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.

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.

Prevalence of Cytauxzoon felis infection in healthy cats from enzootic areas in Arkansas, Missouri, and Oklahoma.

BACKGROUND: Infection with Cytauxzoon felis in domestic cats can cause fever, lethargy, depression, inappetence, icterus, and often death. With a high mortality rate, cytauxzoonosis was historically considered a fatal disease. Within the last 15 years, cats with or without treatment have been recognized as chronically infected survivors of C. felis infection. Our objective was to determine the prevalence of C. felis in healthy domestic cats from Arkansas, Missouri, and Oklahoma. METHODS: Infection with C. felis was determined using DNA extracted from anticoagulated whole blood and PCR amplification using C. felis-specific primers. Chi-square, Fisher's exact tests, and odds ratios were used to compare proportions of cats infected with C. felis. RESULTS: Blood samples were collected from 902 healthy domestic cats between October 2008 and April 2012. DNA from Cytauxzoon felis was detected in 56 of 902 (6.2%; 95% confidence interval, 4.7-7.9) samples. The highest prevalence of C. felis infection (15.5%; 10.3-21.7) was observed in cats from Arkansas, followed by cats from Missouri (12.9%; 6.1-24.0), and cats from Oklahoma (3.4%; 2.2-5.1). Cats sampled in Arkansas and Missouri were 5.1 and 4.2, respectively, times more likely to be chronically infected with C. felis than cats from Oklahoma. CONCLUSIONS: Infection with C. felis is common in domestic cats through Arkansas, Missouri, and Oklahoma. The high prevalence of C. felis reported herein suggests that infected domestic cats are likely reservoirs of infection for naive felines. The high prevalence of C. felis substantiates the importance for the use of approved acaricides on cats to prevent cytauxzoonosis.

Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure.

Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP® 4Dx® and SNAP® 4Dx® Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichia canis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer=128-4096, GMTMAX=548.7) and E. chaffeensis (10/10, maximum inverse titer=1024-32,768, GMTMAX=4096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic pathogens.

Heat treatment prior to testing allows detection of antigen of Dirofilaria immitis in feline serum.

BACKGROUND: Diagnosis of Dirofilaria immitis infection in cats is complicated by the difficulty associated with reliable detection of antigen in feline blood and serum samples. METHODS: To determine if antigen-antibody complex formation may interfere with detection of antigen in feline samples, we evaluated the performance of four different commercially available heartworm tests using serum samples from six cats experimentally infected with D. immitis and confirmed to harbor a low number of adult worms (mean = 2.0). Sera collected 168 (n = 6), 196 (n = 6), and 224 (n = 6) days post infection were tested both directly and following heat treatment. RESULTS: Antigen was detected in serum samples from 0 or 1 of 6 infected cats using the assays according to manufacturer's directions, but after heat treatment of serum samples, as many as 5 of 6 cats had detectable antigen 6-8 months post infection. Antibodies to D. immitis were detected in all six infected cats by commercial in-clinic assay and at a reference laboratory. CONCLUSIONS: These results indicate that heat treatment of samples prior to testing can improve the sensitivity of antigen assays in feline patients, supporting more accurate diagnosis of this infection in cats. Surveys conducted by antigen testing without prior heat treatment of samples likely underestimate the true prevalence of infection in cats.

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.

Disseminated protothecosis diagnosed by evaluation of CSF in a dog.

A 5-year-old female spayed Shetland Sheepdog Mix dog was evaluated for a history of recent seizure activity, progressive hind limb ataxia, polyuria, and polydipsia and no history of gastrointestinal signs. Physical examination findings included conscious proprioceptive deficits, ataxia, and anterior uveitis along with a hypermature cataract in the right eye. Results of a CBC, serum biochemical profile, urinalysis, and computed tomography scan of the brain were unremarkable. Cerebrospinal fluid (CSF) analysis revealed marked eosinophilic pleocytosis and rare organisms consistent with Prototheca spp within neutrophils and macrophages. On postmortem histologic examination, mononuclear inflammation and numerous intralesional algal organisms, similar to those seen on the cytologic preparation of CSF, were found in the brain, eyes, kidneys, and heart. Abnormalities were not detected on gross and histologic examination of the gastrointestinal tract. Cultures of CSF and subdural/olfactory bulb, but not intestinal tract, yielded growth of Prototheca spp, and PCR analysis and DNA sequencing confirmed the organism as Prototheca zopfii genotype 2. We have reported a rare case of disseminated protothecosis that was diagnosed by evaluation of CSF in a dog presented with neurologic signs and no overt enteric disease. Protothecosis should be considered as a rare cause of seizures, even in the absence of obvious enteric signs, and should be included in the differential diagnosis of eosinophilic pleocytosis.

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.

Ehrlichia ewingii infection and exposure rates in dogs from the southcentral United States.

We used PCR and a novel serologic assay to determine infection and exposure rates to Ehrlichia ewingii in dogs from an area of northeast Oklahoma and northwest Arkansas where Amblyomma americanum ticks are abundant. Of 143 dogs assayed, 13 (9.1%) harbored E. ewingii by PCR and 64 (44.8%) had antibodies to E. ewingii detected using a peptide-based microtiter plate ELISA. Dogs were more likely (P=0.001) to be positive by PCR if sampled in August (30.8%) but no association was found between seropositive status and month of collection of sample (P>0.05). Additional testing revealed PCR evidence of Ehrlichia chaffeensis (4/143; 2.8%) and Anaplasma platys (5/143; 3.5%) as well as antibodies reactive to E. chaffeensis (25/143; 17.5%), Ehrlichia canis (2/143; 1.4%), and Anaplasma spp. (8/143; 5.6%). Testing of another 200 dogs from the area revealed additional PCR and/or serologic evidence of E. ewingii, E. canis, E. chaffeensis, and A. platys. None of the 343 dogs evaluated had evidence of Borrelia burgdorferi exposure. These data support the interpretation that E. ewingii may be the primary agent of canine ehrlichiosis in this region, and suggest that diagnostic evaluation of dogs suspected to have a tick-borne disease should include assays targeting this organism.

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.

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.

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.