Lyme borreliosis (LB) is the most common reported tick-borne disease across Europe, North America, and Asia, which has become a worldwide public health problem and remains large challenges (Hao et al., 2011; Leeflang et al., 2016; Schwartz et al., 2017; Bobe et al., 2021; Vandekerckhove et al., 2021). The causative agents of LB fall within the species complex Borrelia burgdorferi sensu lato (BBSL) and are responsible for a wide spectrum of clinical symptoms including skin erythema, arthritis, and multi-system and multi-organ damage such as heart and nerve (Postic et al., 1994; Tamura et al., 2011). BBSL is transmitted by the bites of infected Ixodes tick, and main reservoir hosts include wild vertebrates such as rodents, deer and birds, and livestock animals, which take major contributors to the transmission of this zoonotic parasitosis across the globe (Hussain et al., 2021). More than 20 genospecies of BBSL have been described around the world, 10 of which have been reported to cause human infections (Hao et al., 2011; Hao, 2020).

In China, since LB was first found in Hailin County, Heilongjiang Province, in 1986, there have been documented reports of human cases of LB across almost all provinces in mainland China (Wu et al., 2013; Fang et al., 2015). The epidemic areas of Lyme disease in China are mainly concentrated in northeast, northwest, and parts of North China. However, the pathogen genospecies of most of LB cases were not characterized (Fang et al., 2015). Up to now, at least 100 strains belong to at least seven genotypes including B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. sinica, B. valaisiana, B. bavariensis, and B. bisettii from patients, and ticks vector or host animals have been reported (Hao et al., 2016). Among of them, B. garinii and B. afzelii are the main pathogenic genotypes.

In Yunnan Province, positive serologies against BBSL in human and rodents have been reported over two decades ago (Wan et al., 1991; Pan and Yu, 1992). Other studies examining the prevalence of BBSL are relative lack (Hao et al., 2011; Hao, 2020). Yunnan Province is of particular interest given its wide topographic range and diverse land features including plateaus, low mountains, hills, high mountains, deep valleys, and open river valleys. A high level of small mammal and tick biodiversity exists in this region, often overlapping with largescale agricultural operations. To evaluate the distribution and genetic diversity of BBSL in reservoirs, incidental hosts, and vectors, we performed a broad-range, systematic field investigation of BBSL in Yunnan Province.

A total of 5,044 small mammals, 1,927 livestock samples, and 1,507 ticks were collected, with 302 (3.6%) positive samples of BBSL detected in 21 of 42 sample counties (Figure 1; Table 1). Weixi County had the highest prevalence (9.4%), followed by Deqin (8.8%), and Gongshan (7.3%), with significant differences in positive rates of BBSL by counties observed (χ² = 201.729, p < 0.05). Positive BBSL in small mammals were detected in 15 out of 32 sampled counties, with highest prevalence in Gongshan County (8.6%). The positive sampling points of BBSL in livestock were detected in 8 out of 16 sampling counties with the highest prevalence in Deqin (10.3%). Borrelia burgdorferi s.l. in ticks were detected from 6 out of 14 sampling counties with the highest prevalence in Weixi County (17.8%; Table 1).

Human cases of LB have been confirmed in almost every province found on mainland China, including Yunnan Province. However, most reported patients only have serological evidence indicating previous LB infection, without confirmed specific genotypes information. BBSL have been reported in small mammals trapped in the provinces Qinghai, Hunan, Shanxi, Liaoning, Sichuan, Fujian, Zhejiang, Gansu, Guangdong, Jilin, and Yunnan (Wan et al., 1991; Pan and Yu, 1992; Chen et al., 2000; Shi et al., 2004; Huang et al., 2006; Hou et al., 2010; Meng et al., 2010; Wu et al., 2013), indicating that small mammals are likely the main reservoir hosts in China. This study presents a much larger context of BBSL in Yunnan, including sampling both small mammals, livestock, and ticks across a wide and diverse geographic area. The findings from this survey provide valuable insights about the prevalence, spatial distribution, and genetic diversity of BBSL in Yunnan Province, southwestern China.

We documented BBSL infection in 30 species of small mammals, among which 20 species had not been previously described as potential reservoirs. It is possible these 20 species are incidental hosts that are infected occasionally, requiring additional investigations to determine what, if any, role they play as reservoir hosts. The R. tanezumi (16.5%) was the predominant species trapped in residential areas in Yunnan. Apodemus spp. (24.7%) were the predominant hosts captured in Yunnan, which is consistent with findings from Europe where Apodemus are considered a major reservoir of Borrelia (Sala and De, 2016). Borrelia burgdorferi s.l. was also detected in A. draco and A. chevrieri in Yunnan, with A. draco capable of carrying four pathogenic Borrelia spp. Soriculus leucops had a much higher prevalence (>14.0%) detected in this study, when compared to other provinces in China (Wang et al., 2003; Chu et al., 2006; Huang et al., 2006; Zhang et al., 2015, 2016; Wulantuya and Yin, 2018). Rattus tuekkestanicus, a prominent household species in Yunnan, had a relatively high prevalence (14.3%), although only 7 small mammals were captured. We also found that Sorex cylindricauda tested positive for BBSL DNA (13.0%), requiring further investigation to fully understand their role in maintaining or amplifying infections in nature.

In this study, 52 positive livestock blood samples were detected in 8 counties which were mainly distributed in western Yunnan Province. Cattle, sheep, dogs, and horses were detected positive, with this study reporting the first ever detections of BBSL in Yunnan in cattle, sheep, and horses. The infection rate in dogs was the highest, with three known BBSL genotypes detected including B. afzelii, B. burgdorferi s.s., and B. garinii, which are all pathogenic in humans. Tick samples from 6 out of 9 counties were positive, mainly distributed in the western of Yunnan Province. Six genotypes of Borrelia burgdorferi (B. afzelii, B. garinii, B. japonica, B. valaisiana, B. burgdorferi sensu stricto, and B. sinica) were detected from five tick species (Ha. nepalensis, I. ovatus, I. granulatus, R. microplus, and Ha. kolonini), with B. garinii as the dominant species (47.6%,49/103) and R. microplus was the dominant ticks (41.4%, 624/1507). This study is the first report of B. japonica detected in I. granulosus in China. Borrelia sinica was also detected in I. ovata for the first time in Yunnan Province.

Both Borrelia afzelii and B. garinii were detected in livestock, small mammals, and ticks in Deqin County, B. japonica, B. valaisiana, and B. afzelii were detected in small mammals and ticks in Yunlong, Tengchong, and Weixi County. There are more than three genotypes of BBSL in these three counties located western Yunnan, which warrants further investigation.

Our findings also indicated that detection rates in small mammals were ranked highest to lowest by landscape type as follows: forest landscape > agricultural landscape > residential landscape, which is likely related to vector density and preferred reservoir habitat. Sampling locations in this survey contained a broad range of altitudes from 500 to 4,500 m. Among the three altitude classes, the highest prevalence of BBSL was found above 2,500 m. In the Czech Republic, the distribution of Ixodes ricinus, a known vector of LB, extended toward higher altitudes, which was attributed to warming climates (Daniel et al., 2003). The roles temperature and humidity play in tick reproduction and reservoir preferences require further investigation within these altitude ranges. Additionally, there are no reported human cases at these heights, which might reflect lower populations living in these areas.

No obvious season-associated prevalence of BBSL in the host and vector beside winter was observed, which is different from those in Northern China, where there are four distinct season and temporal dynamic change of BBSL. We also observed the temporal dynamic change via years for BBSL, especially no positive samples after 2017. It is probably because of the deviation in sampling arrangement that most of samples were collected in the sites located northeastern Yunnan and Jinshajiang river low-altitude areas. It also coincides with our general finds that the prevalence of BBSL in Yunnan was mainly in high elevations in the west and northwest areas.

Six genospecies of BBSL were detected in this study, with four of them, excluding B. japonica and B. sinica, previously associated with clinical LB (Diza et al., 2004; Rudenko et al., 2011). The BBSL genotypes with the highest proportion of BBSL positive samples in small mammals, livestock, and ticks were B. afzelii and B. garinii, respectively. There exists a wide distribution and genetic diversity of BBSL in Yunnan, compared to only 1–2 genospecies of BBSL detected in other provinces of China; however, this may also be an artifact of the extent and volume of samples tested in this study. Borrelia afzelii was the predominant genotype (56.5%, 83/147) and was detected in 24 species. According to the sequences detected in this study, most B. afzelii isolates shared 99% identity with clinical samples from northeastern China (Ni et al., 2014), and Borrelia garinii, the most common genospecies elsewhere in China (Fang et al., 2015), was only detected in Deqin County in this study. While B. burgdorferi sensu stricto has previously been detected in Cervus nippon (Sika deer) from Jilin Province and in Caprolagus sinensis from Hunan Province, this study adds a number of detections in small mammals to the literature. Borrelia valaisiana sequences clustered into two clades, with one sharing sequence identity from Guizhou and Zhejiang Province and the other closer to sequences from Europe. While birds are major reservoirs for B. valaisiana in Europe, the transmission cycle maintaining B. valaisiana in Yunan may be different given findings from this study. Finally, B. japonica was detected in Yunlong County, which represents the first documentation of B. japonica in A. draco and Niviventer excelsior in China. At this time, there have been no confirmed patients with PCR confirmed LB in Yunnan Province, requiring further investigation.

Yunnan Province is an important natural focus of BBSL in China. These findings reflect a high level of Borrelia genetic diversity found in a wide range of small mammals, many of which are likely reservoirs for BBSL in Yunnan. Given the absence of reported human cases within this region, efforts to expand clinical surveillance are needed.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: MK333406-MK33427, MZ146346, MZ146350, MZ146351, and OM350159-OM350185.

CD, JJ, and YD conceived and designed the experiments. ZH, BJ, YZ, MY, FW, and SB performed the experiments. JJ, ZH, LH, BJ, and YS analyzed the data. Sample collections were implemented by ZS, XD, HJ, JW, YL, FW, SB, and EP. JJ, ZH, LH, and MF drafted and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

This study was funded by the National Key R&D Program (2019YFC1200501), the Natural Science Foundation of China (U2002219, 81760607, and 81360413), and the State Key Laboratory of Pathogen and Biosecurity (SKLPBS1833). The funders had no role in the study design, data gathering, analysis, interpretation, or writing of the paper. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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