Abstract
To understand the potential role of bats in the epidemiology of Leptospira, we investigated the frequency of Leptospira in bats from Central China in 2018. Sixty bats belonging to four species of two families were captured from rural areas of Xianning City, Hubei Province, China. We extracted DNA from the kidneys of bats, and Leptospira spp. were determined by PCR amplification of the rrs2, flaB, lipL32, and secY genes. We found that 57% (34/60) of bats were positive for Leptospira with at least one of the four genes, and the positive rate of Leptospira in bats was 45% (27/60) with rrs2, 50% (30/60) with flaB, 15% (9/60) with lipL32, and 27% (16/60) with secY. Phylogenetic analysis indicated that bats were infected with two species of Leptospira, including Leptospira interrogans and Leptospira borgpetersenii. A high prevalence of Leptospira spp. in bats suggested that bats were important carriers of Leptospira in China.
Leptospirosis is a global zoonotic disease and is caused by pathogenic spirochetes of the genus Leptospira (Adler 2015). The disease is prevalent in most tropical and sub-tropical regions of the world including Middle East, Europe, South and Central America (Bharti et al. 2003; Bessa et al. 2010), Asia (Victoriano et al. 2009), Australia (Tulsiani et al. 2011), and Africa (Ogawa et al. 2015). Over half a million human cases of leptospirosis are thought to occur each year worldwide with a mortality rate of more than 10% (Dietrich et al. 2015). In China, leptospirosis is caused predominantly by Leptospira interrogans and Leptospira borgpetersenii (Zhang et al. 2012). Leptospirosis is common and widespread in China, and it has been reported in 29 of 34 provinces (Zhang et al. 2012). Previous investigations of animal hosts of Leptospira in China focused on rodents and pigs (Hu et al. 2014). Recently, a growing number of studies highlighted the potential role of bats in the epidemiology of Leptospira (Dietrich et al. 2015). Moreover, a recent study reported a diversity of novel pathogenic Leptospira species in bats from Northern China (Han et al. 2018). However, the ecology of Leptospira in bats from China remains unclear. Therefore, we investigated the prevalence and genetic diversity of Leptospira in bats collected from Central China.
Sixty bats were collected from a karst cave in Xianning City, Hubei Province, China (29°53′N, 114°13′E) from March to July in 2018. We extracted DNA from bat kidney samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. By sequencing the PCR product of the cytochrome B (cytb) gene amplified from bat kidney DNA (Ishii et al. 2014), the 60 captured bats were classified into four species of two families: the Vespertilionidae family (32 Myotis davidii, 17 Myotis adversus, and two Myotis altarium) and the Miniopterus family (nine Miniopterus schreibersii; Table 1).
To identify the species of Leptospira in bats, bat kidney DNA was used as template to amplify the 16S ribosomal RNA gene (rrs2), flagellin B gene (flaB), the outer membrane lipoprotein gene (lipL32), and preprotein translocase gene (secY) as previously described (Ahmed et al. 2006). We analyzed the PCR products by 1.2% agarose gel electrophoresis, stained with ethidium bromide and observed under ultraviolet light. We excised PCR products with expected sizes from the gels and extracted them using a Gel Extraction Kit (Promega, Madison, Wisconsin, USA), and then we inserted them into the pMD 19-T vectors (TaKaRa, Shiga, Japan) for cloning. One colony for each bat sample was selected randomly for Sanger sequencing using M13 universal primers on both DNA strands. Sequences of rrs2, flaB, lipL32, and secY genes were searched using BLAST in the GenBank database (National Center for Biotechnology Information 2018). The rrs2, flaB, lipL32, and secY sequences from this study were aligned with reference Leptospira spp. downloaded from GenBank with ClustalW and phylogenetic trees were constructed using maximum likelihood method of MEGA7.0 (Kumar et al. 2016).
Leptospira were detected in 57% (34/60) of bats with at least one of four genes found positive. The positive rate of Leptospira in bats was 10% (6/60) with all genes, 45% (27/60) with rrs2, 50% (30/60) with flaB, 15% (9/60) with lipL32, and 27% (16/60) with secY. Among four captured bat species, three species were positive for Leptospira, and the frequency of Leptospira was 82% (14/17) in Myotis adversus, 53% (17/32) in Myotis davidii, 33% (1/3) in Miniopterus schreibersii, and 0% (0/2) in Myotis altarium (Table 1).
Phylogenetic analysis of the sequences of rrs2, flaB, lipL32, and secY genes showed that Leptospira strains detected in bats in this study all belonged to the pathogenic group (Fig. 1). Phylogenetic analysis with the rrs2 sequences indicated that Leptospira detected in 27 bats were classified into two clusters; 22 strains were clustered with L. borgpetersenii (support value 70%; Fig. 1A) and five strains were in the same clade with L. interrogans (support value 72%; Fig. 1A). Phylogenetic analysis with the flaB sequences indicated that 30 Leptospira from bats were classified into two clades with 14 sequences closely related to L. borgpetersenii (support value 92%; Fig. 1B) and 16 sequences closely related to L. interrogans (support value 99%; Fig. 1B). Phylogenetic analysis with the lipL32 sequences indicated that the Leptospira detected in nine bats were classified into two clades: three strains clustered with L. interrogans, L. noguchii, and L. kirshneri group with high support value (99%) and six strains were clustered with L. borgpetersenii (support value 96%; Fig. 1C). Phylogenetic analysis with the secY sequences indicated that Leptospira detected in 16 bats were classified into two clades; 14 Leptospira sequences clustered with L. borgpetersenii (support value 53%; Fig. 1D) and two Leptospira sequences with L. interrogans (support value 87%; Fig. 1D). The Leptospira sequences of this study are available in GenBank with accession numbers MH882942–MH882987, MK291978–MK291986, and MH915392–MH915418.
Leptospira infection has been reported in over 50 bat species belonging to eight bat families in the tropics and subtropics as well as in Europe (Dietrich et al. 2015). The prevalence of Leptospira in bats was reported to be 35% (18/52) in Madgascar (Lagadec et al. 2012), 12% (9/77) in Comoros (Lagadec et al. 2012), 19% (7/36) in Tanzania (Mgode et al. 2014), 15% (79/529) in Zambia (Ogawa et al. 2015), 11% (19/173) in Australia (Tulsiani et al. 2011), and 2% (6/343) in Brazil (Bessa et al. 2010). In our study, the prevalence of Leptospira in bats from Central China was 56.7% (34/60), which was comparable to the prevalence in Northern China (62%, 62/124) in a previous report (Han et al. 2018). Myotis spp. bats showed a high prevalence for Leptospira (53%) in our study as well as in a previous study (Han et al. 2018) in Northern China (63%), indicating Myotis bats are a major carrier of Leptospira in China. Our study enriched knowledge on Leptospira in bats from China. It is important to prevent potential spillover events of Leptospira between humans and bats. The positive rates of rrs2, flaB, lipL32, and secY genes were different, which may be caused by the differences of primer sensitivity of different genes and the differences in polymorphism rate of genes. The limitation of our study was that we did not culture Leptospira spp. from the bat kidney samples. The prevalence we determined might have been affected by the limitations of extraction, amplification, and sequencing of Leptospira DNA from clinical samples.
The collection of bats for microbiologic studies was approved by the Ethics Committee of Prevention Medicine of Wuhan University (2018010). This study was supported by a grant from the National Natural Science Funds of China (31570167).
LITERATURE CITED
Author notes
These authors contributed equally to this article;