First Serological Study of Hepatitis E Virus Infection in Backyard Pigs from Serbia
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- Lupulovic, D., Lazic, S., Prodanov-Radulovic, J. et al. Food Environ Virol (2010) 2: 110. doi:10.1007/s12560-010-9033-6
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Hepatitis E virus (HEV) genotype 3 infections in pigs and humans have been lately reported in Europe. In the present study, the prevalence of anti-HEV IgG antibodies in swine was investigated, for the first time, in Serbia by means of an ELISA based on a recombinant open reading frame 2 protein of HEV genotype 3. A total of 315 serum samples from 3 to 4 months-old healthy backyard pigs, collected in 63 herds from 28 towns and villages of 4 different districts of the Vojvodina province, in the northern part of Serbia, were tested. A 34.6% (109/315) of the sera tested were positive. The prevalence of anti-HEV antibodies varied widely between municipalities (range 16.7–75.0%) and herds (range 0–100%). These data indicate that HEV infection is widespread in Serbian backyard pigs.
Hepatitis E virus (HEV) is mostly transmitted by contaminated water and causes serious epidemics in countries with poor sanitary conditions. HEV (family Hepeviridae, genus Hepavirus) is a non-enveloped virus, whose genome is a single-stranded RNA molecule of positive polarity of approximately 7.2 Kb in length, containing three overlapping open reading frames, ORF. ORF-2, which encodes the viral capsid protein and harbors immunoreactive epitopes, is the most widely used antigen for detection of specific antibodies against HEV (Pintó and Saiz 2007).
First isolation of a HEV swine strain was reported in 1997 (Meng et al. 1997). Infected pigs shed virus through feces, but infection is sub-clinical and asymptomatic and viremia is short, which makes difficult viral detection in blood, since, until now, there is no HEV susceptible cell culture system available. RT-PCR amplification of HEV-RNA has shown that HEV is present in pigs from commercial farms around the world (Choi et al. 2003; Di Bartolo et al. 2008; Reuter et al. 2009; Geng et al. 2009; Kaba et al. 2009; Vasickova et al. 2009). This HEV ubiquity in pigs, together with the close genetic relationship of human and swine strains of the same genotype have raised concerns about a possible risk of zoonotic transmission (Pintó and Saiz 2007). Food-borne transmission of HEV was confirmed in people that ate dear, pork, or wild boar uncooked meet and liver (Yazaki et al. 2003; Matsuda et al. 2003), and HEV-RNA has been detected in livers sold in local markets (Bouwknegt et al. 2007; Yazaki et al. 2003).
In Europe, a non endemic area, an increase in the number of autochthonous HEV infection by genotype 3 (gt3) strains have been lately reported in humans (Herremans et al. 2007; Reuter et al. 2009). Several studies have suggested that pig’s handlers, veterinarians, and other workers in close contact with animals present a higher risk of infection (Galiana et al. 2008; Meng et al. 2002), stressing the possible risk of a zoonotic transmission.
A high prevalence of anti-HEV IgG in swine populations around the world has also been reported (Casas et al. 2009; Geng et al. 2009; Kaba et al. 2009; Meng et al. 1997; Reuter et al. 2009). However, most of these studies have been conducted using HEV ELISA that are based on genotype 1 or 2 peptides and are designed for humans, but not for swine (Pintó and Saiz 2007). Actually, these assays sometimes failed to detect specific antibodies in sera from individuals with proven HEV gt3 infections (Herremans et al. 2007; Jiménez de Oya et al. 2009b).
The only study on the presence of HEV in Serbia was conducted in 2008 (Petrovic et al. 2008) and showed that 30 and 45% of the tested stool and tissue samples from commercial farms pigs were positive by RT–PCR, while all samples from backyard pigs and wild boar resulted negative. Here, we have expanded this study to further analyze, for the first time in Serbia, the prevalence of anti-HEV IgG in swine.
Detection of anti-HEV IgG antibodies was performed by means of an ELISA based on the use of recombinant HEV gt 3 ORF-2 expressed in Trichoplusia ni larvae as antigen that has been shown to be highly specific and sensitive when compared with commercial kits (Jiménez de Oya et al. 2009a, b). Briefly, 50 μl/well of antigen (15 ng/well), diluted in 50 mM carbonate/bicarbonate buffer (pH 9.6), was added to each well of microtiter plates (Polysorp., Nunc, Denmark) and incubated overnight at 4°C. All following incubations were carried out for 1 h at 37°C under constant shaking, and plates were washed three times between incubations with PBST (0.5% Tween 20 in PBS). After blocking the plates with 100 μl/well of blocking solution (PBST-3% skim milk), 50 μl/well of sera, diluted 1:10 in blocking solution, was added in duplicate. Next, 50 μl of HRP-conjugated goat anti-swine secondary antibody (MorphoSys, Martinsried-Plangg, Germany) was added to each well. Finally, 50 μl/well of the substrate solution (O-phenylenediamine 30% H2O2) was added and incubated in the dark at room temperature for 10 min before the reaction was stopped by addition of 50 μl/well of stop solution (3 N H2SO4). Optical density of each sample was read at 495 nm in an ELISA microplate reader (Tecan, Vienna, Austria). As controls, previously characterized positive and negative swine sera pools (Jiménez de Oya et al. 2009a) were added to each plate. The cut off of the assay was established as 2.5 times the A495 value of the negative sera pool (0.15, range 0.08–0.14) that was included in each plate. Test samples with optical density equal to or greater than cut off value were considered as positive.
Distribution of anti-HEV antibody in backyard pigs sera from Vojvodina province (Serbia)
Number of towns/villages
Number of tested herds
Number of positive herds (%)
No of tested pigs
No of positive animals (%)
All municipalities tested here had, at least, one positive herd, but seroprevalence was quite variable between municipalities, ranging the number of positive animals from 16.7 to 75% (Table 1). Still, there were no sufficient data regarding the number of tested pigs, to confirm connection between the prevalence of HEV antibodies and geographic location of examined backyard pigs. Similar data (Geng et al. 2009; Yoo et al. 2001) describing a quite variable seroprevalence between relatively close regions have been reported in different provinces of Canada (38.3–88.8%), and China (11–100%).
Prevalence of anti-HEV IgG positive pigs in examined backyard herds
Prevalence of anti-HEV IgG positive pigs in backyard herds (%)
No of examined backyard herds (%)
No of animals in backyard herds
In conclusion, these data support our previous results (Petrovic et al. 2008) reporting a high prevalence of HEV infection among healthy pigs in Serbia, over one-third of the pigs tested were anti-HEV IgG positive. These figures are similar to that found in commercial large-scale farms across Europe, even though the animals analyzed here were raised in family low-scale backyard farms that were disseminated far away one from each other, thus dismissing the risk of contact between animals from different farms; however, backyard pigs are quite often in close contact with the environment, including other wild life species and water courses, which might serve as a source for viral transmission. Since HEV seems to represent a zoonotic risk of transmission and might also be a threat for food safety and public health, further investigation of HEV infection in human and wild life animal species is required to have a better picture of the actual HEV incidence in Serbia.
The study was supported in part by grant (CSD2006-0007) from the Spanish Ministerio de Ciencia e Innovación (MICINN) to JCS and grant (TR 20115) from Serbian Ministry of Science and Technological Development to SL. NJO has been supported by a scholarship from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Two COST-929 short term missions supported training visits of DL and NJO.