Neutrophil gelatinase-associated lipocalin in dogs with chronic kidney disease, carcinoma, lymphoma and endotoxaemia.

OBJECTIVES: To measure serum and urine neutrophil gelatinase-associated lipocalin (NGAL) concentrations in healthy dogs and dogs with chronic kidney disease, neoplasia and endotoxaemia. METHODS: Serum and urine NGAL concentrations were measured in 42 healthy dogs, 11 dogs with chronic kidney disease, 12 dogs with carcinoma, 20 dogs with lymphoma and 15 dogs with lipopolysaccharide-induced endotoxaemia. In dogs with chronic kidney disease, NGAL was measured 3 and 6 months later. RESULTS: Compared with healthy controls, dogs with chronic kidney disease (PÄ0·0008), carcinoma (PÄ0·0072) and lymphoma (PÄ0·0008) had elevated serum and urine NGAL and urine NGAL-to-creatinine ratio. Serum and urine NGAL was not significantly different between dogs with chronic kidney disease, carcinoma or lymphoma (Pê0·12). In dogs with non-progressive chronic kidney disease, NGAL concentrations did not change significantly over the 6-month study period. CLINICAL SIGNIFICANCE: NGAL can be elevated by chronic kidney disease and neoplasia, compared with healthy controls. Further research is needed to determine if uNGAL or uNGAL-to-creatinine ratio is more specific than serum levels to detect chronic kidney disease.

Biomarkers in the assessment of acute and chronic kidney diseases in the dog and cat.

In both human and veterinary medicine, diagnosing and staging renal disease can be difficult. Measurement of glomerular filtration rate is considered the gold standard for assessing renal function but methods for its assessment can be technically challenging and impractical. The main parameters used to diagnose acute and chronic kidney disease include circulating creatinine and urea concentrations, and urine-specific gravity. However, these parameters can be insensitive. Therefore, there is a need for better methods to diagnose and monitor patients with renal disease. The use of renal biomarkers is increasing in human and veterinary medicine for the diagnosis and monitoring of acute and chronic kidney diseases. An ideal biomarker would identify site and severity of injury, and correlate with renal function, among other qualities. This article will review the advantages and limitations of renal biomarkers that have been used in dogs and cats, as well as some markers used in humans that may be adapted for veterinary use. In the future, measuring a combination of biomarkers will likely be a useful approach in the diagnosis of kidney disorders.

The Feline Acute Patient Physiologic and Laboratory Evaluation (Feline APPLE) Score: a severity of illness stratification system for hospitalized cats.

BACKGROUND: Scores allowing objective stratification of illness severity are available for dogs and horses, but not cats. Validated illness severity scores facilitate the risk-adjusted analysis of results in clinical research, and also have applications in triage and therapeutic protocols. OBJECTIVE: To develop and validate an accurate, user-friendly score to stratify illness severity in hospitalized cats. ANIMALS: Six hundred cats admitted consecutively to a teaching hospital intensive care unit. METHODS: This observational cohort study enrolled all cats admitted over a 32-month period. Data on interventional, physiological, and biochemical variables were collected over 24 hours after admission. Patient mortality outcome at hospital discharge was recorded. After random division, 450 cats were used for logistic regression model construction, and data from 150 cats for validation. RESULTS: Patient mortality was 25.8%. Five- and 8-variable scores were developed. The 8-variable score contained mentation score, temperature, mean arterial pressure (MAP), lactate, PCV, urea, chloride, and body cavity fluid score. Area under the receiver operator characteristic curve (AUROC) on the construction cohort was 0.91 (95% CI, 0.87-0.94), and 0.88 (95% CI, 0.84-0.96) on the validation cohort. The 5-variable score contained mentation score, temperature, MAP, lactate, and PCV. AUROC on the construction cohort was 0.83 (95% CI, 0.79-0.86), and 0.76 (95% CI, 0.72-0.84) on the validation cohort. CONCLUSIONS AND CLINICAL IMPORTANCE: Two scores are presented enabling allocation of an accurate and user-friendly illness severity measure to hospitalized cats. Scores are calculated from data obtained over the 1st 24 hours after admission, and are diagnosis-independent. The 8-variable score predicts outcome significantly better than does the 5-variable score.

The acute patient physiologic and laboratory evaluation (APPLE) score: a severity of illness stratification system for hospitalized dogs.

BACKGROUND: Objective risk stratification models are used routinely in human critical care medicine. Applications include quantitative and objective delineation of illness severity for patients enrolled in clinical research, performance benchmarking, and protocol development for triage and therapeutic management. OBJECTIVE: To develop an accurate, validated, and user-friendly model to stratify illness severity by mortality risk in hospitalized dogs. ANIMALS: Eight hundred and ten consecutive intensive care unit (ICU) admissions of dogs at a veterinary teaching hospital. METHODS: Prospective census cohort study. Data on 55 management, physiological, and biochemical variables were collected within 24 hours of admission. Data were randomly divided, with 598 patient records used for logistic regression model construction and 212 for model validation. RESULTS: Patient mortality was 18.4%. Ten-variable and 5-variable models were developed to provide both a high-performance model and model maximizing accessibility, while maintaining good performance. The 10-variable model contained creatinine, WBC count, albumin, SpO(2) , total bilirubin, mentation score, respiratory rate, age, lactate, and presence of free fluid in a body cavity. Area under the receiver operator characteristic (AUROC) on the construction data set was 0.93, and on the validation data set was 0.91. The 5-variable model contained glucose, albumin, mentation score, platelet count, and lactate. AUROC on the construction data set was 0.87, and on the validation data set was 0.85. CONCLUSIONS AND CLINICAL IMPORTANCE: Two models are presented that enable allocation of an accurate and user-friendly illness severity index for dogs admitted to an ICU. These models operate independent of primary diagnosis, and have been independently validated.

Illness severity scores in veterinary medicine: what can we learn?

Illness severity scores are gaining increasing popularity in veterinary medicine. This article discusses their applications in both clinical medicine and research, reviews the caveats pertaining to their use, and discusses some of the issues that arise in appropriate construction of a score. Illness severity scores can be used to decrease bias and confounding and add important contextual information to research by providing a quantitative and objective measure of patient illness. In addition, illness severity scores can be used to benchmark performance, and establish protocols for triage and therapeutic management. Many diagnosis-specific and diagnosis-independent veterinary scores have been developed in recent years. Although score use in veterinary research is increasing, the scores available are currently underutilized, particularly in the context of observational studies. Analysis of treatment effect while controlling for illness severity by an objective measure can improve the validity of the conclusions of observational studies. In randomized trials, illness severity scores can be used to demonstrate effective randomization, which is of particular utility when group sizes are small. The quality of veterinary scoring systems can be improved by prospective multicenter validation. The prevalence of euthanasia in companion animal medicine poses a unique challenge to scores based on a mortality outcome.

Serum acute phase protein concentrations in dogs with autoimmune hemolytic anemia.

BACKGROUND: Serial monitoring of acute phase protein (APP) concentrations in canine autoimmune hemolytic anemia (AIHA) has not been reported. HYPOTHESES: Acute canine AIHA is accompanied by an acute phase response (APR) characterized by increased C-reactive protein (CRP) and alpha1-acid glycoprotein (AAG) concentrations and decreased albumin concentrations. ANIMALS: Twenty-seven dogs with AIHA and 11 control dogs. METHODS: Prospective, cohort study. CRP, AAG, and albumin concentrations, white blood cell (WBC) count, and packed cell volume (PCV) were determined at admission (day 1), every 48 hours until death or discharge, and on days 30, 90, 180, and 365. RESULTS: Compared with controls, CRP and AAG concentrations were increased and albumin concentration was decreased in dogs with AIHA (days 1-7; P < .002) and normalized with disease stabilization (days 9-365; P > .05). APP concentrations on day 1 were not predictive of survival, duration of hospitalization, or number of blood transfusions (P= .153-.940). PCV correlated with APP concentrations over time (CRP r=-.600, AAG r=-.665, albumin r= .533; P < .0001) as did WBC count (CRP r= .253, AAG r= .486, albumin r=-.246; P < .006). Day 1 CRP concentration was lower for dogs that received corticosteroids before referral (115.3 microg/mL) compared with dogs that did not (191.2 microg/mL; P= .02). CONCLUSIONS: An APR occurs in canine AIHA. Initial APP concentrations are not predictive of acute survival, correlate with hematologic markers of remission, and normalize rapidly with disease stabilization.

The effect of glucocorticoids on canine lymphocyte marker expression and apoptosis.

BACKGROUND: Glucocorticoids are commonly administered to dogs for the treatment of inflammatory disorders, autoimmunity and cancers such as lymphoma. Despite evidence of clinical efficacy, understanding of the effects of glucocorticoids on cells of the canine immune system is limited. HYPOTHESIS: Glucocorticoids affect the expression of phenotypic markers on canine lymphocytes and induce apoptosis. ANIMALS: Fifteen healthy mixed breed dogs. METHODS: Prospective randomized study. Prednisone was administered orally for 3 days, and cells aspirated from the popliteal lymph node before prednisone administration, and on days 1, 3, 10, 17, 24, and 38, were labeled with antibodies against canine CD3, CD4, CD8alpha, CD18, CD21, CD45, CD45RA, and CD90 molecules, and analyzed by flow cytometry. Additional samples were cultured in media with prednisolone for 24 hours and analyzed by cytometry for marker expression, and by gel electrophoresis for DNA fragmentation. RESULTS: Treatment of dogs with glucocorticoids resulted in reduced (p < or = .05) proportions of CD3 (days 1, 3, 17, and 24), CD4 (days 3 and 10), CD21 (day 1, 3, and 38), CD45RA (day 17) and CD90 (days 1, 10, and 17) expressing lymphocytes, and reduced intensity of CD18 (day 17) and CD45 (day 17 and 24) molecules on nodal lymphocytes. Culture oflymphocytes with prednisolone for 24 hours caused a significant reduction in the expression of all markers (p < or = .05) and DNA fragmentation. CONCLUSIONS AND CLINICAL IMPORTANCE: Glucocorticoids significantly alter the expression of phenotypic markers on canine lymphocytes, and in vitro induce apoptosis. These findings identify potential mechanisms for clinical immunosuppression from glucocorticoid treatment.

Flow cytometric immunophenotype of canine lymph node aspirates.

Increasing availability of reagents able to distinguish subtypes of lymphocytes and other leukocytes has enabled greater understanding of lymphocyte biology and pathology in the dog. Lymphocytes in circulation most commonly are subjected to immunophenotypic assessment by flow cytometry, but needle aspirates of lymph nodes can be similarly suitable for immunophenotypic examination. In this investigation, the feasibility of immunophenotyping samples obtained by needle aspiration of lymph nodes from 32 dogs with no physical abnormalities and 6 dogs with lymphoma was determined. In addition, samples from 6 dogs were stored overnight at 4 degrees C and reanalyzed 24 hours later. For each sample, stained smear preparations were examined microscopically for lymphocyte morphology, neoplasia, and the presence of inflammatory cells. Expression of antigens on a corresponding sample of aspirated cells was determined by flow cytometric detection of antibody binding on a minimum of 10,000 events. The distribution of data was determined with Anderson-Darling tests, and reference intervals incorporating the central 95% of values were established. Adequate samples were obtained from 30 of 32 clinically normal dogs. Immunophenotypic results after 24 hours of storage were consistent with those obtained immediately after sampling. Reference intervals for lymphocyte subsets from normal dog lymph nodes were similar to the proportions of CD3+, CD4+, CD8+, and CD21+ lymphocytes found in blood. Aspirates of enlarged lymph nodes from dogs with lymphoma were readily classified by this technique. Aspiration of lymph nodes from dogs for comprehensive analysis by flow cytometry is feasible and applicable to immunophenotyping of lymphoma.

The variability of serological and molecular diagnosis of feline immunodeficiency virus infection.

Diagnosis of feline immunodeficiency virus (FIV) infection by polymerase chain reaction (PCR) has recently become available, but little is known about the performance of this assay. The purpose of this study was to determine the sensitivity and specificity of PCR diagnosis of FIV infection. Replicate aliquots of blood samples from cats identified as FIV positive or negative by 2 previous enzyme-linked immunosorbent assay (ELISA) results, and from clinically healthy dogs, were submitted to different laboratories for FIV serologic diagnosis and PCR. The PCR products obtained in 1 laboratory were sequenced to determine the FIV subtype. The PCR assays correctly identified 100%, 80%, and 50% of the FIV-positive samples, and 100%, 90%, and 70% of FIV-negative samples. Each dog sample was reported as FIV PCR positive at least once, and FIV subtypes A, B, and C were identified. It was concluded that PCR tests currently available for FIV infection are unreliable, with highly variable sensitivity and specificity.

Telomerase activity in clinically normal dogs and dogs with malignant lymphoma.

OBJECTIVES: To determine whether telomerase activity was present in lymph nodes, buffy coat, and serum samples from dogs with malignant lymphoma (ML) and in liver, lymph node, buffy coat, and serum samples from clinically normal dogs SAMPLE POPULATION: Tissue specimens and blood samples were obtained from 11 clinically normal adult dogs (age range, 1 to 4 years) and 14 client-owned dogs with ML. PROCEDURE: The telomere repeat amplification protocol assay was used to quantify telomerase activity in the tissues from clinically normal dogs and dogs with ML. RESULTS: Of 11 clinically normal dogs, 8 had lymph node samples, 5 had liver samples, and 1 had buffy coat samples with detectable telomerase activity. None of the serum samples from the clinically normal dogs had detectable telomerase activity. Of 14 dogs with ML, 9 had lymph node samples, 3 had buffy coat samples, and 1 had serum samples with measurable telomerase activity. CONCLUSIONS AND CLINICAL RELEVANCE: Telomerase activity was not specific to tumor cells and overlapped with that found in cells from clinically normal dogs. Telomerase activity in neoplastic lymph nodes was not substantially different from that found in lymph nodes from clinically normal dogs. The determination of telomerase activity cannot be used as a sole diagnostic test for cancer. Therapeutic modalities directed toward the telomerase enzyme may not be feasible in dogs, because somatic tissues from clinically normal dogs possess variable amounts of telomerase activity.

The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs.

In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.

Large granular lymphocytic leukemia in a mixed breed dog.

A mixed breed dog was diagnosed with large granular lymphocytic leukemia. Immunophenotypic analysis indicated the lymphocytes were CD3+, CD8+ T cells expressing the alpha beta T cell receptor and a leukointegrin, alpha d. Chemotherapy and splenectomy resulted in an initial reduction in the lymphocyte count.

Gene therapy for canine alpha-L-iduronidase deficiency: in utero adoptive transfer of genetically corrected hematopoietic progenitors results in engraftment but not amelioration of disease.

Canine alpha-L-iduronidase (iduronidase) deficiency is a model of the human lysosomal storage disorder mucopolysaccharidosis type I (MPS I). We used this canine model to evaluate the therapeutic potential of hematopoietic stem cell (HSC) gene therapy for enzyme deficiencies. In previous studies, iduronidase-deficient dogs infused with autologous marrow cells genetically modified to express iduronidase had long-term engraftment with provirally marked cells, but there was no evidence of proviral iduronidase expression or clinical improvement. The presence of humoral and cellular immune responses against iduronidase apparently abrogated the therapeutic potential of HSC gene therapy in these experiments. To evaluate HSC gene therapy for canine MPS I in the absence of a confounding immune response, we have now performed in utero adoptive transfer of iduronidase-transduced MPS I marrow cells into preimmune fetal pups. In three separate experiments, 17 midgestation fetal pups were injected with 0.5-1.5 x 10(7) normal or MPS I allogeneic long-term marrow culture (LTMC) cells transduced with neo(r)- or iduronidase-containing retroviral vectors. Nine normal and three MPS I pups survived the neonatal period and demonstrated engraftment of provirally marked progenitors at levels of up to 12% for up to 12 months. However, the proportion of provirally marked circulating leukocytes was approximately 1%. Neither iduronidase enzyme nor proviral-specific transcripts were detected in blood or marrow leukocytes of any MPS I dog. Humoral immune responses to iduronidase were not detected in neonates, even after "boosting" with autologous iduronidase-transduced LTMC cells. All MPS I dogs died at 8-11 months of age from complications of MPS I disease with no evidence of amelioration of MPS I disease. Our results suggest that iduronidase-transduced primitive hematopoietic progenitors can engraft in fetal recipients, contribute to hematopoiesis, and induce immunologic nonresponsiveness to iduronidase in MPS I dogs. However, the therapeutic potential of HSC gene transfer in this model of iduronidase deficiency appears to be limited by poor maintenance of proviral iduronidase gene expression and relatively low levels of genetically corrected circulating leukocytes.

Genetically corrected autologous stem cells engraft, but host immune responses limit their utility in canine alpha-L-iduronidase deficiency.

Canine alpha-L-iduronidase (alpha-ID) deficiency, a model of the human storage disorder mucopolysaccharidosis type I (MPS I), is an ideal system in which to evaluate the clinical benefit of genetically corrected hematopoietic stem cells. We performed adoptive transfer of genetically corrected autologous hematopoietic cells in dogs with alpha-ID deficiency. Large volume marrow collections were performed on five alpha-ID-deficient dogs. Marrow mononuclear cells in long-term marrow cultures (LTMCs) were exposed on three occasions during 3 weeks of culture to retroviral vectors bearing the normal canine alpha-ID cDNA. Transduced LTMC cells from deficient dogs expressed enzymatically active alpha-ID at 10 to 200 times the levels seen in normal dogs. An average of 32% of LTMC-derived clonogenic hematopoietic cells were provirus positive by polymerase chain reaction and about half of these expressed alpha-ID. Approximately 10(7) autologous gene-modified LTMC cells/kg were infused into nonmyeloablated recipients. Proviral DNA was detected in up to 10% of individual marrow-derived hematopoietic colonies and in 0.01% to 1% of blood and marrow leukocytes at up to 2 to 3 years postinfusion. Despite good evidence for engraftment of provirally marked cells, neither alpha-ID enzyme nor alpha-ID transcripts were detected in any dog. We evaluated immune responses against alpha-ID and transduced cells. Humoral responses to alpha-ID and serum components of the culture media (fetal bovine and horse sera and bovine serum albumin) were identified by enzyme-linked immunosorbent assay. Cellular immune responses to autologous alpha-ID but not neo(r) transduced cells were demonstrated by lymphocyte proliferation assays. To abrogate potential immune phenomena, four affected dogs received posttransplant cyclosporine A. Whereas immune responses were dampened in these dogs, alpha-ID activity remained undetectable. In none of the dogs engrafted with genetically corrected cells was there evidence for clinical improvement. Our data suggest that, whereas the alpha-ID cDNA may be transferred and maintained in approximately 5% of hematopoietic progenitors, the potential of this approach appears limited by the levels of provirally derived enzyme that are expressed in vivo and by the host's response to cultured and transduced hematopoietic cells expressing foreign proteins.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses.

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

The effects of prednisone and azathioprine on circulating immunoglobulin levels and lymphocyte subpopulations in normal dogs.

This study investigates serum immunoglobulin (SIg) levels and lymphocyte subpopulations in normal dogs in response to putative immunosuppressive doses of prednisone and/or azathioprine. The objectives were to quantify SIg levels and lymphocyte subpopulations, including Thy-1+, CD4+, CD8+ and B cells, in normal dogs both before and after the administration of prednisone and/or azathioprine at 2 mg/kg, PO, each. Eighteen beagles were divided into 3 groups of 6 dogs each. Blood samples for radial immunodiffusion assay of IgG, IgM and IgA, complete blood count (CBC)and flow cytometry were collected prior to the administration of any drugs and again after 14 d of azathioprine, prednisone or azathioprine and prednisone. Peripheral blood mononuclear cells were isolated using density centrifugation and were incubated with monoclonal antibodies reacting with CD4+, CD8+, Thy-1+ and membrane immunoglobulin. Lymphocyte subsets were quantified using flow cytometry. Azathioprine-treated dogs had no significant changes in SIg levels or lymphocyte subpopulations. Prednisone-treated dogs had significant (P < 0.05) decreases in all SIg levels, all lymphocyte subpopulations and erythrocyte numbers, and had an increase in neutrophil counts. Prednisone and azathioprine-treated dogs had significant (P < 0.05) decreases in serum IgG levels and Thy-1+ and CD8+ lymphocyte subpopulations, with an increase in the CD4:CD8. These dogs also had a significant decrease in erythrocyte number and a significant increase in the monocyte count. These findings suggest that azathioprine and prednisone in combination or prednisone alone may be useful for the treatment of T cell-mediated diseases since decreased circulating T cell levels were demonstrated following treatment. The combination of drugs or azathioprine alone may not be appropriate for treatment of acute or autoantibody-mediated immune disease, because SIg levels were minimally affected by treatment.

Fractionation of Gene Modified Hematopoietic Autografts into Multiple Weekly Infusions Does Not Improve Engraftment in Unconditioned Canine Recipients.

Hematopoietic stem cell (HSC) gene therapy will require efficient transfer of genes to HSCs and long term engraftment and proliferation of genetically modified HSCs following adoptive transfer. We evaluated whether fractionation of grafts into 4-5 weekly infusions to non-myeloablated, autologous canine recipients would improve engraftment of genetically modified HSCs. Experimental animals and controls receiving a single infusion had similar levels of engraftment with ∼3-10% of marrow derived progenitors carrying transgene sequences for up to 29 months. There appears to be no improvement of engraftment of genetically modified HSCs in non-myeloablated large animal recipients by dose fractionation.

Blood vessel density in canine osteosarcoma.

Canine osteosarcoma is a prevalent bone neoplasm which has similarities to the human disease. We used a retrospective study to investigate the possibility that tumor vascularity may provide useful prognostic information, indicative of the role of this parameter in progression of this cancer. We quantified microvessel density in 52 histological specimens of primary tumor, immunostained for von Willebrand's Factor to identify vascular endothelium. For the 20 cases not euthanized at presentation or lost to follow-up, we found significantly higher tumor microvascular densities in animals presenting with detectable pulmonary metastases (5 of 20), and significantly lower densities in animals without metastatic disease at presentation, but later surviving to develop pulmonary metastases (7 of 20; P < 0.05). Animals with no evidence of pulmonary metastases at time of death (8 of 20) had intermediate vascular densities in their tumors. The results of this preliminary study suggest that vascularity of the primary tumor may be an indication of tumor progression. Future studies with a larger number of cases should establish whether vascular density can be a useful prognostic parameter for canine osteosarcoma.

A case-control study of verocytotoxigenic Escherichia coli infection in cats with diarrhea.

The objectives of this study were to determine the prevalence of enteric verocytotoxigenic E. coli (VTEC) infection in a population of cats in Ontario, and to determine whether an association exists between the presence of VTEC and feline diarrhea. Fecal samples from 179 cats, representing 113 cats with diarrhea and 66 cats with normal feces, were cultured for E. coli. The fecal cultures were screened for verocytotoxin activity with a Vero cell assay. Confirmation of the presence of verocytotoxin (VT) genes was done with polymerase chain reaction (PCR) amplification; the frequency of occurrence of the genes for generic VT, VT1, and VT2 was determined. VTEC-positive samples were defined as those that demonstrated cytotoxicity on the Vero cell assay and yielded E. coli possessing one or more of the VT genes. All VTEC-positive isolates were serotyped. The overall prevalence of enteric VTEC infection in the cats was 12.3% (22/179). Statistical analysis of the case-control data showed no significant association between VTEC infection and diarrheal illness. The majority of the cats with VT-positive E. coli were positive for the presence of the generic VT, rather than for VT1 or VT2; it is therefore possible that a novel verocytotoxin gene may exist in E. coli isolated from cats. Eight VTEC strains were identified by serotyping; 4 of these serotypes have previously been isolated from humans, and 2 from cattle, suggesting that cats may be capable of acting as reservoirs for human and bovine VTEC serotypes.