Factors related to longevity and mortality of dogs in Italy.

Besides its translational value, an improved understanding of dog longevity and mortality is necessary to guide health management decisions, breed selection, and improve dog welfare. In order to analyse the lifespan of dogs in Italy, identify the most common causes of death, and evaluate possible risk factors, anonymised medical records were collected from 9 veterinary teaching hospitals and 2 public health institutions. Data regarding breed, sex, neuter status, age, diagnosis, and mechanism of death were retrieved. Cause of death (COD) was classified by pathophysiologic process (PP) and organ system (OS). Of the 4957 dogs that died between 2004 and 2020 included in the study, 2920 (59.0%) were purebred, 2293 (46.2%) were female, 3005 (60.6%) were intact, 2883 (58.2%) were euthanised. Overall median longevity was 10.0 years. Median longevity was significantly longer for crossbreds, females, neutered dogs, and small-sized breeds. The breeds with the highest median age at death were the Yorkshire terrier, English cocker spaniel, West Highland white terrier, Italian volpino, and Shih Tzu, whilst the American bulldog, English bulldog, American pit bull terrier, Bernese mountain dog and the Maremma and the Abruzzes sheepdog had the lowest median age at death. The most frequent COD by PP was neoplasia (34.0%), which occurred more frequently in large breeds, namely German shepherd, Labrador retriever and Boxer. Degenerative diseases mostly affected small-sized dogs like Miniature pinscher and Dachshund. Regarding the OS involved, diseases of the renal/urinary system were most frequently responsible for COD (15.0%), prevalently degenerative and inflammatory/infectious. Substantial variation in median longevity according to causes of death by PP and OS was observed. These data are relevant for breeders, veterinary practitioners, and owners, to assist breed selection, facilitate early diagnosis, guide choice when purchasing a purebred dog and making health management decisions, and ultimately improve dog welfare.

Comparative analysis of rare earth elements concentrations in domestic dogs and Apennine wolves of Central Italy: Influence of biological, nutritional, and lifestyle factors.

Rare Earth Elements (REEs) are strategical elements playing a crucial role in the industry, especially in producing high-tech materials. Therefore, REEs are new contaminants of emerging concerns. However, due to the lack of exposure data on REE occurrence in environmental matrices, especially in European countries, it is still tricky to establish environmental background levels to assess the ecotoxicological risk related to REEs exposure. The present study aimed to evaluate the liver concentrations of REEs in domestic dogs (Canis lupus familiaris) and Apennine wolves (Canis lupus italicus) living in the Abruzzo region, Italy. Moreover, for the scope of the present study, the dog's group was divided according to their sex, age, lifestyle, and diet. Wolves were categorized concerning their sex and genetic characteristics. Liver samples from dogs and wolves were collected during diagnostic necropsies from carcasses, sample mineralization was performed by a microwave digestion system with a single reaction chamber, and simultaneous determination of the presence of REEs was performed by Inductively Coupled Plasma Mass Spectrometer (Q-ICP-MS) using standard mode for all rare earth elements except scandium (Sc) which was acquired in kinetic energy discrimination (KED) mode. Hepatic concentrations of REEs were statistically significantly higher in wolves compared to dogs. Moreover, significant differences in REEs concentrations arose also from the genetic type of wolf, since "pure wolves" had higher liver concentrations of REEs compared to wolf-dog hybrids. Female and adult dogs also showed elevated REEs compared to male and juvenile dogs, while no significant differences were demonstrated for dogs' diet and lifestyle. The results of the present study confirm the exposure of domestic and wild carnivores to REEs, showing also the ability of REEs to accumulate in carnivore livers, suggesting the potential role of this species as an alternative bioindicator.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats.  Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

Evaluation of Three Serological Tests for Diagnosis of Canine Brucellosis.

Canine brucellosis caused by Brucella canis, is an infectious disease affecting dogs and wild Canidae. Clinical diagnosis is challenging, and laboratory testing is crucial for a definitive diagnosis. Various serological methods have been described, but their accuracy is uncertain due to limited validation studies. The present study aimed to evaluate the performances of three serological tests for the diagnosis of B. canis in comparison with bacterial isolation (gold standard), in order to establish a protocol for the serological diagnosis of canine brucellosis. A panel of sera from naturally infected dogs (n = 61), from which B. canis was isolated, and uninfected dogs (n = 143), negative for B. canis isolation, were tested using microplate serum agglutination (mSAT), complement fixation performed using the Brucella ovis antigen (B. ovis-CFT), and a commercial immunofluorescence assay (IFAT). The sensitivity and specificity of the three serological methods were, respectively, the following: 96.7% (95% CI 88.8-98.7%) and 92.3 (95% CI 86.7-95.1%) for mSAT; 96.7% (95% CI 88.8-98.7%) and 96.5 (95% CI 92.1-98.2%) for B. ovis-CFT; 98.4% (95% CI 91.3-99.4%) and 99.3 (95% CI 96.2-99.8%) for IFAT. The use in of the three methods in parallel, combined with bacterial isolation and molecular methods, could improve the diagnosis of the infection in dogs.

Brucella abortus Strain RB51 Administered to Prepubescent Water Buffaloes, from Vaccination to Lactation: Kinetics of Antibody Response and Vaccine Safety.

Brucella RB51 is a live modified vaccine. Its use in water buffalo has been proposed using a vaccination protocol different to that used for cattle, but knowledge of the long-term effects of RB51 vaccination in this species remains incomplete. The aim of the study was to evaluate the safety and kinetics of antibody responses in water buffaloes vaccinated according to the protocol described for the bovine species in the WOAH Manual, modified with the use of a triple dose. Water buffaloes were vaccinated with the vaccine RB51. A booster vaccination was administered at 12 months of age. When turning 23-25 months old, female animals were induced to pregnancy. RB51-specific antibodies were detected and quantified using a CFT based on the RB51 antigen. Vaccinated animals showed a positive serological reaction following each vaccine injection, but titers and the duration of the antibody differed among animals. For 36 weeks after booster vaccination, the comparison of CFT values between vaccinated and control groups remained constantly significant. Afterwards, antibody titers decreased. No relevant changes in antibody response were recorded during pregnancy or lactation. In conclusion, results indicated that the vaccination schedule applied is safe and allows for vaccinated and unvaccinated controls to be discriminated between for up to 8 months after booster vaccination.

Listeria monocytogenes Strains Persisting in a Meat Processing Plant in Central Italy: Use of Whole Genome Sequencing and In Vitro Adhesion and Invasion Assays to Decipher Their Virulence Potential.

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.

Detection of Potential Zoonotic Agents Isolated in Italian Shelters and the Assessment of Animal Welfare Correlation with Antimicrobial Resistance in Escherichia coli Strains.

Welfare conditions in shelters, where dogs might be housed for a long period of time, may have a possible correlation with the occurrence of bacterial pathogens and their antimicrobial resistance (AMR). In this study, we assessed the occurrence of AMR in 54 strains of Escherichia coli isolated from dogs housed in 15 Italian shelters and we correlated the resistance patterns to animal welfare. We also aimed to evaluate the presence of specific pathogens with zoonotic potential in sheltered dogs. Thus, nasopharyngeal, rectal, and oral swabs were collected from a group of 20 dogs in each shelter and totaled 758 swabs. We identified 9 Staphylococcus pseudointermedius, 1 Pasteurella multocida, 9 Staphylococcus aureus, 12 Campylobacter spp., 54 Escherichia coli, 2 Salmonella enterica, and 246 Capnocytophaga spp. The antimicrobial susceptibility was assessed for the E. coli isolates using a panel of 14 antibiotics. The highest level of relative AMR was recorded for ampicillin and sulfamethoxazole. The association found between AMR and the levels of animal welfare scores in shelters was evident although not statistically significant. These results support the hypothesis that the good management of shelters can increase the level of animal welfare, thus reducing the use of antibiotics and, as a consequence, the AMR occurrence found in dogs that share their domestic environment with humans.

Lentilactobacillus kefiri SGL 13 and Andrographis paniculata alleviate dextran sulfate sodium induced colitis in mice.

Introduction: Inflammatory bowel diseases (IBD) are chronic inflammatory conditions that typically involve diarrhea, abdominal pain, fatigue, and weight loss, with a dramatic impact on patients' quality of life. Standard medications are often associated with adverse side effects. Thus, alternative treatments such as probiotics are of great interest. The purpose of the present study was to evaluate the effects of oral administration of Lentilactobacillus kefiri (basonym: Lactobacillus kefiri) SGL 13 and Andrographis paniculata, namely, Paniculin 13™, on dextran sodium sulfate (DSS)- treated C57BL/6J mice. Methods: Colitis was induced by administering 1.5% DSS in drinking water for 9 days. Forty male mice were divided into four groups, receiving PBS (control), 1.5% DSS, Paniculin 13™ and 1.5% DSS + Paniculin 13™. Results: The results showed that body weight loss and Disease Activity Index (DAI) score were improved by Paniculin 13™. Moreover, Paniculin 13™ ameliorated DSS-induced dysbiosis, by modulating the gut microbiota composition. The gene expression of MPO, TNFα and iNOS in colon tissue was reduced and these data matched with the histological results, supporting the efficacy of Paniculin 13™ in reducing the inflammatory response. No adverse effects were associated to Paniculin 13™ administration. Discussion: In conclusion, Paniculin 13™ could be an effective add-on approach to conventional therapies for IBD.

Exposure of Mytilus galloprovincialis to Microplastics: Accumulation, Depuration and Evaluation of the Expression Levels of a Selection of Molecular Biomarkers.

Microplastic contamination is a growing marine environmental issue with possible consequences for seafood safety. Filter feeders are the target species for microplastic (MPs) pollution because they filter large quantities of seawater to feed. In the present study, an experimental contamination of Mytilus galloprovincialis was conducted using a mixture of the main types of MPs usually present in the seawater column (53% filaments, 30% fragments, 3% granules) in order to test the purification process as a potential method for removing these contaminants from bivalves intended for human consumption. A set of molecular biomarkers was also evaluated in order to detect any variations in the expression levels of some genes associated with biotransformation and detoxification, DNA repair, cellular response, and the immune system. Our results demonstrate that: (a) the purification process can significantly reduce MP contamination in M. galloprovincialis; (b) a differential expression level has been observed between mussels tested and in particular most of the differences were found in the gills, thus defining it as the target organ for the use of these biomarkers. Therefore, this study further suggests the potential use of molecular biomarkers as an innovative method, encouraging their use in next-generation marine monitoring programs.

Identification and Characterization of Clostridium perfringens Atypical CPB2 Toxin in Cell Cultures and Field Samples Using Monoclonal Antibodies.

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical ß2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.

Chlorinated Persistent Organic Pollutants (PCDD/Fs and PCBs) in Loggerhead Sea Turtles Stranded along the Central Adriatic Coast.

Persistent organic pollutants are widespread in the marine environment. They can bioaccumulate and biomagnify in marine organisms through the food web with a potentially toxic effect on living organisms. The sea turtle Caretta caretta is a carnivorous animal with opportunistic feeding behavior. These turtles tend to bioaccumulate pollutants through food, and hence they can be considered an indicator of chemical pollutants in the marine ecosystem. In this study, 44 loggerhead sea turtles were considered, and liver and fat tissue were sampled from each of them to investigate the levels of dioxins (PCDD/Fs) and polychlorinated biphenyls (PCBs) in sea turtles and their potential correlation with sex and size in terms of curved carapace length (CCL). Results suggested that these contaminants were easily bioaccumulated, and PCBs were predominant compared to dioxins in both liver and fat tissue. The congener patterns were similar to those found in sea fish. Moreover, there were no differences in the contamination levels between females and males, nor was there a correlation with the size. There is a need to harmonize the methodological approaches to better evaluate the results and trends over time and to monitor the species and indirectly the health status of the marine environment.

Development of a Competitive Enzyme-Linked Immunosorbent Assay Based on Purified Recombinant Viral Protein 7 for Serological Diagnosis of Epizootic Haemorrhagic Disease in Camels.

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease.

Genetic diversity of Listeria monocytogenes strains contaminating food and food producing environment as single based sample in Italy (retrospective study).

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.

A Pilot Study to Develop an Assessment Tool for Dogs Undergoing Trap-Neuter-Release (TNR) in Italy. An Overview on the National Implementation of TNR Programmes.

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.

Genetic relationships and biofilm formation of Listeria monocytogenes isolated from the smoked salmon industry.

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.

Intensive Environmental Surveillance Plan for Listeria monocytogenes in Food Producing Plants and Retail Stores of Central Italy: Prevalence and Genetic Diversity.

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.

Survival rate of Escherichia coli O157 in artificially contaminated raw and thermized ewe milk in different Pecorino cheese production processes.

Pecorino is a typical Italian cheese, mostly produced in central and southern Italy regions using ewe raw milk and following traditional procedures. The use of raw milk constitutes a risk linked to the potential survival or multiplication of pathogenic microorganisms, as Shiga toxin-producing Escherichia coli (STEC). The aim of this study was to compare different Italian traditional Pecorino production methods to determine if there were any phases that could influence the Escherichia coli O157 survival rate, but also if they could negatively influence lactic acid bacteria survival rate, during the phases of production and ripening. Therefore batches of Pecorino cheese were prepared using different production methods, representing the real and typical cheese production in southern and central Italy regions: 1) heating the milk at 37 °C for about 40 min before curding, 2) heating the milk at 60 °C (thermization) for 13 min, so that the alkaline phosphatase reaction is still positive before curding, 3) cooking curd at 41 °C and 4) at 45 °C, both for 5 min. Our results demonstrated that traditional milk treatments different from pasteurization can help but do not eliminate serious microbiological treats, as E. coli O157, especially if the raw milk is heavily contaminated. The heat treatment at 60 °C applied to raw milk was able to decrease the concentration of E. coli O157 of 1.7 log10CFU/ml and, according to the inactivation slope, it would be further reduced prolonging the heating treatment. The results obtained also showed that, during the Pecorino cheese ripening, E. coli O157 was always enumerable for 60 days, remaining detectable after 90 days of ripening.

Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy.

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.

Application of the Micro Biological Survey analytical method for the determination of bacterial load in cow raw milk.

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.

A COVID-19 Hotspot Area: Activities and Epidemiological Findings.

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 (χ2 = 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.