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

Identification of nematodes of the genus

The present study delves into a methodological framework aimed at establishing species-specific markers via the utilization of sequences derived from the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA. This method, in conjunction with polymerase chain reaction (PCR) testing, serves as a diagnostic tool for discerning species belonging to the genus Teladorsagia Andreeva et Satubaldin, 1954. These species, constituents of the subfamily Ostertagiinae (Nematoda: Trichostrongylidae), exhibit wide distribution within the gastrointestinal tracts of ruminants across the geographic expanse of Uzbekistan. The heart of this endeavor is the development of species-specific primers, a pioneering creation in its own right. These primers are crafted using sequences emanating from the ITS2 region of the ribosomal DNA, an innovative approach that facilitates the precise identification of morphospecies within the Teladorsagia genus. Notably, the primers exhibit a nucleotide length of 153 base pairs, an attribute instrumental in their capacity to accurately distinguish and diagnose eggs and larvae of three distinct morphspecies: T. circumcincta, T. trifurcata, and T. davtiani. The potential implications of this method are significant, with ramifications reverberating across the field of veterinary diagnostics. Through the application of these primers, practitioners and researchers alike can effectively ascertain the presence of specific Teladorsagia morphospecies in ruminant animals. This holds the promise of not only enhancing diagnostic precision but also contributing to the broader comprehension of the prevalence and distribution of these nematode species within the local ruminant populations

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