High hepatitis E virus prevalence in pig slurry samples from the north-western region of Germany

Pork is recognized as the primary source of transmission for Hepatitis E virus (HEV) genotypes 3 and 4 (gt3/4) in industrialized countries. In Germany, approximately 10 % of retail pork samples test positive for HEV. However, the potential role of pig manure as a reservoir contributing to the environmental and zoonotic transmission of HEV genotype 3 remains insufficiently characterized. To assess HEV contamination in manure, 61 pig slurry samples were collected from various collection points in northwestern Germany and analyzed for HEV and hepatitis A virus (HAV) RNA using quantitative PCR. In line with the "One Health" concept, which emphasizes the interconnectedness of humans, animals, and the environment, this study aimed to evaluate pig manure as a potential source of HEV contamination of agricultural land and food plants, thus posing a risk to both omnivorous and vegetarian populations. HEV RNA was detected in 67 % (41/61) of the samples, with viral loads ranging from 5.2 × 10² to 1.8 × 10⁵ copies/mL (median 6.8 × 10³ copies/mL), whereas HAV RNA was not detected in any sample. Genotyping via nested PCR identified HEV genotype 3c, a subtype frequently detected in human infections in Germany. To further characterize the viral particles, four samples with the highest viral loads were subjected to linear density gradient ultracentrifugation. Two distinct fractions were identified, corresponding to enveloped particles (suggesting urinary origin) and non-enveloped particles (suggesting fecal origin). In conclusion, this study provides evidence for the frequent presence of both enveloped and non-enveloped HEV particles in pig slurry in Germany. These findings highlight manure as a potential environmental reservoir for HEV and underscore the need for further studies to determine HEV infectivity in slurry and to assess its epidemiological significance within the One Health framework.