Diagnosis of rickettsial diseases in dogs and cats.

Rickettsial agents, including those in the genera Anaplasma, Ehrlichia, Neorickettsia, and Rickettsia, are important and common vector-borne pathogens of dogs and cats. Disease induced by these organisms ranges from clinically inapparent to severe and potentially fatal. However, laboratory confirmation of a rickettsial etiology can be complicated by a number of factors, including the wide spectrum of disease induced by these organisms, an often low and widely fluctuating level of organism present in infected animals, cross-reactions on serologic and molecular assays, and the presence of co-infections. Correct diagnosis is most likely to be reached when multiple diagnostic strategies, including careful microscopic examination of stained blood films or tissues, both specific and broad serologic tests, and a suite of molecular detection assays, are used in concert. Accurate interpretation of diagnostic tests requires awareness of the likelihood for multiple agents, including novel organisms, to be responsible for the results seen in a given patient. This review provides an overview of current strategies used to diagnose rickettsial infections in dogs and cats.

Sheep experimentally infected with a human isolate of Anaplasma phagocytophilum serve as a host for infection of Ixodes scapularis ticks.

Anaplasma phagocytophilum, first identified as a pathogen of ruminants in Europe, has more recently been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. A. phagocytophilum is transmitted by Ixodes spp., but the tick developmental cycle and pathogen/vector interactions have not been fully described. In this research, we report on the experimental infection of sheep with the human NY-18 isolate of A. phagocytophilum which then served as a host for infection of I. scapularis nymphs and adults. A. phagocytophilum was propagated in the human promyelocytic cell line, HL-60, and the infected cell cultures were then used to infect sheep by intravenous inoculation. Infections in sheep were confirmed by PCR and an Anaplasma-competitive ELISA. Clinical signs were not apparent in any of the infected sheep, and only limited hematologic and mild serum biochemical abnormalities were identified. While A. phagocytophilum morulae were rarely seen in neutrophils, blood film evaluation revealed prominent large granular lymphocytes, occasional plasma cells, and rare macrophages. Upon necropsy, gross lesions were restricted to the lymphoid system. Mild splenomegaly and lymphadenomegaly with microscopic evidence of lymphoid hyperplasia was observed in all infected sheep. Female I. scapularis that were allowed to feed and acquire infection on each of the 3 experimentally infected sheep became infected with A. phagocytophilum as determined by PCR of guts (80-87%) and salivary glands (67-100%). Female I. scapularis that acquired infection as nymphs on an experimentally infected sheep transmitted A. phagocytophilum to a susceptible sheep, thus confirming transstadial transmission. Sheep proved to be a good host for the production of I. scapularis infected with this human isolate of A. phagocytophilum, which can be used as a model for future studies of the tick/pathogen interface.

Canine intravascular lymphoma with overt leukemia.

A 6-year-old spayed Labrador Retriever Mix dog was evaluated for a 2-week history of progressive generalized weakness and reluctance to stand. Physical examination revealed severe weakness with obtunded mentation, head tilt, bilateral nystagmus, and decreased vision. CBC findings included mild nonregenerative anemia, marked thrombocytopenia, and a few atypical mononuclear cells on the blood film. The cells were 15-30 µm in diameter and had round to oval to reniform centrally placed nuclei with stippled chromatin, prominent nucleoli, and abundant basophilic cytoplasm with numerous discrete vacuoles and, occasionally, small azurophilic granules. Similar cells were found in bone marrow. On histologic examination of tissues collected at necropsy, neoplastic cells were detected in bone marrow, hepatic sinusoids, cerebral and meningeal vessels, and in capillaries of the heart, renal interstitium, small intestinal submucosa, and muscularis, and alveolar septa. A small discrete mass in the right atrium consisted of similar neoplastic cells, and the spleen was diffusely infiltrated. Tissue distribution was suggestive of intravascular lymphoma. Neoplastic cells in tissue sections were immunoreactive for vimentin, CD18, CD45, and granzyme B and lacked immunoreactivity for cytokeratin. Neoplastic cells on bone marrow aspirate smears and blood films lacked immunoreactivity for CD3, CD79a, CD1c, CD11b, CD11c, CD11d, and E-cadherin. In the absence of immunophenotypic evidence for the neoplastic cells being derived from B-cell, T-cell, or histocytic/dendritic lineages and the lack of clonal antigen receptor gene rearrangement(s), along with positive immunoreactivity for granzyme B, a tumor of NK cells was considered likely. Based on current knowledge, this is the first report of canine intravascular lymphoma, of probable NK cell origin, with peripheral blood involvement.

Babesia canis rossi infection in a Texas dog.

A 5-month-old intact male Boerboel dog, imported from South Africa 1 week previously, was presented to a Texas veterinarian for lethargy, anorexia, and labored breathing. The dog was febrile, anemic, leukopenic, thrombocytopenic, and slightly azotemic. Results of the IDEXX SNAP-4Dx enzyme immunoassay were negative for Dirofilaria immitis antigen and antibodies against Ehrlichia canis, Borrelia burgdorferi, and Anaplasma phagocytophilum. An EDTA blood sample analyzed at Oklahoma State University Center for Veterinary Health Sciences revealed nonregenerative anemia, neutropenia, and large protozoal piroplasms in 0.7% of the RBCs. Piroplasms were 2-5µm long and varied in shape from round to oval to piriform; extracellular merozoites were also observed. Nested PCR was performed on DNA extracted from blood using primers that amplify the 18s rRNA gene from all known Babesia species, and the product was sequenced. Basic Local Alignment Search Tool analysis of the 437 base sequence revealed 99-100% similarity to Babesia canis rossi, 92-93% similarity to Babesia canis canis, and 92% similarity to Babesia canis vogeli. The dog responded well to treatment with imidocarb. PCR analysis of a second blood sample 2 weeks later was negative for Babesia spp. DNA. This case represents the first diagnosis of B. canis rossi infection in the United States.

Congenital Pelger-Huët anomaly in a Danish/Swedish Farmdog: case report.

A 13 year old Danish/Swedish Farmdog from Denmark was evaluated in a veterinary clinic in Warsaw, Poland for evaluation of an orthopedic problem. Radiographs revealed spondylosis and degenerative vertebral disease, which responded to treatment with anti-inflammatory medications. A predominance of hyposegmented neutrophils and eosinophils containing condensed chromatin and normal cytoplasm were identified on a routine CBC. Follow-up blood film evaluations over the course of 12 months confirmed that the hyposegmented granulocytes persisted. The majority of neutrophils contained Grade 2 nuclei (slightly indented), and the mean nuclear score varied from 1.9 to 2.3. Pelger-Huët anomaly (PHA), presumably congenital, was diagnosed based on persistent hyposegmented granulocytes in the absence of an underlying cause for acquired PHA; genetically related dogs were unavailable for testing to confirm vertical transmission. To the authors' knowledge this is the first report of PHA in a Danish/Swedish Farmdog.

Appearance of granulated cells in blood films stained by automated aqueous versus methanolic Romanowsky methods.

BACKGROUND: Romanowsky stains are used routinely by veterinary clinical pathology laboratories for cytologic and blood film evaluations. Automated stainers are available for both aqueous and methanolic Romanowsky stains. Mast cell granules and canine distemper virus inclusions are known to stain differently by these 2 methods, but we have noticed differences in the staining characteristics of other granulated cells. OBJECTIVE: The aim of this study was to investigate and document the variable appearance of basophils and large granular lymphocytes in blood films stained using aqueous and methanolic Romanowsky methods. METHODS: Cytologic preparations from 1 canine mast cell tumor and blood films from 8 dogs, 1 cat, 1 rabbit, and 1 ostrich were stained using an automated aqueous stain (Aerospray 7120, with and without a predip fixative) and an automated methanolic stain (Hematek). Staining quality and intensity of the cytoplasmic granules in mast cells, basophils, and large granular lymphocytes was evaluated subjectively. RESULTS: Cytoplasmic granules of mast cells, basophils, and large granular lymphocytes stained poorly or not at all with the automated aqueous stain but stained prominently and were readily identified with the automated methanolic stain. Use of the predip fixative with the Aerospray method improved the visibility of basophil granules but not mast cell granules, and had a variable affect on the visibility of granules in large granular lymphocytes. CONCLUSION: Clinical pathologists should be aware of the staining methodology used on the slides they evaluate to avoid incorrect interpretation of granulated cell populations.

Dendritic cell leukemia in a Golden Retriever.

An 8-year-old castrated male Golden Retriever was evaluated for decreased appetite, lethargy, and labored breathing of 1-week duration. Bilateral pulmonary infiltrates, hepatomegaly, and splenomegaly were present. Results of a CBC revealed marked leukocytosis (62,600/microL; reference interval 4000-15,500/microL) and large numbers of atypical cells (30,700/microL) with abundant cytoplasm. There was no concurrent anemia, neutropenia, or thrombocytopenia. Morphology of the atypical cells was most consistent with a histiocytic origin. Similar cells were identified in bone marrow aspirates, and were morphologically suggestive of the macrophage variant of disseminated histiocytic sarcoma. However, flow cytometry of the abnormal circulating cells revealed CD1c, CD11c, and major histocompatibility complex (MHC) Class II expression without expression of CD11d or lymphoid markers, consistent with myeloid dendritic antigen-presenting cells. At necropsy, the splenic architecture was effaced by neoplastic histiocytes that were also infiltrating lung, liver, an abdominal lymph node, myocardium, an bone marrow. Immunohistochemistry of the splenic neoplastic cells confirmed dendritic cell origin (CD1c+, CD11c+, MHC II+, no expression of CD11d and lymphoid markers). To the authors' knowledge, this is the first report of canine dendritic cell leukemia-in this instance accompanied by marked tissue infiltration.

Canine distemper virus strains circulating among North American dogs.

Canine distemper virus (CDV) is a highly contagious virus that causes multisystemic disease in dogs. We received seven samples from dogs with CD from the United States during 2007. CDV isolates from these samples formed large, multinucleated syncytia in a Vero cell line expressing canine signaling lymphocyte activation molecule (SLAM). Based on the hemagglutinin gene sequences, the CDV isolates from three states (California, Missouri, and Oklahoma) formed two CDV genetic groups: group I (major; six of seven isolates) consisted of CDV isolates closely related to the European wildlife lineage of CDV, and group II (minor; one of seven isolates) was genetically related to the Arctic-like lineage of CDV. However, both CDV groups were genetically different from the current vaccine strains that belong to the American-1 lineage of the old (1930 to 1950) CDV isolates.

Evaluation of the ruminant complete blood cell count.

The complete blood cell count can provide valuable diagnostic and prognostic information when coupled with a thorough physical examination. This article addresses proper sample handling, storage, and interpretation of the erythron, serum iron indices, leukon, and acute-phase proteins for cattle, sheep, and goats.

Sample collection and handling: getting accurate results.

Results of many routine laboratory assays supply important diagnostic information and are an important part of patient care in many situations. Ensuring the accuracy of these results is not only important from a diagnostic standpoint but can prevent the frustration inherent when the effort of collecting and submitting samples does not yield interpretable results. This article discusses some of the routinely encountered problems (and how to avoid them) associated with performing the more commonly requested tests: complete blood cell counts, chemistry profiles, coagulation testing, and cytology specimens. The article presents a general discussion of sample collection and handling and then some specific considerations for the handling of the previously mentioned tests.

Hematology without the numbers: in-clinic blood film evaluation.

Technical advances have made it possible for many private veterinary practices to purchase reasonably priced automated hematology instruments to perform in-clinic blood analyses. Although these instruments can quickly provide "numbers" to the clinician, evaluation of a well-made blood film can often provide information critical to the interpretation of those numbers. Blood film review is essential to identify important abnormalities such as neutrophilic left shifts and toxic change, neoplastic cells, hemoparasites, and erythrocyte morphologic changes that may suggest the cause of an anemia. Additionally, the blood film provides an important quality control measure for the automated hematology results. This article outlines a simple method of blood film evaluation, highlights the most common clinically important abnormalities, and reinforces the importance of blood film evaluation as a quality control measure.

Comparison of core needle biopsy and fine-needle aspiration of enlarged peripheral lymph nodes for antemortem diagnosis of enzootic bovine lymphosarcoma in cattle.

OBJECTIVE: To determine whether antemortem core needle biopsy and fine-needle aspiration of enlarged peripheral lymph nodes could be used to distinguish between inflammation and lymphosarcoma in cattle. DESIGN: Prospective study. ANIMALS: 25 cattle with enlarged peripheral lymph nodes. PROCEDURES: Antemortem biopsies of the selected lymph nodes were performed with an 18-gauge, 12-cm core needle biopsy instrument. Fine-needle aspirates were performed with a 20-gauge, 4-cm needle. Specimens were analyzed by pathologists who were unaware of clinical findings and final necropsy findings, and specimens were categorized as reactive, neoplastic, or nondiagnostic for comparison with necropsy results. RESULTS: Sensitivity and specificity of core needle biopsy ranged from 38% to 67% and from 80% to 25%, respectively. Sensitivity of fine-needle aspiration ranged from 41% to 53%, and specificity was 100%. Predictive values for positive test results ranged from 77% to 89% for core needle biopsy and were 100% for fine-needle aspiration. Predictive values for negative test results were low for both core needle biopsy and fine-needle aspiration. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that core needle biopsy and fine-needle aspiration can aid in the antemortem diagnosis of bovine enzootic lymphosarcoma. Results of fine-needle aspiration of enlarged peripheral lymph nodes were more specific and more predictive for a positive test result than were results of core needle biopsy.

Intracellular success: cytologic findings in an ulcerated submandibular mass from a cat.

A 1-year-old neutered male domestic shorthair cat had an ulcerated, proliferative lesion in the submandibular area that did not respond to antibiotic therapy. Impression smears from the mass revealed septic pyogranulomatous inflammation, with large numbers of pleomorphic bacteria observed intracellularly within macrophages as well as neutrophils. Bacterial culture was consistent with a diagnosis of Rhodococcus equi, a facultative intracellular coccobacillus capable of replicating within macrophages. The cat's lesion resolved after treatment with rifampin and clarithromycin. R equi should be considered as a differential diagnosis when coccobacilli are recognized within macrophages in cytologic samples.

Covert vertical transmission of feline immunodeficiency virus.

Covert vertical transmission of feline immunodeficiency virus (FIV), the feline counterpart of human immunodeficiency virus type 1 (HIV-1), was identified in kittens born to FIV-infected cats. DNA PCR detected FIV gag and env sequences in tissues from kittens nonviable at birth, and in viable kittens monitored postnatally and necropsied at either 11 weeks or 1 year of age. Although FIV DNA was detected in initial blood samples from all 16 viable kittens, viral DNA became increasingly difficult to detect over time and infectious virus could rarely be demonstrated. Only maternal FIV antibody was detected in kitten plasma during the entire postnatal observation period, and kittens remained healthy, with normal CD4:CD8 T cell ratios at >14 months of age. Thus, mother-to-offspring FIV exposure, occurring in utero and postnatally, can result in covert infection in kittens with virus sequestered and contained in tissue sites. These findings appear directly relevant to suspected transient HIV infections and reports of HIV-specific cellular immune responses in highly exposed seronegative adults and uninfected infants born to HIV-positive women.

Feline immunodeficiency virus is concentrated in milk early in lactation.

We studied mother-to-offspring transmission of feline immunodeficiency virus (FIV), focusing on milk-borne virus transmission in order to assess its similarities to perinatal HIV transmission. We also attempted to evaluate the influence of intragestational treatment with 9-[2-(phosphono-methoxy)-propyl]adenine (PMPA) on virus transmission to offspring. Eleven female cats (queens), chronically infected with FIV-B-2542 and bred to an FIV-negative male, produced a total of 25 viable and 18 nonviable term kittens. Overall, the vertical transmission rate by untreated queens was 22%, similar to that for HIV, which unfortunately precluded adequate assessment of PMPA efficacy. However, at delivery 9 of 10 queens (90%) had higher viral RNA loads in milk (4 x 10(4) to 4 x 10(8) viral copies/ml) than in plasma (5 x 10(3) to 2.5 x 10(6) viral copies/ml). Conversely, 10 of 11 queens (91%) had lower proviral loads in milk cells (0 to 10(2) proviral copies/microg DNA) than blood cells (10(2) to 10(4) proviral copies/microg DNA). Thus, FIV is concentrated in early milk despite relatively low proviral loads in milk cells, suggesting that virus may be actively secreted by the mammary gland for dissemination to offspring. FIV provides a model for the study of milk-borne lentivirus transmission and assessment of strategies to reduce postnatal HIV vertical transmission.