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.

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.

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. Graphical abstract

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.

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 (F 4,7785 = 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 (χ 2 = 48.4; df = 1, P < 0.0001) of A. maculatum and D. andersoni (χ 2 = 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.

Successful Cytauxzoon felis transmission studies have occurred using Amblyomma americanum adults acquisition-fed as nymphs on an experimentally infected domestic cat or Dermacentor variabilis adults fed as nymphs on a splenectomized bobcat. Here, we evaluated A. americanum and D. variabilis nymphs acquisition-fed as larvae on a C. felis-infected carrier domestic cat for competence to transmit the protozoan parasite as nymphs to naïve, healthy domestic cats.

Cytauxzoonosis, caused by infection with Cytauxzoon fells, is the most severe tick-borne disease of cats. The purpose of our study was to determine the efficacy of selamectin (6.0 mg/kg) plus sarolaner (1.0 mg/kg) formulated in combination (Revolution (R) Plus / Stronghold (R) Plus, Zoetis) applied topically once a month on cats for three months against induced infestations of Amblyomma americanum adults and to evaluate the effectiveness of the product in preventing the transmission of C. fells. This study was conducted in two phases. Sixteen cats were dosed with selamectin/sarolaner or a placebo (vehicle control) on Days 0, 28, and 56. In phase 1, each cat was infested with 50 (+/- 5) unfed adult A. americanum on Day 4 and tick counts were conducted on Day 6 (48 h post infestation) and Day 7 (72 h post infestation) to evaluate acaricidal efficacy. In phase 2, to confirm acaricidal efficacy and evaluate prevention of C. fells transmission, each cat was infested on Day 60 with 50 (+/- 5) adult A. americanum acquisition fed as nymphs on two C. fells-infected donor cats. Tick counts were conducted on Day 62 (48 h post infestation) and Day 63 (72 h post infestation). Blood samples were collected on Days -9, 60, 70, 76, and 90 and tested for infection with C. fells. Placebo cats were adequately infested on all count days, with least squares (geometric) mean live tick counts ranging from 34.0 (28.8) to 46.1 (46.0). Treatment reduced the least squares (geometric) mean counts compared to placebo by 27.1 (32.1)% and 90.4 (96.8)% on Days 6 and 7, respectively. The corresponding percent reductions were 56.4 (60.6)% and 94.7 (97.3)% on Days 62 and 63, respectively Least squares mean counts were significantly lower in the treated group compared with the placebo group on all count days (P <= 0.0286). All cats were negative for C. fells by PCR prior to study start. In phase 2, seven cats in the control group and no cats in the selamectin/sarolaner group became infected with C. fells (P = 0.0017). Topical treatment with selamectin/sarolaner was > 90% effective in reducing A. americanum tick counts 72 h after infestation and prevented the transmission of C. fells from infected ticks following the third of three monthly treatments. Revolution (R) Plus / Stronghold (R) Plus offers an option for the control of A. americanum infestations on cats and for preventing the transmission of C. fells to cats.

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.

Several commercial Giardia immunoassays were evaluated in baboons for sensitivity and specificity as well as ease of use in a large specific pathogen-free (SPF) colony. An additional objective was to identify the assemblage(s) of Giardia duodenalis present in this baboon colony. A direct immunofluorescent antibody test (IFAT) was used as the reference test. Tests evaluated were a patient-side rapid test for dogs and cats, a human rapid test, and a well-plate ELISA designed for use with humans. Test sensitivities and specificities were compared using the McNemar paired t-test and were further evaluated for agreement using an unweighted Cohen kappa statistic. When compared to the IFAT reference, both human tests were more sensitive than the veterinary test. Based on PCR and sequencing of the G. duodenalis small-subunit ribosomal RNA and glutamate dehydrogenase loci, assemblage AI was present in this baboon colony. We found that 10 of the 110 (9%) baboons in this SPF colony were infected with a zoonotic strain of G. duodenalis.

Objectives The objectives of the present study were to determine the duration of infestation by Amblyomma americanum necessary for transmission of Cytauxzoon felis to domestic cats and to determine if ingestion of C felis-infected A americanum by cats is a route of transmission. Methods Forty-nine cats were assigned to one of seven groups, with seven cats per group. Cats were infested with A americanum adults, acquisition fed as nymphs on a cytauxzoonosis survivor cat, for 12 h (group 1), 18 h (group 2), 24 h (group 3), 36 h (group 4), 48 h (group 5) and to repletion (group 7; control). Cats in group 6 were fed C felis-infected ticks. Thumb counts were performed at the end of the duration of infestation for groups 1–5 and at 48 h for the control group. For group 6, 50 live C felis-infected adult A americanum were mixed with food and fed to each of the cats. Transmission of C felis was determined by examining blood of cats by DNA extraction followed by PCR. Results Of 50 ticks placed on each cat (groups 1–5 and 7), the arithmetic mean attachment ± SEM ranged from 46.9 ± 1.9 in group 3 to 49.3 ± 0.3 in group 1. In group 6, the average number ± SEM of ticks ingested was 46.5 ± 2.3. One cat in group 5 that had been infested for 48 h became infected with C felis. None of the cats in group 6 (ingestion) became infected with C felis. Six of 7 (85.7%) cats in group 7, the control group, became infected with C felis. Conclusions and relevance Our results indicate that transmission of C felis to domestic cats can happen as quickly as >36 h but ⩽48 h of exposure to A americanum infected with C felis and that ingestion of C felis-infected A americanum is not a likely route of transmission.

Trichinella species are zoonotic nematodes that infect wild carnivores and omnivores throughout the world. We examined the prevalence and species of Trichinella infections in cougars (Puma concolor couguar) from Colorado, United States. Tongues from cougars were examined by pepsin-HCl artificial digestion to detect Trichinella spp. larvae. The species or genotype of individual worms was identified by multiplex polymerase chain reaction (PCR). Trichinella spp. larvae were detected in 17 of 39 cougars (43.6% (28.7-59.5%)). Five of the cougars (12.8%) were infected with T. murrelli, 3 (7.7%) were infected with T. pseudospiralis, and 1 (2.6%) had Trichinella genotype T6. Trichinella spp. larvae from eight cougars were not identified at the species level, due to degraded DNA. The high prevalence of Trichinella spp. in cougars from Colorado and reports of the parasite in other populations of Puma spp. suggest that this large predator is a key mammalian reservoir.

Intestinal helminths are common in dogs in the United States, particularly non-treated dogs in animal shelters, but surveys by fecal flotation may underestimate their prevalence. To determine the prevalence of intestinal helminths and evaluate the ability of fecal flotation and detection of nematode antigen to identify those infections, contents of the entire gastrointestinal tract of 97 adult (>1year) dogs previously identified for humane euthanasia at two animal control shelters in northeastern Oklahoma, USA, were screened. All helminths recovered were washed in saline and fixed prior to enumeration and identification to genus and species. Fecal samples from each dog were examined by passive sodium nitrate (SG 1.33) and centrifugal sugar solution (SG 1.25) flotation. Fecal antigen detection assays were used to confirm the presence of nematode antigen in frozen fecal samples from 92 dogs. Necropsy examination revealed Ancylostoma caninum in 45/97 (46.4%), Toxocara canis in 11/97 (11.3%), Trichuris vulpis in 38/97 (39.2%), Dipylidium caninum in 48/97 (49.5%), and Taenia sp. in 7/97 (7.2%) dogs. Passive fecal flotation identified 38/45 (84.4%) A. caninum, 6/11 (54.5%) T. canis, 26/38 (68.4%) T. vulpis, 2/48 (4.2%) D. caninum, and 1/7 (14.3%) Taenia sp. infections, while centrifugal flotation combined with antigen detection assays identified A. caninum in 97.7% (43/44), T. canis in 77.8% (7/9), and T. vulpis in 83.3% (30/36) of infected dogs based on necropsy recovery of nematodes. Taken together, these data indicate that detection of nematode antigen is a useful adjunct to microscopic examination of fecal samples for parasite eggs, and that this approach can improve diagnostic sensitivity for intestinal nematode infections in dogs.

In this study, we evaluated the efficacy of combined treatment with ivermectin and fenbendazole (IVM-FBZ) for treating captive olive baboons (Papio anubis) infected with Strongyloides fülleborni and Trichuris trichiura, 2 common nematode parasites of these NHP. Infected baboons were treated for a total of 9 wk with ivermectin (400 μg/kg IM twice weekly) and fenbendazole (50 mg/kg PO once daily for 3 d; 3 rounds of treatment, 21 d apart). Five baboons naturally infected with both S. fülleborni and T. trichiura (n = 4) or S. fülleborni alone (n = 1) received the combination therapy; an additional baboon infected with both parasites served as a nontreated control. The efficacy of IVM-FBZ was measured as the reduction in fecal egg counts of S. fülleborni and T. trichiura as determined by quantitative fecal flotation examination after treatment of baboons with IVM-FBZ. All baboons treated with IVM-FBZ stopped shedding S. fülleborni and T. trichiura eggs by 8 d after treatment and remained negative for at least 161 d. The nontreated control baboon shed S. fülleborni and T. trichiura eggs throughout the study period. Our results indicate that the IVM-FBZ regimen was efficacious for treating olive baboons infected with S. fülleborni and T. trichiura.

Objectives To determine whether pretreating diagnostic samples with heat increases the detection of Dirofilaria immitis antigen in adult cats, we evaluated feline serum and plasma samples collected in heartworm-endemic areas of the southern United States. Methods Commercial microtiter well assays for detection of D immitis antigen were used to evaluate serum or plasma samples from 385 shelter and free-roaming cats from the southcentral and southeastern United States before and after heat treatment; commercial antibody tests were performed on a subset of samples. Results Prior to sample heat treatment, 1/220 (0.5%) shelter cats and 4/165 (2.4%) free-roaming cats had detectable D immitis antigen. After heat pretreatment, the detection rate increased to 13/220 (5.9%) and 13/165 (7.9%), respectively. Antibody reactive to D immitis was significantly more common ( P <0.001) in the serum of cats that were antigen positive after heat treatment (10/13; 76.9%) than serum from cats that remained antigen negative after heat treatment (22/163; 13.5%). Conclusions and relevance Heat pretreatment of feline samples increased antigen detection by commercial assays for D immitis and improved overall concordance of antigen and antibody test results in antigen-positive samples in this population. Although further work to investigate the specificity of D immitis antigen assays when using pre-treated samples is warranted, this approach may be useful in the diagnosis of heartworm infection in individual cats and may increase the accuracy of surveys based on antigen detection.

Free-roaming domestic cat (Felis catus) populations serve as a valuable resource for studying ectoparasite prevalence. While they share a similar environment as owned cats, free-roaming cats do not receive routine veterinary care or ectoparasiticide application, giving insight into parasite risks for owned animals. We examined up to 673 infested cats presented to a trap-neuter-return (TNR) clinic in the central United States. Ectoparasite prevalences on cats were as follows: fleas (71.6%), ticks (18.7%), Felicola subrostratus (1.0%), Cheyletiella blakei (0.9%), and Otodectes cynotis (19.3%). Fleas, ticks, and O. cynotis were found in all months sampled. A total of 1117 fleas were recovered from 322 infested cats. The predominate flea recovered from cats was Ctenocephalides felis (97.2%) followed by Pulex spp. (2.8%), Cediopsylla simplex (0.6%), and Nosopsyllus fasciatus (0.6%). A total of 373 ticks were recovered from 126 infested cats. The predominate tick species was Amblyomma americanum (65.9%) followed by Ixodes scapularis (32.5%), Dermacentor variabilis (10.3%), and Rhipicephalus sanguineus (0.8%). Immature tick stages accounted for 54.7% of all ticks found, highlighting an under-appreciated source of tick burden on domestic cats. The results of this study emphasize the importance of year-round use of ectoparasiticides with both insecticidal and acaricidal activity on domestic cats.

Parasites of the genus Trichinella are zoonotic nematodes common in carnivores throughout the world. We determined the prevalence and species of Trichinella infections in Florida panthers (Puma concolor coryi).

Infection of cats with Dirofilaria immitis causes seroconversion on antibody tests and pulmonary pathology, often without subsequent development of adult heartworms. Consistent administration of topical 10% imidacloprid-1% moxidectin has been shown to result in sustained plasma levels of moxidectin in cats after three to five treatments, a pharmacokinetic behavior known as “steady state”.

Ascaris eggs are commonly used as a bioindicator to test the success of waste treatment systems. The ability to inactivate this organism's eggs indicates probable destruction of a variety of pathogens. The intent of these experiments was to determine operational parameters for a conical-augur device to treat human solid waste in a time-efficient manner (seconds), without exogenous chemicals (e.g. lime, an alkalizing agent) or an external heat source. Literature reviews and independent viscometry experiments established a target temperature range and residence time within the device. The ability of applied shear force to inactivate Ascaris suum, directly or indirectly, was assessed using viscometry. Shear force alone, as friction, failed to inactivate A. suum eggs. However, shear force was used to generate sufficient heat in a human-fecal simulant to inactivate A. suum eggs under a variety of test conditions. Target operating conditions for the conical-augur device were 70 °C with a 6-s residence time; increasing the temperature allowed for reduced residence time to achieve A. suum inactivation.

Diagnosis of Dirofilaria immitis infection in cats is complicated by the difficulty associated with reliable detection of antigen in feline blood and serum samples.