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Background A variety of tick species infest dogs and cats in North America. Although most of these species also readily feed on people, national data regarding the species and abundance of ticks on dogs and cats are lacking. Here we report a large-scale study of ticks from dogs and cats in the USA over a 12-month period. Methods Tick submissions were invited from veterinary practices in all 50 states. Ticks were submitted with information about the pet and the attachment sites of each tick marked on a biopsy chart. Upon receipt, ticks were identified to species and stage using morphologic keys; when necessary, species identification was confirmed molecularly. Results From February 2018 through January 2019, 10,978 ticks were submitted from 1494 dogs and 336 cats in 49 states and ticks were collected in every month. Dog and cat infestation intensities ranged from 1 to 4765 and from 1 to 38 (median = 1, mean = 6.7 and 2.6), respectively. Dogs were primarily infested with Dermacentor variabilis (532/1494; 35.6%), Ixodes scapularis (409/1494; 27.4%), Amblyomma americanum (345/1494; 23.1%) and Rhipicephalus sanguineus (172/1494; 11.5%). Cats were primarily infested with I. scapularis (156/336; 46.4%), A. americanum (99/336; 29.5%) and D. variabilis (60/336; 17.9%). Other submitted ticks included A. maculatum , Haemaphysalis longicornis , Otobius megnini , and less common Dermacentor spp. and Ixodes spp. Co-infestations were documented in 93 dogs and 14 cats. Reported attachment sites of common tick species differed. In dogs, A. americanum was most commonly attached to the abdomen, axillary, and inguinal regions; D. variabilis and I. scapularis to the head, neck, and back; and R. sanguineus to the head, neck, abdomen, legs, and feet. In cats, I. scapularis was most commonly attached to the head and A. americanum was most commonly attached to the tail and perianal region. Conclusions These data confirm that dogs and cats in the USA are at risk of tick infestation throughout the year and that tick species present in the region have apparent attachment site preferences.

Background Current recommendations for diagnosis of Dirofilaria immitis infection in dogs rely on the detection of antigen produced largely by adult females coupled with the visualization of microfilariae (mf) in the circulation via a microfilaria detection test (MFDT). It is hypothesized that qPCR assays used in parallel with antigen detection tests will perform better in detecting mf than modified Knott’s test (MK), when combined with antigen detection. This study compares probe-based qPCR and MK techniques for mf detection used in parallel with the DiroCHEK ® antigen test to screen for heartworm infection in shelter dogs. Methods Matching blood and serum samples were collected from 300 shelter dogs in Brazos and Harris counties, Texas, USA. Blood was assessed for the presence of mf via MK and the presence of D. immitis DNA by a species-specific probe-based qPCR assay. Serum samples were tested for the presence of heartworm antigen using DiroCHEK ® before and after immune complex dissociation (ICD) via heat treatment. In addition, the performance of each diagnostic test was evaluated via Chi-square test, Cochran’s Q test, and post hoc analysis. Results Qualitatively, MK detected mf in 22.0% (66/300) of samples, 55 of which were morphologically identified as D. immitis and 11 as Acanthocheilonema reconditum . The range of heartworm mf was 28 to 88,803 mf/ml (median: 6627.5). Real-time PCR detected D. immitis DNA in 20.7% (62/300) of samples. Heartworm antigen was detected in 24.7% (74/300) of samples pre-ICD, and in 29.3% (88/300) post-ICD. When comparing tests, the Chi-square and McNemar’s tests showed that the difference between positive and negative proportions was statistically significant. The Cochran test showed the difference in the distributions of cases and non-cases was significant when individual tests were combined ( χ 2  = 62.3, df  = 3, P  < 0.0001) and when parallel methods were combined ( χ 2  = 43.1, df  = 4, P  < 0.0001). Conclusion Considering individual and combined test performances, practicality, and efficient use of bench time, this heartworm-specific probe-based qPCR method is a viable option as a mf detection test to be used in parallel with antigen tests for canine heartworm infection in diagnostic and research settings. Graphical Abstract

Data on vector-borne pathogens infecting dogs from sub-Saharan Africa is limited. In this study, we assessed the prevalence of VBPs, their associated risk factors, and pathogen interactions in domestic dogs. Whole blood samples were obtained for 1202 apparently healthy dogs in Chad from September to October 2021, and nucleic acids were extracted and then subjected to a targeted next-generation sequencing (tNGS) assay for detection of 15 VBPs. Overall, 88.7% of the dogs were positive for at least one pathogen, and 62.9% were coinfected with two or more VBPs. The most frequent pathogen detected was Hepatozoon canis in 62.4% of the dogs, Mycoplasma haemocanis in 59.2%, Anaplasma platys in 29.2%, Candidatus Mycoplasma haematoparvum in 21.2%, Ehrlichia canis in 20.3%, Babesia vogeli in 2.0% and Candidatus Mycoplasma turicensis in 1.5%. While most of the dogs (62.9%) were co-infected with two or more VBPs, having an infection with three pathogens (30.8%) was more common. According to multivariable logistic regression analysis, being a senior dog and residing in Chari Baguirmi south were identified as potential risk factors for infection by most of the pathogens. Network analyses revealed complex interactions suggesting facilitative associations among VBPs. These results are useful in expanding the knowledge of VBPs in Africa and establishing a baseline for downstream studies into hemotropic mycoplasmas.

Background Canine vector-borne diseases (CVBDs) are illnesses caused by pathogens transmitted by blood-feeding arthropods such as ticks and mosquitoes. Many CVBDs, including dirofilariosis, anaplasmosis, and ehrlichiosis, are globally distributed and may cause a variety of clinical signs in dogs. Several CVBD agents are zoonotic, making epidemiological surveillance a joint veterinary and public health effort. In this study, we determined the seropositivity of four pathogens from dogs on Saipan, Northern Mariana Islands, a US Commonwealth located in the western Pacific Ocean. Methods Blood samples ( n  = 443) were collected from client-owned, owner surrendered, and shelter dogs that participated in an island-wide spay-and-neuter event in 2023. All samples were assessed using a commercial, point-of-care enzyme-linked immunosorbent assay (ELISA) test (SNAP ® 4Dx ® Plus, IDEXX Laboratory, Westbrook, Maine, USA) to detect the Dirofilaria immitis antigen and antibodies against Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi sensu lato. Risk factors were assessed for each pathogen through a univariate analysis, followed by a multivariable logistic regression. Results Overall, 66.1% ( n  = 300/443) of the dogs tested positive for at least one pathogen, with the highest prevalence observed for Ehrlichia spp. (58.0%; n  = 246/443), followed by Anaplasma spp. (43.1%; n  = 184/443) and D. immitis (14.8%; n  = 63/443). Among the dogs with a single pathogen detected (30.9%; n  = 137/443), Ehrlichia spp. was most prevalent (64.9%; n  = 89/137), followed by Anaplasma spp. (23.3%; n  = 32/137) and D. immitis (11.6%; n  = 16/137). For co-detection of two or more pathogens (36.7%; n  = 163/443), Ehrlichia spp. +  Anaplasma spp. presented the highest frequency (70.5%; n  = 115/163), followed by Ehrlichia spp. +  D. immitis (6.7%; n  = 11/163), Anaplasma spp. +  D. immitis (3.6%; n  = 6/163), and Ehrlichia spp. +  Anaplasma spp. +  D. immitis (19.0%; n  = 31/163). Age ( P  = < 0.001), residing district ( P  = 0.001), and ownership status ( P  = < 0.001) were significantly associated with D. immitis positive status in a univariable analysis. Age ( P  = < 0.001), residing district ( P  = 0.177), and ownership status ( P  = 0.014) were significant in a univariable analysis with Ehrlichia spp. as an outcome. Finally, Anaplasma spp. had a significant association with ownership status ( P  = < 0.001) as a risk factor in a univariable analysis. Conclusions This study shows high seropositivity for CVBPs in a dog population living in a poorly studied area. The results of this study suggest that strategies for the prevention and control of these CVBDs should be reinforced on the Island of Saipan. Graphical Abstract

African Swine Fever virus (ASFv) is a re‐emerging global swine disease that, if introduced to the United States, would cause severe economic consequences. The widespread presence of feral hogs in addition to the presence of competent tick vectors, specifically Ornithodoros turicata, fosters a greater risk of ASFv establishment. The specific aims of this study were to assess the geographic distribution of O. turicata, the spatial distribution of host species richness, and to identify localities that could be monitored for ASFv as part of a comprehensive surveillance system in the United States moving forward. A systematic literature review and field collections were conducted to identify O. turicata localities. Ticks were collected from 15/16 surveyed Texas counties and were identified using standard morphological keys and confirmed molecularly on a subset of ticks via amplification and sequencing of a 16S rRNA gene fragment. Ecological niche modeling was used to determine the suite of bioclimatic variables associated with the presence of O. turicata based on field collections and the literature review. Six variables of importance were identified: mean temperature of the warmest quarter, mean temperature of the coldest quarter, annual precipitation, precipitation of the wettest month, precipitation of the warmest quarter, and elevation. Subsequently, a map was constructed of the potential distribution of this species, which stretched from southern California to Texas with an allopatric population in Florida. The majority of Texas, with the exclusion of the easternmost quarter of the state, appears to be highly suitable for this species. Host species richness was greatest in Florida and central Texas, suggesting that these regions should be subjected to targeted surveillance priorities. Furthermore, the presence of O. turicata in burrows occupied by feral common warthogs (Phacochoerus africanus) in south Texas is noteworthy, as warthogs are known to sustain ASFv in a sylvatic cycle in Africa. Establishing the current and projected distribution of O. turicata is essential to understanding the potential sylvatic cycle of ASFv and creating long‐term surveillance zones if ASFv is introduced to the United States. Ornithodoros turicata collected from a dry ice baited tick trap.

Background The canine heartworm Dirofilaria immitis, a filarioid nematode of dogs and other carnivores, is widespread in the USA and the world. Over 20 different mosquito species serve as intermediate hosts of D. immitis , but their contribution to transmission varies according to factors like host feeding patterns, geographic locations and climatic conditions. The yellow fever mosquito, Aedes aegypti, is a competent vector of D. immitis but is often dismissed as a vector of veterinary relevance given its anthropophilic feeding behavior. We evaluated the prevalence of D . immitis in pet dogs along the USA-Mexico border and assessed whether Ae . aegypti in the area are naturally infected with heartworm and are potentially acting as a vector. Methods A total of 200 whole blood samples collected from pet dogs in the Lower Rio Grande Valley in south Texas from 2016 to 2019 were included in this study. Canine serum samples for D. immitis were tested using the DiroCHEK® Canine Heartworm Antigen Test Kit pre- and post-immune complex dissociations (ICD) and blood samples were tested using high-resolution melt (HRM) quantitative PCR (qPCR) and a probe-based qPCR. Additionally, mosquito specimens were collected and identified, and Ae. aegypti heads, abdomens and pools were tested using conventional PCR (cPCR) and HRM qPCR. Results Overall, heartworm prevalence in dogs aged > 6 months was 40.8% (64/157) when the results from all testing modalities were considered. Heartworm antigen was detected in 33.5% and 40.7% of the dogs using DiroCHEK® pre- and post-ICD, respectively. By molecular screening, 20.1% of dogs tested positive with probe-based qPCR, while only one tested positive with HRM qPCR. Of the Ae. aegypti abdomens from blood-fed Ae. aeygpti tested, 20 (21.7%) from mosquitoes that fed on dogs and four (7%) from those that fed on humans tested positive for heartworm. Among Ae. aegypti heads from blood-fed Ae. aeygpti , two (1.1%) were positive based on cPCR and four (2.5%) were positive based on HRM qPCR. No D. immitis DNA was detected in the 208 pools of whole bodies (358 individuals) of Ae. aegypti gravid females. Conclusions Our study highlights a high prevalence of heartworm in dogs in south Texas and provides evidence that Ae. aegypti could be contributing to heartworm transmission in canine populations in this region. Graphical Abstract

Abstract Background An evaluation of currently available in-clinic diagnostic tests for Giardia duodenalis infection of dogs and cats has not been performed. In addition, there is discordance among published diagnostic comparisons. The absence of a true gold standard for detecting Giardia duodenalis also complicates diagnostic evaluations. Objectives To evaluate diagnostic tests commercially available in the United States for detecting Giardia duodenalis in dogs and cats, in comparison to a widely used reference test, the direct immunofluorescent assay (IFA), and also to compare the results of 2 methods of analysis: comparison of diagnostic tests to a reference test (IFA) and Bayesian analysis. Animals Fecal samples from a convenience sample of 388 cats and dogs located in Colorado, Oklahoma, and Virginia. Methods Fecal samples were tested for Giardia duodenalis by zinc sulfate centrifugal fecal flotation and 4 different commercial diagnostic immunoassays. Results were analyzed via Bayesian analysis and by comparison to the IFA as the reference test. Results Sensitivity and specificity by comparison to IFA was ≥82% and ≥90%, respectively, for all diagnostic tests in dogs and cats. When analyzed via Bayesian analysis, sensitivity and specificity were ≥83% and ≥95%, respectively. When ZnSO4 centrifugal fecal flotation results were combined with immunoassay results, there was no longer a significant difference between the sensitivities of the commercial in-clinic immunoassays. Conclusion and Clinical Relevance The Bayesian analysis validates using IFA as the reference test. Differences in commercial in-clinic immunoassay sensitivities can be mitigated when the results are combined with ZnSO4 centrifugal fecal flotation results.

An amendment to this paper has been published and can be accessed via the original article.

Throughout North America, Dermacentor spp. ticks are often found feeding on animals and humans, and are known to transmit pathogens, including the Rocky Mountain spotted fever agent. To better define the identity and distribution of Dermacentor spp. removed from dogs and cats in the United States, ticks submitted from 1,457 dogs (n = 2,924 ticks) and 137 cats (n = 209 ticks) from veterinary practices in 44/50 states from February 2018-January 2020 were identified morphologically (n = 3,133); the identity of ticks from regions where Dermacentor andersoni (Stiles) have been reported, and a subset of ticks from other regions, were confirmed molecularly through amplification and sequencing of the ITS2 region and a 16S rRNA gene fragment. Of the ticks submitted, 99.3% (3,112/3,133) were Dermacentor variabilis (Say), 0.4% (12/3,133) were D. andersoni, and 0.3% (9/3,133) were Dermacentor albipictus (Packard). While translocation of pets prior to tick removal cannot be discounted, the majority (106/122; 87%) of Dermacentor spp. ticks removed from dogs and cats in six Rocky Mountain states (Montana, Idaho, Wyoming, Nevada, Utah, and Colorado) were D. variabilis, suggesting this species may be more widespread in the western United States than is currently recognized, or that D. andersoni, if still common in the region, preferentially feeds on hosts other than dogs and cats. Together, these data support the interpretation that D. variabilis is the predominant Dermacentor species found on pets throughout the United States, a finding that may reflect recent shifts in tick distribution. Graphical Abstract

Throughout North America, Dermacentor spp. ticks are often found feeding on animals and humans, and are known to transmit pathogens, including the Rocky Mountain spotted fever agent. To better define the identity and distribution of Dermacentor spp. removed from dogs and cats in the United States, ticks submitted from 1,457 dogs (n = 2,924 ticks) and 137 cats (n = 209 ticks) from veterinary practices in 44/50 states from February 2018-January 2020 were identified morphologically (n = 3,133); the identity of ticks from regions where Dermacentor andersoni (Stiles) have been reported, and a subset of ticks from other regions, were confirmed molecularly through amplification and sequencing of the ITS2 region and a 16S rRNA gene fragment. Of the ticks submitted, 99.3% (3,112/3,133) were Dermacentor variabilis (Say), 0.4% (12/3,133) were D. andersoni, and 0.3% (9/3,133) were Dermacentor albipictus (Packard). While translocation of pets prior to tick removal cannot be discounted, the majority (106/122; 87%) of Dermacentor spp. ticks removed from dogs and cats in six Rocky Mountain states (Montana, Idaho, Wyoming, Nevada, Utah, and Colorado) were D. variabilis, suggesting this species may be more widespread in the western United States than is currently recognized, or that D. andersoni, if still common in the region, preferentially feeds on hosts other than dogs and cats. Together, these data support the interpretation that D. variabilis is the predominant Dermacentor species found on pets throughout the United States, a finding that may refect recent shifts in tick distribution.