Presence and Characterization of Zoonotic Bacterial Pathogens in Wild Boar Hunting Dogs (Canis lupus familiaris) in Tuscany (Italy).

Domestic dogs (Canis lupus familiaris) used for wild boar (Sus scrofa) hunting may represent incidental hosts for several zoonotic pathogens. This investigation aimed to evaluate the presence of anti-Leptospira antibodies and the occurrence, antimicrobial resistance, and virulence of Salmonella spp., Yersinia enterocolitica, and Listeria monocytogenes in sera and rectal swabs collected from 42 domestic hunting dogs in the Tuscany region (Italy). Regarding Leptospira, 31 out of 42 serum samples (73.8%) were positive and serogroup Pomona was the most detected (71.4%) at titers between 1:100 and 1:400. Four Salmonella isolates (9.52%) were obtained, all belonging to serotype Infantis; two of them showed antimicrobial resistance to streptomycin, while pipB and sopE presence was assessed in all but one isolate. Concerning Yersinia enterocolitica, seven isolates (16.7%) were obtained, six belonging to biotype 1 and one to biotype 4. Resistance to amoxicillin-clavulanic acid, cephalothin, and ampicillin was detected. Biotype 4 presented three of the virulence genes searched (ystA, ystB, inv), while isolates of biotype 1 showed only one gene. No Listeria monocytogenes was isolated from dog rectal swabs. The results suggest that hunting dogs are exposed to different bacterial zoonotic agents, potentially linked to their work activity, and highlight the possible health risks for humans.

Coagulase negative staphylococci from ovine bulk-tank milk: Effects of the exposure to sub-inhibitory concentrations of disinfectants for teat-dipping.

Teat-dipping is one of the most effective methods to prevent mammary infections in ruminants, including sub-clinical mastitis caused by coagulase-negative staphylococci (CoNS). Improper disinfectant application could expose microorganisms to sub-inhibitory concentrations leading to phenotypic variations. In this study, 12 chlorhexidine-digluconate (CHDG)-tolerant (of which 4 qac positive) and 12 benzalkonium chloride (BC)-tolerant (of which 7 qac-positive) CoNS isolates from ovine milk were exposed to sub-inhibitory concentrations of CHDG and BC, respectively. Changes in disinfectant susceptibility against BC and CHDG, antibiotic resistance against 12 antibiotics and biofilm production were then assessed for both groups. After CHDG stress, 67 % and 83 % of the CHDG-stressed isolates doubled their MICs for BC and CHDG, respectively and 2 qac-negative isolates showed a four-fold increase of their MBCs for CHDG. After BC stress, MICs for BC and CHDG doubled in 58 % and 83 % of the BC-stressed isolates, respectively, while one qac-positive isolate increased four-fold the MIC for BC. Cross-resistance to antibiotics was assessed by disc diffusion method. Some qac-positive isolates varied their resistance profile, while a blaZ-positive isolate showed a resistant phenotype against ampicillin only after the exposure to the disinfectant. As for qac-positive isolates, one CHDG-stressed and 2 BC-stressed increased their resistance to kanamycin and cefoxitin, respectively. The Congo Red Agar test was carried out to assess the in vitro slime production: all isolates were negative after stress. In conclusion, sub-inhibitory exposure to disinfectants may affect disinfectant and antibiotic susceptibility, the latter in particular for qac-positive isolates and those hosting unexpressed antibiotic resistance genes.

Genital Brucella suis Biovar 2 Infection of Wild Boar (Sus scrofa) Hunted in Tuscany (Italy).

Brucellosis is a zoonosis caused by different Brucella species. Wild boar (Sus scrofa) could be infected by some species and represents an important reservoir, especially for B. suis biovar 2. This study aimed to investigate the prevalence of Brucella spp. by serological and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. From 287 animals, sera, lymph nodes, livers, spleens, and reproductive system organs were collected. Within sera, 16 (5.74%) were positive to both rose bengal test (RBT) and complement fixation test (CFT), with titres ranging from 1:4 to 1:16 (corresponding to 20 and 80 ICFTU/mL, respectively). Brucella spp. DNA was detected in four lymph nodes (1.40%), five epididymides (1.74%), and one fetus pool (2.22%). All positive PCR samples belonged to Brucella suis biovar 2. The results of this investigation confirmed that wild boar represents a host for B.suis biovar. 2 and plays an important role in the epidemiology of brucellosis in central Italy. Additionally, epididymis localization confirms the possible venereal transmission.

Leptospira fainei Detected in Testicles and Epididymis of Wild Boar (Sus scrofa).

Leptospirosis is a re-emerging and worldwide diffused zoonosis. Recently, the high importance of their epidemiology was explained by the intermediate Leptospira strains. Among these strains, Leptospira fainei was the first intermediate strain detected in domestic and wild swine. Wild boars (Sus scrofa) are well known as a reservoir, as well as all swine, for pathogenic Leptospira, but very little information is available concerning intermediate Leptospira infection. The investigation aim was to evaluate if intermediate Leptospira can infect the reproductive systems of wild boars hunted in the Tuscany region (Italy), as previously demonstrated for pathogenic ones. The reproductive system tissue (testicles, epididymides, uteri), and placentas and fetuses, were collected from 200 regularly hunted animals. Bacteriological examination and real-time PCR were performed to detect intermediate Leptospira DNA. Unfortunately, no isolates were obtained. Using real-time PCR, in six (3%) male organs (both testicles and epididymis), intermediate Leptospira DNA was found. The amplification of the 16S rRNA gene identified that all DNA obtained belong to Leptospira fainei. The results of this investigation highlighted for the first time the localization of Leptospira fainei in the male wild boar reproductive system, opening up a new avenue to further investigate.

Prevalence, Virulence and Antimicrobial Susceptibility of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes in European Wild Boar (Sus scrofa) Hunted in Tuscany (Central Italy).

Wild boar is an animal the population of which constantly increases in Europe. This animal plays an important role as a reservoir for several pathogens, including three of the most important zoonoses: salmonellosis, yersiniosis and listeriosis. The aim of this investigation was to evaluate the occurrence of antimicrobial-resistant and virulence factor genes of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolated from wild boar in Tuscany (Central Italy). During two consequent hunting seasons (2018/2019 and 2019/2020), rectal swabs, spleens and livers were collected from 287 hunted wild boar to isolate strains. Each isolate was tested to investigate its antimicrobial resistance and to detect virulence factor genes by PCR. Eighteen Salmonella strains (6.27%) were isolated. Of these, 66.7% were resistant to streptomycin, 13.4% to cephalothin, 6.67% to imipenem and one isolate (6.67%) was resistant simultaneously to five antimicrobials. Moreover, the most detected genes were sopE (73.4%), pipB (66.7%), sodCI (53.3%), spvR and spvC (46.7%). In total, 54 (17.8%) Yersinia enterocolitica were isolated; of them, 26 (48.1%), 9 (16.7%), 17 (31.5%), 1 (1.85%) and 1 (1.85%) belonged to biotypes 1, 2, 3, 4 and 5, respectively. All strains (100%) demonstrated resistance to cephalothin and 70.4% to amoxicillin-clavulanic acid, 55.6% to ampicillin, and 37.0% to cefoxitin. Additionally, the most detected genes were ystA (25.9%), inv (24.1%), ail (22.2%), ystB (18.5%) and virF (14.8%). Finally, only one Listeria monocytogenes isolate (0.35%) was obtained, belonging to serogroup IVb, serovar 4b, and it was found to be resistant to cefoxitin, cefotaxime and nalidixic acid. The results highlighted the role of wild boar as a carrier for pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogens, representing a possible reservoir for domestic animals and human pathogens.

Characterization of Salmonella spp. Isolates from Swine: Virulence and Antimicrobial Resistance.

Salmonella is one of the most important zoonotic pathogens worldwide. Swine represent typical reservoirs of this bacterium and a frequent source of human infection. Some intrinsic traits make some serovars or strains more virulent than others. Twenty-nine Salmonella spp. isolated from pigs belonging to 16 different serovars were analyzed for gastric acid environment resistance, presence of virulence genes (mgtC, rhuM, pipB, sopB, spvRBC, gipA, sodCI, sopE), antimicrobial resistance and presence of antimicrobial resistance genes (blaTEM, blaPSE-1, aadA1, aadA2, aphA1-lab, strA-strB, tetA, tetB, tetC, tetG, sul1, sul2, sul3). A percentage of 44.83% of strains showed constitutive and inducible gastric acid resistance, whereas 37.93% of strains became resistant only after induction. The genes sopB, pipB and mgtC were the most often detected, with 79.31%, 48.28% and 37.93% of positive strains, respectively. Salmonella virulence plasmid genes were detected in a S. enterica sup. houtenae ser. 40:z4,z23:-strain. Fifteen different virulence profiles were identified: one isolate (ser. Typhimurium) was positive for 6 genes, and 6 isolates (3 ser. Typhimurium, 2 ser. Typhimurium monophasic variant and 1 ser. Choleraesuis) scored positive for 5 genes. None of the isolates resulted resistant to cefotaxime and ciprofloxacin, while all isolates were susceptible to ceftazidime, colistin and gentamycin. Many strains were resistant to sulfonamide (75.86%), tetracycline (51.72%), streptomycin (48.28%) and ampicillin (31.03%). Twenty different resisto-types were identified. Six strains (4 ser. Typhimurium, 1 ser. Derby and 1 ser. Typhimurium monophasic variant) showed the ASSuT profile. Most detected resistance genes sul2 (34.48%), tetA (27.58%) and strA-strB (27.58%). Great variability was observed in analyzed strains. S. ser. Typhimurium was confirmed as one of the most virulent serovars. This study underlines that swine could be a reservoir and source of pathogenic Salmonella strains.

Presence of pathogenic Leptospira spp. in the reproductive system and fetuses of wild boars (Sus scrofa) in Italy.

Leptospirosis is a re-emerging and globally spread zoonosis caused by pathogenic genomospecies of Leptospira. Wild boar (Sus scrofa) are an important Leptospira host and are increasing in population all over Europe. The aim of this investigation was to evaluate Leptospira spp. infection in the reproductive systems of wild boar hunted in two Italian regions: Tuscany and Sardinia. From 231 animals, reproductive system tissue samples (testicles, epididymides, uteri) as well as placentas and fetuses were collected. Bacteriological examination and Real-Time PCR were performed to detect pathogenic Leptospira (lipL32 gene). Leptospires were isolated from the testicles and epididymides of one adult and two subadult wild boar. Four isolates from the two subadult males were identified as Leptospira interrogans serogroup Australis by MLST, whereas Leptospira kirschneri serogroup Grippotyphosa was identified from the adult testicles and epididymis. Using Real-Time PCR, 70 samples were positive: 22 testicles (23.16%) and 22 epididymides (23.16%), 10 uteri (7.35%), 3 placentas (6.66%), and 13 fetuses (28.88%). Amplification of the rrs2 gene identified L. interrogans and L. kirschneri species. The results from this investigation confirmed that wild boar represent a potential source of pathogenic Leptospira spp. Isolation of Leptospira serogroups Australis and Grippotyphosa from the male reproductive system and the positive Real-Time PCR results from both male and female samples could suggest venereal transmission, as already demonstrated in pigs. Furthermore, placentas and fetuses were positive for the lipL32 target, and this finding may be related to a possible vertical transmission of pathogenic Leptospira.

Microbial Profile of the Ventriculum of Honey Bee (Apis mellifera ligustica Spinola, 1806) Fed with Veterinary Drugs, Dietary Supplements and Non-Protein Amino Acids.

The effects of veterinary drugs, dietary supplements and non-protein amino acids on the European honey bee (Apis mellifera ligustica Spinola, 1806) ventriculum microbial profile were investigated. Total viable aerobic bacteria, Enterobacteriaceae, staphylococci, Escherichia coli, lactic acid bacteria, Pseudomonas spp., aerobic bacterial endospores and Enterococcus spp. were determined using a culture-based method. Two veterinary drugs (Varromed® and Api-Bioxal®), two commercial dietary supplements (ApiHerb® and ApiGo®) and two non-protein amino acids (GABA and beta-alanine) were administered for one week to honey bee foragers reared in laboratory cages. After one week, E. coli and Staphylococcus spp. were significantly affected by the veterinary drugs (p < 0.001). Furthermore, dietary supplements and non-protein amino acids induced significant changes in Staphylococcus spp., E. coli and Pseudomonas spp. (p < 0.001). In conclusion, the results of this investigation showed that the administration of the veterinary drugs, dietary supplements and non-protein amino acids tested, affected the ventriculum microbiological profile of Apis mellifera ligustica.GABA; beta-alanine; oxalic acid; diet effect; microbiota.

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

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.

Leptospira Survey in Wild Boar (Sus scrofa) Hunted in Tuscany, Central Italy.

Leptospirosis is a re-emerging, worldwide zoonosis, and wild boar (Sus scrofa) are involved in its epidemiology as the reservoir. The aim of this study was to investigate the prevalence of Leptospira with serological, bacteriological, and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. In total, 287 specimens of sera, kidneys, and liver were collected to perform microscopic agglutination tests (MATs), isolation, and RealTime PCR to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene), and saprophytic (23S rRNA gene) Leptospira. Within sera, 39 (13.59%) were positive to the MAT, and Australis was the most represented serogroup (4.88%), followed by Pomona (4.18%), and Tarassovi (3.14%). Moreover, four Leptospira cultures were positive, and once isolates were identified, one was identified as L. borgpetersenii serovar Tarassovi, and three as L. interrogans serovar Bratislava. Pathogenic Leptospira DNA were detected in 32 wild boar kidneys (11.15%). The characterization through the amplification of the rrs2 gene highlighted their belonging to L. interrogans (23 kidneys), L. borgpetersenii (four), and L. kirschneri (one), while nine kidneys (3.14%) were positive for intermediate Leptospira, all belonging to L. fainei. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis among wildlife in Central Italy.

Pathotypes and Antimicrobial Susceptibility of Escherichia Coli Isolated from Wild Boar (Sus scrofa) in Tuscany.

Wild boar are among the most widespread wild mammals in Europe. Although this species can act as a reservoir for different pathogens, data about its role as a carrier of pathogenic and antimicrobial-resistant Escherichia coli are still scarce. The aim of this study was to evaluate the occurrence of antimicrobial-resistant and pathogenic Escherichia coli in wild boar in the Tuscany region of Italy. During the hunting season of 2018-2019, E. coli was isolated from 175 of 200 animals and subjected to antimicrobial resistance tests and PCR for detection of resistance and virulence factor genes. The highest resistance rates were against cephalothin (94.3%), amoxicillin-clavulanic acid (87.4%), ampicillin (68.6%), and tetracycline (44.6%). The most detected resistance genes were blaCMY-2 (54.3%), sul1 (38.9%), sul2 (30.9%), and tetG (24.6%). Concerning genes encoding virulence factors, 55 of 175 isolates (31.4%) were negative for all tested genes. The most detected genes were hlyA (47.4%), astA (29.1%), stx2 (24.6%), eaeA (17.1%), and stx1 (11.4%). E. coli was classified as Shiga toxin-producing E. coli (STEC) (21.7%), enterohemorrhagic E. coli (EHEC) (6.3%), enteroaggregative E. coli (EAEC) (5.1%), and atypical enteropathogenic E. coli (aEPEC) (3.4%). Enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and typical enteropathogenic E. coli (tEPEC) were not detected. Our results show that wild boars could carry pathogenic and antimicrobial-resistant E. coli, representing a possible reservoir of domestic animal and human pathogens.

Crested Porcupine (Hystrix cristata L.): A New Potential Host for Pathogenic Leptospira Among Semi-Fossorial Mammals.

Wildlife plays a pivot role in the epidemiology of leptospirosis and rodents have a reservoir function for several Leptospira serogroups. The crested porcupine is the largest rodent of the Italian fauna and shares the same environment with red foxes, badgers, coypus and wild boars that are known to be infected by Leptospira. Between 2018 and 2019 the seroprevalence of Leptospira in crested porcupine was investigated for the first time. Overall 7 out of 14 sera (50 %) were found positive to Leptospira. Icterohaemorrhagiae resulted as the most detected serogroup (57 %) followed by Pomona, Australis and Sejroe. The highest titer (1:1600) was detected for the serogroup Australis. These results indicate that porcupines could be infected by several serogroup of Leptospira and the role of reservoir or accidental host need to be addressed. Further investigations are necessary in order to clarify the leptospirosis - epidemiology - wildlife framework in light of its potential zoonotic source.

Serological Survey on Bacterial and Viral Pathogens in Wild Boars Hunted in Tuscany.

Wild boar (Sus scrofa) is one of the large mammals most spread worldwide, including Italy. This animal is highly adaptable, and its population has rapidly increased in many areas in Europe. Central Italy, as well as Tuscany region, is an area particularly suitable for wild boar. In order to verify the role of this animal species in the epidemiology of some important infectious diseases for livestock and humans, a seroepidemiological survey on Brucella spp., Leptospira spp., Mycoplasma hyopneumoniae, Pseudorabies virus (PrV), and Hepatitis E virus (HEV) has been performed on 374 sera collected from wild boar during 2015/2016 and 2016/2017 hunting seasons. Overall, 2 out of 374 sera (0.53%) tested positive to Brucella spp., 33 out of 374 sera (8.82%) tested positive for Leptospira spp., while 79 out of 374 (21.12%) were positive for M. hyopneumoniae. Considering viral pathogens, serology indicated that 107 out of 374 (28.60%) samples scored positive for PrV, while 186 out of 374 (49.73%) for HEV. This investigation indicated that wild boar free ranging in the study area are potential hosts for different etiological agents. This animal could contribute to maintaining and/or disseminating some bacterial or viral pathogens to humans (especially hunters) and domestic animals, especially in free range farms.

Antibiotic Susceptibility and Virulence Genes in Enterococcus Isolates from Wild Mammals Living in Tuscany, Italy.

Drug resistance is of great importance to human and animal health, but wild environments are still poorly understood in terms of their function as reservoirs of dangerous microbes and resistance determinants. The aim of the study was to determine the antibiotic susceptibility and virulence factors of Enterococcus bacteria from wildlife in Tuscany, Italy. Of the 36 mammalian fecal samples, 52 isolates were derived and classified as Enterococcus faecium (46% of isolates), Enterococcus hirae (19%), Enterococcus faecalis (13%), Enterococcus gallinarum (8%), Enterococcus casseliflavus (6%), Enterococcus durans (4%), Enterococcus mundtii (2%), and Enterococcus canintestini (2%) using both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and methods based on analysis of genetic material. The isolates tested showed the most frequent resistance to tetracycline (36.5% isolates), ciprofloxacin (36.5%), and erythromycin (25%). Three isolates showed high level of resistance (minimal inhibitory concentration ≥1,024 µg/mL) to vancomycin and teicoplanin, and 15% of the isolates demonstrated multidrug resistance. No isolate resistant to ampicillin, linezolid, or streptomycin was found. Among resistance genes, aac(6)-Ii (50% isolates), msrA/B (48%), msrC (42%), and tetM (31%) were identified most frequently. All E. faecium and E. faecalis isolates were positive for the efaAfm and efaAfs genes, respectively. Other virulence-associated genes, that is, gelE, cylA, asa1, esp, ace, orf1481, ptsD, and sgrA, were found in the majority of E. faecalis and several E. faecium isolates. Multilocus sequence typing analysis performed for selected isolates revealed three new sequence types. The results obtained indicate that wild mammals might act as reservoirs of resistance and virulence determinants that could be transferred between different ecosystems.

Molecular detection of Leptospira spp. in wild boar (Sus scrofa) hunted in Liguria region (Italy).

Leptospirosis is a re-emerging and widespread zoonosis, worldwide distributed, due to a wide variety of wild and domestic animal species able to act as natural or accidental hosts. During last years, in Europe, as in Italy, wild boar (Sus scrofa) population is increased. This animal represents a reservoir for different etiological agents, such as Leptospira. The aim of this investigation was to evaluate the prevalence of Leptospira spp. in wild boar hunted in Liguria region (Italy) during two-year hunting seasons. From 611 hunted wild boar, kidneys were collected. DNA was extracted from each organ and different targets were used to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene) and saprophytic (23S rRNA gene) Leptospira by Taqman-based RealTime-PCR assays. Overall, kidneys were sampled from 282 adults, 155 sub-adults and 174 young wild boar (in total 314 males and 298 females). By RealTime PCR 77 kidneys were positive and, among these, 74 resulted positive for pathogenic (96.10%) and 3 (3.90%) for intermediate Leptospira. No significant differences in pathogenic Leptospira infection ratio were detected between male (11.50%) and female (12.75%). Only 13 sub-adult animals (8.39%) resulted infected by pathogenic Leptospira; 23 young animals (13.22%) and 38 adult animals (13.47%) were positive. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis, which is able to infect other animal species (domestic and wild) including humans. Rarely, intermediate Leptospira could be able to infect wild boar with a renal localization that can contribute to their shedding and circulation.

Epidemiology of leptospirosis in North-Central Italy: Fifteen years of serological data (2002-2016).

Leptospirosis is a re-emerging bacterial zoonosis. North-Central Italy is characterized by a geographic area that promote Leptospira circulation. Data on sero-epidemiological survey carried out from 2002 to 2016 in North-Central Italy were reported and discussed. Overall, 709 out of the 8488 (8.35%) tested sera were positive for Leptospira at the cut-off titer (1:100) and 218 (2.57%) at higher titer (≥1:400). The highest percentages of positivity was recorded for coypus (22.86%), swine (19.74%) and bovine (13.03%). Pomona and Australis resulted the serogroup more often detected, followed by Sejroe and Icterohaemorrhagiae; while, a low number of positive sera was detected for serogroups Ballum, Canicola and Tarassovi. Percentage of positive sera for each year slightly decreased from 2002 to 2008 and rose from 2009. High percentages of positive reactions were recorded in 2014 (17.23%), 2015 (19.61%) and 2016 (38.05%). In conclusion, the results of this investigation reported an increase of leptospirosis in North-Central Italy. Furthermore, several animals resulted infected as accidental hosts by unusual Leptospira serovars. These data could suggest a change in host range for some serovars, that may promote the adaptation to new hosts.

Dietary supplementation of chestnut and quebracho tannins mix: Effect on caecal microbial communities and live performance of growing rabbits.

The interest in antimicrobial compounds as feed additives is currently increasing. Among different options, tannins seem to have several beneficial effects when employed in animals diet. The present study aimed at investigating the influence on caecal microbial communities of the supplementation of a chestnut and quebracho tannins mix in meat rabbit's diet, also considering animals live performances. Four groups of rabbits were fed with a different diet: a control diet (C); a control diet with coccidiostat (CC), and two experimental diets with 0.3% (T0.3) and 0.6% (T0.6) chestnut and quebracho tannins mix. For microbial analysis, culture-dependent and culture-independent methods were employed. Live performances were not significantly affected by tannins mix supplementations, as well as culturable microbial loads of E. coli, Enterobacteriaceae, Bacteroides spp. and Bifidobacterium spp. C. perfringens was always under the detection limit. A consistent result was obtained by qPCR. As for PCR-DGGE analysis, the Richness and evenness (Shannon-Weiner index) of bacterial communities in caecum resulted significantly higher in control samples (C and CC) than in those from rabbit fed with tannin-containing diets. Sequencing analysis revealed that the phylum Firmicutes was less represented in samples from control groups. As for the methanogen archaeal DGGE, no significant differences were found in richness and diversity among different groups, all dominated by Methanobrevibacter spp.. This work highlights the potential antimicrobial effect of chestnut and quebracho tannins mix in an in vivo system revealed by molecular analysis.

Antibiotic Susceptibility and Virulence Factors in Escherichia coli from Sympatric Wildlife of the Apuan Alps Regional Park (Tuscany, Italy).

Today a growing number of studies are focusing on antibiotic resistance in wildlife. This is due to the potential role of wild animals as reservoirs and spreaders of pathogenic and resistant bacteria. This study focused on isolating and identifying Escherichia coli from the feces of wild animals living in the Apuan Alps Regional Park (Tuscany, Italy) and evaluating some of their antibiotic resistance and pathogenicity traits. Eighty-five fecal samples from different species were studied. Seventy-one E. coli were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry analysis, subjected to antibiograms and polymerase chain reaction for the detection of antibiotic resistance genes and pathogenicity factors. The highest resistance rates were found against cephalothin (39.4%) and ampicillin (33.8%), followed by amoxicillin/clavulanic acid (15.5%), streptomycin (12.7%), and tetracycline (5.6%). Regarding resistance genes, 39.4% of the isolates were negative for all tested genes. The remaining isolates were positive for blaCMY-2, sul2, strA-strB and aadA1, tet(B), and tet(A), encoding resistance to beta-lactams, trimethoprim/sulfamethoxazole, streptomycin, and tetracycline, respectively. With regard to virulence factors, 63.4% of the isolates were negative for all genes; 21.1% carried astA alone, which is associated with different pathotypes, 9.9% carried both escV and eaeA (aEPEC); single isolates (1.4%) harbored escV (aEPEC), escV associated with astA and eaeA (aEPEC), astA with stx2 and hlyA (EHEC) or astA and stx1, stx2, and hlyA (EHEC). These results show that wildlife from nonanthropized environments can be a reservoir for antibiotic-resistant microorganisms and suggest the need for a deeper knowledge on their origin and diffusion mechanisms through different ecological niches.

A Serological Survey on Swine Brucellosis Using Standard Procedures, Dot Blot, and Western Blot in Finisher Pigs in Central-North Italy.

In recent years, Brucella suis has been sporadically reported in Italy in domestic and wild swine. Since standard serological tests can determine false positive results, the development of alternative tests with improved sensitivity and specificity is rather essential. We analyzed 1212 sera collected at slaughterhouse from healthy pigs belonging to 62 farms of North-Central Italy. Sera were tested by Rose Bengal Test, Complement Fixation Test, and subsequently by a Dot Blot (DB) and Western Blot assays (WB). Only one serum resulted positive to all tests, indicating that swine brucellosis has a very limited spread. DB and WB could represent a support to the available serological tests; however, further studies to validate these tests are needed. In the presence of reemerging diseases, a prompt and continuous monitoring design is necessary to acquire epidemiological information for the subsequent application of specific health emergency plans.

Mycobacterium aviumsubsp. paratuberculosis isolated from wild red deer (Cervus elaphus) in Northern Italy.

Paratuberculosis (or Johne's disease) is an infectious disease which affects mainly ruminants and it is caused by Mycobacterium avium subsp. paratuberculosis (MAP). During a culling program (years 2011-2015) aimed at controlling the red deer (Cervus elaphus) population in Stelvio National Park (Italian Alps), where paratuberculosis was already described in this species, 382 tissue samples from the Lombardy Region and 102 fecal specimens from the Autonomous Province of Bolzano were analyzed by PCR. Of these, 77 samples (20.16%) from the Lombardy area and 19 specimens (18.63%) from the Bolzano area resulted PCR positive. The cultural test was carried out on PCR positive samples (n = 96), enabling the isolation of 19 MAP field strains which were genotyped using MIRU-VNTR typing and Short Sequence repeats (SSRs). Our results suggest that all isolates share an identical VNTR profile corresponding to the INMV1 genotype. The only variation was on the locus SSR2, but the utility of this last locus has already been questioned because of its instability. Overall, these data suggest a common clonal origin and host adaptation during the diffusion of paratuberculosis in this population. Finally, this profile is the same as that which has already been described in the cattle population in Northern Italy, suggesting a possible inter-species disease transmission pattern from wildlife to domestic ruminants and vice versa.