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.     

Molecular phylogenetics and diagnosis of Anisakis, Pseudoterranova, and Contracaecum from northern Pacific marine mammals

Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.     

Infection status of the sea eel (Astroconger myriaster) purchased from the Noryangjin fish market with anisakid larvae]

Although the sea eel (Astroconger myriaster) is suspected as one of the most important fish host for human anisakiasis in Korea, no report has been made on the infection status of the sea eel with anisakid larvae. In the present study, 26 sea eels (Astroconger myriaster) were purchased from the Noryangjin fish market in Seoul, and anisakid larvae were collected from their viscera, muscle, head and skin. The collected larvae were classified by their morphological types. A total of 1,351 anisakid larvae were collected from 15 of 26 fish examined. Among them, 1,269 were recovered from the viscera, 66 from the muscle, and 16 from the head and skin. Morphologically, most of the anisakids were classified into 6 known larval types, Anisakis type I (564 larvae) of Berland (1961), Contracaecum type A (409) and type D (5) of Koyama et al. (1969), Contracaecum type C' (83) and type D' (117) of Chai et al. (1986), and Contracaecum type V (1) of Yamaguti (1935). Remaining 172 specimens were new in the available literature, hence, designated as Contracaecum type A' (new type). The present results revealed that the sea eels caught in the Korean waters are heavily infected with anisakid larvae, not only in their viscera but also in the muscle, and Anisakis type I was the most common among the 7 larval types.     

The value of external morphology in the identification of larval anisakid nematodes: a scanning electron microscope study

We studied larval nematodes of four genera of the Anisakidae using a scanning electron microscope (SEM). The anterior and posterior extremities and cuticular structures of the 3rd-stage larvae (L3) of Anisakis type I, Pseudoterranova decipiens, Contracaecum type B and Hysterothylacium were examined. The 4th-stage larvae (L4) of Anisakis type I, P. decipiens, recovered after infection into laboratory rats, and the L3 and L4 of Anisakis type I larvae from human were also examined in the same way. There were generic differences in the shape and size of the lip bulges, external papillary structures, the appearance of the boring tooth, the width and depth of the grooves and ridges of the cuticle and the caudal structures of the L3. In Anisakis type I and P. decipiens L3, changes were seen in the anterior extremity, cuticle and posterior extremity after molting to the L4. Similar changes can be expected in larvae infecting man. The L4 of Anisakis type I from rat and man were similar, while the L4 of Anisakis type I and P. decipiens showed differences. These ultrastructural differences might be of value in the identification of fragments recovered during endoscopy in man.     

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.     

Anisakis simplex and Anisakis physeteris: physicochemical properties of larval and adult hemoglobins

To resolve the taxonomic relationship between two types of parasitic nematode larvae (Type I and II) and two species of parasitic nematode adults (Anisakis simplex and A. physeteris) of the aquatic ascarid genus Anisakis, collected in Japanese coastal water, a comparison was made of their hemoglobins' physicochemical properties. The larval hemoglobins were more similar to each other in electrophoretic pattern than to either adult, indeed there were few similarities whatsoever in these patterns of larval and adult hemoglobins. However, isoelectric points were 6.2 for the Type I larva and for A. simplex and 5.4 for the Type II larva and for A. physeteris. All samples showed identical patterns in spectrophotometric scanning. The circular dichroic spectra of the samples were also virtually identical, although slight differences were noted in the oxygenated hemoglobins; the Type II larva and A. physeteris exhibited a small positive peak at 575 nm but the Type I larva and A. simplex exhibited a much smaller peak (negative position). The sedimentation coefficients of the samples possessed essentially identical values (11.2–12.4). The molecular weights of the samples were estimated, roughly, to be in the range 33 to 43 × 10⁴ by thin-layer chromatography on Sephadex G-200. The evidence suggests that a relationship may exist between the Type I larva and A. simplex, and between the Type II larva and A. physeteris.     

Fitness of the marine parasitic nematode Anisakis simplex s. str. in temperate waters of the NE Atlantic.

In temperate waters of the NE Atlantic, third-stage larvae of Anisakis simplex sensu stricto collected from 3 paratenic host species were identified by restriction fragment length polymorphisms. The condition of wild larval infrapopulations was assessed by examining morphometric and growth characteristics. The differentiation patterns and the excretory/secretory products of larvae grown in vitro were also examined. An extensive morphometric, growth and differentiation variability was found between parasite larvae collected from different paratenic host sources. Nematode infrapopulation larvae from the squid comprise those smaller individuals with the lowest values of survival rates and moult success. It may be then concluded that the fitness of A. simplex s. str. larvae is not the best possible in the squid, which impaired the competitiveness of the parasite and its chances of developing into an adult. This suggests that the microenvironments impaired by the paratenic host may provide larval infrapopulations with unique ecological factors probably influencing its recruitment to the final host populations.     

Molecular identification of two strains of third-stage larvae of Contracaecum rudolphii sensu lato (Nematoda: Anisakidae) from fish in Poland

Contracaecum sp. larvae (L3) from fish were identified using nucleotide sequences of the internal transcribed spacers ITS-1 and ITS-2 of the ribosomal DNA. The nematode larvae originated from fish in a freshwater situation (crucian carp Carassius carassius, from Selment Wielki Lake in Mazury, northeastern Poland) and a brackish-water region (Caspian round goby Neogobius melanostomus from the Baltic Sea, Gdafisk Bay at the Polish coast). Two strains (Contracaecum rudolphii A and B) of Contracaecum rudolphii senso lato, a parasite common at the adult stage in fish-eating birds, were identified. In fish from the freshwater site, only the strain temporarily designated C. rudolphii B was identified; in the brackish-water region, both strains were found, suggesting that fish serve as paratenic host for both genotypes. Contracaecum rudolphii sensu lato has been recorded in several species of fish-eating birds in Poland, particularly in the great cormorant, Phalacrocorax carbo, in which the abundance is highest. The results, although based on a restricted number of larvae, suggest that the life cycles of both genotypes can be completed in the Polish region and that at least one of them, C. rudolphii B, can develop both in fresh and brackish water.     

Multiple primer PCR for the identification of anisakid nematodes from Taiwan Strait

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.     

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.     

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.     

Nematodes of elasmobranch fishes from the southern coast of Brazil

New records for nematode species recovered from elasmobranch fishes in Brazil are established and new systematical arrangements proposed. Parascarophis sphyrnae Campana-Rouget, 1955 from the spiral valve of Sphyrna zygaena is referred for the first time in South America as a new host record. Procamallanus (S.) pereirai Annereaux, 1946, from the spiral valve of Raja castelnaui is reported parasitizing an elasmobranch host. Nematode larvae of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are listed from the stomach and spiral valves of several hosts. Anisakidae larvae previously referred in Brazil in the genus Phocanema should be reallocated in Pseudoterranova. Nematodes of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are reported for the first time parasitizing elasmobranchs in Brazil.     

Infection status of larval anisakids in Astroconger myriaster collected from the Southern Sea near Pusan]

A study was presented on the anisakid larvae in Astroconger myriaster which were caught at the Southern Sea and saled at Pusan area. The nematodes were morphologically classified into Anisakis sp. and Contracaecum sp. after fixation in 70% ethanol and clearing in lactophenol. Total of 1,768 larval anisakids were collected from 259 (67.8% positive rate) out of 382 examined A. myriaster. Total 642 larvae of Anisakis sp. were obtained from 94 (24.6%) and 1,126 Contracaecum sp. were recovered from 165 (43.1%) fish. The average number of worms per infected fish was 4.6. The infection rate increased according to the length of fish, and all of the fish over 71 cm were found infected. The numbers (proportions) of recovered worms by the organs were 1,440 (80.5%) in the omentum, 166 (9.4%) in the intestine, 107 (6.0%) in the stomach, 32 (1.8%) in the skin, 18 (1.0%) in the muscle, 13 (0.7%) in the liver, and 2 (0.1%) in the head. The larvae in the muscle may infect the humans who are enjoying raw sliced meat of the fish.     

Helminths parasitizing larval fish from Pantanal, Brazil

Fish larvae of 'corvinas' (Pachyurus bonariensis and Plagioscion ternetzi) from Sinhá Mariana Lagoon, Mato Grosso State, were collected from March 2000 to March 2004, in order to determine the parasitic fauna of fishes. Larvae from the two species were parasitized by the same endoparasites: Contracaecum sp. Type 2 (larvae) (Nematoda: Anisakidae) in the mesentery and Neoechinorhynchus (Neoechinorhynchus) paraguayensis (Acanthocephala: Neoechinorhynchidae) in the stomach and the terminal portion of the intestine. Statistical analysis showed that there was a significant positive correlation between the standard length of hosts and the abundance of acanthocephalans and nematodes, and that the prevalence of nematodes presented a significant positive correlation with the standard length of the two species of hosts, indicating the presence of a cumulative process of infection. The present study constitutes the first record of nematodes and acanthocephalans parasitizing larval fish, as well as the first record of endoparasites in fish larvae in Brazil. In addition, it lists a new locality and two species of hosts for Contracaecum sp. Type 2 (larva) and N. (N.) paraguayensis.     

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.     

RFLP analysis of SLA haplotypes in Swiss large white and American Hampshire pigs using SLA class I and class II probes

Summary

Restriction fragment length polymorphism (RFLP) analyses of swine leukocyte antigen (SLA) class I and class II genes from Swiss Large White and American Hampshire families were performed using porcine DNA probes. Class I and class II RFLPs associated with the serologically‐defined haplotypes SLA H1, H8, H16 and H24 and with serotypes SLA 15, 16; SLA 14; and SLA 6, SB 19, were identified. Seven allelic class I RFLP patterns were observed. For genes in the SLA class II region, six allelic RFLP patterns of DQA and DQB; five allelic RFLP patterns of DRA; and seven allelic RFLP patterns of DRB were observed. The serologically‐defined H8 haplotype was subtyped based on differences in class II RFLPs.     

Diagnosis of a case of gastric anisakidosis by PCR-based restriction fragment length polymorphism analysis

A set of genetic markers, based on PCR-RFLPs of three diagnostic restriction enzymes (Hhal, Hinfl and Taql), which proved to be suitable for the identification of the species of the genus Anisakis, was used for the first molecular identification of a larva obtained by endoscopy in a case of gastric anisakidosis, in a 51 year old woman from Southern Italy. The analysis of the restriction profiles obtained allowed the larva to be identified as Anisakis pegreffii, one of the three sibling species of the A. simplex complex. PCR-RFLP proved to be a cost-effective and reliable tool for the exact identification of Anisakis larvae recovered from infected humans.     

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.     

Insight into the short-finned squid Illex coindetii (Cephalopoda: Ommastrephidae) feeding ecology: is there a link between helminth parasites and food composition?

Squids are especially frequent as paratenic hosts of helminth parasites, particularly to those that have elasmobranchs and mammals as final hosts. Among those parasite species, anisakid nematode larvae and cestode plerocercoids are most effectively transferred through the trophic chain by oegopsid squids. A total of 439 short-finned squids, Illex coindetii (245 males, 190 females and 4 unsexed) were sampled in the central part of the eastern Adriatic Sea in order to assess their helminth component community and parasite dynamics with respect to host sex, maturity, seasonality, and feeding behavior. Two larval helminths were isolated, i.e., larvae of Anisakis pegreffii, characterized by molecular tools at the species level, and plerocercoids of Phyllobothrium sp., with prevalences of 30.5% and 2.3%, respectively. Highly significant seasonal variation in diet consumption, congruent with seasonal variation in anisakid intensity, was observed, underlining the tight role of squid prey in the trophic transmission of parasite. Likewise, the highest helminth prevalence and intensity of infection was recorded in autumn, when the fish prey, mostly Maurolicus muelleri, comprised the greatest proportion of diet. This helped to assign the Adriatic broadtail shortfin squid not as a first, but as a second, paratenic host for the anisakid, unlike as suggested previously. The presence of larval A. pegreffii confirms its previously reported zoogeographical distribution in the Mediterranean and Adriatic Seas. The presence of 2 helminths in I. coindetii describes the feeding patterns of the squid, as well as clearly defined and coevolved predator-prey relationships.     

Allozyme and PCR-RFLP markers in anisakid nematodes,aethiological agents of human anisakidosis

In the framework of the researches granted by MURST COFIN97, studies on anisakid nematodes, aethiological agents of human anisakidosis, were carried out. The project was aimed to implement the knowledge on the systematics, genetics, ecology and epizootiology of species of the genera Anisakis and Pseudoterranova by applying genetic markers obtained from multilocus allozyme electrophoresis and from PCR-based techniques. The results obtained by allozyme studies allowed to extend the geographic distribution and to detect new definitive and intermediate/paratenic hosts of two sibling species of the A. simplex complex, i.e. A. simplex s.s. and A. pegreffii and to characterise the species A. simplex C, a new sibling species within the A. simplex complex as well as a new species belonging to the genus Anisakis, A. ziphidarum. Combined allozyme and morphological analyses provided markers for the identification of the four sibling species of the Pseudoterranova decipiens complex and their nomenclatural designations. New markers based on PCR-RFLP analysis were used for the identification of sibling species of the Anisakis simplex complex and of another four species of the genus. Genetic markers based on three diagnostic restriction enzymes allowed the identification as A. pegreffii of a larva obtained by endoscopy in a case of human anisakidosis in Southern Italy.