The genome of Litomosoides sigmodontis illuminates the origins of Y chromosomes in filarial nematodes.

Heteromorphic sex chromosomes are usually thought to have originated from a pair of autosomes that acquired a sex-determining locus and subsequently stopped recombining, leading to degeneration of the sex-limited chromosome. The majority of nematode species lack heteromorphic sex chromosomes and determine sex using an X-chromosome counting mechanism, with males being hemizygous for one or more X chromosomes (XX/X0). Some filarial nematode species, including important parasites of humans, have heteromorphic XX/XY karyotypes. It has been assumed that sex is determined by a Y-linked locus in these species. However, karyotypic analyses suggested that filarial Y chromosomes are derived from the unfused homologue of an autosome involved in an X-autosome fusion event. Here, we generated a chromosome-level reference genome for Litomosoides sigmodontis, a filarial nematode with the ancestral filarial karyotype and sex determination mechanism (XX/X0). By mapping the assembled chromosomes to the rhabditid nematode ancestral linkage (or Nigon) elements, we infer that the ancestral filarial X chromosome was the product of a fusion between NigonX (the ancestrally X-linked element) and NigonD (ancestrally autosomal). In the two filarial lineages with XY systems, there have been two independent X-autosome chromosome fusion events involving different autosomal Nigon elements. In both lineages, the region shared by the neo-X and neo-Y chromosomes is within the ancestrally autosomal portion of the X, confirming that the filarial Y chromosomes are derived from the unfused homologue of the autosome. Sex determination in XY filarial nematodes therefore likely continues to operate via the ancestral X-chromosome counting mechanism, rather than via a Y-linked sex-determining locus.

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

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

Pathological findings associated with Dipetalonema spp. (Spirurida, Onchocercidae) infection in two species of Neotropical monkeys from Brazil.

Among vector-borne helminths, filarioids of the genus Dipetalonema (Spirurida: Onchocercidae) localize in several tissues and body cavities of several animal species, causing mild to moderate lesions. The pathological findings associated with Dipetalonema spp. infection in Neotropical monkeys from southern Brazil are herein described, along with a fatal case due to filarial polyserositis and entrapment of an intestinal segment. At necropsy, nematodes were observed in abdominal and thoracic cavities, or in the pericardium of 37 (31.3%) out of the 118 individuals examined (i.e., 35 Alouatta guariba clamitans and two Sapajus nigritus). In addition, at histology, 27.0% of positive animals presented microfilarie (inside blood vessels of lung, spleen, liver, and brain) and 8.1% presented adult nematodes in the heart, lung, and liver. In two cases, cross-sections of filarioids were associated with areas of epicardial thickening with intense fibrosis and pyogranulomatous inflammation in the brain, heart, liver, lungs, or spleen. The DNA fragment was amplify using the cox1 gene, sequenced and analyzed to identify the nematode species collected; presence of Wolbachia was assessed in the filarioids using the 16S rRNA gene. At BLAST analysis of the cox1 gene, 10 sequences showed 91.7% nucleotide identity with Dipetalonema gracile, and two with D. gracile (98.5%) and Dipetalonema graciliformis (98.3%). Phylogenetic analyses clustered sequences of the cox1 obtained in this study in two clades corresponding with the host species. Wolbachia sp. endosymbiont was detected in four samples. Data herein reported provide a description of pathological lesions associated with the infection by Dipetalonema spp., suggesting that they may cause disease in Neotropical monkeys. In addition, a better understanding of diversity and biology of Dipetalonema spp. in South America is needed to assess the impact they may cause in native non-human primates from Brazil.

Canine filaria species in selected lymphatic filariasis endemic and non-endemic areas in Sri Lanka.

Subperiodic brugian filariasis and dirofilariasis show a rising trend in Sri Lanka posing a threat to public health. As information was limited on canine filaria species in Sri Lanka, we studied the filaria parasites among dog populations in lymphatic filariasis (LF) endemic and non-endemic regions by microscopy and molecular methods. Thick blood smears (TBSs) were performed among 295 dogs presenting to veterinary clinics for surgical or sterilization procedures in Galle (LF endemic) and Mullaitivu (LF non-endemic) districts, of which 55.6% were positive for any microfilariae. We identified Dirofilaria repens (50.8%) and Brugia spp. (20.6%) by microscopy, which, included mono-infections (D. repens 35.3% and Brugia spp. 5%) and co-infections (15.6%). Infections in Galle and Mullaitivu were 61% and 44.9% respectively. The brugian filariasis rate was significantly higher among canines in LF endemic Galle district (29.9%) than in Mullaitivu (LF non-endemic) (1.1%) (P < 0.001), while D. repens infections were comparable in both districts. Genomic DNA extracted from 10% of microfilariae positive TBSs was amplified using pan-filarial primers targeting the internal-transcriber-spacer region-2 (ITS-2). Sequencing of amplicons confirmed the presence of D. repens (89.28%), Brugia pahangi (7.14%) and B. malayi (3.57%) infections. The phylogeny constructed and analysed in MEGA X indicated genetic variability among D. repens and B. pahangi isolates from Sri Lanka. With this study, we were able to report B. pahangi infections for the first time in Sri Lanka.

Ocular Filariasis in Human Caused by Breinlia (Johnstonema) annulipapillata Nematode, Australia.

We report a human case of ocular filariasis, caused by a species of Breinlia nematode, from Queensland, Australia. Morphological and molecular evidence indicated that the nematode Breinlia (Johnstonema) annulipapillata, or a closely related taxon, likely transmitted from a macropodid marsupial host was involved, which might represent an accidental finding or an emerging zoonosis.

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

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

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.

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.

Species-specific and cross-reactive antigen of filarial worm: Brugia malayi and Setaria digitata.

BACKGROUND & OBJECTIVES: Generally filarial antigens have been found to be cross-reactive in nature. Identification of genus and species-specific antigens has not been successful so far. Due to lack of human adult filarial parasite, researchers have been using other adult worms like Setaria digitata, a cattle parasite or Brugia malayi, a rodent model for their research work. In this situation, specificity of the prepared antigen (S. digitata or B. malayi) to detect the antibodies to Wuchereria bancrofti is questionable. METHODS: In the present investigation, we have tested a panel of human sera (collected from the areas, endemic for bancroftian filariasis) to correlate the immune reactivity against somatic antigens of adult stages and microfilarial stages of S. digitata and B. malayi. Further, using intact microfilariae (mf) from the above two parasites along with W. bancrofti, we have analyzed the antibody response to the sheath antigens. A panel of infected human and cattle sera was tested by immunoperoxidase assay using intact mf of three different parasites, viz. W. bancrofti, B. malayi, and S. digitata. RESULTS: A very significant positive correlation in filarial Igs (polyvalent), IgG, IgM, IgE and IgG4 levels were found between the two adult somatic antigens of B. malayi and S. digitata when tested against human filarial sera. However, such a correlation was not found when mf antigens of B. malayi and S. digitata were tested against a panel of W. bancrofti sera indicating that antigens present in mf could be far less cross-reactive in comparison to those in adult stage parasites. INTERPRETATION & CONCLUSION: The results indicated the differential cross-reactivity of antisheath antibodies to the mf sheath of three different filarial parasites. Soluble antigens of S. digitata could inhibit antisheath antibody reactivity to only S. digitata mf sheath and not to mf sheath of W. bancrofti further confirming the specificity of sheath antigen.

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.

MSProGene: integrative proteogenomics beyond six-frames and single nucleotide polymorphisms.

UNLABELLED: Ongoing advances in high-throughput technologies have facilitated accurate proteomic measurements and provide a wealth of information on genomic and transcript level. In proteogenomics, this multi-omics data is combined to analyze unannotated organisms and to allow more accurate sample-specific predictions. Existing analysis methods still mainly depend on six-frame translations or reference protein databases that are extended by transcriptomic information or known single nucleotide polymorphisms (SNPs). However, six-frames introduce an artificial sixfold increase of the target database and SNP integration requires a suitable database summarizing results from previous experiments. We overcome these limitations by introducing MSProGene, a new method for integrative proteogenomic analysis based on customized RNA-Seq driven transcript databases. MSProGene is independent from existing reference databases or annotated SNPs and avoids large six-frame translated databases by constructing sample-specific transcripts. In addition, it creates a network combining RNA-Seq and peptide information that is optimized by a maximum-flow algorithm. It thereby also allows resolving the ambiguity of shared peptides for protein inference. We applied MSProGene on three datasets and show that it facilitates a database-independent reliable yet accurate prediction on gene and protein level and additionally identifies novel genes. AVAILABILITY AND IMPLEMENTATION: MSProGene is written in Java and Python. It is open source and available at http://sourceforge.net/projects/msprogene/.

First report of Filaria martis Gmelin, 1790 in the European mink, Mustela lutreola (Linnaeus, 1761).

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.

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.

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.

Identification of Avian and Hemoparasite DNA in Blood-Engorged Abdomens of Culex pipiens (Diptera; Culicidae) from a West Nile Virus Epidemic region in Suburban Chicago, Illinois.

Multiple mosquito-borne parasites cocirculate in nature and potentially interact. To understand the community of parasites cocirculating with West Nile virus (WNV), we screened the bloodmeal content of Culex pipiens L. mosquitoes for three common types of hemoparasites. Blood-fed Cx. pipiens were collected from a WNV-epidemic area in suburban Chicago, IL, from May to September 2005 through 2010. DNA was extracted from dissected abdomens and subject to PCR and direct sequencing to identify the vertebrate host. RNA was extracted from the head or thorax and screened for WNV using quantitative reverse transcriptase PCR. Seventy-nine engorged females with avian host origin were screened using PCR and amplicon sequencing for filarioid nematodes, Haemosporida, and trypanosomatids. Filarioid nematodes were identified in 3.8% of the blooded abdomens, Plasmodium sp. in 8.9%, Haemoproteus in 31.6%, and Trypanosoma sp. in 6.3%. The sequences from these hemoparasite lineages were highly similar to sequences from birds in prior studies in suburban Chicago. Overall, 50.6% of blood-fed Culex pipiens contained hemoparasite DNA in their abdomen, presumably from current or prior bloodmeals. Additionally, we detected hemoparasite DNA in the blooded abdomen of three of 10 Cx. pipiens infected with WNV.

Overcoming species boundaries in peptide identification with Bayesian information criterion-driven error-tolerant peptide search (BICEPS).

Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695-716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis.

Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria.

Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link between Wolbachia and this nematode encoded protein. The function of nematode phosphate permease in the endosymbiosis is unknown but could involve transportation of phosphate to Wolbachia, which encode all the genes necessary for de novo nucleotide biosynthesis. Electron microscopic localization of PPE and Wolbachia and RNAi mediated knock-down of PPE in filarial nematodes will bring further insights to the functions of PPE in the Wolbachia-nematode symbiosis.

Cercopithifilaria spp. in dogs in Sardinia Island (Italy).

A survey on Cercopithifilaria spp. was carried out on owned and kennelled dogs in Sardinia, Italy. A total of 180 dogs were sampled and tested by microscopic detection or PCR of dermal microfilariae in skin snip sediments. The overall prevalence for Cercopithifilaria spp. at both microscopy and molecular tests was 9.4 % (17/180), while 8.3 % (15/180) of dogs scored positive at microscopic detection of sediments only. Of the 225 microfilariae measured, 212 were identified as Cercopithifilaria bainae and the remaining as Cercopithifilaria sp. II. All samples were molecularly processed for specific amplification of cytochrome oxidase subunit 1 (cox1) and ribosomal 12S gene fragments. The Basic Local Alignment Search Tool analysis of the cox1 and 12S sequences here obtained showed a high nucleotide similarity (99 and 100 %, respectively) with those of C. bainae available in GenBank. In particular, cox1 haplotype I (HI; n=14), haplotype HXVIII (n=2), and a new haplotype, named HXIX (n=1), differing for a single polymorphism from HI, were detected. This study reports data on the occurrence, distribution, and genetic makeup of C. bainae and Cercopithifilaria sp. II infesting dogs in Sardinia, suggesting that these filarioids are spread in areas where Rhipicephalus sanguineus sensu lato ticks occur.

A filarial parasite potentially associated with the health burden on domestic chickens in Japan.

Chickens in free-range environments are at risk of exposure to various pathogens, such as filarioids transmitted via hematophagous vectors. However, the study of filarioids in poultry has been largely neglected compared to the extensive studies focused on viruses, bacteria, and protozoa. Here, we performed histological and molecular investigations of the filarioids detected in domestic chickens from two different flocks in Hiroshima Prefecture, Japan. In the first case, adult worms were present in the pulmonary artery and right ventricle, and microfilariae were present in multiple organs of deceased chickens. In the second case, similar filarioids were detected in the organs and blood of one necropsied layer. Phylogenetic analysis using 18S rRNA gene fragments positioned the filarioid in the same clade as that of Onchocercidae sp., previously identified in a deceased chicken from Chiba Prefecture, Japan, that is located 500 km away from Hiroshima Prefecture. Based on 28S rRNA and mitochondrial COI gene fragments, the filarioid was positioned distinctly from previously reported genera of avian filarioids. These results suggest that the filarioids are potentially associated with the health burden on domestic chickens and belong to the genus Paronchocerca. Furthermore, we developed a nested PCR assay targeting mitochondrial COI and detected the parasite DNA from the biting midge Culicoides arakawae captured near the flock, suggesting that it serves as a vector. Our findings fill the knowledge gap regarding avian filarioids, laying the groundwork for future studies examining the epidemiology, life cycle, and species diversity of this neglected parasite group.