Leptospira Survey in Wild Boar (Sus scrofa) Hunted in Tuscany, Central Italy

Leptospirosis is a re-emerging, worldwide zoonosis, and wild boar (Sus scrofa) are involved in its epidemiology as the reservoir. The aim of this study was to investigate the prevalence of Leptospira with serological, bacteriological, and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. In total, 287 specimens of sera, kidneys, and liver were collected to perform microscopic agglutination tests (MATs), isolation, and RealTime PCR to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene), and saprophytic (23S rRNA gene) Leptospira. Within sera, 39 (13.59%) were positive to the MAT, and Australis was the most represented serogroup (4.88%), followed by Pomona (4.18%), and Tarassovi (3.14%). Moreover, four Leptospira cultures were positive, and once isolates were identified, one was identified as L. borgpetersenii serovar Tarassovi, and three as L. interrogans serovar Bratislava. Pathogenic Leptospira DNA were detected in 32 wild boar kidneys (11.15%). The characterization through the amplification of the rrs2 gene highlighted their belonging to L. interrogans (23 kidneys), L. borgpetersenii (four), and L. kirschneri (one), while nine kidneys (3.14%) were positive for intermediate Leptospira, all belonging to L. fainei. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis among wildlife in Central Italy.


Introduction
Wild boar (Sus scrofa) is a large ungulate mammal with worldwide distribution. It can live in several types of habitat, including urban and suburban areas [1,2]. Due to their high adaptability, wild boar populations have rapidly increased in number during recent years, in Europe, and especially in Italy [1,3]. In Italy, wild boar is largely spread in all areas, from the Alps to the southern part of the Italian peninsula, including the islands. There is a high density, particularly in specific regions, such as Tuscany [3][4][5]. The abundant presence of wild boar in the Tuscany region, as well in Central Italy, is suggested by the very high number of animals hunted in this area; every year the hunting of about 42,000 specimens is registered [1,[3][4][5]. The massive presence of wild boar in particular areas, other than representing an important source of damage for agriculture [6], can be a severe risk to human and animal health, due to the identification of wild boar as reservoir for many etiological agents; among them typical zoonoses, such as Leptospira [7][8][9].
Leptospirosis is a re-emerging zoonotic disease with worldwide spread. It is caused by Leptospira spp., a Gram-negative spirochetal bacterium [10][11][12]. The genus Leptospira is divided into more than 260 antigenically-different serovars, classified as pathogenic, intermediate, and saprophytic, with different levels of pathogenicity for animals and humans [13,14]. While pathogenic Leptospira cause mild or severe infection, intermediate Leptospira could possibly be pathogenic, causing mild infection, while  Results on distribution of positive sera and kidney for pathogenic Leptospira in relation to hunting season, province, sex, and age class are reported in Table 1.
Results on distribution of positive sera detected by MAT in relation to hunting season, province, sex, and age class are reported in Table 1. No statistical differences (p > 0.05) were reported for the serological positivity considering hunting seasons, provinces, and wild boar sex and age class. Moreover, comparing all parameters, no statistical differences (p > 0.05) were showed in Pisa and Grosseto during the two different hunting seasons.
No statistical differences (p > 0.05) were highlighted comparing province, wild boar sex, or age class. Conversely, the incidence of pathogenic Leptospira-positive kidneys was statically higher (p ≤ 0.01) during 2018/2019 hunting season compared to the 2019/2020 ones.
The detection of pathogenic Leptospira DNA was higher (p ≤ 0.01) during 2018/2019 hunting season in both Pisa and Grosseto provinces compared to the second hunting season. On the contrary, there were no statistical differences (p > 0.05) in the Pisa and Grosseto provinces during the two different hunting seasons, comparing sex and age class of wild boar.
The 3.14% (9 out of 287) of kidneys were positive for intermediate Leptospira.
The positivity in relation to hunting seasons, province, wild boar sex, and age class are showed in Table 1. All the intermediate Leptospira-positive kidneys (4.5%; 9 out of 200) were collected in 2018/2019, highlighting a statistical difference (p ≤ 0.01) in relation to 2019/2020 hunting season. Also, the results showed a statistically-higher infection rate (p ≤ 0.01) in male compared to female, and in Pisa province compared to other provinces. No statistical difference (p > 0.05) were noted among age classes.
No saprophytic Leptospira DNA was detected in kidney samples. No positive reactions were recorded in wild boar livers across all specimens during the two year of investigation.

Leptospira spp. Isolation, Characterization and Genotyping
Four Leptospira cultures were positive after 30 days of incubation. The results, reported in Table 3, show that three isolates were obtained from subadult males hunted in Pisa province, while the other one was from an adult female hunted in Livorno. Through multilocus sequence typing (MLST) analysis, one isolate was identified as Leptospira borgpetersenii serogroup Tarassovi serovar Tarassovi (Sequence Type 153), while the other three were identified as L. interrogans serogroup Bratislava serovar Bratislava (ST 24), as reported in Table 3. Moreover, the amplification of the rrs2 gene from kidney tissue highlights that the species belonged to L. borgpetersenii and L. interrogans, respectively. With regard to characterization of PCR-positive samples, amplification of the rr2 gene highlighted that pathogenic Leptospira belonged to L. interrogans (23 kidneys), L. borgpetersenii (four) and L. kirschneri (one) ( Table 4). Moreover, phylogenetic analysis identified the close relationship to their respective Leptospira species. (Figure 2). Table 4.

Discussion
Leptospirosis is a re-emerging worldwide public health risk, but is underestimated and characterized by a downward trend [34]. Climatic changes, rainfall, modifications of ecological niches, and new potential maintenance hosts all represent important features involved in Leptospira epidemiology.
Wild boar, among wildlife, is an important Leptospira reservoir and, for several areas, represents an appropriate indicator for this zoonotic infectious disease.
In this investigation we reported the results of serological analysis, isolation and molecular investigations performed on 287 hunted wild boar during two hunting seasons (2018/2019 and 2019/2020).
With regard to serological assay, the prevalence of Leptospira infection, recorded in both hunting seasons, was very similar to other studies carried out on wild boar in Tuscany [9,22,26,31,33]. Moreover, the seroprevalence reported in this investigation was very close to other data obtained in different Italian regions [27,[35][36][37][38]. Unfortunately, serological data about leptospirosis in wildlife, especially regarding wild boar, are available just in some regions. It also seems that Leptospira

Discussion
Leptospirosis is a re-emerging worldwide public health risk, but is underestimated and characterized by a downward trend [34]. Climatic changes, rainfall, modifications of ecological niches, and new potential maintenance hosts all represent important features involved in Leptospira epidemiology.
Wild boar, among wildlife, is an important Leptospira reservoir and, for several areas, represents an appropriate indicator for this zoonotic infectious disease.
In this investigation we reported the results of serological analysis, isolation and molecular investigations performed on 287 hunted wild boar during two hunting seasons (2018/2019 and 2019/2020).
With regard to serological assay, the prevalence of Leptospira infection, recorded in both hunting seasons, was very similar to other studies carried out on wild boar in Tuscany [9,22,26,31,33]. Moreover, the seroprevalence reported in this investigation was very close to other data obtained in different Italian regions [27,[35][36][37][38]. Unfortunately, serological data about leptospirosis in wildlife, especially regarding wild boar, are available just in some regions. It also seems that Leptospira serovars/serogroups have a different geographical distribution, suggesting a distinct circulation/epidemiology in other environments/ecosystems. Examining each region, Australis, Pomona, and Tarassovi, are the most-detected serogroups in the Tuscany region [22,26,31,33], In the Lombardy and Emilia Romagna regions it is Bratislava [37][38][39], in the Campania region it is Tarassovi [35], whereas in the Sardinia region it is Pomona and Grippotyphosa [36].
Little information is available on Leptospira isolation in wild boar, especially in Italy [38]. The obtained Leptospira isolates, identified by MLST, confirm the circulation of Tarassovi and Bratislava serogroups within wild boar in Tuscany. Bratislava isolation is commonly performed in wildlife due to the high spectrum of maintenance hosts [45][46][47], while Tarassovi is rarely isolated and detected through serology. Indeed, Tarassovi is strictly a swine-specific serovar; its isolation, reported in this investigation, seems to confirm the hypothesis that wild boar could serve as reservoir of Tarassovi [26,43,48]. Only two of them (Bratislava, isolated from subadult from Pisa province; Table 3) reported correlated serological positivity for serogroup Australis at titer 1:100, while the other two gave negative results in the MAT. No correlation was found between the MAT and PCR-positive results. The seronegativity of Leptospira-positive kidneys has been previously reported for other animal species [49][50][51][52], including swine [47], suggesting an early or chronic infection.
Conversely to serological results, very few studies were performed on pathogenic Leptospira DNA in wild boar kidneys. In spite of this, the prevalence of pathogenic Leptospira infection reported during the years of this investigation was very close to the results obtained in Northern Italy (11.02%) [38] and in the Liguria region (12.13%) [7]. Moreover, prevalences of 10.30% and 15.3% were found in two different investigations performed in Japan [53,54], while 3.40% was reported in the USA [55]. Based on phylogenetic analysis, pathogenic Leptospira DNA in wild boar kidney belong to L. interrogans, L. borgpetersenii, and L. kirschneri. With respect to the serovars in Italy that are more often detected through isolation or serology, [27,38,[56][57][58][59] and the other serovars that are rarely seropositive [28,60], it might be hypothesized that L. kirschneri species found in wild boar kidneys could be related to serogroup Grippotyphosa, while L. borgpetersenii species could be related to the serogroups Tarassovi or Ballum. On the other hand, it is very difficult to infer the serogroup related to L. interrogans positivity, due to the inclusion of Icterohaemorrhagiae, Canicola, Pomona, Australis, and Sejroe serogroups in this species. Probably, in relation to serological and bacteriological results obtained in this study, the identified L. interrogans could belong to serogroup Australis.
The data reported in this investigation suggest that the liver does not seem be a Leptospira target organ in wild boar. Furthermore, it could exclude an early stage of infection (leptospiremia) and confirm that positive animals are only chronic renal carriers, as also suggested by isolation from kidneys.
If very little information is available on pathogenic Leptospira DNA in wild boar, there is even less data on intermediate Leptospira. To the best of these authors' knowledge, it was only in the Liguria region of Italy that 0.49% of wild boar kidneys were positive for intermediate Leptospira DNA in the same year of this investigation [7]. As Liguria and Tuscany are two adjoining Italian regions, a large wild boar movement could be a feature of these regions [61][62][63]. Even though the species of intermediate Leptospira from Liguria were not identified, those found in this investigation belong to L. fainei species. L. fainei was isolated for the first time from fig and was detected in human sera in Australia [64,65] and a human infection with febrile status was reported in France (from a Portuguese citizen) and in two patients in Denmark [66,67]. Considering wild boar behavior and its ability to live in anthropomorphic environment, a transmission between human and wildlife could be possible.
As these are the first determination in European wildlife, more studies are needed to understand the epidemiology of this intermediate Leptospira that could causes severe infection in humans [65][66][67].
The statistical difference presented during the hunting seasons between pathogenic and intermediate Leptospira incidence in wild boar could be related to the temperature and the amount of rainfall recorded in Tuscany during these periods. As reported in literature, rainfall and temperature influence the incidence of leptospirosis in humans and animals [12,[68][69][70][71][72][73][74][75]. Indeed, from 2018 to 2019, the temperatures and the rainfall were both higher than those from 2019 to 2020 [76][77][78][79][80][81], suggesting that these atmospheric phenomena could be involved in these seasonality incidence differences.

Sample Collection
During two hunting seasons (the first from November 2018 to January 2019 and the second from November 2019 to January 2020) hunted wild boar blood, kidney, and liver were sampled. Blood samples were collected by ocular puncture [82]. The boar's age class was determined after assessing the degree of tooth eruption and the wear and tear of teeth of the lower jaw, considering three age classes: young (under 12 months old), sub-adult (between 12 and 24 months), and adult (over 24 months old). The animal' sex was also recorded [83].
All animals were hunted in the Tuscany region during authorized hunting seasons (November-January), following the regional hunting law (Regolamento di attuazione della legge regionale 12 gennaio 1994, n. 3 D.P.G.R. 48/R/2017). No animals were specifically sacrificed for this study purpose. Animals did not present gross lesions related to infectious disease at postmortem examination, performed during sampling operations.

Microscopic Agglutination Test (MAT)
Blood samples were centrifugated at 10,000 rpm for 10 minutes to obtain the serum. In order to detect Leptospira antibodies, sera were tested through microscopic agglutination test (MAT) [84].

Leptospira spp. Isolation
Each wild boar organ was cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Difco, Detroit, MI, USA). Approximately 10 cm 3 from each organ was homogenized with 5 mL of sterile water and 1 mL of homogenate was cultured in 5 mL of EMJH. Cultures were incubated at 30 • C ± 1 • C for 120 days and observed every 10 days under dark-field microscopy to evaluate possible bacterial growth.

Molecular Analysis
From each kidney and liver, DNA was extracted using Quick-DNA Plus Kits (Zymo Research, Irvine, CA, USA) following the manufacturer's instructions.
Two different multiplex Realtime-PCR were employed; the first, targeting Leptospira spp. (16S rRNA gene) and pathogenic Leptospira (lipL32 gene), was performed on all samples [85,86]. The second protocol was only performed on positive Leptospira spp. and negative lipL32 samples, targeting intermediate Leptospira (16S rRNA gene) and saprophytic Leptospira (23S rRNA gene) [16,86]. As a positive control for the lipL32 gene, DNA extracted from a pure culture of Leptospira interrogans serogroup Pomona strain Mezzano was used. As a positive control for the 23S rRNA gene for saprophytic Leptospira, DNA extracted from a pure culture of Leptospira biflexa serogroup Patoc strain Patoc I was used. As a negative control, sterilized saline water was used. A total reaction volume of 15 µL was prepared by using 2x QuantiTect Probe PCR Master Mix (Qiagen, Hilden, Germany), 2 µM of each primer, 500 nM of each probe, and 3 µL of DNA, as previously reported [7]. The RealTime-PCR assay was performed on a Rotorgene Corbett 6000 (Corbett Research, Sydney, Australia) with the following thermal conditions: a holding stage of 95 • C for 5 min and 45 cycles of 95 • C for 15 s and 60 • C for 30 s. Samples with Ct lipL32 < 35 were considered positive and those samples with 35 < Ct lipL32 ≥ 40 were repeated.

Leptospira spp. Characterization and Genotyping
First, serogroups of the isolates were determined through the MAT using a panel of eight polyclonal anti-sera against the eight serovars reported in Section 4.2. The agglutination with specific antiserum was used to identify the presumptive strain's serogroup [84].
Moreover, the Leptospira species were identified from positive pathogenic and intermediate Leptospira PCR reactions, using primer for rrs2 gene and 16S rRNA gene, respectively [86,88].
The amplification of each target gene was realized with HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany), and further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [90]. Phylogenetic analysis was performed by the maximum likelihood method based on the Tamura-Nei model using MEGA 10 software [91].

Statistical Analysis
Data were analyzed with chi-square (X 2 ) test. The statistical test was used to evaluate the Leptospira infection ratio in relationship to sex (male or female), age class (young, sub-adult, or adult), province (Pisa, Lucca, Livorno, Grosseto, or Siena) and hunting season (2018/2019 or 2019/2020). Statistical significance threshold was set at a p value ≤ 0.05 [92].

Conclusions
In conclusion, this investigation confirms through the MAT, isolation, and molecular assays, the role of wild boar in the epidemiology of leptospirosis in Central Italy. Wild boar represents a good indicator of Leptospira circulating in a specific area where many different animal species share the same environment. Furthermore, wild boar populations are able to live in a wide spectrum of habitat types, and, have recently reached sub-urban and urban areas. In Italy, little recent data on human leptospirosis are available; however, some studies investigated the prevalence of infection in risk categories (hunters, farmers, and forestry workers) showing serological positivity to Leptospira [93,94]. Moreover, on the basis of the most recent report on human leptospirosis in Italy [95], a high infection rate was recorded in adult males, and this could indicate that leptospirosis is strictly related to worker activity. Hunters, for example, are usually all male and over 30 years old. In particular, these peoples are exposed to an high risk of infection due to management and slaughtering of dead animals being performed with little health care [96].
Tarassovi and Bratislava are the two main serogroups that circulate within wild boar in Tuscany. Although Bratislava has been more detected, the isolation of Tarassovi suggests that wild boar could be the main reservoir. In addition, as for pathogenic Leptospira, the presence of intermediate species in wild boar kidney underlines the need to perform other studies aimed at understanding the newlyemerging species, L. fainei, in animals and in humans.
Funding: This research was funded by University of Pisa, grant number PRA_2018_56.