| ObjectiveTaking some counties(cities)in Yunnan Province as the spatial scope of this survey,host animals(domestic,small mammals),vector ticks and fever patients as the research objects.The 16 S r RNA,Fla and glpq genes of Borrelia recurrentis were detected and sequenced by molecular epidemiological method.The infection status and gene polymorphism of Borrelia recurrentis in host animals,vector ticks and specific population in this area were investigated to provide scientific basis for the prevention and control of Borrelia recurrentis Method1.Use anticoagulant blood vessels to collect venous blood samples of domestic animals;use night clamp method and cage night method to capture small animals,and dissect and collect liver,kidney and spleen samples;use flag method to collect free ticks and body surface collection method to collect parasitic ticks on animal body surface;collect blood of fever patients with unknown causes in some areas with clues.2.DNA was extracted from the above samples by using blood / tissue genomic DNA extraction kit,and 16 S r RNA gene(353bp)was amplified and screened.16 S r RNA gene(1600bp,Fla(506bp),glp Q(461bp)was amplified in the positive patients.The positive amplification products were sent to sequencing company for sequencing,and the sequences obtained were logged on the website(http://www.ncbi.nlm.nih.gov)for blast comparison,homology analysis,construction of phylogenetic tree for genetic evolution analysis.3.Statistical software SPSS18.0 were used to analyze the data.The statistical method was χ 2 test.Results1.Sample collection: 1836 blood samples(575 cow blood,866 sheep blood,346 dog blood,32 horse blood and 17 donkey blood)of 5 kinds of domestic animals(cattle,sheep,dogs,horses and donkeys)were collected in 15 counties(cities)in western Yunnan Province;4120 viscera samples of small mammals belonging to 5orders,11 families,28 genera and 59 species were collected in 29 counties(cities);1354 samples of ticks belonging to 5 genera and 13 species were collected from 12counties(cities)for single detection.920 ticks and 41 eggs of 7 larva ticks were divided into 260 groups for pathogen isolation and culture.950 ticks from 6 counties and cities were cultured for 260 samples.629 blood samples of patients with fever of unknown cause were collected from 5 counties(cities).(2)The results of domestic animal test: 1836 blood samples of domestic animals,46 were positive in primary screening,the positive rate was 2.51%.It was detected in cattle,sheep and dogs,but not in horses and donkeys.The positive rates of cattle,sheep and dogs were 2.61%(15 / 575),3.46%(30 / 866)and 0.29%(1 / 346),respectively.The positive rate was 10.34%(3 / 29)in Lanping city,7.48%(8 / 107)in Yingjiang city,5.56%(1 / 18)in Weishan city,3.96%(30 / 758)in Tengchong city,2.33%(1 / 43)in Deqin city,2.00%(2 / 100)in Yunlong city and 1.04%(1 / 96)in Shangri La city.3.The results of small mammals: 4120 samples of small mammals,belonging to59 species,collected from 29 counties(cities)were positive only in 1 mus caroli and sorex alpinus.The positive rates of mus caroli from Menghai and sorex alpinus from Gongshan were 0.57%(1 / 175)and 0.15%(1 / 686),respectively.4.The results of tick test:1354 ticks were detected by single test,30 were positive,the positive rate was 2.21%(30 / 1354).Five species of positive ticks were Nepalese Haemaphysalis,coronin Haemaphysalis,Rhipicephalus microphilus,Ixodes ovalis and Rhipicephalus falciformis,The percentage were 66.67%(6 / 9),3.07%(5 /163),2.31%(12 / 518),1.96%(6 / 306)and 1.56%(1 / 64),respectively.The positive rates were 16.67%(1 / 6),10%(7 / 70),2.87%(5 / 174),2.26%(9 / 398),2.11%(6 /284),1.32%(1 / 76)and 0.54%(1 / 184)in Yongde City,Deqin City,Weixi City,Tengchong City,Jianchuan City,Lanping City and Gengma City,respectively.It is mainly distributed in Northwest Yunnan.Twenty four positive samples were detected in group culture,including adult ticks(22),nymphs ticks(1),and ticks eggs(1),all of which were from Tengchong city.A 77.8% sequence homology was detected in a female tick and its eggs.But no pathogens have been isolated.5.Crowd test results: 7 of 629 patients were positive in the initial bloodscreening,including 1 in Jianchuan City,2 in Dali City,2 in Pu’er City and 2 in Tengchong City,but the sequence was not successfully compared by blast homology,and the full-length detection of fla,glpq and 16 Sr RNA was negative.6.Diversity analysis of host vector infection regression: homology comparison,genetic evolution analysis and amino acid sequence analysis of the detected sequence.The results showed that the host and vector infected / carried Borrelia recurrentis in the western region of Yunnan province have high gene diversity characteristics,which are divided into two types.One is similar to the reported B.miyamotoi,which may be a new subtype;the other is quite different from the reported Borrelia recurrentis,which may be a new species,temporarily named Borrelia dali Conclusion1.In the western region of Yunnan Province,there are Borrelia recurrentis spirochetes infection in livestock,host animals of small mammals and vector ticks.The positive rates were 2.51% for domestic animals,0.049% for small mammals and2.21% for ticks.The distribution is concentrated in the northwest of Yunnan.2.The host animals and vector of Borrelia recurrentis spirochetes in western Yunnan Province are complex and diverse.Cattle,sheep,dogs and other livestock,2small mammals,5 ticks were detected.Among them,the infection rate of livestock is the highest,livestock is the main host,2 kinds of small animals are positive,and 5kinds of ticks(1 kind of Ixodes,2 kinds of Haemaphysalis,2 kinds of fanhead ticks)can be positive.3.The results showed that there was high diversity in the area of host and vector infection.The detected sequence showed high gene diversity.Through the analysis of multiple gene loci,the detected Borrelia recurrentis spirochetes were divided into two kinds: B.miyamotoi,a new subtype and a new species,tentatively named B.Dali4.Some areas in the Yunnan province may be the epicenter of Borrelia recurrentis,such as Tengchong City,Deqin City,Lanping City.The homology of the Borrelia recurrentis spirochetes in domestic animals and vector ticks was 98-99%,and there might be a transmission chain of pathogens between hosts and vectors. |
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