Molecular and serological investigation of Hepatitis E virus in pigs slaughtered in Northwestern Italy.

BACKGROUND: Hepatitis E Virus (HEV) is recently considered an emerging public health concern. HEV genotypes 1 and 2 are widely distributed and pathogenic only for humans. In contrast, HEV, genotypes 3 and 4 are observed in swine, deer, wild boars and rabbits and can also be transmitted to humans. The presence of HEV in the liver, muscle, faeces, blood, and bile was detected by real-time RT-PCR in 156 pigs belonging to twenty different farms, ranging from 1 to 8 months of age. The phylogenetic analysis was performed on the viral strain present in the positive biological matrix, with the lowest Ct. HEV-IgG and HEV-IgM in the sera were analysed by two different ELISA kits. RESULTS: Twenty-one pigs, i.e., 13.46% of them (21/156, 95% CI: 8.53%-19.84%), tested positive for HEV in at least one biological matrix by real-time RT-PCR, while phylogenetic analysis revealed the presence of HEV subtypes 3f and 3c. Pig serums analysed by ELISA showed an overall prevalence of 26.92% (42/156, 95% CI: 20.14%-34.60%) for HEV-IgG, whereas the 28.95% (33/114, 95% CI: 20.84%-38.19%) of them tested negative resulted positive for the HEV-IgM. CONCLUSIONS: The faeces are the biological matrix with the highest probability of detecting HEV. The best concordance value (Kappa Kohen index) and the highest positive correlation (Phi index) were observed for the correlation between bile and liver, even when the number of positive liver samples was lower than the positive bile samples. This finding may suggest that a higher probability of HEV occurs in the bile, when the virus is present in the liver, during the stages of infection. Finally, the presence of HEV in muscle was observed in 11 pigs, usually used for the preparation of some dishes, typical of the Italian tradition, based on raw or undercooked meat. Therefore, their consumption is a possible source of infection for final consumer.

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte, Liguria e Valle d'Aosta, Italy.

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016-2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus and Related Risk Factors in Holdings of Veal Calves in Northwest Italy.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have emerged worldwide as zoonotic pathogens. Data on LA-MRSA in veal calf production in Italy are lacking; the aim of this survey was to fill current knowledge gaps in its prevalence and characteristics. Between February 2012 and January 2013 nasal swabs were taken from 1650 three- to six-month-old veal calves on 55 farms in Piedmont (northwest Italy), including gathering-related epidemiological data. S. aureus were screened for methicillin resistance by phenotypic and molecular (mecA gene detection) methods. MRSA were further genotyped by multilocus sequence typing. About 30% of the herds tested positive for MRSA: three different clonal complexes (CC398, CC97, and CC1) and staphylococcal cassette chromosome mec types (IVa, IVb, and V) were detected. Multilevel logistic regression model indicated poor cleaning, importation from Austria, and animal age as risk factors and coagulase-negative staphylococci colonization as a predictive factor for the occurrence of MRSA. The detection of CCs circulating in pigs and dairy cattle in Italy underscores the ability of the LA-MRSA clones to spread among animal production systems. In addition to maintaining preventive control measures for human health, better cleaning procedures need to be implemented, especially after new calves have been introduced into the herd.

Field Evaluation of the Interferon Gamma Assay for Diagnosis of Tuberculosis in Water Buffalo (Bubalus bubalis) Comparing Four Interpretative Criteria.

Bovine tuberculosis (bTB) is a worldwide zoonosis that affects many species of domestic and wild animals. Mycobaterium bovis is the main cause of infection in water buffalo (Bubalus bubalis) and bovines and is of great concern for human health and for buffalo producers in Italy. The bTB eradication programme is based on slaughterhouse surveillance and intradermal skin tests. Other in vivo diagnostic methods such as the interferon-gamma (IFN-γ) assay have been developed and are widely used in cattle to accelerate the elimination of bTB positive animals. The present study is the first to assess the use and performance of IFN-γ assays, which is used as an ancillary test for bTB diagnosis in water buffalo, and presents the results of a field-evaluation of the assay from 2012 to 2019 during the buffalo bTB eradication programme in Italy. The study involved 489 buffaloes with a positive result to the single intradermal tuberculin test (SITT). The IFN-γ assays and single intradermal comparative tuberculin test were used as confirmation tests. Then, a total of 458 buffaloes, reared on officially tuberculosis-free (OTF) herds, that were confirmed bTB-free for at least the last 6 years were subjected to IFN-γ testing. Furthermore, to evaluate the IFN-γ test in an OTF herd with Paratuberculosis (PTB) infection, 103 buffaloes were subjected to SITT and IFN-γ test simultaneously. Four interpretative criteria were used, and the IFN-γ test showed high levels of accuracy, with sensitivity levels between 75.3% (CI 95% 71.2-79.0%) and 98.4% (CI 95% 96.7-99.4%) and specificity levels between 94.3% (CI 95% 91.2-96.50%) and 98.5% (CI 95% 96.9-99.4%), depending on the criterion used. Finally, in the OTF herd with PTB infection, in buffalo, the IFN-γ test displayed high specificity values according to all 4 interpretative criteria, with specificity levels between 96.7% (CI 95% 88.4-99.5%) and 100% (CI 95% 96.2-100%), while SITT specificity proved unsatisfactory, with a level of 45.3% (CI 95% 35.0-55.7%). Our results showed that the IFN-γ test in the buffalo species could reach high Sensitivity and Specificity values, and that the level of Sensitivity and Specificity could be chosen based on the interpretative criterion and the antigens used depending on the health status of the herd and the epidemiological context of the territory. The IFN-γ test and the use of different interpretative criteria proved to be useful to implement bTB diagnostic strategies in buffalo herds, with the possibility of a flexible use of the assay.

Arcobacter spp. in raw milk from vending machines in Piedmont and occurrence of virulence genes in isolates.

Arcobacter spp. has been recognized as an emerging foodborne pathogen and a hazard to human health. In the dairy chain, it has been isolated from different sources, nevertheless data on Arcobacter occurrence in raw milk provided by vending machines are few. This study aimed to identify potentially pathogenic Arcobacter spp. in raw milk intended for human consumption sold through vending machines located in Piedmont. In an 8-month period, 37 raw milk samples were collected from 24 dairy farms: 12 (32,4%) were collected directly in farm from bulk tank milk and 25 (67,6%) from vending machines. Eight (21,6%) out of the 37 milk samples and 7 (29,2%) out of the 24 dairy farms were positive for Arcobacter spp. by culture examination. Four (16%) out of the 25 samples from vending machines and 4 (33,3%) out of the 12 samples from bulk tank milk were positive. All 8 isolates were identified as A. butzleri both by MALDI-TOF MS and multiplex end-point PCR. According to the detection of virulence genes, a total of four Patho-types were highlighted: 5 isolates in P-type 1 and only one isolate for each of the P-types 2-3-4. A. butzleri isolates carrying encoding virulence factors genes were isolated from raw milk intended for human consumption: these findings strengthen the compulsory consumption after boiling as required by current legislation and suggest the need of enlarging the analytical investigations to other microorganisms not yet included in the food safety criteria.

Outbreak of febrile gastroenteritis caused by Listeria monocytogenes 1/2a in sliced cold beef ham, Italy, May 2016.

In May 2016, two separate clusters of febrile gastroenteritis caused by Listeria monocytogenes were detected by the local health authority in Piedmont, in northern Italy. We carried out epidemiological, microbiological and traceback investigations to identify the source. The people affected were students and staff members from two different schools in two different villages located in the Province of Turin; five of them were hospitalised. The epidemiological investigation identified a cooked beef ham served at the school canteens as the source of the food-borne outbreak. L. monocytogenes was isolated from the food, the stools of the hospitalised pupils and the environment of the factory producing the cooked beef ham. All isolates except one were serotype 1/2a, shared an indistinguishable PFGE pattern and were 100% identical by whole genome sequencing (WGS). By combining a classical epidemiological approach with both molecular subtyping and WGS techniques, we were able to identify and confirm a Listeria gastroenteritis outbreak associated with consumption of sliced cold beef ham.

Pseudorabies virus in North-West Italian wild boar (Sus scrofa) populations: prevalence and risk factors to support a territorial risk-based surveillance.

Although  the  eradication  of  Pseudorabies  virus  (PrV)  in  domestic  pigs  is  ongoing,  the circulation  of  this  virus  in  wild  boars  remains  a  threat  in  the  currently  unprotected, 'low prevalence', pig population. In this study, we reported PrV prevalence data and the influence of  possible  risk  factors  in  2  North-West  Italian  wild  boar  populations  (free  and  enclosed) with the goal of supporting the implementation of a risk-based AD surveillance system. Sera from  1,425  wild  boars  were  collected  between  2011  and  2015  and  tested  by  ELISA  for  the presence  of  PrV  antibodies;  the  overall  raw  seroprevalence  was  30.39%  (433/1,425;  CI  95% 28.01-32.85%).  A  significant  difference  was  however  observed  between  the  prevalence rates  of  the  free  range  population  (9.98%;  90/902;  CI  95%;  8.10-12.12%)  and  the  enclosed  population of La Mandria park (Piedmont, Italy) (65.58%; 343/523; CI 95%; 61.51-69.65%). In both populations a significantly higher number of adults and females were found positive to PrV  ELISA.  Specific  territorial  data  on  PrV  circulation  in  wild  boars  should  be  acquired  from other regions for guiding risk-based measures in order to reduce the threat of AD re-infection in a more cost-effective manner.

Control of Mosquito-Borne Diseases in Northwestern Italy: Preparedness from One Season to the Next.

INTRODUCTION: Mosquito-borne diseases (MBDs) are spreading worldwide due to globalization and climate change, representing a threat for both humans and animals. Of great concern are the infections caused by viruses belonging to the Flavivirus genus as West Nile virus (WNV) and Usutu virus (USUV) transmitted by Culex sp. or Dengue virus and Zika virus (ZIKV), transmitted by Aedes sp. This work describes the surveillance protocol enforced in Piedmont (Northwestern Italy) to control MBDs spread, focusing on the activities performed on mosquitoes during the 2015 vector season. MATERIALS AND METHODS: From July to October, mosquitoes were fortnightly sampled in 50 selected sites according to risk factors with CDC dry ice-baited traps and BG-Sentinel traps baited with BG-Lure and dry ice. Adults were counted, identified to species level, pooled, and screened for flaviviruses using different reverse transcription-PCR protocols and sequencing. Finally, phylogenetic analysis was performed on a dataset including 2014 and 2015 WNV sequences and reference sequences retrieved from GenBank. RESULTS AND DISCUSSION: A total of 17,000 mosquitoes, grouped in 730 pools, were tested. Five pools of Culex pipiens were positive for WNV Lineage 2 in Novara, Alessandria, Vercelli, and Torino Provinces. One pool of C. pipiens and one pool of Anopheles maculipennis s.l. were positive for USUV in Vercelli and Alessandria Provinces. In Vercelli Province one pool of C. pipiens resulted positive both for WNV and USUV. Control measures were quickly implemented. Phylogenetic analyses showed that the WNV Lin 2 sequences from Piedmont region cluster with those circulating in Northeastern Italy in the previous years. Given the positive trend in WNV activity compared to 2014 and the emergence caused by other flavivirus as ZIKV, the level of attention for the 2016 vector season may be increased and this surveillance protocol could represent an important tool for public health authorities.

Q fever seroprevalence and risk factors in sheep and goats in northwest Italy.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii; domestic ruminants, mainly goats and sheep, are the main source of Q fever outbreaks in humans. From both a public and an animal health perspective, providing reliable prevalence data is extremely relevant for the decision processes by policymakers and food producer organizations. Information on Q fever seroprevalence in small ruminants in Italy is currently incomplete and largely based on reports of reproductive disorders in livestock farms. To estimate animal and flock seroprevalence of C. burnetii in small ruminants (sheep, goats and mixed flocks), a cross-sectional study with a two-stage design was carried out in northwest Italy. Between January and December 2012, sera from 5738 animals (2553 sheep and 3185 goats) belonging to 411 flocks (206 goats, 111 sheep, and 94 mixed flocks) were examined for specific anti-C. burnetii IgG antibodies by a commercial ELISA kit. A questionnaire investigating possible associations between farm management and C. burnetii seropositivity was administered. At the flock level, the overall true seroprevalence adjusted for test sensitivity and specificity was 31.2% (95% confidence interval [CI]: 24.8-37.7). Sheep-farm and goat-farm true seroprevalence was 38.7% (95% CI 25.5-51.9) and 19.5% (95% CI 11.5-27.6), respectively. Interestingly, the true seroprevalence (48.5%; 95% CI 34.7-62.3) was higher in the mixed flocks (sheep and goats). At the animal level, the overall true seroprevalence was 15.9% (95% CI 15.4-16.4). No difference was found between the two species, but the true seroprevalence was significantly higher (χ(2)=7.49; p<0.007) among the goats in mixed flocks (25.7%; 95% CI 24.4-27.1) than the sheep (16.3%; 95% CI 15.1-17.4), suggesting a potential difference in susceptibility between the two species or the result of factors affecting their immune response or related to the livestock management system as the period of exposure to C. burnetii. A multivariable logistic model that controlled for farm-level clustering identified five main risk factors associated with farm seropositivity (p≤0.05): flock size of more than 12 animals (odds ratio [OR] 4.2; 95% CI 2.6-6.7), contact with other flocks (OR 2.1; 95% CI 1.2-3.6), mixed flock type (OR 2.4; 95% CI 1.4-4.2), farms located in the western area (OR 2.4; 95% CI 1.4-4.2), and infertility during the previous year (OR 2.6; 95% CI 1.2-5.2). The results of this study yielded baseline information that may be useful to set up future epidemiologic, flock management, and public health policies for the prevention and control of Q fever in Italy.

Detection of West Nile and Usutu Viruses in Italian Free Areas: Entomological Surveillance in Piemonte and Liguria Regions, 2014.

West Nile virus and Usutu virus have established in different parts of Italy over the past 10 years. Piemonte and Liguria Regions (Northwestern Italy) are known to be nonendemic areas, despite the presence of competent vectors and environmental conditions conducive to maintaining infection. This work evidences for the first time, through an entomological surveillance implemented on the basis of risk factor approach, the presence of West Nile and Usutu viruses in Piemonte and Liguria Regions (Northwestern Italy).

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).

[Environmental micropollutants in livestock products from Susa Valley, Piedmont]. / La contaminazione ambientale da microinquinanti degli allevamenti della Val di Susa.

OBJECTIVE: to describe the contamination due to dioxins and dioxin-like PCBs of cattle dairy and meat products in cattle herds from Susa Valley (Piedmont) since the second half of 2004. DESIGN: descriptive study based on a biomonitoring campaign on bovine bulk milk and muscle. SETTING AND PARTICIPANTS: the official samplings have been carried out between 2004 and 2010: the dataset refers to 99 mass milk samples and 24 muscle samples from 51 cattle herds based in 23 municipalities of the valley. MAIN OUTCOME MEASURES: counts and frequencies of samples exceeding either the maximum levels or the action levels set by the European legislation; moreover relative distributions of congeners (profiles) were considered. RESULTS: DL-PCBs showed median concentration values double when compared with dioxins values and a couple of congeners (2,3,4,7,8-PeCDF, 118 DL-PCB) were particularly abundant. More than 60% of both the milk samples and herds exceeded the action levels with the involvement of 10 municipalities most of which located nearby a local steel smelter. The DL-PCB values in milk were associated with the distance of each herd from the steel plant. However, the temporal trend highlights a clear decrease of the contamination over the 2004-2010 period, when the plant was renewed and the production levels where negatively impacted by the global economical crisis. CONCLUSION: the findings of the monitoring activity allowed the understanding of the origin and the spatio-temporal evolution of the contamination: this knowledge may be helpful in orienting future controls and the risk mitigation strategies.

A relevant long-term impact of the circulation of a potentially contaminated vaccine on the distribution of scrapie in Italy. Results from a retrospective cohort study.

A sudden increase in the incidence of scrapie in Italy in 1997 was subsequently linked to the use of a potentially infected vaccine against contagious agalactia. The relative risk for the exposed farms ranged between 6 and 40. The aim of this study was to assess the long-term impact of exposure to the potentially scrapie-contaminated vaccine on the Italian classical scrapie epidemic. We carried out a retrospective cohort study, fitting mixed-effects Poisson regression models, dividing national geographic areas into exposure categories on the basis of the vaccine circulation levels. We took into account the sensitivity of the surveillance system applied in the different areas. The population attributable fraction (PAF) was used to assess the impact on the total population of farms associated with the effect of circulation of the vaccine. The provinces where the vaccine was more often sold were noted to have a higher level of disease when compared to those provinces where the vaccine was sold less often (incidence rate ratio [IRR]: 2.7; 95% confidence interval [CI]: 1.1-6.5). The population attributable fraction was high (68.4%). Standardization techniques allowed to account for the potential of geographical variability in the sensitivity of the Italian surveillance system. Although the number of the directly exposed farms was limited, an important long-term impact of the vaccine circulation could be quantified in terms of secondary outbreaks likely due to the exchange of animals from directly exposed flocks.