Infection of Borrelia burgdorferi sensu lato of small mammals in

Abstract


Introduction
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.
. CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted July 25, 2019.; https://doi.org/10.1101/714980doi: bioRxiv preprint burgdorferi sensu lato (BBSL), and is responsible for a wide spectrum of clinical symptoms.
Anti-Borrelia antibodies in rats and humans have been reported in 9 counties and 4 counties of Yunnan, respectively.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].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 systematic field investigation on the prevalence of BBSL infections in a large quantity of rodents sampled in 23 countries in Yunnan Province, and then analyzed the distribution and genetic diversity of BBSL, 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.

Ethics statement
The research protocol for trapping wild small animals and collecting samples was approved

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 in Yunnan Province (Table 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 was 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 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 rodents, environmental landscape, altitude, and testing positive for BBSL 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 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 .CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted July 25, 2019.; https://doi.org/10.1101/714980doi: bioRxiv preprint (573/3659,15.66%)was the predominant species trapped in households in Yunnan.Apodemus draco (579/3659,15.82%)and A. 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 A. chevrieri in Yunnan, with A. 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 BBSL 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, 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 2500m.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 .CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted July 25, 2019.; https://doi.org/10.1101/714980doi: bioRxiv preprint of them except for B. japonica, have previously been associated with LB [24][25].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.the most common genospecies in China, followed by B. afzelii [27].However, 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 and in Caprolagu ssinensis from Hunan, and detected in small mammals in Yunnan within the more populated counties of Gongshan, Deqin, and Weixi (S1, S2, S5) found in northwestern Yunnan.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 BBSL in China, and given the absence of reported human cases within this region, efforts to expand clinical surveillance by the Animal Subjects Research Review Boards at the Yunnan Institute of Endemic Diseases Control and Prevention (2013-003), in accordance with the medical research regulations of China and the Regulation of the People's Republic of China for the Implementation of the Protection of Terrestrial Wildlife.

Fig 1 .
Fig 1. 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 identified in this study.
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, requiring additional study.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

Table 1 .
Prevalence of BBSL in small mammals from different survey sites.

Table 2 .
Prevalence of Borrelia burgdorferi sensu lato in small mammals of different species.

Table 3 .
Risk factors related to Borrelia burgdorferi sensu lato based on univariate analyses.

Table 4 .
Risk factors related to BBSL based on multivariate logistic regression.peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made japonica.Deqin county had a distribution of four Borrelia spp.exceptB.japonicawhichwas only found in Yunlong county (Table1).Additionally, four of five Borrelia spp.were detected in Apodemus draco (Table2).The nucleotide sequences of the B. afzelii sequences . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is madeThe copyright holder for this preprint (which was this version posted July 25, 2019.