Isolation of Leptospira serovar Pomona from a crested porcupine (Hystrix cristata, L., 1758)

Abstract Pathogenic Leptospira is widespread in rodents, the most studied reservoir and the main hosts involved in its transmission. In Italy, among rodents, Hystrix cristata (crested porcupine) is the largest species and it is distributed all over the country. In this paper, the isolation and characterization of pathogenic Leptospira spp. from the kidney of H. cristata is reported for the first time. During Autumn 2018, Leptospira detection by real‐time PCR and isolation were performed from kidneys of two died female porcupines (an adult and a porcupette). Only for porcupette kidney sample, real‐time PCR for pathogenic Leptospira tested positive. The isolated strain was identified as Leptospira interrogans serogroup Pomona serovar Pomona, using the three schemes of multilocus sequence typing. The results show that H. cristata could be a Leptospira host. The infection of serovars Pomona could be related to the habitat shared with wild boar, a typical reservoir host for this serovar.

Italy is the only European country where the crested porcupine live in the wild as naturalized specie (Coppola, Dari, Vecchio, Scarselli & Felicioli, In press, Santini, 1980). Porcupine is the largest rodent of the Italian fauna and is widely distributed in mainland and it is also present in Sicily and Sardinia. (Loy et al., 2019;Mori, Sforzi, Bogliani, & Milanesi, 2018). Recently, Vecchio, Coppola, Scarselli, Giannini, and Felicioli (2018) report the presence of at least one free-ranging crested porcupine in the Island of Elba using camera-trapping rising the question if a population of this rodent is also present in the Island.
Within a more general study on the epidemiology of Leptospira in Italian wildlife, this paper reports the first case of Leptospira serovar Pomona isolation in H. cristata.

| Samples collection
In Autumn 2018, sampling was performed on two crested porcupines died for traumatic impact in a veterinary clinic, in the Grosseto province (Tuscany, Italy). The first one was recovered in the area of Pescia Fiorentina (Capalbio), whereas the second one in the area of Arcille (Campagnatico). At the clinic, animals were treated with enrofloxacin and dexamethasone for about 3 days before the death.
From each carcass, during necropsy, kidneys and a blood sample from the heart cavity were collected. Before the necropsy, both animals were sexed and weighted and the age class was estimated (porcupette < 5 kg; 5 kg ≤ sub-adult < 11 kg; adult ≥ 12 kg; (Coppola, Vecchio, & Felicioli, 2019).

| Microscopic agglutination test
Blood samples were centrifugated at 1,200 g rpm for 10 min to obtain the serum. The sera were tested to detect Leptospira antibodies by MAT (OIE, 2018)

| Leptospira spp. isolation
The kidney samples were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Difco). A portion of 10 cm 3 from each porcupine kidney was homogenized with 5 ml of sterile water.
One ml of homogenate was cultured in 5 ml of EMJH, incubated at 30 ± 1°C for 120 days and checked every 10 days under dark-filed microscopy to assess bacterial growth.
In case of positive cultures, subcultures were performed to maintain the isolated strains alive.

The amplification of each target gene was performed with
HotStarTaq Master Mix Kit (Qiagen). Amplicons were further sequenced (BMR Genomics) using the same primer sets and analysed using BioEdit Software (Hall, 1999).

| Molecular analysis
DNA was extracted from each kidney using the Quick-DNA Plus Kits (Zymo Research) according to the manufacturer's instructions.
The lipL32 gene Taqman RealTime PCR was performed on a Rotorgene Corbett 6000 (Corbett Research) to detect pathogenic leptospires (Stoddard, Gee, Wilkins, McCaustland, & Hoffmaster, 2009). The following thermal conditions were employed: 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 as positive.

| Histopathology and immunohistochemistry
Representative portions of the kidneys collected during necropsy were routinely processed, paraffin-embedded and 5-µm-thick sections were stained with haematoxylin and eosin, Masson trichrome Goldner and Warthin Starry stains. Tissue sections were also submitted to immunohistochemistry. Antigen retrieval was achieved on the slides by placing them in a bath of 10 mmol/L citric acid (pH 6) and boiling for 16 min in an 800-W microwave oven.
The slides were dried at room temperature and washed with running tap water. A peroxidase block was performed, and the slides were incubated with specific rabbit antisera against Leptospira in-

| RE SULTS
Both exanimate porcupines were female, one adult of 11 kg (more than 1 year old) and a porcupette (around 2 months old) of 1.4 kg. The kidney of the porcupette showed small gray-white focal lesions, mainly located in the renal cortex and varying from 1 to 2 mm in diameter. Microscopically, a mild chronic interstitial nephritis was present, characterized by vacuolar degeneration of the tubular epithelium and scattered interstitial foci consisting of lymphocytes and plasma cells (Figure 1a), accompanied by interstitial fibrosis (Figure 1b). In silver-stained sections, leptospires were never detected in the tubular lumen adhering to the luminal surface of tubular cells, whereas Intracytoplasmic spherical bodies within cells of a tubule undergoing regressive changes were observed. In immunoperoxidase-stained sections, using antisera against Leptospira serogroup Pomona an intense immunoreactivity for leptospiral antigen was detectable within the tubular epithelia cells and in cellular debris in tubular lumen (Figure 1c), whereas the absence of immune-labelling was observed when the antiserum against Leptospira serogroup Grippotyphosa was used.
Both sera samples resulted negative to MAT for all Leptospira serogroups tested. Only in porcupette kidney, Leptospira DNA was detected and after 2 months of incubation, the bacterium was isolated.
The isolated strain was identified as L. interrogans serogroup Pomona

| D ISCUSS I ON
Only recently, crested porcupine was investigated as potential vector or host for a wide range of parasites and micro-organisms, such as fleas and hard ticks (Mori et al., 2015;Scaravelli, Senini, & Bonacci, 2017), Giardia duodenalis (Coppola, Maestrini, et al., 2020) and pathogenic Leptospira . For the first time, L. interrogans serogroup Pomona serovar Pomona was isolated from one out of two analysed porcupine kidneys.
Both sampled sera were negative for Leptospira serovars Pomona and for the other Leptospira serovars tested, whereas pathogenic Leptospira DNA was detected only in renal tissue from porcupette.
The seronegativity of Leptospira-positive subjects was previously reported for other species (Agampodi, Matthias, Moreno, & Vinetz, 2012;Hall & Lambourne, 2014;Merien, Baranton, & Perolat, 1995) and for porcupine (S. villosus), as well (Fornazari et al., 2018). The serological negativity of Leptospira-positive porcupette could be related to the quality of blood sample collected from the heart cavity or to an early or chronic infection. However, a chronic infection seems to be unlikely due to the young age of the animal. At the same time, the presence of an early stage infection can be excluded considering the stage of renal lesions, characterized by the presence of inflammatory infiltrates, mild fibrosis and the absence of a large amount of leptospires localized in tubular lumen, typically detectable during early Leptospira infection.
At this stage, the micro-organisms should be numerous, intact and easy to visualize by both the immunohistochemical and silverstaining methods and the perifocal inflammatory infiltrates are scanty or absent (Michna & Campbell, 1969). Subsequently, when the inflammatory cells surround the infected tubules, leptospires are lysed, clumped and leptospiral antigen are taken up by tubular cells. In this case, immunohistochemical studies allow to detect the presence of Leptospira antigen in tubular epithelial cells and to reveal intracytoplasmic spherical bodies within the cells of renal tubules undergoing regressive changes, as previously described in leptospiral nephritis in swine (Scanziani, Sironi, & Mandelli, 1989).
The seronegativity of Leptospira-positive subject could be related to the antibiotic treatment with enrofloxacin, performed during the hospitalization. Activity of enrofloxacin against Leptospira is documented in vitro, but some studies showed increased MIC values in recent isolates (Liegeon, Delory, & Picardeau, 2018;. Carrascosa et al. (2017) showed a low effectiveness of this antibiotic in vivo in order to prevent Leptospira renal colonization in hamster and a decrease in antibiotic effectiveness when the treatment is delayed from the starting of the infection. However, antibiotic treatment could have affect the antibodies response, leading to negative MAT results, as previously reported by Ricaldi, Swancutt, and Matthias (2013) and Courdurie et al. (2017). The antibiotic treatment could have also determined the lack of leptospires in the renal tissue highlighted with specific Warthin Starry staining. The reduced bacterial load has also been highlighted by the long incubation period of the culture before the isolation of the Leptospira strain (Azizi, Kheirandish, & Rahimi, 2014).
Rodents are well known important Leptospira carriers, involved in the infection transmission to animals and humans (Blasdell, Morand, Perera, & Firth, 2019;Mori et al., 2017). The evidence of a possible Leptospira infection in crested porcupine was previously investigated in the same studied area. Seven out of 14 (50%) of porcupine sera resulted positive to anti-Leptospira antibodies detection; Icterohaemorrhagiae resulted the most prevalent serogroup (4 positive sera), followed by serogroup Pomona and Australis (2 sera, respectively). Titres of 1:400 and 1:100 were recorded for serogroup Pomona . At the best of Authors knowledge, excluding H. cristata, among the Hystricomorpha rodents, isolation of Leptospira serovar Pomona from kidney, blood and urine was only performed in one North American porcupine (Mitchell et al., 1966) and no other Leptospira

| CON CLUS ION
Leptospirosis is one of the most widespread and emerging zoonotic disease in the world, and wild animals were known to be reservoir of