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The Anisakis larvae presence in fish for human consumption is a health risk that needs to be monitored. The anchovy is a fish that is highly appreciated by consumers and that can harbour Anisakis. It is thus necessary to periodically evaluate the presence of anisakid larvae in them. So, anchovies from Iberian Peninsula coasts were analysed. Fish examination for macroscopic nematodes showed L3s of both Anisakis type I and Hysterothylacium aduncum. The Anisakis prevalence varies with the catching area and the fish size. The muscle prevalence was 7.45% (mean intensity 1.75; range 1–5). Molecular analysis showed 110 A. simplex s.s. (17 in muscle), 22 A. pegreffii (3) and 7 hybrid genotype individuals (1). Considering that most of the Iberian Peninsula coasts are a sympatry area between these two Anisakis species, it has been observed that A. simplex s.s./A. pegreffii ratio increases from south to north in a clockwise direction. Also, 19 larvae were detected on the fish surface from the Bay of Biscay, indicating the ability of these larvae to migrate after the fish death. The A. simplex s.s./A. pegreffii larvae proportion found on the anchovy surface is similar to the found in viscera and lower than in muscle, suggesting that most of the larvae migrating to the surface must have come from the visceral package. This confirms the importance of removing fish viscera immediately after capture, for those fish species where this is possible. As both species cause anisakiasis/anisakidosis, these data show a real risk to human health, especially in dishes highly prized in Mediterranean countries prepared with raw or semi-raw anchovies., BIO-243 research group of the Junta de Andalucía (Spain), Open access charge: Universidad de Granada / CBUA.

To review the main findings for anisakiasis in the different imaging tests that can be used to diagnose it, based on studies done at our center.The presence of Anisakis species in food consumed in Western countries is becoming more common. Patients with anisakiasis present with acute abdomen; there are no specific clinical signs or laboratory findings. Careful history taking is key to discovering exposure to Anisakis-contaminated food, but this task is hindered by unfamiliarity with the condition and lack of suspicion and is also confounded by the variable latency period after ingestion of Anisakis-contaminated food. Give the nonspecific presentation, patients with anisakiasis often undergo imaging tests to rule out other processes. Thus, radiologists need to be familiar with the spectrum of imaging findings that should lead to the inclusion of anisakiasis in the differential diagnosis, so they can guide clinicians toward directed history taking and specific tests.

The red scorpionfish Scorpaena scrofa (Scorpaenidae) is a high commercial value marine fish species along the Mediterranean coasts. Anisakiasis is a fish-borne parasitic zoonoses caused by Anisakis larvae in consumers. To date, there are only a few epidemiological studies on the presence and molecular identification of Anisakis larvae infecting S. scrofa. A total of 272 S. scrofa captured from the Gulf of Izmir in the Turkish Aegean coasts (FAO 37.3.1) were examined for Anisakis larvae between March 2019 and March 2020. The prevalence, mean intensity and mean abundance of Anisakis larvae were 9.6% (95% CI 6.5-13.7%), 2.8 (95% CI 1.88-5.19), and 0.27 (95% CI 0.15-0.56), respectively. All Anisakis larvae were collected from the viscera and body cavity of S. scrofa. Anisakis pegreffii, A. typica, and A. ziphidarum were genetically identified by RFLP analysis of the ITS region. These species were also confirmed by cox2 sequence analysis. A weak positive and statistically significant correlation between the total length (ρS 0.204; p = 0.001) and total weight (ρS 0.200; p = 0.001) of S. scrofa and the number of Anisakis larvae was observed. This survey presents the first molecular detection of A. typica and A. ziphidarum in S. scrofa. Thus, this fish species is a new host for A. typica and A. ziphidarum. This is also the first report of the presence of A. ziphidarum in the Aegean Sea.

Effects of temperature on development of eggs, recently hatched larvae and L3 larvae of the marine parasitic nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were examined in vitro. The eggs of A. simplex s.s. hatched at 3-25 °C and those of A. pegreffii hatched at 3-27 °C. Days before hatching varied between 2 days at 25 °C and 35-36 days at 3 °C in A. simplex s.s. and between 2 and 3 days at 27 °C and 65 days at 3 °C in A. pegreffii. Hatching rates of A. simplex s.s. were maintained high at temperatures between 3 and 25 °C but decreased to 0% at 27 °C. In contrast, those of A. pegreffii were lowest particularly at 3 °C, but also at 27 °C. The mean 50% survivals of hatched larvae ranged from 5.3 days at 25 °C to 82.3 days at 9 °C in A. simplex s.s., while in A. pegreffii it ranged from 1.2 days at 27 °C to 77.2 days at 9 °C. L3 larvae of A. pegreffii exhibited higher survival rates and activity than those of A. simplex s.s., particularly at 20 and 25 °C. These results suggest that the early stages of A. simplex s.s. are more adapted to lower temperatures whereas those of A. pegreffii are more tolerant to warm environments, which may correspond to their distribution patterns in Japan and Europe.

The infection of blue whiting Micromesistius poutassou from the western Mediterranean Sea, off the eastern coast of Spain, with larvae of Anisakis spp. was studied. Between April 2016 and April 2017, 140 fish were analyzed. Total epidemiological data showed that the prevalence of Anisakis spp. was 29.3% and the mean intensity 1.8. Of the 74 larvae collected, 61% were type I and the remaining 39%, type II. Of the former, 91% were molecularly identified as Anisakis pegreffii (P = 19.3%; MI = 1.4), 2.2% as Anisakis simplex s.s. (P = 0.7%; MI = 1.0), while the rest (6.7%) showed a recombinant genotype between the two (P = 2.1%; MI = 1.0). All the type II larvae analyzed were molecularly identified as Anisakis physeteris (P = 10.0%; MI = 2.1). Three fish (2.1%) were found to have larvae in the muscle, while two were found with 1 larva of A. pegreffii and one with two larvae (1 A. simplex s.s. and 1 A. pegreffii). Statistical analysis showed that the prevalence of Anisakis spp. in blue whiting was higher in spring than in autumn (P 0.001), probably due to the greater size (and age) of the fish and related to factors as diet shift, accumulation with age and higher food intake. Analysis of the data suggested that blue whiting were first infected with Anisakis type I (mean age 2.3 years) and later with Anisakis type II (mean age 2.7 years), probably due to the diet changing with age, with the incorporation of the paratenic/intermediate host species of these parasites. In any case, the public health authorities must continue to emphasize the need for suitable thermal treatment (freezing or cooking) of the fish prior to consumption.

Immunosuppression in sepsis reduces both αβ and γδ T cell subsets. Anisakis sp. is a parasitic nematode with a high prevalence in Spain. Previous contact with the parasite is related to a decrease in γδ T cells. Anti-Anisakis antibodies were measured and related to αβ and γδ T cells in 114 septic patients versus 97 healthy controls. Significant differences were seen with respect to the groups with severe sepsis and septic shock where lower anti-Anisakis levels were observed. A similar decrease appeared in the case of specific IgM with significant differences between the groups of control/uncomplicated sepsis versus severe sepsis and septic shock. These differences were also apparent in the case of specific IgA. The lowest IgE levels were detected in the septic shock group. Anti-Anisakis IgG levels significantly increased in septic shock groups compared with the controls. We observed positive correlations among anti-Anisakis IgA levels and all γδ T cell subsets. There were negative correlations among IgA levels and APACHE and SOFA indices. Greater contact with the parasite (IgG) was directly related with septic shock, inflammation and markers of sepsis severity. A lack of protection in the mucosa (IgA and γδ T cells) was associated with the disease severity.

Atlantic cod ( Gadus morhua L.) is one of the most important fish species in the fisheries industries of many countries; however, these fish are often infected with parasites. The detection of pathogenic larval nematodes is usually performed in fish processing facilities by visual examination using candling or by digesting muscles in artificial digestive juices, but these methods are both time and labor intensive. This article presents an innovative approach to the analysis of cod parasites from both the Atlantic and Baltic Sea areas through the application of rough set theory, one of the methods of artificial intelligence, for the prediction of food safety in a food production chain. The parasitological examinations were performed focusing on nematode larvae pathogenic to humans, e.g., Anisakis simplex, Contracaecum osculatum, and Pseudoterranova decipiens. The analysis allowed identification of protocols with which it is possible to make preliminary estimates of the quantity and quality of parasites found in cod catches before detailed analyses are performed. The results indicate that the method used can be an effective analytical tool for these types of data. To achieve this goal, a database is needed that contains the patterns intensity of parasite infections and the conditions of commercial fish species in different localities in their distributions.

In order to evaluate the infestation by anisakids present in elasmobranchs and their distribution in the Argentine Sea, this study was carried at a regional scale with the following aims: 1) to identify those anisakid species present in skates under exploitation; 2) to characterize quantitatively these infestations and 3) to determine those factors driving the variability in parasite burdens across skate species. A total of 351 skates, belonging to 3 species (218 Sympterygia bonapartii, 86 Zearaja chilensis and 47 Atlantoraja castelnaui) and from different localities of the Argentine Sea were examined for anisakids. Parasites were found in the stomach wall at high prevalence in some samples. Based on morphology and mtDNA cox2 sequences analyses (from 24 larval worms), specimens were identified as Anisakis berlandi, A. pegreffii and Pseudoterranova cattani; the last two known as potentially pathogenic for humans. Differential distribution patterns were observed across parasite and hosts species. In general, fish caught in southern and deeper waters exhibited higher loads of Anisakis spp., whereas infestation levels by P. cattani increase in larger skates. Taking into account that the mere presence of worms or their antigens in fish meat can provoke allergic responses, information on distribution of parasites and their variability is essential for the implementation of food safety practices. Fil: Irigoitia, Manuel Marcial. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina Fil: Braicovich, Paola Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina Fil: Lanfranchi, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina Fil: Farber, Marisa Diana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Timi, Juan Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina

The trehalose-6-phosphate phosphatase (TPP) enzyme is involved in the synthesis of trehalose, the main sugar in the energy metabolism of nematodes. TPP is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. The presence of conserved active sites of TPP in closely related nematodes and its absence in humans makes it a promising target for antiparasitic drugs. In the present study, homology modeling, molecular docking and MD simulation techniques were used to explore the structure and dynamics of TPP. In the active site, a magnesium ion is stabilized by 3 coordinate bonds formed by D189, D191 and D400. The key amino acids involved in ligand binding by the enzyme are C198, Y201,T357, D191 and Y197. This study relied on docking to select potential inhibitors of TPP which were tested in vitro for sensitivity to anthelmintic drugs such as levamisole and ivermectin targeting Anisakis simplex. The higher toxicity of LEV than IVM was demonstrated after 96 h, 30% of larvae were motile in cultures with 100 μg/ml of LEV and 1000 μg/ml of IVM. We identified drug combination of LEV-IVM against in vitro A. simplex as agonistic effect (CI = 1.1). Levamisole appeared to be a more effective drug which inhibited enzyme activity after 48 h and expression of mRNA after 96 h at a concentration of 10 μg/ml. This preliminary study predicted the structure of TPP, and the results of an in vitro experiment involving A. simplex will contribute to the development of effective inhibitors with potential antiparasitic activity in the future.

The genus Anisakis is a well-known group of nematodes that parasitize cetaceans as the final host and cause mucosal damage to their stomach. However, little has been done to precisely identify the nematodes recovered from the final hosts, especially in the Western Pacific, because of taxonomic confusion about the discrimination of sibling species and the difficulties of obtaining specimens from cetaceans. We describe the results of genetic identification and histopathological observations of specimens recovered from an ulcerated lesion and stomach contents in the forestomach of a female harbor porpoise accidentally caught by a set net fishery in Usujiri, southern Hokkaido, Japan. All the specimens arbitrarily collected from the lesion and stomach contents were identified as Anisakis simplex sensu stricto according to their ITS rDNA sequences. The size of the ulcer was approximately 6.3 mm in diameter and it was infected with 119 individual nematodes, mostly consisting of L3 and L4 stage larvae (95.0%). Histological sections were characterized by a locally extensive ulcer with the parasites penetrating into the muscularis externa that caused a thickening of the surrounding mucosa.

Anisakiasis is a parasitic infection caused by larval stages of nematodes of the genus Anisakis, Pseudoterranova and Contracaecum, of the Anisakidae family. The lifecycle of these nematodes develops in aquatic organisms and their final hosts are marine mammals. However, humans can act as accidental hosts and become infected with infective stage larvae (L3) by consuming raw or undercooked fish or shellfish carrying the parasite. Of this group of parasites, the genus Anisakis is the most studied: its presence in humans is associated with non-specific gastrointestinal symptoms or allergic responses that can trigger anaphylactic shock. The lack of studies in anisakiasis and Anisakis in Colombia has resulted in this infection being little-known by medical practitioners and therefore potentially underreported. The objective of this study was to identify anisakid nematodes in the flathead grey mullet fish (Mugil cephalus), caught by artisanal fishing methods and commercialized in Buenaventura. Morphological identification was carried out by classical taxonomy complemented by microscopy study using the histochemical technique Hematoxylin-Eosin. Nematodes of the genus Anisakis were found in the host M. cephalus. The Prevalence of Anisakis larvae in flathead grey mullet fish was 33%. The findings confirm the presence of Anisakis sp. in fish for human consumption in the Colombian Pacific region, a justification for further investigation into a possible emerging disease in this country., Graphical abstract Image 1, Highlights • The research confirmed the presence of anisakids in the fish M. cephalus in Colombia. • First report such anisakids in fish from Colombia's Pacific coast. • Is confirmed the presence of the genus Anisakis in the country. • Anisakis spp. is associated with allergic reactions and gastro-allergies in humans. • The presence the anisakids suggests a possible emerging zoonotic disease in Colombia.

Anisakids are parasitic nematodes responsible for a zoonosis that occurs following the ingestion of fish and fish products infected with larvae belonging to the genera Anisakis and Pseudoterranova. Rarely Contracaecum is found in association with gastric/intestinal illness, while Hysterothylacium is commonly considered not pathogenic. Although Real Time PCR assays have been recently used with the aim to detect and quantify these parasites in food products, methods applied did not undergo through extensive validation process, a feature highly desirable or mandatory in the case of testing laboratories accredited for the ISO EN 17025:2005. Here, a comprehensive study has been performed to validate a commercial kit based on multiplex real time PCR for the qualitative detection of Anisakis and Pseudoterranova. Inclusivity/exclusivity trials were carried out on DNA from species of the genera Anisakis, Pseudoterranova, Contracaecum, Hysterothylacium and Ascaris, on fish intentionally contaminated with Anisakis spp. and Pseudoterranova spp. and on marine organisms as fish, crustacean and squid to test the commercial kit on a large sample. The assay gave positive amplification for several Anisakis and Pseudoterranova species, while providing no signal for the members of the remaining genera. Each sample was correctly assigned either to Anisakis or Pseudoterranova, thus indicating that no cross-reaction occurred. The LOD was determined using two independent standard curves. Robustness was assayed by using two different thermocyclers in three distinct laboratories with different operators. The establishment of a validation dossier will permit the use of the commercial kit for the detection of Anisakis and Pseudoterranova DNA in fish and fish products intended for human consumption by public or private laboratories, following the requirements regarding the quality assurance processes described in the ISO EN 17025:2005.

Gastrointestinal anisakiasis is an uncommon zoonotic parasitic infection caused by consumption of raw or undercooked seafood infected with nematodes of genus Anisakis. Given the non-specific clinical presentation of acute abdomen, nausea, and vomiting these patients are often subject to radiologic imaging. We present ultrasound and computed tomography imaging features in a case of gastric anisakiasis demonstrating characteristic features of diffuse gastric submucosal edema, perigastric stranding and trace ascites that helped to further elaborate the clinical history of uncooked fish consumption prompting timely endoscopic diagnosis and management.

Prevalence, morphological and molecular characterization of Anisakid larvae in smoked herring fish (Clupea harengus) from five governorates that represent Northern, Middle, and Southern Egypt which include; Menofia, Alexandrina, Qaliubiya, Sohag, and Assiut were elucidated in this study, smoked herring fish samples were collected from local markets during 2020, 384 positive (42.7%) out of 815 examined samples, the highest prevalence was observed in middle and southern Egypt (Sohag (139/230, 60.43%), and Menofiya (123/215, 57.20%), they were found to be naturally infected with Anisakis simplex larvae as free in peritoneal cavity, Based on morphological, morphometric by SEM, and molecular analyses of ITS-2 ribosomal DNA targeted gene and phylogenic analysis, these nematodes were identified as third- stage larvae of Anisakis simplex.

Marine fishes represent the important components of the diet in the coastal areas of China and they are also natural hosts of various parasites. However, to date, little is known about the occurrence of ascaridoid parasites in the frequently consumed marine fishes in China. In order to determine the presence of ascaridoid parasites in the frequently consumed marine fishes in the coastal town Huizhou, Guangdong Province, China, 211 fish representing 45 species caught from the South China Sea (off Daya Gulf) were examined. Five species of ascaridoid nematodes at different developmental stages were detected in the marine fishes examined herein, including third-stage larva of Anisakis typica (Diesing, 1860), third and fourth-stage larvae of Hysterothylacium sp. IV-A of Shamsi, Gasser & Beveridge, 2013, adult and third-stage larvae of Hysterothylacium zhoushanense Li, Liu & Zhang, 2014, adults and third-stage larvae of Raphidascaris lophii (Wu, 1949) and adults of Raphidascaris longispicula Li, Liu & Zhang, 2012. The overall prevalence of infection is 18.0%. Of them, Hysterothylacium sp. IV-A with the highest prevalence (17.5%) and intensity (mean=14.6) of infection was the predominant species. The prevalence and intensity of A. typica were very low (1/211 of marine fish infected with an intensity of one parasite per fish). The morphological and molecular characterization of all nematode species was provided. A cladistic analysis based on ITS sequence was constructed in order to determine the phylogenetic relationships of these ascaridoid parasites obtained herein. The present study provided important information on the occurrence and diagnosis of ascaridoid nematodes in the commercially important marine fishes from the South China Sea. The low level of infection and the species composition of ascaridoid nematodes seem to indicate the presence of low risk of human anisakidosis when local population consumed these marine fishes examined herein.

The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved β-tubulin gene (β-TUB). The two cryptic species differed at least in 7 bp in the β-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and β-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.

Background Little is known in the difference of host factors between intestinal and gastric anisakiasis. The aim of this study was to investigate the associated factors of intestinal anisakiasis in patient's characteristics and the subsequent variation compared to gastric anisakiasis. Methods At St. Luke's International Hospital in Tokyo, Japan, a retrospective cohort study was conducted from April 2004 to June 2017. All adult patients who were clinically diagnosed as anisakiasis based on Computed Tomography (CT) scan or endoscopy were included, and anti-Anisakis antibodies (IgG and IgA) were measured for serological validation of anisakiasis, strengthen the diagnosis. Anisakiasis was categorized as either intestinal or gastric depending on its affected site. We compared patients' demographics, social history, and physical and laboratory findings between those with intestinal and gastric anisakiasis by bivariate analyses, followed by multivariate analyses. Results A total of 302 patients were included in this study, where the mean age (SD) was 46.5 (14.4) and 66.6% were male. Ninety-two patients (30.5%) had intestinal anisakiasis. Multivariate regression revealed that patients with intestinal anisakiasis were more 45 years old or older (odds ratio (OR) 3.45, 95% confidence interval (CI): 1.53–7.69), male (OR 2.70, 95% CI: 1.20–6.25) and regular alcohol drinker. In terms of the physical and laboratory findings, patients with intestinal anisakiasis had greater heart rate (OR 2.86, 95% CI: 1.33–6.25), higher total protein (OR 2.86, 95% CI: 1.16–6.67), and higher C-reactive protein (CRP) (OR 11.1, 95% CI: 3.03–33.3). Conclusions Older males who were regular alcohol drinkers were associated with intestinal anisakiasis, and often heart rate, total protein, and CRP were elevated compared to those of patients with gastric anisakiasis.

Anisakid nematodes have been found in a variety of marine fishes throughout the world and they are known to cause anisakiasis in human hosts. The present study investigated the prevalence of potentially zoonotic anisakid larvae in spotted mackerel caught from Taiwanese waters where fish represents an important food sources. Anisakis third-stage larvae (L3, n=502) were isolated from 250 spotted mackerel Scomber australasicus. Anisakis L3 larvae were divided morphologically into two types, Anisakis type I larvae had a longer ventriculus and mucron while type II larvae had a shorter ventriculus and no mucron. Anisakis species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer (ITS) regions of ribosomal DNA and direct sequencing. A simple molecular taxonomic key, utilizing RFLP by two restriction enzymes HinfI and HhaI, enabled the differentiation of the genus Anisakis. The prevalence, mean intensity and mean abundance of Anisakis nematodes recorded for the total specimens were 72.8%, 2.8 (1-15) and 2.0 (0-15), respectively. Anisakis pegreffii was determined to be the dominant species (prevalence=57.2%) and important agent of human anisakiasis. A recombinant genotype (Anisakis simplex sensu stricto × A. pegreffii) was identified as the subdominant species (25.3%) followed by Anisakis typica (10%), Anisakis physeteris (4.0%), Anisakis paggiae (3.0%) and Anisakis brevispiculata (0.5%). The topology of the maximum likelihood and neighbor-joining trees show two well supported clades: one includes the species of A. pegreffii and the other includes A. paggiae, A. physeteris and A. brevispiculata, while A. typica has basal position to all other Anisakis spp. analyzed. This study advances our knowledge of the prevalence of different Anisakis spp. in the spotted mackerel from Taiwanese waters, which is helpful for monitoring the fish populations throughout a diverse array of aquatic ecosystems. More importantly, we provide the concise characterization of multiple Anisakis spp. by PCR-RFLP, which could also be applicable for the rapid diagnosis of human anisakiasis.

Parasitic anisakid nematodes commonly occur in the musculature and visceral organs of many fish species from the North Atlantic. In this respect, the presence of the third stage larvae of Anisakis spp. in the fish musculature may pose a potential consumer hazard due to the parasite's ability to cause anisakidosis. Thus, knowledge on the occurrence and distribution of these potentially zoonotic parasites in the commercially important North Atlantic fish species is crucial in order to evaluate and consequently prevent human infections. In the present study, 300 Sebastes mentella from three North Atlantic fishing grounds (Northern North Sea: Tampen; Barents Sea: off Bear Island; Irminger Sea: off SE Greenland) were examined for anisakid nematodes, with emphasis on occurrence and distribution in the musculature. Overall larval prevalence and mean intensity were significantly higher in redfish from Tampen (94%; 13.5±20.0) and Bear Island (94%; 14.5±19.4) than in fish from SE Greenland (75%; 6.0±5.8; p0.01). The same trend was observed for larval infection in the musculature showing prevalence and mean intensities of 79%, 73%, and 55%, and 5.9±6.6, 5.8±6.5, and 3.2±2.4, in the musculature of redfish from Tampen, Bear Island, and Greenland, respectively. Conventional microscopy and rDNA ITS-gene sequencing of various subsamples of muscle-dwelling nematode larvae of redfish from every catching locality revealed the presence of two anisakid species; Anisakis simplex sensu stricto and the non-zoonotic Hysterothylacium aduncum. Since the larvae of H. aduncum typically occur in or on the viscera of fish, our findings of two specimens in the belly flaps of redfish were unusual. Additionally, more than 92% of the muscle-dwelling larvae occurred in the belly flaps, i.e. the hypaxial part of the musculature surrounding the visceral organs on either fish side. Thus, trimming the fillets of beaked redfish by removal of most of the belly flaps would significantly reduce the probability of anisakid nematode larvae to be present in the final product.

Members of the genera Anisakis and Pseudoterranova are the main causative agents of human anisakidosis: the disease is worldwide distributed, with major impact in countries with a large consumption of raw fish. Because of unspecific symptoms and limited diagnostic tools, incidence and burden of disease are probably underestimated. In Italy, all human infestations where the etiological agent has been properly identified, have been associated to the parasitic species Anisakis pegreffii, the most frequent anisakid in the Mediterranean area. Here, an invasive human case of pseudoterranoviasis is described for the first time in Italy: in 2015, a woman was found infected during a colonoscopy scheduled after the occurrence of nonspecific clinical symptoms. The nematode was found penetrating the ascending colon. The identification was performed by sequencing the mitochondrial region cox2 and by comparison to GenBank retrieved material using the BLAST search tool. The sample showed a 99% identity with Pseudoterranova decipiens sensu stricto. The record underlines the potential risk due to the consumption of raw or undercooked imported fishes.

The prevalence of anisakiasis is rare in the United States and Europe compared with that in Japan, with few reports of its presentation in the emergency department (ED). This study describes the clinical, hematologic, computed tomographic (CT) characteristics, and treatment in gastric and small intestinal anisakiasis patients in the ED.We retrospectively reviewed the data of 83 consecutive anisakiasis presentations in our ED between 2003 and 2012. Gastric anisakiasis was endoscopically diagnosed with the Anisakis polypide. Small intestinal anisakiasis was diagnosed based on both hematologic (Anisakis antibody) and CT findings.Of the 83 cases, 39 had gastric anisakiasis and 44 had small intestinal anisakiasis based on our diagnostic criteria. Although all patients had abdominal pain, the gastric anisakiasis group developed symptoms significantly earlier (peaking within 6 hours) than the small intestinal anisakiasis group (peaking within 48 hours), and fewer patients with gastric anisakiasis needed admission therapy (5% vs 57%, P.01). All patients in the gastric and 40 (91%) in the small intestinal anisakiasis group had a history of raw seafood ingestion. Computed tomographic findings revealed edematous wall thickening in all patients, and ascites and phlegmon of the mesenteric fat were more frequently observed in the small intestinal anisakiasis group.In the ED, early and accurate diagnosis of anisakiasis is important to treat and explain to the patient, and diagnosis can be facilitated by a history of raw seafood ingestion, evaluation of the time-to-symptom development, and classic CT findings.

Chub mackerel (Scomber japonicus) is a pelagic fish species widely distributing in the Indo-Pacific and a commercially important fish species in Korea. It is known to harbor anisakid nematodes larvae, and ingesting the raw or undercooked fish can accidentally cause human infection. In this study, we isolated the nematode larvae in 417 chub mackerel caught from 7 sampling locations around the Korean Peninsula in 2011 and 2012, and identified them by PCR-RFLP of the ITS (internal transcribed spacer) of ribosomal DNA and the direct sequencing of the mitochondrial DNA cox2 gene. The prevalence of infection was 55.4% (231/417) and the mean intensity was 7.0 (1628/231). Most of the nematodes (1523/1628; 93.6%) were found in the body cavity, while 5.5% (89/1628) were found in the gastrointestinal tract. Four different species were identified by PCR-RFLP and direct sequencing. Most of the nematodes (1535/1628; 94.3%) were identified as Anisakis pegreffii, and 2.8% (46/1628) were identified as Hysterothylacium sp. A hybrid genotype (Anisakis simplex sensu stricto×A. pegreffii) and A. simplex sensu stricto were 2.5% (41/1628) and 0.4% (6/1628) of the identified nematodes, respectively. The anisakid nematode assemblage of chub mackerel in Korea was similar to that of chub mackerel from the Tsushima Current stock in Japan, in that A. pegreffii was the dominant species. Since most of the anisakid nematodes were found in the body cavity and most of them were identified as A. pegreffii or Hysterothylacium sp. by PCR-RFLP and direct sequencing, chub mackerel may not greatly contribute to human anisakidosis in Korea. Alternately, A. pegreffii may be responsible for human anisakidosis in Korea, in addition to A. simplex sensu stricto. Further studies, such as the molecular diagnosis of human anisakidosis, are necessary for assessing the epidemiological role of chub mackerel in Korea.

Engraulis encrasicolus and Sardina pilchardus are pelagic fishes of notable economic and gastronomic importance in the northwest Mediterranean (Ligurian Sea, Italy). The consumption of thermally unprocessed or lightly processed, marinated or salted anchovies and sardines presents a potential risk to acquire anisakiasis, a fish-borne parasitic disease in humans. Prevalence and abundance of Anisakis larvae in Engraulis encrasicolus and Sardina pilchardus from the Monterosso fishing grounds (Cinque Terre National Park, Ligurian Sea, Italy) were assessed, and the larvae were identified by morphological and PCR-RFLP methods. Anisakis larvae, all belonging to Anisakis pegreffii spp. were found in the visceral mass of 1050 anchovies (0.8% overall prevalence), whereas no Anisakis larvae were found in the 750 sardines examined. According to these data, the risk of acquiring anisakiasis from the consumption of raw or undercooked anchovies and sardines caught in the fishing area we investigated is very low.

Anisakid nematodes are distributed worldwide in a wide variety of marine fishes and they are known to cause the zoonotic disease, anisakiasis. The temperature control is commonly applied for prevention and control of anisakiasis. To analyze the cellular response to temperature stress in Anisakis, the heat shock protein 90 (Hsp90) was chosen in the present study, as it plays a key role in many cellular processes and responds to stress conditions such as heat or cold shock. Anisakids were sampled from spotted mackerel Scomber australasicus caught from the coastal waters of Yilan, in northeastern Taiwan (25 °N, 121 °E). Anisakid nematodes were pre-identified morphologically and later molecularly by PCR-RFLP. In total, we obtained six species of the genus Anisakis, A. typica, A. pegreffii, A. paggiae, A. brevispiculata, A. physeteris, and a recombinant genotype between A. pegreffii and A. simplex sensu stricto. Thereby we provide new host and locality records for A. paggiae, A. brevispiculata and A. physeteris. The Hsp90 genes of five species (except the recombinant genotype) were cloned by rapid amplification of cDNA ends (RACE) and their deduced amino acid sequences were further characterized. Quantitative real-time PCR and Western blot analysis were used to examine the expression levels of the Hsp90 in A. pegreffii under different temperature conditions. Quantitative RT-PCR showed that Hsp90 transcript levels increased slightly under heat shock (50 °C) treatment, and increased gradually during the first 3h, and thereafter, returned to its baseline value at 37 °C. Under cold shock (4 °C) treatment, the mRNA expression of Hsp90 did not change significantly. In addition, we found a clear time-dependent Hsp90 protein expression pattern of A. pegreffii exposed to high temperature. Our results suggest that the mRNA and protein expression patterns of Hsp90 are related to the temperature, and are especially significantly increased under heat stress.

Human anisakiasis, a disease caused by Anisakis spp. (Nematoda), is often associated with clinical signs that are similar to those associated with bacterial or viral gastroenteritis. With the globalisation of the seafood industry, the risk of humans acquiring anisakiasis in developed countries appears to be underestimated. The importance of this disease is not only in its initial manifestation, which can often become chronic if the immune response does not eliminate the worm, but, importantly, in its subsequent sensitisation of the human patient. This sensitisation to Anisakis-derived allergens can put the patient at risk of an allergic exacerbation upon secondary exposure. This article reviews some aspects of this food-borne disease and explains its link to chronic, allergic conditions in humans.

Anisakiasis is a fish-borne zoonosis caused by third stage larvae of the nematode Anisakis sp. present in fish or cephalopods. This is the first contribution to the molecular identification and epidemiology of Anisakis spp. in commercial fish from the Gulf of Asinara (Sardinia, western Mediterranean Sea). Between April 2006 to November 2011, 777 specimens of 10 fish species (Engraulis encrasicolus, Merluccius merluccius, Micromesistius poutassou, Phycis blennoides, Sardina pilchardus, Sardinella aurita, Scomber colias, Sphyraena viridensis, Trachurus mediterraneus, Trachurus trachurus) were examined for Anisakis sp. larvae. A total of 1286 larvae were found in 218 fish. The great majority of larvae were located in the body cavity, and only a small part (60, 4.7%) in the muscle. All the Type I larvae (1272) were identified as Anisakis pegreffii and all the Type II (14) as Anisakis physeteris, confirming that A. pegreffii is the dominant species and the most important agent of human anisakiasis in the western Mediterranean Sea.

The third-stage larvae of several genera of anisakid nematodes are important etiological agents for zoonotic human anisakiasis. The present study investigated the prevalence of potentially zoonotic anisakid larvae in fish collected on the coastal shelves off Madang and Rabaul in Papua New Guinea (PNG) where fish represents a major component of the diet. Nematodes were found in seven fish species including Decapterus macarellus, Gerres oblongus, Pinjalo lewisi, Pinjalo pinjalo, Selar crumenophthalmus, Scomberomorus maculatus and Thunnus albacares. They were identified by both light and scanning electron microscopy as Anisakis Type I larvae. Sequencing and phylogenetic analysis of the ribosomal internal transcribed spacer (ITS) and the mitochondrial cytochrome C oxidase subunit II (cox2) gene identified all nematodes as Anisakis typica. This study represents the first in-depth characterisation of Anisakis larvae from seven new fish hosts in PNG. The overall prevalence of larvae was low (7.6%) and no recognised zoonotic Anisakis species were identified, suggesting a very low threat of anisakiasis in PNG.

8 páginas, 2 figuras, 3 tablas, During the last 50 years human anisakiasis has been rising while parasites have increased their prevalence at determined fisheries becoming an emergent major public health problem. Although artificial enzymatic digestion procedure by CODEX (STAN 244-2004: standard for salted Atlantic herring and salted sprat) is the recommended protocol for anisakids inspection, no international agreement has been achieved in veterinary and scientific digestion protocols to regulate this growing source of biological hazard in fish products. The aim of this work was to optimize the current artificial digestion protocol by CODEX with the purpose of offering a faster, more useful and safer procedure for factories workers, than the current one for anisakids detection. To achieve these objectives, the existing pepsin chemicals and the conditions of the digestion method were evaluated and assayed in fresh and frozen samples, both in lean and fatty fish species. Results showed that the new digestion procedure considerably reduces the assay time, and it is more handy and efficient (the quantity of the resulting residue was considerably lower after less time) than the widely used CODEX procedure. In conclusion, the new digestion method herein proposed based on liquid pepsin format is an accurate reproducible and user-friendly off-site tool, that can be useful in the implementation of screening programs for the prevention of human anisakiasis (and associated gastroallergic disorders) due to the consumption of raw or undercooked contaminated seafood products, Xunta de Galicia has provided financial support under Projects INCITE-07MMA015CT, IN841C, 10TAL001CT and 10TAL033E. María Llarena-Reino thanks Fundação para a Ciência e a Tecnologia and European Social Fund for financial support under Grant SFRH/BD/45398/2008.

Three salmonid fish species, Oncorhynchus mykiss, Salmo salar and Salmo trutta, were infected experimentally with the parasitic nematode Anisakis simplex (A. simplex) and the difference between in vivo behaviour of the nematode in the three fish species was investigated. Infection success rate differed between species. S. salar (Baltic salmon) showed the highest number of successfully established nematodes, whereas S. trutta (brown trout) and O. mykiss (rainbow trout) had a higher natural resistance. Microhabitat selection of nematodes differed according to fish species. In brown trout, A. simplex larvae were attached to the digestive tract (stomach, pyloric caeca, intestine), while the majority of larvae found in rainbow trout were located between the pyloric caeca. In Baltic salmon, nematodes were dispersed in and on spleen, head kidney, liver, swim bladder and musculature. Encapsulation and inflammatory cellular reactions differed accordingly. Histopathological and immunohistochemical studies using monoclonal antibodies raised against salmonid IgM, CD8 and MHCII were performed to detect the presence of immune cells around the infecting nematodes. None of the three fish species showed positive reactions for IgM-bearing cells in the inflammatory tissue connected with nematodes. CD8+ cells were detected in all three species and MHCII-bearing cells were found associated with encapsulated A. simplex in rainbow trout and brown trout, but not in Baltic salmon. Physiological, immunological and pathological implications of microhabitat differences are discussed.

The accurate identification of anisakid nematodes at any life cycle stage is important both to deepen the knowledge on their taxonomy, ecology, epidemiology and for diagnosis and control, as larval stages cause a clinical disease in humans known as anisakidosis. With the aim to investigate the presence of anisakid larvae, specimens of horse mackerel, Trachurus trachurus (Linnaeus, 1758), silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), European anchovy, Engraulis encrasicolus (Linnaeus, 1758) and opah fish, Lampris guttatus (Brunnich, 1788), were collected by trawling at depths ranging from 50 to 400 m. A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region was used to identify Anisakis spp. larvae recovered in fish samples. Restriction profiles showed three banding patterns, corresponding to Anisakis pegreffii, Anisakis physeteris and to heterozygote pattern between A. pegreffii and Anisakis simplex s.s. Specimens showing the heterozygote restriction pattern were also analyzed by sequencing of the entire ITS region, to confirm the heterozygote status.

A gene coding for a 24 kDa protein (22 U homologous; As22U) was isolated from the Anisakis simplex third-stage larvae cDNA library during expressed sequence tag analysis. As22U was 636 bp long, and was found to code for 212 amino acid residues with a calculated mass of 23.5 kDa and a PI of 9.06. The As22U deduced amino acid sequence harbored a signal peptide region and 16 highly conserved cysteine residues, and it was identified in both the total extracts and excretory secretory (ES) protein of A. simplex. Its molecular weight was measured at 24 kDa via western blot analysis. The expression levels of thymic stromal lymphopoietin, IL-25, and CXCL1 (Gro-α) genes were increased at 6h after recombinant As22U treatment in mouse intestinal epithelial cells. Additionally, thymus and activation-regulated chemokine gene levels were increased at 14 h after treatment. Although we do not currently have sufficient evidence to determine whether As22U plays a role as an allergen, this remains possible. Further in vivo studies may provide some insight as to the allergenic properties of As22U.

The third-stage (L3) larvae of Anisakis, which are the etiological agents of human anisakiasis, have been categorized morphologically into Anisakis Type I larvae and Anisakis Type II larvae. Genetic analysis has allowed easy identification of these larvae: Anisakis Type I larvae include the species Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis simplex C, Anisakis typica, Anisakis ziphidarum, and Anisakis nascettii, whereas Anisakis Type II larvae include the species Anisakis physeteris, Anisakis brevispiculata, and Anisakis paggiae. Since human consumption of raw fish and squid is common in Japan, we investigated Anisakis L3 larvae in 44 specimens of Beryx splendens from Japanese waters. A total of 730 Anisakis L3 larvae collected from B. splendens were divided morphologically into 4 types: Type I, Type II, and 2 other types that were similar to Anisakis Type III and Type IV described by Shiraki (1974). Anisakis Type II, Type III, and Type IV larvae all had a short ventriculus, but their tails were morphologically different. In addition, data from genetic analysis indicated that Anisakis Type II, Type III, and Type IV larvae could be identified as A. physeteris, A. brevispiculata, and A. paggiae, respectively. Therefore, A. physeteris, A. brevispiculata, and A. paggiae can be readily differentiated not only by genetic analysis but also by morphological characteristics of L3 larvae.

Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy.

Anisakis simplex is a representative nematode parasitizing marine organisms, such as fish and squids, and causes not only anisakiasis but also IgE-mediated allergy. Although 10 kinds of proteins have so far been identified as A. simplex allergens, many unknown allergens are considered to still exist. In this study, a chemiluminescent immunoscreening method with higher sensitivity than the conventional method was developed and used to isolate IgE-positive clones from an expression cDNA library of A. simplex. As a result, three kinds of proteins, Ani s 11 (307 amino acid residues), Ani s 11-like protein (160 residues) and Ani s 12 (295 residues), together with three known allergens (Ani s 5, 6 and 9), were found to be IgE reactive. Furthermore, ELISA data showed that both recombinant Ani s 11 and 12 expressed in Escherichia coli are recognized by about half of Anisakis-allergic patients. Ani s 11 and Ani s 11-like protein are characterized by having six and five types of short repetitive sequences (5-16 amino acid residues), respectively. Both proteins share as high as 78% sequence identity with each other and also about 45% identity with Ani s 10, which includes two types of short repetitive sequences. On the other hand, Ani s 12 is also structurally unique in that it has five tandem repeats of a CX(13-25)CX(9)CX(7,8)CX(6) sequence, similar to Ani s 7 having 19 repeats of a CX(17-25)CX(9-22)CX(8)CX(6) sequence. The repetitive structures are assumed to be involved in the IgE-binding of the three new allergens.

In order to investigate the possible use of terpenic derivatives to treat anisakiasis caused by L 3 larvae of Anisakis , we studied the in vitro and in vivo larvicidal activity of three sesquiterpenes (nerolidol, farnesol and elemol). In vitro experiments included the histological study of larval damage and in vivo studies the measurement of myeloperoxidase activity in rat gastrointestinal tract after administration of the sesquiterpenes. In the in vitro assays, the most active compound against the L 3 larvae was nerolidol, followed by farnesol; both caused the death of all nematodes, which showed cuticle changes and intestinal wall rupture. In the in vivo assays, only 20% of infected rats treated with nerolidol or farnesol showed gastric wall lesions in comparison to 86.6% of control animals. According to these results, nerolidol and farnesol are good candidates for further research as biocidal agents against L 3 larvae of Anisakis type I.

To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30 mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.

Ani s 1, the major allergen of Anisakis simplex , is expected to be a useful antigen in allergen-specific immunotherapy of Anisakis allergy. To avoid side-effects such as anaphylactic shock in immunotherapy, it is desirable to use not native allergens but hypoallergenic mutants devoid of IgE-binding epitopes. This study was hence aimed to elucidate IgE-binding epitopes of Ani s 1 toward the development of hypoallergenic Ani s 1 mutants. Mapping experiments using 32 peptides (P1–32) revealed that major IgE-binding epitopes are included in P24 (region 116–130) and P28 (region 136–150). Furthermore, Ala-scanning and truncation experiments established that eight (underlined in the sequence 116- ELF A RE YEGV CK SG K -130) and nine amino acid residues (underlined in the sequence 136- RG S GWMM T IL GKS C D-150) are crucial for the IgE-binding of P24 and P28, respectively. The determined crucial residues of P24 and P28 were assumed to be involved in electrostatic and hydrophobic interactions with IgE, respectively.

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex , hexadecanoic acid in the dichloromethane extract of A. simplex , and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode . The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy: 1 , CoCoPg and Bu0:0B: 6 . The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex . Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite’s inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids – ceramides and sphingoid bases. The signals due to N -octanoyl- d -erythro-octasphinganine ( m/z 288.3) and N -tetranoyl- d -erythro-tetradecasphinganine ( m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.

It has been reported that nearly all cases of anisakiasis in Japan are caused by Anisakis simplex sensu stricto. To elucidate this further, we investigated the presence of Anisakis type I larvae and Pseudoterranova decipiens in 218 Scomber japonicus fish collected from the seas of Japan. Anisakis type I larvae were detected in 74.3% (162/218) of the fish, and 99.8% of the Anisakis type I larvae comprised A. simplex sensu stricto and Anisakis pegreffii. Molecular identification techniques were used for 7.5% (360/4806) of the Anisakis type I larvae. The larvae found in the fish of the Pacific stock (the Pacific coast of Japan) and the Tsushima Warm Current stock (the East China Sea and the Sea of Japan) were primarily A. simplex sensu stricto and A. pegreffii, respectively. In addition, for the first time in Japan, Anisakis simplex C and Anisakis ziphidarum were detected in the fish of the Pacific stock. The average number of A. pegreffii and A. simplex sensu stricto larvae per fish was 47 and 6, respectively. However, the average number (0.61 larvae) of A. simplex sensu stricto in the muscle per fish was 12 times the average number (0.05 larvae) of A. pegreffii. When fish on the purchased day were compared with those held at 4 degrees C and 20 degrees C for 20h, the penetration rates (ratio of the number of larvae detected in the muscle to the total number of larvae detected) of A. pegreffii and A. simplex sensu stricto were as high as 1.8% and 5.8%, respectively. In conclusion, we suggest that anisakiasis in Japan is mainly caused by A. simplex sensu stricto because it penetrates the muscle of the fish at a higher rate than A. pegreffii.

A study was conducted on the parasitization by anisakids of European anchovies (Engraulis encrasicolus) from the Eastern Atlantic (Gulf of Cádiz and Strait of Gibraltar) and Western Mediterranean (Ligurian Sea, Gulf of Lion, Catalonia coast and NW Alborán Sea) throughout 1998 and 1999. The anisakids detected were identified as third larval stages of Anisakis larva type I and Hysterothylacium aduncum. Global prevalence was 9.4% for Anisakis and 24.5% for H. aduncum. Analysis of the origin of the anchovies revealed a higher prevalence of Anisakis than H. aduncum in fish from the Atlantic and vice-versa in fish from the Mediterranean. Analysis of various fishing areas in the Western Mediterranean revealed a prevalence of Anisakis in fish from the Ligurian Sea that was 5-fold or more than in the other three areas, with a significantly greater prevalence of H. aduncum in fish from the NW Mediterranean than from the Spanish Mediterranean coast. The prevalence of infection was found to be significantly related to anchovy length for both Anisakis and H. aduncum. More than 55% of Anisakis larvae were found in the muscle. According to these data, the risk of acquiring anisakiasis/anisakidosis from the consumption of raw or under-cooked anchovies may depend upon the area in which they were caught.