Razvoj i primena različitih laboratorijskih metoda za dijagnostikovanje infekcije izazvane hepatitis E virusom kod svinja i ljudi / Development and application of different laboratory methods for the diagnosis of hepatitis E virus infection in pigs and hu

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E infection in humans. HEV is transmitted through contaminated water and is responsible for the outbreaks of many largescale epidemics in the developing countries of Asia and Africa. Swine HEV was first isolated in 1997, and was later detected in other animal species, such are: wild boar, deer, rabbits, rats, birds and more. The first investigations of HEV infection in domestic and wild pigs in Serbia were carried out in 2008. HEV RNA was detected in 30% of faecal samples and 45% of the tissue samples (Petrovic et al., 2008). Analysing the blood samples of beckyard pigs, the seroprevalence of 34,6% was determined (Lupulovic et al, 2010). The aim of this study was to investigate the prevalence of HEV infection in pigs on farms in Vojvodina, as well as testing the HEV seroprevalence in humans. The methods used for this study were: non-commercial ELISA (in house ELISA), the commercial ELISA, Western blot method, real-time RT-PCR and immunohistochemical method for the detection of HEV antigen. Material for the study were: blood samples of pigs (300) from 3 farms on the territory of South Backa and Srem and blood samples of people (294), as well as faeces, bile, liver and meat collected in slaughterhouses from 95 fatteners and 50 piglets. The presence of specific IgG antibodies against the hepatitis E virus in pigs has been detected on all three examinated farms. Upon the application of in house ELISA, the seroprevalence of 37% was establised on farm A, 31% in farm B and 54% on farm C, while using a commercial ELISA , 40% of seropositive pigs were detected on farm A, 41% of fami B and 65% Farm C. The comparative analysis of the results obtained with both ELISA, determined the average seroprevalence of 40,66% by in house ELISA and 48,66% by commercial ELISA. The research of the presence of specific IgG antibodies against HEV in the serum of blood donors and patients were also conducted. Upon the application of in house ELISA, the seroprevalence of 15% were recorded in blood donors, while blood samples of patients were seronegative. Testing by commercial ELISA, positiv serological findings were diagnosed in 17,86% of blood donors (serums with positive or suspicious results in in house ELISA and a number of seronegative samples were tested), and in patients 2, 12%. As so-called "gold standard" for defining the serological results with suspiciousserological findings, which extinctions were close to cut off values in in house ELISA, we used the Western blot method. The positive result was confirmed In 6 of 11 blood samples of pigs, while in 11 tested human serums, the positive result was established in 7 samples. Samples from slaughterhouses were examined by real-time RT-PCR method, and HEV RNA has been detected in feces (54%), bile (26%), liver (16%) and meat (10%) of piglets. In fatteners, the presence of HEV RNA was confirmed only in feces (7.27%), while all tissue samples were negative. Pathohistological examination has proved the presence of microscopic lesions grade II, which were detected in 3 piglet liver samples (11,53%) of 26 tested samples with positive RT-PCR result. The HEV antigen was not identified in liver samples of piglets by immunohistochemical examination. The laboratory protocols were defined for hepatitis E infection in pigs and humans, as well as in meat and liver samples of pigs in slaughterhouses.