Evidence of Antimicrobial Resistance and Presence of Pathogenicity Genes in Yersinia enterocolitica Isolate from Wild Boars.

Yersinia enterocolitica (Ye) is a very important zoonosis andwild boars play a pivotal role in its transmission. In the last decade, the wild boar population has undergone a strong increase that haspushed them towards urbanized areas, facilitating the human-wildlife interface and the spread of infectious diseases from wildlife to domestic animals and humans. Therefore, it is important to know the serotype, antimicrobial resistance and presence of pathogenicity genes of Yersinia enterocolitica (Ye) isolated in species. From 2013 to 2018, we analyzed the liver of 4890 wild boars hunted in Liguria region; we isolated and serotyped 126 Ye positive samples. A decisive role in the pathogenicity is given by the presence of virulence genes; in Ye isolated we found ystB (~70%), ymoA (45.2%), ail (43.6%) and ystA (~20%). Moreover, we evaluated the susceptibility at various antimicrobic agents (Ampicillin, Chloramphenicol, Enrofloxacin, Gentamicin, Kanamycin, Trimethoprim-Sulfamethoxazole, Sulfisoxazole, Ceftiofur and Tetracycline). The antibiotic resistance was analyzed, and we found a time-dependent increase. It is important to shed light on the role of the wild boars as a reserve of potentially dangerous diseases for humans, and also on the antibiotic resistance that represents a public health problem.

Yersinia enterocolitica-specific modulation of innate immune responses in jejunal epithelial cells.

Gut is often subject to infection by different pathogens like Y. enterocolitica. To date, biotypes (BTs) 1A have been considered as non-pathogenic, because they do not express plasmid of virulence pYV; however, BTs 1A strains present other chromosomic virulence genes and recent studies suggest an implication of this microorganism in reactive arthritis. Although many studies highlighted the molecular basis of pathogenesis of Ye infection, scanty data are available about several environmental BTs 1A strains, often isolated in cases of foodborne disease but not included in pathogenicity studies. The aim of our work was to verify the ability of different Ye 1A strains to adhere and penetrate IPEC-J2 cells and to modulate intestinal innate immunity. Our results showed that all strains under study were able to adhere and penetrate enterocytes, causing inflammatory responses. Indeed, adhesion and invasion of enterocytes is an essential step in Ye pathogenesis (Fàbrega and Vila, 2012). Moreover, our data suggest the possible involvement of strains Ye2/O:9 in reactive arthritis, due to their ability (i) to penetrate enterocytes as pathogenic Ye1/O:8 strains do, and (ii) to increase IL-6, IL-8, IL-12 and IL-18 release. Lastly, our results confirm that IPEC-J2 cells are a very good model to evaluate host-pathogen interaction, and indicate IL-8, TNF-α, TLRs1 and 4 as possible markers of the ability of Ye strains to penetrate enterocytes. Moreover, we showed that Ye strains differently affect the host's innate immune responses.

Characterization of MDCK cells and evaluation of their ability to respond to infectious and non-infectious stressors.

The Madin-Darby Canine Kidney (MDCK) cell line is widely used as epithelial cell model in studies ranging from viral infection to environmental pollutants, and vaccines production. However, little is known about basal expression of genes involved in innate immunity, and the ability to respond to infectious and non-infectious stressors. Therefore, the aims of our study were to evaluate the basal level of expression of pivotal genes in the innate immune response and cell cycle regulation, as well as to evaluate the ability of this cell line to respond to infectious or non-infectious stressors. As surmised in our working hypothesis, we demonstrated the constitutive expression of genes involved in the innate immune response and cell defense alike, including TLRs, Interleukins, Myd88, p65/NF-kB and p53. Moreover, we described the ability of this cell line to respond to LPS and cadmium (Cd2+) in terms of gene expression and cytokine release. These data confirm the possibility of using this cell line as a model in studies of host/pathogen interaction and response to non-infectious stressors.

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.

Effects of thermal treatment on walnut detection and allergenicity.

BACKGROUND: Peanuts and tree nut allergies pose an increasing food safety problem. The aim of our study was to test the accuracy of different commercial enzyme-linked immunosorbent assay (ELISA) kits in the detection of the presence of walnuts in untreated and heat exposed food samples. The effects of thermal treatment of samples were evaluated by exposing walnuts to different heat treatments. All samples were first analysed by two different commercial ELISA assays. Then, we performed a skin prick test (SPT) on nine patients with proven nut allergy using small walnut pieces from raw and treated samples. RESULTS: The presence of nuts proteins in thermally processed foods was not accurately detected by ELISA kits. All patients had a positive SPT reaction with raw walnut, while thermal treatments affected walnut allergenicity. The ELISA test gives a negative result in the case of strong thermal treatment, but at the same time allergic subjects react positively to stimulation with the same sample. CONCLUSION: This study suggests that commercial ELISA kits may not be able to accurately determine the amount of proteins present in thermally processed foods due to changes in the solubility and immunoreactivity of the target proteins. Finally, the clinical results highlight that thermal treatment might induce a reduction in walnut allergenicity. © 2018 Society of Chemical Industry.

Evaluation and validation of an alternative method to detect Campylobacter spp. in dairy products.

Foods implicated in human campylobacteriosis include raw or undercooked poultry and raw dairy products. Because Campylobacter spp. are the most frequently reported cause of bacterial infection in the European Union and because conventional methods are cumbersome, rapid methods for Campylobacter detection and quantification in food are needed. With this study we sought to validate, according to the standard procedure (UNI EN ISO 16140:2003), an alternative to the reference analytical method (UNI EN ISO 10272-1:2006) for official controls of Campylobacter spp. in raw milk and dairy products. Milk samples collected from 16 milk vending machines located throughout the Genoa metropolitan area were analyzed using two different methods, an enzyme-linked fluorescent assay (ELFA) and a real-time PCR assay, and evaluated in parallel against the reference method. In addition, a total of 460 samples of raw milk collected from milk vending machines were analyzed by ELFA. Results obtained with ELFA showed it was compliant with UNI EN ISO 10272-1:2006 criteria and that the immunoassay had 100% sensitivity, specificity, and accuracy. Regarding samples of milk vending machines, 5.0% (23/460) tested positive at ELFA screening and were subsequently confirmed as C. jejuni. Validation according to UNI EN ISO 16140:2003 of the ELFA method suggests it may be a useful alternative to conventional methods for detecting Campylobacter spp. in official controls.

Development and validation of species-specific molecular diagnostic tool for Opisthorchis felineus (Digenea, Opisthorchiidae) metacercariae.

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method.

Tracing Back Clinical Campylobacter jejuni in the Northwest of Italy and Assessing Their Potential Source.

Food-borne 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 fundamental to prioritize food safety interventions and to establish proper 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.

A Salmonella Enterica Subsp. Enterica Serovar Enteritidis Foodborne Outbreak after Consumption of Homemade Lasagne.

In the latest year, and also in 2013, Salmonella was the most frequently detected causative agent in foodborne outbreaks (FBOs) reported in Europe. As indicated in EFSA report (2015) the serotypes mostly associated to FBOs are S. Typhimurium and Enteritidis; while Salmonella Typhimurium is generally associated with the consumption of contaminated pork and beef, FBOs due to Salmonella Enteritidis are linked to eggs and poultry meat. In this study it is described the investigation of a domestic FBO involving four adults and linked to homemade lasagne. Investigations were performed to determine the relatedness of Salmonella strains, identify the sources of infection, and trace the routes of Salmonella contamination in this FBO. Salmonella strains were isolated in 3 out of 4 patient stool samples and from lasagne and all of them were serotyped as S. Enteritidis. Pulsed-field gel electrophoresis (PFGE) analysis revealed the genotypical similarity of all the strains. Although serotyping and PFGE analysis identified the common food source of infection in this FBO, it was not possible to determine how or at what point during food preparation the lasagne became contaminated with Salmonella.

Food Allergens: State of the Art in Piedmont Region in the Period 2011-2012.

The US National Institutes of Allergy and Infectious Diseases defines food allergy as adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food. Undeclared allergens in food label represent a risk for consumers, as there is no therapy for food allergies. According to Directive 2003/89/EC, declaration of all ingredients and derived substances in the label is mandatory. In 2011-2012, in Piedmont region (North-western Italy) 285 food samples were analysed for ß-lactoglobulin and 234 for egg proteins. The aim of this work was to analyse 2 years data in order to assess the presence of undeclared milk and egg allergenic proteins in food placed on the market checking the compliance of labeling of food allergens. Analyses were carried out with ELISA tests, both for the detection of the egg and milk proteins. ß-lactoglobulin was found in 2.8% (8/286) of samples, while egg proteins in 4.7% (11/234).

COX-2 expression in human breast carcinomas: correlation with clinicopathological features and prognostic molecular markers.

OBJECTIVE: COX-2 is implicated in carcinogenesis and tumour progression in many cancers, including breast cancer. Recently, it has been reported that human breast carcinomas aberrantly express COX-2, and that raised tissue levels of COX-2 may have prognostic value. Patients expressing high levels of COX-2 can develop local recurrence, and have reduced disease-free and disease-related overall survival. The aim of this study was to investigate COX-2 expression in human ductal and lobular breast cancers and its possible association with clinicopathological features and prognostic molecular markers. RESEARCH DESIGN AND METHODS: Cytoplasmic COX-2 expression was detected by means of immunohistochemistry in a series of 91 breast carcinomas with ductal (n = 60) and lobular (n = 31) patterns. COX-2 expression was investigated by multivariate analyses and compared with clinicopathological features. RESULTS AND CONCLUSIONS: COX-2 immune positivity and percentage of positive cells correlated significantly with the size, grading, extent of primary tumour and vascular invasion of carcinoma but not with biological parameters (estrogen receptor, progesterone receptor and human EGF receptor 2). The findings of the present study suggest that COX-2 overexpression in lobular and ductal breast cancers, which correlates with traditional clinico-pathological parameters, may be considered as a negative prognostic marker.