Hepatitis E virus in pig faeces, serum, excretory organs, and other edible tissues at slaughter in Spain, 2020-2022.

Foodborne hepatitis E is an emerging zoonosis in Europe, with domestic pigs recognized as the main reservoir of zoonotic hepatitis E virus (HEV) genotype 3. This study evaluated HEV circulation in Spain using a production-representative design (2020-2022). A total of 386 apparently healthy pigs from 11 large slaughterhouses (375 farms, 34 provinces) were analysed. Ten sample types (blood, faeces, liver, heart, kidney and 5 different muscles usually used to elaborate meat products) per animal (n = 3860) were tested for HEV RNA by RT-qPCR. Serum was also analysed for anti-HEV IgG by ELISA. Slaughterhouse wastewater was also screened to assess potential environmental dissemination. Overall seroprevalence reached 68.4%, indicating widespread HEV exposure among pigs. Of the 386 pigs analysed, 213 (55.2%) were positive for HEV RNA in at least one sample. HEV RNA was detected in 8.5% of all samples, most frequently in faeces (36.0%) and liver (16.3%), whereas lean meat and top loin were consistently negative, and only low positivity was found in rib meat samples (9.6%). Multivariable modelling using generalized estimating equations showed that HEV RNA detection was significantly more likely in faeces and liver (OR = 7.4 and 2.3, respectively) compared to kidney, while muscle and fat-rich cuts showed markedly lower odds. HEV RNA was also detected in six of seven wastewater samples, indicating potential environmental release through slaughterhouse effluents. Phylogenetic analysis of partial ORF2 sequences (n = 45) identified five HEV-3 subgenotypes: HEV-3f-A1, 3f-A2, 3c, 3f-B, 3e. These findings demonstrate active, genetically diverse HEV circulation in Spanish pigs, reinforcing the need for strengthened surveillance along the pork production chain.