Leptospira in Slaughtered Fattening Pigs in Southern Italy: Comparison
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Leptospirosis is a zoonosis occurring worldwide, caused by pathogenic spirochaetes of the genus Leptospira, transmitted through direct contact with the urine of infected animals or a urine-contaminated environment. It has a negative economic impact on farm animals, causing economic losses and serious human diseases and mortality.

  • leptospirosis
  • MAT
  • real-time PCR
  • genotyping
  • pigs
  • Sicily

1. Introduction

The genus Leptospira contains 64 named species [2][1]. Leptospira have been classified serologically into more than 250 serovars [1,2,3,4][2][1][3][4]. Leptospires persist for a long time in the kidneys and genital tracts of domestic animals, including pigs, with intermittent shedding in the urine. This causes infections in humans and other animals [5,6,7][5][6][7]. Animal infections are caused by serovars maintained by the same or other species sharing the same geographical location [7].
Swine infections are caused by these pathogenic species: L. borgpetersenii (serovars Sejroe and Tarassovi), L. interrogans (serovars Pomona, Icterohaemorrhagiae, Canicola, and Bratislava), and L. kirschneri (serovars Grippotyphosa and Mozdok). Infections of L. kirschneri serovar Mozdok have been reported in pigs in various European countries [8,9,10][8][9][10] including Italy [11], and this serovar has been shown to be pathogenic for pigs, causing abortion and stillbirth in swine [12]. Serovars Bratislava and Pomona are uniquely adapted to swine; the others occasionally infect swine, being maintained in other species [13]. L. interrogans serovar Hardjo infects pigs sharing the same habitats with cattle. L. interrogans serovar Bratislava is the most frequent swine strain, with a doubtful role as a cause of disease [14].
Porcine leptospirosis imposes economic losses on pig farms, causing abortion, stillborn and weak piglets, and deaths soon after birth [15]. Leptospires cause serious illnesses depending on the serovar and the animal age [16]. When the infective agent enters a farm, its spread is rapid, mostly among fattening pigs [17].
In Italy, swine have been shown to maintain serovar Pomona (Pomona serogroup) and serovar Bratislava (Australis serogroup); serovar Tarassovi has been shown to be responsible for incidental infections [17]. Until 2010, a trivalent vaccine against these serogroups was available, but it was utilized by few swine farmers. In 2011, vaccinations were completely abandoned, due to poor understanding of the risk of leptospirosis and because of the treatments for more virulent diseases [11].
The Office International des Epizooties (OIE) reports the microscopic agglutination test (MAT) as the serological gold standard method [18]. The selection of antigens should include the serogroup strains circulating in the study area annd those known to be maintained by the species to be analyzed [11].
Besides the classical conventional reference methods, over the years, several real-time polymerase chain reaction (PCR) methods and molecular typing techniques have been developed to directly investigate Leptospira DNA in biological samples, to examine individual genomic profiles and to investigate the epidemiology [11,19,20,21,22,23,24,25][11][19][20][21][22][23][24][25]. They provide diagnostic advantages, such as reduced turnaround times, low risk of contamination and greater sensitivity and specificity [25].
Following a protocol published by Weiss et al., 2016 [26], multilocus sequence typing (MLST) avoids pathogen isolation, since it can be directly performed on the biological sample DNA.

2. Serological Survey and Molecular Typing

A high percentage of pigs positive for Australis serogroup (63.64%) followed by Pomona and Sejroe (27.27% and 9.09%, respectively) was observed. AIt similar studywas [17] showed that Pomona, Tarassovi, Bratislava and Muenchen are the most common serovars among swine in Italy. These data indicate swine can act as a reservoir host for these serogroups and that Australis is mainly present in pigs in southern Italy, confirming results from many regions worldwide [16]. AIt studywas conducted in five provinces in Vietnam showed a seroprevalence of 8.17% among fattening pigs [41][27].
The detection of serogroups by MAT depends on the investigation phase [42][28]; the induction of low antibody titers against common antigens of Leptospira spp., as well as cross-reactions of serogroups, are typical of the first phase of infection [42,43][28][29]. Titers of 1:100 or 1:200 may be suggestive of an early stage of infection; higher titers can be considered distinctive of endemic infection [44][30]. The low titers observed in this study in most samples could suggest a recent exposure to Leptospira spp. Moreover, the presence of positive sera reactions, at the same time, with two serovars (Australis-Pomona), indicated cross-reactions and confirmed the first phase of infection, the latter because the induction of antibodies against common antigens of Leptospira is frequent during the acute phase of infection [11]. It has been shown that serovar Mozdok infection causes serological cross-reactions with the Australis, Icteroahemorrhagiae and Grippotyphosa serogroups [12].
In regions where vaccination against leptospirosis has been practiced, including China, Japan, Cuba, and Europe, declines in overall seroprevalence have been reported [34][31]. This decrease has also been connected with improved housing, limiting interactions between animals and the environment [37][32]. A study coInducted in Greece , it was reported a seroprevalence of 17.8% in pig farms [46][33].
The percentages of positivity observed in Sicily compared with the other analyzed regions could be due to particular environmental conditions, potential risk factors and the abundance of reservoirs in the wild fauna More receInt studies in Europe have reported an increase in leptospirosis associated with wetter climatic conditions, promoting the prolonged environmental survival of Leptospira bacteria. Moreover, new climatic conditions have induced a change in herd management in Italy, increasing outdoor activities to improve animal welfare [39,44,50][34][30][35]. In the farms of origin, the bacteria could have been transiently present in water streams, rivers and small pools shared between swine and wildlife, and the pigs could have shared watering spots with the rich local wild fauna (wild pigs, wild boars, foxes, martens, and so etc.on). Among reservoirs, wild boar (Sus scrofa), as well as all swine, are considered the well-known maintenance host to the Tarassovi Leptospira borgpetersenii serogroup and Pomona and Australis Leptospira interrogans serogroups [13]. Moreover, due to their population abundance in all European countries, this animal species could be a suitable indicator of Leptospira prevalence in a specific area and a potential source of leptospires that then infect humans and domestic animals [51,52,53,54][36][37][38][39]. Because of their genetic relationship to domestic swine, wild boars play an important role in the transmission of leptospirosis among free living and domestic species [55][40] and could be identified as a potential source of infection for domestic pigs [56[41][42],57], as well as humans [57][42]. DifferenIt studieswas conducted across Europe on wild boars have shown variable seroprevalence of Leptospira from 65.4% in Portugal, [53][38], 45.8% in Slovenia [46][33] and 31.9% in Croatia [58][43], to 2.6% in Italy [59][44] and 3.1% in Sweden [60][45]. This variation across regions may be due to differences in the populations of wild small mammals acting as maintenance hosts [23]. Slaughterhouses occasionally represent an important surveillance station, mainly for foodborne pathogens (Salmonella, Campylobacter and Trichinella). They can also allow the detection of specific swine infections [50][35]. Moreover, in order to control Toxoplasma gondii infections in the pork supply chain, recommended measures developed by the European Food Safety Authority (EFSA) include serological testing of pigs for this pathogen at the farms or slaughterhouses and on-farm audits for risk factors associated with this infection [61,62][46][47]. For these reasons, slaughterhouses could assume an important epidemiological role in highlighting some important zoonosis not detected in the farms. Moreover, the distribution of serovars in slaughtered pigs could be assumed to reflect the distribution of serovars in pig farms. Swine vaccination against Leptospira in Italy led to a decrease in this infection in the pig population [43][29]. Starting from 2011, vaccinations against Leptospira spp. were no longer practiced, and the management of the breeding herd was adopted as strategy. Strong surveillance systems could improve understanding of the disease epidemiology, and the application of rigorous biosecurity controls and an effective specific prevention strategy (vaccination, slaughterhouse screening) together with farm management could limit pathogen transmission in the herd.

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