Nicolas Rose

Thermal Inactivation of Infectious Hepatitis E Virus in Experimentally Contaminated Food.

ABSTRACT Hepatitis E virus (HEV) infection of zoonotic origin is an emerging concern in industrialized countries. In the past few years, several cases of zoonotic hepatitis E have been identified and the consumption of food products derived from pork liver have been associated with clusters of human cases. More specifically, raw or undercooked pork products have been incriminated. Few data on the effect of heating on HEV inactivation in food products are available. In the present study, the various times and temperatures that are used during industrial processing of pork products were applied to experimentally contaminated food preparations. After treatment, the presence of residual infectious virus particles was investigated using real-time reverse transcription-PCR and an in vivo experimental model in pigs. Results show that heating the food to an internal temperature of 71°C for 20 min is necessary to completely inactivate HEV. These results are very important for determining processing methods to ensure food safety in regard to food-borne hepatitis E.

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome

Abstract Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18–19 weeks) in all eight herds. The seroprevalence in piglets (11–17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Close Similarity between Sequences of Hepatitis E Virus Recovered from Humans and Swine, France, 2008−2009

Frequent zoonotic transmission of hepatitis E virus (HEV) has been suspected, but data supporting the animal origin of autochthonous cases are still sparse. We assessed the genetic identity of HEV strains found in humans and swine during an 18-month period in France. HEV sequences identified in patients with autochthonous hepatitis E infection (n = 106) were compared with sequences amplified from swine livers collected in slaughterhouses (n = 43). Phylogenetic analysis showed the same proportions of subtypes 3f (73.8%), 3c (13.4%), and 3e (4.7%) in human and swine populations. Furthermore, similarity of >99% was found between HEV sequences of human and swine origins. These results indicate that consumption of some pork products, such as raw liver, is a major source of exposure for autochthonous HEV infection.

High prevalence of Hepatitis E virus in French domestic pigs

The importance of the domestic pig reservoir for Hepatitis E virus (HEV) was assessed by estimating the seroprevalence and prevalence of HEV contaminated livers in French slaughter-aged pigs. 6565 sera and 3715 livers were randomly sampled from 186 pig farms throughout the country. Taking the sampling design into account, the farm-level seroprevalence was 65% (95% CI 57-74) and 31% (95% CI 24-38) of the slaughter-aged pigs had antibodies against HEV. The individual prevalence of HEV RNA positive livers was 4% (95% CI 2-6) and 24% (95% CI 17-31) of the farms had at least 1 positive liver. Most isolates were of genotype 3f (76.7%) with smaller amounts of 3c (18.6%) and 3e (4.6%). The high prevalence of HEV in pigs and the similarities between HEV subtypes from pigs and humans corroborates the possible zoonotic origin of some HEV autochthonous infections.

Epidemiology and transmission of porcine circovirus type 2 (PCV2).

PCV2 has been highly prevalent in the pig population for decades, prior to the emergence of associated clinical disease manifestations that severely affected the pig production worldwide in the late 90s. PCV2 can be further subdivided into several genotypes. From descriptive epidemiologic data, there is evidence of a global shift of the main PCV2 genotypes in different countries from PCV2a to PCV2b, which is generally associated with more severe disease. In addition, from analytic epidemiologic studies, the modified within-herd PCV2 dynamics of infection is strongly related to the increased incidence of clinical disorders associated with PCV2 infection. Because PCV2 is shed for a long time by an extremely large variety of routes, it easily spreads within the population both through horizontal and vertical transmission. Even if airborne transmission cannot be formally excluded, direct contact is certainly the most efficient infectious route due to the simultaneous exposure of susceptible pigs to contaminated respiratory, digestive, and urinary secretions since the probability of transmission is strongly limited by the distance between infectious and susceptible animals. Consequently, farm to farm transmission is restricted to the introduction of infected animals or infected animal products such as semen. More information would be required to assess the risk of other vehicles such as vaccines or feed ingredients since the probability of these products to be contaminated by PCV2 is unknown. However, owing to its transmission characteristics, PCV2 is able to be maintained within pig farms for years without any further need for re-introduction due to the population dynamics of modern pig operations, which continually renew the pool of the susceptible population through replacements and pig movements between compartments.

Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: A cross-sectional study

A study was carried out in 125 farrow-to-finish pig herds to assess the relationships between pathogens involved in respiratory disorders and to relate these findings to clinical signs of respiratory diseases and pneumonia and pleuritis at slaughter. Clinical examination and sampling were carried out on four different batches in each herd (pigs aged 4, 10, 16 and 22 weeks). Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, swine influenza viruses (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were detected by serological or PCR tests. Pneumonia-like gross lesions and pleuritis were scored at the slaughterhouse. The results indicate that the percentage of pigs PCR-positive for PCV2 at 4, 10 and 16 weeks old was associated with the percentage of pigs PCR-positive for M. hyopneumoniae at these ages. On the other hand, the percentage of pigs with antibodies against PRRSV at 10, 16 and 22 weeks was positively correlated with the percentage of pigs seropositive for M. hyopneumoniae at 22 weeks, with the percentage of pigs with antibodies against SIV H1N1 and SIV H1N2 and the percentage of pigs sero-positive for A. pleuropneumoniae serotype 2. The findings also indicate that, within the five studied pathogens, M. hyopneumoniae, PRRSV and SIV H1N1 are the major pathogens involved in pneumonia-like gross lesions even though PCV2 may play a role. A. pleuropneumoniae serotype 2, in association with PRRSV, is significantly associated with extensive pleuritis. Respiratory diseases could be significantly reduced by implementing measures including appropriate management practices to control these pathogens.

Complete Genome Sequence of a Porcine Epidemic Diarrhea S Gene Indel Strain Isolated in France in December 2014

ABSTRACT We report the first and only case of a porcine epidemic diarrhea (PED) outbreak occurring in December 2014 in northern France, and we show using the full-length genome sequence of the French PED virus (PEDV) isolate that it was a PEDV indel strain close to German PEDV strains recently isolated.

Estimation of the sensitivity of four sampling methods for Mycoplasma hyopneumoniae detection in live pigs using a Bayesian approach

Four sampling techniques for Mycoplasma hyopneumoniae detection, namely nasal swabbing, oral-pharyngeal brushing, tracheo-bronchial swabbing and tracheo-bronchial washing, were compared in naturally infected live pigs. In addition, a quantitative real-time PCR assay for M. hyopneumoniae quantification was validated with the same samples. 60 finishing pigs were randomly selected from a batch of contemporary pigs on a farm chronically affected by respiratory disorders. Each pig was submitted to nasal swabbing, oral-pharyngeal brushing, tracheo-bronchial swabbing and tracheo-bronchial washing. Nested-PCR and real-time PCR assays were performed on all samples. A Bayesian approach was used to analyze the nested-PCR results of the four sampling methods (i.e. positive or negative) to estimate the sensitivity and specificity of each method. M. hyopneumoniae was detected by nested-PCR in at least one sample from 70% of the pigs. The most sensitive sampling methods for detecting M. hyopneumoniae in live naturally infected pigs were tracheo-bronchial swabbing and tracheo-bronchial washing, as compared to oral-pharyngeal brushing and nasal swabbing. Swabbing the nasal cavities appeared to be the least sensitive method. Significantly higher amounts of M. hyopneumoniae DNA were found at the sites of tracheo-bronchial sampling than in the nasal cavities or at the oral-pharyngeal site (p<0.001). There was no difference between the tracheo-bronchial washing and the tracheo-bronchial swabbing results (p>0.05). Our study indicated that tracheo-bronchial swabbing associated with real-time PCR could be an accurate diagnostic tool for assessing infection dynamics in pig herds.

High Proportion of Asymptomatic Infections in an Outbreak of Hepatitis E Associated With a Spit-Roasted Piglet, France, 2013

BACKGROUND On 11 December 2013, 3 clustered cases of hepatitis E were reported on a French coastal island. Individuals had taken part in a wedding meal that included a spit-roasted piglet. The piglet had been stuffed with a raw stuffing partly made from the liver. Investigations were carried out to identify the vehicle of contamination and evaluate the dispersion of the hepatitis E virus (HEV) in the environment. METHODS A questionnaire was administered to 98 wedding participants who were asked to give a blood sample. Cases were identified by reverse transcription-polymerase chain reaction and serological tests. A retrospective cohort study was conducted among 38 blood-sampled participants after the exclusion of 14 participants with evidence of past HEV infection. Relative risks (RR) and 95% confidence intervals were calculated based on food consumed at the wedding meal using univariate and multivariable Poisson regressions. Phylogenetic analyses were performed to compare the clinical HEV strains. Strains were detected in the liquid manure sampled at the farm where the piglet was born and in the untreated island wastewater. RESULTS Seventeen cases were identified, 70.6% were asymptomatic. Acute HEV infection was independently associated with piglet stuffing consumption (RR = 1.69 [1.04-2.73], P = .03). Of clinical strains from the index cases, veterinary and environmental HEV strains were identical. CONCLUSIONS Our investigation attributed this large HEV outbreak to the consumption of an undercooked pig liver-based stuffing. After infection, the cases became a temporary reservoir for HEV, which was detected in the islands untreated wastewater.

Influenza A virus infection dynamics in swine farms in Belgium, France, Italy and Spain, 2006-2008.

Avian-like H1N1 and reassortant H3N2 and H1N2 influenza A viruses with a human-like haemagglutinin have been co-circulating in swine in Europe for more than a decade. We aimed to examine the infection dynamics of the three swine influenza virus (SIV) lineages at the farm level, and to identify possible regional and seasonal variations in their circulation. Sera were collected from six successive generations of fattening pigs (2006-2008) in a total 80 farrow-to-finish herds in Belgium, Italy, France and Spain and examined for antibodies against the three SIVs in haemagglutination inhibition tests. Overall, in all regions and periods, 9.7% of all farms were negative for SIV, 49% were infected with one subtype, 38% with two subtypes and 3.9% with all three SIVs. We found serological evidence for the circulation of all three subtypes in Belgium, Italy and Spain, while only infections with H1N1 and H1N2 SIVs were detected in France. Despite temporary changes in the circulation of H1N2 in Belgium and in Spain, there was no true seasonal variation. The exact combination of subtypes on the same farm differed in each of the sampling periods. On the other hand, 21 farms were found to be consistently infected with the same SIV subtype throughout the study. This can either be explained by the persistence of the virus in a farm, or by the periodical re-introduction of SIVs of the same subtype.