EVALUATION OF INJURY SEVERITY AND HEMATOLOGIC AND PLASMA BIOCHEMISTRY VALUES FOR RECENTLY CAPTURED...

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EVALUATION OF INJURY SEVERITY AND HEMATOLOGIC ANDPLASMA BIOCHEMISTRY VALUES FOR RECENTLY CAPTURED NORTHAMERICAN RIVER OTTERS (LONTRA CANADENSIS)Author(s): Kevin KimberD.V.M., M.S. and George V. Kollias IID.V.M., Ph.D., Dipl. A.C.Z.M.Source: Journal of Zoo and Wildlife Medicine, 36(3):371-384. 2005.Published By: American Association of Zoo VeterinariansDOI: http://dx.doi.org/10.1638/03-125.1URL: http://www.bioone.org/doi/full/10.1638/03-125.1

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371

Journal of Zoo and Wildlife Medicine 36(3): 371–384, 2005Copyright 2005 by American Association of Zoo Veterinarians

EVALUATION OF INJURY SEVERITY AND HEMATOLOGIC ANDPLASMA BIOCHEMISTRY VALUES FOR RECENTLY CAPTUREDNORTH AMERICAN RIVER OTTERS (LONTRA CANADENSIS)

Kevin Kimber, D.V.M., M.S., and George V. Kollias, II, D.V.M., Ph.D., Dipl. A.C.Z.M.

Abstract: As part of a reintroduction program, blood samples from free-ranging, recently captured Nearctic riverotters (Lontra canadensis) in eastern New York state were collected and analyzed to determine baseline hematologyand plasma biochemistry values for the source population, and to determine whether these values were significantpredictors of trap-injury status. Based on physical exam, each otter was classified as uninjured, moderately injured, orseverely injured. Clinical pathology parameters were compared across sex, age class, and injury classification. Theincrease in likelihood of a change in each parameter in injured versus uninjured otters was determined using logisticregression. Baseline hematology and plasma biochemistry values did not differ significantly from published values forcaptive otters in zoos or other reintroduction programs. Plasma aspartate aminotransferase levels increased as time fromcapture to venipuncture decreased. Some otters in this study showed clinical signs consistent with exertional myopathy,possibly altering our calculation of baseline values. Our results suggest that the hematology and plasma biochemistryvalues obtained in this recently captured population of otters are generally not good predictors of capture-related injury.This could be due to disease processes that are not readily visible upon physical examination or because changes inthese values may be associated with factors independent of capture-related injury.

Key words: River otter, Lontra canadensis, biochemistry, hematology, reintroduction, clinical pathology.

INTRODUCTION

The Nearctic river otter (Lontra canadensis)46

hereafter called river otter, was once found in vir-tually every watershed in North America.45 Habitatdegradation and loss, hunting, and overharvest forthe fur trade have extirpated river otters from muchof their range.25,37 Currently, river otter populationsare expanding their ranges in 20 states and consid-ered stable in 23 other states.9 This expansion hasbeen facilitated by at least 17 reintroduction pro-grams across the United States and one in Cana-da.20,33 For many successful reintroduction pro-grams, veterinary care was provided to the ottersprior to release,23,29,40 but some programs were suc-cessful without published reports of extensive vet-erinary care.8 More intensive medical monitoring,prerelease conditioning, and improved medical careof otters involved in reintroduction programs hasenhanced postrelease survival.16,23,37,40

Baseline hematology and plasma or serum bio-chemical profiles are essential for adequate healthassessment of otters involved in population en-hancement and reintroduction programs. Clinical

From the Wildlife Health Laboratory, Department ofClinical Sciences, College of Veterinary Medicine, Cor-nell University, Ithaca, New York 14853, USA (Kimberand Kollias). Present address (Kimber): Department ofPopulation Medicine and Diagnostic Sciences, College ofVeterinary Medicine, Cornell University, Ithaca, NewYork 14853, USA. Correspondence should be directed toDr. Kimber.

laboratory data for recently captured wild river ot-ters may differ from data for otters held in captivityfor extended periods, born and reared in captivity,or otters that have adjusted to novel surround-ings.38,43 Researchers studying river otters involvedin reintroduction programs obtained blood samplesfrom otters immediately after they were held incaptivity,38 while others did not collect samples for30 or more days following capture.6,16 Serum cre-atine phosphokinase (CK) levels have been inverse-ly correlated with number of days recently capturedotters were held prior to venipuncture.44 Otters un-accustomed to captivity may be exceedingly‘‘stressed’’ during the first few days in novel sur-roundings, so blood samples taken at this time mayproduce different hematology and blood chemistryvalues compared to blood samples from the sameotters after acclimatization to captivity.13 Publisheddata on hematology profiles and plasma or serumbiochemistries in otters generally are small inscale,16,21,37 do not separate injured from uninjuredanimals,6,16 and do not define ‘‘clinically normal’’for those individuals tested.26,38 The single excep-tion used four injury categories for 155 otters inNorth Carolina, USA, but did not test the predictivevalue of complete blood counts and plasma or se-rum biochemistry tests in determining the injurycategory of recently trapped otters using polyto-mous logistic regression techniques.44

The objectives of this study were to determinehematology and plasma biochemistry values fromrecently captured free-ranging river otters used in

372 JOURNAL OF ZOO AND WILDLIFE MEDICINE

a reintroduction program; examine differences inhematology and plasma biochemistry values be-tween otters with significant trap injuries and otterswith minimal or no injuries; examine the effect ofage, sex, and amount of time from capture to ve-nipuncture on hematology and plasma biochemistryvalues in river otters; and compare data on hema-tology and plasma biochemistry values for thisgroup of otters with values for otters publishedelsewhere.

MATERIALS AND METHODS

Otters were captured in eastern New York (pri-marily the Adirondack and Catskill mountains)from September through November of 1995through 2000. Trapping, transport, and captivemaintenance protocols were approved by CornellUniversity’s Institutional Animal Care and UseCommittee guidelines. Otters were held at the NewYork State College of Veterinary Medicine at Cor-nell University, Ithaca, New York (NYSCVM), orthe Seneca Park Zoo, Rochester, New York (SPZ),for at least 3 days prior to release.

Captured otters were placed in specially con-structed transport containers as described previous-ly22,23 and transported in the back of enclosed trucksor small airplanes to the holding and treatment fa-cilities. Water was available ad lib., and cages werecovered with an opaque cloth to minimize stressduring transport.

Upon admission to the holding facility, otterswere transferred from the transport cages to a smallanimal squeeze cage (Model TC-1000A, ResearchEquipment Co., Bryan, Texas 77803, USA),weighed, and visually inspected for evidence of in-juries requiring emergency treatment. If otters didnot need emergency medical care, they were trans-ferred into a stainless-steel primate-type holdingcage (Hoeltge, Inc., Cincinnati, Ohio 45202, USA;36 inch 3 36 inch 3 36 inch) and provided water,food, and a fresh towel. Cages were located in-doors, and lights were set on a timer to match theseasonal photoperiod. Temperature was maintainedbetween 608F and 708F.

Otters were housed singly and fed 1 kg of freshlake trout (Salvelinus namaycush) steaks eachday.23 Small otters were fed twice daily. Otters re-mained in captivity for 3–70 days (x 5 22.3 6 14.0days, median 5 17 days, n 5 93). Seven otters werein captivity for an undisclosed period prior to sam-pling for this study.

Oral medications were embedded in small cubesof fish muscle. All otters received a standardizedset of pharmaceuticals, nutritional supplements, andbiologics as reported previously.23

Fresh fecal samples were obtained within 1–3days of admission and were analyzed within 24 hrof collection for intestinal parasites (using ZnSO4

flotation), anaerobic and aerobic bacterial culture,and Clostridium perfringens Type-A enterotoxin(reverse passive latex agglutination test) by theNew York State Veterinary Diagnostic Laboratory(NYSVDL) at Cornell University. Animals with fe-cal flotations positive for intestinal helminths re-ceived fenbendazol (Panacur, Hoechst-RousselPharmaceuticals, Inc., Somerville, New Jersey10887-1258, USA; 50 mg/kg, s.i.d., p.o.) for 5days.

To facilitate physical examination and placementof a 14-mm intradermal identifying chip (AVID,Inc., Norco, California 91760, USA), otters werefasted for 10 hr and immobilized as previously de-scribed.23 In addition, 13 otters received surgicallyplaced intra-abdominal radiotransmitters.14

Animal ages were estimated by tooth wear andbody size.45 Otters estimated to be more than 2 yrold were considered adults.

Blood was collected via external jugular veni-puncture within 7.8 6 8.6 days (range 1–64 days,n 5 97) of arrival at the holding and treatment fa-cility, and placed into EDTA tubes for completeblood counts (CBC) and Knott’s Microfliaria Test,34

serum clot tubes for viral serology, and lithium hep-arin tubes for plasma biochemistries (Vacutainer,Beckton Dickinson, Franklin Lakes, New Jersey07417-1885, USA). Five otters were sampledtwice; these 10 samples were not included in thecalculation of normal values, and the five repeatsamples were not included in the regression anal-ysis of injury severity. One sample was taken post-mortem and not included in the analyses.

The heparinized plasma was separated within 2hr of sample collection, and all tests were per-formed within 24 hr of sample collection.42 He-matology profiles and plasma chemistries fromsamples taken at Cornell were performed at theClinical Pathology Laboratory, NYSCVM, using aCoulter S 1 IV hematology analyzer (BeckmanCoulter, Inc., Fullerton, California 92834-3100,USA) and a Hitachi 911 chemistry analyzer (RocheDiagnostics Corp., 9115 Hague Road, Indianapolis,Indiana 46250-0457, USA). Samples taken at SPZwere analyzed at Veterinary Laboratory Services ofRochester, 825 Spruce Boulevard, Rochester, NewYork 14623, USA, using a Cell-Dyne 3500 (AbbottLaboratories, 100 Abbott Park Road, Abbott Park,Illinois 60064-3500, USA) for CBC and a COBAS-MIRA biochemistry analyzer (Roche DiagnosticsCorp.). In 1995 and 1996, blood smears were madefrom EDTA-preserved blood shortly after collec-

373KIMBER AND KOLLIAS—OTTER HEMATOLOGY AND PLASMA BIOCHEMISTRY

tion, air-dried, stained with Wright’s Geimsa stain,and examined at 3100 magnification for presenceof microfilaria. A modified Knott’s microfilaria testwas used to determine presence of potential micro-filaria antigens.23 Capture-related wounds werecleaned, flushed with dilute betadine solution in sa-line, debrided, and sutured where appropriate. Post-operative assessment of wound sites was based onthe ability of the otter to use the injured leg or footand by examination of more-severe injuries duringa second immobilization.

After sampling and treatment, otters were movedto a prerelease acclimation facility.22

An otter’s capture-related injury was classified as‘‘severe’’ if it met any of the following criteria: 1)two or more surgeries were required to correct ortreat the problem, 2) amputation of a digit or digitswas required, 3) holding time for medical treatmentexceeded 30 days, 4) the animal was physically de-pressed or showed signs of central nervous systemdepression, or 5) the animal died before release. Aninjury was classified as ‘‘moderate’’ if it met anyof the following criteria: 1) surgery or wound carewas required, 2) holding time for medical treatmentexceeded 21 days, or 3) recent and significant den-tal injury occurred (e.g., loss of more than threeteeth). Injuries were classified as ‘‘minor or insig-nificant’’ when no surgery was required and hold-ing time for medical treatment did not exceed 21days.

Complete necropsies with histopathologic ex-amination of multiple tissues including muscle,lung, liver, and large and small intestines were per-formed at NYSCVM department of pathology onfive otters that died after admission.

Individual samples were selected for the estab-lishment of ‘‘normal’’ values and comparison ofnormal age and gender groups based on the follow-ing cohort inclusion criteria: 1) lack of clinicalsigns of systemic illness such as respiratory dis-tress, diarrhea, lethargy, abscesses, purulent dis-charge from a capture-related wound site or bodyorifice, hyperthermia, hypothermia, dehydration,heavy endoparasite infestation (assessed by fecalflotation) or death; 2) lack of pregnancy or lactation(determined by palpation); 3) blood sampling oc-curred only once; 4) known time interval from cap-ture to sampling was between 1 and 31 days; and5) blood sampling occurred prior to receipt of anytreatment other than the standard admission proto-col as described elsewhere.23

A two-tailed t-test compared each normally dis-tributed (Shapiro–Wilk test of normality) hematol-ogy and plasma biochemistry values for adults ver-sus juveniles and males versus females from clini-

cally normal otters. For two-group comparisons ofnon-normally distributed values, a Wilcoxon ranksum test was used.39 All two-group comparisonswere followed by a Bonferroni multiple-compari-sons adjustment.35 For multiple comparisons, theKruskall–Wallis rank sums test was used.39 A Tu-key–Kramer multiple-comparisons adjustmentbased on the number of categories compared wasfollowed by a Bonferroni multiple-comparisons ad-justment based on the number of times the Krus-kall–Wallis rank sums test was applied to the datain order to control for group-wide Type-1 errors.35

Third-order terms (i.e., permutations of age, sex,and injury classification) were not included in mod-els due to difficulty in interpretation and limitedsample size.17 The number of pairwise and multiplecomparisons was limited a priori because of highcorrelations with another variable using Spearman’srank correlations.17 For variables with correlationsgreater than 0.8, inclusion criteria were based on 1)ease of measurement of the parameter in the field,2) ease of treatment commonly required to correctthe parameter in the field, 3) biologic plausibilityof the relationship to injury level, and 4) breadthof clinical utility. Therefore, erythrocyte count(RBC), hemoglobin (Hgb), unbound iron-bindingcapacity (UIBC), percent saturation of tranferrin(SAT), WBC, mean corpuscular hemoglobin(MCH), Cl, Na/K ratio, aspartate aminotransferase(AST), and indirect bilirubin (IBILI) were not com-pared across age, sex, or injury level, and were notincluded in regression analyses. Magnesium levelswere not included in regression analyses becauseonly 22 samples were measured for this element.Significance was accepted at P , 0.10 and alpha 50.10, except where noted otherwise. (SAS, SAS In-stitute Inc., Cary, North Carolina 27513, USA).39

Three simple linear regressions examined num-ber of days from capture to venipuncture versusplasma AST, CK, and glucose level. A multiple lin-ear regression examined plasma CK versus thenumber of days from capture to venipuncture, AST,blood urea nitrogen (BUN), creatinine, glucose, andK.47

Dichotomous ordinal logistic regressions of in-jury class determined the likelihood of injury givena change in one unit of each regression parameter.Age, sex, and time from capture to venipuncturewere taken into account as confounders.1 For logis-tic regressions, moderately or severely injured ot-ters were stratified into one class, which was named‘‘Injured,’’ and otters with minor or no injurieswere classified as ‘‘Uninjured.’’ Independent vari-ables for logistic regression included each variablein the biochemistry and hematologic profiles, and


the dependent variable was binary injury status.Backward, forward, and step-wise variable-selec-tion techniques were used for each model.17 Rela-tive-risk ratios were calculated for regressions thatindicated a significant predictive value for a specifichematology or blood-chemistry parameter.17

Two otters (identification number CRK4-96 andDK3-96) were included in simple linear regressionanalyses of time from capture to venipuncture andAST and CK but were removed as outliers fromlogistic regression analyses of injury status.

RESULTS

The range, median, 2.5th and 97.5th percentilesfor hematology and plasma biochemistry values forclinically normal river otters in this study areshown in Table 1. No microfilaria were seen under3100 examination of blood smears from 53 otters,and Knott’s tests were negative for all 53 otterssampled. No group among the clinically normal orthe injured otters was more or less likely to haveicteric, hemolyzed, or lipemic samples than anyother group.

Samples collected prior to treatments were dif-ferent from samples collected after treatments forseveral complete blood count and biochemistrytests (Table 2).

There were no statistically significant differencesbetween clinically normal males and females. Cre-atinine was the only variable that showed a statis-tically significant difference between clinically nor-mal adults (n 5 54, median 5 17.68 mmol/L, range8.84–35.36 mmol/L) and juveniles (n 5 12, median5 8.84 mmol/L, range 8.84–17.68 mmol/L) (P 50.0008). There were no statistically significant dif-ferences among subsets of these age and sex cate-gories for clinically normal otters.

Some severely injured otters showed little or noalteration in hematology and plasma biochemistryvalues while other—apparently uninjured—ottersshowed significantly altered values. Values werenot significantly different across each of the threeinjury categories. When stratified into a singlegroup, clinical pathology values of moderately andseverely injured otters were not statistically differ-ent than values for mildly injured otters. Compari-sons of age- and sex-stratified subsets of injuredotters showed statistically significant differences inALT, CK, and Cl among the groups (Table 3). Mostvariation in CK and ALT was attributed to one ju-venile female otter (otter CRK4-96) that was sam-pled within 24 hr of capture and had significantlyelevated levels of these enzymes. All other testsshowed no statistically significant differences based

on injury, sex, age, or any second-order combina-tion of these factors.

Creatine kinase levels (Fig. 1a) and AST levels(Fig. 1b) were reduced in otters that spent a longertime in captivity prior to venipuncture, but this re-lationship was not as strong for glucose levels (Fig.1c).13 When days from capture to venipuncturewere included in a multiple linear regression modelusing ln(BUN), creatinine, glucose and K (enzymesand inorganic analytes commonly affected in clin-ical exertional myopathy [EM]),47 the model ac-counted for only 25.06% of the variation in CK.Creatine kinase and AST were too highly correlatedto include AST in the multiple linear regressionmodel and still assess the impact of the other var-iables. There were no significant differences in thetime from capture to venipuncture based on age,sex, or injury category.

When plasma enzymes commonly associatedwith muscle degeneration (ALP, ALT, CK, andGGT)42 were used to construct a model, femaleswere significantly more likely to be injured thanmales (P 5 0.0022; odds ratio 5 4.51; 95% con-fidence interval 5 1.72–11.83), and adults weremore likely to be injured than juveniles (P 50.0264, odds ratio 5 4.44, 95% confidence inter-val 5 1.19–16.52). Backward, forward, and step-wise variable selection techniques identified thesame variables as significant within the model. Nocombination of these plasma enzymes was astrong predictor of injury status in the otter, thusonly age and sex remained in the model as signif-icant predictors.

Backward selection of parameters that are com-monly altered within days of acute blood loss, softtissue damage, wound infection, and trauma, suchas might occur with capture by toehold traps (he-matocrit, segmented neutrophil count, lymphocytecount, total protein, Fe, and red cell distributionwidth [RDW])42 showed Fe (P 5 0.0261), lympho-cyte count (P 5 0.0276), and RDW (0.0365) valueswere reduced in injured otters after controlling forage, sex, time from capture to venipuncture, he-matocrit, segmented neutrophil count, and total pro-tein levels (Fig. 2a). Removal of otters receivingamoxicillin or subcutaneous fluids (which alteredlymphocyte count) produced a model in which onlylymphocyte count, (P 5 0.0412) and RDW (P 50.0500) were reduced in otters with moderate orsevere injuries.

Using CK, AST, BUN, creatinine, glucose, andK as predictors (parameters used to assess the oc-currence of EM in other mammals)47 showed BUN(P 5 0.022) and glucose (P 5 0.033) were reducedin injured otters, and females were 4.43 (95% con-


Table 1. Hematology and plasma biochemistry values from recently captured clinically normal North Americanriver otters (Lontra canadensis) involved in a reintroduction program.

Parameter n Median 2.5–97.5% Range

Leukocyte (109 cells/L) 53 7.6 7.53–9.68 3.2–19.2Segmented neutrophils (109 cells/L)Band neutrophils (109 cells/L)Lymphocytes (109 cells/L)Monocytes (109 cells/L)Eosiniphils (109 cells/L)Basophils (109 cells/L)

535353535353

5.20.01.40.40.20.0

5.16–7.080.0–0.09

1.34–1.790.41–0.600.27–0.48

0.002–0.02

1.1–15.80.0–1.20.3–3.50.1–1.70.0–1.50.0–0.1

Erythrocyte (1012 cells/L)Nucleated red blood cells (109 cells/L)a

Hemoglobin (g/L)HematocritMean corpuscular volume (fL)

532

535353

7.91.0

118.035.044.0

7.56–8.37NA

113.0–125.133.45–36.9843.61–45.05

4.5–14.30.0–1.0

70.0–216.020.0–65.040.0–51.0

Mean corpuscular hemoglobin (pg)Mean corpuscular hemoglobin concentration (g/L)Red cell distribution widthb

Platelet count (109 cells/L)Mean platelet volume (fL)

5353525050

15.0340.0

16.4522.5

7.55

14.82–15.31334.7–343.516.23–16.82461.0–550.0

7.36–7.94

13.0–18.0292.0–360.0

14.7–20.0272.0–967.0

5.9–12.2Total iron binding capacity (mmol/L)Unbound iron binding capacity (mmol/L)Percent saturation of transferrin (%)Fe (mmol/L)Ca (mmol/L)

6141626267

71.2844.4239.528.12

2.22

69.35–78.1737.51–52.9137.35–48.2026.39–32.49

2.16–2.24

38.51–130.560.0–124.655.0–100.0

5.91–66.981.87–2.50

P (mmol/L)Na (mmol/L)K (mmol/L)Na/K ratioCl (mmol/L)HCO3 (bicarbonate)(mmol/L)

676767666762

1.97150.0

4.1037.0

114.025.0

1.90–2.13149.01–150.64

4.07–4.3734.98–37.40

112.77–114.8423.82–25.47

1.03–3.97141.0–157.0

3.3–6.323.0–45.0

100.0–123.016.0–33.0

Anion gap mmol/L)c

Mg (mmol/L)Alanine aminotransferase (IU/L)Aspartate aminotransferase (IU/L)Alkaline phosphatase (IU/L)

6221676767

15.01.0

180.0134.0153.0

14.53–16.890.98–1.03

195.04–276.12195.81–358.19147.47–208.80

6.0–29.00.9–1.2

57.0–851.043.0–1,695.041.0–591.0

Gamma glutamyl transferase (IU/L)Creatine kinase (IU/L)Amylase (IU/L)Direct bilirubin (mmol/L)Indirect bilirubin (mmol/L)

6767576666

13.0627.0

4.01.713.42

13.63–18.79856.74–1,874.20

4.91–9.060.086–1.20

3.76–5.64

5.0–59.0105.0–9,996.0

1.0–49.00.0–3.420.0–23.95

Total bilirubin (mmol/L)Cholesterol (mmol/L)Total protein (g/L)Globulin (g/L)Albumin (g/L)Albumin/globulin ratio

666769676766

5.134.84

69.034.034.0

1.00

4.79–6.844.77–5.1867.7–69.13.33-35.533.1–35.40.96–1.10

1.71–27.373.21–7.1757.0–83.019.0–4.6024.0–49.00.63–2.58

Urea nitrogen (BUN) (mmol/L)Creatinine (mmol/L)Glucose (mmol/L)

676667

14.2417.68

5.938

13.54–15.4315.03–18.565.403–6.159

6.55–27.788.84–35.36

1.610–9.713

a Two of 76 animals examined showed nucleated red blood cells.b Red cell distribution width 5 (standard deviation of mean corpuscular volume/mean corpuscular volume) 3 100.c Anion Gap 5 (Na 1 K) 2 (Cl 1 HCO3).d NA, not applicable.


Table 2. Complete blood count and plasma biochemistry values affected by treatments administered to North Amer-ican river otters involved in a reintroduction program.

Treatmenta Parameter Mean untreated (n) Mean after treatment (n) P-value

Trimethoprim-sulfab CaCreatinineTotal iron binding

capacity

3.36 mmol/L (29)23.52 mmol/L (29)

61.35 mmol/L (22)

3.55 mmol/L (69)15.12 mmol/L (68)

75.61 mmol/L (68)

0.0016c

0.0003d

0.0006d

Clostridium perfringens

Antitoxine HCO3

KNa : K

25.47 mmol/L (43)4.07 mmol/L (43)

37.42 (43)

23.21 mmol/L (48)4.47 mmol/L (54)

34.12 (53)

0.0012d

0.0019d

0.0014c

Ivermectinf

Amoxicilling

Fenbendazolh

KEosiniphil countLymphocyte countAnion gap

4.00 mmol/L (28)0.478 3 109 cells/L (64)1.506 3 109 cells/L (64)14.61 mmol/L (57)

4.41 mmol/L (60)0.188 3 109 cells/L (17)0.872 3 109 cells/L (17)18.47 mmol/L (34)

0.0007d

0.0018d

0.0001d

0.0001c

Subcutaneous fluidsHCO3

Lymphocyte count25.32 mmol/L (57)

1.30 3 109 cellsi (71)22.53 mmol/L (34)

0.72 3 109 cellsi (10)0.0001c

0.0004j

a No change in values was seen for vitamin B injections, vitamin E/selenium injections, oral metronidazole tablets, oral enrofloxacintablets, oral electrolyte solution, or flunixin injections.

b 20 mg/kg, b.i.d., p.o. for 1–2 weeks.c t-test assuming equal variances.d t-test assuming unequal variances.e Type C and D, equine origin, 3 ml, s.c.f 1% sterile solution, 0.4 mg/kg, s.c.g 11 mg/kg b.i.d., p.o. for 1–2 weeks.h 50 mg/kg, s.i.d., p.o. for 5 days.i Because this is a nonparametric test the median value is listed, not the mean.j Wilcoxon rank sum test.

fidence interval 1.543, 12.72) times more likely tobe injured, after controlling for age, CK, AST, timefrom capture to venipuncture, creatinine, and K(Fig. 2b).

When a backward-selection technique was ap-plied to all significant variables from all threemodels, females were 5.46 (95% confidence inter-val 1.347, 22.11) times more likely to be injuredthan males, and BUN and lymphocyte count weresignificantly inversely correlated with injury (P 50.0297 and P 5 0.0148, respectively), after con-trolling for age, time from capture to venipunc-ture, Fe, RDW, and glucose (Fig. 2c). Removal ofotters receiving subcutaneous fluids or amoxicillinfrom the model eliminated sex, lymphocyte count,and BUN as significant predictors of injury classand rendered the model essentially useless at pre-dicting injury class, with the overall model ac-counting for only 7.8% of the variation in injuryclass.

Twenty-six of 65 otters were positive for Clos-tridium perfringens or other bacteria on fecal cul-ture, and 37 of 65 otters were positive for Creno-soma goblei or other enteric helminthes on fecalflotation.

DISCUSSION

Discussion of logistic regression analysesoverall

Because four logistic regressions were run, andnone indicated statistically significant, confound-ing-free effects on injury classification, it appearsthat hematologic profiles and plasma biochemistryvalues in otters are in general poor predictors ofcapture-related injury. A recently captured wild riv-er otter may show severe injuries from capture, yethematologic profiles and plasma biochemistry val-ues may be completely normal. Conversely, an otterthat appears to have no injuries may have signifi-cant abnormalities in its hematologic and plasmabiochemistry profile.

While every attempt was made to minimizestress to the otters involved in this project, it isimpossible to discount the effects of stress associ-ated with capture, restraint, transport, and healthevaluation on these animals. Chronic stress can in-crease neutrophil counts and reduce lymphocyteand eosiniphil activity; impair humoral immune re-sponse; and cause hypertension, poor wound heal-ing, and increased susceptibility to bacterial infec-


Table 3. Age- and sex-based differences in hematology and plasma biochemistry values from recently capturedclinically normal North American river otters (Lontra canadensis) involved in a reintroduction program.

n Mean Range

Alanine aminotransferase (IU/L)

Mild injuryFemaleMaleAdultJuvenile

5320334112

211.8a

181.2b

230.3c

227.9d

156.6e

46–88946–48456–88946–88972–355

Moderate injuryAdult

Severe injuryFemaleAdultJuvenile

1816231420

3

305.9328.6f

354.3a

374.5b,c

236.2g

1,141.7d,e,f,g

96–1,29196–1,29194–2,61594–2,61594–851

208–2,615

Creatine kinase (IU/L)

Mild injuryFemaleMaleAdult

53203341

1,080.2h

1,195.2i

1,010.5j

953.2k

179–9,789204–9,789179–5,717179–5,717

Juvenile 12 1,513.8l 209–9,789Moderate injury

FemaleMaleAdultJuvenile

1814

416

2

1,837.6m

2,12.9n

874.3o

1,962.6p

837.5q

105–11,253105–11,253260–2,146217–11,253105–1,570

Severe injuryFemaleAdultJuvenile

221419

3

3,727.0h,m

4,736.5i,j,l,n,o

1,565.5r

17,417.0k,l,p,q,r

161–48,240161–48,240161–9,996

1,881–48,240

Cl (mmol/L)

Female adultFemale juvenile

3711

116.1s

108.1s

103–129100–115

a,g,h,i,j,k,l,p,r P , 0.0001.b,c P 5 0.0002.d,e P , 0.0001.f P 5 0.0023.m P 5 0.0006.n P 5 0.0013.o P 5 0.0022.q P 5 0.0005.s P 5 0.0016.

tions.10 Such changes in response to stress are oftenassociated with continuous stimulation of the ad-renal cortex and excessive production of cortisol.10

Other reports suggest that five captive river otterssuccumbed to a syndrome named ‘‘shock disease’’within 2 days of transfer to a new exhibit.4 Thesefive otters were well nourished at the time of death,but gross pathology showed hyperplastic lymphoidtissues, adrenal glands were small with the middleand inner zones showing hypoplasia or atrophy, liv-ers were dark and small, and glycogen stores in theliver were exhausted.4 Examination of the plasma

biochemical profile may provide some indication ofhow stressed an otter is, since severity of visiblecapture-related injury alone does not appear to bestrongly related to many plasma biochemical pa-rameters.

Weaknesses of the approach used in this studyinclude a lack of standardization with respect totiming of sampling vis a vis treatments, fecal flo-tation, fecal culture, or fecal Clostridium perfrin-gens exotoxin results. Future related studies shouldendeavor to systematize these variables and data forcontrol during the analysis phase of the projects.


Figure 1. A 5 regression line, B 5 95% predictioninterval, C 5 95% confidence interval. a. Natural loga-rithm of plasma creatine kinase concentration (IU/L) (CK)in 86 recently captured, free-ranging North American riverotters (Lontra canadensis) as a function of number of daysbetween capture and venipuncture. 1 5 otter CK value.b. Natural logarithm of plasma aspartate aminotransferase

←

concentration (IU/L) (AST) in 84 recently captured, free-ranging North American river otters (Lontra canadensis)as a function of number of days between capture and ve-nipuncture. 1 5 otter AST value. c. Plasma glucose con-centration (mmol/L) in 95 recently captured, free-rangingNorth American river otters (Lotra canadensis) as a func-tion of number of days between capture and venipuncture.1 5 otter glucose value.

Comparison of pretreatment and posttreatmentsamples

Pretreatment samples were different from post-treatment samples for a few of the 52 completeblood count and plasma biochemistry values (Table2), but few biologically plausible explanations forthese differences exist. Trimethoprim sulfadiazine(TMS) was associated with an increase in plasmaCa levels, a decrease in plasma creatinine levels,and an increase in total iron-binding capacity(TIBC). TMS is excreted unchanged renally viaglomerular filtration and tubular secretion and isalso metabolized by the liver, thus the suite ofchanges noted here do not follow a noticeable pat-tern. It is possible that the TMS affected glomerularfiltration by creating a higher osmotic draw acrossthe glomerulus than normal, thereby reducing plas-ma creatinine levels. Also, the TMS may have re-duced diarrheic signs in the otters—thereby reduc-ing loss of dietary Ca from the animal—reflectedin a higher plasma Ca level. Finally, the TMS mayhave been associated with reduced diarrhea—thusfacilitating intestinal absorption of dietary Fe—re-sulting in an increase in TIBC.

Clostridium perfringens antitoxin was associatedwith a decrease in HCO3, an increase in K, and aconcomitant decrease in Na:K ratio. These effectsmay be due to an antidiarrheal effect of the anti-toxin resulting in reduced secretion of HCO3 intothe intestinal lumen. The mode of action of Clos-tridium spp. exotoxin is to bind to the intestinalepithelial cells, increase membrane permeability,and decrease synthesis, resulting in fluid and ionsecretion and epithelial cell death and sloughing.12

Thus, with the antitoxin present, the otters likelyhad reduced epithelial cell damage from Clostridi-um exotoxin, and plasma ion concentration alter-ations consistent with a reduction in diarrheic signswere seen. Similar changes in plasma HCO3 wereseen with fenbendazol administration, possibly dueas well to a reduction in diarrheic signs associatedwith nematode infection.

Otters receiving amoxicillin had reduced eosin-ophil and lymphocyte counts, perhaps in response


Figure 2. a. Relationship between probability of in-jury and concentrations of plasma Fe (mmol/L) (P 50.0261), lymphocyte count (109 cells/L) (P 5 0.0276, andred cell distribution width (P 5 0.0365) in recently cap-tured otters after controlling for age, sex, days from cap-ture to venipuncture, hamatocrit, segmented neutrophilcount (109 cells/L), and total protein (g/L). Red cell dis-tribution width 5 (standard deviation of mean corpuscularvolume/mean corpuscular volume) 3 100. b. Relationship

←

between probability of injury and concentrations of bloodurea nitrogen (mmol/L) (P 5 0.022) and plasma glucose(mmol/L) (0.033) in recently captured otters after con-trolling for age, plasma creatine kinase (IU/L), plasma as-partate aminotransferase (IU/L), days from capture to ve-nipuncture, plasma creatinine (mmol/L), and plasma K(mmol/L). c. Relationship between probability of injuryand concentrations of blood urea nitrogen (mmol/L) (P 50.0297) and lymphocyte count (109 cells/L) (P 5 0.0148)in recently captured otters after controlling for age, daysfrom capture to venipuncture, plasma Fe (mmol/L), redcell distribution width, and plasma glucose (mmol/L). Redcell distribution width 5 (standard deviation of mean cor-puscular volume/mean corpuscular volume) 3 100.

to reduced immunologic challenge. Alternatively,leukopenia has been reported in hypersensitivity re-actions to penicillins,32 and several otters in thetreatment group were more severely leukopenicthan otters in the nontreatment group, althoughthese differences were not statistically significant(P , 0.10). Finally, reductions in circulating lym-phocytes may be in response to increased endoge-nous corticosteroid secretion caused from the stressassociated with capture and transport (i.e., ‘‘stressleukogram’’), and a higher percentage of the 17 ot-ters receiving amoxicillin were more likely to havesevere or moderate injuries, although this trend wasnot statistically significant (P , 0.10).

Subcutaneous fluids were administered to 10 ot-ters, and otters receiving fluids had a reduced lym-phocyte count. As with the amoxicillin, the lym-phopenia could be the result of stress associatedwith capture and transport. However, hemodilutioncould also explain this result.

Comparison with published values

Although this study used plasma biochemistryvalues, the hematology and plasma/serum biochem-ical reference ranges determined here for river ot-ters are similar to reference ranges reported earlierfor this species.6,16,38,44 With the exception of plasmaK, ionized Ca, and albumin levels, plasma bio-chemistry and serum biochemistry values are sim-ilar in domestic canids, thus comparison of our datawith published data for serum values may be rea-sonable in mustelids.3 Our values do not accountfor subclinical disease, instead they present valuesfrom recently captured wild river otters. Hand-raised otters or long-term captive otters probablydo not harbor persistent internal parasites, do nothave recent injuries from toehold traps, are not innovel surroundings, and are not subjected to ad-verse weather conditions as is often the case with


wild trapped otters. Also, method of chemical re-straint, administration of antibiotics, anthelmin-thics, vitamin supplementation, or other treatmentsmay affect hematology and plasma biochemistryvalues in otters.41

Histopathologic findings of skeletal muscle fromseveral otters in this study were consistent withEM13 and suggest that at least three of five otterswith elevated plasma CK, AST, ALT, ALP, or GGThad significant muscle exertion or EM associatedwith capture and transport. There are no reports ofelevated AST levels in North American river otters.However, elevated serum glutamic-oxalacetic trans-aminase (SGOT, analogous to AST) levels havebeen reported as a result of ‘‘muscle exertion’’47 inclinically normal sea otters.48 The SGOT values inclinically normal sea otters did not differ from theAST values for otters in our study and in otherstudies,16,38 therefore otters could have high ASTlevels normally. Alternatively, muscle contractionand fasciculations associated with ketamine, and in-tramuscular injection of the immobilization agentcould artificially increase AST values.2,11

Age-related differences in hematology measure-ments have been reported in North American ot-ters,44 and age- and gender-related differences havebeen reported for European otters (Lutra lutra).29

Trap-injury data from North American river ottershave shown age- and gender-related differences ininjury severity as well.44 Also, WBC counts, neu-trophil counts, and serum Ca levels have been el-evated in recently trapped river otters with closedluxations versus other injuries. Serum Ca levels canbe decreased for river otters with open luxations orfractures.44 In free-ranging sea otters (Enhydra lu-tra), method of capture and method of restraint didnot significantly affect hematology values, but sig-nificant gender- and age-related differences in he-matology measurements were reported.49,50 Elevatedplasma glucose levels in a giant otter (Pteronurabrasiliensis) and southern river otters (Lontra lon-gicaudus) were probably associated with stress pri-or to anesthesia, or to direct effects of the immo-bilizing agent.5 Among these South American ot-ters, elevated levels of SGOT were the suggestedresult of capture stress and transport.5 Thus, it ap-pears that age, sex, capture and restraint technique,and stress may have a significant effect on the he-matology profiles and plasma or serum biochem-istry of otters.

Baseline values for BUN in this study wereslightly higher than other reports,16,19,44 but this dif-ference was still within one standard deviation ofour mean value. Plasma BUN could have been el-evated in this study because the otters were on a

high protein (45% dry weight), high fat (55% dryweight) diet of lake trout.23,28 Our data also providea slightly lower reference value for creatinine thanother studies,6,16,19,44 but most of our values are stillwithin the range of previous studies for recentlycaptured, wild river otters.38,44 Serum or plasma cre-atinine is less likely to be influenced by diet orcatabolism of body tissues than BUN levels.28

Glucose levels in blood from otters in our studycould be artificially reduced because there was aslight (up to 2 hr) time delay between venipunctureand plasma separation. None of the otters involvedin our project showed an increase in glucose com-parable to the increase in glucose found in two Am-azonian otter species (P. brasiliensis and L. longi-caudus),5 and no other study of river otters or seaotters showed such extreme glucose values. Alter-natively, and less likely, captive care and immobi-lization techniques were heavily focused on reduc-ing stress in the otters, and these efforts could haveresulted in a lower median glucose level for thepopulation.

Wide variation in plasma CK levels and plasmaK concentrations has been reported in river ot-ters.16,38,44 Significant increases in blood haptoglo-bin, interleukin-6 ir (IL-6ir), AST, ALT, and CKwere found in 12 river otters exposed to oil fromthe Exxon Valdez spill in Alaska, USA.7 The serumvalues from live-trapped river otters from PrinceWilliam Sound were higher for CK, AST, and he-moglobin and lower for ALT than the correspond-ing plasma values from most otters sampled in ourstudy.7 Differences in the genetic make-up, diet,habitat, and exposure to oil in river-otter popula-tions from Prince William Sound versus easternNew York state probably contribute to the observeddisparity between the two groups. Otters were sam-pled immediately after capture in Prince WilliamSound,7 while we sampled otters as late as 33 daysafter capture. Details of capture techniques, re-straint techniques, and blood-sampling techniquesfrom Prince William Sound were not given,7 sothese cannot be ruled out as potential causes for thedifferences from our plasma chemistry values. Cre-atine kinase levels may also increase if multiplevenipuncture attempts are made prior to successfulphlebotomy, thereby increasing local tissue dam-age. Parasite burden and time from capture to ve-nipuncture also could alter these values. Most otterssampled in our study were being treated with TMSor metronidazole at the time of venipuncture, andhad received s.c. injections of vitamin E/Seleniumand B vitamins upon admission, so these medica-tions could have contributed to differences in plas-ma biochemistry values. All otters received TMS.


Metronidazole administration did not significantlyalter the result of mean plasma biochemistry values(Table 2). These s.c. injections are also unlikely toaffect CK, ALT, or AST levels, although intramus-cular injections used for immobilization could ar-tificially increase CK or AST.28

In most studies, blood was routinely collectedfrom river otters after more than 2 wk in captivity.For the population of otters involved in this study,plasma AST and CK levels declined rapidly be-tween 5–7 days postcapture,13,44 but more strin-gently controlled enzyme kinetic studies in river ot-ters are needed to accurately assess the costs andbenefits associated with timing of blood-samplecollection. Because complete blood count and plas-ma biochemistry values will be different dependingon how close to capture sampling occurs in recentlycaptured otters, our results concur with other stud-ies in supporting the practice of holding otters in-volved in reintroduction programs for a period priorto anesthesia and to release in order to fully assessany disease processes that may not be clinically ev-ident until days after capture and transport.44

Comparison of clinically normal age andsex subsets

Clinically normal juveniles differed from clini-cally normal adults in creatinine values. Clinicallynormal juveniles may have been less likely to havesubclinical disease, or they may have consumedless high-protein food than their adult counterparts.Blood creatinine values are affected minimally bydiet and exercise, but creatinine is formed in themetabolism of muscle, and therefore could be ele-vated after significant muscle exertion or high-pro-tein intake. However, creatinine is generally not re-absorbed in the renal tubules, and the measurementmethod used for creatinine (kinetic Jaffe reaction)can be affected by noncreatinine chromagens suchas acetoacetic acid, glucose, cephalosporin antibi-otics, and bilirubin (Tracy Stokol, pers. comm.).There have been no reports of differences in plasmacreatinine between adult and juvenile otters, butfractional clearances of creatinine have been welldocumented in this species.15 In other mustelids,controlled experiments have shown alterations inserum creatinine levels during the acute phase ofexposure to petroleum products,27 and endogenouscreatinine clearance has been significantly higher infeeding versus fasting Asian small-clawed otters(Aonyx cinerea) with Ca oxalate urolithiasis, al-though plasma creatinine levels for the Asian-smallclawed otters were not reported.31 Thus, there islittle theoretical or empirical evidence to suggest a

mechanism for the observed difference betweenadults and juveniles in our data set.

Logistic regression analyses

Muscle enzymes: Because sex and age were theonly significant predictor variables in a model usingplasma muscle enzymes (AST and CK) and ALT,elevated plasma muscle enzymes and ALT alone orin concert may not be good indicators of muscu-loskeletal injury in these otters, most likely becauseplacement of an otter into an injury class was notlimited to skeletomuscular injury, but includedproblems like Clostridium perfringens exotoxin.

Parameters commonly associated with anemia,injury, or blood loss: Changes in lymphocytecount, Fe, and RDW were highly correlated with achange in likelihood that an otter was injured, whenvariation due to hematocrit, segmented neutrophilcount, total protein concentration, age, and sex wastaken into account. Iron levels are likely to be lowerin animals that have lost significant amounts ofblood.28 Chronic parasitism can cause anemia, andmost river otters in this study were positive for in-testinal or pulmonary parasites using fecal flotation.Additionally, blood loss from injuries incurred dur-ing capture may have contributed to low plasma Felevels. Red-cell distribution width is expected to in-crease during erythrocyte regenerative processes,thus the correlation between high RDW and de-creased probability of injury may reflect an age biasamong otters with severe injuries, although no suchage bias was statistically evident.28 However, theinfluence of lymphocyte count and Fe concentrationon probability of injury is minute (Fig. 2a), sug-gesting that these values may be statistically sig-nificant, but clinically insignificant predictors of in-jury status in this population of otters. Eliminationfrom the model of all otters receiving amoxicillinor subcutaneous fluids prior to blood sampling re-sulted in lymphocyte and RDW as significant pre-dictors of injury classification, suggesting thesemight be more robust parameters than Fe.

Parameters commonly associated with exertionalmyopathy: Glucose and BUN were significant pre-dictors in a model that included parameters com-monly associated with EM (Fig. 2b). A delay be-tween venipuncture and separation of plasma frompacked cells (allowing for catabolism of glucose invitro) could have confounded this model. Thus,glucose levels probably were affected independentof injury status and should not be used as a bio-logically significant predictor of injury in our studyof recently captured otters. Increases in BUN levelsseen here may be the result of the high-protein,high-fat diet the otters were fed while in captivity


during the reintroduction program, and thereforemay also reflect factors unrelated to probability ofinjury.

Combination of all significant parameters fromother logistic regression models: The final model,which included statistically significant parametersfrom each of the previous three models, was simi-larly confounded by acute corticosteroid increasesassociated with recent capture (lymphopenia), andan extremely high-protein, high-fat diet (increasedBUN).

Confounding effects of parasitism and enterotox-emia: All hematology and plasma biochemistryvalues can be affected by heavy parasitism or bac-terial infection. Clostridium perfringens enterotox-emia reported in these otters is probably closelycorrelated with stress, resulting in gastrointestinalovergrowth of the bacteria and subsequent toxinproduction.23,24

Current research needs: Even though the param-eters examined in this study were generally poorpredictors of injury status in this set of river otters,and the values found are similar to other publisheddata on Nearctic river otters, our data contribute tothe growing body of physiologic data for river ot-ters. Differences between values found in river ot-ters in New York and those from other reintroduc-tion programs may be due to biogeographic causesor other ecological factors such as diet, precipita-tion, water quality, etc. Our data suggest a need forfurther evaluation of clinical pathology parameters,such as plasma or serum Amyloid A,30,36 whichmight elucidate subclinical traumatic disease andthereby improve medical management of otters inreintroduction programs. Also, further experimentalor epidemiologic study of toxicologic effects, par-ticularly within a risk assessment or individual an-imal-care framework,18 would prove useful in as-sessing the overall health of wild otter populations.

Acknowledgments: We thank D. Money and themany volunteers of the New York River Otter Pro-ject, Inc., including the participating trappers of theNew York State Trappers Association. Bruce Pen-rod of the New York State Department of Environ-mental Conservation, Division of Wildlife coordi-nated transport and capture of the otters. HussniMohammed provided advice on statistical analyses.The Board of Directors of the New York River Ot-ter Project, Inc., provided financial support for thisstudy.

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Received for publication 29 December 2003

EVALUATION OF INJURY SEVERITY AND HEMATOLOGIC AND PLASMA BIOCHEMISTRY VALUES FOR RECENTLY CAPTURED...

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