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.

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.

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.

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.

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.

Increased prevalence of Dirofilaria immitis antigen in canine samples after heat treatment.

Canine serum samples may contain factors that prevent detection of antigen of Dirofilaria immitis on commercial assays, precluding accurate diagnosis. To determine the degree to which the presence of blocking antibodies or other inhibitors of antigen detection may interfere with our ability to detect circulating antigen in canine samples, archived plasma and serum samples (n=165) collected from dogs in animal shelters were tested for D. immitis antigen before and after heat treatment. Negative samples were also evaluated for their ability to block detection of D. immitis antigen in a sample from a positive dog. All 165 samples were negative prior to heating, but 11/154 (7.1%) became positive after heat treatment, a conversion that was documented and quantified on spectrophotometric plate assays, and 7/165 (4.2%) samples decreased detection of antigen when mixed with a known positive sample, suggesting some blocking ability was present. An additional 103 plasma and serum samples that tested positive prior to heating also were evaluated; the optical density of 14/101 (13.9%) increased by ≥50%, and one sample by as much as 15-fold, after heat treatment. Our results suggest that canine serum and plasma samples from dogs in the southeastern United States can contain inhibitors of D. immitis antigen detection, and that prevalence estimates of heartworm infection based on these assays would benefit from heat treatment of samples prior to testing.

Pre-treatment with heat facilitates detection of antigen of Dirofilaria immitis in canine samples.

Diagnosis of Dirofilaria immitis infection in dogs is largely dependent on detection of antigen in canine serum, plasma, or whole blood, but antigen may be bound in immune complexes and thus not detected. To develop a model for antigen blocking, we mixed serum from a microfilaremic, antigen-positive dog with that of a hypergammaglobulinemic dog not currently infected with D. immitis and converted the positive sample to antigen-negative; detection of antigen was restored when the mixed sample was heat-treated, presumably due to disruption of antigen/antibody complexes. A blood sample was also evaluated from a dog that was microfilaremic and for which microfilariae were identified as D. immitis by morphologic examination. Antigen of D. immitis was not detected in this sample prior to heating but the sample was strongly positive after heat treatment of whole blood. Taken together, our results indicate that blood samples from some dogs may contain factors that inhibit detection of antigen of D. immitis, and that heat treatment of these samples prior to testing could improve the sensitivity of these assays in some patients.

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.