A multi-restriction fragment length polymorphism genotyping approach including the beta-tubulin gene as a new differential nuclear marker for the recognition of the cryptic species Anisakis simplex s.s. and Anisakis pegreffii and their hybridization events.

The detection of Anisakis simplex s.s./A. pegreffii putative hybrids has been a controversial issue in spite of the fact that natural hybridization is an extended process across free living and parasitic organisms. Differential traits of biomedical and ecological importance, such as invasive and allergenic potential have been demonstrated in both cryptic species. Therefore, in this work, we discuss about the potential for hybridization between these anisakid species in sympatric zones, implementing a multi-marker Restriction fragment length polymorphism (RFLP) genotyping approach based on the ribosomal DNA internal transcribed spacer 1 (ITS1), the mitochondrial cytochrome C oxidase 2 (Cox-2) and a new nuclear marker, the highly conserved ß-tubulin gene (ß-TUB). The two cryptic species differed at least in 7 bp in the ß-TUB gene and some larvae with heterozygous genotypes at the 7 diagnostic nucleotide positions were found. Taxonomic, population and genealogical analyses served to support the occurrence of hybridization between both species. Predicted restriction endonucleases enzymes were assayed for Cox-2 and ß-TUB markers. The implemented multi-marker PCR-RFLP allowed us to detect the two pure parental species, F1 hybrids, hybrid backcrossed progeny and individuals with nuclear-mitochondrial discordance, being a useful, simple and reproducible procedure in any laboratory for epidemiological studies.

Evaluation of different Mediterranean essential oils as prophylactic agents in anisakidosis.

CONTEXT: Anisakis Dujardin 1845 (Anisakidae) nematodes can cause gastrointestinal and allergic diseases when humans eat raw or undercooked seafood containing larvae. There is currently no drug available in the market against this parasitic disease, and the study of plant-derived molecules could be useful in the discovery of effective compounds. OBJECTIVE: This research assesses the biocidal activity of a range of essential oils (EOs) from some Mediterranean plants against larvae found in the musculature of fresh fish. MATERIALS AND METHODS: EOs composition was analyzed by gas chromatography-mass spectroscopy. All the EOs were diluted at 5% v/v in olive oil to cover the fish with the solutions for 24 h. The larvae that abandoned the muscle and the larvae obtained from the artificial digestion of the fish were collected. Controls were carried out in parallel. Furthermore, Wistar rats were infected with the live larvae collected from the in vitro trials in order to find any larvae that may have penetrated the gastrointestinal wall. RESULTS: Between 60.8% and 87.6% of parasites treated with EOs abandoned the fish muscle, and the highest in vitro mortality rate was achieved with oregano EO (53.9%). Rats previously treated with oregano, cumin and Spanish lavender EOs showed no detectable lesions in the digestive tract due to the infection with larvae. CONCLUSIONS: Oregano (Origanum vulgare L. Lamiaceae), cumin (Cuminum cyminum L. Apiaceae) and Spanish lavender (Lavender stoechas L. Lamiaceae) EOs could be used as promising ingredients in the development of products for the control of anisakiasis.

Reconocimiento de los antígenos excretores/secretores de Anisakis tipo I y evolución de la IgE en ratas infectadas experimentalmente / Recognition of excretory/secretory antigens of Anisakis type I and evolution of IgE in experimentally infected rats

INTRODUCCIÓN: Anisakis spp., durante la parasitación, libera antígenos de excreción secreción (ES) que, al ponerse en contacto con el sistema inmunológico del hombre, pueden desencadenar una respuesta de hipersensibilidad mediada por la IgE, provocando diversos síntomas alérgicos. OBJETIVOS: Evaluar la respuesta de la IgE en ratas Wistar tras la infección con larvas L3 del parásito. MÉTODOS: Se ha procedido a la obtención de antígenos ES del parásito y suero anti-Anisakis. Se investigan también en este trabajo ciertos factores que intervienen en la técnica de inmunotransferencia, como la concentración de poliacrilamida empleada en la preparación de los geles, la concentración antigénica utilizada y la temperatura requerida para la desnaturalización de las proteínas. RESULTADOS: Las reacciones inmunológicas (Ag-Ac) observadas mediante esta técnica muestran mayor intensidad con los sueros obtenidos después de la reinfección, los cuales han reconocido proteínas que podrían corresponder al antígeno principal Ani s 1 y a otros polipéptidos de interés en el diagnóstico de la anisakiosis humana. CONCLUSIÓN: En este trabajo, se pone de manifiesto que inmunotransferenciala inmunotransferencia es una técnica útil para detectar anticuerpos de tipo IgE frente a proteínas de Anisakis

INTRODUCTION: Anisakis spp., during parasitism, release excretory-secretory antigens that, in contact with the human immune system, can trigger a hypersensitivity response mediated by IgE, causing various allergic symptoms. OBJECTIVES: To evaluate the IgE response in Wistar rats after infection with L3 larvae of the parasite Anisakis spp. METHODS: Some determining factors involved in the technique have been improved in this work, such as: the concentration of polyacrylamide used in the preparation of the gels, the antigen concentration used, and the temperature required for denaturation of proteins. RESULTS: Immune responses (Ag-Ab) observed by the immunoblotting technique showed a greater intensity with serum obtained after reinfection, which have recognized proteins that may correspond to the major antigen Ani s 1 and other polypeptides of interest in the diagnosis of human anisakiasis. CONCLUSION: This paper concludes that immunoblotting is a useful technique to detect IgE antibodies against Anisakis proteins

Actividad de albendazol y los aceites esenciales de menta (Mentha piperita) y manzanilla (Matricaria chamomilla) frente Anisakis tipo I / Activity of albendazole and essential oils of peppermint (Mentha piperita) and chamomile (Matricaria chamomilla) against Anisakis type I

Objetivos: Estudiar la posible actividad sinérgica de los aceites esenciales de Mentha piperita y Matricaria chamomilla frente Anisakis tipo I. Material y métodos: Se ensayó tanto in vitro como in vivo, la actividad larvicida de la mezcla de los aceites esenciales utilizando albendazol como fármaco de referencia y midiéndo su eficacia mediante un modelo estadístico. Resultados: En los ensayos in vitro, a las concentraciones de 250 y 187.5 μg/ml, la mortalidad de las larvas fue del 100% mientras que albendazol no mostró eficacia. In vivo, la mezcla de aceites esenciales fue más eficaz, reduciendo las lesiones en ratas en comparación con las tratadas con albendazol y control. Albendazol no redujo de forma significativa el porcentaje de lesiones producidas por las larvas frente al control. Conclusión: La mezcla de aceite esencial de menta y manzanilla podría ser candidata para su uso en el tratamiento/profilaxis de la anisakiasis humana

Aim: We have studied the possible synergistic activity of the essential oils of Mentha piperita and Matricaria chamomile against Anisakis type I. Materials and methods: the larvicidal activity of the mixture of essential oils was tested in vitro and in vivo, using as a reference the drug Albendazole and effectiveness measured through a statistical model. Results: The in vitro and in vivo experiments both evidenced that the larvicidal activity of essential oils, was higher than the Albendazole activity. In the in vitro assay, at concentrations of 250 and 187.5 μg/ml, the mortality was 100% with the mixture of essential oils while albendazole was ineffective at the concentrations studied. In the in vivo assay, the mixture of essential oils, was significantly more effective in the reduction of numbers of lesions with rats in comparison to albendazole treatments and control. Albendazole did not significantly reduce the percentage of lesions caused by larvae vs. control. Conclusion: Considering the results, we have come to the conclusion that the mixture of peppermint essential oils and chamomile could be used in the treatment/prophylaxis of human Anisakis

[Recognition of excretory/secretory antigens of Anisakis type I and evolution of IgE in experimentally infected rats]. / Reconocimiento de los antígenos excretores/secretores de Anisakis tipo I y evolución de la IgE en ratas infectadas experimentalmente.

INTRODUCTION: Anisakis spp., during parasitism, release excretory-secretory antigens that, in contact with the human immune system, can trigger a hypersensitivity response mediated by IgE, causing various allergic symptoms. OBJECTIVES: To evaluate the IgE response in Wistar rats after infection with L3 larvae of the parasite Anisakis spp. METHODS: Some determining factors involved in the technique have been improved in this work, such as: the concentration of polyacrylamide used in the preparation of the gels, the antigen concentration used, and the temperature required for denaturation of proteins. RESULTS: Immune responses (Ag-Ab) observed by the immunoblotting technique showed a greater intensity with serum obtained after reinfection, which have recognized proteins that may correspond to the major antigen Ani s 1 and other polypeptides of interest in the diagnosis of human anisakiasis. CONCLUSION: This paper concludes that immunoblotting is a useful technique to detect IgE antibodies against Anisakis proteins.