Enterobacteriaceae and Salmonella contamination of wild boar (Sus scrofa) carcasses: comparison between different sampling strategies.

During 2020, a total of 64 wild boar carcasses were tested for Enterobacteriaceae count (EBC), Salmonella and Yersinia enterocolitica in the abdominal region (i) within 5 h after hunting in the game collection point and (ii) before dressing and processing in the game-handling establishment (GHE) (49 carcasses-average time interval between (i) and (ii): 4.3 days). Because of COVID-19 restrictions, 15 carcasses were transported to a near slaughterhouse (average time interval between (i) and (ii): 2.3 days). Mesenteric lymph nodes (MLNs) were collected and tested for Salmonella and Y. enterocolitica. Results are shown in relation to sampling A (49 carcasses-GHE) and sampling B (15 carcasses-slaughterhouse). Sampling A: EBC median values were (i) 2.51 log10 CFU/cm2 and (ii) 2.79 log10 CFU/cm2. EBC increase between (i) and (ii) was statistically significant (p = 0.001). Salmonella prevalence on carcasses varied from (i) 2.0 to (ii) 6.1%. Sampling B: EBC median values were (i) 3.1 log10 CFU/cm2 and (ii) 3.32 log10 CFU/cm2. EBC increase between (i) and (ii) was not statistically significant (p = 0.191). Salmonella prevalence on carcasses varied from (i) 6.7 to (ii) 0.0%. The prevalence (sampling A + B) of lymphatic Salmonella carriers was 7.8% (5/64). From carcasses and/or MNLs, the serovars Enteritidis, Typhimurium, Agama, Zaiman and Diarizonae O:50 (z) were detected. Y. enterocolitica was never isolated. Long chilling periods prior to wild game processing should be avoided, and carcasses should be tested at GHE rather than after shooting to proper reflect the microbial load of wild boar meat entering the food chain.

Is Yersinia bercovieri Surpassing Yersinia enterocolitica in Wild Boars (Sus scrofa)?

Yersiniosis was the fourth reported zoonosis in the European Union in 2018. As well-known, pigs are recognized important reservoirs of Yersinia enterocolitica. The study was focused on Y. enterocolitica detection in mesenteric lymph nodes and faeces of 305 wild boars, but Yersinia bercovieri was more common, being isolated from 108 animals (35.4%). Cold season (p = 1.17 × 10-5) and young age (p = 0.004) significantly increased Y. bercovieri detection. Y. enterocolitica 1A belonging to six serotypes (O:4.32-4.33; O:5; O:6.30-6.31; O:7.8-8-8.19; O:10-34; O:12.25-12.26) was isolated from 8.2% (25/305) of the animals. Cold season significantly affected (p = 0.037) Y. enterocolitica detection.

Geographical restriction of Hepatitis E virus circulation in wild boars (Sus scrofa) in Emilia-Romagna region, Northern Italy.

Hepatitis E virus (HEV) is a singlestrand RNA virus that causes an acute viral hepatitis in humans. Among its eight recognized genotypes, HEV-3 and HEV-4 are zoonotic, infecting humans, pigs and wild boars. Recently, HEV-3 has been also detected in red deer, which represents another reservoir of HEV. Consumption of raw pork products (mainly liver sausages), undercooked wild boar meat, raw wild boar liver and deer meat has been responsible for foodborne HEV human worldwide. From November 2018 to March 2019, liver samples collected from 97 wild boars hunted in Emilia-Romagna region (Northern Italy) were tested for HEV RNA. The hunting area included two territories for an extension of 33 km2, named A (about 13 km2,natural park, deciduous wood) and B (about 20 km2, cultivated fields in proximity of a river) areas. Distance between the two areas ranged between 8 to 10 km. A total of 73 wild boars were hunted in area A, and 24 in area B. HEV RNA was detected by Real-time RT- PCR in 23/73 liver samples of wild boars living in area A only (31.5% - 95% CI: 22.0-42.8%). The HEV sequences (n=13) clustered within genotype 3. The majority of positives belonged to animals < 12 months (12/25; 48%), followed by subadults (13-24 months) (7/16; 43.8%) and adults (4/32; 12.5%). This difference was found to be statistically significant (p=0.0024). In absence of pig farms, the restriction of HEV-positive animals to a well-defined territory of 13 km2 (Boschi di Carrega Regional Park) could hypothetically be related to the presence of red deer (Cervus elaphus), which lived in area A at the beginning of the hunting season. Further studies are needed to confirm or deny our hypothesis.

Limited Exchange of Salmonella Among Domestic Pigs and Wild Boars in Italy.

The study assessed Salmonella carriage in wild boars (Sus scrofa) and compared their isolates with those recovered from the domestic swine population of the same area of northern Italy (Emilia-Romagna), characterized by intensive pig farming and rather high density of wild boars. A total of 189 wild boars hunted during twelve months (2017-2018) were tested for Salmonella in mesenteric lymph nodes (MLN) and faecal samples. Antimicrobial resistance of recovered strains was tested against 14 antimicrobials. Salmonella was detected in 33/189 wild boars (17.5%), specifically from 30/189 MLN (15.9%) and 6/189 faecal samples (3.2%). Three animals were positive in both samples. Thirteen Salmonella serovars were identified, i.e. Typhimurium (the most common), Bovismorbificans, Coeln, Derby, Enteritidis, Gaminara, Hessarek, Houtenae IV, Kottbus, Napoli, Stanleyville, Thompson and Veneziana. Salmonella carriage was higher in warm than in cold months (P = 0.0013). Pregnancy status was never associated with Salmonella carriage, with significant difference in the recovery of the pathogen between non-pregnant and pregnant females (P = 0.003). Only one resistance pattern to streptomycin and tetracycline was found in 15 isolates (41.7%) belonging to Typhimurium (14/14; 100%) and Kottbus (1/3; 33.3%) serovars. Overlap with isolates from farmed pigs was limited at serotype level (Typhimurium, Derby, Enteritis, Bovismorbificans, Kottbus) and absent at PFGE level, and also antimicrobial resistance patterns were substantially different. This evidence indicates a substantial segregation of the two animal populations with regard to infectious contacts, possibly suggesting that biosecurity measures in place at industrial farm level limit the exchange of Salmonella.

Detection of Extended- Spectrum Beta-Lactamase producing Escherichia coli from mesenteric lymph nodes of wild boars (Sus scrofa).

Wild boars (Sus scrofa) are increasing in several European countries, including Italy. In areas with intensive animal farming, like the Italian Emilia-Romagna region, they are likely to be exposed to antimicrobialresistant (AMR) bacteria of livestock origin. In 2017-2018, 108 mesenteric lymph nodes samples were collected from 108 wild boars hunted in Parma province, Emilia-Romagna region, to be tested for ESBL- and carbapenemase-producing Escherichia coli. One isolate (WB-21L) out of 108 (0.9%) was phenotypically confirmed as ESBLproducing E. coli. The strain WB-21L was tested by PCR for the genes bla SHV, bla CTX-M, bla TEM, bla AmpC, bla KPC, bla NDM, bla VIM, bla IMP, bla OXA-48, bla SPM, bla BIC, bla SIM, bla DIM, bla GIM, bla AIM, resulting positive for TEM ß- lactamase. Resistance to ampicillin, amoxicillin/clavulanic acid, streptomycin, sulfasomidine, tetracycline and trimethoprim confirmed the multi-resistance nature of the strain WB-21L. Nine E.coli isolates showed resistance to meropenem by the Kirby Bauer test but none of them showed Meropenem MIC values indicative of resistance. In conclusion, the present study shows the presence of ESBL E. coli in wild boars and the possible risk of transfer to game meat handlers and consumers. Future studies are needed to better evaluate the sources of AMR bacteria in wildlife.