Serological Searching for Hepatitis E Virus Infection Among Pig Liver Transudate from Bulgaria: An Alternative Method for Seroepidemiological Survey

Introduction

For the past 30 years, researchers have shown that the main reservoirs of hepatitis E virus (HEV) infection are domestic pigs and wild boars. In 1990, Balayan et al. did experimental inoculation of HEV from adult patient to four young domestic pigs (Sus scrofa domestica, large white breed) for the first time (Balayan et al. 1990). In 1995, Clayson et al. presented the first survey of HEV among domestic pigs (Clayson et al. 1995).

These authors noted 32.7% (18/55) HEV-specific antibodies among domestic swine living in the Kathmandu Valley of Nepal. In 2004, Sonoda et al. reported HEV seropositivity of 9% (3/35) among wild boars in Japan (Sonoda et al. 2004). In September 2003, Yazaki et al., on the occasion of 10 patients with acute or fulminant HEV infection, 363 samples of raw pig liver were investigated (Yazaki et al. 2003). Identity of 98.5–100% was established between three raw pig liver HEV isolates (swJL145, swJL234, and swJL325) and two human HEV isolates (HE-JA18 and HE-JA4) (Yazaki et al. 2003). These results indicate that raw pig liver may transmit HEV to humans.

Most often foodborne viruses are conventionally processed by quantitative reverse transcription-polymerase chain reaction (RT-PCR), which gives good detection results. Pig liver and liver products are considered the main risk foods for HEV infection in the developed countries and scientific researches focused on the presence of HEV in liver juice are scarce. Feces and bile samples were more likely to test HEV positive compared with liver, pork, and intestine samples (Intharasongkroh et al. 2017). Low viral titers may persist in the liver of nonviremic animals, giving thus the possibility of consumption of contaminated liver of domestic swine and wild boars diagnosed as HEV-negative in serum samples (Gardinali et al. 2012, Risalde et al. 2017).

Nevertheless, there is so far no standardized RT-PCR method for HEV detection available from pork liver and liver products (Zhao and Li 2021). In addition, there is currently no standard diagnostic RT-PCR method in the United States for detecting HEV in food (Harrison et al. 2021). In this regard, in recent years, different investigation teams have made various attempts to optimize diagnostic methods for searching for HEV infection in pig liver (Martin-Latil et al. 2014, Son et al. 2014, Szabo et al. 2015, Hennechart-Collette et al. 2019, Zhao and Li 2021).

Blood serum samples are usually obtained for epidemiological surveys. However, this has one major limitation—the virus can survive short time in blood and is more unstable in blood serum than in the liver (Rutjes et al. 2010). Therefore, the liver is preferred for the detection of HEV. In addition, the application of RT-PCR of liver samples is more expensive and technically more difficult to perform. These are serious difficulties in planning and organizing epidemiological surveys in developing countries and sometimes in developed countries. In this regard, the aim of this research was to perform a seroepidemiological survey of HEV by an alternative method—serological establishment of the virus from pig liver transudate. This is the first such research in Bulgaria. Moreover, no studies have been conducted on HEV in pig liver in our country.

Materials and Methods

All procedures and techniques involving the handling and collection of liver samples from pigs used in this research were approved by the Local Ethics Committee for Animal Experimentation on Faculty of Veterinary Medicine, Trakia University (Stara Zagora, Bulgaria; Approval from April 2022). Verbal consent was obtained from slaughterhouse owners. In relation to animal welfare we complied with the requirements of national and European legislation.

A total of 90 liver samples from healthy pigs were collected in May 2022 from slaughterhouses in three parts of the country (Sofia, Lovech, and Stara Zagora districts). An equal number of samples (n = 30) were obtained from each district. All samples were taken by authorized veterinarians at slaughterhouses after the pigs were slaughtered and were individually placed in a sample bag and transported in refrigerated containers to the Laboratory of Animal Infectious Diseases, Faculty of Veterinary Medicine, Trakia University. Liver samples were stored at −20°C for long term until the detection of antibodies directed against HEV in liver transudate. To obtain liver transudate, samples were frozen and thawed at room temperature. Ten grams of each liver sample were gently squeezed using a pair of tweezers and the resulting liquid was diluted 1:10 with dilution buffer provided by the kit (Navarro et al. 2020).

All liver transudate samples were analyzed for the presence of anti-HEV antibodies using the commercial enzyme-linked immunosorbent assay (ELISA) kit PrioCHECK HEV Ab porcine (Mikrogen GmbH, Neuried, Germany) based on open reading frame (ORF) ORF 2 and ORF 3 of genotypes 1 and 3. Diluted liver transudate samples and controls were added in separate microplate wells and incubated for 60 ± 1 min at 37°C ± 3°C. The wells were washed four times with wash fluid working solution and further ELISA procedure was carried out following manufacturer's recommendations. The plate was read at 450 nm with a microplate reader (Rayto RT-2100C). The cutoff was calculated as mean optical density (OD₄₅₀) of the cutoff control multiplied with 1.2. The results were interpreted according to the instruction defined by the manufacturer. The test has 91.0% sensitivity and 94.1% specificity.

We applied chi-squared test for comparing anti-HEV IgG positivity between different districts. Binary logistic regression was used for evaluation of the risk of HEV positive results according to parts of the country. Statistical analysis has been performed by SPSS Statistics 20.0 (IBM Corporation, Armonk, NY) and Excel 2007 (Microsoft, Redmond, WA). A p value <0.05 was considered statistically significant.

Results and Discussion

The overall HEV seropositivity was 67.7% (mean ± standard deviation: 67.76 ± 33.80) of all 90 tested pig liver transudate samples (Table 1). The highest seroprevalence was found in Stara Zagora district (28/30; 93.3%), and the lowest seroprevalence in Sofia district (6/30; 20.0%). Based on the different districts included in the research, the chi-squared test showed differences in HEV seropositivity between the districts. Two percent of all pig liver transudate samples was assessed as doubtful and consequently these samples were additional testing according to manufacturing.

The odds ratio (OR) in different parts of the country was calculated to estimate the risk for HEV seropositivity using a binary logistic regression. The OR of anti-HEV antibodies occurrence in the Western Bulgaria (Sofia and Lovech districts) was determined comparing with the Eastern Bulgaria (Stara Zagora district) (Table 2). The odds of HEV seroprevalence were 11.46 times higher in the Eastern Bulgaria than in the Western Bulgaria. In this survey, HEV RNA testing was not performed due to financial and technical difficulties.

In the past 5 years, the application of transudate for the diagnosis of HEV has begun. In 2018, Dahnert et al. used body cavity transudate for the diagnosis of HEV in raccoon (Procyon lotor), raccoon dog (Nyctereutes procyonoides), dog (Canis lupus familiaris), and cat (Felis silvestris catus), applying HEV-Ab ELISA test (AXIOM, Burstadt, Germany; designed by Wantai, Bejing, China) and recomLine HEV IgG/IgM ELISA test (Mikrogen GmbH) (Dahnert et al. 2018). These German authors found the following HEV seroprevalence in P. lotor—53.8% (43/80; 95% confidence interval [CI]: 42.8–64.7), N. procyonoides—34.3% (25/73; 95% CI: 23.4–45.1), C. lupus familiaris—56.6% (47/83; 95% CI: 46.0–67.3), and F. silvestris catus—32.3% (21/65; 95% CI: 20.9–43.7).

Two years later, the same research team from Germany used fox cavity transudate for the diagnosis of HEV in red foxes (Vulpes vulpes) from federal state of Brandenburg, applying ELISA tests (AXIOM and Mikrogen) (Eiden et al. 2020). The authors tested 880 fox transudates collected from 1993 to 2012 and reported overall high HEV seroprevalence of 81.2% on mean (range: 48.9–100.0%). In 2020, Navarro et al. for the first time used liver transudate for HEV diagnose in white pigs, Iberian pigs, and wild boars (S. scrofa), performing ELISA kit ID Screen Hepatitis E Indirect Multi-species (IDvet, Montepellier, France) (Navarro et al. 2020). These Spanish researchers reported HEV seroprevalence in white pigs—77.2% (34/44; 95% CI: 63.0–87.1), Iberian pigs—63.0% (29/46; 95% CI: 48.6–75.4), and S. scrofa—62.8% (22/35; 95% CI: 46.3–76.8).

The results from this research showed high overall HEV seropositivity (67.7%) in tested pig liver transudate from Bulgarian swine. These data are similar to our previous serological studies (Tsachev et al. 2019, 2020, 2021a). High overall HEV seroprevalence was recorded from serum samples of industrial pigs—36%, 40%, and 60.3% (Pishmisheva et al. 2018, Tsachev et al. 2019, 2021a), of wild boars—40.8% (Tsachev et al. 2021b), and East Balkan swine (the only aboriginal pig breed in Bulgaria)—82.5% (Tsachev et al. 2020). Consequently, the applying of liver transudate shows similar results such as used of serum samples. In this regard, liver transudate testing could be an alternative method for seroepidemiological survey of HEV in swine.

This study has several limitations. First, a small number of pig liver transudate are enrolled. Second, the study presented results from 3 out of total 28 districts, consequently the study is not nationwide survey. Third, HEV RNA testing was not performed. Despite these limitations, the presented diagnostic tool (liver transudate testing) could be an alternative method for seroepidemiological research. Moreover, our study is the first Bulgarian survey showing the HEV seropositivity in pig liver.

Conclusion

This study shows that liver transudate testing could be a good diagnostic method for serological survey of HEV in pigs. In addition, our research first reported the data for HEV in liver from Bulgarian swine. We found high seropositivity (67.7%), which suggests wide circulation of this infection in our country. These results could be a good starting point for taking adequate measures for the prevention and control of HEV in pigs in Bulgaria. This is a key message for national veterinary organizations dealing with animal health and zoonotic diseases.