Anisakiasis

Rello-Yubero, Francisco Javier; Adroher-Auroux, Francisco Javier; Valero-López, Adela. 2004. An. la Real Acad. Ciencias Vet. Andalucía Orient. 17, 173–198.
Prólogo. Hasta hace poco más de una década, en España no se podía sospechar que algu-nos pescados tradicionales, básicos en la dieta de los españoles, pudieran ser los res-ponsables de graves alteraciones digestivas y alérgicas. Estas patologías están provoca-das por las fases larvarias de ciertos nematodos, llamados anisákidos, con un ciclo de vida marino complejo, en el que distintos mamíferos marinos (focas, ballenas, delfines,...) actúan como hospedadores definitivos, interviniendo además en este ciclo gran variedad de peces, cefalópodos y crustáceos. El hombre sólo se infecta al ingerir accidentalmente larvas vivas de estos nematodos, actuando como un hospedador accidental no óptimo, pues en él no se desarrollan los nematodos adultos. Aunque en España son recientes las primeras denuncias de anisakidosis huma-na, se trata de una parasitación muy estudiada en Japón y Holanda, debido a sus pecu-liares hábitos alimentarios, en los que el consumo de pescado crudo, ahumado o con un tratamiento térmico insuficiente, está muy arraigado. Esto no quiere decir que, anterior-mente, en nuestro país no se diera este tipo de parasitación, sino que no ha sido reco-nocida al confundirla con otros desórdenes gastrointestinales que cursan de manera similar y que conducían a diagnósticos poco precisos en cuanto a su etiología. A un diagnóstico más certero hay que añadir la incorporación a la dieta de platos exóticos preparados con pescado crudo o semicrudo para comprender el aumento del número de casos denunciados en nuestro país. El ciclo de vida de estos parásitos aún no está completamente dilucidado, pero cada vez son más los estudios que implican a un mayor número de hospedadores vertebrados e invertebrados en el mismo. En nuestro país existen un gran número de especies de peces comerciales que pueden actuar como vehículos de transmisión al hombre, entre las que cabe destacar la bacaladilla, la merluza y el boquerón.

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.

Molina-Fernández, D., Benítez, R., Adroher, F.J., Malagón, D., 2019. Acta Trop. 195, 44–50. https://doi.org/10.1016/j.actatropica.2019.04.003
Proteolytic activity was studied in two sibling species of Anisakis (Nematoda: Anisakidae), A. simplex s.s. and A. pegreffii, throughout their in vitro development from third larval stage (L3) from the host fish (L3-0h) to fourth larval stage (L4) obtained in culture. Proteases have a significant role in the lifecycle of the parasite and in the pathogen-host relationship. Proteolytic activity peaks were detected at pH 6.0 and 8.5. Protease activity was detected in all the developmental stages of the two species studied at both pH values. These pH values were used for assaying with specific inhibitors which permitted the determination of metalloprotease activity, and, to a lesser extent, that of serine and cysteine protease. Aspartic protease activity was only detected at pH 6.0. At this pH, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p<0.001), the majority of activity being due to metalloproteases and aspartic proteases, which could be related to nutrition, especially the latter, as occurs in invertebrates. At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p<0.01). At this pH, the majority of activity was due to metalloproteases in all developmental phases of both species, although in L3-0h, the activity of these proteases was significantly higher (p<0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former. Serine proteases have frequently been implicated in the invasive capacity and pathogenicity of some parasites.This may be related to the significantly higher activity (p≤0.05) of serine protease in all the larval stages studied of A. simplex at pH 6.0. In summary, there are interspecific differences in proteases that have been related to pathogenesis in nematodes. These differences could thus be contributing to the previously reported differences in pathogenicity between these two Anisakis species.

In order to evaluate the effect of the high prevalence of parasites Anisakidae family, in fish caught in the fishing area Médano white, Falcon State, Venezuela; consumed by families of fishermen and villagers expended sector Pantano Abajo, municipality Miranda, Falcon state. The participatory diagnosis was made in the fishing area, in the consumer population and the dispenser Health Center (Ambulatory Urban) using unstructured interviews, surveys and review of medical records during the first half of 2013. An average of 1098 patient month has symptoms: asthma allergic diseases, diarrhea and vomiting, with percentages remains in a range that goes from 29.81% to 38.43%. 68% of fishermen dispense fish without sanitary permit. Fishermen and consumers are likely to be accidental hosts of the parasite, not having adequate prevention levels.

Cetaceans are well known definitive hosts of parasitic nematodes of the genus Anisakis (Nematoda: Anisakidae). Anisakid nematodes are also a health hazard for humans, potentially causing gastrointestinal infections or allergic reactions following the consumption of infected fish. In marine mammals, the nematodes develop from third-stage larvae to adults in the stomachs. In the first (or fore-) stomach, these parasites are typically associated with mucosal ulceration; parasites have not been identified in other organs. Two small cetaceans, a bottlenose dolphin Tursiops truncatus and a harbour porpoise Phocoena phocoena, presented marked gastric A. simplex infection, as well as chronic granulomatous and ulcerative dermatitis with intralesional nematodes, bordered by epithelial hyperplasia. Nematodes in the skin of the bottlenose dolphin were morphologically similar to Anisakis spp. Morphology of the parasitic remnants in the skin lesion of the harbour porpoise was indistinct, but mole...

Anisakiasis is a fish-borne zoonosis caused by Anisakis spp. larvae. One challenging issue in the diagnosis of anisakiasis is the molecular detection of the etiological agent even at very low quantity, such as in gastric or intestinal biopsy and granulomas. Aims of this study were: 1) to identify three new cases of invasive anisakiasis, by a species-specific Real-time PCR probe assay; 2) to detect immune response of the patients against the pathogen. Parasite DNA was extracted from parasites removed in the three patients. The identification of larvae removed at gastric and intestinal level from two patients was first obtained by sequence analysis of mtDNA cox2 and EF1 α-1 of nDNA genes. This was not possible in the third patient, because of the very low DNA quantity obtained from a single one histological section of a surgically removed granuloma. Real-time PCR species-specific hydrolysis probe system, based on mtDNA cox2 gene, was performed on parasites tissue of the three cases. I...

Twenty-three adults (only one male) and two fourth-stage larvae of Anisakis, recovered from the stomach of a Blainville&#39;s beaked whale (Mesoplodon densirostris) stranded in Galicia (NW Spain), were studied morphologically and molecularly. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing studies carried out on the ribosomal DNA spacers and on the cox2 mitochondrial gene confirm that these nematodes belong to the taxon Anisakis sp. A, which was previously detected as a larval stage in fishes from Madeiran waters. In addition, our molecular studies demonstrate that Anisakis sp. A and the taxon Anisakis sp., previously found in other Mesoplodon spp., are the same species. The adults of Anisakis sp. A are morphologically similar to A. ziphidarum but with a shorter body length and longer spicules (right spicule 2.42 mm, left spicule 2.30 mm). Since the poor condition of the male tail cuticle prevented a proper description of the caudal p...

Anisakids are fish-borne parasites that are responsible for a large number of human infections and allergic reactions around the world. World health organizations and food safety authorities aim to control and prevent this emerging health problem. In the present work, a new method for the fast monitoring of these parasites is described. The strategy is divided in three steps: (i) purification of thermostable proteins from fish-borne parasites (Anisakids), (ii) in-solution HIFU trypsin digestion and (iii) monitoring of several peptide markers by parallel reaction monitoring (PRM) mass spectrometry. This methodology allows the fast detection of Anisakids in $_amp_$lt;2h. An affordable assay utilizing this methodology will facilitate testing for regulatory and safety applications. The work describes for the first time, the Protein Biomarker Discovery and the Fast Monitoring for the identification and detection of Anisakids in fishery products. The strategy is based on the purification ...

Torralbo-Ramírez, V., Molina-Fernández, D., Malagón, D., Benítez, R., Adroher, F.J., 2019.  Foodborne Pathog. Dis. 16, 744–751. https://doi.org/10.1089/fpd.2019.2633
Humans can contract anisakiasis by eating fish or squid containing live larvae of the third stage (L3) of the parasitic nematodes of the genus Anisakis, majorly from Anisakis simplex s.s. and Anisakis pegreffii, sibling species of the A. simplex s.l. complex. Most cases diagnosed molecularly are due to A. simplex s.s., although A. pegreffii has also been identified in human cases. Cathepsins are mostly lysosomal multifunctional cysteine proteases and can participate in the pathogenicity of parasites. Cathepsin B and L activities were investigated in the two sibling species of Anisakis mentioned. L3 and L4 of both species were collected during their in vitro development, and cathepsin activity was determined in the range of pH 4.0–8.5, using specific fluorogenic substrates. The activity detected with the substrate Z-FR-AMC (N-α-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl-coumarin) was identified as cathepsin L (optimum pH = 5.0, range 4.0–6.0, p < 0.001). Activity was highest in L3 freshly collected from fish, especially in A. simplex s.s., and decreased during development, which could be related to virulence, invasion of host tissues, and/or intracellular digestion. Cathepsin B-like activity was not identified with either of the substrates used (Z-RR-AMC [N-α-benzyloxycarbonyl-L-arginyl-L-arginine-7-amido-4-methyl-coumarin] and Z-FR-AMC). With Z-RR-AMC, cleaving activity was detected almost exclusively in L4 of A. simplex s.s. (p < 0.05) with optimum pH = 8.0 (range 7.0–8.5). Assays with class-specific protease inhibitors showed that this activity was mainly due to serine proteases [up to 90% inhibition with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)], although metalloproteases (up to 40–45% inhibition with 1,10 phenanthroline) and slight cysteine protease activity (<15% inhibition with E64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane]; putative cathepsin B-like) were also detected. These results show differential serine protease activity between sibling Anisakis species, regulated by larval development, at least in A. simplex s.s. The higher cathepsin L and serine protease activities detected in this species could be related to its greater pathogenicity, reported in experimental animals, compared to that of A. pegreffii.

Molina-Fernández, D., Rubio-Calvo, D., Adroher, F.J., Benítez, R., 2018. Int. J. Food Microbiol. 282, 49–56. https://doi.org/10.1016/j.ijfoodmicro.2018.05.026
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, 91.1% 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 2.3years) and later with Anisakis type II (mean age 2.7years),
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.

Gastrointestinal anisakiasis is a parasitic disease acquired by humans after ingestion of raw fish infected with third stage larvae of the nematode Anisakis simplex. Even if considered a rare disease in Western countries, it is underestimated in many countries with large fishing-based activities and widespread consumption of raw sea food. This could also depend on the observation that the nematode is often hidden among gastric folds, and can be confused with gastric mucus. Consequently, gastric detection could be difficult, and the use of narrow band imaging (NBI) has been recently suggested to improve parasite detection at endoscopy [1]. To our knowledge, 11 cases of gastric anisakiasis have been described in Italy [1, 2]. Moreover, some cases have been reported in other European countries, such as Austria, Germany, and Spain [3-5]. We observed a patient complaining of acute epigastric pain and vomiting within 3 hours following the ingestion of uncooked anchovies. Due to persistent...

A total of 1,600 specimens, consisting of 16 different species of marine fish, were dissected and examined for anisakid larvae and adults in visceral organs, abdominal cavity, and muscles. One species of adult-stage nematode was found in two of 16 species of marine fish studied, Johnius carouna and Dendrophysa russelli. No anisakid larvae (third-stage) was found in any of the 16 species of marine fish studied. Morphological study of the adult-stage nematode showed similar morphology to Anisakis simplex. We found that the nematode adult recovered from the marine fish differed from other anisakids in morphology, life cycle and locality of infection in the fish. The anisakid adults recovered were ovoviviparous or larviparous, but not oviparous as is seen in most other anisakids. The intensity and prevalence of nematode infection in Johnius carouna were 2.4 and 31.7%, respectively, and in Dendrophysa russelli 3.9 and 87.5%, respectively.

The endohelminth parasite fauna of a deep water shark, the leafscale gulper shark, Centrophorus squamosus, examined from Madeiran waters, from September 2009 to January 2010, consisted of larval and juvenile cestodes of two orders, namely Trypanorhyncha and Tetraphyllidea, and L3 stages of Anisakis spp. Infection with Anisakis spp. could be due to the shark’s opportunistic feeding on squids and black-scabbard fish, Aphanopus carbo, which is heavily parasitized by Anisakis spp. in Madeira waters. The occurrence of larval and juvenile cestodes only, in this shark, suggests that the leafscale gulper shark features as a paratenic or a dead-end host for the parasites.

A 1,963-bp cDNA was isolated from an Anisakis simplex cDNA library by immunoscreening with a hyperimmune rabbit serum raised against a crude extract of A. simplex L3 larvae. The open reading frame encodes a putative protein of 436 amino acid residues, which exhibits high similarity (70–80%) to enolase molecules from various other organisms, including helminth parasites. After subcloning and expression of the A. simplex cDNA in PGEX-4T-3, the resulting glutathione S-transferase fusion protein, purified by glutathione-Sepharose-4B chromatography, showed functional enolase activity. The immunogenicity of the recombinant A. simplex enolase was analyzed by immunoblotting using sera obtained from (a) mice immunized with crude extracts (CE) of A. simplex, or other nematode species, (b) mice immunized with excretory–secretory (ES) antigens from A. simplex, or (c) mice infected with L3 larvae by the intraperitoneal route. In addition, we used ELISA, to investigate the presence of IgG1 and IgE antibodies against this molecule in sera from patients infected with A. simplex. Mouse sera obtained after infection with L3 or raised against CE antigens, but not sera raised against ES antigens, showed strong reactivity with the recombinant A. simplex enolase. We also obtained good reactivity in Western blotting with sera from mice immunized with CE antigens from Ascaris suum and Toxocara canis, but not with sera from mice immunized with CE antigens from Trichuris muris, Trichinella spiralis or Hysterothylacium aduncum. In contrast to the experimental infections/immunizations in mice, we were unable to detect anti-enolase IgE antibodies in sera from human patients infected with A.simplex (15 sera), and the levels of anti-enolase IgG1 antibodies in these sera were low and apparently nonspecific. These results seem to indicate that, during natural infection in humans, A. simplex larvae do not offer sufficient antigenic stimulus to induce anti-enolase antibodies.