Introduction

Leptospirosis is a globally diffused, re-emerging zoonosis caused by pathogenic bacteria belonging to the genus Leptospira [1–3]. This includes 64 genomospecies; 38 of them are phylogenetically classified as pathogenic [4,5]. Pathogenic leptospires are responsible for infection in both humans and animals [1,6]. The primary site of Leptospira localization is the kidney, resulting in environmental spreading of the bacteria via urine and infection in a wide range of wild and domestics reservoir animals [6,7]. The genital tract could be secondarily affected after bacteremia and renal infection [8,9]. Recently, some studies carried out in cattle and pigs suggested that genital leptospirosis should be considered as a specific syndrome [7,10]. Genital leptospirosis seems to be related to low systemic antibody titers, and chronic uterine infection is often closely associated with a reproductive syndrome characterized by low fertility rates, abortion, and increased perinatal mortality. Sexual transmission has also been demonstrated [7,11–13]. Genital Leptospira infection has unique characteristics, which could be differentiated from renal disease [10]. In the Suidae family, especially in pigs, genital leptospirosis has been observed in sows, sometimes associated with abortion, and in boars [12,14,15]. Moreover, Leptospira infection of the reproductive tract has also been demonstrated in feral swine with the isolation of Leptospira borgpetersenii serogroup Ballum in wild boar aborted fetuses [16].

In Europe, wild boar (Sus scrofa) represent one of the most widespread large mammals [17,18]. This species is very adaptable and is able to survive in different habitats, including suburban and urban areas [17,19,20]. In Italy, the wild boar population is widespread, from the Alps to the southern part of the Italian peninsula, including the islands [18,21].

The reproductive cycle of wild boar is characterized by early puberty (from 5 to 10 months of age), high fertility, and short gestation period (from 115 to a maximum of 122 days) [22–25]. The estrous cycle has a duration of about 22 days, with receptivity for 1–3 days and usually one mating season per year. During the reproductive season, which is in Autumn when the rainfall increases, as well as the leptospirosis infection risk, males generally mate with more than one female [24–26]. Births usually occur in late winter to early spring, with a peak in February or March [26–28]. Furthermore, a bimodal distribution has been observed in some years, as a consequence of genetic breeding with domestic pigs [25,27]. Wild boar reproductive parameters could be affected by habitat, climatic conditions, photoperiods, hunting pressure, and availability of food resources [25,28,29].

The aim of this investigation was to evaluate the possible genital Leptospira infection in male and female wild boar. Moreover, the possibility of vertical transmission to fetuses was evaluated.

Materials and methods

Results

Leptospira spp. isolation, identification, and MLST genotyping

After about 70 days of incubation, six Leptospira isolates were obtained from three testicles and the respective epididymides of an adult male and two subadults (Table 1). Isolates from adult male tissues were identified as Leptospira kirschneri serogroup Grippotyphosa and assigned to Sequence Type (ST) 78, while the remaining four isolates from two subadult males were identified as Leptospira interrogans serogroup Australis and assigned to ST 24 by MLST analysis.

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Table 1. Distribution of positive tissue samples for pathogenic Leptospira in relation to sex and age class.

https://doi.org/10.1371/journal.pntd.0008982.t001

Leptospira spp. detection by multiplex real-time PCR and genomospecies identification

Leptospira DNA was detected in male and female tissues samples. Data reported in Table 1 highlight that 22 out of 95 (23.16%) testicles and the respective epididymides scored positive by Real-Time PCR. Eleven out of 136 (8.08%) uterine samples (five of them from pregnant females, 3.68%), 3 placentas (6.66%), and 13 fetuses (28.88%) out of 45 were positive by Real-Time PCR (Table 1).

Regarding the characterization of PCR-positive samples, amplification of the rrs2 gene highlighted that pathogenic Leptospira belonged to L. interrogans and L. kirschneri. Phylogenetic analysis identified a close relationship between the respective Leptospira species (Fig 1).

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Fig 1. Molecular phylogenetic analysis for PCR-positive tissue samples based on the rrs2 gene of Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri (in the red box with the GenBank Accession Number) by the Maximum Likelihood method based on the Tamura-Nei model.

The branch lengths of the tree measure the number of substitutions per site. The analysis involved 31 nucleotide sequences. There was a total of 452 positions in the final dataset.

https://doi.org/10.1371/journal.pntd.0008982.g001

Discussion

In this study, Leptospira infection in the reproductive tract of male and female wild boar was evaluated by culture-dependent and -independent methods. DNA of pathogenic Leptospira was detected in samples from both males and females, as well as in all age classes, confirming endemic infection among wild boar in the investigated areas. The results of this investigation suggest that both genders and all age classes have the same probability to be infected with Leptospira. However, no suggestive macroscopic lesions were observed during post-mortem examination. During the last decades, the wild boar population has rapidly increased in Italy. This species is adapted to live in contact with humans (i.e., hunters, farmers, etc.) and domestic animals, hence promoting the possibility of interspecies leptospirosis transmission [17,38].

To the best of the Authors’ knowledge, no studies have focused on involvement of the reproductive system in Leptospira infection in wild boar. Based on the obtained data, all positive males presented infected testicles and epididymides, suggesting dual localization. This was confirmed by cultural examinations. Leptospira infection in the reproductive system has already been demonstrated in pigs [7,15]. Leptospira interrogans serovar Bratislava and serovar Muenchen were indeed isolated from swine testicles and urogenital tracts [15]. This investigation suggested that male wild boar could be infected with swine-adapted pathogenic Leptospira interrogans serovar Bratislava.

The presence of Leptospira in the epididymis might be temporary and a consequence of testicular infection; however, there is no available detailed information clarifying this aspect in other domestic or wild animals.

Generally, as reported for bulls and boars, infections in males are often subclinical [7,10,39]. However, during the acute phase of genital infection, male wild boar could present mild and temporary orchitis. Testicular and/or epididymis inflammation due to leptospirosis could cause damage or alter sperm; however the specific damage caused by Leptospira infection to spermatozoa or its influence on sperm viability remains unknown. On the other hand, for other species, the possibility of venereal transmission from males to females is well documented. Pathogenic Leptospira in semen are able to travel up the reproductive tract and reach the uterus and oviduct, as reported in cattle [10,40]. However, although the role of semen in the transmission of bovine leptospirosis is well established [41,42], its role in transmission between wild and domestic swine has not been investigated and can only be hypothesized.

A commercial medium, without modifications, was used for Leptospira detection. After more than 2 months of incubation, serovar Bratislava was successfully isolated. Indeed, it is known that the isolation of serovar Bratislava is challenging, sometimes requiring a longer incubation period and use of a specific medium (EMJH with the addition of Tween 80, Tween 40, and lactalbumin hydrolysate) [12,14,43]. For this reason, the differences between the prevalence obtained by cultural methods and PCR could also be due in part to the medium employed.

In Tuscany, a high seroprevalence of serogroup Grippotyphosa has been recorded among cattle, sheep, and goats [44]. In Sardinia, wild boar are often exposed to Leptospira Bratislava and Pomona, thus potentially acting as a maintenance host for these serovars [45]. Consequently, the observed infection with Leptospira kirschneri serovar Grippotyphosa in an adult male wild boar could be due to free-ranging wild boar sharing their habitat with domestic ruminants.

The wild boar uterus seems to be receptive to Leptospira infection, not only in adults but also in young animals. Some specific Leptospira strains could be adapted to the uterine environment of wild boar, as already hypothesized for cattle. Indeed, it was recently speculated that infection by strains of the Sejroe serogroup in cattle, causing subclinical disease, is associated with strains presenting a certain level of adaptation to the reproductive tract [10]; in this case, a chronic, silent infection occurs and is characterized by agalactia in lactating animals and reproductive failures rather than abortion. It was also suggested that this genital adaptation could promote venereal transmission from females to males [7,10]. Considering that in this investigation no alteration was recorded, the presence of Leptospira in fetuses could suggest a high adaptation level of Leptospira strains and wild boar resistance to infection, which could promote vertical transmission without obvious signs in fetuses. On the other hand, for domestic swine, it was observed that some serovars could influence pregnancy, causing abortions, stillbirths, or birth of weak piglets with reduced viability [7,14]. As suggest for bovines, these events could be related to non-adapted serovars [7,10]. Unfortunately, little information is available clarifying this aspect. The only report concerning this issue reported the isolation of Leptospira borgpetersenii serogroup Ballum from wild boar aborted fetuses. This serogroup was not detected in this present survey by isolation and molecular investigations [16].

While the body of knowledge concerning wild boar is limited, several studies on pathogenic Leptospira from sow reproductive systems are available [7,14,43]. Leptospira belonging to serogroup Australis was isolated from the uterus of sows [14,43], and infections with serovar Bratislava and Muenchen could be associated with abortions in sows [12,14,43].

In this study, no correlation among positive uterus, placenta, and fetus specimens was detected in pregnant females. This is in accordance with a previous study performed on pigs with a history of abortion, where no correlation between positive fetuses and placentas was found [12]. In animals, abortion could be a consequence of uterus inflammation or the fetuses themselves. In this study, the highest prevalence of Leptospira infection was detected in fetuses, followed by the uterus and placenta. As previously suggested for swine [12], Leptospira infection in female wild boar could persist and could be fatal to fetuses during pregnancy. Considering the obtained results, it might be hypothesized that pathogenic Leptospira could infect the kidneys, liver, or other fetal tissues. Unfortunately, based on the present data, it is not possible to establish whether fetuses would be delivered immunized, infected, or would be aborted.

According to phylogenetic analysis, pathogenic Leptospira DNA detected in wild boar reproductive systems belonged to L. kirschneri and L. interrogans. Leptospira kirschneri infection seems to be prevalent in testicles and epididymides, while Leptospira interrogans is present in female genital organs. Based on the serovars more often detected in Italy by cultural examination or serology [46–52], and the rare seropositivity for other serovars [53–55], it is likely that the detected L. kirschneri belonged to the serogroup Grippotyphosa. In this study, L. kirschneri infection was only observed in testicles and epididymides, and this could hypothesize this genomospecies as more often associated with the male reproductive system. Moreover, considering the negativity in females, it could be speculated that venereal transmission of L. kirschneri is unlikely within wild boar. In addition, the ability of this Leptospira species to survive and colonize the uterus compared to other species should be assessed.

As concerns the presence of L. interrogans, it would be very difficult to infer the serogroup since this genomospecies includes several groups, such as Icterohaemorrhagiae, Canicola, Pomona, Australis, and Sejroe. However, in relation to isolates obtained in this investigation and previously published serological results [44,46,51,56], it is likely that serogroup Australis would be prevalent, at least in samples from males. Currently, serovar Bratislava and the closely related serovar Muenchen, belonging to serogroup Australis, are the main emerging swine-maintained pathogenic Leptospira serovars [7]. The epidemiology of these serovars is poorly clarified; however, their strains are grouped into specific pig-adapted strains, strains maintained by pigs, dogs, horses, and hedgehogs, and strains found only in wildlife [7,57]. In pig farms, venereal transmission seems to play an important role in the spread of serovars belonging to the Australis serogroup, especially for Bratislava. In this context, frequent introduction of new male subjects to the farms could represent an important risk factor [9,12,15]. Therefore, genital infection with serovar Bratislava in wild boar could suggest the possibility of venereal transmission among the wild swine population and, on the other hand, increase the risk of transmission to domestic pigs via possible mating with wild boar, especially in areas where semi-extensive breading systems are adopted such as Tuscany or Sardinia.

This is the first investigation focusing on the isolation of pathogenic Leptospira from the reproductive system of healthy wild boar. The impact of Leptospira infection on the reproductive system and performance of wild boar is of great interest since leptospirosis is a neglected and underestimated disease and has an economic impact on the breeding system. The results obtained contribute to understanding the role of wild boar as a pathogenic Leptospira reservoir, and potentially a spreader as well. Leptospira could also cause silent and chronic infection, increasing the possibility of leptospirosis spreading among wild boar population. Unfortunately, in a natural system, the control of genital leptospirosis, and its effects on wild boar reproductive performance, is a challenging task. Under the same conditions experimental infection could be very difficult to perform as well. Conversely, the occurrence of infection is easily assessed in specimens from hunted animals, which is only a snapshot of the entire wild boar population for an investigated area. Data from such investigation could be compared to those on wild boar population dynamics; in this case, only long-term surveillance, also excluding other causes of reproductive syndromes in animals, could probably give exhaustive information.

Leptospira infection in the reproductive system of wild boar could be involved in the epidemiology of human leptospirosis. The hunters’ risk of exposure could be increased during slaughtering, since tissues from the reproductive system are necessarily handled during slaughtering, and the use of personal protective equipment is not commonly adopted among hunters.

In conclusion, the results of this investigation indicate that the genital tract of wild boar could be a target for different Leptospira species. Genital leptospirosis could be associated with reproductive problems, as reported in pigs. Wild boar are more resistant than selected breeding lineages, and they are not subjected to the stresses of intensive farming; for these reasons, the impact of genital leptospirosis on reproductive performance could be lower than in domestic swine, as suggested by the high birth rates reported recently in Italy as well in Tuscany and Sardinia [18,21]. Moreover, the pathogenic Leptospira localization in the reproductive systems could represents a public health risk, especially for hunters, that usually handled these organs during the slaughtering practices without the use of personal protective equipment, favoring the possible contagion.

The possible vertical transmission of Leptospira, suggested by the obtained results, opens new questions on the epidemiology of this disease in wild boar. Further investigation should be performed in order to understand the prevalence of Leptospira in the reproductive system and fetuses, the possible implication of genital infection on reproductive performance, and the effects of vertical transmission and consequent congenital infection, not only in wild boar but also in other wild animal species.

Acknowledgments

We thank Mario D’Incau and his team at the National Reference Centre for Animal Leptospirosis (Istituto Zooprofilattico Sperimentale della Lombardia e dell’ Emilia Romagna “Bruno Ubertini”, via Bianchi 7/9, 25121 Brescia, Italy) for providing the Leptospira live antigens employed in this investigation.