Background Heartworms, Dirofilaria immitis, are known to be widespread in dogs and cats in the USA, but there have been no country-wide prevalence studies performed to date. There have also been no large-scale studies to determine whether the closely related species, Dirofilaria repens, occurs in the USA. Methods To provide this large-scale data, we examined whole blood samples (n = 2334) submitted from around the USA to the Molecular Diagnostic Laboratory at Auburn University between 2016 and 2022. Quantitative PCRs for D. immitis (targeting 16S rRNA) and D. repens (targeting cytochrome c oxidase subunit 1 gene) were performed to determine the presence of Dirofilaria DNA. DNA sequencing was performed to confirm the results. Results Dirofilaria immitis DNA was found in 6.3% (68/1080) of the dogs from 17/39 states, and 0.3% (4/1254) of the cats from 4/42 states. None of the dogs or cats were positive for D. repens. The average 16S rRNA copy number of D. immitis in the dogs was 1,809,604 in 200 µl whole blood, while only a single copy was found in each of the four D. immitis-positive cats. The prevalence of D. immitis in dogs of different ages, sexes, and breeds did not differ significantly, but the prevalence in Southern states (7.5%, 60/803) was significantly higher than in the Western (1.7%, 1/58), Midwest (3.3%, 4/120), and Northeastern states (3.1%, 3/98) (P < 0.05). Dogs positive for D. immitis were identified in each study year (2016: 4.2%, 2/48; 2017: 9.8%, 4/41; 2018: 5.1%, 8/156; 2019: 4.9%, 15/306; 2020: 9.8%, 26/265; 2021: 4.9%, 13/264). Interestingly, dogs infected with Hepatozoon spp. (11.8%, 37/313) were significantly more likely to also be positive for D. immitis than dogs without evidence of Hepatozoon infection (3.9%, 30/760) (P < 0.0001). Conclusions To our knowledge, this is the first nationwide molecular survey of Dirofilaria spp. in dogs and cats in the USA, and the largest molecular survey of canine and feline dirofilariosis worldwide. Further studies are warranted to combine PCR with standard heartworm diagnostics to better understand the prevalence of Dirofilaria spp. and aid in determining the risks posed to dogs and cats in the USA. Graphical Abstract

Costa Rica harbors several flaviviruses, including Dengue (DENV), Zika (ZIKV), West Nile virus (WNV), and Saint Louis encephalitis virus (SLEV). While DENV and ZIKV are hyperendemic, previous research indicates restricted circulation of SLEV and WNV in animals. SLEV and WNV seroprevalence and high transmission areas have not yet been measured. To determine the extents of putative WNV and SLEV circulation, we sampled peri-domestic and domestic animals, humans, and mosquitoes in rural households located in two DENV and ZIKV hyperendemic regions during the rainy and dry seasons of 2017–2018 and conducted plaque reduction neutralization test assay for serology (PRNT) and RT-PCR for virus detection. In Cuajiniquil, serological evidence of WNV and SLEV was found in equines, humans, chickens, and wild birds. Additionally, five seroconversion events were recorded for WNV (2 equines), SLEV (1 human), and DENV-1 (2 humans). In Talamanca, WNV was not found, but serological evidence of SLEV circulation was recorded in equines, humans, and wild birds. Even though no active viral infection was detected, the seroconversion events recorded here indicate recent circulation of SLEV and WNV in these two regions. This study thus provides clear-cut evidence for WNV and SLEV presence in these areas, and therefore, they should be considered in arboviruses differential diagnostics and future infection prevention campaigns.

Arboviruses have two ecological transmission cycles: sylvatic and urban. For some, the sylvatic cycle has not been thoroughly described in America. To study the role of wildlife in a putative sylvatic cycle, we sampled free-ranging bats and birds in two arbovirus endemic locations and analyzed them using molecular, serological, and histological methods. No current infection was detected, and no significant arbovirus-associated histological changes were observed. Neutralizing antibodies were detected against selected arboviruses. In bats, positivity in 34.95% for DENV-1, 16.26% for DENV-2, 5.69% for DENV-3, 4.87% for DENV-4, 2.43% for WNV, 4.87% for SLEV, 0.81% for YFV, 7.31% for EEEV, and 0.81% for VEEV was found. Antibodies against ZIKV were not detected. In birds, PRNT results were positive against WNV in 0.80%, SLEV in 5.64%, EEEV in 8.4%, and VEEV in 5.63%. An additional retrospective PRNT analysis was performed using bat samples from three additional DENV endemic sites resulting in a 3.27% prevalence for WNV and 1.63% for SLEV. Interestingly, one sample resulted unequivocally WNV positive confirmed by serum titration. These results suggest that free-ranging bats and birds are exposed to not currently reported hyperendemic-human infecting Flavivirus and Alphavirus; however, their role as reservoirs or hosts is still undetermined.