Detection of hepatitis E virus RNA in raw sausages and liver sausages from retail in Germany using an optimized method

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Highlights

  • Methods for detection of HEV-RNA in meat products were compared.

  • The optimized method sensitively detects HEV-RNA in liver and raw sausages.

  • A broad distribution of HEV-RNA in pig sausages from Germany was shown.

  • The detected sequences belonged to different subtypes of the zoonotic HEV genotype. 3

Abstract

Hepatitis E virus (HEV) is a pathogen of increasing importance, which can be zoonotically transmitted from domestic pigs, wild boar, and deer to humans. Foodborne transmission by consumption of raw and undercooked liver, meat, or sausages prepared from infected animals has been documented. The aim of this study was to investigate the distribution of HEV in different types of sausages sold in Germany. As no standardized methods for HEV detection in food exist, several techniques of sample homogenization, virus concentration and nucleic acid extraction followed by real-time RT-PCR were compared using artificially contaminated sausages. A method using TRI Reagent® Solution showed the best efficacy of matrix disruption and a treatment with chloroform followed by a silica-based RNA extraction method resulted in the highest HEV detection rates. The detection limit of the method was 2.9 × 103 and 5.3 × 104 genome equivalents per 5 g raw sausage and 2 g liver sausage, respectively. Application of the method to raw and liver sausages from retail in Germany resulted in the HEV genome detection in 14 out of 70 (20%) raw sausages and in 11 out of 50 (22%) liver sausages. The detected HEV sequences showed a high diversity and belonged to different subtypes of HEV genotype 3. The results indicate a broad distribution of HEV-RNA in meat products sold in Germany; however, the infectivity of the detected virus remains to be assessed in future.

Introduction

Hepatitis E virus (HEV) is responsible for large epidemics of acute viral hepatitis in several developing countries in Asia and Africa (Johne et al., 2014a). Outbreaks are linked to fecal–oral transmission mainly associated with the consumption of contaminated water (Aggarwal, 2011). In industrialized countries, hepatitis E may be related to travel to endemic countries; however, sporadic and autochthonous cases of hepatitis E are increasingly reported in many countries in Europe and North America (Dalton et al., 2008, Meng, 2011). Hepatitis E is characterized as a self-limiting acute hepatitis with case fatality rates between 1% and 5% (Pavio et al., 2010). However, the case fatality rates can reach up to 20% in pregnant women and immunocompromised individuals may develop a chronic HEV infection (Péron et al., 2011, Purcell and Emerson, 2008).

HEV is a small, non-enveloped, RNA-virus classified in the family Hepeviridae, which contains the four human pathogenic HEV genotypes 1–4 along with other HEV-related viruses (Johne et al., 2014b). Genotypes 1 and 2 are restricted only to humans, whereas genotypes 3 and 4 are zoonotic and may infect both humans and several animal species (Emerson et al., 2004, Johne et al., 2014a). Especially domestic pigs and wild boars have been identified as reservoir animals for HEV, showing high infection rates in Europe (Ruggeri et al., 2013). However, these animals do not show signs of clinical disease or obvious pathological changes after HEV infection (Halbur et al., 2001).

The presence of HEV in domestic pigs and wild boars increased the public health concern for zoonotic infections through direct contacts with infected animals or the consumption of contaminated animal meat (Deest et al., 2007, Meng, 2010, Pavio et al., 2010). Genomic virus sequences recovered from commercial pig livers are closely related, or even identical to those detected in human hepatitis E patients (Wenzel et al., 2011, Yazaki et al., 2003). Food-borne transmission of HEV via consumption of raw and undercooked liver, meat, or sausages from domestic pigs, wild boar, and deer has been documented in several studies (Bouquet et al., 2011, Colson et al., 2010, Masuda et al., 2005, Matsubayashi et al., 2008, Matsuda et al., 2003, Tei et al., 2003, Yazaki et al., 2003). The presence of HEV in pork has been confirmed in pig liver or pork at retail in the USA (Feagins et al., 2007), the Netherlands (Bouwknegt et al., 2007), UK (Berto et al., 2012), Italy, Spain, the Czech Republic (Di Bartolo et al., 2012) and Germany (Wenzel et al., 2011).

Only few studies have been published on the detection of HEV in pork products, e.g. sausages, so far (Berto et al., 2013a, Bouwknegt et al., 2007, Colson et al., 2010, Di Bartolo et al., 2012, Martin-Latil et al., 2014). The HEV detection rates reported in these surveys are variable and depend on the investigated sample types and the applied detection methods. However, no standardized protocols for the detection of HEV in meat or meat products are available at present and most of the mentioned studies did not assess the efficiency and sensitivity of the applied detection method.

The aim of this study was to investigate the distribution of HEV in meat products in Germany. Several techniques of sample homogenization and virus extraction followed by real-time RT-PCR were compared using artificially contaminated sausages. In contrast to many other foodborne viruses, HEV contamination of meat products is not only restricted to the food surface (Bouwknegt et al., 2009). If the food is prepared from an HEV-infected animal, an internal localization of the virus within the meat product has to be expected. Therefore, different strategies of mechanical sample homogenization as well as different solutions were tested for their ability to disrupt the cells by analysis of released pig DNA. The selected method was then applied to investigate the distribution of HEV in different types of sausages randomly purchased at retail level in Germany.

Section snippets

Preparation of HEV used for contamination trials

A liver sample of a wild boar infected with HEV genotype 3i isolate wbGER27 (Schielke et al., 2009) was used for the contamination trials. Approximately 3 g of the liver tissue was suspended in 10 ml sterile phosphate buffered saline (PBS, pH 7.2, PAN™ Biotech GmbH, Aidenbach, Germany) and the sample was subsequently homogenized using a mechanical disruptor (FastPrep®24, MP Biomedicals, Santa Ana, California, USA) with sterile zirconia beads (1.0 mm, BioSpec Products, Bartlesville, USA). The

Virus recovery rates from raw sausage matrix using different published protocols

In the first set of experiments, the bacteriophage MS2 was used as a surrogate virus and added to the sample which was then processed according to the respective protocol. The resulting recovery rates ranged between 0.04% and 1.92% and are shown in Table 1. Only the method where the sample was homogenized in TRI Reagent® Solution without further precipitation steps resulted in a MS2 recovery rate > 1% and was chosen for further validation. In the second set of experiments, portions of raw

Discussion

Transmission of HEV through consumption of pork and wildboar products has been described repeatedly (Colson et al., 2010, Deest et al., 2007, Matsubayashi et al., 2008, Tamada et al., 2004, Yazaki et al., 2003). However, sensitive and reliable methods for the detection of HEV in meat products such as pig sausages are rare and mostly not well characterized. The meat products chosen in our study are of risk to serve as a transmission vehicle for HEV. Raw sausages like salami are usually eaten

Acknowledgments

We greatly appreciate Dr. Anja Carl for helpful discussions during the project. This study was supported in part by a grant of the German Federal Ministry of Education and Research (BMBF) executed within the framework of the project ZooGloW (FKZ 13 N12697), and by a contract-research-project for the Bundeswehr Medical Service.

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