Prevalence of Anti-Hepatitis E Virus Antibodies and First Detection of Hepatitis E Virus in Wild Boar in Slovenia

Introduction

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and a public health problem. In most cases, the infection is self-limiting; however, in severe cases, the infection can lead to hepatic failure (Crossan et al. 2014). HEV is a small, nonenveloped virus and the only member of the genus Orthohepevirus in the Hepeviridae family (Smith et al. 2014). The genome is a single-stranded, positive-sense RNA molecule consisting of 5′- and 3′-noncoding regions and three open reading frames (Meng 2010). On the basis of genetic variability, genotypes 3 and 4 can infect humans and animals, whereas genotypes 1 and 2 are restricted to humans. HEV is primarily transmitted by the fecal–oral route through contaminated water and food; however, pigs can also play a role as possible source of direct and food borne transmission to humans (Purcell and Emerson 2008, Kim et al. 2011, Sato et al. 2011).

Various animal species, including wild animals, were proven to have serum antibodies to HEV (Tei et al. 2003). HEV RNA and antibodies have been detected in wild boar (Martinelli et al. 2015), sika deer (Tei et al. 2003), and wild mongooses (Nakamura et al. 2006). Recently, a German study determined that the main risk factor for acquiring HEV infection is the consumption of offal and meat from wild boar (Wichmann et al. 2008). Several European countries reported considerably high anti-HEV seroprevalence (40–80%) and HEV RNA detection (20–50%) in farmed pigs and wild boar (de Deus et al. 2008, Adlhoch et al. 2009, Reuter et al. 2009). It has been suggested that wild boar serve as a reservoir for HEV, because strains closely related to human HEV have been detected in this species (Michitaka et al. 2007). In Slovenia, the wild boar population is increasing continuously, and, although swine HEV strains have been detected in domestic pigs in the country, the information on the prevalence of HEV in wild boar is rather scarce.

The objective of this study was to determine the distribution and prevalence of HEV antibodies and HEV RNA in wild boar populations in total territory of Slovenia and to elucidate whether the wild boar may act as a potential reservoir of HEV infections in the region.

Materials and Methods

A total number of 288 wild boar serum samples were collected during 2009–2011: 2009 (n=95), 2010 (n=190), and 2011 (n=3). Animals of both genders were shot as part of the regular hunting bag throughout Slovenia. No ethical/welfare authority approval was required because all samples were collected at postmortem. Wild boar ages ranged between 5 and 60 months, and the animals were selected intentionally as they represented the category usually intended for human consumption. Hunters collected samples from the jugular veins or from the animals' hearts. Prior to the sampling, hunters were instructed regarding the procedures and equipped with field sampling kits. Afterwards, blood samples were transported to the Veterinary Faculty University of Ljubljana, centrifuged for 10 min at 1200×g, and stored at −20°C until testing. Serological and molecular testing was performed at the Central Veterinary Institute of Wageningen Univeristy and Research Centre, Lelystad, The Netherlands.

The final dilutions of serum samples, 1:10 in phosphate-buffered saline (PBS), were mixed and centrifuged for 10 min at 1000 × g. Fifty microliters of supernatant was used for RNA extraction using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany), according to manufacturer instructions. RNA was used immediately in real-time polymerase chain reaction (qPCR) or stored at −70°C until testing. HEV detection by qPCR was performed as described by Jothikumar et al. (2006). Enzyme-linked immunosorbent assay (ELISA) for anti-HEV genotype 3 detection was performed according to Van der Poel et al. (2014).

Results and Discussion

HEV has been described in domestic pigs (Steyer et al. 2011, Raspor Lainšček et al. 2013) and humans (Steyer et al. 2011) in Slovenia. Steyer et al. (2011) reported 20.3% HEV prevalence in stool samples collected from suckling, weanling, and fattening pigs, whereas Raspor Lainšček et al. (2013) reported 19% HEV prevalence in stool of 3-month-old pigs. According to Steyer et al. (2011), 10 serum samples from human patients with confirmed hepatitis E were also tested and three samples turned out to be HEV positive.

Because wild boar might play a role in the HEV epidemiological cycle, we investigated whether the virus is present in the Slovenian wild boar population. From the 288 wild boar serum samples, 87 (30.21%) tested positive for anti-HEV antibodies. One out of 288 (0.35%) serum samples was positive for HEV RNA. Whereas HEV antibodies were frequently detected, i.e., more than 30% of the samples, HEV RNA was detected in just one sample, indicating that in most cases the virus was not circulating in blood. It is possible that the majority of animals were not in the acute phase of infection. The short time viremia of 11 (8–13) days could also explain the low HEV detection rate (Bouwknegt et al. 2009). HEV antibody was detected in all age groups and in all geographical regions.

Similar to the present study, Martinelli et al. (2015) described high seroprevalence and low HEV RNA detection in liver and serum samples from wild boar in Italy. In fact, in their study, despite a 10.2% seroprevalence rate, no HEV RNA was detected. Using the same qPCR method as in the present study, Schielke et al. detected 14.9% of liver samples positive to HEV (Schielke et al. 2009). A similar (29.9%) prevalence of antibody to HEV has been found in Germany (Schielke et al. 2009), whereas Burri et al. (2014) recently reported considerably lower (12.5%) seroprevalence in wild boar in Switzerland. Data on HEV in wild boar in bordering Croatia are scarce. To date, only one infection with HEV was recorded in domestic pig and wild boar in Croatia (Jemeršić et al. 2010). This substantial difference in seroprevalence observed among different geographical areas (Table 1) may be related to different infectivity between HEV strains or to differences in the biology and ecology of the wild boar populations considered (Burri et al. 2014). Even considering that HEV prevalence rate is higher in liver (38.1%) than in serum (15.7%) samples (Adlhoch et al. 2009), the present data compared to data in literature suggest HEV circulation is higher in Germany than in the Slovenian wild boar population. A low prevalence of HEV infection reported in the present study may indicate a lower circulation of HEV in the wild boar population investigated, but differences due to the type of specimens selected and the PCR methods chosen cannot be excluded.

Studies showed occupational activity as a risk factor for hepatitis E (Carpentier et al. 2012). The vast majority of wild boar investigated in this present study were most likely not shedding the virus in feces and were not a source of infection for HEV via the fecal–oral route; however, considering that the viral load in liver and gallbladder are expected to be higher, forestry workers and hunters, which come into contact with the organs such as liver and gallbladder, are under higher risk of infection. Autochthonous cases of HEV are increasing in Europe, and the epidemiology of the disease is not completely understood yet. In Slovenia, it was already described that the virus is circulating in pig populations (Steyer et al. 2011, Raspor Lainšček et al. 2013). In the epidemiology of HEV in the domestic pig population, husbandry conditions could explain the higher seroprevalence observed in the wild animals (Burri et al. 2014).

In the present study, we have shown that wild boar are likely to be part of the HEV epidemiological cycle in Slovenia. Whether wild boars are accidental hosts or an essential part of the cycle still needs to be determined.

Conclusions

In this study, we have described for the first time the presence of HEV RNA and the seroprevalence of HEV in wild boar in Slovenia, suggesting these animals are likely to be part of the HEV epidemiological cycle in Slovenia. To assess the public health risk of HEV in Slovenia, further studies will have to be done in the human population.