Brucellosis is a major public health problem still prevalent as a neglected endemic zoonosis requiring proactive attention in many communities worldwide. The present study involved analysis of Brucella field strains submitted for typing to the Italian National Reference Laboratory for Brucellosis from 2007 to 2015. Strains were identified at the species and biovar levels by classic and molecular techniques according to the World Organisation for Animal Health Manual. In total, 5,784 strains were typed: 3,089 Brucella abortus (53.4%), 2,497 B. melitensis (43.2%), 10 B. ovis (0.2%), 181 B. suis (3.1%), and 7 B. ceti (0.1%). The 2,981 strains from cattle were typed as B. abortus biovars 1, 3, and 6 (90.1%) and B. melitensis biovar 3 (9.9%). The 318 strains from water buffalo were typed as B. abortus biovars 1, 3 (95.9%) and B. melitensis biovar 3 (4.1%). The 2,279 strains from sheep and goats were typed as B. abortus biovars 1 and 3 (4.3%); B. melitensis biovars 1, 3, (95.3%); and B. ovis (0.4%). The 173 strains from wild boar were typed as B. suis biovar 2 (98.3%) and B. melitensis biovar 3 (1.7%). The 11 strains from pigs were typed as B. suis biovar 2. The 13 strains from humans were typed as B. melitensis biovar 3. The two strains from horses were typed as B. abortus biovar 1, while the seven strains from dolphins were typed as B. ceti. This additional knowledge on the epidemiology of brucellosis in Italy may be useful to formulate policies and strategies for the control and eradication of the disease in animal populations. The animal species affected, biovars typed, geographical origins, and spatial distributions of isolates are herein analyzed and discussed.

Campylobacter species are common foodborne pathogens associated with cases of human gastroenteritis worldwide. A detailed understanding of the prevalence, contamination levels and molecular characteristics of Campylobacter spp. in cattle and chicken, which are likely the most important sources of human contamination, is imperative. A collection of 1243 poultry meat samples (665 chicken breasts and 578 chicken thighs) and 1203 bovine meat samples (689 hamburgers and 514 knife-cut meat preparations) were collected at retail outlets, in randomly selected supermarkets located in different Italian regions during one year. Of these samples, 17.38% of the poultry meat and 0.58% of the bovine meat samples tested positive for Campylobacter, of which 131 were Campylobacter jejuni (57.96%) and 95 were Campylobacter coli (42.03%). Campylobacter isolates were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and Campylobacter transmission route to humans. All isolates were molecularly characterized by pulse field gel electrophoresis (PFGE), and further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. Antibiotic resistance was also investigate. The highest levels of resistance among chicken strains were observed for ciprofloxacin (88.25%), nalidixic acid (81.45%) and tetracycline (75.6%). PFGE analysis revealed 73 pulsotypes for C. jejuni and 54 pulsotypes for C. coli, demonstrating the existance of different and specific clones circulating in Italy. MLST of C.jejuni isolates mainly clustered in the CC353, CC354, CC21, CC206 and CC443; while C.coli isolates clustered only in CC828. The most common flaA alleles were 287 for C. jejuni and 66 for C. coli. Our study confirms that poultry meat is the main source of Campylobacteriosis, whereas red meat had a low level of contamination suggesting a minor role in transmission. The high presence of Campylobacter in retail chicken meat, paired with its increased resistance to antimicrobials with several multidrug resistance profiles detected, is alarming and represents a persistent threat to public health.

This study aimed to analyse the diversity of theCampylobacter jejunipopulation in broilers and to evaluate the major source of contamination in poultry meat. Eight rearing cycles over one year provided samples from three different broiler farms processed at the same slaughterhouse. A total of 707 C. jejuniwere isolated from cloacal swabs before slaughter and from the breast skin of carcasses after slaughter and after chilling. All suspectedCampylobactercolonies were identified with PCR assays andC. jejuniwas genotyped by sequence analysis of theflaAshort variable region (SVR) and by pulsed-field gel electrophoresis (PFGE) usingSmaIenzyme. Phenotypic antibiotic resistance profiles were also assayed using minimal inhibitory concentration (MIC). The flocks carried many majorC. jejuniclones possibly carrying over the rearing cycles, but cross contamination between farms may happen. Many isolates were resistant to fluoroquinolones, raising an issue of high public concern. SpecificCampylobacterpopulations could be harboured within each poultry farm, with the ability to contaminate chickens during each new cycle. Thus, although biosecurity measures are applied, with a persistent source of contamination, they cannot be efficient. The role of the environment needs further investigation to better address strategies to controlCampylobacter.

Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Swine are known to be the main reservoir of Campylobacter coli and a possible source infection of humans as a result of carcass contamination at slaughter. The aim of this study was to evaluate the prevalence of C. coli contamination in swine carcasses, the antimicrobial resistance (AMR) patterns of isolates and the genetic diversity between strains obtained from swine and those isolated from humans. The prevalence of contamination was higher on carcasses (50.4%) than in faeces (32.9%). The 162 C. coli isolated from swine were examined by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The results of PFGE indicated a high genetic diversity among the isolates, with 25 different PFGE types. MLST assigned 51 sequence types (STs) to isolates. The most common genotype was ST-854 (16.04%), ST-9264 (10.49 %) and ST-1016 (6.08 %). Results of AMR showed a high resistance to quinolones and fluoroquinolones together with aminoglycosides and tetracycline. Many strains were multi-resistant with predominant R-type TeSCipNa (57%). Five resistance genes were detected along with mutation in the gyrA gene. A strong correlation between phenotypic and genotypic resistance was found for fluoroquinolone and tetracycline. Genetic profiles obtained in swine isolates were compared to those of 11 human strains. All human strains and 64.19% of animal strains (104/162) were assigned to the ST-828 clonal complex.

Foodborne campylobacteriosis is caused mainly by the handling or consumption of undercooked chicken meat or by the ingestion of contaminated raw milk. Knowledge about the contributions of different food sources to gastrointestinal disease is foundamental to prioritize food safety interventions and to realize adequate control strategies. Assessing the genetic diversity among Campylobacter species is essential to our understanding of their epidemiology and population structure. We molecularly characterized 56 Campylobacter jejuni isolates (31 from patients hospitalized with gastroenteritis, 17 from raw milk samples and 8 from chicken samples) using multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) in order to trace the source of the disease. We also used a population genetic approach to investigate the source of the human cases from six different reservoirs of infection. MLST identified 25 different sequence types and 11 clonal complexes (CCs; 21, 658, 206, 353, 443, 48, 61, 257, 1332, 354, 574) and these included several alleles not cited previously in the PubMLST international database. The most prevalent CCs were 21, 206 and 354. PFGE showed 34 pulsotypes divided between 28 different clusters. At the fine scale, by means of PFGE and MLST, only two human cases were linked to raw milk, while one case was linked to chicken meat. The investigation revealed the presence of several genotypes among the human isolates, which probably suggests multiple foci for the infections. Finally the source attribution model we used revealed that most cases were attributed to chicken (69.75%) as the main reservoir in Italy, followed to a lesser extent by the following sources: cattle 8.25%; environment 6.28%; wild bird 7.37%; small ruminant 5.35% and pork 2.98%. This study confirms the importance of correlating epidemiological investigations with molecular epidemiological data to better understand the dynamics of infection.

Brucellosis is a common and chronic disease of cattle and other bovids that often causes reproductive disorders. Natural infection in cattle is caused by Brucella abortus and transmission typically occurs during abortions, calving, or nursing. Brucellosis is also a major zoonotic disease due to contamination of dairy products or contact with the tissues of infected animals. Brucellosis has been eradicated from most of the developed world in the last 40 years but persists in many regions—the disease remains prevalent in portions of Africa, the Middle East, Asia, and Central and South America, as well as in the Mediterranean basin. In Italy, B. abortus has persisted in southern regions in both cattle and water buffalo. Previous attempts at analyzing the phylogenetics of B. abortus in Italy have been challenging due to limited genetic variability and unresolved global population genetic structure of this pathogen. We conducted genome-wide phylogenetic analyses on 11 representative strains of B. abortus from Italy, and compared these sequences to a worldwide collection of publically available genomes. Italian isolates belong to three clades that are basal to the main and global B. abortus lineage. Using six SNP-based assays designed to identify substructure within the Italian clades, we surveyed a collection of 261 isolates and found that one clade predominates throughout endemic districts in the country, while the other two clades are more geographically restricted to portions of southern Italy. Although related strains exist worldwide, B. abortus isolates from Italy are substantially different than those found in much of the rest of Europe and North America, and are more closely related to strains from the Middle East and Asia. Our assays targeting genetic substructure within Italy allowed us to identify the major lineages quickly and inexpensively, without having to generate whole genome sequences for a large isolate collection. These findings highlight the importance of genetic studies to assess the status and the history of pathogens.