Among viral foodborne pathogens, hepatitis E virus (HEV) has been widely detected in different animal origin foodstuffs. Milk, obtained from large and small ruminants, have demonstrated the possibility to detect infectious amounts of virions. Indeed, HEV transmission from blood to the mammary gland, during viremia step, has been observed in different mammalian species (including humans). Small ruminants result receptive to HEV and result also involved in its environmental diffusion through feces and raw milk [1]. In Abruzzo region (Central Italy), the ovine species (Ovis aries) are traditionally farmed following the transhumance method. It means the involvement of many geographical areas (including National Parks) used for grazing also usually shared with wild ruminants. This study aimed to amplify specific HEV RNA regions from 220 ovine unpasteurized milk specimens collected from 3 herds farmed in three provinces (located in Abruzzo region): Teramo, Pescara, and L’Aquila. Successively to the pre-dipping procedures, a volume of 50 mL was individually sampled from each animal. Prior to the RNA extraction, the milk fat layer was removed after centrifugation at 3000X g for 15 minutes at 4°C. The adding of 300 μL of HCl 1M solution was used to facilitate the proteins and nucleic acids precipitation, as described by Dziedzinska et al. [2]. The Trizol LS method was successively used for the viral RNA extraction. Nested RT-PCR and RT-qPCR were performed to amplify specific genetic regions belonging to the HEV ORF-1, ORF-2, and ORF-3. Sanger sequencing and phylogenetic analysis were performed. The IBM® SPSS® Statistics Software was used for the statistical data analysis calculating the chi-square value (with Yates’s correction). Results showed HEV RNA fragments amplification from 5/220 or 2.27% (both ORF-1 and ORF-2 amplicons) milk/subject specimens, and the RT-qPCR detected on average 102 copies/μL from positive specimens. From a geographical perspective, among the discovered positive subjects, 3 animals were farmed in Teramo province and 2 in Pescara one. Sequence analyses and the phylogenetic assays permitted to determine high nucleotide similarities with HEV genotype 3 which is the most diffused one in Central Italy. This investigation discovered for the first time HEV RNA fragments from raw ovine milk in Italy. The scientific explanation of HEV RNA detection can be justified by the transhumance farming method and the environmental sharing with wild animal species, which are natural reservoirs, [3] providing environmental conditions for possible cross-species infections. Basing on the One-health approach, HEV environmental surveillance represents a crucial public health concern.
77° Convegno della Società Italiana delle Scienze Veterinarie (SISVET), svoltosi a Parma dal 12 al 14 giugno 2024
Gianluigi Ferri
Writing – Original Draft Preparation
;Luca PennisiMethodology
;Alberto VergaraWriting – Review & Editing
2024-01-01
Abstract
Among viral foodborne pathogens, hepatitis E virus (HEV) has been widely detected in different animal origin foodstuffs. Milk, obtained from large and small ruminants, have demonstrated the possibility to detect infectious amounts of virions. Indeed, HEV transmission from blood to the mammary gland, during viremia step, has been observed in different mammalian species (including humans). Small ruminants result receptive to HEV and result also involved in its environmental diffusion through feces and raw milk [1]. In Abruzzo region (Central Italy), the ovine species (Ovis aries) are traditionally farmed following the transhumance method. It means the involvement of many geographical areas (including National Parks) used for grazing also usually shared with wild ruminants. This study aimed to amplify specific HEV RNA regions from 220 ovine unpasteurized milk specimens collected from 3 herds farmed in three provinces (located in Abruzzo region): Teramo, Pescara, and L’Aquila. Successively to the pre-dipping procedures, a volume of 50 mL was individually sampled from each animal. Prior to the RNA extraction, the milk fat layer was removed after centrifugation at 3000X g for 15 minutes at 4°C. The adding of 300 μL of HCl 1M solution was used to facilitate the proteins and nucleic acids precipitation, as described by Dziedzinska et al. [2]. The Trizol LS method was successively used for the viral RNA extraction. Nested RT-PCR and RT-qPCR were performed to amplify specific genetic regions belonging to the HEV ORF-1, ORF-2, and ORF-3. Sanger sequencing and phylogenetic analysis were performed. The IBM® SPSS® Statistics Software was used for the statistical data analysis calculating the chi-square value (with Yates’s correction). Results showed HEV RNA fragments amplification from 5/220 or 2.27% (both ORF-1 and ORF-2 amplicons) milk/subject specimens, and the RT-qPCR detected on average 102 copies/μL from positive specimens. From a geographical perspective, among the discovered positive subjects, 3 animals were farmed in Teramo province and 2 in Pescara one. Sequence analyses and the phylogenetic assays permitted to determine high nucleotide similarities with HEV genotype 3 which is the most diffused one in Central Italy. This investigation discovered for the first time HEV RNA fragments from raw ovine milk in Italy. The scientific explanation of HEV RNA detection can be justified by the transhumance farming method and the environmental sharing with wild animal species, which are natural reservoirs, [3] providing environmental conditions for possible cross-species infections. Basing on the One-health approach, HEV environmental surveillance represents a crucial public health concern.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
