Vancomycin resistance and virulence genes evaluation in Enterococci isolated from pork and wild boar meat.

Enterococci are considered valuable sentinel Gram-positive bacteria for monitoring vancomycin antibiotic resistance due to their widespread presence and characteristics. The use of antimicrobials in farming animals has a role in the increasing of Antimicrobial Resistance (AMR) and the anthropogenic transformation of the landscape has forced wildlife into greater contact with humans and their livestock. The transmission of resistant bacteria by their meat products is a significant contributor to AMR development. The present study aimed to assess the prevalence of vancomycin resistant Enterococci spp. In antimicrobial-treated farmed pigs meat and in antimicrobial-free wild boars meat. A total of 341 Enterococci were isolated from 598 pork meat samples (57 %) and 173 Enterococci were isolated from 404 wild boar meat samples (42.8 %). Data found showed that low-resistance was detected more in wild boars meat Enterococci (52.6 %) than in pork meat once (48.4 %). However, the prevalence of resistance genes was at low level (33.9 % in pork meat Enterococci and 4.4 % in wild boar meat ones) and the only gene found was vanC1/C2, related to intrinsic AMR. Normally, Enterococci persist in the normal intestinal flora of animals including humans. However, the presence of resistance genes was frequently linked to the detection of pathogenic genes, mostly gelE in pork meat isolates and asa1 in wild boars meat isolates. Pathogenic bacteria can cause severe infections in human that can become more risky if associated to the presence of AMR. Pathogenic bacteria were characterized and a high presence of E. gallinarum and E. casseliflavus was found. Given the growing interest in wild game meat consumption the monitoring of AMR in these matrices is essential. Further surveillance studies are needed to fully evaluate the emergence and spread of vancomycin-resistant Enterococci (VRE) and pathogenic Enterococci from animal-derived food to humans, including the role of wildlife in this phenomenon. Giving the higher interest in wild animals meat consumption, it is important to better evaluate the spread of AMR phenomenon in the future and intensify hygienic control of wild animals derived food.

Emergence of Group B Streptococcus Disease in Pigs and Porcupines, Italy.

We describe group B Streptococcus linked to disease in farmed pigs and wild porcupines in Italy. Occurrence in pigs was attributed to transmission from nonpasteurized bovine milk whey. Antimicrobial-resistance profiles in isolates from porcupines suggest no common source of infection. Our findings expand the known host range for group B Streptococcus disease.

In vitro susceptibility of Brachyspira hyodysenteriae strains isolated in pigs in northern Italy between 2005 and 2022.

Pleuromutilins (tiamulin and valnemulin) are often used to treat swine dysentery due to recurrent resistance to macrolides and lincosamides. Recently, reduced susceptibility of B. hyodysenteriae to pleuromutilin has been reported. 536 strains of B. hyodysenteriae were isolated from symptomatic pigs weighing 30-150 kg in northern Italy between 2005 and 2022. B. hyodysenteriae was isolated by standard methods and confirmed by PCR. The minimum inhibitory concentration (MIC) to doxycycline, lincomycin, tiamulin, tylosin, tylvalosine and valnemulin was evaluated according to CLSI procedures and MIC data were reported as MIC 50 and MIC 90. The temporal trend of the MIC values was evaluated by dividing the data into two groups (2005-2013 and 2014-2022). Comparison of the distribution in frequency classes in the two periods was performed using Pearson's chi-squared test (p < 0.01). MIC 50 was close to the highest values tested for lincomycin and tylosin, while MIC 90 was close to the highest values tested for all antibiotics. 71.7% of the strains were susceptible to tylvalosin, while 75%-80.4% had reduced susceptibility to valnemulin and tiamulin, respectively. The difference in the distribution of MIC classes was statistically significant in the two periods for doxycycline, tiamulin, tylvalosin and valnemulin, and more MIC classes above the epidemiological cut-off were observed in 2014-2022 compared with 2005-2013. The evaluation of the trends during the period considered shows a decreasing rate of wild-type strains with MIC values below the epidemiological cut-off over time and confirms the presence of resistant strains in northern Italy.

First evidence of widespread positivity to anticoagulant rodenticides in grey wolves (Canis lupus).

Second-generation Anticoagulant Rodenticides (ARs) can be critical for carnivores, due to their widespread use and impacts. However, although many studies explored the impacts of ARs on small and mesocarnivores, none assessed the extent to which they could contaminate large carnivores in anthropized landscapes. We filled this gap by exploring spatiotemporal trends in grey wolf (Canis lupus) exposure to ARs in central and northern Italy, by subjecting a large sample of dead wolves (n = 186) to the LC-MS/MS method. Most wolves (n = 115/186, 61.8 %) tested positive for ARs (1 compound, n = 36; 2 compounds, n = 47; 3 compounds, n = 16; 4 or more compounds, n = 16). Bromadiolone, brodifacoum and difenacoum, were the most common compounds, with brodifacoum and bromadiolone being the ARs that co-occurred the most (n = 61). Both the probability of testing positive for multiple ARs and the concentration of brodifacoum, and bromadiolone in the liver, systematically increased in wolves that were found at more anthropized sites. Moreover, wolves became more likely to test positive for ARs through time, particularly after 2020. Our results underline that rodent control, based on ARs, increases the risks of unintentional poisoning of non-target wildlife. However, this risk does not only involve small and mesocarnivores, but also large carnivores at the top of the food chain, such as wolves. Therefore, rodent control is adding one further conservation threat to endangered large carnivores in anthropized landscapes of Europe, whose severity could increase over time and be far higher than previously thought. Large-scale monitoring schemes for ARs in European large carnivores should be devised as soon as possible.

Eleven Years of Health Monitoring in Wild Boars (Sus scrofa) in the Emilia-Romagna Region (Italy).

In recent years, the growth of wild ungulates has increased the focus on their health monitoring. In particular, the health status of wild boars is relevant for the economic impact on the pig industry. The Emilia-Romagna region activated a wildlife monitoring plan to better evaluate the health status of the wild boar population. Between 2011 and 2021, samples of found dead and hunted wild boar have been examined for trichinellosis, tuberculosis, brucellosis, african swine fever, classical swine fever, Aujeszky's disease, swine vesicular disease, and swine influenza A. Trichinella britovi was identified in 0.001% of the examined wild boars; neither M. bovis nor M. tuberculosis were found in M. tuberculosis complex positive samples; 2.3% were positive for Brucella suis; 29.4% of the sera were positive for Aujeszky's disease virus; and 0.9% of the samples were positive for swine influenza A virus. With an uncertain population estimate, the number of animals tested, the number of positives, and the sampling method do not allow us to make many inferences but suggest the need to implement and strengthen the existing surveillance activity, as it seems to be the only viable alternative for safeguarding animal and human health.

Identification and phylogenetic analysis of Taenia spp. parasites found in wildlife in the Emilia-Romagna region, northern Italy (2017-2022).

The recent expansion of the habitat of several wildlife species, comprising anthropized areas, is a relevant risk factor for many zoonotic diseases and should be considered in national and regional sanitary monitoring systems. We evaluated adult intestinal Taenia spp. parasites isolated from wild carnivores and cystic larval forms isolated from wild mammals analysed at the Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna (IZSLER) as part of the regional wildlife sanitary surveillance plan. Then, we assessed parasite species through molecular analysis (multiplex PCR followed by ribosomal 12S subunit gene sequencing) in order to update the epidemiological situation on Taeniids infection in the Emilia-Romagna wildlife, reporting the prevalence of each isolated species. The most commonly isolated species was Taenia serialis, which we detected in both wolves and foxes as definitive hosts and in roe deer as intermediate host. More attention on the distribution of Taeniids in wildlife should be paid, considering their potential zoonotic role: several Taenia spp. (Taenia solium, Taenia multiceps, Taenia serialis, Taenia brauni, Taenia glomerulatus) are known for causing coenurosis in humans, with possible severe or fatal outcomes.

Wildlife Hosts of Leishmania infantum in a Re-Emerging Focus of Human Leishmaniasis, in Emilia-Romagna, Northeast Italy.

In the last decade, an upsurge of human leishmaniasis has been reported in the Emilia-Romagna region, Northeast Italy. Epidemiologic data have raised doubts about the role of dogs as the main reservoirs for Leishmania infantum. In the present study, a total of 1,077 wild animals were screened for L. infantum DNA in earlobe and spleen samples from 2019 to 2022. The lymph nodes were tested only in 23 animals already positive in the earlobe and/or spleen. A total of 71 (6.6%) animals resulted positive in at least one of the sampled tissues, including 3/18 (16.7%) wolves, 6/39 (15.4%) European hares, 38/309 (12.3%) roe deer, 1/11 (9.1%) red deer, 8/146 (4.9%) wild boars, 13/319 (4.1%) red foxes, 1/54 (1.9%) porcupine, and 1/59 (1.7%) European badger. Most of the infected animals (62/71) tested positive only in the earlobe tissue, only four animals (two roe deer and two wild boars) tested positive only in the spleen, and five animals (three roe deer and two red foxes) resulted positive for both tissues. L. infantum DNA was detected in the lymph nodes of 6/23 animals. L. infantum detection occurred in all seasons associated with low real-time PCR Ct values. Further research is needed in order to clarify the role of wildlife in the re-emerging focus of leishmaniasis in Northeast Italy.

Evaluation of Control Program Against Streptococcus agalactiae Infection in Dairy Herds During 2019-2021 in Emilia-Romagna Region, Northern Italy.

Streptococcus agalactiae (group B Streptococcus, GBS) is a causative agent of mastitis in dairy cattle, mainly causing a subclinical disease associated with a high somatic cell count (SCC), and a consequent decrease in production yield and quality of milk. GBS has been almost eradicated in many Northern European countries, but there are warnings of its re-emergence as a zoonotic threat. In Italy, only two regions carry out a GBS control program: Lombardy and Emilia-Romagna. In Emilia-Romagna, the program has been in place since 2019 and provides for the bacteriological culture of bulk-tank milk (BTM) of all dairy farms every 6 months and the voluntary application of herd eradication programs in the case of positive results. To assess the progress of the program in Emilia Romagna, in terms of herd-level prevalence and GBS transmission between herds, we analyzed the results of 17,056 BTM cultures from 2,831 dairy herds, sampled bi-annually in the period 2019-2021 (six rounds total). The impact of GBS infection on SCC and milk production was also evaluated. The results show a decreasing trend in both the incidence rate (from 3.0 to 1.5%) and apparent prevalence (from 8.9 to 5.2%) of GBS over the study period. By using a susceptible-infected-susceptible (SIS) model for the estimation of the transmission parameters, a basic reproductive number R0 of 1.4 was calculated, indicating an active spread of GBS in the dairy cattle population of the Emilia-Romagna region. GBS infected farms have a consistently higher BTM SCC than negative ones (+77,000 cells/ml), corresponding to a 0.4 kg/cow/day milk loss. Moreover, GBS infected herds resulted in almost three times more likelihood of having non-marketable milk by exceeding the legal SCC limit. This study demonstrates the need to maintain the current control program against GBS to lower its occurrence and prevent significant market losses to farmers.

Influenza A in Wild Boars: Viral Circulation in the Emilia-Romagna Region (Northern Italy) between 2017 and 2022.

A systematic surveillance against influenza A viruses (IAVs) in the Suidae population is essential, considering their role as IAV mixing vessels. However, the viral circulation in wild Sus scrofa species is poorly investigated in comparison to the knowledge of IAV infection dynamics in domestic pigs. This study investigated the circulation and the genetic diversity of wild boars' IAVs detected in the Emilia-Romagna region (2017-2022). A total of 4605 lung samples were screened via an M gene real-time RT-PCR for SwIAV; positive samples were subtyped by multiplex RT-PCR, and viral isolation was attempted. Isolated strains (3 out of the 17 positives) were fully sequenced to evaluate viral genotypic diversity. H1N1 was the most frequently detected subtype, with identification of H1pdm09N1 and H1avN1. Whole-genome phylogenetic analysis revealed SwIAVs belonging to different genotypes, with different genetic combinations, and highlighted the simultaneous circulation of the same genotypes in both pigs and wild boars, supporting the hypothesis of SwIAV spillover events at the wildlife-livestock interface. This study represents an update on the wild boar SwIAV Italian situation, and the strains' complete genome analysis showed an evolving and interesting situation that deserves further investigation.

Salmonella Brandenburg in the pork chain in Italy: Genetic comparison with the human isolates.

Salmonella Brandenburg ranked 16th among the serovars responsible for human infections in EU in 2015 and it was found to be associated with swine. In Emilia- Romagna and Lombardy regions of northern Italy, S. Brandenburg was isolated from mesenteric lymph nodes, fecal matter, carcasses and conveyor belts at pig slaughterhouses in 2014 and 2015. In the same area, S. Brandenburg was detected in pork salami in 2015. In the present study, 12 isolates of S. Brandenburg recovered from the pork food-chain were typed by XbaI PFGE and their three profiles were compared to all human S. Brandenburg isolates processed by the Surveillance System of Emilia- Romagna region from 2012 to 2017 (105 isolates). The most frequent pulsotype of porcine origin (6/12) was the second most frequent in humans (16/105). Of the other two pulsotypes of porcine origine (3/12 each), one was the most frequent in humans (41/105), the other was undetected among human isolates.