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Filariae are parasitic nematodes of high veterinary and medical importance, responsible for some acute tropical diseases. They are transmitted through the bite of hematophagous vectors such as biting midges and blackflies. Filariae are among the most prevalent vector-borne parasitoses in Neotropical primates in which severe infections can cause inflammatory reactions and tissue damage. Given the location inside the host (peritoneal cavity, bloodstream, and lymphatics), the detection of filariid nematodes is challenging and is mostly postmortem; hence the scarcity of studies on the prevalence of filariae in wild primate populations. Here, we report the prevalence of filariid infections in free-ranging populations of Geoffroy's spider (Ateles geoffroyi) and black howler (Alouatta pigra) monkeys across southern Mexico, using a combination of noninvasive sampling and molecular diagnostic techniques. Fecal samples were screened for filariid DNA by qPCR protocols. A total of 88 samples were examined with an overall prevalence of 26%. Filariae were slightly more common in spider monkeys compared to howler monkeys. This study constitutes the first report of the prevalence of infection of filariid nematodes in populations of wild spider monkey across southern Mexico, and the first reporting of filariae in black howler monkeys, as part of a new era of primate parasitology and the diagnostics of parasite infections in light of the everyday more affordable molecular tools., (© 2024 The Author(s). American Journal of Primatology published by Wiley Periodicals LLC.)

Background: Vector-borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited. The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results, assess the potential risk to domestic animals, other wildlife and humans., Methods: Between 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16), Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we performed restriction fragment length polymorphism (RFLP) analysis on piroplasm-positive samples, using HinfI as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp., Results: The pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai (56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematominutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected among the tested samples., Conclusions: The large sample size and diverse study populations in this study provide valuable insights into the distribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wildlife species, domestic animals and human health., (© 2024. The Author(s).)

Vector-borne parasite infections affect both domestic and wild animals. They are often asymptomatic but can result in fatal outcomes under natural and human-induced stressors. Given the limited availability of molecular data on vector-borne parasites in Rhinoceros unicornis (greater one-horned rhinoceros), this study employed molecular tools to detect and characterize the vector-borne parasites in rescued rhinoceros in Chitwan National Park, Nepal. Whole blood samples were collected from thirty-six R. unicornis during rescue and treatment operations. Piroplasmida infections were first screened using nested polymerase chain reaction (PCR) targeting 18S ribosomal RNA gene. Wolbachia was detected by amplifying 16S rRNA gene, while filarial nematodes were detected through amplification of 28S rRNA, COI, myoHC and hsp70 genes. Our results confirmed the presence of Theileria bicornis with a prevalence of 75% (27/36) having two previously unreported haplotypes (H8 and H9). Wolbachia endosymbionts were detected in 25% (9/36) of tested samples and belonged to either supergroup C or F. Filarial nematodes of the genera Mansonella and Onchocerca were also detected. There were no significant association between T. bicornis infections and the age, sex, or location from which the animals were rescued. The high prevalence of Theileria with novel haplotypes along with filarial parasites has important ecological and conservational implications and highlights the need to implement parasite surveillance programs for wildlife in Nepal. Further studies monitoring vector-borne pathogens and interspecies transmission among wild animals, livestock and human are required., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Atypical presentations of filariasis have posed diagnostic challenges due to the complexity of identifying the causative species and the difficulties in both diagnosis and treatment. In this study, we present the integrative histological and molecular analysis of seven atypical filariasis cases observed in regions of nonendemicity of Thailand. All filariasis cases were initially diagnosed based on histological findings. To confirm the causative species, molecular characterization based on both filarial mitochondrial (mt 12S rRNA and COI genes) and nuclear ITS1 markers was performed, together with the identification of associated Wolbachia bacterial endosymbionts. Among the cases studied, Brugia pahangi (N = 3), Brugia malayi (N = 1), Dirofilaria sp. "hongkongensis" (N = 2), and a suspected novel filarial species genetically related to Pelecitus copsychi (N = 1) were identified. By targeting the 16S rRNA gene, Wolbachia was also molecularly amplified in two cases of infection with Dirofilaria sp. "hongkongensis." Phylogenetic analysis further revealed that the detected Wolbachia could be classified into supergroups C and F, indicating the high genetic diversity of this endosymbiont in Dirofilaria sp. "hongkongensis." Furthermore, this study demonstrates the consistency between histological findings and species identification based on mitochondrial loci rather than on the nuclear ITS1. This suggests the utility of mitochondrial markers, particularly COI, as a highly sensitive and reliable diagnostic tool for the detection and differentiation of filarial species in clinical specimens. Precise identification of the causative species will facilitate accurate diagnosis and treatment and is also essential for the development of epidemiological and preventive strategies for filariasis.

Elaeophorosis, infection by the filarial worm Elaeophora schneideri, is a parasitic disease of wild ungulates in North America; however, our understanding of the relevance of E. schneideri to moose (Alces alces) morbidity and mortality is incomplete. Between March 2020 and July 2022, necropsy and histopathology were performed on 61 Shiras moose (Alces alces shirasi) in Idaho, US. Among the 41 adults (greater than 1 yr old), 21 moose were from northern Idaho, and 20 were from southeastern Idaho. Elaeophorosis was diagnosed in 24% (10 of 41). All 10 infected moose were from southeastern Idaho; none of the 21 moose from northern Idaho were infected. No juvenile moose (nine from northern and 11 from southeastern Idaho) were infected. Microfilariae were detected histologically in 9 of 10 infected moose, most consistently in brain tissue associated with lesions indicative of ischemic injury to the neuroparenchyma attributed to occlusion of arterioles and capillaries by microfilariae or fibrin thrombi, including edema, necrosis, and glial nodules. Microfilariae found in other tissues of the head, including the eye, tongue, and pinnae of some animals, as well as in lung, heart, liver, and kidney, typically were associated with inflammation. Three of the 10 infected moose had cropped ears attributed to elaeophorosis, and four exhibited abnormal behavior, which may have been due to neuropathology associated with E. schneideri microfilariae in the brain., (© Wildlife Disease Association 2024.)

Purpose: Medical and veterinary filarial nematodes are transmitted by blood-feeding vectors. In dogs, these parasites are mainly represented by nematodes in which microfilariae dwell in the blood (Dirofilaria spp. and Acanthocheilonema spp.) or skin (Cercopithifilaria spp. and Onchocerca lupi). The aim of this study was to determine the prevalence of these filarial infections in dogs residing in a touristic, heavily populated location in the northeastern region of Brazil., Methods: Blood samples (n = 245) were assessed by a modified Knott test, followed by a qualitative ELISA test (SNAP® 4Dx® Plus, IDEXX Laboratory, Westbrook, Maine, USA) for the detection of antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and antigens of Dirofilaria immitis. Skin samples (n = 71) were microscopically examined and molecularly assessed through a PCR targeting the 12 S rRNA gene., Results: Microfilariae and antigen of D. immitis were detected simultaneously in 15 (6.1%; 95% CI = 3.7-9.8) animals. Nine animals (3.6%; 95% CI = 1.9-6.8) were D. immitis antigen positive but microfilariae negative and nine other animals (3.6%; 95% CI = 1.9-6.8) were microfilariae positive but D. immitis antigen negative. D. immitis positive dogs were found in four different municipalities. No filarioids were detected in the skin after microscopical and molecular analyses., Conclusion: Data from this study demonstrate that D. immitis is the main filarial nematode infecting dogs in coastal areas in northeastern Brazil. Based on the potential risk of infection in which animals are submitted, it is essential to perform tests to detect microfilariae and D. immitis antigen. Preventive measures must be adopted by using microfilaricidal compounds and anti-feeding insecticides to prevent canine infection., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., (© 2022. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.

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., Competing Interests: Mark Bradley currently works for GlaxoSmithKline (GSK). Roy M Anderson is Chairman of Oriole Global Health (OGH) Ltd – OGH played no part in this research.

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.

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.

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.

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.

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., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Published by Elsevier B.V.)

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., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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.

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 10 2 for the cox1-PCR and 10 1 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.

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.

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.

Filaria taxideae is a common subcutaneous filarial parasite of American badgers (Taxidea taxus) that has also been reported in skunks in the Western USA. Dermatitis associated with this parasite has been reported in badgers and skunks; however, many individuals have no gross lesions. With the exception of a captive red panda in California, there have been no reports of F. taxidae infection in domestic or exotic/pet animals. In this case series, we document F. taxideae in domestic ferrets from California, Texas, and Colorado and describe the clinical presentation, gross and histologic lesions, and the molecular characterization of this parasite. In two cases, ferrets were positive on Dirofilaria immitis antigen tests but had no evidence of heartworm infection suggesting that F. taxideae antigens can cross-react. These findings indicate that F. taxideae should be considered in cases of dermatitis in ferrets in areas where this parasite may occur., (Copyright © 2018. Published by Elsevier B.V.)

Some Onchocercidae nematodes such as Pelecitus are parasites of medical and veterinary importance. The adult stage of Pelecitus has been reported infecting birds, and the microfilaria has been associated to human blindness. However, in some of these cases, the nematode was incompletely identified at the species level due to the scarcity of morphological taxonomic keys and, also, to the lack of molecular diagnostic analysis. Here, we report a new Pelecitus species in a crested caracara (Caracara cheriway) producing a severe tenosynovitis and microfilarial dermatitis. It is also the first record of Pelecitus in an American bird of prey. Clinical and histopathological features are described, contributing towards our understanding of the pathogenesis of Pelecitus and the health and conservation of wild bird populations. Our study also provides new information on the molecular diagnosis of this parasite and highlights the potential role of wild birds as Pelecitus reservoirs, and health risk for humans and wildlife.

Rhipicephalus sanguineus sensu lato (s.l.) is one of the most widespread ixodid ticks and is a competent vector of several vector-borne pathogens of veterinary and medical concern. For instance, this tick species transmits nematodes of the genus Cercopithifilaria and protozoa of the genus Hepatozoon to carnivores, including dogs. Here we investigated the occurrence of Cercopithifilaria spp. and Hepatozoon spp. in a population of ticks collected from naturally infested dogs living in rural areas of Northeastern Brazil. From August 2016 to June 2017, 758 tick specimens (mean ticks per month = 68.9 ± 71.4) were sampled from 75 dogs (mean ticks per dog = 10.11 ± 5.2) and dissected under a stereomicroscope in order to visualize Cercopithifilaria spp. larvae and Hepatozoon spp. oocysts and sporocysts. R. sanguineus s.l. was the only species collected, peaking in September (n = 273) and decreasing in February 2017 (n = 39). Different larval stages of Cercopithifilaria bainae were identified in 7 out of 758 (0.93%) ticks. In addition, 4 specimens (0.53%) were positive for oocysts and free sporocysts of Hepatozoon canis. The identity of both species of parasites was molecularly confirmed. These results account for the predominance of R. sanguineus (s.l.) in domestic dogs from rural locations of the study area, as well as for the presence C. bainae and H. canis in these tick populations.

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.

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.

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is an emerging tool for routine identification of bacteria, archaea and fungi. It has also been recently applied as an accurate approach for arthropod identification. Preliminary studies have shown that the MALDI-TOF MS was able to differentiate whether ticks and mosquitoes were infected or not with some bacteria and Plasmodium parasites, respectively. The aim of the present study was to test the efficiency of MALDI-TOF MS tool in distinguishing protein profiles between uninfected mosquitoes from specimens infected by filarioid helminths. Aedes aegypti mosquitoes were engorged on microfilaremic blood infected with Dirofilaria immitis, Brugia malayi or Brugia pahangi. Fifteen days post-infective blood feeding, a total of 534 mosquitoes were killed by freezing. To assess mass spectra (MS) profile changes following filariae infections, one compartment (legs, thorax, head or thorax and head) per mosquito was submitted for MALDI-TOF MS analysis; the remaining body parts were used to establish filariae infectious status by real-time qPCR. A database of reference MS, based on the mass profiles of at least two individual mosquitoes per compartment, was created. Subsequently, the remaining compartment spectra (N = 350) from Ae. aegypti infected or not infected by filariae were blind tested against the spectral database. In total, 37 discriminating peak masses ranging from 2062 to 14869 daltons were identified, of which 17, 11, 12 and 7 peak masses were for legs, thorax, thorax-head and head respectively. Two peak masses (4073 and 8847 Da) were specific to spectra from Ae. aegypti infected with filariae, regardless of nematode species or mosquito compartment. The thorax-head part provided better classification with a specificity of 94.1% and sensitivity of 86.6, 71.4 and 68.7% of D. immitis, B. malayi and B. pahangi respectively. This study presents the potential of MALDI-TOF MS as a reliable tool for differentiating non-infected and filariae-infected Ae. aegypti mosquitoes. Considering that the results might vary in other mosquito species, further studies are needed to consolidate the obtained preliminary results before applying this tool in entomological surveillance as a fast mass screening method of filariosis vectors in endemic areas.

Cercopithifilaria species are tick-transmitted filarial parasites of mammals. In Europe, three Cercopithifilaria spp. are known to parasitize dogs, all occurring mainly in the Mediterranean countries. In Romania, Cercopithifilaria bainae has been reported in a single dog in eastern Romania but the occurrence in other parts of the country is not known. To further elucidate the geographic distribution of Cercopithifilaria spp. infection, 544 ticks were collected from dogs in several locations across Romania. The presence of Cercopithifilaria spp. was investigated with real-time PCR. A single Dermacentor reticulatus female tick was found to be infected with Cercopithifilaria bainae. The finding in the present study is geographically separated from the previous finding in Romania by 800 km, as well as by the Carpathian mountain range. Hence, C. bainae is more geographically widespread in Romania than previously recognized. However, the single detection does suggest that infection is rather uncommon in Romanian dogs. Nevertheless, further studies on Cercopithifilaria spp. distribution and prevalence are needed.

Background: Mosquitoes are arthropods of major importance to animal and human health because they are able to transmit pathogenic agents such as filarioids (Spirurida), vector-borne nematodes, which reside in the tissues of vertebrates. In Europe, recent research has mostly focused on mosquito-borne zoonotic species, while others remain neglected. Mosquitoes are also vectors of avian malaria, which has an almost worldwide distribution, and is caused by several Plasmodium species and lineages, the most common being P. relictum. The Danube Delta region of Romania is one of the most important stopover sites for migratory birds. The local mosquito fauna is diverse and well represented, while filarial infections are known to be endemic in domestic dogs in this area. The aim of the present study was thus to assess the potential vector capacity for various filarial helminths and avian malaria of mosquitoes trapped in the Danube Delta., Methods: In July 2015, mosquitoes were collected at seven sites located in and around a rural locality in the Danube Delta region of Romania, using CO 2 -baited traps and hand aspirators. Additionally, a trap was placed next to a microfilaremic dog co-infected with Dirofilaria repens and D. immitis. All randomly trapped mosquitoes were identified to the species level and pooled according to date, sampling site, and taxon. Three hundred individual mosquitoes sampled next to the microfilaremic dog were processed individually and divided into abdomen and thorax/head. Following DNA extraction, all samples were screened for the presence of DNA of filarioid helminths and avian malaria agents by PCR techniques., Results: All 284 pools (a total of 5855 mosquitoes) were negative for filarioid DNA. One pool of Culex modestus mosquitoes was positive for Plasmodium sp. lineage Donana03. In the individually extracted mosquitoes, one abdomen of Aedes vexans was positive for D. repens DNA, one thorax/head of Ae. vexans was positive for DNA of Setaria labiatopapillosa, and two thorax/head of Cx. pipiens f. pipiens were positive for P. relictum lineage pSGS1., Conclusion: The present study suggests the vector competence of Cx. modestus and Cx. pipiens for avian Plasmodium including pathogenic species P. relictum and Ae. vexans for mammalian filarioids. Moreover, it indicates the role of Cx. pipiens f. pipiens as a potential natural vector of P. relictum lineage pSGS1 in nature.

Background: The filarial nematodes Wuchereria bancrofti (Cobbold, 1877), Brugia malayi (Brug, 1927) and B. timori Partono, Purnomo, Dennis, Atmosoedjono, Oemijati & Cross, 1977 cause lymphatic diseases in humans in the tropics, while B. pahangi (Buckley & Edeson, 1956) infects carnivores and causes zoonotic diseases in humans in Malaysia. Wuchereria bancrofti, W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 and six out of ten Brugia spp. have been described from Australia, Southeast Asia, Sri Lanka and India. However, the origin and evolution of the species in the Wuchereria-Brugia clade remain unclear. While investigating the diversity of filarial parasites in Malaysia, we discovered an undescribed species in the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia)., Methods: We examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Once any filariae that were found had been isolated, we examined their morphological characteristics and determined the partial sequences of their mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes. Polymerase chain reaction (PCR) products of the internal transcribed spacer 1 (ITS1) region were then cloned into the pGEM-T vector, and the recombinant plasmids were used as templates for sequencing., Results: Malayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The Kimura 2-parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews., Conclusions: The newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.

A northern crested caracara (Caracara cheriway) was presented after being found nonambulatory in a field. On physical examination, the bird had severe hind-limb paresis. The bird did not improve after 10 days of hospitalization and was euthanized. Histologic examination of the cerebrum and spinal cord revealed multiple adult filarial nematodes surrounded by granulomatous inflammation with several multinucleated giant cells. These parasites were confirmed to be Chandlerella quiscali with polymerase chain reaction. This is the first report of C. quiscali in a bird of prey.

Worldwide more than 2 billion people are infected with helminths, predominantly in developing countries. Co-infections with viruses such as human immunodeficiency virus (HIV) are common due to the geographical overlap of these pathogens. Helminth and viral infections induce antagonistic cytokine responses in their hosts. Helminths shift the immune system to a type 2-dominated immune response, while viral infections skew the cytokine response towards a type 1 immune response. Moreover, chronic helminth infections are often associated with a generalized suppression of the immune system leading to prolonged parasite survival, and also to a reduced defence against unrelated pathogens. To test whether helminths affect the outcome of a viral infection we set up a filarial/retrovirus co-infection model in C57BL/6 mice. Although Friend virus (FV) infection altered the L. sigmodontis-specific immunoglobulin response towards a type I associated IgG2 isotype in co-infected mice, control of L. sigmodontis infection was not affected by a FV-superinfection. However, reciprocal control of FV infection was clearly impaired by concurrent L. sigmodontis infection. Spleen weight as an indicator of pathology and viral loads in spleen, lymph nodes (LN) and bone marrow (BM) were increased in L. sigmodontis/FV-co-infected mice compared to only FV-infected mice. Numbers of FV-specific CD8+ T cells as well as cytokine production by CD4+ and CD8+ cells were alike in co-infected and FV-infected mice. Increased viral loads in co-infected mice were associated with reduced titres of neutralising FV-specific IgG2b and IgG2c antibodies. In summary our findings suggest that helminth infection interfered with the control of retroviral infection by dampening the virus-specific neutralising antibody response., Competing Interests: The authors have declared that no competing interests exist.

An adult nematode was grossly identified in the meninges of a Malayan sambar (Rusa unicolor equina), with numerous microfilariae associated with encephalitis and vasculitis on histopathology. The nematode was confirmed to be Elaeophora schneideri by sequencing a portion of the 18S rRNA gene. Our report highlights the potential for aberrant migration of E. schneideri in exotic deer species and the use of advanced testing to specifically identify this metazoan parasite, avoiding misidentification of Parelaphostrongylus tenuis., (© 2016 The Author(s).)

The inability to maintain filarial nematodes in long-term in vitro culture greatly limits research into the basic biology of these parasites and hinders in vitro screening of novel anti-filarial agents. In this study, we sought to characterize nutrients that promote the long-term survival of filarial worms in vitro. Using microfilariae (MF) obtained from gerbils infected with Litomosoides sigmodontis, a filarial parasite of rodents, we found that Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) resulted in MF survival of only 5 days. However, co-culturing MF with a mouse endothelial cell line (EOMA) enabled survival for 40 days. Culturing EOMA cells in transwell plates extended MF survival to the same degree as direct co-culture, suggesting that the factors microfilariae require are soluble in nature. Heat inactivation of EOMA conditioned media at 56 °C reduced MF survival by approximately 50%, and heat inactivation at 100 °C reduced survival to 3 days, demonstrating that both heat labile and heat stable factors are involved. EOMA cells require FBS to produce these factors, as conditioned media collected from EOMA cells grown in the absence of FBS failed to prolong survival. The removal of lipids also abrogated survival, indicating MF are likely utilizing lipid factors released by EOMA cells. Dialysis experiments demonstrate that at least some of the required factors are between 0.1 and 1 kDa in size. Importantly, L. sigmodontis adult worms also show significantly extended survival when cultured in EOMA conditioned media. Together, these results suggest that EOMA-produced factors include lipid-containing molecules, heat labile molecules (likely a protein), and micronutrients between 0.1 and 1 kDa in size. These studies have established a cell-free approach to maintaining MF and adult stage filarial worms in long-term in vitro culture and have taken important steps towards biochemically characterizing host-derived nutrients required for parasite survival., (Published by Elsevier Inc.)

Species of filarial nematodes belonging to the genera Dirofilaria and Acanthocheilonema are recognised as common parasites of dogs throughout the world. Recently, other filarioids featured by the presence of dermal microfilariae (e.g., Onchocerca lupi and Cercopithifilaria spp.) have been recognised in Europe. In Brazil, reports of filarioids in dogs are limited to Dirofilaria immitis, Acanthocheilonema reconditum and Cercopithifilaria bainae. To investigate the distribution of filarial infections in dogs living in an endemic region from northeastern Brazil, blood and skin samples (n=104) were microscopically (modified Knott's test and skin snip sediment examination) and molecularly evaluated. Twenty-two dogs (21.15%) were positive at microscopic and/or molecular examination for at least one filarioid species, with 21 (20.19%) animals positive for blood microfilariae at molecular and/or at microscopic examination. Microfilariae of D. immitis were detected in 12 (11.54%) animals, with co-infection of D. immitis and A. reconditum observed in four (3.85%) individuals. One animal was positive for C. bainae at both microscopic and molecular examination. Analysis of sequence obtained in the present study showed significant alignment identity with that of C. bainae from Europe. Considering that in the area of study arthropod vectors (mosquitoes, fleas and ticks) are prevalent throughout the year, preventive measures should be disposed in order to avoid the animal infestation and pathogen infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Moose (Alces alces) are a culturally and economically valued species in Minnesota, where the northeast population has decreased by 60 % since 2006. The cause of the decline is currently unclear; however, parasites, predation, and climate change have all been implicated. Nematode parasites are important pathogens in North American moose, potentially causing severe disease and mortality. Recent spread of Rumenfilaria andersoni, a filarioid nematode of moose, has been documented in Finnish cervids; however, little is known about the epidemiology of this parasite in North America., Methods: To investigate the prevalence and distribution of R. andersoni, 584 blood samples were collected from live-captured and dead animals and screened microscopically for the presence of microfilariae using a modified Knott's test. Microfilariae were identified based on morphological characteristics. A subset of Knott's-positive animals was subjected to polymerase chain reaction (PCR) with filarioid-specific primers targeting the first internal transcribed spacer region (ITS-1) of the rRNA gene cluster., Results: Rumenfilaria microfilariae were present in 20.5 % of Minnesota moose (n = 352), with slight fluctuations observed over four years. Minnesota white-tailed deer (Odocoileus virginianus) (n = 2) and moose (n = 44) from Alaska, Montana, Washington, Maine, and New Hampshire also harbored R. andersoni, suggesting this parasite occurs widely throughout North American moose herds, and white-tailed deer can serve as a patent host. Sequence analysis of cervid blood (moose, n = 15; white-tailed deer, n = 1) confirmed the identity of R. andersoni and revealed the existence of two distinct clades. Genetic comparisons of R. andersoni isolates from North America and semi-domesticated Finnish reindeer found the two groups to be closely related, supporting previous hypotheses that R. andersoni was recently introduced into Finland by the importation of deer from the United States., Conclusions: To the best of our knowledge these observations represent the first report of R. andersoni within the contiguous United States and reveal this nematode as a common parasite of North American moose and white-tailed deer. Although the implications of R. andersoni infection on moose health is unclear, increased awareness of this parasite will help prevent unintentional introduction of R. andersoni into naïve populations via the translocation of wild and captive cervids.

:  During a long-term study on biodiversity of bats in the Yungas and Entre Ríos provinces, 1,304 specimens of bats included in the families Noctilionidae, Phyllostomidae, Vespertilionidae, and Molossidae were collected and checked for filarioids. Litomosoides molossi Esslinger, 1973 was recovered from the thoracic and abdominal cavities of Molossus molossus (prevalence [P] = 6.4%); Litomosoides chandleri Esslinger, 1973 from Artibeus planirostris (P = 6.9%), Sturnira oporaphilum (P = 66.6%), Sturnira erythromos (P = 23.8%), Sturnira lilium (P = 7.2%), and Eumops perotis (P = 50%); and Litomosoides saltensis n. sp. was collected from Eptesicus furinalis (P = 1.7%). In this paper, we emend the description of L. molossi; describe a new species, Litomosoides saltensis n. sp., on the basis of 1 female specimen; and report for the first time L. molossi and L. chandleri parasitizing Argentinean bats, expanding the host and locality records. Litomosoides molossi exhibits a slender buccal capsule, with an anterior segment transparent, and the posterior chitinous portion displays 2 thickenings in the first third; possesses 1 dorsal prominent cephalic papilla and 4 labial papillae distributed around the mouth; cuticle with lateral punctuations all along the hypodermic chords in both sexes; and male with area rugosa and tail without cloacal papillae. In L. chandleri, the lateral punctuations are distributed on the posterior extremity of the body in both sexes. Litomosoides saltensis n. sp. displays a thick buccal capsule with a posterior segment well cuticularized, possessing 2 thickenings in the anterior half; 4 labial and 2 ventral cephalic papillae; a globular vulva located anterior to the esophagus-intestine junction; cuticle with lateral punctuations in the posterior extremity of the body; and tail with salient phasmids. We also provide a taxonomic key for the identification of the Litomosoides sp. of bat dwelling. Long-term studies and large sample sizes are needed to detect the presence of Litomosoides sp. in bats, in contrast to the findings in cricetid rodents, which seem to be more frequently collected.

Dirofilaria repens is endemic in eastern and southern European regions but was recently found in Germany in dogs, mosquitoes and one human patient. Since some of the positive dog and mosquito samples were collected in Brandenburg, it was aimed to systematically assess the prevalence of D. repens and other canine vector-borne pathogens in Brandenburg. Dog owners also received a questionnaire and were asked to provide more information about the dogs including travel history. In total, 1023 dog blood samples as well as 195 fox spleen and 179 fox blood samples were collected. DNA was analysed by PCR for the presence of filariae, piroplasms, anaplasmataceae and Rickettsia spp. Filariae were detected in six dogs (0.6%), two were positive for DNA from D. repens, two from Dirofilaria immitis and two from Acanthocheilonema reconditum. One of the D. repens positive dogs originated from an animal shelter in Brandenburg, but the origin of the other one remained unknown. Interestingly, both D. repens ITS-1 sequences showed 100% identity to a D. repens sample obtained from a Japanese woman that travelled in Europe and were 97% identical to a newly proposed species Dirofilaria sp. 'hongkongensis' described from Hong Kong. However, identity to other D. repens sequences from Thailand was considerably lower (81%). Identity of 12S rRNA and cytochrome oxidase I to D. repens samples from southern Europe was 99%. Due to the low number of Dirofilaria spp. positive dogs and since the origin of these was unknown, endemic occurrence of Dirofilaria in Brandenburg could not be confirmed. Anaplasma phagocytophilum was found in 15 dogs (1.5%), Candidatus Neoehrlichia mikurensis in three dogs (0.3%) and E. canis in one dog (0.1%), which was co-infected with D. repens. Rickettsia spp. were detected in 8 dogs (0.8%), seven were Rickettsia raoultii and one was Rickettsia felis. To the author's knowledge, R. raoultii DNA was detected for the first time in dogs in Germany in this study and Candidatus N. mikurensis for the second time. In spleen samples of red foxes with 47.5% a high prevalence of piroplasms was found. Sequencing of 11 samples identified 10 as Theileria annae. Despite the high prevalence of this pathogen in its reservoir host, it was absent in dog samples. In one dog (0.1%), Babesia canis was detected but there was no further information about the dog's origin. Evaluation of the questionnaire identified a high proportion of dogs (74.2%, n=233) which was not protected by ectoparasiticides. Moreover, 21.2% (n=236) of the dogs originated from inland or abroad shelters, and therefore might potentially come from areas endemic for dirofilariosis or babesiosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The genus Micipsella comprises three species of filariae to date identified in lagomorphs only, whereas the other genera belonging to the subfamily Splendidofilariinae are described as parasites of birds, reptiles and mammals. In the present study seven specimens of Micipsella numidica (Seurat, 1917), collected from the hare Lepus europaeus in Italy, were characterized genetically by molecular amplification of the mitochondrial genes (12S rDNA; cox1) and the 5S rDNA gene spacer region. Phylogenetic trees inferred using available sequences from filariae and those identified in this study evidenced a close relationship between M. numidica and Splendidofilariinae of other mammals and reptiles (Rumenfilaria andersoni and Madathamugadia hiepei). The present findings, apart from adding new data about the hosts in Italy, support the taxonomic position of M. numidica and highlight the substantial biological and molecular differences existing between Splendidofilariinae and other Onchocercidae. The study also contributes to our knowledge of the molecular/genetic diagnosis of filarial parasites of veterinary and medical concern in any vertebrate or invertebrate host.

The riparian European mink (Mustela lutreola), currently surviving in only three unconnected sites in Europe, is now listed as a critically endangered species according to the IUCN. Habitat loss and degradation, anthropic mortality, interaction with the feral American mink (Neovison vison), and infectious diseases are among the principal causes of its decline. Surveys of helminth parasites of this host that also include focus on subcutaneous potentially pathogenic helminths such as those belonging to the genus Filaria are very scarce. We report here the presence of specimens of Filaria martis in the subcutaneous connective tissues of three M. lutreola individuals from Spain. This is the first finding of a subcutaneous nematode in a representative of the genus Mustela. The report also enlarges the known range of the definitive hosts of this nematode. These worms were mainly located in the dorsal region of mink and more rarely in the knees, elbows, and hips. Skin sloughing was only observed in one M. lutreola with both septicaemia and an associated high burden of F. martis. Therefore, more attention should be paid to potentially pathogenic helminths when designing conservation programs dedicated to M. lutreola.

Background: Leishmaniosis caused by the protozoan Leishmania infantum and dirofilariosis caused by the nematodes Dirofilaria immitis or Dirofilaria repens are vector-borne zoonoses widely present in the Mediterranean basin. In addition, some studies reported that the endosymbiont Wolbachia spp. play a role in the biology and pathogenesis of filarial parasites. The aim of this work was to evaluate the frequency of mono- and co-infections by L. infantum, filariae and Wolbachia spp. and their association with clinical signs in dogs from the south of Portugal. Leishmanial, filarial and Wolbachia spp. DNA were evaluated by specific real-time polymerase chain reaction (qPCR) assays in blood samples from 230 dogs., Findings: One hundred and thirty-nine (60.4 %) dogs were qPCR-positive for L. infantum and 26 (11.3 %) for filariae (24 for D. immitis only, one D. immitis and for Acanthocheilonema dracunculoides and another one for Acanthocheilonema reconditum only). Wolbachia spp. DNA was amplified from 16 (64.0 %) out of the 25 D. immitis-positive dogs. Nineteen (8.3 %) dogs were co-infected with L. infantum and D. immitis, including the one (0.4 %) A. drancunculoides-positive animal. In dogs without clinical signs consistent with leishmaniosis and/or dirofilariosis, L. infantum prevalence was 69 %, whereas in those dogs with at least one clinical manifestation compatible with any of the two parasitoses prevalence was 42.7 %. Leishmania prevalence was significantly higher in apparently healthy mongrels (77.2 %) and pets (76.9 %) than in defined-breed dogs (including crosses; 58.8 %) and in dogs with an aptitude other than pet (i.e. farm, guard, hunting, shepherd or stray), respectively, whereas in those dogs with at least one clinical sign, the detection of L. infantum DNA was higher in males (53.3 %) and in those dogs not receiving insect repellents (52.8 %)., Conclusions: The molecular detection of canine vector-borne disease (CVBD) agents, some of which are zoonotic, reinforces the need to implement efficient prophylactic measures, such as insect repellents and macrocyclic lactones (including compliance to administration), in the geographical areas where these agents are distributed, with the view to prevent infection and disease among mammalian hosts including humans.

Ticks collected in 2011 were screened for the presence of filarial nematode genetic material, and positive samples were sequenced for analysis. Monanema-like filarial nematode DNA was recently discovered in Amblyomma americanum in northern Virginia, marking the first time genetic material from this parasite has been discovered in ticks in the state. Phylogenetic analysis revealed that this material was directly related to a previously discovered filarial nematode in A. americanum populations in Maryland as well as recently identified parasites in Ixodes scapularis from southern Connecticut. Further study is warranted to visually confirm the presence of these nematodes, characterize their distribution, and determine if these ticks are intermediate hosts., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Lymphatic filariasis (LF) is a chronic disease and is caused by the parasites Wuchereria bancrofti (W. bancrofti), Brugia malayi (B. malayi) and Brugia timori (B. timori). In the present study, Setaria cervi (S. cervi), a bovine filarial parasite has been used. Previously, it has been reported that the S. cervi shares some common proteins and antigenic determinants with that of human filarial parasite. The larval stages of filarial species usually cannot be identified by classical morphology. Hence, molecular characterization allows the identification of the parasites throughout all their developmental stages. The genomic DNA of S. cervi adult were isolated and estimated spectrophotometrically for the quantitative presence of DNA content. Screening of DNA sequences from filarial DNA GenBank and Expressed Sequence Tags (EST's) were performed for homologous sequences and then multiple sequence alignment was executed. The conserved sequences from multiple sequence alignment were used for In Silico primer designing. The successfully designed primers were used further in PCR amplifications. Therefore, in search of a promising diagnostic tool few genes were identified to be conserved in the human and bovine filariasis and these novel primers deigned may help to develop a promising diagnostic tool for identification of lymphatic filariasis.

This paper reports the findings of a study on the presence of various species of filarial nematodes in dogs in Liguria, north-west Italy, a region traditionally considered free from the disease. Between 2009 and 2012 blood samples were taken from 365 dogs in rural areas in Liguria. The blood samples were then submitted to Knott's test, histochemical staining, polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis antigens. Overall, 35 of the 365 dogs were positive using Knott's test for microfilariae (prevalence 9.6%; 95% confidence interval (CI): 6.6-12.6%). Acanthocheilonema reconditum was the most prevalent species (8.0%), while Dirofilaria repens (1.4%) and Dirofilaria immitis (0.6%) were less common. One co-infection by D. repens and A. reconditum was observed. All morphological identifications were confirmed by histochemical staining and PCR. In addition, a retrospective analysis of data on D. immitis antigens in 11,363 samples of canine sera was carried out. Sera were collected and analysed for D. immitis antigens by the Istituto Zooprofilattico Sperimentale (IZS) of Piedmont, Liguria and Aosta Valley (Imperia section) between 2004 and 2013 during annual tests for leishmaniasis on autochthonous dogs throughout Liguria. Serological data from IZS showed an overall seroprevalence of 0.65% (95% CI: 0.50-0.80%) for D. immitis throughout the region. The present study updates the epidemiological map of canine filarial infections in Italy and suggests the need for surveillance and prophylaxis in Liguria.

The neglected tropical disease onchocerciasis affects more than 35 million people worldwide with over 95% in Africa. Disease infection initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious L3 larvae. New treatments and diagnostics are required to eradicate this parasitic disease. Herein, we describe that a previously discovered biomarker for onchocerciasis, N-acetyltyramine-O-glucuronide (NATOG) is also present in urine samples of jirds infected with the onchocerciasis model nematode Litomosoides sigmodontis. Increased NATOG values paralleled a progressing infection and demonstrated that quantification of NATOG in this rodent model can be utilized to track its infectivity. Moreover, our findings suggest how NATOG monitoring may be used for evaluating potential drug candidates., (Copyright © 2015 Elsevier Ltd. All rights reserved.)