Fabio Ostanello

Hepatitis E Virus in Pork Production Chain in Czech Republic, Italy, and Spain, 2010

Processing does not substantially abate endogenous virus.

Widespread Diffusion of Genotype 3 Hepatitis E Virus among Farming Swine in Northern Italy

Hepatitis E virus (HEV) causes acute hepatitis in humans, and infects several animal species, mostly asymptomatically. Swine and human HEV strains are genetically related suggesting both a zoonotic and a possible foodborne transmission. The prevalence of swine HEV was investigated in 274 randomly selected pigs from six different swine farms of Northern Italy, testing viral RNA in stools by nested reverse-transcription-polymerase chain reaction. HEV genome was detected in 115 stools (42%). All farms resulted positive for HEV, with a prevalence ranging between 12.8% and 72.5%. HEV-positive pigs were detected in all age groups and production stages tested, although infection was more prevalent in weaners than in the older fatteners (42.2% vs. 27.0%). Genetic characterization of swine strains identified was performed by sequencing and database alignment. Phylogenetic analysis on the nucleotide sequences from 16 positive PCR products indicated that all strains belonged to genotype 3. In particular, one group of seven Italian strains clustered close (91.6-96.2% identity) to human and swine European HEV strains.

Prevalence and transmission of hepatitis E virus in domestic swine populations in different European countries

BackgroundHepatitis E virus (HEV) genotype 3 and 4 can cause liver disease in human and has its main reservoir in pigs. HEV investigations in pigs worldwide have been performed but there is still a lack of information on the infection dynamics in pig populations.FindingsThe HEV transmission dynamics in commercial pig farms in six different European countries was studied. The data collected show prevalence in weaners ranging from 8% to 30%. The average HEV prevalence in growers was between 20% and 44%. The fatteners prevalence ranged between 8% and 73%. Sows prevalence was similar in all countries. Boar faeces were tested for HEV only in Spain and Czech Republic, and the prevalence was 4.3% and 3.5% respectively. The collected data sets were analyzed using a recently developed model to estimate the transmission dynamics of HEV in the different countries confirming that HEV is endemic in pig farms.ConclusionsThis study has been performed using similar detection methods (real time RT-PCR) for all samples and the same model (SIR model) to analyse the data. Furthermore, it describes HEV prevalence and within-herd transmission dynamics in European Countries (EU): Czech Republic, Italy, Portugal, Spain, The Netherlands and United Kingdom, confirming that HEV is circulating in pig farms from weaners to fatteners and that the reproductive number mathematical defined as R0 is in the same range for all countries studied.

Detection of hepatitis E virus in pork liver sausages

Hepatitis E infection is regarded as an emerging public-health concern. The disease is normally self-limiting (mortality rate 1%), but chronic infections have recently been observed in transplanted patients. The etiological agent HEV is a small RNA virus infecting both humans and animals. In humans, the disease may be food-borne and pig is a main reservoir for zoonotic strains. In the present study, we evaluated the presence of HEV and swine fecal cross-contamination in pork liver sausages sold at a grocery store in Italy. HEV genome detection was performed by RT-qPCR, using harmonized protocols that included a process control (murine norovirus) and an internal amplification control. Swine fecal cross-contamination was assessed by determination of the ubiquitous porcine adenovirus. Overall, HEV genome belonging to genotype 3 was detected in both raw (10 out of 45 slices, 250 mg each, 22.2%) and dry (1 of 23 slices, 4.3%) liver sausages, but infectivity of the virus was not demonstrated. This pilot study fosters more investigations on HEV presence in pork-derived food, to assess the possible risk for the consumers.

Quantitative risk assessment of verocytotoxin-producing Escherichia coli O157 and Campylobacter jejuni related to consumption of raw milk in a province in Northern Italy.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

Porcine Circovirus 2 Replication in Colostrum-deprived Piglets Following Experimental Infection and Immune Stimulation Using A Modified Live Vaccine against Porcine Respiratory and Reproductive Syndrome Virus

Porcine circovirus type 2 (PCV2) infection is now recognized as the major factor in the development of post‐weaning multisystemic wasting syndrome (PMWS). Although Koch’s postulates have been fulfilled for PCV2 and PMWS, the severe clinical expression of the disease observed in field cases has been difficult to reproduce experimentally. Some studies have demonstrated that immune stimulation associated with the use of some commercially available swine vaccines may trigger progression of PCV2 infection to disease and lesions characteristic of PMWS. Here we describe the effects on PCV2 infection in an experimental model following the use of a commercially available modified live vaccine to porcine respiratory and reproductive syndrome virus (PRRSV). Although none of the piglets infected with PCV2 developed clinical PMWS, the severity of microscopical lesions and the PCV2 antigen load associated with these lesions were higher in the PRRSV‐vaccinated piglets compared with those detected in the PCV2 only infected animals.

Detection of Hepatitis E Virus (HEV) in Italian pigs displaying different pathological lesions

In this study we investigated the HEV prevalence in Italian pigs displaying different pathological lesions, possible risk factors related to the infection, and the possible relations occurring between HEV and other concomitant pig pathogens. Genetic characterization of some of the identified strains was also performed. Detection of HEV RNA was accomplished using a nested reverse-transcription polymerase chain reaction on bile samples from 137 pigs of 2-4months of age submitted for diagnostic purposes. Forty-one of the 137 examined pigs (29.9%) tested positive for HEV RNA. Animals of 80-120days of age showed a higher prevalence of HEV infection (46.9% against 20% of younger animals). No statistically significant correlations between HEV positivity and the presence of other pathological conditions detected at necropsy, or concomitant coinfections with PCV2 and/or PRRSV were detected. All identified strains belonged to genotype 3, and were similar to other HEV subtypes 3e, 3f, 3c circulating in Europe.

Presence of Hepatitis E Virus in a RED Deer (Cervus elaphus) Population in Central Italy.

&NA; Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free‐living red deer (Cervus elaphus) population in central Italy by detecting virus‐specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti‐HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.

Detection of hepatitis E virus in Italian pig herds

HEPATITIS E is a public health concern in many developing countries, where it is primarily transmitted by the faecal-oral route through contaminated water and food (Emerson and Purcell 2003). The disease is caused by a small, non-enveloped single-stranded RNA virus classified as the separate genus Hepevirus. Although hepatitis E disease occurs only sporadically in countries with good health care systems, the seroprevalence in healthy individuals can be high (Emerson and Purcell 2003). Porcine hepatitis E virus (HEV) is not pathogenic to general pig populations, but there is evidence that the virus may be a zoonotic agent and that animal reservoirs may exist. Experimental interspecies transmission of HEV between non-human primates and pigs has been demonstrated (Meng and others 1998), and seroepidemiological studies have shown that pig handlers are at higher risk of HEV infection than the general population (Meng and others 2002). In Japan, studies have supported the possibility of zoonotic transmission, as consumption of undercooked pig organs or meat and, in one case, of deer meat, was closely linked to cases of hepatitis E in human beings (Tei and others 2003, Yazaki and others 2003). The first porcine strain of HEV was characterised in the USA in 1997 (Meng and others 1997). Since then, several other porcine strains have been described worldwide. In the past few years, sporadic cases of autochthonous hepatitis E in human beings have been reported in several European countries, including Italy (Zanetti and others 1999). In many of these cases, the infecting HEV strain showed a high degree of homology with porcine strains of HEV detected in the same country (van der Poel and others 2001, Clemente-Casares and others 2003, Banks and others 2004a). In recent years in Europe, HEV in pig herds has been reported only in Spain (Clemente-Casares and others 2003), the UK (Banks and others 2004b) and the Netherlands (van der Poel and others 2001). This short communication describes the detection, by a nested reverse transcriptase-PCR (nested RT-PCR), of HEV in two Italian pig farms, and the phylogenetic analysis of the viral strains. Thirty-four faecal and 22 serum samples were collected from five different farrow-to-finish farms located in northcentral Italy. Samples were collected from healthy pigs between two and five months of age. Faecal samples represented pools of faeces from animals of the same age group. Total RNA was extracted from 140 μl of faecal suspension or serum using a QIAmp viral RNA kit (Qiagen), according to the manufacturer’s instructions. Template cDNA was reverse transcribed using random hexamers, according to standard protocols. A 145 base pair (bp) fragment of the open reading frame 2 of HEV was amplified from the prepared cDNA by nested PCR, as described by Erker and others (1999). A faecal suspension from a human patient with hepatitis E was used as a positive control. Nested RT-PCR products were visualised on 2 per cent agarose gel, and bands of the correct size were excised and purified with the QIAquick Gel Extraction Kit (Qiagen). Nucleotide sequencing was performed using the ABI PRISM BigDye Terminator kit, version 2·0 (Applied Biosystems). Sequences were assembled with SEQMAN (DNASTAR), and alignment was performed using the ClustalX algorithm. The HEV genome was detected in two faecal pools (5·9 per cent) collected at two different farms, but all the serum samples were negative. The positive faecal pools were obtained from groups of pigs aged 4·5 and 2·5 months, respectively. Phylogenetic analysis of the two viral sequences (113 bp), termed HEVBO/01 and HEVPI/01, was performed by the neighbour-joining method using PHYLIP 3·6. Bootstrap confidence values (500 replicates) were calculated by using the Seqboot and Consense programs. A phylogenetic tree (Fig 1) was created with the Treeview software using an avian HEV isolate as out group. Phylogenetic analysis of the viral sequences showed that the two Italian strains, HEVBO/01 and HEVPI/01, belonged to genotype 3, as did other porcine and human HEV strains indigenous to Europe. However, they differed significantly from each other, being only 84 per cent identical (18 nucleotide changes). The two Italian strains clustered with strains from countries where HEV is considered non-endemic. In particular, HEVPI/01 was related (with 90 per cent identity) to a human strain (AY540113) detected in a sporadic case of acute autochthonous hepatitis E in Spain (Buti and others 2004). In conclusion, this report represents the first description of HEV in Italian pig herds, and confirms the presence of the virus in apparently healthy pigs. These findings are important, because of the potential risk of transmission of porcine HEV to human beings, either by contact with infected pigs or by ingestion of contaminated undercooked meat. However, further studies are needed to address the true zoonotic potential of HEV in pigs. Studies are in progress to evaluate the prevalence of the infection in Italian pigs. The study of a high number of viral strains will be necessary to assess intraspecies and interspecies HEV homologies and to understand whether zoonotic transmission of HEV may occur in Italy.

Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein

BackgroundThe hepatitis E virus (HEV) has been detected in both humans and animals, particularly pigs, worldwide. Several evidences, including human infection following consumption of raw contaminated meat, suggest a zoonotic transmission of HEV. In Italy, large circulation of genotype 3 HEV has been reported in swine, and recent studies have confirmed the involvement of this genotype in autochthonous human cases.ResultIn this study 111 sera collected from healthy pigs in two Italian regions were tested for anti-HEV IgG antibodies. For specific HEV antibody detection in swine, we developed ELISA and Western blotting methods, using a truncated capsid (ORF2) protein lacking the first 111 amino acids of a swine HEV genotype 3 strain. The ORF2-based ELISA revealed anti-HEV antibodies in 104 out of 111 pigs compared with 102 detected with a commercial ELISA kit. A lower number of sera reacted with the recombinant ORF2 protein in a Western blotting format (81/111). Using a Latent class analysis (LCA), the estimated sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 and 0.936, respectively, whereas specificities were 0.599 and 0.475. The estimated sensitivity of Western blotting was 0.775, and the specificity was 0.944.ConclusionsThe overall results confirm the high prevalence of HEV seropositive healthy pigs in Italy. Through comparisons with a commercial ELISA test, the swine genotype 3 HEV antigen produced in this study was proven suitable to detect anti-HEV antibodies in pig sera by both ELISA and Western Blotting.