Dermal microfilariae of dogs, jackals and cats in different regions of Iran.

BACKGROUND: Due to the complexity of retrieving skin-dwelling microfilariae, filarioids of dogs presenting dermal microfilariae (e.g. Cercopithifilaria spp., Onchocerca lupi) are relatively unknown compared to Dirofilaria spp. and Acanthocheilonema spp. whose microfilariae circulate in the blood. Although Cercopithifilaria spp. and O. lupi filarioids are distributed worldwide, there is a paucity of information on their occurrence in Iran. The aim of this study was to investigate these filarioids in a large population of dogs from different regions of Iran. METHODS: From October 2018 to September 2020, skin biopsies were obtained from dogs housed in shelters (n = 557) and privately owned dogs (n = 26) in seven provinces of Iran (Hamedan, Kermanshah, Yazd, Mazandaran, Khuzestan, Lorestan, Esfahan), as well as from three road-killed jackals (Canis aureus) and three cats (Felis catus) in Hamedan province. The skin biopsies were first soaked in saline solution at room temperature overnight, and examined for dermal microfilariae under the microscope. Positive skin specimens and sediments were tested by PCR for a 304-bp region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and amplicons were sequenced. RESULTS: Microfilariae of Cercopithifilaria spp. were found in skin biopsies of 32 of the 583 (5.5%) dogs tested, with infection rates of up to 25% in Kermanshah. No microfilariae were recovered from skin biopsy samples collected from dogs in Khorramabad and Ahvaz, nor from the examined jackals and cats. None of the privately owned dogs were found to be infected. Morphologic and morphometric characteristics of the microfilariae were consistent with C. bainae. Eighteen skin samples were positive for the cox1 gene, of which 15 sequences showed a nucleotide identity of 100% and three of 93.4% with the reference sequence of C. bainae available in GenBank (haplotype I; GenBank accession number: JF461457). CONCLUSIONS: The data from this study broadens current knowledge on the geographical distribution of C. bainae in dogs in Middle Eastern countries. Further studies on different wild canine species in the country (e.g. jackal, fox, wolf) could provide further information on the epidemiology of these filarioids. A particular focus should be put on zoonotic O. lupi given the reports of its presence in human patients from this country.

Multiple vector-borne pathogens of domestic animals in Egypt.

Vector Borne Diseases (VBDs) are considered emerging and re-emerging diseases that represent a global burden. The aim of this study was to explore and characterize vector-borne pathogens in different domestic animal hosts in Egypt. A total of 557 blood samples were collected from different animals using a convenience sampling strategy (203 dogs, 149 camels, 88 cattle, 26 buffaloes, 58 sheep and 33 goats). All samples were tested for multiple pathogens using quantitative PCR and standard PCR coupled with sequencing. We identified Theileria annulata and Babesia bigemina in cattle (15.9 and 1.1%, respectively), T. ovis in sheep and buffaloes (8.6 and 7.7%, respectively) and Ba. canis in dogs (0.5%) as well as Anaplasma marginale in cattle, sheep and camels (20.4, 3.4 and 0.7%, respectively) and Coxiella burnetii in sheep and goats (1.7 and 3%; respectively). New genotypes of An. centrale, An. ovis, An. platys-like and Borrelia theileri were found in cattle (1.1,3.4, 3.4 and 3.4%, respectively), An. platys-like in buffaloes (7.7%), An. marginale, An. ovis, An. platys-like and Bo. theileri in sheep (3.4, 1.7, 1.7 and 3.4%, respectively), An. platys, An. platys-like and Setaria digitata in camels (0.7, 5.4 and 0.7%, respectively) and Rickettsia africae-like, An. platys, Dirofilaria repens and Acanthocheilonema reconditum in dogs (1.5, 3.4, 1 and 0.5%, respectively). Co-infections were found in cattle, sheep and dogs (5.7, 1.7, 0.5%, respectively). For the first time, we have demonstrated the presence of several vector-borne zoonoses in the blood of domestic animals in Egypt. Dogs and ruminants seem to play a significant role in the epidemiological cycle of VBDs.

Molecular and morphological characterization of three new species of avian Onchocercidae (Nematoda) with emphasis on circulating microfilariae.

BACKGROUND: Blood parasites have been the subject of much research, with numerous reports of the presence of microfilariae in the peripheral blood (circulating microfilariae) of birds belonging to many orders. Current limitations in molecular characterization methods and species identification using morphological characters of circulating microfilariae are major obstacles to improving our understanding the biology of Filarioidea species, particularly in wildlife. The aim of this study was to partially fill these gaps, with particular emphasis on morphological features of microfilariae, which are the most readily accessible stages of these pathogens. METHODS: Peripheral blood samples of 206 birds belonging to genera Acrocephalus (five species) and Sylvia (five species) were examined using the buffy coat method to process the blood samples for the presence of microfilariae. Positive birds were dissected to collect adult nematodes. Microfilariae and adult nematodes were described, and sequences of their mitochondrial cytochrome c oxidase subunit I and nuclear 28S rDNA gene fragments were obtained and used for molecular characterization and Bayesian phylogenetic inferences. RESULTS: Overall prevalence of microfilariae was 2.9%. Microfilariae were found in the blood samples from six birds (2 Acrocephalus scirpaceus and 1 each of A. arundinaceus, Sylvia atricapilla, S. borin and S. curruca), which were dissected. All parasite species observed were new. Eufilaria acrocephalusi sp. n. and Eufilaria sylviae sp. n. were present in subcutaneous, peritracheal and periesophageal connective tissues in A. scirpaceus and S. borin, respectively. Splendidofilaria bartletti sp. n. was found in finger joins of S. atricapilla. Illustrations of microfilariae and adult nematodes are shown, and morphological and phylogenetic analyses identified the DNA barcode haplotypes that are associated with these species. Phylogenetic analysis places the parasites of different genera in different closely related clades. CONCLUSIONS: Adult nematode morphological characters, which have been traditionally used in the taxonomy of Filarioidea species, have a phylogenetic value. Importantly, in our study parasites of different genera were readily distinguishable based on the morphology of their microfilariae. The link between molecular and morphology data requires more study in Filarioidea species research, particularly because this approach provides new knowledge on species identity using only readily accessible blood stages (microfilariae), thereby avoiding host dissection and thus minimizing harm to wildlife during research.

Molecular detection of filarial nematode parasites in Japanese black bears (Ursus thibetanus japonicus) from Iwate Prefecture, Japan.

This study aimed to detect filarial parasites in blood samples of Japanese black bears (Ursus thibetanus japonicus) collected from Iwate Prefecture, Japan. Positive amplicons were obtained from 26 out of 30 samples by nested PCR targeting 18S ribosomal RNA gene and first internal transcribed spacer regions. DNA sequences of Mansonella sp. close to M. ozzardi and Dirofilaria sp. were detected for eight and 11 positive amplicons, respectively. Co-infection was detected for the remaining seven amplicons. Dirofilaria sp. was identified as D. ursi by further genetic analysis of 5S ribosomal RNA gene sequence. The results of this study will contribute to further investigations of Japanese black bears for monitoring their risk as a reservoir of possible zoonotic filarial parasites.

Mosquito-borne parasites in the Great Plains: searching for vectors of nematodes and avian malaria parasites.

Vector-borne diseases in the United States have recently increased as a result of the changing nature of vectors, hosts, reservoirs, parasite/pathogens, and the ecological and environmental conditions. While most focus has been on mosquito-borne pathogens affecting humans, little is known regarding parasites of companion animal, livestock and wildlife and their potential mosquito hosts in the United States. This study assessed the prevalence of mature infections of Dirofilaria immitis and avian malaria parasites (Haemosporida) within urban mosquito (Diptera, Culicidae) communities in Oklahoma. 2,620 pools consisting of 12,686 mosquitoes from 13 species collected over two summers were tested for the presence of filarioid and haemosporidian DNA. Dirofilaria immitis-infected mosquitoes were detected only in Aedes albopictus (MIR=0.18-0.22) and Culex pipiens complex (MIR=0.12) collected in cities in central and southern Oklahoma. Two other filarioid nematode species with 91-92% similarity with Onchocerca spp. and Mansonella spp. were also detected. Haemosporidian DNA was detected in 13 mosquito pools (0.9% of pools tested) from seven mosquito species out of 13 species tested. Plasmodium DNA in four species (Cx. coronator, Cx. pipiens complex, Cx. tarsalis, and Psorophora columbiae) had high homology with published sequences of avian Plasmodium species while DNA in four other species (Cx. nigripalpus, Ps. columbiae, Anopheles quadrimaculatus, and An. punctipennis) were closely related to Plasmodium species from deer. One pool of Cx. tarsalis was positive with a 100% sequence identity of Haemoproteus sacharovi. This study provides a baseline concerning the diversity of parasites in different mosquito species present in the southern Great Plains. These studies provide important information for understanding the factors of transmission involving the mosquito community, potential hosts, and different mosquito-borne parasites in this important region involved in livestock management and wildlife conservation.

Phylogenetic characterization of Setaria equina and its association with other filarids.

Molecular characterization studies on Setaria equina are limited. The present study aimed to characterize S. equina at the cytochrome c oxidase gene and to examine its phylogenetic relationships with other filarid species. Sequence analysis showed 100% nucleotide homology with an S. equina sequence from Italy (AJ544873). However, both sequences exhibited 7 nucleotide substitutions from a S. equina donkey isolate from Egypt (MK541847). Overall, S. equina formed a monophyletic sister group to Setaria tundra. All Setaria spp. examined formed a separate group on the phylogenetic tree that was related to corresponding Onchocerca spp. and Dirofilaria spp. clades. Human filarid worms-Brugia spp. and Wuchereria spp. grouped in a separate clade alongside Theilezia spp. Dipetalonema spp.-formed a separate group at the top of the tree.

New host and geographical records for Parafilaroides normani (Nematoda: Filaroididae) Dailey, 2009 in South American fur seal, Arctocephalus australis, from southern Brazil.

Lungworms are a common finding in seals and fur seals around the world. However, from existing records, the biogeographical distribution of filaroid helminths appears to be restricted, and these parasites are endemic in only certain areas and species, mainly in the Northern Hemisphere. The occurrence of infection in pinniped species in the Southern Hemisphere is scarce. The objective of this work is to verify the prevalence of lungworms in Arctocephalus australis in waters off the southern coast of Brazil. Twenty subadult specimens of A. australis found recently dead on the southern coast of Brazil were necropsied and their lungs were examined. Parasitic cysts were found in only one specimen (prevalence of 5%). The helminths were morphologically identified as Parafilaroides normani (Metastrongyloidea: Filaroididae). This helminth species has been reported in pinnipeds from Australia, New Zealand and South Africa. This is the first record of P. normani in A. australis and for the western South Atlantic, providing additional data regarding the biogeographic distribution of the parasite.

Defining a prevalence level to describe the elimination of Lymphatic Filariasis (LF) transmission and designing monitoring & evaluating (M&E) programmes post the cessation of mass drug administration (MDA).

The global decline in prevalence of lymphatic filariasis has been one of the major successes of the WHO's NTD programme. The recommended strategy of intensive, community-wide mass drug administration, aims to break localised transmission by either reducing the prevalence of microfilaria positive infections to below 1%, or antigen positive infections to below 2%. After the threshold is reached, and mass drug administration is stopped, geographically defined evaluation units must pass Transmission Assessment Surveys to demonstrate that transmission has been interrupted. In this study, we use an empirically parameterised stochastic transmission model to investigate the appropriateness of 1% microfilaria-positive prevalence as a stopping threshold, and statistically evaluate how well various monitoring prevalence-thresholds predict elimination or disease resurgence in the future by calculating their predictive value. Our results support the 1% filaremia prevalence target as appropriate stopping criteria. However, because at low prevalence-levels random events dominate the transmission dynamics, we find single prevalence measurements have poor predictive power for predicting resurgence, which suggests alternative criteria for restarting MDA may be beneficial.

Cercopithifilaria species in dogs and ticks from Greece.

Filarioids of the genus Cercopithifilaria (Spirurida, Onchocercidae) are parasites of wild and domestic animals in tropical and subtropical regions being transmitted by ixodid ticks. Though this filarioid species have been studied in canine and tick populations in Europe, data on their species diversity and geographical distribution in Greece is scant. Thus, the aims of this study were to investigate the presence of Cercopithifilaria spp. in dogs and ticks across Greece and to assess the possible risk factors. A total of 500 skin biopsies were collected from dogs, while 508 ticks were collected from 180 infested animals and examined. Sediments from skin biopsies were microscopically screened for detection of dermal microfilaria (mfs). Skin samples (n = 115) and tick specimens (n = 153) were molecularly subjected by PCR. Overall, 70 samples (14%) scored positive for mfs. Specifically, 68 samples (13.6%) were positive for Cercopithifilaria bainae and two (0.4%) were co-infected with C. bainae and Cercopithifilaria sp. II. Molecular analyses revealed that all sequences obtained belong to C. bainae. Haplotype I was the most frequent (92.6%), followed by haplotype XVIII (3%) and haplotypes II and IX (1.5%). Three new haplotypes of C. bainae, named XIX, XX, and XXI, were also identified. Among the risk factors examined, habitat, dog use, body weight, tick infestation history, and the use of acaricides were associated with the presence of C. bainae. The estimated prevalence of Cercopithifilaria spp. demonstrates that these filarioids are common in dogs and ticks in Greece. Finally, the identification of 7 haplotypes for C. bainae confirms their genetic variability.

Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum.

BACKGROUND: Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. METHODS: We developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearman's correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections. RESULTS: The detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10-1 to 1.5 × 10-4 mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98-1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively. CONCLUSIONS: The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.

Giant cutaneous cyst in a dog infected by Cercopithifilaria bainae.

Cercopithifilaria bainae is the most prevalent species of filarioids within the genus. This parasite localizes in the skin, sometimes causing erythematous dermatitis. Herein, the authors describe a case of giant cutaneous cyst in a dog infected by Cercopithifilaria bainae. A 9-year-old male mixed-breed dog presented to a veterinary facility in Dourados, Mato Grosso do Sul (Midwest Brazil) with a mass in the lumbosacral region. On clinical examination, the mass was observed to be approximately 15 cm in diameter with a floating consistency and conspicuous presence of viscous fluid; the lesion, however, was non-ulcerated and non-adherent. Cytological examination revealed the presence of moderate lymphocyte cellularity and foamy macrophages, erythrophagocytosis and the presence of numerous microfilariae. After morphological and molecular analysis of the 12S ribosomal RNA gene, the microfilariae were identified as C. bainae, exhibiting 99-100% identity with DNA sequences available from Genbank. Surgery was recommended and after resection of the giant cyst, the dog was treated with ivermectin for two weeks and the clinical condition was completely resolved. Based on the presence of microfilariae in the cyst fluid the role of this filarioid in the determinism of the lesion has been discussed.

Filarial worms: a systematic review and meta-analysis of diversity in animals from Iran with emphasis on human cases.

Current systematic review and meta-analysis demonstrate the prevalence reports of filariasis in animals in Iran along with human cases. Studies were screened, relevant papers were selected and the random-effect model was used by forest plot with 95% confidence interval (CI). Of 17 records of human case-reports, particularly from Khuzestan province (5 cases), Dirofilaria repens was the most detected parasite (10 cases) with higher involvement of the right eye (7 cases) than other organs. Eleven animal species were reported to be parasitised by filarioids in Iran. The prevalence of Dirofilaria immitis in canids was 14.69% (95% CI: 10.33-19.67), with highest rates (20.92%; 95% CI: 13.84-29.03) in free-ranging dogs. Male (10.07%; 95% CI: 5.10-16.47) and more than 1-year old (20.77%; 95% CI: 8.66-36.42) dogs were more likely to be found infected. The frequency of other filarioids of zoonotic interest was: Acanthocheilonema reconditum in dogs 2.15% (95% CI: 0.71-4.33), Dipetalonema evansi in camels 10.16% (95% CI: 4.73-17.34), Onchocerca cervicalis in horses 3.63% (95% CI: 1.44-6.75%) and Onchocerca fasciata 16.57% (95% CI: 10.12-24.24%) in camels. Still, our knowledge on parasitic filariae in Iran is limited and more investigation is needed in both human and animal populations.

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.

Neofoleyellides boerewors n. gen. n. sp. (Nematoda: Onchocercidae) parasitising common toads and mosquito vectors: morphology, life history, experimental transmission and host-vector interaction in situ.

Anuran filarial nematodes are restricted to two comparatively small subfamilies (Icosiellinae and Waltonellinae) of the filariae that currently comprise six genera and 41 recognised species. However, the life histories of only five anuran filarial nematodes, proposed as an ancestral group based on molecular phylogenetic studies, have been elucidated. Furthermore, data on the natural vectors (in situ) and parasite transmission is limited. In the current study we elucidate the life history of Neofoleyellides boerewors n. gen. n. sp. parasitising the guttural toad, Sclerophrys gutturalis and the mosquito vectors Uranotaenia (Pseudoficalbia) mashonaensis and Uranotaenia (Pseudoficalbia) montana. Additionally, we report on the unique host-seeking behaviour of the mosquito vectors which locate their toad hosts using their calls. The complex host-vector relationship and specialised host-seeking behaviour by these mosquitoes indicate biases towards host species and male toad infections.

First report of the isolation and phylogenetic characterization of equine Setaria digitata from India based on mitochondrial COI, 12S rDNA, and nuclear ITS2 sequence data.

Equine ocular setariasis arising mainly from ectopic infestation of Setaria digitata is a common vision impairing ophthalmic disease in India, and the identification of this filarial nematode is based solely on morphology. However, morphological characters alone are inadequate to detect and differentiate S. digitata from its congeners. The present communication reports the first phylogenetic characterization of equine S. digitata from India based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), and the nuclear internal transcribed spacer 2 (ITS2). Three isolates were characterized for each gene, and respective sequences were submitted to NCBI database (MN078131, MN078132, and MN095798). The sequences were also compared with the other related sequences available from PubMed around the globe, and phylogenetic analysis was carried out in conjunction with nucleotide homologies. There was no intraspecific variation among the Indian isolates. The phylogenetic analysis of S. digitata, inferred from these genes, showed that the isolate sequences obtained from different host species created a separate monophyletic clade within the genus Setaria with minor sequence variations revealing similar molecular characteristics of S. digitata isolates throughout the globe. In addition, the studied Indian isolates were found closer to Sri Lankan isolates. The S. digitata and S. labiatopapillosa appeared as sister species.

Diagnosing bovine parafilariosis: utility of the cytochrome c oxidase subunit 1 gene and internal transcribed spacer region for PCR detection of Parafilaria bovicola in skin biopsies and serohemorrhagic exudates of cattle.

BACKGROUND: Parafilaria bovicola (Nematoda: Filariidae) causes cutaneous bleedings in bovine species. Flies serve as intermediate hosts. In recent years, reports on bovine parafilariosis have become more frequent, corroborating the necessity of reliable diagnostic interventions especially since no molecular or serological test has been available. We aimed to establish a polymerase chain reaction assay to detect DNA of P. bovicola in flies, skin biopsies and serohemorraghic exudates of bleeding spots. METHODS: PCRs targeting the cytochrome c oxidase subunit 1 (cox1) gene and the internal transcribed spacer region (ITS) of the ribosomal RNA gene cluster were evaluated for their diagnostic sensitivity as well as performance and specificity on biopsy and serohemorrhagic exudate samples from P. bovicola-infected cattle. RESULTS: Using serohemorrhagic exudates (n = 6), biopsies (n = 2) and flies (n = 1), the PCR targeting the cox1 gene resulted in a gel band of almost 700 bp. Cloning, sequencing, and removal of primer sequences yielded a 649-bp fragment of the P. bovicola cox1 gene. The PCR targeting the ITS region showed a band of about 1100 bp. Cloning, sequencing, and removal of primer sequences resulted in a 1083 bp stretch of the P. bovicola ITS region. Testing samples from presumably affected animals, the cox1-PCR resulted in bands with the expected size and they were all confirmed as P. bovicola by sequencing. In contrast, the ITS-PCR proved to be less sensitive and less specific and additionally amplified the ITS region of Musca domestica or buttercup DNA. When analysing for sensitivity, the cox1-PCR yielded visible bands up to 2 ng of genomic DNA, whereas the ITS-PCR produced bands up to 3 ng. In a plasmid dilution series, the minimum number of target DNA copies was 102 for the cox1-PCR and 101 in the ITS-PCR. CONCLUSIONS: The evaluated cox1-PCR enables reliable detection of P. bovicola DNA in skin biopsies and serohemorrhagic exudates. This PCR and, to a limited extent, the ITS-PCR, may help evaluate different therapeutic approaches. Furthermore, the cox1-PCR may be useful for epidemiological studies on the geographical distribution of P. bovicola. Further understanding of the epidemiology of this parasite will help develop and implement effective control strategies.

Molecular detection of vector-borne pathogens from mosquitoes collected in two zoological gardens in Germany.

In Germany, knowledge of disease agents transmitted by arthropods in zoological gardens is scarce. In the framework of ecological studies, mosquitoes were therefore collected in German zoological gardens and examined for mosquito-borne pathogen DNA and RNA. In total, 3840 mosquitoes were screened for filarial nematodes and three groups of viruses (orthobunyaviruses, flaviviruses, alphaviruses) while 405 mosquitoes were tested for avian malaria parasites. In addition to the filarial nematode species Dirofilaria repens (n = 1) and Setaria tundra (n = 8), Sindbis virus (n = 1) and the haemosporidian genera Haemoproteus (n = 8), Leucocytozoon (n = 10) and Plasmodium (n = 1) were demonstrated. Identified pathogens have the potential to cause disease in zoo and wild animals, but some of them also in humans. Positive mosquitoes were collected most often in July, indicating the highest infection risk during this month. Most of the pathogens were found in mosquito specimens of the Culex pipiens complex, suggesting that its members possibly act as the most important vectors in the surveyed zoos, although the mere demonstration of pathogen DNA/RNA in a homogenised complete mosquito is not finally indicative for a vector role. Outcomes of the study are not only significant for arthropod management in zoological gardens, but also for the general understanding of the occurrence and spread of mosquito-borne disease agents.

Culicoides species community composition and infection status with parasites in an urban environment of east central Texas, USA.

BACKGROUND: Despite their importance as vectors of zoonotic parasites that can impact human and animal health, Culicoides species distribution across different habitat types is largely unknown. Here we document the community composition of Culicoides found in an urban environment including developed and natural sites in east central Texas, a region of high vector diversity due to subtropical climates, and report their infection status with haemoparasites. RESULTS: A total of 251 individual Culicoides were collected from May to June 2016 representing ten Culicoides species, dominated by C. neopulicaris followed by C. crepuscularis. We deposited 63 sequences to GenBank among which 25 were the first deposition representative for six Culicoides species: C. arboricola (n = 1); C. nanus (n = 4); C. debilipalpis (n = 2); C. haematopotus (n = 14); C. edeni (n = 3); and C. hinmani (n = 1). We also record for the first time the presence of C. edeni in Texas, a species previously known to occur in the Bahamas, Florida and South Carolina. The urban environments with natural area (sites 2 and 4) had higher species richness than sites more densely populated or in a parking lot (sites 1 and 3) although a rarefaction analysis suggested at least two of these sites were not sampled sufficiently to characterize species richness. We detected a single C. crepuscularis positive for Onchocercidae gen. sp. DNA and another individual of the same species positive for Haemoproteus sacharovi DNA, yielding a 2.08% prevalence (n = 251) for both parasites in this species. CONCLUSIONS: We extend the knowledge of the Culicoides spp. community in an urban environment of Texas, USA, and contribute to novel sequence data for these species. Additionally, the presence of parasite DNA (Onchocercidae gen. sp. and H. sacharovi) from C. crepuscularis suggests the potential for this species to be a vector of these parasites.

Avian Filariasis in Backyard Chickens in Japan.

In May 2017, a hen in a backyard chicken flock in Japan exhibited mild clinical signs, and the bird was examined for diagnosis. Unexpectedly, many microfilariae were observed in the lung by histologic examination, although no adult worms were detected within the body. In a blood test performed in July, microfilaremia was confirmed in a few clinically normal chickens of the same flock. Molecular analysis of the nematode partial 18S ribosomal RNA gene revealed that the gene detected in the lung of the necropsied hen was positioned in the group of the family Onchocercidae in the phylogenetic tree. These data show that avian filarioids that can infect chickens inhabit Japan.

Emergence of the arterial worm Elaeophora schneideri in moose (Alces alces) and tabanid fly vectors in northeastern Minnesota, USA.

BACKGROUND: Moose (Alces alces) are a culturally and economically valued species in Minnesota. However, the moose population has experienced a sudden, marked decline in their range, including extirpation in the northwest and a 66% decline in the last decade in the northeast portions of the state. Although the exact cause of this decline is unclear, parasitic metastrongylid and filarioid nematode infections are known causes of morbidity and mortality in moose across North America. METHODS: To determine if these parasitic nematodes could be contributing to the Minnesota moose population decline, we molecularly examined banked tissues obtained from moose that died of known and unknown causes for the presence of nematode DNA. Extracted brain DNA of 34 individual moose was amplified utilizing primers targeting the 18S rRNA gene and internal transcribed spacer regions of nematodes. RESULTS: DNA sequencing revealed that PCR products obtained from 15 (44.1%) of the moose were 99% identical to Parelaphostrongylus tenuis, a metastrongylid known to cause neurological disease and death. Additionally, brain tissue from 20 (58.8%) individuals yielded sequences that most closely aligned with Elaeophora schneideri, a parasite associated with neurological impairment but previously unreported in Minnesota. Setaria yehi, a common filarioid parasite of deer, was also detected in the brain tissue of 5 (14.7%) moose. Molecular screening of 618 captured tabanid flies from four trapping sites revealed E. schneideri was present (6%) in the Minnesota environment and transmission could occur locally. Prevalence rates among the flies ranged between 0-100% per trapping site, with Chrysops spp. and Hybomitra spp. implicated as the vectors. CONCLUSIONS: Ultimately, these data confirm that P. tenuis is widespread in the Minnesota moose population and raises the question of the significance of E. schneideri as a contributing factor to morbidity and mortality in moose.