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PATHOGENESIS OF LEUKOPENIA IN CALVES EXPERIMENTALLY INFECTED WITH NON-CYTOPATHTC TYPE II BOVINE VIRAL DIARRHEA VIRUS A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by ROBERT DARREN WOOD In partial fiilfilIrnent of requirements for the degree of Doctor of Veterinary Science August, 2000 0 Darren Wood, 2000

Transcript of PATHOGENESIS OF LEUKOPENIA IN CALVES EXPERIMENTALLY ... · PDF filepathogenesis of leukopenia...

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PATHOGENESIS OF LEUKOPENIA IN CALVES EXPERIMENTALLY INFECTED WITH

NON-CYTOPATHTC TYPE II BOVINE VIRAL DIARRHEA VIRUS

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

ROBERT DARREN WOOD

In partial fiilfilIrnent of requirements

for the degree of

Doctor of Veterinary Science

August, 2000

0 Darren Wood, 2000

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PATHOGENESIS OF LEUKOPENIA IN CALVES EXPERIMENTALLY INFECTED WITH

NON-CYTOPATHTC TYPE II BOVINE VIRAL DIARRHEA VIRUS

Robert Darren Wood

Universiq of Guelph, 2000

Advisor:

Robert M. Jacobs

This study is an investigation of the pathogenesis of the hematological abnonnalities

observed in calves experimentally infected with non-cytopathic (ncp) type II bovine viral diarrhea

virus (BVDV). Calves were intransally inoculated with a virulent strain, 2451 5, or a low

virulence strain, 1 1 Q. Calves inoculated with the virulent strain developed anorexia, diarrhea and

respiratory signs. Calves inoculated with the low virulence strain exhibited no overt clinical

signs. Fevers were significantly higher in 245 1 5-inoculated catves. Serial peripheral blood and

bone marrow samples were obtained before and after virus inoculation and monitored for changes

in the circulating and bone marrow ce11 populations, respectively. Bone m q o w samples were

evaluated for the presence of vira1 antigen in hematopoietic cetls with irnrnunocytochemistry

using the monoclonaL antibody 15C5. Calves inoculated with both viruses developed leukopenia,

neutropenia, lymphopenia and thrombocytopenia to varying degrees. White blood ce11 counts

were significantly lower and persisted Ionger with the virulent 245 15 strain. The bone marrow

myeloid maturation pool decreased concurrently with development of leukopenia in anirnaIs

- inoculated with either virus but depletion persisted Ionger in 245 15-inoculated anirnals. The bone

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marrow proliferation pool increased in proportion to the decrease in the maturation pool in 1 lQ-

inoculated calves, but took 4 days longer to increase in 245 15-inoculated animals, Viral antigen

in bone marrow cells was sparse and transient in 245 15-inocuiated animals but was present when

peripheral blood counts were lowest. Viral antigen was never demonstrated in 1 1 Q-inoculated

calves. Megakaryocytes were the predominant ce11 type exhibiting positive staining. These

experiments demonstrated that infection with ncp type II BVDV strains of varying virulence

result in leukopenia. The virulent isolate caused a delay in the production of myeloid cells which

could potentially compromise the ability of the host to satisfy tissue demands for neutrophils and

contribute to secondary bacterial infections. Infection of myeloid precursor cells appeared to

contribute to this delay, AbiIity to delay production of bone marrow myeloid celIs and infection

of megakaryocytes may enhance virulence, contributing to the ability of certain ncp type 11

BVDV strains to induce severe disease.

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ACKNOWLEDGEMENTS

Many people have contributed in various ways to the completion of this thesis. Without

them, it would not have been possible.

Firstly, thank you very much to my advisor, Dr- Robert Jacobs, who encouraged me to

start the project very early in rny prograrn, which has allowed me to finish within three years. His

guidance and advice has been greatly appreciated. The other members of my advisory

committee, Dr. Tim Lumsden, Dr. Ian Barker and Dr- Ken Bateman gave valuable insight and

encouragement along the way.

I am deeply indebted to the technologists in the histology and clinical pathology labs for

countiess months of sample processing. 1 hope they know 1 realize it was not easy work, but that

it was greatfy appreciated. As welI, the great support of Dave, Tony and Jackie in the isoIation

facility ensured that our animais received the best of care.

Without the invaluable help of Barb Jefferson, 1 would never have been able to

learn to function at a basic level in a research Iaboratory. Her assistance with immunostaining

was especially appreciated.

Thanks are also in order for my fellow graduate students who helped me in many ways

during my degree. Denise Goens, who collaborated on the project with me, taught me so much

that 1 cannot possibly thank her adequately. My fellow clinical pathology graduate students,

Cathy Thom, Paula Krimer, Jatain Sondhi, and Felipe Reggeti have been great sources of

knowledge, challenged my thinking and kept me smiling.

1 would also like to acknowledge the contributions of Dr. Judy Talyor and Dr, Dorothee

Bienzle to my training as a clinical pathologist. The countless hours at the microscope discussing

excellent cases has prepared me well for my future endeavors.

1 would also like to thank the faculty and graduate students in anatomic pathology for

helping me become somewhat competent at interpreting surgical biopsies during my program.

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Finally, without the constant support, love and devotion of my wife Chantelle, 1 would

never have reached this point She and Gavin, our son, have kept me grounded during diffkult

times and encouraged me that 1 would succeed, reminding me that they were the ultimate reason

for any success 1 obtained, This thesis is dedicated to them.

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4 . Discussion ........................................................................................ 5 . Sources and Manufacturers ..................................................................... 6 . References ........................................................................................

APPENDIX 1 ............................................................................................

APPENDIX 4 ............................................................................................ ............................................................................................ APPENDIX 5

APPENDIX 6..... ....................................................................................... APPENDIX 7 ............................................................................................

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LIST OF TABLES

Page

Table 1. List of treatment groups with m i n of BVD virus, purification status,

volume and titer administered intranasally.. ........................................ 45

Table 2. Upper and lower threshold values calculated fiom pre-inoculation samples.. . 46

Table 3. Non-cytopathic type II BVDV isolation from b u w coat cells of alternate

..................................................................... day bIood samples. 47

Table 4. Prevalence of bIood cytopenia, mean day of onset of cytopenia post-

inoculation and rnean duration of cytopenia arnong groups of calves

.................... inoculated with 11Q and 24515 strains of ncp type II BVDV 48

Table 5. Repeatability study for bone marrow cytology. Twenty samples were

......... randomly re-evaluated and the data for three variables are presented.. 49

Table 6. Immunostaining of sequential bone marrow sarnples. BVDV-positive

staining in cells of the megakaryocyte series (M) and the myeloid series 0

are indicated.. .......... .. ... .... .................................................. 50

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LIST OP FIGURES

Page

Figure 1. Bone marrow cellularity assessed fiom core bone marrow biopsy samples for

strain 1 1Q and strain 245 15 (n=9) inoculated calves pre- and post-inoculation

with virus.. 51 ................................................................................

Figure 2. Mean blood leukocyte counts for I 1Q-, p245 15- and up245 15-inoculated

cahes.. ................................................................................... 52

Figure 3. Mean blood neutrophi1 counts for 1 1Q-, p245 15- and up24515-inoculated

calves ... ................................................................... 53 .............

Figure 4. Mean blood lymphocyte counts for 1 1 Q-, pz45 15- and up245 15-inocuiated

calves. ........................ .. .......................................................... 54

Figure 5. Mean blood platelet counts for 1 1Q-, p24S 15- and up245 15-inoculated

calves ...................................................................................... 55

Figure 6. Comparison of differences between the mean pre-inoculation value and the

post-inoculation nadir for leukocytes, neutrophils and lymphocytes for l1Q-

and 245 15-inoculated calves.. 56 ..........................................................

Figure 7. Comparison of day of onset and duration of leukopenia, neutropenia,

Ipphopenia and thrombocytopenia for 1 1Q- and 245 15-inoculated calves.. ..

Figure 8. Mean percentage of myeloid proliferation and maturation pools fiom senal

bone marrow cytoIogy samples of calves inoculated with strain 1 lQ.. 58 ..........

Figure 9. Mean percentage of myeloid proliferation and maturation pools fiom serial

bone marrow cytology samples of calves inoculated with strain 245 15.. 59 ........

Figure 10. M:E ratios fkom bone rnarrow cytology pre- and pst-inoculation for 1 1Q-

and 245 15-inoculated calves.. 60 ........................................................

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Figure Il, Bone rnarrow cytoiogy from an animal inoculated with virulent 245 15 non-

cytopathic type II bovine viral diarrhea virus on day 8 post-inoculation, ........ 61

Figure 12. Scatterplot cornparhg mean difference (delta) between repeat

values fiom 20 samples and the mean of 40 values collected from the 20

repeat samples for the proliferation pool- 62 ............................................

Figure 13. Acetone-fixed cultures Madin-Darby bovine kidney (MDBK) cells used as

controIs for immunocytochemistry of fiozen bone marrow aspirates,

Mayer's hematoxylin, ....... ,., ......................................................... 63

Figure 14. Acetone-fixed cultures Madin-Darby bovine kidney (MDBK) cells used as

controls for immunocytochemistry of frozen bone marrow aspirates,

Mayer's hematoxylin 63 ..........................................................................

Figure 15. BS-fixed cultured Madin-Darby bovine kidney (MDBK) cells used as

uninfected negative controls for immunohistochernistry of paraffin-

embedded bone marrow sections.. 64 ..................................................

Figure 16. BS-fixed cultured Madin-Darby bovine kidney (MDBK) cells used as

positive controls for immunohistochernistry of paraffin-embedded

bone marrow sections.. ...............................................................

Figures 17a and 17b, Positive-staining megakaryocytes fiom acetone-fxed fkozen

bone marrow aspirate (a) and BS-fixed bone marrow sarnple (b) fiom

245 15-inoculated calves, Mayer's hematoxylin (a), Harris' hematoxylin (b).. 65

Figure 18, Positive staining myefoid cell from a BS-fixed bone marrow sample fiom

a 245 15-inoculated calf', Harris' hernatoxyiin.. .................................... 66

Figure 19. Positive staining myeloid cells fiom a BS-fixed bone marrow sarnple fiom

a 245 15-inoculated caIf, Harris' hematoxylin.. ..................................... 67

vii

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CHAPTER 1

Literature Review and Study Objectives

1. Introduction

Examination of peripheral blood is commonIy performed during evaluation of illness in

cattle and is used fiequentiy as part of the database that leads to discovery of the underlying

process. Although specific diseases are rarely diagnosed solely using a complete blood count

(CBC), viral diseases produce a wide range of alterations in blood parameters, which may suggest

considering some diseases over others. In addition, the CBC ofien is helpful in distinguishing

infectious fiom non-infectious disease.

Understanding the pathogenesis of specific alterations in blood parameters contributes to

an understanding of the disease process and therefore irnproves ability to treat and offer

prognosis. Serial observations are important when determining prognosis. Leukopenia can

exacerbate the primary disease process by predisposing the animal to secondary infections. This

can prolong recovery fiom disease and can contribute to ongoing losses frorn decreased

production and fertility. Furthermore, predisposed by leukopenia, secondary infections may

result in the death of an animal that might have recovered ftom the initial disease process.

Thrombocytopenia occurs fiequently in viral diseases and if severe enough c m result in

hernorrhage. Severe hemorrhage obviously can contribute to the outcome of the disease. Anemia

is uncommonly observed in most viral infections unless severe hemorrhage occurs. Furthermore,

because of the 160 day mean lifespan of a bovine red blood cell', anemia due to infection of

erythroid precursors is usually not appreciated during the course of an acute viral infection.

Exceptions include the more chronic viral infections such as equine infectious anemia virus2 and

B 19 parvovirus of people?

Concurrent examination of a bone marrow sample can provide an explanation for

alterations in peripheral blood parameters and may be useful in diagnosing disease. There may be

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no changes, non-disease-speci fic reactive changes or disease-specific, i-e. diagnostic,

abnormalities in the bone marrowm4 Most viral diseases induce non-disease-specific alterations in

bone rnarrow ce11 populations,

2. Pathogenesis of Leukopenia

Leukopenia is a decrease in the circulating population of leukocytes, and rnay involve

changes in the number of neutrophils or lymphocytes depending on the relative size of the two

populations.' Many inammal ian viral diseases induce at least a transient leukopenia"

Lyrnphopenia can result fiom increased destruction of cells, decreased production of cells

or redistribution of cells within and outside of vascular spaces. Lymphocytes (and other infected

cells) can be destroyed as a result of cytotoxic products induced by virusesb Vimses rnay also

directly invade lymphocytes, which rnay result in direct cytotoxicity. Alternatively, vims-

infected cells rnay be eliminated by the immune system after recognition of altered surface

molecules. This would be reflected as increased apoptosis.

Decreased production of lymphocytes in the Iymphopoietic organs rnay occur as viruses

infect progenitor cells, which do not develop into mature lymphocytes, or develop incorrectly and

are removed.

Lymphocytes norrnally circulate between the primary and secondary lymphoid organs

and the peripheral blood. Viral infection rnay cause a redistribution o f these cells, such that fewer

are in circulation because they are required either in the lymphoid organs or in other tissues as

part of viral defense and dimination.'

Lyrnpliopenia ficirn virai infection may be partially due to stress of disease, causing

increased blood cortisol levels. Hypercortisolemia causes lymphopenia, due to lympholysis in

blood and lymphoid tissues and increased margination or emigration of lymphocytes fiom

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circulation: Alterations in the relative proportions of subsets of lymphocytes occur in several

viral diseases?

Neutropenia results from similar mechanisms. Cells can be destroyed directly or

indirectly by viruses or their products resulting in decreased numbers in circulation. Neutrophils

also can enter tissues or be Iost to a body cavity if there is chernotactic dernand. This results in a

decreased transit time in the peripheral blood.

Decreased production of neutrophils may occur in the bone marrow, either as a direct

result of viral infection of precursor cells, or by removal of these cells as a result of surface

molecule alterations. Mature neutrophils themselves are rarety infected with virus. Neutrophils

have a short lifespan and antimicrobial mechanisms that do not easily allow infection.' Viral

particles c m be coated by antibody and phagocytosed as immune complexes by neutrophils and

other ~ e l l s . ~

Neutrophils can be redistributed, moving fiom the circulating pool to the marginated

pool as demand in tissues or body cavities increases. This latter effect tends to be an acute

change, as seen with endotoxicosis. Neutrophils leave the circulating pool and enter the marginal

pool, sequestering in the microvasculahire, especially in the lung?*I0

CI.iemicals such as epinephrine and cortisol actually cause increases in circulating

neutrophils and are not a cause of neutropenia, Neutrophilia due to epinephrine results fiom an

increase in the circulating pool because of a decrease in the marginal pool.11 Excess exogenous

or endogenous cortisol causes an increased mobilization of marrow granuIocyte reserves to the

peripheral blood, as well as decreased margination.ll

3. Neutropenia in Viral Infection

Neutrophils often are found in very early virus-induced lesions but are quickly replaced

by mononuclear tells.'* Neutropenia is a common finding in viral diseases, and may be associated

3

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with decreased production in the bone r n a ~ o w . ' ~ Cytologically, this is seen as myeloid

hypop Iasia-

Many viruses are cytocidal and cause lysis of the target ce11 via induction of ce11

membrane damage, and by akering protein synthesis, DNA replication and cytoskeletal

Viruses can result in bone marrow failure by one or more of three mechanisms: 1) direct

inhibition of or cytotoxicity to hematopoEetic progenitor cells, 2) direct inhibition of or

cytotoxicity to marrow stromal cells required for hematopoiesis, or, 3) stimulated production of

cytokines or cytotoxic lymphocytes that ixh-ibit the production of or destroy hematopoietic

ce11s.14-16

The first mechanism is demonstrated by the parvovimses, especially feline

panleukopenia virus, which are well known for their tropism for hematopoietic cells. and often

result in decreases in circulating neutrophils and lymphocytes.'7 The pathogenesis of feline

panleukopenia involves direct infection amd destruction of mature cells, and inhibition of bone

marrow myeloid progenitors, resulting in marrow hypocellularity~*'8

Infection with dengue virus resuIts in bone marrow suppression by al1 three mechanisms.

Dengue viral antigen is easily detected o n the surface of infected myeloid cells. Therefore, these

cells may be targeted as "innocent bystanders" in an attempt to elirninate virus.'g A study of

dengue-2 infection in long-term bone marrow cultures showed infection of marrow stroma1

cel lso This suggested that viral infection o f cells of the hematopoietic inductive

microenvironment might result in decreased production of the myeloid series, at least transiently.

Furthermore, dengue-specific T-cells reswlt in the release of bone marrow suppressive

cytokines. l6

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4, Special Considerations in the Evaluation of Bovine Hematology

Calves and other young ruminants are born with a neutrophi1:lymphocyte (N:L) ratio

similar to most other species, but by the fourth or fifkh day of Iife, lymphocytes begin to

predominate?' However, in the mature animal, the blood N:L ratio is much lower than observed

in many other domestic anirnals; lymphocytes outnurnber neutrophils. As a result, severe

neutropenia and left shifts can deveIop more quickly than in most other species. Therefore,

severe neuiropenia with a lefi shift does not irnply the same severity of the inflammatory response

in cattle as in other species-

Some authors suggest that bovine bone rnarrow has less marrow granulocyte reserve than

other a n i r n a l ~ . ~ This may explain why cattle quickly develop a left shifi when there is a severe

peripheral demand for neutrophils. In cattle, leukopenia that persists for more than 3 to 4 days

indicates inadequate rnyelopoiesis to meet peripheral dernand?'

5. Thrombocytopenia in Viral Infection

Thrombocytopenia results fiom increased destruction or use of platelets, fiom decreased

production, or by sequestration. Platelets can be consumed rapidly during disseminated

intravascular coagulation (DIC) or because of immune-mediated destruction. Both these

phenornena may occur secondary to viral infection. For example, platelet-associated IgG and IgM

were demonstrated in foals infected with equine infectious anernia virus?

Bone marrow production of pIatelets may be decreased if viruses infect megakaryocytes,

resulting in either ce11 death or ineffective thrornbopoiesis. Splenic sequestration can result in

lower numbers in the peripheral blood. Sequestration is rarely, if ever observed with viral

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infection. As with the myeloid series, more than one mechanism may contribute to

thrombocytopenia d u h g viral infection.

6. Hematology of Flavivirus Infections

Flaviviruses are single-stranded, positive-sense RNA viruses.~ There are three genera in

the farnily Flaviviridae; genus FZmivirus, genus Pestivirur and genus ~e~acivi-?' Dengue

virus and yellow fever virus are important pathogenic flaviviruses. The only virus classified in the

genus Hepacbim is hepatitis C . The genus Pestivim contains three viruses, each important in

veterinary medicine: classical swine fever virus (hog choiera); border disease virus; and bovine

viral diarrhea virus (BVDV), Infection of cells by viruses in the family Flaviviridae usually

results in ~ ~ t o l ~ s i s . ~ Non-cytopathic strains of BVDV are an exception, at least in vitro.

Hepatitis C virus of the genus Nepacivim is associated with mild transient bone marrow

suppression and rarely, aplastic anemia26

in contrast, dengue fever virus uniforrnly produces thrombocytopenia and neutropenia in

people.'627 Mechanisms of hematosuppression include both direct viral cytolysis and immune-

mediated destruction of progenitor cells, resulting in decreased production?7 During dengue

hemorrhagic fever, hypocellularity of al1 bone marrow ce11 lines occurs from days 1-4 post-

infection, with increasing cellularity from days 5-8, and a return to normal bone marrow

cellularity after day 1 O? A lympliocytic infiltrate in the bone marrow, which likely represents

immune system activation against virus infected cells, is aiso present from days 5-8 post-

infection.

The p n m q cell targets of dengue virus are those of the monocyte-macrophage system,

but other hematopoietic cells often also are infe~ted.~' Four dengue serotypes exist that Vary in

their ability to infect different ce11 lines. For example, dengue 2 is much less eff~cient than

dengue 4 at infecting erythroid cells, but infects ce11 lines of both the myelomonocytic and

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lymphocytic lineages.lg Simultaneous suppression of both rnegakaryocytes and myeloid cells

suggests that there is infection of a pluripotential stem cell, or concurrent infection of both

1ineages. " Alterations in stroma1 marrow cells and their secretions, in addition to direct viral cellular

destruction, maycontribute to hematosuppression. Furthemore, only low numbers of stromal

cells may need to be infected to signal the entire marrow environment to decrease proliferation in

an attempt to minimize infection of rapidly dividing cells by virus. This rnay be considered a

protective mechanism fiom the perspective of limiting spread of virus and means that very

quickly (3 to 5 days post-infection) production of hernatopoietic cells can be down-regulated.I6

As infected cells are eliminated by specific immune responses, the signals inhibiting production

are reduced and a rebound in mitosis occurs.

7- Hematology of Pestivirus Infections

Pestiviruses are known for their ability to produce marked hernatologic abn~rmali t ies .~~

Classical swine fever virus (hog cholera) results in rnarked leukopenia3* and in thrombocytopenia

which rnay be severe enough to result in overt hernorrhage?' Border disease of sheep also may

result in lymphopenia and n e u t r ~ ~ e n i a ? ~ ~ The hernatology associated with BVDV infection will

be discussed in detail in the next sections.

In hog cholera and border disease, mechanisms for the development of some

hematologicai abnormalities have been studied. Classical swine fever viral antigen can be

demonstrated by immunohistochemistry in 259.0% of rnegakaryocytes in the bone marrow,

along with morphological changes suggestive of necrosis?' However, infection of

rnegakaryocytes could not account for the severe thrombocytopenia induced by the disease, since

overall megakaryocyte production in the bone marrow was unimpaired. Alterations in the

myeloid series associated with classical swine fever virus infection were not assessed.

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Lymphocyte subsets have been evaluated in sheep with border diseaseS2 and in cattle with

BVDV infe~tion?~ The destination of lymphocytes leaving the peripheral blood dunng viral

infection is still unclear. However, reductions in both T- and B-celIs occurred during infection

with both vir~ses.'~' Early in infection, CD4+ cells may be diminished3= while CD8+ cells

may also decrea~ed)~ or may remain relatively unchanged?* Towards the end of active infection

(10-12 days post-infection) CD4+ cells returned to pre-inoculation levels, while CDS+ cells

increased significantly over pre-infection values. Neutrophiis and lymphocytes may leave the

circulation and migrate to sites of viral replication?'

Pestivimses have been documented to infect lymphocytes, which rnay be killed by

natural killer cells or cytotoxic lymphocytes?

The kinetics of ce11 production during infection has not been closely exarnined.

Characterization of the hematological and bone marrow kinetics of domestic anirnals with viral

diseases is poorly understood.

8. Introduction to Bovine Viral Diarrhea Virus (BVDV)

Bovine viral diarrhea virus (BVDV) is a pestivirus in the family ~~av~ir idoe." BVDV

has recently assumed increased significance in the bovine industry due to outbreaks of a

seemingly new form of the disease that began in 1993 in Ontario, Quebec and the Great Lakes

tat tes.'^'' The disease occurred in veal calves as well as in older a n i r n a l ~ ~ ~ and caused morbidity

and mortality significant enough to result in alarming production oss ses?^

BVDV has been classified into two biotypes, non-cytopathic (ncp) and cytopathic (cp),

based on the effect in cultured ce~ls?"~ Cytopathic effect can range fiom minor changes in the

ce11 structure to ce11 dysfunction, ce11 lysis or ce11 transformation-4' Cytopathogenicity in cultured

cells has no bearing on the ability of BVDV to cause disease in an animal. Non-cytopathic strains

typically have been associated with a subclinical self-Iimiting disease, but they also are

8

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responsible for persistent infections and reproductive problems in infected cattle. Cytopathic

strains, associated with mucosal disease, are considered to result by mutation fiom a non-

cytopathic strain persistently infecting an These strains have an additional protein, p80,

generated by proteolysis of the Iarger non-structural protein, p 125, permitting differentiation of

biotypes by molecular rnethod~.'~

More recently, BVDV has been divided into two genotypes, types 1 (a and b subtypes43

and II, based on differences in the 5'-untranslated region of the gen~me."~~"* Type 1 strains have

been used in diagnostic tests. in research and for most vaccine pr~ductionP5*46 Type II strains are

isoIated predominantly fiom fetal bovine serum, persistently infected animals and animals with

acute BVD and hemorrhagic d i~ease?~*~ ' There also is a vaccine based on a type II strain.

BVDV infection may result in a number of distinct clinical syndromes, depending on the

host, the virus and epiderniologic circurnstances. Manifestations rnay include: 1) subclinical

infection with viral clearance, 2) acute disease of the intestinal, respiratory andor hematopoietic

organs, 3) chronic disease with multiple organ involvement, 4) fetal infections and reproductive

disorders, 5) persistent infections and 6) mucosal disease. 39.47-52

Type 11 strains have been known since the early 1980s?' However, it wasn't until the

Iate 1980s that animals with signs resembling mucosal disease or a hemorrhagic diathesis were

associated with ncp BVDV." In 1993 in Ontario, outbreaks of acute diarrhea, anorexia, fever,

depression and overt hemorrhage were linked to ncp type II strains of BVDV?~ The outbreak

appeared to be due to an evolution in virdence of strains that previously caused only subclinical

or mild disease and did not result in appreciable morbidity or mortality. Outbreaks of severe

acute BVDV began to increase and cause unprecedented losses fiom death of animals, abortions

and decreased rnilk production?6 Many herds experïenced widespread disease in animals of

different age groups, including neonatal calves, older calves and adult cattle. Affected older

calves (> 6 months) and adult cattle tended to exhibit more gross and microscopie alirnentary

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l e~ ions?~ Type II strains seem variable in their ability to cause disease, since ncp type II viruses

also can be isolated fiom animals that have not shown signs of overt disease.

9, HernatoIogy in BVDV Infection

There are numerous case reports and experimental studies documenting the hematologic

abnormalities associated with BVDV infection?3J7 Even the earliest descriptions of the disease

include leukopenia as part of the clinical Subclinical BVDV infections are

associated with a mild fever and transient leukopenia of a few days duration? Severe acute

BVDV infection typically causes mild to severe leukopenia characterized by moderate to severe

neutropenia and mild to moderate lymphopenia, MiId to marked thrombocytopenia is a fiequent

feature of animals affected with this form of B V D V . ~ ~ ~ ~ ~ ~ ~ If the platelets are sufficiently

decreased to result in hemorrhage, a regenerative, blood-loss anemia can be found. Non-

regenerative anemia also has been observed with ncp type II BVDV infections." Not oniy are

absolute numbers of cells decreased, but fbnction of both lymphocytes, neutrophils and platelets

may be affectedP1"

Immunosuppression fiom leukopenia rnay permit secondary or opportunistic infections,

or prolong the course of disease." The mechanisms are not well understood, but may involve

viral replication in cells of the immune ~ ~ s t e r n . ~ Concurrent infections fiequently include bovine

respiratory syncytial virus ( B R S V ) ~ ~ ~ ~ ~ , parainfluenza-3 virus (PI-316' and bovine hecpesvims-1 P7

There is ongoing debate about the role of BVDV in causing or contributing to respiratory disease

in cattfe.

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10. Pathogenesis of Hematological Abnormalities in BVDV Infection

BVDV infection is established by inhalation, with viral replication initially in the

oronasal mucosa and oropharyngeal lymphoid tissue." Virus is disseminated widely via

Ieukocytes circulating in lymph and blood. Viral replication continues in cells in lymphoid

tissues, in circulating leukocytes and in the bone marrow." The bone marrow may be an

important site of viral replication and subsequent dissemination throughout the body. Ce11

tropism may vary with viral strain. Most of the genetic diversity is from variation within the

major envelope glycoprotein 53 (gp53) which likely accounts for different ce11 preferences.'

Leukopenia appears to coincide with episodes of fe~er .~ ' As discussed previously, several

mechanisms can contribute to the development of these abnormalities. No detailed studies have

been performed to detemine the reason for the leukopenia that develops in BVD. An assessment

of bone marrow production during infection would heIp elucidate the mechanism(s) involved.

The few previous studies have produced conflicting conclusion^.^^""^ These reports are Iimited

by examination of the bone marrow only at post-rnortem or at a few specified intervals during

infection. in one report, bone rnarrow at post-mortem examination (1 0-12 days post-inoculation)

was hypopiastic, with necrosis and minimal granulopoiesis, with a predominance of immature

for~ns.'~ Another study found a large number of BVDV-positive myeloid precursor cells in

several calves at necropsy on day 10 post-infection!8 Bone rnarrow cellular kuietics during

infection has not been adequately studied to determine if there is a change in production of

myeloid cells.

Much work has been done to answer the same questions about platelet numbers.

RecentIy, a mode1 of a type II ncp BVDV infection that results in overt hemorrhage has been

reportedS and the same authors suggest that there may be a defect in platelet function which

could contribute to h e r n ~ r r h a ~ e . ~ ~ However, decreased production in the bone marrow as a

mechanism for thrombocytopenia has not been thoroughly exmined.

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The current Iiterature is confirsing with respect to the causes of thrombocytopenia in

BVlDV infection. Megakaryocyte hyperplasia, normal megakaryocyte populations, necrosis and

dysmegakaryopoiesis have been reported?3-55'7.68

Walz et al suggested that assessment of mean platelet volume (MPV) can heIp

discriminate between thrombocytopenia due to peripheral destruction or lack of production as

reported in humans with W infection and swine with classical swine fever virus infection."

They demonstrated a significant decrease in MPV in BVDV-infected versus control animals.

Immunoglobulin was not demonstrated on the surface of pIatelets of BVDV-infected

calves, suggesting that thrombocytopenia was not due to immune-mediated destruction."

Megakaryocyte hyperplasia in other experimentally-infected calves suggested peripheral

consumption of platelets and leukocytes as a mechanism for cytoPenia?' But, a correlative study

of peripheral blood platelet counts and concurrent bone rnarrow cytology and histopathology has

not been reported.

Infection of calves with a cytopathic isolate did not result in a significant decrease in

platelets, compared to the effect of several ncp i so~a tes .~~ Those authors suggested that only ncp

BVDV strains have the ability to induce thrombocytopenia,

Disseminated intravascular coagulation has been evaluated as a cause for

thrornbocytopenia during BVDV infection." Coagulation parameters assessed included

prothrombin time, partial thromboplastin time and fibrin degradation products. Obsewed values

were within reference intervals in al1 animals. Therefore, DIC was unlikely as a mechanism for

thrombocytopenia in infected calves.

11. Effect of Age

The age of the animal may have an important impact on the outcome of disease. Most

experimental BVDV studies use neonatal calves or those < 3 months of age."Hns9*6sn Minimal

. 12

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evaluation of BVDV infection in older calves and mature animals has been done. Very young

animals sometimes are more susceptible to pathogens since their immune systems have not yet

matured." Yet with acute BVD, al1 age groups may develop severe clinical disease.

12. ViruIence Factors

Virulence refers to the relative capacity to cause disease."' Individual virulence factors of

a particular virus may contribute to the developrnent of severe disease. There are usually multiple

factors for any one virus, in those viral systems vrhere virulence factors have been characterized.

The susceptibility or resistance of the host (usually multifactorial) is also a very important factor

in the outcorne of viral infection." It is the interaction of specific virulence factors of the agent

with host factors that results in the overall outcome of viral infection- A comparison of virulence

factors requires that variables such as infecting dose, age, sex, health of animals and immune

status must be controlled. Commonly, viral virulence has been assessed using lethal dose (LD)SO

as an index.75 Assessrnent of the relative severity of histopathologic lesions also is fiequently

used in evaluations of virulence.7s

The classic virulence factor in BVDV is the production of the protein p80/NS3,

characteristic of a cytopathic strain that mutates in an animal persistently infected with a ncp

train?^ Type II non-cytopathic strains vary in their ability to cause disease but the most

important virulence factors are poorly understood?' Potential variability at the genomic level and

ce11 tropism also likely are important virulence factors. Genomic differences contributing to

virulence have been identified in many vir~ses?~

In order to infect a specific ce11 iype, a virus must express attachment proteins

complementary to receptors on target ~ells?"~ These proteins can be host and ce11 specific or

may permit the invasion of many ce11 types?' Carbohydrates, Iipids and proteins al1 can serve as

attachment molec~les?~ If a virus has the capability to infect multiple ce11 types, this is an

13

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obvious advantage to the virus, since it provides for effi~cient dissemination throughout the body.

Flavivimses circulate free in and as cell-associated virus. Variations in virus-

macrophage interactions also may account for differences in virulence between viral strains and

for differences in host resistance? For example, feline infectious peritonitis (FiP) virus infection

relies on the ability of the virus to replicate in macrophages.*

There are a few reports exarnining the virulence of non-cytopathic strains of BVDV. One

group infected neonatal calves with two different ncp type II BVDV, which resulted in only mild

clinical disease, proving that not al1 ncp type II strains were highly Another study

found differences in disease produced by two different ncp strains, as weIl as variability in the

severity of clinical signs, hematologic abnormalities and lesions induced among calves infected

with the more virulent strain."

13. Celi Tropism of BVDV in Bone Marrow

Irnmunocytochemical studies on bone marrow have been perforrned in an attempt to

delineate ce11 tropism of BVDV. Monoclona1 antibodies to various glycoprotein components of

the virion have been used to detect the presence of virus, and 15CS is a monoclonal antibody in

wide use for both research and diagnostic purposes. .It labels glycoprotein 48(E0), a viral

structural protein important in construction of the capsid and other components of the virus.""

Ouring infection, type II ncp-BVDVs have a wide tissue distribution, including cells of

the bone marrow, where megakaryocytes express viral antigen, as do 'mononuclear cells',

stroma1 cells and myeloid cells. 53,54,68.69,72 Infection of megakaryocytes may be a mechanism for

viral dissemination, since virus has been demonstrated in platelets in the peripheral blood." The

ability of a particular BVDV strain to adhere to and infect rnegakaryocytes could be considered a

virulence factor, This has not been extensively studied imrnunohistochemically and observations

tend to be based upon a single post-mortem sample. However, Spagnuolo et al demonstrated that

14

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myeloid cells expressed BVDV antigen 3 to 10 days post-inoculation and that megakaryocytes

expressed viral antigen o n al1 but day 3 post-inocu~ationd9

14. Summary

To date, no reports document which celIs are infected in type II ncp BVDV throughout

infection and at what t ime the cells become infected- A study o f the kinetics of the

hematological abnorrnalities, concurrent with bone marrow cytology, would heIp delineate the

rnechanisms o f Ieukopenia associated with BVDV infection. Serial immunocytochernica1 studies

on bone marrow could help determine when bone marrow ceIIs become infected and what ceII

Iines are susceptible. A cornparison of BVDV strains o f varying virulence could determine if ce11

tropism in the bone marrow is related to development of severe disease.

Therefore, in an attempt to better understand the pathogenesis of the hernatological

changes in severe acute ncp Pype II BVDV, the following objectives were outlined:

1) Documentation of sequential changes in the peripheral blood leukocytes during infection

2) Description o f sequential changes in the bone marrow ce11 types and how they relate to the

circulating populations of leukocytes

3) Show if the appearance o f virus in various bone marrow ce11 types relates to observations on

the cells o f the bone marrow and leukocytes in peripheral circulation

4) Discover if the virulence of BVDV strains influence bone marrow morphology and

circulating leukocytes

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76. Donis RO: 1995, Molecular biology of bovine viral diarrhea virus and its interactions

with the host- Veterinary CIinics of North America (Food Animal Practice)

I1:393-423-

77. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: 1999, Detenninants of viral

virulence and host resistance/susceptibiIity. In: Veterinary Virology, eds. Murphy

FA, Gibbs EPJ, Horzinek MC, Studdert MJ, 3rd Ed., pp. 1 1 1- 125. Academic

Press, San Diego,

78. Welsh RM, McFarland HI: 1993, Mechanisms of viral pathogenesis. In: Viruses and

Bone Marrow: Basic Research and CIinical Practice, ed, Young NS, pp. 3-30.

Marcel Dekker, Inc., New York.

79- Zheng L, Zhang S, Xue W, Kapil S, Minocha HC: 1998, Expression of a 50 kDa putative

receptor for bovine viral diarrhea virus in bovine feta1 tissues. Canadian Journal

of Veterinary Research 62: 156-1 59.

80. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: 1999, Viral replication. In:

Veterinary Virology, eds. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ,

3rd Ed., pp. 43-59. Academic Press, San Diego,

81. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: 1999, Mechanisms of infection and

viral spread through the body. In: Veterinary Virology, eds. Murphy FA, Gibbs

EPJ, Horzinek MC, Studdert MJ, 3rd Ed., pp, 93-109. Academic Press, San

Diego.

82. Brock KV: 1995, Diagnosis of bovine viral diarrhea virus infections. Veterinary Clinics

of North America (Food Animal Practice) 1 1 549-56 1.

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83. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: 1999, The nature of vimses as

etiologic agents of veterinary and zoonotic diseases. In: Veterinary Virology, eds.

Murphy FA, Gibbs EPJ, Horzinek MC, S tuddert MJ, 3 rd Ed., pp. 3-2 1. Academic

Press, San Diego.

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Pathogenesis of leukopenia in calves experimentally infected with non-cytopathic type II bovine viral diarrhea virus

Introduction

Bovine viral diarrhea virus (BVDV), a pestivirus in the family ~~aviviridae', is a

comrnon bovine pathogen and responsible for worldwide economic losses. The virus is

categorized into non-cytopathic and cytopathic biotypes based on effects on cuItured cells2 and

into genotypes I and II based on genomic d~erences. '~ Genotype 1 has fùrther been subdivided

into types [a and ~ b . ~

BVDV is well known for its association with mild acute diarrhea, mucosal disease,

persistent infection and reproductive dis or der^-'^*^** S ince the early 1 WOs, it has been implicated

as the cause of severe acute signs, including fever, anorexia, diarrhea and overt hemorrhage in

young calves i d adult cattle in North ~merica.~*'* This severe acute manifestation is caused

primarily by non-cytopathic type II strains of BVDV. Previously, these strains were associated

with a self-limiting mild disease, and the more recent association with severe clinical signs

appears to represent an evolution toward increasing virulence. Subsequently, an a l m i n g

increase in morbidity and mortality due to BVDV has been associated with the appearance of

these more virulent strains.

The pathogenesis of acute infection with non-cytopathic type TI BVDV is incompletely

understood. Hematological abnormalities are a consistent feature during infection and contribute

to clinical signs and disease outcorne." An understanding of the pathogenesis of these

abnormaIities would enhance understanding of virus-host interactions.

Flavivinises are capabIe of inducing rnarked hernatological changes during infection.

Dengue is well known to cause severe leukopenia and thrombocytopenia in people.'z" Classical

swine fever virus (hog cholera) and border disease of sheep also induce leukopenia and

thrornb~c~to~enia.~~*'~~'~ A delay in adequate production of hernatopoietic precursor czlls in the

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bone marrow rias been implicated as a major cause of Ieukopenia and thrombocytopenia in these

diseases. ""

Non-cytopathic type II acute BVDV infections aIso are associated with the development

of leukopenia and t h r o m b ~ c ~ t o ~ e n i a " ~ ' ~ Leukopenia is characterized by neutropenia and

fiequently lymphopenia. White much work has been done, our understanding of the pathogenesis

of thrornbocytopenia is still u n ~ l e a r . ' ~ ' ~ The pathogenesis of leukopenia has received little

study.18 Severe neutropenia can increase an animal's susceptibility to secondary bacterial

infections, while lymphopenia may contribute to irnmunosuppression and prolong morbidity.

Since other related viruses have been implicated in decreased bone marrow production, it

is suspected that BVDV may have a similar effect. Here, we examined the role of increased

peripheral utilization of leukocytes as an alternative, or complement to, decreased production in

rnediating teukopenia The development of leukopenia during experimental infection with two

non-cytopathic type II BVDV strains of differing vinilence is initially described. Secondly, serial

bone marrow cytology and histopathology was used to examine the effect of BVDV infection on

myeloid cells as an explanation for observed leukopenia. Finally, identification of virus in

hematopoietic cells using immunohistochemical methods was performed in an attempt to detect

an association with leukopenia. Experiniental studies often use a purified viral product for animal

inoculation, but we sought differences between the effects of unpurified virus stock and purified

vims. Furthermore, an evaluation of strains of differing virulence provided an opportunity to

identie if variation in tropism for cells of the hematopoietic system is an important factor in the

development of severe disease.

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Materiais and Methods

1. Anirnals and Experimental Design

Six to eight-month-old Holstein steers (n=2 1) were housed in groups of three in an

isolation facility. CaIves were obtained fiom a commercial supplier" and were negative for BVD

virus and antigen. Routine virological studies were performed according to standardized methods

at a centralized laboratoryb. Experiments were conducted in accordance with the Guidelines For

ne Cure And Use Of Experimental AnimaZs established by the Canadian Council on Animal

Care under protocols approved by the University of Guelph Animal Care Cornmittee. Anirnals

considered healthy on physical examination after 5 to 7 days of acclimation in isolation were

admitted to the study,

Tbe steers were assessed daily for temperature, puIse, respiration and a series of

subjective clinical parameters inciuding attitude, appetite, hydration sta tu and fecal consistency

(Appendix 1). Blood sarnples were taken on alternate days for 8 to 10 days prior to the day of

virus inoculation (day 0) for development of baseline complete blood count (CBC) parameters.

M e r blood sarnples were obtained, calves were given an intrarnuscular injection of xylazine

hydrochioridec at a dose of 0.25rn~/kg?~ Once sedated to the point of immobility when placeci in

lateral recumbency, the sternum was shaved and surgicaily prepared using a

chlorhexidine/isopropyl alcohol solutiond. The ventral rnidline was located by palpation and a

stab incision through the skin was made with a #15 scalpel blade. A Jamshidi bone marrow

biopsy needle was introduced and 4 to 6 ml of bone rnarrow was aspirated into a syringe

containing 0.5 ml 1% EDTA. The needle was re-introduced and a core biopsy was obtained

ftom the opposite side of the sternum and fured in B5. Four to £ive bone marrow sarnples were

obtained prior to virus inoculation to establish baseline cytology and histology for each cal..

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On day 0, each calf was given an intranasal inoculation (1 mVnostri1) of medium (Earle's

minimal essential medium (EMEM)' supplemented with 10% horse serumc and antibioticsf)

containing virus. Control animals received the sarne volume of medium without virus. The

treatment groups, virus strain and titer administered are given in TabIe 1. Bone rnarrow sarnples

were not collected on the day of virus inoculation. Blood and bone rnarrow samples were

collected every second day untii euthanasia on day 14- This resulted in seven seria1 blood

samples and six seriai bone marrow samples for each calf post-inocuiation (pi). Bone rnarrow

samples were not cotlected for treatrnent group 7; these calves were only included in the

hematological evaluation, Semm fiom clotted blood samples and EDTA-anticoagulated blood

were collected at the same intervais for viral serology and virus isolation, respectively,

Calves were assessed daily throughout the infection period for the clinicai parameters

previously mentioned on page 30. Calves were euthanized on day 14, or earlier if certain criteria

were fulfilled. These criteria included pyrexia 2 41°C, complete anorexia or diarrhea for 2 days

without improvement, or inability to rise for more than six hours. Cafves were kiIled humanely by

an intravenous barbiturate overdose. Complete necropsies were performed immediately on al1

calves. Gross lesions were recorded and tissues (Appendix 2) were coIIected and fixed in 10%

neutral buffered formalin for histological evaluation.

2. Virus

Two type II non-cytopathic BVDV strains were chosen for use in these experiments;

virulent 245 15 and less virulent 1 1 Q. The 245 15 strain was isolated fiom the fetus of a morbid

cow during the outbreak of acute BVDV in Ontario in 1993. The 1 1Q strain was isolated fiom a

persistently infected feedlot animal in Manitoba. This herd was experiencing intermittent

diarrhea and pneumonia in calves and adult animals.

Both vimses were passaged three times in Madin-Darby bovine kidney (MDBK) cells

cultured in EMEM to produce an unpurified (up) virus stock. Unpurified virus stock was purified

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(p) by limiting dilutions to result in a single viral clone." Titres of 1 O' TCID5dml (groups 2,3,5,7)

and 10' TCIDsdml (groups 4,6) were obtained. Supernatant containing virus at the above titres

was used to inoculate calves intranasally (Table 1).

3. Hematology

SampIes for complete blood counts were processed within 4 hours of collection,

Automated analysis with a hematology analyzefi and a manual 100 ce11 differential count were

performed. CBC parameters rneasured are listed in Appendix 3. Values for total leukocyte,

segrnented neutrophil, lymphocyte and platelet counts were cornpiled. The daily means for each

group were plotted. The pre-inoculation and post-inoculation values were examined to evaluate

alterations during disease, Mean values fiom al1 calves prior to inoculation with virus (n=21)

were used to establish age-specific threshold values for the different ce11 types (Table 2). For the

purposes of this study, cytopenia was defined as a statistically significant difference (px0.05) in

the post-inoculation absolute ce11 count compared to the mean of the individual animal's pre-

inoculation values and/or as being outside the above established threshold values,

For each animal, the difference between the mean of pre-inoculation values and the post-

inoculation nadir was calculated as the reduction in absolute ce11 count These data were used to

assess differences in the severity of cytopenia between viruses and groups of animals.

4. Bone Marrow Cytology

Bone marrow aspirates obtained in EDTA coated syringes w&e used to make smears for

cytological analysis and immunocytochemical staining.

Bone marrow was ejected ont0 slides, excess blood was tipped off, and marrow granules

were captured and spread on another slide. Three smears from each sample were rapidiy air-

dried, Wright's stainedh, covenlipped and stored until cytological evaluation was performed.

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Each sample of three slides was encoded for blind analysis. A bone rnarrow differential

count o f 1000 cells was performed on each Wright's stained sample. Cells were identified based

on rnorphologic criteria described elsewhere? Cells of the megakaryocyte, erythroid and myeloid

lineages, other celis, and subjective assessments of granule cellularity and marrow granulocyte

reserve (MGR) were recorded (Appendix 4). A ratio of myeIoid to erythroid cells (M:E ratio)

was catculated based on the total number of cells/1000 in each lineage. The cells of the myetoid

series were divided into the proliferation (myelob~asts, promyelocytes and neutrophilic

myelocytes) and maturation (neutrophilic metamyelocytes, neutrophilic bands and segmented

neutrophils) pools for analytical purposes. Pre- and post-inoculation bone marrow cytology

parameters were compiled and compared for treatrnent effects.

A repeatability test was perforrned on a subset of bone marrow cytology data. Twenty

samples were randomly selected fiom the originaI samples and given a new number. After a

1000 ceIl differential count was performed, slides were decoded and the results compared with

the original differential ce11 count.

5. Bone Marrow Histology

Bone marrow core biopsies were fixed in B5 (Appendix 5) and 10% buffered formalin,

decalcified and embedded in paraffh. Sections were cut at 3 microns, stained with Harris'

hematoxylini and eosid (E-I&E)~' and coverslipped with a xylene-based mounting mediumk. Any

bone marrow samples remaining after preparing slides for cytology and irnmunostaining were

centrifiged and the pellet fixed in B5, embedded and paraffinized. These sections were cut at 5

microns and siides were prepared the same as the core biopsies.

Core biopsies were assessed for architecture, megakaryocyte numbers, cellularity and any

abnonnalities. Megakaryocyte numbers were counted in an area of representative cellularity free

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of bone, cartilage and fat. Counts were made at 1Ox magnification in a 1000 prn diameter using

an ocular m icrometer.

6. Bone Marrow Immunocytochemistry

The presence of viral antigen in bone marrow was assessed in acetone-fixed aspirate

smears and in BS-fixed, paraffin-embedded sections for each individual sarnple. Ten bone

marrow slides were prepared at each sampling, fixed in cold acetone for 30 seconds and stored at

-70 degrees Celcius until imrnunocytochemistry was peri?ormed- In addition, unstained paraffin

ernbedded core biopsies and marrow clots were affixed to Snowcoat X-tra slidesi and allowed to

dry - The monoclonal antibody 15C5, developed against glycoprotein 48 (gp48) in a structural

region of the BVD virus, was used for immunostaining procedures.'* This monoclonal antibody is

widely used for BVDV diagnosis and re~earch.~*

Controls for the aspirate srnears were prepared fiom cultured MDBK ceIIs in LabTek 2-

weli chamber slides" , Sarnples were either uninfected (negative control) or infected with

unpurified strain 1 1 Q, purified strain I IQ, unpurified strain 245 15 or punfied strain 245 15

(positive controls). Preliminary staining of these samples revealed that each virus resulted in

similar staining patterns. An irrelevant monoclonal antibody, mouse anti-rabies", was also used

as a negative control. Negative, positive and irrelevant controls were used with each batch of

slides.

Controls for BS-fixed, paraffm-embedded sections were prepared fiorn uninfected or

virus-infected MDBK cells. Cells were harvested, centrifuged and the pellet fixed and processed

identically to the bone marrow core biopsy samples. Irrelevant antibody and pre-inoculation bone

marrow samples were used as fitrther negative controls.

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Positive conirols for formalin-fixed paraffin-embedded sections were prepared fiom

sirnilady processed tissues from an animal with confinned ncp type II BVDV infection, Infection

was confirmed by virus isolation and a type-specific antibody assay. Again, irrelevant antibody

and pre-inoculation samples were used as negative controls.

Paraffin-embedded sections were deparafinized and rehydrated through graded xylene

and alcohol solutions prior to staining. BS-fixed sections were additionally immersed in Gram's

iodine for 10 minutes and dipped in sodium thiosulfate three times for removal of mercuric

chloride and decolorization, respectively. Slides were immersed in an endogenous peroxidase

inactivating solution of 4ml3O% H20 in 100ml methanol for 12 minutes and rinsed three times

in automation buffer solution0- Working automation buffer solution was prepared by adding

lOOml of the 10X concentrate and lOml 15% Brij-3SP to 800ml of sterile water. This solution

was thoroughly mixed, lOOm1 of acetone added, and the solution mixed again.

The sections were digested in a solution of 50mg protease X I V pet lOOrnl of phosphate

buffered saline (PBS) at 37OC for 20 minutes and rinsed three times in automation buffer

solution. Sections were outlined with a PAP to prevent subsequent solutions fiom running

off the slides while incubating.

Three or four drops of a blocking solution made of PBS supplemented with 4% normal

horse serum and 2% normal bovine serum (BVDV antibody-fiee) was applied to the slides to

decrease any non-specific binding. Slides were incubated in a humidified chamber for 10

minutes at room temperature before removing excess blocking solution and applying the primary

antibody, rnouse anti-BVDV 1 5C5, or the irrelevant antibody, mouse anti-rabies. Al1 antibodies

were diluted in blocking solution. An optima1 dilution of 1 :3200 was detennined for the primary

antibodies in preliminary studies. Slides were incubated in a humidifed chamber at room

temperature for 2 hours o r ovemight at 4 O C.

After rinsing three times with automation buffer solution, the secondary antibody,

biotinylated horse anti-mouser, at a I:800 dilution in blocking solution, was applied and incubated

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for 30 minutes at room temperature in a humidified chamber, After rinsing three times with

automation buffer solution, an avidin-biotin peroxidase solutions made in PBS was then applied

for 45 minutes, Afier rinsing three times with automation buffer solution, three to four drops of a

solution of 100 pl of the chromogen 3,3-diaminobenzidine @AB)" in 1 Oml PBS was applied and

the slides incubated for 5 minutes. Just before the DAB solution was applied, 3 . 5 ~ 1 of 30% &O2

was added to develop the chromogen. After rinsing witli deionized water, sections were stained

with Harris' hematoxylin and dehydrated through graded alcol101 and xylene solutions before

coverslipping with xylene-based mounting medium.

Acetone-fixed aspirate smears were processed in a sirnilar manner with the following

exceptions. On removal fiom the -70°C fieezer, slides were irnmediately immersed in cold

acetone for 5 minutes. Rehydration through alcohols and xyienes and protease digestion were

unneccessary (persona1 communication, D. Haines, 1998). The inactivator solution contained

lml 10% sodium azide and 2m130% HzOz in lOOml methanol. Slides were stained in Mayer's

hematoxylin solutionq for 20 seconds, rinsed in water and air-dried prior to coverslipping.

Slides were coded in a manner similar to those for cytological analysis, 1000 cells were

exarnined and classified as to ce11 type and staining. A positive staining cell was identified by the

presence of prominent d i f i se , red-brown cytoplasmic granular stippling (Figure 14).

7. Virus Isolation and Serology

Virus isolation was performed by standard methods.' Briefly, bufQ coat ce11 preparations

were fkozen and inoculated ont0 embryonic bovine spleen cells. Noncytopathic isolates were

detected by immunofluorescence using the monoclonal antibody 1 5 ~ 5 ~ ~ and were typed as type 1

or II, using type-specific monoclonal a n t i b o d i e ~ . ~ ~ ~ *

For serology, anti-BVDVantibodies in serum were titrated by virus neutralization

using BVDV type II strain 125 according to standardized methods by a centralized laboratoryb.

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8. Statistical Analysis

Data were analyzed using the SAS System for Windows version 6-12' and Analyse-Itu.

Differences with a p-value S 0.05 were considered statistically significant.

Peripheral blood totat leukocyte, neutrophil, lymphocyte and platelet counts were

assessed for significant decreases (cytopenia) by comparing mean absolute values before and

afier inoculation with virus. Bone marrow data were similarly analyzed. Results were examined

for differences among virus treatrnents and among groups of animals. The difference of the

absolute mean value before inoculation and the post-inoculation nadir were also compared

between viruses and groups of animals for hematology and bone marrow data. One-way

ANOVA aiialyses were performed on these data. Rectal temperatures were also compared

between viruses and groups by one-way ANOVA.

Cornparison of day of onset and duration of cytopenia or changes in the proliferation and

maturation pooIs in bone marrow between viruses was evaluated by an unpaired Student's t-test.

Three parameters (proliferation pool, maturation pool and M:E ratio) were exarnined for

differences between the first and second determination using the Wilcoxon signed-ranks test and

a Spearman rank correlation.

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1. Animals

A11 six calves (100%) infected with purified (n=3) or unpurified (n=3) 1 IQ virus

exhibited no overt clinical signs- Mild fever (39-5 - 40°C) developed in 316 (50%) of these

animals on day 3 or 4 but quickly subsided. AI1 six calves ( I 00%) again had fevers of 2 to 3 days

duration by day 7 or 8 post-inoculation (pi). During this latter febrile period, rectal temperatures

ranged fiom 40-41°C. By day 14 pi (day of euthanasia), al1 six calves had normal temperatures,

Calves inoculated with 245 15 strains also deveioped pyrexia- Mild fever (39-5-40 OC)

occurred initially by day 3 or 4 pi but subsided to some degree after 1 to 2 days, Fever returned

by day 7 or 8 pi and persisted for 4 to 6 days. During the second febrile period, ternperatures

were 41-42 OC. However, 7/12 (58%) calves had normal temperatures by the end of the study.

The second fever peaks in calves inoculated with 245 15 strains were significantly higher (p<O.OS)

than in calves inoculated with the 11Q strains.

A11 twelve calves infected with purified or unpurified 245 15 virus developed clinical

signs commonly observed with acute BVDV infection. Diarrhea was observed in al1 calves with

an onset at days 6 to 8 pi- The character of the diarrhea varied from profise and watery to scant

with hematochezia in two calves in group 6. Melena was observed in a calf in group 7.

Respiratory signs, including coughing, increased respiratory rate and serous nasal discharge,

developed in 9/12 (75%) calves. Calves in groups 6 and 7 tended to have more severe respiratory

signs, including dyspnea and mucopurulent nasal discharge. Most caIves developed partial

inappetence, and 4/6 (67%) infected with upî45lS were completely anorexic from day 12 pi until

termination. These four calves also became lethargic and responded minimally to external

stimulation. Subjective evahation of hydration status suggested mild dehydration in several

caives by days 12-14 pi. Of calves in groups 6 and 7,2/6 (33%) developed rare multifocal

erythematous erosions of the nasal mucosa by day 12 pi. Al1 three calves in group 7 fulfilled the

38

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criteria for euthanasia by day 13 and were immediatety euthanized. One calf in group 6 fulfilled

the criteria for euthanasia at day 14 pi. Other calves were not deteriorating or were actually

showing some clinical improvement by day 14 pi.

2- Virology and SeroIogy

Type II non-cytopathic BVDV was isolated from bufQ coats of 17/18 (94%) virus-

inoculated calves. The one caIf in group 4 that did not develop detectable viremia did develop

anti-BVDV antibodies on day 12 pi. In al1 virus-inoculated cakes, detectable viremia was first

demonstrated on day 4 or 6 pi and was undetectable by day 12 pi (Table 3).

Al1 virus-inoculated calves developed neutralizing antibodies to BVDV type II strain 125

on day 12 or 14 pi.

3. Gross and Kistological Findings

Gross lesions were not cornmon with either virus, but were more pronounced in calves

inoculated with strain 245 15. In groups 2 and 3, inoculated with strain 1 lQ, Iesions were limited

to mild to moderate lymph node enlargement with occasional petechial hemorrhages, and

infiequent patchy mucosal congestion in the small intestine.

Calves inoculated with strain 245 15 had mildly enlarged interna1 and peripherai Iyrnph

nodes, with fiequent petechial hemorrhages. Retropharyngeal lymph nodes were markedly

enlarged. The serosal surfaces of Peyer's patches were occasionally raised and hernorrhagic.

Intestinal lesions were generaily Iimited to patchy mucosal congestion with occasional petechial

hemorrhages; however, intestinal congestion and petechial hemorrhage were severe in one calf in

group 6. Rarely, 3-8mm diameter mucosal erosions were observed on the ventral surface of the

tongue and in the esophagus. Mild to moderate cranioventral bronchopenumonia was observed in

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most calves inoculated with strain 24515 and one calf in group 6 developed marked f ibrino~s

bronchopneumonia. This same calf had muitifocal petechial to ecchymotic hemorrhages in most

tissues.

Histological lesions associated with both strains were primarily limited to the intestinal

tract, lungs and lymphatic tissues. They also were more prominent in calves inoculated with strain

245 15,

Calves inoculated with strain 11Q had no substantial intestinal lesions- However, 245 15-

inoculated calves had mild to severe multifocaI mucosal ulceration with crypt necrosis. Peyer's

patches were frequently completely depleted o r there was evidence of ongoing lymphocyte

necrosis. Follicular remnants were present in many sections. Congestion was present in most

Iymph nodes. Intemal and peripheral lymph nodes were similarly affected and were often

completely depleted of lymphocytes. Follicular necrosis and neutrophilic lymphadenitis were

occasionally observed. Similar but milder lymph node lesions were occasionally observed in

calves inoculated with strain 11Q.

No substantial lesions were noted in the lungs of 11Q-inoculated calves, afthough there

was mild microscopic congestion and interstitial thickening in severd calves. Calves inoculated

with strain 245 15 had more severe but similar Iesions as well as mild to severe cranioventral

fibrinopunilent bronchopneumonia.

Ristology of bone marrow samples revealed that architecture was uniformly retained

after inoculation in al1 groups. Cellularity ranged from 20-70% in pre-inoculation samples and

did not Vary significantly after infection, although there was a tendency for ceilularity to increase

mildly in calves inoculated with 1lQ and to decrease mildly in calves inoculated with 245 lS(Fig.

1). One animal in group 6 developed a marked decrease in bone marrow cellularity towards the

end of the study period.

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Megakaryocyte numbers were significantly higher post-inoculation for groups 2 through

5. Al1 three calves in group 6 had significantly lower megakaryocyte nurnbers post-inoculation.

Rare focal bone marrow necrosis was observed in group 6 cafves,

Data collected fiom calves in groups 2 and 3 (1 1Q strains) were never statiçtically

different so results are based on the combined six~animals. In some instances, there were

differences between purified and unpurified 245 15 strains, and these differences are indicated

when relevant. Therefore, results may be based on six or twelve calves. There were never

significant differences when comparing groups given difierent titers of virus, so results were

pooled and therefore minimum group size is six animals.

Mean leukocyte counts were significantly decreased after viral inoculation cornpared to

the pre-inoculation mean values in 17/18 (94%) animals(Fig. 2). Only one calf in group 2 did not

develop significant leukopenia. Calves inoculated with strain 245 15 developed significantly

lower post-inoculation mean leukocyte counts than calves inocuiated with strain 1 IQ.

Leukopenia was characterized by moderate to severely decreased neutrophil nurnbers and

mild to moderately decreased numbers of lymphocytes. Mean neutrophil counts post-inoculation

were significantly decreased compared to pre-inoculation mean values in 18/18 (100%) infected

calves (Fig. 3). There were no differences when comparing neutropenia between 11Q infected

animals and 24515 infected animals except for days 8 and 14.

AI1 groups of calves developed a significant decrease in the mean lymphocyte counts

post-inoculation (Fig. 4)- Calves inoculated with strain 245 15 developed significantly lower post-

inoculation lymphocyte counts than strain 1 1Q infected calves, and up245 15 calves had

significantly lower counts than pz45 15 calves on days 10 to 14.

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Mean platelet counts also decreased significantly post-inoculation in al1 infected calves

(Fig. 5). Calves infected with strain 245 15 developed significantly greater decreases in platelet

counts than strain 1 IQ inoculated calves and calves given up245 15 had significantly lower

platelet counts than other groups fiom days 10 to 14.

For each animal, the difference of the pre-inoculation mean absolute ce11 count and the

post-inoculation nadir value were calculated for leukocytes, neutrophils and lymphocytes for each

group of animals (Fig. 6). The mean leukocyte nadir of 1 IQ inoculated calves was 4.8 x 109/L

(range 3.5-6.4) and of 24515 inoculated calves was 3.4 x log& (range 0.9-4.8). Significant

decreases in Ieukocyte count post-inoculation were observed in al1 infected groups compared to

control anirnals, The decrease was significantly greater in 245 15-inoculated calves (n=12)

compared to 1 lQ infected anirnals (n=6),

Reductions in neutrophil count were significantly different post-inoculation compared to

control animals in 3/6 (50%) groups (3,6,7). The rnean neutrophil nadir of 1 1Q inocutated calves

was 0.9 x 1 09/L, (range 0.6- 1. L) and that of 245 15 inocuiated calves was 0.4 x 109/L (range 0.03-

1.1). There were no differences in the magnitude of the decrease in neutrophil count between al1

1 1Q infected calves and alI 245 15 infected calves. There was a significant difference however,

between animals inoculated with punfied or unpurified 245 15 virus.

Decreases in lymphocyte count post-inoculation were significantly different fiom control

animals in al1 groups except group 3- The mean lymphocyte nadir of I 1 Q inoculated calves was

3.4 x 109/L (range 2.4-4.3) and that of 245 15-inoculated calves was 2.5 x lo9& (range 0.8-4.2).

Calves inoculated with strain 245 15 had significantly greater decreases than did calves inoculated

with strain 1 1 Q.

Differences in the day of onset and duration of cytopenia up to euthanasia (day 14) were

studied. Results are shown in Figure 7 and significant differences are summarized in TabIe 4.

There were no significant changes in red blood ce11 parameters afier virus inoculation in

any group (data not shown).

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5. Bone Marrow Cytology

Differential ce11 counts on sequential bone rnarrow myeloid cells are shown in Figures 8

and 9. The percentage of cells in the proliferation pool before and after virus inoculation was

significantly greater in anirnals given strain 245 15 (Fig. 9) but not with animals given strain 11Q

(Fig. 8). However, there were no significant differences when cornparhg the severity of change

between 1 1 Q and 245 15 viruses.

Similar findings were present in the myeloid maturation pool, The percentage of cells

after virus inoculation was significantly decreased in anirnals given strain 24515 but not with

animals given strain 1 1Q. Furthemore, when comparing strains 1 lQ and 245 15, there was a

significant difference in the degree of change in the rneans after inoculation.

Animals infected with the 11Q strains developed a maximal decrease in the maturation

pool at a mean of day 8 pi (range day 6 - day 12 pi) and a maximal increase in the proliferation

pool at a mean of day 8 pi (range day 8 - day 12 pi). Both pools were not different from pre-

inoculation values by the end of the study period.

Conversely, animals infected with strain 245 15 developed a maxima1 decrease in the

maturation pool at a rnean of day 6 pi (range day 2 - day 14 pi) and a maximal increase in the

proliferative pool at a mean of day I O pi (range day 6 - day 12 pi),

Myeloid to erythroid (ME) ratios were calculated for each cytology sample in groups

2,3,4,5, and 6 (IFig, 10). Mean M:E ratios were calculated for catves inoculated with 11Q and

with 245 15 strains. There was no significant difference in the mean M:E ratios before and after

virus inoculation except for day 6 pi for 245 15 inoculated calves,

An assessment of marrow granulocyte reserve (MGR) was performed on each sample.

1 IQ-inoculated animals tended to have adequate MGR until day 8 pi when there was a decrease

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which persisted until day 10 pi. Animals inoculated with 245 15 tended to have decreased MGR

by day 4 pi, and it persisted until day 10 pi (Fig. 1 1). Two animals in group 6 did not have a

rebound to normal MGR by the end of the study period.

Bone marrow Iyrnphocyte percentages were evaluated but there were no trends or

abnonnalities noted in experiments with either virus.

Results of a repeatability study on bone marrow cytology are shown in Table 5 and

Figure 12,

Immunostaining was performed on bone marrow aspirates and fixed core biopsies.

Controls used for staining procedures are depicted in Figures 13 to 16. Results were simiiar for

aspirate and core samples- No positive staining was observed in any samples fiom animals

infected with strain 1 1Q. In animals inoculated with strain 245 15, positive staining was very

dispersed and transient. However, 6/9 (67%) animals exhibited at least some positive staining,

including al1 three animals in group 5, two animals in group 4 and one animal in group 6. Viral

antigen was detected in bone marrow cells primarily from day 6 to day IO pi (Table 6).

Megakaryocytes were the most common ce11 type with virus (69%) followed by early myeloid

cells (28%) (Figures 17 to 19). The other 3% of cells were not identifiable morphologically. Of

the six animais that exhibited positive staining, two had positive staining cells on only one day.

One animal had positive staining on two consecutive sampling days and three had positive

staining on three sarnpling days. The percentage of cells staining positive ranged f?om O. 1% to

0.8% with a mean of 0.32% (n=9), OnIy 9/f 05 (8.5%) of post-inoculation samples (al1 1 1 Q and

245 15) exhibited positive staining for viral antigen and only 9/63 (14%) of 245 15 samples had

positive staining.

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Table 1, List of treatrnent groups with strain o f BVD virus, purification statu*, volume? -

and titer administered intranasally.

Treatrnent Groupz Virus Volume and Titer

- 2mI medium onIy

PI lQ 2ml x 1 O' TCIDS0

U P ~ IQ 2ml x 10' TCIDSO

p245 15 2ml x 1 0' TCIDSo

p245 15 2mI x 1 o7 TCIDSO

up245 15 2mI x 10' TCIDso

up245 15 2ml x lo7 TCIDso

* p = purified; up = unpurified 7 lm1 per nostril $ n=3 in al1 groups

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Table 2. Upper and lower threshold values for hernatologic parameters calculated from pre-

inoculation samples*.

Threshold Parameter Valuest Units Mean SDZ

Leukocytes

Neutrophils

Lyrn p hocytes

Platelets

RBC

Hernatocrït

* n = 2 1, six to eight month old steers t Range o f mean +/- 2SD $ SD = standard deviation

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Table 3. Isolation o f non-cytopathic type II BVDV from bufi coat cetls fiom

btood sarnples collected on alternate days.

Day post-inoculation* Group Calf 2 4 6 8 10 12 f4f-

- - - - -- - - - - - - -

* - = no virus isolated; + = ncp type II BVDV isolated, t The last blood sample for animals in group 7 was day 13 post-inoculation.

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Table 4, Prevalence of blood cytopenia, rnean day of onset of cytopenia post-inoculation

(pi) and mean duration of cytopenia among groups of calves inocuIated with 1 1Q and

245 15 strains of ncp type II BVDV.

Prevalence of Mean day of onset Mean duration cytopenia (pi) (days) of cytopenia

CeII type 1 1Q

Total leukocytes 516 (83 %)

Neutrophils 6/6 (1 00%)

Lymphocytes 3/6 (50%)

P latelets 5/6 (83%)

Significant differences (pc0.05) between 1 1 Q and 245 15 inoculated caIves are indicated by *.

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Table 5. Repeatability study for bone marrow cytology. Twenty samples were randomly

re-evaluated and the data for three variables are presented-

Statistic Proliferation Pool Maturation Pool M:E Ratio - ppppp

Mean* 4.6 24.6 0.73 5

S m 1.7 6-9 0.264

CvX 37.7 28.4 35.9

Mean delta5 1.6 5.4 15.9

Wilcoxon signed ranks test[[ 0.473 0.812 0.9 19

Spearman rank correlationfi 0.009 0.036 0.002

* mean of the 40 values derived fiom original and repeat of 20 samples; (%) for maturation and prolifzration pools

standard deviation $ coefficient of variation (%) 5 mean difference between repeat values of 20 samples (%) II p vaiue, repeat samples not significantly different if >O.Os fi p value, repeat samples significantly correlated if <O.OS

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Table 6. Immunostaining o f sequential bone marrow sampIes. BVDV-positive

staining in cells o f the megakaryocyte series (M) and myeloid series (N) are indicated.

Day Post-Inoculation

Group* Animal 2 4 6 8 t O 12 14

* Groups 2 and 3 inoculated with 1 IQ, groups.4 to 6 inoculated with 245 15. For further group identification see Table 1.

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Days (pre- and post-inoculation)

Figure 1. Bone marrow cellularity assessed fiom core biopsy sarnples for strain 1 1Q (*) (n=6)

and strain 245 15 (n=9) inoculated calves pre- and post-inoculation with virus. Open syrnbols

represent significant differences compared to pre-inoculation values. Asterisks indicate

differences between viruses.

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Days (pre- and post-inoculation)

Figure 2. Mean blood leukocyte counts for control (+, n=3), il Q (I, n=6), p24515 (A,

n=6) and up24515 (O, n=6) inoculated calves. Day O is the day of inoculation. Standard

error bars are shown. Dotted Iines indicate the threshold values (mean +/- 2 SD).

Significant differences between pre- and post-inoculation values are indicated by open

syrnbols. Significant differences between groups are indicated by an asterisk.

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Days (pre- and post-inoculation)

Figure 3. Mean blood neutrophil counts for control (e l n=3), 11 Q (I, n=6), p24515 (A,

n=6) and up24515 (el n=6) inoculated caives. Day O is the day of inoculation. Standard

error bars are shown. Dotted lines indicate the threshold values (mean +/- 2 SD).

Significant differences between pre- and post-inoculation values are indicated by open

symbols. Signïfïcant differences between groups are indicated by an asterisk,

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Days (pre- and post-inoculation)

Figure 4. Mean blood lymphocyte counts for control (+, n=3), 1lQ (I, n=6), pz451 5

(A, n=6) and up24515 (O, n=6) inoculated calves. Day O is the day of inoculation.

Standard error bars are shown. Dotted lines indicate the threshold values (mean +/- 2

SD). Significant differences between pre- and post-inoculation values are indicated by

open symbols. Significant differences between groups are indicated by an asten'sk.

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Days (pre- and post-inoculation)

Figure 5. Mean blood platelet counts for control (+, n=3), I 1Q (Il n=6), p24515 (A,

n=6) and up24515 (a, n=6) inoculated caives. Day O is the day of inoculation. Standard

error bars are shown. Dotted lines indicate the threshold values (mean +/- 2 SD).

Significant differences between pre- and post-inoculation means are indicated by open

symbols- Significant differences between groups are indicated by an asterisk.

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2 3 4 5 6

Group

Group

Figure 6- Comparison of differences between the mean pre-inoculation value and

the post-inoculation nadir for leukocytes (A), neutrophils (6) and lymphocytes (C)

for 1 1 Q- and 2451 5-inoculated calves- Each bar represents the mean of three

animals. For group details see Table 1 .Results were compared to the control

group (group 1) to assess for significant differences which are indicated by an

asterisk. Inter-grou[p differences are indicated by different column shading.

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al neut

C - - plate !

O 2 4 6 8 10 12 14 16

Day after inoculation

leuk 1 - 1 1

O 2 4 6 8 10 12 14 16

Day after inoculation

lmph

al n % neut C - -

plate

leuk

O 2 4 6 8 10 12 14 16

Day after inoculation

leuk 1 - O 2 4 6 8 10 12 14 16

Day after inoculation

Figure 7. Cornparison of day of onset and duration of leukopenia, neutropenia, lympho-

penia and thrombocytopenia for 11 Q- and 24515-inoculated calves. Cytopenia was de-

fined as falling below the Iower threshold value caIculated from pre-inoculation samples

(see Table 2, page 45)

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Days (pre- and post-inoculation)

Figure 8. Percentage myeloid proliferation (+) and maturation (I) pcoIs fiom serial bone

marrow cytology sarnples fiom calves inoculated with strain I 1Q. Each data point represents the

mean of six animals. Day O is the day of inoculation, Standard error bars are shown. Significant

differences between pre- and post-inoculation means are indicated by open symbols.

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Days ( pre- and post-inoculation)

Figure 9. Percentage myeloid proIiferation (+) and maturation (I) pools frorn serial

bone marrow cytoIogy samples of calves inoculated with strain 2451 5. Each data point

represents the mean of nine anirnals. Day O is the day of inoculation. Standard error

bars are shown. Significant differences between pre- and post-inoculation rneans are

indicated by open symbois,

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0.00 1 1 1 l I I I 1 I i 1 1 1

-10 -8 -6 -4 -2 . 2 4 6 8 10 12 14

Days (pre- and post-inoculation)

Figure 10. M:E ratios fiom bone marrow cytoIogy pre- and post-inoculation for 1 IQ-

(+, n=6) and 245 15- (1, n=9) inoculated calves. Dotted Iines represent the mean +/- 2

SD of the mean pre-inoculation M:E ratios of 15 animais. Standard error bars are s h o w .

Significant differences fiom the mean pre-inoculation values are indicated by open

synibols. Significant differences between groups are indicated by an asterisk.

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Figure 11. Bone marrow cytology fiom an animal inoculated with virulent 245 15 non-cytopathic

type II bovine viral diarrhea virus on day 8 post-inoculation. Marrow granulocyte reserve is

depleted, there is a shifi to early myeloid precursors and the M:E ratio is increased favoring

myelopoiesis. Wright's stain. 200x

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O 0.02 0.04 0.06 0.08 0.1

Mean values (Y,

Figure 12. Scatterplot of the mean of the difference (delta) between repeat vaiues from 20

samples and the mean o f 40 values co1Iected fkom the 20 repeat sarnples for the proliferation

pool. As the mean value increased, delta values also increased.

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Figure 13. Acetone-fmed cultured Madin-Darby bovine kidney (MDBK) cells used as controls

for immunocytochemistry of fkozen bone marrow aspirates- This image is fiom an uninfected

sarnple used as a negative control- Stained with Mayer's hematoxylin. 200x

Figure 14. Acetone-fixed cultured Madin-Darby bovine kidney (MDBK) celk used as controls

for irnrnunocytochemistry of f h e n bone marrow aspirates. This image is fiom a sarnple infected

with non-cytopathic type II bovine viral diarrhea virus. Positive staining for BVDV antigen is

identified as red-brown cytopIasmic stippling. Stained with Mayer's hematoxylin. 200x

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Figure 15. BS-fixed cultured Madin-Darby bovine kidney (MDBK) cells used as controk for

immunohistochemistry of paraffm-embedded bone marrow sections. This image is fi-orn an

uninfected sample used as a negative control. Stained with Harris' hematoxylin. 200x

g-b QI - aF *-Y& - eJt ) as Po,, 5&" a-- *-

VI&* = Yi-

-- 91, k

z L r a4) 7 cts > -

1

Figure 16. B5-fixed cultured Madin-Darby bovine kidney (MDBK) cells used as controls

immunohistochemistry of paraffrn-embedded bone rnarrow sections. This image is fkorn a

for

sample infected with non-cytopathic type II bovine viral diarrhea virus. Positive staining is

identified as red-brown cytoplasmic stippling. Stained with Harris' hematoxylin. 200x

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Figure 17a. Positive-staining megakaryocytes from an acetone-futed fiozen bone marrow

aspirate sample fkorn a 245 15-inoculated animal. Positive staining is identified as red-brown

cytoplasmic stippling. Mayer's hematoxylin. lOOx

Figure 17b. Positive-staining megakaryocyte &om a B5-fixed bone marrow sarnple fiom a

245 15-inoculated animal. Positive staining is identified as red-brown cytoplasmic stippling.

Harris' hematoxylin. 400x

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Figure 18. Positive-staining myeloid ce11 fkom a BS-fwed bone marrow sample fkom a 245 15-

inoculated animal. Positive staining is identified as red-brown cytoplasmic s t ipphg (arrow).

Harris' hematoxylin. 400x

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Figure 19. Positive-staining myeloid cells fkom a BS-fmed bone marrow sarnple f?om a 2451 5-

inocdated animal, Positive staining is identified as red-brown cytoplasmic stippIing (arrow).

Harris' hematoxylin. 400x

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Discussion

Experirnental infection of Holstein steers with two strains of ncp type II BVDV resulted

in a varied clinical presentation. TypicaI clinical signs of severe acute BVDV were observed in

al1 animals inoculated with the virulent 2451 5 strains. Most importantly, al1 animais inoculated

with the lower virulence 1 1 Q strains Iacked overt chnical syrnptoms.

The 11Q virus has not previously been evaluated in experimental studies. It was one of

several strains isolated from cattle in a feedlot in Manitoba (persona1 communication, Gopi

Nayar). Calves in this herd had been experiencing intermittent diarrhea and pneumoniâ.

Furthemore, abortions were being atenbuted to BVDV infection. This strain was fortuitously

chosen as the first to evaluate for clinical signs in anirnals. The goal was to isolate a low

virulence ncp type ï I BVDV for rnolecular evaluation. Pyrexia was the only abnorrnality noted

during daily physical examination and observation during the fourteen day study period- Fever is

common during BVDV infection but in a herd situation, the febrile period induced by 1 I Q would

likely go unnoticed. Although bi-phasic fever has been reported with virulent BVDV strains," it

is interesting that the animals with otherwise inapparent disease also developed pyrexia. The

fever induced by this lower virulence strain, however, was significantiy lower and resolved more

quickly than with the more virulent strain 245 15. Experirnental infection with m i n 1 1Q was

characteristic of the traditional self-limiting, clinically inapparent ncp type II BVDV infections?'

The virulent strain 245 15 has been used in previous experiniental infection studies of

neonatal animals and the clinical signs have been documented." In the fieid situation, infection

in older calves and adult animals is also important? However, it was unknown if older animals

with mature immune systems, though naïve to BVDV, would develop severe disease when

infected with this strain. These experiments prove that older calves experimentally infected with

the strain 245 15 develop severe disease, which closely resembles the field situation. The

severity of clinical signs varied substantially arnong the twelve animals inoculated with 245 15

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strains. Al1 calves deveIoped diarrhea by day six or eight pi but only three calves developed

hemorrhagic diarrhea, This strain can cause severe thrombocytopenia with subsequent

development of hemorrhagic syndrome." In the present study, hemorrhagic diarrhea developed

only in animals inoculated with unpurified strains and even then was found in only 50% of

calves. The effects of purification are discussed below,

Respiratory signs were very cornmon in these experimental infections and were observed

only with 245 15 strains. Coughing and nasal discharge often were noted, but three calves from

groups 6 and 7 also developed severe fibrinous bronchopneumonia The role of BVDV in the

developrnent of bovine pneumonia is an ongoing debate. Irnmunosuppression secondary to

BVDV infection may play a role in predisposing animals to opportunistic infection^.'^-^

The viruses dif5ered in the range of gross Iesions present at necropsy at day 14 pi and on

histopathologic evaluation. Previous reports of Iesions induced by virulent ncp m e II BVDV

infections found that in animals which develop severe clinical disease, post-mortem Iesions may

be minimal to quite Gross and histological Iesions in 245 15-inoculated calves 14

days after inoculation were found primarily in the lymphoid tissues, intestinal tract and lungs,

similar to other reports- There is one report of minimal lesions induced by a low virulence strain,

but the strain was a type I virus.)6 In this study, the only abnormalities found were enlarged

lyrnph nodes containing scattered petechiai hemorrhages. Lesions at day 14 pi in animals

inoculated with 11Q were also Iimited to lymphoid tissues and similar in character- Necropsy at

an earlier tirne during the infection period (during pyrexia and virernia) may have resulted in a

greater fkequency and severity of gross and histoiogical lesions that may have resolved by day 14

pi when animals were afebrile and clinically improving. This rnay also have been the case with

animals inoculated with purified 24515, that were a1so improving by the end of the study period.

Lesions induced by virulent 245 15 were more extensive and severe than those caused by 1 IQ.

Cliemotactic demand for neutrophils in tissues can contribute to the development of

neutropenia. Neutrophil accumulation in tissues of the intestinal tract was minimal and limited to

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occasionaI necrotic crypts. However, neutrophil loss into the intestinal lumen could not be

demonstrated as migration through the wall of the gut can be quite rapid, Again, analysis of

lesions during peak pyrexia and viremia may have revealed this Iesion if it occurred. In animals

with severe bronchopneumonia, areas of necrosis frequently were accompanied by a neutrophilic

infiltrate.

Some animals had sinusoidal and cortical neutrophitic infiltrates of both peripheral and

intemal lymph nodes. The cause of this lesion is unknown and has not previously been described

with BVDV infection. In sorne lymph nodes, it rnay have been a response to lyrnphoid necrosis,

but it was present in other lymph nodes that had no evidence of necrosis. It is impossible to

directly quanti@ the contribution of neutrophil tissue loss to the development of ieukopenia in

this experiment but indirect support is provided by the sequential examination of blood and bone

marrow.

Thrombocytopenia and leukopenia characterized by neutropenia and lyrnphopenia have

been described in 24515-inoculated calves based upon a single blood sarnple collected pnor to

euthanasia-l' Other studies have docurnented the changes in blood parameters during infection

with other ncp type II BVDV ~ t r a i n s . ' * * ~ ~ ~ ~ One study evaluated two ncp BVDV strains and

showed that one induced severe clinical disease (BVDV-890) and caused rnarked hematological

abnormalities and that the other (BVDV-TGAN) did not result in clinically apparent disease or

cause changes in hernatological parameters?6 This was done in 2-9 week old calves, but

immunological and hematological function differs fiom older animals." Furthemore, BVDV-

890 is a type II virus while BVDV-TGAN is a type 1 Therefore, this is the f r s t m d y

comparing virulence of two ncp type II BVDV strains.

The experiments perfonned in this study document sequential changes in CBC

parameters in ncp type LI BVD viruses of differing virulence in BVDV-naïve animals with mature

immune systems. An advantage of this study is that pre- and post-inoculation sarnples could be

cornpared. These values could also be used to develop age-specific threshold values to further

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characterize cytopenias. Not only did animals inoculated with virulent 245 15 strains develop

Ieukopenia and thrornbocytopenia, but animais inoculated with the less vimlent 1 1Q strain also

developed similar, albeit Iess severe, hematological abnormalities.

Despite the fact that strain L 1Q inoculation did not result in clinically apparent disease,

significantly decreased post-inoculation neutrophiI and lymphocyte counts occurred for most

animals. Platelet counts dropped slightly but rarely below the pre-inoculation established lower

threshold value. This is in contrast to an earlier study tliat evaluated a low virulence strain in

which no hematological changes occurred during infection." However, this virus was not a type

II strain making it difficult to compare the two studies.

When comparïng al1 strain 1 I Q-inoculated calves to al1 strain 245 15-inoculated calves,

there wai no difference in the degree of change in neutrophil counts. However, the magnitude of

decreased lymphocyte num bers in 245 1 5-inocuiated animals was larger than any animal infected

with an 11Q strain. This suggests that one or more of the mechanisms contributing to

neutropenia in virulent BVDV also occur during subclinical disease, white this is not the case for

fymphopenia. Hence, the ability of a BVDV strain to cause neutropenia done may not be an

important virulence factor.

There was no difference in the day of onset of Ieukopenia or lymphopenia for eittier

virus, and neutropenia occurred only a day earlier in 245 15-inoculated calves. However,

decreased ce11 counts persisted 2-5 days longer in calves inoculated with strain 24515. This

suggests that the reason for onset of decreased ce11 count is similar regardless of virulence, but

due to other reasons, these changes persist longer with a more virulent strain. There may be a

delay before production of myeloid precursors increases or there may be a larger peripheral

demand beyond the capacity of the marrow granulocyte reserve.

Although platelet counts did decrease, severe thrombocytopenia was not a prominent

feature of infection with 1 1Q strains and platelet numbers never were low enough to resutt in

hemorrhage. in contrast, 2451 5 infection caused significantly greater thrornbocytopenia and was

7 1

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occasionaIly associated with hemorrhage. This suggests that the ability to cause a large decrease

in platelet counts rnay enhance virulence,

Sequential bone marrow sarnples were examined to further define the reasons for

hematological alterations A single sample of bone rnarrow is thought to be representative o f the

organ and therefore there should be minimal variation due to sample site. However, there is

within-sample variation depending on the area of the smear examined and there can also be

variation in rnorphologic evaluation of cells between and within observers- To show variation in

evaluation of bone marrow cytoiogy for the observer in this study, a repeatability assay was

perfonned. The results of this study suggest that for blinded re-evaluations, the observer was

capable of producing a similar differential ce11 count. It was noted that as the percentage of a ce11

type increased in a sarnple, there was more variation between deterrninations.

In calves inoculated with 1 1Q strains, peripheral blood neutrophil counts were beginning

to decrease on day 2 pi and were even lower by day 4 pi. Concurrently, the maturation pool in

the bone marrow also decreased and reached a nadir by day 8 pi (Figure 8). This appeared to

represent increased peripheral demand, as there was no delay apparent for myeloid precursor

increases. In fact, the percentage of ceils in the proliferation pool was increasing by day 4 or 6 pi

and peaked by day 8 pi, at the same time that the peripheral neutrophil counts were lowest. Bone

marrow cellularity was not decreased, Given that some time is necessary for up-regulation of

stem cells to commit to the myeloid developmental pathway, this would indicate that even before

there was much drain fiom the maturation pool, there was already stimulus to increase myeloid

production. Considering the kinetics of neutrophil production, the stimulus to increase marrow

production must have occurred by the first or second day pi. This is interpreted as an appropriate

and tirnely response by the bone marrow to increased peripheral demand. By the end of the study

period, the peripheral blood leukocyte counts were returning to pre-inoculation values. In

addition, the maturation and proliferation pools in the bone marrow were also returning to pre-

inoculation values, indicating an end to the increased peripheral demand and return toward

72

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homeostasis. It is clear that lack of production in the bone rnarrow does not contribute to

leukopenia in calves infected with the1 1Q strain.

In calves inocuIated with 245 15 strains, neutrophil counts were beginning to decline by

day 2 pi, and were drarnatically reduced by day 4 pi- This was similar to 1 1Q inoculated calves.

The bone marrow maturation pool was concurrently diminishing and actually reached its low

point by day 4 to 6 pi in many animals and continued to rernain Iow until day 10 pi if recovery

did occur (group 6) (Figure 9). Therefore, there appeared to be an abrupt drain of granulocyte

reserves that did not rebound as quickly as with inoculation with the 1 IQ strains. This was

primarily because of a slow increase in the proliferation pool. In most animals, it was increasing

by day 8 or 10 pi, but did not peak until day 12 pi. At this point, it was also difficult to project

whether a few animals were going to effectively replenish the maturation pool and restore the

neutrophil count.

Bone marrow cellularity was also significantly lower in 2451 5 inoculated animals at the

end of the study period (Figure 1). It could be hypothesized that because this was a more virulent

strain there was a larger peripheral dernand and therefore the bone marrow maturation pool was

more quickly and extensiveiy exhausted. When demands are large, because of tissue necrosis or

inflammation, there should be a very prompt increase in bone marrow precursor cells and

subsequently in the pool of mature ~ells.'~"~ If a large dernand persisted for some time however,

cells would continue to leave the bone marrow as soon as they mature or even sooner (seen as a

left shift in peripheral blood).

Most studies on the kinetics of cells of the myeloid series in the bone marrow and

neutrophils in the peripheral blood and tissues occurred in the 1960s and 1970s:'"~ In the bovine,

it takes about seven days for a segrnented neutrophil to develop fiom a myeloblast? In other

species, this maturation interval may be shorter when production is accelerated due to acute

peripheral d e r n a ~ ~ d . ~ ~ If this was the case in mature cattle, it rnight be possible to recruit

segmented neutrophils more quickly to meet the chernotactic dernand, whereas a neonatal calf

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may be unable to mount such a response. However, it is suspected that cattle cannot mount a

response to peripheral demand as quickly as some other speciesP4

Regardless, if the bone marrow was responding appropnately, there should have been at

l e s t a similar onset in increased proliferation pool numbers in the 245 15-inoculated calves as in

the 1 IQ-inoculated calves. Furthermore, if increased peripheral dernand was the only

mechanism, there should have been an early and persistent shift to immature myeIoid cells and a

subsequent equal increase in both bone marrow pook However, this was not the case, Instead,

production was significantly delayed by about 4 days. Furthermore, in one group (group 5) there

was a decrease in numbers in the proliferation pool percentage cells before they increased,

Therefore, virulent ncp type II BVDV infection, like other Flaviviridae, does appear to

cause a decrease or delay in bone marrow production, as opposed to less virulent strains- Still, in

immunocompetent animals this bone marrow suppression usuaIly is transient and chnical

recovery corresponds with elimination of virus and virus-infected cells and an increase in bone

marrow cellularity. The onset of bone rnarrow suppression occurred at day 2 to 4 pi and persisted

for 5 to 6 days. Bone marrow suppression with dengue fever has been well described and

similady occun at 3 to 4 days pi.12

Delayed production and larger peripheral demand contribute to longer duration of

leukopenia in calves inoculated with strain 245 15, which provides a window of opportunity for

secondary pathogens to establish infection, further increasing animal morbidity and risk of

mortality.

In al1 instances, there were no significant differences in hematologic parameters between

groups 2 and 3 (1 1Q strains), indicating that there was no effect of purification or iower viral

titer. However, since changes in blood parameters were not as severe as with the virulent 245 15

strain, a reduction in virulence may have been more difftcult to appreciate.

It was interestkg that 4/6 (67%) calves inoculated with the unpurified 245 15 strains

(groups 4 and 5) required euthanasia towards the end of the study period, but al1 six calves

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inoculated with punfied 245 15 virus were showing cIinicaI improvement by day 14 pi. There

was also considerable variability of clinical signs arnong those inoculated with purified strains.

The reasons for this apparent difference are probably multifactorial. Firstly, virulence rnay be

attenuated during the purification process. Multiple passaging o f virus has been shown to result

in decreased virulence compared to the unpurified state? During the purification process, a

single viral clone is isolated- This isolate rnay not represent the sum virulence of the multiple

clones present in an unpurified stock. It is possible that if unpurified virus stock were punfied

again, a more virulent clone might be isolated due to random seIection, Secondly, individual

virus-host interactions are very important, To a certain extent, each animal rnay respond

differently to an infecting strain due to biological variation,

Tlie titer of virus inoculated did not appear to influence disease severity. Calves in group

6 deveioped similar and occasionaIIy more severe signs than the tfiree calves in group 7 which

were exposed to the higher titer. A difference in titer of 102 TCIDso in the inocutum, within the

range of titers used, does not appear to affect ability to cause disease. This is not surprking,

considering that initial vira1 replication occurs in lymphoid tissues of the oropharynx.

Afterwards, viral amplification occurs throughout the body. Therefore, the number of infecting

particles rnay be greatly amplified regardless of the infecting dose.

Viral infection rnay alter ce11 fùnction or shorten life-span. Therefore, bone rnarrow stem

c e k harboring viral antigen rnay not mature into differentiated cells. No BVDV-positive staining

was demonstrated in any bone marrow sarnples collected fiom 1 IQ-inoculated calves, suggesting

that infection of bone marrow precursor cells is not an important contributor to the transient

leukopenia that developed during infection.

Calves inoculated with 245 15 strains did have demonstrable viral antigen in

hematopoietic cells, however, the number of cells involved was very low. Still, it is possible that

virus-induced interference with progenitor ce11 maturation is a major contributory mechanisrn to

the development of marked leukopenia (and thrornbocytopenia) in animals infected with virulent

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ncp type II BVDV strains- To further characterize the ce11 types infected, virus isolation could be

attempted fiom subsets of hematopoietic cells.

Other factors rnay contribute to bone marrow suppression, inctuding production of

cytokines that interfere with normal granulopoiesis and thrombopoiesis, inhibition or cytolysis of

stromal cells necessary for hernatopoiesis, or 'apparent' suppression because of large peripheral

dernand from intestinal and lung lesions. No viral antigen was definitively dernonstrated in

rnarrow stromal cells in these experiments. But, as with hernatopoietic ceIIs, only a few stromal

cells may need to be affected to pr0duce.a major change in bone marrow dynamics. In support of

this theory, an in vitro study of dengue-infected bone marrow progenitors did not show direct

cytotoxicity, but proliferation of bone marrow cells was decreased.12 Other experiments by the

sarne authors showed that the earliest progenitor cells did not becorne infected until they had

differentiated for at least 7 days, and even then positive staining was rare. This could be a

protective rnechanism since non-dividing hematopoietic cells would be less susceptible to

infe~tion.'~ Infection of marrow stromal cells, although few in number, ~red0rninated.l~

Therefore, since s-in 245 15 was present in bone marrow cells but 1 1Q was not, tropism for

hernatopoietic celIs may be an important virulence factor.

As with most biological systems, one mechanism rarely is the sole reason for production

of an alteration in a body system dunng disease. However, if one mechanism predominates,

altering or eliminating this pathway might Iargely change the outcorne. This scenario may apply

to the reasons for alterations in hematologic parameters during BVDV infection. Although

decreased bone marrow production probably contributes to the development of leukopenia, as

may direct cytolysis of infected precursor cells, increased peripheral demand is probably very

important. The role of endotoxin as a contributor to neutropenia early in infection was not

evaluated in this study. In calves inoculated with 1 lQ, there was less peripheral demand, no viral

antigen in the bone marrow and no delay in bone marrow response. Calves inoculated with

245 15 however, had a larger peripheral demand, viral antigen in hematopoietic cells and a

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delayed bone marrow response. Tissue demand was not easily demonstrable in histology sections

obtained at post-mortem, but many animals were actually recovering and therefore the most

severe lesions including neutrophilic infiltrate in the intestinal rnucosa, might have been resolved.

In conclusion, it appears that ncp type II BVDV strains of differing virulence induce

cytopenias proportional to severity of infection. Severity of neutropenia does not appear to be an

important vindence factor, but neutropenia persisted significantly longer with the more virulent

strain providing potential increased risk of opportunistic infections. Bone marrow response

during infection with a virulent strain was delayed. The deIay could be mediated.by an altered

hematopoietic inductive microenvironment or direct infection of hematopoietic myeloid cells.

Tnfection of megakaxyocytes also appears to be an important virulence factor. Future work

should explore alterations in local bone marrow cytokines, virus isolation fiom subsets o f

hematopoietic cells, and demonstration of histological lesions at peak fever and viremia.

Radiolabelling and tracking of neutrophils could be perforrned in the live animal, to further

characterize their kinetics and distribution during infection-

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Sources and Manufacturers

a. Gil Technologies, Orangeville, ON, Canada,

b. Animal Health Laboratory, University of Guelph, ON, Canada.

c- Xylamax 100mg/ml, Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada,

d. Steri-Stat, Ingram & Bell, Don Milis, ON, Canada.

e. Gibco-BRL, Life Technologies, Burlington, ON, Canada.

f. Penicillin-Streptomycin, Life Technologies, Burlington, ON, Canada.

g- H*1 Technicon, Tanytown, PA.

h- Hema-Tek Slide Stainer, Ames Co., Elkhart, IN

i. Fisher Scientific, Fairlawn, NJ.

j. AFP method.

k. CytoSeal60, Stephens Scientific, Kalamazoo, MI.

1. Surgipath, Winnipeg, MB, Canada.

m. NaIge Nunc International, Naperville, IL.

n. Cedarlane Laboratories Ltd., Homby, ON, Canada.

o. Biomeda Corp., Foster City, CA.

p. ICN Biomedicals Inc., Aurora, OH.

q. Sigma Chernical Co., St. Louis, MO.

r. Vector Horse Biotinyiated Anti-Mouse IgG, Vector Laboratories Inc., Burlingarne, CA.

S. ABC Vectastain Eiite, Vector Laboratories Inc, Burlingarne, CA.

t. SAS Institute, Caxy, NC.

u. Analyse-It Software Ltd., UK.

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APPENDIX 1

Daily Physical Examination Sheet

lated

From a Distance

Inquisitive to VisuaVNoise Yes=O Mildly=I No=2

Standing or Stands Up on Enûy Yes=O Eventually=l No=2

Breathing Norm=O SI. Labored=l V.Labored=2

Appetite Yes=O Some food left--i No=2

Oculo-Nasal Discharge No=O Mild=l Severe=2

I I

Physical Examination I I

Responds to Physical Stimuli 1 1 Yes=O Mildly=l No=2 Temperature (OC)

Heart Rate / minute

Respiratory h t e / minute

I

Blood Drawn 3xlOml EDTA, 3xlOmi Red Top Comments

Dehydration ' No=O 5-7%=1 7-10%=2 Forrned Feces Yes=O Sorne Anirnals=l N0=2 Score

.

Date Euthanized

I circulatory signs, muscle tremors

I 5%= doughy skin, conjunctival congestion; 7%= prolonged skin tent, sunken eyes; 1

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APPENDIX 2

List of tissues examined by histopathology

Oral mucosa

Pharynx

Esophagus

Rumen

Reticulum

Omasum

Abomasum

Duodenum

Jejunum

Ileum

Cecum

Spiral colon

Rectum

Trachea

Cranioventral lung

Middle lung

Caudodorsal lung

Retropharyngeal Iyrnph node

HiIar lyrnph node

Superfkial cervical lymph node

Mesenterie lymph node

Prefemoral lymph node

Thymus

Liver

Spleen

Pancreas

Adrenal gland

Kidney

B ladder

Urethra

Skin

E Y ~

Cerebrum

Bone marrow

Right auricle

Lefi auricle

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APPENDIX 3

CBC Parameters

Leukocyte count

Red blood ce11 count

Hemoglo b in

Hematocrit

Mean corpuscuiar volume

Mear, corpuscuiar hernoglobin

Mean corpuscular hemoglobin concentration

Red ce11 distribution width

Mean platetet volume

Neutrophil count

Lymphocyte count

Monocyte count

Eosinophil count

Basophil count

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APPENDM 4

Bone Marrow Cytology DifferentiaI Sheet

1 Code: Animal: Date: Vims:

Ce11 Type Rubnblast Proru bricyte

1 I

Polychromatophilic rubricyte 1 Eosinophilic myelocyte 1

Total

I 4

( Progranulocyte 1

Basophilic mbricyte Neutrophilic myelocyte

Ce11 Type Mveloblast

Normochromic rubricyte

Total

Basophilie rnyelocyte

Metmbricyte Neutrophilic metamyelocyte

Mitotic cells TOTAL ERYTHROCYTIC CELLS =

Eosinophiiic rnetarnyelocyte Basophilie metamyelocyte

Hematogones

Mitotic cells Eosinophil Megakaryocytes Basophil Osteoclasts TOTAL GRANULOCYTIC CELLS =

I

Neutrophilic band I

Lymphocytes Plasma cells Monocytes .

I - - - - - - -- - -

Eosinophilic band Baçophilic band Segmented neutrophil

Macrophages Unclassifed cells Degenerated cells TOTAL OTHER CELLS = Other comment5

M:E Ratio Granule cellularity MGR (- N +) Mega/granule/ 1 Ox

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APPENDIX 5

B5 Fixative

Ingred ien ts:

60 grams mercuric chioride

12.5 grarns anhydrous sodium acetate

900 ml hot distilled water

Mix and refngerate untiI use.

Before use mix:

9 parts B5 solution

1 part 37% formaldehyde

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APPENDIX 6 Individual Animal Hematology Data

Group 1 - Control

O t I 1 1 I i 1 1 1 i i 1 t

-10 -9 -7 4 1 O 3 5 7 I O 12 14

Days (Pre- and Post-Inoculation)

4- Leukocytes +Lymphocytes Neutrophils

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Group 2 - p l 1 Q

Days (Pre- and Post-Inoculation) -C Leukocytes +Lymphocytes +Neutrophils

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Group 3 - up1 IQ

20 -

1 5 - O

x I O - V) - - d 5 -

-10 -8 -6 -4 -2 O 2 4 6 8 I O 12 14

Days (Pre- and Post-Inoculation)

+ Leukocytes -)lLymphocytes +Neutrophils

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Group 4 - ~24515x5

82-22

-17 -15 -13 -11 -9 O 2 4 6 8 10 12 14

Days (Pre- and Post-Inoculation)

Leukocytes +Lymphocytes * Neutrophils

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Group 5 - ~24515x7

-15 -13 -11 -9 -7. -5 -2 O 2 4 6 8 I O 12 14

Days (Pre- and Post-Inoculation)

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Group 6 - ~~24515x5

B5-32

V I I I i I I I I I I 1 I 1 I

.-Il -9 -7 -5 -3 O 2 4 6 8 10 12 14

Days (Pre- and Post-Inoculation)

-C Leukocytes -C Lymphocytes +Neutrophils

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Group 7 - ~ ~ 2 4 5 1 5 x 7

Al -02

20 -

'.,$ 1 5 - O F

x I O - tn - - a 5 - O

-27 -20 -16 -8 O 2 4 6 8 'IO 12 13

Days (Pre- and Post-Inoculation)

+ Leukocytes l l y r n p h o c y t e s Neutrophils

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Appendix 7 lndividual Animal Bone Marrow Cytology

Group 2 - p l l Q

A7018

Days (Pre- and Post-Inoculation) + Proliferation Pool +Maturation Pool

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APPENDIX 7 (con't) Individual Animal Bone Marrow Cytology Data

Group 3 - upl1Q

-10 -8 -6 -4 -2 2 4 6 8 10 12 14 Days (Pre- and Post-Inoculation)

Proliferation Pool +Maturation Pool

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APPENDIX ? (con't) Individual Animal Bone Marrow Cytology Data

Group 4 - p24515

82-22

-11 -9 -7 -5 -2 2 4 6 8 10 12 14 Days (Pre- and Post-f noculation)

Proliferation Pool +Maturation Pool

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Appendix 7 (con't) Individual Animal Bone Marrow Cytology

Group 5 - ~24515x7

O I 1 1 1 I I 1 1 1 1 1 1

-15 -13 -11 -9 -7 -2 2 4 6 8 I O 12 14 Days (Pre- and Post-Inoculation)

P r o l i f e r a t i o n Pool +Maturation Pool

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APPENDIX 7 (con't) Individual Animal Borie Marrow Cytology Data

Group 6 - up24515x5

Days (Pre- and Post-Inoculation) + Proliferation Pool +Maturation Pool