Pathogenic Leptospira Infections in Hubei Province, Central China

Leptospirosis is an important zoonosis that is caused by pathogenic Leptospira, which is considered to be a re-emerging infectious disease in many countries. Rodents are the most important reservoirs for both human and animal infection. An epidemiological survey of pathogenic Leptospira in rodents is important for the prevention and control of leptospirosis. In this study, a total of 964 rodents were captured from six cities in Hubei Province, and two pathogenic Leptospira species (L. interrogans and L. borgpetersenii) were detected using nested PCR with an overall prevalence of 4.8%. L. interrogans was distributed in five sampling sites, which may be the dominant species of pathogenic Leptospira in Hubei Province. In addition, Rattus norvegicus showed a relatively high infection rate, which may play an important role in the transmission and infection of pathogenic Leptospira. This study reveals the prevalence of pathogenic Leptospira in wild rodents in Hubei Province, suggesting that the risk of leptospirosis infection in Hubei Province still exists.


Introduction
Leptospirosis caused by the pathogenic Leptospira spp. is one of the most significant zoonotic diseases, mainly distributed in tropical and subtropical countries [1,2]. It is estimated that there are 1.0 million cases and 58,900 deaths due to leptospirosis worldwide every year [3]. The genus Leptospira can be classified into pathogenic, saprophytes and intermediate species according to their virulence status [2,4]. Among the pathogenic group, L. interrogans, L. borgpetersenii and L. kirschneri are the main pathogenic bacteria responsible for human leptospirosis worldwide [5][6][7].
Leptospira is excreted from the kidneys of host animals into urine and then contaminates soils and rivers. Human infection may result from direct contact with the urine of infected animal hosts or the ingestion of contaminated water [8,9]. At least 200 species of animals have been confirmed as natural carriers of pathogenic Leptospira, especially rodents, which are considered the main reservoir for Leptospira infection in humans [9][10][11][12]. Therefore, the surveillance and investigation of pathogenic Leptospira in wild rodents will contribute to understanding animal-to-human transmission and tracking for leptospirosis.
Leptospirosis, a category B notifiable infectious disease in China, was first reported in 1934 and has been a mandatory notifiable disease since 1955 [13]. To date, more than 2.5 million cases and over 20,000 deaths have been reported in China [14]. Hubei Province is known as the "land of a thousand lakes", and most areas possess a subtropical humid monsoon climate [15,16]. Abundant rainfall, rice cultivation and high density of rodents provide a favorable environment for the survival and transmission of Leptospira. According to incidence of leptospirosis, China can be divided into four regions [12]. Hubei belongs to region B (the temperate region located in the middle and lower areas of the Yangtze River) with the highest human leptospirosis incidence [12]. Over the past few decades, China has dramatically reduced the incidence of human leptospirosis through a series of measures [17]. However, some studies have shown that there are persistent high risk leptospirosis clusters along the Yangtze River basin, suggesting that the risk of leptospirosis infection in Hubei Province should not be ignored [18]. According to the data available from the official departments in mainland China, the total number of confirmed leptospirosis cases was 403 in 2021 in China (http://www.nhc.gov.cn/jkj/s3578/202204/4fd88a291d914 abf8f7a91f6333567e1.shtml, accessed on 5 November 2022), while from 2007 to 2018, the total incidence in Hubei Province was 0.4022 cases per 100,000 people, which was at a high level [19]. Therefore, it is of great significance to monitor and investigate the prevalence of pathogenic Leptospira in rodents in Hubei Province for the prevention and control of human leptospirosis.

Rodent Collection and Sample Processing
Rodents were captured with cage traps loaded with food bait in Wuhan, Shiyan, Huangshi, Jingzhou, Xiangyang and Xianning cities in Hubei Province in 2021 ( Figure 1). The topography of Hubei is generally high in the west and low in the east. Shiyan is located in the northwestern Hubei mountain region with an average altitude of 736.9 m, whereas Xiangyang, Jingzhou, Wuhan, Xianning and Huangshi are in the hilly plain area, with an average altitude of 347.6, 42.7, 37.1, 189.6 and 109.4 m, respectively. Rodent species were identified using morphological examination and further confirmed using mitochondrial cyt-b gene sequence analysis. All rodents were captured alive and euthanized based on humanitarian principles. The kidney tissue samples were collected and stored at −80 • C until further use.

Screening and Molecular Characterization of Pathogenic Leptospira
Total DNA was extracted from kidney tissue samples according to the instructions of the DNA extraction kit (Omega, Doraville, CA, USA). All samples were screened for the presence of Leptospira using nested PCR, and the primers targeting the conserve region of 16S rRNA (rrs) gene in pathogenic Leptospira species were selected [20]. Furthermore, the partial LipL32 and secY genes, which are widely used in phylogenetic analyses [21][22][23], were also recovered from the positive samples to better identify and characterize different species in positive samples. The PCR products with expected size were sent for sequencing (Wuhan Gene Create Biological Engineering Co., Ltd., China). To prevent contamination, dedicated pipets and filtered tips were used, and each operation, including PCR mixture preparation, template addition and agarose gel electrophoresis were performed in separate rooms. The primer sequences used in this study are listed in Table S1.

Genetic and Phylogenetic Analysis
All the nucleotide sequences were assembled and edited using SeqMan program (DNASTAR, Madison, WI, USA) and then compared with reference sequences using basic local alignment search tool (BLAST, https://blast.ncbi.nlm.nih.gov, accessed on 15 July 2022). The sequences obtained in this study and the reference sequences downloaded from Gen-Bank were aligned and calibrated using Clustal W in MEGA7. The nucleotide (nt) sequence identities were calculated using the MegAlign program available within the Lasergene software package (version 7.1). The maximum likelihood method (ML) and general time reversible (GTR) model was used to reconstruct the phylogenetic trees with bootstrap support values calculated from 1000 replicates implemented in MEGA7. All the sequences obtained in this study have been submitted to GenBank under the accession numbers OP860838-OP860883 (rrs), OP874962-OP875007 (secY) and OP875008-OP875053 (LipL32).

Statistical Analysis
The statistical analysis was performed using the Statistical Package for Social Sciences Version 27.0 software (SPSS, Chicago, IL, USA). Fisher exact test was performed to determine the differences in Leptospira positivity rates between different collected locations and rodent species [24,25], and significance was defined at a p value of 0.05. The 95% confidence interval was calculated using the Epitools (https://epitools.ausvet.com.au/trueprevalence, accessed on 26 October 2022).

Detection of Pathogenic Leptospira
A total of 964 rodents belonging to 6 different species were captured from six cities in Hubei Province in 2021 (Figure 1), including 389 Rattus norvegicus, 192 Apodemus agrarius, 107 Mus musculus, 270 Rattus tanezumi, 5 Niviventer confucianus and 1 Micromys minutus (Table 1). Nested PCR targeting a conserve region of 16S rRNA (rrs) gene was performed to detect pathogenic Leptospira. We found 46 Leptospira-positive rodents, including 40 Rattus norvegicus and 6 Apodemus agrarius, with an overall positive rate of 4.8% (95% confidence interval: 3.6-6.3%).  R. norvegicus showed a significantly highest infection rate of 10.3% among all of the rodent species. Geographically, Leptospira detected in this study showed variable prevalence in the six cities, ranging from 0 to 20%. Fisher's exact test revealed highly significant differences in the distribution of leptospiral prevalence across the rodent species and collected locations among these pathogenic Leptospira (p < 0.001). In addition, Leptospira was detected in R. norvegicus in five of the six sampling areas, with positive rates ranging from 5.7 to 25.4%, and the infection rates of R. norvegicus collected in Xianning city were the highest.

Molecular Characterization of Pathogenic Leptospira
The LipL32 and secY genes of pathogenic Leptospira were amplified from all positive samples to better characterize the species of the detected pathogenic Leptospira strains. BLAST analysis of the rrs gene sequences showed that the Leptospira detected in our study were identified as L. interrogans (n = 44) and L. borgpetersenii (n = 2).
The ML phylogenetic tree constructed based on the rrs (1057 bp), LipL32 (587 bp) and secY (833 bp) gene sequences showed a similar topology; all the 46 Leptospira strains detected in this study belonged to the pathogenic group and clustered into two clades, L. interrogans and L. borgpetersenii (Figure 2).

Geography and Host Analysis of Pathogenic Leptospira
The Fisher's exact test revealed significant differences in the distribution of leptospiral species prevalence across the collected sites (p = 0.032) and rodent species (p = 0.014) among the 46 strains (Table 2). L. interrogans was widely distributed in Wuhan, Xianning, Jingzhou, Xiangyang and Huangshi. Moreover, L. interrogans was detected in both R. norvegicus and A. agrarius, whereas L. borgpetersenii was found only in A. agrarius.  In this study, R. norvegicus showed a high positive rate in the captured rodents, and L. interrogans showed a high infection rate and a wider prevalence among the two Leptospira species, indicating that R. norvegicus may be the main carrier of pathogenic Leptospira, and L. interrogans was the dominant species, followed by L. borgpetersenii.

Discussion
Leptospirosis is an important but neglected zoonotic disease with insufficient research attention in relation to burden, and the incidence rates are significantly underestimated due to a lack of epidemiological work, insufficiently rapid diagnostics and misdiagnosis [26,27]. It has caused serious public health problems worldwide, especially in East and Southeast Asian countries such as China, South Korea and Vietnam [12,28,29]. In recent decades, China has effectively reduced the incidence and mortality of leptospirosis nationwide which is attributed to public education, vaccination, rural environmental improvements and better sanitation [30,31]. Although the incidence of leptospirosis has significantly decreased, local outbreaks are still frequently reported in certain areas [31,32]. Rodents are considered to be the main carriers of Leptospira and are also an important contributor to leptospirosis in humans [33,34]. Hubei Province is one of the main endemic areas of leptospirosis [12]; however, studies on the epidemiology of pathogenic Leptospira in rodents in Hubei Province are very limited.
In this study, 46 pathogenic Leptospira strains were detected, including 44 L. interrogans and 2 L. borgpetersenii with positive rates of 4.6 and 0.2%, respectively. It is worth noting that these two Leptospira strains are the main causes of leptospirosis in China, of which L. interrogans has caused at least 60% of the human cases of leptospirosis in China historically [12,14]. The high prevalence of L. interrogans has also been reported in previous studies [20,24,25,35]. All these suggest that L. interrogans may be the main pathogenic Leptospira circulating in nature.
Studies have shown that the prevalence of Leptospira varies in different geographical ecological environments [24,36]. In our study, the prevalence rate of rodents carrying Leptospira is high in hilly and plain areas, such as Wuhan, Jingzhou, Xianning, etc., whereas no rodent was found to be infected with Leptospira in Shiyan, located in a mountain area. This observation is consistent with a previous study, which found that all Leptospirapositive rodents were detected in low-altitude locations, whereas none of the rodents in higher locations hosted Leptospira [23]. Rodents may migrate between habitats, and these movements may involve rodents infected with Leptospira [36]. The differences in topography and altitude in these areas in this study may affect this migration and thus the distribution of Leptospira.
Due to their abundance and close association with human habitats, rodents play an important role in the transmission of Leptospira. Among them, Rattus norvegicus is the main source of Leptospira infection in humans [10,37,38]. Previous studies have reported that R. norvegicus is the main host of L. interrogans in urban areas [39]. It was also found that R. norvegicus accounted for 38.1% of Leptospira infected animals, although it accounted for only 15.6% of the captured animals [40]. In our study, most of the Leptospira detected in rodents captured in Hubei Province were also from R. norvegicus. All these indicated that R. norvegicus was the main species involved in Leptospira transmission.
It is widely accepted that the presence of rivers near human settlements increases the infection risk of Leptospoira, and flooding has also been linked to outbreaks of leptospirosis [41][42][43][44]. In this study, we sampled rodents in Hubei Province and found the prevalence of two pathogenic Leptospira species in wild rodents, with L. interrogans as the dominant species and R. norvegicus as the main host. The prevalence of Leptospira varied in Wuhan, Huangshi, Jingzhou, Xiangyang and Xianning, which may be related to the region, representativeness and density of the samples we captured. Although based on limited rodent samples, these results reveal that genetically diversified pathogenic Leptospira are disseminating among wild rodents in Hubei Province and indicate a potential risk of rodent-derived leptospirosis in Hubei Province.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11010099/s1, Table S1: Primers used in this study. Reference [20] is cited in the supplementary materials.

Institutional Review Board Statement:
Our study belongs to the National Natural Science Foundation project of China: Pathogenic and epidemiological study of important pathogens carried by rats and bats in Hubei Province based on reverse etiology (Project identification code: U20A20396). On 15 May 2020, it was approved by the Ethics Committee of Wuhan University to strictly implement the relevant rules on "ethics and biosafety".