Molecular Typing of Pathogenic Leptospira Species Isolated from Wild Mammal Reservoirs in Sardinia

Simple Summary Leptospirosis is caused by pathogenic spirochaetes of the genus Leptospira. Humans can become infected with these bacteria through direct contact with urine from infected animals or indirectly through interaction with a urine contaminated environment. Among wildlife species, rodents are considered the primary reservoir hosts for leptospirosis in rural and urban environments. Epidemiological data, regarding leptospirosis in various wild species in Europe, suggest that these animals play a different role in leptospiral persistence. Unfortunately, studies on the presence and typing of Leptospira species in wild mammals are lacking in Sardinia. The aim of the present study was to investigate the prevalence of Leptospira species in wild mammals. Kidneys collected from carcasses were analyzed by culture and molecular testing. Greater positivity was found in hedgehogs, followed by weasels and rodents. The results obtained suggest that Sardinian fauna may play a possible sentinel role in the transmission cycle of leptospirosis to humans. Gathering this information in different wildlife species is crucial for better understanding of the epidemiology of leptospirosis and for the development of appropriate prevention measures. Abstract Leptospirosis is a global zoonosis caused by pathogenic species of Leptospira that infect a large spectrum of domestic and wild animals. This study is the first molecular identification, characterization, and phylogeny of Leptospira strains with veterinary and zoonotic impact in Sardinian wild hosts. All samples collected were cultured and analyzed by multiplex real time polymerase chain reaction (qPCR). Sequencing, phylogenetic analyses (based on rrs and secY sequences), and Multilocus Sequence Typing (MLST) based on the analysis of seven concatenated loci were also performed. Results revealed the detection of Leptospira DNA and cultured isolates in 21% and 4% of the samples examined, respectively. Sequence analysis of Leptospira positive samples highlighted the presence of the interrogans and borgpetersenii genospecies that grouped in strongly supported monophyletic clades. MLST analyses identified six different Sequence Types (ST) that clustered in two monophyletic groups specific for Leptospira interrogans, and L. borgpetersenii. This study provided about the prevalence of leptospires in wild mammals in Sardinia, and increased our knowledge of this pathogen on the island. Monitoring Leptospira strains circulating in Sardinia will help clinicians and veterinarians develop strategic plans for the prevention and control of leptospiral infections.


Introduction
Leptospirosis, which is caused by pathogenic spirochaetes of the genus Leptospira, is a re-emerging zoonotic disease with veterinary and public health importance due to its cross-over between humans, domestic animals, and wildlife. Leptospirosis cases have been reported worldwide, in particular in the tropical regions of South and Southeast Asia [1], Africa [2], Western Pacific [3], and Central and North America [4]. It has been estimated that more than one million cases occur each year, including about 60,000 deaths [5].
The most recent advances in the description of Leptospira phylogeny have resulted from the study of Vincent et al. [6] in which new species of Leptospira belonging to subclades P1 and P2 have been classified on the basis of their pathogenicity. To date, at least 64 different Leptospira species have been validated worldwide, based on the average nucleotide identity (ANI) values of their genomes.
The main reservoirs of pathogenic Leptospira species are rodents; however, more recently, an increasing number of vertebrate and invertebrate hosts have also been reported to shed this pathogen in their urine. Among rodents, rats represent the main reservoir of pathogenic Leptospira, while wild and domestic mammals [7,8], livestock [9,10], amphibians [11], reptiles [12], ticks [13], and bats [14] also appear to play an important role in the spread of the leptospires. Most human infections occur following exposure to soil or water contaminated with the urine of reservoir animals [15].
In Italy, molecular studies have highlighted the presence of pathogenic Leptospira in wild boars [16] and pigs [17], porcupines [18], rodents [19][20][21], horses [22], dogs [23], and humans [24]. In Sardinia, recent isolations of Leptospira Bratislava and Pomona from wild boars [25] and marine mammals [26] suggest that these animals could potentially act as reservoirs of these serovars. To date, the current literature lacks information on the presence and spread of Leptospira species in Sardinian wildlife or the possible role that wild mammals play as maintenance or accidental hosts for these bacteria. The purpose of the present study was to (i) investigate the potential presence of Leptospira genomospecies and their sequence types (ST) in wild vertebrates, (ii) identify and genotype leptospires by 16S rRNA, secY, and Multilocus Sequence Typing (MLST), and (iii) reconstruct the phylogeny of the obtained sequences.

Sample Collection
Carcasses of small mammals, whose cause of death was related to traffic accidents or attacks from wildlife or domestic animals, were collected between January 2011 to December 2019 from 6 collection sites of North Sardinia (Sassarese, Angola, Gallura, Mantacuto, Nurra, and Goceano). Sardinia, the second largest island in the Mediterranean Sea with an area of 23,821 km 2 , is a region with a great naturalistic importance. The island is characterized by a wide diversity in geology, vegetation and landscape features, surrounded by mountains, forests, and green valleys covered by the typical Mediterranean maquis with Cistus, lentisk, myrtle, and rosemary shrubs. The landscape is also characterized by cultivated coastal plains, watercourses and rocky sheer coasts. Many areas are dedicated to rearing and grazing of sheep, goats, bovines, swine, and horses. Sardinia is also an extraordinary habitat for wild animals such as mouflons, Sardinian deer, wild pigs and foxes, and many birds. The field researchers were trained to sample the dead animals following predetermined guidelines. The carcasses were trans-ported at 4 • C to the laboratory where the general body condition of each carcass was evaluated, and only those animals that did not show obvious signs of deterioration were analyzed. Additionally, nutritional conditions, size, age, and sex of each animal was evaluated before the necropsy. Decomposed carcasses were not included in this study. A total of 387 carcasses were identified phenotypically by an expert veterinarian and included in the study [27]. Dead animals were necropsied and the kidneys were collected under sterile conditions from each animal. Specifically, 25 mg of tissue extracted between the cortical and medullary areas was immediately used for culture, the other was frozen at −20 • C for molecular investigation and characterization of leptospires.

Leptospira spp. Isolation
In order to evaluate the presence or the potential growth of leptospires, collected kidneys were homogenized by using a Stomacher bag (bag filter Avantor ® , Lutterworth, UK) and inoculated into a sterile tube (Corning-Falcon ® , Corning, NY, USA) (by using a sterile pipette) containing the commercial semi-solid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium with EMJH enrichment (DifcoTM, BD, Franklin Lakes, NJ, USA), supplemented with 5-Fluorouracil (5-FU; 2 g/L). The media was incubated aerobically at 28 • C and the growth of leptospires was examined under a dark-field microscope weekly, over a period of 3 months for the presence or potential growth of leptospires. Samples that failed to show any evidence of growth after 3 months were considered negative and were discarded [28].
Positive cultures were subjected to purification. Briefly, exceeding nucleotides and primers were inactivated by using the Applied Biosystems™ CleanSweep™ PCR Purification Reagent (Life Technologies Europe BV, Monza MB, Italy), according to vendor's recommendations. Pure isolates, free of contaminants, were used for serological and molecular identification.

Genomic DNA Extraction
DNA from the kidney was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Due to validate the extraction processes and all downstream steps, nuclease-free water and 10 fg of DNA extracted from Leptospira interrogans serovar Copenhageni (Fiocruz L1-130) were used as positive and negative controls, respectively. DNA extracted from each sample was stored at −20 • C until use.

Molecular Detection of Leptospira spp. by Multiplex qPCR, and Amplification of rrs and secY Genes
To discriminate between both pathogenic and non-pathogenic leptospires, all DNA samples were tested by multiplex qPCR using lipL32 and 16S rRNA partial target genes. More specifically, primers LipL32-45F (5 -AAGCATTACCGCTTGTGGTG-3 ), LipL32-286R (5 -GAA CTCCCA TTT CAG CGA TT-3 ), and the probeLipL32-189P (FAM-5 -AA AGC CAG GAC AAG CGCCG-3 -BHQ1) [29], were combined with primers 16S-P1 forward (5 -TAGTGAACGGGATTAGATAC-3 ), and 16S-P2 reverse (5 -GGTCTACTTAATCCGTTAGG-3 ) and probe 16S-Prob (Cy5-5 -AATCCACGCCCTAAACGTTGTCTAC-3 -BHQ2) that amplify 242 and 104 bp of the lipL32 and 16S rRNA genes, respectively. An internal control consisting of exogenous DNA added to the sample before the extraction phase. The qPCR was performed in a total volume of 20 µL containing 50 ng of Leptospira spp. genomic DNA, 250 nM of each of the forward and reverse primers, and 10 µL of 5× Master Mix QuantiFast Pathogen PCR + IC Kit (Qiagen, Milan, Italy). All the reactions were performed in duplicates on a 7500 Fast Real-Time PCR System (Applied Biosystems) under the following conditions: 95 • C for 5 min, followed by 45 cycles of denaturation at 95 • C for 15 s, and annealing and elongation for 30 s at 60 • C. A negative control (DNA extracted from water) and a positive control (DNA extracted from the reference strain of L. interrogans ATCC ® BAA1198D5TM) were included in each PCR test.
Among all positive samples obtained by qPCR, only samples with a threshold cycle (Ct) values lower than or equal to 32 were tested for further analyses. Specifically, 6 kidney samples and 17 Leptospira isolates (Table 1) were analyzed with a set of primers that amplified a fragment of 541 bp of the 16S rRNA gene, and of 549 bp of the secY partial gene [30]. Negative and positive controls were included in each test, with a negative and a positive for every 20 samples tested. The PCR reactions were performed by using a T100 Thermal Cycler (Bio-Rad apparatus). PCR products were visualized by electrophoresis in 1.5% agarose gel stained with SYBR-Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA), and examined under UV transillumination.

Sequencing and Phylogenetic Analyses
All rrs (16S rRNA gene) and secY positive amplicons were purified and directly sequenced by using an BigDye terminator cycle sequencing ready reaction kit (Life Technologies, UK). Sequences were edited with Chromas 2.2 (Technelysium, Helensvale, Australia), then aligned with Clustal X [31] in order to assign them to unique sequence types, and checked against the GenBank database with nucleotide blast (BLASTn) [32]. Multiple sequence alignments and sequence similarities were calculated using the Clustal W [33] and the identity matrix options of BioEdit [34], respectively. For phylogenetic analyses, the sequence types obtained in this study were aligned with a set of 22 sequences representing rrs and secY variability of the different species belonging to the genus Leptospira.

Characterization of Leptospira Isolates
Sequencing results performed on the rrs and secY amplicons obtained from the 17 kidney cultures and the 6 samples (23 positive samples in total) from qPCR analyses produced clear sequencing signals, with an identity superior to 99% (Table 3). Among the rrs positive samples, the resulting BLASTn analysis revealed that 9 sequences were members of L. borgpetersenii group, 13 belonged to the L. interrogans group, and 1 sequence exhibited the highest homology with the intermediate L. johnsonii (100% identity). Samples positive for the Leptospira species by using 16S rRNA target gene were also positive when tested with the set of primers targeting the protein translocase secY subunit present in Leptospira species. Sequencing of the 23 secY amplicons revealed that 14 (61%; 95% CI: 41-81%) and 9 (39%; 95% CI: 19-59%) sequences were 99-100% similar to L. interrogans and L. borgpetersenii strains, respectively. The rrs and secY sequence types, the host origin of all sequences and BLASTn identity are shown in Table 3. The MLST analysis of the 23 Leptospira strains, allowed to obtain 6 different sequence types (ST), belonging to ST149 (derived from 3 Apodemus sylvaticus, 1 Rattus rattus, 4 Erinaceus europaeus, and 1 Martes martes), ST198 (found in 1 Apodemus sylvaticus, 4 Erinaceus europaeus, 1 Martes martes, and 1 Vulpes vulpes). ST36 (from 1 Rattus rattus and 1 Rattus norvegicus), ST24 (from 1 Mustela nivalis), ST17 (from 1 Rattus rattus), and ST140 (from 1 Vulpes vulpes). MLST based on 7-loci scheme results obtained from the 23 Leptospira isolates are shown in Table 4.

Phylogenetic Analysis
Phylogenetic analysis based on the alignment of the 14 rrs sequence types obtained in this study with the 22 Leptospira reference sequences (Figure 1), identified 3 main groups representative of the pathogenic, intermediate, and saprophytic Leptospira species.
Animals 2021, 11, x FOR PEER REVIEW Figure 1. rrs-based phylogenetic analyses of the sequence types generated in this study and of 22 sequences represent of the different species of the genus Leptospira. Evolutionary history was inferred using the Neighbor-Joining method optimal tree with the sum of branch length = 0.41105704 is shown. The percentage of replicate trees in which the associ taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.
More specifically, sequence types named Seqrrs2, Seqrrs3, Seqrrs4, Seqrrs5 Seqrrs7, Seqrrs8, Seqrrs9 Seqrrs10, Seqrrs11, Seqrrs12, and Seqrrs13 grouped in a supported clade with pathogenic Leptospira strains while Seqrrs1 was inclu separate clade including the intermediate Leptospira species. The phylogene obtained by aligning the six secY sequence types and the 6 STs resulted fro analyses with the Leptospira reference strains, indicated that all isolates from th grouped with reference strains representative of L. interrogans and L. borgp respectively. It indicated that all sequences here detected can be classified w pathogenic Leptospira group. The sequence clusters obtained were statistically su by bootstrap analyses (Figures 2 and 3). More specifically, sequence types named Seqrrs2, Seqrrs3, Seqrrs4, Seqrrs5, Seqrrs6, Seqrrs7, Seqrrs8, Seqrrs9 Seqrrs10, Seqrrs11, Seqrrs12, and Seqrrs13 grouped in a strongly supported clade with pathogenic Leptospira strains while Seqrrs1 was included in a separate clade including the intermediate Leptospira species. The phylogenetic trees obtained by aligning the six secY sequence types and the 6 STs resulted from MLST analyses with the Leptospira reference strains, indicated that all isolates from this study grouped with reference strains representative of L. interrogans and L. borgpetersenii, respectively. It indicated that all sequences here detected can be classified within the pathogenic Leptospira group. The sequence clusters obtained were statistically supported by bootstrap analyses (Figures 2 and 3).

Discussion
The importance of rodents as reservoirs for a variety of Leptospira serovars has been widely studied in the world [37]. There is an increasing interest in monitoring of Leptospira spp. hosts, and studies on the prevalence of this pathogen in wild mammals are increasing over Europe. In Germany, 6% of animals tested positive for L. kirschneri and L. interrogans [38]. In France, studies conducted on reported serological positivity for L. interrogans, L. kirschneri, and L. borgpetersenii in 24 different mammalian species [39]. In Sardinia, studies on presence and typing of Leptospira species in wild mammals are still lacking.
This report examined circulating Leptospira strains in 15 different wild species, including rodents, using culture and DNA characterization tools. Our results indicate that all wild species examined are carriers of pathogenic Leptospira species in Sardinia. Pathogenic Leptospira were found with a frequency of 54% (95% CI: 38-70%) in hedgehogs, followed by mustelids with 40% (95% CI: 0-83%), end wild rodents with 21% (95% CI: 10-33%). The detection of L. interrogans serovar Australis and L. borgpetersenii serovar Ballum in hedgehogs was in agreement with other studies conducted in several European countries, including France [40], Italy [41], the Netherlands [42], and Scotland [43], which showed the presence of these Leptospira spp. from Erinaceomorphs. Moreover, pathogenic Leptospira were isolated from hedgehogs in France, as well as in China [44] recently. These findings show that hedgehogs could act as important source of pathogenic Leptospira spp. serogroup Australis and outline the importance of leptospirosis surveillance in this species. In this study, the presence of L. interrogans in the carnivore group was

Discussion
The importance of rodents as reservoirs for a variety of Leptospira serovars has been widely studied in the world [37]. There is an increasing interest in monitoring of Leptospira spp. hosts, and studies on the prevalence of this pathogen in wild mammals are increasing over Europe. In Germany, 6% of animals tested positive for L. kirschneri and L. interrogans [38]. In France, studies conducted on reported serological positivity for L. interrogans, L. kirschneri, and L. borgpetersenii in 24 different mammalian species [39]. In Sardinia, studies on presence and typing of Leptospira species in wild mammals are still lacking.
This report examined circulating Leptospira strains in 15 different wild species, including rodents, using culture and DNA characterization tools. Our results indicate that all wild species examined are carriers of pathogenic Leptospira species in Sardinia. Pathogenic Leptospira were found with a frequency of 54% (95% CI: 38-70%) in hedgehogs, followed by mustelids with 40% (95% CI: 0-83%), end wild rodents with 21% (95% CI: 10-33%). The detection of L. interrogans serovar Australis and L. borgpetersenii serovar Ballum in hedgehogs was in agreement with other studies conducted in several European countries, including France [40], Italy [41], the Netherlands [42], and Scotland [43], which showed the presence of these Leptospira spp. from Erinaceomorphs. Moreover, pathogenic Leptospira were isolated from hedgehogs in France, as well as in China [44] recently. These findings show that hedgehogs could act as important source of pathogenic Leptospira spp. serogroup Australis and outline the importance of leptospirosis surveillance in this species. In this study, the presence of L. interrogans in the carnivore group was demonstrated for the first time on the island. Among the species analyzed was Vulpes vulpes ichnusae, a Sardinian species endemic to urban and peri-urban areas, including human environments. The hunting of this species is allowed on the island, and is regulated by the Regional Law number 23 of 1998. Foxes are known to be an important source of Leptospira in Europe, where the Grippotyphosa serogroup is known as the most frequently reported in Germany [45], and the Poi and Saxkoebing serogroups, and Sejroe are the most common in Poland [46]. The presence of pathogenic Leptospira in Sardinian wild foxes needs further investigation to clarify the role of wild carnivores as a reservoir of pathogenic Leptospira serovars on the island, as well as their epidemiological role in the zoonotic cycle. Still within the carnivore group, we also report the first molecular detection of L. borgpetersenii and L. interrogans from two species of Sardinian Mustela and one Martens. Together with the hedgehog and the fox, it is among the mammals most commonly hit by cars. Additionally, for these species the results obtained are in agreement with the studies conducted in France which show that the mustelid species have the highest risk of being infected by L. interrogans, L. borgpetersenii, and L. kirschneri [40]. This study also reveals the presence of L. interrogans in Sardinian rodents (Rattus norvegicus, Rattus rattus, and Apodemus sylvaticus). It has been known that wild rats (Rattus spp.) Are the most important sources of Leptospira infection, as they are abundant in urban and peri-domestic environments [39]. The brown rat is reported to be the primary host of L. interrogans related to the serogroup Icterohaemorrhagiae, which is responsible for the most severe forms of the disease in humans [47,48]. With the identification of 14 different rrs sequences, 6 for the secY gene, and 6 MLST profiles, our study reflects the wide diversity of Leptospira genotypes circulating in wildlife. Only 6 samples from biological matrix were added to the molecular analysis, the remaining 74 positive sample for Leptospira could not be amplified, most likely due to low DNA concentrations.
In the present study, the use of the rrs and secY genes represented a useful tool for detecting the Leptospira genomospecies in wild mammals and allows differentiating members of the pathogenic, intermediate and saprophytic Leptospira group. The 3 types obtained showed an obvious phylogenetic divergence from all recognized species belonging to the 3 clades of Leptospira and it was in accordance with previous studies [6,49]. Results obtained by using rrs and secY genes are the same as those obtained by MLST analyses except for those obtained from 1 Mustela nivalis. The partial ribosomal 16S gene sequence detected in this mammal hosts, was identical to that of a species already isolated from soil in Japan namely L. johnsonii and belonging to the intermediate clade [50]. However, the presence of this P2 intermediate species were not confirmed with secY and MLST analyses, indicating that the molecular identification of Leptospira strains needs the use of further genetic markers in order to confirm these results.

Conclusions
Our results show that several pathogenic strains of Leptospira are circulating in Sardinian fauna. Information on the possible role as sentinel or reservoirs of wild mammals is critical to understand the possible zoonotic potential of Leptospira. Therefore, the characterization of the genetic diversity of Leptospira strains is fundamental to designing epidemiological studies and control strategies for leptospirosis in the same area. Further studies are needed to better characterize isolates by analyzing more discriminative genes, and to identify the main reservoirs of Leptospira strains in Sardinia island.