Complete Blood Cell Count and White Blood Cell Counting Method Comparison in 49-day-old Bobwhite Quail (Colinus virginianus).

The northern bobwhite quail (Colinus virginianus) is a game bird experiencing decline throughout much of its range. There are limited species-specific and age-specific hematologic data for bobwhite quail. Complete blood cell counts in eighteen 49-day-old captive-raised quail of unverified sex were used to contribute data to reference intervals for the species as juvenile animals. Values for packed cell volumes, total solids, red and white blood cell counts, and white cell differential calculations were recorded for each animal. Bobwhite quail of this age were found to be primarily lymphocytic, but considerable variability was observed. White blood cell counts were obtained by estimates from blood smears and Phloxine B stain. White blood cell counts performed with ×1000 magnification did not compare well with other white blood cell methods, including Phloxine B stain.

Efficacy of an imidacloprid 10 % / flumethrin 4.5 % collar (Seresto®, Bayer) for preventing the transmission of Cytauxzoon felis to domestic cats by Amblyomma americanum.

Infection of Cytauxzoon felis in domestic cats produces a severe disease characterised by fever, lethargy, inappetence, anorexia, depression, dehydration, icterus and often death. Transmission of C. felis to cats is dependent on being fed upon by infected Amblyomma americanum (lone star ticks). The purpose of the present study was to determine if application of a 10 % imidacloprid/4.5 % flumethrin collar (Seresto®, Bayer) on cats prevents transmission of C. felis by repelling ticks. Twenty cats were randomised to either a treated (n = 10) or non-treated control group (n = 10) based on their susceptibility to ticks. Cats of high, medium and low tick susceptibility were represented in both groups. Treated cats were fitted with 10 % imidacloprid/4.5 % flumethrin collars on study day 0 and both groups were then infested with C. felis-infected A. americanum on study day 30. Tick thumb counts were performed at 24 and 48 hours post infestation. Transmission of C. felis was determined by examining blood of cats by DNA extraction followed by PCR amplification with piroplasm-specific primers. Ticks did not attach to any of the 10 % imidacloprid/4.5 % flumethrin- treated cats. However, ticks attached and fed on all the non-treated control cats. The geometric mean number of ticks attached to the non-treated control cats at 24 and 48 hours was 15.3 and 14.2, respectively. Cytauxzoon felis was transmitted to 9 of 10 (90 %) non-treated control cats; C. felis was not transmitted to any of the treated cats. Transmission of C. felis to the non-treated cats was first detected between 8 and 16 days post infestation. Our results indicate that application of the 10 % imidacloprid/4.5 % flumethrin collar to cats prevented ticks from attaching, feeding and transmitting C. felis.

A Novel Vaccine Strategy to Prevent Cytauxzoonosis in Domestic Cats.

Cytauxzoonosis is caused by Cytauxzoon felis (C. felis), a tick-borne parasite that causes severe disease in domestic cats in the United States. Currently, there is no vaccine to prevent this fatal disease, as traditional vaccine development strategies have been limited by the inability to culture this parasite in vitro. Here, we used a replication-defective human adenoviral vector (AdHu5) to deliver C. felis-specific immunogenic antigens and induce a cell-mediated and humoral immune response in cats. Cats (n = 6 per group) received either the vaccine or placebo in two doses, 4 weeks apart, followed by experimental challenge with C. felis at 5 weeks post-second dose. While the vaccine induced significant cell-mediated and humoral immune responses in immunized cats, it did not ultimately prevent infection with C. felis. However, immunization significantly delayed the onset of clinical signs and reduced febrility during C. felis infection. This AdHu5 vaccine platform shows promising results as a vaccination strategy against cytauxzoonosis.

Cytauxzoonosis in North America.

Cytauxzoonosis is an emerging tick-borne disease of domestic and wild felids produced by infection of Cytauxzoon felis, an apicomplexan protozoan similar to Theileria spp. Transmitted by Amblyomma americanum, lone star tick, and Dermacentor variabilis, American dog tick, infection of C. felis in cats is severe, characterized by depression, lethargy, fever, hemolytic crisis, icterus, and possibly death. Cytauxzoonosis occurs mainly in the southern, south-central, and mid-Atlantic United States in North America, in close association with the distribution and activity of tick vectors. Infection of C. felis, although severe, is no longer considered uniformly fatal, and unless moribund, every attempt to treat cytauxzoonosis cats should be made. Herein we review cytauxzoonosis, including its etiology, affected species, its life cycle and pathogenesis, clinical signs, diagnosis, and epidemiology, emphasizing clinical pathology findings in cats infected with this important emerging tick-borne disease in North and South America.

Confirmation of Amblyomma americanum (Acari: Ixodidae) as a vector for Cytauxzoon felis (Piroplasmorida: Theileriidae) to domestic cats.

Amblyomma americanum was confirmed as a competent vector in the transmission of Cytauxzoon felis to domestic cats. Infection with C. felis was produced and replicated in four domestic felines by the bite of A. americanum adults that were acquisition fed as nymphs on a domestic cat that survived cytauxzoonosis. Numerous attempts to transmit C. felis with Dermacentor variabilis at the same time were not successful. All cats upon which infected A. americanum were transmission fed exhibited disease typical of cytauxzoonosis, and the eitiologic agent's presence was confirmed. Clinical signs including fever, inappetence, depression, and lethargy were observed beginning 13 d postinfestation. Pale mucus membranes, splenomegaly, icterus, and dyspnea were also observed during the course of the disease. Rectal temperatures of the C. felis-infected principal cats fluctuated from high to subnormal before returning to the normal range. Clinical signs of cytauxzoonsis improved by 24 d postinfestation in all but one cat, with survivors remaining parasitemic and subclinically infected with C. felis. Unengorged A. americanum and D. variabilis were collected from wild habitats to determine the minimum infection rate of C. felis in ticks from an enzootic area. Infection of C. felis was found only in wild-collected A. americanum. The minimum infection rate of C. felis in A. americanum was 0.5% (one of 178) in males, 0.8% (three of 393) in nymphs, and 1.5% (three of 197) in females. We found no wild-collected D. variabilis infected with C. felis. Our results confirm that A. americanum is a primary vector of C. felis.

Transmission of Cytauxzoon felis to a domestic cat by Amblyomma americanum.

Cytauxzoon felis was transmitted to a domestic cat by Amblyomma americanum. The infection was produced by the bite of A. americanum adults that were acquisition fed as nymphs on a domestic cat that naturally survived infection of C. felis. Fever, inappetence, depression, and lethargy were first noted 11 days post-infestation (dpi). Pale mucus membranes, splenomegaly, icterus, and dyspnea were also observed during the course of the disease. The body temperature of the experimentally infected C. felis cat was subnormal from 16 dpi until 24 dpi when it returned to within normal limits. All clinical signs of cytauxzoonsis began to resolve by 23 dpi when the cat became subclinically infected with C. felis. The cat developed a marked, regenerative anemia beginning by 13 dpi and reached a nadir at 20 dpi before recovering. A moderate neutrophilia and marked lymphocytosis also developed between 18 and 26 dpi. Schizonts of C. felis were observed in spleen aspirates of the infected cat at 15 dpi. DNA of C. felis was amplified by real-time PCR starting 17 dpi and piroplasms of C. felis were first noted by light microscopy 18 dpi. Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus were also tested in a similar manner at the same time but did not transmit C. felis. Prior to the present study, only D. variabilis had been shown experimentally to transmit infection of C. felis. This is the first report of C. felis being transmitted by A. americanum. The transmission of C. felis infection from one domestic cat to another indicates that domestic cats subclinically infected with C. felis may be a reservoir of infection for naive domestic cats.

Detection of Babesia gibsoni and the canine small Babesia 'Spanish isolate' in blood samples obtained from dogs confiscated from dogfighting operations.

OBJECTIVE: To determine the prevalence of Babesia gibsoni infection in dogs that were confiscated from dogfighting operations. DESIGN: Cross-sectional study. ANIMALS: 157 pit bull-type dogs that were confiscated as part of dogfighting prosecution cases in Iowa, Michigan, Mississippi, Ohio, Pennsylvania, Virginia, and Washington and 218 randomly selected animal shelter dogs with no known history of dogfighting. PROCEDURES: Blood samples collected from confiscated dogs were tested for infection with B gibsoni by use of a nested PCR assay. Samples that yielded positive results underwent DNA sequencing to confirm infection with B gibsoni. Control blood samples collected from 218 randomly selected dogs in animal shelters (ie, dogs that had no known involvement in dogfighting events) were also analyzed. RESULTS: Results of nested PCR assays indicated that 53 of 157 (33.8%) confiscated dogs were infected with B gibsoni; 1 (0.6%) dog was infected with the canine small Babesia 'Spanish isolate' (also known as Theileria annae). To the authors' knowledge, this is the first report of infection with this small Babesia 'Spanish isolate' in a North American dog. Dogs with scars (indicative of fighting) on the face, head, and forelimbs were 5.5 times as likely to be infected with B gibsoni as were dogs without scars. Of the control dogs, 1 (0.5%) pit bull-type dog was infected with B gibsoni. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that B gibsoni is a common parasite of dogs confiscated from dogfighting operations and suggested that dogs with a history of fighting should be evaluated for infection with B gibsoni.

Human Mast Cell Development from Hematopoietic Stem Cells in a Connective Tissue-Equivalent Model.

Mast cells (MCs) play critical roles in the pathogenesis of IgE- and non-IgE-mediated immune responses, as well as host defense against parasites, bacteria, and viruses. Due to the effect of extracellular matrix components on tissue morphogenesis and cell behavior, utilizing a tissue model that mimics MC microenvironmental conditions in vivo has greater relevance for in vitro studies. For this work, MCs were developed within a connective tissue-equivalent model and cell function was examined in response to an allergen. MCs are located in proximity to fibroblasts and endothelial cells (ECs) that play a role in MC development and maturity. Accordingly, MC progenitors isolated from human peripheral blood were co-cultured with human primary fibroblasts in a 3D collagen matrix to represent the connective tissue. The matrix was coated with type IV collagen and fibronectin before seeding with primary human ECs, representing the capillary wall. The stem cell-derived cells demonstrated MC characteristics, including typical MC morphology, and the expression of cytoplasmic granules and phenotypic markers. Also, the generated cells released histamine in IgE-mediated reactions, showing typical MC functional phenotype in an immediate-type allergenic response. The created tissue model is applicable to a variety of research studies and allergy testing. Impact Statement Mast cells (MCs) are key effector and immunoregulatory cells in immune disorders; however, their role is not fully understood. Few studies have investigated human ex vivo MCs in culture, due to the difficulties in isolating large numbers. Our study demonstrates, for the first time, the generation of cells exhibiting MC phenotypic and functional characteristics from hematopoietic stem cells within a connective tissue-equivalent model with ancillary cells. Utilizing the 3D matrix-embedded cells can advance our understanding of MC biological profile and immunoregulatory roles. The tissue model can also be used for studying the mechanism of allergic diseases and other inflammatory disorders.

Feline histoplasmosis presenting with bone and joint involvement: clinical and diagnostic findings in 25 cats.

OBJECTIVES: The aim of this study was to describe clinical and diagnostic findings in cats with bone and joint disease associated with histoplasmosis. METHODS: Medical records from between 2011 and 2017 were reviewed. Inclusion criteria required: (1) diagnosis of histoplasmosis by cytology, histology, urine or serum Histoplasma antigen testing, or culture; and (2) lameness or joint effusion as a presenting complaint or physical examination finding. RESULTS: Twenty-five cases met the inclusion criteria. Four had incomplete records, but available data were included when applicable. Lameness was a presenting complaint in 17/21 cats and was the only complaint in 9/21 cats. Initial diagnosis was made by cytology in 22/25 cats and by culture, urine antigen and necropsy in one case each. Diagnostic cytology samples included synovial fluid (n = 13), lymph node (n = 5), skin (n = 2), lung (n = 1) and bone (n = 1). Two additional cases had synovial fluid examined but no organisms present. Inflammation was present in all synovial fluid samples examined. Biopsy was obtained in two cats and histologic diagnoses included osteomyelitis with no infectious organisms identified and severe lymphoplasmacytic synovitis suggestive of feline periosteal proliferative polyarthritis. Histoplasma urine antigen test was positive in 7/12 cats. CONCLUSIONS AND RELEVANCE: Inflammatory arthritis is common in cats with histoplasmosis, with lameness a common presenting complaint. Organisms are found in synovial fluid cytology in most cases. If not, appropriate additional diagnostics must be pursued.

Metastatic disease in a dog with a well-differentiated perianal gland tumor.

Fine-needle aspirates from a perianal mass on an 8-year-old, intact male, Miniature Poodle presenting for tenesmus showed a uniform population of well-differentiated hepatoid cells with no notable criteria of malignancy. The cytologic diagnosis was a perianal gland tumor, with adenoma likely given the cytomorphology. The abdominal ultrasound revealed multiple, markedly enlarged, intra-abdominal lymph nodes. LN aspirates also showed well-differentiated polygonal, hepatoid cells displaying no notable cellular atypia. The presence of the metastasis led to the interpretation of a well-differentiated, malignant perianal gland tumor despite the benign cellular appearance. Histopathology of the surgically excised perianal mass and one enlarged abdominal lymph node revealed lobules of uniform polygonal hepatoid cells arranged in organized islands and trabeculae surrounded by a single layer of uniform reserve cells. Few mitotic figures were present. The only histopathologic indication of malignancy within the primary mass was the presence of small islands of well-differentiated hepatoid cells infiltrating into adjacent tissue and possible lymphatic invasion. The histopathologic diagnosis was perianal gland adenocarcinoma. Most textbooks describe perianal gland adenocarcinomas as showing increased cellular atypia including pleomorphism, disorganization of hepatoid cells, and increased numbers of pleomorphic reserve cells with mitotic figures. This case is an example of the occurrence of a well-differentiated perianal gland tumor with metastasis and highlights the importance of realizing that with these tumors, a benign cytologic and histologic appearance may not correlate with biologic behavior. To the authors' knowledge, this is the first case reporting both the cytologic and histologic appearance of a well-differentiated metastatic hepatoid gland tumor.

Influx of neutrophils and persistence of cytokine expression in airways of horses after performing exercise while breathing cold air.

OBJECTIVE: To determine effects of exercise performed while breathing cold air on expression of cytokines and influx of neutrophils in airways of horses. ANIMALS: 9 adult horses. PROCEDURES: In a crossover study, bronchoalveolar lavage fluid (BALF) was obtained 24 and 48 hours after each of 2 submaximal exercise sessions performed by horses while breathing warm (25 degrees C) or cold (-5 degrees C) air. Total and differential nucleated cell counts were determined for each BALF sample. Relative mRNA expression of cytokines in BALF cells was quantified by use of a reverse transcription-PCR assay. RESULTS: Horses had a modest but significant influx of neutrophils into the airways 24 hours after a single exercise session while breathing cold air. No other cell types were increased at 24 or 48 hours after exercising while breathing cold air. Continued increases in expression of cytokines interleukin (IL)-5 and-10 as well as proinflammatory cytokines IL-1, -6, and -8 were detected 24 hours after exercising while breathing cold air. Forty-eight hours after exercising while breathing cold air, expression of IL-10 was still higher than that for IL-10 after horses exercised while breathing warm air. Expression of tumor necrosis factor-alpha was significantly increased at 48 hours after exercising while breathing cold air. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of intrapulmonary airways to cold air alters immunologic responses of horses for at least 48 hours. The increased expression of cytokines that suppress cell-mediated immunity may predispose athletes to viral infections of the respiratory tract following exercise in cold weather.

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.

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.

Efficacy of the early administration of valacyclovir hydrochloride for the treatment of neuropathogenic equine herpesvirus type-1 infection in horses.

OBJECTIVE To determine whether prophylactic administration of valacyclovir hydrochloride versus initiation of treatment at the onset of fever would differentially protect horses from viral replication and clinical disease attributable to equine herpesvirus type-1 (EHV-1) infection. ANIMALS 18 aged mares. PROCEDURES Horses were randomly assigned to receive an oral placebo (control), treatment at detection of fever, or prophylactic treatment (initiated 1 day prior to viral challenge) and then inoculated intranasally with a neuropathogenic strain of EHV-1. Placebo or valacyclovir was administered orally for 7 or 14 days after EHV-1 inoculation or detection of fever (3 horses/group). Effects of treatment on viral replication and clinical disease were evaluated. Plasma acyclovir concentrations and viremia were assessed to determine inhibitory concentrations of valacyclovir. RESULTS Valacyclovir administration decreased shedding of virus and viremia, compared with findings for control horses. Rectal temperatures and clinical disease scores in horses that received valacyclovir prophylactically for 2 weeks were lower than those in control horses. The severity of but not the risk for ataxia was decreased by valacyclovir administration. Viremia was decreased when steady-state trough plasma acyclovir concentrations were > 0.8 µg/mL, supporting the time-dependent activity of acyclovir. CONCLUSIONS AND CLINICAL RELEVANCE Valacyclovir treatment significantly decreased viral replication and signs of disease in EHV-1-infected horses; effects were greatest when treatment was initiated before viral inoculation, but treatment was also effective when initiated as late as 2 days after inoculation. During an outbreak of equine herpesvirus myeloencephalopathy, antiviral treatment may be initiated in horses at various stages of infection, including horses that have not yet developed signs of viral disease.

More than meets the eye: subretinal aspirate from an acutely blind dog.

A 14-year-old, spayed female Cocker Spaniel was presented to the Boren Veterinary Medical Teaching Hospital at Oklahoma State University with acute loss of vision in the right eye and a history of intermittent bloody diarrhea of unknown duration. Small, white, plaque-like lesions in the retina and subretina were visualized by direct ophthalmic examination, and aspirated with ultrasound-guidance. A direct smear of the subretinal fluid was highly cellular and contained large numbers of pleomorphic organisms consistent with Prototheca sp. The structures were round, oval, or elongated, 4-6 microm width and 8-16 microm in length and surrounded by a thin, clear cell wall. Small, central, pink to purple nuclei were observed in some organisms, but in most, the nuclei were obscured by a deeply basophilic, granular cytoplasm. Some organisms contained endospores. Negatively-stained structures of similar size and shape were considered to be empty casings (theca) of ruptured sporulating and nonsporulating forms of the organism. Protothecosis usually is a disseminated, fatal disease in dogs. The Prototheca organisms observed in this case showed characteristic morphology, illustrating the ability to diagnose protothecosis in cytologic samples.

Cytologic identification of immature endospores in a dog with rhinosporidiosis.

An 8-year-old, intact, male Labrador Retriever was presented to the Boren Veterinary Medical Teaching Hospital at Oklahoma State University with a 2-month history of severe sneezing episodes that resulted in epistaxis and bilateral sanguineous discharge. Rhinoscopy revealed a small polypoid mass, and specimens were obtained for histopathology. Microscopic examination of formalin-fixed tissue specimens revealed organisms consistent with Rhinosporidium seeberi. The mass was surgically excised and impression smears were made for cytology examination. Smears revealed high numbers of endospores, typical of those previously described for R seeberi. In addition, numerous smaller structures, presumed to be immature endospores, were noted. The immature endospores were morphologically distinct from mature endospores and have not been described previously. Recognition of immature forms of Rhinosporidium may help prevent misidentification of the organism or misdiagnosis of a dual infection.

A new species of Cytauxzoon from Pallas' cats caught in Mongolia and comments on the systematics and taxonomy of piroplasmids.

DNA was extracted and the 18S ribosomal RNA gene was amplified and sequenced from the blood of 2 Pallas' cats (Otocolobus manul) infected with small intraerythrocytic piroplasms. Sequences of the parasite were found to be identical with that of a previously reported Cytauxzoon-like piroplasm from a Pallas' cat. Phylogenetic analyses of the parasite DNA sequences obtained from the 3 Pallas' cats to other piroplasms revealed a sister group relationship to C. felis. The mean corrected percent sequence divergence between the Pallas' cat parasite and C. felis was 1.490%, which is greater than that for most other piroplasms in which species status has been accepted. On the basis of the sequence variation, we propose to name the Pallas' cat parasite C. manul. Phylogenetic analyses of C. manul also revealed a close relationship with the Spanish Cytauxzoon-like isolate because they exhibited only 0.389% sequence divergence, yet these sequences exhibit a mean of 1.690% sequence divergence from the New World isolate of C. felis. Our phylogenetic analyses also revealed several taxonomic problems that have impeded the development of a classification that accurately reflects evolutionary history of piroplasms. As currently arranged, Babesia and Theileria are paraphyletic taxa and are in need of reorganization.

Feline cytauxzoonosis.

Cytauxzoon felis is a protozoan hemoparasite of wild and domestic cats. In domestic cats, it causes severe clinical disease with high mortality.