Detection of P1 and P2 Subclades of Leptospira carriage in swine in Sri Lanka

Abstract

Background

Leptospirosis, caused by pathogenic Leptospira spp., is an important zoonotic disease that is found globally, with recent outbreaks posing a major public health challenge. This disease affects a wide range of mammalian species. While rodents, dogs, cattle, and buffaloes are recognized reservoirs of Leptospira, the role of swine in transmission in Sri Lanka remains unclear. This study aimed to investigate the carrier status of Leptospira spp. in swine raised for human consumption in Sri Lanka, and to assess potential zoonotic risks.

Results

Analysis of livestock data revealed the highest density of pigs in the Gampaha, Colombo, and Puttalam districts. Kidney (n = 181) and blood (n = 25) samples were collected from swine slaughtered for human consumption in the Colombo and Kegalle districts. Leptospiral DNA was detected in 25 of the kidney samples (13.8%), identifying Leptospira interrogans (n = 21), P1 (pathogenic) subclade, and L. licerasiae (n = 4), P2 (intermediate) subclade. Serological analysis revealed seropositivity to the serogroups Autumnalis, Icterohaemorrhagiae, Canicola, and Sejroe.

Conclusions

This study demonstrates that swine reared for human consumption in Sri Lanka serve as an important reservoir of Leptospira spp. All pigs sampled were clinically healthy at ante-mortem inspection, confirming their role as asymptomatic carriers of Leptospira spp. The detection of strains identical to those previously reported in human infections suggests a potential zoonotic threat exacerbated by inadequate farm hygiene and environmental contamination. These findings highlight the need for comprehensive research from a One Health perspective to gain a deeper understanding of the epidemiology of leptospirosis in Sri Lanka.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-05171-3.

Introduction

Leptospirosis, caused by pathogenic spirochetes of the genus Leptospira, is a zoonotic disease that has serious consequences for human and animal health [1]. Approximately 1.03 million cases of human leptospirosis and 58,900 deaths attributed to leptospirosis are estimated to occur annually worldwide [2]. Human leptospirosis was first reported in Sri Lanka in 1953, and since then, cases have been reported in almost all regions of the country [3]. Furthermore, leptospirosis outbreaks have been frequent throughout the year in the last decade [4]. In 2020, the country experienced an unprecedented leptospirosis outbreak characterized by 8,579 confirmed cases and more than 800 estimated deaths [5]. In addition, the Ministry of Health reported 6946 and 7001 suspected human leptospirosis cases islandwide in 2021 and 2022, respectively [6].

Rodents are the primary reservoir animals for Leptospira spp., but domestic animals such as cattle, buffalo, pigs, and dogs have also been reported to act as reservoirs [7–15]. Leptospira spp. colonize the proximal convoluted tubules of the kidneys of these reservoir hosts, which excrete leptospires in the urine [16, 17]. The shedding of leptospires in the urine can persist for prolonged periods, varying by animal species [1, 8]. The transmission of leptospirosis to humans occurs through direct contact with the urine of reservoir animals or exposure to contaminated soil and water [18].

Swine play an important role in the transmission of Leptospira spp. and serve as a common reservoir for the disease [19, 20]. Various studies worldwide have identified swine as maintenance hosts for several Leptospira serovars, including Pomona, Bratislava, and Tarassovi [21–23]. Molecular analyses of swine kidney samples have confirmed the persistent shedding of leptospires [24, 25]. In addition, a study on feral swine populations in Alabama, USA, demonstrated the prevalence of Leptospira spp., highlighting the zoonotic risk [26]. The clinical manifestations of swine leptospirosis are nonspecific and include fever, lethargy, anorexia, and jaundice [27]. In addition to the usual routes of transmission through damaged skin or mucous membranes, genital exposure represents another important route of transmission in swine. Infected boars can transmit pathogenic leptospires to clean gilts or sows during mating, whereas infected sows can carry Leptospira spp. in the uterus, leading to reproductive failures such as low fecundity, abortion, still birth, birth of runt piglets, agalactia, increased weaning-to-oestrus interval, and so-called SMEDI syndrome (Stillbirth; Mummification/Maceration; Embryo Death; Infertility) [28–30]. In addition, infected sows can transmit bacteria to healthy males during mating, further perpetuating the cycle [29, 31, 32].

In Sri Lanka, small peridomestic rodents and domestic animals such as dogs, captive elephants, cattle, and buffaloes have been identified as carriers of leptospires in several studies [7, 10, 33–36]. However, there is only a single report dating back to 1971 which described leptospirosis infection in swine population in Sri Lanka [37]. Consequently, the current status of swine leptospirosis in Sri Lanka remains unknown. Therefore, this study aimed to determine the carrier status of Leptospira spp. in domestic swine reared for human consumption in Sri Lanka, and to assess the potential zoonotic risks associated with swine leptospirosis. We compared the frequency of Leptospira infection in pigs from Colombo, a high swine-density area, and Kegalle, a low swine-density area (Supplementary Table 1). This comparison was conducted by detecting leptospiral DNA from swine kidney samples using PCR followed by sequencing, as well as the detection of anti-leptospiral antibodies using the microscopic agglutination test (MAT).

Results

An analysis of livestock data collected from the Department of Census and Statistics revealed that 100,250 pigs were distributed on 6,343 farms island-wide in 2022. The highest density of pigs was found in the Gampaha district, followed by farms in the Colombo and Puttalam districts (Supplementary Table 1).

In the initial screening, 156 kidney tissue samples were collected from 19 farms across both districts: 80 from 14 farms in Colombo and 76 from 5 farms in the Kegalle district. Pathogenic leptospiral DNA was detected in 17 samples (10.9%) by the initial screening. Of the seventeen, 13 (76.5%) belonged to the P1 subclade, and four (23.5%) belonged to the P2 subclade (Table 1). Among the 17 PCR-positive cases, ten were from a single farm in Colombo, and the remaining seven were from a farm in Kegalle. In the follow-up sampling conducted after a 2-month interval, a total of 25 kidney tissue and blood samples were collected from the two farms positive for initial screening: 13 from the farm in Colombo and 12 from the farm in Kegalle. Eight of the 13 follow-up samples from Colombo were positive for the P1 subclade, whereas none of the Kegalle farm samples were positive (Table 1).

Table 1.

Detection of Leptospira DNA from swine kidney samples by flaB-nested PCR

District	No. of samples collected			No. of PCR positive (%)				Leptospira species (no.)
	1st	2nd	Total	1 st screening		2nd screening
				P1 subclade	P2 subclade	P1 subclade	P2 subclade
Colombo	80	13	93	10 (12.5%)	0	8 (61.5%)	0	L. interrogans (18)
Kegalle	76	12	88	3 (3.9%)	4 (5.2%)	0	0	L. licerasiae (4) L. interrogans (3)
Total	156	25	181	13 (8.3%)	4 (2.5%)	8 (32.0%)	0	L. interrogans (21) L. licerasiae (4)

DNA sequencing of the amplicons revealed that all the P1 subclade PCR-positives were L. interrogans, whereas all the P2 subclade PCR-positive species were L. licerasiae, showing identical sequences (Fig. 1). The 18 L. interrogans sequences from Colombo were identical, and the three L. interrogans sequences from Kegalle were also identical, but the Colombo and Kegalle sequences were different. All 18 identical L. interrogans sequences were detected in pigs from the same farm in Colombo during both the initial and follow-up sampling rounds conducted two months apart, indicating persistence of infection within this farm. The L. interrogans sequence from Kegalle was identical to those from several previous Sri Lankan human isolates [38], while the L. licerasiae sequences were identical to sequences previously detected in a human and a dog clinical sample from Sri Lanka [7, 39] (Fig. 1).

Fig. 1.

Phylogenetic tree constructed using the neighbor-joining method based on partial Leptospira flaB gene sequences. Sequences identified in this study are shown in boldface with accession numbers. Reference sequences used for alignment were retrieved from GenBank (see Supplementary Table 4 for details of accession numbers, host species, and references). *Swine herd represents 18 identical sequences detected from the farm in Colombo (LC830882)

Out of 25 swine serum samples tested by Microscopic Agglutination Test (MAT), 18 (72%) were seropositive at the ≥ 1:100 cut-off (Table 2). These seropositive animals included ten pigs from the Colombo district and eight from the Kegalle district. Antibodies were detected against four serogroups: Autumnalis, Icterohaemorrhagiae, Canicola, and Sejroe. Among the 18 seropositive pigs, six were also PCR-positive for the P1 subclade, whereas the remaining 12 had antibodies without detectable renal Leptospira colonization. This may reflect a past cleared infection or a transient leptospiremia.

Table 2.

Distribution of MAT titers among swine serum samples (n = 25)

Serogroups	Serovar	Strain	No. of Positive serum samples
			1:100		1:200		1:400		Total Positive samples (≥ 1:100)
			Colombo	Kegalle	Colombo	Kegalle	Colombo	Kegalle
Autumnalis	Autumnalis	Akiyami A			2	1	2	3	8
Icterohaemorrhagiae	Icterohaemorrhagiae	Ictero No. 1			1		2	1	4
Canicola	Canicola	Hond Utrecht IV				1	2		3
Sejroe	Hardjo	Hardjoprajitno	1			1		1	3

Cross-reactivity was observed in several samples. All five sera with Autumnalis titers of 1:400 also cross-reacted with Hardjo (1:100), and four of these additionally reacted with Canicola (1:100). Two of the three sera positive for Autumnalis at 1:200 also cross-reacted with Hardjo at 1:100. These results suggest Autumnalis as the predominant reacting serogroup, although interpretation should take into account cross-reactivity, particularly with Hardjo and Canicola.

Discussion

This study revealed the renal colonization of pathogenic Leptospira spp. in pigs reared for human consumption in the Colombo and Kegalle districts of Sri Lanka, highlighting the risk of the dissemination of zoonotic pathogens to other livestock species, livestock farmers, and the environment. Indeed, the partial flaB sequences obtained in this study were identical to those identified in human isolates or canine clinical samples (Fig. 1, Supplementary Table 3). In addition, the detection of identical L. interrogans sequences from the same Colombo farm at two time points, two months apart, strongly suggests persistent contamination and possible pig-to-pig transmission on the farm. This finding highlights the potential for long-term maintenance of leptospiral infection in swine populations. It is noteworthy that all pigs included in this study were clinically healthy at ante-mortem inspection, yet several were carriers of pathogenic and intermediate Leptospira spp. This finding highlights the asymptomatic carrier status of swine and the associated risk of transmission to humans.

In this study, L. interrogans (P1) was detected in kidney samples, which is consistent with the global trends observed in swine populations. In 2015, Miraglia et al. reported the isolation of L. interrogans serovar Pomona from Brazilian swine. Similarly, in 2017, another study detected congruent L. interrogans serogroup Australis in Brazil, highlighting the widespread prevalence of this species [40, 41]. In addition, this pathogenic strain was also identified in porcine kidney samples as part of an extensive investigation in the USA [24]. In the context of Sri Lanka, it is interesting to note that L. interrogans has been detected in a variety of animals, including dogs, captive elephants, and livestock such as cattle and buffalo [7, 10, 33, 35].

L. licerasiae was also identified in four swine kidney samples in the present study. This species was first identified in Peruvian Amazon residents [42]. In addition, L. licerasiae was detected in a traveler returning to Japan from Brazil in 2017 [43]. The detection of L. licerasiae in swine has recently been reported by Steinrigl et al. [44]. Our findings further support the role of swine as potential hosts of L. licerasiae, underscoring the need for continued surveillance [44]. A closely related intermediate pathogenic species (P2), L. venezuelensis, which shares 99% identity with L. licerasiae, has also been detected in cattle urine samples from Venezuela, South America [45].

The finding that 6 of the 18 seropositive pigs were also PCR-positive, whereas 12 had antibodies but no detectable renal colonization of Leptospira, is consistent with patterns reported in other studies and provides insight into infection dynamics. In pigs and other reservoir hosts, seropositivity without detectable renal colonization or shedding may indicate a past infection that has been cleared, or a transient phase of leptospiremia during which bacteria are present in the blood but have not yet colonized the kidneys. For example, experimental infection studies have shown that animals may seroconvert (i.e., develop antibodies) even in the absence of consistent detection of organisms in tissues or excretions [46]. Similarly, in wild pigs, Poudel et al. reported a higher proportion of animals with antibodies than with detectable Leptospira DNA in renal tissue, suggesting that many exposures may be transient or that infection is not always persistent [26].

The 72% seroprevalence observed in this study is higher than that reported in other regions. For example, Santos et al. (2023) reported 68.8% in pigs in Paraná, Brazil, while Gomes de Araújo et al. (2023) reported 36.4% in South America and 34.1% in North America [47, 48]. These comparisons suggest that swine exposure to Leptospira is widespread across geographic regions, although prevalence varies depending on environmental and livestock management conditions. It should also be noted that the 25 blood samples analysed in this study were collected during follow-up sampling from two farms where infected pigs had been identified in the initial collection, which may explain the higher seropositive percentage.

The MAT results showed that Autumnalis was the predominant reacting serogroup, followed by Icterohaemorrhagiae, Canicola and, Sejroe. Cross-reactivity was also observed, particularly between Autumnalis and Hardjo, as well as Autumnalis and Canicola. These findings are consistent with previous reports highlighting the challenge of identifying the infecting serovar based solely on MAT results due to antigenic similarities among serovars [49].

The predominance of Autumnalis in the present study aligns with findings from other countries where this serogroup has been identified in swine populations, such as Greece and Brazil [50, 51]. Similarly, a study conducted in Germany reported seropositivity to Autumnalis (3.7%), Icterohaemorrhagiae (4.0%), and Canicola (2.0%), which are comparable to the serogroups identified here [46]. This indicates that the serogroups detected in the current study are also circulating in swine across different regions.

In Sri Lanka, the serogroups Autumnalis, Icterohaemorrhagiae, Canicola, and Sejroe have previously been identified in clinical samples from patients admitted with acute febrile illness at the Teaching Hospital Peradeniya [52, 53]. The overlap between the serogroups identified in swine in this study and those reported in humans suggests that pigs may serve as a potential reservoir for human infections in the country. Furthermore, serogroups such as Canicola and Icterohaemorrhagiae have also been detected in dogs in Sri Lanka [7], supporting the possibility of transmission between domestic animals and humans. Together, these findings highlight the importance of swine and other companion animals in the epidemiology of leptospirosis in Sri Lanka and emphasize the need for integrated surveillance within a One Health framework.

The farms where Leptospira spp. were detected lacked wastewater treatment systems, resulting in the direct discharge of animal excreta into the environment. In addition, poor hygienic practices, such as inadequate use of personal protective equipment during farm cleaning and animal or meat handling, were observed. These practices reflect a lack of awareness of disease transmission among workers in the swine industry. This, combined with the absence of a comprehensive leptospirosis vaccination program for swine in Sri Lanka, significantly increases the risk of infection for individuals working in farms, slaughterhouses, and abattoirs. Therefore, to better understand and control the spread of the disease, a One Health approach is essential to identify the potential transmission pathways of the pathogen among pigs, the environment, and humans.

This study has several limitations. Although Gampaha had the highest pig density overall (Supplementary Table 1), logistical considerations and accessibility of slaughterhouses limited our sampling to Colombo and Kegalle, and therefore, the findings may not represent all pig-rearing areas of Sri Lanka. In addition, the serovars Pomona and Bratislava, which are well known to be associated with pigs, were not included in our MAT panel. This limitation may have led to an underestimation of seropositivity.

Conclusions

This study confirms that swine raised for human consumption in Sri Lanka are significant carriers of Leptospira spp., including both P1 and P2 subclades. The detection of strains identical to those found in human cases highlights the zoonotic risk posed by these animals. Poor farm hygiene practices and environmental contamination further increase the threat to farm workers, nearby residents, and wildlife. These findings underscore the urgent need for comprehensive studies with a One Health perspective to better understand the epidemiology of leptospirosis in Sri Lanka. Additionally, strategies should be implemented to improve biosecurity, promote proper hygiene practices, and ensure the use of personal protective equipment by workers in pig farming and related industries. Establishing vaccination programs for livestock is particularly crucial for mitigating disease transmission and ensuring public health security.

Methods

Study site and sample collection

The study was conducted in the Colombo and Kegalle districts of Sri Lanka (Fig. 2). Supplementary Table 1 shows the variation in pig population density across districts in Sri Lanka. Colombo was selected as a representative high-density swine-rearing district, while Kegalle represented a low-density district. Samples were collected from healthy pigs slaughtered for human consumption between April and October 2022. Sampling was conducted as convenience sampling at slaughter. The number of pigs collected per farm depended on the availability of animals at the time, rather than being proportional to farm size. Veterinarians performed an ante-mortem inspection to ensure apparently healthy pigs. During the initial screening, kidney samples were collected from slaughtered pigs for Leptospira DNA detection to identify disease-endemic farms in the selected study areas. Kidney and blood samples were subsequently collected from the farms with more positive results in the initial screening. Kidney tissue samples were collected in sterile plastic bags, and blood samples were collected in plain blood collection tubes. All the samples were transported to the laboratory of the University of Peradeniya in a cool box. The serum was separated from the blood samples by centrifugation at 1500 × g for 10 min. All the samples were stored at −20 °C until laboratory testing.

Fig. 2.

Map of Sri Lanka indicating the sampling districts (green) and areas where samples were collected (red)

National pig population data were obtained from the Department of Census and Statistics. The raw data were tabulated and pig population density was calculated directly from census data [54].

Detection of Leptospira DNA from swine samples

DNA was extracted from the kidney tissue samples using the DNAzol™ reagent (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. The extracted DNA was subjected to nested PCR targeting the Leptospira flaB gene using the primers to detect P1 and P2 subclades of Leptospira spp. (Supplementary Table 2). The PCR amplicons were sequenced using the dideoxynucleotide chain termination method using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The partial flaB sequences obtained in this study, as well as representative reference strains and previously reported sequences from humans and dogs in Sri Lanka, were aligned in MEGA-X using CLUSTALW. Accession numbers and corresponding references are listed in Supplementary Table 4.

The phylogenetic distance was calculated in MEGA-X using the neighbor-joining method. The nucleotide sequences of flaB have been deposited in a public database (accession numbers LC830693 and LC830882-LC830888).

Serological identification of anti-Leptospira antibodies

The microscopic agglutination test (MAT) was performed to detect anti-Leptospira antibodies in the serum samples using a live antigen panel containing 14 serovars representing 13 serogroups: serovars Autumnalis (serogroup Autumnalis), Bataviae (Bataviae), Canicola (Canicola), Grippotyphosa (Grippotyphosa), Hardjo (Hardjoprajitno strain, Sejroe), Hebdomadis (Hebdomadis), Icterohaemorrhagiae (Icterohaemorrhagiae), Javanica (Javanica), Manilae (Pyrogenes), Panama (Panama), Patoc (Semaranga), Shermani (Shermani), Tarassovi (Tarassovi), and Wolffi (Sejroe). These strains were maintained on liquid Ellinghausen–McCullough–Johnson–Harris (EMJH) media and passaged weekly. Four to seven-day-old cultures containing approx. 2 × 10⁸leptospires/mL were used for performing MAT. As vaccination against Leptospira is not practiced in pigs in Sri Lanka, a titer of ≥ 1:100 was considered positive, in accordance with WOAH recommendations.

Abbreviations

Leptospira spp.

Leptospira Species

MAT

Microscopic Agglutination Test

flaB

Flagellin protein subunit B

Authors’ contributions

N.R.: Investigation, Data curation, Visualization, Writing—original draft, Writing—review & editing. D.M.: Investigation, Data curation, Visualization, Writing—original draft. R.V.: Investigation. P.S.: Investigation. V.B.: Investigation. Y.S.: Investigation. R.N.: supervision. K.S.: Resources. K.Y.: Resources. S.R.: Supervision. N.K.: Methodology, Supervision, Fund acquisition, Writing—review & editing. C.G.: Conceptualization, Methodology, Supervision, Writing—review & editing. All authors reviewed the manuscript and approved the final version for publication.

Funding

This study was partly supported by the Research Program on Emerging and Re-emerging Infectious Diseases (JP24fk0108683) from the Japan Agency for Medical Research and Development (AMED) (Grant received by N.K.).

Data availability

The nucleotide sequences obtained in this study are available in the DNA Data Bank of Japan (DDBJ) repository under the accession numbers LC830693 and LC830882–LC830888 (https://getentry.ddbj.nig.ac.jp/getentry/na/LC830693/, https://getentry.ddbj.nig.ac.jp/getentry/na/LC830882/, https://getentry.ddbj.nig.ac.jp/getentry/na/LC830883/, https://getentry.ddbj.nig.ac.jp/getentry/na/LC830884/, https://getentry.ddbj.nig.ac.jp/getentry/na/LC830885/, https://getentry.ddbj.nig.ac.jp/getentry/na/LC830886/, https://getentry.ddbj.nig.ac.jp/getentry/na/LC830887/, https://getentry.ddbj.nig.ac.jp/getentry/na/LC830888/). Although the essential data are provided in the tables, additional data are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Review Committees of the Postgraduate Institute of Science, University of Peradeniya, Sri Lanka, and Cardiff Metropolitan University, UK (UG-6118).

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.