巴尔通体在滇西南蝙蝠中高度流行并具有丰富的遗传变异特征

蝙蝠是很多病原微生物的自然宿主, 全球多项研究表明蝙蝠是巴尔通体(Bartonella species)的主要宿主。为了解滇西南地区蝙蝠中巴尔通体的流行特征, 我们于2015-2017年间在云南省4个地区应用网捕法捕获蝙蝠3种305只。经种类鉴定后采集肝脾组织, 提取核酸, 通过TaqMan实时荧光定量PCR方法检测巴尔通体的tmRNA基因ssrA, 并进行测序鉴定和系统发育分析。结果发现172只蝙蝠检出该基因, 总感染率为56.4%; 其中临沧、西双版纳、保山和瑞丽4个采样点的蝙蝠感染率分别为50.0% (22/44)、61.7% (29/47)、62.1% (18/29)和55.7% (103/185)。中菊头蝠(Rhinolophus affinis)、小菊头蝠(R. blythi)和棕果蝠(Rousettus leschenaultii)的感染率分别为50.0% (22/44)、62.1% (18/29)和56.9% (132/232), 差异没有统计学意义(χ² = 1.135, P = 0.567), 表明巴尔通体在云南当地的蝙蝠种群中高度流行。定量PCR扩增产物2次扩增后测序获得37个巴尔通体ssrA序列, 属于10个系统发育分支, 其中1个为伊丽莎白巴尔通体(B. elizabethae)、特利波契巴尔通体(B. tribocorum)和克拉斯诺夫巴尔通体(B. krasnovii)的近缘种。其余序列与已知巴尔通体距离较远, 与亚洲、欧洲和美洲等其他地域来源于蝙蝠的巴尔通体近缘。遗传多样性分析显示, ssrA基因的核苷酸多样性指数(π)为0.11381 ± 0.00928, 基因型多样性指数(Hd)为0.985 ± 0.010, 形成29个基因型(单倍型), 说明云南蝙蝠巴尔通体具有丰富的遗传多样性。通过对本研究标本与全球相关序列的系统发育网络重建, 分析全球蝙蝠巴尔通体的地理和宿主分布特征, 可以看出巴尔通体与蝙蝠之间存在显著的宿主特异性关联。因此可初步确定蝙蝠-巴尔通体具有协同进化特征, 同时受到地理隔离的影响。

High prevalence and genetic variation of Bartonella species inhabiting the bats in southwestern Yunnan

Aims: Bats are natural reservoir hosts of many zoonotic viruses and non-viral pathogens. Several lines of evidence indicate that bats harbor diverse groups of Bartonella species and genotypes. Little is still known about how prevalence varies and the genetic diversity of Bartonella spp. in bats in China.
Methods: Between 2015 and 2017, bats were captured in four areas in southwestern Yunnan by the net trapping method. Liver and spleen tissues of bats were collected, and nucleic acids were extracted after mixing and grinding. The ssrA gene of Bartonella spp. was detected by using the TaqMan real-time fluorescent quantitative PCR assay (qPCR). The positive products of qPCR were amplified and sequenced to identify the species of Bartonella. The phylogenetic relationships were constructed by binary and network methods.
Results: We present the results of the investigation of bat-borne Bartonella in four areas of Yunnan Province, which have shown a high prevalence of Bartonella and high genetic diversity in the local bat population. Bartonella gene ssrA was detected by using qPCR in 56.4% (172/305) bats captured from four sampling sites, including 22 Rhinolophus affinis, 18 Rhinolophus blythi, and 132 Rousettus leschenaultii. There was no statistically significant difference (χ² = 1.135, P = 0.567) in the infection rates among species. The infection rates of Lincang, Xishuangbanna, Baoshan, and Ruili were 50.0% (22/44), 61.7% (29/47), 62.1% (18/29), and 55.7% (103/185), respectively. Thirty-seven ssrA sequences of Bartonella were obtained by second amplification from qPCR and sequencing. The binary tree revealed 10 phylogroups, one of which was related to B. elizabethae, B. tribocorum and B. krasnovii, the other sequences were far from the known Bartonella spp. and were closely related to bat-borne Bartonella from bats in Asia, Europe, and America. The genetic diversity analysis showed that the nucleotide diversity index (π) was 0.11381 ± 0.00928, and the genotypic diversity index (Hd) was 0.985 ± 0.010, which brought about 29 genotypes. The genetic networks constructed by 115 ssrA sequences exhibited that Bartonella spp. from global bats had an incomplete geographical distribution and host specificity.
Conclusion: We found that the bat-borne Bartonella in Yunnan was genetically diverse, geographically widespread, and endemic and that bats play a part in transmitting Bartonella species locally. The analysis of phylogenetic networks based on the global dataset of ssrA sequences of bat-borne Bartonella points to an evolutionary association of Bartonella spp. with their bat hosts and the effect of geographical isolation.