Dentition patterns and molecular diversity of Mastophorus muris (Gmelin, 1790) (Nematoda: Spiruroidea) support a host-associated subdivision.

Mastophorus muris (Gmelin, 1790) is a globally distributed parasitic nematode of broad range mammals. The taxonomy within the genus Mastophorus and the cryptic diversity among the genus are controversial among taxonomists. This study provides a detailed morphological description of M. muris from Mus musculus combined with a molecular phylogenetic approach. Moreover, descriptions and molecular data of M. muris from non-Mus rodents and wildcats complement our findings and together provide new insights into their taxonomy. The analysis of M. muris was based on light microscopy and scanning electron microscopy. The morphological description focused on the dentition pattern of the two trilobed pseudolabia. Additionally, we described the position of the vulva, arrangement of caudal pairs of papillae, spicules and measured specimens from both sexes and the eggs. For the molecular phylogenetic approach, we amplified the small subunit ribosomal RNA gene and the internal transcribed spacer, and the cytochrome c oxidase subunit 1. Mastophorus morphotypes based on dentition patterns and phylogenetic clustering indicate a subdivision of the genus in agreement with their host. We recognize two groups without a change to formal taxonomy: One group including those specimens infecting Mus musculus, and the second group including organisms infecting non-Mus rodents. Our genetic and morphological data shed light into the cryptic diversity within the genus Mastopohorus. We identified two host-associated groups of M. muris. The described morphotypes and genotypes of M. muris allow a consistent distinction between host-associated parasites.

First report of molecular and phylogenetic analysis of Physaloptera praeputialis in naturally infected stray cats from India.

Nematodes of the genus Physaloptera are globally distributed and infect a multitude of hosts. Their life cycle involves orthopterans and coleopterans as intermediate hosts. The morphological characters alone are inadequate to detect and differentiate Physaloptera spp. from its congeners. Moreover, molecular studies are limited to compare them precisely. The present communication reports the first molecular phylogenetic characterization of feline Physaloptera spp. from India based on mitochondrial cytochrome c oxidase subunit 1 (COX1) and small subunit ribosomal DNA (18S rDNA). The nematodes were first isolated from the stomach of adult stray cats during necropsy examination. Based on the gross and microscopic characters, the worms were identified as P. praeputialis. Morphological identification was further confirmed through PCR targeting the barcode region of the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene, using nematode-specific primers cocktail followed by species specific primers targeting partial COX1 and 18S rRNA genes. Generated sequences were submitted in NCBI GenBank (MW517846, MW410927, MW411349), and phylogenetic trees were constructed using the maximum likelihood method. When compared with other sequences of Physaloptera species across the globe, the present isolates showed 85.6-97.7% and 97.3-99% nucleotide homology based on COX1 and 18S rRNA gene, respectively. BLASTn analysis revealed a strong identity to other Physaloptera spp., and the phylogenetic tree placed all Physaloptera spp. in the same cluster. This study again indicates the usefulness of molecular techniques to substantiate the identity of species that may lack adequate descriptions and impart new insight for the potentially overlooked significance of P. praeputialis infections in felines.

Molecular phylogenetic study in Spirocercidae (Nematoda) with description of a new species Spirobakerus sagittalis sp. nov. in wild canid Cerdocyon thous from Brazil.

The nematode family Spirocercidae Chitwood and Wehr, 1932, comprises three subfamilies, Spirocercinae Chitwood and Wehr, 1932; Ascaropsinae Alicata and McIntosh, 1933; and Mastophorinae Quentin, 1970, which occur worldwide. Spirocercids infect canids and can cause severe illness. The crab-eating fox, Cerdocyon thous (Linnaeus, 1766), is a canid that inhabits most of South America, including Brazil and is a host for several parasitic worms, in particular, nematodes. However, few reports or genetic data are available on the spirocercids found in this host. In the present study, we describe a new species of Spirobakerus Chabaud and Bain 1981, from the intestine of two crab-eating foxes from two different biomes in Brazil. Spirobakerus sagittalis sp. nov. presents a) unequal spicules, with a long, thin left spicule with a lanceolated shape at the tip; b) a pair of sessile papillae and a median unpaired papillae located anteriorly of the cloaca, and c) a tuft without spines at the tip of the tail in females. The molecular phylogenetic analysis indicated that S. sagittalis sp. nov. is basal to the other species of the subfamily Ascaropsinae, which was not recovered as monophyletic. Our phylogenies also indicated that Spirocercidae is paraphyletic, given that Mastophorinae did not group with Ascaropsinae and Spirocercinae. We provide the first molecular data on the genus Spirobakerus and expand the molecular database of the spirocercids. However, further studies, including the sequences of other spirocercid taxa, are still needed to infer the relationships within this family more accurately.

Morphological and Molecular Evidence for a New Rhabdochonid Species (Nematoda: Rhabdochonidae) Parasitizing Profundulus oaxacae (Profundulidae) in the State of Oaxaca, Mexico.

The genus Rhabdochona Railliet, 1916 is one of the most speciose group of nematodes, parasites of freshwater fishes, with approximately 103 species described worldwide. Twenty-two species have been recorded in the Americas, 14 of them in Mexico. In this paper we describe a new species of Rhabdochona on the basis of light and scanning electron microscopy and molecular tools. Rhabdochona adentata n. sp. was recovered from the gallbladder of the freshwater Oaxaca killifish, Profundulus oaxacae (Meek, 1902) (Profundulidae) captured in the Río Grande, State of Oaxaca, Mexico. Rhabdochona adentata n. sp. differs markedly from its congeners by possessing an unusual prostom lacking anterior teeth, small simple deirids, and the location of the excretory pore at the level of the union of the muscular and glandular esophagus. Sequences of cytochrome c oxidase subunit I (COI) from mitochondrial deoxyribonucleic acid and the D2 + D3 domains of the large ribosomal subunit (28S) were obtained from 3 specimens and were analyzed using maximum likelihood and Bayesian inference analyses. Phylogenetic analyses using COI and 28S genes recovered 1 new lineage of Rhabdochona. The new species is described on the basis of a detailed morphological study. This parasite represents the first species of Rhabdochona without prostomal teeth and with a different site of infection, the gallbladder.

The Occurrence of Physaloptera hispida and a Mastophorus Sp. in Pulmonary Vessels of Hispid Cotton Rats (Sigmodon hispidus) from Georgia, U.S.A.

During 2017-2018, a survey for the rat lungworm, Angiostrongylus cantonensis (Nematoda: Metastrongyloidea), in rodents from Piedmont and Lower Coastal Plains physiographic regions of Georgia was conducted. On 4 occasions, a single worm was recovered from the pulmonary vessels of a single cotton rat (Sigmodon hispidis). One of these worms was identified as a Physaloptera sp. and the remaining 3 as a Mastophorus sp. by morphology. No A. cantonensis were found. Physaloptera (Nematoda: Physalopteroidea) and Mastophorus species (Nematoda: Spiruroidea) are stomach parasites of many wild and domestic animals. This is the first report of these species in the pulmonary vessels of a definitive host. To better characterize these parasites, representative specimens were collected from cotton rat stomachs and identified morphologically and molecularly. Based on partial cytochrome c oxidase subunit 1 (COI) gene sequences, Physaloptera hispida from stomachs were identical to the Physaloptera sp. from the pulmonary vessels. The COI sequences from the Mastophorus sp. from the stomach exhibited a higher degree of variability but confirmed that the pulmonary worms were the same Mastophorus species. Furthermore, sequences of Mastophorus from a coastal site clustered separately from a clade of Mastophorus sequences from cotton rats from a Piedmont site. Our data show that adult worms recovered from pulmonary vessels of cotton rats could be either Physaloptera or Mastophorus sp., indicating that these parasitic worms are not always restricted to the stomach and that worms from pulmonary vessels must be carefully examined to obtain a definitive diagnosis of A. cantonensis infection.

Incidence of cutaneous habronemosis in Manipuri ponies in India.

Information pertaining to parasitic fauna and parasitic diseases in Manipuri ponies in India is not available. Moreover, no systematic studies have been undertaken on cutaneous habronemosis in Manipuri ponies which is a common skin problem of Manipuri ponies as reported by pony owners. Keeping in the view of the importance of parasitic infections in veterinary health coverage particularly in Manipuri ponies, the present study was planned. A survey of natural cases of cutaneous habronemosis followed by molecular confirmation of species involved and treatments were done. Out of 200 ponies examined, nine cases (4.5%) of cutaneous habronemosis was recorded. Gross examination revealed raised and ulcerated wounds with necrotic tissues covered with yellowish-tan granulation. Histopathological study revealed eosinophilic granuloma and in the center of the granuloma with necrotic debris. Remnants of the Hebronema larvae with infiltrating neutrophils surrounded by proliferating fibrous tissue with numerous eosinophils, macrophages and lymphocytes were also observed. Molecular detection of Habronema sp. was confirmed by semi-nested PCR. Sequence analysis revealed larvae of H. muscae was the common spirurid species responsible for producing cutaneous habronemosis in Manipuri ponies. Subsequently, sequence submitted to NCBI GenBank and accession number obtained (MH038181). Surgical removal of necrotic tissue, ivermectin injection along with antibiotics successfully cured all the lesions in infected ponies.Results confirmed occurrence of cutaneous habronemosis in Manipuri ponies in India.

A New Species of Ascarophis (Nematoda: Cystidicolidae) Parasitizing Clinocottus analis (Pisces: Cottidae) from Baja California, Mexico.

A new species of nematode, Ascarophis morronei n. sp. (Cystidicolidae), is described from the stomach wall of the woolly sculpin Clinocottus analis (Cottidae) collected in the rocky intertidal from northwestern Baja California, Mexico. Collected nematodes were studied using both light and scanning electron microscopy. Sequence fragments for 18S rDNA molecular markers were obtained from the new nematode species, in order to test its position within the family Cystidicolidae under a phylogenetic context. Main characters distinguishing this new species include the reduced labia and the morphology of the eggs, distances of nerve ring and excretory pore from the anterior end, and left spicule of males. The new species described here is the second for the genus Ascarophis reported as adult in the Southern California Bight, and the first one recorded for the fish genus Clinocottus.

First case of human Gongylonema pulchrum infection in Slovenia.

Nematodes of the genus Gongylonema infect a wide range of mammals worldwide but are only sporadically reported in humans. We describe a case of human infection with Gongylonema pulchrum in a 41-year-old man. The patient extracted the nematode from the submucosa under his tongue and correctly self-diagnosed the infection with the help of the Google search engine. In the laboratory, the collected nematode was confirmed as G. pulchrum microscopically by morphological analysis and genetically by amplifying and sequencing the parasite's rDNA. This is the first report of human G. pulchrum infection in Slovenia.

Taxonomy of Physaloptera mirandai (Nematoda: Physalopteroidea) based in three-dimensional microscopy and phylogenetic positioning.

Gastrointestinal nematodes are important ecological assets for the maintenance of the biodiversity in the Atlantic Forest in Brazil. They parasitize a number of animals of the local fauna, in which some species can promote serious injuries in the stomach wall of their hosts, which may lead to death. Among these nematodes, parasites of the genus Physaloptera are known to parasitize mammals (particularly carnivores and small rodents), birds and reptiles, being important for the local biodiversity. In this work, three hundred and sixty-two nematodes were recovered from the stomach of twenty-one Metachirus nudicaudatus (Didelphimorphia: Didelphidae) collected in Duas Bocas Biological Reserve, State of Espírito Santo, one of the largest Atlantic Forest remnants and important wildlife refuge of the Atlantic Forest in Brazil. Analysis using fluorescence and scanning electron microscopy as well as phylogenetic assessment using the mitochondrial cytochrome c oxidase subunit I gene showed that the parasites belong to the Physaloptera. Our results show details of the nematode morphology including the cloacal papillae distribution, cuticular topography details, 2D and 3D measurements of the structures with taxonomic importance. Molecular data confirmed the validity of P. mirandai and the phylogeny supported the monophyly of the assemblage formed by Physaloptera and Turgida. The use of a combination of quantitative and multidimensional microscopy tools, such as 3D reconstruction and modeling, allied to phylogenetic analysis may provide grounds for a new approach on helminth taxonomy and structural characterization.

Diversity of Rhabdochona mexicana (Nematoda: Rhabdochonidae), a parasite of Astyanax spp. (Characidae) in Mexico and Guatemala, using mitochondrial and nuclear genes, with the description of a new species.

Among fish parasitic nematodes Rhabdochona is one of the most speciose genera, with c. 100 species. Twelve congeneric species occur in Mexican freshwater fishes, in a region located between the Nearctic and Neotropical biogeographical regions. Host association and biogeographical history have determined the high species richness of Rhabdochona in Mexico. One of these species, Rhabdochona mexicana, is highly specific to the characid genus Astyanax. Characids are a group of freshwater fish with Neotropical affinity. In this paper, we explore the genetic diversity of R. mexicana through samples obtained from populations of Astyanax spp. across river basins of Mexico and Guatemala. Sequences of one mitochondrial and two ribosomal genes were obtained from 38 individuals and analysed using Maximum Likelihood and Bayesian Inference analysis. Phylogenetic analyses using cox1, and a concatenated alignment of 18S + 28S + cox1 recovered two genetic lineages. One of them corresponded with R. mexicana sensu stricto; this lineage included three reciprocally monophyletic subgroups; the other lineage was highly divergent and represented a putative candidate species. A detailed morphological study was conducted to corroborate the molecular findings. We describe a new species herein and discuss the implications of using molecular tools to increase our knowledge about the diversity of a speciose genus such as Rhabdochona.

Taxonomic status of Rhabdochona ictaluri (Nematoda: Rhabdochonidae) based on molecular and morphological evidence.

The genus Rhabdochona includes more than 100 species infecting freshwater fishes in all zoogeographical regions of the world. In Mexico, 12 nominal species of Rhabdochona have been recorded. Of these, Rhabdochona ictaluri was originally described as a parasite of endemic catfishes of the family Ictaluridae; however, the species was later considered on morphological grounds as a junior synonym of Rhabdochona kidderi. In this study, newly sampled specimens of R. ictaluri were obtained from the type host and type locality and were used to perform a detailed morphological analysis and molecular phylogenetic inferences through one mitochondrial and two nuclear genes; data were used in an integrative taxonomy context to test the taxonomic status of R. ictaluri. This approach proved to be very useful to confirm the validity of this species, and robust species limits were established between these two putative species considering morphology, molecular data, host association, and biogeography.

Gongylonema infection of wild mammals in Japan and Sardinia (Italy).

The gullet worms, classical Gongylonema pulchrum and newly differentiated Gongylonema nepalensis, are prevalent in various mammals in Japan and Sardinia, Italy, respectively. The former species is cosmopolitan in distribution, dwelling in the mucosa of the upper digestive tract of a variety of domestic and wild mammals, and also humans. At present, the geographical distribution of G. nepalensis is known in Nepal and Sardinia, with the nematode having been recorded from the oesophagus of water buffaloes (Nepal), cattle, sheep, goats and wild mouflon (Sardinia). To clarify their natural transmission cycles among domestic and wild mammals, the present study analysed the ribosomal RNA gene (rDNA) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) of worms of various origins: G. pulchrum worms from sika deer, wild boars, Japanese macaques, and feral alien Reeves's muntjacs in Japan, and G. nepalensis worms from a red fox and a wild boar in Sardinia. Although the internal transcribed spacer (ITS) regions of rDNA and partial cox1 nucleotide sequences of G. pulchrum from native wild mammals in Japan were distinct from those of the worms in cattle, the worms from feral alien Reeves's muntjacs showed the cattle-type ITS genotype and cox1 cattle-I and II haplotypes. The rDNA and cox1 nucleotide sequences of G. nepalensis from a red fox in Sardinia were almost identical to those of the worms from domestic and wild ruminants on the island. The ecological interaction between domestic and wild mammals and their susceptibility to different Gongylonema spp. must be considered when trying to elucidate this spirurid's transmission dynamics in nature.

Molecular identification of Physaloptera sp. from wild northern bobwhite (Colinus virginianus) in the Rolling Plains ecoregion of Texas.

Physaloptera spp. are common nematodes found in the stomach and muscles of mammals, reptiles, amphibians, and birds. Physaloptera spp. have a complicated life cycle with multiple definitive hosts, arthropod intermediate hosts, aberrant infections, and possible second intermediate hosts or paratenic hosts. For example, Physaloptera sp. larvae have been found within the tissues of wild northern bobwhite quail (Colinus virginianus), and it is suspected that quail may serve as paratenic or secondary hosts of these parasites. However, because it is not known what role quail play in the life cycle of Physaloptera spp. and descriptions of Physaloptera spp. larvae are limited, molecular tools may be beneficial when identifying these helminths. In this study, we generated primers using universal nematode primers and obtained a partial mitochondrial cytochrome oxidase 1 (COX 1) sequence. Morphological identification of Physaloptera sp. in bobwhite was confirmed via polymerase chain reaction (PCR), and a phylogenetic tree was constructed using the maximum likelihood method. BLAST analysis revealed a strong identity to other Physaloptera spp. and the phylogenetic tree placed all Physaloptera spp. in the same cluster. We also documented a marked increase in Physaloptera infections in bobwhite from 2017 to 2018, and the similarity of these parasites to Onchocerca volvulus and Wuchereria bancrofti may give insight into the increased prevalence we observed. This study demonstrates the usefulness of molecular techniques to confirm the identity of species that may lack adequate descriptions and provides new insight for the diagnosis and potentially overlooked significance of Physaloptera sp. infections of bobwhite in the Rolling Plains ecoregion of Texas.

Molecular genetic diversity of Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914) (Spirurida: Gongylonematidae) from rodents in Southeast Asia.

More than a dozen Gongylonema spp. (Spirurida: Spiruroidea: Gongylonematidae) have been described from a variety of rodent hosts worldwide. Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914), which dwells in the gastric mucosa of rats such as Rattus norvegicus (Berkenhout) and Rattus rattus (Linnaeus), is currently regarded as a cosmopolitan nematode in accordance with global dispersion of its definitive hosts beyond Asia. To facilitate the reliable specific differentiation of local rodent Gongylonema spp. from the cosmopolitan congener, the genetic characterisation of G. neoplasticum from Asian Rattus spp. in the original endemic area should be considered since the morphological identification of Gongylonema spp. is often difficult due to variations of critical phenotypical characters, e.g. spicule lengths and numbers of caudal papillae. In the present study, morphologically identified G. neoplasticum from 114 rats of seven species from Southeast Asia were selected from archived survey materials from almost 4,500 rodents: Thailand (58 rats), Cambodia (52 rats), Laos (three rats) and Philippines (one rat). In addition, several specimens from four rats in Indonesia were used in the study. Nucleotide sequences of the ribosomal RNA gene (rDNA) (5,649 bp) and the cytochrome c oxidase subunit 1 gene (cox1) (818 bp) were characterised. The rDNA showed little nucleotide variation, including the internal transcribed spacer (ITS) regions. The cox1 showed 24 haplotypes, with up to 15 (1.83%) nucleotide substitutions regardless of parasite origin. Considering that Rattus spp. have been shown to originate from the southern region of Asia and G. neoplasticum is their endogenous parasite, it is reasonable to propose that the present study covers a wide spectrum of the genetic diversity of G. neoplasticum, useful for both the molecular genetic speculation of the species and the molecular genetic differentiation of other local rodent Gongylonema spp. from the cosmopolitan congener.

Redescription and genetic characterization of Cystidicoloides vaucheri, including first description of male and current status on the phylogeny of Cystidicolidae (Nematoda: Habronematoidea).

Cystidicoloides vaucheri collected in the stomach of the redtail catfish Phractocephalus hemioliopterus from River Acre, State of Acre, Brazil is redescribed, including the first description of males and the first genetic characterization based upon 18S and 28S genes of the rRNA. Newly collected females were biometrically smaller than those reported in the original description, but similar morphology shared by the two samples revealed that they belong to the same species. Scanning electron micrographs showed the accurate structure of the cephalic region, described here in detail. Furthermore, the morphology of males completed the specific diagnosis, strengthening the validity of the species. The three other congeners differ from C. vaucheri mainly as follows: in C. dlouhyi the area rugosa is absent, the cephalic structures in C. fischeri are completely distinct, and in both species the spicules have membranous outgrowths, absent in C. vaucheri. Despite the dubious generic assignment of C. izecksohni, it differs from C. vaucheri in several biometrical and morphological features. Because of data availability, only sequences of the 18S were used for phylogenetic reconstructions. Results showed that the genus Ascarophis and the families Cystidicolidae and Physalopteridae are not monophyletic. Cystidicoloides vaucheri formed an independent branch clustering with representatives of Cystidicolidae, confirming its validity. The inclusion of Salmonema and Spinitectus within Cystidicolidae should be reviewed, since they formed an assemblage with species from Rhabdochonidae. In fact, current classification of some taxa belonging to Habronematoidea, Physalopteroidea and Thelazioidea need to be re-evaluated, mainly based on molecular data from different genes.

First record of Gongylonema nepalensis in domestic and wild ruminants in Europe.

The gullet worm, Gongylonema pulchrum, is cosmopolitan in distribution, infecting a variety of mammals including domestic and wild ruminants. Gongylonema nepalensis recently collected from the esophageal epithelium of water buffaloes in Nepal was separated from G. pulchrum based on its distinctly shorter left spicule relative to body length and unique nucleotide sequences of the ribosomal RNA gene (rDNA) and the mitochondrial cytochrome c oxidase subunit 1 gene (cox-1). During meat inspections at four abattoirs on Sardinia Island, Italy, 25 Gongylonema worms were collected from one each individual of cattle (n=8), sheep (n=7), goats (n=4), and mouflon (Ovis aries musimon; n=6), and characterized morphologically and genetically. Intriguingly, all of the collected worms from these ruminants were G. nepalensis, exhibiting comparable body lengths to G. pulchrum in cattle from other regions but with significantly shorter left spicules like G. nepalensis (less than 20.9% of the entire body length in contrast to 21.8-65.6%, the reported proportion of G. pulchrum). Furthermore, the rDNA nucleotide sequences of these worms from different ruminant species on Sardinia Island were almost identical to each other and to Nepalese G. nepalensis isolates. With the exception of one worm from a sheep (displaying a single nucleotide substitution), the 369-bp cox-1 nucleotide sequences of all the Sardinian G. nepalensis isolates from the different host sources were absolutely identical, forming a clade with Nepalese G. nepalensis isolates and not G. pulchrum isolates. The present study reveals that G. nepalensis is not a local parasite in the Indian subcontinent (Nepal), but instead has a certain geographical distribution in Europe and takes several ruminant species as a definitive host.

Morphological and molecular identification of Habronema spp. (Nematoda: Habronematidae) from donkeys in Xinjiang, China, and notes on the taxonomical status of Habronema majus (Creplin, 1849) and H. microstoma (Schneider, 1866).

Habronematid nematodes were collected from the stomachs of donkeys, Equus asinus L., in the Tarim Basin, Xinjiang, China. After examination by light and scanning electron microscopy, Habronema muscae (Carter, 1861) and H. majus (Creplin, 1849) were identified. The morphology of our specimens representing H. muscae (Carter, 1861) agreed well with previous redescriptions in the shape of the lateral lips, origin of the lateral alae, ratio of left and right spicules, and number and arrangement of caudal papillae. However, H. majus (Creplin, 1849) differs from H. microstoma (Schneider, 1866) in the arrangement of the caudal papillae in the male. Moreover, molecular analysis also showed interspecific differences of 26.2-28.2% in ITS2 and 8.6-8.9% in cox1 between H. majus and H. microstoma, a divergence much higher than the known intraspecific variation of Habronema spp. (6.6-8.7% in ITS2; 0.2-2.2% in cox1). The results indicate that both H. microstoma (Schneider, 1866) and H. majus (Creplin, 1849) are valid species.

Intraspecific and interspecific genetic variation of Gongylonema pulchrum and two rodent Gongylonema spp. (G. aegypti and G. neoplasticum), with the proposal of G. nepalensis n. sp. for the isolate in water buffaloes from Nepal.

The gullet worm (Gongylonema pulchrum) has been recorded from a variety of mammals worldwide. In an earlier study, we demonstrated two separate transmission cycles in cattle (Bos taurus) and wild mammals in Japan based on nucleotide sequences of the ribosomal RNA gene (rDNA) and cytochrome c oxidase subunit I (cox-1) region of mitochondrial DNA of multiple isolates of different origins. Our earlier study additionally demonstrated two major cox-1 haplotypes of G. pulchrum prevalent in cattle in Japan. In the present study, we collected G. pulchrum from cattle and goats (Capra hircus) in Alashan League, Inner Mongolia, China; Gongylonema aegypti from spiny mice (Acomys dimidiatus) in the Sinai Peninsula, Egypt; and Gongylonema neoplasticum from a black rat (Rattus rattus) in Okinawa Island, Japan, to analyze their genetic relationships with G. pulchrum in Japan. The gullet worms from Alashan League had almost identical rDNA nucleotide sequences and two cox-1 haplotypes as seen in G. pulchrum from the cattle in Japan. The two rodent Gongylonema spp. had distinct rDNA nucleotide sequences compared with those of G. pulchrum; only the 18S and 5.8S rDNA sequences showed high identities at 97.2-98.7%, while the remaining sequences were less than 75% identical. The 18S, 5.8S, and 28S rDNA sequences of the two rodent Gongylonema spp. showed nucleotide identities of 99.8% (1811/1814), 100% (158/158), and 98.9% (3550/3590), respectively. The cox-1 regions showed 91.6% (338/369)-92.1% (340/369) identities, with completely identical amino acid sequences. The genetic diversities of three distinct Gongylonema spp. and their possible intraspecific genetic variation may allow us to resolve the taxonomic position of Gongylonema spp. which display few obvious morphological differences from their congeners. Consequently, the Gongylonema isolate from water buffaloes (Bubalus bubalis) in Nepal reported in our previous study is concluded to be a new species, and Gongylonema nepalensis n. sp. is erected for it.

Molecular genomics resource for the parasitic nematode Spirocerca lupi: Identification of 149 microsatellite loci using FIASCO and next generation sequencing.

Understanding genetic diversity and movement patterns in parasitic organisms is paramount to establish control and management strategies. In this study we developed a microsatellite resource as well as a diagnostic multiplex for the cosmopolitan parasitic nematode Spirocerca lupi, known to cause spirocercosis in canids. A combination of microsatellite enrichment and 454 sequencing was used to identify 149 unique microsatellite loci in S. lupi. Twenty loci were characterized further in two sampling sites in South Africa, with 10 loci identified as polymorphic (allele ranges from 4 to 17). These loci were designed into a single diagnostic multiplex suitable for species identification and population genetics studies. The markers were also successful in cross-species amplification in Cylicospirura felineus, Philonema oncorhynchi and Gongylonema pulchrum. Our resource provides a large set of candidate loci for a number of nematode studies as well as loci suitable for diversity and population genetics studies of S. lupi within the South African context as well as globally.

The complete mitochondrial genome of the gullet worm Gongylonema pulchrum: gene content, arrangement, composition and phylogenetic implications.

BACKGROUND: Gongylonema pulchrum (Nematoda: Gongylonematidae), a thread-like spirurid gullet worm, infects a range of mammalian definitive hosts, including cattle, pigs, equines, goats, primates and humans, and can cause gongylonemiasis. METHODS: In the present study, the complete mitochondrial (mt) genome of G. pulchrum was obtained using Long-range PCR and subsequent primer walking. The phylogenetic position of G. pulchrum within the Spiruromorpha was established using Bayesian analyses of the protein-coding genes at the amino acid level. RESULTS: The length of this AT-rich (75.94%) mt genome is 13,798 bp. It contains 12 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and one non-coding region. The gene arrangement is the same as those of Thelazia callipaeda (Thelaziidae) and Setaria digitata (Onchocercidae), but distinct from that of Heliconema longissimum (Physalopteridae). Phylogenetic analyses, based on the concatenated amino acid sequence data for all 12 protein-coding genes using Bayesian inference (BI) method, showed that G. pulchrum (Gongylonematidae) was more closely related to Spirocerca lupi (Spiruroidea) than other members of the infraorder Spiruromorpha. CONCLUSIONS: The present study represents the first mt genome sequence for the family Gongylonematidae, which provides the opportunity to develop novel genetic markers for studies of epidemiology, population genetics and systematics of this nematode of human and animal health significance.