Pet dogs as sentinels for environmental contamination.

The presence of environmental contaminants in air, water and food may pose significant health risks to the exposed human population. However, problems associated with assessing chronic exposure to low doses of environmental chemicals, multiple exposure routes, diseases with long latency periods, and non-specific health outcomes make it difficult to conduct the appropriate human epidemiologic studies. It may be useful to complement human epidemiology with animal studies. Animals monitored or evaluated in situ for the appropriate suite of endpoints can provide information about both exposure levels and potential adverse health effects. Animals have served as sentinel indicators for health effects associated with a number of environmental exposures, including pesticides and asbestos. Pet dogs may be particularly valuable sentinels because they share the human environment. In addition, dogs respond to many toxic insults in ways analogous to humans, they have physiologically compressed life spans, and they are free from some important lifestyle risk factors for disease. An example of how pet dogs may be used as sentinels for potential human health hazards involves a study of the genotoxic effects resulting from exposure to a mixture of chemicals from nearby Superfund sites. We conducted a cross-sectional study of exposed dogs (living in the community with the Superfund sites) and controls (living in a nearby community). The pet owners completed a questionnaire, and we collected a blood sample from each dog. The blood samples were analyzed for standard clinical parameters and assays for possible genotoxic effects (peripheral blood lymphocyte micronucleus frequency and lymphocyte subtyping). Pet dogs living near the Superfund sites had a higher micronucleus frequency than control animals, suggesting that the dogs may have been exposed to environmental contaminants from these sites.

Platelet-associated immunoglobulin (antiplatelet antibody) in canine Rocky Mountain spotted fever and ehrlichiosis.

Antiplatelet antibodies were detected in the sera of dogs with naturally occurring and experimentally induced Rickettsia rickettsii and Ehrlichia canis infections. This is the first known report documenting elevated platelet-associated immunoglobulin (PAIg) titers in Rocky Mountain spotted fever (RMSF) infections. In the naturally occurring RMSF infections and ehrlichiosis, the antibodies persisted for weeks or months, even when the platelet counts had normalized. Results of this study indicate an immunological component for rickettsial thrombocytopenia. Therefore, current therapeutic recommendations, especially regarding avoiding the use of immunosuppressive drugs in patients with rickettsial diseases, need to be critically reviewed.

Evaluation of microcytosis in 18 Shibas.

OBJECTIVE: To determine whether microcytosis is a typical finding in Shibas. DESIGN: Prospective study. ANIMALS: 18 Shibas. PROCEDURE: Blood and serum samples were obtained for automated hematologic analyses (18 dogs) and for determination of ferritin concentration, using ELISA (14 dogs). Blood samples from 30 clinically normal dogs of various other breeds was analyzed to establish a reference range for ferritin concentration. RESULTS: Erythrocyte mean corpuscular volume in Shibas ranged from 55.6 to 69.1 fl (mean+/-SD, 61.2+/-4.3 fl; median, 60.6 fl; reference range, 63 to 73 fl). Microcytosis was identified in 12 of 18 dogs. Males and females were affected equally. Mean corpuscular hemoglobin concentration was slightly low (range, 32.0 to 33.9%; reference range, 34 to 38%) in 6 dogs, 4 of which had microcytic RBC. Serum ferritin concentrations ranged from 61.2 to 277.0 ng/ml (mean+/-SD, 110.6+/-51.4 ng/ml; median, 106 ng/ml). Reference range for serum ferritin concentration was 50.7 to 440.0 ng/ml (mean+/-SD, 121.2+/-67.1 ng/ml; median, 111.5 ng/ml). Thrombocytopenia (range, 110,000 to 196,000 platelets; reference range, 200,000 to 450,000 platelets) was found in 7 dogs, 6 of which also had microcytic RBC. CLINICAL IMPLICATIONS: Microcytosis can be a typical finding in Shibas. Common origin of Shibas and Akitas, a breed predisposed to microcytosis, suggests a hereditary basis for this finding.

Granulocytic ehrlichiosis in dogs from North Carolina and Virginia.

Medical records of 3 dogs from North Carolina and 3 dogs from Virginia with ehrlichial morulae in circulating neutrophils were studied retrospectively. Two clinically distinct disease syndromes, including chronic, moderate to severe anemia (n = 3) and polyarthritis (n = 2) were associated with canine granulocytic ehrlichiosis (CGE) in these dogs. One dog was clinically healthy, and abnormalities were not detected during physical examination. Clinical signs were nonspecific and included fever, lethargy, anorexia, vomiting, and diarrhea. The most frequent laboratory abnormalities were normocytic normochromic nonregenerative anemia, moderate thrombocytopenia with large platelets, lymphopenia, and eosinopenia. Considerable variability was found in the serologic responses to Ehrlichia equi, Ehrlichia canis, and Ehrlichia chaffeensis antigens among the 5 dogs for which stored sera were available for indirect fluorescent antibody testing. Polymerase chain reaction amplification and sequencing of portions of the 16S rRNA gene from blood (collected in ethylenediaminetetraacetic acid) of 1 severely anemic dog (dog 3) and 1 polyarthritic dog (dog 4) resulted in DNA sequences nearly identical to the GenBank accessions for Ehrlichia ewingii. The DNA sequence from a 3rd dog (dog 5) was most similar to that of E. canis. Serologic or molecular results support the possibility of E. ewingii, E. equi, and E. canis coinfection or serologic cross-reactivity among canine granulocytic and monocytic Ehrlichia species in dogs from North Carolina and Virginia. Variability in response to tetracycline or doxycycline treatment was noted in these dogs, with more rapid resolution of signs in dogs with polyarthritis. We report the 1st cases of CGE in dogs from North Carolina and Virginia, including recognition of CGE in a healthy dog.

Flow cytometric method for detecting thiazole orange-positive (reticulated) platelets in thrombocytopenic horses.

OBJECTIVE: To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. ANIMALS: 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-negative horses. PROCEDURE: Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms. RESULTS: Healthy ponies (n = 9) had 1.28 to 2.83% (mean +/- SD, 2.03 +/- 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 +/- 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41% (26.51 +/- 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 +/- 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 +/- 20,500 and 39,300 +/- 13,500 platelets/microliters, respectively (reference range, 94,000 to 232,000 platelets/ microliters). CONCLUSION: Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses. CLINICAL RELEVANCE: Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses.

Immunophenotypic characterization of canine lymphoproliferative disorders.

One hundred ninety six dogs with spontaneously occurring lymphoproliferative disorders were immunophenotyped. Dogs with lymphoma (175) were determined to be derived from B-cells in 134/175 (76%), T-cells in 38/175 (22%) and 3/175 (2%) were null cells (non-reactive with any canine-specific lymphocyte antibody). Dogs with T-cell lymphomas were at significantly higher risk of relapse and early death compared with B-cell lineage lymphoma following therapy (52 vs. 160 days; p < 0.001 and 153 vs. 330 days; p < 0.001, respectively). Hypercalcemia was associated only with CD4+ lymphomas. A nonimmunoglobulin B-cell marker (B5), expressed in 95% of nonneoplastic lymphocytes, was expressed at a reduced level in 63% (64/104) of dogs with B-cell lymphoma. Dogs with lymphoma in which the B5 antigen was expressed below normal levels experienced shorter progression free survival (125 vs. 202 days; p < 0.05) and overall survival times (203 vs. 385 days; p < 0.05) than dogs with B-cell lymphoma in which the B5 antigen was expressed normally. Chronic lymphocytic leukemia in dogs was primarily associated with a CD8+ phenotype (8/12) and acute lymphoblastic leukemia was determined to be of either null cell (4/9) or T-cell (3/9) phenotype. Although canine and human non-Hodgkin's lymphoma are phenotypically similar, canine leukemia is phenotypically distinct from human leukemia. The development of canine-specific probes has facilitated a priori assessment of treatment outcome in dogs with lymphoma and may in the future contribute to the comparative understanding of leukemo- and lymphoma-genesis in these species.

Prednisolone at anti-inflammatory or immunosuppressive dosages in conjunction with doxycycline does not potentiate the severity of Rickettsia rickettsii infection in dogs.

Dogs were experimentally inoculated with Rickettsia rickettsii to determine if anti-inflammatory or immunosuppressive dosages of prednisolone, when administered in conjunction with an antirickettsial antibiotic (doxycycline), induced therapeutically relevant pathophysiological consequences that ultimately influence disease outcome. Although the duration of rickettsemia was prolonged in dogs receiving immunosuppressive, but not anti-inflammatory, corticosteroids, concurrent administration of doxycycline and corticosteroids conferred no other detected detrimental effects. Treatment with doxycycline or doxycycline in conjunction with prednisolone resulted in decreased R. rickettsii-specific antibody titers; however, examination of appropriately timed acute- and convalescent-phase serum samples would have facilitated an accurate diagnosis of Rocky Mountain spotted fever (RMSF) in all 16 dogs. We conclude that the concurrent use of anti-inflammatory or immunosuppressive doses of prednisolone in conjunction with doxycycline, early in the course of experimental RMSF, confers no clinically relevant detrimental effects and that additional studies might be indicated to detect possible beneficial effects in cases of severe or potentially fulminant RMSF. However, because the illness induced in these dogs was of mild to moderate severity, the results of this study should definitely not be construed as supporting the safety or efficacy of prednisolone for treatment of severe canine or human RMSF.

Blood cell markers.

Cytochemistry and immunocytochemistry are important adjunctive technologies to the morphologic characterization of blood cells and hematopoietic neoplasms. Although cytochemistry is briefly discussed, the emphasis of this article is on the clinical application of flow cytometry and leukocyte monoclonal antibodies in veterinary medicine. Classification and significance of immunophenotyping in canine and feline lymphomas and immunodeficiencies such as feline immunodeficiency virus and feline leukemia virus are discussed.

Flow cytometric analysis of punctate and aggregate reticulocyte responses in phlebotomized cats.

Five cats were made anemic by one-time phlebotomy, and their reticulocyte responses were monitored daily for 20 days, using manual enumeration and a standardized feline reticulocyte protocol developed and validated in our laboratory. The reticulocyte responses of 38 clinically normal client-owned cats also were analyzed manually and cytometrically to determine clinical reference ranges. Increases in the percentage of aggregate reticulocytes over the reference range were detected in 5 of 5 phlebotomized cats, using the cytometric protocol. Only 4 of the 5 cats had an increase by results of manual enumeration. Manual aggregate counts had considerable daily variation and often fluctuated in and out of reference range, whereas cytometric aggregate counts remained consistently increased for distinct periods. Increased numbers of aggregate cells could also be detected for longer periods when evaluated by flow cytometry. Increased numbers of punctate reticulocytes were detected in 4 of 5 cats, using the cytometric protocol. None of the cats had increased numbers of punctate cells when evaluated by use of the manual technique. Aggregate reticulocytes in the 38 clinically normal cats ranged from 0.1 to 0.5%, which corresponded to 8,487 to 42,120 cells/microliter. Punctate reticulocytes ranged from 2 to 17%, which corresponded to 225,400 to 1,268,584 cells/microliter. Flow cytometry, using a standardized analysis protocol, was a more reliable and sensitive technique for detection and evaluation of feline reticulocytosis than was manual enumeration. The sensitivity of the flow cytometer to small amounts of intracellular nucleoprotein makes this assay especially valuable for detection of punctate reticulocytosis and low degrees of aggregate reticulocytosis in cats.

Erythrocyte fragility and chronic intermittent pigmenturia in a dog.

A 2-year-old spayed female Shetland Sheepdog had recurrent episodes of discolored urine. Treatments administered for presumed urinary tract infection did not prevent recurrence. Episodes of pigmenturia appeared to correlate with stressful situations or excessive activity. Examination of urine sediment consistently revealed that RBC were not evident, despite a positive result for blood on urinalysis. This was suggestive of hemoglobinuria, and diagnostic testing was instituted to determine the underlying cause. Results of alkaline and osmotic fragility tests were useful in determining that an increase in erythrocyte fragility was the underlying cause of the recurrent pigmenturia. Erythrocyte fragility testing should be considered in animals that do not respond to appropriate treatments for pigmenturia.

Thrombocytopenia associated with neoplasia in dogs.

Ten percent (214/2,059) of all dogs with cancer at North Carolina State University Veterinary Teaching Hospital had thrombocytopenia. The thrombocytopenia was associated with infectious/inflammatory etiologies in 4%, miscellaneous disorders (therapy, bone marrow failure, disseminated intravascular coagulation) in 35%, and neoplasia without identifiable secondary factors in 61% of cancer-bearing dogs. Classifying these dogs by tumor groups revealed the following proportionate ratios: lymphoid, 29%; carcinoma, 28%; sarcoma, 20%; hemic neoplasia, 7%; multiple, 5%; unclassified, 3%; benign, 3%; brain, 3%; and endocrine, 3%. Dogs with hemangiosarcoma, lymphoma, and melanoma were at increased risk of developing thrombocytopenia. Cytotoxic therapy was the major factor increasing the risk of thrombocytopenia in dogs with melanoma. Golden Retrievers were the only breed recognized with a predisposition to develop thrombocytopenia. If thrombocytopenia is identified in a dog with cancer, we recommend thorough evaluation of the coagulation system before surgery or therapy, and careful consideration of the risks and potential benefits of myelosuppressive or L-asparaginase therapy.

Thrombocytopenia in cats: a retrospective study of 41 cases.

The prevalence of feline thrombocytopenia (< 200,000 platelets/microL) at North Carolina State University, College of Veterinary Medicine Teaching Hospital, from January 1985 to March 1990, was 1.2% (41/3300). Cats were divided into six categories based on clinical diagnoses: 29% (12/41) had infectious disease, 20% (8/41) had neoplasia, 7% (3/41) had cardiac disease, 2% (1/41) had primary immune-mediated disease, 22% (9/41) had multiple diseases, and 20% (8/41) had disorders of unknown etiology. The mean platelet count for all thrombocytopenic cats was 52,000/microL +/- 46,000/microL (1 SD) with a range of 1000-190,000/microL. No significant differences were found between groups with respect to platelet count, packed cell volume, or white blood cell count, though anemia and leukopenia were common among the cats as a whole. Bleeding disorders (hemorrhage or thrombosis) were observed in 29% (12/41) of thrombocytopenic cats and were more likely to be associated with neoplasia, cardiac disease, and platelet counts less than or equal to 30,000/microL. Disseminated intravascular coagulopathy was diagnosed in 12% (5/41) of the cats. Infections and/or neoplasia affecting the bone marrow were the most common diseases associated with thrombocytopenia. Feline leukemia virus and myeloproliferative neoplasia accounted for approximately 44% (18/41) of the specific diagnoses in thrombocytopenic cats.

Human recombinant interleukin-2 induces maturation and activation signals for feline eosinophils in vivo.

Immunotherapy, with interleukin-2 (IL-2) or IL-2 plus lymphokine-activated killer (LAK) cells, has been used to treat cancer and acquired immunodeficiency syndrome (AIDS) in man. Similarities between feline leukemia virus (FeLV) infection in the cat and human immunodeficiency virus (HIV) infection in man have prompted immunotherapeutic studies in the cat. To develop baseline data on hematological responses to infused IL-2, cats were given daily (1-14 days) i.v. injections of 5 x 10(4) U/kg of recombinant human IL-2 (rHulL-2). Complete blood cell (CBC) counts were done weekly. Red blood cell (RBC), neutrophil, and lymphocyte numbers did not change appreciably over the course of the study. In contrast, rHulL-2 caused an eosinophilia in all but the 1 day treatment group. Treatment for 3 days generated a transient eosinophilia on day 7 that returned to baseline by 3 weeks. Five day and 7 day treatments generated an eosinophilia by day 7 that peaked on day 14 and returned to normal values by day 28. Treatment of cats for 14 days did not increase the magnitude or duration of the eosinophilia beyond the 5 or 7 day treatments. Bone marrow (BM) biopsies from rHulL-2-treated cats revealed a marked selective hyperplasia of eosinophil precursors. In the 5 day treatment group, all maturation stages of eosinophils were elevated by week 1 of treatment. By week 2, the early stages had returned to normal, whereas the late stage cells remained elevated, suggesting an ordered maturation response. Numbers of all eosinophil precursors approximated pretreatment numbers by weeks 3-4. Thus the BM hyperplasia preceded the blood eosinophilia by 1 week, suggesting that an enhanced maturation response of BM eosinophil precursors is a major contributor to the rHulL-2-induced blood eosinophilia. In addition to a maturation signal, rHulL-2 induces a potent activation signal for eosinophils as measured by a decrease in density and an increase in longevity in culture. The significance of the activated eosinophil in the therapeutic or toxicologic response to rHulL-2 infusion is discussed.

Peritoneal fluid values from healthy foals.

Peritoneal fluid was analysed from 17 foals, aged 13 to 134 days with a mean age of 68 days. Cytologically, the peritoneal fluid was characterised by a mean total cell count of 0.45 x 10(9)/litre (range 0.06 to 1.42 x 10(9)/litre), rare eosinophils, rare cytophagia and variable percentages of neutrophils and mononuclear cells. These data indicate that peritoneal fluid nucleated cell counts over 1.50 x 10(9)/litre in the foal should be interpreted as elevated. Biochemical evaluation revealed a mean biuret protein level of 12 g/litre, mean refractive index protein level of 16 g/litre and urea nitrogen concentration of 1.96 mmol/litre. There was no correlation between the foals' white blood cell and peritoneal fluid nucleated cell counts. Results of this study indicate that adult horse reference values for evaluation of peritoneal fluid are of questionable validity for foals. Diagnostically, the most important observation was that maximum peritoneal fluid nucleated cell counts in healthy foals were much lower than reported maximal reference values for adult horses (1.5 x 10(9)/litre versus 5.0 x 10(9)/litre or 10.0 x 10(9)/litre).

Risk factors for Haemobartonella felis infection in cats.

A seroepidemiologic survey for Haemobartonella felis infection in cats of Wake County, NC was undertaken. To help assess risk factors, cat owners completed a 10-item questionnaire. Additionally, blood samples were obtained for determination of H felis presence, FeLV infection, and anemia. Prevalence rates for H felis presence were as follows: all cats, 4.9% (6/123); healthy cats, 3.6% (3/83); and ill cats, 7.5% (3/40). The estimated relative risk for haemobartonellosis was also increased in cats with any of the following: anemia, FeLV-positive status, lack of vaccinations, history of catbite abscesses and/or anemia, age less than or equal to 3 years, or outdoor-roaming status. The sex, breed, number of cats in the household, or presence of fleas were not significant factors, although ill male cats had a greater estimated relative risk for haemobartonellosis.

Platelet aggregation in dogs experimentally infected with Rickettsia rickettsii.

Platelet aggregation studies were performed on nine Beagle dogs experimentally infected with Rickettsia rickettsia. Platelets from dogs with Rocky Mountain spotted fever tended to be more aggregable than controls.

Bone marrow biopsy and evaluation.

Bone marrow evaluation provides valuable diagnostic and prognostic information about neoplastic, metabolic, and inflammatory diseases. Bone marrow biopsies should be done only after examination of peripheral blood, to avoid performing unnecessary biopsies. A blood sample should be taken at the time of the bone marrow biopsy, for complete hematopoietic evaluation. It is preferable to take both an aspiration and core biopsy simultaneously. A good sample is mandatory for accurate evaluation and interpretation. The method of evaluation should be systematic, complete, and cover the following points: adequacy of specimens; estimation of cellularity; identification of number, maturation pattern, and morphology of megakaryocytes, myeloid cells, and erythroid cells; estimation of M:E ratio; and identification of abnormal cells, cellular reactions, infectious agents, or abnormal stromal reactions. Bone marrow findings should be interpreted in conjunction with signalment, history, physical findings, and laboratory results. Reference or institutional laboratories should be contacted for proper handling of bone marrow specimens for special procedures, such as histopathology, cytochemistry, immunopathology, and electron microscopy.

Cytogenetic analysis in nine leukaemic cats.

Eight of nine leukaemic cats had chromosomal abnormalities. The major differences between the healthy, FeLV-negative control cats and the leukaemic cats were the increased number of hyperdiploid cells and the presence of double minute or morphologically abnormal chromosomes in the leukaemic cats. Three leukaemic cats had cells with double minute chromosomes, while no normal cats had cells with double minute chromosomes. Tetrasomy or trisomy most frequently involved chromosomes C2 and B4. Monosomy was most common in chromosomes A3, C2, D4, E3 and F2. Chromosome abnormalities were also observed in bone marrow cells from two of three healthy, FeLV-positive cats and both cats with thymic lymphosarcoma. Increased numbers of hyperdiploid cells, double minute chromosomes and trisomy of C2 were the most important findings. No correlations between cytogenetic findings and diagnosis or prognosis were found. Additional research is necessary to determine the significance of chromosome aberrations in cells from healthy, FeLV-positive cats. If these changes prove to represent early neoplastic transformation, chromosome analysis could provide valuable diagnostic information and identify patients that might benefit from early chemotherapy. Results of this limited study indicate (1) chromosome aberrations are common in leukaemic cats, (2) chromosome changes are not completely random, (3) direct bone marrow technique for chromosome analysis is generally preferable in leukaemic cats and (4) the cat appears to be a good model in which to study chromosomal abnormalities in leukaemia, though more research is needed.