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PubMed2001

In vitro cultivation of anisakis simplex: pepsin increases survival and moulting from fourth larval to adult stage.

This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3.8 and 6.5 cm long and the females between 4.5 and 8.0 cm. Some individually cultivated females laid eggs which had an average size of 44.4 x 50.5 microm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4.0 and a temperature of 37 degrees C, and in air atmosphere with 5% CO2. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1.9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4.2 times, from 22.9 to 95.6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites.

Animals↗
PubMed2003

Altered autonomic control in rat intestine due to both infection with Anisakis simplex and incubation with the parasite's crude extract.

Anisakis simplex IgE may bring on allergic responses such as angioedema, vomiting, and urticaria from eating seafood, but it is not the only etiology. Induced cholinergic hyperreactivity or adrenergic blockade in the target tissue can cause these diseases nonimmunologically also. Here we studied the effects on normal intestinal motility of brief A. simplex infections and in vitro exposures to the parasite's extract (CE). Each approach was evaluated according to its ability to induce cholinergic hyperreactivity or adrenergic blockade in rat duodenum (RD), jejunum (RJ), and ileum (RI) in vitro. Additionally, bolus propulsion in RD, RJ, and RI was evaluated with time in vivo utilizing animals infected 4 h previously with A. simplex larvae (L3) vs sham animals. Tissues, after inoculation of 1, 5, 10, and 20 L3, exhibited time- and dose-dependent motility changes after carbachol (Ch) and noradrenaline (NA), justifying our using herein rats from the fourth hour of infection with 20 L3. We observed a persistent, yet differential effect of the infection on RD, RJ, and RI responses to Ch or NA. It caused cholinergic (muscarinic) hyperreactivity in RD only, and adrenergic blockade in all other parts, and consequently increased the transit index in RD, not in RJ or RI. In contrast, exposing RD, RJ, and RI to CE persistently increased both parameters, amplitude of twitches and muscular tone, in all, albeit that, here also, responses to Ch and NA were CE dose dependent. Interestingly, sensitivity to CE was in the order RI > RJ > RD, the reverse situation of that observed during active infection. Thus, previously viable A. simplex L3, after digestion, can exert bystander disturbance in autonomic control in the whole intestine. Our findings demonstrate that A. simplex L3, alive or dead, can induce cholinergic hyperactivity and adrenergic blockade in the whole small intestine and, as a consequence, gastrointestinal symptoms. Significantly, they may do so long before parasite-specific IgE is detectably induced or despite the occurrence of such IgE.

Adrenergic alpha-Agonists↗
PubMed2000

A double-blind, placebo-controlled oral challenge study with lyophilized larvae and antigen of the fish parasite, Anisakis simplex.

BACKGROUND: The third-stage larvae of Anisakis simplex may be a hidden source of allergens in fish. The objective was to determine whether the ingestion of lyophilized A. simplex larvae, or antigen, induces clinical symptoms in a group of A. simplex-sensitized patients. METHODS: Double-blind, placebo-controlled oral challenges were conducted in 11 individuals who had experienced allergic reactions after eating fish. Another patient had chronic urticaria unrelated to the ingestion of fish. All patients had positive skin tests and specific IgE determinations for A. simplex and negative skin tests to a battery of fish species. Conjunctival tests with A. simplex extracts were conducted in all patients and in five controls. The 12 patients received capsules containing either lactose or one, five, or 25 lyophilized larvae of A. simplex at 2-h intervals in a double-blind fashion. The highest single dose was 100 larvae. ECP and tryptase levels in serum were measured before and after the last oral challenge. Lyophilized antigen was also given to five patients. RESULTS: None of the 12 patients experienced a positive reaction after the ingestion of the placebo, the lyophilized larvae, or the antigen. Tryptase and ECP levels before and after challenges did not change significantly. Conjunctival provocation tests were positive in 11 out of the 12 patients and in none of the controls. CONCLUSIONS: The ingestion of 100 lyophilized A. simplex larvae, or its equivalent in antigen, does not induce clinical symptoms in individuals with a clinical history and laboratory findings of hypersensitivity to A. simplex. The data suggest that only the ingestion of live larvae may be capable of inducing allergic manifestations.

Administration, Oral↗
PubMed2001

Allergenic cross-reactivity between the nematode Anisakis simplex and the dust mites Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, and Dermatophagoides pteronyssinus.

BACKGROUND: The nematode Anisakis simplex is a common parasite on fish and other seafood. It is considered to be a food allergen and to induce IgE-mediated reactions. Allergenic cross-reactivity between A. simplex and other nematodes has been reported, as has cross-reactivity with arthropods: red mosquito larvae and German cockroach. We have here studied the allergenic relationship between A. simplex and four different dust-mite species. METHODS: Serum samples collected from 69 farmers allergic to dust mites were analyzed for IgE to A. simplex by CAP FEIA. Allergenic cross-reactivity between A. simplex and dust mites was studied in two of the sera by CAP FEIA and immunoblotting inhibition. RESULTS: We found that 14/69 farmers had detectable levels of IgE antibodies to A. simplex. The IgE response in CAP FEIA to A. simplex was inhibited to various degrees in the two studied sera by extracts of the dust mites Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, and Dermatophagoides pteronyssinus. In the reverse inhibition experiment, extract of A. simplex inhibited the response in both sera to A. siro and T. putrescentiae, but not to L. destructor. The IgE binding to D. pteronyssinus was inhibited in one of the two sera. In blotting inhibition experiments, the IgE binding to several allergens in A. simplex was inhibited by each of the four mite extracts, especially by A. siro and T. putrescentiae, which completely inhibited the IgE binding to several allergens. CONCLUSION: The results show allergenic cross-reactivity between several allergens in A. simplex and four dust-mite species. The clinical significance of this cross-reactivity remains to be evaluated.

Animals↗
PubMed2001

Prevalence of and risk factors for IgE sensitization to Anisakis simplex in a Spanish population.

BACKGROUND: The number of allergic reactions to A. simplex reported in Spain has increased dramatically in the last decade. Nevertheless, there have been no studies of the prevalence of and possible risk factors for IgE sensitization to this parasite, possibly because suitably specific diagnostic methods have only recently become available. The objective was to investigate the prevalence of and risk factors for IgE sensitization to A. simplex in Galicia, a region of northwestern Spain with a population of about 3 million and high average fish consumption (78.5 g/person per day). METHODS: The study was performed with a random sample of 2801 healthy blood donors distributed in 53 geographic areas, proportional to the density of donors. IgE sensitization to A. simplex was tested by a capture ELISA method that has proved to be the most specific method currently available. RESULTS: The results showed a total of only 12 positive subjects, of whom five also showed IgG1 sensitization. All positive subjects and 101 randomly selected seronegative subjects were then included in a case-control study of risk factors for sensitization to A. simplex, based on a telephone interview about fish consumption (especially raw and undercooked fish). All seropositive subjects (but only 25% of seronegative subjects) reported consumption of undercooked fish or homemade raw-fish products. CONCLUSIONS: Our results strongly suggest that sensitization to A. simplex is caused only by live larvae, and not by allergens contained in fish tissues, and that ingestion of homemade boquerones (anchovies [Engraulis encrasicholus] in vinegar), and to a much lesser extent of undercooked fish, are the main risk factors for IgE sensitization to Anisakis in this region.

Adolescent↗
PubMed2001

Presence of IL-4-like molecules in larval excretory-secretory products and crude extracts from Anisakis simplex.

Acute symptoms of anisakidosis are caused by a type I allergic reaction in the gastrointestinal wall with elevated specific immunoglobulin (Ig)E. The purpose of this study was to investigate the presence of interleukin (IL)-4 in the larval antigens of Anisakis simplex. We detected concentrations of IL-4 in pg/ml when larval excretory--secretory products and crude extract from A. simplex were investigated by ELISA. Specific antibodies were obtained by immunization of rabbits with mouse IL-4 and tested in ELISA against A. simplex antigens obtaining higher values of optical density, that were confirmed by western blot analysis. The absorption of these sera with A. simplex antigen resulted in a 70--80% inhibition of antigen binding when retested in ELISA. We demonstrated that A. simplex antigens react with antibodies raised against vertebrate IL-4. The results obtained by us support the hypothesis that A. simplex shares several epitopes with IL-4, important for the Th2 response development in human anisakiasis, where the parasite may modulate the Th1--Th2 dichotomy for its own benefit by mucosal inflammation control in an attempt to avoid the larval expelling.

Animals↗
PubMed2003

Characterization of allergens of Anisakis simplex.

BACKGROUND: Anisakis simplex is an intestinal parasite of sea mammals. The larvae infect crustaceans, cephalopods and fish. Humans may consume A. simplex third stage larvae (L3) when eating infected raw or under-cooked fish. Consumed larvae cause an inflammatory reaction when they penetrate the digestive mucosa. The larvae or their secretory/excretory products can sensitize humans and induce an immunoglobulin E (IgE)-mediated allergic reaction. This parasite is now being implicated in numerous cases of allergic reactions after eating fish. The purpose of this study was to evaluate the allergenicity of proteins present in an extract of the third stage larva. METHODS: Rabbit antiserum raised to A. simplex somatic extract (L3) was reacted by crossed immunoelectrophoresis (CIE) with the same somatic extract. Crossed radioimmunoelectrophoresis (CRIE) was also performed by incubating CIE gels first in the sera of 13 individuals with positive immunoCAP to A. simplex and then in radiolabeled anti-human IgE. RESULTS: Twelve to 16 antigen-antibody precipitin peaks were visualized on Coomassie blue stained CIE gels in which somatic extract was reacted with somatic-antiserum. Autoradiography of CRIE gels showed that 18 different proteins bound IgE in patient sera. Individual patients had serum IgE directed at two to 10 different allergens. Five of these allergens were recognized by >/=50% of the patients. No allergen was recognized by every patient and no patient had serum IgE directed at all 18 allergens. CONCLUSION: Somatic extracts of A. simplex L3 larva contain a large number of allergenic molecules and there is significant variability between patients in their sensitivity and reactivity to these allergens.

Adult↗
PubMed2000

Isolation and characterization of a major allergen from the fish parasite Anisakis simplex.

BACKGROUND: Ingestion of raw or undercooked fish can lead to infection of human subjects by the fish parasite Anisakis simplex, a disease known as anisakiasis or anisakidosis. Patients sensitized to this fish parasite show high levels of total and specific IgE. Cross-reactions seem to explain the fact that specific IgE antibodies are also found in a high number of normal subjects, as reported in other parasitoses. OBJECTIVE: We sought to purify and characterize a major IgE-binding protein from the parasite. METHODS: A protein was purified from the crude parasite extract by means of ethanol precipitation and reversed-phase HPLC. Its clinical relevance was tested on 20 parasite-positive sera by using IgE and IgG4 immunoblotting. A monospecific human serum was used to study its localization in the parasite body. RESULTS: A 24-kd protein was purified, to which only 45% of the sera had specific IgG4, but 85% of sera had specific IgE. The protein was present only in the excretory gland, as shown by immunohistochemistry. N-terminal amino acid sequence (17 residues) showed no homology to previously described proteins. CONCLUSION: A simplex contains a potent allergen in the excretory gland. This major parasite allergen, named Ani s 1, could have important clinical relevance, as shown by the high number of positive sera in the specific IgE immunoblotting.

Allergens↗
PubMed2004

Action of different monoterpenic compounds against Anisakis simplex s.l. L3 larvae.

Different natural monoterpenes (geraniol, citronellol, citral, carvacrol, cuminaldehyde and eugenol) are studied in vitro against Anisakis simplex s.l. L3 larvae, employing perillaldehyde as a reference substance. Final concentrations used were: 12.50, 6.25 and 3.12 microg/ml for each of the tested products. The parameters average survival, survival 50 and maximum survival were determined at 4, 8, 24 and 48 hours after the start of the experiment. All tested products, except eugenol, were active at the highest concentration (12.50 microg/ml). The damage caused to A. simplex s.l. L3 was by examining histological sections. The antioxidant activity of the tested products by DPPH free radical scavenging does not appear to be associated with their larvicide activity against A. simplex s.l. L3.

Animals↗
PubMed2002

Immunoglobulins anti-Anisakis simplex in patients with gastrointestinal diseases.

The nematode Anisakis simplex causes anisakidiasis, a disease that often mimics other gastrointestinal diseases. Patients with digestive haemorrhaging, Crohn's disease, digestive cancer and appendicitis were analysed for antibodies to A. simplex. Antibody detection was carried out by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using crude extract (CE) antigen and excretory-secretory (ES) products. Total immunoglobulin (Igs), IgG, IgM, IgA and IgE were studied. The highest percentage was obtained when Igs were tested against CE antigen. A higher percentage of positivity was observed with the appendicitis group. The Crohn's disease group showed the highest levels of IgG against the ES antigen. Using immunoblotting, 24% and 48% of sera from patients with symptoms of Crohn's disease and digestive haemorrhaging, respectively, showed a positive immunorecognition pattern of CE antigen. The prevalence of detectable antibodies against A. simplex is higher in patients with digestive disorders than in the healthy population. A linear correlation was observed between prothrombin activity and Igs-CE, IgA-CE and IgA-ES but not between IgE-CE and the other immunoglobulin levels. Specific IgA is associated with a higher activity index of Crohn's disease. Specific antibodies were observed against A. simplex in patients with appendicitis and gastrointestinal cancer, indicating a higher rate of positivity for IgA.

Adult↗
PubMed2002

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.

Animals↗
PubMed2003

Cross-reactivity induced by Anisakis simplex and Toxocara canis in mice.

The aim of this study was to verify whether cross-reactivity appeared between Toxocara canis and Anisakis simplex in an experimental rodent model. No cross-reactions were detected using sera from mice infected with T. canis eggs. When responses obtained against T. canis ES antigen using sera from BALB/c and C57BL/10 mice infected with T. canis eggs were compared with those obtained by testing sera from mice infected with one A. simplex L3, an increase in cross-reactions was observed using the C57BL/10 strain.

Animals↗
PubMed2005

Molecular characterization of Anisakis pegreffii larvae in Pacific cod in Japan.

It is now recognized that the morphospecies Anisakis simplex is not a single species but a complex composed of three sibling species, A. simplex sensu stricto, A. pegreffii and A. simplex C. In Japan, A. simplex-like larvae have been isolated from a variety of fish and humans, but the larvae collected have been identified as A. simplex by only light microscopy. Therefore, the epidemiology of the A. simplex complex, composed of three sibling species, is still unclear in Japan. In the present study, 26 A. simplex-like larval isolates were obtained from two Pacific cod landed in Hokkaido, Japan, and examined genetically by PCR-RFLP and direct sequencing of the ITS region of rDNA. Among the 26 isolates, 24 were identified as A. simplex sensu stricto, the other two as A. pegreffii. The present study is the first to confirm the distribution of A. pegreffii in Japan, and to detect A. pegreffii larvae in Pacific cod.

Animals↗
PubMed1998

Effects of Anisakis simplex on nitric oxide production in J774 macrophages.

Since nitric oxide (NO) was recognized as a potent microbicidal agent, its role in host defence against intracellular parasites has been widely demonstrated. Recent evidence suggests a role for NO in combating extracellular and multicellular pathogens. This defence activity has been demonstrated toward the larvae of Schistosoma mansoni, microfilariae of Onchocerca linealis, several stages of Brugia malayi and protoscoleces of Echinococcus multilocularis. Many parasites suppress Th1 lymphocytes and directly inhibit NO production by inducing cytokines, such as IL-4, IL-10 and TGF-beta. In this study, we have investigated the effects of Anisakis simplex, an enhancer of Th2-dominant responses, on NO production. We studied the effect of crude extracts (CE) and excretory-secretory (ES) products on the induction of inducible nitric oxide synthase (iNOS) in bacterial lipopolysaccharide (LPS)-treated J774 macrophages. Stimulation of macrophages by LPS (1 microg/ml) increased nitrite concentrations in the culture medium at 24 h. Co-administration of A. simplex products with LPS, dose-dependently reduced the accumulation of nitrite. Nitrite production is due to induction of iNOS, and both L-NAME (N(G)-nitro-L-arginine methyl ester) (50 microM) and dexamethasone (10 microM) inhibited nitrite accumulation (54.2 and 92.1% inhibition, respectively). The inhibition of nitrite production by A. simplex was 42.1-97.8% in the range 4.75-76 microg/well (CE products) and 37.2-61.5% in the range 5-20 microg/well (ES products). Cell viability assayed by the mitochondrial-dependent reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) verified that the inhibition was not due to general cellular toxicity. However, the effects of A. simplex, were reduced when NOS had been induced by prior exposure to LPS and any possible further induction was blocked by cycloheximide, an inhibitor of protein synthesis.

Animals↗
PubMed1994

Hyaluronidases of the gastrointestinal invasive nematodes Ancylostoma caninum and Anisakis simplex: possible functions in the pathogenesis of human zoonoses.

The gastrointestinal invasive stages of two parasitic nematodes, Ancylostoma caninum and Anisakis simplex, were each found to release in vitro a hydrolytic enzyme that degrades the glycosaminoglycan hyaluronic acid. The parasite hyaluronidases were partially purified by ion-exchange chromatography and biochemically characterized. The hyaluronidase from A. caninum adult worms had a molecular weight of 65,000 and a pH optimum of 6 with activity at neutral pH, while the hyaluronidase from A. simplex larvae had a molecular weight of 40,000 and a pH optimum of 4 with no activity at neutral pH. Both parasite hyaluronidases also degraded the glycosaminoglycan chondroitin sulfate A. Cupric sulfate and high concentrations of sodium chloride were inhibitory. The nematode hyaluronidases are postulated to have a role in tissue histolysis and mucosal invasion; their distinct biochemical properties have relevance to the pathogenesis of the zoonoses anisakiasis and eosinophilic enteritis.

Ancylostoma↗
PubMed2000

Digestive haemorrhage in patients with anti-Anisakis antibodies.

OBJECTIVE: Anisakis simplex parasite causes anisakidosis, a disease that often mimics other gastrointestinal diseases such as peritonitis, appendicitis, Crohn's disease, ulcer, etc. Patients with digestive haemorrhage caused by ulcers, varices or Mallory syndrome were analysed for antibodies to the worm A. simplex. METHODS: Antibody detection was carried out by enzyme-linked immunosorbent assay (ELISA) and immunoblot using crude extracts of antigen and excretory/secretory products. Total immunoglobulin (Ig), IgG, IgM, IgA and IgE were studied. RESULTS: Eighty-seven patients were studied. The following prevalence rates were found with crude antigen: total Ig 30% (95% confidence interval 21-40), with values for IgG, IgM, IgA and IgE of 22 (CI 14-31), 17 (CI 10-26), 37 (CI 27-47) and 12% (CI 6-20), respectively. Twenty-four positive sera for total Ig response and crude products were selected for determination of specific antibodies with excretory/secretory antigens. We obtained 8, 13, 3 and 16 positive cases for total Ig, IgG, IgM and IgA respectively. The percentages of positivity within the varices and Mallory groups of patients were higher although differences were not significant (35 and 50% respectively). In a healthy population, the prevalence for total Ig is much lower (6%). Twenty-five positive sera for total Ig response were tested by means of immunoblot analysis against crude larval antigen. Concerning total Ig antibody response, 12 of the sera showed an immuno-recognition pattern concordant with the human anisakidosis reference serum (E17). Specific IgG bands were visualized in 30 sera; specific IgM and IgA in 6 and 12, respectively. Different clinical variables of these patients were studied: leucocytes, eosinophils, haemoglobin, prothrombin activity, thromboplastin time, fibrinogen, platelets and erythrocyte sedimentation rate. There were a few significant differences: for total Ig in prothrombin activity and platelets, and for IgM in eosinophils. CONCLUSIONS: The prevalence of detectable antibodies against A. simplex is higher in patients with digestive haemorrhage than in the healthy population.

Adult↗
PubMed2005

Anisakis simplex: sensitization and clinical allergy.

PURPOSE OF REVIEW: Whereas gastric anisakiasis has been known for several decades, the implications of Anisakis simplex-related allergic disorders had not been thoroughly studied until the late 1990s. This article reviews recent knowledge of allergic disorders ascribed to A. simplex contact or parasitism. RECENT FINDINGS: Gastroallergic anisakiasis describes an acute hypersensitivity reaction emerging in the context of an acute parasitism by the nematode A. simplex. But other frequent allergic disorders like chronic urticaria are now being studied for a possible relationship with A. simplex parasitism. In recent investigations, non-IgE mediated mechanisms, such as the involvement of other immunoglobulin isotypes (IgG4), or non-immunological events are discussed. SUMMARY: The experience of the last several years shows that allergic hypersensitivity symptoms in gastroallergic anisakiasis are clinical events accompanying a wide range of immunologic reactions as a host response against a ubiquitous parasite. The discussed and reviewed studies should motivate allergists around the world to search for this entity. Further studies in the field of allergy could benefit from the experience of this peculiar food-related disorder.

Animals↗
PubMed2004

Characterization of allergens secreted by Anisakis simplex parasite: clinical relevance in comparison with somatic allergens.

BACKGROUND: Diagnostic methods for the study of allergic reactions to Anisakis simplex (A.s.) based on whole-body extracts of the larva are clearly insufficient. OBJECTIVES: To study the allergenicity of the proteins secreted by the parasite. Comparison with somatic antigens and determination of their clinical importance in allergic patients were also addressed. METHODS: An excretory/secretory (E/S) extract was produced by culturing third-stage A.s. larvae. It was used to perform immediate skin tests and to determine specific IgE in 10 patients diagnosed with allergy to A.s. Both tests were compared with the results obtained with the whole-body extract (somatic (S)). The molecular weight (MW) of their allergens was determined by immunoblotting, and a single-blind placebo-controlled oral challenge with E/S proteins was performed. Finally, allergens' resistance to gastric pepsin and acid pH was explored. RESULTS: A.s. larvae secreted allergens more potent than those present in the S extract. The skin prick test wheal area produced by E/S molecules and the absorbance obtained in the determination of specific IgE with these allergens (ELISA) were 5.8 times bigger than those obtained with S extract. MW allergens of 72 and 56 kDa in E/S extracts and those of 56, 48 and 43 kDa in S extract were recognized by more than 50% of the patients. Partial cross-reactivity between them was revealed by immunoblotting inhibition studies. Oral challenge with E/S extract (up to 479 microg) was negative in all the patients. Treatment of E/S proteins with gastric pepsin inhibited the binding of the E/S allergens for specific IgE. The acid pH did not affect the overall binding of IgE to E/S extract. It decreased by 15.23% and 19.96% at pH 4 and 2, but the difference was not statistically significant. CONCLUSION: A.s. secretes allergens more potent than somatic antigens and should be used in the diagnostic procedures. These allergens are inactivated by the pepsin, which supports the theory that live larva is necessary to induce an allergic reaction in most of the patients.

Adult↗