Seroprevalence, DNA isolation, and genetic characterization of Toxoplasma gondii from black bear (Ursus americanus) sera collected in Eastern Oklahoma.

Black bears (Ursus americanus) are commonly exposed to Toxoplasma gondii. However, there are no reports of exposure or infection with T. gondii in black bears from Oklahoma. The purpose of our project was to determine the seroprevalence of T. gondii antibodies in black bears collected in Oklahoma. Additionally, since only serum was available from these bears, we sought to determine if DNA extraction and PCR amplification for T. gondii was possible on serum samples from bears with positive titers. Seroprevalence was determined using modified agglutination test (MAT). Serum was collected from 44 live-trapped bears in southeastern Oklahoma; 32 (73% ± 58-84%) had antibodies against T. gondii. Seroprevalence in adult bears (85% ± 67-95%) was significantly higher (p = 0.028) than yearlings (33.0% ± 56-80%). Adult bears were 3.4 times more likely to have antibodies to T. gondii than yearlings. From the bears with positive titers, T. gondii DNA was detected in 12 of the 32 seropositive samples by PCR of the B1 gene, with two of the samples showing variation in two nucleotide positions when compared with available sequences. Multilocus PCR-RFLP genotyping of these 12 samples revealed three ToxoDB genotypes, including #2 (type III, haplogroup 3), #4 (type XII, haplogroup 12), and #74 (haplogroup 12). To the best of our knowledge, this is the first report of T. gondii seroprevalence in black bears from Oklahoma. Our results indicate that exposure and infection with T. gondii in black bears from Oklahoma is common.

Nymphal engorgement weight predicts sex of adult Amblyomma americanum, Amblyomma maculatum, Dermacentor andersoni, Dermacentor variabilis, and Rhipicephalus sanguineus ticks.

The engorgement weights of laboratory-raised nymphs of five common ticks in the USA, Amblyomma americanum, Amblyomma maculatum, Dermacentor andersoni, Dermacentor variabilis, and Rhipicephalus sanguineus, experimentally fed to repletion on an adult sheep (Ovis aries) were evaluated to determine the sex of molted adult ticks. Length of feeding period of nymphs, molting success and durations, and sex ratios between tick species were also compared. Individual replete nymphs were weighed and allowed to molt to adults in a humidity chamber. Length of feeding duration was different by species (F4,7785 = 1963.79; P < 0.0001); R. sanguineus nymphs became replete fastest, followed by A. americanum, D. variabilis, A. maculatum, and D. andersoni. Significant difference in molting success was not detected. The mean body weight of engorged nymphs of A. americanum (t = 32.3; df = 662), A. maculatum (t = - 9.70; df = 255), D. variabilis (t = 15.7; df = 751), and R. sanguineus (t = 5.17; df = 560; all P < 0.0001) molting into females was greater than that of nymphs molting into males, whereas heavier D. andersoni engorged nymphs became males (t = 8.71; df = 480; P < 0.0001). Amblyomma maculatum nymphs that molted to females fed to repletion faster (t = 3.33; df = 265; P ≤ 0.001) than nymphs that molted to males and a higher proportion (χ2 = 48.4; df = 1, P < 0.0001) of A. maculatum and D. andersoni (χ2 = 8.19; df = 1, P = 0.004) molted to females than males. Our study demonstrated biological and behavioral differences in and between engorging nymphs of five ixodid species. These findings may aid in studies evaluating the role of tick sex in transmission of tick-borne pathogens.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.

BACKGROUND: Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development. METHODS: Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model. RESULTS: Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis. CONCLUSIONS: Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.

Cytauxzoonosis in Indiana, USA: a case series of cats infected with Cytauxzoon felis (2018-2022).

CASE SERIES SUMMARY: This case series describes six cases involving seven cats naturally infected with Cytauxzoon felis in Indiana, USA. Medical records were retrospectively reviewed and all available information on signalment, history, clinical and diagnostic findings, treatment, outcome and pathology was reported. Cats infected with C felis were domestic shorthairs, were aged between 2 and 9 years and all but one of the cats were male. The seven infected cats originated from five counties in southwestern Indiana. Six of seven cats were found to have acute cytauxzoonosis based on clinical signs, gross pathologic lesions, observation of C felis in tissues and/or detection of C felis DNA. One cat was identified as a subclinical survivor cat with no known clinical history of cytauxzoonosis. RELEVANCE AND NOVEL INFORMATION: The reported cases are the first confirmed reports of acute and chronic cytauxzoonosis in cats from Indiana and document an expansion in the range of C felis. Veterinary practitioners in Indiana should consider infection with C felis as a differential diagnosis for cats that present with fever, inappetence, lethargy, depression, dehydration, dyspnea, hemolytic crisis, anorexia or icterus. Administration of approved acaricides to cats currently offers the best protection and control against C felis infection.

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.

Differential gene expression response to acute and chronic Cytauzxoon felis infection in domestic cats (Felis catus).

Cytauxzoonosis is a severe tick transmitted protozoan disease of domestic cats, caused by Cytauxzoon felis. The disease is characterized by acute onset of high fever, depression, lethargy, inappentence, anorexia, icterus, dehydration, hemolytic anemia, and alteration of immune response. The aim of our study was to further detail the immune response of domestic cats to C. felis infection by comparing the differential expression of feline immune transcriptional elements during acute and chronic cytauxzoonosis. True single molecule sequencing (tSMS) was used to analyze the whole genome of acutely and chronically infected C. felis cats, focusing on the analysis of genes involved on the immune response. Two C. felis donor cats were infested with Amblyomma americanum nymphs, which after repletion were collected and kept in humidity chambers until they molted. The resulting A. americanum were randomly selected to infest three C. felis naïve principal cats. Infection of these cats was confirmed by nested PCR of the 18S rRNA C. felis gene and clinical signs. RNA was extracted from whole blood at different time points and used for tSMS analyses, the results revealed overexpression in transcripts involved in type I interferon signaling, cellular and cytokine responses during the acute stage of infection, while cell cycle, and metabolic processes were downregulated. Genes involved in cell adhesion increased their expression in the chronic infected cats, whereas inflammatory and apoptotic related genes were downregulated. This study provided information on the host immune response to C. felis in domestic cats, demonstrating that inflammatory, apoptotic, and cell adhesion are some of the pathways altered during acute and chronic infection.

Cytauxzoon felis in salivary glands of Amblyomma americanum.

Cytauxzoon felis is a tick-borne piroplasmid hemoparasite that causes life-threatening disease in cats. Despite the critical role that ticks play in pathogen transmission, our knowledge regarding the C. felis life cycle remains limited to the feline hosts. Specific life stages of C. felis within the tick host have never been visualized microscopically and previous investigations have been limited to molecular detection by polymerase chain reaction (PCR). Sporozoites are the infectious stage of piroplasmids that are transmitted by ticks. In other tick-borne piroplasmids, sporozoite-based vaccines play a key role in disease prevention and management. We believe sporozoites have similar potential for cytauxzoonosis. Therefore, the objective of this study was to use different molecular and microscopic techniques to detect and evaluate C. felis sporozoites in tick salivary glands (SG). A total of 140 Amblyomma americanum adults that were fed on C. felis-infected cats as nymphs were included for this study. Specifically, dissected SGs were quartered and subjected to C. felis RT-PCR, RNAscope® in situ hybridization (ISH), histology, direct azure staining, and transmission electron microscopy (TEM). Cytauxzoon felis RT-PCR was also performed on half tick (HT) carcasses after SG dissection. Cytauxzoon felis RNA was detected in SGs of 17/140 ticks. Of these, 7/17 ticks had microscopic visualization via ISH and/or TEM. The remaining 10/17 ticks had only molecular detection of C. felis in SGs via RT-PCR without visualization. Cytauxzoon felis RNA was detected solely in HT carcasses via RT-PCR in 9/140 ticks. In ISH-positive tick SGs, hybridization signals were present in cytoplasms of SG acinar cells. TEM captured rare C. felis organisms with characteristic ultrastructural features of sporozoites. This study describes the first direct visualization of any developing stage of C. felis in ticks. Forthcoming studies should employ a combination of molecular and microscopic techniques to investigate the C. felis life cycle in A. americanum.

Prevalence and genetic characterization of Toxoplasma gondii strains isolated from 31 wild Passeriformes collected in North-Central Oklahoma.

Passerine birds are widely distributed and adapted to various habitats, therefore they are commonly exposed to, and infected with Toxoplasma gondii. The purpose of our project was to determine the prevalence and genotypes of T. gondii in 31 different species of passerines collected as mortalities due to window collisions in North-Central Oklahoma. DNA was extracted from breast tissue and subjected to PCR with primers that amplify a portion of the T. gondii B1 gene. Genotyping was based on a portion of the infected birds based on a multiplex PCR followed by RFLP of 12 T . gondii markers. Of 103 birds comprising 31 species, the overall prevalence (95% confidence interval) of T. gondii infection was 33.0% (24.1‒42.6%). Significant differences in the proportion of T. gondii in birds according to sex or weight were not observed. However, sample sizes of each species were small and prevented a robust analysis of T. gondii according to those biological variables. Genotyping of T. gondii in a subset of 13 infected individuals of 7 species revealed 4 genotypes, according to the Toxoplasma Data Base: #54, #139, #20, and #220. Our results, while hampered by a small sample size for each bird species, suggest that infection with T. gondii in Oklahoma, is common in both migrant and resident passerines.

HIGH PREVALENCE OF CYTAUXZOON FELIS IN BOBCATS (LYNX RUFUS) ACROSS OKLAHOMA AND OCCURRENCE IN WEST TEXAS, USA.

Cytauxzoonosis is a fatal tick-borne disease in domestic cats caused by infection with the apicomplexan Cytauxzoon felis. Bobcats are the natural wild-vertebrate reservoirs for C. felis, and infections are typically subclinical and chronic in this species. The present study was done to determine the prevalence and geographic distribution of C. felis infection in wild bobcats from Oklahoma and the occurrence in northwestern Texas. Tongue samples from 360 bobcats were collected from 53 counties in Oklahoma and 13 samples from three counties in Texas. For DNA extracted from each tongue sample, a probe-based droplet digital PCR assay was performed targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Prevalence of C. felis infection was calculated for each county sampled, and data from individual counties were combined according to geographic regions and compared using chi-square tests. Overall prevalence of C. felis in bobcats from Oklahoma was 80.0% (95% confidence interval [CI], 75.6-83.8). The prevalence of infection was >90% for bobcats from central, northeastern, south-central, and southeastern regions of Oklahoma, but <68% for bobcats from northwestern and southwestern regions. Bobcats from central counties in Oklahoma were 25.693 times more likely to be infected with C. felis compared to all other bobcats sampled from the state. Higher prevalence estimates of C. felis in bobcats appeared to be in counties where known tick vectors are most common. Occurrence of C. felis in bobcats from northwestern Texas was 30.8% (95% CI, 12.4%-58.0%) based on 13 samples. Results of this study support the utilization of bobcats as sentinel animals to identify geographic areas with risk of C. felis infection to domestic cats.

Transmission of Cytauxzoon felis by injection of Amblyomma americanum salivary glands.

BACKGROUND: Cytauxzoonosis is a life-threatening disease of cats, caused by the tick-borne piroplasmid hemoparasite, Cytauxzoon felis. Current experimental models for cytauxzoonosis rely on either tick transmission or direct injection of infected cat tissues. These models require researchers to directly work with infected ticks or use cats with acute cytauxzoonosis. To improve the feasibility and accessibility, there is a need to establish sharable resources among researchers. In related piroplasmid parasites, sporozoite-based inoculums are routinely produced from tick salivary glands, cryopreserved and distributed to other investigators and facilities. For these parasites, sporozoites have been the basis for vaccine development and in vitro cultivation, both of which remain lacking for C. felis research. If infectious sporozoites can be similarly isolated for C. felis, it would significantly broaden our capabilities to study this parasite. Aims of this study was to determine if C. felis sporozoites inoculums collected from the salivary glands of Amblyomma americanum ticks were capable of inducing cytauxzoonosis in naïve cats. MATERIALS AND METHODS: A. americanum nymphs were acquisition-fed on a donor cat chronically infected with C. felis and allowed to molt to adults. Four groups of adult ticks (n = 50/group) were either stimulation-fed for 4 days on naïve cats or were heated at 37 °C for 4 days. After these treatments, salivary glands (SG) of each group of ticks were collected to create inoculums. Infectivity of these inoculums was then tested by subcutaneous injection into naïve cats. RESULTS: The two naïve cats used for stimulation feeding and as controls both developed cytauxzoonosis, indicating these groups of ticks were capable of producing infectious sporozoites. Of the 2 cats that were injected with SGs from the stimulation-fed ticks, one cat developed cytauxzoonosis and C. felis infection was confirmed by both light microscopy and PCR. The other cat did not develop cytauxzoonosis and only had equivocal evidence of infection. Neither cat injected with SGs from the heated ticks developed cytauxzoonosis. One of these cats had equivocal evidence of infection and one had no evidence of infection. CONCLUSION: This study validates the feasibility of collecting infectious sporozoites from C. felis-infected ticks that can be used to infect naïve cats. While this model requires further optimization, it has the potential to expand resources to study C. felis and further advance research in this field.

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.

Babesiosis and Theileriosis in North America.

Babesia and Theileria are apicomplexan parasites that cause established and emerging diseases in humans, domestic and wild animals. These protozoans are transmitted by Ixodid ticks causing babesiosis or theileriosis, both characterized by fever, hemolytic anemia, jaundice, and splenomegaly. In North America (NA), the most common species affecting humans is B. microti, which is distributed in the Northeastern and Upper Midwestern United States (US), where the tick vector Ixodes scapularis is established. In livestock, B. bovis and B. bigemina are the most important pathogens causing bovine babesiosis in tropical regions of Mexico. Despite efforts toward eradication of their tick vector, Rhipicephalus microplus, B. bovis and B. bigemina present a constant threat of being reintroduced into the southern US and represent a continuous concern for the US cattle industry. Occasional outbreaks of T. equi, and T. orientalis have occurred in horses and cattle, respectively, in the US, with significant economic implications for livestock including quarantine, production loss, and euthanasia of infected animals. In addition, a new species, T. haneyi, has been recently discovered in horses from the Mexico-US border. Domestic dogs are hosts to at least four species of Babesia in NA that may result in clinical disease that ranges from subclinical to acute, severe anemia. Herein we review the pathogenesis, diagnosis, and epidemiology of the most important diseases caused by Babesia and Theileria to humans, domestic and wild animals in Canada, the US, and Mexico.

TOXOPLASMA GONDII PREVALENCE, PARTIAL GENOTYPES, AND SPATIAL VARIATION IN NORTH AMERICAN RIVER OTTERS (LONTRA CANADENSIS) IN THE UPPER PENINSULA OF MICHIGAN, USA.

Toxoplasma gondii is a ubiquitous parasitic protozoan that poses a health threat to wildlife and human health worldwide. Oocysts shed into the environment in felid host feces may persist for several years. Runoff from rainfall and snowmelt may carry the oocysts into waterways. Semiaquatic mammals such as the Northern American river otter (Lontra canadensis) are particularly at risk of exposure, as they may encounter infective stages in both terrestrial and aquatic environments. Despite this risk, only a small number of studies have examined the prevalence of T. gondii in US river otter populations. Tongue tissue was sampled from 124 otters from the Upper Peninsula of Michigan submitted by trappers to the Michigan Department of Natural Resources in the 2018-19 harvest season. Following DNA extraction, a portion of the B1 T. gondii gene was amplified with PCR. A subset of positive samples was genotyped for comparison with known T. gondii sequences. Of the 124 tongue samples, 35 (28%) were positive for T. gondii. Prevalence did not differ significantly between sexes or age classes across the entire study area. Most (53.8%) of the genotyped samples were type 4 (type 12), a genotype commonly found in North American wildlife. Genotypes 127 and 197 were also found. Three clusters of T. gondii prevalence were identified through SaTScan analysis, although they were not significant. When modeling prevalence of T. gondii with covariates at individual otter locations, the top three models included the presence of Sarcocystis, area of exotic plants, area of agriculture, and sex of the otter. Our results suggest that T. gondii is widespread in otter populations in the Upper Peninsula of Michigan.

Direct injection of Amblyomma americanum ticks with Cytauxzoon felis.

Cytauxzoon felis is a tick-borne hemoprotozoan parasite that causes life-threatening disease in domestic cats in the United States. Currently, the platforms for C. felis research are limited to natural or experimental infection of domestic cats. This study aims to develop an alternative model by infecting Amblyomma americanum ticks with C. felis via direct injection. Amblyomma americanum adults were injected with C. felis-infected feline erythrocytes through two routes: directly into the digestive tract through the anal pore (IA injection), or percutaneously into the tick hemocoel (IH injection). RNAscope® in situ hybridization (ISH) was used to visualize the parasites within the ticks at different time points after injection. Four months after injection, ticks were divided into 3 infestation groups based on injection methods and inoculum type and fed on 3 naïve cats to assess the ticks' ability to transmit C. felis. Prior to the transmission challenge, selected ticks from each infestation group were tested for C. felis RNA via reverse transcription-PCR (RT-PCR). In both IA- and IH-injected ticks, ISH signals were observed in ticks up to 3 weeks after injection. The number of hybridization signals notably decreased over time, and no signals were detected by 4 months after injection. Prior to the transmission challenge, 37-57% of the sampled ticks were positive for C. felis RNA via RT-PCR. While the majority of injected ticks successfully attached and fed to repletion on all 3 cats during the transmission challenge, none of the cats became infected with C. felis. These results suggest that injected C. felis remained alive in ticks but was unable to progress to infective sporozoites after injection. It is unclear why this infection technique had been successful for other closely related tick-borne hemoprotozoa and not for C. felis. This outcome may be associated with uncharacterized differences in the C. felis life cycle, the lack of the feeding or molting in our model or absence of gametocytes in the inoculum. Nonetheless, our study demonstrated the potential of using ticks as an alternative model to study C. felis. Future improvement of a tick model for C. felis should consider other tick species for the injection model or utilize infection methods that more closely emulate the natural infection process.

A Serodiagnostic IgM ELISA to Detect Acute Cytauxzoonosis.

Cytauxzoonosis is a tick-borne infectious disease affecting domestic cats with high mortality and limited treatment modalities. Because early diagnosis and therapeutic intervention are crucial to survival of infected cats, the objective of this study was to develop an ELISA capable of detecting cytauxzoonosis and differentiating acute vs. chronic infection in clinical feline blood samples. A microsphere immunoassay (MIA) was developed to evaluate the production of Cytauxzoon felis-specific IgM and IgG antibodies in serial plasma samples from cats with experimental C. felis infection by targeting a C. felis-specific transmembrane protein (c88). Recombinant c88 protein was utilized to develop indirect ELISAs to detect IgM and IgG antibodies in clinical plasma samples from: PCR-positive cats with acute C. felis infection (n = 36), C. felis-negative cats with pyrexia (n = 10), healthy C. felis-negative cats (n = 22), and chronic C. felis carriers (n = 4). Anti-c88 IgM antibodies were detectable at day 12 post-tick infestation in cats with experimental C. felis infection (within 24 hours of developing clinical signs), while anti-c88 IgG was detectable at day 15 post-tick infestation - indicating IgM could be used to detect early infection. Using a cut-off value of 19.85 percent positive, the C. felis IgM ELISA detected acute cytauxzoonosis in 94.44% (34/36) of cats presented with clinical signs of acute cytauxzoonosis with 100% specificity (indicating a "Strong Positive" result). When a lower cutoff of 8.60 percent positive was used, cytauxzoonosis was detected in the 2 remaining PCR-positive cats with 87.88% specificity (indicating of a "Weak Positive" result). One C. felis-negative, febrile cat had high IgG, and chronic carriers had variable IgM and IgG results. Combined interpretation of IgM and IgG ELISAs did not reliably differentiate acute vs. chronic infection. While further validation on assay performance is needed, the C. felis IgM ELISA is a promising test to detect acute cytauxzoonosis and can be utilized to develop a point-of-care test for clinical use.

Species Distribution Model of Trichinella Species in Cougars (Puma concolor) for the Southwestern Region of Colorado, USA.

Little information exists on the occurrence of Trichinella spp. in cougars (Puma concolor) throughout North and South America. However, species distribution models (SDM) can be used to predict suitable habitat for elusive species with limited occurrence data. In 2018, we used existing occurrence data to estimate the suitable habitat and ecological drivers of Trichinella species in cougars from the southwestern region of Colorado, US from winter months. Environmental layers were constructed and an SDM was then created for the southwestern region of Colorado. The final model (area under the curve=0.73) found areas of suitable habitat for Trichinella spp. to be associated with lower elevation, lower temperature, and higher proportions of evergreen needleleaf forests and grasslands. These results might indicate potential transmission hotspots for Trichinella spp. in the southwestern region of Colorado for where cougars are consuming infected hosts and where cougars can transmit to other hosts, including humans. However, due to limited occurrence data, more suitable habitat likely exists.

Ticks infesting dogs and cats in North America: Biology, geographic distribution, and pathogen transmission.

A diverse array of ixodid and argasid ticks infest dogs and cats in North America, resulting in skin lesions, blood loss, and disease. The ticks most commonly found on pets in this region are hard ticks of the genera Amblyomma, Dermacentor, Ixodes, and Rhipicephalus, as well as the more recently established Haemaphysalis longicornis. Soft tick genera, especially Otobius and Ornithodoros, are also reported from pets in some regions. In this review, we provide a summary of the complex and diverse life histories, distinct morphologies, and questing and feeding behaviors of the more common ticks of dogs and cats in North America with a focus on recent changes in geographic distribution. We also review pathogens of dogs and cats associated with the different tick species, some of which can cause serious, potentially fatal disease, and describe the zoonotic risk posed by ticks of pets. Understanding the natural history of ticks and the maintenance cycles responsible for providing an ongoing source of tick-borne infections is critical to effectively combatting the challenges ticks pose to the health of pets and people.

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.

Emergence of Babesia conradae infection in coyote-hunting Greyhounds in Oklahoma, USA.

BACKGROUND: Babesia species are intraerythrocytic Apicomplexan parasites that infect a wide range of vertebrate hosts. These pathogens are typically transmitted either by tick vectors or by direct blood-to-blood contact, and may cause life-threatening clinical disease, such as thrombocytopenia, hemolytic anemia and acute renal failure, in canine hosts. While Babesia vogeli and Babesia gibsoni infections have both been reported in Oklahoma, reports of Babesia conradae infections have been limited to California. METHODS: Four separate kennels of coyote-hunting dogs were identified in Oklahoma after the kennels had consulted with Oklahoma State University Boren Veterinary Medical Teaching Hospital (antemortem cases) or the Oklahoma Animal Disease Diagnostic Lab (postmortem cases). Upon owner consent, every accessible dog from each of the four kennels was briefly examined for ectoparasites, particularly ticks, and whole blood samples were collected in EDTA tubes. Clinically ill dogs were examined by a practicing veterinarian, and clinical signs included anorexia, vomiting, lethargy, fever and anemia. DNA was extracted from each blood sample, and a nested PCR was performed using general apicomplexan primers for the partial 18S rRNA gene. PCR products were electrophoresed in agarose matrix, and appropriately sized amplicons were sequenced. Sequences were compared to reference 18S rRNA gene sequences available in GenBank, and samples with > 98% homology to B. conradae (GenBank: AF158702) were considered positive. Babesia conradae-positive dogs were then treated with atovaquone (13.5 mg/kg three times per day) and azithromycin (10 mg/kg once daily) for 10 days and retested at 30 and 60 days post-treatment by PCR. RESULTS: Of 40 dogs tested, 15 (37.5%) were positive for B. conradae with 98-99% sequence homology to B. conradae from California. All positive cases were coyote-hunting Greyhounds. Ectoparasites were not identified on any of the dogs at the time of blood collection. Treatment of clinically ill dogs with atovaquone and azithromycin resulted in complete clinical recovery in all treated dogs with negative follow-up PCR at 30 and 60 days post-treatment. CONCLUSIONS: Collectively, this study (i) documents the occurrence of B. conradae in Oklahoma, (ii) highlights this pathogen as a differential to be considered when clinical signs are present, (iii) supports the use of atovaquone and azithromycin as effective treatment in these cases and (iv) demonstrates chronic subclinical carrier dogs serving as potential reservoirs of B. conradae infection to naïve dogs.

A probe-based droplet digital polymerase chain reaction assay for early detection of feline acute cytauxzoonosis.

Cytauxzoonosis is a tick-borne disease of domestic cats with high mortality and narrow therapeutic window, particularly in the southcentral and southeastern United States. The causative agent is the apicomplexan protozoal parasite Cytauxzoon felis and is primarily transmitted by Amblyomma americanum, the lone star tick. Currently there is no vaccine available to prevent cytauxzoonosis and treatment is often ineffective if not initiated early enough in the course of disease. Early diagnosis and therapeutic intervention are therefore crucial for the survival of infected cats. Several methods are available for diagnosis of cytauxzoonosis, with PCR being the most sensitive. However, current PCR assays, which employ double-stranded DNA intercalating dyes to detect C. felis infection, have inherent limitations such as the potential for false positive detection of non-specific amplification products and inability to provide absolute quantification of parasite load. The objective of this study was to develop a probe-based droplet digital PCR (ddPCR) assay capable of detection and quantification of C. felis load over time and during treatment. The C. felis ddPCR assay was able to (i) reliably detect and quantify C. felis DNA in clinical blood samples from cats with acute cytauxzoonosis and (ii) monitor clinical parasite load in response to anti-protozoal treatment through absolute quantification of C. felis DNA over time. When tested on blood samples from cats with experimental C. felis infection, the assay was able to detect infection in cats as early as 24 h prior to the development of clinical signs. In addition, we demonstrate that this probe-based design can be utilized in traditional real-time PCR systems, with similar detection capabilities as compared to ddPCR. The C. felis probe-based ddPCR was also able to detect infection in samples with lower parasite loads when compared to existing nested PCR assays, although these results were not significant due to small sample size. To the author's knowledge, this is the first reported probe-based ddPCR assay to detect Cytauxzoon felis infection in domestic cats.