The Infection Status And Influencing Factors Of Bartonella In Wild Small Mammals On The Plague Natural Foci Of Yunnan Province

ObjectiveTo identify the infection status and influencing factors of Bartonella in wild small mammals on the plague natural foci of Yunnan province,and genotypes of Bartonella epidemic strains were analyzed.Meanwhile,this paper can also be used to provide basic data for Bartonellosis to effectively prevent and control the occurrence and epidemic on natural plague foci of Yunnan province.Methods1.Sample sources: From 2015 to 2016,2611 wild small mammals were captured in Yulong plague foci,Jianchuan plague foci and Lianghe plague foci of Yunnan province.The small mammals of captured were translated back to the laboratory for classificating and identificating,and their collection sites,seasons,species of small mammals,gender,altitude and habitats were recorded.The livers and spleens of the captured small mammals were collected by aseptic operation and stored in 2.0 m L cryopreservation tube,numbered and labeled,and stored at-40℃.2.Sample sampling: A total of 1605 samples from 2549 samples of liver and spleen was selected by stratified sampling as the object for detecting Bartonella infection.3.DNA extraction: DNA was extracted by automatic nucleic acid extractor in accordance with the procedure of magnetic bead microcell/tissue genomic DNA extraction kit.4.Determination of nucleic acid concentration: If the nucleic acid concentration is <50 ng/μl or the A260/A280 ratio is <1.8 or >2.1,the sample DNA is re-extracted.5.Polymerase chain reaction(PCR)and sequence analysis: The Bartonella was detected by polymerase chain reaction.Firstly,the Bartonella non-coding RNA gene(ssr A)was amplified by real-time PCR.According to the standard curve,if quantification cycle(C q)< 35 was positive and Cq≥35 was negative.Then,the ssr A gene fragment of positive samples was amplified by ordinary PCR.The amplified products were directed to the company for sequencing,and the sequence was compared by homologyin NCBI.Phylogenetic tree was constructed by MEGA7.0 and evolutionary relationship was analyzed.6.Statistical analysis: All data were entered by Epi Data 3.0 and were analyzed under R software.The infection rates were described using descriptive statistics and were compared using Chi-square test.Logistic regression analysis was used to analyze influencing factors that the wild small mammals were infected Bartonella.Results1.Distribution of wild small mammals: 1605 samples of liver and spleen of wild small mammals were 550 in Yulong,648 in Jianchuan and 407 in Lianghe.Among them,Apodemus chevrieri,Eothenomys miletus,Apodemu sdraco and Rattus tanezumi were dominant,with 471,305,157 and 104 samples,respectively.The dominant species were different in different areas.Apodemus chevrieri,Eothenomys miletus and Apodemus draco were dominant species in Yulong plague foci and Jianchuan plague foci,while Rattus tanezumi,Rattus steini and Rattus norvegicus were dominant species in Lianghe plague foci.2.Infection rate of Bartonella in wild small mammals: Among 1605 samples of 30 species,18 genera,6 families,3 orders,25 wild small mammals were infected with Bartonella and the infection rate of Bartonella in wild small mammals was 47.85%(768/1605).Among them,the infection rate of the genus Anourosorex aquamipes(Anourosorex aquamipes)was 80.00%(20/25),the genus Rattus coxigiandersoni(Rattus fulvescins,Rattus niviventer,Rattus coxigiandersoni)was 59.55%(53/89),the genus Apodemus(Apodemus chevrieri,Apodemus draco,Apodemus latronum)was 55.01%(383/697),the genus Mus(Mus pahari)was 54.55%(24/44),the genus Mus(Rattus tanezumi,Rattus steini,Rattus norvegicus,Rattus nitidus)was 51.22%(105/205),the genus Dremomys(Dremomys pemyi)was 47.62%(10/21),the genus Sorex(Sorex alpinus,Suncus murinus)was 45.95%(17/37),the genus Eothenomys(Eothenomys miletus,Eothenomys proditor,Eothenomys eleusis,Eothenomys melanogaster)was 36.34%(129/355),the genus Crocidura(Crocidura attenuata,Crocidura draculadracula)was 32.35%(11/34),the genus Hylomys(Hylomyssuillus)was 32.14%(9/28),the genus Cricetomys(Berylmys bowersi)was 11.11%(1/9),the genus Tupaia(Tupaia belangeri)was 2.17%(1/46).Two Vernaya fulva and Bandicota indica were infected Bartonella.Micromys minutus,Sorex alpinus,Callosciurus erythraeus,Sciurotamias forresti,Eothenomys eleusis were not infected Bartonella.3.Areas: The infection rates of Bartonella in wild small mammals in Yulong,Jianchuan and Lianghe plague foci were 50.91%(280/550),39.97%(259/648)and 56.27%(229/407),respectively.The infection rates of Bartonella in wild small mammals in Lianghe plague foci were significantly higher than Yulong and Jianchuan plague foci(c2=29.74,(49)<0.05).4.Season: The wild small mammals were infected with Bartonella in four seasons and the infection rates were 47.63%(171/359)in spring,56.52%(208/368)in summer,44.99%(211/469)in autumn and 43.52%(178/409)in winter.The infection rates in summer were higher than autumn and winter(c2=15.71,(49)<0.05).5.Gender: The infection rate of Bartonella in male small mammals(50.12%)was higher than female small mammals(45.11%)(c2=3.99,(49)<0.05).6.Habitat environment: The wild small mammals were captured from Five habitats,including forest land,cultivated land,forestland-cultivated land junction,cultivated land shrub junction and shrub.The infection rates of Bartonella were 51.16%(530/1036)in forest land,39.66%(92/232)in cultivated land,42.54%(77/181)in forestland-cultivated land junction,48.33%(58/120)in cultivated land shrub junction,30.56%(11/36)in shrub.The highest infection rate was found in forest land(c2=17.16,(49)<0.05).7.Altitude: With the increase of altitude,the infection of Bartonella in wild small mammals has an increasing trend,but it has certain adaptability to one of the wave gradients.The specific conditions were as follows: the infection rates of Bartonella in wild small mammals in Yulong plague foci were 31.15% in 2400~2600m,36.84% in 2600~2800m,61.54% in 2800~3000m and 48.87% over 3000 m,and the infection rates of Bartonella in wild small mammals between 2800 and 3000 m was the highest(c2=18.44,(49)<0.05).the infection rate of wild small mammals with Bartonella in Jianchuan plague foci were 35.56% in 2250~2450m,38.05% in 2450~2650m,47.97% in 2650~2850m.There was no significant difference in the infection rate of Bartonella in wild small mammals for the different altitude gradients in Jianchuan plague foci(c2=5.31,(49)>0.05).The infection rates of Bartonella in wild small mammals in Lianghe plague foci were 48.72% in 1000~1200m,39.50% in 1200~1400m,78.18% in 1400~1600m and 63.93% in above 1600 m.The infection rates of Bartonella in wild small mammals were the highest at altitude of 1400~1600m(c2=35.76,(49)<0.05).8.influencing factor: The infection of Bartonella in wild small mammals on the plague natural foci of Yunnan province are related to the factors sampling area,season,altitude and species of small mammals.Compared with Jianchuan plague foci,the infection rate of wild small mammals in Yulong plague foci increased by 1.44 times and that of wild small mammals in Lianghe plague foci increased by 9.16 times.Compared with spring,the infection rate of wild small mammals in summer increased by 1.32 times,that of wild small mammals in autumn decreased by 30%,and that of wild small mammals in winter decreased by 25%.Compared with Apodemus chevrieri,the infection rate of Bartonella in Apodemus draco reduced by 5%,the infection rate of Bartonella in Eothenomys miletus reduced 49%,the infection rate of Bartonella in Rattus tanezumi reduced by 45%,the infection rate of Bartonella in Rattus steini was reduced by 59%,the infection rate of Bartonella in other species of small mammals was reduced by 64%.Compared with the altitude range of between 1000 m and 1500 m,the infection rate of Bartonella in wild small mammals increased by 2.21 times in the altitude range of between 1500 m and 2000 m,the infection rate of Bartonella in wild small mammals increased by 1.27 times at over 3000 m altitude and the infection rate of Bartonella in wild small mammals increased by 1.07 times in the altitude range of between 2500 m and 3000 m altitude,and the infection rate of Bartonella in small mammals reduced by 33% from the altitude range of between 2000 and 2500 m.9.Genetic evolution analysis of Bartonella: Bartonella genotype were detected in small mammals including Bartonella rochalimae,Bartonella japonica,Bartonella tribocorum,Bartonella sylvatica,Bartonella rattimassiliensis,Bartonella washoensis.Conclusion1.The infection rate of Bartonella in wild small mammals is 47.85% in Yulong plague foci,Jianchuan plague foci and Lianghe plague foci in Yunnan province,and the host of Bartonella is diverse.2.The infection of Bartonella in small mammals is affected by four factors: sampling area,the species of small mammals,season and altitude.Bartonella infection rate of small mammals was the highest in Lianghe plague foci,followed by Yulong plague foci,and the lowest is in Jianchuan plague foci.In the four seasons,there are wild small mammals infected with Bartonella and the infection rate was highest in summer.With the increase of altitude,the infection rate of wild small mammals showed an upward trend.3.There are diversity of Bartonella genes in wild small mammals on the plague natural foci of Yunnan province,three of them are pathogenic to human.