Serological and Molecular Evidence of Pathogenic Leptospira spp. in Stray Dogs and Cats of Sicily (South Italy), 2017–2021

Leptospirosis is a zoonosis of public health concern. Its prevalence in stray animals in the South of Italy is unknown. This study aimed to investigate Leptospira spp. prevalence in 1009 stray animals. Out of them, 749 were alive animals, including 358 dogs (316 from Palermo and 42 from Ragusa) and 391 cats (359 from Palermo and 32 from Ragusa), and 260 were corpses (216 dogs and 44 cats) randomly collected in Sicily. Dogs and cats underwent a serological screening by Microscopic Agglutination Test and a molecular investigation by Real-Time PCR targeting lipL32. Corpses were subjected to Real-Time PCR. Serological analyses showed a prevalence of 1.12% (4/358) for dogs and 0.26% (1/391) for cats, with the only positive cat coming from Palermo. Serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni, followed by Canicola and Bratislava, were the most spread among dogs, while the serological positive cat reacted with Hardjo serogroup. Two urine (2/32, 6.25%) and one blood (1/391, 0.26%) samples of cats, all from Ragusa, were positive at Real-Time PCR for pathogenic Leptospira spp. Sequencing analyses showed the presence of L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni in one of the positive urine samples and in the positive blood sample. Analyses on corpses showed a prevalence of 1.85% (4/216) in Sicilian dog kidney samples, while all corpses of cats resulted in negative. Genotyping analysis showed a genetic relatedness between cat and human isolates. Results show Leptospira spp. circulation among Sicilian stray animals. The genetic relatedness between cat and human isolates suggests a possible common infection source.


Introduction
Leptospirosis is an important zoonotic disease caused by infection with pathogenic spirocheaete bacteria belonging to Leptospira genus. Traditionally, the classification of Leptospires was based on their antigenic phenotype: more than 250 pathogenic Leptospira serovars are known. These are classified into 25 different serogroups. More recently, the biomolecular approach based on DNA homology has permitted us to identify 17 pathogenic species of Leptospira spp., seven of which (L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri, L. alexanderi) are the most common agents causing disease in human and a wide variety of wild and domestic animals [1][2][3][4].
The infection is spread worldwide, particularly in Tropical areas, where many environmental and cultural conditions favorable for its transmission occur, such as the close coexistence between people and wild or domestic animals, as well as a predominantly warm-humid climate [5]. However, it can also occur in urban environments of industrialized countries or in temperate rural contests -with a seasonal trend characterized by a maximum incidence in warm and rainy months. Reservoir animals harbor Leptospires in their kidneys for a long time, often without symptoms, shedding them in the environment through urinary elimination and playing an important role as a source of infection [6,7]. Animals can develop variable clinical signs such as fever, renal and hepatic failure, and reproductive disorders [8]. As concerning the association of serovar-reservoir, many epidemiological studies reported specific animal species might act as the reservoir for particular Leptospira serovars [9].
Rodents are the main reservoir hosts of pathogenic Leptospira serovar Icterohaemorrhagiae, along with other marsupial and mammalian species [10]. Dogs are susceptible to a wide range of serovars. In Europe, the major ones detected in dogs are Icterohaemorrhagiae, Grippotyphosa, Australis, Sejroe, and Canicola [11]. Infection with one of these serovars frequently leads to severe clinical symptoms [12,13]. However, it is difficult to determine if susceptibility in dogs can be related to age, breed, or sex [14]. Among the main serovars detected in dogs, Canicola recognizes the dog as a reservoir host, representing a major concern in a One Health vision. On the other hand, mass vaccination of dogs has contributed to the decrease of infection by Leptospira interrogans serogroup Canicola serovar Canicola in Europe [12]. Different kinds of dog vaccines are available in Italy: bivalent vaccines containing two antigen strains, belonging respectively to two serovars (Canicola and Icterohaemorrhagiae), a trivalent vaccine containing three antigen strains, belonging to three serovars (Canicola, Icterohaemorrhagiae, and Grippotyphosa), and tetravalent vaccines containing four antigen strains, belonging to four serovars (Canicola, Icterohaemorrhagiae, Grippotyphosa, and Bratislava).
In cats, clinical signs are rare. The main serovars reported in Europe in these animals are Icterohaemorrhagiae, Canicola, Grippotyphosa, Pomona, Hardjo, Autumnalis, Ballum, and Bratislava. Probably cats become infected by catching reservoir animals, such as rats, mice, and voles infected with pathogenic strains of Leptospira [15]. However, no association has been found between serological positivity and sex or breed. Several studies reported instead an association with age, being a serological response to leptospira more frequent in old cats living outdoors or sharing the same household with another cat [16].
Moreover, both infected dogs and cats can shed Leptospires in the environment through their urine [16][17][18][19], potentially contributing to the infection spreading.
The Microscopic Agglutination Test (MAT) has been widely employed for the serodiagnosis of acute disease and is considered the gold standard to confirm leptospiral infection [20]. However, its partial inability to identify the infecting serovar and to differentiate the vaccine from natural exposure-induced titers have limited its use in a clinical setting [21]. PCR-based techniques have been successfully used to confirm infection at the early stages of the disease, as well as to identify infecting species by further sequencing of PCR amplicons. Currently, genotyping by multi-locus sequence typing (MLST) has been also used to establish Leptospira presence from various clinical samples [22][23][24]. However, PCR may not provide reliable results in the convalescent phase of the disease [25].
Leptospirosis still remains a major health problem and dogs and cats can play a sentinel role or can highlight the risk of exposure to humans. The aim of this study was to carry out a serological and molecular investigation to improve knowledge about the circulation of Leptospira strains in stray dogs and cats in Sicily.

Ethical Statement
All methods were carried out in accordance with relevant guidelines and regulations. The study did not involve any animal experiments. Sample collection was carried out for laboratory analyses and did not involve any suffering of the sampled animals. This study was conducted as part of the IZS SI 09/15 RC research project approved by the Italian Ministry of Health on 29 July 2016 (DGSAF-0018379-P-29/07/2016) and of the IZS SI 11/16 RC research project approved by the Italian Ministry of Health on 9 August 2017 (DGSAF-0018913-P-09/08/2017).

Study Design and Sample Collection
A total of 1009 biological samples from 574 stray dogs and 435 stray cats, were tested for this study between November 2017 and December 2021 (Table 1). Particularly, these two groups included: 358 asymptomatic alive dogs, out of which 316 samples were from Palermo city and province (North West Sicily) and 42 samples from Ragusa city and province (South Sicily); 391 asymptomatic alive cats, out of which 359 from Palermo area and 32 from Ragusa area. All the dogs from Ragusa were unvaccinated, like most of those from Palermo. Cats were all not vaccinated.
In addition, samples included 216 dog corpses and 44 cat corpses, randomly collected throughout the Sicilian territory and transferred to the Laboratory of Istituto Zooprofilattico Sperimentale (IZS) of Sicily for the diagnosis (Table 1). In particular, among dog corpses, one showed clinical signs attributable to leptospirosis. The signs included the typical jaundice color of the skin, and kidney and liver failure.
Ethylenediaminetetraacetic acid (EDTA)-treated and untreated blood samples and urine samples (only from Ragusa) were obtained from each animal by public veterinarians during the sterilization, as part of the National Control Plan for the prevention of stray animals; while the kidney samples were collected from carcasses.
The blood samples were stored at 4 • C and centrifugated at 3000 rpm for 10 min at room temperature for serum separation. Serum samples were frozen at −20 • C until further serological analyses.
Samples from dogs and cats were subjected to both molecular and serological analyses, while only molecular investigations were made possible on corpses collected from the whole region.
Each antigen was a reference strain obtained by the National Centre for Leptospirosis IZS of Lombardia and Emilia Romagna "Bruno Ubertini" (IZSLER, Brescia, Italy) or by the Amsterdam University Medical Centers and was maintained in Leptospira Medium Base Ellinghausen-MacCullough-Johnson-Harris (EMJH-Difco, Becton, Dickinson and Company, Sparks, MD, USA), subcultured every 7-10 days and checked for purity, mobility and agglutination power before the use. The MAT was performed according to the World Organization for Animal Health (WOAH) guidelines [26,27]. Briefly, the MAT consists of a screening and a confirmation phase. In the first screening phase, all sera were diluted 1:100 using phosphate-buffered saline (PBS) at a pH of 7.6 and incubated for 2 to 4 h at 30 • C ± 2 • C with each Leptospira serovar in 1:1 proportion. The presence of microagglutination (positivity index) was verified by microscope observation in a dark field, with a 10× magnification, (Leica Microsystems, Wetzlar, Germany). Samples were considered positive when 50% or more Leptospires were agglutinated. MAT cut-off was 1:100. Therefore, each positive sample was successively titrated performing a series of double dilutions (from 1:100 to 1:6400) and incubating each dilution with the reactive serovar at 30 • C ± 2 • C for 2 h. The analysis was performed by microscopy observation. The titration was used to verify the last useful dilution for serological positivity. The serovar presenting the highest titer was considered the reactive serovar. Both in the screening and titration phase, four different controls were used: positive control (100% microagglutination) with positive sera for antibodies against the strains used; negative control (0% microagglutination) with negative serum for antibodies against Leptospira spp.; 50% microagglutination reading control for each antigen; antigen control (0% microagglutination) with only the antigens diluted 1:2 with physiological solution.

DNA Extraction and Real-Time PCR Detection
For DNA extraction from kidneys, the surface was flamed, and 1 g of tissue was withdrawn and homogenized in 9 mL of sterile physiological solution with Stomacher ® 80 Biomaster (Seward Limited, London, UK). For the urines, a volume of 2 mL was centrifuged at the rate of 12,000 g for 30 min and the pellet was resuspended with 0.2 mL of ultrapure distilled water.
DNA was extracted from 0.2 mL of blood or homogenized kidney or resuspended pellet from urines. Lysozyme 10 mg/mL (Roche, Basel, Switzerland) was added to each sample and incubated at 37 • C for 30 min as previously described [28]. DNA extraction was carried out using the PureLink Genomic DNA kit (Invitrogen, Paisley, UK), according to the manufacturer's instructions. The extraction internal control (IC) included in the Quantifast Pathogen + IC Kit (Qiagen, Hilden, Germany) was used (0.1 µL per µL of elution volume). The DNA was stored at −80 • C until use.
A multiplex Taqman-based Real-time PCR assay targeting the Leptospira genus-specific 16 S ribosomal RNA gene (rRNA gene) and the pathogen-specific lipL32 gene present on the external membrane of pathogenic Leptospira species was performed, using primers described previously [29,30].
The PCR was performed in a 25 µL final volume, using 5 µL of extracted DNA, 5 µL of 5× Mastermix Quantifast (Quantifast Pathogen + IC Kit, Qiagen, Hilden, Germany), 700 nM of primers, and 200 nM of the probe. All extraction sessions included a negative control (water) and all amplification sessions included both a negative (water) and a positive control (DNA of Leptospira interrogans serogroup Australis serovar Bratislava strain supplied by the National Reference Center for Leptospirosis, IZS LER, Brescia, Italy and maintained at our laboratories). The assay was performed on a Bio-Rad CFX96 System using 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.
Partial rpoB gene sequencing was performed for the identification of Leptospira species [31]. Analyses were conducted using the commercial GoTaq ® G2 DNA Polymerase (Promega Corporation, Milan, Italy) in a 25 µL reaction mix consisting of 5 µL of a 5× GoTaq ® Reaction Buffer, 1 µL of a dNTP mix (200 µM), 0.6 µL of each primer (0.5 µM), 0.125 µL of GoTaq ® G2 DNA Polymerase, and 5 of extracted DNA. Amplification was conducted under the following thermal conditions: 95 • C for 2 min to activate TaqPol followed by, 35 cycles of 94 • C for 30 s, 51 • C for 30 s, 72 • C for 30 s, and a final extension of 72 • C for 7 min and further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software version 4.0 [32].
The sequences of primers and probes are presented in Table 2.
Confidence intervals (CI 95%) of the positive results were calculated for proportions.

MLST Genotyping Analysis
Real-time PCR-positive samples were submitted to the IZSLER, Brescia for MLST analyses [33].
A phylogenetic tree was built using MEGA X [35] on the concatamers of the seven MLST genes. The evolutionary history was inferred using the Neighbor-Joining method [36]. The evolutionary distances were computed using the Maximum Composite Likelihood method [37].
In particular, all 32 cats from the Ragusa area were negative at MAT for Leptospira pathogenic strains. One of 359 (0.28% CI 95% −0.002-0.008%) cats from the Palermo area resulted positive at MAT for Leptospira interrogans serogroup Sejroe serovar Hardjo, even if only with a titer 1:100 (Table 3).

Molecular Detection and Genotyping Analyses
All blood and urine samples collected from dogs both in Palermo and Ragusa areas resulted in negative by Real-Time PCR.

Sequencing Analysis
For cats, sequencing analysis performed on one of the positive urine samples and on the positive blood showed the presence of L. interrogans serogroup Icterohaemorrhagiae. Sequencing analysis performed on the positive kidneys from the dog corpse showed the presence of L. interrogans serogroup Icterohaemorrhagiae.

Genotyping Analysis
Genotyping analysis was carried out on the DNA of Leptospira extracted from the urine (Cat B, ID 3598/2019) and the blood (Cat D, ID 3606/2019) of two cats and from the kidneys of the dog (Dog H, ID 79788/2021) revealed the presence of strains belonging to ST17, referred to L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni as showed in Figure 1.
Nucleotide sequences were submitted to the GenBank database under the following accession numbers: OP598830-OP598850.
Phylogenetic analysis carried out using the concatemer of the seven MLST genes showed that Leptospira strains circulate on Sicilian cats, clustered with the serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae (or Copenhageni).
Genotyping carried out on the positive dog kidneys revealed the identity of L. interrogans Icterohaemorrhagiae as shown in Figure 2, confirming MAT results.

Genotyping Analysis
Genotyping analysis was carried out on the DNA of Leptospira extracted from the urine (Cat B, ID 3598/2019) and the blood (Cat D, ID 3606/2019) of two cats and from the kidneys of the dog (Dog H, ID 79788/2021) revealed the presence of strains belonging to ST17, referred to L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni as showed in Figure 1.
Nucleotide sequences were submitted to the GenBank database under the following accession numbers: OP598830-OP598850.
Phylogenetic analysis carried out using the concatemer of the seven MLST genes showed that Leptospira strains circulate on Sicilian cats, clustered with the serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae (or Copenhageni).
Genotyping carried out on the positive dog kidneys revealed the identity of L. interrogans Icterohaemorrhagiae as shown in Figure 2, confirming MAT results.
The identified genotype ST17 indicated the presence of L. interrogans serogroup Icterohaemorrhagiae both in cats and dogs (Figure 3).   Reference strains are indicated with: the sequence type number, the species, the serogroup, the serovar, and the name of the strain. The evolutionary history was inferred using the Neighbor-Joining method [38]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [38]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree [39]. There were a total of 3111 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [35]. Reference strains are indicated with: the sequence type number, the species, the serogroup, the serovar, and the name of the strain. The evolutionary history was inferred using the Neighbor-Joining method [38]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [38]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree [39]. There were a total of 3111 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [35]. The identified genotype ST17 indicated the presence of L. interrogans serogroup Icterohaemorrhagiae both in cats and dogs (Figure 3).

Discussion
This study investigated the seroprevalence of Leptospira antibodies and identified by molecular methods the infective Leptospira serovars in stray dogs and cats in southern Italy (Sicily).
Serological positivity emerged by using MAT in 4 of the 358 dogs in Sicily with titers ranging from 1:100 to 1:3200; serovars Copenhageni, Bratislava, and Canicola were prevalent, including the same strains of the tetravalent vaccine formulation in trade. The MAT is considered the serological test of choice for leptospirosis [40], despite its technical limitations, such as the inability to distinguish between present and past infection [41] and non-specific cross-reactions, such as those linked to a recent dog vaccination or recent infection [42]. A serological positivity with a minimum 1:100 antibody titer leads to a leptospirosis sospicious.
In Italy, recent surveys conducted by MAT on dogs reported seroprevalence values ranging from less than 10% to over 20% [43,44]. In these studies, the most prevalent serogroups were Icterohaemorrhagiae, Grippotyphosa, Australis, and Canicola, and a low number of positive sera were also observed for Pomona and Sejroe serogroups.
Leptospira spp. presence on dogs was confirmed by Real-Time PCR: pathogenic strain DNA was detected in 4/216 kidneys from dog carcasses.
Sequencing analysis showed the presence of L. interrogans serogroup Icterohaemorrhagiae on the positive kidneys from the corpse sent with the clinical suspect of leptospirosis. Our results corroborate previous serological findings showing that Icterohaemorrhagiae is likely the main infecting serogroup in dogs [25,45], reinforcing the hypothesis that dogs are highly exposed to environmental contamination promoted by rodents. Serogroup Icterohaemorrhagiae is also a causative agent for human leptospirosis [46] and

Discussion
This study investigated the seroprevalence of Leptospira antibodies and identified by molecular methods the infective Leptospira serovars in stray dogs and cats in southern Italy (Sicily).
Serological positivity emerged by using MAT in 4 of the 358 dogs in Sicily with titers ranging from 1:100 to 1:3200; serovars Copenhageni, Bratislava, and Canicola were prevalent, including the same strains of the tetravalent vaccine formulation in trade. The MAT is considered the serological test of choice for leptospirosis [40], despite its technical limitations, such as the inability to distinguish between present and past infection [41] and non-specific cross-reactions, such as those linked to a recent dog vaccination or recent infection [42]. A serological positivity with a minimum 1:100 antibody titer leads to a leptospirosis sospicious.
In Italy, recent surveys conducted by MAT on dogs reported seroprevalence values ranging from less than 10% to over 20% [43,44]. In these studies, the most prevalent serogroups were Icterohaemorrhagiae, Grippotyphosa, Australis, and Canicola, and a low number of positive sera were also observed for Pomona and Sejroe serogroups.
Leptospira spp. presence on dogs was confirmed by Real-Time PCR: pathogenic strain DNA was detected in 4/216 kidneys from dog carcasses.
Sequencing analysis showed the presence of L. interrogans serogroup Icterohaemorrhagiae on the positive kidneys from the corpse sent with the clinical suspect of leptospirosis. Our results corroborate previous serological findings showing that Icterohaemorrhagiae is likely the main infecting serogroup in dogs [25,45], reinforcing the hypothesis that dogs are highly exposed to environmental contamination promoted by rodents. Serogroup Icterohaemorrhagiae is also a causative agent for human leptospirosis [46] and serovars Icterohaemorrhagiae and Copenhageni are reported as the main leptospiral causative agents in humans [47].
The vaccination against serogroup Icterohaemorrhagiae has been used for many years in Italy, contributing to preventing the onset of the disease in animals, however, as shown also by this study, the circulation of the pathogen is still high among dogs due to the role of rats as reservoirs. On the contrary, vaccination against Leptospira interrogans serogroup Canicola serovar Canicola allowed reducing notably the circulation of this serovar in Italy [48,49] as well as in the rest of Europe [12].
Regarding cats, one of 359 samples from Palermo resulted positive at MAT for Leptospira interrogans serogroup Hardjo serovar Sejroe, even if only with a 1:100 titer. In a previous study carried out in the Sicilian territory, Grippi et al., 2017 [50] found one cat sample (0.1%) positive for antibodies against Leptospira spp. with a MAT titer 1:100 for Leptospira Australis. Formerly, cat resistance against infections caused by spirochaetes was thought of without considering feline leptospirosis in the differential diagnosis in cats [13]. Antibodies detection in cats has shown infection can occur also among cats and these can be incidental hosts of some Leptospira serovars prevalent in wildlife or in other domestic animals [15], such as Ballum, Copenhageni, Hardjo, Icterohaemorragiae, Rachmati, Bratislava, Bataviae, Canicola, Autumnalis and Grippotyphosa [51].
The identified genotype ST17, indicating the presence of L. interrogans serogroup Icterohaemorrhagiae, was confirmed as the major cause of leptospirosis in cats and dogs. Previous serological surveys have consistently shown Icterohaemorrhagiae as the most reactive serogroup in dogs suspected of having leptospirosis [53].
The results also confirm the usefulness of using multiple diagnostic approaches to confirm leptospirosis. Indeed, the used Real-time PCR method can detect all pathogenic Leptospira species currently known [54,55] with efficiency, sensitivity, and specificity, allowing to overcome some limits of serological investigation (e.g., failure of an early diagnosis, low sensitivity). On the other hand, routine PCR does not allow for identifying the species/serovar involved, so serology has still a relevant role in epidemiological studies also allowing for cost containment. The importance of using multiple diagnostics is further strengthened by the occurrence of different phases during leptospirosis infection (leptospiraemia, leptospiruria, the last often intermittent), so PCR can lead to false negative results.

Conclusions
The results of this study show Leptospira spp. circulation among Sicilian stray animals. In particular, serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae and Copenhageni, followed by Canicola and Bratislava, were found in dog samples. One serological-positive cat reacted with the serogroup Hardjo, even if at a low titer. In addition, we detected two cat urine samples and a blood sample positive for pathogenic Leptospira spp. DNA. Sequencing analysis identified L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni in one of the positive urine samples as well as in the blood sample. Genotyping analysis showed a genetic relatedness between cat and human isolates. Among corpses, Leptospira spp. DNA was detected in the kidneys of four dogs, including in the one with leptospirosis clinical signs. Genotyping carried out on this last sample revealed identity to L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni.
To the best of our knowledge, this is the first report of Leptospira interrogans serogroup Icterohaemorrhagiae in cats in Italy. Further investigations will help understand the true role of Leptospira in cats.