EMERGING PATHOGEN ARCOBACTER SPP. IN FOOD OF ANIMAL ORIGIN

7.1 Foreword The genus Arcobacter, previously classified as “aerotolerant campylobacter”, from 1991 is classified as a new genus member of the family of Campylobacteriaceae, belonging to the epsilon-proteobacteria. The genus Arcobacter contains eight described species: Arcobacter butzleri, A. cryaerophilus, A. nitrofigilis; A. skirrowii ,A. cibarius, A. thereius, A. halophilus and 46 A. mytili . The bacteria are Gram negative, non-sporeforming, motile, curved, occasionally straight, rods which may appear as spiral. The most important differences between Arcobacter and Campylobacter are the ability of Arcobacter to grow at 15-25°C and its marked aerotolerance. Arcobacter spp. have been considered as potential zoonotic foodborne and waterborne agents (. Arcobacter spp. can be found in meat (veal, beef, pork and poultry), milk and water. Nevertheless the real occurrence of these potential pathogens in food is largely unknown. 7.2 Objectives The aim of this study is to evaluate prevalence and distribution of Arcobacter spp. in food of animal origin (raw meat and milk) in Northern Italy and to test the antimicrobial susceptibility of collected strains. 7.3 Trial 1: Prevalence and Distribution of Arcobacter spp. In Veal Calves in Northern Italy In this study the prevalence and distribution of Arcobacter spp. in samples taken from feces and carcasses of healthy veal calves, raised in Northern Italy, were investigated. A membrane filtration technique with a non-selective blood agar was employed after enrichment in Arcobacter selective broth to isolate a wide range of Arcobacter spp. In addition, the same samples were tested at the species level by using a multiplex-PCR assay. Samples from feces (50 during a summer sampling, 50 during a winter sampling) and carcasses (50 during a summer sampling a 50 during a winter sampling) were collected at the slaughterhouse from 50 veal calves originating from five different farms. Of the fecal samples examined, 36 (72%) were found positive for at least one species of Arcobacter during summer sampling and 38 (76%) were found positive during winter sampling. Of the sampled carcasses examined, 50 (100%) were found positive for at least one species of Arcobacter in summer sampling and 20 (40%) in winter sampling. From feces, 23 (46%), 17 (34%) and 5 (10%) samples were found positive by m-PCR for A. cryaerophilus, A. skirrowii and A. butzleri, respectively in summer. On the other side in winter 15 (30%), 12 (24%) and 2 (8%) samples were found positive by m-PCR for A. cryaerophilus, A. skirrowii and A. butzleri, respectively. From carcasses, 47 (94%), 17 (34%) and 5 (10%) samples were positive for A. cryaerophilus, A. skirrowii and A. butzleri, respectively in summer sampling whereas 5 (10%), 4 (8%) and 8 (16%) samples were positive for A. cryaerophilus, A. skirrowii and A. butzleri, respectively in winter sampling . Some sampled carcasses (34% in summer and 6% in winter) and some fecal samples (18% both in summer and in winter) resulted contaminated with multiple Arcobacter species. The present study indicates that veal calves can harbour a variety of Arcobacter spp. in the intestinal tract and that the presence of Arcobacter spp. may represent a source of contamination and dissemination in slaughterhouse. 7.4 Trial 2: Isolation and Characterization of Arcobacter spp. in Bulk Tank Milk Bulk tank milk (BTM) from 50 dairy herds in Northern Italy was examined for the presence of foodborne pathogen: Arcobacter spp. A membrane filtration technique with a non-selective blood agar was employed after enrichment in Arcobacter selective broth to isolate a wide range of Arcobacter spp. In addition, the same samples were tested at the species level by using a multiplex-PCR assay. Arcobacter butzleri and A. cryaerophilus were detected in 40 and 8% of bulk tank milk samples, respectively. One bulk tank milk sample contained both Arcobacter butzleri and A. cryaerophilus. No BTM sample resulted positive to A. skirrowii. No significant association between the presence of Arcobacter spp. in BTM and herd management was observed. The findings of the study demonstrated the presence Arcobacter spp. as a potential milkborne pathogen and warrant the need for educational programs for dairy producers about the risks associated with consumption of raw milk. 7.5 Trial 3: Antimicrobial Susceptibility of Arcobacter spp. Isolated from Food of Animal Origin Arcobacter spp. is a human pathogen and a potential animal pathogen. Thus is important to study antimicrobial susceptibility of strains collected from food of animal origin testing the most common antimicrobial agents used in human and veterinary medicine. In the present study, we examined the antimicrobial susceptibility of 50 Arcobacter spp. strains collected in previous studies from veal calves carcasses and bulk tank milk (BTM), testing 8 antimicrobial agents by disk diffusion testing. Antibiotics and the concentrations of discs (μg) were kanamycin (30), streptomycin (10), gentamicin (10), tetracycline (30), cephalothin (30), ciprofloxacin (5), nalidixic acid (50) and sulfamethoxazole–trimethoprim (23.75 and 1.25, respectively). The plates were cultured at 37 °C and after 48 h of incubation, the diameter of the inhibition zones was measured with a slide caliper. All the tested Arcobacter strains showed resistance to cephalotin, sulfamethoxazole+trimethoprim and nalidixic acid. A. cryaerophilus from veal calf carcasses showed a 100% susceptibility to tetracycline. A. butzleri from BTM showed 100% susceptibility to gentamycin. 7.6 Conclusions This study shows that Arcobacter spp. is largely diffused in healthy livestock and in food of animal origin, both raw meat and raw milk. Although their pathogenicity remains to be fully elucidated, arcobacters are potential foodborne pathogens and their prevalence should be carefully investigated to guarantee public human and animal health.