abstracts europeansocietyofveterinaryclinical pathology...

A B S T R A C T S

European Society of Veterinary ClinicalPathology (ESVCP) 14th Annual Congressand International Society for Animal ClinicalPathology (ISACP) 15th Biennial Congress.

Ljubljana, Slovenia – July 3-7, 2012DOI: 10.1111/j.1939-165X.2012.00465.x

Oral Platform Presentations: Abstracts 1–45Poster Presentations: Abstracts 46–121

1 EFFECT OF TIME RELATED TO CALVING AND RUMEN PROTECTED CHOLINE (RPC) SUPPLEMENTATION

ON BLOOD ANALYTES AND ON LIVER LIPID AND GLYCOGEN. T. Gaal1, P. Elek2, F. Husveth3. 1School of

Veterinary and Biomedical Sciences, Faculty of Health Sciences, Murdoch University, Perth, Australia; 2Provimi

Hungary Co. Zichyujfalu, Hungary; 3Department of Animal Science, Georgikon Faculty of Agriculture, University

of Pannonia, Keszthely, Hungary.

2 ASSOCIATION BETWEEN HEMATOLOGICAL AND ELECTROPHORETIC PROFILES AND PREVALENCE OF

POST-PARTUM DISEASES IN DAIRY COWS. S. Paltrinieri1, P. Moretti2, M. Venturini2, N. Morandi3,

A. Giordano4. 1University of Milan, Milan, Italy; 2Central Laboratory, Large Animal Hospital, Milan, Italy; 3Parco

Tecnologico Padano, Lodi, Italy; 4Department of Veterinary Pathology, Hygiene and Public Health,Milan, Italy.

3 HEMATOLOGICAL CHANGES ASSOCIATED WITH RETAINED PLACENTA IN CATTLE. P. Moretti1,

A. Giordano2, M. Venturini3, N. Morandi4, S. Paltrinieri2. 1Large Animal Hospital, Lodi, Italy; 2Department

of Veterinary Pathology, University of Milan, Milan, Italy; 3Central laboratory, Large Animal Hospital, Lodi, Italy;4Parco Tecnologico Padano, Lodi, Italy.

4 SURVEY OF VITAMIN A, BETA-CAROTENE, CALCIUM AND PHOSPHORUS CONCENTRATIONS IN SERUM

OF EWES WITH RETAINED PLACENTA. A. Davasaztabrizi. Department of Clinical Sciences, Tabriz Branch,

Islamic Azad University, Tabriz, Iran.

5 URINE PROTEOME IN DOGS AFFECTED BY LEISHMANIASIS.A. Buono1, F. Dondi1, E. Ferlizza1, G. Isani1,

J. Duque2, P. Tapia2, C. Zaragoza2, R. Barrera3. 1University of Bologna, Ozzano Emilia, Italy; 2University of

Extremadura, Caceres, Spain; 3Department of Internal Medicine, University of Extremadura, Caceres, Spain.

6 EVALUATION OF THE REVISED QUANTITATIVE INSULIN SENSITIVITY CHECK INDEX IN DAIRY COWS

SHOWING VARIOUS FORMS OF KETONE PATTERN AND PUERPEAL METRITIS. M. Kerestes1, V. Faigl1,

M. Kulcsar1, O. Balogh1, J. Foldi2, H. Febel3, Y. Chilliard4, G. Huszenicza1. 1Faculty of Veterinary Science,

Budapest, Hungary; 2Intervet, Budapest, Hungary; 3Research Institute for Animal Breeding and Nutrition, Herce-

ghalom, Hungary; 4INRA, Saint-Genes-Champanelle, France.

7 DIAGNOSTIC VALUE OF ACUTE PHASE INDEX OF MILK IN DETECTION OF SUBCLINICAL MASTITIS IN

DAIRY COWS. S.H. Shirazi-Beheshtiha1, S. Safi2, V. Rabbani1,M.B. Bolourchi3. 1Karaj Branch, Islamic Azad

University, Karaj, Iran; 2Science and Research Branch, Islamic Azad University, Tehran, Iran; 3Faculty of Veteri-

naryMedicine, University of Tehran, Tehran, Iran.

Vet Clin Pathol 0/0 (2012) 1–47�2012 American Society for Veterinary Clinical Pathology 1

Veterinary Clinical Pathology ISSN 0275-6382

8 A CASE OF CANINE SURRA IMPORTED INTO GERMANY. M. Defontis1, J. Richartz1, N. Engelmann1,

C. Bauer1, V.M. Schwierk2, P. Buscher3, A. Moritz1. 1Justus-Liebig University, Giessen, Germany;2Pulheim-Stommeln, Germany; 3Antwerpen, Belgium.

9 CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL CELL LYMPHOMA IN A HORSE. F. Cian1, G. Tyner2,

V. Martini3, S. Comazzi3, J. Archer1. 1Department of Veterinary Medicine, University of Cambridge,

Cambridge, UK; 2Chiltern Equine Clinic, Chalfont St Giles, UK; 3Department of Veterinary Pathology, University

ofMilan,Milan, Italy.

10 CASE REPORT: CUTANEOUS T-CELL LYMPHOMA. B. Rutgen1, I. Flickinger2, B. Wolfesberger2,

B. Litschauer2, A. Fuchs-Baumgartinger3, S.E. Essler4, A. Saalmuller4, I. Schwendenwein5. 1University

of Veterinary Medicine Vienna, Vienna, Austria; 2Clinic for Internal Medicine, 3Institute of Pathology, 4Institute

of Immunology, and 5Central Laboratory, Department for Pathobiology, Universtiy of Veterinary Medicine,

Vienna, Austria.

11 CHANGES IN THE CONCENTRATIONS OF SERUM AMYLOID A (SAA) FOLLOWING SURGERY, TRAUMA

OR SNAKE ENVENOMATION IN DOGS: A STUDY OF THE KINETICS OF SAA. M. Christensen1, E. Moldal1,

R. Langhorn1, A. Goddard2, A. Tvarijonaviciute3, M. Kjelgaard-Hansen1. 1Department of Small Animal

Clinical Sciences, University of Copenhagen, Frederiksberg, Denmark; 2Department of Companion Animal

Clinical Studies, University of Pretoria, South Africa; 3Department of Animal Medicine and Surgery, University of

Murcia, Murcia, Spain.

12 EVALUATION OF A CANINE SPECIFIC QUANTITATIVE POINT-OF-CARE IMMUNOASSAY FOR

MEASURING C-REACTIVE PROTEIN IN DOGS. M. Costa, K. Papasouliotis. School of Veterinary Sciences,

University of Bristol, Bristol, UK.

13 PCR-BASED DETECTIONOF A. PHAGOCYTOPHILUMDNA IN PARAFFIN-EMBEDDED SKIN BIOPSIES FROM

DOGS SEROPOSITIVE AGAINST A. PHAGOCYTOPHILUM. I. Berzina1, N. Muller2, C. Krudewig3, I. Matise1,

R. Ranka4, M. Welle3. 1Faculty of Veterinary Medicine, Latvia University of Agr, Jelgava, Latvia; 2Institute of

Parasitology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland; 3Institute of Animal Pathology, Vetsuisse-

Faculty, University of Bern, Bern, Switzerland; 4Latvian Biomedical Research and Study Center, Riga, Latvia.

14 PRELIMINARY REFERENCE INTERVALS FOR FLUOROGENIC MEASUREMENT OF THROMBIN GENERA-

TION IN THE BEAGLE DOG.M. Defontis1, S. Cote2, M. Stirn2, D. Ledieu2. 1Justus-Liebig University, Giessen,

Germany; 2Basel, Switzerland.

15 FLOW CYTOMETRIC EVALUATION OF DRUG RESISTANCE IN CANINE LYMPHOMA SHORT-TERM CELL

CULTURES. P. Vajdovich. Szent Istvan University, Faculty of Veterinary Science, Budapest, Hungary.

16 MEASUREMENT OF SOLUBLE MESOTHELIN RELATED PEPTIDES (SMRP) IN CANINE BODY CAVITY

EFFUSIONS. M. Costa1, K. Murphy2, D. Wilson1, K. Papasouliotis1. 1School of Veterinary Sciences, Univer-

sity of Bristol, Bristol, UK; 2Bath Veterinary Referrals, Bath, UK.

17 PREDICTIVE VALUE ADVISOR: SOFTWARE FOR THE EVALUATION OF THE IMPRECISION OF PREDICTIVE

VALUES OF TEST RESULTS ACCORDING TO IMPRECISION OF SENSITIVITY, SPECIFICITY AND PRE-TEST

PROBABILITY.N. Bourges-Abella1, P. Bourdaud’hui2, A. Geffre1, D. Concordet2, J.P. Braun1, O. Dossin1,

C. Trumel1. 1Institut National Polytechnique-Ecole Nationale Veterinaire de Toulouse, Toulouse, France;2UMR331 Toxalim, Institut National Polytechnique-Ecole Nationale Veterinaire, Toulouse, France.

18 REFERENCE VALUE ADVISOR (V.2) NOW PERFORMS REGRESSION-BASED REFERENCE INTERVALS

WITH CONTINUOUS COVARIABLES. N. Bourges-Abella1, A. Geffre1, D. Concordet2, J.P. Braun1,

C. Trumel1. 1Institut National Polytechnique-Ecole Nationale Veterinaire de Toulouse, Toulouse, France;2UMR331 Toxalim, Institut National Polytechnique-Ecole Nationale Veterinaire, Toulouse, France.

Vet Clin Pathol 0/0 (2012) 1–47�2012 American Society for Veterinary Clinical Pathology2

2012 ESVCP and ISACP Congress Abstracts

19 ACTIVATION OF LIPID METABOLISM IN CANINE BABESIOSIS CAUSED BY BABESIA CANIS. N. Kucer1,

V.Mrljak1, R. Rafaj Baric2, J. Kules2, I. Smit1, J. Selanec1,M. Crnogaj2. 1Clinic for Internal Diseases, Faculty

of Veterinary Medicine, University of Zagreb, Croatia; 2Department for Chemistry and Biochemist, Faculty of

VeterinaryMedicine, Zagreb, Croatia.

20 LACTATE DEHYDROGENASE ISOENZYMES IN DOGS AFFECTED BY LYMPHOMA. A. Giordano,

D. Stefanello,M. Pastore, R. Ferrari, L. Giori, P.Moretti, S. Paltrinieri.University ofMilan,Milan, Italy.

21 EFFECTS OF PLATELET-ACTIVATING FACTOR RECEPTOR ANTAGONIST (PAFRA) ON SELECTED INFLAM-

MATORY AND BIOCHEMICAL PARAMETERS IN A RAT ENDOTOXEMIA MODEL. R. Col, E. Keskin. Selcuk

University, Konya, Turkey.

22 URINEPROTEOMEFROMHEALTHYDOGS, CATS, HORSESANDCOWS.E. Ferlizza, E.Armuzzi, A. Buono,

E. Carpene, G.Andreani, F. Dondi, G. Isani.University of Bologna, Ozzano Emilia, Italy.

23 EVALUATION OF THREE URINE DIPSTICKS FOR DOGS, CATS, AND COWS. S. Klenner1, M. Defontis1,

N. Bauer1, K. Failing2, A. Moritz1. 1Central Laboratory, University (JLU) Giessen, Hannover, Germany; 2Unit

for Biomathematics and Data Processing, Giessen, Germany.

24 CLINICAL RELEVANCE OF TGF-ß1 IN DOGS. S. Neumann, S. Gaedcke. Institute of Veterinary Medicine,

Goettingen, Germany.

25 LDH IN BODY CAVITY FLUIDS: IS IT DIAGNOSTICALLY USEFUL? C. Smuts, J. Mills, T. Gaal. Murdoch

University, Perth, Australia.

26 CANINE PANCREATIC LIPASE (SPEC CPL) AND C-REACTIVE PROTEIN (CRP) IN DOGS TREATED WITH

ANTICONVULSANTS (PHENOBARBITALANDPOTASSIUMBROMIDE). J. Pastor1, V.Albarracin1,A.Melendez-

Lazo1,J. Rodon2. 1Universitat Autonoma de Barcelona, Bellaterra, Spain; 2IDEXXSpain, Barcelona, Spain.

27 IMMULITE 1000 MEASUREMENTS IN VETERINARY SAMPLES. R. Pato1, R. Pena2, Y. Saco2, A. Bassols2.1Universitat Autonoma de Barcelona, Bellaterra, Spain; 2Servei de Bioquımica Clınica Veterinaria, Departament

de Bioquımica, Bellaterra (Barcelona), Spain.

28 KINETICS AND CHARACTERISTICS OF FELINE PLATELET AGGREGATION IN VITRO. B. Riond1,

A.K. Waßmuth1, S. Hartnack2, R. Hofmann-Lehmann1, H. Lutz1. 1Clinical Laboratory, Vetsuisse Faculty,

University of Zurich, Zurich, Switzerland; 2Section of Epidemiology, Vetsuisse Faculty, University of Zurich,

Zurich, Switzerland.

29 EFFECTIVE PREVENTION OF PSEUDOTHROMBOCYTOPENIA IN FELINE EDTA BLOOD SAMPLES WITH

THE PROSTAGLANDIN I2-ANALOGUE ILOPROST. B. Riond1, A.K. Waßmuth1, S. Hartnack2, R. Hofmann-

Lehmann1, H. Lutz1. 1Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; 2Section

of Epidemiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.

30 EXPLORATIVE REFERENCE INTERVALS FOR BIOCHEMISTRY AND HAEMATOLOGY DATA IN

HEALTHY PIGLETS DURING THE FIRST 27 DAYS OF LIFE. B. Rutgen1, E. Hooijberg2, L. Schwarz1,

H.L. Worliczek1, I. Schwendenwein1. 1University of Veterinary Medicine Vienna, Vienna, Austria; 2Labor

InVitro, Vienna, Austria.

31 THE “QUATREFOIL” ERYTHROCYTES:UN-EXPECTEDPATTERN INCANINEREDBLOODCELLMORPHOLOGY.

A. Gavazza, G. Lubas, M. Ricci, B. Gugliucci, A. Pasquini. Dept Veterinary Clinic, University of Pisa, Pisa,

Italy.



32 PLATELET INDICES IN VIRULENT CANINE BABESIOSIS AND THEIR ASSOCIATION WITH OUTCOME.

A. Goddard1, A. Kristensen2, A. Leisewitz1, P. Thompson1, J. Schoeman1. 1University of Pretoria, Pretoria,

South Africa; 2University of Copenhagen, Copenhagen, Denmark.

33 D-DIMER MEASUREMENT BY LATEX AGGLUTINATION; COMPARISON OF A SEMIQUANTITATIVE AND

QUANTITATIVE METHOD AND EVALUATION OF THE CLINICAL VALUE OF THE TEST IN DOGS. J. Archer,

A. Pastorello, P. Monti, P. Kuczybska. Department of Veterinary Medicine University of Cambridge,

Cambridge, UK.

34 DIAGNOSTIC PERFORMANCES OF FLOW CYTOMETRIC DETECTION OF BLOOD AND BONE MARROW

INFILTRATION IN DOGS WITH LARGE B-CELL LYMPHOMA. V. Martini1, M.E. Gelain2, S. Comazzi1.1University ofMilan,Milan, Italy; 2University of Padua, Padua, Italy.

35 DOUBLE ERYTHROCYTE POPULATIONS: RETROSPECTIVE STUDY ON CASES OBSERVED WITH SYSMEX

XT-2000iV (2008-2012). N. Bourges-Abella, B. Cuq, F. Granat, A. Geffre, C. Hanot, C. Trumel. Institut

National Polytechnique-Ecole Nationale Veterinaire de Toulouse, Toulouse, France.

36 LEEK DIET MAY CAUSE HEMOLYTIC ANEMIA: A CASE REPORT IN A CAT.N. Bourges-Abella, F. Granat,

A. Geffre, C. Trumel. Institut National Polytechnique-Ecole Nationale Veterinaire de Toulouse, Toulouse,

France.

37 GASTRIC DILATATION AND VOLVULUS SYNDROME – ARE THERE ANY CLUES IN THE HEMATOLOGIC

EXAMINATION? I. Uhrikova, L. Rauserova-Lexmaulova, K. Rehakova, I. Hajek, J. Doubek. University of

Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic.

38 EFFECT OF STORAGE CONDITIONS ON KAOLIN/ NON-KAOLIN-ACTIVATED THROMBOELASTOGRAPHY

AND HEMOSTATIC PARAMETERS IN CATS. R. Col1, C.M. Iazbik2, G. Couto2. 1Selcuk University, Konya,

Turkey; 2Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, OH, USA.

39 PRELIMINARY REPORT OF HEMOSTATIC PARAMETERS IN THE EXPERIMENTAL GOTTINGEN MINIPIG.

M. Defontis1, S. Cote2, M. Stirn2, D. Ledieu2. 1Justus-Liebig University, Giessen, Germany; 2Basel,

Switzerland.

40 AFLATOXINS IN SLOVENE MILK AND FEED SAMPLES. B. Jakovac-Strajn, G. Tavcar-Kalcher,

I. Ujcic-Vrhovnik, K. Pavsic-Vrtac, K. Fon-Tacer, A. Vengust. Veterinary faculty, Ljubljana, Slovenia.

41 IS ALKALINE PHOSPHATASE ACTIVITY IN BOVINE NASAL SECRETION A RESULT OF LOCAL SYNTHESIS?

M.F. Ghazali, N.N. Jonsson, P.D. Eckersall.University of Glasgow, Glasgow, UK.

42 SERUM CALCIUM AND INORGANIC PHOSPHATE EVALUATION IN HERMANN’S TORTOISES (Testudo

hermanni). M. Stvarnik1, M. Klinkon2, A. Dovc3. 1Veterinary faculty, Ljubljana, Slovenia; 2Clinic for

Ruminants, Veterinary faculty, Ljubljana, Slovenia; 3Institute for the Health Care of Poultry, Veterinary faculty,

Ljubljana, Slovenia.

43 HEMATOLOGY REFERENCE INTERVALS IN CYNOMOLGUS MONKEYS. N. Bourges-Abella1, A. Geffre1,

E. Moureaux2, M. Vincenti1, J.P. Braun1, C. Trumel1. 1Institut National Polytechnique-Ecole Nationale

Veterinaire de Toulouse, Toulouse, France; 2BioPRIM, Bazieges, France.

44 EVALUATION OF SERUM AMYLOID A, HAPTOGLOBIN, NUCLEATED CELL COUNT, TOTAL PROTEIN AND

HEMOLYSIS IN PERITONEAL FLUID FOR DIFFERENTIATION OF MEDICAL AND SURGICAL COLIC IN

HORSES. E. Scheepers1, T. Holberg Pihl2, M. Sanz1, P. Page1, A. Goddard1, S. Jacobsen2. 1University of

Pretoria, Pretoria, South Africa; 2University of Copenhagen, Taastrup, Denmark.



45 MEASUREMENT OF SERUM ALBUMIN BY PROTEIN ELECTROPHORESIS AND BROMOCRESOL GREEN

METHODS IN CANINE AND EQUINE PATIENTS. E. Ramery, F. Bureau. Liege University, Faculty of Veterinary

Medicine, Liege, Belgium.

46 THE EFFECT OF PARITY ON BONE FORMATION DURING LATE PREGNANCY AND EARLY LACTATION IN

SAANEN GOATS. M. Belic1, V. Kusec2, M. Robic1, J. Grizelj1, A. Svetina1, Z. Vrbanac1, R. Turk1. 1Veteri-

nary Faculty, Zagreb, Croatia; 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia.

47 SUBCLINICAL MASTITIS IN SHEEP: NOVEL DIAGNOSTIC FINDINGS. A. Miglio1, M.T. Antognoni1,

L. Moscati2, C. Maresca2, A. Valiani2, E. Scoccia2, V. Mangili1, G. Fruganti1. 1University of Perugia, Faculty

of VeterinaryMedicine, Perugia, Italy; 2Istituto Zooprofilattico Sperimentale Dell’Umbria E DelleMarche, Perugia,

Perugia, Italy.

48 MEASURING GLUCOSE CONCENTRATION IN BLOOD OF COWS WITH THE OPTIUM XCEED TM METER

AND THE BIOCHEMICAL ANALYSER – COMPARISON OF METHODS. J. Jezek, J. Staric, M. Klinkon,

M.Nemec.University of Ljubljana, Veterinary Faculty, Clinic for ruminants, Ljubljana, Slovenia.

49 CHEEK TOOTHABNORMALITIES IN SHEEP FLOCKS.V. Erjavec. Veterinary Faculty, Ljubljana, Slovenia.

50 GLUTAVAC® TEST SET (GLUTARALDEHYDE BLOOD TEST) – DIAGNOSTIC SUPPORT FOR FOREGIN BODY

DISEASE. T. Zadnik1, S. Rakulic Zelov2. 1Veterinary Faculty, Ljubljana, Slovenia; 2Veterinary station, Zuzem-

berk, Slovenia.

51 MONITORING SUBCLINICAL KETOSIS IN TRANSITION COWS PREGNANT WITH A SINGLE CALF OR

TWINS. T. Zadnik1, R. Lombar2. 1Veterinary Faculty, Ljubljana, Slovenia; 2Veterinary Station, Tenetise,

Slovenia.

52 LOGISTIC REGRESSION MODELING FOR THE PREDICTION OF LEFT DISPLACED ABOMASUM IN DAIRY

COWS. S. Safi1, N. Basiri1, A. Rahimi Foroushani2. 1Faculty of Veterinary Medicine, Science and Research

Branch, Islamic Azad University, Tehran, Iran; 2Department of Epidemiology and Biostatistics, Faculty of Public

Health, Tehran University, Tehran, Iran.

53 CANINECUTANEOUSHISTIOCYTOMA: ARETROSPECTIVE STUDYOF 55 CASES (2002-2011).C. Pressanti,

F. Granat, A. Geffre, M.C. Cadiergues. Toulouse Veterinary School, Toulouse, France.

54 CANINE NEOPLASTIC EFFUSION: MORPHOMETRIC ANALISYS OF CYTOLOGICAL SAMPLES.G. Ghisleni,

V. Pizzatti Sertorelli, A. Forlani, V. Bronzo,M. Caniatti.DIPAV, University ofMilan,Milan, Italy.

55 EFFECT OF NEEDLE GAUGE ON THE QUALITY OF CANINE SPLEEN FINE-NEEDLE ASPIRATION SPECI-

MENS – A PRELIMINARY STUDY. N. Bourges-Abella, C. Layssol, A. Geffre, C. Trumel. Institut National

Polytechnique-Ecole Nationale Veterinaire de Toulouse, Toulouse, France.

56 DETERMINATION OF PROLIFERATIVE ACTIVITY IN CANINE LEUKEMIAS BY FLOW CYTOMETRY.

A. Poggi1, B. Miniscalco1, L. Aresu2, M.E. Gelain2, S. Comazzi3, V. Martini3, E. Morello1, F. Gattino1,

F. Riondato1. 1Dept. of Animal Pathology, University of Torino; 2Dept. of Public Health, Comparative Pathology

and Vet. Hygiene, University Of Padua; 3Dept. of Veterinary Pathology, Hygiene and Health, Unit of General

Pathology, University ofMilan, Italy.

57 PROGNOSTIC VALUE OF KI67 IN CANINE LYMPHOMA DETERMINED BY FLOW CYTOMETRY. A. Poggi1,

B. Miniscalco1, L. Aresu2, M.E. Gelain2, S. Comazzi3, V. Martini3, E. Morello1, F. Gattino1, F. Riondato1.1Dept. of Animal Pathology, University of Turin; 2Dept. of Public Health, Comparative Pathology and Vet.

Hygiene, University of Padua; 3Dept. of Veterinary Pathology, Hygiene and Health, Unit of General Pathology,

University ofMilan, Italy.



58 “WATER DIABETES” IN A GREYHOUND ASSOCIATED WITH CORTICOSTEROIDS. L. Blythe. Oregon State

University, Corvallis, OR, USA.

59 ACUTE PHASE PROTEIN RESPONSE IN PIGS AFTER EXPERIMENTAL INFECTION WITH DIFFERENT

STRAINS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS. Y. Saco1, M. Cortey2,

J. Segales3, R. Pato4, R. Pena4, F. Martınez-Lobo5, C. Prieto5, A. Bassols6. 1Universitat Autonoma de Barce-

lona, Bellaterra, Spain; 2Unite de Virologie Moleculaire, Emergence et Co-Evolution Virale UMR 6578, Marseille,

France; 3CReSA, UAB-IRTA, Dept. Sanitat i Anatomia Animals, Fac. Veterinaria, UAB, Barcelona, Spain; 4Servei

de Bioquımica Clınica Veterinaria, Fac. Veterinaria, UAB, Barcelona, Spain; 5Departament de Sanidad Animal,

Fac. Veterinaria, Univ. Complutense deMadrid,Madrid, Spain; 6Departament de Bioquımica i BiologiaMolecular,

Fac. Veterinaria, UAB, Barcelona, Spain.

60 DIAGNOSTIC VALUE OFMILK ACUTE PHASE INDEX IN DETECTION OF BOVINE SUBCLINICALMASTITIS.

S. Safi1, S.H. Shirazi-Beheshtiha2, V. Rabbani2, M. Bolourchi3. 1Science and Research Branch, Islamic Azad

University, Tehran, Iran; 2Department of Clinical Sciences, Faculty of Veterinary Medicine, Islamic Azad Univer-

sity, Karaj, Iran; 3Department of Clinical Sciences, Faculty of Veterinary Medicine, Tehran University, Tehran,

Iran.

61 SERUM AMYLOID A AS A MARKER OF INFLAMMATION IN CANINE JOINTS. J. Francuski1, N. And-

ric2, M. Lazarevic Macanovic3, D. Markovic4, A. Radovanovic4, M. Kovacevic Filipovic1. Departments

of 1Pathophysiology and Biochemistry, 2Equine, Small Animals, Poultry and Wild Animal Diseases, 3Radiology

and Radiation Hygiene, 4Histology and Embriology, Faculty of Veterinary Medicine, University of Belgrade,

Serbia.

62 PRO-INFLAMATORY CYTOKINE QUANTIFICATION IN PIGS EXPERIMENTALLY INOCULATED WITH ACTI-

NOBACILLUS PLEUROPNEUMONIAE. M. Quezada¹, A. Islas¹ A. Ruiz¹, T. Casanova¹, C. Lecoq ², D. Munoz².¹Faculty of Veterinary Sciences, University of Concepcion, Chile; 2Agricultural Service of Agricultural Ministery of

Chile.

63 EVALUATIONOF LUNG LESIONSOF PIGS INOCULATEDWITHA LOWPATHOGENIC SEROTYPE 6OFACTI-

NOBACILLUS PLEUROPNEUMONIAE.A. Islas1, M. Quezada1, A. Ruiz1, D. Munoz2, C. Lecocq2. 1Faculty

of Veterinary Sciences, University of Concepcion, Chillan, Chile; 2Agricultural and Livestock Service, Ministry of

Agriculture, Chile, Santiago, Chile.

64 CRAYFISH PLAGUE IN SLOVENIA: FIRST REPORT OF THE PLAGUE AGENT APHANOMYCES ASTACI.

D. Kusar1, A. Vrezec2, M. Ocepek1, V. Jencic1. 1Veterinary Faculty, Ljubljana, Slovenia; 2National Institute of

Biology, Ljubljana, Slovenia.

65 PROTEINURIA INDOGSNATURALLY INFECTEDWITH THEBACTERIUMANAPLASMA PHAGOCYTOPHILUM.

U. Ravnik1, B. Premrov Bajuk2, K. Babnik2, N. Tozon1. 1Small Animal Clinic and 2Institute of Physiology,

Pharmacology and Toxicology, Veterinary Faculty, Ljubljana, Slovenia.

66 EPIDEMIOLOGY OF IBR/IPV, MB/BVD AND SOME RESPIRATORY VIRUSES IN SLOVENIAN AUTOCHTHO-

NOUS BREEDS. M. Nemec1, P. Hostnik2, Z. Klinkon3, T. Zadnik1, M. Klinkon1. 1Veterinary Faculty, Clinic

for Ruminants, Ljubljana, Slovenia; 2Veterinary Faculty, National Veterinary Institute, Virology Laboratory,

Ljubljana, Slovenia; 3Veterinary Clinic Klinkon, Homec, Slovenia.

67 FEASIBILITY OF DETECTION OF TESTOSTERONE AND ESTRADIOL IN HAIR USING COMMERCIAL ELISA

KITS. T. Snoj1, A. Nemec Svete2, N. Cebulj-Kadunc1, S. Kobal1. 1Institute of Physiology, Pharmacology and

Toxicology, Veterinary Faculty, Ljubljana, Slovenia; 2Clinic for Surgery and Small Animal Medicine, Veterinary

Faculty, Ljubljana, Slovenia.



68 ELECTRONMICROSCOPIC EXAMINATIONOF BUFFY COAT: SIMPLEMETHOD USING PLASTICMEMATO-

CRIT TUBE AND APPLICATION FOR DIAGNOSIS OF LYSOSOMAL STORAGE DISEASES IN DOGS AND CATS.

A. Yabuki, S. Yoneshige, K. Mizukami,M.M. Rahman, M.M. Uddin, O. Yamato. Kagoshima University,

Kagoshima, Japan.

69 LYMPHOCYTE ANTIGENS: STABILITY OF EXPRESSION AMONG HEALTHY DOGS AND UPON STORAGE.

F. Riondato, A. Poggi, A. Rota, B.Miniscalco.Dept. of Animal Pathology, Grugliasco (TO), Italy.

70 DIAGNOSTIC MEASUREMENTS OF CANINE SERUM AMYLOID A USING AUTOMATED LATEX AGGLUTI-

NATION TURBIDIMETRY: AN INTERLABORATORIAL COMPARISON. M. Christensen1, J. Ceron2,

A. Tvarijonaviciute2, M. Kjelgaard-Hansen1. 1Department of Small Animal Clinical Sciences, University of

Copenhagen, Frederiksberg, Denmark; 2Department of Animal Medicine and Surgery, University of Murcia,

Murcia, Spain.

71 ELEVATED PLASMA ADIPONECTIN LEVEL AND PERIPHERAL BLOOD LEUKOCYTE ADIPONECTIN RECEP-

TOR EXPRESSION IN INSULIN DEFICIENT DOGS. N. Mori. Nippon Veterinary and Life Science University,

Tokyo, Japan.

72 COMPARING THE USE OF BODY FAT PERCENTAGE VERSUS 5-POINT BODY CONDITION SCORE FOR

ASSESSING OVERWEIGHT STATUS IN DOGS BY SCREENING PLASMA METABOLITIC PROFILES. G. Li,

P. Lee, N.Mori, I. Yamamoto, K. Koh, T. Arai.Nippon Veterinary and Life Science University, Tokyo, Japan.

73 cDNA CLONING AND mRNA EXPRESSION OF DOG CDKAL1. I. Yamamoto, A. Mori, H. Takemitsu,

N.Mori, G. Li, K. Kawasumi, T. Arai.Nippon Veterinary and Life Science University, Tokyo, Japan.

74 DETECTIONOF PROTEINURIA IN DOGSAND CATS –AGREEMENT BETWEENAN IN-HOUSE DRY CHEMIS-

TRY SYSTEM (AUTION MICRO ™) AND CONVENTIONAL WET CHEMISTRY. B. Rutgen, I. Schwendenwein.

University of VeterinaryMedicine Vienna, Vienna, Austria.

75 HEME-MEDIATED BINDING OF ALPHA-CASEIN TO FERRITIN: EVIDENCE OF ALPHA-CASEIN BINDING

TO FERROUS IRON.K.Orino, Y. Yoshikawa, K.Watanabe.Kitasato University, Towada-shi, Japan.

76 C-REACTIVE PROTEIN AND SOME BIOCHEMICAL PARAMETERS IN FEMALE DOGS AFTER OVARIOHYS-

TERECTOMY.N.Hadzimusic. Veterinary Faculty Sarajevo, Sarajevo, Bosnia-Herzegovina.

77 COMPARATIVE STUDY OF PLASMA LIPIDS IN HEALTHY DOGS AND DOGS WITH SKIN DISEASES.

R. Barrera1, P. Ruiz1, C. Zaragoza1, F.J. Duque1, A. Buono2. 1University of Extremadura, Caceres, Spain;2University of Bologna, Bologna, Italy.

78 RELIABILITY OF A SINGLE COLLECTION OF URINE SAMPLE IN DETERMINATION OF URINE PROTEIN TO

CREATININE RATIO. G. Rossi1, A. Zatelli2, S.Paltrinieri3. 1University of Milan, Milano, Italy; 2Clinica Veteri-

naria Pirani, Reggio Emilia, Italy; 3Department of Veterinary Pathology, Hygiene and Public Health,Milan, Italy.

79 MEASUREMENT OF SERUM PROGESTERONE IN MARES: A METHOD COMPARISON STUDY. G. Lubas,

A. Gavazza, A. Rota, D. Panzani, S. Demi. Dept Veterinary Clinic, University of Pisa, San Piero a Grado, Pisa,

Italy.

80 ISCHEMIA-MODIFIED ALBUMIN IN HORSES EXERCISED AT DIFFERENT INTENSITIES ON A TREADMILL.

L.A. Yonezawa1, T.S. Barbosa2, M.J. Watanabe2, J.L. Knaut2, C.L. Marinho2, L.E.S. Michima3,

A. Kohayagawa2. 1School of Veterinary Medicine and Animal Science, Sao Paulo State University, Sao Paulo,

Brazil; 2School of Veterinary Medicine and Animal Science, Sao Paulo State University, Botucatu, Brazil; 3School

of VeterinaryMedicine and Zootechny, University of Sao Paulo, Sao Paulo, Brazil.



81 HIGH MOBILITY GROUP BOX 1 AS A PREDICTOR OF GASTRIC NECROSIS. I. Uhrikova, L. Rauserova-

Lexmaulova, K. Rehakova, I. Hajek, J. Doubek. University of Veterinary and Pharmaceutical Sciences Brno,

Brno, Czech Republic.

82 COMPARISON OF THE SCHMIDT HAENSCH AND ATAGO PAL-USG REFRACTOMETERS FOR DETERMINA-

TION OF URINE SPECIFIC GRAVITY. H. Tvedten1, A. Noren2. 1Swedish University of Agric. Sciences, Vange,

Sweden; 2Stromsholm Specialist Animal Hospital, Stromsholm, Sweden.

83 EFFECT OF PHENOBARBITAL THERAPY ON SELECTED LABORATORY PARAMETERS: A RETROSPECTIVE

STUDY (2007-2012). I. Hajek, P. Schanilec, I. Uhrikova.Veterinary and Pharmaceutical University Brno, Brno,

Czech Republic.

84 APPLICATIONOF PROTEOMICS TO SERA: DIFFERENCESBETWEENVACCINATEDANDNONVACCINATED

SHEEP. J. Mavromati1, G. Mazzuchelli2. 1Veterinary Medicine Faculty, Tirana, Albania; 2University of Liege,

Mass Spectrometry Laboratory GIGA, LIEGE, Belgium.

85 COMPARISON OF URINE PROTEIN / CREATININE RATIO IN URINE SAMPLES COLLECTED BY CYSTOCEN-

TESIS ANDMANUAL COMPRESSION IN CATS – PRELIMINARY RESULTS.H. Vilhena1, R. Santos2, T. Sargo3,

T. Lima4, S. Dias4, F.L. Queiroga3, A.C. Silvestre-Ferreira3. 1Escola Universitaria Vasco da Gama, Oliveirinha

- Aveiro, Portugal; 2Escola Universitaria Vasco da Gama, Coimbra, Portugal; 3Departamento de Ciencias Veter-

inarias - Universidade Tras-os-Montes e Alto Douro, Vila Real, Portugal; 4Policlınica Veterinaria de Aveiro, Aveiro,

Portugal.

86 CHARACTERIZATION OF CANINE BONE MARROW MESENCHYMAL STEM CELLS. C. Tejero1,

L.K. Ostronoff2, M.L. Fermın3, C. Fragio3, L.G. Leon4, E. Kremmer5. 1Universidad Complutense de Madrid,

Facultad de Veterinaria, Madrid, Spain; 2Department of Biochemistry and Molecular Biology IV, Veterinary

Faculty Madrid, Madrid, Spain; 3Department of Animal Medicine and Surgery, Veterinary Faculty Madrid,

Madrid, Spain; 4Institute of Bio-Organic (IUBO-AG), La Laguna, La laguna, Spain; 5Institut fur Immunologie,

GSF-ForschungszentrumUmwelt und Gesundheit, Munchen, Germany.

87 FIBRINOGEN CONCENTRATION MEASUREMENT IN DOGS: A COMPARISON BETWEEN THE QBC-VET

AUTOREADER AND MC1 PLUS SEMIAUTOMATIC COAGULOMETER. L.V. Athanasiou1, T.A. Petanides2,

M.K. Chatzis1, M.N. Saridomichelakis1. 1Faculty of Veterinary Medicine, University of Thessaly, Karditsa,

Greece; 2Veterinary Center of Thessaloniki, Thessaloniki, Greece.

88 REFERENCE INTERVALS FOR HEMATOLOGIC PARAMETERS OF ARABIAN HORSES. A. Rocky, M. Razi

Jalali, S. Gooraninejad, M. Pourmahdi, F. Naghashpour. Faculty of Veterinary Medicine, Shahid Chamran

University, Ahvaz, Iran.

89 A NOVEL POINT MUTATION IN THE BETA1-TUBULIN GENE IN ASYMPTOMATIC MACROTHROMBOCY-

TOPENIC NORFOLK AND CAIRN TERRIERS. M.E. Gelain1, W. Bertazzolo2, G. Tutino2, E. Pogliani2,

F. Cian3, M.K. Boudreaux4. 1University of Padua, Padua, Italy; 2Veterinary Clinic “Tibaldi”, Milan, Italy;3Department Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom; 4Department of

Pathobiology, College of VeterinaryMedicine, Auburn University, Auburn, AL, USA.

90 DETERMINATION OF COMPLETE BLOOD COUNT REFERENCE INTERVALS IN PYRENEAN MOUNTAIN

DOGS.A. Pasquini, I. Annibali, G. Biagi, B. Gugliucci, G. Lubas, A. Gavazza. University of Pisa, San Piero a

Grado (PI), Italy.

91 CANINE BONE MARROW CYTOLOGICAL EXAMINATION: A RETROSPECTIVE STUDY OF 295 CASES.

V. Turinelli1, A. Gavazza2, G. Lubas2. 1Idexx Laboratories Italy, Livorno, Italy; 2Dept. Veterinary Clinic,

University of Pisa, Pisa, Italy.



92 RETROSPECTIVE STUDY OF INCIDENCE AND CLASSIFICATION OF ABNORMALITIES IN FELINE BONE

MARROW BY CYTOLOGICAL EXAMINATION.A. Gavazza1, V. Turinelli2, G. Lubas3. 1University of Pisa, San

Piero a Grado, Pisa, Italy; 2Idexx Laboratories Italy, Milan, Italy; 3Dept Veterinary Clinic, University of Pisa, Pisa,

Italy.

93 USEOF THEMCDhSTAINING FORHAEMATOLOGYOFDOGS, CATS, HORSESANDCATTLE: PRELIMINARY

RESULTS.N. Bourges-Abella, C. Hanot, C. Marche, J.P. Braun, C. Trumel. Institut National Polytechnique-

Ecole Nationale Veterinaire de Toulouse, Toulouse, France.

94 INCREASED ERYTHROCYTE OSMOTIC RESISTANCE IN DOGS WITH A SINGLE EXTRAHEPATIC PORTO-

SYSTHEMIC SHUNT.K. Rehakova1, I. Uhrikova1, L. Rauserova–Lexmaulova1, J. Lorenzova1, L. Stehlik1,

O. Skor2, J. Doubek1. 1University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic;2Veterinary Clinic, Bila Hora, Prague, Czech Republic.

95 COMPARISON OF THE THROMBOTIC MICROSCOPIC PLATELET COUNTING METHOD WITH THE LEUKO-

PLATE MICROSCOPIC, ADVIA 2120 AND IDEXX VETAUTOREAD™ PLATELET METHODS.H. Tvedten1,

J. Ljusner2, I. Lilliehook2. 1Swedish University of Agricultural Sciences, Vange, Sweden; 2Swedish University of

Agricultural Sciences, Uppsala, Sweden.

96 FACTOR VIII LEVELS IN A GROUP OF BELGIAN SHEPHERD MALINOIS DOGS IN ITALY. G. Lubas1,

A. Gavazza2, M. Caldin3. 1University of Pisa, San Piero a Grado, Pisa, Italy; 2University of Pisa, Dept. Veterinary

Clinic, San Piero a Grado, Pisa, Italy; 3SanMarco Veterinary Laboratory, Padova, Italy.

97 PROTHROMBIN TIME MEASUREMENTS USING A NOVEL POINT-OF-CARE DEVICE IN CANINE WHOLE

BLOOD. P. Vajdovich1, J. Antal2. 1Szent Istvan University, Faculty of Veterinary Science, Budapest, Hungary;2Diagon Kft. Hungary, Budapest, Hungary.

98 RETICULOCYTE PARAMETERS IN DOGS WITH LEISHMANIASIS: A PRELIMINARY STUDY. J. Pastor,

A. Melendez-Lazo, C. Miret, M. Planellas, M. Mesalles. Universitat Autonoma de Barcelona, Bellaterra,

Spain.

99 BLOOD PARAMETERS OF CIKA CALVES AND BULLS.M. Klinkon1, G. Gorjanc2, Z. Klinkon3, J. Jezek1,

M. Simcic2. 1Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia; 2Biotechnical Faculty, University of

Ljubljana, Department of Animal Science, Domzale, Slovenia; 3Veterinary Clinic Klinkon, Radomlje, Slovenia.

100 COMPARISON OF THE ABC VET AND BC 2800 VET VETERINARY HAEMATOLOGICAL ANALYZERS.

M.Nemec,M.Klinkon, J. Jezek. Veterinary Faculty, Clinic for Ruminants, Ljubljana, Slovenia.

101 CANINE CBC ANALYSIS WITH THE LASERCYTE AND PROCYTE DX AUTOMATED HEMATOLOGY ANA-

LYZERS.D. DeNicola1, M. Haynes1, C. Lee1, J. Hammond1, M. Wellman2, G. Couto2. 1IDEXX Laboratories,

Inc., Westbrook, United States of America; 2The Ohio State University, Columbus, OH, USA.

102 CANINE RETICULOCYTE ANALYSIS WITH THE LASERCYTE AND PROCYTE DX AUTOMATED HEMATOL-

OGY ANALYZERS. D. DeNicola1, C. Lee1, M. Haynes1, J. Hammond1, J. Christian2, J. Knoll3, M. Scott4,

H. Wamsley5. 1IDEXX Laboratories, Inc., Westbrook, ME, USA; 2Purdue University, West Lafayette, INB, USA;3Tufts University, North Grafton, MA, USA; 4Michigan State University, East Lansing, MI, USA; 5University of

Florida, Gainesville, FL, USA.

103 PORCINE HEMATOLOGY EVALUATION ON THE IDEXX PROCYTE DX HEMATOLOGY ANALYZER.

D.DeNicola, J. Chase, J. Hammond, J. Russell. IDEXX Laboratories, Inc., Westbrook,ME, USA.

104 AGE-ASSOCIATED ALTERATIONS IN A PERIPHERAL LEUKOCYTE SUBPOPULATION AND RESPONSE

OF CYTOKINE SYNTHESIS TO INSULIN IN BEAGLE DOGS. M. Fujiwara1, H. Otsuka2, T. Yonezawa3,



I. Yamamoto1, T. Arai1. 1Nippon Veterinary and Life Science University, Musashino-shi, Tokyo, Japan;2Hiromichi, Kitasato University, Musashino-shi, Tokyo-to, Japan; 3Kitasato University, Towada-shi, Aomori-ken,

Japan.

105 HEME-MEDIATED BINDING OF ALPHA-CASEIN TO FERRITIN: EVIDENCE OF ALPHA-CASEIN BINDING

TO FERROUS IRON.K. Orino, Y. Yoshikawa, K. Watanabe. Laboratory of Veterinary Biochemistry, School of

VeterinaryMedicine and Animal Sciences, Kitasato University, Aomori, Japan.

106 AMELIORATIVE EFFECTS OF DIETARY ALUMINOSILICATES ON BIOCHEMICAL CHANGES IN BROILER

CHICKENS DURING AFLATOXICOSIS.D. Prvulovic1, D. Kojic2, M. Popovic1, G. Grubor-Lajsic2. 1Faculty of

Agriculture, Novi Sad, Novi Sad, Serbia; 2Department of Biology and Ecology, Faculty of Science, Novi Sad, Serbia.

107 DETERMINATION OF ATRAZINE IN SERUM AND URINE OF CATTLE BY HPLC, USING MOLECULAR

IMPRINTED POLYMERS (MIP). S.Z. Peighambarzadeh1, S. Safi2, S.J. Shahtaheri3, M. Javanbakht4,

A. Rahimi Forushani5. 1Department of Veterinary Medicine, Islamic Azad University, Shoushtar Branch,

Shoushtar, Iran;2Department of Clinical Pathology, Faculty of Specialized Veterinary Sciences, Science and

Research Branch, Islamic Azad University, Tehran, Iran;3Department of Occupational Health, School of Public

Health, Center for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran;4Department of

Chemistry, Amirkabir University of Technology, Tehran, Iran;5 Department of Biostatistics, School of Public

Health, Tehran University ofMedical Sciences, Tehran, Iran.

108 CHARACTERIZATION OF STEM CELL MARKERS ON BONE MARROW- AND ADIPOSE TISSUE-DERIVED

CANINE MESENCHYMAL STEM CELLS. Y. Takemitsu, D. Zhao, I. Yamamoto, T. Arai. Veterinary and Life

Science University, Musashino-shi Tokyo, Japan.

109 RARELY USED METHODS FOR INVESTIGATING PORTOSYSTEMIC SHUNTS IN DOGS. C. Smuts1,

M. Bennett1, M. Sharman2, J. Mills1, T. Gaal1. 1Murdoch University, Perth, Australia; 2Melbourne University,

Melbourne, Australia.

110 RELATIONSHIP BETWEEN COLLIE EYE ANOMALY AND IVERMECTIN TOXICOSIS-ASSOCIATED MUTA-

TIONS: ANALYSIS ACROSS MULTIPLE DOG BREEDS. K. Mizukami1, H-S. Chang1, D. Endoh2, A. Yabuki1,

O. Yamato1. 1Kagoshima University, Kagoshima, Japan; 2Rakuno Gakuen University, Ebetsu, Japan.

111 ABERRANT ANTIGEN EXPRESSION IN CANINE LYMPHOMA: A RETROSPECTIVE STUDY. V. Martini1,

A. Pasqua1,M.E. Gelain2, S. Comazzi1. 1University ofMilan,Milan, Italy; 2University of Padua, Padua, Italy.

112 CLINICAL PRESENTATION CURRICULUM IN VETERINARY EDUCATION: A COGNITIVE PERSPECTIVE.

S. Safi1, P. Hemmati2, S.H. Shirazi-Beheshtiha3. 1Department of Clinical Pathology, Faculty of Specialized

Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran; ²Ministry of Health and

Medical Education, Center for Disease Control, Senior Officer of Center for Disease Control, Tehran, Iran; ³Depart-ment of Clinical Sciences, Faculty of VeterinaryMedicine, Karaj Branch, Islamic Azad University, Karaj, Iran.

113 STATISTICAL STUDY ON PREVALENCE OF TUMORS IN INSURED DOGS IN JAPAN. A. Shimamura,

T. Arai.Nippon Veterinary and Life Science University, Tokyo, Japan.

114 N-ACYLHOMOSERINE LACTONES OF PSEUDOMONAS AERUGINOSA IN CLINICAL SAMPLES FROM

CANINE OTITIS EXTERNA: VALIDATION OF AN ANALYTICAL METHOD USING LIQUID CHROMATOGRAPHY

WITH TANDEM MASS SPECTROSCOPY (LC-MS/MS). K. Srimpf, J. Hosseini, T. Kotnik, D. Kusar,

M. Vengust, G. Tavcar-Kalcher.University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia.

115 PATHOLOGY OF LEGWEAKNESS IN BROILER CHICKENS. I. Dinev. Dept of General & Clinical Pathology,

Faculty of VeterinaryMedicine, Trakia University, Stara Zagora, Bulgaria.



116 PATHOMORPHOLOGICAL INVESTIGATIONS OF THE INCIDENCE OF AXIAL SKELETON PATHOLOGY

ASSOCIATED WITH POSTERIOR PARALYSIS IN COMMERCIAL BROILER CHICKENS. I Dinev. Department of

General and Clinical Animal Pathology, Faculty of VeterinaryMedicine, Trakia University, Stara Zagora, Bulgaria.

117 HISTOPATHOLOGIC FINDINGS IN ABOMASA OF FALLOW DEER FROM THE BRIJUNI REGION IN

CROATIA. M. Robic, B. Artukovic, A. Beck, R. Turk, M. Belic, Z. Grabarevic. Veterinary Faculty, Zagreb,

Croatia.

118 COMPARISON OF TOTAL LEUKOCYTE RESPONSES OF HORSES SUBJECTED TO EXERCISE ON HIGH

SPEED TREADMILL – PRELIMINARY RESULTS. T. Barbosa, L. Yonezawa, C. Marinho, M.J. Watanabe,

A. Kohayagawa. School of Veterinary Medicine and Animal Science - Sao Paulo State University, Botucatu,

Brazil.

119 EFFECT OF EXERCISE ON A HIGH-SPEED TREADMILL ON DNA DAMAGE IN PERIPHERAL LEUKOCYTES

OF HORSES. T. Barbosa, L. Yonezawa, C. Marinho, J. Knaut, M.J. Watanabe, D. Salvadori, D. Almeida,

A. Kohayagawa. School of Veterinary Medicine and Animal Science - Sao Paulo State University, Botucatu,

Brazil.

120 REFERENCE VALUES FOR HAEMATOLOGICAL PARAMETERS IN THE LUSITANOHORSE.A.C. Silvestre-

Ferreira1, M. Cotovio1, M.Maia1, F. Queiroga2, M.J. Pires2, A. Colaco2. 1Department of Veterinary Sciences

– Veterinary Hospital – University of Tras-os-Montes e Alto Douro, Vila Real, Portugal; 2Department of Veterinary

Sciences –CECAV - University of Tras-os-Montes e Alto Douro, Vila Real, Portugal.

121 MONONUCLEAR CELL CONCENTRATIONS IN FRACTIONATED SAMPLES OF BONE MARROW

ASPIRATES FROM HORSE STERNUM. F. Correa, J. Galecio, H. Bustamante, F. Wittwer, A. Ramirez,

B.Menarim.Universidad Austral de Chile, Valdivia, Chile.

.Oral Platform Presentations

1EFFECT OF TIME RELATED TO CALVING AND RUMENPROTECTED CHOLINE (RPC) SUPPLEMENTATION ONBLOOD ANALYTES AND ON LIVER LIPID AND GLYCO-GEN. T. Gaal1, P. Elek2, F. Husveth3. 1School of Veteri-nary and Biomedical Sciences, Faculty of Health Sciences,Murdoch University, Perth, Australia; 2Provimi HungaryCo. Zichyujfalu, Hungary; 3Department of Animal Sci-ence, Georgikon Faculty of Agriculture, University ofPannonia, Keszthely, Hungary.

Background: High producing dairy cows have temporary

changes in blood analytes and may develop fatty liver after calv-

ing. The latter might be prevented by supplementation of the diet

with RPC. Objective: The aim of this study was to compare

some blood and liver parameters in both control and RPC-supple-

mented transition cows to their pre-calving values and textbook

reference ranges. Methods: Blood and liver biopsy samples

were obtained from 16Holstein Friesian cows supplementedwith

RPC and from 16 control animals 21 days before and 7, 21, 35,

and 60 days after calving. Blood glucose, triglycerides, choles-

terol, nonesterified fatty acids, beta-hydroxybutyrate, urea,

ammonia, and AST with liver total lipids, triglycerides and glyco-

gen were measured. Effects of time-to-calving interval and RPC

supplementation on all analytes were statistically evaluated.

Results: Significant time-related changes in all blood parame-

ters were found in both groups after calving with the highest

increase in blood NEFA, BHB, AST and ammonia (8x, 3.5x, 2x,

and 1.7x, respectively) on d7/d21 when compared to pre-calv-

ing values. RPC cows had relatively lower NEFA, BHB, and

ammonia with higher TG post-partum than controls. Liver TL

and TG increased and liver glycogen decreased in both groups

after calving, with milder fatty infiltration of the liver in

RPC-cows. Liver glycogen was not influenced by RPC supple-

mentation. Conclusion: Due to significant changes post-

partum, textbook reference ranges for adult cows can only be

used with care in transition cows. Choline supplementation of

the diet may also influence these results.

2ASSOCIATION BETWEEN HEMATOLOGICAL ANDELECTROPHORETIC PROFILES AND PREVALENCE OFPOST-PARTUM DISEASES IN DAIRY COWS. S. Paltri-nieri1, P. Moretti2, M. Venturini2, N. Morandi3,A.Giordano4. 1University of Milan, Milan, Italy; 2CentralLaboratory, Large Animal Hospital, Milan, Italy; 3ParcoTecnologico Padano, Lodi, Italy; 4Department of Veteri-nary Pathology, Hygiene and Public Health, Milan, Italy.

Background: Opportunistic bacteria can induce post-partum

diseases (e.g., metritis, mastitis, digital dermatitis, retained pla-

centa, etc.) in dairy cows. Metabolic disturbances can predispose



to these diseases and the association between the prevalence of

disease and changes in chemical metabolites is well known.

Conversely, little is known about the association between the

prevalence of disease and haematological or electrophoretic

profiles. Objective: To investigate the possible presence of

haematological and electrophoretic profiles associated with sub-

clinical inflammation potentially predisposing to post-partum

diseases. Methods: The health status of 3 dairy herds was

monitored over a 3-month period. Routine hematology and

serum protein electrophoresis were performed on samples

collected 3 days after parturition from 110 animals (herd 1 = 40;

herd 2 = 36; herd 3 = 34). Clinical events occurring in the transi-

tion period were recorded. Haematological and electrophoretic

results recorded in the 3 herds from animals that remained

clinically healthy were statistically compared to each other.

Results: The prevalence of post-partum diseases in herd 2

(22.2%) was significantly lower (P<0.05) than that recorded in

herd 1 (42.5%) and in herd 3 (47.1%). No significant differences

were found between leukograms and electrophoretograms

recorded in herd 2 compared with herds 1 and 3. Conversely,

RBC, Hb and Ht were significantly higher in herd 2 compared

with other herds. Conclusions: The prevalence of post-partum

disease is not associated with leukogram abnormalities or protein

profiles potentially predisposing to infections. Conversely, the

different erythrograms suggest that lower prevalence of disease is

associatedwith a bettermetabolic profile.

3HEMATOLOGICAL CHANGES ASSOCIATED WITHRETAINED PLACENTA IN CATTLE. P. Moretti1,A. Giordano2, M. Venturini3, N. Morandi4,S. Paltrinieri2. 1Large Animal Hospital, Lodi, Italy;2Department of Veterinary Pathology, University ofMilan,Milan, Italy; 3Central Laboratory, Large Animal Hospital,Lodi, Italy; 4Parco Tecnologico Padano, Lodi, Italy.

Background: Retained placenta (RP) occurs frequently in cattle

but little is known about the pathogenic or prognostic role of

hematological changes in this disease. Objective: To describe

hematological changes associated with RP and to assess their pos-

sible prognostic relevance. Methods: Blood samples were col-

lected 2 to 4 days after calving in cattle from two different herds

(A: n=45; B: n=45). Seventeen cows were affected by RP in herd

A and 27 cows in herd B. Results from RP-affected cows were

compared with those of clinically healthy controls (28 from herd

A; 18 from herd B). The follow-up of RP-affected cows was

monitored during the whole lactation period and they were

then subgrouped based on the presence (8 in herd A; 8 in herd

B) or absence (9 in herd A; 19 in herd B) of further health prob-

lems. Results: In both herds, RP-affected cows had lower neu-

trophil counts (associated with lower total leukocyte counts in

herd B) and higher RBC, Ht and Hb values.Cattle showing com-

plications after pregnancy (metritis, mastitis, digital dermatitis)

had significantly lower RBC, leukocyte, and lymphocyte counts

compared with cattle that did not have complications. Conclu-

sions: Leukopenia and neutropenia are associatedwith RP. Leu-

kopenia and lymphopenia in the early postpartum period could

predispose to further complications during transition and lacta-

tion. The possible association between leukopenia and RP will be

further investigated within the ProZoo project, focused on the

identification of the genetic basis of resistance/susceptibility to

diseases.

4SURVEY OF VITAMIN A, BETA-CAROTENE, CALCIUMAND PHOSPHORUS CONCENTRATIONS IN SERUM OFEWESWITH RETAINED PLACENTA.A.Davasaztabrizi.Department of Clinical Sciences, Tabriz Branch, IslamicAzad University, Tabriz, Iran.

Background: Retained placenta (RP) may result from a num-

ber of factors, such as abortion, induced labor, delayed gestation,

early parturition, uterine atony, infections, and seasonal and hor-

monal disorders. In addition, it is well known that deficiencies of

some vitamins and minerals induce or predispose animals to RP.

Objective: The objective of the present study was to investigate

the relationship between blood serum concentrations of vitamin

A, beta-carotene, calcium and phosphorus and the development

of RP in ewes. Method: 25 ewes had retained placenta; they

had not expelled their placenta after parturition. 20 others had

expelled their placenta normally after parturition. Blood samples

were obtained from all study animals from the jugular vein by

venipuncture. Samples were centrifuged, sera were prepared,

and concnetrations of vitamin A, beta-carotene, calcium and

phosphorus were measured. Results: Data showed that there

was no significant difference in vitamin A and b-carotenebetween the two groups (P>0.05), whereas there was a signifi-

cant difference in mean value of calcium and posporous between

the two groups.Conclusion: This research indicated that imbal-

ance of calcium and phosphorous and their deficiency can induce

retained placenta in ewes in the Tabriz region of Iran.

5URINE PROTEOME INDOGSAFFECTEDBY LEISHMAN-IASIS. A. Buono1, F. Dondi1, E. Ferlizza1, G. Isani1,J. Duque2, P. Tapia2, C. Zaragoza2, R. Barrera3. 1Uni-versity of Bologna, Ozzano Emilia, Italy; 2University ofExtremadura, Caceres, Spain; 3Department of InternalMedicine, University of Extremadura, Caceres, Spain.

Background: Leishmaniasis is a frequent cause of nephropathy

in dogs. Evaluation of the urine proteome is a useful and nonin-

vasive method to monitor therapy and discover novel biomar-

kers. Objective: To compare different electrophoretic methods

for urine protein separation and monitor therapy in dogs affected

by leishmaniasis. Methods: Urine samples were collected from

43 dogs with leishmaniasis. UP/C, UA/C and high-resolution

electrophoresis (HRE) were performed on all samples. Moreover,

SDS-PAGE (26 samples) and a semi-automated SDS-AGE (12

samples) were performed. Results: UP/C and UA/C median

(range) values were 1.45 (0.06-20.08) and 0.38 (0.01-14.8),

respectively. With HRE different electrophoretic patterns were

evident and urinary albumin concentrations (median 32 mg/dL;

range 5-463 mg/dL) and percentage (mean±SD; 39±13.7) were

determined. With SDS-AGE different patterns of proteinuria

were found: 9 dogs had mixed proteinuria (complete or incom-

plete) and 3 had nonselective glomerular proteinuria. Upon SDS-

PAGE, 35-40 different bands were commonly visualized.The

most abundant proteins were at molecular weights of 71, 66, 55,

46, 37, 26, 22, 14 kDa. Conclusion: HRE is a useful and rapid

method to monitor renal function and therapeutic effective-

ness. In addition, urinary albumin concentration can also be

quantified. SDS-AGE, according to literature, allows the locali-

zation of nephronal damage. SDS-PAGE, due to its high sensi-



tivity, permits visualization of many urine proteins, which

could be studied as novel biomarkers of nephropathy.

6EVALUATION OF THE REVISED QUANTITATIVE INSU-LIN SENSITIVITY CHECK INDEX IN DAIRY COWSSHOWING VARIOUS FORMS OF KETONE PATTERNAND PUERPERAL METRITIS. M. Kerestes1, V. Faigl1,M. Kulcsar1, O. Balogh1, J. Foldi2, H. Febel3,Y. Chilliard4, G. Huszenicza1. 1Faculty of VeterinaryScience, Budapest, Hungary; 2Intervet, Budapest,Hungary; 3Research Institute for Animal Breeding andNutrition, Herceghalom, Hungary; 4INRA, Saint-Genes-Champanelle, France.

Background: In dairy cows, insulin resistance may play a piv-

otal role in the pathogenesis of metabolic diseases. The revised

quantitative insulin sensitivity check index (RQUICKI ) was eval-

uated previously in healthy cows. There are no data on changes

in RQUICKI, in periparturient or hyperketonemic cows. Objec-

tive: The aim of the study was to evaluate the RQUICKI in hy-

perketonemic cows without or with puerperal metritis.

Methods: Blood samples were collected from 28 Holstein

cows from -18 d before through 70 d after calving for ß-hy-

droxybutyrate (BHB), non-estherified fatty acids, glucose, and

insulin. Animals were subjected to intravenous glucose (GTT)

and insulin tolerance test (ITT) at 3 time points. Animals were

assigned into four groups: normoketonemic (NK; n=9); tran-

siently hyperketonemic (transient HK; n=7); continuously HK

(continuous HK, n=7); and continuously HK with puerperal

metritis (continuous HK+PM; n=6). Results: Insulin area

under the curve and insulin response to glucose were lower in

the early postpartum period than in late pregnancy or mid-lac-

tation (P<0.001) and in cows affected by continuous HK

(P<0.001). Insulin-stimulated blood glucose decrease was lower

in HK cows with/without PM. There was a strong time effect

on RQUICKI value (P<0.001). There was no difference in the

RQUICKI in cows with different forms of HK, and no correla-

tion between the RQUICKI and tolerance tests was observed.

Conclusion: The RQUICKI is affected by the considerable

changes of plasma metabolic concentrations around calving and

has low discrimination power in diagnosing decreased insulin

sensitivity in cows affected by variousmetabolic diseases.

7DIAGNOSTIC VALUE OF ACUTE PHASE INDEX OFMILK IN DETECTION OF SUBCLINICAL MASTITIS INDAIRY COWS. S.H. Shirazi-Beheshtiha1, S. Safi2,V. Rabbani1, M.B. Bolourchi3. 1Karaj Branch, IslamicAzad University, Karaj, Iran; 2Science and ResearchBranch, Islamic Azad University, Tehran, Iran; 3Faculty ofVeterinaryMedicine, University of Tehran, Tehran, Iran.

Background: Currently, somatic cell count (SCC) and bacterial

culture are considered the reference standard methods for diag-

nosis of subclinical mastitis. However, SCC has a low diagnostic

accuracy and bacterial culture is time-consuming. For the identi-

fication of animals with subclinical mastitis, new biomarkerswith

high diagnostic accuracy are needed.Objective: The aim of the

present study was to compare the clinical accuracy of milk amy-

loid A, whey proteins and acute phase index (API) with SCC and

bacteriology for the diagnosis of bovine subclinical mastitis.

Methods: A total of 90 clinically healthy cows were randomly

selected. Of these, 56 cows were considered to have subclinical

mastitis based on a SCC higher than 1009 1,000 cells/mL of milk

and positive bacterial culture results of milk samples obtained

from at least one of the quarters. Milk amyloid A and whey pro-

teins were measured in the samples and API was calculated.

Receiver-operating characteristic analysis was used to assess the

performance of each test using SCC and bacterial culture as the

reference methods. Results: API was the most accurate test

with a diagnostic sensitivity of 98.2%, specificity of 94.1% and

clinical accuracy of 0.994 at cutoff point of > 0.105. Conclu-

sion: Based on the high sensitivity and specificity of milk API

and milk amyloid A in diagnosis of subclinical mastitis observed

in this study, we recommend these methods be used as reliable

alternative or complementary tests to the routine SCC for early

diagnosis of subclinical mastitis in order to minimize the eco-

nomic losses due to this disease.

8A CASE OF CANINE SURRA IMPORTED INTOGERMANY. M. Defontis1, J. Richartz1, N. Engelmann1,C. Bauer1, V.M. Schwierk2, P. Buscher3, A. Moritz1.1Justus-Liebig University, Giessen, Germany; 2Pulheim-Stommeln, Germany; 3Antwerpen, Belgium.

A 9-year-old male Jack Russell Terrier with a travel history to

Thailand was presented with chronic lethargy, weight loss and

unilateral ocular opacity. On blood smear evaluation numerous

trypomastigotes were observed. Molecular identification of the

parasite revealed infection with Trypanosoma evansi, the causative

agent of Surra. The patient was treatedwith suramin and suffered

a relapsewith central neurological symptoms after 88 days. Histo-

pathological evaluation revealed marked non-purulent menin-

goencephalitis and PCR testing in CSFwas positive for T. evansi.

9CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL CELLLYMPHOMA IN A HORSE. F. Cian1, G. Tyner2,V. Martini3, S. Comazzi3, J. Archer1. 1Departmentof Veterinary Medicine, University of Cambridge,Cambridge, UK; 2Chiltern Equine Clinic, Chalfont StGiles, UK; 3Department of Veterinary Pathology, Univer-sity of Milan,Milan, Italy.

A 16-year-old Irish Draft mare was admitted to the Chiltern

Equine Clinic for an annual health check. The mare was in excel-

lent body condition. Mild generalized lymphoadenomegaly was

identified. Rectal palpation and transrectal ultrasonography

revealed prominent mesenteric lymph nodes. A transcutaneous

abdominal ultrasonographic evaluation was unremarkable.

Hematology revealed a marked leukocytosis (63.06 x10^9/L)

with lymphocytosis (58.2 x10^9/L) with themajority being small

lymphocytes. No evidence of anemia or thrombocytopenia was

identified. The serum chemistry profile showedmild hyperglobu-

linemia. Severe hypoglycemia and mild elevation in phosphate

were likely to be due to delayed analysis. Serum protein electro-

phoresis was unremarkable. Fine-needle aspirates of multiple

lymph nodes and a bone marrow aspirate were performed, both

revealing the presence of a monomorphic population of small

lymphocytes similar to those observed in the peripheral blood

indicating the presence of a leukemic small cell lymphoma/



chronic lymphocytic leukemia (CLL). Considering the

lymphoadenomegaly was identified at the same time as the

lymphocytosis, the distinction between these two conditions

could not be made. The peripheral blood was examined by flow

cytometry and immunocytochemistry and the results suggested a

T cell phenotype. As the horse remained clinically normal no

treatment was performed though regular re-examinations were

undertaken. Small cell lymphoma and leukemia are rare in horse

and according to literature they are most commonly of T cell

phenotype. To the authors’ knowledge this is the first report of

combined use of immunocytochemistry and flow cytometry in a

horse with leukemic lymphoma/CLL.

10CASE REPORT: CUTANEOUS T-CELL LYMPHOMA.B. Rutgen1, I. Flickinger2, B. Wolfesberger2, B. Litsc-hauer2, A. Fuchs-Baumgartinger3, S.E. Essler4,A. Saalmuller4, I. Schwendenwein5. 1University ofVeterinary Medicine Vienna, Vienna, Austria; 2Clinic forInternal Medicine, 3Institute of Pathology, 4Institute ofImmunology, and 5Central Laboratory, Department forPathobiology, Universtiy of Veterinary Medicine, Vienna,Austria.

A 7-year-oldmale Boxer with a 3.5 year history of atopy and food

hypersensitivity was presented with multiple diffuse nodules and

macules of the skin and tongue and jaundiced mucosal mem-

branes. Cytologic and histologic examination of the skin lesions

revealed cutaneous epitheliotropic lymphoma. Cells showed

expression for CD3+ and CD8+ in flow cytometry. CBC showed a

moderate leukocytosis with 16% atypical lymphocytes with

irregularly cleaved nuclei. In peripheral blood FCM showed an

elevated proportion of the CD8+T lymphocyte subpopulation,

indicating a malignant population of T cell origin and a monoclo-

nal band for TCRc in the PCR for antigen receptor rearrangement.

Liver enzyme activity was markedly elevated and abdominal

ultrasound showed increased echogenicity of the liver and

lymphadenomegaly. Fine-needle aspirates of the liver confirmed

infiltration with malignant lymphocytes showing the same mor-

phology as the cells detected in skin and peripheral blood. Treat-

ment was induced with L-asparaginase, CCNU and prednisone.

Partial clinical remission of the skin and tongue lesions was

achieved within 10 days and hematological abnormalities

resolved. Despite further treatment with L-asparaginase and

CCNU the dog relapsedwithin 1month andwas euthanized.

11CHANGES IN THE CONCENTRATIONS OF SERUMAMYLOID A (SAA) FOLLOWING SURGERY, TRAUMAOR SNAKE ENVENOMATION IN DOGS: A STUDY OFTHE KINETICS OF SAA. M. Christensen1, E. Moldal1,R. Langhorn1, A. Goddard2, A. Tvarijonaviciute3,M. Kjelgaard-Hansen1. 1Department of Small AnimalClinical Sciences, University of Copenhagen, Frederiks-berg, Denmark; 2Department of Companion Animal Clin-ical Studies, University of Pretoria, South Africa;3Department of Animal Medicine and Surgery, UniversityofMurcia, Murcia, Spain.

Background: Serum amyloid A (SAA) has proven useful for

diagnostic purposes in dogs, but advanced knowledge about the

kinetics is needed if follow-up measurements should be used for

monitoring purposes. Objective: To investigate the kinetics of

SAA in dogs by measuring the concentrations at different inter-

vals after castration/ovarioectomy/ovariohysterectomy, trauma

(blunt accidental traumas), and snake envenomation (vipers or

cobras) in dogs. Material: Serum was collected prior to surgery

and 6, 12, 24, 48, 72, and 96 hours after (n=5-30); <12, 12-24,

36-48, and 60-72 hours post-trauma (n=3-9), and <6, 6-18, 24-

30, and 36-48 hours post-envenomation (n=19-40).Method: A

validated latex agglutination turbidimetric assay was used for

measurements of SAA. Changes in concentrations were tested for

significance using Kruskal-Wallis and Dunns multiple compari-

son tests (significance level P<0.05). Results: Significant 2 to

337-fold increases of SAA were observed 24 hours post surgery

(n=21). Similar results were observed in snake envenomed dogs

showing significant 1 to 13-fold increases in SAA between <6

hours and 6-18 hours (n=13) and 24-30 hours (n=35), respec-

tively. Significant 7 to 50-fold increases in SAA were observed

post-trauma <12 and 12-24 hours (n=6). No additional increases

after 24 hours were observed in any of the groups. SAA decreased

significantly 96 hours post-surgery towards pre-surgical concen-

trations (n=5). Conclusion and perspectives: Significant

increases in SAAwere observed in the first 24 hours post-surgery,

trauma and envenomation, representing three etiologically dif-

ferent causes of systemic inflammation. Additional increases after

24 hours should, however, not be expected in uncomplicated

cases.

12EVALUATION OF A CANINE SPECIFIC QUANTITATIVEPOINT-OF-CARE IMMUNOASSAY FOR MEASURINGC-REACTIVE PROTEIN IN DOGS. M. Costa, K. Papaso-uliotis. School of Veterinary Sciences, University of Bris-tol, Bristol, UK.

Background: Measurement of C-reactive protein (CRP) aids in

the diagnosis and management of various infectious, inflamma-

tory, immune mediated or neoplastic diseases. Objective: To

evaluate a new canine specific point-of-care (POC) immunoassay

(TECOmedical, Switzerland). Methods: CRP was measured in

40 canine serum clinical samples by a previously validated

canine immunoturbidometric assay (Reactivlab, Scotland), a

new canine ELISA (TECOmedical, Switzerland) and the POC.

The POC was evaluated with intra- and inter-assay precision

studies (seven to ten consecutive runs of low and high concen-

tration samples) and with linearity studies (equal step dilutions

of a high concentration sample [111.2 mg/L]). Results: The

intra-assay coefficient of variation (CV) was 18.2% with a low

(15.6 mg/L) and 37.8% with a high concentration (111.2 mg/L)

sample. The inter-assay CVwas 52.5%with a low (6.9 mg/L) and

13.6%with a high concentration (145.1 mg/L) sample. Linearity

was acceptable. The Spearman’s correlation coefficient (rs) a)

between the POC (median: 13.4, range: 5.1-194.6 mg/L) and

TECO ELISA (median: 8.6, range: 0–148 mg/L) was 0.89

(p<0.0001) and b) between the POC and Reactivlab (median:

21.9, range: 0.1 – 367 mg/L) was 0.81 (p<0.0001). Agreement

studies (Deming regression and Bland-Altman difference plots)

between the POC and Reactivlab methods revealed a negative

bias (mean:-33, 95% limits of agreement:-169 to 103 mg/L),

mainly due to samples with high concentrations. Agreement

studies between the POC and ELISA showed a positive bias

(mean: 13.9, 95% limits of agreement:-46 to 74 mg/L).



Conclusions: The POC has an acceptable performance for clini-

cal purposes, but results obtained by the POC and the other two

methodologies cannot be used interchangeably.

13PCR-BASED DETECTION OF A. PHAGOCYTOPHILUMDNA IN PARAFFIN-EMBEDDED SKIN BIOPSIES FROMDOGS SEROPOSITIVE AGAINST A. PHAGOCYTOPHI-LUM. I. Berzina1, N. Muller2, C. Krudewig3, I.Matise1, R. Ranka4, M. Welle3. 1Faculty of VeterinaryMedicine, Latvia University of Agr, Jelgava, Latvia;2Institute of Parasitology, Vetsuisse-Faculty, University ofBern, Bern, Switzerland; 3Institute of Animal Pathology,Vetsuisse-Faculty, University of Bern, Bern, Switzerland;4Latvian Biomedical Research and Study Center, Riga,Latvia.

Background: Canine granulocytic anaplasmosis (CGA) is

caused by the rickettsial microorganism A. phagocytophilum. CGA

is mostly characterized by fever, joint problems, lethargy, anor-

exia, thrombocytopenia and other nonspecific clinical signs. Skin

lesions have been described in naturally infected lambs and

humans. The pathobiology of CGA is not entirely clear and the

persistence of the organism after the resolution of clinical signs

has been questioned. Objective: To investigate if A. phagocyto-

philum is associated with skin lesions in dogs seropositive against

the bacterium. Methods: Tissue blocks from skin biopsies of 12

seropositive dogs were evaluated. Histology of the skin lesions did

not indicate an apparent cause. DNA was extracted from the par-

affin-embedded tissues and a conventional PCR for partial 16s

rRNA genewas performedwith the carry-over elimination by the

use of uracil-DNA glycosylase. Results: In skin biopsies from 3

dogs A. phagocytophilum DNA was amplified, and two of the

resulting sequences were 100% identical to the prototype

A. phagocytophilum human strain (accession number at the

GenBank U02521). Titers of the PCR-positive dogs were 1:320,

1:800 and 1:2048. Time period between serology and biopsy

sampling ranged between 4-90 days. Histopathologically skin

lesionswere characterized bymoderate to severe edema and vari-

able hemorrhage. In addition a superficial and periadnexal mixed

cell infiltrate andmultifical nodules composed of neutrophils and

histiocytes were seen. No obvious signs of vasculitis were found.

Conclusion: A. phagocytophilum DNA can be recovered from

skin lesions in dogs seropositive against A. phagocytophilum and

may be causative for the skin disease.

14PRELIMINARY REFERENCE INTERVALS FOR FLUORO-GENIC MEASUREMENT OF THROMBIN GENERATIONIN THE BEAGLE DOG. M. Defontis1, S. Cote2,M. Stirn2, D. Ledieu2. 1Justus-Liebig University,Giessen, Germany; 2Basel, Switzerland.

Background: The serine protease thrombin plays a crucial role

in the coagulation cascade. Fluorogenic measurement of its for-

mation after tissue factor stimulation using the Calibrated Auto-

mated Thrombogram (CAT) is a more sensitive tool to detect

thrombotic or hemorrhagic tendencies compared with the plas-

matic coagulation times PT and aPTT.Objective: The aim of this

study was to determine preliminary reference intervals for the

CAT variables in platelet-poor plasma collected from healthy

Beagle dogs. Methods: Citrated blood was taken from 20

healthy Beagle dogs (10 males and 10 females between 12 and 20

months of age) by jugular venipuncture and platelet-poor plasma

was immediately prepared and stored at -80°C for a maximum 2

weeks. The animals were kept under controlled housing condi-

tions and had not received any medication for the last 15 days.

For each animal, CAT measurements were performed in tripli-

cate. The results were displayed on histograms for visual inspec-

tion and a 90% double-sided reference interval was calculated

for the following parameters: Lagtime, Time to peak, Peak height,

ETP (Endogenous Thrombin Potential) and Start Tail using the

MedCalc® software and a robust method (CLSI Guidelines C28-

A3). Results: The calculated 90% double-sided reference inter-

vals were as follow: Lagtime=1.1-1.9 min, Time to peak=2.5-3.6

min, Peak height=83.9-155.4nM, ETP=241.5-375.3nM/min and

Start Tail=5.0-15.2min.Conclusion: The Calibrated Automated

Thrombogram can be used in the Beagle dog to quantify throm-

bin generation.

15FLOW CYTOMETRIC EVALUATION OF DRUG RESIS-TANCE IN CANINE LYMPHOMA SHORT-TERM CELLCULTURES. P. Vajdovich. Szent Istvan University, Fac-ulty of Veterinary Science, Budapest, Hungary.

Background: Chemotherapy resistance is a critical factor in re-

ponse to treatment. Prediction of response of the neoplastic cells

to various cytostatic drugs can help in choosing the appropriate

therapy. Objective: The aim of the study was to evaluate the

apoptotic response of the cells derived from lymph nodes of dogs

with lymphoma to cytostatic drugs.Methods: Six dogs with dif-

fuse large B-cell lymphoma were included in the study. Lympha-

tic cells were separated, a cell suspension was made for

immunophenotyping and a short-term cell culture was also pre-

pared. Immunophenotype of cells was evaluated by flow cytome-

ter. The followingmarkers were examined: CD3, CD4, CD5, CD8,

CD14, CD21, CD34, CD45, and MHC II. Lymph nodes were eval-

uated by immunohistochemistry. Proliferation ability was evalu-

ated by the Ki67 marker. Cell cultures were treated with the

following drugs: vincristine (Vin), cyclophasphamide (Cyc),

doxorubicin (Dox), methylprednisoslone (Mpred), and etoposide

(Eto). Etidium bromide treatment of cells was used to differenti-

ate apoptotic or viable cells. Results: The apoptotic rate

increased in cells according to the following course Cyc, Vin,

Mpred, Dox, Eto. Apoptotic rate in cell cultures correlated

between Dox and Cyc (r=0.974), and between Vin and Mpred

(r=0.968). The cell suspensions rich in CD14-positive cells

(monocyte marker) and CD34-positive cells (haemopoietic stem

cell marker) negatively correlated with the apoptotic rate in cell

cultures treated by Vin (CD14, r=-0.994, CD34, r=-0.995) and

Mpred (CD14, r=-0.980, CD34, r=-0.981). Conclusion: Flow

cytometric evaluation of cytostatic resistance can help in proper

drug selection for the treatment of canine lymphoma.



16MEASUREMENT OF SOLUBLE MESOTHELIN RELATEDPEPTIDES (SMRP) IN CANINE BODY CAVITY EFFU-SIONS.M.Costa1, K.Murphy2, D.Wilson1, K. Papaso-uliotis1. 1School of Veterinary Sciences, University ofBristol, Bristol, UK; 2Bath Veterinary Referrals, Bath, UK.

Background: In humans, measurement of Soluble Mesothelin

Related Peptides (SMRP) has been considered useful in differenti-

ating mesothelioma from other malignant or benign effusions. It

has been shown that human and canine mesothelins share the

same open reading frame. Objective: To measure SMRP in

canine body cavity effusions by the MesoMARK™, an ELISA

designed for humans that uses the monoclonal antibodies OV569

and 4H3 (Fujirebio Diagnostics, Inc, USA). Methods: Thirty-

two canine effusions (19 pleural, 11 peritoneal, 2 pericardial) col-

lected over 18months were included in the study. Final diagnosis

was based on clinical, cytological, clinicopathological, imaging

and histopathological findings. Aliquots of the fluids were stored

at -20°C until measurement of SMRP concentrations with the

MesoMARK™. According to manufacturer’s instructions a

calibration curve was constructed using six SMRP standards

(0-32nmol/L). Results: 10 effusions were neoplastic (mesothe-

lioma [n=4], lymphoma [n=2], carcinoma [n=3], undifferenti-

ated [n=1]), 16 were non-neoplastic (chylous [n=1], septic

[n=1], transudate/modified transudate [n=14]) and 6 contained

suspicious or atypical mesothelial cells on cytology. During the

study, 3 calibration curves were constructed; optical densities

(absorbance) for the 0, 2, 4, 8, 16 and 32 nmol/L standards ranged

from -0.003-0.003, 0.086-0.113, 0.165-0.199, 0.298-0.350,

0.493-0.594and 0.804-1.024, respectively. The absorbances

generated by all clinical samples ranged from -0.004 to 0.010.

Conclusions: SMRP was undetectable in all clinical samples.

Lack of cross-reactivity of the monoclonal antibodies with canine

SMRP and/or the absence of SMRP from canine effusions may

explain our findings. The MesoMARK™ is not a useful test in

caninemedicine.

17PREDICTIVE VALUE ADVISOR: SOFTWARE FOR THEEVALUATION OF THE IMPRECISION OF PREDICTIVEVALUES OF TEST RESULTS ACCORDING TO IMPRECI-SION OF SENSITIVITY, SPECIFICITY AND PRE-TESTPROBABILITY. N. Bourges-Abella1, P. Bour-daud’hui2, A. Geffre1, D. Concordet2, J.P. Braun1,O. Dossin1, C. Trumel1. 1Institut National Polytech-nique-Ecole Nationale Veterinaire de Toulouse, Toulouse,France; 2UMR331 Toxalim, Institut National Polytech-nique-Ecole Nationale Veterinaire, Toulouse, France.

Background: It is commonly accepted that the imprecision of

criteria of diagnostic efficiency needs to be reported as the 95%

confidence interval of sensitivity, specificity, likelihood ratio or

odds ratio. Moreover, the imprecision in evaluation of pre-test-

probability is not precisely quantifiable. The effects of all these

imprecisions are not taken into account in the estimation of pre-

dictive values.Objective: To evaluate the possible effects of the

all imprecision factors on predictive values and design freeware

allowing clinicians to make simulations. Methods: A web-

based calculator was devised to compute the 95% CIs of PVP &

PVN as a function of pre-test probability, according to the number

of animals plus either Se and Sp or the numbers of true and false

negatives and positives. Moreover, based on the uncertainty of

pre-testprobability proposed by the user, histograms representing

the range of PVP and PVN are displayed. A test version is available

at: http://www.biostat.envt.fr/ppvnpv/. Evaluation: This soft-

ware allows clinicians to test different hypotheses/imprecisions

for a given test and to appreciate the uncertainty of the PVs that

can be estimated for the tests they have performed. Conclu-

sion: Imprecision of PVP and PVN aids in support of the

clinician’s medical decision giving fast quantification of

uncertainty, which is too long to calculate without specialized

tools and is usually underestimated. It cannot be used for each

case but in some instances. Even retrospectively, it can help to

make correct use of the data published, which are too frequently

not used except formeeting publication requirements.

18REFERENCE VALUE ADVISOR (V.2) NOW PERFORMSREGRESSION-BASED REFERENCE INTERVALS WITHCONTINUOUS COVARIABLES. N. Bourges-Abella1,A. Geffre1, D. Concordet2, J.P. Braun1, C. Trumel1.1Institut National Polytechnique-Ecole Nationale Veteri-naire de Toulouse, Toulouse, France; 2UMR331 Toxalim,Institut National Polytechnique-Ecole Nationale Veteri-naire, Toulouse, France.

Background: Reference intervals determined using Reference

Value Advisor v.1, freeware running with Excel 2003, are

obtained rapidly but do not allow testing possible effects of cova-

riates.Objective: To develop a tool that helps identify effects of

continuous factors of variation (e.g., age, weight) and represents

regression-based reference limits with their 90% confidence

intervals (CIs) as reference bands on graphs. Methods: A set of

macros was designed for Excel 2007 and 2010 to compute regres-

sion-based reference limits and their 90% CIs as graphs. These

reference bands can be built using homoscedastic and heteros-

cedastic polynomial parametric models for a single variable or

several covariables. The computation of the optimal Box-Cox

transformation of the analyte concentration is proposed. Non-

parametric reference bands can also be computed when the sam-

ple size is large enough. Results: This software allows clinical

pathologists to easily check whether or not a covariate can be

considered as a valid factor of variation and to automatically

derive the corresponding reference limits. Conclusion: Most of

the factors of variation are demographic characteristics. Taking

these factors into account can substantially reduce the width of

standard reference intervals. This is helpful in veterinary clinical

pathology as it improves the information for medical decisions

when reference limits are used as a decision threshold.

19ACTIVATION OF LIPID METABOLISM IN CANINEBABESIOSIS CAUSED BY BABESIA CANIS. N. Kucer1,V.Mrljak1, R.Rafaj Baric2, J.Kules2, I. Smit1, J. Selan-ec1, M. Crnogaj2. 1Clinic for Internal Diseases, Faculty ofVeterinary Medicine, University of Zagreb, Croatia;2Department for Chemistry and Biochemist, Faculty ofVeterinaryMedicine, Zagreb, Croatia.

Background: Development of medicine, biochemistry and

immunology has provided a more complete insight into factors



that influence the pathogenesis of canine babesiosis. Today it

is known that inflammation has a major role in complicated

and severe forms of disease. Lipid metabolism is of importance

in babesiosis involving the acute phase response as part of a

complex process regulated by nervous and endocrine systems.

Because the majority of hemoparasites including Babesia can-

not synthesize their own lipids, they take them up from host

plasma, predominantly from the high density lipoprotein frac-

tion. Objective: The aim of this research was to determine if

there are changes in lipid metabolism during babesiosis and

when they occur during the course of disease. Methods:

Experiments were conducted on 17 healthy and 26 naturally

infected dogs. Blood samples were taken on the1st day (at admis-

sion) and on the 3rd and 7th day after admission. Biochemical

parameters were obtained using an automated biochemical ana-

lyzer (Olympus AU 600) according to standard methods with ori-

ginal reagent kits. The biochemical profile included triglycerides,

high density lipoproteins, low density lipoproteins and choles-

terol.Results: Our data show that the cholesterol concentration

was significantly increased on day 3 in sick dogs, while the high

density lipoprotein concentration had significantly decreased at

day 1. There were nomajor alterations in the triglyceride and low

density lipoprotein concentrations. Conclusion: This

study confirms that lipid metabolism is being activated during

babesiosis.

20LACTATE DEHYDROGENASE ISOENZYMES IN DOGSAFFECTED BY LYMPHOMA. A. Giordano, D. Stefa-nello, M. Pastore, R. Ferrari, L. Giori, P. Moretti,S. Paltrinieri.University of Milan,Milan, Italy.

Background: Lactate dehydrogenase (LDH) is a marker of

cell damage of many tissues. Total LDH is used for the diagno-

sis of skeletal and cardiac muscle damage, or of hepatic and

renal disorders. In people, LDH has also been used as a marker

of neoplasia, since the isoenzymatic pattern of LDH changes in

neoplastic tissues. Increased total LDH activity has been

observed in dogs with lymphoma, but little is known about

the possible diagnostic or prognostic role of LDH isoenzymes

in canine lymphoma. Objectives: To provide information

about total activity and isoenzymatic patterns of LDH in dogswith

lymphoma at first diagnosis and during the course of disease or

following chemotherapy. Methods: After preliminary valida-

tion, 108 sera from 31 dogs (20 with neoplasia and 11 healthy

controls) were examined. Neoplastic dogs were sampled at first

presentation and during follow-up. Based on clinical and labora-

tory findings, sick dogs were grouped as follows: L= lymphoma

(n=13); Lth= lymphoma already undergoing therapy (n=3); O=

neoplasia other than lymphoma (n=4). Results: The percent-

age of isoenzymes was less influenced than total LDH by antico-

agulant and storage. At first presentation, dogs with neoplasia

had higher LDH activity than controls. Dogs with lymphoma

showed the highest values for LDH-2 and LDH-3 with different

patterns between T-and B-lymphoma. No specific changes were

recorded during follow-up. Conclusions: Increases of total LDH

activity and of LDH-2 and LDH-3 could support a diagnosis of

lymphoma. Further data are needed to verify the prognostic role

of LDH isoenzymes.

21EFFECTS OF PLATELET-ACTIVATING FACTOR RECEP-TOR ANTAGONIST (PAFRA) ON SELECTED INFLAM-MATORY AND BIOCHEMICAL PARAMETERS IN A RATENDOTOXEMIAMODEL.R. Col, E. Keskin. Selcuk Uni-versity, Konya, Turkey.

Background: Platelet-activating factor (PAF) is a potent auta-

coid mediator produced by a variety of cells involved in septic

inflammatory reactions. PAFRA was demonstrated to have

potent anti-inflammatory activity and survival benefit. During

endotoxemia, lipopolysaccharide (LPS, endotoxin) and proin-

flammatory cytokines are involved in the pathogenesis of sep-

sis and play a pivotal role in the initiation of tissue damage,

multiple organ failure (MOF) and haemostatic disturbances.

Objective: In this experimental study, our aim was to investi-

gate the role of PAFRA on inflammatory and biochemical distur-

bances in LPS-treated rats. Methods: A total of 32 adult male

Wistar rats (weight range: 200– 250 g) were divided into four

equal groups: Group 1 served as a negative control (C). Ani-

mals in Group 2 were given LPS (Escherichia ooli LPS, 0.111:B4)

intravenously (1.6 mg/kg). In Group 3, PAFRA (10mg/kg) was

injected intraperitoneally. In Group 4, PAFRA (10mg/kg IP)

and LPS (1.6 mg/kg IV) were administrated simulta-

neously. Blood samples were collected 6 h after treatment.

Results: LPS caused statistically significant increases in serum

TNF-a, IL-6 and IL1b levels and C-reactive protein, AST, ALT,

LDH, creatinine, BUN, cholesterol, triglyceride concentrations,

and caused statistically significant decreases in glucose, total pro-

tein and albumin levels, compared with the control group. In

group 4, PAFRA inhibited serum TNF-a and IL1b levels compared

with group 2 (P<0.05). However, there were no significant

differences in the other values between the two groups. Conclu-

sions: At the administered dose and route, PAFRA had a slight

effect in the pathogenesis of endotoxemia.

22URINE PROTEOME FROM HEALTHY DOGS, CATS,HORSES AND COWS. E. Ferlizza, E. Armuzzi, A. Buon-o, E. Carpene, G. Andreani, F. Dondi, G. Isani.University of Bologna, Ozzano Emilia, Italy.

Background: Data on the urinary proteome of domestic ani-

mals are limited; as a consequence the study of urinary proteome

of healthy domestic animals is the first step to discover new

biomarkers of nephropathy. Objective: To study of the

urine proteome from healthy dogs, cats, horses and cows.

Methods: Urine samples were collected from clinically healthy

dogs (15), cats (10), horses (37) and cows (10). For all samples,

UP/C was calculated and SDS-PAGE with silver staining was per-

formed. Results: All samples had UP/C lower than 0.2. After

SDS-PAGE, all samples showed many bands, which were unde-

tectable by routine methods. Dogs had 15-19 bands, cats: 17-20,

horses: 13-15 and cows: 15-18. On the basis of the apparent

molecular weight (MW) reported in kDa and comparing with

human urine proteome, we tried to identify the most common

proteins: P90 (Tamm-Horsfall), P76 (transferrin), P64 (albumin),

P54 (immunoglobulin heavy chain), P27 (immunoglobulin light



chain), and P14 (lysozyme). All samples contained putative albu-

min, transferrin, IG heavy and light chains; Tamm-Horsfall was

clearly evident in dogs, horses and cats. Dogs and cows presented

proteins mainly at MW lower than albumin, cats presented pro-

teins mainly at MWhigher than albumin, while horses presented

proteins at both high and low MW. Conclusion: SDS-PAGE

with silver staining is a very sensitive method to detect low abun-

dance proteins in urine of healthy domestic animals, allowing the

identification of different species-specific electrophoretic profiles.

Work is in progress to confirm the identity of the different

proteins.

23EVALUATION OF THREE URINE DIPSTICKS FOR DOGS,CATS, AND COWS. S. Klenner1, M. Defontis1,N. Bauer1, K. Failing2, A.Moritz1. 1Central Laboratory,University (JLU) Giessen, Hannover, Germany; 2Unit forBiomathematics and Data Processing, Giessen, Germany.

Background: Urine evaluation is performed using dipsticks

usually designed for human use. Objective: The aim of this

study was to evaluate the CombiScreenVet11Plus (CS), the Com-

bur10UX (CB) and Multistix10SG (MS) for urinalysis in dogs,

cats, and cows. Methods: Urine samples from 101 dogs, 50 cats

and 100 cows were evaluated by all dipsticks using their auto-

mated reflectometer testing devices comparedwith the respective

reference methods. The Spearman rank correlation coefficient

and sensitivity and specificity for detection of proteinuria, glucos-

uria, and isosthenuria were calculated.Results: Between auto-

mated reading and reference methods, correlation was poor in

dogs and cows for all sticks for RBC, WBC, and specific gravity

as well as for glucose (dogs), protein (cows), and WBC (cats)

and was poor/fair for specific gravity (cats). Fair/good correla-

tion was present for glucose (cats, cows), protein (dogs), and

RBC (cats). All sticks showed good/excellent correlation for all

species for pH. Detection of glucosuria (cut-off 50mg/dl) was

sensitive (dog 80(MS)-100%, cat 85(MS)-93%, cow 76(MS)-

100%) and specific (dog 96-98%, cat 96-100%, cow 87(CB)-

96%). CS and CB were sensitive (dog 83+92%, cat 67+100%,

cow 91%) but dominantly nonspecific (dog 65+39%, cat 93

+53%, cow 47+20%) for detection of isosthenuria while speci-

ficity of the MS was excellent in all species (97-98%). Evalua-

tion of proteinuria (cut-off 30mg/dl) lacks specificity (CS+MS

dog 81+38%, cat 79+32%, cow 43+27%) except for CB (dog

91%, cat 95%, cow 89%). Sensitivity for detection of protein-

uria was lacking in dogs (CS 74%) and cows (CB 37%). Con-

clusion: Detection of WBC and specific gravity are hampered in

all dipsticks and all species.

24CLINICAL RELEVANCE OF TGF-ß1 IN DOGS.S. Neumann, S. Gaedcke. Institute of Veterinary Medi-cine, Goettingen, Germany.

Background: Cytokines are mediators of disease mechanism,

so their serum concentrations could be of clinical relevance.

Objective: he aim of this study was to prove the stability of

TGF-ß1 in different sample collections, to observe the clinical rel-

evance in different internal diseases of dogs and to assess in this

way the impact of TGF-ß1 as diagnostic tool.Methods: TGF-ß1

concentration was assessed using an enzyme-linked

immunosorbant assay (ELISA) in 169 dogs. Stability under

controlled storage conditions as well as correlation of serum and

plasma levels from 55 dogs was evaluated.Results: TGF-ß1 was

stable at a storage temperature of -70°C for a minimum of three

month in plasma and serum. The serum concentration was in

accordance with the platelet count. Plasma concentrations had

no relation to the platelet count. Different reference values were

found for plasma and serum TGF-ß1. Acute inflammatory dis-

eases had no influence on TGF-ß1 concentration in serum or

plasma. Diseases that hadan influencewere those with fibrosis

during their course. Dogs with lung fibrosis, hepatic fibrosis and

diabetes mellitus had significantly increased concentrations of

TGF-ß1 in serum or plasma compared with healthy controls.

Conclusion: Assessment of TGF-ß1 in a daily clinical practice is

feasible and offers potential diagnostic utility as marker for fibro-

sis in dogs.

25LDH IN BODY CAVITY FLUIDS: IS IT DIAGNOSTICALLYUSEFUL? C. Smuts, J. Mills, T. Gaal. Murdoch Univer-sity, Perth, Australia.

Lactate dehydrogenase (LDH), an enzyme of the anaerobic glyco-

lytic pathway with several isoforms, is found intracellularly in

most tissues. Increased LDH is frequently used in human medi-

cine to distinguish pleural exudates from transudates (Light’s cri-

teria), and therefore may also be useful to help differentiate body

cavity effusions in animals. Because LDH is a large molecule that

does not pass readily though intact endothelial surfaces, concen-

trations in pleural or peritoneal fluid do not equilibrate rapidly

with plasma values, and high effusion LDH is therefore likely to

originate from inflammatory or damaged cells in that cavity. We

tested pleural, peritoneal and pericardial effusions from dogs, cats

and horses (n=50) using three different LDH tests – RANDOXwet

chemistry testing both the lactate-to-pyruvate (L-P) and pyru-

vate-to-lactate (P-L) reaction and dry chemistry (IDEXX) mea-

suring P-L. Although there is a good correlation between all the

tests (r2= 0.98), there are significant differences between their

absolute values. IDEXX chemistry produces the highest values

(approximately 4x Daytona L-P) followed by Daytona P-L

(approximately 2x L-P values) and Daytona L-P usually the low-

est. Therefore it is necessary to establish different cut-off values

for differentiating between transudates and exudates depending

on which method is used. Results to date show that LDH activity

tends to be highest in exudates and modified transudates in all

methods evaluated and lowest in transudative effusions. This test

may be useful for general practice where fluid analysis is not

immediately available.

26CANINE PANCREATIC LIPASE (SPEC cPL) ANDC-REACTIVE PROTEIN (CRP) IN DOGS TREATED WITHANTICONVULSANTS (PHENOBARBITAL AND POTAS-SIUM BROMIDE). J. Pastor1, V. Albarracin1,A.Melendez-Lazo1, J. Rodon2. 1Universitat Autonomade Barcelona, Bellaterra, Spain; 2IDEXX Spain, Barcelona,Spain.

Background: Animals treated with anticonvulsants (potassium

bromide and/or phenobarbital)may be predisposed to pancreatitis.



Canine pancreatic specific lipase (Spec cPL) is a sensitive test for

the diagnosis of pancreatitis. Elevations in C-reactive protein

(CRP) are associated with the clinical severity of pancreatitis.

Objective: To evaluate Spec cPL, CRP and liver enzyme concen-

trations in dogs treatedwith phenobarbital and/or potassium bro-

mide. Methods: Serum samples from dogs (n = 337) treated

with anticonvulsants (potassium bromide and/or phenobarbital)

were included in the study. Serum concentrations of potassium

bromide, phenobarbital, Spec cPL, triglycerides, cholesterol,

GGT, ALP, ALT, AST and CRP were determined. Results: Ani-

mals treated with potassium bromide and phenobarbital (n=55)

had statistically higher values of ALP compared with animals

with single treatments (bromide n=21 and phenobarbital

n=257). Animals treated with potassium bromide had statistically

significant lower values of CRP compared with other groups.

6.8% (23/337) of the animals showed an increase of Spec cPL

over the reference range and showed a statistically significant

correlation between this parameter and concentration of triglyce-

rides, ALP and ALT. There were no statistically significant differ-

ences in CRP values among the three groups of Spec cPL (less

than 200 μg/L, 201-399 μg/L and > 400 μg/L). Only 2 of 337 ani-

mals (0.6%) showed a Spec cPL > 400mg/L and CRP above refer-

ence range suggesting pancreatitis. Conclusions: Animals

treated with phenobarbital and/or potassium bromide may have

an increase in serum concentrations of Spec cPL; however, pan-

creatitis is rare in these animals.

27IMMULITE 1000 MEASUREMENTS IN VETERINARYSAMPLES. R. Pato1, R. Pena2, Y. Saco2, A. Bassols2.1Universitat Autonoma de Barcelona, Bellaterra, Spain;2Servei de Bioquımica Clınica Veterinaria, Departamentde Bioquımica, Bellaterra (Barcelona), Spain.

Background: The measurement of hormones by immuno-

analysis in serum of veterinary species is a common problem

due to the lack of specific reagents. IMMULITE® Enzyme-

Amplified Chemiluminescence methods have been validated

basically in human samples, but not for veterinary use in the

majority of the available assays. The aim of this study was to

test cross reactivity of the specific IMMULITE® 1000 reagents

for human samples in veterinary species. Methods: Veteri-

nary serum samples were tested using an IMMULITE® 1000 sys-

tem (Siemens Healthcare Diagnostics) and the following human

kits: Total T4 and Third Generation TSH kits for pig serum; ACTH

kit for horse and rat serum; Estradiol and Progesterone kits for

female Catalan donkey serum and Progesterone kit for bovine

serum. Results: In serum obtained from umbilical blood of

newborn piglets (n=35), total T4 values ranged from <1-11 μg/dLand TSH values ranged from 0.006-0.097 μUI/mL. In horse serum

from newborn foals (n=27), ACTH values of the control group

ranged from 15.3-31.1 pg/mL. ACTH was also determined in

healthy rats, and basal values ranged from 10.7-64.8 pg/mL

(n=16). In serum obtained in different phases of the reproductive

cycle from female Catalan donkeys, 17-b-estradiol values rangedfrom <20-126 pg/mL (n=64) and progesterone values ranged

from 0.421-36.8 ng/mL (n=16). In bovine serum (n=60), proges-

terone ranged from <0.2 to 10.9 ng/mL. Conclusions: Values

obtained were similar to those described in the bibliography.

Good linearity under dilution was obtained for each assay. No

interferences were detected.

28KINETICS AND CHARACTERISTICS OF FELINEPLATELET AGGREGATION IN VITRO. B. Riond1,A.K. Waßmuth1, S. Hartnack2, R. Hofmann-Lehmann1, H. Lutz1. 1Clinical Laboratory, VetsuisseFaculty, University of Zurich, Zurich, Switzerland;2Section of Epidemiology, Vetsuisse Faculty, University ofZurich, Zurich, Switzerland.

Background: Feline platelets (PLT) are prone to aggregation in

vitro after blood collection rendering difficult the determination

of accurate PLT counts in the cat. No information is available

about the kinetics of PLT aggregate formation in feline EDTA

blood samples and the course of PLT counts over a clinically rele-

vant time period of 24 hours after collection. Objectives: The

aim of this study was to investigate the kinetics of PLT aggregate

formation and the course of PLT andWBC counts in feline EDTA

blood samples with respect to the variables “time after blood col-

lection”, “anesthesia”, “bleeding conditions” and “glucose con-

centration”. Methods: From 30 clinically healthy cats blood

samples were analyzed over a period of 24h on the Sysmex

XT-2000iV, the Mythic 18, by the use of a counting chamber

and by blood smear evaluation. Results: Fourteen (Sysmex

XT-2000iV) and 12 (Mythic 18), respectively, of 30 samples were

thrombocytopenic at one to eight time points after collection.

Subsequently all thrombocytopenic samples returned to normal

PLT counts. Seventeen of 30 samples had PLT aggregates in the

counting chamber. Significant differences in PLT counts were

associated with the presence and size of aggregates, time after

bleeding, and glucose concentration, but not with quality of

blood collection and anesthesia. Conclusion: PLT aggregation

and pseudothrombocytopenia occurred in 57% of the samples

at different time points and with different duration. Deaggre-

gation of feline PLT aggregates could be demonstrated as a

reversible effect of PLT aggregation. Quality of blood collection

seems not to be causative for PLT aggregation.

29EFFECTIVE PREVENTION OF PSEUDOTHROMBO-CYTOPENIA IN FELINE EDTA BLOOD SAMPLES WITHTHE PROSTAGLANDIN I2-ANALOGUE ILOPROST.B. Riond1, A.K. Waßmuth1, S. Hartnack2, R. Hof-mann-Lehmann1, H. Lutz1. 1Clinical Laboratory,Vetsuisse Faculty, University of Zurich, Zurich, Switzer-land; 2Section of Epidemiology, Vetsuisse Faculty,University of Zurich, Zurich, Switzerland.

Background: Prostaglandin I2 (PGI2) is the most powerful

endogenous inhibitor of platelet (PLT) aggregation but is unsta-

ble. Iloprost is a stable PGI2 analogue. Objectives: The aims of

this study were to (1) evaluate the antiaggregatory effect of Ilo-

prost on feline PLT in EDTA blood samples from clinically healthy

cats; (2) investigate the effect of Iloprost on hematological param-

eters; and (3) determine the stability of Iloprost in K3-EDTA tubes

for up to 16 weeks. Methods: From 20 clinically healthy cats

blood was drawn from the jugular vein and immediately aliquot-

ed into a 1.3 ml K3-EDTA tube and two 1.3 ml K3-EDTA tubes

containing 20 ng and 200 ng Iloprost, respectively. A CBC was



performed on the Sysmex XT-2000iV and the Mythic 18 at eight

consecutive time points after collection. Blood smears were eval-

uated for the presence of PLT aggregates. Results: In the

absence of Iloprost, pseudothrombocytopenia was observed in

50% of the investigated samples and led to significantly

decreased optical PLT counts by a mean of 105 x10³/μl. Pseudo-thrombocytopenia was prevented by the addition of Iloprost,

which led to an increase in PLT counts by a mean of 108 x10³/μl.Conclusion: A strong antiaggregatory effect of Iloprost in feline

EDTA blood was demonstrated. In all cats, pseudothrombocy-

topenia was prevented by adding Iloprost to EDTA tubes prior to

blood collection. Iloprost also prevented falsely increased WBC

counts in samples with PLT aggregates analyzed on impedance-

based instruments. Other hematological parameters were not

influenced to a clinically significant degree.

30EXPLORATIVE REFERENCE INTERVALS FOR BIO-CHEMISTRY AND HAEMATOLOGY DATA IN HEALTHYPIGLETS DURING THE FIRST 27 DAYS OF LIFE.B. Rutgen1, E. Hooijberg2, L. Schwarz1, H.L.Worliczek1, I. Schwendenwein1. 1University of Veteri-nary Medicine Vienna, Vienna, Austria; 2Labor InVitro,Vienna, Austria.

Background: Pigs are kept primarily for meat production but

also serve as models in medical research. Object: Data derived

from serial blood sampling of a sentinel group of piglets in an

experimental setting were compiled to establish exploratory ref-

erence intervals for healthy piglets during their first 27 days of

life.Methods: Twelve piglets from two crossbred strains were

serially sampled on day 0 before first colostrum intake and

then on days 1, 7, 14, 21 and 27. Bodyweight was recorded

simultaneously. CBC was performed on the ADVIA120™,

complemented by microscopy. Glucose, fructosamine, urea,

creatinine, TP, fibrinogen, albumin, AST, ALT, GLDH, GGT,

lipase, CK, electrolytes, minerals and iron were measured by

standardized methods. Statistical analysis was performed us-

ingAnalyze-it™ software. Results: Body weight followed the

expected growth curve confirming health status. RBC parameters

showed a significant decrease from day 0 to day one followed by a

constant increase. TWBC showed a constant increase. A highly

significant increase in glucose, fructosamine, urea, TP, albumin

and fibrinogen from day 0 to day 1 was observed. Urea decreased

again from day 1 to day 7 and remained stable thereafter. Creati-

nine was high at birth and decreased significantly on day 1 to

remain stable until day 27. Liver and pancreatic enzymes, AST,

ALT, GGT, CK and lipase, showed a significant peak after colos-

trum intake whereas GLDH was stable throughout the study per-

iod. Conclusion: These data confirm previously published data

from young piglets and follow the expected patterns that occur

due to colostrum intake, growth and physical activity.

31THE “QUATREFOIL” ERYTHROCYTES: UNEXPECTEDPATTERN IN CANINE RED BLOOD CELL MORPHOL-OGY. A. Gavazza, G. Lubas, M. Ricci, B. Gugliucci,A. Pasquini. Dept Veterinary Clinic, University of Pisa,Pisa, Italy.

Background: A variety of causes canmodify RBC shape includ-

ing regenerative response, altered metabolism (iron and lipid

above all, but also electrolyte depletion), oxidative injury,

immune-mediated damage, mechanical fragmentation, sepsis,

toxins and drugs. Objective: The aim of this study was to con-

firm and investigate the occurrence of an unusual RBC pattern

(so-called “quatrefoil RBCs” or “Pisa cross”) in dog blood smears.

Methods: 3,958 blood smears from 2,016 dogs, stained with

Diff-Quick, were examined microscopically from May 2009 to

October 2010. To exclude the possibility of EDTA artifact, some

smears were prepared from whole blood without anticoagulant.

Results: “Quatrefoil RBCs” were found in 3.89% canine blood

smears (154 of 3958) and 6.89%of dogs (139 of 2016with 14 dogs

out of 139 presenting with this RBC pattern in replicate samples).

Statistically significant differences (p< 0.05) including increased

age, anisocytosis, and polychromasia, were found in dogs with

quatrefoil RBCs than from dogs with normal morphology. The

effective presence of this new erythrocyte pattern was confirmed

both by optical and scanning electron microscopy (SEM).

Conclusion: Quatrefoil RBCs representadhesion between

two erythrocytes, which is different from both rouleaux (orga-

nized linear arrays or chains of RBCs) and agglutination (disorga-

nized clumping or clusters). It was not possible to identify the

etiology and clinical relevance of “quatrefoil” morphology. Some

hypotheses were advanced to explain this particular RBC

arrangement.

32PLATELET INDICES IN VIRULENT CANINE BABESIOSISAND THEIR ASSOCIATION WITH OUTCOME.A. Goddard1, A. Kristensen2, A. Leisewitz1,P. Thompson1, J. Schoeman1. 1University of Pretoria,Pretoria, South Africa; 2University of Copenhagen,Copenhagen, Denmark.

Background: Thrombocytopenia, without clinical evidence of

hemorrhage, is a consistent finding in virulent canine babesio-

sis. Objective: To investigate platelet indices in babesiosis,

caused by Babesia rossi, and their association with outcome.

Method: Admission EDTA whole blood samples were analyzed

on the ADVIA 2120 from 68 Babesia-infected and 6 healthy con-

trol dogs. Indices included platelet count (PLT), mean platelet

volume (MPV), platelet volume distribution width (PDW),

thrombocrit (PCT), mean platelet dry mass (MPM), mean platelet

component concentration (MPC) and platelet component distri-

bution width (PCDW). Indices were also determined at 24 and 48

hours in admitted dogs that survived. Results: Of the Babesia-

infected dogs, 30 were treated as outpatients and 38 admitted

to the hospital of which 9 died within 24 hours (13%). The

following indices were significantly different between Babesia-

infected and control dogs: median PLT (32 vs. 259 9109/L;

p<0.001); median MPV (15.7 vs. 9.8 fL; p<0.0001); median

PDW (60.9 vs. 55.9%; p=0.005); median PCT (0.05 vs. 0.24%;

p<0.001); mean MPM (2.3 vs. 1.8 pg; p=0.003) and mean

MPC (19.3 vs. 21.6 g/dL; p<0.0001). There were no significant

differences between outpatient and admitted dogs, or dogs that

died and dogs that survived. PLT and MPC showed significant

increases at 24 hours (p=0.012 and p=0.009, respectively) and

48 hours (p<0.0001 and p=0.01, respectively). Conclusion:

Large, degranulated platelets may play a significant procoagu-

lant role in the lack of clinical hemorrhage, despite severe

thrombocytopenia, observed in canine babesiosis. Platelet activa-

tion as measured by MPV and MPC does not appear to be associ-

atedwith outcome.



33D-DIMER MEASUREMENT BY LATEX AGGLUTINA-TION; COMPARISON OF A SEMIQUANTITATIVE ANDQUANTITATIVE METHOD AND EVALUATION OF THECLINICAL VALUE OF THE TEST IN DOGS. J. Archer,A. Pastorello, P. Monti, P. Kuczybska. Departmentof Veterinary Medicine University of Cambridge, Cam-bridge, UK.

D dimer measurement is widely used to exclude thromboembolic

disease in dogs. Recently several kits for semiquantitative

measurement of D-dimers have been discontinued. Objec-

tives: 1) Comparison of a quantitative automated method and a

manual semiquantitativemethod. 2) Restrospective study of clin-

ical outcomes. Methods: D-dimer concentrations in the same

plasma sample from 45 dogs weremeasuredwith a semiquantita-

tive (SQ) latex agglutination kit and an automated quantitative

(Q) particle enhanced immunoturbidimetric assay.Results and

clinical outcome in 700 dogs were analyzed retrospectively.

Results: SQ values of <250ng/mL corresponded to a range of

0.03-0.11 ug/mLQ;<1000 ng/mL SQ to 0.21-1.07 ug/mL Q;

2000->2000 ng/mL SQ to 0.24-1.17 ug/mLQ, demonstrating that

there was correlation between SQ and Q with some outliers. In

the retrospective study of 700 dogs, dogs with thromboembolic

disease and IMHA had the highest D-dimer values (>1000 ng/

mL), 23% of those with thromboembolic disease had values

>2000 ng/mL, while 33% of dogs with malignant neoplasms had

values above 2000 ng/mL. Conversely, of the dogs with thrombo-

embolic disease 18%had values <250 ng/mL and of the dogswith

coagulopathies 23% had values <250 ng/mL. Of dogs with renal

and respiratory diseases 68%-73%had values <250 ng/mL.Con-

clusions: Moderate correlation between SQ and Q D-dimer

assays was found with weak correlation between D-dimers and

clinical condition.

34DIAGNOSTIC PERFORMANCES OF FLOW CYTOMET-RIC DETECTION OF BLOOD AND BONE MARROWINFILTRATION IN DOGS WITH LARGE B-CELLLYMPHOMA. V. Martini1, M.E. Gelain2, S. Comazzi1.1University of Milan, Milan, Italy; 2University of Padua,Padua, Italy.

Background: Canine lymphoma is classified as “stage V” by the

WHO clinical staging system when neoplastic cells are present in

blood, bone marrow (BM) or any other non-lymphoid tissue.

Cytological examination is commonly used, but procedures are

not standardized. Some authors prefer more sensitive techniques

such as flowcytometry (FC) or molecular techniques, but no offi-

cial cut-off for positive samples has been defined. Recently, some

authors found that the presence of >10% large lymphocytes on

blood smear examination had a 60% sensitivity and a 87% speci-

ficity in detecting >10% BM infiltration. Objective and meth-

ods: The purpose of this work was to determine if FC is a more

sensitive tool compared with morphology alone in assessment of

blood and BM involvement in 60 dogs with large B-cell lym-

phoma. Agreement betweenBM infiltration assessed via cytology

and FC was evaluated and a cut-off value for cytometric blood

involvement suitable for predicting >10% bone marrow infiltra-

tion was calculated. Results: oderate agreement between BM

infiltration via cytology and FC was found; 13% circulating neo-

plastic cells was identified as the ideal cut-off with 65% sensitiv-

ity and 100% specificity to predict BM infiltration.

Conclusion: Based on these results, flow cytometry could be

considered not only a more specific but also a more sensitive tool

than morphological evaluation alone for canine large B-cell lym-

phoma staging. Further clinical studies are needed, including

more cases, other lymphoma subtypes and follow-up informa-

tion, to establish the possible prognostic value of bone marrow

involvement and a clinically useful cut-off.

35DOUBLE ERYTHROCYTE POPULATIONS: RETROSPEC-TIVE STUDY ON CASES OBSERVEDWITH THE SYSMEXXT-2000iV (2008-2012). N. Bourges-Abella, B. Cuq,F. Granat, A. Geffre, C. Hanot, C. Trumel. InstitutNational Polytechnique-Ecole Nationale Veterinaire deToulouse, Toulouse, France.

Background: The Sysmex XT-2000iV is based on impedance

for measuring erythrocyte count and indices. The standard ery-

throgram is a right-skewed curve. In rare cases, abnormal curves

have been observed with two well-separated peaks that seemed

to represent two different erythrocyte subpopulations.

Objective: The aim of this study was to determine the

frequency of this abnormality and the associated outcomes.

Methods: Hematology reports of the Laboratoire Central de

Biologie Medicale were reviewed (four-year period) for well-

separated double peaks on erythrograms as the only inclusion cri-

teria. Smears and clinical data from the cases were then studied

using the National Veterinary School database. Results: Nine

cases were identified out of the 19,266 CBCs performed: five cats,

three dogs and a cow. Two animals (one cat and the cow) had

been previously transfused. The remaining cats and the dogs had

a marked regenerative anemia, but the reticulocyte count did not

fit within the second peak. In cats, one was positive for FIV and

Mycoplasma haemofelis, one had cholangiohepatitis, and two

had immune-mediated haemolytic anemia of unknown origin.

In dogs, this erythrogram abnormality was associatedwithmicro-

cytosis. One was a weanling puppy and the others were cases of

chronic bleeding. Only one animal died: a cat transfused with

canine blood. Conclusion: This retrospective study shows that

a two-peak erythrogram is very uncommon, associated with

marked regenerative anemia or transfusion errors, and has no

prognostic significance.

36LEEK DIET MAY CAUSE HEMOLYTIC ANEMIA: A CASEREPORT IN A CAT. N. Bourges-Abella, F. Granat,A. Geffre, C. Trumel. Institut National Polytechnique-Ecole Nationale Veterinaire de Toulouse, Toulouse,France.

Case presentation: A 7-year-old neutered female domestic

shorthair cat was presented because of ingestion of a bone. No

abnormalities were noted at clinical examination, and abdominal

radiography showed the presence of a bone in stomach. Medical



treatment based on a leek-based high-fiber diet and nonsteroidal

anti-inflammatory drugs was chosen to try natural elimination of

the bone. At the following presentation eight days later, no

abnormality in the behavior of the cat was reported by the own-

ers. At clinical presentation, the cat did not have abdominal pain,

but the mucosa were pale. Abdominal radiography and ultrason-

ograpgy suggested that the bone had been eliminated. CBC,

blood film examination, biochemical profile, hemostatic testing,

and urinalysis were performed. Results showed an intense hemo-

globinuria and hemoglobinemia, and an anemia associated with

the presence of many Heinz bodies on the blood smear. A diagno-

sis of intravascular hemolytic anemia induced by a severe oxida-

tive stress with formation of Heinz bodies was made. Leek

ingestion was stopped and medical antioxidant treatment was

prescribed. Anemia and hemoglobinuria had disappeared fifteen

days later. Interpretation: Interpretation ws very high

suspicion of intravascular hemolytic anemia induced by a severe

oxidative stress due to the ingestion of leek. Discussion & con-

clusion: The leek is a plant of the same family as the onion,

known to induce oxidative stress in animals. Practitioners should

be aware of leek toxicity before long-term prescription of a high-

fiber diet based on leeks to facilitate elimination of foreign bodies.

37GASTRIC DILATATION AND VOLVULUS SYNDROME –ARE THEREANY CLUES IN THEHEMATOLOGIC EXAM-INATION? I. Uhrikova, L. Rauserova-Lexmaulova,K. Rehakova, I. Hajek, J. Doubek. University of Veter-inary and Pharmaceutical Sciences Brno, Brno, CzechRepublic.

Background: Gastric dilatation and volvulus syndrome (GDV)

is a life-threatening disease associated with gastric rotation and

dilatation. Prognosis depends on duration of disease, degree of

gastric rotation and presence of complications, but final outcome

before surgical treatment.cannot be determined. Objective:

Hematologic examination is inexpensive and is a widely available

laboratorymethod. The aim of our study was to evaluate changes

in hematologic examination in patients with GDV in connection

to final outcome and selected proteins. Methods: Blood was

collected from 39 dogs with GDV before surgery (A), after surgery

(B), and 6-10 hours (C) and 20-24 hours (D) after surgery. Rou-

tine hematologic examination with blood smear evaluation and

measurement of C-reactive protein (CRP) and high mobility

group box 1 (HMGB1) were performed.Results: Analyses were

performed on 142 samples obtained from 31 survivors and 8 non-

survivors. Frequent hematologic abnormalities such as leukope-

nia (0-29%), leukocytosis (3-66%), anemia (28-47%) and

thrombocytopenia (56-82%) were present. Significant differ-

ences between survivors and non-survivors were found in total

leukocyte counts (A, mean 11.1 vs. 16.1x109/L), neutrophils

(A, 9.6 vs. 13.7x109/L), eosinophils (A, B; 0.14 vs. 0.03x109/L,

0.17 vs. 0.06x109/L) and platelet counts (B, 144 vs. 85x109/L).

Dacryocytes decreased significantly in survivors, whilst in

non-survivors were not significantly changed (A vs. B). HMGB1

was moderately correlated with segmented neutrophils (A,

r=0.45, p<0.01) and CRP with bands (D, r=0.44, p<0.05).

Conclusion: Hematologic examination can provide additional

information about patient prognosis. Study was supported by

Grant No. 24/2011/IGAVFUBrno.

38EFFECT OF STORAGE CONDITIONS ON KAOLIN/NON-KAOLIN-ACTIVATEDTHROMBOELASTOGRAPHYAND HEMOSTATIC PARAMETERS IN CATS. R. Col1,C.M. Iazbik2, G. Couto2. 1Selcuk University, Konya,Turkey; 2Department of Veterinary Clinical Sciences, TheOhio State University, Columbus, OH, USA.

Background: Fresh-frozen plasma (FFP) and frozen plasma

(FP) are used therapeutically in cats; however, there are no

reports regarding the hemostatic activity of these plasma prod-

ucts. Objective: In this study, our aim was to evaluate the

dynamics of hemostasis in fresh and stored plasma using TEG, he-

mostasis assays, and activities of factors V, VIII, IX, and X.Meth-

ods: Whole blood was collected in CPDA from cats (n=45) and a

plasma extractor was used to obtain plasma. We evaluated fresh

plasma (FR) (<1 hour storage), FFP (<1 year storage), and

FP (>1 year storage). TEG was done using calcium chloride or ka-

olin as activators on a TEG-5000. OSPT, APTT, fibrinogen con-

centration (FIB), and factor V, VIII, IX, and X activities were

measured on an automated coagulation analyzer. Results: The

R and MA in FFP and FP were significantly different from FR

when using kaolin or CaCl2. The OSPT and APTT were signifi-

cantly longer in FFP and FP than in FR, and the FIB was signifi-

cantly lower in both; interestingly, the activity of factor V (but

not VIII and X) was significantly lower in FP than in FR, and in FP

than in FFP; factor IX activity was significantly lower in FP than

in FR. Conclusions: Storage of feline plasma for transfusion

had a significant effect on hemostasis variables as measured by

TEG and conventional assays. Although, as expected, factor V

activity decreased significantly over time, factor VIII activity did

not. Factor IX activity did not decrease significantly in FFP, but

was significantly lower in FP, suggesting this may be a labile fac-

tor in cats.

39PRELIMINARY REPORT OF HEMOSTATIC PARAME-TERS IN THE EXPERIMENTAL GOTTINGEN MINIPIG.M. Defontis1, S. Cote2, M. Stirn2, D. Ledieu2. 1Justus-Liebig University Giessen, Giessen, Germany; 2Basel,Switzerland.

Background: The animal model, the Gottingen minipig, is

gaining popularity in toxicity testing, however, more information

concerning hemostatic parameters is needed. Objective: The

aim of this study was to optimize whole bloodMultiplate® aggre-

gometry and to provide preliminary values for TEG variables in

the Gottingen minipig. Methods: Citrated and heparinized

whole blood sampleswere obtained from20 healthymale Gottin-

gen minipigs (5-8 months of age) who were kept in controlled

housing conditions and had not received any medication known

to have an effect on platelet function for 15 days. ForMultiplate®aggregometry, the agonists ADP, collagen, arachidonic acid (AA),

Par-4 agonist and TRAP-6 were used in a wide concentration

range to determine their EC50 in heparinized blood. TEG mea-

surements were performed in duplicate on calcium-activated ci-

trated blood. Mean and range values (minimum-maximum) are

reported for the following TEG variables: R, K, MA and G. For the

parameter angle, median and range values are given. Intra-assay

repeatability was calculated as the overall coefficient of variation.



Results: The concentrations of TRAP-6 and Par-4 agonists

tested failed to induce an aggregation response in all animals. The

calculated EC50 in heparinized blood were 0.8μM ADP, 0.25μg/ml collagen and 0.06mM AA. Preliminary values for the TEG

variables were: R= 5.3 (2.0-9.9) min; K= 1.35 (0.8-2.5) min;

angle= 72.6 (49.8-78.4) degrees; MA= 74.6 (66.5-80.9) mm and

G= 15.2 (9.9-21.2) d/sc. Overall CVs ranged between 1.8% and

13.6%. Conclusion: The platelet agonists TRAP-6 and Par-4

agonist cannot be used for whole blood aggregometry in the

Gottingenminipig.

40AFLATOXINS IN SLOVENE MILK AND FEED SAMPLES.B. Jakovac-Strajn, G. Tavcar-Kalcher, I. Ujcic-Vrhovnik, K. Pavsic-Vrtac, K. Fon-Tacer, A. Vengust.Veterinary Faculty, Ljubljana, Slovenia.

Background: Aflatoxins are secondary metabolites of

moulds, predominantly Aspergillus sp., contaminating diverse

food and feed materials. Among the aflatoxins (B1, B2, G1,G2)

aflatoxin B1 is the most toxic, both for humans and animals,

and is a potent carcinogen. Its metabolite aflatoxin M1 appears

in milk and milk products as the result of intake of aflatoxin

B1-contaminated feed. Objective: The aim of this work is to

present the results of analyses where for the first time the Slo-

vene feed and milk samples contained aflatoxins in concentra-

tions above limits given in the legislation. Methods: For the

determination of aflatoxin B1 and M1, procedures consisting of

sample immunoaffinity column cleanup and liquid chromatogra-

phywere used. 243milk samples and 53 feed samples were inves-

tigated. Results: The maximum level for aflatoxin M1 in

consumable milk given in the legislation (50 ng/kg) was

exceeded in 23 samples of milk. The concentrations were from 50

to 3554 ng/kg. 47 samples contained aflatoxin M1 in the concen-

trations from 5 to 50 ng/kg. Among 53 different feed samples ana-

lyzed, 20 samples contained aflatoxin B1 in concentrations

higher than maximum permitted levels (from 0.02 to 0.663 mg/

kg). 24 samples contained from 0.0002 to 0.020 mg/kg aflatoxin

B1. Conclusion: Although exposure to aflatoxins is generally

considered to occur mainly from importedmaterials, the possibil-

ity of contamination of European agricultural products cannot be

entirely excluded. In consideration of the carcinogenic properties

of aflatoxin B1, human exposure should be reduced to levels as

low as is reasonably achievable.

41IS ALKALINE PHOSPHATASE ACTIVITY IN BOVINENASAL SECRETION A RESULT OF LOCAL SYNTHESIS?M.F. Ghazali, N.N. Jonsson, P.D. Eckersall.Universityof Glasgow, Glasgow, UK.

Background: Investigation of bovine nasal secretion has

revealed a higher activity of alkaline phosphatase (ALP) than that

found in serum, but it is not clear whether ALP is synthesised in

the nasal tissue. Objective: The aim of this study was to mea-

sure the level of ALP activity in bovine nasal secretion and in

extracts of relevant bovine tissues. Methods: Nasal secretions

were collected onto absorbent material from thirty-eight clini-

cally healthy Holstein-Friesian cows aged 2-5 years and were

extracted from the absorbent by centrifugation. The nasal secre-

tions were examined for ALP activity and other analytes using

standard clinical biochemistry methods. Samples of heart, liver,

kidney, small intestinal mucosa and nasal epithelium were

obtained from cows during necropsy. Two grams of each sample

were washed with isotonic saline solution and homogenized

mechanically in 5ml saline containing 20% (v/v) n-butanol. The

ALP activity in the aqueous extract was determined as above.

Results: ALP activity was higher in the nasal secretion samples,

with amean (± SD) of 1193 ± 500 U/L, compared wtih the labora-

tory reference range for bovine serum (20-280 U/L). Other bio-

chemical analytes were within or near or below the reference

range for serum. The nasal epithelium extract had the highest

ALP activity of 1855 U/L followed by kidney (1086 U/L), small

intestinal mucosa (832 U/L), liver (668 U/L) and heart (377 U/L).

Conclusion: Bovine nasal secretion has a higher ALP activity

than bovine serum. The nasal ALP results from local synthesis

and secretion of the enzyme.

42SERUM CALCIUM AND INORGANIC PHOSPHATEEVALUATION IN HERMANN’S TORTOISES (TESTUDOHERMANNI). M. Stvarnik1, M. Klinkon2, A. Dovc3.1Veterinary Faculty, Ljubljana, Slovenia; 2Clinic forRuminants, Veterinary Faculty, Ljubljana, Slovenia;3Institute for the Health Care of Poultry, Veterinary Fac-ulty, Ljubljana, Slovenia.

Background: Blood values of calcium and phosphorus might

be influenced in Testudo hermanni by life-style, season, and habi-

tat diversity. T. hermanni is one of the indigenous species

imported to Slovenia. Objective: The aim of this study was to

evaluate differences in serum calcium and inorganic phosphate

values and Ca/P ratio in the post-hibernation period, at different

times throughout the active season, and during mating and

hatching.Methods: From a paddock, sixteen males and sixteen

females were chosen for the study. Bloodwas taken from the dor-

sal coccygeal vein. Serum was analysed with an RX Daytona bio-

chemical analyser. Results: Mean values for calcium were 2.64

(0.59-4.88) mmol/L and for inorganic phosphate were 0.95

(0.15-5.62) mmol/L. The Ca/P ratio in females in the spring was

2.1:1; the ratio changed to 1:1 inMay. After the egg-laying period

in July, the ratio increased to 2.6:1. At the end of May the ratio

was 0.97:1, which was 0.03 lower than the ratio in males. After

the egg-hatching period in July the ratio increased to 2.8:1. Con-

clusion: We consider it important to take samples throughout

the active season to account for oscillation in serum calcium and

inorganic phosphate values due to external environmental

factors.

43HEMATOLOGYREFERENCE INTERVALS IN CYNOMOL-GUS MONKEYS. N. Bourges-Abella1, A. Geffre1,E.Moureaux2,M. Vincenti1, J.P. Braun1, C. Trumel1.1Institut National Polytechnique-Ecole Nationale Veteri-naire de Toulouse, Toulouse, France; 2BioPRIM, Bazieges,France.

Background: Currently available hematology reference values

in Cynomolgus monkeys do not meet CLSI-IFCC and ASVCP

recommendations. Objective: The aim of this study wasa poste-

riori determination of reference intervals in this species. Meth-

ods: Data were collected between 2009 and 2011 during routine



health examinations at BioPRIM center (AAALAC accredited).

Animals originated mainly from Mauritius and also from Philip-

pines and Vietnam. Results were included according to health

status and full control of pre-analytical and analytical conditions.

Analyses had been performed in 3-mL K3-EDTA specimens with

a Sysmex KX-21 analyzer at BioPRIM. Reference intervals were

calculated by the nonparametric method, and effects of covari-

ables were tested by a general linear model. Calculations were

performed with Reference Value Advisor 21 and Systat 13.

Results: From the 450 cases available, 272 animals were finally

included. Most (85%) were born in captivity; age ranged from

17.3 to 120monthswith amajority being 2 to 3 years old. The ref-

erence intervals were [4.85-7.00]x1012/L for RBC, [104.0-136.0]

g/L for HGB, [0.365-0.470]L/L for HCT, [62.7-79.1]fL for MCV,

[17.3-23.7]pg for MCH, [264-310]g/L for MCHC, [31.9-44.0]fL

for RDW, [4.7-25.7]x109/L for WBC, [2.00-20.60]x109/L for

neutrophils, [1.15-5.23]x109/L for lymphocytes, [0.2-1.6]x109/L

for mixed cells, [224 -562]x109/L for PLT, [8.8-12.8]fL for MPV,

and [10.3-18.7]fL for PDW. Conclusion: The reference limits

and their confidence intervals have been determined according

to international recommendations and thus can be used for trans-

fer and/or validation. However, they are only a basis for further

extensive prospective studies usingmoremodern analyzers.

44EVALUATION OF SERUM AMYLOID A, HAPTOGLOBIN,NUCLEATED CELL COUNT, TOTAL PROTEIN ANDHEMOLYSIS IN PERITONEAL FLUID FOR DIFFERENTI-ATION OF MEDICAL AND SURGICAL COLIC INHORSES. E. Scheepers1, T. Holberg Pihl2, M. Sanz1,P. Page1, A. Goddard1, S. Jacobsen2. 1University ofPretoria, Pretoria, South Africa; 2University of Copenha-gen, Taastrup, Denmark.

Background: Horses with severe medical colic caused by duo-

denitis-proximal jejunitis, colitis or peritonitis that do not need

surgery can be difficult to differentiate from horses with strangu-

lating obstructions or displacements that need surgery. Objec-

tive: To evaluate the usefulness of serum amyloid A (SAA) and

haptoglobin (Hp) measured in peritoneal fluid (PF) to differenti-

ate between horses with mild medical colic (group 1, n=138),

severe medical colic (group 2, n=46) and surgical colic (group 3,

n=124), compared with the classic PF markers: nucleated cell

count (NCC), total protein and hemolysis. Method: Admission

PF concentrations of SAA, Hp, NCC, total protein and hemolysis

were compared among the three groups with ANOVA and chi-

squared test. The effect of duration of colic pre-admission was

included in the analysis. Results: Hemolysis was present in

36% of horses in group 3, 24% in group 2 and 10% in group 1.

All markers were significantly higher in group 2 and 3 compared

to group 1. NCC was the only marker with a difference (p=0.015)

between group 2 (mean: 5.2 x 109/L) and 3 (mean: 1.6 x 109/L).

SAA (p<0.001) and Hp (p=0.009) were increased after 12 hours

of colic duration, whereas NCC was only increased (p=0.01) after

24 hours. Conclusion: Not all horses with hemolysis in PF

require surgery. Measuring a panel of traditional and novel

inflammatory markers in PF can assist in segregating horses

with colic in three groups that require fundamentally different

treatments. When interpreting levels of a measured marker,

duration of disease should be considered.

45MEASUREMENT OF SERUM ALBUMIN BY PROTEINELECTROPHORESIS AND BROMOCRESOL GREENMETHODS IN CANINE AND EQUINE PATIENTS.E. Ramery, F. Bureau. Liege University, Faculty ofVeterinaryMedicine, Liege, Belgium.

Background: The bromocresol green method (BCG) is rou-

tinely used in veterinary laboratories to measure albumin. Good

agreement between BCG and protein electrophoresis (SPE) in

healthy domestic animals is reported. However, in human medi-

cine, at low albumin and high globulin concentrations, BCG also

binds to a- and b-globulins fractions. Surprisingly, there is no

recent literature evaluating the BCGmethod in diseased domestic

animals. Objective: The purpose of the present study was

therefore to compare the serum albumin values obtained by SPE

and BCG in canine and equine patients. Methods: Albumin

measured by BCG and SPE was compared in the serum from 30

canine and 30 equine patients. Results: Good correlation was

found (Spearman´s rho rs = 0.91 in dogs and 0.84 in horses).

However, Altman and Bland analysis of results showed a positive

bias of BCG with SPE (3.41 g/L in horses and 0.41 g/L in dogs)

that increased when only hypoalbuminemic patients were

considered (4.17 g/L in horses and 1.31 g/L in dogs). Conclu-

sion: Results confirm that, despite good correlation, BCG

method tends to overestimate albumin, especially in hypoalb-

uminemic patients. This is particularly evident in horses.

Poster Presentations

46THE EFFECT OF PARITY ON BONE FORMATION DUR-ING LATE PREGNANCY AND EARLY LACTATION INSAANEN GOATS. M. Belic1, V. Kusec2, M. Robic1,J. Grizelj1, A. Svetina1, Z. Vrbanac1, R. Turk1. 1Veter-inary Faculty, Zagreb, Croatia; 2Clinical Institute of Labo-ratory Diagnosis, Clinical Hospital Centre, Zagreb,Croatia.

Background: Bone alkaline phosphatase (BALP) activity

changes in different physiological conditions of the organism, so

in pregnancy and lactation it can have a significant role in evalua-

tion of the rate of bone turnover. There are gaps in our knowl-

edge about changes in bone metabolism during pregnancy and

lactation in small ruminants. Objective: The purpose of this

research was to assess the effect of parity on bone formation dur-

ing pregnancy and lactation in goats. Methods: The research

was conducted on 35 goats of the Saanen breed, which were

divided into 3 groups regarding the number of previous deliveries

(primiparous, biparous and multiparous goats). Blood samples

were obtained 15 days before and 15 days after delivery. The

activity of BALP was measured with a commercial enzyme

immunoassay kit. Differences between study groups were tested

by Student’s t-test. Results: In primiparous goats BALP activity

was significantly lower (p<0.001) 15 days after delivery

compared with the activity measured 15 days before delivery. In

biparous and multiparous goats, BALP activity was higher



post-delivery than in late pregnancy, but not significantly. There

was a statistically significant (p<0.001) negative correlation

between BALP and age in biparous and multiparous goats.

Conclusions: Results demonstrated active bone formation in

late pregnancy and early lactation in goats indicating increased

bone turnover during transition period.

47SUBCLINICAL MASTITIS IN SHEEP: NOVEL DIAGNOS-TIC FINDINGS.A.Miglio1, M.T. Antognoni1, L. Mosc-ati2, C. Maresca2, A. Valiani2, E. Scoccia2,V. Mangili1, G. Fruganti1. 1University of Perugia,Faculty of Veterinary Medicine, Perugia, Italy; 2IstitutoZooprofilattico Sperimentale Dell’Umbria E DelleMarche,Perugia, Perugia, Italy.

Background: Subclinical mastitis (SM) negatively affects yield

and milk quality. The udder of sheep with SM is clinically

healthy, but milk has an increased somatic cell count (SCC)

(>500,000cells/mL) and mastitis bacteria. Objective: To

compare conventional methods of diagnosis of SM (physical

examination of udder and cyto-bacteriological analysis of milk)

with markers of inflammation measured in blood, serum and

milk. Furthermore, we investigated changes of some laboratory

parameters in sheep affected by SM. Methods: Udder-half

milksamples and bloodsamples were collected from 132 clinically

healthy lactating Lacaune ewes. We performed SCC, bacteriolog-

ical analysis, neutrophil-granulocyte count (MNG) and concen-

tration of milk amyloidA (MAA) in milk as well as blood

leukocyte count (WBC) and concentrations of bloodserum total

proteins and electrophoretic protein fractions (PT), iron (Ir), zinc

(Zn), urea (Ur), lysozyme (Lys), haptoglobin (Hp) and serum

amyloid A (SAA). The data were submitted to retrospective

epidemiological study and descriptive and univariate analysis.

Results: MNG and MAA were positively correlated with SCC

and also were significantly higher (P� 0.05) in mastitic samples

(MNG: mean±SD=4,236,505±764/mL and 95% CI=1,079,448-

7,393,562/mL; MAA: 114.37±41.14μg/mL and 98.24-130.50μg/mL) compared with healthy samples (MNG: 8070±434/mL and

6358-9783/mL; MAA: 29.68±27.98μg/mL and 23.84-35.52 μg/mL). Blood parameters were not correlated with SM and

SCC>500,000cells/mL. Conclusions: This report, the first to

evaluate MAA in ewe milk with a milk ELISAkit, suggests MNG

and MAA are potential markers of SM in sheep. Future studies,

includingmore sheep breeds, are needed to confirm the reference

limits detected. WBC, PT, Ir, Zn, Ur, Lys, Hp and SAA are not

useful to identify SM.

48MEASURING GLUCOSE CONCENTRATION IN BLOODOF COWS WITH THE OPTIUM XCEED TM METER ANDTHE BIOCHEMICAL ANALYSER – COMPARISON OFMETHODS. J. Jezek, J. Staric,M.Klinkon,M.Nemec.University of Ljubljana, Veterinary Faculty, Clinic forRuminants, Ljubljana, Slovenia.

Background: Measuring blood glucose concentration

under field conditions is useful in farm animal practice. Objec-

tive: The aim of our research was to compare two different

methods for measuring blood glucose concentration, a reference

biochemical analyser and an on-site method for measuring glu-

cose concentration. Methods: The measurements were per-

formed on 29 bovid blood samples. An appropriate blood volume

for antiocoagulantwas drawn into sodiumfluoride and potassium

oxalate evacuated tubes. The glucose concentration was mea-

sured with the Optium Xceed TM meter in whole blood, directly

on-site. The results were compared with the measurements pre-

formed by the biochemical analyser, a CobasMira. The data were

processedwith SPSS (Ver. 15). The correlation coefficient by Pear-

son was calculated and for assessment of agreement the method

by Bland and Altman (1986) was used.Results: The mean con-

centration of glucose established with Optium Xceed TM was

(3.61 ± 1.47 mmol/L). It was slightly lower than the mean con-

centration established by the Cobas Mira biochemical analyser

(3.62 ± 1.17 mmol/L). The correlation coefficient was 0.984. The

calculation of agreement confirmed that, on average, Optium

Xceed TM gave 0.02 mmol/L (SD = 0.41 mmol/L) lower values

than the biochemical analyser. Conclusion: Based on our

results, we think that this hand-held meter is appropriate for

measuring glucose concentration in the field conditions.

49CHEEK TOOTH ABNORMALITIES IN SHEEP FLOCKS.V. Erjavec.Veterinary Faculty, Ljubljana, Slovenia.

Background: Dental problems are one of the main causes of

premature culling of sheep, i.e., long before they have reached

the end of their natural reproductive life. Objective: To deter-

mine the range of dental pathology occurring in both clinically

healthy sheep and those showing loss of condition within a flock

of sheep. Specimens submitted for investigation of unexplained

death at a pathology centre were also examined. The literature

tends to concentrate on a syndrome often referred to as ‘broken

mouth’, affecting the incisor teeth. However, the posterior or

cheek teeth are rarely considered and their disease, wear

and function during mastication is poorly understood. Meth-

ods: Farm records were studied to determine losses from known

or suspected dental disease. Post-mortem examinations were per-

formed when feasible on slaughtered animals and any that died

naturally to try to determine whether dental disease was a factor.

When lesions were found, the skulls were prepared to show den-

tal abnormalities.Results: Over the 5 years of the study, 34% of

the flockwas found to have visible incisor abnormalities (gingival

recession, excessive mobility and tooth loss). 10% of the flock

showed non-specific signs such as weight loss, breeding failure or

unexpected death. All animals of this latter group were con-

firmed to have cheek tooth disease in addition to their incisor

abnormalities. Advanced cheek tooth disease was found in 84%

of the sheep submitted to the pathology laboratory, whilst only

28% had advanced incisor disease. Conclusion: The results

show that dental disease is a serious problem for sheep flocks.

50GLUTAVAC® TEST SET (GLUTARALDEHYDE BLOODTEST) – DIAGNOSTIC SUPPORT FOR FOREGIN BODYDISEASE. T. Zadnik1, S. Rakulic Zelov2. 1VeterinaryFaculty, Ljubljana, Slovenia; 2Veterinary Station, Zuzem-berk, Slovenia.

Background: Traumatic peritonitis and reticulopericarditis are

consequences of perforated reticula with sharp objects. The glu-



taraldehyde test is used on whole blood for the diagnosis of acute

and chronic inflammation and infection. Objective: We have

used the Glutavac®test as diagnostic support in cows that showed

symptoms of rumen stasis.Methods: 70 cowswith acute rumen

stasis were analyzed using ready-to-use vacutainers with the

Glutavac® test. Venipuncture was performed at the vena coccyg-

ica. The vacutainer was inverted3-times and then left for 15 min-

utes. If the blood coagulates in 5 minutes after it has been mixed

with reagents, the result is highly positive (+++). If coagulation is

finished in 10minutes, the result is positive (++). If coagulation is

finished in 15 minutes, the test is poorly positive (+). In all cows

that had positive test reactions, exploratory laparorumenotomy

was performed.Results: Of Glutavac®-assayed blood of 70 cows

with acute rumen stasis, 54 (77.2 %) bovid samples had highly

positive reactions (49 samples were associated with traumatic

inflammation and 5with liver fluke infestation); 9 (12.8%) cows

had positive (++) reactions (7 cows had reticuloperitonitis traum-

atica and 2 had liver fluke infestation); 7 (10.0 %) cows had

poorly positive (+) results (1 cow had traumatic indigestion and 6

had liver fluke infestation). Conclusion: Based on results, we

recommend the use of the Glutavac® test as a simple valuable

paraclinical confirmative diagnostic method to be used in field

conditions.

51MONITORING SUBCLINICAL KETOSIS IN TRANSITIONCOWS PREGNANT WITH A SINGLE CALF OR TWINS.T. Zadnik1, R. Lombar2. 1Veterinary Faculty, Ljubljana,Slovenia; 2Veterinary Station, Tenetise, Slovenia.

Background: The 7-day close-up period and 15-day fresh per-

iod are critical transitions for optimal health in dairy cows. The

gold standard test for monitoring subclinical ketosis is serum

beta-hydroxybutyrate (BHB). Clinical ketosis is typically defined

as 2.6 mmol/L BHB or more. At concentrations above 1.40

mmol/L BHB, cows are at risk for metabolic disorders. Objec-

tive: The aim of this study was to compare BHB concentrations

in the whole blood of cows in the early pre- and early post-

calving period and pregnant with a single calf or twins. Meth-

ods: Blood BHB concentration was measured with the Opti-

mum XceedTM meter. The results were compared within groups

of transition cows (n = 80) that were pregnant with a single calf

(group 1) or with twins (group 2) (n = 42). All data were statisti-

cally analyzed with SPSS 17.0 (one-way ANOVA).Results: Sta-

tistical analyses showed that mean (1.12 ± 0.93 mmol/L) BHB

concentration in group 1 (single calf) and mean (1.19 ± 1.23

mmol/L) BHB values in group 2 (twins) were not statistically sig-

nificantly different (P>0.05). However, cows with twins were

3times more prone to fetal membrane retention, metritis, masti-

tis, and abomasal dislocation.Conclusion: Based on the results,

we think this BHB method is appropriate for the detection of

ketosis in field conditions. The advantage of the method is that it

is simple and quick. This test is a precise tool to monitor subclini-

cal ketosis and is useful for periodic assessment of subclinical

ketosis in a herd.

52LOGISTIC REGRESSION MODELING FOR THE PREDIC-TION OF LEFT DISPLACED ABOMASUM IN DAIRYCOWS. S. Safi1, N. Basiri1, A. Rahimi Foroushani2.1Faculty of Veterinary Medicine, Science and ResearchBranch, Islamic Azad University, Tehran, Iran; 2Depart-ment of Epidemiology and Biostatistics, Faculty of PublicHealth, Tehran University, Tehran, Iran.

Left displaced abomasum (LDA) is one of themajormetabolic dis-

eases in dairy cattle, which causes large economic losses in the

dairy industry. Thus, predicting the occurrence of LDA, especially

in early weeks post-partum, is very important. In the present

study, 14 biochemical analytes of cows with LDA before and after

parturition were compared with those of healthy cows (the con-

trol group) using the logistic regression statistical model. Changes

in six analytes, including beta-hydroxybutyrate (BHB), non-

esterified fatty acids (NEFA), aspartate aminotransferase (AST),

calcium, sodium and potassium, were found to have statistically

significant differences (p<0.05) between groups and were

included in the prediction model. The model was proposed as:

Log (odds) = 12.778 + 0.053 (NEFA) - 0.031(Na) - 0.550 (Ca) -

0.721(K) + 0.988 (BHB) + 0.008 (AST) = X; Odds = exp (X).

53CANINE CUTANEOUS HISTIOCYTOMA: A RETROSPEC-TIVE STUDY OF 55 CASES (2002-2011). C. Pressanti,F. Granat, A. Geffre,M.C. Cadiergues. Toulouse Veter-inary School, Toulouse, France.

Background: Canine cutaneous histiocytoma (CCH) is a com-

mon benign neoplasm, usually found in young dogs and present-

ing as a solitary lesion that undergoes spontaneous remission.

Objective: The purpose of this study was to compare features of

the disease in a specific population with the small amount of data

available in the literature. Methods: The medical records of

dogs diagnosedwith CCH at Toulouse Veterinary School between

2002 and 2011 were reviewed. Collected information included

breed, age, sex, location and size of the tumor, clinical and cyto-

logical examination, and progression. Diagnosis was made at

minimum by cytological examination. The data obtained were

compared with a reference baseline population (Chi-2 test, p

0.05). Results: Fifty-five dogs were included. Boxers, Bull-

dogs and crossbred dogs were significantly overrepresented

compared with the reference population. Mean age was 2.4

years. The average size was 1.1 cm. Preferential distribution of

the tumor was extremities (19/55), head (17/55), trunk (14/

55), and neck (3/55). Cytological characteristics were quite

homogeneous: clumps of round cells with a variable amount

of pale, often vacuolated basophilic cytoplasm and round

nuclei, occasionally indented, usually eccentric with finely

granular chromatin. Twenty tumors regressed spontaneously

and 12 were surgically excised. Discussions: CCH affects all

breeds with overrepresentation of Boxers and Bulldogs. It most

commonly affects dogs less than 3 years old although in this study

5 dogs were over 6. The extremities, the face and particularly the



pinnae and eyelids were most often affected. All cases were

benign neoplasms. Neither recurrent/persistent CCH nor the

severe Langherhans cell histiocytosis was reported.

54CANINE NEOPLASTIC EFFUSION: MORPHOMETRICANALISYS OF CYTOLOGICAL SAMPLES. G. Ghisleni,V. Pizzatti Sertorelli, A. Forlani, V. Bronzo, M.Caniatti.DIPAV, University ofMilan,Milan, Italy.

Background: Cytology is the best way to detect the presence of

malignancy in body cavity fluids. The differential diagnosis bee-

tween mesothelioma and carcinomas is difficult and may require

additional diagnostic techniques, such as immunocytochemistry.

Objective: The purpose of this study was to evaluate the mor-

phology of neoplastic cells in body cavity fluids using image anal-

ysis software to identify their mesothelial or epithelial origins.

Materials and methods: Ten canine neoplastic effusion

(5 carcinomas and 5 mesotheliomas) were included in this

study. Immunocytochemistry with a panel of 6 antibodies

(CKAE1/AE3, CK20, vimentin, desmin, HBME1, CK5/6) was

used to support the cytologic diagnosis of mesothelioma vs. car-

cinoma. For image analysis, 50 neoplastic cells and 10 clusters

of cells on May Grunwald-Giemsa stained smears were evalu-

ated. Both cellular and nuclear area, diameter (maximum,

mean, minimum), perimeter and roundness of neoplastic cells

were calculated. Area, diameter (maximum, mean, minimum),

perimeter and roundness of larger clusters of cells were

also measured. Statistical analysis of data was performed.

Results: On morphometry, values for mean area of cellular

clusters in carcinomas were 66,234.68 μm2, while for mesotheli-

oma were 21,813.93 μm2, (P < 0.006, highly significant). Signifi-

cant differences were also noted among carcinomas and

mesotheliomas for all other measurements, except roundness.

Conclusion: These results suggest that automated cytodiagno-

sis may aid in the diagnosis of carcinoma andmesothelioma. Fur-

ther studies evaluating stained archived cytology samples

(retrospective studies) and development of systems of automated

cytodiagnosis are warranted.

55EFFECT OF NEEDLE GAUGE ON THE QUALITY OFCANINE SPLEEN FINE-NEEDLE ASPIRATION SPECI-MENS – A PRELIMINARY STUDY. N. Bourges-Abella,C. Layssol, A. Geffre, C. Trumel. Institut NationalPolytechnique-Ecole Nationale Veterinaire de Toulouse,Toulouse, France.

Background: Fine-needle aspiration biopsy (FNAB) of the

spleen is a valuable tool to assess hemolymphatic disorders, infec-

tious diseases and tumorinfiltration. Needle diameters used for

spleen FNAB are variable in literature and have never been com-

pared, nor has animal pain been assessed. Objectives: The aim

of this study was to compare the pain and quality of spleen FNAB

specimens obtained with three different needle diameters: 23, 25

and 27gauge needles on dogs with splenic abnormalities for

which clinicians had prescribed FNAB. Methods: 21 dogs were

included in the study and spleen FNAB was performed according

to the procedure followed routinely, with the three needles in

randomized order for each dog. The 63 slides were indepen-

dently, randomly and blindly studied by two clinical pathologists

(CT and AG) and gradedwith a subjective scoring system. Animal

pain was subjectively evaluated by the operator who performed

spleen FNAB. Results: Agreement between the two observers

was good. Samples obtained with the 27G needle method had

lower cellularity (p = 0.046), poorer stroma (p = 0.02) and less

mesothelial cells (p = 0.001). No difference was found between

the three needle gauges for the 17 other criteria, nor for animal

pain. Conclusion: These preliminary results suggest that 27G

needles may be less valuable than 23 and 25G needles for spleen

FNAB.

56DETERMINATION OF PROLIFERATIVE ACTIVITY INCANINE LEUKEMIAS BY FLOW CYTOMETRY. A.Poggi1, B. Miniscalco1, L. Aresu2, M.E. Gelain2,S. Comazzi3, V. Martini3, E. Morello1, F. Gattino1,F. Riondato1. 1Dept. of Animal Pathology, University ofTorino; 2Dept. of Public Health, Comparative Pathologyand Vet. Hygiene, University Of Padua; 3Dept. of Veteri-nary Pathology, Hygiene and Health, Unit of GeneralPathology, University ofMilan, Italy.

Background: Nuclear antigen Ki67 is widely used as a prognos-

tic indicator in many human tumors, while in veterinary

medicine only few studies on solid tumours exist and no datacon-

cerningleukemias are present. Objective: To evaluate Ki67-

positive cells in peripheral blood samples fromleukemic dogs by

flow cytometry. Methods: 21 canine leukemias were evalu-

ated. Based on clinical aspects, CBC, immunophenotype and

cytologic features, leukemias were grouped as acute lympho-

blastic (ALL, n=2), acute myeloblastic (AML, n=4), acute

undifferentiated (AUL, n=3), chronic lymphocytic (CLL, n=8)

and CLL/lymphoma complex (n=3). EDTA blood samples were

labeled with Ki67-FITC (clone MIB-1) with a rapid fixation/

permeabilizationmethod using methanol.Proliferative activ-

ity was expressed as percentage of Ki67-positive cells.

Results: Percentage of Ki67-positive cellswas significantly

higher in acute than in chronic leukemias (P<0.01).In particular,

decreasing values were recorded in AML, ALL, AUL and CLLwith

statistically significant difference between AML and CLL

(P<0.05).B-CLL presented higher proliferative activity compared

with T-CLL (13% and 4.8%, respectively), although without

statistical significance.No differences between aberrant and

non-aberrant immunophenotypes were detected among CLL.

Conclusion: The higher proliferative activity recorded in acute

canine leukemiascompared with chronic leukemiasconfirms the

role of Ki67 antigen as areliable indicator of biological behavior.A

larger series of samples is neededto check for differences between

immunophenotypesamong AML, ALLand CLL. Follow-up infor-

mationwill help determine if Ki67 could be used to provide

prognostic information.



57PROGNOSTIC VALUE OF KI67 IN CANINE LYMPHOMADETERMINED BY FLOW CYTOMETRY. A. Poggi1,B. Miniscalco1, L. Aresu2, M.E. Gelain2, S. Comazzi3,V. Martini3, E. Morello1, F. Gattino1, F. Riondato1.1Dept. of Animal Pathology, University of Turin; 2Dept. ofPublic Health, Comparative Pathology and Vet. Hygiene,University of Padua; 3Dept. of Veterinary Pathology,Hygiene and Health, Unit of General Pathology, Univer-sity of Milan, Italy.

Background: Percentage of Ki67-positive cells (%Ki67) is

related to malignancy and survival in human nonHodgkin’s lym-

phoma. Little data from immunohistochemical studies are avail-

able for canine lymphoma (LSA). Objective: To investigate the

prognostic significance of flow cytometric %Ki67 in canine LSA.

Methods: 47 canine LSAswere classified by flow cytometry and

cytology, according to the updated Kiehl classification as: 33

high-grade (HG) and 1 low-grade (LG) B-LSA; 10 HG and 3 LG

T-LSA. Lymphnode aspirateswere labeledwith Ki67-FITC (clone

MIB1) using a methanol-based method. Differences in %Ki67

were assessed by a t-test. Kaplan-Meier curves were used to eval-

uate prognostic significance of%Ki67 for complete remission and

survival in 16 dogs treated with the University of Wisconsin-

Madison protocol. Results: %Ki67 was significantly higher in

HG compared with LG LSA (p<0.001). No significant difference

between B- and T-LSA was detected. Treated LSA with

Ki67>40% showed a significantly shorter time to complete

remission (CR) and a significantly lower survival at the end of

chemotherapy (day 175) and overall survival compared to LSA

with Ki67<40%, regardless of grade of malignancy and lineage.

Ki67>40% was also associated with a shorter time to CR among

HG LSA, B-LSA and HG B-LSA and with a lower overall survival

among HG LSA. Conclusion: Flow cytometric %Ki67 is useful

in differentiating HG vs LG LSA using a cutoff value of 12%.

Regardless of cell lineage, %Ki67 can also be considered as a

prognostic marker for time to remission and survival using a cut-

off value of 40%.

58“WATER DIABETES” IN A GREYHOUND ASSOCIATEDWITH CORTICOSTEROIDS. L. Blythe. Oregon StateUniversity, Corvallis, OR, USA.

Background: Greyhounds differ from other breeds of dogs in a

number of ways. Corticosteroids have been known to cause tran-

sient polyuria and polydipsia in normal dogs, which is usually

manageable by the owner.Objective: The aim of this case study

was to describe the use of corticosteroids in a Greyhound. Case

Study: An 8-year-old neutered male Greyhound presented to

the Teaching Hospital with lethargy, anorexia, and neck pain fol-

lowing an episode of chasing a skunk under a building. A CT scan

revealed a ruptured disc at cervical 4 and 5 vertebra with disc

material present in the vertebral canal. A ventral slot surgical pro-

cedure removed the disc material. Following surgery 30 mg/kg of

methylprednisolonewas given. Upon release, excessive urination

was noted. Water was restricted to 1.5 liters per day, but polyuria

continuedwith urination every 30 to 40minutes. The dogwas re-

admitted to the Teaching Hospital where a renal panel and uri-

nalysis revealed urine specific gravity of 1.007. Skin turgor

showed 3% dehydration. The polyuria was treated with continu-

ous IV therapy. By day 4, urine specific gravity was 1.032.

Results: Severe polyuria was induced in this Greyhound by a

single dose of 30mg/kgmethylprednisolone. Four days of contin-

uous intravenous fluids, water restriction, and vasopressin was

needed to return urine concentrating ability to normal. Conclu-

sion: Greyhounds should not be given corticosteroids; when-

ever possible avoid their use or, when given, use of the lowest

possible dose should be considered.

59ACUTE PHASE PROTEIN RESPONSE IN PIGS AFTEREXPERIMENTAL INFECTION WITH DIFFERENTSTRAINS OF PORCINE REPRODUCTIVE AND RESPIRA-TORY SYNDROME VIRUS. Y. Saco1, M. Cortey2,J. Segales3, R. Pato4, R. Pena4, F. Martınez-Lobo5,C. Prieto5, A. Bassols6. 1Universitat Autonoma deBarcelona, Bellaterra, Spain; 2Unite de Virologie Molecu-laire, Emergence et Co-Evolution Virale UMR 6578, Mar-seille, France; 3CReSA, UAB-IRTA, Dept. Sanitati Anatomia Animals, Fac. Veterinaria, UAB, Barcelona,Spain; 4Servei de Bioquımica Clınica Veterinaria, Fac.Veterinaria, UAB, Barcelona, Spain; 5Departament deSanidad Animal, Fac. Veterinaria, Univ. Complutense deMadrid, Madrid, Spain; 6Departament de Bioquımicai Biologia Molecular, Fac. Veterinaria, UAB, Barcelona,Spain.

Background: Porcine reproductive and respiratory syndrome

virus (PRRSV) is a major porcine pathogen, and infection is char-

acterized by reproductive failure in sows and respiratory disease

in growing pigs. The lack of heterologous protection by PRRSV

vaccines is currently a major problem in the field and better

knowledge in the pathogenesis of this infection is necessary for

developing more efficacious vaccines. Objective: The aim of

this work was to characterize and compare the acute phase

response of pigs experimentally infected with different strains of

PRRSV. Methods: 105 3-week-old piglets were divided in 7

groups of 15 animals, of which 4 groups were exposed to geno-

type 1 (European) strains, 2 to genotype 2 (American) strains and

1 served as a non-inoculated, negative control. Clinical signs

were recorded daily. On days 7, 14 and 21 post-inoculation (PI) 5

animals per group were euthanized and pathologically investi-

gated. Blood samples were taken before challenge and on days 3,

6, 9, 12, 15, 18 and 21 PI. The levels of acute phase proteins

(APPs), haptoglobin (Hp), Pig-MAP and CRP were quantified.

Kruskal-Wallis orMann-Whitney tests were performed to investi-

gate the relationship between levels of APPs versus PRRSV strain

and time after infection. Results: Hp and CRP, but not Pig-

MAP, were significantly discriminatory between infected and

control pigs. Pigs challenged with genotype 2 strains showed

significantly higher levels of APPs than those infected with geno-

type 1. Conclusions: APP levels differ between genotypes; the

higher production of APPs induced by American strains

correlatedwith themore pathogenic properties of this strain.

60DIAGNOSTIC VALUE OFMILK ACUTE PHASE INDEX INDETECTION OF BOVINE SUBCLINICAL MASTITIS.S. Safi1, S.H. Shirazi-Beheshtiha2, V. Rabbani2,M. Bolourchi3. 1Science and Research Branch, IslamicAzad University, Tehran, Iran; 2Department of ClinicalSciences, Faculty of Veterinary Medicine, Islamic AzadUniversity, Karaj, Iran; 3Department of Clinical Sciences,



Faculty of Veterinary Medicine, Tehran University, Teh-ran, Iran.

Currently, somatic cell count (SCC) and bacterial culture are con-

sidered the gold standard methods for diagnosis of bovine sub-

clinical mastitis. However, SCC has a low diagnostic accuracy.

Therefore, new biomarkers with high diagnostic accuracy are

needed for identification of infected animals. The objective of this

study was to determine the diagnostic value of milk APPs and

acute phase index for the diagnosis of subclinical mastitis in dairy

cows. Ninety Holstein cows were randomly selected from 5 dairy

farms in Tehran province, Iran. Milk samples were analyzed for

milk amyloid A, albumin, beta-lactoglobulin, alpha-lactalbumin,

immunoglobulins concentrations and milk acute phase index.

Receiver-operating characteristic analysis (ROC) was used to

assess the clinical accuracy of each test using SCC as the reference

method. At a cutoff value of 130,000 for SCC, milk acute phase

index was the most accurate analysis, with a sensitivity of 96.2%

and specificity of 92.1% at values > 0.225. Furthermore, the sen-

sitivity and specificity of milk acute phase index in bovine sub-

clinical mastitis caused by prevalent types of microbial pathogens

in Iran were 100% and its clinical accuracy was 1. In conclusion,

acute phase index has a high sensitivity and specificity in the

diagnosis of subclinical mastitis and may replace SCC so early

diagnosis of subclinical mastitis will become easier and economic

losses will decrease.

61SERUM AMYLOID A AS A MARKER OF INFLAMMA-TION IN CANINE JOINTS. J. Francuski1, N. Andric2,M. Lazarevic Macanovic3, D. Markovic4, A. Radova-novic4,M.Kovacevic Filipovic1. 1Departments of Path-ophysiology and Biochemistry, 2Equine, Small Animals,Poultry andWild Animal Diseases, 3Radiology and Radia-tion Hygiene, 4Histology and Embriology, Faculty of Vet-erinaryMedicine, University of Belgrade, Serbia.

Background: Gross and cytological synovial fluid (gcSF)

evaluation enables differentiation between infectious and

noninfectious clinically visible inflammation, but is not useful in

distinction between subclinical inflammatory and degenerative

changes of the joints. In dogs, serum amyloid A (SAA) is a major

acute phase protein and could be a useful tool in diagnosis of sub-

clinical inflammatory joint diseases. Objective: The aim of this

study was to compare clinical signs, gross anatomy, radiology and

gcSF findings with SF SAA concentration in canine knee joints.

Methods: Examination was performed on four military work-

ing dogs (11 years old) and two nonworking dogs (8 months old)

euthanized for different reasons. Knee joints were xrayed and

gross anatomy changes were assessed bymodifiedMaking’s grad-

ing system. GcSF evaluation and SF total protein (biuret method)

were done. SAA concentration was determined by ELISA (TRID-

ELTA, Ireland). Results: Three military dogs without signs of

lameness, no xray and gross anatomy changes and no gcSF

abnormalities did not have detectable SF SAA. The fourth

military dog had lameness and visible erosive osteoarthritis

(Manking scale 4), no gcSF abnormalities, but had a detectable

SAA concentration. Two young dogs (8 months) euthanized

because of idiopathic epilepsy (no signs of lameness) and the

crush syndrome (no visible knee joint changes) did not have gcSF

abnormalities but had increased SF SAA concentrations.Conclu-

sion: SF SAA concentrationmight be an important parameter in

diagnosis of inflammatory joint diseases even in cases without

lameness or xray, gross anatomy visible changes and gcSF

abnormalities.

62PRO-INFLAMATORY CYTOKINE QUANTIFICATIONIN PIGS EXPERIMENTALLY INOCULATED WITHACTINOBACILLUS PLEUROPNEUMONIAE. M. Quezada¹,A. Islas1, A. Ruiz1, T. Casanova1, C. Lecoq2, D.Munoz2. ¹Faculty of Veterinary Sciences, University ofConcepcion,Chile; 2Agricultural Service of AgriculturalMinistery of Chile.

Background: Actinobacillus pleuropneumoniae (App) is the agent

of Porcine Contagious Pleuropneumonia. App is characterized by

its membrane lipopolysaccharide that is able to induce an

inflammatory response with stimulation of pro-inflammatory

cytokines. This study evaluated the inflammatory response

induced by App through the quantification of IL-1b, IL-6 and

TNF-a in tonsils, retropharyngeal and mediastinal lymph nodes.

Methods: Thirty pigs eight weeks of age were inoculated with

an isolated field strain of App.The control group (n=10) was inoc-

ulated with sterile culture medium and the infected group

(n=20) was inoculated with the isolated field strain of App. The

pigs were euthanized in batches of 5 animals per group, for the

control group at 0 and 48 hours post-inoculation (hpi) and for

the infected group at 4, 8, 24, and 48 hpi. Tissues were obtained

for mRNA extraction and cytokine quantification by qPCR.

Results: Significant differences in cytokine expression were

revealed. IL-1b increased at 4 and 24 hpi in the tonsil; at 8 and 48

hpi in the retropharyngeal lymph nodes; at 8 hpi in the mediasti-

nal lymph nodes. For TNF-a, a significant increase was observed

in the mediastinal lymph nodes. Conclusions: These results

helped to confirm that the patterns of expression of cytokines are

consistent with the pathogenesis of PCP. Support: DIUC Nº209.152.019-1-0

63EVALUATION OF LUNG LESIONS OF PIGS INOCU-LATED WITH A LOW PATHOGENIC SEROTYPE 6 OFActinobacillus pleuropneumoniae. A. Islas1, M. Quezada1,A. Ruiz1, D.Munoz2, C. Lecocq2. 1Faculty of VeterinarySciences, University of Concepcion, Chillan, Chile; 2Agri-cultural and Livestock Service, Ministry of Agriculture,Chile, Santiago, Chile.

Objectives: To study the macroscopic and histological lesions

caused byA. pleuropneumoniae (App), serotype 6, inoculated intra-

nasally in susceptible pigs. Methods: The research was done

with 40 pigs 8 weeks old serologically negative to App. Pigs

were subdivided in 2 groups (G1 and G2) of 20 animals each

and housed in 2 different separate units. Animals were moni-

tored daily for 15 days to control their temperature and clinical

signs. G1 (control group) was inoculated intranasally with 5

mL of sterile PPLO medium + NAD. Pigs in group G2 were

inoculated intranasally with 5 mL of PPLO + NAD culture med-

ium containing 9.3 x 109 CFU/mL of App. Five pigs from each



group were sacrificed at 6, 24, 48 and 72 hours post-inocula-

tion (hpi). Lung score was evaluated and tissue samples were

taken for microbiological culture, PCR and histopathology.

Results: G1 did not show any lung injury, lung score 0. In G2,

injury was observed from 6 hpi; the lung score was 1.0, 1.4, 11.2

and 4.4 at 6, 24, 48 and 72 hpi, respectively. Histopathologically,

in G2 characteristic lesions of the disease after 48 hpi were

observed, with accumulations of PMNs andmacrophages in alve-

oli and ducts, and necrotic lesions. Isolation of App from lung tis-

sue was 0, 20, 80 and 100% at 6, 24, 48 and 72 hpi, respectively.

Conclusions: The lesions were progressive with characteristics

lesion from 48 hrs, which was coincident with the App isolates

from lung samples.Support: FONDECYT 1111045 Project, Chile.

64CRAYFISH PLAGUE IN SLOVENIA: FIRST REPORT OFTHE PLAGUE AGENT APHANOMYCES ASTACI.D.Kusar1, A. Vrezec2,M. Ocepek1, V. Jencic1. 1Veter-inary Faculty, Ljubljana, Slovenia; 2National Institute ofBiology, Ljubljana, Slovenia.

Background: Freshwater crayfish suffer from many diseases,

but the crayfish plague caused by parasitic oomyceteAphanomyces

astaci is the most detrimental for indigenous crayfish species

(ICS), leading to complete extinctions of affected populations. In

Slovenia, the disease was reported at the beginning of the 20th

century, but not in recent times; however, the invasion of resis-

tant non-indigenous crayfish species (NICS) was first recorded in

2003.Objective: The aim of our studywas to inspect all ICS and

NICS populations that inhabit Slovenian freshwaters and show

no clinical signs of infection for the presence of A. astacias

determined by a molecular assay, which represents a reliable and

fast alternative to formerly established diagnostic tools.

Methods: After extraction of DNA from the crayfish cuticle

using the commercial DNeasy Blood & Tissue Kit (Qiagen) pre-

ceded by mechanical tissue disintegration in the MagNA Lyser

Instrument (Roche Diagnostics), highly specific TaqMan MGB

real-time PCR was performed to detect A. astaci in crayfish

material. Results: Of 67 analyzed crayfish, two specimens of

Austropotamobius torrentium (stone crayfish, ICS) and four of

Pacifastacus leniusculus (signal crayfish, NICS) tested positive.

Conclusions: Crayfish plague has not yet become a consider-

able problem in Slovenia. However, for the first time results

revealed the presence of A. astaci in the ICS population of

A. torrentium, which presumably was not in contact with the

NICS carrier, raising new questions about the recent crayfish

plague expansion in Europe.

65PROTEINURIA IN DOGS NATURALLY INFECTED WITHTHE BACTERIUM ANAPLASMA PHAGOCYTOPHILUM.U.Ravnik1, B. PremrovBajuk2,K.Babnik2,N. Tozon1.1Small Animal Clinic and 2Institute of Physiology, Phar-macology and Toxicology, Veterinary Faculty, Ljubljana,Slovenia.

Background: Tick-transmitted infections are frequent triggers

of immune-mediated diseases in animals. Infection gives rise to

immune complexes of microbial antigen-antibody-complement

that may lodge in capillary beds of different organs.Objective:

The aim of this study was to investigate the occurence and origin

of proteinuria in dogs naturally infected with A. phagocytophilum.

Methods: Urine samples were obtained from 45 dogs seroposi-

tive or PCR positive forA. phagocytophilum and 17 control dogs.

Microscopic evaluation of urine sediment, urine protein to creati-

nine ratio (UPC) and SDS-PAGE electrophoresis of urine proteins

were performed. Results: Proteinuria was detected in 36 (58.1

%) samples. Proteins were divided into low molecular weight

(LMW; < 66 kDa), middle molecular weight (MMW; 66-76 kDa),

and highmolecularweight (HMW; > 76 kDa) proteins. LMWpro-

teins with bands at 10, 15, 25 and 55 kDa were frequently

observed in control dogs, so those were considered physiological.

In 11 samples LMW and MMW or LMW, MMW and HMW

together with UPC above the reference interval and casts in

urine sediment were found, all in seropositive dogs. Conclu-

sion: Infection with A. phagocytophilummay lead to the develop-

ment of immune-mediated glomerulonephritis characterised by

the presence of glomerular non-selective and tubular protein-

uria, which prompts follow-up of seropositive patients for early

signs of chronic renal disease.

66EPIDEMIOLOGY OF IBR/IPV, MB/BVD AND SOMERESPIRATORY VIRUSES IN SLOVENIAN AUTOCHTHO-NOUS BREEDS.M. Nemec1, P. Hostnik2, Z. Klinkon3,T. Zadnik1, M. Klinkon1. 1Veterinary Faculty, Clinic forRuminants, Ljubljana, Slovenia; 2Veterinary Faculty,National Veterinary Institute,Virology Laboratory,Ljubljana, Slovenia; 3Veterinary Clinic Klinkon, Homec,Slovenia.

Background: We studied the epidemiology of infectious bovine

rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV),

bovine viral diarrhea (BVD), bovine respiratory syncytial virus

(BRSV), parainfluenza 3 virus (PI-3) and adenovirus 3 (Adeno 3)

infections in autochthonous Cika cattle. Objective: The pur-

pose of the study was to collect preliminary epidemiologic data in

order to maintain a healthy herd.Methods: The study included

94 Cika cattle from different Slovenian regions. Blood samples for

haematology, biochemistry and serology, including detection of

antibodies against IBR/IPV, BVD, BRSV, PI-3 and Adeno 3, were

collected and divided according to results of positive and negative

serological investigations. Significant differences between the

studied values were determined using the Student’s t-test.

Results: We found a positive serological reaction to IBR/IPV in

2 Cika cows. In 36.67 % of the tested animals antibodies to BVD

were serologically detected; 21.59%of the animals were serologi-

cally positive for the presence of BRSV, 47.73% of tested samples

were seropositive for the PI-3 virus and more than half (62.50%)

for the adenovirus type 3. The results of hematological and bio-

chemical investigations showed that the most significant differ-

ences were observed for BVD and only one blood analysis was

significantly different for BRSV. Conclusion: This preliminary

study showed minimal exposure to IBR/IPV and the greatest

prevalence of exposure to adenovirus type 3 based on serologic

evaluation.



67FEASIBILITY OF DETECTION OF TESTOSTERONE ANDESTRADIOL IN HAIR USING COMMERCIAL ELISA KITS.T. Snoj1, A. Nemec Svete2, N. Cebulj-Kadunc1,S. Kobal1. 1Institute of Physiology, Pharmacology andToxicology, Veterinary Faculty, Ljubljana, Slovenia;2Clinic for Surgery and Small Animal Medicine, Veteri-nary Faculty, Ljubljana, Slovenia.

Background: Detection of steroids in hair can be performed by

several methods; however, the use of commercial ELISA kits has

not been described for this purpose. Objective: In the present

study, detection of hair testosterone and estradiol after intra-

muscular administration of testosterone propionate and estra-

diol valerate with commercial ELISA kits was described.

Methods: Wistar rats were divided into two experimental (16

animals) and two control (14 animals) groups. Eight female rats

were subjected to IM administration of testosterone propionate

(20 mg/kg) and eight male rats to estradiol valerate (10 mg/kg).

Animals in the control groups received placebo (0.2 mL of sterile

olive oil). Hair samples were collected before treatment and 3, 6

and 9 days after treatment. Extraction of steroids from hair was

performed by methanol. Commercial testosterone and estradiol

ELISA kits were used to determine testosterone and estradiol

concentrations in hair. Results: A significant increase (P<.05)

in hair testosterone was observed 3, 6 and 9 days after the

administration of testosterone propionate. Estradiol valerate

administration resulted in a significant increase (P<.05) in hair

estradiol concentrations on days 3 and 9 after treatment. In

comparison with the control group, hair testosterone and

estradiol concentrations were significantly higher (P<.01) at all

three sampling times after treatment. Conclusion: Results

indicate that detection oftestosterone and estradiol in hair can be

achieved by use of commercial ELISA kit. Up to 0.80 % of the

administrated testosterone and 0.06%of the administrated estra-

diol were present in hair.

68ELECTRON MICROSCOPIC EXAMINATION OF BUFFYCOAT: SIMPLE METHOD USING PLASTIC MEMATO-CRIT TUBE AND APPLICATION FOR DIAGNOSISOF LYSOSOMAL STORAGE DISEASES IN DOGS ANDCATS. A. Yabuki, S. Yoneshige, K. Mizukami,M.M. Rahman, M.M. Uddin, O. Yamato. KagoshimaUniversity, Kagoshima, Japan.

Background: Electron microscopic (EM) observation of the

buffy coat helps for diagnosis of lysosomal storage diseases, but it

is not easy to prepare EM samples from the buffy coat.

Objective: The aim of the present study was to develop a simple

method to prepare EM samples from the buffy coat and evaluate

the diagnostic efficacy of this method.Methods: Blood samples

were obtained from dogs and cats, including clinical cases with

lysosomal storage diseases. Blood was drawn into the plastic

hematocrit tubes and centrifuged. Short pieces containing the

buffy coatwere cut off andfixedwith glutaraldehyde and osmium

tetraoxide. The buffy coat was expelled from the tubes in a rehy-

dration step and embedded in epoxy resin using flat molds. After

polymerization, sample blocks were attached to the other blocks

formed with a capsule mold so that longitudinal sections could be

obtained. Thin sections were stained with toluidine blue, and ul-

trathin sections cut from leucocyte layers were stained with ura-

nyl acetate and lead. Results: The present method provided a

microscopic view of the arrangement of the layers, which con-

sist of platelets, leukocytes and red blood cells, and target leu-

kocytes could be easily found within the layers. In clinical

cases, ultrastructural features of inclusion bodies in leukocytes

were identified without difficulty. Morphological quality of the

cells was successfully preserved. Conclusion: Using the pres-

ent method, buffy coat is conveniently prepared for EM, and this

method is useful for ultrastractural examination of the buffy coat

in lysosomal storage diseases.

69LYMPHOCYTE ANTIGENS: STABILITY OF EXPRESSIONAMONG HEALTHY DOGS AND UPON STORAGE.F. Riondato, A. Poggi, A. Rota, B. Miniscalco. Dept.of Animal Pathology, Grugliasco (TO), Italy.

Background: Reliable detection of fluorescence intensity (FI)

by flow cytometry is fundamental. FI is dependent on instrument

settings, and measurement should be done using controls with

known FI, preferably microbeads or cell subsets with stable mean

FI (MFI) within the population. Data about the stability of anti-

gen expression in healthy dogs do not exist, both among different

subjects and after sample storage. Objective: The aim was to

evaluate MFI stability of the main lymphocyte antigens among

different subjects and after storage (fresh vs 24h). The final aim

was to detect antigens to be used as internal controls in flow anal-

yses of FI. Methods: Fresh (n=14) and stored (n=20) EDTA

peripheral blood samples from healthy dogs were submitted for

flow MFI analysis of CD3, CD5, CD4, CD8, CD21, and cyCD79b

using conjugated MAbs. Adding FlowCheck microbeads (Coul-

ter) MFI was calculated as MFIratio (CD/beads). Stability among

subjects was assessed as CV of MFIratio (fresh samples). For each

CD, CV of MFIratios and mean CV of fluorescence CVs were con-

sidered. Comparison between MFIratio and CV of fresh vs stored

samples was carried out (t-test). Results: Less variation

between subjects was reported for CD79b, CD4 and CD21hi.

Minor variation between lymphocytes of single subjects was

reported for CD21hi and CD4. No CDs reported significant MFI

differences between fresh and stored samples; a significant differ-

ence in CV was detected for CD5 and CD21. Conclusion: Con-

sidering that CD79 needs permeabilization and that a consistent

CD21hi population is not constantly recognizable, CD4 is the best

internal control for CDMFI evaluation.

70DIAGNOSTIC MEASUREMENTS OF CANINE SERUMAMYLOID A USING AUTOMATED LATEX AGGLU-TINATION TURBIDIMETRY: AN INTERLABORA-TORIAL COMPARISON. M. Christensen1, J. Ceron2,A. Tvarijonaviciute2,M.Kjelgaard-Hansen1. 1Depart-ment of Small Animal Clinical Sciences, University ofCopenhagen, Frederiksberg, Denmark; 2Department ofAnimal Medicine and Surgery, University of Murcia,Murcia, Spain.

Background: Canine serum amyloid A (SAA) has proven use-

ful as an inflammatory marker, and a latex agglutination turbidi-

metric immunoassay (LAT) (Eiken, Japan) has been validated for

use in dogs. For an assay to be useful for routine diagnostic pur-



poses, it should be applicable across different laboratories.Objec-

tive: The aimof the studywas to evaluate the performance of the

LAT across two laboratories. Method: An automated clinical

chemical analyser was used at the University of Murcia (Olympus

AU600) for comparison of the initial evaluations previously

obtained at the University of Copenhagen (Advia 1800). Intraas-

say coefficients of variations (CV) were calculated from replicate

determinations of serum pools. The lower limit of quantification

(LLOQ) was the concentration where CV exceeded 20%. Detec-

tion limit (DL) was determined from replicate determinations of

blanks (n=7). Inaccuracy was investigated by dilution of pooled

serumwith high SAA concentration (linear regression). Interlab-

oratorial method comparison was made by measuring SAA in 20

samples in both settings (linear regression and Bland-Altman

plot). Results: Imprecision was 1.4% when measuring high

concentrations of SAA and increasing CVswere observed towards

LLOQ=18mg/L. DLwas 0.0mg/L. Acceptable linearity under dilu-

tion was observed from 0-2700mg/L. A systematic disagreement

was observed between the laboratories (Y-intercept 22.45). Con-

clusion: Comparable performance was obtained in the two lab-

oratories and comparable overlap performances can be expected.

However, clinical decision levels should be estimated locally.

71ELEVATED PLASMA ADIPONECTIN LEVEL ANDPERIPHERAL BLOOD LEUKOCYTE ADIPONECTINRECEPTOR EXPRESSION IN INSULIN DEFICIENT DOGS.N. Mori. Nippon Veterinary and Life Science University,Tokyo, Japan.

The aim of this study was to investigate the effects of obesity or

insulin deficiency on plasma adiponectin level and peripheral

blood leukocyte (PBL) adiponectin receptor 1 and 2 (ADIPOR1

and –R2) expression in dogs. Obese and insulin deficient (ID) dogs

demonstrated a significant reduction (60% lower) and significant

increase (2x higher) in circulating adiponectin level, respectively,

as comparedwith normal controls. PBL ADIPOR1 and -R2mRNA

expression was also significantly higher in obese (R1=333 mean

fold higher, R2=64 mean fold higher) and ID dogs pre-insulin

treatment (R1=48 mean fold higher, R2=16 mean fold higher) as

compared with normal control PBL. Insulin treatment reduced

ADIPOR1 (3 mean fold higher) and -R2 (1.5 mean fold higher)

expression to near control PBL levels in ID animals. The upregula-

tion of adiponectin receptor expressionmight reflect an increased

need for adiponectin signaling; however, the increasemay have a

different implication between obese and ID dogs. This may be due

to adiponectin’s contradictory paradoxical dual role, having both

anti-inflammatory and pro-inflammatory effects on PBL, espe-

cially on monocytes, depending on the biological context and

adiponectin isoform. Adiponectin receptors have the potential to

become importantmetabolic parameters for dogs.

72COMPARING THE USE OF BODY FAT PERCENTAGEVERSUS 5-POINT BODY CONDITION SCORE FORASSESSING OVERWEIGHT STATUS IN DOGS BYSCREENING PLASMA METABOLITIC PROFILES. G. Li,P. Lee, N.Mori, I. Yamamoto, K.Koh, T. Arai.NipponVeterinary and Life Science University, Tokyo, Japan.

Currently, the 5-point BCS system is commonly used by veteri-

narians and clinicians to assess adiposity in dogs in Japan. How-

ever, assigning a BCS score to an animal is subjective in nature.

The use of body fat percentage (BF%) is more objective and may

be more relevant for assessing increasing adiposity, especially in

overweight animals. The sensitivity of BF % versus 5-point BCS

for detecting increasing adiposity in overweight dogs via altera-

tions to plasma metabolitic values was compared against healthy

animals. Overall, healthy BF % range was determined to be

15-20% for non-neutered male dogs and 15-25% for female

(non-spayed/spayed) and neutered male dogs. BCS-categorized

overweight animals displayed significantly higher levels of non-

esterified fatty acids (NEFA; p=.005), whereas significantly

higher levels of NEFA (p=.006), total cholesterol (T-Cho;

p=.029), and triglycerides (TG; p=.001) were observed in BF

%-categorized overweight animals as compared with healthy

animals. BF % positively correlated with plasma insulin, NEFA,

T-Cho, TG, blood urea nitrogen, creatinine, and total protein

levels, which all tend to increase as a result of increasing adipos-

ity. BF% appears to be more sensitive than 5-point BCS to detect

alterations in plasma metabolitic values, especially lipid metabo-

lites, induced as a result of increasing adiposity in dogs. Therefore,

5-point BCS and BF % should complement one another and be

used in tandem, in order to better detect and diagnose overweight

status in dogs, allowing for possible early intervention and pre-

vention of development of more advanced stages of obesity.

73cDNA CLONING AND mRNA EXPRESSION OF DOGCDKAL1. I. Yamamoto, A. Mori, H. Takemitsu,N. Mori, G. Li, K. Kawasumi, T. Arai. Nippon Veteri-nary and Life Science University, Tokyo, Japan.

Background: CDK5 regulatory subunit-associated protein 1 -

like 1 (CDKAL1) gene encodes a protein that regulates insulin

secretion through the regulation of CDK5 activity.Objective: In

this study, we performed cDNA cloning of dog CDKAL1 to deter-

mine the gene structure and mRNA expression profile in tissues.

Methods: Total RNAwas extracted from dog tissue using TRIzol

reagent. The full cDNA cloning was performed using the 5’ and 3’

RACE PCRmethod. The mRNA expression profile was performed

by realtime PCR analysis. Results: Dog CDKAL1 cDNA con-

sisted of 160 bp of 5’ –UTR, 1728bp ofORFand 953 bp of 3’ –UTR.The predicted dog CDKAL1 amino acid sequence was compared

with other animals and revealed high sequence similarity to cow

(92.2 %), human (91.7 %), mouse (88.5 %), frog (83.6 %), sal-

mon (80.3 %) and chicken (80.2 %). Based on BLAST Genome

analysis, dog CDKAL1 gene consisted of 15 exons and revealed

the presence of an approximately 630 kb intron. mRNA expres-

sionwas detected in all examined tissueswith high levels in heart,

skeletal muscle and testis in 2 year old male beagles. Conclu-

sion: These results suggest that dog CDKAL1 gene is well con-

served and may have tissue specific mRNA expression

mechanisms in dogs.

74DETECTION OF PROTEINURIA IN DOGS AND CATS –AGREEMENT BETWEEN AN IN-HOUSE DRYCHEMISTRY SYSTEM (AUTION MICRO™) ANDCONVENTIONAL WET CHEMISTRY. B. Rutgen,I. Schwendenwein. University of Veterinary MedicineVienna, Vienna, Austria.

Background: Proteinuria is an early symptom of renal disease

and is crudely assessed by dipstick or precipitation methods.



Determination of the urinary protein creatinine ratio (UPCR)

allows quantification of urinary protein losses without 24h urine

collection. In-house ssays for assessment of UPCR are not widely

available. Objective: The purpose of this study was to deter-

mine agreement between the UPCR results derived by the Aution

Micro™an in-house dry chemistry system with conventional

methods on a Cobas 501c autoanalyzer. Methods: The Aution

Micro™ (Menarini Diagnostics) measures urine creatinine and

urine albumin by reflectance photometry and calculates UPCR.

Creatinine is determined by a palladium-dye complex reaction

and albumin by a dye-binding method (TCTIF), which forms a

pale pink complex. The reference methods were an enzymatic

method for creatinine and a turbidimetric method with benze-

thoniumchloride for protein on a Cobas 501c. Simultaneous

analysis of 100 fresh canine urine samples and 81 feline samples

was performed. Statistical evaluation of agreement was per-

formed by a kappa test as only semiquantitative results are

obtained by the test system. The cutoff for the reference method

in dogs was 0.3 and 0.5in cats.Results: 87% of dogs and 81% of

cats were correctly classified by the test, which had a sensitivity

of 85% in dogs and 80% in cats. Specificity was 89% in dogs and

81% in cats. Conclusions: The Aution Micro shows adaequate

agreement, sensitivity and specificity compared with conven-

tional methods for the semiquantitative assessment of protein-

uria in dogs and cats.

75HEME-MEDIATED BINDING OF ALPHA-CASEIN TOFERRITIN: EVIDENCE OF ALPHA-CASEIN BINDINGTO FERROUS IRON. K. Orino, Y. Yoshikawa, K.Watanabe.Kitasato University, Towada-shi, Japan.

Background andObjective: The iron-storage protein, ferritin,

is not only present in all kinds of cells but also in extracellular flu-

ids, such as serum, synovia, andmilk. Ferritin binds heme as well

as iron. Bovine a-casein was identified as a ferritin-binding pro-

tein. This study was done to reveal the binding mechanism of

a-casein with ferritin. Methods: Casein protein-coated plates

(20 pmol/well) were incubated with bovine spleen ferritin

(1 pmol/well) followed by detection using biotinylated anti-

bovine spleen ferritin antibody and ALP-labeled avidin. To

examine the binding mechanism of a-casein with hemin and

metal ions, biotinylated hemin was used. Casein protein-

coatedplates (4 pmol/well) were incubated with biotinylated

hemin (32 pmol/well) followed by detection using ALP-labeled

avidin. Results: The binding of a-casein to bovine spleen

ferritin was blocked by hemin, but not by protoporphyrin IX or

zinc-protoporphyrin IX. The binding of a-casein to ferritin and

biotinylated hemin was inhibited by ferrous ammonium sulfate

(FAS). FAS-mediated inhibition of a-casein to biotinylated

hemin was neutralized with Ferrozine, but not with NTA, while

FAS- as well as ferric chloride-mediated inhibition was neutral-

ized by NTA. The following ions also inhibited a-casein-biotiny-lated hemin binding in order of potency of inhibition: Fe2+, Fe3+,

Cu2+, Zn2+, Mn2+, Ca2+, Mg2+. Conclusion: These results

suggest that the binding of a-casein to ferritin is heme-mediated

through direct binding of a-casein to heme iron on the surface of

the ferritin molecule, and that a-casein preferentially binds Fe2+

comparedwith any othermetal ions, including Fe3+.

76C-REACTIVE PROTEIN AND SOME BIOCHEMICALPARAMETERS IN FEMALE DOGS AFTER OVARIOHYS-TERECTOMY.N. Hadzimusic. Veterinary Faculty Saraj-evo, Sarajevo, Bosnia-Herzegovina.

Background: An increase in serum CRP concentration as well

as changes in some biochemical parameters have been described

after different surgical procedures in dogs. Ovariohysterectomy is

widely used as a method of contraception and a large number of

female dogs are subjected to surgery for neutering each year.

Objective: The aim of this studywas to investigate CRP concen-

trations before and after ovariohysterectomy, as well as the corre-

lation between CRP concentrations and some biochemical

parameters (total protein, albumin, alkaline phosphatase, alanine

transaminase, aspartate transaminase and lactate dehydroge-

nase).Methods: Ten clinically healthy female dogs were admit-

ted for elective ovariohysterectomy. Blood samples were

collected from the distal cephalic vein just before ovariohysterec-

tomy (baseline values) and after 24 hours (day 1), 48 hours (day

2), 72 hours (day 3) and 7 days (day 7). Results: Means-

erumlevelsofCRPonday1(85.15 ±16.2 lg/mL) and day 3 (31.27 ±12.06 lg/mL) were significantly higher (P<.001 for day 1 and

P<.05 for day 3)than the baseline value (15.35±13.77lg/mL). No

statistical difference was found between the baseline value and

day 7 (12.08 ± 4.91 lg/mL). There was no significant relationship

between CRP levels and investigated biochemical parameters.

Conclusions: The present study showed that CRP changes rap-

idly, with maximum CRP concentration detected on the first day

after surgery, while on the 7th postoperative day serumCRP con-

centration was within physiological ranges. During the postoper-

ative period changes in CRP concentration and investigated

biochemical parameters showed no significant correlation.

77COMPARATIVE STUDY OF PLASMA LIPIDS INHEALTHY DOGS AND DOGS WITH SKIN DISEASES.R. Barrera1, P. Ruiz1, C. Zaragoza1, F.J. Duque1,A. Buono2. 1University of Extremadura, Caceres, Spain;2University of Bologna, Bologna, Italy.

Background: The diagnostic protocol in canine dermatology

includes determination of plasma lipids, which is also used in the

diagnosis of endocrine diseases. However, these parameters can

also increase in dogs with nonendocrine skin disorders. Objec-

tive: The aim of this study was to contribute to the interpreta-

tion of the levels of plasma lipids in the diagnosis of skin diseases

in canine medicine. Methods: 142 dogs were studied including

Group I (20 healthy dogs) and Group II (122 dogs with skin dis-

ease). Diseases included: hyperadrenocorticism (16), hypothy-

roidism (7), demodectic mange (18), leishmaniasis (28), primary

pyoderma (14) and allergy (39). The plasma concentrations of

total cholesterol, HDL-cholesterol, LDL-cholesterol and tri-

glycerides were determined. Results: When we compared the

results obtained in the different diseases with those observed in

healthy dogs we found statistically higher concentra-

tions (P <.001) for all parameters in both endocrine diseases, a

significant increase (P <.05) of LDL-c in the subgroup with

demodectic mange, a significant increase (P <.05) in the con-

centrations of total cholesterol, triglycerides and LDL-c in dogs

with leishmaniasis and, finally, a significant increase in the



concentrations of total cholesterol (P <.001), triglycerides,

HDL-c and LDL-c (P < 0.05) in the dogs with allergy. Conclu-

sions: Increases of the plasma concentrations of lipids and

lipoproteins are very frequent in skin diseases and not only in dis-

eases of endocrine origin. The study of these lipid alterations is

very useful to diagnose the diseases affecting the skin. Study

funded by Junta de Extremadura (Spain).

78RELIABILITY OF A SINGLE COLLECTION OF URINESAMPLE IN DETERMINATION OF URINE PROTEIN TOCREATININE RATIO. G. Rossi1, A. Zatelli2, S. Paltrini-eri3. 1University ofMilan,Milano, Italy; 2Clinica Veterina-ria Pirani, Reggio Emilia, Italy; 3Department of VeterinaryPathology, Hygiene and Public Health, Milan, Italy.

Background: Several studies have stressed the importance of

correctly identifying and treating proteinuria in dogs. The per-

sistence of proteinuria is a key element in the assessment of

proteinuria. Therefore, proteinuric dogs should be repeatedly

tested to be classified as “persistently proteinuric”. Neverthe-

less, no studies about the biological variability of proteinuria in

dogs are available. Objective: The aim of the study wad to

assess the biological variability of proteinuria and its possible

influence on classification of patients based on the International

Renal Interest Society (IRIS) staging. Methods: Urine samples

were collected from 36 dogs. Exclusion criteria were: poorly con-

trolled renal disease, recent changes of treatments or diet, and

presence of concurrent diseases. Urine samples were collected

twice, at least two weeks apart, and centrifuged. Sediment analy-

sis and measurement of the urinary protein:creatinine (UPC)

ratio were performed. Results of first and second sampling were

compared using a Wilcoxon test either on the whole population

or after grouping dogs according to the IRIS stage.Results: Dogs

were substaged as N.23=non-proteinuric, N.6=borderline pro-

teinuric, and N.7=proteinuric. No significant differences were

detected between the UPC ratio recorded at first and second sam-

pling either in the whole population (P=.905) or in groups based

on IRIS staging (Stage I: p=.821; Stage II: p=.687; Stage III: .468).

Nevertheless, in 8/36 cases the biological variability was high

enough to induce a shift from non-proteinuric to borderline pro-

teinuric. Conclusions: The biological variability of the UPC

ratio in dogs with stable renal conditions is low, but it could affect

the IRIS classification of patients.

79MEASUREMENT OF SERUM PROGESTERONE INMARES: A METHOD COMPARISON STUDY. G. Lubas,A. Gavazza, A. Rota, D. Panzani, S. Demi.Dept Veteri-nary Clinic, University of Pisa, San Piero a Grado, Pisa,Italy.

Background: Serum progesterone (P4) concentration in the

mare can be used to determine the phase of the estrous cycle and

adds useful information in the establishment andmaintenance of

early pregnancy. Objective: A comparison study used several

methods/techniques to assess P4 values, including fluorescence

enzyme immunoassay (FEIA) (2 assays), chemiluminescence (2

assays), and liquid chromatography tandem mass spectrometry

(LC/MS). Methods: Forty serum samples from mares in differ-

ent stages of estrus cycle or pregnancy were collected and stored

at -20°C until evaluation. A P4 recovery study measured low,

medium and high concentrations using the FEIA marketed by

Tosoh Bioscience (TB). All samples were initially assayed by

FEIA-TB. Fifteen samples including low, medium and high P4

values were used for comparison studies (regression analysis, r2).

Results: The recovery study for FEIA-TB showed results over

100% especially when testing high P4 values. FEIA-TB had

very good agreement with the two chemiluminescence assays

(r2 >.95), whereas the two ELFA assays showed a lower r2

(.91). The lowest r2 (.87) was found between the FEIA-TB and

LC/MS values. FEIA0TB had a positive bias in comparison with

all other methods. Conclusion: The P4 measurement using

FEIA-TB showed good to excellent agreement when compared

with different methods/techniques. It should be pointed out that

the FEIA-TB produced results consistently slightly higher than

other methods/techniques. A test-specific reference interval is

recommended for clinical use in equine reproduction.

80ISCHEMIA-MODIFIED ALBUMIN IN HORSES EXER-CISED AT DIFFERENT INTENSITIES ON A TREADMILL.L.A. Yonezawa1, T.S. Barbosa2, M.J. Watanabe2,J.L. Knaut2, C.L. Marinho2, L.E.S. Michima3,A. Kohayagawa2. 1School of Veterinary Medicine andAnimal Science, Sao Paulo State University, Sao Paulo,Brazil; 2School of Veterinary Medicine and AnimalScience, Sao Paulo State University, Botucatu, Brazil;3School of Veterinary Medicine and Zootechny, Univer-sity of Sao Paulo, Sao Paulo, Brazil.

Background: Ischemia-modified albumin (IMA) is a marker of

myocardial ischemia in humans in contrast to other biomarkers

released only after cardiac necrosis. Little is known about release

conditions of IMA in exercise and this is the first report in equine

species. Objectives: This study determined IMA for evaluation

of possible myocardial ischemic conditions induced by high- and

low-intensity exercise in horses. Methods: Ten clinically

healthy horses were submitted to a high-intensity test (HIT) and

a low-intensity test (LIT). Blood samples were taken before, dur-

ing and immediately after exercise, and 15 and 30min thereafter.

Serum IMA, lactate, albumin, and plasma malondialdehyde

(MDA) concentrations were determined. Results: There were

no significant changes (P>.05) in IMA concentration in any of

exercise tests. IMA levels ranged from 0.242±0.071 to

0.292±0.056 ABSU in HIT and from 0.246±0.026 to 0.277±0.021ABSU in LIT. There was also a negative correlation between IMA

and albumin levels in both tests, and between IMA and lactate

levels in LIT, suggesting possible assay interferences. Conclu-

sions: HIT and LIT did not promote significant changes in IMA

concentration in horsesin this study. Though literature data sug-

gest that exercise does not produce myocardial ischemia, we

believe that it occurs. Further studies should be undertaken to

determine if IMA is sensitive for ischemic evaluation in equids in

situations of exercise. Financial support: Sao Paulo State

Research Foundation (FAPESP), Brazil.



81HIGH MOBILITY GROUP BOX 1 AS A PREDICTOR OFGASTRIC NECROSIS. I. Uhrikova, L. Rauserova-Lexmaulova, K. Rehakova, I. Hajek, J. Doubek.University of Veterinary and Pharmaceutical SciencesBrno, Brno, Czech Republic.

Background: Gastric dilatation and volvulus syndrome (GDV)

is an acute disease with high mortality. Gastric rotation and dila-

tation leads to decreased venous return with development of

shock and decreased perfusion of the stomach wall. Gastric

necrosis is a serious complication of the disease and one of the

major negative prognostic factors. High mobility group box 1

(HMGB1) is a nuclear protein that is released during cell necrosis.

Objective: The aim of this study was to evaluate HMGB1 as an

indicator of gastric necrosis.Methods: Bloodwas collected from

36 dogs with GDV before surgical treatment. Serum was stored

and HMGB1 analysis was performed according to manufacturer

instructions. Results: Twenty-nine dogs with GDV survived

and seven died due to gastric necrosis. From the group of survi-

vors two dogs with gastric necrosis underwent partial gastrec-

tomy. There was no significant difference in HMGB1

concentration between survivors and non-survivors, with mean

HMGB1 concentrations of 6.0 ng/ml and 35.3 ng/ml, respec-

tively. A significant difference in HMGB1 concentration was

found between patients with gastric necrosis (30.4 ng/ml) and

others (5.5 ng/ml, p=.05). A cutoff value of 10 ng/ml had a sensi-

tivity of 88% and specificity of 92% for the presence of gastric

necrosis. Sensitivity reached 100% at cutoff value of 6 ng/ml but

specificity was only 63%. Conversely, 100% specificity was

obtained at a cutoff value of 28 ng/ml but sensitivity dropped to

44%. Conclusion: HMGB1 is a candidate marker for detection

of gastric necrosis in patients with GDV. This studywas supported

by Grant No. 24/2011/IGAVFUBrno.

82COMPARISON OF THE SCHMIDT HAENSCH ANDATAGO PAL-USG REFRACTOMETERS FOR DETERMI-NATION OF URINE SPECIFIC GRAVITY. H. Tvedten1,A. Noren2. 1Swedish University of Agric. Sciences,Vange, Sweden; 2Stromsholm Specialist Animal Hospital,Stromsholm, Sweden.

Background: Our veterinarians suggested that urine specific

gravity results from our new Atago digital PAL-USG refractome-

ter (Atago) were too low. Materials and Methods: Specific

gravity results of 60 paired patient urine samples from dogs and

cats analyzed with the Atago were compared with those of a

Schmidt + Haenschmodel HR12P refractometer (S+H). Addition-

ally, specific gravity of dilutions of 10% glucose or 10% NaCl

solutions or highly concentrated patient urine samples was deter-

mined with both hand-held refractometers. Dilutions were

100% to 10% of original concentration. Results: Both refrac-

tometers reported 1.000 with distilled water. Atago had a propor-

tional negative error of a mean of 0.006 units. The Atago’s

negative bias at an S+H urine specific gravity of 1.040 was 0.012

units. Of 35 urine samples with readings from both instruments,

10 samples had an S+H reading of > 1.030, whereas paired Atago

results were 1.023-1.028. S+H results with the glucose solution

dilution matched exactly expected values, but Atago results were

clearly less. S+H results with the saline solution dilutionwere clo-

ser to expected values but lower. Atago saline results were clearly

less than expected or S+H results. Discussion and conclu-

sions: Atago had a negative proportional error that would often

adversely affect determination of a clinically concentrated urine

sample (> 1.030 dog, > 1.035 cat). The highest S+H reading was

1.040; thus dilution of urine would be needed to determine

higher values.

83EFFECT OF PHENOBARBITAL THERAPY ON SELECTEDLABORATORYPARAMETERS:ARETROSPECTIVESTUDY(2007-2012). I. Hajek, P. Schanilec, I. Uhrikova. Vet-erinary and Pharmaceutical University Brno, Brno, CzechRepublic.

Background: It is well known that specific drugs alter clinical

biochemistry results. Phenobarbital as an antiepileptic medica-

tion significantly increases levels of liver enzymes in humans as

well as in dogs and cats. The magnitude of these changes is vari-

ously described in dogs and is unknown in cats.Objective: The

aim of this study was to evaluate the effect of phenobarbital ther-

apy on hematological and biochemical profiles. Methods: Data

from hematology and clinical biochemistry of patients medicated

with phenobarbital due to epilepsy were collected. The total

number of 32 dogs and 4 cats was divided into two groups. Group

A consisted of 20 dogs and 4 cats with blood collected at the

beginning of medication and one or more repeated blood

collections from 1 week to 9 months of medication, and group B

of 12 dogs treated with phenobarbital for more than one year.

The dose of phenobarbital was from 1.6 to 6.8 mg/kg/day.

Results: In group A, a significant effect of phenobarbital medi-

cationwas noticed fornALP activity (p=.02)with amean increase

of 1.4 ukat/L and platelet count (p=.03) with mean increase of

19.109/L. No significant change was found in ALT activity, con-

centrations of albumin, total protein, creatinin, urea, or total leu-

kocyte and erythrocyte counts. Mean ALT and ALP activities in

dogs in group B were 1.46 and 3.67 ukat/L, respectively. In cats

only albumin concentration significantly decreased (p=.01), but

all samples were collected within 2 months after beginning of

medication. Conclusion: Clinically relevant changes in bio-

chemical profile were found only in alkaline phosphatase activity

in dogs.

84APPLICATION OF PROTEOMICS TO SERA: DIFFER-ENCES BETWEEN VACCINATED AND NONVACCI-NATED SHEEP. J. Mavromati1, G. Mazzuchelli2.1Veterinary Medicine Faculty, Tirana, Albania; 2Univer-sity of Liege,Mass Spectrometry Laboratory GIGA, LIEGE,Belgium.

Background: The proteomes of laboratory-grown strains Rev

1 and 16M of Brucella melitensis have been investigated, but no

proteomic study about sheep serum after vaccination has been

conducted yet. Objective: The main purpose of this study was

to identify protein biomarker candidates of the sheep vaccination

process in serum.Methods: The study was conducted in a flock

of sheep free of brucellosis and in another flock vaccinated with

Rev. 1B. melitensis through the conjunctival route in Albania.



After preparation of the serum samples in the mass spectrometry

laboratory–GIGA - Proteomics, University of Liege, Belgium dur-

ing November 2011, a differential proteomic study using a label

free LC-MSe method was conducted (nano-2D UPLC coupled to

a Synapt TMHDMS TMMSe,Waters).Results: The numbers of

identified proteins were about 50 and 110, respectively, in each

sample pool using the sheep NCBI database and reviewed mam-

malian data base. The results, especially using the homology

identification method against the mammalian database, showed

interesting findings. Proteins were identified from other taxono-

mies indicating that these protein sequences are not yet available

in the NCBI sheep database, but most probably are present in the

sheep serum. Conclusion: Some protein activity seems to

have been modified between the two sheep populations. This

was shown by the fact that numbers of proteins partners from

the same biochemical pathway were found to be overexpressed

or modulated only in the group of vaccinated sheep. These

results need to be further investigated.

85COMPARISON OF URINE PROTEIN/CREATININERATIO INURINE SAMPLES COLLECTEDBYCYSTOCEN-TESIS AND MANUAL COMPRESSION IN CATS – PRELI-MINARY RESULTS.H. Vilhena1, R. Santos2, T. Sargo3,T. Lima4, S. Dias4, F.L. Queiroga3, A.C. Silvestre-Ferreira3. 1Escola Universitaria Vasco da Gama, Oliveir-inha - Aveiro, Portugal; 2Escola Universitaria Vasco daGama, Coimbra, Portugal; 3Departamento de CienciasVeterinarias - Universidade Tras-os-Montes e Alto Douro,Vila Real, Portugal; 4Policlınica Veterinaria de Aveiro,Aveiro, Portugal.

Background: The urine protein/creatinine (UPC) ratio is gen-

erally considered reliable only in urine samples collected by cy-

stocentesis. In dogs, previous studies demonstrated the efficacy of

quantification of proteinuria in urine samples obtained by free

catch when compared with samples collected by cystocentesis.

Objective: The aim of this study was to determine if, in cats, the

results for UPC ratios determined in urine samples collected

by manual compression provided similar diagnostic information

to that obtained in samples collected by cystocentesis. Meth-

ods: Twenty-five client-owned cats were included in the study.

Three ml of urine from the midstream phase of micturition were

collected by manual compression, and 3 ml of urine were

obtained by ultrasound-guided cystocentesis in all cats. A com-

plete urinalysis was performed in all samples, and pre- and post-

renal proteinuria was excluded. Determination of concentration

of protein and creatinine was performed by Biuret and Jaffe

methods, respectively. Cats were classified as non-proteinuric

(UPC < 0.2, n = 6), borderline proteinuric (UPC 0.2 – 0.4; n = 9)

or proteinuric (UPC > 0.4; n = 10), according to the IRIS (sub)

staging system. Results: The correlation between UPC ratios in

urine samples collected bymanual compression and cystocentesis

was strong (0.99). All cats had UPC ratios from both methods of

collection that resulted in classification in the same IRIS substage.

Conclusions: The results obtained suggest that collection of

urine samples bymanual compression in cats is a reliable alterna-

tive to cystocentesis in determination of UPC ratios. Nonetheless,

more studies are needed due to the low number of animals

analyzed.

86CHARACTERIZATION OF CANINE BONE MARROWMESENCHYMAL STEM CELLS. C. Tejero1, L.K. Ostro-noff2, M.L. Fermın3, C. Fragio3, L.G. Leon4, E. Krem-mer5. 1Universidad Complutense de Madrid, Facultad deVeterinaria, Madrid, Spain; 2Department of Biochemistryand Molecular Biology IV, Veterinary Faculty Madrid,Madrid, Spain; 3Department of AnimalMedicine and Sur-gery, Veterinary FacultyMadrid,Madrid, Spain; 4Instituteof Bio-Organic (IUBO-AG), La Laguna, La Laguna, Spain;5Institut fur Immunologie, GSF-ForschungszentrumUmwelt und Gesundheit , Munchen, Germany.

Background: Adult mesenchymal stem cells (MSC) were ini-

tially described as an adherent non-hematopoietic cell type that

could form fibroblastic colonies in vitro. Currently, these cells can

be defined as multipotent cells able to differentiate into osteo-

genic, adipogenic and condrogenic lineages under appropriate

conditions. Objective: Our aim was to expandand assess cell

surface markers of canine bone marrow-derived mesenchymal

stem cells. Methods: Four healthy 2-year old female Beagle

dogs were used as marrow donors. Mononuclear cells were cul-

tured in IMDMmediumwith 10%DS and incubated at 38ºCwith

5%CO2, and after 48hours the nonadherent cells were discarded.

After obtaining a confluent MSC layer, adherent cells were har-

vested with trypsin/EDTA and replated at 700 cells/cm2 with or

without SCF. Cells were harvested at first, second and third pas-

sages and incubated with FITC-conjugated CD90, FITC-conju-

gated CD34, FITC-conjugated CD45 and FITC-conjugated

MHCII. Results: In vitro growth of MSC proceeded as an initial

lag phase, followed by rapid expansion. Between the first and sec-

ond passage the cell number was multiplied by approximately

28-fold. A dot plot of stained MSC after incubation with primary

antibodies revealed that these cells had positive results for CD90

and were negative for MHCII. The lack of expression for CD34

and CD45 showed that cells of hematopoietic origin had been

excluded during the cell expansion process. Conclusion: In

conclusion, we have isolated and characterized a canine bone

marrow-derived mesenchymal stem cell population. Further

investigations will make them promising candidates for use in

regenerativemedicine.

87FIBRINOGEN CONCENTRATION MEASUREMENT INDOGS: A COMPARISON BETWEEN THE QBC-VETAUTOREADER AND MC1 PLUS SEMIAUTOMATICCOAGULOMETER. L.V. Athanasiou1, T.A. Peta-nides2, M.K. Chatzis1, M.N. Saridomichelakis1. 1Fac-ulty of Veterinary Medicine, University of Thessaly,Karditsa, Greece; 2Veterinary Center of Thessaloniki,Thessaloniki, Greece.

Background: Fibrinogen is a glycoprotein that plays a key role

in blood clotting and is a non-specific indicator of inflammation.

The most widely used techniques for measuring plasma fibrino-

gen concentration include heat precipitation (Millar’s technique)

and modified thrombin clotting time measurement (Clauss

method). Both have been automated in a haematology analyzer

(QBC-Vet Autoreader, IDEXX;Millar’s technique) and in a newly

developed semi-automatic mechanical and optical detection co-

agulometer (MC1 Plus, DIASys Greiner GmbH; Clauss method).



Objective: Our aimwas to compare the above-mentioned tech-

niques for the measurement of plasma fibrinogen concentration,

to establish a reference interval for the new coagulometer, and to

detect canine diseases associated with hyperfibrinogenemia.

Methods: Fibrinogen concentration was measured in the blood

of 85 clinically normal dogs and 43 dogs presented with clinical

and/or clinicopathological abnormalities that have been previ-

ously associated with dysfibrinogenemia. The results of the two

techniques were compared using Passing-Bablock’s regression

and Bland Altman difference plots. Results: No correlation was

found between the results of the two techniques. A reference

interval (112-688 mg/dl) was calculated for the Clauss method

following NCLS standards. Hyperfibrinogenemia was observed in

dogs with various diseases, either alone or in combination,

including leishmaniosis, monocytic ehrlichiosis, dirofilariasis,

autoimmune diseases (e.g., pemphigus foliaceous, immune-

mediated hemolytic anemia), inflammatory conditions (e.g., uri-

nary tract infection, pyometra, discospondylitis, peritonitis, fever

of unknown origin), chronic liver disease and neoplasia.Conclu-

sion: The two techniques cannot be used interchangeably; thus

a different reference interval should be used for assessment of

fibrinogen concentration by the MC1 Plus using the Clauss

method.

88REFERENCE INTERVALS FOR HEMATOLOGICPARAMETERS OF ARABIAN HORSES. A. Rocky, M.Razi Jalali, S. Gooraninejad, M. Pourmahdi, F.Naghashpour. Faculty of Veterinary Medicine, ShahidChamran University, Ahvaz, Iran.

Background: Laboratory results are interpretated by using spe-

cies-specific reference intervals, but breed, age, and sex may

influence the interpretation of results. Objective: The aim of

this study was to establish reference intervals for hematologic

parameters of clinically healthy Arabian horses and to evaluate

the influence of age and sex on these parameters. Methods: A

total of 255 clinically healthy female and male Arabian horses

from the Khuzestan province, Iran were included in this study.

Blood samples taken from jugular vein were collected into tubes

containing EDTA and immediately transferred to the Clinical

Pathology Laboratory. Blood samples were analyzed using a

BC-2800Vet hematology analyzer (Mindray, China). The ani-

mals were divided into 3 age groups: < 2 years, 2-5 years, and > 5

years old. Reference intervals for 17 hematologic analytes were

expressed as 2.5th and 97.5th percentiles. Results: Significant

differences (P<.05) primarily between < 2 and > 5 year old

horses were seen for 9 hematologic analytes: WBC, lympho-

cyte, and RBC counts, hemoglobin, hematocrit, MCV, MCH,

MCHC and MPV. Significant differences (P<.05) were also seen

between male and female horses for 4 hematologic analytes:

WBC count, lymphocyte count, hemoglobin and MPV. Con-

clusion: Breed-specific reference intervals for Arabian horses

will better represent normal characteristics of this breed. The

results of this study showed that age has a major influence on he-

matologic parameters of Arabian horses and may affect interpre-

tation of CBC results in this breed.

89A NOVEL POINT MUTATION IN THE BETA1-TUBULINGENE IN ASYMPTOMATIC MACROTHROMBOCYTOPE-NIC NORFOLK AND CAIRN TERRIERS. M.E. Gelain1,W. Bertazzolo2, G. Tutino2, E. Pogliani2, F. Cian3,M.K. Boudreaux4. 1University of Padua, Padua, Italy;2Veterinary Clinic “Tibaldi”, Milan, Italy; 3DepartmentVeterinary Medicine, University of Cambridge,Cambridge, UK; 4Department of Pathobiology, College ofVeterinary Medicine, Auburn University, Auburn, AL,USA.

Asymptomatic inherited macrothrombocytopenia is a well-

recognized condition in Cavalier King Charles Spaniels

(CKCS) due to a point mutation in the beta1-tubulin gene

that affects proplatelet formation and platelet production.

Recently, phenotypically similar asymptomatic macrothromb-

ocytopenia was described in Norfolk terriers (NT) and isolated

cases were also reported in Cairn terriers (CT). Our aim was

to evaluate the presence of a genetic defect in the beta1-

tubulin gene in macrothrombocytopenic Norfolk and Cairn

terriers. Genomic DNA was harvested from peripheral EDTA-

anticoagulated blood samples from 20 healthy NT (n=13) and

CT (n=7) collected at different institutions in Italy (n=8), the

United Kingdom (n=3) and the United States (n=9). Primers

were designed to assess all coding areas and exon–intronsplice sites on the canine beta1-tubulin gene. PCR products

were electrophoresed on agarose gels and target bands were

sequenced. None of the samples had the CKCS mutation.

Twelve dogs (9NT and 3CT) showed a single nucleotide poly-

morphism (SNP) in exon 1 at nucleotide position 5 (G5A)

that would result in the change of an arginine to a histidine

at amino acid position 2 (R2H). Four dogs (3NT and 1CT)

were heterozygous for the SNP and 4 (1NT and 3CT) showed

a normal sequence. All dogs with the SNP were macro-

thrombocytopenic, while heterozygous and normal dogs had

normal platelet counts and morphology. The tubulin N-termi-

nal amino acids form the nucleotide binding domain, and it

is possible that the amino acid change at position 2 (R2H)

could affect GTP binding enough to influence platelet forma-

tion only in homozygous dogs.

90DETERMINATION OF COMPLETE BLOOD COUNT REF-ERENCE INTERVALS IN PYRENEAN MOUNTAINDOGS.A. Pasquini, I. Annibali, G. Biagi, B. Gugliucci,G. Lubas, A. Gavazza. University of Pisa, San Piero aGrado (PI), Italy.

Background: Though important diagnostically, few studies of

the different haematological characteristics in different dog

breeds exist. The purpose of this study was to examine the com-

plete blood count (CBC) of Pyrenean Mountain Dogs (PMD) and

to establish breed-specific RIs as recommended in the Clinical

and Laboratory Standards Institute (CLSI) guidelines. Materials

and methods: 133 CBCs of healthy subjects were examined.

Analyses were carriedout using an impedance cellcounter (He-

CoVet, SEAC, Firenze, Italy), and the leukocyte differential count



and morphological evaluation were performed by microscopic

examination of modified Wright–stained blood smears. RBC

count, HGB, HCT, MCV, MCHC, RDW, PLT count, WBC count,

differential leukocyte count, and RBC morphology were quanti-

tatively measured and PLT count estimated. Data were analyzed

by multifactorial ANOVA post hoc and Bonferroni multiple com-

parison, including sex and age effects. Haematological RI values

for PMDs were obtained in accordance with the CLSI recom-

mended validation procedure Results: BCs of PMD showed

some peculiarities concerning a few haematologic measurements

and erythrocyte morphology (poikilocytosis). Standard RIs were

validated for all CBC parameters except hematocrit (HCT), MCV

and eosinophil counts (PMD vs. standard RI, respectively, 34.8-

49.5 vs 37-55 (%), 59-70 vs 60-76 (fL) and 0.0-2.1 vs 0.1-1.7

(9 109/L). Conclusions: New reference intervals partitioned by

breed should be used for the correct clinical interpretation of

laboratory data in dogs.

91CANINE BONE MARROW CYTOLOGICAL EXAMINA-TION: A RETROSPECTIVE STUDY OF 295 CASES.V. Turinelli1, A. Gavazza2, G. Lubas2. 1Idexx Laborato-ries Italy, Livorno, Italy; 2Dept. Veterinary Clinic, Univer-sity of Pisa, Pisa, Italy.

Background and objective: Cytological bone marrow evalua-

tion is a simple exam, indicated when peripheral blood abnor-

malities are detected, to stage neoplastic conditions, and to search

for parasitic infection or occult diseases in animals with fever of

unknown origin, weight loss and unexplainedmalaise. A suitable

sample is necessary for accurate assessment of the bone marrow.

Methods: This retrospective studywas conducted between Jan-

uary 2010 and March 2012. Two hundred and ninety-five bone

marrow smears were classified using cytological and numerical

criteria. Samples were collected from dogs referred for several

disorders from Italy (71%), Germany (22%), the Netherlands,

Switzerland, Spain, Poland, Norway and Sweden (7%).

Results: Cytological exam allowed classification of the bone

marrow samples as normal 19.3%; hyperplasia 25.1% (general-

ized 1.4%; megakaryocytic 6.8%; erythroid 33.8%; granulocytic

58.1%); hypo/aplasia 7.8% (aplastic anemia 47.8%; megakary-

ocytic 8.7%; erythroid 30.4%; granulocytic 13%); dysplasia

1.7% (myelodysplastic syndrome 20%; dysmegakariopoiesis

20%; dyserithropoiesis 40%; dysgranulopoiesis 20%); lympho-

plasmacytic hyperplasia 1.4%; hematopoietic malignancy

12.9%; metastasis 0.3%; miscellaneous 1%; and nondiagnostic

30.5%. Conclusion: This study outlines the high incidence of

both hyperplasia (granulocytic) and malignant hemopoietic dis-

ease (leukemia and lymphoma) and the rarity of metastasis. It is

noteworthy to observe the high number of nondiagnostic results

due to several causes (poor quality of aspiration, hemodilution,

smears damaged by formalin fumes). Cytological bone marrow

examination can provide information about thepathogenesis of

abnormalities found in blood and often the etiological diagnosis

can be made. Final conclusions and interpretations must include

other laboratory results and peripheral blood smear examination.

92RETROSPECTIVE STUDY OF INCIDENCE AND CLASSI-FICATION OF ABNORMALITIES IN FELINE BONEMAR-ROW BY CYTOLOGICAL EXAMINATION. A. Gavazza1,V. Turinelli2, G. Lubas3. 1University of Pisa, San Piero aGrado, Pisa, Italy; 2Idexx Laboratories Italy, Milan, Italy;3Dept Veterinary Clinic, University of Pisa, Pisa, Italy.

Background: Bone marrow cytological evaluation is often per-

formed in cats affected by non-regenerative anemia, persistent

neutropenia, and thrombocytopenia, to assess involvement in

neoplastic conditions, or to identify suspected infectious agents

(e.g.,Toxoplasma gondii). Peripheral blood cytopenias are fre-

quently observed in FIV/FeLV-infected cats, where a loss of nor-

mal cellular composition of bone marrow is documented.

Objective and methods: This study was conducted between

January 2010 and March 2012. Seventy-one bone marrow

smears were classified using cytological and numerical criteria.

The majority of the cases were from Italy (97%) and a few were

from Germany (3%). Results: The cytological exam allowed

classification of the bone marrow samples as normal 5.6%;

hyperplasia 36.6% (generalized 3.8%; megakaryocytic 3.8%;

erythroid 33.8%; granulocytic 58.1%); hypo/aplasia 12.7%

(aplastic anemia 66.7%; megakaryocytic 0%; erythroid

22.2%; granulocytic 11.1%); dysplasia 7% (myelodysplastic

syndrome 40%; dysmegakaryopoiesis 0%; dyserythropoiesis

20%; dysgranulopoiesis 40%); lympho-plasmacytic hyperplasia

1.4%; hematopoietic malignancy 7%; metastasis 0%; miscella-

neous 2.8%; and nondiagnostic 26.8 %. Conclusion: These

findings outline the high incidence of hyperplasia (granulocytic)

and of hypo/aplasia. Granulocytic hyperplasia occurs most fre-

quently in response to bacterial infections. Ineffective granulo-

cytic hyperplasia, with increased numbers of immature

granulocyte precursors and decreased mature neutrophils, fre-

quently occurs in myelodysplastic disorders and is especially

common in neutropenic cats with FIV/FeLV infections. In order

to make a proper conclusive report from bone marrow examina-

tion a CBC should be performed the same day of bone marrow

aspiration.

93USE OF THE MCDhSTAINING FOR HAEMATOLOGY OFDOGS, CATS, HORSES AND CATTLE: PRELIMINARYRESULTS. N. Bourges-Abella, C. Hanot, C. Marche,J.P. Braun, C. Trumel. Institut National Polytechnique-Ecole Nationale Veterinaire de Toulouse, Toulouse,France.

Background: Micro Chromatic Detection for haematology

(MCDh) (RAL Diagnostics) is a new stain used in human

haematology. Although it is a fast-acting stain, it gives red meta-

chromatic staining to azurophilic granules. Moreover, MCDh is

methanol-free, which ensures higher safety for users.

Objective: The aim of this prospective double-blind study was

to evaluate if MCDh gives a range of colors similar to May-

Grunwald Giemsa staining and permits identification of morpho-



logical abnormalities commonly encountered in veterinary

haematology. Methods: After determination of a standard

staining procedure for smears from pets, horses and cattle, valida-

tion of the MCDh staining was performed in 3 steps for each spe-

cies (dog, cat, horse and cattle) by two experienced operators: 1)

validation of staining times; color and morphology of cellular

components were assessed by comparison with data from the lit-

erature and ranked on a scale of 0 to 3; 2) method comparison

between MCDh and May-Grunwald Giemsa staining for WBC

differential and platelet counts, according to CLSI recommenda-

tions; and 3)method comparison betweenMCDh andMay-Grun-

wald Giemsa staining for usual morphological abnormalities of

blood cells (such as polychromasia, parasites, toxic neutrophils).

Results: Mean scores (n=46) were significantly higher (p<.05)

for MCDh-stained smears for platelet morphology and

counts than for May-Grunwald Giemsa-stained smears. Conclu-

sion: Preliminary results suggest that MCDh may improve

platelet assessment.

94INCREASED ERYTHROCYTE OSMOTIC RESISTANCEIN DOGS WITH A SINGLE EXTRAHEPATIC PORTO-SYSTHEMIC SHUNT. K. Rehakova1, I. Uhrikova1, L.Rauserova–Lexmaulova1, J. Lorenzova1, L. Stehlik1,O. Skor2, J. Doubek1. 1University of Veterinaryand Pharmaceutical Sciences Brno, Brno, CzechRepublic; 2Veterinary Clinic, Bila Hora, Prague, CzechRepublic.

Background: Alterations in red blood cell deformability due to

modification ofmembrane lipid composition occurring in chronic

liver disease are well known from human medicine but in dogs

are not described. Objective: The aim of the study was to

investigate changes in erythrocyte osmotic resistance in dogs

with congenital portosystemic shunts (PSS). Methods: Twelve

dogs with a single extrahepatic PSS confirmed by surgical explo-

ration were included. Osmotic fragility tests with complete blood

counts and liver biochemistry profiles including ammonia and

total bile acid concentrations were compared with those of 30

healthy dogs. Absorbance in osmotic fragility test was assessed by

the ELISA reader ELX 808.Results: Dogs with a single extrahe-

patic PSShad significantly increased (p<.001) erythrocyte osmo-

tic resistance with 50% and 90% hemolysis corresponding to

0.35% and 0.3% NaCl solution, respectively. Target cells related

to the more resistant cell populations were seen directly on the

blood smear in 50% of dogs, while increased osmotic resistance

against NaCl solution causing 50% hemolysis was found in 75%

of dogs. Moreover, none of 12 dogs with PSS had increased

osmotic fragility for any degree of hemolysis. No correlation was

found between MCV, ammonia, bile acid or cholesterol levels

and erythrocyte osmotic fragility. Conclusion: Due to altera-

tions in lipid metabolism in atrophic liver, dogs with congenital

PSS have significantly more resistant red blood cells against

hypotonic hemolysis suggesting their impaired deformability

functions.

95COMPARISON OF THE THROMBOTIC MICROSCOPICPLATELET COUNTING METHOD WITH THE LEUKO-PLATE MICROSCOPIC, ADVIA 2120 AND IDEXXVETAUTOREAD™ PLATELET METHODS. H. Tvedten1,J. Ljusner2, I. Lilliehook2. 1Swedish University of Agri-cultural Sciences, Vange, Sweden; 2Swedish University ofAgricultural Sciences, Uppsala, Sweden.

Introduction: The thrombotic microscopic platelet counting

method is stated to cause disaggregation and rounding of platelets

for more precise microscopic enumeration. Methods: Throm-

botic platelet counts (PLT) of 21 feline blood samples were com-

pared with PLT determined with a Leucoplate stain method or

the Advia 2120. Additionally PLT were determined in 8 canine

bloodsamples with the IDEXX VetAutoread™ (QBC). Precision

was determined with 1 canine blood sample analyzed 10 times.

Results: Precision (CV %) was: Advia 3.2 %, QBC 15.3 %,

Thrombotic 7.8 %, and Leucoplate 9.0 %. Median PLT of 21

feline blood samples were 202 x 109/L Thrombotic, 238 x 109/L

Leucoplate, and 156 x 109/L Advia. Median canine PLT were 277

x 109/L Thrombotic, 241 x 109/L Leucoplate, 267 x 109/L Advia,

and 289 x 109/L QBC. In only 5 of 21 feline samples were no

aggregates seen in either of the 2 counting chambers or on blood

smears. More aggregates were seen in Thrombotic counting

chambers with 8 of 21 feline bloodsamples compared with the

Leucoplate. In no sample were more aggregates seen in the Leu-

coplate method. Conclusions: Precision was best with the Ad-

via and acceptable for the 2 manual methods. The QBC had

worse precision but actually measures platetcrit, which is then

converted to a platelet count. Platelet counts with the Thrombotic

method were comparable to the Leucoplate manual method. The

Thrombotic method had more feline platelet aggregates than the

Leucoplate method. Manual methods gave higher PLT than the

Advia, which suggests manual methods may be somewhat more

accurate with blood containing platelet aggregates.

96FACTOR VIII LEVELS IN A GROUP OF BELGIANSHEPHERD MALINOIS DOGS IN ITALY. G. Lubas1,A. Gavazza2, M. Caldin3. 1University of Pisa, San Piero aGrado, Pisa, Italy; 2University of Pisa, Dept. VeterinaryClinic, San Piero a Grado, Pisa, Italy; 3San Marco Veteri-nary Laboratory, Padova, Italy.

Occurrence of hemophilia A in the Belgian Shepherd Malinois

(BSM) dog has been recently described. This is the most common

and severe inherited canine coagulopathy. Diagnosis is based on

clinical signs, prolonged aPTT, and reduction in factor VIII activ-

ity. The study aim was to assess the level of FVIII in BSM dogs

in Italy. Fifty-four studbook-registered BSMs werestudied

(34 males, 20 females), in which hemophilia A was a possible

condition. For each BSM, genealogical data and, whenever possi-

ble, history and other clinical data were collected. All BSMs were

tested for FVIII concentration using two reference laboratories.

When possible, the vWF concentration in females was also

assessed (carrier FVIII/vWF<0.60). Most of the data are displayed

in a free accessible website (www.malinemo.net). Ten male dogs



had a low level of FVIII (range 5-21%, consistent with hemo-

philia A), four females showed FVIII between 28-55% (carrier

range), and 21 dogs had FVIII level >70%. Nineteen dogs had a

FVIII level thatwas borderline either for the carrier or clear range.

The present work supports evidence for hemophilia A in BSM

dogs with a relatively high frequency. Unfortunately, the level of

FVIII alone does not allow a clear distinction between carrier and

clear dogs. Only a complete clinical examination, together with

laboratory data, can ruleout concurrent disorders (i.e., liver fail-

ure or acquired coagulopathy). Further studies on factor VIII

gene mutations using offspring to correctly identify hemophilia

A in BSMdogs are in progress.

97PROTHROMBIN TIME MEASUREMENTS USING ANOVEL POINT-OF-CARE DEVICE IN CANINE WHOLEBLOOD. P. Vajdovich1, J. Antal2. 1Szent Istvan Univer-sity, Faculty of Veterinary Science, Budapest, Hungary;2Diagon Kft. Hungary, Budapest, Hungary.

Background: A point-of-care device was developed for in-

house prothrombin time (PT) measurement using whole blood.

This device usesa turbidimetric method to detect PT and whole

blood can be used.Objective: The aim of the study was to eval-

uate the applicability of this point-of-care device in canine

plasma and whole blood samples. Methods: Samples from 15

dogs with various PT values were analysed using the point-of-

care device and the results were comparedwith those of a 2 chan-

nel semi-automatic photo optical ball coagulometer using

Thromborel S (Behring) reagent. Results: Statistically signifi-

cant correlation was found between the reference method

(mean: 11.6 sec) and plasma (mean: 7.5 sec, r=.845) and whole

blood (mean: 8.6 sec, r=.924) results of the point-of-care device.

Plasma and whole blood results measured by the point-of-care

device were also correlated (r=.935). Precision CV was 4.2%, and

the bias of plasma results was -35.4%, and of whole blood results

was -25.6%. Conclusion: Point-of-care devices can be used if

supported by evidence-based medicine. Whole blood analysis of

coagulation is suitable before any surgical intervention or sam-

pling method together with bleeding time measurement. The

device that we used for the analysis has to be adapted to the

required reference standard results.

98RETICULOCYTE PARAMETERS IN DOGS WITHLEISHMANIASIS: A PRELIMINARY STUDY. J. Pastor,A.Melendez-Lazo, C.Miret,M. Planellas,M.Mesalles.Universitat Autonoma de Barcelona, Bellaterra, Spain.

Background: The pathophysiological mechanisms of anemia in

animals with clinical leishmaniasis are not completely under-

stood and may be multifactorial. Reticulocyte parameters such as

reticulocyte Hgb content (CHr) and reticulocyte MCV (rMCV)

have proven more useful in detecting early iron deficiency ane-

mia than conventional hematologic and biochemical indices.

Few reports have been published studying CHr and rMCV

changes in infectious and chronic inflammatory diseases.Objec-

tive: The aim of this studywas to compare erythrocyte and retic-

ulocyte parameters (RBC count, HGB, HCT, MCV, RDW, HDW,

reticulocyte count, rMCV, CHr) between dogs with clinical leish-

maniasis and healthy dogs. Methods: EDTA blood samples

were obtained from 9 dogs with clinical leishmaniasis not treated

previously and 23 healthy Beagle dogs serologically and PCR-

negative for Leishmania sp. Blood samples were analyzed with the

ADVIA 120 automated hematology analyser within 12 hours of

blood collection.Results: Of ill animals 5/9 werepresented with

nonregenerative anemia (HCT<35%). Statistically significant

differences between ill and healthy animals were found for RBC

count, HGB, HCT, RDW, reticulocytes count and rMCV (p<0.05).

Animals with leishmaniasis had lower RBC count, HGB, HCT,

and reticulocyte count and higher RDW and rMCV than healthy

dogs. Conclusion: Animals with leishmaniasis have statistically

significant increases in RBC anisocytosis and have inhibition of

erythropoiesis that cause a decrease in reticulocyte release and

reticulocyte-relatedmacrocytosis. This may be due to the effect of

inflammatory cytokines on erythropoiesis.

99BLOOD PARAMETERS OF CIKA CALVES AND BULLS.M. Klinkon1, G. Gorjanc2, Z. Klinkon3, J. Jezek1,M. Simcic2. 1Veterinary Faculty, University of Ljubljana,Ljubljana, Slovenia; 2Biotechnical Faculty, University ofLjubljana, Department of Animal Science, Domzale,Slovenia; 3Veterinary Clinic Klinkon, Radomlje, Slovenia.

Background: Cika is the only indigenous cattle breed in Slove-

nia that was mainly used for milk productionin the past. Cur-

rently, this breed’s milk yield is too low compared with that of

modernmilk breeds. Therefore, farmers rear Cika cows mainly in

the cow-calf system. Objective: The aim of this study was to

compare blood valuesof Cika cattle partitioned by age. Meth-

ods: The study included 36 blood samples from18 Cika bulls.

Each animal was sampled twice in its life: at weaning (calf) and at

slaughter (fattened bull). Calves originated from different farms,

but were fattened together in the Educational Research Centre

Logatec. The average age of calves at sampling was 7.5 months,

whereas the average age of bulls at slaughter was 2 years. RBC,

WBC, and platelets (PLT) counts and values for haemoglobin

(Hb), haematocrit (Ht), MCV, MCH, and MCHC were measured

with the haematological analyser, ABC Vet. Collected data were

analyzed with a MIXED procedure (SAS) considering age by lin-

ear regression and animal to account for repeatedmeasurements.

Results: Mean values showed that Hb, Ht, MCV, and MCH sig-

nificantly increased, whereas RBC and PLT counts significantly

decreased from weaning (calves) to slaughter time (bulls). WBC

count and MCHC were unchanged. Conclusion: Despite the

fact that age significantly affected some blood values, the values

werewithin laboratory reference values for cattle.

100COMPARISON OF THE ABC VET AND BC 2800 VETVETERINARY HAEMATOLOGICAL ANALYZERS. M.Nemec, M. Klinkon, J. Jezek. Veterinary Faculty,Clinic for Ruminants, Ljubljana, Slovenia.

Background: Two veterinary haematological analyzers made

by differentmanufacturers have the same principles of operation.

Objective: The aim of the study was to evaluate the useful-



ness of the BC 2800 Vet analyzer in veterinary medicine. Meth-

ods: The ABC Vet was used as a reference analyzer; it is in rou-

tine use, regularly calibrated and controlled, and included in the

international inter-laboratory comparison. Five consecutive

analyses of control samples were performed at low, normal and

high levels to determine the repeatability of measurements on

the BC 2800 Vet analyzer. To determine the reproducibility of the

analyzer, we used daily measurements of control blood samples.

To compare analyzers, we collected 76 blood samples from differ-

ent animals (13 cows, 12 sheep, 13 pigs, 11 horses, 11 dogs, 9 cats

and 7 rabbits), and comparative analyses were performed. Agree-

ment between results was assessed with the statistical method

developed by Bland and Altman. Results: For the BC 2800

Vet good repeatability and reproducibility were established at

all levels for most parameters (CV <5 %). Applying the statis-

tical method of agreement, we found appropriate comparabil-

ity between analyzers in the normal operating range for most

parameters; less comparable results were found for haemoglo-

bin and platelets. On average, the ABC Vet analyzer gave

slightly higher results for most parameters whereas the WBC

count was slightly lower than that of the BC 2800 Vet. Con-

clusion: The BC 2800 Vet has proven to be suitable for use in

veterinarymedicine for domestic species.

101CANINE CBC ANALYSIS WITH THE LASERCYTEAND PROCYTE DX AUTOMATED HEMATOLOGYANALYZERS. D. DeNicola1, M. Haynes1, C. Lee1,J. Hammond1, M. Wellman2, G. Couto2. 1IDEXXLaboratories, Inc., Westbrook, ME, USA; 2The Ohio StateUniversity, Columbus, OH, USA.

Background: The IDEXX LaserCyte and ProCyte Dx Hematol-

ogy Analyzers provide CBC results including a 5-part differential

and reticulocyte count. As a component of constant product

development/improvement, LaserCyte performance enhance-

ments related to optimized fluidic sequence modifications have

been developed. Objective: The aim of this study was to evalu-

ate the LaserCyte optimized sequence enhancements. Meth-

ods: 30 fresh canine samples were analyzed on the LaserCyte

and ProCyte Dx within four hours of collection and within one

hour between instruments.Results: Correlation factors (R) and

slopes (m)were calculated for each least squares regression as fol-

lows: RBC (R=0.99; m=0.99), HGB (R=0.97; m=1.00), HCT

(R=0.98; m=0.99), PLT (R=0.98; m=1.00), WBC (R=0.99;

m=1.00) and NEU (R=0.99; m=0.88). Remaining parameters

were evaluated by%bias and results were as follows:MCV (1%),

RETIC (6%), LYM (8%), MONO (12%) and EOS (3%). Conclu-

sion: Strong correlations were shown for RBC, HGB, HCT, PLT,

WBC and NEU. Calculated slope values indicated accurate results

between instruments. MCV, RETIC, LYM, MONO, and EOS all

had datasets with small dynamic range. Least squares regression

analysis performs poorly with this type of input; therefore % bias

calculations were performed showing strong performance for

MCV, RETIC and EOS and a slight bias for LYM and MONO.

Further analysis with a larger dataset will provide greater insight

into each of these parameters.

102CANINE RETICULOCYTE ANALYSIS WITH THE LASER-CYTE AND PROCYTE DX AUTOMATED HEMATOLOGYANALYZERS. D. DeNicola1, C. Lee1, M. Haynes1,J. Hammond1, J. Christian2, J. Knoll3, M. Scott4,H. Wamsley5. 1IDEXX Laboratories, Inc., Westbrook,ME, USA; 2Purdue University, West Lafayette, IN, USA;3Tufts University, North Grafton, MA, USA; 4MichiganState University, East Lansing, MI, USA; 5University ofFlorida, Gainesville, FL, USA.

Background: Automated in-house hematology analyzers have

been used to enumerate reticulocytes for many years. The IDEXX

LaserCyte analyzer relies on stained cells with precipitated RNA

to selectively scatter light while the ProCyte Dx analyzer utilizes a

specific RNA fluorescence stain for reticulocyte identification.

Each method requires specific algorithm optimization. Objec-

tive: The aim of this study was to compare a newly developed

canine reticulocyte algorithm for the LaserCyte with the ProCyte

Dx florescencemethod.Methods: Two LaserCyte analyzers and

one ProCyte Dx analyzer were installed at each of the following

veterinary colleges: Tufts University, University of Florida, Pur-

due University, and Michigan State University. 100 fresh canine

samples were analyzed at each locationwithin four hours of draw

and within one hour between instrument runs. A least squares

regression analysis, fixed at the origin, was performed to

determine correlation between the two analyzer responses.

Results: Integrating the LaserCyte response for all eight Laser-

Cyte analyzers and the correlated ProCyte responses from the

four ProCyte analyzers yielded an aggregated correlation coeffi-

cient, R=0.86. The newly optimized LaserCyte canine reticulo-

cyte algorithm yields results that closely match the ProCyte Dx

with an aggregate regression slope of 0.9 across all analyzers.

Conclusion: The newly optimized LaserCyte canine reticulo-

cyte algorithm provides results that correlate well to the fluores-

cent RNA-specificmethodology of the ProCyte Dx.

103PORCINE HEMATOLOGY EVALUATION ON THE IDEXXPROCYTE DX HEMATOLOGY ANALYZER.D. DeNicola,J. Chase, J. Hammond, J. Russell. IDEXX Laboratories,Inc., Westbrook, ME, USA.

Background: The IDEXX ProCyte Dx Hematology Analyzer is

approved for the following species: canine, feline, equine,

bovine, and mustaline. This study aimed to support the addition

of the mini-pig as a newly supported species. Objective: The

objective of this study was to evaluate the results of 100 Yucatan

mini-pigs on the IDEXX ProCyte Dx as compared with the Sys-

mex XT-V analyzer.Methods: 100 porcine EDTA-anticoagulat-

ed samples were evaluated on four ProCytes and one XT-V. In

addition, manual packed cell volume measurements were per-

formed and compared with LaserCyte results. Results: Correla-

tion coefficients were calculated from least squares regression

analysis. Correlation factor (R) and slope (m) results from one

representative LaserCyte compared with the XT-V were as fol-



lows: RBC (R = 0.98; m= 1.01), HCT (R= 0.98; m=1.01), HGB

(R=0.99; m=0.99), MCV (R= 0.99; m=1.05), WBC (R=0.98;

m=0.82), PLT (R=0.96; m=0.89), and PCV (R=0.83; m=1.00.).

Conclusion: Correlations greater than 0.95 for CBC parameters

demonstrate strong correlation between the ProCyte DX and XT-

V as well as between the ProCyte Dx HCT andmanual PCV. Based

on slope results, no significant biases were noted for any of the

parameters evaluated. These findings support the addition of this

species on the ProCyte Dx analyzer.

104AGE-ASSOCIATED ALTERATIONS IN A PERIPHERALLEUKOCYTE SUBPOPULATION AND RESPONSE OFCYTOKINE SYNTHESIS TO INSULIN IN BEAGLE DOGS.M. Fujiwara1, H. Otsuka2, T. Yonezawa3, I. Yamam-oto1, T. Arai1. 1Nippon Veterinary and Life Science Uni-versity, Musashino-shi, Tokyo, Japan; 2Hiromichi,Kitasato University, Musashino-shi, Tokyo-to, Japan;3Kitasato University, Towada-shi, Aomori-ken, Japan.

Background: The immune system is considered to be affected

by aging, which is linked with various immune disorders. Insulin

plays an important role in maintaining energy metabolism in T

lymphocytes and may have an effect on age-related change of

immune dynamics. Objective: We observed changes in cyto-

kine production and insulin reactivity of PBMCs to determine the

response of the age-associated immune alterationsto insulin.

Methods: We analyzed the relationship between peripheral

leukocyte subpopulations and age. Dogs were divided into 3

groups: young (< 4 yr), middle-aged (4-8 yr), and older dogs (> 8

yr). After incubation of PBMCs with concanavalin A (Con A, 5.0

mg/ml) and/or insulin (1 mg/ml), mRNA levels of various cyto-

kine genes were analyzed usingreal-time PCR. Results: There

were significant negative correlations between age and the num-

ber of PBMCs. Notably, the number of naıve lymphocytes

decreased significantly with age. When Con A was added, the

mRNA level for IL-2, IL-2Ra and IL-4 was significantly increased

in the young group, whereas only IFNg and/or IL-4 were

increased with aging. When insulin and Con A were added, IL-2

mRNA was decreased in the young group. Furthermore, IL-4

mRNA increased and IFNg/IL-4 ratio decreased with aging. Con-

clusion: Taken together, results of this study demonstrated that

the number of naıve lymphocytes decreased and cytokine syn-

thesis from Th1 to Th2 agedependently. Moreover, insulin-

induced Th2 conversion became more effective with aging, and

these changes could be involved in age-dependent immune

degradation in dogs.

105HEME-MEDIATED BINDING OF ALPHA-CASEIN TOFERRITIN: EVIDENCE OF ALPHA-CASEIN BINDING TOFERROUS IRON. K. Orino, Y. Yoshikawa, K. Wa-tanabe. Laboratory of Veterinary Biochemistry, School ofVeterinary Medicine and Animal Sciences, KitasatoUniversity, Aomori, Japan.

Background and objective: The iron-storage protein, ferritin,

is not only present in many types of cells but also in extracellular

fluids such as serum, synovial fluid, and milk. Ferritin binds

heme as well as iron. Bovine a-casein was identified as a ferritin-

binding protein. Te aim of this study was investigate the binding

mechanism of a-casein with ferritin. Methods: Casein protein

coated-plates (20 pmol/well) were incubated with bovine spleen

ferritin (1 pmol/well) followed by detection using an biotinylated

anti-bovine spleen ferritin antibody and ALP-labeled avidin. To

examine the binding mechanism of a-casein with hemin

and metal ions, biotinylated hemin was used. Casein protein-

coatedplates (4 pmol/well) were incubated with biotinylated

hemin (32 pmol/well) followed by detection using ALP-labeled

avidin. Results: The binding of a-casein to bovine spleen ferri-

tin was blocked by hemin, but not by protoporphyrin IX or zinc-

protoporphyrin IX. The binding of a-casein to ferritin and bioti-

nylated hemin was inhibited by ferrous ammonium sulfate

(FAS). FAS-mediated inhibition of a-casein to biotinylated

heminwas neutralized with ferrozine, but not NTA, whereas FAS

as well as ferric chloride-mediated inhibition in their interaction

was neutralized by NTA. The following ions also inhibited a-casein-biotinylated hemin binding in order of potency of inhibi-

tion: Fe2+<<Fe3+<Cu2+<Zn2+<Mn2+<Ca2+<Mg2+. Conclu-

sion: These results suggests that the binding of a-casein to

ferritin is heme-mediated through direct binding of a-casein to

iron in the heme on the surface of fthe erritin molecule, and that

a-casein preferentially binds Fe2+ compared with any other

metal ions, including Fe3+.

106AMELIORATIVE EFFECTS OF DIETARY ALUMINOSILI-CATES ON BIOCHEMICAL CHANGES IN BROILERCHICKENS DURING AFLATOXICOSIS. D. Prvulovic1,D. Kojic2, M. Popovic1, G. Grubor-Lajsic2. 1Facultyof Agriculture, Novi Sad, Novi Sad, Serbia; 2Departmentof Biology and Ecology, Faculty of Science, Novi Sad,Serbia.

Background: Aflatoxicosis in poultry causes histopathological

and biochemical changes in major organs that can assist in the

diagnosis of intoxication. Producers and researchers desire to

develop an effective decontamination technology dealing with

this feed-borne toxin. Non-nutritive aluminosilicates such as

clays and zeolites are preferred because of their high binding

capacities against aflatoxin and their ability to reduce aflatoxin

absorption from the gastrointestinal tract. Objective: The pur-

pose of the present study was to evaluate the toxic effects of afla-

toxin B1 (AFB1) by biochemical examination of liver, kidney, and

pancreas of broilers, and to determine the possible preventive

role of dietary aluminosilicates (mixture of clinoptilolite and ben-

tonite) on these biochemical values.Methods: In total, 84 broi-

ler chicks were divided into two treatment groups: control-basal

diet and basal diet plus 5 g aluminosilicate kg-1. After 21 days,

twelve hours prior to sacrifice, 21 chicks from each group

received one dose of AFB1 orally. Enzymes and parameters of

lipid peroxidation were analyzed. Results: The activities of

gamma-glutamyltransferase and glutathione-S-transferase in

liver, and superoxide dismutase, guaiacol peroxidase, and pyro-

gallol peroxidase in kidneys decreased after oral administration of

a single dose of AFB1 to broilers. Lipid peroxidation was signifi-

cantly increased in both organs. Supplementation with alumi-

nosilicates partially decreased these effects. No effects due to

AFB1 were observed in enzyme activities in the pancreas. Con-

clusion: These data suggest that a single dose of AFB1 caused a

toxic effect on biochemical indices of liver and kidney in broilers.

Aluminosilicates alleviated these effects and protected broilers

against the harmful effects of AFB1.



107DETERMINATION OF ATRAZINE IN SERUM ANDURINE OF CATTLE BY HPLC, USING MOLECULARIMPRINTED POLYMERS (MIP). S.Z. Peighambar-zadeh1, S. Safi2, S.J. Shahtaheri3, M. Javanbakht4,A. Rahimi Forushani5. 1Department of VeterinaryMed-icine, Islamic Azad University, Shoushtar Branch, Sho-ushtar, Iran; 2Department of Clinical Pathology, Facultyof Specialized Veterinary Sciences, Science and ResearchBranch, Islamic Azad University, Tehran, Iran; 3Depart-ment of Occupational Health, School of Public Health,Center for Environmental Research, Tehran University ofMedical Sciences, Tehran, Iran; 4Department of Chemis-try, Amirkabir University of Technology, Tehran, Iran;5Department of Biostatistics, School of Public Health, Teh-ran University of Medical Sciences, Tehran, Iran.

Background: Atrazine is a major herbicide of the s-triazine

family and has been heavily used, especially in corn-growing

areas. The molecular imprinted solid phase extraction method

has been developed to measure the residue of pollutants in bio-

logical samples. Objective: The objective of the present study

was to determine the residual concentrations of atrazine in serum

and urine samples of cattle fed atrazine-treated feed using MIP.

Methods: Blood and urine samples were collected from 45 Hol-

stein cows. The cows were fed corn silage from farms in which

atrazine was used at 3 kg/hectare. The Supel MIP Triazine10 was

used as solid phase extractionmedia and atrazine was detected in

samples by HPLC. Results: The mean ± SD concentrations of

atrazine in serum and urine samples of the study group (0.739 ±0.567 ppm and 1.389±0.633 ppm, respectively) were higher

(P<.05) than the concentrations of atrazine in serum and urine

samples of the control group (0.002 ± 0.005 ppm and 0.012 ±0.026 ppm, respectively). Males and younger cows had higher

concentrations of atrazine in the studied samples (P<.05). Con-

clusions: The results of this study showed that atrazine in feed

ingredients ingested by cattle could be transferred to biological

samples and consequently can be considered a potential hazard

for human beings.

108CHARACTERIZATION OF STEM CELL MARKERS ONBONE MARROW- AND ADIPOSE TISSUE-DERIVEDCANINE MESENCHYMAL STEM CELLS. Y. Takemitsu,D. Zhao, I. Yamamoto, T. Arai. Veterinary and Life Sci-ence University, Musashino-shi Tokyo, Japan.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) and

adipose tissue-derived mesenchymal stem cells (AT-MSCs) are

potential cellular sources of therapeutic stem cells. MSCs are a

multipotent population of cells capable of differentiating into a

number of mesodermal lineages. Treatment using MSCs appears

to be a helpful approach in regenerativemedicine. Correct identi-

fication of these cells is necessary, but there is inadequate infor-

mation on the canine MSC profile of cell surface markers and

mRNA expression. In this study, we performedmolecular charac-

terization of canine BM- and AT-MSC using stem cell markers.

Samples were confirmed to be multipotent based on their differ-

entiation, and these cells were checkedas stem cells using hema-

topoietic and embryonic stem cell markers by flow cytometry.

BM- and AT-MSCs showed high expression of CD29 and CD44

and moderate expression of CD90; others were negative. Quanti-

tative real-time PCR revealed expression of Oct3/4, Sox2, and

Nanog in BM- and AT-MSCs. There was no significant difference

in expression of Oct3/4 and Sox2 between BM-MSCs and

AT-MSCs. However, Nanog expression was 2.5-fold higher in

AT-MSCs than in BM-MSCs. Using immunocytochemical analy-

sis, Oct3/4 and Sox2 proteins were observed in BM- and

AT-MSCs. These results provide a foundation for more reproduc-

ible and reliable quality control in the identification of canine

BM-MSCs and AT-MSCs.

109RARELYUSEDMETHODS FOR INVESTIGATING PORTO-SYSTEMIC SHUNTS IN DOGS. C. Smuts1, M. Bennett1,M. Sharman2, J. Mills1, T. Gaal1. 1Murdoch University,Perth, Australia; 2Melbourne University, Melbourne,Australia.

Background: In dogs with portosystemic shunting (PSS) or

chronic liver disease metabolic functions of the liver are dis-

rupted, resulting in increased concentrations of ammonia,

amino acids and uric acid in the blood. This is associated with

hepatic encephalopathy and often excretion of ammoniumu-

rate crystals in the urine. Objective: The aim of this study

was to apply seldom-used methods such as electron dispersive

analysis (EDA) for urinary crystal investigation and measure-

ment of serum aromatic amino acids (AAA) and blood

ammonia concentrations to investigate a case with PSS. Meth-

ods: Scanning electron microscopy with EDAwas used to deter-

mine crystal type from urine sediment. Blood ammonia levels

were measured using a portable ammonia meter. AAA concen-

trations were analysed in a human laboratory and comparedwith

an age-matched control dog.Results: AAA and blood ammonia

concentration were elevated in the patient. EDA revealed the

presence of sodium and potassium urate crystals in the urine

sediment. Conclusions: Blood ammonia and serum AAA are

useful tests for investigation of PSS providing additional infor-

mation for clinicians assessing dogs with hepatic dysfunction.

Measurement of serum AAA concentrations are routinely per-

formed for people but infrequently applied in animals, although

testing can be available through human laboratories. EDA as a

new, semi-quantitative method for examining urinary crystals

can be effectively used to identify a morphologically unidentifi-

able urinary crystal type in PSS dogs. Because many urinary

crystals are morphologically similar, crystal analysis can be use-

ful to determine their content and hence their pathogenesis.

This information is especially useful when the typical ammo-

nium biurate crystals are absent.

110RELATIONSHIP BETWEEN COLLIE EYE ANOMALYAND IVERMECTIN TOXICOSIS-ASSOCIATED MUTA-TIONS: ANALYSIS ACROSS MULTIPLE DOG BREEDS.K. Mizukami1, H-S. Chang1, D. Endoh2, A. Yabuki1,O. Yamato1. 1Kagoshima University, Kagoshima, Japan;2Rakuno Gakuen University, Ebetsu, Japan.

Background: Collie eye anomaly (CEA) is an inherited ocular

disorder, which is associatedwith a 7.8-kbp deletion in the canine

NHEJ1 gene. A 4-bp deletion in the canine MDR1 gene causes



neurotoxicosis after administration of P-glycoprotein substrates

such as ivermectin. It is known that both of these genetic disor-

ders are common in Collie-related breeds. However, we recently

demonstrated that Hokkaido dogs have a high prevalence of the

CEA-associated mutation.Objective: The aim of this study was

to investigate whether the 2 mutations are actually related or not

in the population of multiple dog breeds. Materials and Meth-

ods: Collies, Border Collies, 3 Japanese breeds (Hokkaido, Ry-

ukyu, and Shiba dogs), Korean Jindo dogs, and 2 non-Collie

breeds (Toy Poodles and Miniature Dachshunds) were used. The

genotypes of each mutation were determined using real-time

PCR methods. Results: Collies had a very high prevalence of

the 2 mutations and Border Collies had a high prevalence in CEA

but a low prevalence in the MDR1 mutation. Hokkaido dogs did

not have the MDR1 mutation although this breed had a high

prevalence of CEA. Ryukyu dogs did not have CEA, but some

dogs had theMDR1mutation. The other breeds did not have the 2

mutations. Conclusion: These data suggest that these genetic

disorders arose before the establishment of Collie-related breeds

but there was no apparent relationship in non-Collie breeds.

Therefore, these tests should be performed separately for each

breed. Then, based on the data gathered in the present study, it

should be determined which testing is indicated for individual

dogs.

111ABERRANT ANTIGEN EXPRESSION IN CANINELYMPHOMA: A RETROSPECTIVE STUDY. V. Martini1,A. Pasqua1, M.E. Gelain2, S. Comazzi1. 1University ofMilan,Milan, Italy; 2University of Padua, Padua, Italy.

Background: Distinguishing B- or T-cell phenotype provides

important prognostic information for dogs with lymphoma. The

presence of different aberrant phenotypes has been widely

reported and recently some authors identified a more aggressive

clinical behavior for aberrant lymphomas, when compared with

nonaberrant ones. Objective: The aim of this study was to ret-

rospectively investigate the prevalence of different aberrations in

B- or T-cell lymphomas, and their possible prognostic signifi-

cance. Methods and Results: Among 371 lymphoma cases in

our database, 70.1% were B-cell, 29.6% T-cell, and 0.3% were

biphenotypic; 15.1%had at least one aberration,most commonly

among T-cell lymphomas (35.5%). Low expression of CD45 was

the most common aberration among T-cell lymphomas and

CD34 positivity among B-cell lymphomas. Although treatment

was not standardized, a significantly shorter survival was found

for dogs with CD4+CD8+ T-cell lymphoma when compared with

other aberrant T lymphomas. CD34+ B-cell lymphomas had a

shorter survival compared with other B-celllymphoma cases,

although the differencewas not statistically significant. Dogswith

T-cell lymphoma with CD45 expression had a longer survival,

although not statistically different from the other T-cell cases, but

this aberration was frequently associated witha “small clear cell”

morphology that is known to have a better prognosis. Conclu-

sion: Although preliminary, this study suggests that some

abnormalities may have prognostic significance. Further studies

are needed, with a larger number of dogs and introducing non-

aberrant control cases. Furthermore, a prospective study with

standardized chemotherapeutic treatment should be performed

to assess the real prognostic value of the different aberrations.

112CLINICAL PRESENTATION CURRICULUM IN VETERI-NARY EDUCATION: A COGNITIVE PERSPECTIVE.S. Safi1, P. Hemmati2, S.H. Shirazi-Beheshtiha3.1Department of Clinical Pathology, Faculty of SpecializedVeterinary Sciences, Science and Research Branch, Isla-mic Azad University, Tehran, Iran; ²Ministry of Healthand Medical Education, Center for Disease Control,Senior Officer of Center for Disease Control, Tehran, Iran;³Department of Clinical Sciences, Faculty of VeterinaryMedicine, Karaj Branch, Islamic Azad University, Karaj,Iran.

There is a discrepancy between the knowledge of veterinarymed-

icine graduates and professional and society expectations. The

main reason for this discrepancy is the conventional curricula,

which rely on backward reasoning or disease-centered medical

education. The aim of the present study was to develop a scheme

as the most important step in creation of a full Clinical Presenta-

tion (CP) module. Vomiting was chosen in this study as a com-

mon CP in dogs. Three series of focused questions were

developed. The physiological mechanisms (visceral, cortical, ves-

tibular, metabolic and chemical) were chosen to reconstruct the

top layer of the scheme. To reconstruct the second and third lay-

ers of the scheme anatomical concepts were applied to define the

subcategories. To design the fourth layer, the disease classes that

result in vomiting in each of the physiological schemes were con-

sidered.We believe that introducing “vomiting in a dog” to veter-

inary medical students as a leading presentation meets the

students’ requirements because it uses clinical presentations as

the organizing core structure.

113STATISTICAL STUDY ON PREVALENCE OF TUMORS ININSURED DOGS IN JAPAN. A. Shimamura, T. Arai.Nippon Veterinary and Life Science University, Tokyo,Japan.

Objective: Anicom, the leading pet insurer in Japan, started its

operation about 10 years ago, and currently almost 400,000 pets

have insurance policies. Insurance claim data could provide epi-

demiological information. In this study, we tried to investigate

the prevalence of tumors with analysis of pet insurance data.

Methods: All insurance claims data for 252,414 dogs (males

133,775 and females 118,639) contractedwith Anicom pet insur-

ance from April 12008 to March 312008 were used for the

research. Results: The incident rate of tumors was 7.9% in

females, higher than that of males (6.4%). Among 144 insured

dogs reported as dead by 10 years of age, 16.9% claimed reim-

bursement because of tumor just before the death. Incident rate

of tumors in Golden Retrievers (15.4%) was the highest among

the top 17 insured breeds. The reason why the rate in the females

was higher than that in the males seemed to be the high inci-

dence of mammary tumors in the females. The rate of mammary

tumors was very low (almost 0) in the males but was 1.4% in the

females. Conclusion: Pet insurance data can provide informa-

tion about disease trends in dogs. It is suggested that we develop

more effective sampling methods for research and introduce bet-

ter treatment and prevention bymore effective use of these data.



114N-ACYLHOMOSERINE LACTONES OF PSEUDOMONASAERUGINOSA IN CLINICAL SAMPLES FROM CANINEOTITIS EXTERNA: VALIDATION OF AN ANALYTI-CAL METHOD USING LIQUID CHROMATOGRAPHYWITH TANDEM MASS SPECTROSCOPY (LC-MS/MS).K. Srimpf, J. Hosseini, T. Kotnik, D. Kusar,M. Vengust, G. Tavcar-Kalcher. University of Ljublj-ana, Veterinary Faculty, Ljubljana, Slovenia.

Background: Bacteria regulate their phenotype, including vir-

ulence, by a type of intercellular communication (i.e., quorum

sensing) via the production of autoinducers, e.g.,N-acylhomoser-

ine lactones (AHLs). Pseudomonas aeruginosa associated with

severe otitis externa in dogs produces mostly N-3-oxododeca-

noyl-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-ho-

moserine lactone (C4-HSL); other AHLs, e.g.,N-hexanoyl-

homoserine lactone (C6-HSL), are less abundant. For detection

of AHLs, highly sensitive analytical methods are needed because

concentration of AHLs in clinical samples is very low. Objec-

tive: The aim of our study was to validate an analytical proce-

dure for detection of 3-oxo-C12-HSL, C4-HSL and C6-HSL in ear

washes from dogs. Methods: According to the validation

scheme, ear washes from healthy dogs were spiked with 20 ng

each of the three selected AHLs. After dichloromethane extrac-

tion and evaporation, AHLs were re-dissolved in a methanol-

water mixture and measured by LC-MS/MS. Results: The limit

of detection was 0.5 ng/mL for all three AHLs. The repeatability

of results for 3-oxo-C12-HSL, C4-HSL and C6-HSL at 20 ng/mL

represented by the relative standard deviation (RSDr) was 10%,

13% and 15%, respectively. The relative standard deviation for

the within-laboratory reproducibility (RSDR) was 10%, 35% and

22% and the mean recoveries were 72%, 53% and 87%, respec-

tively. Conclusions: Results indicated that the procedure is

suitable for determination of the three selected AHLs in canine

ear washes. Monitoring of AHLs in P. aeruginosa-affected dogs

could serve as an alternative diagnostic tool that enables clinical

follow-up and appropriate therapy.

115PATHOLOGY OF LEG WEAKNESS IN BROILER CHICK-ENS. I. Dinev. Dept of General & Clinical Pathology, Fac-ulty of Veterinary Medicine, Trakia University, StaraZagora, Bulgaria.

Background: Thewelfare of broiler chickens and breeder flocks

continues to be closely related to skeletal pathology of the legs.

Although some reports establish that most abnormalities related

to legs in chickens were largely overcome by selection work and

improvement of production systems by the end of the 20th cen-

tury, the problems still occupy an important place in broiler pro-

duction. Objective: The aim of the present work was to review

the literature concerning the incidence of some major skeletal

pathologies of legs in broiler chickens and broiler breeders related

to poor animal welfare. Methods: On the basis of data on the

incidence of skeletal pathology of legs published by us and other

researchers, this work reviewed some reports on femoral head

necrosis (FHN), tibial dyschondroplasia (TD), rupture of the gas-

trocnemius tendon (RGT) and rickets. Results: Cases of TD

were related to significantly younger chickens, approximately

24 days, among modern hybrid broiler chickens and were pre-

dominantly associated with insufficient Ca. Subclinical and

clinical rickets formed within broiler flocks were a possible

prerequisite for a number of pathological conditions, such as

FHN, osteomyelitis, fractures, TD, and vertebral column defor-

mations. RGT was the most common problem related to the

skeleton of the legs among parent flocks of broiler breeders.

Conclusion: Analysis of the overview indicates that the most

common problems were related to skeletal leg pathology among

modern hybrid broiler chickens, namely FHN, TD and rickets.

FHN was most commonly caused by osteomyelitis, which was

caused predominantly by E. coli infection.

116PATHOMORPHOLOGICAL INVESTIGATIONS OF THEINCIDENCE OF AXIAL SKELETON PATHOLOGY ASSO-CIATED WITH POSTERIOR PARALYSIS IN COMMER-CIAL BROILER CHICKENS. I Dinev. Department ofGeneral and Clinical Animal Pathology, Faculty of Veteri-naryMedicine, Trakia University, Stara Zagora, Bulgaria.

Background: Most reported data are on the prevalence of

lameness in broiler chickens and broiler breeders associated with

pathology of the hind limb. Data on axial skeleton abnormalities,

mostly spondylolisthesis and scoliosis, which could be asymp-

tomatic, can be found in separate reports.Objective: The aim of

the current pathomorphological study was to determine the

prevalence of various pathological conditions of the axial skele-

ton associated with posterior paralysis in commercial broiler

chickens at a farm over a prolonged period. Methods: Cases of

clinical posterior paralysis at a commercial broiler chicken farm

were registered over a one-year period. A total of 151 vertebral

columns from chickens that had exhibited posterior paralysis

were randomly collected from 6 isolated chicken carcasses from

each of the observed flocks at the processing plant. The samples

were submitted to gross, histological, radiographic, andmicrobio-

logical examinations. Results: Of the 36 broiler flocks, signs of

posterior paralysis were found in a total of 1480 (0.21%) chickens

from 23 flocks. Spondylolisthesis� 64 (42.3%), followed by cases

of spondylosis with ankylosis, osteosclerosis and fractures� 54

(35.8%), vertebral osteomyelitis � 19 (12.6%), and osteochon-

drosis with scoliosis and fractures - 14 (9.3%) were the most pre-

valent conditions. Conclusion: Summarizing the results of our

research, most of the established pathological conditions, which

are manifested by posterior paralysis, were related to anatomical

features and the level of maturity of the axial skeleton among

broiler chickens, whereas the observed vertebral osteomyelitis

was spontaneouswith proven etiology.

117HISTOPATHOLOGIC FINDINGS IN ABOMASA OF FAL-LOW DEER FROM THE BRIJUNI REGION IN CROATIA.M. Robic, B. Artukovic, A. Beck, R. Turk, M. Belic,Z. Grabarevic. Veterinary Faculty, Zagreb, Croatia.

Background: Histopathologic examination was performed on

abomasal samples of fallow deer (Dama dama) from the Brijuni

region in Croatia. Objective: The aim of the study was to

determine histopathologic changes and the possible presence of

gastric Helicobacter-like organisms in abomasa of fallow deer.

Method: Paraffin embedded, H&E-stained samples of 23 abo-

masa taken from fallow deer were histopatholgically examined.

Samples were stained with toluidine blue, Giemsa and the War-

thin Starry method for detection of Helicobacter-like organisms.



Results: In all samples examined, chronic inflammatory

changes were visible. It was noteworthy that histopathologic

findings were similar, indicating the epizootical nature of disease.

All samples were negative for Helicobacter-like organisms using all

stains. Conclusion: Abomasa of fallow deer from the Brijuni

region revealed chronic inflammatory changes morphologically

similar to those described in dogs with Helicobacter infection.

However, these changes could not be attributed to gastricHelicob-

acterinfection, because these organisms were not detected after

appropriate special stains were applied. The underlying etiology

of chronic inflammatory lesions in the abomasa of these deer is

still to be determined.

118COMPARISON OF TOTAL LEUKOCYTE RESPONSES OFHORSES SUBJECTED TO EXERCISE ON HIGH SPEEDTREADMILL – PRELIMINARY RESULTS. T. Barbosa,L. Yonezawa, C. Marinho, M.J. Watanabe,A. Kohayagawa. School of Veterinary Medicine andAnimal Science - Sao Paulo State University, Botucatu,Brazil.

Background and objectives: The objective was to observe dif-

ferences regarding response of leukocytes in horses subjected to

high intensity and short duration (HI) and low intensity and long

duration (LO) exercise on a high-speed treadmill. Five Arabian

horses performed two exercise tests. Individual maximum oxy-

gen uptake (VO2max) was determined to establish speeds for each

horse.Methods: The HI test consisted of 5 min at 50% VO2max,

5 min at 1.5min and 90 s at 105%VO2max set at +6% slope, and 5

min at 3.0 m/s (0% slope). The LO test consisted of 60 min at

35% VO2max (+6% slope). Blood samples were collected before

exercise (M0), immediately after exercise, and 15 and 30min and

6, 12, 24, 48, 72 and 96 hours after each test for determination of

white blood cell (WBC) using an automated cell counter, the

Cell-Dyn®. Results: Only at times M0 and after 15 and 30 min-

utes were there significant differences between the two types of

exercise (p < .05). Possibly the reason for the higher value at M0

was due to apprehension of horses when entering the exercise

place, which caused neutrophil demargination, whereas at the

following times lymphocytes had a greater mobilization from

spleen to circulation, but the values did not exceed the reference

values. In both types of exercise, values up to 12 hours were

above the baseline and after 24 hours remained below the base-

line.We conclude that the response of leukocytes did not differ in

high and low intensity exercises. Financial support: Sao Paulo

Research Foundation (FAPESP).

119EFFECT OF EXERCISE ON A HIGH-SPEED TREADMILLON DNA DAMAGE IN PERIPHERAL LEUKOCYTESOF HORSES. T. Barbosa, L. Yonezawa, C. Marinho,J. Knaut, M.J. Watanabe, D. Salvadori, D. Almeida,A. Kohayagawa. School of Veterinary Medicine andAnimal Science - Sao Paulo State University, Botucatu,Brazil.

Background: Recently, there has been growing interest in

exercise-induced DNA damage due to its potential involvement

in organ dysfunction. However, it has not been reported in horses

submitted to strenuous exercise on a high-speed treadmill. The

comet assay is a relatively new method used to measure single

DNA strand breaks in peripheral leukocytes, and the tail moment

is the product of tail length and percentage DNA in the tail.

Objectives: The purpose of this study was to determine the

effects of exercise on DNA damage in peripheral leukocytes of

horses submitted to exercise on a high-speed treadmill. Meth-

ods: Ten untrained horses were subjected to exercise at high

intensity and short duration on a high-speed treadmill. The

comet assay was used to assess DNA damage in circulating leuko-

cytes (mononuclear cells – MN and polymorphonuclear cells -

PMN) at rest before exercise (M0) and 12 h, 48 h and 72 h after

exercise. Results: The tail moment of DNA damage in MN was

8.27% at M0, 7.64% at 12h, 7.84% at 48h and 7.58% at 72h,

whereas in PMN was 7.86% at M0, 6.71% at 12h, 9.17% at 48h

and 12.39% at 72h. There were no significant differences (P>.05)

among all analyzed times. Conclusions: This protocol of exer-

cise did not result in DNA damage in peripheral leukocytes of

horses as detected by the comet assay. Financial support: Sao

Paulo State Research Foundation (FAPESP), Brazil.

120REFERENCE VALUES FOR HAEMATOLOGICALPARAMETERS IN THE LUSITANO HORSE. A.C. Silves-tre-Ferreira1, M. Cotovio1, M. Maia1, F. Queiroga2,M.J. Pires2, A. Colaco2. 1Department of VeterinarySciences – Veterinary Hospital – University of Tras-os-Montes e Alto Douro, Vila Real, Portugal; 2Department ofVeterinary Sciences – CECAV - University of Tras-os-Montes e Alto Douro, Vila Real, Portugal.

Background: The Lusitano horse is an autochthonous Portu-

guese horse breed with growing worldwide expansion. Objec-

tive: The aim of this study was to establish reference values for

haematological parameters using the haematological cell coun-

ter, LaserCyte (IDEXX). Methods: One-mL EDTA blood sam-

ples were obtained from 75 healthy horses of both sexes and 2 to

23 years of age. Reference values were estimated by bootstrap

and nonparametric methods with Reference Value Advisor soft-

ware. Results: The reference values were 6.3-10.7 x1012/L for

red blood cells, 30.7-46.6% for hematocrit, 12.7-17.3 g/dL for

haemoglobin, 42.1-53.3 fL for mean corpuscular volume, 15.5-

20.9 pg for mean corpuscular haemoglobin, 35.3-39.5 g/dL for

mean corpuscular haemoglobin concentration, 17.8-20.6% for

red cell distribution width, 4.8-10.0 x109/L for white blood cells,

2.3-6.0 x109/L for neutrophils, 0.8-4.7 x109/L for lymphocytes,

0.2-0.5 x109/L for monocytes, 0.1-0.6 x109/L for eosinophils,

0.0-0.1 x109/L for basophils, 78.9-164.5 K/mmL for platelets,

4.3-9.1 fL for mean platelet volume, 18.7-24.5% for platelet dis-

tribution width and 0-0.1% for plateletcrit.Conclusion: Except

for leukocytes and platelets, reference values obtained in this

study were close to data reported for other hot-blooded horse

breeds and provide important clinical information for clinicians

to use.

121MONONUCLEAR CELL CONCENTRATIONS IN FRAC-TIONATED SAMPLES OF BONE MARROW ASPIRATES-FROM HORSE STERNUM. F. Correa, J. Galecio,H. Bustamante, F.Wittwer, A. Ramirez, B.Menarim.Universidad Austral de Chile, Valdivia, Chile.

Background: The use of bone marrow mononuclear cells

(MNCs) has been described as an alternative procedure to graft-



ing mesenchymal stem cells (MSCs) in injured tissues. Objec-

tive: The aim of this study was to compare the concentration of

MNCs of bone marrow samples obtained in 5 steps. Methods:

Bone marrow aspirates were obtained from the sternum of 5

Chilean Criollo horses and five sequential 5-mL fractions were

recovered from each horse. Once collected, the mononuclear

fraction of each sample was isolated through Ficoll density

centrifugation and subsequently submitted to a cell count and

viability test using epifluorescence microscopy. Results: The

total number (median ± QD) of MNCs per fraction was 13.4 ±12.6 x 106 for the first fraction, 28.6 ± 5.8 x 106 for the second,

114.8 ± 51.2 x 106 for the third, 6.6 ± 6.1 x 106 for the fourth, and

4.8 ± 4.2 x 106for the fifth fraction. The first three fractions repre-

sented 93.2% of the total 25mL (the five fractions together). The

cell viability was 97.5 ± 1.2 %. Conclusions: The major finding

of this studywas that there was a heterogeneous concentration of

MNCs within five fractions consecutively obtained from different

horses. Furthermore, we suggest the need to obtain at least the

first 15mL of bone marrow aspirated from the sternum when the

sample is intended to be used as a concentrate of bone marrow

MNCs.



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