1.3.2.1. Nematoda

The most relevant zoonotic nematodes include parasites belonging to the Anisakidae family, encompassing some species of the genera Anisakis, Phocanema ⁴ (recently resurrected genus to encompass six species formerly placed in genus Pseudoterranova) (see Bao et al., 2023) and Contracaecum. In the latter genus, only species of the C. osculatum (s. l.) complex maturing in pinnipeds are considered zoonotic.

These anisakid species share some general life cycle traits in that they all use planktonic or semi‐planktonic crustaceans as intermediate hosts and fish and/or squid (Anisakis) as paratenic/transport hosts carrying the infective larval stage (L3). Marine mammals, i.e. cetaceans (Anisakis) or pinnipeds, act as definitive hosts where the nematodes reach maturity and from which the eggs are released.

Concerning human zoonosis, the term anisakidosis is related to the disease caused generally by anisakids, while the term anisakiasis refers to the disease caused by Anisakis spp. Generally speaking, anisakidosis and anisakiasis are underestimated because they are misdiagnosed and there is no compulsory notification at EU level. An overview of human anisakidosis was also recently provided by Shamsi and Barton (2023).

Anisakis : The two species A. simplex (s. s.) and A. pegreffii are the most common zoonotic nematodes associated with the consumption of raw or mildly thermally processed seafood. The L3 larval stages are mostly located in the visceral body cavity and outside the internal organs, but they are also found in the musculature of commercially important fish species (reviewed in Mattiucci et al., 2018; Shamsi & Barton, 2023) that are infected intra vitam and/or post‐mortem. The latter phenomenon is temperature dependant and occurs in the species A. pegreffii infecting anchovy (Engraulis encrasicholus) above 2°C (Cipriani et al., 2016). Storage temperature and time influence the post‐mortem motility of A. simplex (s. s.) larvae in herring (Clupea harengus) and blue whiting (Micromesistius poutassou) (Cipriani et al., 2024). Keeping the temperature at ≤2°C prevents larval post‐mortem migration into the flesh during fish storage, handling and transportation (Cipriani et al., 2024). Several studies have reported that the ventral (as compared to the dorsal) flesh is the most infected part of the fish (reviewed in Mattiucci et al., 2018; Levsen et al., 2018). Humans may become infected with Anisakis L3 larvae when they consume raw or undercooked fish, but these larvae do not develop to the adult stage. However, in a few cases, fourth‐stage larvae were isolated from human patients (Suzuki et al., 2021). When the larvae are viable and infective, the L3 of Anisakis penetrates the mucosal layers of gastric and/or the intestinal tract within a few hours of being ingested causing tissues damage. This acute gastrointestinal form of Anisakis infection represents an invasive disease (i.e. gastric anisakiasis (GA) or intestinal anisakiasis (IA)). The ulceration is accompanied by an eosinophilic inflammatory response with subsequent granuloma formation surrounding the penetrated larva. Clinical symptoms include severe stomach or abdominal pain, nausea, vomiting and intestinal obstruction, and mimic other common gastrointestinal diseases, such as acute appendicitis, peptic ulcer, etc. Basic diagnosis requires an anamnestic survey of the symptomatic patient and endoscopy, if possible, supported by serological diagnosis. Early removal and identification of the worm is essential to avoid the chronic infection developing into gastrointestinal eosinophilic granulomatosis.

The symptoms may also be complicated by a mild to strong allergic response (gastroallergic anisakiasis, GAA). It is clearly documented that ingestion of viable L3 larvae is required for the induction of allergic manifestations (Mattiucci et al., 2013). However, it is still unknown if an allergic reaction can occur because of exposure to allergens from dead parasites, e.g. present in canned or other heavily processed fishery products. Some allergens are relatively resistant to enzyme digestion or heat treatment and can trigger IgE reactions even when L3 larvae are killed by freezing and/or cooking of the fish (Carballeda‐Sangiao et al., 2014). Since allergens related to zoonotic parasites will not be specifically covered by the current assessment, this aspect will not be further discussed in this opinion.

The majority (72%) of human cases of illness caused by above‐mentioned species of Anisakis are GA, while 26% are IA cases, with 2% being extra‐gastrointestinal or ectopic anisakiasis (for a review, Mattiucci et al., 2018). A. simplex (s. s.) has been identified by molecular methods causing invasive gastric and IA (Arai et al., 2014; EFSA BIOHAZ Panel, 2010; Roca‐Geronès et al., 2020). Although associated with allergic anisakiasis, in some European countries, the aetiological agent has not been identified at species level (Daschner et al., 2021; EFSA BIOHAZ Panel, 2010; Mattiucci et al., 2018). Anisakis pegreffii causes GA, IA, GAA and ectopic anisakiasis, being identified in cases of invasive anisakiasis, detected by observation and molecular identification of the viable larvae during endoscopy (Arai et al., 2014; Lim et al., 2015; Mattiucci et al., 2011, 2013; Mattiucci, Colantoni, et al., 2017; Mattiucci, Paoletti, et al., 2017), colonoscopy (D'Amelio et al., 2023; Mattiucci, Paoletti, et al., 2017), or in surgically removed eosinophilic granulomas (Mattiucci et al., 2011; Mattiucci, Paoletti, et al., 2017; Mladineo et al., 2016). A. pegreffii infections have also been identified in Italy (D'Amelio et al., 2023; EFSA BIOHAZ Panel, 2010; Mattiucci et al., 2011, 2013; Mattiucci, Colantoni, et al., 2017), Croatia (Mladineo et al., 2016), South Korea (Lim et al., 2015) and Japan (Arai et al., 2014). A. simplex (s. s.) has been linked to invasive GA and IA in Japan (Arai et al., 2014; Auer et al., 2007; EFSA BIOHAZ Panel, 2010). The first case of human anisakiasis due to A. simplex (s. s.) occurring in Poland has recently been described (Kołodziejczyk et al., 2020). In Portugal, the first case was reported in 2017, but the larvae were only identified as Anisakis sp. (Carmo et al., 2017). Finally, records of human anisakiasis have been documented in recent years from Iran (Najjari et al., 2022), although it was not possible to identify the Anisakis species, despite the application of the real‐time PCR (qPCR) methodology. Similarly, the first case of human ectopic anisakiasis due to Anisakis sp. was recently described in Greece, by histological and molecular diagnosis (Dinas et al., 2024). It is still unclear if A. pegreffii and A. simplex (s. s.) differ in their pathogenic characteristics in human anisakiasis (Suzuki et al., 2010). The finding of Anisakis sp. larvae in patients suffering from gastric and/or colon carcinoma has also been reported (García Pérez et al., 2015), although the demonstration of potential tumour‐provoking capacity exerted by Anisakis spp. larvae has not yet been demonstrated.

All cases of human anisakiasis that were confirmed by molecular methods have been caused by infections with A. pegreffii or A. simplex (s. s.). However, one cannot exclude that other Anisakis species such as A. typica (s. l.) may act as aetiological agents of human anisakiasis in areas where these parasites commonly occur in food fish. For example, A. typica (s. l.) has been found in the muscle of commercially important fish species in the Indian ocean (Cipriani et al., 2022).

Phocanema : The Phocanema decipiens (s. l.) complex (see Bao et al., 2023; Paggi et al., 1991) consists of at least six sibling species: Ph. decipiens (s. s.), Ph. azarasi, Ph. cattani, Ph. krabbei, Ph. bulbosum and Ph. decipiens sp. E. As adults, they live in pinnipeds, with distinct gene pools and different geographical distribution (Mattiucci & Nascetti, 2008). So far, some of these species are reported as zoonotic. The first human case caused by these nematodes was described by Lee et al. (1985) in Korea, but the aetiological agent was not identified to species level by molecular markers. In Europe, in the recent years, cases caused by Ph. decipiens (s. s.) were identified by molecular markers in Italy (Cavallero et al., 2016), and in France (Brunet et al., 2017) most likely related to imported fish consumed raw or only lightly processed. A further infection due to Ph. decipiens (s. s.) was detected in the nasal cavity of a patient from Denmark with rhinitis symptoms (Nordholm et al., 2020). Live larvae of Ph. decipiens (s. l.) were also found in the pharynx, vomit or faeces in patients from Iceland in the years 2004 and 2005, after the consumption of fresh fish such as wolffish and cod (Skirnisson, 2022). A human case caused by Ph. azarasi was reported from Japan (Arizono et al., 2011). Several cases caused by Ph. decipiens (s. s.) from South Korea were reviewed by Song et al. (2022), while cases linked to the species Ph. cattani were described in Argentina (Menghi et al., 2020; Weitzel et al., 2015).

Contracaecum: Contracaecum osculatum (s. s.) identified both by morphology and molecular markers was found to infect pigs and elicit gastric eosinophilic granulomas suggesting the zoonotic potential of this anisakid (Strøm et al., 2015). Two cases of human infection, likely due to C. osculatum (s. l.), were reported from Japan (Nagasawa, 2012); however, no molecular identification was performed. Additionally, a human case of Contracaecum sp. infection acquired from eating locally caught fish was described in Australia (Shamsi & Butcher, 2011). All of these cases in Japan and Australia elicited milder symptoms compared to anisakiasis, including vomiting, gastric or intestinal pain, and in some cases, spontaneous expelling of larva in the stool (reviewed in Mattiucci, Cipriani, et al., 2017). The zoonotic role of Contracaecum species maturing to the adult stage in fish‐eating birds, e.g. C. rudolphii (s. l.), has not been demonstrated to date.

1.3.2.2. Trematoda

Zoonotic trematodes are present in the families Opisthorchiidae, Cyathocotylidae and Heterophyidae. Clonorchis sinensis and taxa belonging to the genera Opisthorchis, Metorchis and Pseudamphistomum (family Opisthorchidae) may cause cholangitis, choledocholithiasis, pancreatitis and cholangiocarcinoma (EFSA BIOHAZ Panel, 2010; Kondrashin et al., 2023). Their prevalence in wild fish ranges from less than 1% to greater than 90%, depending on the region of the world. It is noteworthy that human cases caused by species within these three genera occur sporadically in various parts of Italy (Pozio et al., 2013, and more recent cases) and Russia (Kondrashin et al., 2023). The metacercariae of Paracoenogonimus ovatus (family Cyathocotylidae) commonly occur in the flesh of several wild freshwater fish species including various cyprinids and pike (Esox lucius) in central and south‐eastern Europe. The zoonotic ability of P. ovatus has been demonstrated experimentally in infection trials using laboratory rats which showed intestinal lesions that were causatively linked to the infection (Goncharov & Soroka, 2016).

The metacercariae of the heterophyid species Cryptocotyle lingua, inducing black spot disease in fish, commonly occur in many finfish species from the temperate and subarctic seas of the NE Atlantic (Duflot et al., 2023). C. lingua develops to maturity in fish‐eating birds but also in some terrestrial mammalian species, while periwinkles (Littorina spp.) act as first the intermediate host from which the fish‐infective cercaria stage emerge. Human infections with C. lingua have been reported from Greenland and Alaska (Babbott et al., 1961; Rausch et al., 1967). Marine cage‐reared fish are at risk of infection with C. lingua metacercariae if the cages are sufficiently close to the shoreline. Some C. lingua cercariae may penetrate the skin of the fish host and encyst in the superficial layers of the fillets, thus posing a zoonotic risk if infected fillets are consumed raw or only lightly processed.

1.3.2.3. Cestoda

Cestodes of the genus Dibothriocephalus (syn. Diphyllobothrium) include at least 13 species that infect humans. These cestodes are widely distributed in fish, mammal and bird hosts (EFSA BIOHAZ Panel, 2010; Gustinelli et al., 2016) and are generally associated with intermediate and definitive hosts living in temperate or cold‐water habitats. In recent years, several reports have suggested an expansion of the geographic area of distribution of this genus, due to host switching events (Kuchta et al., 2019). The broad fish tapeworm Dibothriocephalus latus has been considered the principal species infecting humans in Europe (Kuchta et al., 2015). Human diphyllobothriosis due to D. latus has reemerged in the subalpine region of Italy (Lago Maggiore and Lago di Como), Lake Geneva and from France while it shows a decrease in other historical endemic areas, such as the Baltic countries (Kuchta et al., 2015). Raw fillets of perch (Perca fluviatilis) represent the major source of infection. Sporadic cases of human diphyllobothriosis have also been reported from some countries of central and western Europe, which have been considered free of the parasite. These cases seem to be due to the consumption of imported fish. This is the case of D. pacificum and D. nikonkaiensise from Pacific region (reviewed in Kuchta et al., 2015). The occurrence of the broad fish tapeworm, D. latus, in, e.g. wild European perch (Perca fluviatilis) is well documented (e.g. Gustinelli et al., 2016; Králová‐Hromadová et al., 2021), and even non‐endemic areas, such as Spain, report infections in fish (Esteban et al., 2014). The older literature suggested widespread infections of wild fish in the Danube River. However, recent studies on European perch from the river did not detect this parasite, and it was suggested that the infection was no longer as widespread as previously considered (Radačovská et al., 2019).

In recent years, the occurrence of larval cestodes (Trypanorhyncha) of the genera Molicola and Gymnorhynchus has been reported in the edible parts (muscle) of fish of commercial importance (i.e. scabbard fish, Lepidopus caudatus) (Palomba, Marchiori, et al., 2023). These larval parasites are maturing to the adult stage in elasmobranchs in European waters. Although these larval parasites are harmless to humans, a heavy infection could negatively impact the perceived quality of seafood in particular due to the visible larval stages of trypanorhynch species in the edible parts of infected fish. Indeed, their occurrence has been also documented in RASFF notifications (see Figure 1). There is also speculation that Trypanorhyncha larvae, i.e. Gymnorhynchus gigas and Molicola horridus, infecting the fish host musculature, could release proteins that might trigger allergic reactions in humans (Gòmez‐Morales et al., 2008). However, these proteins have not yet been identified in marine metacestodes.

Open in a separate window

FIGURE 1

Number of RASFF notifications of parasite infections in fish and fishery products from 2010 to end of 2023. (a) Includes parasites reported as nematodes without further specifying genus and species (i.e. Pseudoterranova). (b) Includes parasites other than nematodes (i.e., Trypanorhyncha, Opisthorchis, Acanthocephala, etc.). Unknown: notifications from unknown parasitic infections. Source: Graphic built by EFSA using data provided by RASFF (Annex B, last update on January 30 2024, data included until December 2023).

1.3.2.4. Acanthocephala

Acanthocephalans, using marine mammals, particularly whales, as the definitive hosts, use various fish species as paratenic hosts, in which the infective larval stage resides. Genera Bolbosoma and Corynosoma include species which are zoonotic. Two species within the genus Bolbosoma (B. capitatum and C. nipponicum) have caused human infections involving severe clinical symptoms. The cases, all in Japan, were all associated with prior ingestion of fish products (e.g. sashimi) (Mathison et al., 2021).

The genus Corynosoma, using primarily seals (pinnipeds) and to some extent whales (cetaceans) as definitive hosts, comprises two species, C. villosum and C. validum, which have been connected to human infections. Ingestion of undercooked fish products was suspected to be the source of infection (Mathison et al., 2021). Since various species of Corynosoma occur in wild populations of e.g. Atlantic cod (Setyawan et al., 2020), the zoonotic potential of European species cannot be excluded. However, no records from aquacultured fish infected with this parasite genus exist.

1.3.2.5. Cnidaria (Myxozoa)

At least two species within the myxosporidian genus Kudoa, i.e. K. thyrsites and K. islandica, are of concern to the European fishery and aquaculture industries since they may induce ‘soft flesh’ which is a rapid and severe post‐mortem enzymatic degradation of the fish body musculature. Both species can infect commercially important wild fish species such as Atlantic mackerel (Scomber scombrus) and wolffish (Anarhichas spp.) in NE Atlantic waters, respectively (Giulietti et al., 2022; Kristmundsson & Freeman, 2014). Kudoa thyrsites has been identified as the causative agent of ‘soft flesh’ in farmed Atlantic salmon in the Pacific, inflicting significant losses for the industry. There have only been two reports of K. thyrsites in farmed Atlantic salmon from Spain and Ireland (Barja & Toranzo, 1993; Palmer, 1994). The parasite has not been identified from other farmed fish species in the EU/EFTA. This may change, however, since climate‐related alterations in the ecosystem may facilitate the spread of K. thyrsites into northern European aquaculture systems.

Two Kudoa species, K. septempunctata and K. hexapunctata, have been identified as the causative agents of food poisoning following consumption of raw farmed or wild fish in Japan and Korea (Kang et al., 2020; Kawai et al., 2012; Sung et al., 2023; Suzuki et al., 2015). Other species may be involved in hitherto unclarified episodes of adverse reactions after raw fish consumption. Hence, in a Spanish study, anti‐Kudoa sp. antibodies were detected in persons showing allergic reactions after fish consumption (Martínez de Velasco et al., 2007). Although neither K. thyrsites nor K. islandica seem to cause food poisoning in humans, a hypersensitisation to the parasites cannot be ruled out.

1.3.2.6. Contamination of fish with other parasites

Occasionally, protozoan parasites that are not naturally infecting fish may contaminate fishery products. For example, the oocyst of Cryptosporidium, Toxoplasma and Blastocystis species, originating from terrestrial vertebrates, have been detected in oysters, mussels and marine coastal fish (Aco‐Alburqueque et al., 2022; Moratal et al., 2022; Santoro et al., 2020; Tedde et al., 2019). Oocysts of these parasites are washed by rainfall from the anthropogenically or livestock‐contaminated terrestrial areas, or sewage water and are accidentally retained by bivalves or fish species that feed by filtering seawater for plankton and microorganisms. Moreover, farmed seabass can be infected with the zoonotic species Cryptosporidium ubiquitum, thus representing a potential risk for consumers (Moratal et al., 2022). However, its typical infection site in fish, i.e. the gastrointestinal tract, is normally not consumed which largely prevents human infections. However, the possible risk of infection during the fish cleaning and food processing cannot be excluded. As stated in the interpretation of ToRs (Section 1.2), these parasites will not be considered in this assessment.

3.3.2.1. Bioassays

Bioassays are considered as the gold standard (Franssen et al., 2019) and experimental laboratory animals have been used to study the pathological reactions related to Anisakis, Phocanema and C. osculatum infections. Recent examples include studies of Anisakis larvae (Morsy et al., 2017; Romero et al., 2013; Zuloaga et al., 2013) or third‐stage C. osculatum larvae (Strøm et al., 2015). Bioassays have also been used for the study of C. sinensis metacercariae and Dibothriocephalus spp. (syn. Diphyllobothrium) plerocercoid larvae (Franssen et al., 2019) and O. viverrini metacercariae (Onsurathum et al., 2016). However, to reduce the use of experimental animals, in vitro assays have been adopted to replace the in vivo methods (Franssen et al., 2019). Indicators of behaviour, physiology, metabolism or physical damage of the parasite can be good alternatives for assessing inactivation.

3.3.2.2. Loss of larval mobility – Visual determination

The survival of anisakid larvae to different treatments is usually measured by their viability. A viable larvae is physically intact and motile measured by its ability to move spontaneously or after stimulation with tweezers and a needle (EFSA BIOHAZ Panel, 2010). This has been assessed under natural or UV light (Vidaček et al., 2011), at 37°C or at room temperature (Łopieńska‐Biernat et al., 2020; Sánchez‐Alonso et al., 2020), immediately or 24 or 48 h after a treatment (Onitsuka et al., 2022; Sánchez‐Alonso et al., 2020). Some authors specify the number of assessments (i.e. times showing no movement after stimulation) for a larva to be considered as dead (Lee et al., 2016), or use a video camera to record movements for a subsequent visual observation (Vidaček et al., 2011), while others have adopted viability scores (e.g. Trabelsi et al., 2019). With the exception of a few examples, where motility has been monitored directly, i.e. by candling (Pascual et al., 2010), the assays are usually performed on isolated larvae, either after manual extraction from the tissues, or by means of the artificial digestion method (Section 3.2.1.1). The latter may underestimate the number of mobile larvae and this limitation needs to be taken into account when designing inactivation protocols (Sánchez‐Alonso, Rodríguez, Tejada, et al., 2021).

3.3.2.3. Loss of larval mobility – Instrumental methods

Slow movements exerted by the larvae may not be detected by the observers (Kroeger et al., 2018) and visual inspection can be tiring and time consuming, since larva may remain motionless for long periods (Guan, Usieto, Cobacho Arcos, et al., 2023).

One technological solution that may be used instead of visual observations was developed for anisakid nematodes by Kroeger et al. (2018). Using near‐infrared imaging, the device measures elastic curvature energies and geometric shape parameters determined from contours, and these are used as a measure of viability. Authors reported that the device differentiated individual living and dead larvae isolated from marinated, deep‐frozen and salted products.

The need for the discovery of anthelminthic drugs for treating several parasitic infections has led to the development of medium‐ to high‐throughput phenotypic screening platforms which can assess not only worm motility but also a series of physiological, metabolic and morphological indicators (for review, see e.g. Herath et al., 2022). In particular, a ‘worm tracking’ commercial system which measures the interference of an infrared light beam by worm movement has been developed (Simonetta & Golombek, 2007), adapted for faster data acquisition (Taki et al., 2021) and applied to study Anisakis larvae (Ogata and Tagishi, 2021; Guan, Usieto, Cobacho Arcos, et al., 2023). Although these methods need to be tested on a larger number of samples including different anisakids or zoonotic parasites species, they may be used for high‐throughput screening in the future.

3.3.2.4. Loss of larval function other than mobility

The agar penetration test can be applied to substitute in vivo assays as there are good correlation of this in vitro method with bioassays (Ruitenberg, 1970). A similar observation was made in a study examining the differences in the penetrative ability of two Anisakis species (Arizono et al., 2012; Suzuki et al., 2010), which were independently corroborated with in vivo studies (Romero et al., 2013). Over the years, some modifications of the method have been adopted (Arizono et al., 2012; López et al., 2019; Sánchez‐Alonso et al., 2018; Suzuki et al., 2010). Other authors measured mobility, as the total distance that the larva migrated inside the agar medium, and motility, as the in situ movements of the larvae into the agar (Guan et al., 2021). In addition, indicators such as the mortality of the larvae upon incubation in an artificial gastric fluid (Arizono et al., 2012; Sánchez‐Alonso, Carballeda‐Sangiao, Rodríguez, et al., 2021), respiratory analysis of intact larvae (Sánchez‐Alonso et al., 2019), the use of dyes, the determination of physiological stress indicators, morphological features and integrity by microscopic examination (Franssen et al., 2019) could all be adopted as complementary measurements, but this would require research on their relationship with parasite infectivity.

At present, the relationship between the survival of the larvae, after a given treatment, and their infection capacity in humans remains unclear (EFSA BIOHAZ Panel, 2010), and as stated in the previous opinion, a precautionary principle is adopted (i.e. all larvae should be dead) which gives a higher margin of safety. However, the new approaches for the measurement of parasite inactivation discussed in this section could lead to the development and application of new indicators of treatment efficacy. High‐throughput methods that assess mobility (or the loss thereof), e.g. will enable operators to rapidly test high numbers of parasites thus facilitating research and the development of monitoring activities that could be applied in industry.

New studies on the factors affecting human host tissues penetration (in the case of infective A. pegreffii L3 in the gastrointestinal tract) will be an important step in understanding the pathogenesis of the disease (Palomba, Rughetti, et al., 2023) and may also provide the scientific basis for new strategies against anisakiasis, including the development of new indicators, e.g. by advanced molecular approaches.

3.3.3.2. Microwave treatment

The method of heating of solid foods by microwaves involves dielectric and ionic mechanisms, water in the food being the primary component responsible for dielectric heating since as water oscillates at the high frequencies of the microwave ovens, heat is produced. The oscillatory migration of ions in the food is the second mechanism that generates heat under the influence of the oscillating electric field (Datta & Davidson, 2000; Ozkoc et al., 2014). As for microorganisms, it is thought that temperature/time history at the coldest location will determine the safety of the process (Datta & Davidson, 2000). However, uneven distribution of the heat may occur in standard domestic microwave ovens so that turning food during cooking, covering food and adding liquid as it cooks all help to even out the heating (Datta & Davidson, 2000; Ozkoc et al., 2014).

Several studies show that temperatures ≤74°C combined with different times (i.e. 74°C, 15 s was the temperature/time combination recommended in EFSA BIOHAZ Panel (2010)) are effective in killing A. simplex (s. l.) L3 larvae. For example, Vidaček et al. (2009) microwaved water suspensions of ovaries and viscera tissues heavily infected with larvae at 900 W for 30 s, and no survivors were found. The maximum temperatures of the samples ranged between 67.8°C and 69.2°C. In hake sandwiches processed in a commercial 900 W microwave oven for 3 min, 66.9°C was reached in the thermal centre of the sandwiches immediately after treatment and no larvae survived (Tejada et al., 2006). This was also the case for larvae in a microwave digestion oven which allowed control of the time to reach a preset temperature as well as optimal distribution of the microwaves (Vidaček et al., 2011). Thus, in conditions where the target temperature was reached in 1 min, 60°C (800 W) or 70°C (1,000 W) for a holding time of 1 min was lethal to Anisakis larvae immersed in distilled water. In hake sandwiches, 5 min and 3 min were needed, respectively, for killing all tested larvae under these microwave conditions. In another study, fish sandwiches spiked with Anisakis spp. were placed in a microwave oven at 600 W. Samples were neither covered nor rotated. After 1 min, a temperature sensor was inserted into the sample without removing it from the oven. Once the temperature was registered, the sample was removed and maintained at room temperature for 15 min. Surviving larvae were not detected when the registered temperature was 75.6°C (Lanfranchi & Sardella, 2010) (Table 7).

The U.S. Food and Drug Administration (FDA, 2023) recommends, for raw animal foods, an internal temperature of 74°C in all parts of microwaved animal‐derived foods plus an additional 2 min of standing time for the covered food to achieve temperature equilibrium, although this is not specifically for zoonotic parasites. In addition, food should be rotated or stirred throughout or midway during cooking to ensure a more even distribution of heat and covered to retain surface moisture.

Results of survival after microwave treatment vary among studies owing to the lack of standardisation of wattage in standard domestic microwave ovens (usually 800–1200 W), uneven distribution of the microwaves, differences in size, shape, composition and dipolar components in the food. The effect of moisture and salt content in the products is significantly higher using microwaves as compared to conventional processing due to their effect on dielectric properties (Vidaček et al., 2011). More data should be gathered in order to define more specific lethal conditions for parasites in microwaved fishery products.

3.3.3.3. High‐pressure processing

High‐pressure processing (HPP) is a non‐thermal treatment in which foods are subjected to isostatic pressures. Traditionally, this is an in‐batch process that can be applied to both solid and liquid prepacked foods. Pressure is transmitted rapidly and uniformly in an isostatic manner so that all parts of the food are subjected to the same pressure simultaneously.

An EFSA scientific opinion on the efficacy and safety of high‐pressure processing of food has been recently published (EFSA BIOHAZ Panel, 2022). Within the industrial context, pressures of between 400 and 600 MPa are most often applied for microbial inactivation in foods, with common holding times ranging from 1.5 to 6 min. The water used as pressure‐transmitting fluid is often prechilled at 4–8°C.

HPP ranging from 100 to 350 MPa (1–5 min) was applied to A. simplex larvae and parasitised hake (Merluccius merluccius) muscle. The larvae were killed at pressures ≥200 MPa and times ≥1 min, producing alterations in the larva body and ruptures in the cuticle when observed by scanning electron microscopy (Vidacek et al., 2009). In this study, the increased shear resistance and changes in colour of the fish muscle were also detected. However, visual changes in appearance of the fish muscle were observed only at 300 MPa.

In studies undertaken since 2010, Lee et al. (2016) exposed the white spotted conger flesh containing 20 live larvae to different pressures (150 and 200 MPa for 1 and 5 min; 250 and 300 MPa for 1 min). The optimum HPP inactivation conditions for A. simplex L3 in the flesh were found to be 200 MPa at 5 min and 300 MPa for 1 min, since they rendered complete mortality of the tested larvae but the former was preferred since it resulted in no significant changes in colour and sensory analysis. In another study, infected hake sandwiches were treated at 200 MPa for 15, 30, 60–90 s. In these conditions, all A. simplex larvae survived, but none of them were able to penetrate into agar, thus suggesting an impaired infectivity after HPP application (Parasite Consortium, 2016). As indicated in the previous EFSA BIOHAZ Panel (2010) opinion, the changes in muscle colour and appearance may limit the application of HPP for the treatment of raw fish, but this treatment may be suitable for particular fish species or for processed fish (Table 7).

3.3.3.4. Pulsed electric fields

Pulsed electric field (PEF) treatments consist of subjecting a product placed between two electrodes, usually immersed in an aqueous solution, to high‐intensity electric fields (between 0.5 and 30 kV/cm) by applying intermittent pulses of short duration (microseconds to milliseconds) without increasing the product's temperature (Martínez et al., 2023).

Onitsuka et al. (2022) applied PEF to fillets of horse mackerel (Trachurus japonicus) spiked with Anisakis, presumably A. pegreffii. The fish fillets were immersed in a saline solution, and pulsed power was applied. They observed optimal inactivation at NaCl solutions with electrical conductivity of 5 mS/cm, charging voltage 15 kV, capacitance 80 μF, frequency 1 Hz, the distance between electrodes 11 cm and 500 pulses. Sensory evaluation after the treatment showed that, although slightly lower than the control, the fish retained their quality as sashimi.

Abad et al. (2023) evaluated the efficacy of PEF on the inactivation of Anisakis spp. larvae (n=10 larvae per condition in triplicate, total 480 larvae in the calibration experiment) in an aqueous solution. Response surface methodology was used and a central composite design was constructed to evaluate parameters such as electric field strength (1–3 kV/cm), specific energy (3–50 kJ/kg) and pulse width (3–100 μs) on Anisakis survival and data obtained were described by a second‐order polynomial equation. The viability of Anisakis was highly dependent on field strength. The loss of survival as a function of specific energy was higher at the highest field strength (3 kV/cm). On the other hand, at the lowest field strength (1 kV/cm) pulse width exerted a significant influence, whereas no effect was found at the highest field strength (3 kV/cm). These results were validated with an independent experiment in aqueous solution with very good relationship between the predicted and experimental data. Maximum inactivation (i.e. 90%–100%) was obtained at 3 kV/cm and 50 kJ/kg.

In hake spiked with Anisakis (n=10 larvae per condition, total of 600 larvae), Abad et al. (2023) used pulses of 30 μs and 50 kJ/kg specific energies, with varying field strengths (1 to 3 kV/cm) and compared the percentage of survivors with the expected mortality according to the polynomial model developed in isolated larvae. A good agreement was found with the model, confirming that a treatment condition using 30 μs, 50 kJ/kg and 3 kV/cm is capable of inactivating a significant percentage of Anisakis present in pieces of hake (i.e. 80%–100%). The authors observed that the application of this optimal PEF treatment to hake fillets affected moisture, water‐holding capacity and cooking loss to a lesser extent than if these fillets were frozen at −18°C for 48 h and then thawed.

In another study, Onitsuka et al. (2024) studied: (a) the effect of water conductivity, (b) the capacitance, (c) charging voltage of the energy storage capacitor, (d) the input energy density into the treatment electrode, (e) the distance between parallel plate electrodes, (f) the position of the larvae in the fish fillet, or (g) in whole fish, and (h) the fish species in spiked and in naturally parasitised fish. They observed that the higher the input energy density per pulse (J/cc/pulse), the higher the inactivation effect of Anisakis larvae. However, the inactivation efficiency was affected by the position of the larva in the fish flesh owing to the effect of the shape and composition on current distribution. The authors did not find differences between naturally parasitised and spiked fish. Unlike previous studies (Abad et al., 2023; Onitsuka et al., 2022), they achieved complete immobility even at about total input energy density of 10 J/cc, suggesting that Anisakis can be inactivated at relatively low energy. According to this study, the applied energy was significantly lower than that used in their previous work (260 J/cc) (Onitsuka et al., 2022) and of Abad et al. (2023) (3 kV/cm; 50 J/cc) (Table 7). The authors suggested that more studies need to be performed to improve efficiency and to standardise conditions (Onitsuka et al., 2024).

Martínez et al. (2023) reviewed the potential application of PEF technology in the control of food‐borne parasites. The authors highlighted the need to continue research related to the execution and scaling of PEF treatments to facilitate the development of industrially competitive processes. They considered that the presence of bubbles, the need to design new treatment chambers and the absence of sufficiently powerful equipment are problems to be solved for the treatment of solid products such as fish or fish fillets. The electrical parameters must also be carefully optimised to avoid alterations in quality and a case‐by‐case analysis of the PEF efficiency in each of the matrices is necessary.

3.3.4.2. Marinating

Marinating is a traditional process in which fish is immersed in vinegar and other ingredients, with the aim of inhibiting or retarding bacterial growth by lowering the pH and also conferring desired organoleptic characteristics on the product (EFSA BIOHAZ Panel, 2010; Sampels, 2015). Since 2010, different marinating solutions have been investigated by exposing isolated Anisakis spp. larvae in the solution, which included salt and sugar, lemon juice, acetic acid, alcohol, wine and/or apple vinegar. The survival of larvae immersed in these solutions was about 5days in lemon juice and lemon juice with acetic acid. In alcohol vinegar, larvae survived for only 48 h (Šimat & Trumbić, 2019). Other authors have included citric acid, acetic acid and NaCl at different concentrations and observed inactivation of isolated larvae at times ranging from 12–17 h to 22–32 h, and reported a complex relation between larval size and different marinating solutions in Anisakis type I larvae (Giarratana et al., 2012). However, these studies may not reflect what happens when the fish is marinated. Carpaccio and wine vinegar marinades, for example, failed to kill Anisakis spiked in anchovies during the marinating process, which were only inactivated after 60days of storage (Šimat & Trumbić, 2019).