• Background: Among zoonotic foodborne pathogens, Hepatitis E virus (HEV) is recognized for its environmental persistence. The Suidae family are the primary target hosts for HEV genotype 3. Based on these considerations, this study aimed to molecularly screen effluent samples collected from a swine slaughterhouse in Central Italy. • Methods: Between January and December 2025, a total of 72 effluent samples (1.0 liter each) were collected every 10 days over one year. More in detail, 36 samples were collected from the collection tank at the stocking yard point (V1), and the remaining 36 at the wastewater treatment plant (WWTP) level (V2) after the depuration processes. The number of slaughtered pigs per each sampling day was also registered. All samples were centrifugated at 180,000 x g for 2 hours and the obtained pellets were resuspended in 500 µL of RNase-free water and stored at -80 °C. RNA was extracted from all samples and molecularly screened using real time RT-qPCR and nested RT-PCR assays. The obtained amplicons were sequenced and successively published on the GenBank platform. Finally, a phylogenetic tree was constructed based on the Neighbor Joining method and realized using the iTOL Software. • Results: From a general perspective, HEV RNA was detected in 26.39% of the total samples. More in detail, it was found in 27.78% of V1 and 25.00% of V2 ones. The highest viral load, 105 GE/mL, was observed in two V1 samples collected in December and January months, corresponding to the periods with the highest slaughtering volumes. Statistically significant differences of HEV RNA loads were observed between V1-V2 (p<0.001) and cold-warm seasons (p=0.02). Finally, the obtained sequences were deposited and published on the GenBank platform with the following accession numbers: PX925942, PX925944, PX925945, PX925946, and PX925948. • Conclusions: The detection of HEV RNA in effluent samples collected at the swine slaughterhouse represents the first report of this finding in Italy. Similar approaches have been applied in Brazil and Spain, with reported prevalences of 50.0% and 0.4%, respectively. The demonstrated ability of HEV to overcome the WWTP highlights the need for further investigation and the development to find new solutions to this environmental issue.
Hepatitis E virus detection in effluent samples collected from a swine slaughterhouse: A one-year molecular surveillance
Gianluigi Ferri
Writing – Original Draft Preparation
;
2026-01-01
Abstract
• Background: Among zoonotic foodborne pathogens, Hepatitis E virus (HEV) is recognized for its environmental persistence. The Suidae family are the primary target hosts for HEV genotype 3. Based on these considerations, this study aimed to molecularly screen effluent samples collected from a swine slaughterhouse in Central Italy. • Methods: Between January and December 2025, a total of 72 effluent samples (1.0 liter each) were collected every 10 days over one year. More in detail, 36 samples were collected from the collection tank at the stocking yard point (V1), and the remaining 36 at the wastewater treatment plant (WWTP) level (V2) after the depuration processes. The number of slaughtered pigs per each sampling day was also registered. All samples were centrifugated at 180,000 x g for 2 hours and the obtained pellets were resuspended in 500 µL of RNase-free water and stored at -80 °C. RNA was extracted from all samples and molecularly screened using real time RT-qPCR and nested RT-PCR assays. The obtained amplicons were sequenced and successively published on the GenBank platform. Finally, a phylogenetic tree was constructed based on the Neighbor Joining method and realized using the iTOL Software. • Results: From a general perspective, HEV RNA was detected in 26.39% of the total samples. More in detail, it was found in 27.78% of V1 and 25.00% of V2 ones. The highest viral load, 105 GE/mL, was observed in two V1 samples collected in December and January months, corresponding to the periods with the highest slaughtering volumes. Statistically significant differences of HEV RNA loads were observed between V1-V2 (p<0.001) and cold-warm seasons (p=0.02). Finally, the obtained sequences were deposited and published on the GenBank platform with the following accession numbers: PX925942, PX925944, PX925945, PX925946, and PX925948. • Conclusions: The detection of HEV RNA in effluent samples collected at the swine slaughterhouse represents the first report of this finding in Italy. Similar approaches have been applied in Brazil and Spain, with reported prevalences of 50.0% and 0.4%, respectively. The demonstrated ability of HEV to overcome the WWTP highlights the need for further investigation and the development to find new solutions to this environmental issue.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
