Experimental anisakid infections in mice.

Anisakidosis is a human parasitic disease caused by infections with members of the Anisakidae family. Accidental infection after fish intake affects the gastrointestinal tract as a consequence of mechanical damage caused by migrating larvae. Infections can also trigger allergies, hives, severe asthma or anaphylaxis with angioedema. Although mouse models of intraperitoneal antigenic stimulation exist, enabling immunological studies, few models using gastric introduction of live larvae are available for the study of immunological and gastrointestinal damage in mice. This study was designed to characterize serum reactivity against Anisakis spp. and Contracaecum spp. in Balb/c mice following orogastric inoculation and to assess gastrointestinal damage. These anisakid species were classified at the Universidade Federal Fluminense (UFF) School of Veterinary Medicine and materials for live larval inoculation were developed at the UFF Immunobiology laboratory. Live larvae were inoculated following injection with a NaCl solution. Blood samples were collected and sera screened for immunoglobulin (Ig)E and IgG anti-larva responses to both nematodes, specific for somatic and excretory/secretory antigens, by enzyme-linked immunosorbent assay (ELISA). The means of the optical densities were analysed using analysis of variance (ANOVA) with Tukey's post-hoc test and the general linear model. This analysis identified the presence of anti-IgG seroreactivity to both somatic and excretory/secretory Anisakis antigens in inoculated animals compared with controls (P< 0.001), and no gastric or intestinal damage was observed. These experiments demonstrated that introduction of live Contracaecum spp. into the gastrointestinal tract did not elicit serum sensitization in animals.

Intestinal anisakiasis treated successfully with conservative therapy: importance of clinical diagnosis.

Intestinal anisakiasis is not only a rare parasitic disease, but is also difficult to diagnose. The symptoms are not specific and are often very severe and abrupt, and the findings of clinical imaging are very remarkable. Therefore, intestinal anisakiasis is often misdiagnosed as acute abdomen or intestinal obstruction and is treated surgically. However, if intestinal anisakiasis could be diagnosed correctly, it is well treated conservatively. We experienced three cases of intestinal anisakiasis, which were diagnosed correctly and treated successfully with conservative therapy. A correct clinical history and imaging interpretation helped us diagnose intestinal anisakiasis correctly and thus treat the patients successfully with conservative therapy.

Anisakiasis and gastroallergic reactions associated with Anisakis pegreffii infection, Italy.

Human cases of gastric anisakiasis caused by the zoonotic parasite Anisakis pegreffii are increasing in Italy. The disease is caused by ingestion of larval nematodes in lightly cooked or raw seafood. Because symptoms are vague and serodiagnosis is difficult, the disease is often misdiagnosed and cases are understimated.

A phage display system for the identification of novel Anisakis simplex antigens.

Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaining a primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereas the insert sizes varied from 0.35 to 1.2kb. Both libraries were subsequently analyzed by enrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera from patients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinized detecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promising novel A. simplex allergen. Phage display technology has to date not yet been applied to the identification of new A. simplex allergens, and the present work opens up new avenues to the understanding of the Anisakis allergenic process.

Multiple acute parasitization by Anisakis simplex.

Hypersensitivity to Anisakis is an increasingly prominent medical problem throughout the world, due to a better understanding of diseases induced by parasites and to modern culinary habits of eating raw or undercooked fish. We describe the case of a patient who presented epigastric pain, wheals, erythema, and pruritus 3 hours after the ingestion of fish. More than 200 larvae were obtained by endoscopy. However, the patient only developed an immune response with specific immunoglobulin E and eosinophilia peaking at day 18 and decreasing during the 17-month follow-up. Only eosinophilia reached normal limits.

Anisakiasis, an underestimated infection: effect on intestinal permeability of Anisakis simplex-sensitized patients.

Anisakis simplex is a parasite that, if present in uncooked and contaminated saltwater fish, can invade the human gut. Two different clinical situations are recognized: the first, known as a gastrointestinal disease, varying from an asymptomatic episode to vomiting and diarrhea, and the second, classified as an adverse reaction to food, characterized by a wide spectrum of allergic reactions like rhinitis, conjunctivitis, or even anaphylaxis causing hypotension and/or shock. The intestinal epithelium, the major defense system against external molecules, represents an open gate for toxins and allergens if its protective function is compromised. Previous data have demonstrated a strict relationship between an altered intestinal permeability (I.P.) and worsening of the clinical manifestations in patients with adverse reactions to the food. In this article we evaluated the sensitization to A. simplex among patients who referred clinical symptoms of allergy. All subjects underwent commonly used alimentary skin prick test for food allergens, to which Ani s1, an A. simplex allergen, was added. In addition, in A. simplex-sensitized subjects, I.P. was determined upon their enrolment to the study (time 0) and after 6 months of consuming a raw fish-free diet (time 6). Five hundred and forty subjects were screened, and 170 had a positive skin prick test, 87 (51.2%) of whom were positive to Ani s1. Increased I.P. was evidenced in A. simplex-sensitized subjects with worse clinical symptoms, which receded after 6 months' elimination of raw seafood. With our data we demonstrated that the alimentary habit to eat raw fish represents a high risk for the integrity of the intestinal mucosa, and we suggest that this pathological situation may constitute an ideal, under-estimated, open gate for molecules that predispose to other, more important pathologies.

Expression of a recombinant protein immunochemically equivalent to the major Anisakis simplex allergen Ani s 1.

BACKGROUND: Anisakis simplex is a nematode which can parasitize humans, producing anisakiasis and can induce immunoglobulin-(Ig)-E-mediated allergic symptoms. Parasite recombinant proteins, such as the major allergen Ani s 1, may be useful tools to avoid misdiagnosis of A simplex allergy due to cross-reactivity when whole parasite extracts are used. OBJECTIVE: To obtain Ani s 1 allergen as a recombinant protein with IgE-binding properties similar to its natural counterpart. METHODS: Ani s 1-encoding cDNA was amplified by polymerase chain reaction and cloned. The allergen was expressed in Escherichia coli as a nonfusion protein. Natural and recombinant Ani s 1 were investigated by means of Western blotting, enzyme allergosorbent test, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition using sera from 53 patients with A simplex allergy. RESULTS: Residues of the amino acid sequence of the encoded protein were 99.4% identical to the reported one. Purified rAni s 1 was obtained with a yield of 2 mg/L of culture while the yield of the natural counterpart was only 50 micro/g of larvae. rAni s 1 reactivity was not significantly different from that of the natural allergen; the correlation was excellent (p = 0.92, P < .001). ELISA-inhibition experiments showed that the dose-response inhibition curve obtained with rAni s 1 overlapped with that of nAni s 1. In an enzyme allergosorbent analysis, 86.8% of the A simplex-allergic patient sera reacted to rAni s 1. CONCLUSION: Recombinant Ani s 1 is immunochemically equivalent to its natural counterpart and therefore might be useful for the in vitro diagnosis of anisakiasis and A simplex-mediated allergy.

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.

Immunopathogenesis of human gastrointestinal infection by Anisakis simplex.

BACKGROUND: Anisakis simplex is a parasite of fish, and in the case of human infestation, it should be considered as a possible cause of gastrointestinal disease, especially in countries where raw or undercooked fish is a frequent food. Clinical features of anisakiasis may simulate acute abdominal pain, such as that found in patients with gastric ulcers, appendicitis, and Crohn's disease. Furthermore, many cases of anisakiasis are diagnosed as eosinophilic gastroenteritis, which is a broad term for a specific disease. OBJECTIVE: The purpose of this study was to investigate the immunopathogenesis of human gastrointestinal infestation by A simplex. METHODS: Thirteen intestinal biopsy specimens from patients with anisakiasis were analyzed for the presence of messenger (m)RNA for different cytokines and inflammatory mediators by RT-PCR. Specific IgE, eosinophil cationic protein, eosinophil protein X, and tryptase levels were measured in each patient's serum. Also, cell cultures were set up with lymphocytes from some patients and stimulated in vitro with Anisakis and Ascaris antigens. RESULTS: We performed immunologic phenotyping in 13 patients. All patients underwent biopsy after emergency surgery caused by episodes of acute abdominal pain. In all cases inflammatory infiltrate composed of eosinophils and lymphocytes was found in the intestinal wall. We demonstrated that after infestation, a T(H2)-type immune response occurred. Also, major basic protein, nitric oxide, and eotaxin were found in the tissue, and eosinophil cationic protein and eosinophil protein X levels were elevated in sera. CONCLUSION: These data and in vitro lymphocyte cultures indicate that a T(H2) mechanism plays an important role in the inflammatory infiltrate produced by the anchorage of parasites in the gastrointestinal wall.

Laboratory determinations in Anisakis simplex allergy.

BACKGROUND: Anaphylactic reactions caused by the fish nematode, Anisakis simplex, after ingestion of parasitized fish, have been described. OBJECTIVE: This study was undertaken to confirm, by histamine release tests, that A. simplex is able to trigger IgE-mediated reactions and to describe the serologic profiles in this sensitization. METHODS: Twelve patients who had anaphylactic symptoms after ingestion of cooked fish and positive prick test results and determinations of IgE to A. simplex were studied by indirect IgG ELISA and IgG and IgE immunoblotting. Sera from subjects parasitized with other nematodes, patients with fish allergy, and healthy donors were included as controls. A histamine release test was performed in a representative case. RESULTS: IgE immunoblotting was a specific test to detect A. simplex allergy. IgE-reacting bands were found in serum samples from 11 of our patients. Specific IgG antibodies were found by ELISA and immunoblotting, but this response was less specific. Histamine release was positive with A. simplex extract and negative with fish. CONCLUSION: A specific and intense immune response to an A. simplex extract was found in our patients. A. simplex is able to elicit anaphylactic reactions, and A. simplex allergy should be suspected in patients with allergic symptoms after ingestion of fish. A positive prick test response to A. simplex and a negative response to fish is a good indication for a diagnosis of A. simplex allergy.

Detection of antibodies to Anisakis simplex larvae by enzyme-linked immunosorbent assay and immunoelectrophoresis using crude or purified antigens.

The enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis (IEP) were used for the serodiagnosis of larval Anisakis simplex infections in man and immunized rabbits. Sephacryl S-300 gel filtration was used for separating crude antigen. Four fractions were obtained. Sera from patients with other helminth infections sometimes cross reacted with Anisakis larval antigens. With IEP, crude antigen is more sensitive than purified antigens. With ELISA, the third fraction is the most sensitive for detecting antibodies to Anisakis larvae in the sera of humans and immunized rabbits.

Haematological and morphological changes in experimental anisakiasis in pigs.

The aim of this paper is to establish the haematological and organic changes in halothane-sensitive and halothane-resistant pigs in the course of experimental anisakiasis. Experiments were carried out on two groups of pigs (3 animals each). The pigs from the first group were given fifty A. simplex B larvae, the pigs from the second one received ten larvae and then again fifteen larvae each after the 5th and 6th days. The number of leucocytes, neutrophils, lymphocytes, monocytes and eosinophils was greater--different in both series, but similar in halothane (stress)-resistance and -sensitive pigs. In the case of sensitive pigs much greater reactive changes were found in the stomach submucosa than in that of resistant pigs. In this group of pigs nematode larvae have also been traced in the submucosa of the same organ.