(PDF) Anisakiasis and Anisakis: An underdiagnosed emerging disease and its main etiological agents

Adroher-Auroux, F.J., Benítez-Rodríguez, R., 2020. Res. Vet. Sci. 132, 535–545. https://doi.org/10.1016/j.rvsc.2020.08.003Anisakiasis or anisakiosis is a human parasitic infection caused by the third-stage larvae (L3) of nematodes of the genus Anisakis, although the term is also used in medical literature for the much less frequent (<3% of cases) infection by L3 of other genera of anisakids, particularly Pseudoterranova. These parasites have a marine lifecycle. Humans are infected by the L3 through ingesting of fish and squid, the intermediate/paratenic hosts. The live larvae generally penetrate the wall of the stomach or intestine causing, among other symptoms, intense pain or allergic symptoms. These are emerging, cosmopolite illnesses. Diagnosis and treatment is usually by endoscopy and extraction and identification of the larvae. Allergic forms are usually diagnosed by prick-test and/or allergen-specific IgE detection and treated with a suitable anti-allergy treatment. The patient is also warned against further consumption of marine fish or squid, as these may be infected with Anisakis. The most common method of prevention is thermal treatment of the entire fish or squid prior to consumption (>60 °C, >1 min or − 20 °C, >24 h). Useful measures for the control of anisakiasis would be to establish a national register of cases, to initiate educational campaigns for the general public and consciousness-raising and training campaigns for health professionals. These would be complemented by control measures for the relevant sectors of the economy: fish operators, fish farming, fishermen, fishmongers, fish industry and catering facilities. Possible genetic predisposition for allergy to Anisakis and the possible relationship between anisakiasis and cancer would also require further investigation.

This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 1 For: Research in Veterinary Science, Special Issue: “Foodborne and Waterborne Zoonotic 2 Parasites of Veterinary and Medical Importance” 3 4 Title: Anisakiasis and Anisakis: an underdiagnosed emerging disease and its main etiological 5 agents 6 Authors: Francisco Javier Adroher-Auroux* and Rocío Benítez-Rodríguez# 7 8 Address: Departamento de Parasitología, Facultad de Farmacia, Universidad de Granada, 9 18071-Granada, Spain 10 *Corresponding author: F.J. Adroher (fadroher@ugr.es). ORCID ID https://orcid.org/0000- 11 0002-7969-6658 12 13 Highlights 14 1.- Anisakiasis is an emergent, cosmopolite, subdiagnosed seafood-borne parasitic zoonosis. 15 2.- Awareness and training of health personnel is decisive for the diagnosis of anisakiasis. 16 3.- Anisakiasis prevention and control measures are effective if implemented. 17 4.- The development of less invasive and more specific diagnostic methods is required. 18 5.- The relationship of anisakiasis with cancer and other diseases needs to be clarified. 1 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 19 Abstract 20 Anisakiasis or anisakiosis is a human parasitic infection caused by the third-stage larvae (L3) of 21 nematodes of the genus Anisakis, although the term is also used in medical literature for the 22 much less frequent (<3% of cases) infection by L3 of other genera of anisakids, particularly 23 Pseudoterranova. These parasites have a marine lifecycle. Humans are infected by the L3 24 through ingesting of fish and squid, the intermediate/paratenic hosts. The live larvae generally 25 penetrate the wall of the stomach or intestine causing, amongst other symptoms, intense pain 26 or allergic symptoms. These are emerging, cosmopolite illnesses. Diagnosis and treatment is 27 usually by endoscopy and extraction and identification of the larvae. Allergic forms are usually 28 diagnosed by prick-test and/or allergen-specific IgE detection and treated with a suitable anti- 29 allergy treatment. The patient is also warned against further consumption of marine fish or 30 squid, as these may be infected with Anisakis. The most common method of prevention is 31 thermal treatment of the entire fish or squid prior to consumption (>60 ºC, >1 min or -20 ºC, 32 >24 h). Useful measures for the control of anisakiasis would be to establish a national register 33 of cases, to initiate educational campaigns for the general public and consciousness-raising and 34 training campaigns for health professionals. These would be complemented by control 35 measures for the relevant sectors of the economy: fish operators, fish farming, fishermen, 36 fishmongers, fish industry and catering facilities. Possible genetic predisposition for allergy to 37 Anisakis and the possible relationship between anisakiasis and cancer would also require 38 further investigation. 39 Keywords: Anisakiasis; allergy; seafood-borne disease; Anisakis; diagnosis; control. 2 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 40 Introduction. 41 Anisakidosis or anisakiasis is an infradiagnosed, emerging, cosmopolite illness. It entails the 42 accidental infection of humans by the third larval stage (L3) of parasitic nematodes of the 43 family Anisakidae (genera Anisakis, Pseudoterranova and, very rarely, Contracaecum), which, 44 with an aquatic, mainly marine, lifecycle have marine mammals and fish-eating birds as 45 definitive hosts and crustaceans, cephalopods and fish as intermediate/paratenic hosts. It has 46 also been related to anisakiasis, although rarely, to raphidascarid Hysterothylacium aduncum 47 (Yagi et al., 1996), which is very close to anisakids (Fig. 1). Cases of allergy to these parasites 48 are also considered as anisakiasis (Audícana and Kennedy, 2008). 49 Infection by larvae of the genus Anisakis is specifically classified as anisakiosis, although the 50 term anisakiasis is the term most commonly employed in medical literature, perhaps as a 51 result of the etiological agent of 97% of cases of anisakiasis being L3 of the complex A. simplex 52 sensu lato, specifically the species A. simplex sensu stricto and A. pegreffii. 53 The presence of Anisakis in fish has been known at least since the 13th century (Myers, 1976). 54 In 1767, Linnaeus named it Gordius marinus, while, in 1809, Rudolphi described larvae in fish 55 and adults in porpoise, but without relating them (Rudolphi, 1809). Finally, in 1845, Dujardin 56 created the genus Anisakis, in which this parasite is included with the name Anisakis simplex 57 (Rudolphi, 1809). 58 It was not known that it could affect humans. Hitchcock (1950) observed a larva, probably 59 Anisakis, in the faeces of an Inuit in Alaska. The first cases in which symptoms were associated 60 with the presence of Anisakis larvae were diagnosed by Dr. Straub in the Netherlands in 1955- 61 59 (van Thiel et al., 1960). 62 The L3 of the genus Anisakis are very similar morphologically (Fig. 1), traditionally classified as 63 type I or type II larvae , with the species within each type being morphologically 3 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 64 indistinguishable (Berland, 1961). They can however be differentiated molecularly and have 65 been grouped into 4 clades (Mattiucci and Nascetti, 2008) (Table 1). 66 Transmission and epidemiology 67 Humans are infected on ingesting viable L3 found in host fish and squid, especially by those in 68 the muscle tissue since those in the visceral cavity are eliminated when the fish is gutted for 69 culinary purposes. However, some small fish are consumed whole, in which case the worm 70 larvae in the body cavity can also cause anisakiasis. Almost all species of teleost fish studied 71 throughout the oceans can act as hosts for Anisakis larvae. Consequently, the consumption of 72 fish or some species of squid is a source of infection in humans. 73 Each year thousands of cases are reported globally, particularly in developed countries with a 74 significant fishing industry, high per capita consumption of fish and where there is a culinary 75 tradition of dishes featuring raw fish or squid. Japan is the country where most cases are 76 diagnosed, over 500 annually (Suzuki and Murata, 2011), but estimated to be approximately 77 7,000 (Sugiyama et al., 2013, cited in IARS, 2017). In other countries with high fish 78 consumption, such as Spain, the number of reported cases is much lower, at around 150/year 79 (Herrador et al., 2019). Although increased information and raising awareness in health 80 professionals results in more effective diagnosis (Castán et al., 2002), some authors have 81 expressed concern regarding the continued infradiagnosis. For example, in Spain, Bao et al. 82 (2017) used a quantitative risk assessment model to predict around 8000 cases/year of 83 anisakiasis due to consumption of anchovies, while Herrador et al. (2019), working with 84 hospitalization data, calculated 10,000-20,000 cases. However, only around 500 cases are 85 reported annually in the whole of Europe. The global frequency of infection is estimated at 86 0.32 cases/100,000 inhabitants (Orphanet, 2020), with cases of anisakiasis diagnosed in more 87 than 20 countries. 4 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 88 The lifecycle of Anisakis (Fig. 2) can start in the stomach chambers of odontocete cetaceans, 89 definitive hosts, in which adult worms are free-living (Fig. 3). The female is fertilized by the 90 male and lays eggs which are passed in the host’s faeces. The eggs develop in the sea up to L3 91 which then hatch (Køie et al., 1995). The L3 (200-300 μm) is enclosed in a sheath, the L2 92 cuticle, and can survive for up to 3-4 months (Højgaard, 1998) in seawater until predated by 93 the first suitable intermediate host, a crustacean, generally euphausiids (Smith, 1983, 1971) or 94 large calanoid copepods (Klimpel et al. 2004), although smaller plankton crustaceans could act 95 as paratenic hosts, prior to ingestion by the first intermediate host (Klimpel et al., 2004; Køie, 96 2001; Shimazu, 1974). The L3, after losing its sheath, passes from the digestive system of the 97 crustacean into the coelomic cavity. Here it undergoes a period of growth, which can reach up 98 to 32.7 mm in these hosts (Smith, 1983). These larvae are now infective for the definitive host. 99 However, in order to reach an odontocete cetacean, the cycle must continue through a second 100 suitable intermediate host, in this case squid (rarely any other cephalopod) or teleost fish 101 which predate on euphausiids. When the infected crustacean is ingested, the L3, in order to be 102 infective for the host, must have attained a minimum size (L3 from 8.8 mm have been 103 recorded in host fish; Smith, 1983). The L3 then passes from the host’s digestive system to the 104 visceral cavity (Wootten and Smith, 1975). Occasionally, L3 pass from the visceral cavity to the 105 fish musculature. This L3 will be infective for the definitive host, for which it must apparently 106 have a suitable size (Asami and Inoshita, 1967; Iglesias et al., 1997; Kikuchi et al., 1967; vs 107 Beverley-Burton and Pippy, 1977; Hays et al., 1998). Next, when the intermediate host is 108 ingested by a cetacean the L3 occupy the stomach chambers, most often the first, and attach 109 themselves to the gastric mucosa, normally in groups, where they cause an ulcer. The L3 moult 110 to L4 which remain together in the ulcers until the final moult to adult when they generally 111 free themselves and grow and mature in preparation for mating while roaming throughout the 112 stomach chambers (Højgaard, 1999; Kikuchi et al., 1967; Smith, 1989, 1983; Young and Lowe, 113 1969). The adult female (4.5-15.0 cm) is usually larger than the male (3.5-7.2 cm), with the 5 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 114 cycle being completed when the male fertilizes the female (Grabda, 1976; Iglesias et al., 2001; 115 van Banning, 1971). 116 In nature this lifecycle is complicated by the undefined number of paratenic hosts which can 117 potentially intervene (Fig.2). If an L3 can to infect the next host but its paratenic host is then 118 ingested by a host from the same level the larva will again occupy the visceral cavity, either 119 free or encapsulated, and will not progress. This host will thus be paratenic. As the 120 intermediate and paratenic hosts are the same they are normally known as 121 “intermediate/paratenic hosts” as they can perform both functions according to the larval 122 development. This large number of potential hosts for the genus Anisakis, especially with 123 regard to intermediate/paratenic hosts, allows the parasite to follow alternative lifecycles, 124 depending on both biotic and abiotic factors, for its adaptation and survival at different marine 125 latitudes (Klimpel et al., 2004; Kuhn et al., 2016). However, many aspects of the Anisakis 126 lifecycle remain unclear and must be studied to clarify them. 127 Despite the large number of hosts that may be involved in the lifecycle of these parasites not 128 all cetaceans are suitable definitive hosts for all species: Delphinidae are ideal for species of 129 the complex A. simplex s.l. and for A. typica, Ziphiidae for A. ziphidarum and A. nascetti, 130 Physeteridae for A. physeteris and Kogiidae for A. paggiae and A. brevispiculata (Klimpel et al., 131 2010; Mattiucci et al., 2018; Mattiucci and Nascetti, 2008). Other cetaceans, such as misticetes 132 (Raga et al., 1986), can serve as occasional definitive hosts, but not other marine mammals, 133 such as the pinnipeds, in which the parasites do not attain maturity (Skrzypczak et al., 2014). 134 Clinics and pathogenesis 135 Infection in wild and farmed fish. 136 Fishermen have been aware of the presence of these parasites in fish for centuries, as 137 commented. In some fish, such as herring, they were so abundant that that they were believed 138 to be the main food source for these fish (see comment in van Beneden, 1870, p. 65). 6 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 139 Templeman et al. (1957) drew attention to the commercial problem represented by the 140 presence of larvae of Porrocaecum (=Pseudoterranova) and Anisakis in fish, particularly cod, 141 suggesting that a reduction in the number of seals (definitive hosts of Pseudoterranova) at the 142 fishing grounds could reduce the parasite load of these fish. To date more than 200 species of 143 fish, including many of commercial value, have been reported as hosts of Anisakis, with more 144 added each year. Such valuable fish as cod, salmon, hake, saithe, redfish, blue whiting, 145 pouting, horse mackerel, sardine, anchovy, mackerel, herring, etc. are habitual hosts, showing 146 high prevalence and intensity, with these being affected by capture zone and the size/age of 147 the fish (Adroher et al., 1996; Beck et al., 2008; Levsen and Lunestad, 2010; Molina-Fernández 148 et al., 2018, 2015; Rello et al., 2009, 2008; Strømnes and Andersen, 1998; Valero et al., 2006). 149 The presence of cetaceans in and around the fishing grounds may help to perpetuate the 150 lifecycle of the parasite (Rello et al., 2009), while, since L3 of Anisakis can live within a fish for 3 151 years or more (Smith, 1984), parasites tend to accumulate as the fish grows (Bussmann and 152 Ehrich, 1979; Valero et al., 2000). Nonetheless, in some cases older fish may show a reduction 153 in parasite intensity, possibly due to a better immune response from the host (Dezfuli et al., 154 2016; Serrat et al., 2019) or to the death of the hosts with the highest parasite load (Levsen 155 and Berland, 2012; Strømnes and Andersen, 1998). 156 The passage of Anisakis larvae through the stomach wall of the fish results in the formation of 157 ulcers, although these do not seem to affect the normal performance of the organ (Jones, 158 1994) and nor does the presence of the parasites in the visceral cavity (Fig. 4A) appear to 159 significantly affect the host’s health, regardless of whether they are free or encapsulated on 160 the liver (Wootten, 2012). The effects of Anisakis on fish have rarely been considered and are 161 worthy of further study, particularly as Anisakis has recently been related to red-vent 162 syndrome in Atlantic salmon (Beck et al., 2008). In any case, Fulton’s condition factor (CF) 163 (Fulton, 1904) for fish is a general indicator of their health (Monstad, 1990). According to this, 164 a parasitized fish should show a lower value of CF to one free from parasites. However, this is 7 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 165 open to debate. Some authors have found CF in parasitized fish to be unaffected (Molina- 166 Fernández et al., 2018, 2015; Mouritsen et al., 2010). On the other hand, other authors have 167 suggested that CF is not only affected when the intensity of parasites is high but also by other 168 factors such as season, age/length or maturity of the fish (Richards, 1977; see Rohde, 1984, 169 and references therein). However, Serrat et al. (2019) suggest that the CF will only be affected 170 if the parasite load affects the availability of energy for the fish. This controversial topic 171 requires further study. 172 In marine fish-farming, although the feed is processed to avoid infection, it is still possible for 173 small prey animals (crustaceans, squid and small fish) infected with Anisakis to pass through 174 the netting of the cages and to be ingested by the fish. However, although a few cases of 175 caged fish being infected with Anisakis (reviewed by Lima dos Santos and Howgate, 2011; Mo 176 et al., 2014) have been reported prevalence is generally very low or nil (Brooker et al., 2016; 177 Cammilleri et al., 2018; Peñalver et al., 2010). Other practices such as the capture of wild fish 178 such as cod and tuna for subsequent growing-on in cages present a greater risk as these may 179 already be infected when captured and may cause anisakiasis when sold (Heuch et al., 2011; 180 Smrzlić et al., 2012). Consequently, the possibility of farmed fish being parasitized should 181 always be taken into consideration (Rückert et al., 2008). 182 Infection in cetaceans. 183 There are many species of cetaceans in which Anisakis has been detected in their stomach 184 chambers (Fig. 4B). As described previously, these nematodes form clusters on the gastric 185 mucosa forming an ulcer to which they remain attached until moulting to adults (Højgaard, 186 1999; Young and Lowe, 1969), some 3-5 weeks later (Iglesias et al., 2001), by penetrating the 187 mucosa with their anterior end, even reaching the submucosa (Kikuchi et al., 1967; Young and 188 Lowe, 1969). These ulcers may be associated with oedemas, haemorrhages and alterations 189 such as eosinophilic and granulomatous inflammation with giant cells, hemosiderosis, fibrosis 8 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 190 and areas of necrosis associated with location of parasites within the gastric mucosa (Motta et 191 al., 2008). Fernández-Maldonado (2016) suggested that the presence of Anisakis in cetaceans 192 may be related to a state of immunosupression associated with microbial systemic infections 193 in addition to significant digestive disorders. Dermal symptoms have also been described (van 194 Beurden et al., 2015). Recent studies seem to show that Anisakis populations in cetaceans 195 could be increasing over recent decades (Pons-Bordas et al., 2020). At least, captive marine 196 mammals can be treated, with varying degrees of success, using thiabendazole or levimasole 197 (Smith and Wootten, 1978). 198 Infection in humans. 199 Man is an accidental host of Anisakis in whom the live L3, ingested on consuming raw or 200 under-cooked fish or squid, tend to attach to the gastric mucosa, and, on occasions, the 201 intestinal mucosa (Fig. 4C). Their attempts to penetrate the mucosa are generally unsuccessful. 202 A single larva can cause symptoms. This L3 very rarely moults to L4 in humans (van Thiel et al., 203 1960). The type of anisakiasis produced depends on the location of the larva and the 204 symptoms it is causing: 205 Gastric anisakiasis: the attaching of the larva to the gastroduodenal mucosa gives rise to 206 intense epigastric pain which may be accompanied by other symptoms such as nausea, 207 vomiting, urticaria and diarrhoea (Furuya et al., 2018) which develop between 2 to 6 hours 208 after ingestion of the larva. The symptoms last as long as the larva is alive. This is the most 209 common form of anisakiasis, accounting for 72% of cases (Valls Sánchez et al., 2009). 210 Intestinal anisakiasis: when the larva attaches to this part of the digestive system symptoms 211 start to appear 2 to 3 days after ingestion, typically severe abdominal pain which may be 212 accompanied by nausea, vomiting and/or diarrhoea. Occasionally, a chronic form develops, 213 resulting in the formation of granulomas or abscesses. These may resemble episodes of 9 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 214 appendicitis or intestinal obstruction accompanied by an oedema with fibrinous exudate (Valls 215 Sánchez et al., 2009). 216 It is not clear why the larva attaches to the gastric or intestinal mucosa. However, in Japan the 217 intestinal form has been related to factors pertaining to the human host such as sex, habitual 218 consumption of alcohol or cardiac risk (Yamamoto et al., 2020). Geographical factors, 219 potentially related to culinary traditions, may also be involved. In both cases, in the zone of 220 attachment, the organism reacts by generating an eosinophilic granuloma which normally kills 221 the larva in 1 to 2 weeks, at which point the symptoms disappear. 222 Ectopic or extraintestinal anisakiasis: is rare, occurring when the larva passes through the 223 digestive wall giving rise to ‘visceral larva migrans syndrome’. There are no specific symptoms 224 and these depend on the organ affected. 225 Allergic anisakiasis or ‘allergy to Anisakis’: occurs when the presence of larval allergens (due to 226 live or dead parasite larvae) triggers an allergic response in the host, with symptoms ranging 227 from urticaria (Kasuya et al., 1990) and/or angioedemas to anaphylaxis (Audícana et al., 1995; 228 see section 3.2.1 in EFSA-BIOHAZ, 2010). These usually appear within the first hour after 229 consuming the parasitized fish. Although it is not absolutely clear whether a dead larva 230 (following cooking or freezing of the fish) is capable of sensitizing the subject, there is evidence 231 to suggest that human sensitization can only be produced by contact with live larvae, although, 232 once sensitized, the subject will also exhibit an allergic response to dead larvae (Alonso-Gómez 233 et al., 2004; Audícana et al., 2002; Daschner et al., 2012). 234 Gastroallergic anisakiasis: The requirement for contact with live larvae for sensitization to 235 occur has given rise to the name of this type of anisakiasis, in which digestive and allergic 236 symptoms are combined (Alonso et al., 1997; Daschner et al., 2000), being defined as a severe 237 case of IgE-mediated allergy, typically accompanied by gastric digestive symptoms (Valls 10 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 238 Sánchez et al., 2009). This acute form may evolve, in some cases, into chronic urticaria 239 (Petithory, 2007). 240 Occupational anisakiasis: there is evidence for occupational allergy (dermatitis, asthma, 241 conjunctivitis) caused by A. simplex in fishermen, fishmongers or other fish industry workers 242 (Añíbarro and Seoane, 1998; Armentia et al., 1998; Carretero Añíbarro et al., 1997; 243 Nieuwenhuizen et al., 2006; Purello-D’Ambrosio et al., 2000). 244 Diagnosis 245 Diagnosis of anisakiasis is not easy due to the lack of specificity of the symptoms. An 246 anamnesis is necessary (Del Rey-Moreno et al., 2008) to establish the consumption, in the 72 247 hours previous to the appearance of symptoms, of sea fish, either raw, marinated, pickled in 248 vinegar, in brine or seared, that is, inadequately cooked such that it contains live larvae of 249 Anisakis, for patients with acute epigastric or abdominal pain. Following confirmation there 250 are several diagnostic tests to aid the doctor’s decision-making. 251 Diagnosis of infection by Anisakis. 252 From the first cases of anisakiasis to be identified up to the end of the 20th century diagnosis 253 was generally by identification of the larvae in the eosinophilic granulomas or in segments of 254 the digestive tract affected (Fig. 5), which were surgically resected. However, advances in 255 imaging techniques, especially in endoscopy, have made them more easily available and 256 financially viable for patients suspected of infection following the anamnesis. In this sense, 257 endoscopy is currently the technique of choice for diagnosing acute gastric or intestinal 258 anisakiasis (Bucci et al., 2013). In addition to being able to observe the larvae (Fig. 4C) or 259 fragments of these using gastroscopy or colonoscopy it is also possible to extract them for 260 posterior identification by their morphological features and thus avoid unnecessary surgery 261 (Fig. 6). Furthermore, it has been suggested that the computed tomography, performed before 262 endoscopy, is useful for the diagnosis (Ashida et al., 2017; Shibata et al., 2014). 11 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 263 On occasions, in the absence of a precise diagnosis, particularly in the case of intestinal 264 anisakiasis, exploratory surgery is required. This enables observation and extraction of the 265 larvae from either the abscess or the eosinophilic phlegmon. The anatomopathological study 266 of these samples may reveal sections of the larvae: In the transverse sections we will observe a 267 fine cuticle without wings, a well-developed polymyarian subcuticular musculature, lateral 268 nerve cords in a “Y” shape and smaller dorsal and ventral cords. If the section is of the first 269 third of the larva the muscular esophagus with a tri-radiate lumen can be observed, as can the 270 excretory cell or the excretory tubule (Fig. 6). If the section is of the posterior two thirds the 271 intestine can be observed, as can its epithelium with columnar cells with the nucleus at the 272 base of each cell. Saggital or oblique sections are more difficult to identify. If the larva cannot 273 be identified due to its poor state, a molecular diagnosis can be carried out following DNA 274 extraction (D’Amelio et al., 2012; Paoletti et al., 2018). 275 Diagnosis of allergy to Anisakis. 276 When allergic symptoms are present a prick-test with Anisakis L3 antigen can be performed. 277 Other techniques may be used to determine specific IgE in the patient’s serum (Petithory, 278 2007) such as ImmunoCAP® with total antigen or with selected purified or recombinant 279 antigens from the parasite, the most widely utilized being Ani s 1, responsible for the majority 280 of cases of sensitization to Anisakis. Some authors have raised doubts concerning these 281 techniques due to possible cross reactions leading to false positive results (Mattiucci and 282 D’Amelio, 2014). Radioallergoabsorbence (RAST) permits differentiation between subclinical 283 allergy and anaphylaxis (Desowitz et al., 1985), but is an expensive technique. 284 Currently, the most highly-rated techniques are those based on microarrays (Armentia et al., 285 2017) such as ImmunoCAP® ISAC (Immuno Solid-phase Allergen Chip) to detect, above all, the 286 thermostable allergens Ani s 1 and Ani s 3 as these permit differentiation between patients 287 who could experience anaphylaxis (sensitive to Anis s 1) and those only having a subclinical 12 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 288 allergy (sensitive to Ani s 3). Furthermore, the former is detected in 88% of patients affected 289 by gastroallergic anisakiasis (Shimakura et al., 2004; Subiza, 2020). 290 Anadón et al. (2010) believed the recombinant allergens rAni s 1 and rAni s 7 to be the best 291 option for serological diagnosis of anisakiasis in terms of sensitivity and specificity (Trisakis-170 292 ELISA kit) since Ani s 3 (tropomyosin) is a panallergen and gives cross reactions with 293 tropomyosins from other invertebrates such as shrimps, cockroaches or mites (Daschner et 294 al., 2012; Guarneri et al., 2007). 295 Although these techniques are considered valid for the diagnosis of allergy to Anisakis, the fact 296 that part of the healthy population show positive responses to the allergens of Anisakis (Del 297 Rey Moreno et al., 2006) raises doubts regarding the choice of suitable techniques or allergens 298 (Table 2). In this sense, Mazzucco et al. (2018) emphasized that the estimates of 299 hypersensitivity varied greatly according to geographical zone, population characteristics, 300 diagnostic criteria and laboratory assays. 301 Treatment 302 The treatment for infection by Anisakis consists of extracting all the larvae found in the 303 digestive tract during the endoscopic examination (Fig. 7), either by gastroscopy or 304 colonoscopy, depending on the zone affected (Audícana Berasategui et al., 2007). There is no 305 completely efficacious drug available for the treatment of anisakiasis. However, since 306 endoscopy permits the immediate removal of the larvae, leading to an improvement in the 307 patient’s condition within a few hours, no pharmacological treatment is required in these 308 cases. The most widely-used drug is albendazole with other anthelmintics such as 309 thiabendazole, flubendazole or ivermectine also used, although none is completely efficacious 310 (Shimamura et al., 2016). Current studies are focusing on natural substances such as wood 13 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 311 creosote (Sekimoto et al., 2011) and essential oils of plants and their components (reviewed by 312 Valero et al., 2015). 313 When the larva is located in zones of the intestine which are difficult to access, some authors 314 recommend a conservative treatment with fluid therapy and antibiotics, which usually improve 315 the patient’s condition (Audícana Berasategui et al., 2007; Shrestha et al., 2014). Occasionally, 316 however, laparotomy and resection of the affected fragment of the intestine is required. 317 ‘Allergy to Anisakis’ should be treated immediately, with the recommendation that the patient 318 should avoid consuming marine fish (Petithory, 2007). In allergic patients suspected of 319 suffering anaphylactic shock, they must be provided with an injectable dose of adrenaline to 320 cope with the contingency and, if it occurs, immediate referral to hospital (Audícana 321 Berasategui et al., 2007). 322 Prevention and control 323 The prevention of anisakiasis is based on avoiding the ingestion of live larvae of Anisakis 324 through consumption of raw or under-cooked fish or squid. These should have undergone 325 some form of termal treatment since preparation with vinegar or lemon juice, smoking, 326 brining, pickling, marinating, etc. does not always inactivate all the larvae (EFSA-BIOHAZ, 327 2010). It is generally considered that fish and squid should be cooked at >60 ºC for >1 min or 328 frozen whole at -20 ºC for >24 h (or -35 ºC, >15 h) to ensure the death of the larvae (EU, 2011). 329 This eliminates the possibility of infection by Anisakis and sensitization to its allergens. 330 However, if the subject is already sensitized it is still possible for them to develop allergic 331 symptoms (Alonso-Gómez et al., 2004; Audícana et al., 2002; Daschner et al., 2012). 332 Anisakis is difficult to control due to its complex lifecycle and the large number of potential 333 hosts, particularly paratenic hosts. However, there is a variety of measures to reduce the 14 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 334 incidence of human anisakiasis. The first is health education, which should principally consist 335 of raising awareness regarding prevention in consumers. This education should be broadened 336 and adapted to different economic sectors of the population, such as catering or fishing. Public 337 health agencies (FDA, 2019) and experts committees (EFSA-BIOHAZ, 2010) have recommended 338 a series of measures to become part of a countries’ legislation. In the European Union (EU, 339 2019, 2011, 2004) and other countries as Japan in 2012, most of these measures are now 340 included in current legislation. These measures require: 341 - establishments serving food or selling prepared food, to freeze fish and squid that are to be 342 consumed raw or under-cooked. 343 - high seas fish processors, to gut fish and squid to avoid migration from body cavity to muscle 344 tissues (Borderías and Sánchez-Alonso, 2011) and to destroy these viscera rather than to 345 dispose of them in the sea where they would disseminate parasites. 346 - boats, not to fish in zones where the parasites are known to be abundant, for example, those 347 close to areas with high numbers of cetaceans, definitive hosts of Anisakis, where prevalence 348 of the parasite in fish is usually higher than elsewhere (Rello et al., 2009). 349 - fish farms, to feed fish with products that are free of viable parasites capable of affecting 350 human health. 351 - food business operators, to guarantee that fish products are inspected visually to detect 352 visible parasites before going on sale and that fish with visible parasites are destroyed. 353 - food industry businesses selling fish products intended to be consumed raw, marinated, 354 salted or other treatments that are not sufficient to inactivate the parasites, to treat them, in 355 their entirety, at >60 °C for >1 min or, alternatively, to freeze them at -20 °C for >24 hours, or - 356 35 °C for >15 hours, to ensure inactivity of the larvae. 15 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 357 Countries should incorporate these measures into their legislation. It may also be useful to 358 establish the prevalence of anisakiasis in each country with a view to implementing and 359 managing public health resources effectively. This would require that every country have a 360 mandatory national register of all diagnosed cases. Finally, the recommended thermal 361 treatment measures, applicable to many foods, prevent not only anisakiasis but many 362 foodborne pathogens. 363 Future studies and research needed. 364 While much progress has been made, our knowledge of Anisakis and anisakiasis is still limited. 365 Here, we will only address two important aspects: commercial and sanitary. 366 From a commercial viewpoint the presence of Anisakis in fish products is an aesthetic issue 367 since worms visible to the naked eye repel the consumer. Furthermore, the well-informed 368 consumer is also aware that this parasite can cause health problems. Consequently, the great 369 challenge for the fishing industry is to provide the consumer with a risk-free product, i.e. free 370 from Anisakis. The current parasite detection methods, based on visual inspection and 371 candling, are far from precise, despite being endorsed by European Union legislation, as some 372 worms may not be detected. The development of effective detection methods (Smaldone et 373 al., 2020), accompanied by clear and precise legislation, must be a priority and both are vital 374 for the industry (Fig. 8). 375 From a sanitary point of view, the cosmopolitan nature of Anisakis and the increased 376 awareness and knowledge of anisakiasis and its diagnosis by health professionals are 377 responsible for the increase in the number of cases reported in an increasing number of 378 countries, along with cultural and commercial globalization, since gastronomic products 379 prepared with raw or under-cooked fish or squid are consumed worldwide. So, dishes such as 380 sushi, sashimi, gravlax, groene haring, ceviche/cebiche, boquerones en vinagre, etc., can be 16 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 381 sources of viable parasites and thus anisakiasis if not properly pre-frozen. However, it seems 382 clear that there is an infradiagnosis in many countries (Seal et al., 2020), related both to 383 awareness among health professionals and to diagnostic methods (Bao et al., 2017; Herrador 384 et al., 2019). Therefore, it is necessary to search for target molecules or infection markers that 385 can be identified with the development of new more sensitive, specific and cheaper diagnostic 386 techniques that are more reliable and accessible to laboratories that must unify diagnostic 387 criteria (Mazzucco et al., 2018). 388 A. simplex s.s. seems more pathogenic than A. pegreffii (Jeon and Kim, 2015; Quiazon et al., 389 2011; Romero et al., 2013; Suzuki et al., 2010). However, this requires confirmation, while 390 possible differences between the two should be identified (Cavallero et al., 2020, 2018; Llorens 391 et al., 2018; Molina-Fernández et al., 2019; Torralbo-Ramírez et al., 2019), including allergens 392 (Arcos et al., 2014; Baird et al., 2016), as they may affect diagnostic methods. As it has also 393 been suggested that anisakiasis constitutes a risk factor for stomach and colon cancers 394 (Corcuera et al., 2018; García-Pérez et al., 2015), studies to determine its relationship with 395 these and other diseases are especially relevant. Furthermore, Sánchez-Velasco et al. (2000) 396 suggested a genetic predisposition to allergy to Anisakis, whose study could help prevent 397 hypersensitivity in at-risk subjects. Likewise, the study of immunogenic molecules for vaccine 398 development and the design allergen-specific immunotherapy are options for the future. 399 Finally, there are many potentially useful topics for research and development in the field of 400 anisakiasis and new omic tools will undoubtedly facilite the task (D’Amelio et al., 2020). Here, 401 we have only mentioned those that we consider most urgent regarding the prevention, 402 diagnosis and control of anisakiasis. 403 17 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 404 Acknowledgements 405 Funding: This research did not receive any specific grant from funding agencies in the public, 406 commercial, or not-for-profit sectors. 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Public Heal. 62, 13–24. 848 Suzuki, J., Murata, R., Hosaka, M., Araki, J., 2010. Risk factors for human Anisakis infection and 849 association between the geographic origins of Scomber japonicus and anisakid 36 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 850 nematodes. Int. J. Food Microbiol. 137, 88–93. 851 https://doi.org/10.1016/j.ijfoodmicro.2009.10.001 852 853 854 Takei, H., Powell, S.Z., 2007. Intestinal anisakidosis (anisakiosis). Ann. Diagn. Pathol. 11, 350– 352. https://doi.org/10.1016/j.anndiagpath.2006.03.018 Templeman, W., Squires, H.J., Fleming, A.M., 1957. Nematodes in the fillets of cod and other 855 fishes in Newfoundland and neighbouring areas. J. Fish. Res. Board Canada 14, 831–897. 856 https://doi.org/10.1139/f57-037 857 Torralbo-Ramírez, V., Molina-Fernández, D., Malagón, D., Benítez, R., Adroher, F.J., 2019. 858 Differential cleaving of specific substrates for cathepsin-like activity shows cysteine and 859 serine protease activities and a differential profile between Anisakis simplex s.s. and 860 Anisakis pegreffii, sibling species major etiologic agents of anisakiasis. Foodborne Pathog. 861 Dis. 16, 744–751. https://doi.org/10.1089/fpd.2019.2633 862 Valero, A., López-Cuello, M.M., Benítez, R., Adroher, F.J., 2006. Anisakis spp. in European hake, 863 Merluccius merluccius (L.) from the Atlantic off north-west Africa and the Mediterranean 864 off southern Spain. Acta Parasitol. 51, 209–212. https://doi.org/10.2478/s11686-006- 865 0032-6 866 Valero, A., Martín-Sánchez, J., Reyes-Muelas, E., Adroher, F.J., 2000. Larval anisakids 867 parasitizing the blue whiting, Micromesistius poutassou, from Motril Bay in the 868 Mediterranean region of southern Spain. J. Helminthol. 74, 361–364. 869 https://doi.org/10.1017/S0022149X00000536 870 Valero, A., Romero, M.C., Gómez-Mateos, M., Hierro, I., Navarro, M.C., 2015. Natural products: 871 Perspectives in the pharmacological treatment of gastrointestinal anisakiasis. Asian Pac. J. 872 Trop. Med. 8, 612–617. https://doi.org/10.1016/J.APJTM.2015.07.017 873 874 Valls Sánchez, A., Daschner, Á., De Frutos Moreno, C., 2009. Anisakiasis gastroalérgica. JANO 19–23. 37 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 875 876 877 878 van Banning, P., 1971. Some notes on a successful rearing of the herring-worm, Anisakis marina L. (Nematoda: Heterocheilidae). J. Cons. Int. Exploration Mer 34, 84–88. van Beneden, P.-J., 1870. Les poissons des côtes de Belgique, leurs parasites et leurs commensaux. Mém. Acad. Roy. Sci. Lettr. Beaux-Arts Belgique 38, xx+100+6 plaques. 879 van Beurden, S.J., IJsseldijk, L.L., Cremers, H.J.W.M., Gröne, A., Verheije, M.H., Begeman, L., 880 2015. Anisakis spp. induced granulomatous dermatitis in a harbour porpoise Phocoena 881 phocoena and a bottlenose dolphin Tursiops truncatus. Dis. Aquat. Organ. 112, 257–263. 882 https://doi.org/10.3354/dao02818 883 884 van Thiel, P.H., Kuipers, F.C., Roskam, R.T., 1960. A nematode parasitic to herring, causing acute abdominal syndromes in man. Trop. Geogr. Med. 2, 97–113. 885 WHO/IUIS Allergen Nomenclature Sub-Committee (2020) Allergen Nomenclature. Retrieved 886 from http://www.allergen.org/search.php?Species=Anisakis%20simplex (accessed 887 04/03/2020). 888 889 890 Wootten, R., 2012. The Parasitology of Teleosts. Fish Pathol. 292–338. https://doi.org/10.1002/9781118222942.ch7 Wootten, R., Smith, J.W., 1975. Observational and experimental studies on the acquisition of 891 Anisakis sp. larvae (Nematoda: Ascaridida) by trout in fresh water. Int. J. Parasitol. 5, 892 373–378. https://doi.org/10.1016/0020-7519(75)90087-9 893 Yagi, K., Nagasawa, K., Ishikura, H., Nakagawa, A., Sato, N., Kikuchi, K., 1996. Female worm 894 Hysterothylacium aduncum excreted from human: a case report. Japanese J. Parasitol. 895 45, 12–23. 896 Yamamoto, K., Takahashi, O., Kobayashi, D., 2020. Comparison of risk factors between human 897 intestinal and gastric anisakiasis. Parasitol. Int. 75, 102024. 898 https://doi.org/10.1016/j.parint.2019.102024 38 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 899 Young, P.C., Lowe, D., 1969. Larval nematodes from fish of the subfamily Anisakinae and 900 gastro-intestinal lesions in mammals. J. Comp. Pathol. 79, 301–313. 901 https://doi.org/10.1016/0021-9975(69)90043-7 902 39 This is a preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 Table 1.- Molecular classification of known Anisakis species in clades (after Mattiucci et al., 2018). Clades Clade 1 Clade 2 Clade 3 Clade 4 Anisakis species A. simplex sensu stricto A. pegreffii A. berlandi A. ziphidarum A. nascetti L3 typea Type I Type I Type I Type I Type I Anisakiasis Humanb Humanb ?c ? ? Observations A. physeteris A. brevispiculata A. paggiae A. typica Type II Type II Type II Type I Lab animalsb,d ? Lab animalsb,d ? Human anisakiasis by Anisakis larva type II or A. physeteris (morphological diagnosis) reportede. Low frequency in fish muscle tissue a Low frequency in commercial fish Low frequency in commercial fish Low frequency in commercial fish ( ) Sensu Berland (1961), larvae of the same type (I or II) are morphologically indistinguishable. (b) Confirmed by molecular diagnosis. (c) ?, Human and experimental infections have not been proved. (d) Experimental infection by Romero et al. (2014). (e) See references in MolinaFernández et al. (2018a). 865 37 This is a preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 Table 2.- Characteristics of the described allergens of third larval stage (L3) of Anisakis simplex s.l. Allergena Origin Protein features Mw (kDa)a References Ani s 1 ES Kunitz-type serine-protease inhibitor; domain 21/24 Moneo et al., 2000 lustrine-type cystein-rich. Major allergen. Shimakura et al., 2004 Thermostable. Ani s 2 Somatic Paramyosin. Cross-reactivity with other helminth 97/100 Pérez-Pérez et al., 2000 and arthropod paramyosins. Ani s 3 Somatic Tropomyosin. Cross-reactivity with arthropod 41 Asturias et al., 2000a, 2000b tropomyosins. Ani s 4 ES Cysteine protease inhibitor. Thermostable. 9/12.7 Moneo et al., 2005 Resistant to pepsin digestion. Rodríguez-Mahillo et al., 2007 Ani s 5 ES SXP/RAL-2 family protein. Thermostable. 15 Kobayashi et al., 2007 Caballero et al., 2008 Ani s 6 ES Serine protease inhibitor. Resistant to pepsin 7 Kobayashi et al., 2007 digestion. Ani s 7 ES Glycoprotein with tandem repeat sequences. 139 Rodríguez et al., 2008 Anadón Major allergen. Specific for Anisakis-infected et al., 2009 patiens. Ani s 8 ES SXP/RAL-2 family protein. Thermostable. 15 Kobayashi et al., 2007b Crossreactivity with Ani s 5. Ani s 9 ES SXP/RAL-2 family protein. Thermostable. Share 14 Rodríguez-Pérez et al., 2008 identity partial with Ani s 5 and Ani s 8. Ani s 10 Somatic Unknown function. With repeat sequences. 21 Caballero et al., 2011 Thermostable. Ani s 11 ? Unknown function. With repeat sequences. Ani s 27 Kobayashi et al., 2011 11-like 78% similarity (17 kDa) Carballeda-Sangiao et al., 2016 Ani s 12 ? Unknown function. With repeat sequences. 31 Kobayashi et al., 2011 Ani s 13 ? Haemoglobin. No cross-reactivity to Ascaris suum 37 González-Fernández et al., 2015 haemoglobin. Ani s 14 ? Unknown function. Share identity partial with Ani 24/27 Kobayashi et al., 2015 s 7 and Ani s 12. a Allergens nomenclature according to WHO/IUIS Allergen Nomenclature Sub-Committee (2020). ES, excretory-secretory. ?, no reported. 38 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 903 Legends to Figures: 904 Figure 1.- Differential morphological features among third stage larvae (L3) of nematodes that 905 frequently occur in fish and that can potentially cause anisakidosis. Left: A freshly collected L3 906 from the body cavity of a blue whiting, host fish. Right: Drawings of the cephalic end, 907 ventricular area and caudal end of the larvae showing its external morphology and digestive 908 system. Note the differences between species, especially in the number of cecums in the 909 ventricular area. Also, note the position of the excretory pore to differentiate Contracaecum 910 and Hysterothylacium. Finally, note the differences between Anisakis type I (elongated 911 ventriculus with oblique join to the intestine, tail with mucron) and type II larvae (shorter, 912 thicker ventriculus with straight join to the intestine, conical tail). Abbreviations: a, anus; bt, 913 boring tooth; e, esophagus; ep, excretory pore; i, intestine; ic, intestinal caecum; m, mouth; nr, 914 nerve ring; p, papilla; r, rectum; rg; rectal gland; s, spine or mucron; v, ventriculus; va, 915 ventricular appendage. Drawing by Irene Adroher-Benítez. 916 917 Figure 2.- Lifecycle of Anisakis spp. L1, L2 and L3 are the first, second and third larval stages of 918 these parasitic nematodes into the eggs. L3 are also found in the body cavity of all 919 intermediate and paratenic hosts throughout the lifecycle of the worms. The fourth larval 920 stage (L4) and adults develop in the digestive tract of the final hosts. Note that L3 is the 921 infective stage for all hosts. Drawing by Lola Molina-Fernández. 922 923 Figure 3.- Anisakis simplex s.l., female adult obtained from in vitro culture described by Iglesias 924 et al. (2001). Scale in cm. 925 926 Figure 4.- Infection by Anisakis simplex s.l.: Worms (L3) in the body cavity of blue whiting, 927 Micromesistius poutassou (A), adults in the first gastric chamber of the bottlenose dolphin, 40 This is the accepted preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 928 Tursiops truncatus (B), and L3 in the human stomach (C). Credits: Image B courtesy of 929 Fernández-Maldonado (2016), and C from Shimamura et al. (2016). 930 931 Figure 5.- Anisakis simplex s.l. L3 section in human. A) L3 penetrating the digestive wall 932 surrounded by acute inflammatory cells (bar, 1 mm). B) Cross section of L3 surrounded by a 933 thick cuff of acute inflammatory cells with numerous eosinophils “showing transmural 934 infiltration of acute inflammatory cells with diffuse subserosal (ss) involvement” (bar, 500 µm). 935 From Takei and Powell (2007), courtesy from Elsevier ©. 936 937 Figure 6.- Cross section through the L3 of Anisakis simplex s.l. (M: polymyarian muscle layer; 938 LEC, lateral nervous cord; EG, excretory cell; DT, esophageal lumen). From Takei and Powell 939 (2007), courtesy from Elsevier ©. 940 941 Figure 7.- Extraction of a larva of Anisakis simplex s.l. from human digestive tract (after 942 Shimamura et al., 2016). 943 944 Figure 8. Observation of pressed and frozen fillets of blue whiting in UV-light allows the 945 detection of Anisakis larvae as bright fluorescent spots. The straight lines mark the boundary 946 between the epiaxial (dorsal) and hypoaxial (belly) muscles of each side of fish. Photo by A. 947 Levsen (EFSA-BIOHAZ, 2010). 41 This is a preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 Figures protected by copyright. Figure 1. Figure 2. 41 This is a preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 Figures protected by copyright. Figure 3. Figure 4. Figure 5. 42 This is a preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 Figures protected by copyright. Figure 6. Figure 7. 43 This is a preprint. Article published in Research in Veterinary Science 132: 535-545 (2020). © 2020 Elsevier Ltd. All rights reserved. Downloadable from: https://linkinghub.elsevier.com/retrieve/pii/S0034528820309759 or https://doi.org/10.1016/j.rvsc.2020.08.003 Figures protected by copyright. Figure 8. 44

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