Size characteristics of asbestiform fibers in lung tissue of animals that grazed in areas rich in ophiolitic outcrops in Central Calabria (Southern Italy).

In Calabria (Southern Italy) naturally occurring asbestos (NOA) mainly occurs in the ophiolitic sequences cropping in the Mount Reventino area. The most common type of asbestos detected was the amphibole tremolite; fibrous antigorite and minor chrysotile were also found. The development of asbestos-related diseases depends on, among other things, the morphological characteristics of fibers, length and width, affecting the durability of asbestos fibers in the lung. In this work fifteen lung samples of sheep, goats and wild boars, grazing around the Mount Reventino area were collected and asbestos fibers analysed. Observed fibers (357), of which 97 % were tremolite and 3 % antigorite fibers, were grouped according to species, grazing area and age of the animals. The aim of this work was to highlight any differences among the groupings and to compare our size results with data in literature related to exposed populations. Principal Component Analysis (PCA) highlighted a positive correlation between tremolite fiber length and width and revealed groupings in terms of animal age. The Kruskal-Wallis test showed statistically significant differences between fiber mean widths in young and old animals. 63 % observed asbestiform fibers were longer than 5 µm and 7 % of the fibers were longer than 20 µm (critical fiber length connected to the frustrated phagocytosis by the macrophage). Fibers conforming to the Stanton Hypothesis size (predictor of the carcinogenic potency of fibers) were 1 %. Our size parameters of fibers detected in the animal lungs were in fairly good agreement with literature data for human asbestos exposure to tremolite. These results confirmed that an animal-sentinel system could be used to monitor the natural background of the airborne breathable fibers exposure. In addition, the size correlation of animal-human breathed fibers could be useful to study their potential toxicity. Additional data are necessary for improving the agreement with human exposure data.

Evaluation of the Presence and Viability of Mycobacterium bovis in Wild Boar Meat and Meat-Based Preparations.

The aim of the present study is to provide information about the ability of Mycobacterium bovis to survive within wild boar (Sus scrofae) meat and meat-based preparations and the duration of this survival, and to consider the preservation of its infectious potential toward humans and animals. Meat samples were artificially contaminated with an M. bovis field strain and then stored at -20 °C, while two sausages batches were contaminated with the same field strain at two different concentrations, 105 CFU/g and 103 CFU/g, before storing them in proper conditions to allow for their ripening. A third sausage batch was contaminated by adding 2 g of wild boar lymph nodal tissue with active tuberculous lesions to the meat mixture. Bacteriological and biomolecular (PCR) methods were used to test the meat and sausage samples every 60 days and every 7-10 days, respectively. M. bovis was detected as still alive and viable on the frozen meat for the last test on the 342nd day, while from the sausage samples, M. bovis was isolated until 23 days after contamination. Our results indicate that M. bovis can stay alive and be viable for 23 days within sausages prepared with contaminated meat from infected wild boars. These products are usually eaten as fresh food after grilling, often cooking at a temperature that does not ensure complete inactivation of the pathogenic microorganisms present, which can pose a risk for humans to develop zoonotic tuberculosis.

Seroprevalence and phylogenetic characterization of hepatitis E virus in pig farms in Southern Italy.

Hepatitis E virus (HEV) infection is an emerging public health problem in industrialized countries. The infection is associated with waterborne epidemics and transmitted via faecal-oral route. Zoonotic cases of HEV in humans have increased in Europe, and HEV genotype 3 (HEV-3) is the most frequent among humans and animals. Nevertheless, HEV surveillance in the Italian pig farming industry is patchy. Here, HEV prevalence in pig farms located in the Calabria region in Southern Italy was investigated. A total of 692 serum samples were collected from 26 farms and tested for anti-HEV IgG antibody detection. The percentage of HEV-seropositive pigs was 56.8 %. Small farm size, farrow-to-finishing production, and infrequent cleaning procedures were associated with higher HEV seroprevalence. In 12 of the HEV-seropositive farms, 67 faecal samples were collected and 10 of these (10.6 %) tested positive for HEV RNA. Seven of 10 viral RNA sequences were genotyped for phylogenetic analysis, five of which belonged to subtype HEV-3f and two to subtype HEV-3e. The high HEV seroprevalence and the circulation of HEV-3 strains among domestic pigs in the Calabria region pose a risk for the zoonotic transmission of HEV from pigs to occupational exposed workers.

Serotyping and Evaluation of Antimicrobial Resistance of Salmonella Strains Detected in Wildlife and Natural Environments in Southern Italy.

Wild animals are potential vectors of antibiotic-resistant bacteria in the environment. The present study aimed to investigate the occurrence of antimicrobial resistance among Salmonella serovars isolated from wildlife and the environment in Italy. A total of 164 Salmonella isolates were analyzed, and six different subspecies and 64 serovars were detected. High proportions of Salmonella isolates proved resistant to streptomycin (34.1%), followed by trimethoprim-sulfamethoxazole (23.2%), tetracycline (17.7%), ciprofloxacin (14.63%) and ampicillin (11.59%). By source, the lowest level of resistance was observed in Salmonella serovars isolated from a water environment, while antimicrobial resistance was frequent in strains collected from shellfish, reptiles and birds. Multidrug-resistant strains were recovered from seafood (n = 11), mammals (n = 3) and water (n = 1). Three S. Typhimurium monophasic variant strains showed asimultaneous resistance to ampicillin, streptomycin, tetracycline and trimethoprim-sulfamethoxazole, which represents a recognized alert resistance profile for this serovar. These data indicate the environmental dissemination of resistant strains due to anthropogenic activities, which, in southern Italy, probably have a higher impact on marine ecosystems than on terrestrial ones. Moreover, as most of the animals considered in the present study are usually consumed by humans, the presence of resistant bacteria in them is a matter of great concern.

Presence of Mycobacterium bovis in slaughterhouses and risks for workers.

An investigation was carried out to detect the presence of Mycobacterium bovis in slaughterhouses where intradermal tuberculin test positive cattle were slaughtered, and to evaluate the risk of contamination by M. bovis among exposed slaughterhouse workers. Swabs were taken from the carcasses of slaughtered animals showing autoptic signs of non-generalized forms of tuberculosis, thus authorized for free consumption. Swabs were also taken from the hands and clothes of the staff employed in the butchery production line. Environmental samplings were conducted on the slaughterhouse air using filters and air aspiration devices, and on water used to wash the carcasses after slaughter. Samples from the carcasses of healthy animals were also taken on a following slaughtering session. The swabs were analysed by means of Polymerase Chain Reaction for the detection of mycobacteria. M. bovis was detected on meats, on the hands of one worker, and in the washing water. The results obtained from this study confirm that workers are highly exposed to infection by zoonotic tuberculosis, and that cleaning procedures were ineffective in our setting.

Environmental contamination by naturally occurring asbestos (NOA): Analysis of sentinel animal lung tissue.

Ophiolites are known sources of naturally occurring asbestos (NOA). In Calabria (Southern Italy) NOA are mainly concentrated in the ophiolitic sequences cropping in the Mount Reventino area, in the southern part of the Sila massif, and along the Coastal Chain. The most common type of asbestos identified in the rocks of these areas belongs to the tremolite-actinolite series. Another identified asbestiform mineral is fibrous antigorite belonging to the serpentine mineral group with a minor amount of chrysotile. The purpose of the present study is to evaluate the diffusion of natural asbestiform fibers from NOA using sentinel animals. Fifteen lung samples of sheep, goats and wild boars from Mount Reventino area and two from an area free from NOA were collected. The lung samples were subjected to anatomopathological examination and lung fiber burden analysis by electron microscopy. Abundant tremolite and few antigorite fibers were detected in the lung samples coming from the NOA area. No corpuscle of asbestos was observed. No fiber was found in the two lung samples of sheet from the area free from NOA. These concentrations of fibers per gram of dry weight of lung tissue (f/gdw) ranged from 104 to 106 f/gdw. The asbestos fibers detected in the lungs of the examined animals reflect the geological features of the areas where they grazed and lived. The anatomopathological analysis showed that 60% of the examined animals had macroscopic lesions affecting their lungs. The presence of tremolite fibers in the lungs confirms the diffusion of mineral fibers in the environment and the real advantage of using animal populations in the study areas.

Dispersal and molecular characterisation of the Echinococcus granulosus (Batsch, 1786) complex isolated from various intermediate hosts in the Calabria region, southern Italy.

Cystic echinococcosis (CE) is a zoonotic disease caused by the tapeworms of the Echinococcus granulosus sensu lato complex, which have worldwide distribution. No data on the circulation of genotypes of the E. granulosus complex in intermediate hosts in endemic areas in Calabria are available. The aims of our study were to evaluate the dispersal of genotypes of the E. granulosus complex in Calabria and to characterise parasite isolates by Sanger sequencing and phylogenetic analysis. We collected 71 animal samples from pigs, wild boars, sheep, cattle and goats. The first PCR screening analysis targeted three partial genomic regions: the cytochrome c oxidase subunit 1 (cox1), calreticulin protein (cal) and NADH dehydrogenase subunit 1 (nad1); this identified 28 parasitic cysts. Bidirectional sequencing of cox1 amplicons and phylogenetic analysis allowed us to characterise all isolates. Molecular analyses of 28 newly generated cox1 sequences revealed that most wild boars (n = 16) and three pigs were parasitised by the larval stage of Taenia hydatidena Pallas, 1766, called cysticercus tenuicollis. Two isolates from wild boars were identified as Echinococcus canadensis Webster and Cameron, 1961 (G7), while five sheep and two goats were infected with E. granulosus G1 (sheep strain) and G1 microvariant (previously reported as G2 genotype or Tasmanian sheep strain), respectively. These molecular findings should prompt further and more extensive studies, to elucidate regional transmission patterns and to guide control programs.

Local and Transboundary Transmissions of Methicillin-Resistant Staphylococcus aureus Sequence Type 398 through Pig Trading.

Livestock-associated methicillin-resistant Staphylococcus aureus sequence type (ST) 398 (LA-MRSA ST398) is a genetic lineage for which pigs are regarded as the main reservoir. An increasing prevalence of LA-MRSA ST398 has been reported in areas with high livestock density throughout Europe. In this study, we investigated the drivers contributing to the introduction and spread of LA-MRSA ST398 through the pig farming system in southern Italy. Whole-genome sequencing (WGS) of LA-MRSA ST398 isolates collected in 2018 from pigs (n = 53) and employees (n = 14) from 10 farms in the Calabria region of Italy were comparatively analyzed with previously published WGS data from Italian ST398 isolates (n = 45), an international ST398 reference collection (n = 89), and isolates from Danish pig farms (n = 283), which are the main suppliers of pigs imported to Italy. Single-nucleotide polymorphisms (SNP) were used to infer isolate relatedness, and these data were used together with data from animal trading to identify factors contributing to LA-MRSA ST398 dissemination. The analyses support the existence of two concurrent pathways for the spread of LA-MRSA ST398 in southern Italy: (i) multiple introductions of LA-MRSA ST398 through the import of colonized pigs from other European countries, including Denmark and France, and (ii) the spread of distinct clones dependent on local trading of pigs between farms. Phylogenetically related Italian and Danish LA-MRSA ST398 isolates shared extensive similarities, including carriage of antimicrobial resistance genes. Our findings highlight the potential risk of transboundary transmission of antimicrobial-resistant bacterial clones with a high zoonotic potential during import of pigs from countries with high LA-MRSA prevalence.IMPORTANCE Over the past decade, livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 (LA-MRSA ST398) has spread among pig holdings throughout Europe, in parallel with the increased incidence of infections among humans, especially in intensive pig farming regions. Despite the growing prevalence of LA-MRSA ST398 in Italian pig farms, the transmission dynamics of this clone in Italy remains unclear. This work provides genome-based evidence to suggest transboundary LA-MRSA ST398 transmission through trading of colonized pigs between European countries and Italy, as well as between farms in the same Italian region. Our findings show that both international trading and local trading of colonized pigs are important factors contributing to the global spread of LA-MRSA ST398 and underscore the need for control measures on and off the farm to reduce the dissemination of this zoonotic pathogen.

Foodborne Pathogen Assessment in Raw Milk Cheeses.

General hygienic parameters and selected foodborne pathogens in raw milk cheeses at the retail level were evaluated. A total of 245 raw milk cheese samples were analysed for total bacterial count, Enterobacteriaceae, E. coli, Salmonella spp., Listeria monocytogenes, coagulase-positive Staphylococci, and staphylococcal enterotoxin. Results showed only 3 samples that were not compliant with European rules on staphylococcal enterotoxin, but coagulase-positive Staphylococci were evidenced in all samples tested. Salmonella spp. and Listeria monocytogenes were never detected whereas E. coli was evidenced in 20 samples. Results suggest a need for improvement of good manufacturing practice and milking operation.

Identification of pmrB mutations as putative mechanism for colistin resistance in A. baumannii strains isolated after in vivo colistin exposure.

Colistin resistance among extensively-resistant Acinetobacter baumannii isolates is a serious health-care problem. Alterations in PmrA-PmrB two-component system have been associated with resistance to colistin. We investigated three pairs of colistin-susceptible and colistin-resistant A. baumannii, sequentially isolated from three patients before and after colistin treatment, respectively. The pmrA and pmrB genes were sequenced by Sanger method. Amino acidic positions and their effect on protein were predicted by InterPro and PROVEAN tools. Expression of pmrA, pmrB and pmrC genes was assessed by semi-quantitative reverse transcription-PCR (qRT-PCR). We found three different nonsynonymous substitutions P233T, E301G and L168K in pmrB coding region, each one in a different colistin resistance strain. The E301G and L168K substitutions represent novel mutations in pmrB, not previously described. Relative expression of pmrA, pmrB and pmrC mRNA increased in all colistin resistant strains. In our study, pmrB substitutions were associated with pmrC over-expression and colistin resistance. Further studies are necessary to understand their impact on modification of lipid A components.

Unidirectional animal-to-human transmission of methicillin-resistant Staphylococcus aureus ST398 in pig farming; evidence from a surveillance study in southern Italy.

Background: Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 398 is recognized as an occupational hazard for workers employed in intensive animal husbandry, especially in the swine-breeding chain. In this study, we compared the prevalence and epidemiological type of MRSA isolates from swine and farm workers in a large area of southern Italy. Methods: Between January and March 2018, 88 workers from 32 farms where we had previously performed a survey for MRSA colonization of farmed pigs, were sampled by nasal swabbing. A follow-up investigation was conducted on seven workers 1 year after primary screening. MRSA isolates were characterized by MLST, spa and SCCmec typing, and tested for susceptibility to 15 antimicrobials. Epidemiological correlations between human and swine MRSA isolates were supported by Rep-MP3 and RAPD PCR fingerprinting, and whole-genome sequencing. Results: The overall colonization rate of MRSA in swine farm workers was 21.6%, being significantly higher in intensive farms and in workers with direct animal contact. All human MRSA isolates were multi-drug resistant, belonged to the ST398 livestock clade, and did not carry Panton-Valentine leukocidin and enterotoxin genes. Notably, 94.1% of human MRSA isolates belonged to the same epidemiological type as swine MRSA isolates from the corresponding farm. Persistent MRSA carriage was documented in some workers 1 year after primary sampling. Conclusions: We report a high prevalence of MRSA among swine farm workers, with higher colonization rates associated with intensive breeding and animal exposure. Our findings suggest unidirectional animal-to-human transmission of LA-MRSA and denote the high zoonotic transmissibility of the ST398 livestock clade.

Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carriage and pattern of antibiotic resistance among sheep farmers from Southern Italy.

PURPOSE: We conducted a cross-sectional study to measure the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization, with a particular focus on livestock associated (LA)-MRSA in farmers working in contact with livestock (sheep) in one Italian region. Furthermore, we have assessed the antimicrobial resistance pattern of isolates and the association of carriage with specific characteristic of farms and working tasks. PATIENTS AND METHODS: Demographic data, occupational history, and contact with animals information was collected. Nasal and oropharyngeal swabs were collected and all samples were tested for the isolation and identification of S. aureus. Isolates were examined for antimicrobial susceptibility and all MRSA strains underwent molecular analyses through multiple-locus variable number of tandem repeat analysis (MLVA). RESULTS: A total of 115 sheep farms and 275 sheep farmers were enrolled. MRSA colonized workers were found in three farms; S. aureus was isolated in 97 workers (35.5%), whereas MRSA was isolated in 3 (1.1%) workers. All MRSA isolates were classified as multidrug resistant. Two of the MRSA isolates were resistant to quinupristin/dalfopristin (QDA), mupirocin, erythromycin, and tetracycline. Among methicillin-susceptible S. aureus (MSSA), 32 (34%) were resistant to tetracycline, 31 (33%) to erythromycin, 26 (27.6%) to QDA, and 22 (23.4%) to linezolid and clindamycin. One MRSA belonged to MLVA complex (MC) 001, found to colonize both humans and animals. CONCLUSION: The picture of MRSA transmission among sheep farmers does not seem to be critical, although there is the need to improve adequate control measures to prevent and minimize any biological risk in sheep farms for both animal and human health. Specific monitoring/surveillance programs would help in better understanding the epidemiology of resistant strains.

Clinical, Virological Characteristics, and Outcomes of Treatment with Sofosbuvir/Ledipasvir in Two Pediatric Patients Infected by HCV Genotype 4.

Direct-acting antiviral drugs to cure infections with Hepatitis C virus (HCV) achieve a sustained virological response (SVR) in more than 90% of adult patients. At present, clinical trials are ongoing and real-life data are still limited in children. Herein, we report two cases of pediatric patients treated with fixed-dose combination of sofosbuvir/ledipasvir, already approved to treat HCV4 genotype. Both young girls achieved SVR even though HCV4 isolates carried L28M and M31L NS5A resistance-associated substitutions (RASs). Therefore, possible effects of these RASs merit further study, especially in children.

Malignant peritoneal mesothelioma in a boar who lived in Calabria (Italy): Wild animal as sentinel system of human health.

Mesothelioma is a tumor of the serosal membranes described both in human and veterinary medicine. While in humans the relationship between mesothelioma and exposure to asbestos and some other asbestiform minerals is well known, in animals it is still difficult to establish. In this paper a case of malignant peritoneal mesothelioma probably related to asbestos exposure in a wild boar is described. At post-mortem evaluation the peritoneum, diaphragm and serosal surface of liver and kidneys showed isolated to coalescent multiple nodular lesions. Samples from diaphragm, liver and lung were collected to perform microbiological and histological investigations. To assess the presence of asbestos and/or other asbestiform minerals, SEM-EDS investigations were performed on organs and soil samples collected from the area where the wild boar lived. Microbiological investigations were negative for Mycobacterium species. Gross and histological examination were compatible with a biphasic mesothelioma, with nodules composed of epithelioid and sarcomatoid elements with high pleomorphism. Immunohistochemistry revealed only multifocal scattered positivity for WT-1 and D2-40. Asbestos fibres were detected in all samples (organs and soil) by SEM-EDS, demonstrating a potential relationship between the neoplasia and the exposure to naturally occurring asbestos (NOA). In conclusion, the results of the present study are further confirmation that wild animals, such as the boar, are suitable sentinels to indicate the risk of environmental exposure to asbestos for human populations.

Prevalence, molecular epidemiology, and antimicrobial resistance of methicillin-resistant Staphylococcus aureus from swine in southern Italy.

BACKGROUND: Colonization by livestock-associated MRSA (LA-MRSA) has increasingly been reported in the swine population worldwide. The aim of this study was to assess the prevalence of MRSA nasal carriage in healthy pigs, including the black (Calabrese) breed, from farms in the Calabria Region (Southern Italy). Between January and March 2018, a total of 475 healthy pigs reared in 32 farms were sampled by nasal swabbing. MRSA isolates were characterized by spa, MLST and SCCmec typing, and susceptibility testing to 17 antimicrobials. RESULTS: 22 of 32 (66.8%) pig farms resulted positive for MRSA. The prevalence of MRSA was 46.1% (219 MRSA culture-positive out of 475 samples). MRSA colonization was significantly higher in intensive farms and in pigs with a recent or ongoing antimicrobial treatment. All 219 MRSA isolates were assigned to ST398. The most common spa types were t011 (37.0%), t034 (22.4%) and t899 (15.1%). A novel spa type (t18290) was detected in one isolate. An insertion of IS256 in the ST398-specific A07 fragment of the SAPIG2195 gene was detected in 10 out of 81 t011 isolates. Nearly all isolates carried the SCCmec type V element, except 11 isolates that carried the SCCmec type IVc. None of the isolates was positive for the Panton-Valentine leukocidin. All isolates were resistant to tetracycline. High resistance rates were also found for clindamycin (93.1%), trimethoprim/sulfamethoxazole (68.4%), fluoroquinolones (47.9-65.3%) and erythromycin (46.1%). None of the isolates was resistant to vancomycin and fusidic acid. Overall, a multidrug resistant phenotype was observed in 88.6% of isolates. CONCLUSIONS: We report a high prevalence of MRSA among healthy swine in Southern Italy farms, with higher isolation frequency associated with intensive farming. The epidemiological types identified in our study reflect those reported in other European countries. Our findings underscore the importance of monitoring the evolution of LA-MRSA in pig farms in order to implement control measures and reduce the risk of spread in the animal population.

Development and evaluation of two multi-antigen serological assays for the diagnosis of bovine tuberculosis in cattle.

There is currently an increased interest in the use of serological approaches in combination with traditional cell-mediated immunity-based techniques to improve the detection of tuberculosis (TB)-infected animals. In the present study, we developed and validated two different serological TB-detection assays using four antigens, MPB70, MPB83, ESAT6 and CFP10, and the tuberculin PPDb. A conventional multi-antigen TB-ELISA method and a novel TB multiplex test, based on Luminex technology, were developed to detect antibodies to multiple antigen targets. The performance levels of the two tests were evaluated and compared using selected panels of samples having known TB states. The TB-ELISA test (containing five antigens, including PPDb) had a sensitivity (Se) of 74.2% and a specificity (Sp) of 94.9%, while the TB-Luminex test had higher Se (79.0%) and Sp (99.1%) rates even when only one reactive antigen was used to classify the test as positive. If a more restrictive criterion, requiring two positive antigens to classify the test as positive, was used, then the TB-ELISA's Sp rate increased to 99.8% but the Se decreased to 61.3%, while the TB-Luminex test's Sp rate increased to 100% but the Se decreased to 51.2%. TB-ELISA and TB-Luminex were applied to a panel of 257 sera collected from bTB-positive herds, as determined by a post-mortem inspection. They showed good performance levels, identifying 49 (80.3%) and 48 (78.7%), respectively, of 61 samples that had tested positive by the intradermal tuberculin (IDT) test and/or interferon-gamma assay. In addition, TB-ELISA and TB-Luminex were able to identify 60 and 42 samples as positive, respectively, out of the 196 samples that tested negative to IDT and interferon-gamma at the time of serum collection. Subsequent IDT tests performed after 1-2 months, confirmed the positivity of 18 samples, indicating the strategic value of having two serological assays to detect TB-infected herds that were not reactive to initial IDT testing, thereby allowing for the rapid control of outbreaks and eradication of the disease.

Phenotypic Antimicrobial Resistance Profile of Isolates Causing Clinical Mastitis in Dairy Animals.

Mastitis is the most frequent and costly disease of lactating animals and is associated with a significant reduction in milk yield, increased cost and culling. Early and specific antibiotic based treatment reduces the severity of the disease. Over the years the extensive use of antimicrobials has led to increase antimicrobial resistance. The present study was designed to investigate the prevalence of microorganisms responsible for mastitis and their antimicrobial resistance pattern. A total of 282 milk samples were collected from different animal species (sheep, cows and goats) with clinical mastitis. Antimicrobial resistance was evaluated for Streptococcus spp. and Staphylococcus spp. In cow samples Streptococcus spp. represented the most frequently isolated genus (33.84%), while Staphylococcus spp. was the most prevalent genus in sheep and goat samples (44.4 and 73.86%, respectively). Gentamicin and chloramphenicol were found to be the most effective drugs against the tested isolates, while the highest resistance rates were observed for amoxicillin, ampicillin, tetracycline, trimethoprim-sulfamethoxazole.

Contamination of Bovine, Sheep and Goat Meat with Brucella Spp.

A study was conducted in order to evaluate the contamination by Brucella spp. of meat from animals slaughtered because they had resulted positive for brucellosis at some time during their life. After slaughter and before delivery to market outlets, swab samples were taken from 307 carcasses of infected animals: 40 cattle, 60 sheep and 207 goats. The swabs were subsequently analysed by means of polymerase chain reaction (PCR) tests. In addition, bacteriological tests were carried out on the lymph nodes and internal organs of the same animals. Brucella spp. was detected by means of PCR in 25/307 carcasses (8%): 1 bovine (2.5%), 9 sheep (15%) and 15 goats (7.2%) and was isolated by means of a cultural method in 136/307 carcasses (44%). Moreover, additional analysis, performed on lymph nodes from the same carcasses that had proved positive by PCR, allowed highlighting type 3 Brucella abortus in the bovine carcass and type 3 Brucella melitensis in the sheep and goat carcasses. The study shows that cattle, sheep and goats meat of animals slaughtered because they had tested positive for brucellosis may be contaminated by Brucella spp. As this could constitute a real risk of transmission to both butchery personnel and consumers, the meat of animals infected by Brucella spp. should be analysed before being marketed. In this respect, PCR technique performed on swabs proved to be more useful, practical and faster than the traditional bacteriological method.

Molecular Typing of Staphylococcus Aureus Isolate Responsible for Staphylococcal Poisoning Incident in Homemade Food.

In October 2012, two persons fell ill with symptoms consistent with staphylococcal food poisoning after eating home-canned tuna fish and tomatoes. Laboratory investigation detected the enterotoxins in the home-canned tuna and molecular analysis of the isolated Staphylococcus aureus confirmed it carried toxin genes. Qualitative enzyme-linked immunosorbent assay and enzime linked fluorescent assay methods and quantitative assay identified the enterotoxins in the food leftovers, specifically staphylococcal enterotoxins type A (SEA) and D (SED), respectively 0.49 and 2.04 ng/g. The laboratory results are discussed considering the relation to the fish in oil, survival and heat resistance of S. aureus, and presumptive microbial contamination due to improper handling during home-canning procedures. This is the first reported cluster of foodborne illnesses due to staphylococcal enterotoxins in tuna in Italy. In this study, we reported cases described and analysed for their spa-type. Showing a high heterogeneity of isolates, spa-type t13252 is correlated in a node of the minimum spanning tree and it has never been reported as responsible for foodborne outbreak. This case underlines the importance of risk communication and dissemination of home-canning guidelines to reduce the incidence of foodborne outbreaks caused by homemade conserves.

Serological and Molecular Investigation of Swine Hepatitis E Virus in Pigs Raised in Southern Italy.

Hepatitis E virus (HEV) infection is a common acute hepatitis transmitted by the fecal-oral route. In developed countries, the virus has a zoonotic potential, and domestic pigs and wild boars are considered main reservoirs. To assess the prevalence of HEV-positive animals in the Calabria region (southern Italy) on a serological and molecular level, a total of 216 autochthonous healthy pigs (Apulo-Calabrese breed) were sampled. Both sera and feces were collected. Pigs were grouped based on age: 117 pigs <6 months and 99 pigs >6 months. By using a commercial enzyme-linked immunosorbent assay system, a total of 173 (80%) of the 216 pigs tested seropositive. In all sampled farms (n = 8), pigs with antibodies (immunoglobulin G) against HEV were detected at a level higher than 60%, with a significant difference among age groups (P < 0.0001). Moreover, 16 fattening pigs were found to be nested reverse transcription PCR positive and thus to shed viral genomes in their feces. These positive findings resulted in a prevalence of 48.4% on the farm level (16 of 35 pigs) and an overall prevalence of 7.4% at the animal level (16 of 216 pigs). Based on the present study, HEV seems to circulate among the autochthonous domestic pig population of southern Italy with a low sharing rate. Further studies exploring the origin of infection are needed to minimize the risk of human exposure and to reduce consequences for public health.