HEMATOLOGY, SERUM CHEMISTRY, AND SEROLOGY OF GALA

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<ul><li><p>HEMATOLOGY, SERUM CHEMISTRY, AND SEROLOGY OF</p><p>GALAPAGOS PENGUINS (SPHENISCUS MENDICULUS) IN THE</p><p>GALAPAGOS ISLANDS, ECUADOR</p><p>Erika K. Travis,1,2,6,7 F. Hernan Vargas,3 Jane Merkel,1,4 Nicole Gottdenker,5 R. Eric Miller,1</p><p>and Patricia G. Parker1,4</p><p>1 Saint Louis Zoo, One Government Drive, Saint Louis, Missouri 63110, USA2 College of Veterinary Medicine, University of Missouri, 203 Veterinary Medicine Building, Columbia, Missouri 65211,USA3 Wildlife Conservation Research Unit, University of Oxford, Tubney House, Abingdon Road, OX13 5QL, UK4 Department of Biology, University of MissouriSaint Louis, 8001 Natural Bridge Road, Saint Louis, Missouri 63121,USA5 Institute of Ecology, The University of Georgia, Athens, GA 30602, USA6 Current address: Utahs Hogle Zoo, 2600 E. Sunnyside Avenue, Salt Lake City, Utah 84108, USA7 Corresponding author (email: erikazoovet@yahoo.com)</p><p>ABSTRACT: The Galapagos penguin (Spheniscus mendiculus) is an endangered species endemicto the Galapagos Islands, Ecuador. In 2003 and 2004, 195 penguins from 13 colonies on the islandsof Isabela and Fernandina in the Galapagos archipelago were examined. Genetic sexing of 157penguins revealed 62 females and 95 males. Hematology consisted of packed cell volume (n5134),white blood cell differentials (n583), and hemoparasite blood smear evaluation (n5114).Microfilariae were detected in 22% (25/114) of the blood smears. Female penguins hadsignificantly higher eosinophil counts than males. Serum chemistry on 83 penguins revealed nosignificant differences between males and females. Birds were seronegative to avian paramyxovirustype 13, avian influenza virus, infectious bursal disease virus, Mareks disease virus (herpes),reovirus, avian encephalomyelitis virus, and avian adenovirus type 1 and 2 (n575), as well as toWest Nile virus (n587), and Venezuelan, western and eastern equine encephalitis viruses (n526).Seventy-five of 84 (89%) penguins had antibodies to Chlamydophila psittaci but chlamydial DNAwas not detected via polymerase chain reaction in samples from 30 birds.</p><p>Key words: Chemistry, filarid, Galapagos Islands, hematology, hemoparasite, penguin,serology, Spheniscus mendiculus.</p><p>INTRODUCION</p><p>The Galapagos Islands straddle theequator and are 1,000 km west of conti-nental Ecuador in the Pacific Ocean.Galapagos avifauna is comprised of 58resident species, of which 52% are en-demic (Tye et al., 2002). The Galapagospenguin (Spheniscus mendiculus) is anendemic species with the majority of thepopulation residing on two western islandsof the archipelago, Isabela and Fernan-dina; smaller populations reside on Flor-eana, Bartolome, and Santiago Islands(Mills and Vargas, 1997). The Galapagospenguin is one of the worlds smallestpenguin species and it is the only speciesfound in the tropics, with part of itspopulation inhabiting the northern hemi-sphere (Boersma, 1977). It is consideredendangered due to its small range and</p><p>fluctuating population (BirdLife Interna-tional, 2000). Galapagos penguin popula-tions were dramatically reduced duringthe 198283 (Valle and Coulter, 1987) and199798 El Nino events (Vargas et al.,2006). Since 1998, the adult populationhas been relatively stable whereas thejuvenile population has been variable(Vargas and Wiedenfeld, 2004). In 2004,the population was estimated at 1505individuals (Vargas and Wiedenfeld,2004).</p><p>No avian species have become extinctfrom the Galapagos Islands (Wikelski etal., 2004), but the possibility of diseasecausing significant morbidity and mortalityis of great concern. Avian health studies inthe Galapagos Islands have been ongoingsince 2001, and to date, 16 avian specieson 11 islands have been sampled. The firstcomprehensive health assessment of the</p><p>Journal of Wildlife Diseases, 42(3), 2006, pp. 625632# Wildlife Disease Association 2006</p><p>625</p></li><li><p>penguin population occurred in August2003 and March 2004. Serologic testswere determined from previous penguindisease surveys (Austin and Webster,1993; Wallace et al., 1997; Clarke andKerry, 2000; Gauthier-Clerc et al., 2002;Tuttle et al., 2005). Penguins also weretested for exposure to multiple poultrypathogens that could be associated withchickens in the Galapagos Islands (Gott-denker et al., 2005). Potential spillover ofpoultry pathogens to penguins exists viapoultry waste or other infective material infreshwater runoff, aerosol transmission ofviruses from inhabited coastal regionsadjacent to penguins, discarded poultryproducts from commercial (fishing andtour) boats in areas where penguinsreside, and contact with volant birds (i.e.,yellow warblers or finches) that mighttransmit pathogens of poultry origin topenguin nesting sites. Serologic test selec-tion was also directed by positive serologicresults from other endemic Galapagosavian species (Padilla et al., 2003, 2004).In order to establish a baseline for futuremonitoring of the endangered Galapagospenguin, hematology and serum chemistryparameters also are reported.</p><p>MATERIALS AND METHODS</p><p>Study area and sample collection</p><p>Galapagos penguins were studied on thecoasts of Isabela (0u259300S, 91u79W) andFernandina (0u22900S, 91u319200W) Islands.All sampling procedures were in accordancewith Saint Louis Zoo institutional animal careand use committee standards. Eighty-four and112 penguins were examined in August 2003and March 2004, respectively. On FernandinaIsland, 27 penguins in 2003 and seven in 2004were sampled, and on Isabela Island, 57penguins in 2003 and 105 in 2004 wereevaluated. Information on capture technique(Vargas et al., 2005), and morphometric mea-surements and aging (Boersma, 1977), haspreviously been described. During manualrestraint, a transponder (AVID Microchip,Folsom, Louisiana, USA) was placed subcuta-neously over the left dorsal mid phalangeal areaand the skin defect sealed with tissue glue (3MVetbond, St Paul, Minnesota, USA). Jugular</p><p>venipuncture was performed with 20 gaugeneedles and syringes to collect up to 12 ml ofblood per bird. Several drops of blood wereimmediately placed into cryogenic vials con-taining a lysis buffer preservative solution(Longmire et al., 1988) for genetic testing ofgender, as well as preliminary hemoparasitemolecular analysis. During the 2003 fieldseason, whole blood was placed in lithiumheparin (Vacutainer PST gel, Becton Dick-inson, Franklin Lakes, New Jersey, USA);during the 2004 field season a small amountof whole blood was placed in ethylenediamine-tetraacetic acid (EDTA) (Microtainer gel,Becton Dickinson), and the remainder placedin serum separator tubes (Vacutainer SST geland clot activator, Becton Dickinson). Bloodtubes were kept on ice packs until processing. Asingle sterile swab (Copan Diagnostics, Corona,California, USA) was used to collect a combinedconjunctival, choanal, and cloacal swab per bird(n597). The swabs were stored in cryogenicvials (Nalge Nunc International, Rochester,New York, USA). No ectoparasites wereobserved on feathers during either year. Fecalsamples from 11 individual penguins from 2004were collected opportunistically from freshexcreta during restraint. Feces were preservedin cryogenic vials with potassium dichromateand/or formalin and stored at room tempera-ture for later microscopic examination.</p><p>Sample processing</p><p>The timing of full sample processing variedfrom less than 1 hr up to 6 hr after collection.Most samples were centrifuged within 3 hr ofcollection. Two blood smears were made fromwhole blood (lithium heparin in 2003, EDTAin 2004), and air dried and fixed with methanolin the field. Microhematocrit tubes (n5134)were centrifuged (Mobilespin Model 128,Vulcon Technologies, Grandview, Missouri,USA) for 20 min, and the packed cell volumes(PCV) determined. The blood in lysis buffersolution was held at ambient temperature. Thelithium heparin whole blood samples (n574)and serum separator samples (n597) werecentrifuged for 20 min, and the plasma orserum decanted and stored in cryogenic vials.The plasma, serum, and swabs were stored inliquid nitrogen in the field and in thelaboratory.</p><p>Sample and data analysis</p><p>Blood smears were stained using a modifiedWrightGiemsa stain (JorVet Dip-Quick, Jor-gensen Laboratories, Loveland, Colorado,USA) at the Saint Louis Zoo for white bloodcell (WBC) differentials based on 100 cells</p><p>626 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 3, JULY 2006</p></li><li><p>counted at 10003 magnification. Bloodsmears were evaluated for thrombocytes andhemoparasites. Genetic testing for sex de-termination (n5157) was performed at theUniversity of MissouriSaint Louis (UMSL)via polymerase chain reaction (PCR) (Fridolfs-son and Ellegren, 1999). Ninety of the 2004samples were processed for serum biochemis-try (AVL Veterinary Laboratory, Saint Louis,Missouri, USA), in one batch on the AceClinical Chemistry System (Alfa Wassermann,Inc., West Caldwell, New Jersey, USA). Sevenresults were not included in the analysis due tohemolysis or lipemia.</p><p>Serology testing was restricted to the 2004samples. Testing for antibodies to Chlamydo-phila psittaci was done by the Texas VeterinaryMedical Diagnostic Laboratory, College Sta-tion, Texas, USA using direct complementfixation (DCF) on serum (n584); combinedconjunctival, choanal and cloacal swabs from 30of the antibody positive penguins were alsotested for C. psittaci DNA by real-time PCR.</p><p>Serologic tests for viral antibodies, exceptfor WNV, were conducted at the NationalVeterinary Services Laboratory, US Depart-ment of Agriculture, Ames, Iowa, USA.Hemagglutination inhibition (HI) testing wasused to test for antibodies to avian para-myxovirus type 13 (n575; titers $8 wereconsidered positive) and Venezuelan (VEE),western (WEE), and eastern (EEE) equineencephalitis viruses (n526; titers $10 wereconsidered positive). Agar gel immunodiffu-sion (AGID) testing was used to test forantibodies to avian influenza virus, infectiousbursal disease virus, Mareks disease (herpes)virus, avian adenovirus types 1 and 2, andavian encephalomyelitis virus (n575). Indirectfluorescent antibody (IFA) testing was used totest for antibodies to reovirus (n575). Serumwas tested for antibodies to West Nile virus byplaque reduction neutralization (n587) at theAnimal Health Diagnostic Center, CornellUniversity, Ithaca, New York, USA. Fecalanalysis from fecal floatation and sedimenta-tion was conducted with microscopy at theSoutheastern Cooperative Wildlife DiseaseStudy, College of Veterinary Medicine, TheUniversity of Georgia, Athens, Georgia, USA.</p><p>A statistical software package (NCSSH,Kaysville, Utah, USA) was used for dataanalysis. Results were inspected for normalityusing a Shapiro-Wilk W test and t-tests wereperformed. Mann-Whitney U-tests were usedon samples where normality was rejected. Astatistical significance was determined asP#0.05 except for the serum chemistry valueswhere a Bonferroni-corrected P#0.004 (0.05/12) was used.</p><p>RESULTS</p><p>Genetic sexing, body weight, hematolo-gy, and microscopic hemoparasite evalua-tions were performed on the 2003 and2004 samples, counting each individualonce because some individuals weresampled both years. Serum chemistries,serology, and fecal analysis were con-ducted only for the 2004 samples. Allpenguins examined were in good bodycondition, as determined both by palpa-tion of pectoral muscles and body weightmeasurement. There were no externalsigns of disease. In 2003, 84 penguinswere sampled; eight (10%) nestlings, five(6%) juveniles, and 71 (84%) adults. In2004, 112 penguins were sampled; four(4%) nestlings, 28 (25%) juveniles, and 80(71%) adults. Twenty-five penguins in2004 were recaptured from the previousyear. Genetic sexing of 157 individualsrevealed 62 females (39.5%) and 95 males(60.5%). Body weight and hematologyresults are provided in Table 1. Becausethere was a significant difference betweenthe sexes for body weight and PCV,separate gender means are listed. Therewere no differences in PCV or differen-tials between heparin or EDTA blood.The mean PCV of the combined male andfemale sampled population was 44.1%(SD 6.8%, n5134). Males had significant-ly higher body weights (P,0.00001) andPCV results (P,0.006) compared to fe-males. There were no differences in WBCdifferentials between genders except thatfemales had elevated eosinophils(12.269.1%, n529) compared to males(5.360.7%, n554) (P,0.0002). Micro-scopic evaluation of blood smears revealedmicrofilariae in 21.9% (25/114) of thepenguins. Thrombocytes were adequatefor both genders because at least two tothree thrombocytes were seen on eachhigh power field (Pierson, 2000).</p><p>Results of serum chemistries are pro-vided in Table 2; there were no significantdifferences between female and malepenguin biochemistry values. All penguins</p><p>TRAVIS ET AL.HEMATOLOGY, CHEMISTRY, SEROLOGY GALAPAGOS PENGUINS 627</p></li><li><p>were serologically negative to all viruses.Seventy-five of 84 penguins (89.3%) wereseropositive to C. psittaci with titers of 8 to64. Combined swabs were submitted forC. psittaci PCR on 30 of the 75 seropos-itive penguins; all were negative. Hema-tology and biochemistry results did notdiffer between C. psittaci or filarialpositive penguins and those that werenegative. Fecal samples were negative forhelminth eggs and larval nematodes, butone penguin (1/11) had three Eimeriaoocysts detected (17316 mm).</p><p>DISCUSSION</p><p>These are the first published hematol-ogy, serum chemistry, and large-scaleserology results for the Galapagos pen-guin. The Humboldt penguin (S. hum-</p><p>boldti) is closely related to the Galapagospenguin (Robert Fleischer, pers. comm.);therefore, blood work from a free rangingHumboldt population (Wallace et al.,1995) was used for comparison. Becausethe methodology of the studies wasdifferent, trends between the penguinspecies is discussed instead of statisticaldifferences. The Galapagos penguins hadhigher chloride, calcium, uric acid, andaspartate aminotransferase values, butlower PCV, glucose, and albumin com-pared to the Humboldt penguins. Labo-ratory results can be affected by manyvariables such as species, gender, age,nutritional status, or geographic location(Dein, 1986), and the difference betweenthe Galapagos and Humboldt penguinslikely are not clinically significant.</p><p>A change in the Galapagos penguin</p><p>TABLE 1. Weight, packed cell volume, and white blood cell differentials for free-ranging Galapagos penguins.</p><p>Parameter Mean6SD Range n</p><p>Female weight (kg)a 1.960.3 1.42.5 62Male weight (kg)a 2.360.3 1.73.5 103Female packed cell volume (%)b 41.767.0 28.052.0 53Male packed cell volume (%)b 45.366.8 23.058.0 79Heterophils (%) 57.6616.0 8.085.0 83Lymphocytes (%) 31.5615.5 6.084.0 83Monocytes (%) 2.462.1 0.09.0 83Eosinophils (%) 8.067.5 0.037.0 83Basophils (%) 0.460.7 0.03.0 83</p><p>a Significant difference P,0.00001.b Significant difference P,0.006.</p><p>TABLE 2. Serum chemisty results for free-ranging Galapagos penguins (n583).</p><p>Parameter (SI units) Mean6SD Range</p><p>Aspartate aminotransferase (U/l) 282.5689.8 132.0525.0Total protein (g/l) 56.06 7.7 36.077.0Albumin (g/l) 12.261.5 9.017.0Phosphorus (mmol/l) 1.660.8 0.84.2Calcium (mmol/l) 2.560.2 2.23.7Glucose (mmol/l) 12.161.9 5...</p></li></ul>

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