Acute intestinal anisakiasis in Spain: a fourth-stage Anisakis simplex larva

A case of acute intestinal anisakiasis has been reported; a nematode larva being found in the submucosa of the ileum of a woman in Jaén (Spain). The source of infection was the ingestion of raw Engraulis encrasicholus. On the basis of its morphology, the worm has been identified as a fourth-stage larva of Anisakis simplex. In Spain, this is the ninth report of human anisakiasis and also probably the first case of anisakiasis caused by a fourth-stage larva of A. simplex.

Interleukin-4 production in BALB/c mice immunized with Anisakis simplex

We investigated the interleukin (IL-4) levels in BALB/c mice immunized with Anisakis extract in single or multiple doses and in mice orally infected with a larva. From animals immunized maximum responses were obtained with the multiple doses with an only IL-4 peak. Conversely, in the mice inoculated with a larva per os, the IL-4 levels showed two peaks of different rates.

Evaluation by ELISA of Anisakis simplex Larval Antigen Purified by Affinity Chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 µg/ml of antigenic concentration.

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

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.

Sensitization to Anisakis simplex species in the population of northern Morocco.

OBJECTIVE To investigate sensitization to third-stage Anisakis simplex larvae in a randomly selected population in northern Morocco. METHODS We studied sera obtained from clinical analysis laboratories in Tangier and Tetuouan and from fishermen at Tangier port. The age of the study population ranged from 6 to 83 years. ImmunoCAP and immunoblotting techniques were used to determine total and specific immunoglobulin (Ig) E values and the chi2 and Fisher exact tests were applied to analyze relationships between study variables. RESULTS A seroprevalence of 5.1% was found, with a higher percentage of positive sera in the 31-to-43-year age group. Sensitization was not significantly associated with the origin, sex, occupation, or age of the individuals studied. In sera positive by InmunoCAP, immunoblotting studies detected numerous bands of between 7 kDa and >209 kDa, with a predominance of bands in the approximately 20-kDa to 24-kDa range. CONCLUSIONS Although no cases of human anisakiasis have been reported in Morocco to date, part of a randomly selected population in Northern Morocco shows sensitization to A simplex proteins.

Cellular immune responses in mice immunized with Anisakis simplex larval antigens

Cellular immune responses to Anisakis simplex L3 antigens were investigated in BALB/c mice injected subcutaneously with a homologous crude extract (CE). Popliteal lymph nodes (PLN) were found to be increased in size and weight after A. simplex CE footpad injection. The effects of A. simplex CE in vitro proliferation were assayed with non-fractionated PLN cells or nylon-wool purified T cells derived from pooled lymph node cells of mice subcutaneously injected with CE. Spleen cells from immunized animals (antigen alone, or larva alone, or antigen plus larva) were studied by flow cytometry. The immunization induced a high proportion of CD4 + and TCR alpha beta + T cells. The number of B cells (CD45 + and TCR alpha beta-) in pre-immunized and infected mice was lower than that observed in animals subjected to infection only. The number of CD4 + T cells increased in the infected and in the pre-immunized and infected mice. In the latter, a decrease of CD8a + T cells was noted. The greatest increase in CD8a+ and TCR alpha beta- T cells was found in mice that had been subjected to infection only. Histological analysis showed that the most prominent lesions were gastric and intestinal in animals infected orally with one larva.

Anisakis infection in allis shad, Alosa alosa (Linnaeus, 1758), and twaite shad, Alosa fallax (Lacépède, 1803), from Western Iberian Peninsula Rivers : zoonotic and ecological implications

Acknowledgments The authors would like to thank M. N. Cueto and J.M. Antonio (ECOBIOMAR) for their excellent technical support and also Rodrigo López for making the map of the study area. We also thank the personal of the Vigo IEO, for providing information about shad captures at sea collected on the basis of national program (AMDES) included in the European Data Collection Framework (DCF) project. We are also grateful to Comandancia Naval de Tui for providing fishing data. M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial support of the contract from the EU Project PARASITE (grant number 312068). This study was partially supported by a PhD grant from the Portuguese Foundation for Science and Technology (FCT) SFRH/BD/44892/2008) and partially supported by the European Regional Development Fund (ERDF) through the COMPETE—Operational Competitiveness Programme and national funds through Foundation for Science and Technology (FCT), under the project BPEst-C/MAR/ LA0015/2013. The authors thank the staff of the Station of Hydrobiology of the USC BEncoro do Con^ due their participation in the surveys. This work has been partially supported by the project 10PXIB2111059PR of the Xunta de Galicia and the project MIGRANET of the Interreg IV BSUDOE (South-West Europe) Territorial Cooperation Programme (SOE2/P2/E288). D.J. Nachón is supported by a PhD grant from the Xunta de Galicia (PRE/2011/198)

Survey on parasites of Rutilus frisii Kutum in South west of Caspian Sea with the emphasis of pathology of Dactylogyrus frisii and morphological identification of Paradiplozoon sp and Anisakis sp

Rutilus frisii Kutum is one of the most precious fish in the Caspian Sea. Investigation of the various aspects of its biocharactristics. Including its parasite fauna and ecological aspects are of prime importance. In this study the farmed kutum fry were on the focus of investigation in various seasons of the year and prior to their being released in the sea. This included also the study on the kutum spawners caught both from liver and the sea. The results were that 17 external and internal parasite species were distinct within different organs which were further identified down to genus and species. The single celled parasites identified included Ichthyophthirius multifilils, Chilodonella hexastica, Chilodonella pisicola, Trichodina sp Along with the monogene parasites that included Paradiplozoon chazaricum, D. rarissimus, D. turaliensis, D. nybelini, Dactylogyrus frisii. Meanwhile Diplostomum spathaceum constituted the single eyed parasites and the intestinal termatode were Aspidogaster limacoides, Asymphyoldora kubanicum as well as Bothriocephalus gowkongeniss as the sestads. The nematodes defrentiated were Raphidascaris acus, Dioctophyma renale, and Eustrongylides excisus followed by Lernaea cyprinacea as a crustacean. In this study, infestations by single celled parasites, crustaceans and sestod were found to be present only among the farmed kutum fry which varied in terms of percentage and intensity of infection as well as the parasite species and season of the year. The highest percentage of infection among kutum fry and spawners in both fresh water and in the sea during all seasons belonged to monogene parasites (33%). This was up to 100% among spawners. Infection caused by nematodes was exclusively detected among riverine spawners (7.5-5%) and the infection by Asymphyoldora kubanicum and Aspidogaster limacoides among Spawners caught at Sea and rivers varied within different seasons of the year. The infestation of Metacercer diplostomum spathaceum among kutum fry was 12% which compared to spawners was in slightly higher level. The study could identify Dioctophyma renale for the first time in the country and Eustrongylidis excisu was also detected among Rutilus frisii kutum.

Helminths of Sotalia guianensis (Cetacea: Delphinidae) from the South and Southeastern Coasts of Brazil

From May 1997 to October 2000, 49 Sotalia guianensis (tucuxi dolphin) incidentally caught in fishing nets or stranded in Sao Paulo (SP) and Parana (PH) states in Brazil were necropsied. In total, 17 lungs, 35 stomachs, and 30 intestines were analyzed. Contents were washed through a sieve (mesh, 150 mm) and examined under a stereoscopic microscope for parasites. Histopathologic analyses were performed in the lungs of five infected dolphins. The nematode Halocereus brasiliensis was found in 88% of all lungs examined, inducing moderate-to-severe pneumonia. Braunina cordiformis, Anisakis sp., and acanthocephalans were found in the stomachs. The trematode Synthesium tursionis was the only parasite found in the intestines, and it was identified in 73% of the animals necropsied. No macroscopic lesions were seen due to parasites in the stomachs and intestines analyzed.

Helmintofauna de cavalas, Scomber japonicus Houtt, do Rio de Janeiro

Os autores identificaram as seguintes espécies de helmintos, coletados de 50 cavalas, Scomber japonicus, no Rio de Janeiro: Kuhnia scombri (Kuhn, 1829) e Grubea cochlear (Diesing, 1858) (Monogenea); Opechona orientalis (Layman, 1930), Lecithocladium harpodontis Srivastava, 1942 e Nematobothrium scombri (Taschenberg, 1879) (Digenea); plerocercos de Trypanorhyncha Scolex pleuronectis Müller, 1788 e Rhinebothrium sp. (Cestoda); Bolbosoma sp. (Acanghocephala) e Anisakidae larvares (Nematoda), identificados aos tipos larvares Raphidascaris, Phocanema, Contracaecum e Anisakis tipo 1. Os digenéticos foram os de maior incidência, 84% dos peixes mostraram-se parasitados por uma ou mais espécies. Quanto às espécies, a de maior incidência foi Nematobothrium scombri (Digenea, Didymozoidae), em 46% dos peixes. São pela primeria vez assinalados Scomber japonicus larvas de Phillobothiidae, possivelmente Rhinebothrium, além de larvas de Anisakis do tipo 1. São pela primeira vez assinaladas no Brasil as espécies, Grubea cochlear, Kuhnia scombri, Nematobothrium scombri e Opechona orientalis.

Helminofauna da sarda (Scomber scombrus L. ) peixe da Costa Continental Portuguesa

Dos primerios 80 espécimens da sarda examinados (de janeiro a maio de 1982), apenas seis (7,5%) estavam livres de helmintos, apresentando os demais as espécies de parasitos seguintes, por ordem de freqüência dentro de cada grupo taxonômico: Kuhnia scombri (Kuhn, 1829) e Grubea cochlear (Diesing, 1858) (classe Monogenea); Lecithocladium excisum (Rudolph, 1819) e Opechona basillaris (Molin, 1859)(classe Digenea); pelrocercoides de Lacistrorhynchus tenuis (Beneden, 1858) de Scolex pleuronectis (Muller, 1788) e de Echeneibothrium sp. (classe Cestoda); Rhadinorhynchus tenuicornis (Linton, 1891) (filo Acanthocephala); formas dos tipos larvares Anisakis e Contracaecum e ainda, larvas de Goezia sp. (classe Nematoda). São referidas, pela primeira vez, neste hospedeiro, formas larvares do cestóide Echeneibothrium sp. e do nematóide Goezia sp.

Parasites of the flatfish Paralichthys adspersus (Steindachner, 1867) (Pleuronectiformes) from northern Chile

Three species of protistan and 22 species of metazoan parasites were obtained from a sample of 179 flatfish, (Paralichthys adspersus) taken-off Antofagasta, northern Chile. Prevalence of infection of seven parasites (Protista: 1, Copepoda: 2, Digenea: 1, Acantocephala: 1, Nematoda: 2) was significantly and positively correlated with host size. Host's sex do not seem to affect prevalence of infection, except for Nybelinia surmenicola, Capillaria sp. and Anisakis sp. (prevalence of infection significantly greater in males than females) and Philometra sp. (prevalence higher in females). Mean abundance is correlated with size in nine species (Protista: 1, Copepoda: 2, Digenea: 3, Acantocephala: 1, Nematoda: 2). Host's sex do not affect mean abundance, except for Cainocreadium sp. and Philometra sp.(mean abundance higher in females) and Nybelinia surmenicola, Capillaria sp. and Anisakis sp. (mean abundance higher in males).