2009 ACVP Abstract Awards

• ACVP/AAVLD Diagnostic Travel Award
• ACVP Young Investigator Awards
• STP Student Speaker Award
• Veterinary Student Poster Awards

MICROCHIP-ASSOCIATED SOFT TISSUE SARCOMA AND MASSIVE MULTIORGAN EXTRAMEDULLARY HEMATOPOIESIS IN A HOUSE MUSK SHREW (SUNCUS MURINUS).
L.K. Schutt, P.V. Turner. Department of Pathobiology, University of Guelph, Guelph, ON, Canada.

   A 16 month-old female house musk shrew (Suncus murinus) with a 2-month history of a rapidly growing subcutaneous mass on the dorsum was euthanized and submitted from a research colony to the Laboratory Animal Diagnostic Service for necropsy. Macroscopic examination identified an irregular, well-demarcated, solid, tan-white subcutaneous mass on the interscapular dorsum. A small cavity containing a microchip was present at the center of the mass. Massive splenomegaly was also noted grossly. Histologically, the subcutaneous mass was comprised of spindle cells arranged in a storiform pattern of interweaving bundles consistent with a high-grade soft tissue sarcoma with multifocal necrosis. Preliminary immunohistochemical investigation suggested the neoplastic cells were positive for vimentin, neuron-specific enolase, and rarely smooth muscle actin and negative for cytokeratin and desmin, most suggestive of a diagnosis of fibrosarcoma. To our knowledge, this is the first report of a microchip-associated soft tissue sarcoma in a shrew. Neoplastic growths at the site of microchip implants have been described in both dogs and laboratory rodents. Additionally, the degree and extent of organ involvement with extramedullary hematopoiesis noted histologically was impressive in this shrew, involving the hepatic portal tracts, splenic red pulp, renal cortical interstitium, and adrenal medulla. Splenic extramedullary hematopoiesis has been previously reported in healthy musk shrews and may be physiologically normal in this species.

ACVP Young Investigator Awards

Diagnostic Pathology – First Place
Chantelle Bozynski, University of Missouri

VASCULAR ANOMALY OF THE VERTEBRAL AND SPINAL ARTERIES OF A DOG ASSOCIATED WITH CERVICAL SPINAL COMPRESSION.
C. Bozynski1, L. Vasquez2, G. Johnson1, D. O’Brien2. 1Veterinary Medical Diagnostic Laboratory and 2Veterinary Medicine and Surgery, University of Missouri, Columbia, MO.

   A 4-year-old, male neutered Catahoula leopard dog mix was referred to the Veterinary Medical Teaching Hospital, University of Missouri at Columbia, for acutely progressive non-ambulatory tetraparesis and cervical hyperesthesia of 48 hour duration. Neuro-localization identified C6-T2 myelopathy. Survey radiographs and magnetic resonance imaging of the spine revealed bilaterally symmetrical tubular structures, associated with markedly enlarged intervertebral foramina at C4-5 and C5-6 vertebrae causing severe compression of the associated spinal cord segments. The C4 and C5 vertebral bodies were irregular and sclerotic. At necropsy, both vertebral arteries were severely ectatic and entered the vertebrae lateroventrally at C6. The cervical spinal cord was ventrally compressed, and was red and soft in texture. On histopathologic examination, the ectatic branches of the vertebral arteries and ventral spinal arteries were surrounded by fibrosis, hemorrhage and mononuclear cell infiltrates. There was severe bone loss in C4 and C5 vertebrae. Branches of the vertebral arteries had severe, focally extensive reduction in the tunica media, and a large fibrinocellular thrombus was present in one. Several smaller neighboring vessels showed fibrinoid degeneration and were surrounded by fibrinocellular fluid. The white matter of the affected cervical spinal cord had dilated myelin sheaths, digestion chambers and spheroids, vascular congestion, and focally extensive malacia. Multifocally, both the dorsal and ventral nerve roots were moderately edematous, with axonal degeneration and demyelination. Vascular malformations resulting in myelopathy are infrequently reported in dogs. In this case, severe ectasia of the vertebral and spinal arteries, severe loss of cervical vertebral bone, and associated compression of the cervical spinal cord led to degenerative radiculomyelopathy, contributing to clinical signs.

Diagnostic Pathology – Second Place
Adam Stern, Oklahoma State University

DISSEMINATED CUTANEOUS MAST CELL TUMORS WITH EPITHELIOTROPISM AND SYSTEMIC MASTOCYTOSIS IN A DOMESTIC CAT.
A.W. Stern1, C.G. Lamm2, A.J. Smith2, E.J. Cooper2, S.W. Ullom3, G.A. Campbell2. 1Department of Pathobiology, Oklahoma State University, Stillwater, OK, 2Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, OK, 3Timbers Vet Clinic, Tahlequah, OK.

   A 15-year-old, female, domestic medium-haired cat presented to the referring veterinarian with a 2 month history of nodular skin lesions. A biopsy of one of the lesions was submitted to the Oklahoma Animal Disease Diagnostic Laboratory for histopathologic examination. Histologically, there were multiple dermal nodules composed of sheets of neoplastic round cells. Multifocally within the epidermis, these neoplastic cells formed multiple small clusters of 3-5 cells. Within the neoplastic cells there were distinct cytoplasmic granules when stained with toluidine blue and Giemsa stains. Immunohistochemistry was performed; the neoplastic cells were immunoreactive for c-KIT and lacked immunoreactivity for CD3. Based on these findings, multiple epitheliotropic cutaneous mast cell tumors were diagnosed. The cat’s health declined rapidly despite aggressive treatment and the animal was humanely euthanized. Necropsy revealed sheets of similar neoplastic mast cells within the spleen and liver with individual neoplastic cells scattered within the bone marrow. Exon 11 of the c-KIT mRNA from one of the cutaneous masses and the spleen was amplified with RT-PCR, sequenced, and compared with the published c-KIT mRNA sequence from fetal cat tissues. The maximum identity was 100% for both tissue samples. This report is the first report of disseminated cutaneous mast cell tumors with epitheliotropism and systemic mastocytosis in a domestic cat.

Diagnostic Pathology – Third Place
Heather Tillman, Michigan State University

APPLICATION AND CLINICAL RELEVANCE OF THE WHO HISTOLOGIC CLASSIFICATION SYSTEM TO CANINE THYMOMAS.
H. Tillman1, J.C. Zitz3, S.J. Birchard3, G.C. Couto3, V.F. Samii3, S.E. Weisbrode2, G.S. Young3, M Kiupel1. 1Department of Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, MI, 2Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, 3Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, OH.

   Historically canine thymomas are classified as a relatively uniform benign to low-grade malignant neoplastic entitie, with surgical excision considered curative. However, studies indicate canine thymomas often display a more complex biological behavior similar to stage I and II disease in humans. Human thymomas are classified microscopically according to the World Health Organization (WHO) into distinct morphologic subtypes. Subtypes are based on the morphologic appearance of neoplastic epithelial cells and their relative numbers compared to non-neoplastic lymphocytes. The WHO classification is currently used as a prognostic tool for human thymomas. Similar to human thymomas, canine thymomas exhibit marked variation in histologic appearance. We hypothesized that the WHO system is applicable to canine thymomas. In this retrospective case series we classified canine thymomas according to the WHO system and evaluated the clinical significance of the defined subtypes. Biopsies were reviewed from 35 canine patients, 5-15 years of age, over a twenty-four year period from two veterinary teaching institutions. Our classification resulted in the following subtypes: 3 type A, 5 type AB, 7 type B1, 15 type B2, 4 type B3, and 1 carcinoma. Prognostic relevance of the WHO histologic classification was further correlated with available clinical and survival data with the following categorical variables: age, gender, paraneoplastic syndromes, stage, and completeness of surgical resection. Application of the WHO system provided a consistent microscopic classification scheme that may be useful for more accurate prognostication for specific subtypes.

Experimental Disease – First Place
Jessica Grieves, Ohio State University

AN INTRANASAL VACCINE CANDIDATE PROTECTS THE UPPER AIRWAY FROM INFECTION WITH RESPIRATORY SYNCYTIAL VIRUS.
J. Grieves1,2, L. Martinez-Sobrido3, A. Garcia-Sastre4, L. Bakaletz1, J. Durbin1. 1The Research Institute at Nationwide Children’s Hospital Columbus, OH, 2The Ohio State University College of Veterinary Medicine, Columbus, OH, 3University of Rochester, Rochester, NY, 4Mount Sinai School of Medicine, New York, NY.

   Respiratory Syncytial Virus (RSV) is an important cause of upper and lower airway disease in children worldwide. The adaptive immune response to RSV is ineffective and re-infection occurs throughout life. Type I interferons (IFNs), which are generally induced by viral infection, act as an important link between the innate and adaptive immune systems. Importantly, RSV is not only a poor stimulator but also a direct inhibitor of IFN production. Based on the hypothesis that delivery of RSV antigen in an environment of robust IFN production would prevent RSV infection, we constructed a Newcastle Disease Virus (NDV) vector that expresses the RSV F glycoprotein (NDV-F). NDV is a strong inducer IFN production and is non-pathogenic in mammals. Chinchillas were vaccinated intranasally (i.n.) with NDV-F, NDV, or allantoic fluid followed 28 days later by i.n. RSV challenge. Viral load was reduced 10-fold in nasopharyngeal lavage fluids and many fewer virus-infected cells were detected by immunohistochemistry in the nasal mucosa of NDV-F vaccinated animals compared to controls. Additionally, NDV-F vaccination resulted in earlier production of serum RSV-specific antibodies compared to controls. Finally, the nasal mucosa of NDV-F vaccinated animals contained a significant plasma cell infiltrate that was not detected in the nasal mucosa of control animals, suggesting that NDV-F vaccination mediated induction of a mucosal immune response. These data provide important mechanistic information regarding RSV infection and support future work toward clinical development of this vaccine.

Experimental Disease – Second Place
Katherine Gailbreath, USDA-ARS

INITIAL REPLICATION IN THE LUNG AND SYSTEMIC DISSEMINATION OF OVINE HERPESVIRUS 2 IN AMERICAN BISON AFTER INTRANASAL NEBULIZATION.
K.L. Gailbreath1,2, C.W. Cunha1, N.S. Taus1, J.L. Oaks2, D. O’Toole3, D.P. Knowles1,2, H. Li1. 1ADRU, USDA-ARS, and 2Dept. of Vet Micro-Path, WSU, Pullman, WA, 3WSVL, Laramie, WY.

   Malignant catarrhal fever caused by ovine herpesvirus 2 (OvHV-2) is an important infectious disease in American bison with high mortality rates during natural outbreaks. Virus-host interactions in bison are essentially unknown. Recent experimental infection of sheep by intranasal nebulization with OvHV-2 from sheep nasal secretions showed that a brief period of viral replication in the lungs is followed by systemic dissemination of latent virus. This conclusion is based, in part, on the observation that expression of ORF25 (capsid protein), a marker for lytic replication, is detected only in the lungs during early infection. In contrast, studies in bison during late preclinical and clinical stages have demonstrated ORF25 transcripts in most tissues. In order to compare early events in the viral life cycle between sheep and bison, 24 bison infected with OvHV-2 were euthanized in pairs at two to three day intervals between 1 and 26 days post-nebulization (DPN). ORF25 expression was detected in lungs between 7 and 16 DPN. A concurrent increase in OvHV-2 genome copy number in lungs began at 9 DPN, peaked at 12 DPN and declined by 14 DPN. Viral DNA was first detected in tracheal-bronchial lymph node at 21 DPN and in up to 65% of tissues by 23 DPN. We conclude that a brief period of viral replication in bison lungs is similar to what occurs in sheep but is followed by dissemination and continued lytic replication. This suggests that the development of MCF in bison is due to a failure to control lytic virus following systemic dissemination of OvHV-2. 

Experimental Disease – Third Place
Stacey Fossey, Ohio State University

THE NOVEL CURCUMIN ANALOG FLLL32 EXHIBITS BIOLOGIC ACTIVITY AGAINST HUMAN AND CANINE OSTEOSARCOMA.
S. Fossey1, M. Bear1, J. Lin2,4, C. Li3, E. Schwartz3, P. Li3,4, J. Fuchs3, C. London1,4. 1Department of Veterinary Biosciences, 2Department of Pediatrics, 3Division of Medicinal Chemistry, and 4Comprehensive Cancer Center, The Ohio State University, Columbus, OH.

   Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Unfortunately, 30% of children and over 90% of dogs will die of their disease necessitating the development of new therapies. In our previous work we found that canine and human OSA cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. We further demonstrated that siRNA mediated downregulation of STAT3 in OSA cell lines decreased expression of survivin, VEGF, and MMP2 (all transcriptional targets of STAT3), reduced cell viability, and promoted caspase-3/7 mediated apoptosis. Curcumin, a naturally occurring compound found in the spice tumeric, inhibits STAT3 in vitro but does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin that possesses superior biochemical properties. The purpose of this work was to evaluate the effects of FLLL32 mediated STAT3 inhibition in OSA. Canine and human OSA lines were treated with FLLL32 and assayed for cell viability, apoptosis, downregulation of pSTAT3 and loss of transcriptional target expression. FLLL32 promoted loss of cell viability leading to caspase-3 dependent apoptosis as evidenced by PARP cleavage and caspase 3/7 activity. FLLL32 blocked STAT3 phosphorylation and STAT3 DNA binding, ultimately inducing downregulation of total STAT3. Lastly, treatment of OSA cells decreased expression of survivin, VEGF, and MMP2 at both message and protein levels. These data demonstrate that FLLL32 exhibits biologic activity against OSA cell lines.

Natural Disease – First Place
Jennifer Luff, University of California-Davis

POLYMERASE CHAIN REACTION AND NUCLEOTIDE SEQUENCE ANALYSIS OF CANINE PAPILLOMAVIRUS-ASSOCIATED LESIONS WITH PHYLOGENETIC COMPARISON TO KNOWN PAPILLOMAVIRUSES.
J.A. Luff, V.K. Affolter, B. Yeargen, P.F. Moore. Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA.

   Papillomaviruses are epitheliotropic viruses within the family Papillomaviridae that are associated with various epithelial proliferations. Canine papillomavirus (PV)-associated lesions are classified based on morphological criteria into exophytic papilloma, endophytic papilloma and pigmented plaques. Alternatively they are categorized based on location; canine oral papilloma versus lesions in the skin. The complete viral genome of four canine PVs has been identified, including canine oral papillomavirus (COPV) in association with oral papillomas, canine papillomavirus-2 (CfPV-2) from an endophytic papilloma, and both canine papillomavirus-4 (CPV-4) and canine papillomavirus-3 (CPV-3) from pigmented plaques. The aim of this study was to investigate whether different PV-induced lesions in dogs were associated with specific canine PVs and to identify any potentially novel PV sequences. This investigation encompassed PCR amplification of a highly conserved 450 base pair region of the L1 gene from 7 endophytic, 8 exophytic, and 6 oral papillomas, and 27 pigmented plaques. All endophytic and exophytic papillomas revealed 99-100% sequence identity with CfPV-2; all oral papillomas revealed 100% sequence identity with COPV. 48% (13/27) of the pigmented plaques revealed 98-100% sequence identity with CPV-4; one pigmented plaque revealed 100% sequence identity with CPV-3. The remaining 13 pigmented plaques revealed 8 putatively novel PV sequences. A phylogenetic tree comparing these novel sequences and published papillomavirus genomes was constructed using CLC Bio’s free CLC Work Bench. Based upon these results, there are potentially eight novel PVs types most closely related to CPV-3 and CPV-4. Identification and future cloning of these novel PVs will generate a foundation for further research on the malignant potential of these unique canine PVs.

Natural Disease – Second Place
Gillian Shaw, Johns Hopkins University

METABOLIC BONE DISEASE IN A COLONY OF COMMON MARMOSETS (CALLITHRIX JACCHUS).
G.C. Shaw, K.K. Brennan, T.L. Southard, M.C. Zink. Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD.

   Bone lesions were detected in 18 of 76 (24%) common marmosets (Callithrix jacchus) submitted for necropsy to Johns Hopkins University between 2001 and 2009. The ages of affected marmosets ranged from 3 days to 7 years and included 10 males and 8 females. The clinical histories included: no noted illness, fracture, diarrhea, anorexia, wasting, weakness, constipation, failure to thrive (neonates) and respiratory distress. Diarrhea and wasting were the most common clinical findings in affected animals. Morphological diagnoses relevant to the skeletal system included: myelofibrosis, osteopenia, fibrous osteodystrophy and/or pathological fracture. Myelofibrosis was characterized by replacement of the bone marrow by fibroblasts and collagen. Osteopenia was diagnosed when there was an apparent decrease in the mass of structurally normal cortical bone; this often was accompanied by myelofibrosis. Fibrous osteodystrophy was diagnosed when there was evidence of increased osteoclastic bone resorption, replacement of metaphyseal and cortical bone with fibrous connective tissue and reduced mineralization. Together, these lesions likely represent a spectrum of abnormalities referred to as metabolic bone disease (MBD) and suggest a dietary imbalance of vitamin D3, calcium and/or phosphorous. Historically, simian bone disease has been associated with diets deficient in vitamin D3, which is an absolute dietary requirement for new world primates. The marmosets in this study received a commercial marmoset diet containing vitamin D3, but rations often were supplemented with treats, which might have altered the mineral balance. Also, several affected animals were closely related, possibly suggesting a familial predisposition within the breeding colony. Current studies are investigating whether affected animals have abnormal levels of vitamin D3 metabolites consistent with previous reports of competitive blockade of target receptors.

Natural Disease – Third Place
Leah Zadrozny, North Carolina State University

SPONTANEOUS HEPATIC NEOPLASMS IN CAPTIVE LEMURS.
L. Zadrozny, C. Williams, A. Remick, J. Cullen. North Carolina State University, College of Veterinary Medicine, Raleigh, NC, Duke Primate Center, Duke University, Durham, NC, Biotechnics, Hillsborough, NC.

   There are few reports of primary hepatic neoplasia in prosimians. We report a review of hepatic neoplasia from a large collection of prosimians maintained in captivity between 1999 and 2009. Of the 145 lemurs necropsied between the ages of 6 to 40 years old, 14/145 (9.7%) had hepatocellular carcinoma characterized histologically by both trabecular and pseudoglandular patterns in most tumors along with prominent pleomorphism of neoplastic hepatoctyes. These tumors had an unusually aggressive growth pattern for animal species, as metastasis to the lungs was evident in 7/145 (50%) of these animals; 1 of which also had metastasis to the vessels of the stomach, heart and kidney. In addition to having lung metastasis, one of these animals may have had a post-operative abdominal implant rather than naturally occurring metastasis to the mesentery. Five animals had hepatocellular adenomas, one had a preneoplastic hepatocellular lesion and 1 animal had a biliary carcinoma. In order to investigate possible pathogenic mechanisms, we evaluated the relationship between hepatic iron stores and other metals and the presence of hepatocellular carcinoma. Increased iron levels were not associated with higher rates of hepatocellular carcinoma, suggesting that iron is not a key element in liver tumor formation, although there was an association with liver lead concentration and hepatocellular tumors. Analysis of 23 serum samples indicated that there was no evidence of active infection with hepadnavirus or the hepatitis B virus family, in the livers of normal animals or those with liver tumors. Hepatic neoplasia is relatively common in captive lemurs, although the cause remains unclear.

STP Student Speaker Award
Stacey Fossey, Ohio State University

THE NOVEL CURCUMIN ANALOG FLLL32 EXHIBITS BIOLOGIC ACTIVITY AGAINST HUMAN AND CANINE OSTEOSARCOMA.
S. Fossey1, M. Bear1, J. Lin2,4, C. Li3, E. Schwartz3, P. Li3,4, J. Fuchs3, C. London1,4. 1Department of Veterinary Biosciences, 2Department of Pediatrics, 3Division of Medicinal Chemistry, and 4Comprehensive Cancer Center, The Ohio State University, Columbus, OH.

   Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Unfortunately, 30% of children and over 90% of dogs will die of their disease necessitating the development of new therapies. In our previous work we found that canine and human OSA cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. We further demonstrated that siRNA mediated downregulation of STAT3 in OSA cell lines decreased expression of survivin, VEGF, and MMP2 (all transcriptional targets of STAT3), reduced cell viability, and promoted caspase-3/7 mediated apoptosis. Curcumin, a naturally occurring compound found in the spice tumeric, inhibits STAT3 in vitro but does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin that possesses superior biochemical properties. The purpose of this work was to evaluate the effects of FLLL32 mediated STAT3 inhibition in OSA. Canine and human OSA lines were treated with FLLL32 and assayed for cell viability, apoptosis, downregulation of pSTAT3 and loss of transcriptional target expression. FLLL32 promoted loss of cell viability leading to caspase-3 dependent apoptosis as evidenced by PARP cleavage and caspase 3/7 activity. FLLL32 blocked STAT3 phosphorylation and STAT3 DNA binding, ultimately inducing downregulation of total STAT3. Lastly, treatment of OSA cells decreased expression of survivin, VEGF, and MMP2 at both message and protein levels. These data demonstrate that FLLL32 exhibits biologic activity against OSA cell lines.

Veterinary Student Poster Awards

Experimental Disease
Stephanie Montgomery, North Carolina State University

DEVELOPMENT OF A MOUSE MODEL OF HUMAN CHIKUNGUNYA VIRUS INFECTION
T. Heise3, and Thomas E. Morrison4
1College of Veterinary Medicine, North Carolina State University, 2Cellular and Molecular Pathology Branch, NIEHS and NTP, 3Department of Genetics and Carolina Vaccine Institute, University of North Carolina-Chapel Hill, and 4Department of Microbiology, University of Colorado, Denver

Chikungunya virus (CHIKV) is an emerging mosquito-borne Alphavirus that causes debilitating acute and persistent arthritis/myositis in humans.  In 2005-2007, a CHIKV outbreak in the Indian Ocean region affected millions of people, yet no licensed vaccines or therapies are available.  The pathogenesis of CHIKV is poorly understood, and a major challenge to the study of CHIKV disease has been the lack of an animal model that recapitulates the major outcomes of human infection.  In this study, the pathogenesis of CHIKV in C57BL/6J laboratory mice was investigated utilizing low passage CHIKV isolated directly from human serum.  CHIKV inoculation of 14 day-old mice in the rear footpad caused reduced weight gain and significant swelling and loss of function in the inoculated limb.  At 4 days post-infection (dpi), virus was isolated from tissue from both tarsi, but was undetectable by viral plaque assay in serum, spleen, and brain tissue.  Examination of hematoxylin and eosin stained hindlimb sections at 14 dpi revealed a severe necrotizing myositis in both the inoculated and contralateral limb with fibrosis and myocyte regeneration, and a severe chronic active tenosynovitis with coagulative necrosis.  Other notable findings included a mixed inflammatory cell arthritis, sporadic necrotizing vasculitis, and inclusion-like bodies within myocytes and macrophages.  This work demonstrates the development of a mouse model of human CHIKV infection in which the clinical manifestations and histopathological findings are consistent with the disease signs and symptoms experienced by CHIKV-infected humans.

Natural Disease
Cynthia Willson, North Carolina State University

EFFECT OF ESTROUS CYCLE PHASE ON CLINICAL PATHOLOGY PARAMETERS IN BEAGLE DOGS
Cynthia J. Willson1,2, Sundeep Chandra2, Carie Kimbrough2 and Holly Jordan2
1North Carolina State University College of Veterinary Medicine, Raleigh, NC and  2Safety Assessment, GlaxoSmithKline, Research Triangle Park, NC

The duration of diestrus in dogs is considerably longer (about 75 days) than in most species and is characterized by high circulating progesterone and growth hormone levels that may influence clinical pathology parameters. The influence of the phase of the reproductive cycle on clinical pathology parameters has been reported in many species, but there is a relative paucity of information in the dog. Such differences may potentially confound interpretation of data in toxicity studies, which often have small group sizes. The objective of this retrospective study was to investigate differences in clinical pathology data in dogs in diestrus relative to dogs in other phases of the estrous cycle. For 86 healthy control females from 23 toxicity studies (age range: 11-22.5 months), estrous cycle stage was determined by histological examination of reproductive tissues. Serum chemistry, hematology, and urinalysis parameters were compared using two-tailed t-tests. For Beagles in diestrus (n = 38), serum cholesterol was 35.0% higher (P < 0.0001), AST was 14.0% lower (P = 0.0011), chloride was 0.9% lower (P = 0.0224), eosinophils were 45.8% higher (P = 0.0035), hemoglobin and red blood cell count were 7.8% lower (P < 0.0001), and hematocrit was 7.6% lower (P < 0.0001) than dogs in all other stages combined (anestrus, proestrus, estrus, and immature; n = 48). Urine parameters did not differ significantly between the two groups. Interpretation of clinical pathology data in female dogs should be performed with due consideration given to the stage of the estrous cycle.