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Nematodes of the Onchocercidae family, such as Pelecitus spp., are filarial parasites of medical and veterinary importance. Although infections are widely distributed among avian species, only 2 cases of human Pelecitus ocular infection, both in South America, have been reported. We describe a 61-year-old man in northeast Thailand diagnosed with an ocular infection. Morphologic characteristics suggested the causative agent was a female Pelecitus nematode: coiled body, rounded anterior and posterior extremities, a distinct preesophageal cuticular ring, lateral alae, a postdeirid, and a protuberant vulva. Sequences of the 12S rDNA gene indicated 95%-96% identity and cox1 gene 92%-96% identity with published P. copsychi sequences. P-distance for cox1 sequences between the causative agent and P. copsychi was 6.71%. Phylogenetic trees of 12S rDNA and cox1 genes indicated the species differed from but is closely associated with P. copsychi. Healthcare providers should be aware of the threat of ocular infection from Pelecitus spp. nematodes.

Background Filarioses are common nematode infections in camels ( Camelus spp.). The most significant disease is caused by Deraiophoronema evansi , which impacts camel reproductive function, working ability, and productivity. The taxonomy of this onchocercid is equivocal, and its phylogenetic relationships within Onchocercidae are ambiguous. Methods We analyzed D. evansi specimens from camels and examined their morphology. For comparative material, we analyzed fresh specimens from camels in Iran and specimens from Egypt deposited in the Muséum National d’Histoire Naturelle (MNHN) collections. Multi-locus sequence analyses based on seven genes (two mitochondrial genes cox1 and 12S ribosomal DNA (rDNA) and five nuclear genes 18S rDNA, 28S rDNA, MyoHC , rbp1 , and hsp70 ) of these filarioids and six genes of Wolbachia ( 16S rDNA, ftsZ , dnaA , coxA , fbpA , and gatB ) were analyzed. Results Deraiophoronema evansi (Lewis, 1882) Romanovitch 1916 combinatio nova (n. comb.)(syn: Dipetalonema evansi ) was described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with Setaria sp. Wolbachia endosymbiont was not present in D. evansi . Conclusions We provide new molecular and morphological data on D. evansi , increasing the number of valid genera of Setariinae to four and setting up the taxonomic information regarding this species of veterinary importance. Graphical abstract

Background In camels, thelaziosis is mainly caused by Thelazia leesei Railliet & Henry, 1910, a little-known eyeworm species. Given the paucity of scientific data, this study aimed to provide new insights into the morphology, molecular characterization, and phylogenetic relationship of T. leesei and its occurrence in camels from Iran, where animals suffer from the high burden of eyeworms. Methods From December 2020 to November 2022, slaughtered camels ( n  = 400) of different sex and age groups were examined in Sistan-va-Baluchestan province in Southeast Iran’s local abattoirs. Adult eyeworms were fixed and stored for morphological identification by light and scanning electron microscopy (SEM). Polymerase chain reaction (PCR) products corresponding to the partial sequences of the mitochondrial cytochrome c oxidase subunit I ( cox 1) of eyeworms were Sanger sequenced and analyzed phylogenetically. Results A total of 118 (29.5%) camels from all five counties examined were infected with eyeworms, with an abundance of 0.9 and a mean intensity of 3.2 (i.e., up to 18 worms from a single animal). The infection rate was higher in camels older than 4 years of age ( P  = 0.01901). Lachrymation was associated with infection in animals ( P  < 0.00001). The morphology of our specimens resembled that of T .  leesei , with the exception of the position of the nerve ring and esophagus length. Genetic analysis showed that the cox 1 partial sequences of our T. leesei specimens had genetic distances of 8.8% to 13.5% compared with other Thelazia species. Conclusions On the basis of the morphometrics and morphological characteristics, we identified our specimens as T .  leesei . In the phylogenetic tree, T. leesei herein isolated formed a monophyletic group together with its congeners, and T .  leesei formed a sister clade to T. lacrymalis . In addition, we demonstrated the epidemiology of the infestation of T .  leesei in camels in the endemic areas of southeastern Iran. The data presented are crucial for better understanding the pathogenic role of T. leesei and developing effective treatment strategies. In particular, studies on the intermediate host(s) of T. leesei in these regions will support effective control strategies for this parasitosis. Graphical Abstract

Wolbachia is an alpha-proteobacterial symbiont widely distributed in arthropods. Since the identification of Wolbachia in certain animal-parasitic nematodes (the Onchocercidae or filariae), the relationship between arthropod and nematode Wolbachia has attracted great interest. The obligate symbiosis in filariae, which renders infected species susceptible to antibiotic chemotherapy, was held to be distinct from the Wolbachia -arthropod relationship, typified by reproductive parasitism. While co-evolutionary signatures in Wolbachia -arthropod symbioses are generally weak, reflecting horizontal transmission events, strict co-evolution between filariae and Wolbachia has been reported previously. However, the absence of close outgroups for phylogenetic studies prevented the determination of which host group originally acquired Wolbachia . Here, we present the largest co-phylogenetic analysis of Wolbachia in filariae performed to date including: (i) a screening and an updated phylogeny of Wolbachia ; (ii) a co-phylogenetic analysis; and (iii) a hypothesis on the acquisition of Wolbachia infection. First, our results show a general overestimation of Wolbachia occurrence and support the hypothesis of an ancestral absence of infection in the nematode phylum. The accuracy of supergroup J is also underlined. Second, although a global pattern of coevolution remains, the signal is derived predominantly from filarial clades associated with Wolbachia in supergroups C and J. In other filarial clades, harbouring Wolbachia supergroups D and F, horizontal acquisitions and secondary losses are common. Finally, our results suggest that supergroup C is the basal Wolbachia clade within the Ecdysozoa. This hypothesis on the origin of Wolbachia would change drastically our understanding of Wolbachia evolution.

Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals. We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected. The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions.

Background Onchocerca fasciata is a prevalent filarial species in camelids of Asia and Africa forming nodules in the skin of dromedary and Bactrian camels. In spite of recent advances in the biology and epidemiology of this nematode species, a relatively scant number of studies have focussed on the morphology of this parasite. The main objective of this study was to describe morphological characteristics of adults, microfilariae and eggs of O. fasciata by scanning electron microscopy (SEM), staining and histology. Methods From April 2016 to March 2017 dromedary camels ( n  = 456) were inspected for infection with O. fasciata in a slaughterhouse in Kerman (south of Iran). Adult worms in nodules were isolated by digestion of nodules in collagenase and used for SEM. Skin nodules were also fixed, sectioned and stained with hematoxylin and eosin for histopathology. Skin microfilariae that were isolated from tissues surrounding the nodules were confirmed as O. fasciata by sequencing of the cytochrome c oxidase subunit 1 ( cox 1) and 12S rRNA genes and used for SEM and Giemsa staining. Results Single or multiple O. fasciata nodules (1.2–2.2 cm in diameter and 507–845 mg in weight) were found in 30.3% of the examined camels. SEM analysis helped identify 18 papillae in the caudal region of the male. Discontinuous longitudinal cuticular crests were observed in the posterior region of the male. In female nematodes, the ridges had a rounded shape with a height/width ratio of 7/16 in longitudinal sections. Unsheathed skin microfilariae with a rounded anterior extremity measured 210.7 × 2.5 μm on average. Developed eggs containing microfilariae measured 35.9 × 31.0 μm and their smooth shell surface had characteristic tongue-like appendages. In addition to inflammatory reactions surrounding the parasites, accumulation of intracellular ceroid pigment, golden-yellow to brown in colour, was observed within macrophages upon histopathological examination. Conclusions We found longitudinal crests on the surface of the posterior region of the male nematode. Measurements of the main morphological features of microfilariae and eggs, and the shape index of ridges (height/width) in female nematodes are described for the first time.

Infections by gastrointestinal parasites are found in a variety of animals worldwide. For the diagnosis of such infections, the flotation method is commonly used to detect parasitic microorganisms, such as oocysts or eggs, in feces. Instead of adding a flotation solution after the final centrifugation step and using a cover slip to collect the parasites, the method using a wire loop for the recovery of the organisms has been reported as one of alternative methods. However, the recovery rates of microorganisms from the flotation method have not been analysed. In the present study, the utility of a flotation method with the use of a wire loop of 8 mm in diameter (the loop method) was evaluated using different numbers of E. tenella oocysts and Heterakis gallinarum eggs, and chicken fecal samples collected at the farms. Consequently, we found that the oocysts and eggs in tubes could be collected at a ratio of 2.00 to 3.08. Thus, our results indicate that the loop method is a simple and time saving method, implicating the application for the estimated OPG/ EPG (Oocysts/Eggs per gram) of the samples. Graphical ► Utility of a flotation method with the use of a wire loop of 8 mm in diameter was evaluated. ► E. tenella oocysts and Heterakis gallinarum eggs in tubes could be collected at a ratio of 2.00 to 3.08. ► Our results may implicate the application for the estimated OPG/ EPG of the samples as a simple and time saving method.

Background We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers ( coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms. Results DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species. Conclusion An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.

Background Dirofilaria ursi is a filarial nematode that parasitizes the subcutaneous tissues of the American black bear ( Ursus americanus ) and Japanese black bear ( Ursus thiabetanus japonicus ). D . ursi that has parasitized black bears has the potential to subsequently infect humans. In addition, extra-gastrointestinal anisakiasis is less common in Japan. Case presentation We report a case of ventral subcutaneous anisakiasis and dorsal subcutaneous dirofilariasis that was acquired in Fukushima, in the northern part of Japan. The patient was an 83-year-old Japanese female, and subcutaneous parasitic granulomas were present on her left abdomen (near the navel) and left scapula. A pathological examination of the surgically dissected tissue sections from each region demonstrated eosinophilic granulomas containing different species of parasites. To enable the morphological and molecular identification of these parasites, DNA was extracted from paraffin-embedded sections using DEXPAT reagent, and the cytochrome oxidase 2 (COX2), internal transcribed spacer 1 (ITS1), 5.8S and ITS2 regions of the Anisakis larvae, and the 5S rRNA region of the male Dirofilaria were sequenced. The PCR products were examined and compared with DNA databases. Molecular analysis of the COX2 and 5S rRNA sequences of each worm revealed that the nematode found in the ventral region belonged to Anisakis simplex sensu stricto (s.s.) and the male Dirofilaria found in the dorsal region was classified as D. ursi . Conclusion The present case showed a combined human case of D. ursi and A. simplex s.s. infections in subcutaneous tissues. The results of this study will contribute to the identification of unknown parasites in histological sections.

Background The genus Onchocerca Diesing, 1841 includes species of medical importance, such as O. volvulus (Leuckart, 1893), which causes river blindness in the tropics. Recently, zoonotic onchocercosis has been reported in humans worldwide. In Japan, O. dewittei japonica Uni, Bain & Takaoka, 2001 from wild boars is a causative agent for this zoonosis. Many filarioid nematodes are infected with Wolbachia endosymbionts which exhibit various evolutionary relationships with their hosts. While investigating the filarial fauna of Borneo, we discovered an undescribed Onchocerca species in the bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae). Methods We isolated Onchocerca specimens from bearded pigs and examined their morphology. For comparative material, we collected fresh specimens of O. d. dewittei Bain, Ramachandran, Petter & Mak, 1977 from banded pigs ( S. scrofa vittatus Boie) in Peninsular Malaysia. Partial sequences of three different genes (two mitochondrial genes, cox 1 and 12S rRNA, and one nuclear ITS region) of these filarioids were analysed. By multi-locus sequence analyses based on six genes ( 16S rDNA, ftsZ , dnaA , coxA , fbpA and gatB ) of Wolbachia , we determined the supergroups in the specimens from bearded pigs and those of O. d. dewittei . Results Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. is described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with O. d. dewittei . Calculated p-distance for the cox 1 gene sequences between O. borneensis n. sp. and O. d. dewittei was 5.9%, while that between O. d. dewittei and O. d. japonica was 7.6%. No intraspecific genetic variation was found for the new species. Wolbachia strains identified in the new species and O. d. dewittei belonged to supergroup C and are closely related. Conclusions Our molecular analyses of filarioids from Asian suids indicate that the new species is sister to O. d. dewittei . On the basis of its morphological and molecular characteristics, we propose to elevate O. d. japonica to species level as O. japonica Uni, Bain & Takaoka, 2001. Coevolutionary relationships exist between the Wolbachia strains and their filarial hosts in Borneo and Peninsular Malaysia.