Leptospira interrogans Serogroup Pomona in a Dairy Cattle Farm in a Multi-Host Zootechnical System

Bovine leptospirosis is an infectious zoonotic disease causing reproductive problems and economic losses in livestock. This work reports, for the first time in Sicily (South Italy), an outbreak of Leptospira interrogans serogroup Pomona that occurred in cattle farms within the Nebrodi Park and was mainly characterized by full-term abortion. Blood and urine samples were collected at different time points from animals of six different farms (Farms A–F) sharing the same grazing area. Research of antibodies against pathogenic Leptospira species in serum samples was carried out via Micro Agglutination Test (MAT). Urine samples were subjected to pathogen isolation and molecular analyses via TaqMan Real Time-PCR. Genotyping of Leptospira species was obtained by Multi-locus sequence typing. MAT detected antibodies against Leptospira interrogans serogroup Pomona in serum samples of all the farms. Pathogenic Leptospira spp. DNA and culture isolation was obtained from urine samples. Genotyping confirmed the excretion of L. interrogans serogroup Pomona. This study describes clinical manifestations, diagnostic implications and epidemiological characteristics of an outbreak in cattle due to L. interrogans Pomona in a protected multi-host area, where domestic and wild animals share the same habitat, suggesting a role of wild species in transmission and persistence of Pomona serogroup among cattle.


Introduction
Leptospirosis is a zoonosis with a worldwide distribution [1,2]. It is caused by pathogenic helical spirochetes of the Leptospira genus (family Leptospiraceae, order Spirochaetales). The pathogen may affect several species of domestic and wild animals as well as humans [3][4][5][6][7]. In susceptible hosts, the clinical manifestations range from severe conditions to mild febrile symptoms or asymptomatic conditions [8,9]. After the bacteremia, the pathogen is able to colonize the kidneys and it is released in the urine, which thus represent the most common contamination route for Leptospira species [10].

Farms
Outbreak identification occurred in January 2019 in a dairy herd (Farm A) in the Nebrodi Park, a protected area located in Northeastern Sicily (South Italy), following the report of abortions and fertility disorders. The farm was included in a livestock production facility consisting of four municipal housing structures shared among different breeders, located at a distance of 500 m from each other (Figure 1). Vet. Sci. 2022, 9, x FOR PEER REVIEW 3 of 15

Farms
Outbreak identification occurred in January 2019 in a dairy herd (Farm A) in the Nebrodi Park, a protected area located in Northeastern Sicily (South Italy), following the report of abortions and fertility disorders. The farm was included in a livestock production facility consisting of four municipal housing structures shared among different breeders, located at a distance of 500 m from each other (Figure 1). Farm A consisted of 33 Simmental cattle and extended over an area of about 1000 square meters, separated from the other structures by a difference in height and walls. The farm had an independent water supply and a biosecurity plan. Lactating animals were sheltered at night and led to pasture during the day in a surrounding 3 km radius Farm A consisted of 33 Simmental cattle and extended over an area of about 1000 square meters, separated from the other structures by a difference in height and walls. The farm had an independent water supply and a biosecurity plan. Lactating animals were sheltered at night and led to pasture during the day in a surrounding 3 km radius area. Contacts with other species, such as dogs, domestic pigs and wild boars, the latter endemic in the area, were likely.
The other farms, B (52 heads), C (17 heads), D (2 heads) and E (2 heads), sited in separate sheds of the same zootechnical complex, were considered as the same epidemiological unit. All farms had a semi-wild farming system of cow/calf for meat production, grazed in promiscuity with other animal species and sheltered in the coldest periods of the year. The unit did not present biocontainment plans. The grazing land and the water sources fall within the same perimeter as farm A, but watering points were not shared, although the presence of small ponds in the field, where all animals could drink from, or water streams shared between cattle and wildlife were transiently present, especially during the winter season, when the rains were more frequent. The last herd, Farm F (66 heads), was located about 1.2 km from the livestock complex, but it was included in the same epidemiological unit since its grazing areas were contiguous with herd A. The herd contained a nucleus of Angus breed cattle from Eastern Europe introduced about 2 years before.
No vaccination protocols for leptospirosis were adopted in the farms.

Sampling
Blood samples were obtained by venipuncture of the coccygeal vein. In farm A, 33 cows were sampled at three different time points: on day 0 (T0), which occurred seven days after the first signs of abortion were noticed, on day 90 (T1), which occurred after antibiotic treatment, and on day 120 (T2). In farm B, the 52 cattle were sampled at T0 and T1 without having received any antibiotic treatment, while in farms C, D, E and F a unique blood sampling was carried out on all the animals (T0).
Urine samples were collected by spontaneous urination to perform Leptospira species detection by culture and molecular methods. In detail, for farm A, 16 and 24 urine samples were collected at T0 and T1, respectively; for farm B, 2 and 20 urine samples were collected at T0 and T1; in farms C, D and E, respectively, 3, 2 and 2 urine samples were taken, while no urine samples were collected in farm F. No urine samples were collected at T2.
Milk samples were collected from all the cows of farm A and were subjected to Leptospira spp. research by culture examination and molecular methods. In addition, two aborted fetuses (heart, lung, brain, spleen, liver), together with the placenta and uterovaginal discharge, were collected from herd A and subjected to culture examination and DNA research of Leptospira species.
Soil and water samples and the blood of a dog present in farm A were also sampled and subjected to molecular analyses.

Differential Diagnosis
In order to determine the cause of abortion, different serological tests against the main abortion agents were performed.
The serological response to Brucella spp. was assessed by Rose Bengal Test (RBT), and Complement Fixation Test (CFT), according to standard OIE procedures [35].
All serum samples were tested for antibodies against Coxiella burnetii, Neopsora caninum, Infectious Bovine Rhinotracheitis or bovine herpesvirus (IBR or BHV) and Bovine Viral Diarrhoea (BVD) by enzyme-linked immunosorbent assays (ELISA) using commercial test kits and following the manufacturer's instructions. The corresponding values for optical density were recorded by a 96-well microplates reader. Specific details of the ELISA kits, along with the sensitivities and specificities of the assays, are shown in Table 1.

Serological Test for Leptospirosis
The gold standard microscopic agglutination test (MAT) was carried out on all the serum samples according to the OIE Manual of Terrestrial Animals [2,36].
Cultured Leptospira spp. strains belonging to the eight pathogenic serogroups circulating in Italy were provided by the National Center for Leptospirosis (IZS LER, Brescia, Italy) and were used for sample agglutination [16]. In particular, they included L. interrogans A MAT cut-off of 1:100 was used to identify positive samples and two-fold serial dilutions starting from 1:100 up to 1:6400 were used for titration of positive sera. The titer showing at least 50% agglutination of leptospires was considered the sample titer.

Culture Examination/Isolation of Leptospira spp.
The isolation procedure was performed according to the OIE [36], using selective liquid EMJH and selective semisolid EMJH. Each urine sample was collected in EMJH medium (dilution 1:10) selective for Leptospira species and delivered to the Laboratory at room temperature within six hours from collection. Each urine inoculum was further diluted with selective liquid EMJH (dilution 10 −2 ) and with selective semisolid EMJH (dilutions 10 −3 and 10 −4 ). Abortion tissue samples were transported at room temperature, the outer part was flamed and 1 g of sample was withdrawn, and 9 mL of sterile physiologic solution was added and manually homogenized. Subsequently, 1 mL of homogenate (dilution of 10 −1 ) was subjected to further dilutions (up to 10 −4 ) as described for urine samples.
Inocula were checked every 20 days under a darkfield microscope with a 10 × objective. To define a negative sample, inocula were further renewed in liquid and semi-solid selective EMJH, up to a maximum of six months. Leptospira positive isolations were subjected to molecular investigation.

Molecular Tests for Leptospirosis
Molecular investigations were carried out on urine samples, urine inocula, placenta, utero-vaginal discharge, milk, soil and water samples. For DNA extraction from placenta, the surface was flamed and 1 g of tissue withdrawn and homogenized in 9 mL of sterile physiological solution with Stomacher ® 80 Biomaster (Seward Limited, London, UK).
Lysozyme 10 mg/mL (Roche, Linscott, USA) was added to each sample and incubated at 37 • C for 30 min. DNA extraction was carried out using the Purelink Genomic DNA Kit (ThermoFisher Scientific, Rodano, Italy) according to the manufacturer's instructions. For each sample, the extraction internal control (IC) included in the Quantifast Pathogen + IC Kit (Qiagen, Hilden, Germany) was used. A Taqman-based Real Time-PCR was carried out to detect pathogenic Leptospira species by amplifying a lipL32 gene fragment, which encodes the outer membrane protein Lipoprotein L32, present only in pathogenic Leptospira species [37].
Primers and probe sequences are reported in Table 2. Sterile physiological solution was used as a negative extraction control. Sterile MilliQ water and DNA extracted from one of the eight pathogenic Leptospira cultured strains were used as negative and positive amplification control, respectively. Table 2. Nucleotide sequences of primers and hydrolysis probe used for the amplification of a lipL32 gene fragment from pathogenic Leptospira species.

Oligonucleotide
Target The amplification program included a denaturation step (95 • C for 5 min) and 45 amplification cycles (denaturation at 95 • C for 15 s and annealing and extension at 60 • C for 30 s).
A phylogenetic tree was built using the concatemer of the seven MLST genes linked in the followed order: glmU-pntA-sucA-tpiA-pfkB-mreA-caiB. The evolutionary history was inferred using the Neighbor-Joining method [42]. The optimal tree with the sum of branch length = 0.18381177 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [43]. 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. The evolutionary distances were computed using the Maximum Composite Likelihood method [44] and are in the units of the number of base substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X [45].

Differential Diagnosis
Two animals from farm A resulted positive for N. canimun and one for C. burnetii. None of these showed signs of abortion.
Six animals showed a positive reaction for IBR specific gB and a negative reaction for IBR specific gE.
All the animals resulted negative for Brucella spp. and BVD.

Serological Results for Leptospirosis
In farm A, nine cows showed full-term abortion, followed by placental retention and reduced milk production. Out of the 33 animals, 11 (33.3%) resulted positive at MAT at T0,  (Table 3). Table 3. MAT results in cows from Farm A at T0, T1 and T2 (Cut-off ≥ 100). Abbreviations: M, male; Neg, negative; Antib, subjected to antibiotic treatment. The arrows ↑↓ indicate an increase (↑) or a reduction (↓) of the antibody titer respect to the previous sampling, the (=) is used in the case of no variation, and the (I) sign indicates the first detection at T1 of Pomona serogroup for the serologically negative animals at T0.  In herd B, serological investigation carried out at T0 detected 5 cows with antibodies against Leptospira species (Table 4) Table 4, results of MAT are summarized for farm B obtained in the T0 and T1 sampling. In Table 5, positive results at MAT are summarized for the farms C, D, E and F obtained in the only sampling, T0.

Leptospiral Molecular Detection and Isolation
For farm A, the molecular investigation carried out on urine samples collected from serologically positive cows detected pathogenic Leptospira spp. DNA in all 11 samples (100%) analyzed at T0, and in 6 out of 24 samples (25%) at T1. Pathogenic Leptospira species were isolated from 5 positive urine samples (two from T0 and three from T1). Genotyping carried out in seven positive urine samples collected at T0 confirmed the excretion of L. interrogans serogroup Pomona. Phylogenetic analysis carried out using the concatemer of the seven MLST genes showed that Sicilian Leptospiral DNA samples clustered with the Pomona serovar (serogroup Pomona) reference strain (Figure 2).
(100%) analyzed at T0, and in 6 out of 24 samples (25%) at T1. Pathogenic Leptospira species were isolated from 5 positive urine samples (two from T0 and three from T1). Genotyping carried out in seven positive urine samples collected at T0 confirmed the excretion of L. interrogans serogroup Pomona. Phylogenetic analysis carried out using the concatemer of the seven MLST genes showed that Sicilian Leptospiral DNA samples clustered with the Pomona serovar (serogroup Pomona) reference strain (Figure 2). Neither culture examination nor Real Time-PCR showed evidence of Leptospira spp. presence in samples of milk, water, soil, utero-vaginal discharge, placenta, aborted fetuses or from the dog serum.
For herd B, at time T0, one urine sample was positive at the isolation of Leptospira species; at T1, renal excretion of pathogenic Leptospira spp. DNA was detected in 5 out of 20 urine samples.
For breeding unit C, one urine sample out of the three collected ones allowed Leptospira species isolation. In the other farms, no detection by Real Time-PCR nor isolation of Leptospira spp. were showed.
In Table 6, results of Real Time-PCR, MLST and isolation in the acute phase are summarized.  Neither culture examination nor Real Time-PCR showed evidence of Leptospira spp. presence in samples of milk, water, soil, utero-vaginal discharge, placenta, aborted fetuses or from the dog serum.
For herd B, at time T0, one urine sample was positive at the isolation of Leptospira species; at T1, renal excretion of pathogenic Leptospira spp. DNA was detected in 5 out of 20 urine samples.
For breeding unit C, one urine sample out of the three collected ones allowed Leptospira species isolation. In the other farms, no detection by Real Time-PCR nor isolation of Leptospira spp. were showed.
In Table 6, results of Real Time-PCR, MLST and isolation in the acute phase are summarized.

Discussion
Numerous bacterial, viral, protozoan and fungal pathogens have been associated with infertility and abortion in cattle.
Determining the cause of abortion in cattle is difficult and a major challenge to the herd owner and veterinarian. Infectious agents represent the leading etiology, and the majority of diagnosed abortions are attributed to infections with the bacteria Brucella abortus, endemic in Sicily, and Leptospira interrogans, the protozoa Neospora caninum and the two viruses IBR and BVD [46]. Moreover, Coxiella burnetii, the causal agent of Q fever, which is a zoonotic disease, has been related to stillbirth, aborted fetuses and the delivery of weak and nonviable neonates in ruminants. Yet, the correlation between Coxiella seropositivity and abortion risk in bovines is far less understood [47,48].
These pathogens can result in substantial economic losses, indicating the need for control measures to prevent infection or disease.
This study describes an outbreak in cattle in Sicily, mainly characterized by full-term abortions. Although, from the differential diagnosis, two animals resulted positive for N. canimun and one for C. burnetii, none of them showed signs of abortion. Moreover, the animals with a positive reaction for only specific IBR gB indicated they were vaccinated with gE-deleted marker vaccines and not infected. All the animals resulted negative for Brucella spp. and BVD. Numerous animals tested positive to leptospiral diagnosis, even among those who had aborted. Therefore, the study focused on clinical manifestations, diagnostic implications and epidemiological characteristics of this outbreak in cattle associated with L. interrogans serogroup Pomona. The outbreak occurred in the Northeast of Sicily (Italy), in a protected natural area within the Nebrodi Park. The area is characterized by a multi-host breeding system with the simultaneous presence of several animal species (cattle, sheep, goats, pigs, donkeys, equines) and by the uncontrolled and increased proliferation of wild pigs and wild boars.
The high number of positive animals obtained could be related to the semi-extensive or extensive breeding, promoting contacts with wildlife.
The acute phase of the disease (T0), both in farm A and in farm B, was characterized by antibody reaction to different serogroups (Australis Icterohaemorhagiae serogroup, Sejroe serogroup, Pomona serogroup). These results are consistent with the MAT method, as reported by other authors [49]. This can be explained by the test ability to detect both IgG and IgM immunoglobulins simultaneously. IgM (early antibodies) are present mainly in the first weeks of the disease or the acute phase, and interact with different antigens, some shared by several leptospires, thus showing a reduced specificity towards a single serogroup. Because of the lower specificity of antibodies in the acute phase and cross reactions, the attention in this phase was more focused on leptospiral isolation and molecular detection. Specificity increases in the subsequent convalescence/chronicization phase of the infection (T1 and T2) with the IgG predominance (late antibodies). In this phase, the test is better suited to identify the exact serogroup involved in the outbreak.
Even in farms C and F, subjected to a single sampling, MAT detected the presence of Leptospira Pomona with a prevalence of 66% and 29.5%, respectively, confirming the circulation of the Pomona serogroup in four of the six farms during the epidemic outbreak. Furthermore, in farm F a significant circulation of the Tarassovi serogroup occurred with a prevalence of 50%, followed by Sejroe and Grippotyphosa, as a further indicator of the presence of Leptospira serovars of swine and other wild animal origin [21]. Farms D and E (with farm consistency of two animals each) showed antibody positivity towards serogroups Ballum and Sejroe, respectively, with low antibody titers. However, they did not show any epidemiological significance due to the small number of animals reared and the low antibody titer.
Although congenital jaundice in aborted fetuses has been included among the clinical signs of leptospiral abortion, the fetuses were not subjected to necropsy because they were in an advanced state of degradation, having been recovered 24 h after the abortion, and after any jaundice was visible.
In Italy, a higher prevalence of serogroup Sejroe serovar Hardjo has been detected in cattle, confirming that cattle represent the main maintenance-host for Leptospira belonging to this serogroup [30,34] and, in particular, some strains isolated from urine samples were classified as Hardjobovis. In addition, previous studies showed a relatively high number of positive reactions to serogroups Pomona, Grippotyphosa and Bratislava (serogroup Australis) in cattle. Severe infections in cattle due to Pomona serogroup are uncommon and usually occur in young animals. Nevertheless, in Italy, Pomona resulted in the second most commonly isolated serovar in cattle [24,30]. Although this serogroup has been associated mainly with leptospirosis in pigs, considered its natural carriers [50], other species can also be affected, such as dogs, cattle and sheep [51][52][53][54]. Clinical signs in cattle caused by Pomona are generally different from both Hardjo and Hardjobovis infection and, especially in producing cows, fever and lethargy are milder and usually go unnoticed. At the same time, a transient reduction in milk production and/or agalactia may be detected. In pregnant cows, serogroup Pomona is generally associated with abortion [55].
Many wildlife species have been implicated as reservoirs for the bacteria, including red fox (Vulpes vulpes) and wild boar (Sus scrofa). Among wildlife, wild boar is an important Leptospira reservoir and could represent an appropriate indicator for this zoonotic infectious disease. In Sicily, a study of free-roaming semi-wild black swine demonstrated leptospires by PCR targeting the 16S rRNA gene with prevalence of 40% [56].
True foxes are well recognized as Leptospira reservoirs, in particular red foxes (Vulpes vulpes), but no isolation was performed among them [57].
Red foxes prey on small rodents, notably Rattus norvegicus, which is known to be the main reservoir of L. Icterohaemorhagiae serovar.
Surveys conducted throughout Europe have shown differences in the prevalence of leptospirosis in foxes: 26.3% in central and eastern Poland [58], 31.3% in Croatia [59].
Due to their predatory behavior and their varied diet, mainly composed of small mammals, red foxes could also be considered sentinel animals of environmental contamination with leptospires.
Because these "unconventional" hosts share the environment with cattle, the object of this study, they could have played an important role in leptospirosis spread, and further knowledge of them could give new insights into the epidemiology of this infection.

Conclusions
This study provides the first description of a Leptospira outbreak in cattle due to Pomona serovar in a protected natural geographical area of Northeastern Sicily, characterized by a multi-host environment with the presence of different animal species, domestic and wild, sharing pastures and food and water sources. In this ecosystem, where several domestic and wild mammals are natural reservoirs of pathogenic leptospires, and where appropriate management of wild pigs and boars is lacking, further investigations are required to confirm the role of domestic and wild species in the transmission, diffusion and persistence of the Pomona serovar among cattle farms.

Institutional Review Board Statement:
The study did not involve any animal experiments. Blood and urine samples were taken from animals suspected of leptospiral infection. Blood sampling was necessary in order to perform laboratory analysis and did not involve any suffering of the animals sampled.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.