Understanding the range of vectors and pathogen diversity in sub-Saharan Africa is crucial for efficient disease prevention and control efforts. Domestic dogs are susceptible to many vector-borne pathogens (VBPs) and serve as sentinels for several human pathogens. Yet, critical surveillance gaps in canine VBPs persist in Central Africa, where the tropical climate and pastoral agro-livelihoods create unique transmission risks. We conducted a cross-sectional survey from September to October 2021, collecting 4238 ticks from 1254 domestic dogs across 56 villages in Chad, Africa. All ticks were identified morphologically and/or by sequence analysis of 16S rDNA, 12S rDNA, and cytochrome c oxidase subunit I (COI) gene regions. A representative subset (n = 618) was subsequently screened for comprehensive, simultaneous detection of 21 VBPs using a targeted next-generation sequencing (tNGS) assay. Logistic regression models were used to assess associations between potential risk factors and pathogen detection. Five tick species belonging to four genera were identified: Rhipicephalus linnaei (89.8

Anthelmintic resistance (AR) in gastrointestinal nematodes (GIN) is an emerging concern in grazing livestock, including the North American bison (Bison bison). Nevertheless, little is known about routine parasite control practices in ranched bison herds. This cross-sectional survey of bison producers in 22 states across the southern and central United States characterized herd demographics, pasture management, nutrition, animal sourcing, and anthelmintic use. Seventy-nine producers from 15 states responded, most managing herds on fewer than 300 acres. Parasite control was predominantly drug-centric: 87.3% (69/79) dewormed at least once annually, 31.6% (25/79) treated in both warm and cool seasons, and nearly one-third dewormed more than twice per year. Macrocyclic lactones (ivermectin: 65.7%, moxidectin: 29.9%) and benzimidazoles (fenbendazole: 50.8%, albendazole: 28.4%) were widely used, frequently in rotation or combination; 28.1% (18/64) administered multiple dewormers simultaneously. Only 34.8% (23/66) dosed based on individual body weights, while most relied on visual estimates or herd averages, and fewer than 10% (7/70) intentionally left animals untreated as refugia. Although 64% (46/72) and 65% (47/72) reported moderate-to-high concern (≥3/5), about internal nematodes and AR, respectively, routine diagnostics (fecal egg counts, fecal egg count reduction tests, targeted selective treatment) were rarely employed. These findings reveal that intensive whole-herd deworming, minimal diagnostics, and limited refugia are common within the US bison industry, likely increasing selection pressure for AR and underscoring the need for bison-specific, evidence-based, diagnostic-driven parasite control guidelines and education.

Abstract Background Dirofilaria immitis , the causative agent of canine heartworm disease, is a mosquito-borne filarial nematode, endemic to most of North America. Clinical signs may include coughing, exercise intolerance, abnormal heart sounds, and lethargy, which can lead to heart failure and death in severe cases. For accurate diagnosis, the American Heartworm Society (AHS) recommends testing dogs annually for the detection of heartworm antigens and microfilariae. In this study, we assessed the prevalence and potential risk factors for D. immitis in dogs from Albuquerque, New Mexico, using antigen and microfilariae detection tests. Methods In 2023, serum and whole-blood samples ( n = 402) were collected from dogs in Albuquerque, New Mexico, as part of a larger study to detect Onchocerca lupi . Serum samples were screened for D. immitis using a commercially available antigen-detection ELISA (DiroCHEK ® , Zoetis) with pre- and post-immune complex dissociation (ICD) via heat treatment, although this is not currently recommended by AHS. Following genomic DNA extraction, we also screened whole blood using a newly optimized probe-based real-time polymerase chain reaction (qPCR) assay. To assess the potential association between D. immitis presence and risk factors, including age, sex, breed group, and coat color, a univariate analysis was performed using Fisher’s exact test or the chi-squared test. Results Serum of 4 (0.99%) dogs tested antigen-positive pre-ICD, while 14 (3.47%) dogs tested antigen-positive post-ICD. We did not detect any positive dogs for D. immitis microfilariae by the probe-based qPCR. No risk factors were associated with the detection of the D. immitis antigen in this dog population. Conclusions Although microfilariae were not detected in this study, it is possible that infections were in the early stages of disease progression, with D. immitis detected in pre- and post-ICD via antigen testing. Low seroprevalence and the absence of microfilariae DNA detected highlight the importance of using both pre- and post-ICD antigen detection alongside molecular-based microfilariae tests, particularly for companion animals living in low-endemic regions and for shelter populations that are less likely to be on heartworm prevention.

Tick-borne filarial nematodes are a complex and understudied group of parasites that rely on ticks for transmission in vertebrates. This review examines how intrinsic and extrinsic factors may influence the successful transmission of filarial nematodes in tick vectors, drawing insights from extensively studied haematophagous dipteran vector systems. We explore how different tick life stages contribute to potential nematode transmission to the host and the influence of abiotic factors on nematode survival and development within ticks. Understanding nematode life stage periodicity and enhancement phenomena in tick-borne filarial systems is equally important for determining transmission dynamics and co-infection patterns. Also important to nematode transmission are vertebrate host movement and tick feeding patterns, which create opportunities for parasite exchange across ecological environments. Knowledge gaps include the physical and molecular mechanisms of transmission, the potential influence of tick genetic variation on vector competence, immune responses across different tick-filarial associations, and epidemiological studies of host and tick patterns of nematode infection. Comprehensive empirical data are also needed to model transmission patterns, including temperature and humidity thresholds for nematode development and survival, field-based tick infection rates, and how tick immune responses affect these processes. Understanding these factors requires integrating expertise from multiple disciplines and adopting an ecosystem-based perspective that considers the interconnected nature of vertebrate hosts, vectors, and environment in the transmission of filarial nematodes.

Gastrointestinal (GI) parasites of dogs, including helminths and protozoans, are of substantial relevance to veterinary medicine and public health. Nevertheless, epidemiological data are scarce worldwide, especially in remote locations. The emergence of novel technologies and diagnostic platforms facilitates comprehensive screening of multiple GI parasites. Our study aims to establish a baseline prevalence for GI parasites in dogs from Saipan, Northern Mariana Islands. Fecal samples were collected from dogs (n = 420) from May to June 2023 during a spay-neuter campaign. Age, sex, ownership status, and residing location were recorded. Following genomic extraction, samples were screened using the KeyScreen™ GI Parasite PCR (Antech Diagnostics), a real-time PCR panel that detects 20 endoparasite infections, detects benzimidazole resistance in Ancylostoma caninum, and determines the zoonotic potential of Giardia duodenalis. If inconclusive results for Ancylostoma spp. were obtained, conventional PCR and Sanger sequencing were performed, targeting the ITS-1 region for species identification. Additionally, demographics (i.e. age, sex, ownership, and residing location) were evaluated as potential risk factors for each pathogen as the outcome with an initial univariate analysis, followed by multivariable logistic regression with backward stepwise selection. Overall, parasites were detected in 267/420 (63.5

Canine leishmaniosis (CanL) is caused by Leishmania species. While vertical transmission is the primary route of CanL transmission in the USA, the role of sand fly vectors in its transmission and the prevalence in the broader pet population remain under-investigated. Diagnosis of CanL may include serology, PCR, or histopathology. However, ELISA tests are seldom used for the diagnosis of CanL in the USA despite their preference in endemic regions. Firstly, a serological and molecular survey was conducted in Oklahoma and Texas using the SNAP™ Leishmania Test (IDEXX Laboratories Inc.) on canine blood or serum, while serum only was used for commercial indirect immunofluorescence assay (IFA) testing. Molecular testing for Leishmania spp. included targeting segments of the small subunit rRNA gene by qPCR, the minicircle kinetoplast using a dPCR assay (QIAGEN), and a confirmatory Vector-borne Disease Panel (Antech Diagnostics). Secondly, sand fly trapping and testing were conducted in Oklahoma and Texas with morphological and molecular identification targeting the ITS-2 gene, as well as qPCR for Leishmania spp. Finally, a nationwide serological survey of apparently healthy dogs was conducted using the SNAP Leishmania Test and confirmatory testing with a commercial IFA test. In the regional survey, seroprevalence was 0.61

Canine heartworm, a parasitic zoonosis caused by Dirofilaria immitis, is expanding geographically. This study investigated the prevalence, associated risk factors, and molecular characteristics of D. immitis in dogs from Ilhéus, Bahia, a coastal tourist region in northeastern Brazil previously considered non-endemic. In a cross-sectional study with 481 domestic dogs, blood samples were tested for adult D. immitis antigens (SNAP® 4Dx® Plus) and microfilariae (modified Knott's test). Risk factors were analyzed using logistic regression and spatial analysis, including the association with the Normalized Difference Vegetation Index (NDVI). Positive samples underwent polymerase chain reaction (PCR), sequencing, and phylogenetic analysis targeting the cox1 gene. The overall prevalence of D. immitis, defined as positive in at least one test, was 19.6% (84/428; 16.0%-23.7%). Seroprevalence was 16.0% (77/481; 12.7%-19.3%), while microfilaremia was 14.3% (61/428; 11.2%-17.9%). Most infected dogs were asymptomatic (71.4%). Larger dogs, those kept outdoors, and those exposed to environmental risk factors (e.g., lack of doghouse or standing water) showed significantly higher odds of seropositivity. The presence of dense vegetation (NDVI median = 0.51) and a rural environment were also associated with increased risk. Spatial analysis revealed a cluster of cases in the southern area of the municipality. Phylogenetic analysis indicated high genetic similarity between local isolates and those from other regions worldwide. These findings confirm the circulation of D. immitis in Ilhéus and highlight the role of environmental and management factors in transmission. The high proportion of asymptomatic carriers reinforces the need for regional surveillance and prevention strategies.

Heterobilharzia americana is a trematode parasite that causes canine schistosomiasis, a disease capable of causing significant morbidity and mortality in dogs. Reliable diagnosis is essential for limiting disease progression. This study aimed to optimize and validate a previously developed TaqMan qPCR assay for detection of H. americana DNA in feces. The assay targets a highly repetitive non-coding DNA sequence located across multiple loci within the H. americana genome. PCR optimization involved gradient thermocycling and serial dilutions to refine annealing temperature and efficiency. PCR products were cloned and sequenced to confirm target specificity. Analytical sensitivity was assessed using serial two-fold dilutions of H. americana eggs spiked into feces and matrix-free samples, with PCR detection across replicates. Diagnostic sensitivity was assessed in 111 fecal samples from infected dogs using fecal sedimentation as gold standard. Analytical and diagnostic specificities were assessed by testing 54 fecal samples containing non-target parasites and 100 fecal samples from H. americana-uninfected dogs, respectively. Target specificity was confirmed by BLAST. Assay efficiency was 107 %. Matrix-dependent analytical sensitivity was 3 eggs/g feces (100 % replicates) and 1.5 eggs/g feces (60 % replicates); matrix-free sensitivity was 3 eggs/mL in saline. Diagnostic sensitivity was 98.2 % (95 % CI: 93.7-99.8 %). Both analytical and diagnostic specificities were 100 % (95 % CI: 93.6-100 % and 96.4-100 %, respectively). The assay demonstrated low intra-assay and inter-assay variability and minimal inter-operator variability across the dilution range tested. These findings support the optimized TaqMan qPCR assay as a highly sensitive and specific tool for detecting H. americana DNA in dog feces.

The current American Heartworm Society guidelines recommend the concomitant use of an antigen detection test and a microfilariae detection test (MFDT) for diagnosing heartworm infection in dogs. The modified Knott’s (MK) test is the preferred MFDT for determining the morphological characteristics of Dirofilaria immitis microfilariae, but it requires extensive microscopy training and can be time-consuming in a clinical setting. The Pluslife Mini Dock is a point-of-care diagnostic platform that uses RNase HII-assisted amplification (RHAM) to eliminate the need for DNA extraction, with results available within 30 min. This study aimed to assess the performance of the Pluslife Mini Dock duplex Dirofilaria immitis/Dirofilaria repens assay in dog blood compared with the MK. Archival, frozen whole-blood samples collected from 250 dogs at shelters in central Texas, USA, were used. Samples were subjected to the MK on the day of collection and stored at 2°C until further processing. The samples were then thawed and subjected to the Pluslife Mini Dock D. immitis/D. repens duplex assay. The results were analyzed using Cohen’s kappa coefficient and McNemar’s Chi-squared test. Overall, 93.6

Dracunculiasis, or Guinea worm disease (GWD), was targeted for eradication in 1986 after which, the annual incidence decreased by over 99.9%. As human cases of GWD near elimination, dogs have become the primary reservoir for the parasite and remain a challenge for eradication efforts. The high burden of GWD in dogs relative to humans highlights the need for therapeutic interventions in conjunction with behavioral interventions. Laboratory experiments in ferrets demonstrated that flubendazole is partially effective in inhibiting the infectivity of D. medinensis larvae. However, the implementation of large clinical field trials in dogs has proven challenging in settings where access to study areas is unreliable throughout the year and attrition is high. Alternative study design approaches are required to address these challenges. This paper outlines challenges encountered during a previous field trial and ways in which these challenges were addressed in the study design and implementation for a second clinical trial of flubendazole in dogs. The results of the second clinical trial are outlined in a separate manuscript.

The family Onchocercidae, or filarioid nematodes, comprises common vector-borne parasitic worms that can infect, among other vertebrates, wild North American ruminant ungulates. Infection is a product of the available hosts and vectors distributed in viable ecosystems that permeate throughout the North America (i.e., Canada, United States, and Mexico) and Central America (i.e., Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, and Panama). The formation of these host-parasite assemblages can produce serious to no apparent clinical signs for the infected host animal depending on a multitude of factors. Overall, the scope of our knowledge regarding filarioid nematode infection of North American wild ruminant ungulates remains spotty and neglected throughout the scientific literature. However, reviewing these details provides a foundation for important biological, historical, ecological, and one health relevant questions. Analysis of the current scientific literature reveals that, at this time, nine out of fourteen wild North American ruminant ungulates can be infected with at least one species of filarioid worm. This includes four genera of known Onchocercidae species circulating within the North and Central American ecosystems: Elaeophora, Onchocerca, Rumenfilaria, and Setaria.

Black skimmers (Rynchops niger niger) are New World coastal seabirds that breed along the Gulf of Mexico, southern Atlantic and Pacific coasts of the United States. Black skimmer populations are declining in North America, and little is known about health and diseases of this species. An outbreak of mortality causing the death of approximately 160 juvenile black skimmers occurred in the West Galveston and East Matagorda Bay populations during the 2022 and 2023 breeding seasons. Necropsy, histopathology, and molecular genetic methods were utilized to investigate the cause of morbidity in six chicks that died or were euthanized. The main gross findings included bilateral renomegaly with multiple pale tan foci. Characteristic histologic findings were granulomatous nephritis and cloacitis with intralesional coccidia. Various developmental stages including immature gamonts, microgamonts, macrogamonts, and oocysts were present extracellularly and in the cytoplasm of epithelial cells of distal tubules, medullary collecting ducts, ureters, and cloacal epithelium. A few oocysts were in the cytoplasm of multinucleated giant cells. Additionally, schizonts were observed in the small intestine of one case. Genetic analysis of partial 18S rRNA gene revealed a previously uncharacterized Eimeria lineage affecting kidney and cloaca and another Eimeria lineage affecting the small intestine. To the best of our knowledge, this is the first report of Eimeria infection in the genus Rynchops. The significance of renal and intestinal coccidiosis in black skimmers is unknown; however, in the present work it appeared to cause morbidity and likely contributed to the recent mortality outbreak.

Giardia duodenalis is a protozoal parasite that infects a wide range of animal hosts including dogs and humans. Although social determinants of health (SDOH) are well characterized in human disease, their role in shaping animal health outcomes remains poorly understood. This ecological study, guided by One Health principles, evaluated temporospatial trends in canine Giardia infection in Texas and assessed associations with county-level human socioeconomic indicators. Veterinary diagnostic test results from commercial laboratories during 2012-2022 were analyzed to characterize temporal and geographic variation in test positivity. Negative binomial regression models were used to examine associations between Giardia positivity and county-level measures of poverty, unemployment, income, educational attainment, and veterinary care accessibility scores (VCAS). Cumulatively, 4.6% of canine tests were positive at the state level, with substantial county-level variation (median 4.6% [range 0.6-12.1%]; mean 4.9% [CI 4.1-5.8%]). Higher positivity was associated with limited veterinary care access (uPR 2.0 [CI 1.4-3.0], P value < 0.001) and a lower proportion of college graduates (uPR 1.6 [CI 1.2-2.1], P value < 0.001). Temporal increases in positivity were observed in Cameron County and Lubbock County, whereas decreases were noted in El Paso County and Parker County. These findings suggest that socioeconomic factors related to veterinary access and education are associated with canine Giardia infection risk and may help identify regions where targeted interventions are warranted. However, further studies are needed to elucidate the underlying mechanisms and zoonotic implications.

Onchocerca lupi, a zoonotic filarial nematode documented in southwestern North America and the Old World, is associated with ocular onchocercosis in dogs and cats. Clinical signs range from ocular discharge and conjunctivitis to chronic nodular lesions involving the conjunctiva, sclera, and retrobulbar space. Many infections remain subclinical and undiagnosed. Current diagnostic approaches rely on microscopy and conventional PCR (cPCR) to confirm adult worms in nodules or microfilariae in skin snips. Real-time PCR (qPCR) has been applied primarily in large-scale epidemiological studies. However, amplification of O. lupi DNA may be inhibited, leading to false-negative results. Highly sensitive techniques such as droplet digital PCR (ddPCR) can enable the detection of parasite DNA in various biological samples through absolute quantification. The objectives of this study were to: (I) validate a novel ddPCR assay that detects O. lupi, and (II) assess the performance of the ddPCR assay compared to cPCR and qPCR in suspected clinical cases. Following assay validation and optimization, 202 suspected clinical cases from 11 US states were assessed. Of these clinical cases, 97

Moose are significant ecological, economical and cultural animals for the stakeholders of Alaska, USA. Thus, the impact of pathogens, like filarial nematodes, is a critical area of moose research. These vector-borne parasites, including Setaria yehi, and Rumenfilaria andersoni, can lead to severe health consequences (e.g., peritonitis). However, little is known about filarial nematode distribution, diversity and its associated life cycle with Alaskan moose hosts. Newly developed next-generation sequencing techniques offer the ability to efficiently screen multiple species of co-infecting filarial nematodes in a single sample and thus improve our ability to monitor and understand these parasites. Blood collected from wild moose in the Kenai Peninsula, AK, was screened using deep amplicon sequencing (DAS) with filarial nematode primers targeting the cytochrome oxidase c subunit 1 (cox1) gene. In addition, samples subjected to DAS were also screened using the Modified Knott's Test (MKT). Setaria yehi and R. andersoni were detected by both diagnostic methods. Overall, 190 moose samples were tested via DAS, with filarioid DNA being detected in 51.58% (98/190) of these. Out of a subset of 138 samples, filarioid nematodes were found in 50.72% (n = 70) and 57.25% (79/138) via DAS and MKT, respectively. However, 18 (13.04%) co-infections were detected by DAS compared to 12 (8.70%) identified via MKT. A DAS molecular tool for surveillance has several advantages when paired with host blood collection metadata (i.e., years, season, region, host age) to better understand filarial nematode life cycle and ecology.

Echinococcus multilocularis is a zoonotic cestode that uses canids as definitive hosts and rodents as intermediate hosts. In humans, this parasite is the causative agent of alveolar echinococcosis. Recently, its range has been expanding across the Northern Hemisphere, and it is increasingly detected in wild canids, domestic dogs, and humans across Canada and the United States. While this expansion has been documented in isolated studies across the continent, a lack of routine sampling in wildlife hinders our ability to anticipate and mitigate further spread of E. multilocularis. We confirmed the presence of E. multilocularis in Washington State, USA, using a combination of morphological and molecular techniques across carcasses and field-collected scats of coyotes (Canis latrans), this region's most common wild canid. Morphological identification of adult worms was confirmed by next-generation sequencing. Over a third of all samples tested positive for E. multilocularis when all methodologies were combined. Sequencing revealed a haplotype of E. multilocularis matching a documented haplotype originally of European origin in British Columbia, Canada. Our study provides the first confirmation of E. multilocularis in a wild host on the west coast of the contiguous United States and provides additional haplotype information crucial to tracking the geographical expansion of the parasite. We also provide a new next-generation sequencing primer targeting cestodes of canids. The difference in amplification between intestinal and fecal samples suggests that non-invasive fecal sampling using DNA metabarcoding - a popular method of helminth surveillance - may lead to underestimation of prevalence, hindering control measures. The global significance of these findings extends beyond North America; E. multilocularis is a major public health concern in Europe and Asia, where alveolar echinococcosis is increasingly diagnosed in humans. Our study highlights the urgent need for increased surveillance and improved diagnostic strategies worldwide, particularly in regions with significant human-wildlife contact.

Canine babesiosis is an emerging tick-borne disease of worldwide distribution, caused by protozoa of the genus Babesia, and characterized by hemolytic anemia. In the United States (US), Babesia vogeli, Babesia gibsoni, Babesia conradae, and Babesia vulpes have been recognized as the main species causing babesiosis in dogs, the latter species also infecting foxes and raccoon dogs across Europe, Asia, and North America. In this study, we evaluated the prevalence of Babesia spp. infections in shelter dogs from Central Texas. Genomic DNA was extracted from blood samples (n = 446) of dogs, followed by conventional PCR targeting the 18S ribosomal RNA gene and sequencing of positive amplicons. The association between demographic variables (i.e., age, sex, breed group) and the outcome (B. vulpes positivity) was evaluated through univariate Pearson's chi-squared tests. We found an overall prevalence of 6.28% (n = 28; CI 95% 4.39-9.20) within this shelter dog population. A total of 12 distinct haplotypes were identified among these isolates. No statistical association between the positivity of B. vulpes and demographic variables was identified. This study highlights the haplotypic diversity of B. vulpes isolates circulating in a single regional population of domestic shelter dogs. These findings suggest a heterogenous transmission dynamic which may involve ticks associated with owned, sheltered, and wild canids, as well as other transmission routes. Finally, data herein corroborates the current geographical distribution of B. vulpes in Central Texas, reinforcing the need to monitor shelter dogs for vector-borne pathogens and deploy the use of tick preventive products year-round.

Dirofilaria immitis is a vector-borne filarioid nematode distributed worldwide and endemic across most of North America. In dogs, it is considered the most clinically significant parasite and a known cause of acquired cardiomyopathy. Clinically, canine heartworm disease can manifest acutely as caval syndrome and chronically as heart failure, often resulting in death. Different types of diagnostic tests, such as antigen-detection and microfilariae-detection tests (MFDTs), including modified Knott’s test and molecular assays, may be used to diagnose D. immitis infection. Although some molecular tools, such as real-time PCR (qPCR), exhibit high accuracy, cross-reactivity with other Dirofilaria species found in companion animals remains a concern. Following our laboratory’s previously published protocol, cross-reactivity was found with Dirofilaria striata isolated from a subcutaneous nodule of a cat from Texas. The objectives of this study were to: (i) evaluate the performance of three newly designed probe-based qPCR assays for detecting D. immitis and (ii) assess overall detection with the newly designed protocols compared with the previously published protocol and other diagnostic tests. We designed three new probes using accessioned D. immitis cytochrome c subunit 1 sequences from various countries worldwide to increase reliability. These new probes were then tested with genomic DNA extracted from D. immitis and D. striata. Following this, we further assessed the performance of these newly designed probes and previously developed probe using 136 archival shelter dog samples previously found to be positive for D. immitis by at least one diagnostic test. Out of the three probes tested, two (probe 2 and probe 3) specifically detected D. immitis and showed no cross-reactivity with D. striata. Probe 3 showed the highest prevalence of D. immitis among all qPCR assays (68.4

Heartworms (Dirofilaria immitis) are parasitic nematodes that cause significant cardiopulmonary-associated morbidity and mortality in canids worldwide. The global spread of heartworms is believed to have occurred alongside the dispersal of modern domesticated dogs over the past few hundred years. However, this conclusion has been based on limited sampling, both geographically and numerically. To test this theory more rigorously, we analyse the whole genomes of 127 adult heartworm specimens collected from mammalian carnivore hosts across Australia, the USA, Central America, Europe, and Asia. Here we show distinct genetic differences between heartworms from different continents, indicating a more ancient dispersal in canid hosts than previously recognised. Using genetic diversity and admixture analyses, we find an Asian origin for Australian heartworms, aligning with the arrival of dingoes into Australia via Asia thousands of years ago; however, we cannot exclude the alternate hypothesis that heartworms were also introduced from Asia in post-colonial times. Finally, the genetic relatedness between European and Central American heartworms suggests that modern dispersal, potentially associated with human colonisation of the Americas by Europeans, occurred with domesticated dogs. This work sheds light on the population dynamics and deep evolutionary history of a globally widespread parasite of veterinary significance.

The American Heartworm Society (AHS) recommends diagnosing canine heartworm (HW) infection using both antigen and microfilaria detection tests. Antigen–antibody immune complexes may block antigen detection. Therefore, the AHS recommends immune complex dissociation (ICD) via heat treatment for discordant cases. The trūRapid™ FOUR antigen test kit (Antech Diagnostics) is a novel commercially available point-of-care (POC) device. This study evaluated the performance of trūRapid™ FOUR compared to other commercially available POC tests, namely SNAP® 4Dx® Plus (IDEXX), VETSCAN® Flex4 (Zoetis), and a plate-based assay, DiroCHEK® (Zoetis), for early detection of HW infection in experimentally infected dogs. Archival frozen serum samples from six purpose-bred beagles experimentally infected with Dirofilaria immitis were collected weekly for 5.5 months, from week 1 to week 22 post-infection. Sera were tested for the detection of HW antigen, pre- and post-ICD, via heat treatment (103 °C for 10 min in a heat block) using each of the commercial antigen tests. DiroCHEK® results were interpreted using optical density readings. HW antigen was not detected in samples collected from week 1 through week 19. In week 20, the trūRapid™ FOUR, SNAP® 4Dx® Plus, and VETSCAN® Flex4 detected HW antigen in one out of six samples (16.7