甘肃省甘南州家畜细粒棘球蚴病例特征及其病原遗传多样性分析

Study on characteristics of cystic echinococcosis and genetic diversity of livestock in Gnanan Profecure,Gansu Province

目的分析甘肃省甘南州细粒棘球蚴(Echinococcus granulosus)基因型、遗传多样性及群体间遗传差异,为甘南州细粒棘球溯源及制定预防控制策略提供参考。方法收集2016-2022年甘南州玛曲县及碌曲县屠宰场家畜肝脏或肺脏感染的囊型棘球蚴病(cystic echinococcosis,CE)包囊,提取包囊基因组DNA,PCR扩增线粒体NADH脱氢酶2(nad2)基因,测序剪接后进行Blast比对,DnaSPv5软件分析nad2序列的基因型,利用Mega11软件采用邻接法构建系统进化树,再使用PopArt 1.7软件建立细粒棘球蚴nad2基因网络图,最后使用Arlequin 3.5.2.2分析nad2不同群体间的遗传差异。结果PCR结果显示,家畜CE感染率为9.97%(64/642),绵羊(9.14%,48/525)和牛(13.68%,16/117)感染率差异极显著(χ^(2)=175.6,P<0.01)。nad2基因条带大小为882 bp,将测序结果与GenBank中发表的细粒棘球蚴的序列进行比对,相似度均达到99%以上。序列分析结果显示,分为G1(占90.63%,58/64)和G3(占9.37%,6/64)两个基因型,序列相似度可达100%;共有12个单倍型,其中Hap1(占50.00%,32/64)是最主要的单倍型,Hap2(占14.06%,9/64)次之,其中Hap3是和Hap4属于G3基因型(占9.37%,6/64)。系统发生树结果显示,G1和G3基因型处在两个小的分支上,在基因网络图中以Hap1单倍型为中心,其他11个单倍型呈散射状分布;序列单倍型多样性范围为0.7086~0.7706,核苷酸多样性范围为0.00321~0.00452。甘南州两个县细粒棘球蚴种群田岛检验(Tajima's D)均为负值,表明甘南州的细粒棘球蚴种群处于扩张或净化选择的趋势,中性检验(Fu's F)推测两个县的细粒棘球蚴种群也处于扩张趋势,且存在一些特有的基因型,FST结果表明,甘南州细粒棘球蚴不同种群间遗传分化差异不显著(P>0.05)。结论本研究发现甘南州家畜表现出较高的遗传多样性,并处于扩张趋势,存在地区特有的单倍型。共有G1和G3两个基因型,12个单倍型,其中Hap1占50%,其近缘单倍型在全世界均有分布,是甘南州乃至其他主要流行区的主要致病单倍型。

Objective The objective of this study was to analyze the genotypes,genetic diversity,and inter-population genetic differences of Echinococcus granulosus in Gannan Prefecture,Gansu Province.The findings aim to inform the development of tracing,prevention,and control strategies for E.granulosus in Gannan Prefecture.Methods Cystic echinococcosis(CE)infected with liver or lung in slaughterhouses were collected in Maqu County and Luqu County,Gannan Prefecture from 2016 to 2022.The DNA of the cysts was extracted and the mitochondrial NADH dehydrogenase 2(nad2)gene was amplified by PCR.After sequencing and splice,Blast comparison was performed.The genotype of nad2 sequences were analyzed by DnaSPv5 software.Mega11 software was applied to construct a phylogenetic tree using the adjacency method,and PopArt 1.7 software was used to construct a nad2 gene network map of E.granulosus.Finally,Arlequin 3.5.2.2 was used to analyze the genetic differences among nad2 populations.Results The PCR results showed that the infection rate of CE in livestock was 9.97%(64/642),with significant differences in infection rates between sheep(9.14%,48/525)and cattle(13.68%,16/117)(χ^(2)=175.6,P<0.01).The size of the nad2 gene band was 882 bp,and the similarity between the sequencing results and the published sequences of E.granulosus in GenBank was above 99%.The analysis of the sequencing results showed that the genotypes were divided into G1(90.63%,58/64)and G3(9.37%,6/64),with sequence similarity of 100%,there were 12 haplotypes,with Hap1(50.00%,32/64)being the most dominant haplotype,Hap2(14.06%,9/64)being the second,and Hap3 and Hap4 belonging to G3 genotype(9.37%,6/64).Phylogenetic tree showed that G1 and G3 genotypes were distributed in two small branches,with Hap1 haplotype as the center and the other 11 haplotypes scattered,the range of haplotype diversity was 0.7086-0.7706,and the range of nucleotide diversity was 0.00321-0.00452.The Tajima's Dtest for the two Counties in Gannan Prefecture E.granulosus populations was negative,indicating that the E.granulosus opulation in Gannan Prefecture was expanding or undergoing purifying selection.The Neutral's F test(Fu's F)also suggested that the two County populations were expanding and had some unique genotypes.Pairwise fixation index(F_(ST))results showed that the genetic differentiation between the E.granulosus populations in Gannan Prefecture was not significant(P>0.05).Conclusion This study found that livestock in Gannan Prefecture exhibited high genetic diversity and in an expanding trend,with region-specific haplotypes.There are two genotypes,G1 and G3,as well as 12 haplotypes,among which Hap1 accounts for 50%.Its closely related haplotypes are distributed worldwide and represent the main pathogenic haplotype in Gannan Prefecture and other major endemic areas.