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Characterization of benzimidazole resistance in Haemonchus contortus: integration of phenotypic, genotypic and proteomic approaches

Tan TK, Lim YAL, Chua KH, Chai HC, Low VL, Bathmanaban P, et al.

Parasitol Res, 2020 Sep;119(9):2851-2862.
PMID: 32651637 DOI: 10.1007/s00436-020-06790-5

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 β-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.

Matched MeSH terms: Haemonchus/genetics
Fulltext Genetic diversity of Haemonchus contortus isolated from sympatric wild blue sheep (Pseudois nayaur) and sheep in Helan Mountains, China

Shen DD, Wang JF, Zhang DY, Peng ZW, Yang TY, Wang ZD, et al.

Parasit Vectors, 2017 Sep 19;10(1):437.
PMID: 28927469 DOI: 10.1186/s13071-017-2377-0

BACKGROUND: Haemonchus contortus is known among parasitic nematodes as one of the major veterinary pathogens of small ruminants and results in great economic losses worldwide. Human activities, such as the sympatric grazing of wild with domestic animals, may place susceptible wildlife hosts at risk of increased prevalence and infection intensity with this common small ruminant parasite. Studies on phylogenetic factors of H. contortus should assist in defining the amount of the impact of anthropogenic factors on the extent of sharing of agents such as this nematode between domestic animals and wildlife.
METHODS: H. contortus specimens (n = 57) were isolated from wild blue sheep (Pseudois nayaur) inhabiting Helan Mountains (HM), China and additional H. contortus specimens (n = 20) were isolated from domestic sheep that were grazed near the natural habitat of the blue sheep. Complete ITS2 (second internal transcribed spacer) sequences and partial sequences of the nad4 (nicotinamide dehydrogenase subunit 4 gene) gene were amplified to determine the sequence variations and population genetic diversities between these two populations. Also, 142 nad4 haplotype sequences of H. contortus from seven other geographical regions of China were retrieved from database to further examine the H. contortus population structure.
RESULTS: Sequence analysis revealed 10 genotypes (ITS2) and 73 haplotypes (nad4) among the 77 specimens, with nucleotide diversities of 0.007 and 0.021, respectively, similar to previous studies in other countries, such as Pakistan, Malaysia and Yemen. Phylogenetic analyses (BI, MP, NJ) of nad4 sequences showed that there were no noticeable boundaries among H. contortus populations from different geographical origin and population genetic analyses revealed that most of the variation (94.21%) occurred within H. contortus populations. All phylogenetic analyses indicated that there was little genetic differentiation but a high degree of gene flow among the H. contortus populations among wild blue sheep and domestic ruminants in China.
CONCLUSIONS: The current work is the first genetic characterization of H. contortus isolated from wild blue sheep in the Helan Mountains region. The results revealed a low genetic differentiation and high degree of gene flow between the H. contortus populations from sympatric wild blue sheep and domestic sheep, indicating regular cross-infection between the sympatrically reared ruminants.

Matched MeSH terms: Haemonchus/genetics*
Sympatric species distribution, genetic diversity and population structure of Haemonchus isolates from domestic ruminants in Pakistan

Hussain T, Periasamy K, Nadeem A, Babar ME, Pichler R, Diallo A

Vet Parasitol, 2014 Dec 15;206(3-4):188-99.
PMID: 25468018

Haemonchus species are major gastro-intestinal parasites affecting ruminants across the world. The present study aimed to assess the sympatric species distribution, genetic diversity, population structure and frequency of β-tubulin isotype 1 alleles associated with benzimidazole resistance. Internal transcribed spacer 2 (ITS2) sequences revealed three sympatric species of Haemonchus, H. contortus, H. placei and H. longistipes with 12 distinct genotypes circulating among ruminant hosts in Pakistan. High genetic variability was observed in Pakistani Haemonchus isolates at nicotine amide dehydrogenase subunit 4 (ND4) and cytochrome oxidase subunit 1 (COI) gene loci. Intra-population diversity parameters were higher in H. contortus isolates than H. placei. Phylogenetic analysis of ND4 and COI sequences did not reveal clustering of haplotypes originating from a particular host indicating high rate of gene flow among Haemonchus parasites infecting sheep, goat and cattle in Pakistan. ND4 and COI haplotypes from Pakistan were compared to sequences of Haemonchus isolates from 11 countries to elucidate the population structure. Multidimensional scaling (MDS) plot of pairwise FST derived from 531 ND4 haplotypes revealed clustering together of H. contortus from Pakistan, China, Malaysia and Italy while the isolates from Yemen and United States were found to be genetically distinct. With respect to H. placei, isolates from Pakistan were found to be genetically differentiated from isolates of other countries. The tests for selective neutrality revealed negative D statistics and did not reveal significant deviations in Pakistani Haemonchus populations while significant deviation (P < 0.05) was observed in Brazilian and Chinese H. contortus populations. Median Joining (MJ) network of ND4 haplotypes revealed Yemenese H. contortus being closer to H. placei cluster. β-tubulin isotype 1 genotyping revealed 7.86% frequency of Y allele associated with benzimidazole resistance at F200Y locus in Pakistani Haemonchus isolates.

Matched MeSH terms: Haemonchus/genetics*
Fulltext Genetic variability within and among Haemonchus contortus isolates from goats and sheep in China

Yin F, Gasser RB, Li F, Bao M, Huang W, Zou F, et al.

Parasit Vectors, 2013 Sep 25;6(1):279.
PMID: 24499637 DOI: 10.1186/1756-3305-6-279

BACKGROUND: Haemonchus contortus (order Strongylida) is a common parasitic nematode infecting small ruminants and causing significant economic losses worldwide. Knowledge of genetic variation within and among H. contortus populations can provide a foundation for understanding transmission patterns, the spread of drug resistance alleles and might assist in the control of haemonchosis.
METHODS: 152 H. contortus individual adult worms were collected from seven different geographical regions in China. The second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA and mitochondrial nicotinamide dehydrogenase subunit 4 gene (nad4) were amplified by polymerase chain reaction (PCR) and sequenced directly. The sequence variations and population genetic diversities were determined.
RESULTS: Nucleotide sequence analyses revealed 18 genotypes (ITS-2) and 142 haplotypes (nad4) among the 152 worms, with nucleotide diversities of 2.6% and 0.027, respectively, consistent with previous reports from other countries, including Australia, Brazil, Germany, Italy, Malaysia, Sweden, the USA and Yemen. Population genetic analyses revealed that 92.4% of nucleotide variation was partitioned within populations; there was no genetic differentiation but a high gene flow among Chinese populations; some degree of genetic differentiation was inferred between some specimens from China and those from other countries.
CONCLUSIONS: This is the first study of genetic variation within H. contortus in China. The results revealed high within-population variations, low genetic differentiation and high gene flow among different populations of H. contortus in China. The present results could have implications for studying the epidemiology and ecology of H. contortus in China.

Matched MeSH terms: Haemonchus/genetics*
Genetic variation of Haemonchus contortus (Trichostrongylidae) in sheep and goats from Malaysia and Yemen

Gharamah AA, Azizah MN, Rahman WA

Vet Parasitol, 2012 Sep 10;188(3-4):268-76.
PMID: 22538095 DOI: 10.1016/j.vetpar.2012.04.003

The large stomach worm, Haemonchus contortus, commonly known as "the barber's pole worm", is a blood-sucking nematode found in the abomasa of sheep and goats. This work is the first documentation on the ND4 sequences of H. contortus from sheep and goats in Malaysia and Yemen and the results provide a preliminary insight on the genetic differences of H. contortus found in the two countries. In general, this study showed a high degree of diversity and low population structure of this species within the same country in comparison with higher genetic structuring at a wider geographical scale. The results also showed that the majority of genetic variance was within H. contortus populations. The Malaysian sheep and goat populations investigated appeared to share the same isolate of H. contortus while different isolates may be found in Yemen which must be taken into account in the design of an effective control strategy. Analysis of the internal transcribed spacer-2 (ITS-2) confirmed that all samples investigated in this study belonged to H. contortus. However presence of other Haemonchus species parasitizing these two hosts can only be confirmed by further detailed studies.

Matched MeSH terms: Haemonchus/genetics*