Serological and molecular characteristics of pathogenic Leptospira in rodent populations in Fujian Province, China, 2018–2020

Background Leptospirosis is a significant emerging infectious disease worldwide. Rodents are considered to be the most critical hosts of Leptospira spp. Fujian Province is a region highly endemic for leptospirosis in China. However, the genetic diversity of leptospires circulating among rodents in Fujian is limited. Results The carrier status of rodents for Leptospira spp. was investigated by culture and serological detection in Fujian during 2018–2020. A total of 710 rodents, including 11 species, were trapped, with Rattus losea being the dominant trapped species (50.56%). Fourteen pathogenic Leptospira strains were obtained. Seven L. borgpetersenii serogroup Javanica strains belonging to ST143, 4 L. interrogans serogroup Icterohaemorrhagiae strains belonging to ST1 and ST17, 2 L. interrogans serogroup Bataviae strains belonging to ST96 and ST333, and 1 L. interrogans serogroup Pyrogenes strains belonging to ST332 were identified using 16S rDNA gene sequencing, microscopic agglutination test (MAT) and Multilocus sequence typing (MLST). L. borgpetersenii serogroup Javanica belonging to ST143 was the dominant type (50.00%). A total of 387 rodent serum samples were tested by MAT. Serum were considered positive for seroreactivity at a titer ≥ 1:160 against at least one serovar. A total of 90 (23.26%) serum samples tested positive, and four serogroups were identified, with Javanica being the dominant serogroup (87.78%), which was similar to the dominant serogroup isolated from rodents. This study demonstrates a high prevalence of leptospirosis in rodents and public health education among high-risk workers is highly recommended. Conclusions R. losea was the dominant trapped rodent, and L. borgpetersenii serogroup Javanica ST143 was widely distributed among rodents in Fujian from 2018 to 2020. Despite the low number of isolates obtained from rodents, this study suggests that continuous epidemiological surveillance of the aetiological characteristics of pathogenic Leptospira in wild animal reservoirs may help reduce the possible risk of disease transmission. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-022-02566-2.

Humans and animals can be infected primarily through direct or indirect exposure to urine or tissues of infected mammalian hosts and urine-contaminated soil or water [9]. Among wildlife species, rodents are considered the most critical reservoir of pathogenic Leptospira worldwide due to their close contact with humans, infected livestock and companion animals [1,10]. Therefore, longterm active surveillance and investigations of the carriage status of rodents will contribute to understanding animal-to-human transmission, investigating outbreaks, and tracking sources of leptospirosis and will be instrumental in designing better bacterin vaccines for leptospirosis. Although leptospirosis has significantly decreased over the past few years worlwide, leptospirosis is still recognized as a critical zoonosis in China. Based on an investigation from 2018 to 2020 in Fujian Province, the leptospirosis disease incidence was between 0.0381 and 0.0639 cases per 100,000. This incidence was slightly higher than the national incidence (between 0.0113 and 0.0212 cases per 100,000 during 2018-2020), showing that Fujian Province was a significant epidemic region for leptospirosis (data are available from the National Notifiable Infectious Disease Surveillance System in China with login authorization: https:// 10. 249.6. 18: 8881/ cdc/ login). The average incidence rates in some regions of Fujian Province, especially Pucheng (0.334 cases per 100,000), Luoyuan (0.778 cases per 100,000), and Wuping (0.361 cases per 100,000) city in Fujian, are markedly higher than the national incidence in China (0.0159 cases per 100,000). Unfortunately, people living in Fujian Province have a poor level of knowledge about potential risk factors for leptospirosis. Our previous study reported two predominant pathogenic Leptospira named L. interrogans serogroup Icterohaemorrhagiae and L. borgpetersenii serogroup Javanica, with sequence type (ST) 1 and ST143 being the dominant STs circulating in rodent populations in Jiangxi Province in China [11]. However, there is little information regarding the most crucial rodent hosts and transmission to humans in the highly endemic district of Fujian Province. Therefore, this study aimed to investigate the prevalence of pathogenic Leptospira in potential reservoir animals of rodents and to identify the predominant species/serogroups/genotypes circulating in rodent populations in Fujian Province. The detailed molecular and serological characteristics of Leptospira in this area will improve diagnoses and provide deeper insights into the molecular characterization, epidemiology, and prevention and control of leptospirosis.

Rodent distribution from Fujian Province
A total of 9,136 traps were placed, and 710 rodents belonging to 11 different species were successfully captured between 2018 and 2020 in Fujian Province. The species identifications of the trapped rodents and the numbers of rodents with positive cultures are listed in Table 1 Fisher's exact test revealed highly significant differences in the species distribution of trapped rodents across different collected sites (P < 0.001) (Table S1). These species represented most of the trapped rodent diversity in Fujian Province. R. losea was the most commonly trapped species in Fuqing (24/27, 88.89%), Luoyuan (76/81, 93.83%), and Wuping (217/428, 50.70%), R. flavipectus was most common in Changtai (55/100, 55.0%), N. fulvescens was most common in Jianyang (25/37, 67.57%), and A. agrarius was most common in Pucheng (11/37, 29.73%).

Results of species identification of 14 isolates based on 16S rDNA gene sequencing
Among the 14 isolates, species identification revealed that L. interrogans and L. borgpetersenii accounted for 50%, respectively ( Fig. 1 and Table S2). Fisher's exact test revealed highly significant differences in the distribution of leptospiral species prevalence across the collected sites among the 14 strains (p < 0.001) (Table S3), but no differences across rodent species (Table S4). L. interrogans was widely represented in Jianyang, Pucheng, Fuqing and Changtai, while L. borgpetersenii was only distributed in the remaining two cities of Luoyuan and Wuping (Table S3). Furthermore, L. interrogans and L. borgpetersenii were present every year between 2018 and 2020 (Table S2).
There were some significant regional differences across the six collected sites in the distribution of the dominant serogroups among the 14 strains according to Fisher's exact test (p < 0.05) (Table S5), but there were no differences across rodent species (Table S6). Icterohaemorrhagiae was the only predominant serogroup in Jianyang and Pucheng, Bataviae in Fuqing, and Javanica in Luoyuan and Wuping, and Serogroups Bataviae and Pyrogenes were identified in Changtai. In addition, serogroup Javanica was present every year between 2018 and 2020 in Wuping (Table S2).

Genetic diversity of 14 isolates from Fujian using MLST analysis
A total of 6 different STs, including 2 new STs (ST332 and ST333), were identified from 14 pathogenic Leptospira isolates in Fujian Province (Table S2). The most predominant ST was L. borgpetersenii ST143 (7/14), followed by L. interrogans ST1 (2/14) and L. interrogans ST17 (2/14). The remaining 3 isolates belonged to 3 different STs (Table S2). The UPGMA dendrogram of STs showed that the strains isolated from the same trapping sites clustered closely together except for the two strains belonging to two different STs from Changtai (Fig. 2). The dendrogram revealed that the 14 pathogenic Leptospira strains belonged to five major clades. Fisher's exact test revealed highly significant differences in the distribution of ST prevalence across collected sites/host species among the 14 strains (p < 0.01) (Table S7 and Table S8). ST143 was distributed in R. flavipectus and R. losea, ST1 was distributed in A. agrarius, ST17 was distributed in N. fulvescens and N. nivivente, and ST322 and ST323 were distributed in R. flavipectus and ST96 in R. losea. In addition, there were some significant regional variations in the distribution of the dominant STs. ST143 was the only typical ST in Luoyuan and Wuping, ST17 was identified in Jianyang, ST1 was identified in Pucheng, and ST96 was identified in Fuqing, while ST332 and ST333 were identified in Changtai (Fig. 2).

MAT of rodent sera
Three hundred twenty-three rodents had insufficient serum. Therefore, a total of 387 rodent serum samples from the trapped rodents were tested for Leptospira antibodies by MAT with a panel of 10 local serovars. A total of 90 (90/387, 23.26%) serum samples were tested positive at a MAT titer ≥ 1:160 and four leptospiral serogroups were identified (Table 2 and Table S9). The major circulating leptospiral serogroup was Javanica (79/90, 87.78%),    (Table  S9). The highest titer detected was 1:2,560 for serogroups Javanica and Pomona, followed by the titer of 1:1,280 for Javanica, Pomona, and Icterohaemorrhagiae.
The seroprevalence was significantly different across species of rodents (P < 0.05) (

Discussion
Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp. and is considered endemic in Fujian Province. The role of rodents as a source of Leptospira infection has long been suggested, particularly as rodents are ubiquitous worldwide [1,12]. However, systematic studies on the primary vector, rodents, and the serological and molecular characteristics of Leptospira circulating among rodent populations in Fujian have been limited. In this study, we provided the systematic distribution of rodents, the carrier status of Leptospira spp. in rodents by culturing and serological detection, and the molecular characteristics of pathogenic Leptospira circulating in potential reservoirs of rodent populations in Fujian Province during 2018-2020, which plays an important role in the epidemiology of leptospirosis.
Four serogroups of Javanica, Icterohaemorrhagiae, Pyrogenes, and Bataviae were identified in these 14 strains isolated from rodents in Fujian, with serogroup Javanica being the most prevalent (7/14, 50.00%). In addition, the serological analysis showed that the seroprevalence was 23.26%, and the four serogroups Javanica, Pomona, Australis, and Icterohaemorrhagiae were found in serum samples in rodent populations by MAT. Among these serogroups, Javanica was the major circulating leptospiral serogroup (79/90, 87.78%). Therefore, Javanica was the most prevalent serogroup in Fujian, which was similar to our previous report in Jiangxi [11]. The lower diversity of leptospirosis serogroups/STs in Fujian may be due to a low number of isolates obtained from the rodent population. In this study, the two most abundant species, L. interrogan, and L. borgpetersenii, were identified in Fujian, which is consistent with a previous epidemiological investigation of leptospirosis in humans and potential small animal reservoirs in Jiangxi and worldwide [11]. It was reported that four species, L. borgpetersenii, L. interrogans, L. kirschneri and L. weilli, were detected in rodents and human isolates in Thailand, Lao PDR, and Cambodia, with L. interrogans and L. borgpetersenii widely distributed among rodent populations [13]. It was reported that L. interrogans, L. kirschneri and L. borgpetersenii were the most widespread and prevalent species detected in Russia [14]. Together, these data revealed that the major Leptospira species from these neighbouring countries were similar.
In this study, 6 different STs were obtained from 14 pathogenic Leptospira isolates in Fujian Province, with ST143, ST1, and ST17 being the most prevalent STs (Table S2). It was reported that the five most predominant STs ( ST1, ST143, ST105, ST37, and ST17) were identified as long-term and ubiquitous virulent strains throughout Jiangxi Province [11]. ST143, as the most prevalent ST in Fujian and Jiangxi, was also identified in Malaysia [15]. ST17 was also identified in 90 strains of serogroup Icterohaemorrhagiae in Sao Paulo [16]. Five common STs (ST37, ST17, ST199, ST110, and ST146) were reported to have a long-term and ubiquitous distribution in Russia [14]. ST17 was isolated from rodents, pigs, dogs, and humans in Argentina and China [17][18][19]. ST143 was dominant in rodents in Malaysia and China [18,20]. Therefore, these prevalent STs (ST17 and ST143) reported in Fujian and Jiangxi in China were also the same prevalent STs in the rest of the world. Generally, there is a complex phylogenetic structure and different distribution of STs of Leptospira worldwide. These predominant isolates are likely to have adaptive selective advantages in the environment or potential reservoirs, allowing them to circulate worldwide. These results highlight the importance of understanding the epidemiology and ecology of Leptospira worldwide. Further studies are needed to explore the molecular mechanisms that allow them to develop into more prevalent pathogenic strains.
In this study, the density of field rodents was measured to be between 4.81-16.77% at different sampling sites in Fujian Province, which is similar to the rates in Jiangxi Province in China (4.76-12.56%) determined by our previous surveys [11]. However, the most abundantly trapped species differed in these two provinces, with R. losea, N. fulvescens and R. flavipectus dominating in Fujian and A. agrarius and R. losea being most common in Jiangxi. The overall carriage rate (14/710, 1.97%) in rodents detected in this investigation based on culture isolation in Fujian was much lower than previous studies based on isolation in other countries such as Malaysia (6.7%) [21], Barbados(16% to 19%) [22], and Brazil (80.3%) [23]. It is important to note that different methods were performed for trapping and determining the presence of leptospires in other labs. The carriage rates of the 6 different species of rodents in Fujian were between 0.71-18.18%. The highest carriage rates based on culture isolation were those of A. agrarius (2/11, 18.18%) in Fujian, which is consistent with previous studies showing that A. agrarius was the major small animal reservoir in Jiangxi and Guizhou Provinces in China [11,24].
The infection rates of Leptospira in rodents vary in different areas around the world. In this study, the seropositivity of pathogen leptospiral renal carriage in trapped rodents using MAT was 23.26%. It was reported that 8% of 266 rodents were seropositive, primarily for the serovars Australis and Grippotyphosa, in southern Germany [8]. It was reported that 27.1% of R. norvegicus were seropositive and that 70% of the positive serum samples reacted with serogroup Bataviae in northern Vietnam, Hanoi, and Bac Giang [25]. It was reported that 68.1% of the 142 captured R. norvegicus were seropositive with a MAT titer of ≥ 1:100 in Brazil [23]. The seroprevalence in rats was 7.1% in Andaman Islands [26]. Leptospirosis infection is commonly associated with exposure to contaminated environmental sources, such as those caused during flooding events or high rainfall seasons, by working in rice, pineapple, banana or cane fields, or by exposure to river water during recreational activities or adventure tourism [1,27], including caving [28], canoeing, rafting, kayaking [29,30] and triathlons [31]. Our study found that rodents had a relatively high degree of infection with Leptospira in Fujian, indicating a potential risk of rodent-borne leptospirosis in this region. Fujian Province is a significant epidemic region for leptospirosis, and rice, banana, cane and tea are the leading local crops. Additionally, Changtai is famous for rafting and kayaking. Therefore, agricultural workers, water sportsmen, and travellers with possible exposures in this epidemic region are at high risk for leptospirosis in Fujian. Leptospirosis ranges in severity from a mild, self-limited febrile illness to a life-threatening illness including renal failure, hypotension, haemorrhage and respiratory failure [27]. The signs and symptoms of leptospirosis are usually mistaken for other causes of an acute febrile syndrome, such as influenza [27]. Unfortunately, people living in Fujian Province have a poor level of knowledge about potential risk factors for leptospirosis. Therefore, increased self-protection awareness and appropriate personal protective measures such as rubber boots, gloves, protective eyewear, or prophylaxis with doxycycline are needed to prevent Leptospira infections in Fujian.
In this study, the leptospiral diversity of STs was significantly different across the different species of captured rodents in Fujian. ST143 was distributed in R. flavipectus and R. losea, and ST1 was distributed in A. agrarius, which was consistent with our previous epidemiological investigation of leptospirosis in Jiangxi [18]. In addition, the leptospiral diversity of species/ serogroups/STs was significantly different across the six other regions. The leptospiral diversity may be due to the difference in special geographic areas. In this study, Jianyang and Pucheng are located in the northern mountain region. Wuping and Changtai were hills and plain sites, and Luoyuan and Fuqing were eastern coastal areas. The differences in ecological factors, such as altitude, geomorphology, climate among these areas, and various local animal host species may influence leptospiral diversity.
This study revealed the prevalence rates and genetically diverse pathogenic Leptospira circulating among rodent populations in Fujian Province, China, suggesting that rodents could be a critical Leptospira source. Based on the high infection rate present in this population, these findings highlight the risk of proximity of the infection reservoirs to humans. Furthermore, the various distributions of genotypes of pathogenic Leptospira may provide a clue regarding species-specific antigens for vaccine design in different epidemic regions. Therefore, active and long-term monitoring of infections in wild rodents and constructing a suitable database in epidemiological tracing analysis in outbreaks of leptospirosis are necessary. These results may be useful in developing guidelines for the accurate and rapid diagnosis, prevention, and control of leptospirosis in Fujian Province.

Conclusions
This study detailed a systematic epidemiological investigation, including the molecular characteristics and seroprevalence of pathogenic Leptospira in potential reservoirs of rodent populations in Fujian Province during 2018-2020. Two pathogenic species, four serogroups and 6 STs were identified in these 14 isolates by 16S rDNA gene sequencing, MAT, and MLST analysis. Significant geographic variations in the distribution of dominant serogroups, species and STs were found among the Leptospira isolates in Fujian. The serological analysis revealed a relatively high seroprevalence rate of leptospirosis among rodent populations in Fujian, indicating a potential risk of rodent-borne leptospirosis in this region. Thus, our present study provides a blueprint for further disease prevention and control.

Rodent collection from Fujian Province
This study and research protocols were approved by the Ethical Committee of Fujian Center for Disease Control and Prevention, China (No: FJCDCNT1811-2015). The trapping, handling, and euthanasia of wild rodents in this study were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition). Furthermore, this study was carried out in compliance with the ARRIVE guidelines.
Six different trapping sites representing different geographical locations, including Jianyang (27. Rodents were trapped using the trap-night method from April to October in 2018 through 2020 within the framework of the CERoPath project (www. cerop ath. org) [32]. Trap placement was based on indicators of rodent activity such as rice field environments, open sewers, tracks, or faeces. Within each sample site, rodents were trapped over an area of approximately 10 square kilometres. Trapping was conducted with peanut bait on the first afternoon and collected in the following early morning during a period of five days and four nights. At each trapping site, 10 trapping lines, including 10 locally hand-made wire traps (approximately 40 × 12 × 12 cm), were placed, with at least five metres of spacing between traps [32]. First, the gender, genus and species of the trapped rodents were identified based on phenotypic characteristics (body, ears, tail, fur colour, and sex) [33]. The rodent density was calculated using the formula: (Number of rodents trapped in each region/Number of total traps successfully placed for each region * 100).

Leptospiral isolation from Fujian Province and DNA extraction from Leptospira isolates
After transportation to the laboratory, the rodents were euthanized by isoflurane inhalation, and kidney tissue was collected following the Guide for the Care and Use of Laboratory Animals (8th edition). For the isolation of leptospires, approximately 1 g of fresh kidney tissue samples from rodents were aseptically inoculated into 10 ml of liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Difco Laboratories, USA) with 5-fluorouracil (Merck, Germany) at 28 °C in the laboratory. Within 48 h, 1 mL of the initial tube was transferred into a second EMJH culture bottle at a dilution of approximately 1:100 to reduce the inhibitory effects of anaerobic conditions created by autolysis of the tissues. The cultures were observed weekly using dark-field microscopy to determine the presence of Leptospira for up to 3 months. Positive cultures were immediately subcultured in fresh liquid EMJH medium. Samples with no growth of Leptospira after 3 months were considered negative [34]. The isolates were stored long-term at − 70 °C and were passaged every six months. When needed, the isolated colonies were subcultured at 28 °C in EMJH liquid medium for 7 days and DNA was extracted from the cultures of Leptospira isolates using Wizard Genomic DNA Purification Kit (Promega, Madison, USA) according to the manufacturer's directions.

Species identification of pathogenic Leptospira strains isolated in Fujian Province
Species identification of the isolates was conducted based on 16S rDNA gene sequencing with lengths ranging from 1,422 to 1,432 bp for leptospires as previously described by Morey [35]. A total of 68 reference sequences representing the major reported pathogenic, intermediate and saprophytic Leptospira species worldwide were obtained from the GenBank database (Table S12) [35][36][37][38]. The 16S rDNA gene sequences of Turneriella parva NCTC 11395 T and Leptonema illini NCTC 11301 T were set as the outgroup (Table S12) [35]. The 16S rDNA gene sequences from leptospiral isolates from Fujian were compared with the 68 reference sequences, and phylogenetic analysis was performed using Clustal W. A neighbor-joining tree was constructed using Mega software version 6.00 with a bootstrap value of 1,000 replicates.

Serogroup identification of leptospiral strains isolated from Fujian Province
MAT was performed for serogroup identification of the leptospiral isolates by a panel of 15 Chinese prevalent serogroup-specific rabbit antisera representing 15 serogroups provided by the National Institutes of Food and Drug Control, China (Table S13). All reference rabbit antisera were serially diluted with a starting dilution of 1: 50 and up to a dilution of 1: 6, 400 in sterile saline using twofold dilutions and tested with leptospiral isolate suspensions. MAT endpoint titers were determined until the last well showing 50% agglutination in free-moving leptospires was observed. The serogroup scoring the highest MAT titer was defined as the corresponding serogroup.

MLST analysis of isolated leptospiral strains
MLST analysis of the isolates was conducted using seven housekeeping genes (glmU, pntA, sucA, tpiA, pfkB, mreA and caiB) as previously described [39]. The available PubMLST Leptospira database (http:// pubml st. org/ lepto spira/) was used for assigning alleles and STs. Phylogenetic analysis was conducted using the unweighted pair group method with average linkages provided in BioNumerics software version 5.10.

Serological antibody detection in rodents using MAT.
Serum samples from the trapped rodents were collected, and MAT analyses were performed for Leptospira-specific antibodies with a panel of 10 live pathogenic Leptospira antigens representing 10 different local serogroups (Icterohaemorrhagiae, Javanica, Autumnalis, Australis, Pomona, Hebdomadis, Grippotyphosa, Pyrogenes, Bataviae and Sejroe) as previously described [40]. All sera were serially diluted with a starting dilution of 1: 10 and reaching a screening dilution of 1: 80 in sterile saline using twofold dilutions and tested against antigen suspensions to establish the antibody titer. Following incubation at 30 °C for 2 h, each suspension was observed by a dark field microscope (Nikon, Eclipse 50i) at 400X magnification. The sera with positive screening results (1:160) were further diluted to determine the endpoint titer for each sample. The MAT endpoint titer was defined as the highest dilution showing ≥ 50% agglutination in free-moving leptospires [41] compared to a negative control obtained by using sterile saline instead of serum. Sera were considered positive for seroreactivity at a MAT titer ≥ 1:160 against at least one serovar [42].

Statistical analysis
Fisher's exact test was performed to compare the differences in the distribution of dominant Leptospira across species, serogroups and STs between rodent species and six different collected sites. In addition, the statistical chi-square test was used to determine if there were differences in Leptospira positivity rates between different collected sites and rodent species. The p value was computed by Monte Carlo simulation. All statistical analyses were conducted using R software (R version 3.5.1, https:// www.r-proje ct. org/) [43], considering a significance level of 0.05.
Additional file 1: Table S1. Species distribution of the captured rodents among the six different regions in Fujian. (XLS). Table S2. Fourteen pathogenic Leptospira strains isolated from Fujian Province were used in this study. (XLS). Table S3. Species distribution of the 14 pathogenic Leptospira strains among the six different regions in Fujian. (XLS). Table S4. Species distribution of the 14 pathogenic Leptospira strains among the different species of captured rodents in Fujian. (XLS). Table S5. Serogroup distribution of the 14 pathogenic Leptospira strains among the six different regions in Fujian. (XLS).