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.

HOST-SWITCHING EVENTS IN LITOMOSOIDES CHANDLER, 1931 (FILARIOIDEA: ONCHOCERCIDAE) ARE NOT RAMPANT BUT CLADE DEPENDENT.

The genus Litomosoides Chandler, 1931, includes species that as adults occur in the thoracic and abdominal cavity of mammalian hosts and are presumably vectored by mites. The vertebrate hosts include a variety of Neotropical mammals such as phyllostomid and mormoopid bats; cricetid, sciurid, and hystricognath rodents; and didelphid marsupials. It has been suggested that Litomosoides is not a monophyletic group and that rampant horizontal transfer explains their presence in disparate groups of mammals. Herein we present a phylogenetic reconstruction including mitochondrial genes of 13 vouchered species. This phylogeny is used to reconstruct the evolutionary history of these parasites and the ancestral states of key characters used in species classification, namely, the configuration of the spicules. The historical association of these filarioids with 6 groups of mammals, as well as their ancestral geographic distributions, were reconstructed using Bayesian statistical approaches comparing alternative models of biogeography and evolution and fossil states in selected nodes of the phylogeny. The optimal reconstruction suggests a model of dispersal, extinction, and cladogenesis (DEC) driving the evolution of Litomosoides; the results suggest an origin of Litomosoides in South America and association of ancestors with phyllostomids, and strong evidence of at least 2 host-switching events: 1 of these involving cricetid rodents and the other mormoopid bats. The latter event included a simultaneous geographic expansion of the parasite lineage across South and North America. The host-switching event from phyllostomid bats into cricetid rodents occurred once these rodents diversified across South America; subsequent diversification of the latter clade resulted in 2 branches, each showing expansion of the parasites back into North America. This result suggests that both parasites and cricetid rodents established an association in South America, underwent diversification, and then dispersed into North America. Further, this clade of cricetid-dwelling species includes parasites featuring the "sigmodontis" spicule type. The identification of a single host-switching event involving the disparate lineages of Chiroptera and Rodentia offers a framework to reconstruct the gene evolution and diversification of this lineage after the host-switching event. This will help in predicting the ability of these parasites to infect sympatric mammals.

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.

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.

First report of an Onchocercidae worm infecting Psychodopygus carrerai carrerai sandfly, a putative vector of Leishmania braziliensis in the Amazon.

Sandflies are insects of public health interest due to their role as vectors of parasites of the genus Leishmania, as well as other pathogens. Psychodopygus carrerai carrerai is considered an important sylvatic vector of Leishmania (Viannia) braziliensis in Amazonia. In this study, sandflies were collected in a forested area in the Xapuri municipality, in the State of Acre (Northern Brazil). Two Ps. carrerai carrerai females were found parasitized with a larval form of a filarial worm, one in the labium of the proboscis, the other after the head was squashed, suggesting they were infective larvae. Sandflies were identified through morphological characters as well as amplification and sequencing of the cytochrome oxidase gene (COI). This was the first sequence obtained for Ps. carrerai carrerai for this marker. The obtained nematodes were also characterized through direct sequencing of a fragment of COI and 12S genes, both mitochondrial, and ITS1, a nuclear marker. Phylogenetic analyses revealed that the filarial nematodes belong to a species without sequences for these markers in the database, part of family Onchocercidade and closely related to genus Onchocerca (12S tree). Although sandfly infection with nematodes including members of the Onchocercidae has been reported in the Old World, this is the first report of sandfly infection by a member of the Onchocercidae family in the New World, to the best of our knowledge. Considering that the phylogenetic relationships and location in the insect, it can be expected that this is a parasite of mammals and the transmission cycle should be clarified.

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.

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.

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.

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.

Screening of Cercopithifilaria bainae and Hepatozoon canis in ticks collected from dogs of Northeastern Brazil.

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.

New insights into the clinico-histopathological and molecular features of Pelecitus (Filarioidea: Onchocercidae) from a raptor bird.

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.

Human filarial proteins attenuate chronic colitis in an experimental mouse model.

Encouraged by our earlier results of promising therapeutic effect of filarial recombinant proteins BmALT2, BmCys and WbL2 individually in the mouse model of acute ulcerative colitis, in this study, these proteins have been explored individually and in different combinations for their therapeutic potential in dextran sulphate sodium (DSS)-induced chronic colitis mice. These mice, treated with filarial proteins, showed reduced disease parameters including body weight loss, disease activity index, macroscopic and histopathological scores of colon and myeloperoxidase activity in colonic mucosa. Among various treatment schemes, rBmALT2 + rBmCys which showed most pronounced therapeutic implication was found to downregulate the mRNA expressions of IFN-γ and TNF-α and upregulate IL-10 and TGF-ß expression in the splenocytes. Also, increase in level of IgG1 and IgG2a isotypes in the sera of rBmALT2 + rBmCys-treated colitis mice was noted. Activated NF-κB level was found to be reduced in the colon of treated colitis mice compared to untreated one. In conclusion, filarial proteins in combination have been shown to improve the clinicopathologic status of chronic colitis through suppression of pro-inflammatory immune response most possibly in NF-κB-dependent manner. We propose this therapeutic strategy to be tested further to be considered as an effective option in chronic colitis.

GRANULOMATOUS FILARIAL ENCEPHALOMYELITIS CAUSED BY CHANDLERELLA QUISCALI IN A NORTHERN CRESTED CARACARA (CARACARA CHERIWAY).

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.

Filarioids infecting dogs in northeastern Brazil.

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.

Emended Description of Litomosoides molossi (Nematoda: Onchocercidae) and First Records of Litomosoides Species Parasitizing Argentinean Bats.

: 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.

Diversity, Host Specialization, and Geographic Structure of Filarial Nematodes Infecting Malagasy Bats.

We investigated filarial infection in Malagasy bats to gain insights into the diversity of these parasites and explore the factors shaping their distribution. Samples were obtained from 947 individual bats collected from 52 sites on Madagascar and representing 31 of the 44 species currently recognized on the island. Samples were screened for the presence of micro- and macro-parasites through both molecular and morphological approaches. Phylogenetic analyses showed that filarial diversity in Malagasy bats formed three main groups, the most common represented by Litomosa spp. infecting Miniopterus spp. (Miniopteridae); a second group infecting Pipistrellus cf. hesperidus (Vespertilionidae) embedded within the Litomosoides cluster, which is recognized herein for the first time from Madagascar; and a third group composed of lineages with no clear genetic relationship to both previously described filarial nematodes and found in M. griveaudi, Myotis goudoti, Neoromicia matroka (Vespertilionidae), Otomops madagascariensis (Molossidae), and Paratriaenops furculus (Hipposideridae). We further analyzed the infection rates and distribution pattern of Litomosa spp., which was the most diverse and prevalent filarial taxon in our sample. Filarial infection was disproportionally more common in males than females in Miniopterus spp., which might be explained by some aspect of roosting behavior of these cave-dwelling bats. We also found marked geographic structure in the three Litomosa clades, mainly linked to bioclimatic conditions rather than host-parasite associations. While this study demonstrates distinct patterns of filarial nematode infection in Malagasy bats and highlights potential drivers of associated geographic distributions, future work should focus on their alpha taxonomy and characterize arthropod vectors.

Molecular and phylogenetic analysis of the filarial nematode Micipsella numidica from the hare Lepus europaeus in Italy.

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.

Canine filarial infections in Liguria, north-west Italy.

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.