Harboring & co-harboring of viral RNA belonging to zoonotic enteric pathogens in caprine unpasteurized milk samples

This study aims to discover viral RNA genetic fragments, belonging to the following zoonotic enteric foodborne pathogens: astrovirus (Ast), hepatitis A virus (HAV), hepatitis E virus (HEV), norovirus genogroups I and II (NoV-GI and NoV-GII), and rotavirus (Rtv), amplified from unpasteurized caprine milk samples collected from animals farmed in Central Italy. For this purpose, a population of 383 primaparous and plutiparous goats (Capra aegagrus hircus) with average age of 2.80.5 years and weight ranging between 508 kg was involved, and animals were farmed in six sites located in L’Aquila province (Abruzzo region, Italy). Between 2024-2025, 50.0 mL of raw milk were collected from each animal (following pre- and post-dipping procedures) during their lactation peaks and transported under refrigerated conditions at laboratory level. Milk fat layer was separated by performing centrifugation at 3,000x g for 15 minutes at 4 °C, and finally removed. A volume of 200 µL of HCl 1M solution (pH 3.5-4.0) was added to each sample to facilitate milk protein and viral RNA precipitation. These ones were finally centrifugated at 4,000x g for 10 minutes at 4 °C (removing fat layers and supernatants), and the obtained pellets were resuspended in 5.0 mL of PBS (pH 7.2), in agreement with Dziedzinska et al. [1]. The TRIzol LS method was used for the viral RNA extractions permitting to obtain final pellets of 50 µL resuspended with RNase-free water and conserved at -80 °C until biomolecular screenings. For all pathogens (Ast, HAV, HEV, NoV-GI, NoV-GII, and Rtv), real time RT-qPCR assays were performed using the GENE UP® System (bioMérieux, Paris, France) and viral RNA fragments were expressed as genome equivalents (GE/mL). The commercial kits KQASV (Ast), KQHAV (HAV), KQHEV (HEV), KQNVGI (NoV-GI), KQNVG (NoV-GII), KQRV (Rtv) (ceeramTools—Thermo Fisher ScientificTM) were used, and the quantitative nucleotide concentrations were determined comparing the obtained curves to the standard ones (concentrations from 107 to 102 GE/mL). Nested RT-PCR assays represented the qualitative confirmations, and the suspected positive bands, to the electrophoresis runs, were finally purified and sequenced. Results showed that 59/383 (15.40%) of the screened animals harbored at least the RNA fragments of one out of the six considered pathogens. More in detail, 20/383 (5.22%) were positive to HEV RNA detection (102 GE/mL), 16/383 (4.17%) for NoV-GI (101 GE/mL), and 10/383 (2.61%) for Ast (102 GE/mL). Thirteen out of fifty-nine (22.03%) positive animals co-harbored more than one viral RNA fragment belonging to different pathogens; indeed, 11/13 (86.41%) of the screened raw milk samples detected both Ast-NoV GI genetic regions and Ast-HEV, and in 2/13 (15.38%) of positive specimens were discovered three amplicons referred to Ast, HEV, and NoV GI. On the contrary, HAV, NoV-GII, and Rtv were not detected. Concerning HEV RNA detection, the obtained prevalence (5.22%) was in line with the observed prevalence of 2.80% by Dziedzinska et al. [1] in Czech Republic, but lower than 18.46% described by Demirci et al. [2] in Turkey. Ast (2.61%) and NoV-GI (4.17%) RNA fragments detection represents the first Italian data. The scientific explanation is based on three different variables: possible cross-species infections among domestic and wild receptive hosts, farming methods, and the viral environmental resistance typical of naked or quasi-enveloped viruses. The required One-health approach based on biomolecular surveillance are mandatory to guarantee safe products for consumers.