CO04 | PROCESS VALIDATION OF FREEZING AND HOT-SMOKING TREATMENTS FOR THE INACTIVATION OF OPISTHORCHIS FELINEUS METACERCARIAE IN NATURALLY INFECTED TENCH (TINCA TINCA) FILLETS

Raffaella Franceschini¹, David Ranucci², Francesco Agnetti³, Eleonora Radicchi³, Rossana Roila², Raffaella Branciari² | ¹Dipartimento di Medicina Sperimentale, Università degli Studi di Roma Tor Vergata, Roma, Italy; ²Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia, Perugia, Italy; ³Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”, Perugia, Italy.

Opisthorchis felineus is a foodborne zoonotic trematode transmitted to humans through the consumption of raw or undercooked freshwater fish, belonging to the Cyprinidae family. Human opisthorchiasis represents a relevant public health concern in endemic areas of Central Italy, where outbreaks have been repeatedly associated with the consumption of tench (Tinca tinca) from lake environments. In this context, the validation of effective mitigation strategies is essential to support food business operators and competent authorities in the management of parasitic risk in local freshwater fish supply chains. The aim of this study was to evaluate the efficacy of a company freezing and hot-smoking protocol for the inactivation of O. felineus metacercariae in naturally infected tench fillets. The study was designed as a process-validation challenge test using naturally infected fish, in order to reproduce real contamination conditions occurring in the local production chain. Three independent batches of tench naturally infected with O. felineus were included. For each batch, 15 fillets were analysed, giving a total of 45 fillets. Within each batch, untreated fresh fillets were used as positive controls to confirm the presence of viable metacercariae before treatment, while the remaining fillets were assigned to the freezing or hot-smoking protocols. The freezing protocol consisted of treatment at −26°C for 22 h followed by storage at −20°C for 24 h, 3 days or 6 days. The hot-smoking protocol consisted of treatment at 72°C for 1–3 min, followed by refrigerated storage at <4°C. The presence and viability of O. felineus metacercariae were assessed by peptic–hydrochloric acid digestion performed by an accredited laboratory. The treatment was considered effective when no viable metacercariae were detected in treated samples, while viable metacercariae were present in the corresponding untreated controls. Viable metacercariae were detected in untreated fresh tench fillets, confirming the natural infection of the raw material and the suitability of the selected batches for the challenge test. Conversely, no viable O. felineus metacercariae were found in any treated samples from the three independent batches, including frozen fillets analysed after 24 h, 3 days and 6 days of storage at −20°C, as well as hot-smoked products. Dead metacercariae were observed after treatment, supporting the inactivation effect of both processing procedures. These findings are consistent with current evidence indicating that freezing and heating remain among the most effective methods for killing viable parasites in fishery products. The results indicate that the company freezing and hot-smoking protocols were effective in inactivating O. felineus metacercariae in naturally infected tench fillets under the tested conditions. The use of three independent batches and a defined number of fillets per batch strengthens the scientific robustness of the validation, accounting for variability in parasite burden and fish matrix. This process-validation challenge test provides practical evidence to support the implementation of preventive measures in freshwater fish supply chains and may contribute to improving the safety of traditional fish products from endemic areas.