Introduction

Leptospirosis is a reemerging zoonosis with worldwide distribution and a significant impact on livestock production. The disease is frequently associated with reproductive disorders, including embryonic resorption, fetal mummification, stillbirths, or neonatal mortality causing significant economic losses. Furthermore, leptospirosis in farmers, slaughterhouse workers, and veterinarians is very common and occurs through direct contact with urine or tissues from infected animals, or indirectly through contaminated soil and water [1]. Unfortunately, knowledge about the disease in livestock is biased towards intensive breeding industries in developed regions of the world, while the epidemiological characteristics of leptospirosis in developing nations remain unclear [2].

In most low-income tropical countries, leptospirosis is endemic and common. Ownership of a small number of peridomestic livestock is frequent in poor rural areas, and pigs are often part of this community. In these situations, pigs are often raised under poor sanitary conditions without veterinary guidance [3,4]. Importantly, the social and geoclimatic characteristics of these areas are conducive to the transmission and maintenance of pathogenic Leptospira species. Indeed, in endemic regions, leptospires can persist in the urogenital tract of asymptomatic animals that excrete the bacteria in urine and genital fluids, providing a source of infection for susceptible hosts [5]. In these settings, spillover to humans is likely as humans and animals live in close proximity [2]. Although vaccination can reduce leptospira transmission, efficacy is commonly reduced due to low immunity against strains that are not represented in vaccines [6]. This is especially problematic in countries where very little information on circulating serovars is available and when local isolates are not available for inclusion in MAT tests for disease diagnosis. Given the high, uncharacterized diversity of pathogenic Leptospira in tropical developing countries, understanding the local epidemiology of leptospirosis is paramount for disease mitigation.

Recent studies in rural communities on the coast of Ecuador show that leptospirosis is one of the most important febrile diseases in the region. Analysis of blood samples show that a high percentage of febrile patients (between 14% and 40%) are infected with pathogenic species of Leptospira [7,8]. In these communities, people are frequently exposed to cattle, cows, and pigs in the peridomestic environment. A high prevalence of Leptospira from these animals and pathogen genotypes shared with clinical cases suggest that all of these livestock types are potential sources of transmission to humans [7–9].

Swine leptospirosis has historically been linked to exposure to urine from carrier animals, but there have been no links to the reproductive system in disease transmission [7,8,10,11]. In this report, we present the case of a domestic boar raised in a rural area of Ecuador, that was found to excrete leptospires in its semen. The boar had been vaccinated and showed no clinical signs of disease, but laboratory analysis identified a pathogenic Leptospira species that had not been previously described in the reproductive tract of pigs. We present serologic, molecular, and histopathologic data from this rare case of porcine genital carriage of pathogenic Leptospira. Our results reaffirm the need for a thorough understanding of the epidemiology of leptospirosis in endemic regions at the local level in order to implement appropriate preventive vaccination and improve disease control programs [12].

Materials and methods

Results

MAT on serum samples gave positive results with titters of 1:100 for serovar Canicola in sample S1, and 1:100 for serovars Pomona and Hardjo in sample S2 (S1 Table).

Leptospira DNA was detected in semen (spermatic and post-spermatic fraction), and kidney and testicle tissues in different time points (Table 1). Sequences obtained from the semen sample were indicative of L. santarosai, most similar to L. santarosai strain U164 (Sequence ID: CP028370.1) with 98.05% identity and 99% coverage. Other Leptospira santarosai sequences shared between 97.75% to 94.75% identity across 99% of coverage (Sequence IDs: CP028377.1, CP006694.1, CP027843.1, CP022887.1). The next closest species was L. noguchii with 78% coverage. (Data available at https://www.ncbi.nlm.nih.gov Bioproject ID: PRJNA741491, Biosample ID: SAMN1989635, Barcodes 12–14). Concatenated consensus sequence is shown in S1 File.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Positivity of pathogenic Leptospira (amplification of lipL32 gene) in urine, semen, and tissue samples.

https://doi.org/10.1371/journal.pone.0274362.t001

Interestingly, histopathology from kidney, testicle, and epididymis stained with hematoxylin-eosin, showed intact tissue with no microscopic lesions (Fig 1). Also, no gross lesions were observed in the organs during necropsy.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Histopathology of the boar organs.

(A) Histology of the kidney presenting normal glomeruli (arrows) and tubules (arrowheads), showing no sign of inflammation. (B) Normal histology of the epididymis, exhibiting abundant spermatozoa in the lumen (arrow). (C) Normal histology of the testicle, physiological development of cell populations and normal architecture are maintained. No signs of inflammation are evident.

https://doi.org/10.1371/journal.pone.0274362.g001

Discussion

Genital leptospirosis can go unnoticed, compromising the reproductive productivity of herds over long periods of time [20]. However, the severity of the disease varies depending on the infecting strain and the affected species [2]. This research is the first record of L. santarosai in the reproductive system of a boar; specifically in testes and semen. We were also able to detect the bacteria in kidney tissue, but not in urine, probably because the animal was not excreting detectable amounts of leptospires at the time of urine collection and the focus of infection seems to have been in the reproductive tract. Leptospira santarosai have been reported in pig urine samples from Ecuador [7]. Our current findings are of particular interest because they indicate that reproductive and urinary aspects of the urogenital track of boars can both be colonized with L. santarosai, and present two routes of shedding and transmission.

Our research was carried out after observing that some sows, inseminated with semen from the same donor, had reproductive failures. The donor boar did not show clinical signs of leptospirosis, and the histopathological samples did not show evidence of tissue damage in the testes or epididymis, despite presence of blood in the semen. However, PCR and sequencing identified L. santarosai in testicles and semen samples. As previously reported for bulls [21], rams [22] or stallions [23] without apparent clinical signs, the presence of pathogenic Leptospira species DNA in semen suggests the potential venereal transmission of this pathogen. Leptospira interrogans and L. kirschneri have also been isolated from the reproductive tract of boars in Italy [24]. In the present case, the history of failed pregnancies is consistent with leptospirosis and, coupled with infected semen of the boar, strongly suggest that leptospires were transmitted from the boar to sows via semen during artificial insemination (AI). In fact, L. santarosai has previously been associated with AI-transmitted bovine genital leptospirosis in other Latin American countries [20]. AI is a useful tool to introduce superior genes into herds and reduces the risk of injury and disease transmission through natural mating. However, semen can be contaminated with pathogens like Leptospira spp. [25]. The best strategy to prevent diseases transmitted by AI is to use pathogen-free boars, regularly monitoring animals and semen, and maintaining biosecurity strategies such as rodent control [26]. Indeed, the risk of leptospira occurrence in the semen of boars from large commercial farms is low [27]. However, as sanitary conditions can affect AI, this method is a risk factor associated with leptospirosis on pig farms [28,29]. To our knowledge, prior to our study, the boar never tested positive for leptospirosis, however the presence of rodents in and around the farm put the animal at risk of infection.

Vaccination is one of the main strategies used to limit the spread of leptospires in herds. Currently, polyvalent vaccines include the most frequent serovars. However, vaccines are less effective in sites where local serovars are non-included in the vaccine [30]. This is consistent with the fact that the boar under study was infected despite being vaccinated. Specifically, the vaccine used on this boar includes six serovars (Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae and Pomona), while 15 different serovars have been identified in L. santarosai (Alice, Atlantae, Babudieri, Bananal, Batavidae, Beye, Canalzonae, Georgia, Guaricura, Kremastos, Peru, Pyrogenes, Shermani, Szwajizak and Tabaquite) but were not contained in the vaccine used on the farm [31–33].

Identification and removal of infected animals is used to control many infectious diseases, but is of limited value when the subclinical form is the most common presentation, and tests do not reliably identify carrier animals [2]. The MAT is the most common serological screening method used for leptospirosis. It is performed by incubating patient serum with various serovars of leptospires with any reacting serovar being indicative of the infecting serovar. Confirmation of an active infection is made by testing a second sample and demonstrating an increase in antibody titer. The MAT has the advantage of detecting antibodies that are specific to the infecting serogroup/serovar, but is prone to false negative or non-specific results if the panel does not contain representative antigens of local strains [6]. Furthermore, the MAT results must be interpreted with caution as it cannot discriminate between antibodies resulting from infection or vaccination, and high titers are not necessarily indicative of infection [34]. In our case, the boar MAT results show titers of 1:100 against three different serovars (Canicola, Pomona, and Hardjo), but this is undoubtedly due to vaccine-based immunity [6]. It is impossible to know if the animal would have shown any response to a serovar of L. santarosai because the reference diagnostic laboratory, where the MAT was performed, does not use L. santarosai serovars or any characterized local isolates. Molecular methods are also important tools for diagnosis in animals that do not show serological responses [35]. As previously reported for other domestic species [21–23], our results show that PCR and sequencing are important tools for the detection and characterization of leptospires in semen at swine artificial insemination centers. However, PCR and amplicon sequencing typically provides only species level identification and does not allow for serovar identification, limiting translational utility. This information is crucial for a better understanding of the epidemiology, the utility of diagnostic tests, and the development of new vaccines [36]. Based on these findings, we consider that the combined use of MAT as a screening test, followed by PCR and amplicon sequencing for the direct detection of, and characterization of Leptospira spp. was adequate for the identification of carrier animals, but bacteriological isolation of local serovars is critical for increasing the accuracy of MATs and improvement to vaccination strategies.

Leptospirosis is considered an underreported occupational disease, especially in developing countries [37]. Transmission among animals and humans through direct contact or indirectly through contaminated environments in low-tech peridomestic pig farms may be relatively common [38,39]. Leptospira santarosai have been previously identified in cases of human leptospirosis [33,40,41], and our findings reflect the potential risk of pigs on rural farms in low-income countries as a possible source of human and animal leptospirosis. Prevention and control measures for leptospirosis must be approached from a one-health perspective, however, a major limiting factor has been the lack of communication and cooperation between the human and animal healthcare communities [42]. This is the case of Ecuador, where leptospirosis is a notifiable human disease, but not a notifiable animal disease. In fact, the Ministry of Public Health reported 643 cases of human leptospirosis between 2016 and 2021, but there are no official reports of leptospirosis in livestock [43]. However it is clear from our past work that pathogenic Leptospira species infect both, animals and humans in Ecuador [7,8]. This information gap contributes to the lack of knowledge of the epidemiology of leptospirosis in the region.

Conclusion

This is the first report on the detection and identification of a pathogenic L. santarosai from the reproductive system of a boar. The finding of L. santarosai in testicles and semen, coupled with evidence of failed pregnancies in recipient sows is significant because it strongly suggest venereal transmission of leptospirosis. Furthermore, the silent and chronic spread of L. santarosai or any other species represents additional risks to public health, which needs to be approached from a one-health perspective by effective communication between animal and human health surveillance sectors.

Supporting information

Acknowledgments

We are grateful to Belen Prado-Vivar (Centro de Bioinformatica, Universidad San Francisco de Quito USFQ) for her technical assistance during sequencing.