| Ixodes persalcatus is a kind of tick that exists widely in the northern forests of China,and is one of the dominant tick species in Heilongjiang province.It can carry and transmit a variety of pathogens,seriously threatening the health of human and animals.However,there are few studies on molecular classification of Ixodes persalcatus.Therefore,it is of great significance to study population differentiation and genetic polymorphism of Ixodes persalcatus in Heilongjiang province.In order to provide reliable data for the prevention and control of piriformiasis and Lyme disease,the cases of piroplasm and borrelia burgdorferi carried by Ixodes persalcatus were investigated.In the present study,816 free Ixodes persalcatus were collected from 12 sites in five forest areas of Greater Khingan Mountains,Lesser Khingan Mountains,Wanda Mountains,Zhangguangcai Range and Daqing Mountains in Heilongjiang Province using the cloth flag method from April to August in 2020.Ten ticks were randomly selected from each site and DNA was extracted.The 16 S r DNA sequence,cox1 sequence and ITS2 sequence were amplified by PCR,respectively,and cloned and sequenced for further analysis of sequence polymorphism,population genetic structure and phylogeny.At the same time,based on the 18 S r DNA sequence of piroplasm and the 5S-23 S r DNA fragment of Borrelia burgdorferi,all the collected Ixodes persalcatus were detected by PCR,followed by sequence analysis and statistical analysis of positive samples.The results showed that:(1)A total of 9 haplotypes were obtained based on the16 S r DNA sequence of Ixodes tick,including 1 shared haplotype;25 haplotypes were found based on the cox1 sequence,including 2 shared haplotypes;73 haplotypes were found based on the ITS2 sequence,one of which was a shared haplotype.(2)Under different gene markers,the sources of genetic variation and the degree of population differentiation are different in Ixodes persalcatus,and the distribution of haplotypes is not related to regions.Most populations follow the principle of neutral evolution,and only some regions have experienced population expansion and evolved under selection pressure.(3)The 16 S r DNA,cox1 and ITS2 sequences were used as markers in Ixodes persalcatus,and the haplotypes of the isolates were all on the same branch,and there was no geographical correlation.(4)In the study,the positive rate of Ixodes persalcatus carrying piriformis was 4.66%(38/816),of which the positive rate of Babesia spp.was4.17%(34/816)and the positive rate of Theileria spp.was 0.49%(4/816),the overall positive rate of Borrelia burgdorferi was 18.26%(149/816),and the mixed infection was 0.613%(5/816).(5)A total of two Babesia species were detected in this study,a total of one Theileria and a total of two Borrelia burgdorferi,namely Babesia venatorum,Babesia microti,and Theileria sinensis,Borrelia afzelii and Borrelia garinii.This study was the first to analyze the genetic polymorphisms of Ixodes persalcatus in Heilongjiang forest area.Based on the three sequences of cox1,ITS2 and16 S r DNA,the genetic polymorphism analysis showed that the differentiation degree of Ixodes persalcatus populations was unequal,and the distribution of haplotypes was not correlated with the geographical area.Most populations followed the principle of neutral evolution,and only some regions experienced population expansion and evolved under selection pressure.PCR methods were used to investigate the situation of piroplasm and Borrelia burgdorferi carried by Ixodes persalcatus from 12 collection sites.Two species of Babesia(Babesia venatorum,Babesia microti),one species of Theileria(Theileria sinensis)and two species of Borrelia burgdorferi(Borrelia garinii,Borrelia afzelii)were detected,of which Babesia venatorum,Babesia microti,Borrelia garinii,Borrelia afzelii were zoonotic pathogens. |
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