Molecular characterization of Anisakis larvae from fish caught off Sardinia

Anisakis spp. larvae are parasitic, and potentially zoonotic, nematodes transmitted by marine fish and cephalopods, which are the main intermediate hosts of the third larval stage. The accidental consumption of infected raw or poorly cooked fish may cause gastroenteric diseases and allergies in humans. The aim of the present study was to use polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to define the occurrence, species variability, and host preferences of Anisakis spp. larvae in fish caught off the coast of Sardinia. Necropsy was used on 285 samples; 552 Anisakis spp. L3 larvae were isolated from 87 fish that tested positive for this nematode. Anisakis pegreffii was most frequently encountered (90.6%), with a primary preference for Scomber scombrus, Zeus faber, and Trachurus mediterraneus. In contrast, the prevalence of Anisakis physeteris was only 1.3%. A hybrid genotype of Anisakis simplex sensu stricto and Anisakis pegreffii was also observed, which confirms the results of previous studies carried out in the western Mediterranean. Interestingly, no Anisakis simplex s.s. larvae were recovered. These results indicate that the diversity of Anisakis species is low in Sardinia waters, probably because of its geographic position.     

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.     

Identification of the secreted neutral proteases from Anisakis simplex

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.     

Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder

The third-stage larvae of Anisakis simplex sensu lato (s.l.) are found in many marine fishes. To ensure food safety, it is important to determine whether these larvae are present in the body muscle of commercial fish species. However, there is little information regarding the tissue specificity of Anisakis and two of its sibling species, A. simplex sensu stricto (s.s.) and Anisakis pegreffii, that are common in marine fish in Japanese waters. We orally challenged rainbow trout (Oncorhynchus mykiss (Walbaum)), and olive flounder (Paralichthys olivaceus (Temminck and Schlegel)) with L3 larvae of these two sibling species and monitored infection for 5weeks. In rainbow trout, A. simplex s.s., but not A. pegreffii larvae, migrated into the body muscle. A small number of freely moving A. pegreffii larvae were recovered within the body cavity. In olive flounder, A. simplex s.s. larvae were found in both the body cavity and body muscle. A. pegreffii larvae were found only in the body cavity and primarily encapsulated in lumps. Our results indicate that there are differences in the sites of infection and host specificity between the two sibling species of A. simplex s.l.     

Molecular identification of Anisakis species from Pleuronectiformes off the Portuguese coast

Anisakid nematodes belonging to the Anisakis simplex complex are highly prevalent in several fish species off the coast of Portugal and are an important zoonotic problem in the Iberian Peninsula. Two reproductively isolated sibling species of the Anisakis simplex complex were identified from Pleuronectiformes inhabiting the Portuguese coast using restriction fragment length polymorphism (RFLP). Recombinant genotypes corresponding to presumptive Anisakis simplex sensu stricto and Anisakis pegreffii hybrids were also detected by this technique, as well as the species Anisakis typica. Although 25 species of Pleuronectiformes were investigated, Anisakis spp. larvae were only found in seven: Arnoglossus imperialis, Arnoglossus laterna, Lepidorhombus boscii, Citharus linguatula, Platichthys flesus, Dicologlossa cuneata and Solea senegalensis. The occurrence of hybrids in relatively sedentary fishes such as the Pleuronectiformes suggests that the Portuguese coast may constitute an area of hybridization and, therefore, is of particular interest for the study of the process of hybridization and speciation for these anisakids.     

Anisakis - from the platter to the microfuge

Anisakiasis is a disease caused by the ingestion of anisakid nematodes in raw or improperly prepared fish dishes. Invading larvae penetrate the mucosa and submucosa of the gastrointestinal tract and produce lesions characterized by a marked inflammatory response. Judy Sakanari describes the biochemical and molecular studies on the proteins excreted and/or secreted by Anisakis larvae that are currently underway to help elucidate the role these proteins may play in the invasion process and the pathogenesis of anisakiasis.     

Genetic identification of Anisakis larvae in European hake from Atlantic and Mediterranean waters for stock recognition

The occurrence of seven species of the larval parasitic nematode Anisakis , which can be used as a biological tag for hake Merluccius merluccius stocks throughout their geographical range, is reported. Hake were collected from 14 localities in the Mediterranean Sea and the Atlantic Ocean. Anisakis larvae ( n  = 1950), which were recovered, were identified to species by means of genetic markers (allozymes). Within Anisakis type I, the larvae of A. pegreffii , A. simplex s.s ., A. typica and A. ziphidarum were detected, while within Anisakis type II, A. physeteris , A. brevispiculata and Anisakis sp. were identified. There were significant differences in the relative proportions of the various Anisakis species identified in hake samples from the Mediterranean Sea and Atlantic Ocean, suggesting the existence of different stocks of M. merluccius in European waters.     

Anisakis pegreffii Larvae in Sea Eels (Astroconger myriaster) from the South Sea,Republic of Korea

Anisakis simplex sensu stricto (s.s.), Anisakis pegreffii, Anisakis berlandi (=A. simplex sp. C), and Anisakis typica are the 4 major species of Anisakis type I larvae. In the Republic of Korea (Korea), A. pegreffii, A. berlandi, and A. typica larvae in fish hosts has seldom been documented. In this study, molecular analysis was performed on Anisakis larvae from the sea eels (Astroconger myriaster), the major source of human anisakiasis in Korea, collected from Tongyeong City, a southern coastal area of Korea. All 20 sea eels examined were infected with Anisakis type I larvae (160 larvae; 8 per fish). Their species were analyzed using PCR-RFLP patterns and nucleotide sequences of internal transcribed spacers (ITS1, 5.8 subunit gene, and ITS2) and mitochondrial cytochrome c oxidase 2 (cox2). Most (86.8%; 112/129) of the Anisakis type I larvae were A. pegreffii, and 7.8% (10/129) were A. typica. The remaining 5.4% (7/129) was not identified. Thus, A. pegreffii is the major species of anisakid larvae in sea eels of the southern coast of Korea.     

The identity of Anisakis type II larvae with Anisakis physeteris confirmed by restriction fragment length polymorphism analysis of genomic DNA.

The identity of Anisakis type II larvae with adult A. physeteris was confirmed by comparison of restriction fragment length polymorphisms (RFLPs) of 25S ribosomal DNA (rDNA). Patterns of RFLPs in larvae were almost identical with those in adult worms. Directly labelled 25S rDNA might serve as an appropriate probe with highly specific activity for examining RFLPs of larvae and adult worms.     

Electrophoretic identification of Anisakis sp. larvae (Ascaridida: Anisakidae) from Clupea harengus L. in Baltic Sea

On the basis of electrophoretic studies carried out on 15 gene-enzyme systems, 80 Anisakis sp. larvae from the herring Clupea harengus, fished in South Baltic Sea and Gdanisk Bay, were identified as Anisakis simplex B. This is the first record of Anisakis simplex B in the Baltic Sea. The spawning migration of the herrings from the North Sea to the Baltic Sea and the distribution of A. simplex B are briefly discussed.     

Ani s 10, a new Anisakis simplex allergen: cloning and heterologous expression

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.     

Radiolabelling of the excretory-secretory and somatic antigens of Anisakis simplex larvae.

Anisakis simplex larvae were cultured in vitro in medium containing 35S-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immuno-complexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera.     

The response of Anisakis larvae to freezing

Anisakis third stage larvae utilize a variety of fish as intermediate hosts. Uncooked fish are rendered safe for human consumption by freezing. Larvae freeze by inoculative freezing from the surrounding medium but can survive freezing at temperatures down to -10 degrees C. This ability may be aided by the production of trehalose, which can act as a cryoprotectant, but does not involve recrystallization inhibition. Monitoring of fish freezing in commercial blast freezers and under conditions which simulate those of a domestic freezer, indicate that it can take a long time for all parts of the fish to reach a temperature that will kill the larvae. This, and the moderate freezing tolerance of larvae, emphasizes the need for fish to be frozen at a low enough temperature and for a sufficient time to ensure that fish are safe for consumption.     

Cross-reactivity between antigens of Anisakis simplex s.l. and other ascarid nematodes

A study of the cross-reactivity among somatic and excretory-secretory antigens of the third stage larvae of Anisakis simplex s.l. and somatic antigens of other ascarid nematodes (Ascaris lumbricoides, A. suum, Toxocara canis, Anisakis physeteris, Hysterothylacium aduncum and H. fabri) was carried out by immunoblotting. It was revealed a high degree of cross-reactivity among ascarids in the 30 and > 212 kDa range by using sera against somatic and excretory-secretory antigens of A. simplex s.l. It has been revealed also specific components of the Anisakis genus (< 7.2, 9, 19 and 25 kDa) that will be interesting in diagnosis.     

Electrophoretic identification of larvae and adults of Anisakis (Ascaridida:Anisakidae)

The relationships between larvae and adults of Anisakis from the Mediterranean Sea and North-East Atlantic Ocean were analysed by multilocus electrophoresis. The correspondence of type I larvae with the A. simplex complex, including the sibling species A. simplex A and B, and of type II larvae with A. physeteris is confirmed. 19 of the 22 loci studied discriminated between the two larval types. Biochemical keys are given for the electrophoretic identification of A. simplex A, A. simplex B and A. physeteris, at both the larval and adult stages.     

Anisakis simplex (s.s.) larvae in wild Alaska salmon: no indication of post-mortem migration from viscera into flesh

The prevalence, mean intensity and distribution of Anisakis nematode third-stage larvae (L3) in the muscle and viscera of wild-caught chum salmon Oncorhynchus keta, pink salmon O. gorbuscha and sockeye salmon O. nerka were compared immediately after catch. Salmon were collected during the fishing season in July 2007 in Bristol Bay and Prince William Sound close to Cordova, Alaska (USA). All fish were infected, and more than 90% of the nematode larvae were found in the edible muscle meat. The isolated anisakid L3 were genetically identified as A. simplex (s.s.). The distribution of nematodes in the muscle meat of fresh-caught salmon was examined in 49 O. keta, 50 O. nerka and 12 O. gorbuscha from Cordova. Most of the larvae were detected in the muscle parts around the body cavity, but nematodes were also found in the tail meat and epaxial muscle (loins). The mean intensity of Anisakis larvae in the edible part was 21 individuals for O. gorbuscha, 62 individuals for O. keta and 63 individuals for O. nerka. No difference in the intensity of Anisakis larvae in the hypaxial muscle was found between fresh-caught and immediately gutted salmon and individuals stored ungutted for 24 h either on ice or in refrigerated sea water.     

Occurrence and survival of the larval nematode Anisakis sp. in the flesh of fresh, frozen, brined, and smoked pacific herring, Clupea harengus pallasi

Two hundred and twenty Pacific herring (Clupea harengus pallasi) from Yaquina Bay, Oregon, were divided into five groups to test the effects of various methods of handling and processing (fresh, frozen, brined, cold smoked, and cold smoked-gibbed) on the occurrence and survival of Anisakis larvae in the flesh. Viscera and flesh were digested separately using a pepsin-HC1 technique, and counts were taken of Anisakis larvae. Parasite loads were statistically largest in the flesh of frozen, brined, and smoked fish. Smoked fish harbored the greatest proportions of larvae in the flesh. Human consumption of brined or cold smoked Pacific herring represents a potential public health hazard.     

Distribution of Anisakis larvae, identified by genetic markers, and their use for stock characterization of demersal and pelagic fish from European waters: an update

In the present paper, recent results obtained on the use of different distributions observed in larval species of Anisakis, genetically identified by means of allozyme markers, for stock characterization of demersal (Merluccius merluccius), small (Trachurus trachurus) and large pelagic (Xiphias gladius) finfish species in European waters, are reviewed and discussed. Several species of Anisakis were identified in the three fish hosts: A. simplex (s.s.), A. physeteris, A. typica, A. ziphidarum, A. pegreffii, A. brevispiculata and A. paggiae. Canonical discriminant analysis performed on all the samples of the three fish species collected in areas comprising their geographical range, according to the different species of Anisakis identified, showed distinct fish populations in European waters. In all the three fish hosts, the pattern of distribution of Anisakis larvae allowed discrimination of Mediterranean stocks from Atlantic stocks. In the case of swordfish, the possible existence of a southern Atlantic stock separated from a northern one is also suggested. Congruence and discordance with the population genetic data inferred from allozyme markers on the same samples of the three fish species are also discussed.     

Radioresistance of Anisakis simplex third-stage larvae and the possible role of superoxide dismutase

The radioresistance of Anisakis simplex third-stage larvae and the possible role of sublethal radiation on superoxide dismutase (SOD) were investigated. Larvae were isolated from the viscera of the sea eel Anago anago; irradiated with 10, 100, 200, 500, or 1,000 Gy; and then given orally to rats. Worms were recovered at 16 hr postinoculation. Most larvae were found to have invaded the gastric wall, omentum, and abdominal cavity, suggesting that their viability and infectivity were not controlled by irradiation with the doses used. To determine the relationship between SOD activities in parasites and their radiosensitivities, the larvae of A. simplex and the metacercariae of Neodiplostomum seoulense (a radiosensitive control) were irradiated with 0, 30, 100, or 500 Gy, and parasite SOD levels were measured. In nonirradiated A. simplex larvae, the average SOD level was 38.9 U/mg, and this increased to 51.3 U/mg at 500 Gy. However, at all radiation doses applied, SOD activities of N. seoulense metacercariae were significantly (P < 0.05) lower than those of A. simplex larvae. Our results demonstrate that A. simplex third-stage larvae are radioresistant, and suggest that SOD plays a role in this radioresistance.