Genetic Variability Of Haemonchus Contortus In China

Haemonchus contortus is a trichostrongyloid nematode and one of the major pathogens affecting small ruminants. The adult female of this species produces large numbers of eggs. The blood-feeding activity of adults causes anaemia, oedema, diarrhoea and even death, consequently causing serious production and economic losses. As there is no commercial vaccine against haemonchosis, the control of H.contortus relies largely on anthelmintics. Drug resistance and the extremely high prevalence of multi-drug resistance in some regions both attribute to the unlimited long-term use of drugs. Knowledge of genetic variation of H. contortus can provide a foundation for understanding the spread of drug resistance alleles and making the strategy of the control of haemonchosis. But it is unknown about the genetic diversity of H.contortus in China.Mitochondrial DNA is a genetic marker outside of the nucleus, with the characteristics of faster mutation rate and maternal inheritance. Microsatellites are inherited in a Mendelian as codominant markers and tend to be distributed in the genome widely, with high polymorphism. In the present study, we explored genetic variation within and among populations of H. contortus from different geographical regions of China, employing the mitochondrial nad4gene and microsatellites as markers.(1) Genetic diversity analysis based on mitochondrial nad4geneSeven populations including Suizhou, Yidu, Yunnan, Shaanxi, Guangxi, Liaoning and Heilongjiang were applied to genetic diversity analysis. From the152nad4amplicons,142distinct haplotypes were defined. Nucleotide diversities ranged from0.0178to0.0369, and the haplotype diversity ranged from0.993to1.000. The highest nucleotide diversity estimated was for the H. contortus population from Yunnan, which also showed the higher pairwise FST than other populations indicating moderate level of genetic differentiation. AMOVA analysis revealed high within-population variation. Tajima’s and Fu’s test implied no occurred population expansion phenomenon.Phylogenetic analysis showed random distribution of sequences representing different locations and no obvious boundaries among individuals, with weak support (<50%) for some nodes. The random distribution of142nad4haplotypes across the parsimony network also did not support any particular genetic structure among H. contortus specimens. Ten nad4sequences were chosen randomly from the Chinese sample set and from each of the four other H. contortus populations representing four other countries (Italy, Malaysia, the USA and Yemen) to construct phylogenetic tree revealed there was no clear grouping according to country. FST values were also calculated between samples from China and those from each of the other four countries revealed that the highest level of genetic differentiation was recorded between China and the USA, and the lowest level of differentiation was found between China and Malaysia.These findings indicated that on a global scale, there is no or a low level of genetic differentiation among H. contortus populations from the same continent due to high gene flow, and significantly higher levels of genetic differentiation among those from different continents was infected by geographical isolation(2) Genetic diversity analysis based on microsatellitesSeven populations including Suizhou, Yunnan, Shaanxi, Guangxi, Liaoning, Heilongjiang and Inner Mongolia were applied to genetic diversity analysis. There were eight markers (Hcms15, Hcms28, Hcms40, Hcms22co3, Hcms19, Hcms33, Hcms36, Hcms37) identified from published research, which have the characteristics of good stability, high polymorphism and amplification efficiency. Parameters of genetics such as allele number, heterozygosity and PIC implied these markers which could accurately reflect the information of population genetic were polymorphism locus. All seven populations showed varying degrees departure from Hardy-Weinberg equilibrium at eight microsatellite locus. After Bonferroni correction (P<0.002381), all seven populations still showed significant departure from Hardy-Weinberg equilibrium at five microsatellite locus (Hcms15, Hcms28, Hcms40, Hcms22co3, Hcms37) with heterozygote deficiency or heterozygote excess.AMOVA analysis also revealed high within-population variation. FST values showed the Inner Mongolia population has higher population variation than other populations indicating moderate level of genetic differentiation. Phylogenetic analysis indicated seven populations was divided into three clusters consistent with sampling geography patterns, and in agreement with the results of phylogenetic analysis based on mitochondrial nad4gene. Structure analysis showed no obvious population genetic structure attributed to high gene flow. Mantel test revealed no significant correlation between genetic distance and geographic distance indicating the absence infected by geographical isolation. Population dynamics test under LAM, TPM and SMM three modes showed no bottleneck event occurred.In the present study, we explored genetic variation of H. contortus from different geographical regions of China, employing the mitochondrial nad4gene and microsatellites as markers. The results revealed high within-population variation, low population genetic differentiation and high gene flow of H. contortus in China. These findings can provide valuable information for predicting the spread of anthelminitic resistance and further studying the molecular epidemiology.