Persistence of hepatitis E virus (HEV) subtypes 3c and 3e: Long-term cold storage and heat treatments.

Hepatitis E virus (HEV) is the causative agent of foodborne infections occurring in high income countries mainly by consumption of undercooked and raw pork products. The virus is zoonotic with pigs and wild boars as the main reservoirs. Several studies proved the presence of HEV-RNA in pork liver sausages, pâté and other pork by-products. However, the detection of HEV nucleic acids does not necessary correspond to infectious virus and information on the persistence of the virus in the food is still limited. To which extent and how long the virus can survive after conventional industrial and home-made conservation and cooking procedures is largely unknown. In the present study, we investigated the persistence of two subtypes of HEV-3, by measuring the viral RNA on cell supernatant of infected A549 cells, after long-term storage at +4 °C and -20 °C and after heating for short or long-time span. Results confirmed that either low temperature storage (+4 °C) or freezing (-20 °C) do not influence the survival of the virus, and only a moderate reduction of presence of its RNA after 12 weeks at +4 °C was observed. To the other side, heating at 56 °C for long time (1 h) or at higher temperatures (>65 °C) for shorter time inactivated the virus successfully.

Investigation of Salmonella, hepatitis E virus (HEV) and viral indicators of fecal contamination in four Italian pig slaughterhouses, 2021-2022.

In the pork production chain, the control at slaughterhouse aims to ensure safe food thanks to proper hygienic conditions during all steps of the slaughtering. Salmonella is one of the main foodborne pathogens in the EU causing a great number of human cases, and pigs also contribute to its spreading. Pig is the main reservoir of the zoonotic hepatitis E virus (HEV) that can be present in liver, bile, feces and even rarely in blood and muscle. The aim of this study was to assess the presence of both Salmonella and HEV in several points of the slaughtering chain, including pig trucks. Other viruses hosted in the gut flora of pigs and shed in feces were also assayed (porcine adenovirus PAdV, rotavirus, norovirus, and mammalian orthoreovirus MRV). Torque teno sus virus (TTSuV) present in both feces, liver and blood was also considered. Four Italian pig abattoirs were sampled in 12 critical points, 5 of which were the outer surface of carcasses before processing. HEV and rotavirus (RVA) were not detected. Norovirus was detected once. Salmonella was detected in two of the 4 abattoirs: in the two lairage pens, in the site of evisceration and on one carcass, indicating the presence of Salmonella if carcass is improper handled. The sampling sites positive for Salmonella were also positive for PAdV. MRV was detected in 10 swabs, from only two abattoirs, mainly in outer surface of carcasses. TTSuV was also detected in all abattoirs. Our study has revealed a diverse group of viruses, each serving as indicator of either fecal (NoV, RVA, PAdV, MRV) or blood contamination (TTSuV). TTSuV could be relevant as blood contamination indicators, crucial for viruses with a viremic stage, such as HEV. The simultaneous presence of PAdV with Salmonella is relevant, suggesting PAdV as a promising indicator for fecal contamination for both bacterial and viruses. In conclusion, even in the absence of HEV, the widespread presence of Salmonella at various points in the chain, underscores the need for vigilant monitoring and mitigation strategies which could be achieved by testing not only bacteria indicators as expected by current regulation, but also some viruses (PAdV, TTSuV, MRV) which could represent other sources of fecal contamination.

Hepatitis E Virus (HEV) in Heavy Pigs in Slaughterhouses of Northern Italy: Investigation of Seroprevalence, Viraemia, and Faecal Shedding.

Hepatitis E virus (HEV) is considered an emerging threat in Europe, owing to the increased number of human cases and the widespread presence of the virus in pigs at farms. Most cases in industrialized countries are caused by the zoonotic HEV-3 genotype. The main transmission route of HEV-3 in Europe is foodborne, through consumption of raw or undercooked liver pork and wild boar meat. Pigs become susceptible to HEV infection after the loss of maternal immunity, and the majority of adult pigs test positive for IgG anti-HEV antibodies. Nonetheless, HEV-infected pigs in terms of liver, faeces, and rarely blood are identified at slaughterhouses. The present study aimed to investigate the prevalence of HEV-positive batches of Italian heavy pigs at slaughterhouses, assessing the presence of animals still shedding HEV upon their arrival at the slaughterhouse by sampling faeces collected from the floor of the trucks used for their transport. The occurrence of viraemic animals and the seroprevalence of anti-HEV antibodies were also assessed. The results obtained indicated the presence of anti-HEV IgM (1.9%), and a high seroprevalence of anti-HEV total antibodies (IgG, IgM, IgA; 89.2%, n = 260). HEV RNA was not detected in either plasma or faecal samples. Nevertheless, seropositive animals were identified in all eight batches investigated, confirming the widespread exposure of pigs to HEV at both individual and farm levels. Future studies are needed to assess the factors associated with the risk of HEV presence on farms, with the aim to prevent virus introduction and spread within farms, thereby eliminating the risk at slaughterhouse.

Porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus alone or associated are frequent intralesional detected viruses in porcine respiratory disease complex cases in Northern Italy.

Methods: This study aimed to examine the pathological impact of Porcine Circovirus type 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) through histological and immunohistochemical analysis of 79 cases of Porcine Respiratory Disease Complex (PRDC) collected from 22 farms in Northern Italy. Lung tissue and several lymphoid organ samples were deployed to associate PCV2-positive stain with Circovirus-associated Diseases (PCVD). Results: The most common lung lesion observed was interstitial pneumonia, alone or combined with bronchopneumonia. By immunohistochemistry, 44 lungs (55.7%) tested positive for PCV2, 34 (43.0%) for PRRSV, 16 (20.3%) for both viruses and in 17 cases (21.5%) neither virus was detected. Twenty-eight out of 44 (63.6%) PCV2-positive cases had lymphoid depletion or granulomatous inflammation in at least one of the lymphoid tissues examined; thus, they were classified as PCV2 Systemic Diseases (PCV2-SD). In the remaining 16 out of 44 cases (36.4%), PCV2-positive lung lesions were associated with hyperplastic or normal lymphoid tissues, which showed PCV2-positive centrofollicular dendritic cells in at least one of the lymphoid tissues examined. Therefore, these cases were classified as PRDC/PCV2-positive. In the PCV2-positive animals, 42.9% of the PCV2-SD cases (12/28) showed immunohistochemistry (IHC) positivity for PRRSV in the lung tissue, while 25.0% of PRDC/PCV2-positive cases (4/16) showed double positivity for PCV2 and PRRSV. Discussion: In light of the caseload presented in this study, characterized by the high proportion of PCV2-SD cases alongside the overall respiratory symptomatology, it is imperative to emphasize the crucial role of a comprehensive sampling protocol. This is critical to avoid underestimating the harm caused by PCV2 in farms, particularly with respect to the systemic form of the disease. PCV2 and PRRSV remain the primary infections associated with PRDC in Italy that can significantly impact farm health and co-infections in the field can worsen the pathology, thus the selection of appropriate preventive measures is critical.

Cross-sectional study of hepatitis E virus (HEV) circulation in Italian pig farms.

Foodborne transmission is considered the main way of spreading zoonotic hepatitis E virus (HEV) infection in Europe. In recent years, the human cases of hepatitis E in subjects without history of travel in endemic areas have raised, suggesting that domestic HEV transmission is increasing. Pork products with or without liver, are often indicated as the source of many human foodborne HEV cases as well as small outbreaks. Pigs are recognized as the main reservoir of the zoonotic HEV-3 genotype, the most frequently detected in human cases in the EU. In the absence of a harmonized surveillance of HEV circulation, data on prevalence are heterogeneous but confirm a widespread circulation of HEV-3 in pig herds across EU. HEV-3 can pass through the food chain from farm to fork when infected animals are slaughtered. In Italy, several studies reported the circulation of HEV-3 in pig farms, but results are heterogeneous due to different methodologies applied. In the present study, we performed a survey over 51 pig herds belonging to three main types of farms: breeding, fattening and farrow-to-finish. HEV-RNA was analyzed by broad range Real-time RT-PCR on 20 samples for each farm, obtained by pooling together feces from 10 individuals. Overall, HEV RNA was confirmed on 150 fecal pooled samples out of 1,032 (14.5%). At least one positive pooled sample was detected from 18 farms out of 51 tested (35.3%). By lowering the number of infected pigs at primary production, the risk of HEV-3 entering into the food chain can be reduced. Hence, information on HEV circulation in herds is highly relevant for choosing preventive measures and deserves development of a monitoring program and further investigations.

Porcine Lawsonia intracellularis Ileitis in Italy and Its Association with Porcine Circovirus Type 2 (PCV2) Infection.

The objective of this study was to employ a diagnostic algorithm, which involves detecting positive farms by stool PCR followed by PCR and histology/immunohistochemistry on ileum samples, for diagnosing Lawsonia intracellularis proliferative enteritis in Northern Italy. The primary aim was to examine the relationship between the gold standard of L. intracellularis diagnostics, namely histology and immunohistochemistry, and PCR in acute and chronic cases of L. intracellularis enteritides. An additional goal was to investigate the coinfection of L. intracellularis with porcine circovirus type 2 (PCV2). Twenty-eight ileum samples, including four from acute cases and 24 from chronic cases, were collected. PCR yielded positive results in 19 cases (four acute and 15 chronic cases). In comparison, immunohistochemistry was positive in 16 cases (four acute and 12 chronic cases), with an observed agreement of 89%. The findings suggest that performing the two tests in series can increase the specificity of the causal diagnosis. PCR may be used as a screening tool to identify the presence of the microorganism, and only positive cases will be examined by histology and immunohistochemistry to confirm the causative role of L. intracellularis. Co-infection with PCV2 was demonstrate in two out of four acute cases and in two out of 24 chronic cases, providing further evidence to support the hypothesis that when the infection starts with ubiquitous pathogens such as L. intracellularis, it may boost the possibility of PCV2 replication, especially in acute cases. As a result, this may trigger a transition from subclinical to clinical forms of PCV2 disease.

Absence of Hepatitis E Virus (HEV) in Italian Lagomorph Species Sampled between 2019 and 2021.

The zoonotic hepatitis E virus genotype 3 (HEV-3) causes most autochthonous human hepatitis E cases in Europe, which are due to the consumption of raw or undercooked food products of animal origin. Pigs and wild boars are considered the main reservoirs of this genotype, while rabbits are the reservoir of a distinct phylogenetic group named HEV-3ra, which is classified within the HEV-3 genotype but in a separate clade. Evidence for the zoonotic potential of HEV-3ra was suggested by its detection in immunocompromised patients in several European countries. HEV-3ra infection was found in farmed and feral rabbit populations worldwide and its circulation was reported in a few European countries, including Italy. Furthermore, Italy is one of the major rabbit meat producers and consumers across Europe, but only a few studies investigated the presence of HEV in this reservoir. The aim of this study was to assess the presence of HEV in 328 Italian hares and 59 farmed rabbits collected in 3 Italian macro-areas (North, North-Central, and South-Central), between 2019 and 2021. For this purpose, liver samples were used to detect HEV RNA using broad-range real-time RT-PCR and nested RT-PCR. Using 28 liver transudates from hares, the ELISA test for anti-HEV IgG detection was also performed. Neither HEV RNA nor anti-HEV antibodies were detected. Further studies will be conducted to assess the HEV presence in Italian lagomorphs to establish the role of this host and the possible risk of transmission for workers with occupational exposure, to pet owners and via food.

In Vitro Replication of Swine Hepatitis E Virus (HEV): Production of Cell-Adapted Strains.

The hepatitis E caused by the virus HEV of genotypes HEV-3 and HEV-4 is a zoonotic foodborne disease spread worldwide. HEV is currently classified into eight different genotypes (HEV-1-8). Genotypes HEV-3 and HEV-4 are zoonotic and are further divided into subtypes. Most of the information on HEV replication remains unknown due to the lack of an efficient cell cultivation system. Over the last couple of years, several protocols for HEV cultivation have been developed on different cell lines; even if they were troublesome, long, and scarcely reproducible, they offered the opportunity to study the replicative cycle of the virus. In the present study, we aimed to obtain a protocol ready to use viral stock in serum free medium that can be used with reduced time of growth and without any purification steps. The employed method allowed isolation and cell adaptation of four swine HEV-3 strains, belonging to three different subtypes. Phylogenetic analyses conducted on partial genome sequences of in vitro isolated strains did not reveal any insertion in the hypervariable region (HVR) of the genomes. A limited number of mutations was acquired in the genome during the virus growth in the partial sequences of Methyltransferase (Met) and ORF2 coding genes.

Efficacy of Five Disinfectant Products Commonly Used in Pig Herds against a Panel of Bacteria Sensitive and Resistant to Selected Antimicrobials.

The growing threat of antimicrobial resistance worldwide has led to an increasing concern in the human, veterinary, and environmental fields, highlighting the need for strategies to effectively control bacterial contamination. Correct biosecurity practices, including the appropriate use of disinfectants, play a crucial role in controlling bacterial contamination. This study aimed to verify whether the recommended concentrations defined according to the Defra General Orders concentration (GO, published by the UK Department for Environment, Food and Rural Affairs' disinfectant-approval scheme) of five commercial disinfectant preparations (peroxygen-based, phenol-based, two halogen-releasing agents, and glutaraldehyde/quaternary ammonium compound-based; disinfectants A to E, respectively) were sufficient to inhibit growth and inactivate selected bacterial strains, including some that carry known phenotypic patterns of multidrug resistance. The effectiveness of each disinfectant was expressed as the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, determined by the broth-microdilution method. The results indicate that the type of disinfectant and its concentration influence the inhibitory and bactericidal efficacy. The glutaraldehyde/quaternary ammonium compound-based (disinfectant D) and chlorocresol-based products (disinfectant B) were the most effective, and the GO concentration was bactericidal in all the strains tested. The efficacy of the other compounds varied, depending on the bacterial species tested. The GO concentrations were at least able to inhibit the bacterial growth in all the products and bacterial strains tested. A greater tolerance to the compounds was observed in the strains of E. coli with multidrug-resistance profiles compared to the strains that were sensitive to the same antimicrobials.

Dynamic of Hepatitis E Virus (HEV) Shedding in Pigs.

Genotype 3 of hepatitis E virus (HEV-3) is the most common in Europe in both humans and pigs. HEV-3 strains are zoonotic, and foodborne cases associated with consumption of raw and undercooked pork products, mainly liver sausages, have been described. HEV-3 circulates largely in European pig farms, maybe due to its long persistence in the environment. Animals get infected around 3-4 months of age; shortly after, the infection starts to decline up to the age of slaughtering (8-9 months of age in Italy). With the purpose to understand the duration in farmed pigs of the shedding of the virus and its quantity, HEV-RNA detection was performed by Real-time RT-PCR from feces collected individually from two groups of 23 pigs. Sampling was conducted for 4 months shortly before slaughtering age. At 4-months-old, all animals were shedding HEV-3 to high load around 105 genome copies per gram (GC/g). Prevalence was higher in growers than in fatteners, with most of the pigs still positive around 166 days of age. Beyond some difference among individual pigs, the amount of HEV in feces decreased with the age of animals. The longest fattening period should ensure a lower risk of HEV shedder animals at slaughter, reducing the risk of food contamination.

Detection of antibodies against influenza D virus in swine veterinarians in Italy in 2004.

Influenza D virus (IDV) was first isolated from a swine with respiratory disease symptoms in 2011 in the United States. Epidemiological and serological studies support the hypothesis that cattle represent the natural reservoir of IDV with periodical spillover events to other animal hosts. Little is known about the seroprevalence in humans and in specific target groups such as veterinarians in Italy. This study was designed to assess the prevalence of antibodies against two influenza D lineages (D/660 and D/OK) in Italy in archived serum samples from veterinarians working with swine collected in 2004. Serum samples were tested by haemagglutination inhibition (HI) and virus neutralization (VN) assays. Results showed that 4.88% (4/82) of tested samples were positive for D/660 and 2.44% (2/82) for D/OK by HI assay. Three out of four samples showed positivity when tested by VN assay. Our data suggest undetected IDVs might have circulated and/or been introduced in Italy as early as 2004 at least in some animal species such as swine. In addition, it seems that the virus was circulating among veterinarians before the first isolation in 2011. This finding highlights the importance to continue monitoring the IDV spread in animals and humans for more detailed surveillance.

Hazard Identification Related to the Presence of Vibrio spp., Biogenic Amines, and Indole-Producing Bacteria in a Non-Filter Feeding Marine Gastropod (Tritia mutabilis) Commercialized on the Italian Market.

Tritia mutabilis is a carrion-feeder edible marine gastropod with an open circulatory system. Therefore, biological, and chemical contaminants associated with the feed can reach all body tissues. The aim of the present study was to investigate the possible association of these characteristics with some food safety hazards. Vibrio spp. load, and the prevalence of pathogenic V. parahaemolyticus, V. vulnificus, and V. cholerae, were investigated. Moreover, biogenic amines (BAs) and indole-producing bacteria (IPB), markers of seafood decomposition, were quantified for the first time in an edible carrion-feeder. Overall, 49 batches were analyzed (38 from retail, and 11 from primary production). The Vibrio spp. load resulted of 5.64 ± 0.69 log10 CFU g-1 at retail, and 5.27 ± 0.74 at harvest but all batches resulted negative for pathogenic Vibrio. Histamine, putrescine, cadaverine, and tyramine were detected both at harvest and at the retail level. Their sum (BAs Index) showed a mean value of 50.45 and 65.83 mg Kg-1 in batches at harvest and at retail, respectively. IPB were detected at harvest and upon refrigeration for three days (T1-T3). The mean load resulted in 2.52 ± 0.85 log10 MPN g-1 at T0, 3.31 ± 1.23 at T3 in batches immediately refrigerated, and 3.22 ± 1.18 at T3 in batches previously immersed in clean seawater. Our results contribute to identifying food-borne hazards for T. mutabilis that may be related to the retention of biogenic amines and indole-producing bacteria due to carrion feeding.

The Role of Pathology in the Diagnosis of Swine Respiratory Disease.

The definition "porcine respiratory disease complex" (PRDC) is used to indicate the current approach for presenting respiratory pathology in modern pig farming. PRDC includes pneumonias with variable pictures, mixed with both aerogenous and hematogenous forms with variable etiology, often multimicrobial, and influenced by environmental and management factors. The notion that many etiological agents of swine respiratory pathology are ubiquitous in the airways is commonly understood; however, their isolation or identification is not always associable with the current pathology. In this complex context, lung lesions registered at slaughterhouse or during necropsy, and supplemented by histological investigations, must be considered as powerful tools for assigning a prominent role to etiologic agents. In recent years, the goal of colocalizing causative agents with the lesions they produce has been frequently applied, and valid examples in routine diagnostics are those that indicate pulmonary involvement during porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) infections.

Evaluation of Prognostic Factors, including Duodenal P-Glycoprotein Expression, in Canine Chronic Enteropathy.

The aim of this retrospective single-center study was to evaluate which factors, including expression of P-glycoprotein (P-gp), a membrane-bound protein involved in multiple drug resistance, could predict the response to treatment in canine immunosuppressant-responsive enteropathy (IRE). Dogs with IRE or non-responsive enteropathy (NRE) that were examined from 2005 to 2014 were included and were divided into two groups (IRE vs. NRE). Signalment, history, and clinical and laboratory findings were collected. P-glycoprotein immunohistochemistry was carried out on duodenal biopsies of both groups stored in our biobank, and immunophenotyping and molecular clonality were performed on the NRE samples. Ninety-two dogs were enrolled, 73 IRE (79.3%) and 19 NRE (20.7%), with a prevalence of pure breed (78.3% vs. 21.7%) and male dogs (p < 0.001). Factors associated with a worse prognosis were previous treatment with steroids (p = 0.033) and lower serum total protein concentration (p = 0.005). Clonality testing on the NRE duodenal biopsies showed 5/16 clonal responses, assuming a latent undiagnosed lymphoma as a possible cause of the NRE.

Pilot Investigation of Anti-Salmonella Antibodies in Oral Fluids from Salmonella Typhimurium Vaccinated and Unvaccinated Swine Herds.

Oral fluid (OF) can be a simple, cheap and non-invasive alternative to serum or meat juice for the diagnosis and surveillance of important pathogens in pigs. This study was conducted on four Salmonella Typhimurium-positive farrow-to-finish pig farms: two Salmonella-vaccinated (V) and two non-vaccinated (NV). Gilts and sows in the V farms were vaccinated with a live, attenuated vaccine prior to farrowing. Pooled faecal and OF samples were collected from the sows and their offspring. Salmonella was isolated according to ISO6579-1:2017. In parallel, IgG and IgA levels were assessed in OF samples using a commercial ELISA assay. Salmonella was detected in 90.9% of the pooled faecal samples from the NV farms and in 35.1% of the pooled faecal samples from the V farms. Overall, a higher prevalence was observed in the pooled faecal samples from the offspring (76.3%) compared to the sows (36.4%). IgG antibodies measured in V farms are likely to be related to vaccination, as well as exposure to Salmonella field strains. The detection of IgA antibodies in OF was unreliable with the method used. The results of this study show that IgG is the most reliable isotype for monitoring Salmonella-specific antibody immunity in vaccinated/infected animals via OF.

Canine Placenta Histological Findings and Microvascular Density: The Histological Basis of a Negative Neonatal Outcome?

Placenta is essential for the development of the fetus, and its impaired function can lead to a negative outcome (i.e., neonatal mortality). In dogs, investigations on placenta histology and neonatal outcome in healthy bitches are lacking, and a contribution is provided in this study to emphasize the use of placenta histology in practice. Fifty-one placentas from 11 litters were collected during cesarean section, classified according to the litter size (large (L) or small (S)) and the outcome, this latter as healthy (Group 1) or dead within 7 days (Group 2). The placenta/puppy weight ratio (PPR) was calculated, and specimens were formalin-fixed and paraffin-wax embedded, and on the resulting histological slides, capillary density (CD) was quantified. Among necrosis, calcification, and intravascular leucocytes, only the presence of multifocal-confluent necrosis (significantly more frequent in Group 2) was associated with a higher risk of death within 7 days (odds ratio = 30.7). Mixed logistic regression ruled out the effect on death both of a bitch and cesarean type (programmed vs. emergency). PPR and CD values were associated with litter size; large litters had lower PPR (p < 0.01) and higher CD (p < 0.05) than small litters. The relationship between PPR and CD was negative and significant (p < 0.01). Necrosis was a frequent finding in canine placentas, but only when multifocal-confluent was it associated with a poor outcome. The litter size influenced PPR (lower in L) and CD (higher in L), and this is likely due to the plasticity of placenta adaptation.

Long-term surveillance for hepatitis E virus in an Italian two-site farrow-to-finish swine farm.

In humans, hepatitis E virus (HEV) is responsible for an acute enterically transmitted hepatitis, which can become chronic in immune-compromised patients. Genotypes 3 and 4 (HEV-3 and HEV-4) are zoonotic, and domestic pigs and wild boar are the main reservoirs. The occurrence of autochthonous cases in Europe, which have been increasing over the last 10 years, has been associated with food-borne zoonotic transmission of HEV-3, mainly linked to consumption of undercooked or raw pork products (sausages containing liver) and wild boar meat. Zoonotic HEV-3 strains are widespread on pig farms, but little information is available on the dynamic of HEV-3 infection within farms, among pigs. The aims of this study were to evaluate the prevalence of the infection among pigs of different ages along the production chain by the zoonotic HEVs, and to evaluate how long the virus may persist in the farm environment. The presence of HEV-RNA was investigated by real-time reverse transcription PCR (RT-PCR) in 281 test faecal pools over 19 months (2017-2019) on a two-site farrow-to-finish farm (about 1,000 sows), in Northern Italy. A total of 67/281 test faecal pools (23.8%) resulted positive for the presence of HEV-RNA (site 1: 59/221, 26.7%; site 2: 8/60, 13.3%). Nucleotide sequencing revealed a unique HEV-3 viral variant circulating during 19 months of surveillance. The same HEV-3 strain was detected in the same farm on 2012, indicating the persistence of the same virus over 7 years, and highlighting the role of the environment as a continuous source of infection on pig farms. The results confirmed the circulation of the zoonotic genotype HEV-3 in pigs before slaughtering.

Hepatitis E Virus Occurrence in Pigs Slaughtered in Italy.

In Europe, foodborne transmission has been clearly associated to sporadic cases and small clusters of hepatitis E in humans linked to the consumption of contaminated pig liver sausages, raw venison, or undercooked wild boar meat. In Europe, zoonotic HEV-genotype 3 strains are widespread in pig farms but little information is available on the prevalence of HEV positive pigs at slaughterhouse. In the present study, the prevalence of HEV-RNA positive pigs was assessed on 585 animals from 4 abattoirs located across Italy. Twenty-one pigs (3.6%) tested positive for HEV in either feces or liver by real-time RT-PCR. In these 21 pigs, eight diaphragm muscles resulted positive for HEV-RNA. Among animals collected in one abattoir, 4 out of 91 plasma tested positive for HEV-RNA. ELISA tests for the detection of total antibodies against HEV showed a high seroprevalence (76.8%), confirming the frequent exposure of pigs to the virus. The phylogenetic analyses conducted on sequences of both ORF1 and ORF2 fragments, shows the circulation of HEV-3c and of a novel unclassified subtype. This study provides information on HEV occurrence in pigs at the slaughterhouse, confirming that muscles are rarely contaminated by HEV-RNA compared to liver, which is the most frequently positive for HEV.

Pilot Investigation on the Presence of Anti-Hepatitis E Virus (HEV) Antibodies in Piglet Processing Fluids.

Identifying Hepatitis E virus (HEV)-positive pig farms is important to implement surveillance programs for this emerging zoonotic agent. The aim of this study was to evaluate the use of serosanguineous fluids obtained as part of castration practice (processing fluids (PFs)) to detect anti-HEV antibodies in newborn piglets. Ninety-five paired serum and PF samples were collected from piglets of 29 different litters and tested with a commercial ELISA kit. A significant positive correlation (Spearman's rho: 0.600; p < 0.01) was found between anti-HEV antibodies in serum and PF samples. In 26 out of 29 litters (89.7%), there was at least one positive piglet in the serum. Sixteen litters out of 29 (55.2%) were also positive in PFs. To simulate the use of PF as pooled samples, the limit of detection of the ELISA was assessed mixing the PF sample with strong, medium, medium-weak and weak ELISA titres with 3, 4, 5 and 6 negative PF samples. Our results suggest that it is still possible to identify a positive PF pool when at least one individual PF sample with medium or strong antibody levels is mixed with 5 or 6 individual negative PF samples. The detection of anti-HEV maternal-derived antibodies in PF confirms a past exposure of sows to the virus. PF may represent a rapid, noninvasive and economical tool to identify HEV-positive farms.

Seroprevalence of Hepatitis E Virus in Forestry Workers from Trentino-Alto Adige Region (Northern Italy).

People with some occupational or recreational activities, such as hunters and veterinarians, may have increased risk to be infected by the hepatitis E virus (HEV). The aim of the present study was to establish whether forestry workers could be considered at a higher risk of HEV infection than a control group. One hundred and fifty sera from forestry workers and a control group of 85 sera were analysed by anti-HEV IgG antibodies detection using a commercial ELISA kit. The anti-HEV IgG seroprevalence was 14% for forestry workers and 9.4% for the control group. Comparing the risk of HEV infection in the two groups, there was no difference in the odds ratio. However, the seroprevalence in older subjects was higher in the forestry workers than in the control group. Two sera from forestry workers were also positive for anti-HEV IgM, and, in one of them, HEV-RNA was detected. Our findings showed an increase of seroprevalence with age, which is likely to reflect cumulative exposure to HEV over time. The occupation of forestry workers did not seem to be associated with a higher risk of HEV infection. The study provided new insights into the risk of acquiring HEV in occupational exposure workers with open-air activities.