SIX- YEARS PERSISTENCE OF HEV IN A PIG FARM IN NORTHERN ITALY

Background: Hepatitis E virus (HEV) is the causative agent of an acute hepatitis, generally selflimited which can become chronic. In Europe, an increase of cases has been observed in the last 10 years and the main route of transmission is foodborne. The HEV-3 and HEV-4 are zoonotic for which pigs are considered the main reservoir. In pigs HEV-3 is widespread and several studies reported detection of the virus in farms worldwide. The infection is transmitted through the fecal oral route but it is not clear how long the virus can persist in animals and which environmental and epidemiological parameters influence the transmission process. In the present study, we investigated the occurrence and persistence of HEV in a farrow-to-wean farm (about 1000 sows with moderate external replacement) in Northern Italy surveilled twice in a 6 years period. Materials and Methods: In 2012 and 2018, a total of 44 and 224 stool samples were collected respectively from the same pig farm. Pooled fecal samples were collected from the pen floor (a mean of 10 pools for every 20 animals). RNA was extracted by a QiampViral extraction kit (Qiagen) and analysed by Real-time RT-PCR and by nested RT-PCR amplifying three genomic regions (Methyltransferase, RNA dependent RNA polymerase, capsid). Amplicons obtained were sequenced and phylogenetic analysis was conducted (MEGA6). Results: The first survey was conducted in 2012 in which HEV was detected in 6 out of 44 pools tested and only animals belonging to the age class >90 days were positive (6/16). Six years later, HEV was still detected on the same farm, in 69 out of 224 fecal pools. Samples from pigs aged between 2 and 4 months were found positive (69/95), neither younger (<60 days; no. 39) nor older (4-40 months, no. 90) animals were infected by HEV. Sequence analysis was conducted on 8 fecal pools from animals sampled in both 2012 and 2018. The subtype of the HEV-3 strain detected was not assignable by comparison with subtypes defined so far. The sequences obtained shared high nucleotide identities with similar identity rates for all the diagnostic fragments investigated (Methyltransferase 98.63%; RdRp 99.19%; capsid 98.96%), suggesting that the same HEV-3 strain was circulating on the farm in both 2012 and 2018. Conclusion: The survey conducted showed that animals aged between 2-4 months are more frequently infected by HEV and that there is a clearance of the infection in older animals (gilts and sows). None of the animals younger than 2 months (including the group of suckling) were infected by HEV. The detection of the same HEV strain after 6 years leads to hypothesize that the virus may persist for a long time in the environment. Infected animals are housed in pens with solid floors which are not easily cleaned. Animals of 1-2 months that may already be susceptible to HEV infection resulted negative. These animals are housed in small groups, the pen floor is 60 cm above the ground, ensuring better hygienic conditions.