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In this study, we used both a WGS and an in vitro approach to study the virulence potential of nine Listeria monocytogenes (Lm) strains belonging to genetic clusters persisting in a meat processing plant in Central Italy. The studied clusters belonged to CC1-ST1, CC9-ST9, and CC218-ST2801. All the CC1 and CC218 strains presented the same accessory virulence genes (LIPI-3, gltA, gltB, and aut_IVb). CC1 and CC9 strains presented a gene profile similarity of 22.6% as well as CC9 and CC218 isolates. CC1 and CC218 showed a similarity of 45.2% of the same virulence profile. The hypervirulent strains of lineage I (CC1 and CC218) presented a greater ability to adhere and invade Caco-2 cells than hypovirulent ones (CC9). CC1 strains were significantly more adhesive and invasive compared with CC9 and CC218 strains, although these last CCs presented the same accessory virulence genes. No statistically significant difference was found comparing CC218 with CC9 strains. This study provided for the first time data on the in vitro adhesiveness and invasiveness of CC218-ST2801 and added more data on the virulence characteristics of CC1 and CC9. What we observed confirmed that the ability of Lm to adhere to and invade human cells in vitro is not always decipherable from its virulence gene profile.

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Simple Summary This paper describes the development of a tool to assess the welfare of dogs recruited in trap-neuter-release (TNR) programmes and the Italian situation involving the implementation of these programmes. The TNR approach has been proposed as an alternative to long-term sheltering to control the rising population of free-roaming dogs. The protocol was developed on the basis of a shelter quality protocol (SQP). The measures included in the protocol were integrated with other welfare indicators proposed in the scientific literature. Nine Italian regions out of 20 (all from central and southern Italy) prescribe by law the implementation of TNR programmes. A varied scenario and some critical issues related to the TNR approach for the management of the dog population emerged. The findings, although preliminary, suggest that the protocol could be a useful tool for the assessment of dog welfare. Abstract A descriptive analysis, inter-observer and test–retest reliability of the animal-based measures (ABMs) included in the protocol were performed. This study aimed at the development of a welfare assessment protocol for dogs recruited in the trap-neuter-release (TNR) programmes and the description of the implantation of these programmes in Italy. Nine Italian regions carried out TNR programmes. A varied scenario, along with some critical issues, emerged. Fifty dogs were recruited and assessed simultaneously by two assessors to determine the reliability of ABMs included in the protocol. A subsample of ten dogs were assessed three times to assess test–retest reliability. All females were neutered against 36% of males. Most dogs were adults (58%) and of a large size (68%). Vaccine prophylaxis and parasitic prevention were regular in 13% and 76% of dogs, respectively. Few dogs showed lameness, evidence of pain, other clinical problems, or thermal discomfort. Overall, 82% of dogs did not show fear or aggression to unfamiliar people. The level of agreement between the two assessors was quite high, ranging from substantial (0.61–0.80) to perfect (1) for the majority of measures. This study highlighted some critical issues in TNR implementation and the suitability of the protocol as a tool for animal welfare assessment.

Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level, 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

Human listeriosis outbreaks are often associated with food products, which could be contaminated, at the same time, also by different clones of Listeria monocytogenes. This emphasize the need to type more than one L.monocytogenes isolate found in a single food or environmental sample. The purpose of the present study was to evaluate the presence of different L.monocytogenes strains in food and food production environment in order to understand if there is need to type more isolates from the same sample in case of presence of L.monocytogenes. Between 2011 and 2015, at the Italian National Reference Laboratory for L.monocytogenes, for each positive sample, from two to twenty-three isolates of L.monocytogenes were collected. All the isolates were characterized by conventional serotyping and pulsed field gel electrophoresis (PFGE). Moreover, isolates from the same sample, having indistinguishable PFGE profile, were subjected to whole genome sequencing in order to perform core genome Multi Locus Sequence Typing (cgMLST). Within each sample, more than one serotype and one pulsotype were found in 11.9% and 27.5%, respectively. For indistinguishable PFGE patterns the cgMLST analysis showed 96.2% of concordance demonstrating the added value of new sequencing technologies. This study has demonstrated the need to select and type more than one L.monocytogenes colony in one food or food environmental sample to detect the diversity of L.monocytogenes strains and facilitate downstream investigations and effective source attribution in foodborne outbreak inquiry.

Shelf‐life studies in ready‐to‐eat (RTE) modified atmosphere packaged (MAP) precut iceberg lettuce (minimally processed) were carried out in order to evaluate the natural microflora of the product and survival or multiplication of Listeria monocytogenes (L. monocytogenes), taking into consideration the impact of the production steps resulting in a reduction of the shelf life of the fresh‐cut produce, due to the accelerated enzymatic activity, moisture loss, and microbial proliferation. The research first aimed to evaluate the characteristics of the natural microflora of the product, and then, L. monocytogenes dynamics were studied via challenge tests. L. monocytogenes concentration was studied at 8 and 12°C storage temperature for 10 days, 6 days longer than their shelf life. The number of L. monocytogenes in samples stored both at 8°C and 12°C increased gradually, more evidently in samples stored at 12°C. L. monocytogenes dynamics were studied to define maximum growth rate (μmax) at 8°C (0.0104 log10CFU/g/h) and 12°C (0.0183 log10CFU/g/h). Data obtained from the study were used to develop and validate a specific predictive model able to predict the behavior of L. monocytogenes in RTE MAP iceberg lettuce. According to the model, an increase in storage temperature of 6°C (e.g., from 8 to 14°C) would lead to an increase in L. monocytogenes concentration of more than 6 log10CFU/g at the 10th day of the challenge test (12th days of shelf life). Storage at 4°C allowed to increase L. monocytogenes enumeration from 3.30 log10CFU/g at D0 to 3.60 log10CFU/g at D10. The model could be applied to microorganisms other than L. monocytogenes, modifying the coefficients of the polynomial equation on which it is based., Shelf‐life studies in ready‐to‐eat (RTE) modified atmosphere packaged (MAP) precut iceberg lettuce were carried out in order to evaluate the natural microflora of the product and survival or multiplication of Listeria monocytogenes. Data obtained from the study were used to develop and validate a specific predictive model able to predict the behavior of L. monocytogenes in this product.

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments, however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm.

The aim of this study was to evaluate the performance of “Micro Biological Survey – MBS Test” in the enumeration of bacterial load in cow raw milk. The MBS test is based on a colorimetric method recently developed and patented by “Roma Tre” University, Italy. The evaluation of the performance of the MBS method was carried out by comparison with plate count at 30°C (gold standard) and flow cytometry. Thirteen independent set of experiments were performed analyzing a total of 104 samples of cow raw milk with the selected methods. Results obtained using the MBS method are comparable with those obtained with the plate count method at 30°C (CFU/mL) and flow cytometry technology; in particular, the results obtained with the MBS method are very close to plate count’s at 30°C. On the other hand, there are statistically significant differences between these two methods’ and flow cytometry technology’s results that could be due to the different experimental conditions.

The Abruzzo Regional Law Nr. 47/2013, following a circular from the Italian Ministry of Health and OIE recommendations, allows the local municipalities to release free-roaming dogs (FRDs) caught on the territory once the local veterinary services (LVSs) have rated the dogs as unowned and not aggressive, have neutered them, as well as identified them through a microchip and a visible collar. The responsibility of these &ldquo, community-owned dogs&rdquo, (CODs) falls under the mayor of the local municipality that can entrust their custody to qualified people. The present study was conducted in the Abruzzo region, located in central Italy, to investigate public perception towards CODs, and in particular whether their presence is perceived as a problem or a benefit by the local communities. The data were collected by both direct interviews and an online survey, based on a questionnaire developed by a multidisciplinary team. The questionnaire was distributed in 31 municipalities sampled on the basis of the urbanization rate, and 497 people were interviewed over a 9-month period. More than half of the respondents (54%) stated that CODs can contribute to the control of stray dogs. The majority (83%) believed that a greater commitment is needed to involve the local communities on issues regarding CODs. The findings of this study highlighted the general difficulty for people to distinguish stray dogs from CODs that are not fully known, as evidenced by the fact that 59% of the respondents did not know the aforementioned Regional Law that defines and regulates the presence of the CODs.

Pecorino di Farindola is a typical cheese produced in the area surrounding the village of Farindola, located in the Abruzzo Region (central Italy), unique among Italian cheese because only raw ewe milk and pig rennet are used for its production. In the literature it is well documented that raw milk is able to support the growth of pathogenic microorganisms such as Listeria monocytogenes. Predictive microbiology can be useful in order to predict growth-death kinetics of pathogenic bacteria, on the basis of known environmental conditions. Aim of this study was to compare predictions obtained from a model, originally designed to predict the kinetics of L. monocytogenes in the dynamic growth-death environment of drying fresh sausage, with the results of challenge tests performed during the ripening of Pecorino di Farindola produced from artificially contaminated raw ewe milk. A challenge test was carried out using ewe raw milk inoculated with L. monocytogenes, in order to produce Pecorino di Farindola cheese stored at 18 °C for 149 days of ripening. During the ripening period, pH and aw values decreased in all samples analysed; lactic acid bacteria become the prevailing microbial population, while for L. monocytogenes a period of stability (neither growth nor death) followed the initial situation. The growth inhibition and the following inactivation may mostly be due to competition with the autochthonous microbiota and to the reduction of water activity. Mathematical modelling was used in order to predict microbial kinetics in the dynamic ripening environment, joining growth and death patterns in a continuous way, and including the highly uncertain growth/no growth range separating the two regions. The effect of lactic acid bacteria on the growth of pathogens was also included. Predicted microbial kinetics were satisfactory, as confirmed by the absence of statistically significant difference between observed and predicted values (p > 0.05). The present study proved, via challenge tests, that a dynamic growth/death model, previously used for a meat product, can be fruitfully used in cheese characterized by active competitive microbiota and progressive drying during ripening.

In this study, the estimate of chlorophyll "a" and the dispersion of sediment in the sea, calculated from Sentinel-2, was compared with real data acquired in situ by a multiparametric probe, along the Abruzzo coast. The ultimate goal is to optimize parameters and algorithms to be able to derive concentration maps of chlorophyll and suspended solids from satellite, taking advantage of the high time frequency and high spatial resolution of the detections. This information is of particular relevance for aquaculture activities, for monitoring water quality and for analyzing sedimentary processes.

Among pathogens, L. monocytogenes has the capability to persist on Food Processing Environment (FPE), first of all posing safety issues, then economic impact on productivity. The aim of this work was to determine the influence of biofilm forming-ability and molecular features on the persistence of 19 Listeria monocytogenes isolates obtained from FPE, raw and processed products of a cold-smoked salmon processing plant. To verify the phenotypic and genomic correlations among the isolates, different analyses were employed: serotyping, Clonal Complex (CC), core genome Multi-Locus Sequence Typing (cgMLST) and Single Nucleotide Polymorphisms (SNPs) clustering, and evaluation of the presence of virulence- and persistence-associated genes. From our results, the biofilm formation was significantly higher (*P 0.05) at 37 °C, compared to 30 and 12 °C, suggesting a temperature-dependent behaviour. Moreover, the biofilm-forming ability showed a strain-specific trend, not correlated with CC or with strains persistence. Instead, the presence of internalin (inL), Stress Survival Islet (SSI) and resistance to erythromycin (ermC) genes was correlated with the ability to produce biofilms. Our data demonstrate that the genetic profile influences the adhesion capacity and persistence of L. monocytogenes in food processing plants and could be the result of environmental adaptation in response to the external selective pressure.

By late March 2020, Villa Caldari, a small village of the municipality of Ortona (Abruzzo region), was registering an incidence rate of COVID-19 cases ten times greater than the overall municipality and was declared a hotspot area. Twenty-two days later, epidemiological investigation and sampling were performed, to evaluate SARS-CoV-2 circulation and the presence of SARS-CoV-2 antibodies. Overall, 681 nasopharyngeal swabs and 667 blood samples were collected. Only one resident of the village resulted in being positive for RNA viral shedding, while 73 were positive for SARS-CoV-2 antibodies. The overall seroprevalence was 10.9%. The difference between the seroprevalence of infection in asymptomatic and symptomatic individuals was significant (&chi, 2 = 14.50 p-value = 0.0001). Amongst the residents positive for antibodies, fatigue and/or muscle pain, fever and anosmia were the most experienced symptoms, whose most frequent onset was observed during the first two weeks of March. Familial and habit-related clusters were highlighted. Nevertheless, the investigations showed a low SARS-CoV-2 circulation in the village at the time of the sampling, demonstrating virus transmission could be limited when strict emergency measures are followed. Given the favorable results, the emergency measures were then lifted.

The acceptance of serology data instead of challenge for market release of new batches of commercial vaccine is under evaluation by regulatory agencies in order to reduce the use of animals and costs for manufacturers. In this study two vaccines for Bluetongue virus serotype 8 were submitted to quality controls required by the European Pharmacopoeia and tested on sheep in comparison with a commercial inactivated vaccine. Body temperature, antibody titres and viraemia of vaccinated and controls sheep were recorded. In addition IL4 and IFNγ in sera and supernatant derived from in vitro stimulation of blood cells were also quantified using two commercial ELISA kit. The outer-capsid protein VP2 contained in vaccine formulations was quantified using a home-made capture-ELISA. Results obtained indicates that in-lab evaluation of cell-mediated and humoral immune response are useful parameters to predict the efficacy of BTV inactivated vaccines avoiding the challenge phase required to release new batches of vaccines with proven clinical efficacy and safety. The correlation observed between serology data and VP2 protein concentration of final product could be useful in-process control to predict if a new vaccine batch of BTV must be discarded or released to the market.

Stress factors and poor animal welfare can increase the susceptibility of food-producing animals to diseases, posing microbial risks to consumers. Animal welfare levels, objectively measured with the application of the Welfare Quality® protocol, were assessed in thirteen broiler flocks, including organic ones, to evaluate the presence of statistically significant differences in relation to Campylobacter and Salmonella faecal shedding and consequent microbiological contamination of broiler carcases at slaughterhouse. Each flock underwent animal welfare evaluation the day before slaughtering, followed by Campylobacter and Salmonella detection in faeces (caecal content) and neck skin at slaughterhouse. A total of 1040 samples (520 caecal contents; 520 neck skins) were included in the study. Campylobacter enumeration and Salmonella serotyping were also carried out. The highest welfare scores were reported in organic flocks. Significantly lower Campylobacter concentrations both in caecal content and neck skins (P This study provides new evidence that high animal welfare standards at farms, other than an ethical issue and a value-add for the end product, could also improve the microbiological safety of poultry meat, ultimately contributing to the protection of consumers.

Four hybridoma cell lines (C4B, 10C2G5, 6C5F4C7, 2D10G11) were adapted to grow in serum-free conditions. Cell proliferation, viability, and antibody production in Nutridoma SP and Ex-Cell HSF 610 media were evaluated and results compared with those obtained using DMEM containing 10% fetal bovine serum (control medium). Clone C4B showed the best IgG productivity in control medium, but viability and number of cells per milliliter were similar for the three media. For clone 10C2G5, the highest values of cell viability were obtained with both control medium and Nutridoma SP; no significant differences in IgG yields and number of cells/mL were observed among the three media. Clone 6C5F4C7 provided no significant differences when grown in the two serum-free media and in control medium. Clone 2D10G11 showed the best IgG productivity in control medium and in Ex-Cell HSF 610, even if Ex-Cell HSF 610 provided the lowest number of cells/mL; no significant differences among the three media were obtained for viability. Purified antibodies produced from each hybridoma cell line grown in serum-free media showed a higher degree of purity than those produced by the same cell lines grown in control medium. Purified MAbs were also titrated by ELISA to test MAb-antigen affinity.

A telephone survey was carried out on 13,486 randomly selected households located in the Abruzzo region (central Italy). Three questionnaires were specifically designed in relation to three different groups of foods (Meat and meat products, Fishery products, Fruit and vegetables). Questions were mainly focused on the amount and number of purchases, type of retailer, and home food handling practices with respect to the week prior to the interview. Data were classified according to a multilingual thesaurus system (LanguaL). Results allowed to estimate domestic purchases (in kg) per capita for different food categories. The category “ Red meat / Cattle ” accounted for a large part of purchases in the “meat and meat products” group (10.9 kg per capita/year; 32.2% of purchases in the group) while the category “ Fish or related organism / Fish ” was the most purchased in the “fishery products” group (6.9 kg per capita/year; 63.3%). The aggregation of more detailed characteristics enabled the identification of popular categories of foods, such as “ Red meat / Cattle / Divided into pieces ” (25% of the “meat and meat products” group). Significant differences ( p Poultry / Chicken ” and “ Red meat / Cattle ” categories. Householders were also asked about post-purchase food handling practices that might be hazardous to food safety. More than 20% of those surveyed stated that they thawed frozen meat at room temperature. The degree of doneness after cooking of different food categories was generally high: over 90% of products purchased in the majority of meat and fish categories were properly cooked. However a noticeable proportion of householders (about 15%) reported medium or rare cooking of “ Red meat / Cattle ” and “ Red meat / Swine ”. Differences ( p The survey was carried out in a specific geographic area and on a statistically significant sample of households, thus allowing a collection of data on domestic habits relating to food purchases and home food handling practices. This information should be included in the framework of quantitative microbial risk assessment (QMRA) models as a measure of the actual exposure of consumers to pathogenic microorganism. The LanguaL system also proved to be a practical language-independent method useful not only to identify and describe food items but also to classify them according to specific food safety characteristics.

African horse sickness (AHS) is a non-contagious, insect-borne disease of equids caused by a RNA virus (AHSV), which belongs to the genus Orbivirus, family Reoviridae. The disease is endemic in sub-Saharan and western Africa, where prevention strictly depends upon vaccination. The present paper aims at evaluating the serological response and the occurrence of AHS in horses bred under field condition and regularly immunized using the commercially available live attenuated vaccine (LAV) produced by Onderstepoort Biological Products. The study was carried out in a farm located in the district of Windhoek (Namibia), where the disease is endemic. A total of 72 cross-breed horses, out of the 150 housed on the farm, were subdivided in six age groups, from 2 to 7 years-old. Each group consisted of 12 heads which were born during the same breed- ing season and had undergone from four to nine vaccination courses. AHSV specific immune response was evaluated by serum-virus neutralization test. Data about the clinical occurrence of the AHS from 2006 to 2011 were made available. The immune response, in terms of number of seropositive horses and serum neutralizing titers, was quite variable among horses and against different serotypes. Neutralizing antibodies against all serotypes were recorded in all the horses only after eight vaccination courses at 6 years of age onwards. Immune response to AHSV-5 and 9, which are not included in the LAV formulation, were also established. A severe AHS epidemic occurred in Namibia in 2011. On the farm under study, a total of 32 animals were clinically affected, 12 died, 11 of them were 2 year-old or younger. Our data confirm that vaccination with LAV is a useful tool to reduce the severity of the disease in ende- mic areas. However, clinical and sometimes fatal AHS can still affect young vaccinated horses, thus high- lighting the necessity to better understand the immune response to AHSV and to dispose of more effective vaccines. 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

Epizootic haemorrhagic disease (EHD) is a vector-borne infectious viral disease of domestic and wild ruminants. EHD could spread from infected northern African countries in free territories like the EU; therefore, the availability of diagnostic assays would represent key components for adequate surveillance and control programs. In this study, the gene encoding the VP7 protein of EHD virus (EHDV) was expressed into a baculovirus-infected insect cell system. With this unpurified protein we developed a home-made competitive ELISA (cELISA) and a total number of 275 serum samples, originating from domestic and wild ruminants, were tested. 74/275 were previously shown to be positive for EHDV antibodies by a commercially available ELISA kit. A "very good" agreement was demonstrated when compared to a commercial ELISA kit (Cohen's kappa value=0.832). Samples which caused disagreement between the two assays originated from wildlife which highlights the need for further validation by using serum samples from wild animals.

Bluetongue, Schmallenberg and African horse sickness viruses are transmitted by vectors belonging to the genus Culicoides (Diptera: Ceratopogonidae). Among this genus, species of the Obsoletus complex play a crucial role in Europe. In Italy the complex includes three species: Culicoides obsoletus sensu strictu, Culicoides scoticus and Culicoides montanus. These three sibling species were identified in 80 collection sites across Italy through a multiplex PCR test. Sixty‑four sites were classified with a predominant species through a Bayesian approach. Environmental, topographic and climatic factors together with landscape metrics were investigated in each species group using the non‑parametric Mann‑Whitney test. Culicoides obsoletus s.s. resulted the most widely distributed species across the peninsula (51 sites). This species was collected in many eco‑climatic conditions, at a wide range of temperature and altitudes, mainly in anthropogenic landscape. Conversely C. scoticus collection sites were dominated by natural vegetated areas or forest, at medium higher altitudes, preferably in a wilder and more pristine environment (predominant species in 8 sites). Culicoides montanus was a rarer species, statistically predominant in only 3 sites located in Southern Italy, characterized by temperatures higher than the other species. These results, together with other on‑going researches on the vector competence of the species belonging to the Obsoletus complex, and with investigation of larval and breeding habitats, could greatly improve epidemiological knowledge of possible spreading Culicoides‑borne viruses.

Vaccination is the most effective strategy for controlling Bluetongue virus (BTV) spread and economic consequences thereof. In this study we verified in sheep, using one commercially available inactivated vaccine for BTV-8 (BTVPUR AlSap 8), when, during the recommended vaccination schedule, animals start to be effectively protected against challenge with wild-type strain. To this aim, sheep were challenged at different time points shortly after the first vaccine injection. Twenty-four Sarda sheep were divided into four groups vaccinated two weeks before challenge (Group A), one week before challenge (Group B) and concurrently with challenge (Group C). A second vaccine was performed twenty-eight days later with respect the first vaccine administration in each experimental group. The last group consisted of six non vaccinated-infected animals (NVIA). Virological and serological examinations were performed before and after challenge up to 42 and 77days post challenge, respectively. The results of the study show that vaccination commenced as little as two weeks before challenge (Group A) prevented viremia and RNAemia in challenged sheep altogether. Conversely, Group B was partially protected from challenge and Group C showed viraemia and RNAemia similar to NVIA. This study indicates that the first administration of inactivated vaccine performed two weeks before challenge was able to prevent viraemia. Overall, our findings may have direct consequences for the management of an unexpected BTV-8 outbreak in sheep and for the legislation on sheep trade from BTV restriction areas.

Traditional Italian pork products can be consumed after variable drying periods, where the temporal decrease of water activity spans from optimal to inactivating values. This makes it necessary to A) consider the bias factor when applying culture-medium-based predictive models to sausage; B) apply the dynamic version (described by differential equations) of those models; C) combine growth and death models in a continuous way, including the highly uncertain growth/no growth range separating the two regions. This paper tests the applicability of published predictive models on the responses of Listeria monocytogenes and Yersinia enterocolitica to dynamic conditions in traditional Italian pork sausage, where the environment changes from growth-supporting to inhibitory conditions, so the growth and death models need to be combined. The effect of indigenous lactic acid bacteria was also taken into account in the predictions. Challenge tests were carried out using such sausages, inoculated separately with L. monocytogenes and Y. enterocolitica , stored for 480 h at 8, 12, 18 and 20 °C. The pH was fairly constant, while the water activity changed dynamically. The effects of the environment on the specific growth and death rate of the studied organisms were predicted using previously published predictive models and parameters. Microbial kinetics in many products with a long shelf-life and dynamic internal environment, could result in both growth and inactivation, making it difficult to estimate the bacterial concentration at the time of consumption by means of commonly available predictive software tools. Our prediction of the effect of the storage environment, where the water activity gradually decreases during a drying period, is designed to overcome these difficulties. The methodology can be used generally to predict and visualise bacterial kinetics under temporal variation of environments, which is vital when assessing the safety of many similar products.

The present study evaluated the occurrence of zoonotic parasites in feces and on fur of stray and private dogs living in Italy. Individual fecal samples collected from 117 and 385 kenneled (i.e., rescue shelter) and privately owned dogs, respectively, were examined by conventional copromicroscopy. Samples positive for Giardia duodenalis and Cryptosporidium spp. were subjected to further genetic characterization. Additionally, 174 fur samples of private and kenneled dogs living in the same area, 27 of which were also subjected to copromicroscopical examinations, were examined for the presence and viability of parasite elements. Out of 502 fecal samples, forty-one (8.2 %) scored positive for G. duodenalis cysts while one (0.2 %) for Cryptosporidium oocysts. Nine private dogs were molecularly positive for G. duodenalis assemblage C (n. 7), assemblage D (n. 1), and assemblages C + D (n. 1), while 11 kenneled dogs were positive for assemblage C (n. 8), assemblage D (n. 1), and assemblages C + D (n. 2). Cryptosporidium spp. were identified as C. canis in a private dog. One hundred and seventy-two dogs (34.3 %), i.e., 92 private and 80 kenneled, were positive for zoonotic nematodes: Toxocara canis (6.6 %), Ancylostoma caninum (6.8 %), and Capillaria aerophila (0.6 %). Unviable eggs of Toxocara spp. were found on the hair of five (2.9 %) private dogs. The present findings indicate that canine feces from both private and kenneled animals may contain zoonotic parasites and may be a potential risk for humans and other animals, especially when they contaminate the environment. The role of dog fur as source of human infections should be further investigated.

Background Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is widespread in sub-Saharan Africa. The current live vaccine T1/44 has limited efficacy and occasionally leads to severe side effects in the animals. A better understanding of the immune responses triggered by Mycoplasma mycoides subsp. mycoides and their role in disease progression will help to facilitate the design of a rational vaccine. Currently, knowledge of cytokines involved in immunity and immunopathology in CBPP is rather limited. The aim of this study was to characterize the in vivo plasma concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10 and the overall role of CD4+ T cells in the development of cytokine levels during a primary infection. Plasma cytokine concentrations in two groups of cattle (CD4+ T cell-depleted and non-depleted cattle) experimentally infected with Mycoplasma mycoides subsp. mycoides were measured and their relationship to the clinical outcomes was investigated. Results Plasma cytokine concentrations varied between animals in each group. Depletion of CD4+ T cells did not induce significant changes in plasma levels of TNF-α, IL-4, and IL-10, suggesting a minor role of CD4+ T cells in regulation or production of the three cytokines during the time window of depletion (1-2 weeks post depletion). Unexpectedly, the IFN-γ concentrations were slightly, but statistically significantly higher in the depleted group (p < 0.05) between week three and four post infection. Three CD4+ T cell-depleted animals that experienced severe disease, had high levels of TNF-α and IFN-γ. Only one severely diseased non-depleted animal showed a high serum concentration of IL-4 post infection. Conclusions Comparison of most severely diseased animals, which had to be euthanized prior to the expected date, versus less severe diseased animals, irrespective of the depletion status, suggested that high TNF-α levels are correlated with more severe pathology in concomitance with high IFN-γ levels.

Naples and Caserta provinces are extensively affected by the illegal dumping of hazardous and urban wastes, which were periodically set to fire. Several studies were made on the possible health impact of this illegal waste management. The aim of the study was to detect dioxins levels in breast milk of volunteer primiparae and to assess the possible source of dioxins in the affected areas. The authors determined dioxins levels in breast milk from 100 primiparae from the study area and collected anamnestic information on donors. We determined dioxins levels in breast milk from 100 primiparae from the study area and collected anamnestic information on donors. As a measure of environmental risk of dioxins (EDR) we used the interpolated values of dioxins concentration in buffalo milk samples collected in the study area. Correlations between the EDR, age of the mother, smoking habit, cheese consumption, occupation in activity at risk, presence of plants for the disposal of toxic waste or illegal burning of solid waste near the residence of the donor and dioxin level in breast milk were investigated. The dioxin level in breast milk is significantly correlated to the EDR, the age of the sampled women and the presence of illegal burning of solid waste.

Italian style fresh sausage is a traditional pork product that is frequently consumed raw, usually after drying. So far, the prediction of the behaviour of microorganisms in varying environments has been carried out through separate growth or death models. The aim of this study is to show how to combine these, in order to describe the kinetics of Yersinia enterocolitica and Listeria monocytogenes in sausage during the drying period. During this time, the storage temperature and the pH are fairly constant, while the water activity decreases, being the primary cause of shifting the organisms’ kinetics from growth to inactivation. The water activity takes up values from growth (RG), uncertainty (R0) and death (RD) regions. In the uncertainty region, neither growth nor death is predicted. Among the native flora, lactic acid bacteria may have significant effect on these kinetics. Here we show how to generate predictions for such scenarios, from data that are generated in constant environments. Thus the predictions in our dynamic environments are extrapolations and could be problematic if the environmental fluctuation is extreme. Our approach can be used to predict bacterial growth/death kinetics under temporal variation of storage environments, which is vital when assessing the safety of fresh sausage. We envisage similar applications to other RTE meat products, too.

The overall aim of the project was to evaluate the use of routinely collected animal based measures (ABMs) for an evaluatio n of the overall animal welfare in dairy cow herds. ABMs being able to detect worst adverse effects in relation to animal welfare were identified based on the existing literature and expert opinion. The validity and robustness of these ABMs were evaluated and cow mortality, somatic cell count and lameness were selected for further study. A number of factors of variation were selected using expert opinion and used in a model to collate routinely collected data from Italy, Belgium and Denmark on selected ABMs . The routinely collected data was uploaded to the Data Collection Framework platform at EFSA and the data management in this process was evaluated. Five research datasets from Italy, Belgium, Denmark and France including information on ABMs as well as a m easure of ’overall animal welfare’ at herd level were analysed to evaluate the association between the ABMs (individually or in combination) and overall welfare. The measure of ’overall animal welfare’ were not the same for all datasets. Except from the It alian data, the association between the ABMs and the different overall welfare measures were generally weak. Likewise, combining more than one ABM only improved the prediction of the overall welfare in the Italian dataset. Analyses of the other datasets co uld not confirm this finding. Finally, suggestions for future recordings of ABMs not routinely collected at the moment were given with a special focus on lameness. In conclusion, t he relationship between selected ABMs and overall welfare at the herd level is complex and still not sufficiently studied. Therefore, a system using routinely collected ABMs to predict the overall welfare at herd level in dairy herds does not seem realistic based on the results from the present study