A broad-range survey of Borrelia burgdorferi sensu lato infection in small mammals in Sino-Burmese border area, Yunnan Province, China CURRENT STATUS: UNDER REVIEW

Background: Lyme disease is caused by Borrelia burgdorferi sensu lato which is usually found in wild and domestic mammals worldwide. In China, Human cases of B. burgdorferi infections have been identified in a wide distribution, but little direct surveillance of potential rodent reservoirs has been performed in Yunnan Province, the tropical region, Sino-Burmese border area, southwestern China. Here we report a thoroughly investigation of B. burgdorferi sensu lato in small mammals collected in 2011 to 2016 in the region. Methods: A nested PCR was done for the 5S-23S rRNA intergenic spacer gene of Borrelia burgdorferi sensu lato. The PCR-positive amplicons were directly sequenced. Sequence analysis was carried out using a FASTA search on the Genbank database, with phylogenetic trees constructed using MEGA software, version 6.06. Statistical Analysis were conducted using SPSS.version 17.0. Results: Totally 57 species, 3659 mammals were captured at 159 sample sites located in 23 counties in Yunnan Province. Thirty species, 146 (3.99%) mammals were tested positive for B. bourgdorferi s.l . by nested PCR based on 5S-23S rRNA intergenic spacer gene. 20 species mammals were first reported infected with B. burgdorferi s.l. Sequence analysis revealed that five genotypes of B. burgdorferi s.l. were detected, including B. afzelii , B. burgdorferi sensu stricto, B. japonica , B. garinii and B. valaisiana. Conclusions: Significant differences in prevalence rates of B. bourgdorferi s.l 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 5S-23S rRNA intergenic spacer gene revealed that there were five genotypes of B. burgdorferi s.l. detected, indicating 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), and were responsible for a wide spectrum of clinical symptoms. While there have been documented reports of human cases of Lyme disease in southwestern China [4], the only information on the prevalence of BBSL in rodent reservoirs came from one study, where a majority of rodents were trapped indoors [5]. IFA was reported to detect BBSL infection in rodents and humans from Yunnan. Yunnan Province is of particular interest given its wide topographic range and high level of small mammal biodiversity, many of which may potential reservoirs for BBSL. We performed a broad-range, systematic field investigation on the prevalence of BBSL infections in rodents sampled in 23 counties in Yunnan Province, and then analyzed the distribution and genetic diversity of the pathogen, as well as the association between infections and suspected risk factors. This study aims to evaluate the role that small mammals play in the transmission of BBSL across 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 at 159 sample sites from 23 counties ranging from 530 to 4300m altitude in Yunnan Province ( Figure 1). Two hundred snap traps per sample site were placed for three consecutive nights and checked daily. Mammal species were identified 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 identification of species, spleen tissues were removed from the animals and stored in liquid nitrogen until tested. For unidentified species in the field, the craniums were brought to the laboratory for further identification.

DNA Extraction and PCR Analysis
DNA was extracted from spleen tissue using the DNA blood and tissue kit (Tiangen Biotechnique, Beijing, China) according to the manufacturer's instruction. A nested PCR for the 5S-23S rRNA intergenic spacer gene of BBSL was done as previously described [6].
The PCR-positive amplicons were directly sequenced with an automated DNA sequencer (ABI PRISM 373; Perkin-Elmer, Norwalk, CT). Sequence analysis was carried out using a FASTA search on the Genbank database, with phylogenetic trees constructed using MEGA software, version 6.06 [7]. The 5S-23S rRNA intergenic spacer gene of BBSL obtained in this study was 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 rodents, environmental landscape, altitude, and testing positive for B. burgdorferi s.l. using a chi-square test. All variables with a P-value of <0.05 from univariate analysis were entered into a multivariate forward stepwise logistic regression analysis. All analyses were conducted using SPSS (version 17.0, SPSS Inc. Chicago, IL).

Discussion
Human cases of LB have been confirmed in almost every province found on mainland China including Yunnan Province. However, most of patients only had serological evidence and were not confirmed for specific genotypes. BBSL has been reported in small mammals trapped in the provinces Qinghai, Hunan, Shanxi, Liaoning, Sichuan, Fujian, Zhejiang, Gansu, Guangdong, Jilin and Yunnan [8][9][10][11][12][13][14][15][16], suggesting that small mammals are likely the main reservoir hosts in China. This study presents a large sample size extending over a wide geographic area, which provides insight into the prevalence, spatial distribution and genetic diversity of BBSL in small mammals collected in Sino-Burmese border area, Yunnan Province.
We documented BBSL infection in 30 species of small mammal, among which, 20 species had not been previously documented. These species may be infected occasionally, whether they serve as reservoir hosts need a further study. The Rattus tanezumi (573/3659, 15.66%) was the predominant species trapped in residential area in Yunnan.
Apodemus draco (579/3659, 15.82%) and Apodemus chevrieri (402/3659, 10.99%) were the predominant hosts species in Yunnan, which was consistent with results from Europe where Apodemus are considered a major reservoir of Borrelia [ 17]. BBSL was detected in Apodemus draco and in Apodemus chevrieri in Yunnan, with Apodemus draco capable of carrying four Borrelia spp. The Ochotona gloveri, Soriculus leucops and Rattus tuekkestanicus also had a much higher prevalence (> 14%) with much larger sample sizes in this study than in other provinces in China [12; 18; 19; 20; 21; 22]. Rattus norvegicus is the prominent household species in Yunnan, which had a high prevalence (12.50%) and was detected positive for pathogenic genotypes (B. afzelii and B. burgdorferi sensu stricto). We also found that the uncommon species Sorex cylindricauda in this study tested positive for B. burgdorferi s.l DNA, requiring further investigation to fully understand their role in maintaining or amplifying infections in nature.
Our findings indicated that prevalence rates in rodents are ranked highest to lowest by landscape type as follows: forest landscape > agricultural landscape > residential landscape ( Table 2), which is likely related to tick vector density and preferred habitat.
This reiterates the need for individuals traveling into potential tick habitats, like the forest, to take proper protective measures to limit tick bite exposure. Sampling locations in this survey contained a broad range of altitudes from 500 meters to 4500 meters.
Among the three altitude classes, small mammals with the highest prevalence of BBSL were found above 2500 m. It was reported that Ixodes ricinus distribution in Sumava National Park 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 reflect lower populations living in these areas.
Our study found five genospecies of BBSL in small mammals in Yunnan Province, four of them except for B. japonica, have previously been associated with LB [24][25]. B. afzelii was the prominent genotype (83/146, 56.84%) in this study, which 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 sequence analysis carried out in this study, most of the B.
afzelii sequences shared 99% identity with clinical isolates from patients in northeastern China [26]. Most of the B. burgdorferi sensu stricto sequences were identical to the sequence from a human case reported in France (KY594010.1). At this time, there have been no confirmed patients with registered sequence of Lyme disease spirochetes 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 within the sequence from Guizhou and Zhejiang province, the other three 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. B. japonica have only been found in Yunlong county, with this representing the first report documenting B. japonica in Apodemus draco and Niviventer excelsior in China. B. garinii is the most common genospecies in China, followed by B. afzelii [ 27].
However, B. garinii was detected from different species of small mammals only in Deqin County. We found that B. afzelii was the main genospecies detected in Yunnan, which is consistent with previous reports [4]. B. burgdorferi sensu stricto has been detected in Sika deer from Jilin province and in Caprolagu ssinensis 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 (Fig. 1). These findings reflect that Yunnan Province is of particular interest given its diverse topographic range and high level of biodiversity in small mammals that are potential reservoirs for BBSL.
In conclusion, Yunnan Province is an important natural foci of Borrelia burgdorferi sensu lato in China, and given the absence of reported human cases within this region, efforts to expand clinical surveillance are urgently needed.

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