Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens of animals and humans.

BACKGROUND: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. 'hongkongensis'. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies' (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott's test (MKT). RESULTS: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. 'hongkongensis'. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections. CONCLUSIONS: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT' small and portable MinION™ means that such methods could be deployed for field use.

Haemosporidian parasite infections of Malagasy Philepittidae and Nectariniidae are driven by phylogeny rather than ecology.

The nectarivorous common sunbird asity (Neodrepanis coruscans) is phylogenetically closely related to the frugivorous velvet asity (Philepitta castanea), yet it shares similar habitat and foraging behaviour as the Malagasy sunbirds (Cinnyris spp.). As ecological factors have been shown to influence blood parasite prevalence, it should be tested whether parasite abundance, prevalence and diversity of N. coruscans are more similar to the sunbirds than to its relative. Therefore, blood samples (n = 156) and smears (n = 60) were tested for different blood parasites (Haemosporida, trypanosomes, filarioid nematodes) using molecular and microscopic methods. High prevalence of haemosporidian parasites was observed in all bird taxa, with rates ranging from 23% in N. coruscans to 84.6% in C. notatus. The Malagasy Cinnyris spp. exhibited a high occurrence of mixed haemosporidian infections (>76%) with various specialized lineages. Within the Philepittidae family, no Haemoproteus infection was detected and just a few cases of mixed infections. Nectariniidae species predominantly had specialized haemosporidian lineages, while Philepittidae had infections mainly caused by generalist lineages. These findings emphasize the diverse range of blood parasites in Nectariniidae, while additionally highlighting the high diversity of trypanosomes and filarioid nematodes in Philepittidae. Additionally, several newly discovered haemosporidian lineages, Trypanosoma isolates and filarioid nematode isolates were identified. Notably, Philepittidae exhibited a lower prevalence of avian haemosporidian parasites compared to Nectariniidae, possibly due to potential resistance mechanisms. Despite N. coruscans sharing similar habitat and behavioural ecology with both Cinnyris spp., it closely resembles its relative, P. castanea, in all aspects of haemosporidian parasitism.

The first report of Setaria tundra (Issaitshikoff & Rajewskaya, 1928) in Slovakia by using of molecular methods.

Setaria tundra is a filarioid parasite occurring in the northern hemisphere. Adult forms of helminths are located free in the peritoneal cavity of its definitive host - cervids, while microfilariae are presented in the host's bloodstream. Intermediate hosts are represented by several mosquito species, mainly of the genus Aedes.Nematode S. tundra is well adapted to roe deer (Capreolus capreolus) and therefore is the infection usually asymptomatic. In this study we present the first report of S. tundra in Slovakia. During a period 2022 a total of 6 roe deer coming from eastern Slovakia (Trebisov district) were examined. Nematodes were found during the evisceration process in the abdominal cavity of 3 specimens Intensity of infection was in range from 5 to 38 helminths per host. Mean intensity of infection reached 18.3 parasites per host. The helminths were identified as S. tundra by morphological examination and molecular typing of the COI gene. This study is the first report of S. tundra in Slovakia.

Extensive Aberrant Migration of Onchocerca lupi in a Dog.

A 13-year-old Labrador retriever mixed breed dog from Arizona was presented for evaluation of an acute onset of a head tilt as well as vocalization and head shaking upon palpation of the ears. The dog was previously treated for ocular onchocercosis associated with the right eye 10 years earlier. Ophthalmic examination at presentation revealed irregular, tan-colored, masses on the sclera of both eyes. Otoscopic evaluation of the left ear was limited because the canals were stenotic and inaccessible. Cytology did not reveal any infectious etiologies and the dog was subsequently treated with an anti-inflammatory dose of prednisone for 10 days. Two weeks later the dog developed a mild dysphonia and stridor that eventually progressed to include difficulty breathing. The dog was euthanized and postmortem examination revealed white-to-tan nodules identified in the episclera, trachea, subcutis around the nares, external ear canals, and within the fascia overlying the temporalis muscle, as well as in the parietal pleura, and pericardium. There was also a large mass that obliterated the laryngeal cartilage that partially occluded the laryngeal opening. Microscopically, the described nodules consisted predominately of lakes of abundant mineralized debris, admixed with granulomatous inflammation centered around degenerate nematodes that were subsequently confirmed by PCR and sequence analysis to be Onchocerca lupi. The veterinary literature is comprised of only 2 reports that describe aberrant O. lupi migration to the trachea and larynx. Here, we provide the first detailed description of a dog with extensive aberrant onchocercosis.

Setaria tundra in a roe deer (Capreolus capreolus) in the Donau-Ries district of Bavaria, Germany.

INTRODUCTION: Setaria tundra is known as a common parasite of sylvatic ungulates in Northern latitudes. Although mostly considered of low pathogenicity, severe disease outbreaks and remarkable economic losses have been observed in reindeer (Rangifer tarandus tarandus). Host density and climatic factors are major drivers of the expansion of Setaria spp. facilitating their development and spread. Five adult specimens of S. tundra were retrieved from a male roe deer in Bavaria, Germany. Deoxyribonucleic acid (DNA) barcoding confirmed morphological identification. Cyclooxygenase 1 gene sequences showed 98,73-99,68 % similarity to sequences of other S. tundra specimens found in deer (Cervidae) and mosquitoes (Culicidae). The results raise awareness for the presence of S. tundra in a hitherto unkown endemic region and represent a starting point for broader investigations to understand the biology and distribution of this parasite in roe deer as well as other potential definitive hosts.

INTRODUCTION: Setaria toundra est connu comme un parasite commun des ongulés sylvatiques aux latitudes septentrionales. Bien que généralement considéré comme faiblement pathogène, de graves épidémies et des pertes économiques significatives ont été observées chez le renne (Rangifer tarandus tarandus). La densité d'hôtes et les facteurs climatiques sont les principaux moteurs de l'expansion de Setaria spp., facilitant leur développement et leur diffusion. Cinq spécimens adultes de S. toundra ont été récupérés sur un chevreuil mâle en Bavière, en Allemagne. Le séquençage de l'acide désoxyribonucléique (ADN) a confirmé l'identification morphologique. Les séquences du gène de la cyclooxygénase 1 ont montré une similarité de 98,73 à 99,68 % avec les séquences d'autres spécimens de S. toundra trouvés chez les cerfs (Cervidae) et les moustiques (Culicidae). Les résultats sensibilisent à la présence de S. toundra dans une région endémique jusqu'alors inconnue et représentent un point de départ pour des investigations plus larges pour comprendre la biologie et la distribution de ce parasite chez le chevreuil ainsi que d'autres hôtes définitifs potentiels.

Molecular detection of Cercopithifilaria bainae in brown dog ticks collected from dogs across the United States.

Cercopithifilaria bainae is a filarioid nematode of dogs shown to use Rhipicephalus sanguineus sensu lato (s.l.), the brown dog tick, as the vector. Previously in the United States, C. bainae infections have been reported in a dog from Florida, and in dogs and ticks in Oklahoma, but data are lacking from other areas of the country. Here, we tested brown dog ticks from across the United States for C. bainae DNA to assess the geographic distribution of where this novel parasite may be cycling in ticks and dogs. Archival brown dog ticks were available for testing through the national tick survey Show Us Your Ticks. Ticks were morphologically identified, dissected, and tested by PCR to detect filarioid mitochondrial DNA. A total of 1400 brown dog ticks were tested from 321 separate animals from 23 states, with 5.7 % (80/1400) of the ticks testing positive for C. bainae DNA. At least one positive tick was detected in submissions from 9 states in addition to Florida and Oklahoma. Cercopithifilaria bainae DNA was detected in larval, nymphal, and adult stages of brown dog ticks and only in ticks removed from dogs. Of all dogs with brown dog ticks collected from them, 17.6 % (55/312) were infested with at least one tick that harbored C. bainae DNA. Findings from this study demonstrate a wider geographic range of C. bainae than previously known, and that dogs are commonly infested with brown dog ticks with molecular evidence of C. bainae infection.

Co-occurrence of Eutrombicula alfreddugesi and Oswaldofilaria chabaudi in Tropidurus torquatus and first report of microfilariae in the chigger mite: possible evidence of a lifecycle pathway?

While much attention has been paid to vector-borne filariasis, diseases that threaten millions of people in tropical and subtropical countries, the literature on host-parasite associations and transmission strategies of filarial nematodes in wildlife is scarce. Here, we report the co-occurrence of chigger mites (Eutrombicula alfreddugesi) and onchocercid nematodes (Oswaldofilaria chabaudi) parasitizing the lizard Tropidurus torquatus in the State of Minas Gerais, Brazil. Examination of chiggers established, for the first time, the occurrence of microfilariae in trombiculid mites (Trombiculidae). These larvae were morphologically similar to those recovered from adult females of O. chabaudi. The current evidence suggests that chiggers do not play a role in the transmission of filarioid nematodes, but rather act as accidental or dead-end hosts. Nevertheless, considering the polyphagous nature of trombiculid mites, similar to blood-sucking insects involved in the transmission of several infectious diseases, further studies may shed light on the potential role of chiggers as vectors of filarioids.

Vector-Borne Blood Parasites of the Great-Tailed Grackle (Quiscalus mexicanus) in East-Central Texas, USA.

Great-tailed grackles (Quiscalus mexicanus) have dramatically expanded into North America over the past century. However, little is known about the blood that parasites they support. Here, for the first time, we document an assemblage of trypanosome, haemosporida, and filarial nematodes co-circulating in invasive great-tailed grackles. Between February and July, 2015, 61 individuals were captured in an urban environment of College Station, Texas. Field microscopy and molecular diagnostics indicate that 52% (24/46) were visually infected with filarioid nematodes, 24% (11/46) with avian trypanosomes, and 73% (n = 44/60) with haemosporida parasites, such as Haemoproteus (Parahaemoproteus) and Plasmodium cathemerium. Overall, 87% of great-tailed grackles were infected with blood parasites. Although 50% of individuals hosted parasites from multiple phylum, no patterns of parasite assembly were observed. Results indicate that great-tailed grackles can support a relatively high level of blood parasitism. However, the consequences for avian health remain to be determined.

Detection of Cercopithifilaria bainae infection in shelter dogs and ticks in Oklahoma, USA.

BACKGROUND: Cercopithifilaria bainae is a filarioid nematode of dogs. Infection with the parasite was not reported in the USA until 2017, when a dog with skin lesions in Florida was diagnosed. Brown dog ticks, Rhipicephalus sanguineus (sensu lato), are the purported tick vectors, and are widespread in the USA. Therefore, C. bainae is likely present in additional states. Here, we tested dogs and ticks in Oklahoma for evidence of C. bainae infection. METHODS: Dermal punch biopsies were opportunistically collected from municipal shelter and client-owned dogs. Multiple skin samples collected from interscapular and head regions were tested by saline sedimentation to recover live microfilariae for morphometric identification and by PCR to amplify a 330 bp region of the filarioid 12S rRNA gene. Also, ticks observed on surveyed dogs were collected, identified to species level, and tested for filarioid DNA. RESULTS: A total of 496 saline sedimentations were performed on 230 shelter and 20 client-owned dogs. Cercopithifilaria bainae infections were identified in 2.6% (6/230) of shelter dogs by morphometry of microfilariae in sedimentations and/or amplification of DNA from skin. DNA sequences amplified from PCR positive skin samples were 99-100% identical to C. bainae reported in Italy. All skin samples from client-owned dogs were negative for filarioid infection by saline sedimentation and PCR. A total of 112 ticks, comprised of four species, were collected. Two of 72 R. sanguineus (s.l.), both engorged females found attached to a C. bainae infected dog, harbored C. bainae DNA (99-100% identity). One attached R. sanguineus (s.l.) male on the same dog harbored filarioid DNA sequence which was difficult to interpret at numerous base-pair locations, but was closest in identity (~80%) to C. bainae. CONCLUSIONS: The distribution of C. bainae is more widespread than previously known. To our knowledge, we document C. bainae infections in dogs and DNA in brown dog ticks in Oklahoma for the first time. As brown dog ticks are commonly found throughout the USA, veterinarians in this region should consider C. bainae infection as a differential diagnosis in canine patients with dermatitis or polyarthritis.

Massive microfilaremia in a dog subclinically infected with Acanthocheilonema dracunculoides.

Canine filarioids are worldwide distributed nematodes transmitted by arthropods with variable virulence depending on the species. Dirofilaria immitis is the most virulent and serological antigen tests are commonly employed to detect it. This study reports on the heaviest cavity filariasis recorded so far in a dog, which showed no apparent clinical signs of infection. The 6-year-old male was positive to a D. immitis antigen test. Blood samples collected and analyzed with the modified Knott's test for microfilariae revealed 264,367 microfilariae/ml. In a post-mortem examination 791 adult filarial nematodes were found in the dog's thoracic and peritoneal cavities. Morphological and molecular analysis identified the nematode as Acanthocheilonema dracunculoides and no other species were present. This is evidence that massive A. dracunculoides infections in dogs may not be clinically evident, they may cause serologic cross-reaction with D. immitis infection and become a life-threatening condition if dogs are treated with a microfilaricidal treatment without previously performing an adequate diagnosis.

Heartworm Disease (Dirofilaria immitis) in Two Roaming Dogs From the Urban Area of Castel Volturno, Southern Italy.

The zoonotic filarioid nematode Dirofilaria immitis is transmitted by bloodsucking mosquitoes and causes heartworm disease in dogs and wild canines. In the last decade, D. immitis has spread in southern Europe including Italy. Few autochthonous foci of infection have been reported in previously non-endemic areas of southern Italy based only on the identification of microfilariae, antigen and serological tests, and polymerase chain reaction assay from both the blood of dogs and mosquito vectors with no description of cases of heartworm disease in both domestic and wild canines. Here, we report first on two cases of heartworm disease found at post-mortem examination in two roaming dogs from the urban area of Castel Volturno in Campania region of southern Italy. Immunological analyses of 11 roaming dogs from the same pack of those two submitted for necropsy and available necropsy data from the dogs recovered from the Campania region along the past 10 years were both negative for D. immitis infection. Although rare in southern Italy, these two cases are noteworthy because D. immitis may cause serious human infection. We highlight the need to identify the mosquito vectors of heartworm disease in this area using sensitive molecular assay for D. immitis DNA for predicting and controlling the spread of infection. We strongly recommend the control and systematic treatment of the domestic and roaming dogs that could constitute the most important infection reservoir.

Screening of mosquitoes for filarioid helminths in urban areas in south western Poland-common patterns in European Setaria tundra xenomonitoring studies.

In recent years, numerous studies screening mosquitoes for filarioid helminths (xenomonitoring) have been performed in Europe. The entomological monitoring of filarial nematode infections in mosquitoes by molecular xenomonitoring might serve as the measure of the rate at which humans and animals expose mosquitoes to microfilariae and the rate at which animals and humans are exposed to the bites of the infected mosquitoes. We hypothesized that combining the data obtained from molecular xenomonitoring and phenological studies of mosquitoes in the urban environment would provide insights into the transmission risk of filarial diseases. In our search for Dirofilaria spp.-infected mosquitoes, we have found Setaria tundra-infected ones instead, as in many other European studies. We have observed that cross-reactivity in PCR assays for Dirofilaria repens, Dirofilaria immitis, and S. tundra COI gene detection was the rule rather than the exception. S. tundra infections were mainly found in Aedes mosquitoes. The differences in the diurnal rhythm of Aedes and Culex mosquitoes did not seem a likely explanation for the lack of S. tundra infections in Culex mosquitoes. The similarity of S. tundra COI gene sequences found in Aedes vexans and Aedes caspius mosquitoes and in roe deer in many European studies, supported by data on Ae. vexans biology, suggested host preference as the most likely cause of the mosquito genus-biased infections. High diversity of the COI gene sequences isolated in the city of Wroclaw in south western Poland and the presence of identical or almost identical sequences in mosquitoes and roe deer across Europe suggests that S. tundra has been established in most of Europe for a very long time.

Litomosoides silvai (Nematoda: Onchocercidae) parasitizing Akodon montensis (Rodentia: Cricetidae) in the southern region of Brazil / Litomosoides silvai (Nematoda: Onchocercidae) parasitando Akodon montensis (Rodentia: Cricetidae) na região Sul do Brasil

Abstract In the present study, Litomosoides silvai parasitizing Akodon montensis in the southern region of Brazil is reported for the first time. New morphological information is provided for some structures of this nematode species, such as a flattened cephalic extremity, presence of two dorsal cephalic papillae, female tail with a constriction at its tip, "s" shaped vagina, spicules characteristic of the carinii species group and microfilaria tail constricted at the tip. This nematode was found parasitizing the thoracic cavity with a prevalence of 10% (2/20), mean intensity of 4 (6/2), mean abundance of 0.4 (8/20) and range of infection of 2-6 specimens per host, in southern Brazil. This occurrence of L. silvai in A. montensis is a new geographical record for southern Brazil, in the Upper Paraná Atlantic Forest ecoregion of the northwestern region of Rio Grande do Sul, which is part of the Atlantic Forest biome.

Resumo No presente estudo é relatado pela primeira vez Litomosoides silvai parasitando Akodon montensis coletados na região Sul do Brasil. Foram fornecidas novas informações morfológicas para algumas estruturas desta espécie de nematódeo, tais como extremidade cefálica achatada, a presença de duas papilas cefálicas dorsais, cauda das fêmeas com uma constrição na ponta da cauda, vagina em forma de "s", espículas de característica do grupo de espécies de carinii e cauda da microfilária com constrição na ponta. Este nematódeo parasitava a cavidade torácica com uma prevalência de 10% (2/20), intensidade média de 4 (8/2) e abundância média de 0,4 (8/20), e intervalo de infecção de 2-6 espécimes por hospedeiro no Sul do Brasil. A ocorrência de L. silvai em A. montensis é um novo registro geográfico, no sul do Brasil, a noroeste do estado do Rio Grande do Sul, na ecorregião da Mata Atlântica do Alto Paraná, parte do bioma da Mata Atlântica.

Litomosoides silvai (Nematoda: Onchocercidae) parasitizing Akodon montensis (Rodentia: Cricetidae) in the southern region of Brazil

Abstract In the present study, Litomosoides silvai parasitizing Akodon montensis in the southern region of Brazil is reported for the first time. New morphological information is provided for some structures of this nematode species, such as a flattened cephalic extremity, presence of two dorsal cephalic papillae, female tail with a constriction at its tip, s shaped vagina, spicules characteristic of the carinii species group and microfilaria tail constricted at the tip. This nematode was found parasitizing the thoracic cavity with a prevalence of 10% (2/20), mean intensity of 4 (6/2), mean abundance of 0.4 (8/20) and range of infection of 2-6 specimens per host, in southern Brazil. This occurrence of L. silvai in A. montensis is a new geographical record for southern Brazil, in the Upper Paraná Atlantic Forest ecoregion of the northwestern region of Rio Grande do Sul, which is part of the Atlantic Forest biome.

Resumo No presente estudo é relatado pela primeira vez Litomosoides silvai parasitando Akodon montensis coletados na região Sul do Brasil. Foram fornecidas novas informações morfológicas para algumas estruturas desta espécie de nematódeo, tais como extremidade cefálica achatada, a presença de duas papilas cefálicas dorsais, cauda das fêmeas com uma constrição na ponta da cauda, vagina em forma de s, espículas de característica do grupo de espécies de carinii e cauda da microfilária com constrição na ponta. Este nematódeo parasitava a cavidade torácica com uma prevalência de 10% (2/20), intensidade média de 4 (8/2) e abundância média de 0,4 (8/20), e intervalo de infecção de 2-6 espécimes por hospedeiro no Sul do Brasil. A ocorrência de L. silvai em A. montensis é um novo registro geográfico, no sul do Brasil, a noroeste do estado do Rio Grande do Sul, na ecorregião da Mata Atlântica do Alto Paraná, parte do bioma da Mata Atlântica.

First report of Setaria tundra in roe deer (Capreolus capreolus) from the Iberian Peninsula inferred from molecular data: epidemiological implications.

BACKGROUND: Filarioid nematode parasites are major health hazards with important medical, veterinary and economic implications. Recently, they have been considered as indicators of climate change. FINDINGS: In this paper, we report the first record of Setaria tundra in roe deer from the Iberian Peninsula. Adult S. tundra were collected from the peritoneal cavity during the post-mortem examination of a 2 year-old male roe deer, which belonged to a private fenced estate in La Alcarria (Guadalajara, Spain). Since 2012, the area has suffered a high roe deer decline rate (75 %), for unknown reasons. Aiming to support the morphological identification and to determine the phylogenetic position of S. tundra recovered from the roe deer, a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from the two morphologically identified parasites was amplified, sequenced and compared with corresponding sequences of other filarioid nematode species. Phylogenetic analyses revealed that the isolate of S. tundra recovered was basal to all other formely reported Setaria tundra sequences. The presence of all other haplotypes in Northern Europe may be indicative of a South to North outbreak in Europe. CONCLUSIONS: This is the first report of S. tundra in roe deer from the Iberian Peninsula, with interesting phylogenetic results, which may have further implications in the epidemiological and genetic studies of these filarioid parasites. More studies are needed to explore the reasons and dynamics behind the rapid host/geographic expansion of the filarioid parasites in Europe.

A new case of zoonotic onchocercosis in northern Kyushu, Japan.

A case of zoonotic onchocercosis has been found in a resident who lived in Iizuka City, Fukuoka Prefecture, Japan for some time. A 24-year-old male developed a painful nodule on the middle finger of his right hand. The nodule was surgically removed from the vagina fibrosa tendinis of the finger at Beppu Medical Center, Beppu City, Oita Prefecture in 2012. The causative agent was identified as a female Onchocerca dewittei japonica based on its histopathological characteristics. The identity of the filarioid has been confirmed by sequencing the cox1 gene. The present study indicates that the zoonotic onchocercosis caused by O. dewittei japonica has been concentrated in northeast Kyushu.

Prevalence of filarioid nematodes and trypanosomes in American robins and house sparrows, Chicago USA.

Hosts are commonly infected with a suite of parasites, and interactions among these parasites can affect the size, structure, and behavior of host-parasite communities. As an important step to understanding the significance of co-circulating parasites, we describe prevalence of co-circulating hemoparasites in two important avian amplification hosts for West Nile virus (WNV), the American robin (Turdus migratorius) and house sparrow (Passer domesticus), during the 2010-2011 in Chicago, Illinois, USA. Rates of nematode microfilariemia were 1.5% of the robins (n = 70) and 4.2% of the house sparrows (n = 72) collected during the day and 11.1% of the roosting robins (n = 63) and 0% of the house sparrows (n = 11) collected at night. Phylogenetic analysis of nucleotide sequences of the 18S rRNA and cytochrome oxidase subunit I (COI) genes from these parasites resolved two clades of filarioid nematodes. Microscopy revealed that 18.0% of American robins (n = 133) and 16.9% of house sparrows (n = 83) hosted trypanosomes in the blood. Phylogenetic analysis of nucleotide sequences from the 18s rRNA gene revealed that the trypanosomes fall within previously described avian trypanosome clades. These results document hemoparasites in the blood of WNV hosts in a center of endemic WNV transmission, suggesting a potential for direct or indirect interactions with the virus.