope of the programand were therefore not considered.

Of the 15 applications submitted to the Review Panel, 10 were favor-ably appraised and nine applicants received awards (one candidate was unableto accept for personal reasons). Two of the grants have been extended foran additional year. The successful candidates' fields of study includedhematology (1), electron microscopy (1), perinatology (2), endocrinology (2),immunology (2), and cytogenetics (1). The five unfavorable appraisals weremainly due to lack of adequate research background. The ages of the candi-dates ranged from 26 to 40 with an average of 33 years.

SIGNIFICANCE

The program is specifically designed to take advantage of trainingopportunities of Latin American institutions whose research is well recog-nized. By orienting the trainees toward problems and conditions they arelikely to encounter on their return home, the program can be expected tocontribute to moderating the emigration of scientific workers from LatinAmerica and the Caribbean area.

PUBLICATIONS

1. Grants Program for Research Training in Clinical Medicine.PAHO Pamphlet, 1969.

2. The PAHO Research Training Program. PAHO/ACMR 10/8, June 1971.

OTHER DATA

Grantee: Dr. H. Torloni (Project Coordinator), Departmentof Research Development and Coordination, PanAmerican Health Organization, Washington, D.C.

Funded by: The Wellcome Trust, and the Pan American HealthOrganization


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