First case of human anisakiasis in Poland

Consumption of raw or inadequately processed marine fish may result in anisakidosis – a zoonotic disease caused by larvae of the parasitic nematodes of the family Anisakidae (anisakiasis when caused by members of the genus Anisakis (Nematoda: Anisakidae)), commonly found in a variety of marine fish species all over the world. Most cases of anisakidosis have been detected in the residents of Japan and South Korea, which results from the tradition of eating raw and semi-raw fish dishes. However, the disease is now increasingly often diagnosed in other parts of the world, including Europe (mainly in Spain and Italy). In Poland, no cases of human infection with anisakid nematodes have been detected so far. In this study, we report the first case of gastric anisakiasis in Poland, in a 59-year-old female patient, after eating raw Atlantic salmon (Salmo salar). The parasite was identified as the third-stage larva of Anisakis simplex sensu stricto on the basis of morphology and molecular analysis. The larva was still alive and causing pain until it was removed, which occurred more than 5 weeks after infection. The described case prove that anisakiasis should be considered as a potential cause of gastrointestinal tract ailments following the consumption of seafood in countries where no cases of this zoonosis have been reported to date.     

A case of acute gastric anisakiasis provoking severe clinical problems by multiple infection

Acute gastric anisakiasis with multiple anisakid larvae infection is reported. A 68-year-old woman residing in Busan, Korea, had epigastric pain with severe vomiting about 5 hours after eating raw anchovies. Four nematode larvae penetrating the gastric mucosae in the great curvature of the middle body and fundus were found and removed during gastro-endoscopic examination. Another one thread-like moving larva was found in the great curvature of upper body on the following day. On the basis of their morphology, the worms were identified as the 3rd stage larvae of Anisakis simplex. This case is acute gastric anisakiasis provoking severe clinical problems by the multiple infection and the greatest number of anisakid larvae found in a patient in Korea.     

Intestinal anisakiasis in Italy: case report

A case of intestinal anisakiasis caused by Anisakis sp. larva type I in a woman from Italy who consumed raw marinated anchovies, is reported. The diagnosis was based on the morphological features characteristic of anisakid larval stages, which were readily recognized in a large granuloma removed after emergency surgical treatment.     

A Case of Anisakidosis Caused by Pseudoterranova decipiens Larva

Pseudoterranova decipiens larva is a rare cause of anisakiasis. Indeed, prior to the present study, there had been only 12 reports of larval P. decipiens infection in the Republic of Korea. In June 2011, an anisakid larva, 32.1 mm in length and 0.88 mm in width, and finally identified as the third stage larva of P. decipiens owing to the presence of an intestinal cecum but lacking ventricular appendage, was discovered in a 61-year-old woman during the course of endoscopy executed as a part of routine physical examinations. The patient had eaten raw a rockfish 13 hr prior to the endoscopy, but showed no symptoms of anisakiasis. This paper is the 13th report of P. decipiens infection in Korea.     

A case of anisakiasis causing intestinal obstruction

A 31-year old salesman living in Seoul developed suddenly abdominal pain due to intestinal obstruction. Exploratory laparotomy exhibited segmental jejunal cellulitis caused by penetrating Anisakis larva. The patient had eaten raw fish. The typical history of intestinal anisakiasis was presented with a short review of Korean patients of anisakiasis.     

Molecular identification of the etiological agent of the human anisakiasis in Japan

Anisakis simplex complex presently comprises three sibling species, A. simplex sensu stricto, A. pegreffii and A. simplex C. A. simplex is a common parasite in fishes and cephalopods and capable of causing anisakiasis in humans. Therefore, identification of sibling species of A. simplex was important for human health. In this study, one hundred Anisakis type I larvae isolated from eighty five patients with anisakiasis in Hokkaido and Kyushu in Japan were analyzed by adapting the new molecular method that can identify the sibling species of A. simplex complex. Based on the restriction fragment length polymorphism (RFLP) pattern of ITS regions including 5.8 subunit rRNA gene, we identified two sibling species, A. simplex s. str. and A. pegreffii. However, the infection rate of A. simplex s. str. was significantly higher than that of A. pegreffii. Eighty four (98.8%) out of the eighty five patients were infected with A. simplex s. str. On the contrary, one patients (1.2%) in Kyushu infected with A. pegreffii. This study provided basic information about human infection with A. simplex complex. Furthermore, we suggested that A. simplex s. str. is the most important etiological agent in Japan.     

Anisakiasis: Report of 15 Gastric Cases Caused by Anisakis Type I Larvae and a Brief Review of Korean Anisakiasis Cases

The present study was performed to report 15 anisakiasis cases in Korea and to review the Korean cases reported in the literature. Total 32 Anisakis type I larvae were detected in the stomach of 15 patients by the endoscopy. Single worm was detected from 12 cases, and even 9 larvae were found from 2 cases. Epigastric pain was most commonly manifested in almost all cases, and hemoptysis and hematemesis were seen in 1 case each. Symptom manifestations began at 10-12 hr after eating fish in 73.3% cases. Endoscopy was performed 1-2 days after the symptom onset in most cases. The common conger, Conger myriaster, was the probable infection source in 7 cases. In the review of Korean anisakiasis cases, thus far, total 645 cases have been reported in 64 articles. Anisakis type I larva was the most frequently detected (81.3%). The favorable infection site of larvae was the stomach (82.4%). The common conger was the most probable source of human infections (38.6%). Among the total 404 cases which revealed the age and sex of patients, 185 (45.8%) were males, and the remaining 219 (54.2%) were female patients. The age prevalence was the highest in forties (34.7%). The seasonal prevalence was highest in winter (38.8%). By the present study, 15 cases of gastric anisakiasis are added as Korean cases, and some epidemiological characteristics of Korean anisakiasis were clarified.     

Intestinal myiasis due to Eristalis tenax: report of a new case in Spain

We describe a new case of accidental intestinal myiasis by Eristalis tenax in Spain. Only about 20 cases have been reported worldwide, two of them occurring in Spain. A 51-year-old patient with nonspecific abdominal pain and occasional diarrhoea expelled larvae in her stool. Macroscopic analysis of these larvae revealed morphology compatible with that of Eristalis tenax. The larva analysis showed its autofluorescence as parasitological feature described for the first time.     

Immune reactions and allergy in experimental anisakiasis

The third-stage larvae (L3) of the parasitic nematode, Anisakis simplex, have been implicated in the induction of hyperimmune allergic reactions in orally infected humans. In this work, we have conducted a review of an investigation into immune reactions occurring in animals experimentally infected with A. simplex L3. The patterns of serum antibody productions in the experimental animals against excretory-secretory products (ESP) of A. simplex L3 contributed to our current knowledge regarding specific humoral immune reactions in humans. In our review, we were able to determine that L3 infection of experimental animals may constitute a good model system for further exploration of immune mechanisms and allergy in anisakiasis of humans.     

Onchocerca volvulus: biochemical and morphological characteristics of the surface of third- and fourth-stage larvae

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.     

Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.     

The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island Wallaby (Macropus eugenii): The parasitic stages

Smales L. R. 1977. The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island Wallaby (Macropus eugenii): the parasitic stages. International Journal for Parasitology7: 457–461. Labiostrongylus eugenii infective larvae ingested by the Kangaroo Island Wallaby exsheathe on the saccular stomach wall, then invade the mucosa causing one or two chronic irritative hyperpastic nodules, each containing a number of larvae. Six to eight weeks later the larvae leave the stomach wall and moult to fourth-stage. The fourth-stage larva is characterized by eight lips, while the oesophagus and anterior intestine assume the adult form. The posterior ends of late fourth-stage larvae become sexually differentiated. Moulting to adult occurs after 6–8 weeks with the first gravid females being observed 3 months later. The prepatent period appears to last about 6 months. Reasons for failure of experimental infection trials and the epidemiological significance of the life cycle were discussed.     

Paracuaria hispanica n. sp. (Nematoda: Acuariidae), a stomach parasite of the pyrenean desman Galemys pyrenaicus Geoffr. (Insectivora: Talpidae), with a redefinition of the genus Paracuaria Rao, 1951

Adult and fourth-stage larvae of Paracuaria hispanica n. sp., from the stomach of the Pyrenean desman Galemys pyrenaicus Geoffroy (Insectivora: Talpidae) in northern and central Spain, are described. The new species differs from the other members of the genus Paracuaria (P. adunca and P. soricis), among other morphological details, in its smaller body and spicule sizes, the presence of a cuticular ring around the tip of the female tail, and the existence of lateral alae running longitudinally along its body from the cervical region to the tail. In view of the latter feature, the genus Paracuaria is redefined. The fourth stage larva of the new species is distinguished from that of P. adunca by its monocuspid deirids. P. hispanica occurred in 45% of the 20 host specimens examined.     

A case of abdominal syndrome caused by the presence of a large number of Anisakis larvae.

A 58-year-old woman living in Shizuoka Prefecture, Japan, complained of severe epigastric pain and nausea about 8 h after having eaten 'Sashimi'. A gastrocamera examination undertaken as a result of her clinical history of gastric anisakiasis indicated that a large number of worms had penetrated the posterior walls. Fifty-six larval nematodes were directly removed from the greater curvature of the stomach with a gastroendoscopic biopsy clipper, and were identified as the larvae of Anisakis simplex.     

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.     

Ani s 1, the major allergen of Anisakis simplex: purification by affinity chromatography and functional expression in Escherichia coli

Third stage larvae of the nematode Anisakis simplex often infect marine fish and invertebrates. When the larvae are ingested orally via seafood, they can cause IgE-mediated allergic reactions as well as anisakiasis. Of the known A. simplex allergens, Ani s 1 (Kunitz/bovine pancreatic trypsin inhibitor family protein) has been demonstrated to be a major allergen, being expected to be a useful tool for diagnosis of A. simplex allergy. For a diagnostic purpose, sufficient amounts of either natural Ani s 1 (nAni s 1) or recombinant Ani s 1 (rAni s 1) with an IgE-binding capacity should be stably supplied whenever needed. In this study, therefore, we first developed a simple and rapid purification method for Ani s 1 that is based on affinity chromatography using anti-Ani s 1 antibodies as ligands. The method was shown to produce nAni s 1 with a higher yield than the previously reported methods. Then, an attempt was made to express rAni s 1 in Escherichia coli as a His-tagged protein. rAni s 1 obtained as an inclusion body was solubilized in a solvent containing denaturing and reducing reagents and purified by nickel-chelate chromatography. Refolding of rAni s 1 was accomplished by dialysis in the presence of arginine, followed by that in the absence of arginine. Fluorescence ELISA and inhibition ELISA data revealed that rAni s 1 is IgE reactive enough to be used as a diagnostic tool.     

Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI‐TOF/TOF analysis or de novo sequencing. Seventy‐five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty‐eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 ( http://proteomecentral.proteomexchange.org/dataset/PXD000662 ).     

Distribution of Anisakis species larvae from fishes of the Japanese waters

Human anisakiasis is caused by the consumption of raw, marinated or undercooked fish and squid infected with nematodes of the genus Anisakis Dujardin, 1845. In view of food safety, this study was carried out to examine the distribution of Anisakis species in marine fishes within Japanese waters. Seven fish species from six localities were collected and examined for Anisakis infection. Morphological and molecular (ITS region and mtDNA cox2 gene) characterization revealed the presence of two, among the three sibling species of Anisakis simplex, viz. A. simplex sensu stricto (s.s.) and A. pegreffii. Distribution data were collated with the results from the previous researches to better understand Anisakis distribution in Japanese waters. Distributions of Anisakis species were found to be locality-specific rather than host-specific, particularly between the two major species, A. simplex s.s. and A. pegreffii. Anisakis simplex s.s. is mainly found in fishes from northern Japan to Pacific sides, whereas A. pegreffii is in fishes from the Sea of Japan to East China Sea sides.     

Population structure of Anisakis simplex (Nematoda) in harbor porpoises Phocoena phocoena off Denmark

The population structure and habitat selection of Anisakis simplex in 35 harbor porpoises off Denmark are described. The nematodes were collected from the stomach and duodenal ampulla and were categorized as third-stage larvae, fourth-stage larvae, subadults, and adults. The porpoises harbored 8,043 specimens of A. simplex. The proportion of adults and subadults increased with infrapopulation size. The number of development stages across infrapopulations covaried significantly (Kendall's test of concordance). Concordance was higher in hosts with the highest intensities than in those with low and medium intensities. All stages occurred mainly in the forestomach, but this trend was stronger for the adults. Adult and subadult sex ratios did not depart significantly from 1:1. Our data suggested that recruitment and duration of each stage were the main factors accounting for infrapopulation structure. The preference of A. simplex for the forestomach conformed with previous studies, but the narrower distribution of adults relative to other stages might indicate a strategy to enhance mating opportunities. Information on sex ratios of A. simplex is scarce and contradictory. We suggest that the discrepancies might partly reflect differences in categorization criteria and statistical methods.     

Morphological and molecular characterization of Anisakis larvae (Nematoda: Anisakidae) in Beryx splendens from Japanese waters

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.