Molecular analysis of polymorphic species of the genus Marshallagia (Nematoda: Ostertagiinae)

Background: The genus Marshallagia (Family Haemonchidae, subfamily Ostertagiinae) contains multiple species of nematodes parasitising the abomasum (or duodenum) of ruminants, in particular of Caprinae. Male specimens have been described to be polymorphic with the frequent/major morphotype initially described in the genus Marshallagia while the minor/rare morphotype was initially often placed in the genus Grossospicularia. Due to common morphological features, certain pairs of morphotypes were suggested to belong to the same species such as Marshallagia marshalli/M. occidentalis. However, molecular evidence to confirm these pairs of morphotypes belonging to the same species is missing. Methods: In the present study, Marshallagia sp. were collected from domestic sheep in Uzbekistan. Male specimens were morphologically described with particular emphasis on the structure of the bursa copulatrix. After DNA isolation from morphologically identified specimens, PCRs targeting the ribosomal internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) regions were conducted. After Sanger sequencing, maximum likelihood phylogenetic analyses and pairwise identities between sequences were calculated. Results: The major morphotypes of M. marshalli, M. schumakovitschi and M. uzbekistanica and the minor morphotypes M. occidentalis, M. trifida and M. sogdiana were identified and their morphology was documented in detail. ITS2 sequences showed little variation and did not allow diagnosing species. In contrast, phylogenetic analysis of cox1 sequences identified highly supported clusters and verified that M. marshalli, M. occidentalis and M. uzbekistanica are different morphotypes of the species M. marshalli while M. schumakovitschi and M. trifida represent distinct morphotypes of M. trifida. For M. sogdiana no corresponding major morphotype could be identified in the present study. Due to a large barcoding gap, comparison of cox1 sequences in terms of percent identity was sufficient to reliably assign the sequences to a particular species without phylogenetic analysis. Conclusions: The data presented here create a framework that will allow the classification of other members of the genus in the future and underline that parallel morphological and molecular analysis of specimens is crucial to improve the taxonomy of polymorphic species

Occurrence of Larval Dicrocoelium dendriticum and Brachylaima sp. in Gastropod Intermediate Hosts from Fergana Valley, Uzbekistan

The occurrence of larval Dicrocoelium dendriticum and Brachylaima sp. is described with molecular evidences in gastropod intermediate hosts from Fergana Valley, Uzbekistan. Larvae of D. dendriticum were detected in 28 (10.7%) out of 262 Xceropicta candacharica, and 8 (9.7%) of 82 Angiomphalia gereliana. Brachylaima sp. larvae were found in 3 (1.6%) of 95 Pseudonapaeus sogdiana. The total number of larvae per snail varied from 8 to 110 individuals. Alignment of the first four sequences of 28S rDNA was revealed a 99-100% similarity to D. dendriticum. Larvae from P. sogdiana snails were 98% similar to Brachylaima sp. In this study, it was confirmed that 2 species of terrestrial snail, X. candacharica and A. gereliana, act as the first intermediate hosts of D. dendriticum, and P. sogdiana snail play a role of intermediate host of Brachylaima sp. in the Fergana Valley, Uzbekistan

ウズベク共和国における Protostrongylidae 科線虫の中間宿主 : 腹足類

Protostrongylid species are causative agents of pulmonary protostrongyliasis in caprine species in Uzbekistan. These nematodes typically require one intermediate host, terrestrial mollusks, to complete their life cycle. In this study, eight species of gastropods were found to be positive for protostrongylid larvae. Haplotypes of larvae corresponding to sequences of Protostrongylus rufescens and Muellerius capillaris were detected. Morphological identification of gastropods, based on shell characteristics, revealed six different morphotypes. Anatomic-morphological and molecular results confirmed the membership of these gastropods to the Buliminidae, Hygromiidae, Agriolimacidae and Parmacellidae and revealed eight different species: Pseudonapaeus maydanika, P. sogdiana, P. albiplicatus, Pseudonapaeus sp., Angiomphallia regeliana, Xeropicta candacharica, Candaharia levanderi and Deroceras reticulatum. This study displays the first report of P. maydanika, C. levanderi and D. reticulatum as natural intermediate hosts of M. capillaris. The infection rate of snails with protostrongylids was 27.9%(616/2,207) and the infection rate of slugs was 6.5%(18/279).ウズベク共和国における羊の肺虫症の原因寄生虫プロトストロンギルス科線虫は陸棲腹足類を中間宿主としているので、この線虫症の疫学では腹足類の調査が必須であった。そこで、同国において貝殻の形態で 6種に分類された軟体類を対象にプロトストロンギルス科の感染幼虫の保有状況を調査した。その結果、分子生物学的にProtostrongylus rufescens および Muellerius capillaris のハプロタイプに一致した感染幼虫を得た。また、今回、幼虫が得られた腹足類について解剖学および分子生物学的な検討によりこれらは 4つの科 Buliminidae, Hygromiidae, Agriolimacidae あるいは Parmacellidae の次のような 8種に同定された; Pseudonapaeus maydanika, P. sogdiana, P. albiplicatus, Pseudonapaeus sp. Angiomphallia regeliana, Xeropicta candacharica, Candaharia levanderi, Deroceras reticulatum. 本研究により P. maydanika, Candaharia levanderi および D. reticulatum が M. capillaris の自然中間宿主であることが証明された。全体的なプロトストロンギルス科線虫の感染幼虫の保有率はカタツムリ類で 27.9%(616/2,207)、一方、ナメクジ類では 6.5%(18/279)と低かった