A broad-range survey of Borrelia burgdorferi sensu lato infection in small mammals in Yunnan Province, China

Background: Lyme disease, caused by Borrelia burgdorferi sensu lato (BBSL), is commonly found in wild and domestic mammals worldwide. In China, human cases of B. burgdorferi infections have been identied across a wide distribution, but little direct surveillance of potential small mammal reservoirs has been performed in Yunnan Province, a tropical region in southwestern China. Here we report a thorough investigation of BBSL in small mammals collected from 2011 to 2016 from this region. Methods: Small mammals were captured using snap traps in 23 counties located in Yunnan Province. DNA was extracted from spleen tissue using DNA blood and tissue kits. A nested PCR targeting the 5S-23S rRNA intergenic spacer gene of BBSL was used for pathogen detection. Amplicons of 252bp expected sizes were sequenced directly and analyzed using BLAST algorithm. A phylogenetic tree was constructed using MEGA software and statistical analysis were conducted using SPSS version 17.0. Results: Overall, 3659 mammals belonging to 57 species were captured at 159 sample sites located in 23 counties in Yunnan Province. Borrelia burgdorferi s.l. was found in 146 mammals (3.99%), from 30 different species, 20 of which represent the rst reported detection in that species. Sequence analysis revealed ve genotypes of B. burgdorferi s.l., including B. afzelii, B. burgdorferi sensu stricto, B. japonica, B. garinii and B. valaisiana. Conclusions: Signicant differences in prevalence rates of BBSL were observed at varying landscape types and altitudes. Small mammals in forested areas had higher prevalence rates than other landscape types, as did small mammals found at altitudes greater than 2500 meters. The ve genotypes of BBSL detected, suggests high genetic diversity within this province.


Background
Lyme borreliosis (LB) is the most commonly reported vector-borne disease across Europe, North America and Asia [1][2][3][4]. The causative agents of LB fall within the species complex B. burgdorferi sensu lato (BBSL) mainly including B. garinii, B. afzelii, B. japonica, and B. valaisiana, and are responsible for a wide spectrum of clinical symptoms. While there have been documented reports of human cases of LB in 30 provinces in China through serum epidemiological investigation as well as molecular gene-typing research [4,5]. A little bit information on the prevalence of BBSL in human in Yuanan province [4,5]. Also, the report on BBSL in small mammal reservoirs were about indoors small mammals or some limited local small-scale area. Yunnan Province is of particular interest given its wide topographic range, where the elevation is high in the northwest and low in the southeast, and land features include plateaus, low mountains, hills, high mountains, deep valleys and open river valleys. A high level of small mammal biodiversity exists within this region, many of which may serve as potential reservoirs for BBSL. To evaluate the distribution and genetic diversity of BBSL, as well as the role that small mammals play in transmission, we performed a broad-range, systematic eld investigation of BBSL in Yunnan Province.

Collection of Small Mammal Samples
From 2011 to 2016, small mammals were captured using animal snap traps set at agricultural, forested, and residential areas, with elevation ranging from 530 to 4300m. A total of 159 sample sites located in 23 counties across Yunnan Province ( Figure 1) were surveyed. Two hundred snap traps per sample site were placed for three consecutive nights and checked daily. Mammal species were identi ed according to external morphology, fur color, measurements, and visible characters of dentition. Each animal's sex, developmental stage, and location were recorded at the time of sample processing. After identi cation of species, spleen tissues were removed from the animals and stored in liquid nitrogen until tested. For unidenti ed species in the eld, craniums were brought to the laboratory for further identi cation.

DNA Extraction and PCR Analysis
DNA was extracted from spleen tissue using the DNA blood and tissue kits (Tiangen Biotechnique, Beijing, China) according to the manufacturer's instruction. A nested PCR for the 5S-23S rRNA intergenic spacer gene of BBSL, with the rst-round primers (5'-CGACCTTCTTCGCC TTAAAGC-3' and 5'-TAAGCTGACTAATACTAATTACCC-3') and second round primers (5'-TCCTAGGCATTCACCATA-3' and 5'-CTGCGAGTTCGCG GGAGA-3' was performed as previously described [6]. The 252bp PCR-positive amplicons were directly sequenced using an automated DNA sequencer (ABI PRISM 373; Perkin-Elmer, Norwalk, CT). Sequence analysis was carried out using a FASTA search of the GenBank database, with phylogenetic trees constructed using MEGA software, version 6.06 [7]. The 5S-23S rRNA intergenic spacer gene sequences of BBSL obtained in this study were deposited in GenBank under accession numbers MK333406-MK33427 and KP677523.1 respectively.

Statistical Analysis
Univariate analysis was used to access the association between gender, developmental stage of small mammals, environmental landscape, altitude, and positive detection rate of BBSL. using a chi-square test. All variables with a P-value of <0.05 from univariate analysis were included in a multivariate forward stepwise logistic regression. All analyses were conducted using SPSS (version 17.0, SPSS Inc. Chicago, IL).

Discussion
We documented BBSL infection in 30 species of small mammal, among which, 20 species had not been previously documented. These species may be incidental hosts that are infected occasionally, further study is required to determine what, if any, role the play as reservoir hosts. The Rattus tanezumi (15.66%) was the predominant species trapped in residential areas in Yunnan. Apodemus spp. (31.35%) were the predominant hosts captured in Yunnan, which was consistent with results from Europe where Apodemus are considered a major reservoir of Borrelia [17]. BBSL was detected in A. draco and in A. chevrieri in Yunnan, with A. draco capable of carrying four pathogenic Borrelia spp. Soriculus leucops had a much higher prevalence (>14%) with a larger sample sizes in this study than in other provinces in China [12; 18-22]. Rattus norvegicus, the prominent household species in Yunnan, had a relatively high prevalence (2/16, 12.50%), although only 16 small mammals were captured, and tested positive for pathogenic genotypes B. afzelii and B. burgdorferi sensu stricto. We also found that Sorex cylindricauda tested positive for BBSL DNA (14.28%), requiring further investigation to fully understand their role in maintaining or amplifying infections in nature.
Our ndings 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 tick vector density and preferred habitat. Sampling locations in this survey contained a broad range of altitudes from 500 meters to 4500 meters. Among the three altitude classes, the highest prevalence of BBSL was found above 2500m. In the Czech Republic, the distribution of Ixodes ricinus, a known vector of LB, extended toward higher altitudes, probably in relation to warming climates [23]. The roles temperature and humidity play in tick reproduction and reservoir preferences requires further investigation within these altitude ranges. Additionally, there are no reported human cases at these heights, which might re ect lower populations living in these areas.
Our study found ve genospecies of BBSL in small mammals in Yunnan Province, four of them, excluding B. japonica, have previously been associated with LB [24][25]. Borrelia afzelii was the prominent genotype (56.84%) in this study, which was detected in 24 species. There exists a wide distribution and genetic diversity of BBSL in Yunnan, compared to only 1-2 genospecies of BBSL in most provinces in China, such as Qinghai, Zhejiang, Guizhou and Guangxi. According to the sequences detected in this study, most B. afzelii shared 99% identity with clinical isolates from patients in northeastern China [26]. The B. burgdorferi sensu stricto sequences were identical to the sequence from a human case reported in France. At this time, there have been no con rmed patients with PCR con rmed LB in Yunnan province, requiring further investigation in the near future. The sequence of B. valaisiana obtained from small mammals cluster into two clades, one cluster fell within the sequence identities from Guizhou and Zhejiang province, the other cluster fell within close proximity to sequences from Europe. Birds are major reservoirs for B. valaisiana in Europe, however the transmission cycle maintaining B. valaisiana in Yunan may be different from other areas. Borrelia japonica was only detected in Yunlong county, with this representing the rst report documentation of B. japonica in Apodemus draco and Niviventer excelsior in China. Borrelia garinii is the most common genospecies in China [27], but was only detected in Deqin County in this study. We found that B. afzelii was the main genospecies detected in Yunnan, which is consistent with previous reports [4]. Borrelia burgdorferi sensu stricto has been detected in Sika deer from Jilin province and in Caprolagus sinensis from Hunan province, and detected in small mammals in Yunnan province within the more populated counties of Gongshan, Deqin, and Weixi (S1, S2, S5) found in northwestern Yunnan (Figure 1). These ndings re ect 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.
In conclusion, Yunnan Province is an important natural focus of BBSL in China. Given the absence of reported human cases within this region, efforts to expand clinical surveillance are urgently needed.  Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Phylogenetic tree of 252bp of 5S-23S rRNA intergenic spacer gene of Borrelia burgdorferi sensu lato.
Maximun Likelihood phylogenetic tree based on a comparison of Borrelia burgdorferi sensu lato 5S-23S rRNA intergenic spacer gene sequences obtained from Yunnan small mammals with Borrelia burgdorferi sensu lato reference strains. The number on each branch shows the percent occurrence in 1000 bootstrap replicates. Black circles stood for novel sequences identi ed in this study.