Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of the E198A SNP in the isotype-1 ß-tubulin gene of Haemonchus contortus populations in China.

Haemonchus contortus is one of the most important gastrointestinal nematodes of small ruminants around the world, seriously hampering the healthy development of the sheep industry. The control of this parasite mainly depends on anthelmintics, however, drug resistance of H. contortus has become a serious problems worldwide. Previous studies demonstrated that the E198A (GAA to GCA), a single nucleotide polymorphism (SNP) in the isotype-1 ß-tubulin gene is associated with benzimidazole resistance in H. contortus. However, only PCR-RFLP and ARMS-PCR methods have been previously used for the detection of the E198A mutation. In the present study, a loop-mediated isothermal amplification (LAMP) assay was established for rapid detection of the E198A SNP in H. contortus. The results showed that optimization of LAMP reaction reagents and conditions could achieve this. The resulting amplicons were visualized by adding hydroxynaphthol blue dye (HNB) prior to amplification. The color of LAMP products amplified without DNA or from DNA from worms with the E198A homozygous susceptible genotype was still violet, but the products with DNA from worms with the E198A heterozygous genotype or the E198A resistant homozygous genotype changed to sky blue. The specificity of this method was further verified by sequencing, which confirmed the successful LAMP detection of the E198A mutation with high specificity. In conclusion, the developed LAMP method has high specificity and good reproducibility for screening the E198A SNP of isotype-1 ß-tubulin gene of H. contortus of field samples without using sophisticated equipment, providing useful technique for the rapid detection and thus prevention and control of benzimidazole resistant H. contortus infections.

A DAF-3 co-Smad molecule functions in Haemonchus contortus development.

BACKGROUND: The Smad proteins function in TGF-ß signalling transduction. In the model nematode Caenorhabditis elegans, the co-Smad, DAF-3 mediates R-Smads and performs a central role in DAF-7 signal transduction, regulating dauer formation and reproductive processes. Considering the divergent evolutionary patterns of the DAF-7 signalling pathway in parasitic nematodes, it is meaningful to explore the structure and function of DAF-3 in parasitic nematodes, such as Haemonchus contortus. METHODS: A daf-3 gene (Hc-daf-3) and its predicted product (Hc-DAF-3) were identified from H. contortus and characterised using integrated genomic and genetic approaches. In addition to immunohistochemistry employed to localise Hc-DAF-3 within adult worm sections, real-time PCR was conducted to assess the transcriptional profiles in different developmental stages of H. contortus and RNA interference (RNAi) was performed in vitro to assess the functional importance of Hc-daf-3 in the development of H. contortus. RESULTS: Hc-DAF-3 sequences predicted from Hc-daf-3 displayed typical features of the co-Smad subfamily. The native Hc-DAF-3 was localised to the gonad and cuticle of adult parasites. In addition, Hc-daf-3 was transcribed in all developmental stages studied, with a higher level in the third-stage larvae (L3) and adult females. Moreover, silencing Hc-daf-3 by RNAi retarded L4 development. CONCLUSION: The findings of the present study demonstrated an important role of Hc-DAF-3 in the development of H. contortus larvae.

Characterization of the Complete Mitochondrial Genome of a Whipworm Trichuris skrjabini (Nematoda: Trichuridae).

The complete mitochondrial (mt) genome of Trichuris skrjabini has been determined in the current study and subsequently compared with closely related species by phylogenetic analysis based on concatenated datasets of mt amino acid sequences. The whole mt genome of T. skrjabini is circular and 14,011 bp in length. It consists of a total of 37 genes including 13 protein coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNAs) genes, and two non-coding regions. The gene arrangement and contents were consistent with other members of the Trichuridae family including Trichuris suis, Trichuris trichiura, Trichuris ovis, and Trichuris discolor. Phylogenetic analysis based on concatenated datasets of amino acids of the 12 PCGs predicted the distinctiveness of Trichuris skrjabini as compared to other members of the Trichuridae family. Overall, our study supports the hypothesis that T. skrjabini is a distinct species. The provision of molecular data of whole mt genome of T. skrjabini delivers novel genetic markers for future studies of diagnostics, systematics, population genetics, and molecular epidemiology of T. skrjabini.

Characterization of the Complete Mitochondrial Genome of Ostertagia trifurcata of Small Ruminants and its Phylogenetic Associations for the Trichostrongyloidea Superfamily.

The complete mitochondrial (mt) genome of Ostertagia trifurcata, a parasitic nematode of small ruminants, has been sequenced and its phylogenetic relationship with selected members from the superfamily Trichostrongyloidea was investigated on the basis of deduced datasets of mt amino acid sequences. The entire mt genome of Ostertagia trifurcata is circular and 14,151 bp in length. It consists of a total of 36 genes comprising 12 genes coding for proteins (PCGs), 2 genes for ribosomal RNA (rRNA), 22 transfer RNA (tRNA) genes and 2 non-coding regions, since all genes are transcribed in the same direction. The phylogenetic analysis based on the concatenated datasets of predicted amino acid sequences of the 12 protein coding genes supported monophylies of the Haemonchidae, Dictyocaulidae and Molineidae families, but rejected monophylies of the Trichostrongylidae family. The complete characterization and provision of the mtDNA sequence of Ostertagia trifurcata provides novel genetic markers for molecular epidemiological investigations, systematics, diagnostics and population genetics of Ostertagia trifurcata and its correspondents.