Simplex Sensu Lato Research Articles

Occurrence and prevalence of third-stage larvae of the Anisakidae family in Macrouridae species captured from bathyal depths of Sagami Bay, Japan

Anisakiasis is the prevailing form of foodborne illness in Japan, with Anisakidae larvae, specifically Anisakis simplex sensu stricto, serving as its causative agents. These larvae are commonly found in mackerel, sardines, and skipjacks retailed at fresh fish establishments. While the infection rates of Anisakidae larvae in these surface-dwelling fish have been extensively studied, their prevalence in deep-sea fish species remains inadequately understood. This investigation aims to determine the infection rates of Anisakidae larvae in three Macrouridae species—namely Coryphaenoides acrolepis, Coelorinchus japonicus, and Coelorinchus macrochir—which represent the most significant inhabitants of the deep-sea realm. Employing artificial digestion techniques, we successfully retrieved all internal contents of the fish, including the nematode larvae. Some Anisakidae larvae were morphologically identified as A. simplex sensu lato and further identified to species by PCR-RFLP analysis. Two larvae showed a band pattern that appeared to be a hybrid between A. simplex sensu stricto and A. berlandi, which after sequence analysis was considered to be an intraspecific variation of A. berlandi. On the other hand, species considered to be unknown were sequenced and phylogenetic analysis was carried out along with identified larvae. The findings indicate that A. simplex sensu stricto was notably more abundant in C. acrolepis, obtained at a depth of 1000 m. In C. japonicus and C. macrochir, captured at a depth of 400 m, a considerable number of Skrjabinisakis paggiae and Pseudoterranova ceticola, were detected in the deep-sea dwelling Kogia breviceps and K. sima as definitive hosts, were detected. Notably, a single C. macrochir harbored seven species of Anisakidae larvae, with an infection tally of nearly 200 individuals of S. paggiae. Thus, a substantial prevalence of Anisakidae larvae in the deep-sea-dwelling Macrouridae species potentially plays a significant role in the transmission pathway to cetaceans as their final hosts.

On survival of the larvae of marine parasitic nematode <i>Anisakis simplex</i> sensu lato (Rudolphi, 1809) in the fresh waters

Survival of the larvae of marine parasitic nematode Anisakis simplex sensu lato (Rudolphi, 1809) in the fresh waters was monitored. In total, 37 live larvae of A. simplex s.l. were sampled from charr Salvelinus malma caught in the lower reaches of the Kamchatka River. The larvae were kept in the freshwater refreshed everyday, without food, their viability was examined daily at 9 a.m. All larvae were still alive in the 4 th day (100 %); 2.7 % were alive after 9 days in freshwater, but all of them were died after 10 days. Dependence of the number of dead larvae on the time of their exposition in the freshwater is well approximated by von Bertalanffy equation (R 2 = 0.996). Besides, viability of A. simplex s.l. larvae in the bodies of dead sockeye spawners was examined at the spawning grounds in Lake Azabachye: the larvae were alive in the body cavity of fish that died at least 3–5 days ago and remained alive at least 3 days more being placed in the freshwater. The larvae of marine nematode A. simplex s.l. are able to enter to the fresh waters with migratory fish. Following to our results, they can survive there for several days. So, measures for disinfecting against A. simplex s.l. are recommended for the freshwater fish caught in the water bodies connected with the marine environment.

First report on molecular identification of Anisakis simplex in Oncorhynchus nerka from the fish market, with taxonomical issues within Anisakidae.

Alive anisakids cause acute gastrointestinal diseases, and dead worms contained in food can provoke sensibilization and allergic reactions in humans. Detected in the purchased minced salmon Oncorhynchus nerka nematodes were identified as Anisakis simplex sensu stricto (Anisakidae). We found that recently published phylogenetic trees (reconstructed using different ribosomal and mitochondrial genetic markers) showed independent clusterization of species recognized in the A. simplex sensu lato species complex. This prompted us to undertake this full-fledged molecular genetics study of anisakids from Kamchatka with phylogenetic reconstructions (NJ/ML) and calculated ranges of interspecific and intergeneric p-distances using ITS1-5.8S-ITS2 sequences. We confirmed that molecular markers based on the ITS region of rDNA were able to recognize ‘pure’ specimens belonging to the cryptic species. We offer new insights into the systematics of anisakids. The genus Anisakis sensu stricto should include Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis berlandi, Anisakis ziphidarum, and Anisakis nascettii. Presumably, two genera should be restored in the structure of the subfamily Anisakinae: Skrjabinisakis for the species Anisakis paggiae, Anisakis brevispiculata, and Anisakis physeteris; and Peritrachelius for the species Anisakis typica. In addition, we provide the short annotated list of some genera of the family Anisakidae, including their diagnoses.

Anisakis and Hysterothylacium species in Mediterranean and North-East Atlantic fishes commonly consumed in Spain: Epidemiological, molecular and morphometric discriminant analysis

The consumption of raw fish parasitized with larval ascaridoid nematodes of the family Anisakidae can cause anisakiasis, provoking gastrointestinal and/or allergic symptomatology. The main causative agents in the Anisakis genus are the sibling species Anisakis simplex sensu stricto (s.s.) and A. pegreffii of the A. simplex sensu lato (s.l.) complex. Larvae of A. simplex (s.l.) are frequently detected in fish commonly consumed in Spain, as are larvae of the genus Hysterothylacium of the family Raphidascarididae, associated with allergic reactions but not considered pathogenic. Reported here are the results of an epidemiological survey of ascaridoid larvae in three commonly consumed fish species in Spain, horse mackerel (Trachurus trachurus) (n = 52), blue whiting (Micromesistius poutassou) (n = 93) and anchovy (Engraulis encrasicolus) (n = 69), caught in the North-Eastern Atlantic, West Mediterranean and Adriatic Sea. The larvae found in the dissected fish were identified in the following order of abundance: A. simplex (s.l.) (n = 2003), Hysterothylacium aduncum (n = 422), H. fabri (n = 180) and A. physeteris (n = 15). Binomial regression analysis showed a correlation between A. simplex (s.l.) and Hysterothylacium larvae abundance and the host geographical location, the North-Eastern Atlantic being the area with the highest parasitation. Fish length and weight and Fulton's condition factor were correlated with A. simplex (s.l.) abundance only in horse mackerel. There was a significant presence of A. simplex (s.l.) and H. aduncum larvae in the musculature of North-Eastern Atlantic blue whiting, the most parasitized part being the anteroventral region, followed equally by the anterodorsal and central sections. The ITS rDNA of larvae of the sibling species A. simplex (s.s.) and A. pegreffii was identified by PCR-RFLP, and a binary logistic regression model was developed to study their morphometric differentiation. Anisakis simplex (s.s.) was detected in the North-Eastern Atlantic and A. pegreffii in all the areas studied. The morphometric analysis discriminated between the two species at the third and fourth larval stages (L3 and L4), the latter obtained by in vitro culture in RPMI-1640 medium. Two discriminant functions were obtained for the L3 and L4 larvae, the ventricle being a key parameter for specific differentiation in both stages, providing taxonomical criteria that could be used besides molecular identification. The present study reveals differences in the parasitation of the studied fish, including the distribution of larvae in the musculature, related to the host species and its geographical origin.

Genome-wide analysis of Anisakis simplex sensu lato: the role of carbohydrate metabolism genes in the parasite’s development

Anisakis simplex sensu lato is a parasitic nematode which can cause gastric symptoms and/or allergic reactions in humans who consume raw and undercooked fish. Anisakiasis poses a growing health problem around the globe because it causes non-specific symptoms and is difficult to diagnose. This genome-wide study was undertaken to expand our knowledge of A. simplex s.l. at the molecular level and provide novel data for biological and biotechnological research into the analyzed species and related nematodes. A draft genome assembly of the L3 stage of A. simplex s.l. was analyzed in detail, and changes in the expression of carbohydrate metabolism genes during the parasite's life cycle were determined. To our knowledge, this is the first genome to be described for a parasitic nematode of the family Anisakidae to date. We identified genes involved in parasite-specific pathways, including carbohydrates metabolism, apoptosis and chemo signaling. A total of 7607 coding genes were predicted. The genome of A. simplex s.l. is highly similar to genomes of other parasitic nematodes. In particular, we described a valuable repository of genes encoding proteins of trehalose and glycogen metabolism, and we developed the most comprehensive data set relating to the conversion of both saccharides which play important roles during the parasite's life cycle in a host environment. We also confirmed that trehalose is synthesized at the expense of glycogen. Trehalose anabolism and glycogen catabolism were the predominant processes in stages L4 and L5, which could confirm our and other authors’ previous reports that trehalose is synthesized at the expense of glycogen. The A. simplex s.l. genome provides essential data for post-genomic research into the biology of gastrointestinal and allergic anisakiasis in humans and the biology of other important parasitic helminths.

Population genetic analysis of Anisakis simplex s.l. and Anisakis pegreffii (Nematoda, Anisakidae) from parapatric areas and their contact zone

Genetic markers (ribosomal DNA and mitochondrial DNA) were used for molecular dissection of the Anisakis simplex sensu lato (s.l). complex populations. Host fish were caught off Moroccan coasts, where only Anisakis pegreffii is present, the sympatric area comprising Spanish coasts, and the Little Sole Bank fishing area from Nordeast Atlantic Ocean where the only present species is A. simplex sensu stricto(s.s.). Sequence variations in the amplification products were then assessed indirectly by digestion with restriction endonucleases or directly by sequencing for 623 L3 larvae. The sequences were used to infer the relationships between the two species under study using various methodological approaches. We reveal the high genetic diversity of Anisakis simplex s.s. and A. pegreffii in both mitochondrial and nuclear genes. We detected 10 and 2 fixed differences between A. simplex s.s and A. pegreffii in the Cox2 and ITS1, respectively. We found a proportion of putative hybrids below 20% with similar figures on the Atlantic and Mediterranean coasts. Moroccan hybrids were more similar to A. pegreffii reflecting backcrosses between these mixed genotypes and his ancestor A. pegreffii. We discuss the possible interpretation of these putative hybrids.

Haemoglobin, a new major allergen of Anisakis simplex

Gastro-allergic anisakiasis and Anisakis sensitisation associated chronic urticaria are diseases which differ in their IgE and IgG4 responses against both crude extract and specific allergens. Anisakis and Ascaris are closely related nematodes that usually cause problems with specificity in immunodiagnostics. In this study we measured IgE and IgG4 antibodies against Anisakis simplex sensu lato (s. l.) and Ascaris suum haemoglobins in sera of 21 gastro-allergic anisakiasis and 23 chronic urticaria patients. We used a capture ELISA with the anti-Anisakis haemoglobin monoclonal antibody 4E8g, which also recognises Ascaris haemoglobin. In addition, we determined specific IgE and IgG4 to both nematodes by indirect ELISA and immunoblotting. Anti-A. simplex s. l. haemoglobin IgE and IgG4 levels were higher in gastro-allergic anisakiasis than in chronic urticaria patients (P=0.002 and 0.026, respectively). Surprisingly, no patient had detectable IgE levels against A. suum haemoglobin. Finally, we carried out an in silico study of the B-cell epitopes of both haemoglobin molecules. Five epitopes were predicted in Anisakis pegreffii and four in A. suum haemoglobin. The epitope propensity values of Anisakis haemoglobin in the equivalent IgE binding region of the allergenic haemoglobin Chi t 1 from Chironomus thummi, were higher those of the Ascaris haemoglobin. In conclusion, we describe A. simplex haemoglobin as a new major allergen (Ani s 13), being recognised by a large number (64.3%) of sensitised patients and up to 80.9% in patients with gastro-allergic anisakiasis. The presence of a specific epitope and the different values of epitope propensity between Anisakis and Ascaris haemoglobin could explain the lack of cross-reactivity between the two molecules. The absence of IgE reactivity to Ascaris haemoglobin in Anisakis patients makes Anisakis haemoglobin (Ani s 13) a potential candidate for developing more specific diagnosis tools.

Increase in parasite burden and associated pathology in harbour porpoises (Phocoena phocoena) in West Greenland

Knowledge about parasitism in harbour porpoises and their health status around Greenland is scarce. This study provides knowledge about the poorly studied cetacean in its rapidly changing environment. Parasites and pathological findings in 20 harbour porpoises (Phocoena phocoena) hunted in waters around West Greenland are presented. Carcasses were dissected, and parasitological, histological and bacteriological investigations were carried out. Protozoa (Sarcocystis sp.), Nematoda (Halocercus invaginatus, Stenurus minor, Anisakis simplex sensu lato (s.l.), Crassicauda sp.), Trematoda (Campula oblonga) and Cestoda (Phyllobothrium delphini, Monorygma grimaldii) were found. Parasitic infection of the peribullar cavity and lung with pseudaliid nematodes was found in most animals. Sixty per cent of the porpoises were infected with stomach worms, and trematodes were present in liver and pancreas of 90 and 30 % of the porpoises, respectively. Crassicauda sp. was isolated from perimuscular fascia in 45 % of the animals. This is the first record of tetraphyllidean merocercoids in harbour porpoises. M. grimaldii and P. delphini were found in blubber layer of 15 % and abdominal cavity of 50 % of the porpoises. Bronchopneumonia, gastritis, cholangitis, pancreatitis and panniculitis were almost exclusively associated with parasitic infection and usually mild. Compared with a previous study of Greenlandic porpoises from 1995, a significant increase in severity of parasitic infections and the emergence of new parasite species were observed, most likely associated with changes in diet, influenced by increasing sea temperatures and receding ice cover.

Molecular characterization and comparison of four Anisakis allergens between Anisakis simplex sensu stricto and Anisakis pegreffii from Japan

It remains unclear whether allergens are the same among the sibling species of Anisakis simplex sensu lato. This study was carried out to compare the amino acid sequences of three major (Ani s 1, 2 and 12) and one minor (Ani s 9) Anisakis allergens between A. simplex s.s. and Anisakis pegreffii. We found 2 (out of 163), 1 (out of 869), and 29 (out of 266) amino acid variable sites for Ani s 1, 2, and 12, respectively. However, as both intra- and inter-species variations were present at the same amino acid positions, no amino acid variations clearly distinguished the two sibling species. IgE-binding epitopes (Ani s 1) and a binding motif of human leukocyte antigen (Ani s 2 and 9) demonstrated by previous studies were conserved. The similarities of the amino acid sequences of the allergens indicate possible similar allergy-associated health risks in humans infected with or accidentally ingesting either Anisakis species.

A polyphasic approach with molecular phylogeny for the characterization of Anisakis pegreffii (Anisakidae: Nematoda) in fishes from Adriatic Sea

Abstract In this study we investigated the morphometric and molecular characterization of a liver encapsulated third-stage larval population of Anisakis spp. infecting Merluccius merluccius and Lophius piscatorius caught in the Adriatic Sea waters (southern Italy). A polyphasic approach based on PCR-RFLP profiles of the ITS region, mitochondrial COI (cytochrome c oxidase subunit 1), sequencing and molecular phylogeny of ITS and mitochondrial COI was used to identify Anisakis larvae collected from fish samples. PCR-RFLP analysis showed three banding pattern corresponding to the peculiar pattern of A. pegreffii. Sequence data from ribosomal ITS and mitochondrial COI were analysed by Neighbour Joining, Minimum Evolution and Maximum Parsimony methods to evaluate the phylogenetic relationships among A. simplex sensu lato. The phylogenetic trees obtained for both ITS and COI revealed the existence of three distinct clades for A. simplex sensu sricto, A. simplex C and A. pegreffii and the sequences obtained in this study clearly clustered together with A. pegreffii sequences present in the database. Histopathological observations of anisakid nematode specimens detected on the liver surface of M. merluccius are illustrated. Encapsulated specimens of the L3 stage of the nematode were similar in size and morphometry to those found into the peritoneal cavity. Anisakis larvae encapsulated on the liver surface within dense and pearl coloured envelops caused host hepatic tissue necrosis, large cavities and oedematous liver spots to the host.

Genetic characterisation and taxonomy of species of Anisakis (Nematoda:Anisakidae) parasitic in Australian marine mammals

Three species of Anisakis from Australian marine mammals, including Anisakis brevispiculata, A. simplex C and A. pegreffii, are described and characterised genetically on the basis of sequence data for the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear rDNA. Parasite specimens were collected from Delphinus delphis, Tursiops truntatus and Kogia sima in Australia. A. brevispiculata is reported for the first time in Australia. However, analyses of sequence data suggests that A. brevispiculata in Australia is genetically distinct from specimens considered to represent the same species from other parts of the world. Fourth larval and adult stages of A. pegreffii were found in dolphins. Assigning larvae to A. pegreffii was based on the ITS-1 and ITS-2 sequences. A description of these larvae also is provided. Furthermore, fourth-stage larvae of A. simplex C were found in Kogia sima. Alignments of ITS-1 and ITS-2 sequences for members of A. simplex sensu lato revealed that nucleotide differences in ITS-1 can be used to differentiate among members of A. simplex sensu lato. This study reinforces the use of a combined molecular and morphological approach for the specific identification of anisakid nematodes.

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 5 weeks. 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.

Anisakis simplex sensu lato associated with red vent syndrome in wild adult Atlantic salmon Salmo salar in England and Wales

Wild Atlantic salmon Salmo salar with swollen and/or bleeding vents (red vent syndrome) have been observed in rivers across England and Wales. Low numbers of salmon with this condition have previously been recorded, but reports have increased in 2006 and 2007. Affected and non-affected salmon were sampled from fish traps and their vent pathology was examined. Third stage larvae of the nematode Anisakis simplex sensu lato were observed in the tissue surrounding the vent in all affected fish. Histology of the vent showed gross lesions with an inflammatory response associated with non-encapsulated nematode worms in the connective tissue and skeletal muscle. The presence of the parasite in the unusual location of the vent areas, with associated haemorrhaging, is possibly a new emerging pathological association with the nematode A. simplex infection in Atlantic salmon. The reason for this unusual tissue tropism by larval A. simplex and the significance on already depleted salmon stocks are currently unknown.

A Preliminary Study on the Infection of Anisakid Larvae in Juvenile Greater Amberjack Seriola dumerili Imported from China to Japan as Mariculture Seedlings

Juvenile greater amberjack Seriola dumerili (fork length: 39.5-43.0 cm) imported from China to Japan as mariculture seedlings were found infected with larval anisakid nematodes in the spring of 2005. The parasite was morphologically identified as Anisakis type I larva causing human anisakiasis. Based on the nucleotide sequence of ITS1-5.8S rRNA-ITS2 region, the parasite was tentatively identified as A. pegreffii, one of the species comprising A. simplex sensu lato. The main infection site was the wall and serous membrane of the stomach. No worms were found in the ventral side of the body muscle of fish. This is the first documented case of Anisakis infection in cultured marine fishes.

Anisakis spp. in European hake, Merluccius merluccius (L.) from the Atlantic off north-west Africa and the Mediterranean off southern Spain

AbstractWe studied the parasitization by Anisakis spp. in European hake (Merluccius merluccius) of 40–52 cm from the Atlantic off north-west Africa and the Mediterranean off southern Spain. Infection parameters differed: The fish from the Atlantic showing a prevalence of 87.97% and a mean intensity of 4.69, while, in those from the Mediterranean, these were 41.27% and 1.73, respectively. In both samples the two-third larval stage types were isolated: Anisakis simplex sensu lato and Anisakis larvae type II, with prevalence of 85.71% and 30.83% in fish from the Atlantic and 41.27% and 1.59% for those from the Mediterranean, respectively. In both samples, the prevalence of larvae in viscera was clearly higher than in the muscle tissue. We also observed an increase in parasitization with increasing host length, those ≥ 46 cm having the highest prevalences (94.87% for those from the Atlantic and 58.33% for those from the Mediterranean; p<0.03).

Accumulation of heavy metals in the whaleworm Anisakis simplex s.l. (Nematoda: Anisakidae)

Third-stage larval and adult infrapopulations of the whaleworm Anisakis simplex sensu lato (s.l.) (Nematoda: Anisakidae) from their respective fish, cephalopod and cetacean hosts caught or stranded in the Galician coast (north-west Spain), were analysed for heavy metals by atomic absorption spectrometry. Results suggest that the whaleworm may accumulate lead and copper to levels far in excess of their host tissues.

Element concentration variability in the whaleworm Anisakis simplex s.l.

Concentrations of the elements C, N, Fe, Na, K, Ca and Mg were analysed by chromatography and atomic absorption spectrometry in the whaleworm Anisakis simplex sensu lato. Nearly all mean inorganic element concentrations measured were higher in adult worms than in larval forms. Similarly, adults contained significantly higher nitrogen (i.e. lower C/N ratio values) than either eggs or larvae. Evidence for the significance of the parasitic life cycle stage and the systematic position of their hosts on the element concentration variability is discussed.