Serum IgA contributes to the comprehension of Anisakis simplex associated chronic urticaria.

The phenotype of allergic diseases associated with Anisakis determines the pattern of cytokines related to antibody production. However, the role of serum IgA and the immunomodulatory mechanisms exerted by active infection of L3 or passive mucosal contact with A. simplex specific antigens has not been studied before. We measured serum cytokine by flow cytometry (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, TGF-ß1) and antibody levels (IgE, IgG4, IgA) by ELISA against total and excretory-secretory (ES) antigens, Ani s 3,and the group of major allergens Ani s 1, Ani s 7, and Ani s 13 in sera from 10 patients with gastro-allergic anisakiasis (GAA), 11 Anisakis sensitization associated chronic urticaria (CU+) as well as 17 non-Anisakis-sensitized patients with chronic urticaria (CU-), compared with the urticaria control group (18 subjects). Specific IgE, IgG4 and IgA were high in the GAA, but IgA levels were significantly higher in the CU+ with respect the CONTROL group. We observed higher levels of the ratio IgA/IgG4 in CU+ than GAA group for Ani s 1, Ani s 7, Ani s 13 and ES. Furthermore, chronic urticaria (CU) patients showed significant lower levels of IL-10, IFN-γ and IL-17A than patients without CU. The anti-Ani s 13 IgA/IgG4 ratio correlated positively with pro-inflammatory cytokines and ratios (TNF-α, IL-17A, Th17/Th2, Type1/Type2 and TNF-α/IL-10) in CONTROL group. In general, Anti-Anisakis IgA/G4 ratio was high in CU patients. In conclusion, this study demonstrates the importance of serum IgA because it is associated with chronic urticaria independently of Anisakis sensitization.

Association between anti-Anisakis simplex antibodies and interleukin-7 levels.

IL-7 is a crucial factor for the development of lymphocytes, and it is absolutely necessary for γδ T cells. Mice deficient in L-7 have a deficit of B and αß T lymphocytes, and an absence of mature γδ TCR cells. IL-7 is essential for the survival, development and maturation of Schistosoma sp., although its production is associated with protection against intestinal helminths. The presence of anti-Anisakis simplex antibodies, especially IgA, is related to a lower frequency in CD3 + CD56 + αß + lymphocytes and all subpopulations of γδ T cells. In this work, the relationship of IL-7 with humoral and cellular responses against A. simplex in 100 healthy subjects was studied. We have found significantly higher IL-7 levels in anti-A. simplex IgA-positive subjects (p < 0.001). The positivity of anti-A. simplex IgA was associated with a significant reduction in the frequency of CD3 + αß+ (p < 0.01), CD3 + CD4 + αß+, CD3 + CD8 + αß+, CD3 + CD56 + αß+, CD3 + Î³Î´+, CD3 + CD4-CD8-γδ+ and CD3 + CD56 + Î³Î´+ (p < 0.05) cells. In the case of NKT cells, this same phenomenon was also associated with IgE positivity. There was a weak inverse correlation (Spearman) of IL-7 levels with the frequencies of CD3 + CD4 + αß+ (-0.125, p = 0.047), CD3 + CD8 + αß+ (-0.204, p = 0.032), CD3 + CD56 + αß+ (-0.247, p = 0.007), CD3 + Î³Î´+ (-0.267, p = 0.007), CD3 + CD4-CD8-γδ+ (-0.266, p = 0.003), and CD3 + CD8 + Î³Î´ + (-0.302, p = 0.002) cells. The role of NKT cells in the anti-A. simplex response was confirmed and an association between IL and 7 levels and specific antibodies, especially IgA, was demonstrated. The higher production of IL-7 would represent a compensatory mechanism in response to the reduction in lymphocyte populations associated with the response against this parasite.

Anisakis simplex: Immunomodulatory effects of larval antigens on the activation of Toll like Receptors.

AIMS: The objective of this investigation is to evaluate the mechanisms Anisakis simplex employs to modify its host immune system, regarding the larval antigens interactions with Toll-Like-Receptors (TLRs). METHODS AND RESULTS: In a previous study, we described that the stimulation of bone marrow derived dendritic cells (BMDCs) with A. simplex larval antigens drive an acute inflammatory response in BALB/c mice, but a more discrete and longer response in C57BL/6J. Moreover, when A. simplex larval antigens were combined with TLR agonists (TLR 1/2-9), they modified mainly TLR2, TLR4 and TLR9 agonists responses in both mice strains, and also TLR3, TLR5 and TLR7 in BALB/c. Antigen-presenting ability was analyzed by the detection of CD11c + cells expressing surface markers (CD80-86, MHC I-II), intracellular cytokines (IL-10, IL-12, TNF-α) and intracellular proteins (Myd88, NF-κß) by Flow Cytometry. Secreted IL-10 was measured by ELISA. CONCLUSION: Our findings confirm not only that the host genetic basis plays a role in the development of a Th2/Th1/Treg response, but also it states A. simplex larval antigens present specific mechanisms to modify the innate response of the host. As allergies share common pathways with the immune response against this particular helminth, our results provide a better understanding into the specific mechanisms of A. simplex allergy related diseases.

Expansion of T regulatory lymphocytes by murine bone marrow dendritic cells previously stimulated with Anisakis simplex larval antigens.

BACKGROUND: Anisakis simplex antigens present immunomodulatory properties by the induction of tolerogenic dendritic cells (DCs) in mice. OBJECTIVES: To study the capacity of DCs stimulated with A. simplex excretory-secretory (ES) or crude extract (CE) to generate Tregs. To investigate in vitro effects of antigens on the metabolic activity of splenocytes induced by LPS or CpG. METHODS: Phenotypic and functional characterization of T cells co-cultured with A. simplex-pulsed DCs was performed by flow cytometry. Lymphocyte mitochondrial respiratory activity was estimated by the Alamar Blue® Assay. FINDINGS: In C57BL/6J, CD4+CD25-Foxp3+ and CD8+CD25-Foxp3+ populations increased by CE-stimulated-DCs. In BALB/c, CE-stimulated-DCs caused the expansion of CD4+CD25+Foxp3+IL-10+ and CD8+CD25+Foxp3+IL-10+. IFN-γ expression raised in BALB/c CD4+CD25+ and CD4+CD25- for CE and ES, respectively. ES-stimulated-DCs increased CD4+CD25+ Foxp3+ and CD8+CD25- Foxp3+ expression in T cells. The association of ES or CE with LPS produced the increase in splenocyte activity in C57BL/6J. The association of CE with CpG decreased the proliferation caused by CpG in C57BL/6J. MAIN CONCLUSIONS: A. simplex increase the frequency of Tregs, which in turn produce IL-10 and IFN-γ. The host genetic base is essential in the development of anti-Anisakis immune responses (Th2, Th1, Treg).

Modulation by Anisakis simplex antigen of inflammatory response generated in experimental autoimmune encephalomyelitis.

The impact of immunization with Anisakis simplex larval antigen on the occurrence and progression of experimental autoimmune encephalomyelitis (EAE) induced in mice was studied. C57BL/6J mice were immunized with the MOG35-55 peptide and one batch was treated with A. simplex total larval antigen on days 1, 8, 10 and 12 after EAE induction. Significantly higher values were obtained in the EAE clinical parameters of the antigen-treated group. Likewise, there was a significant decrease in the weights of the animals. Anisakis-treatment produced a significant decrease in anti-MOG35-55 specific IgG1 on day 21. On day 14 there was an increase in serum IL-2, IL-6, IL-10, IL-17A, and TGF-ß in the treated group. On day 21, a decrease in IL-4, IL-6, TNF-α, TGF-ß was observed. All brain determinations were made on day 21. The treatment decreased values of IL-6, IL-10, IL-17A and TNF-α. A. simplex antigen caused a significantly higher incidence of EAE and an advance in the appearance of the disease manifestations. However, treatment with the antigen was able to cause a decrease in proinflammatory cytokines (IL-6, IL-17A, and TNF-α) in nervous tissue that could establish a future preventive scenario for myelin damage.

Expansion of T regulatory lymphocytes by murine bone marrow dendritic cells previously stimulated with Anisakis simplex larval antigens

BACKGROUND Anisakis simplex antigens present immunomodulatory properties by the induction of tolerogenic dendritic cells (DCs) in mice. OBJECTIVES To study the capacity of DCs stimulated with A. simplex excretory-secretory (ES) or crude extract (CE) to generate Tregs. To investigate in vitro effects of antigens on the metabolic activity of splenocytes induced by LPS or CpG. METHODS Phenotypic and functional characterization of T cells co-cultured with A. simplex-pulsed DCs was performed by flow cytometry. Lymphocyte mitochondrial respiratory activity was estimated by the Alamar Blue® Assay. FINDINGS In C57BL/6J, CD4+CD25-Foxp3+ and CD8+CD25-Foxp3+ populations increased by CE-stimulated-DCs. In BALB/c, CE-stimulated-DCs caused the expansion of CD4+CD25+Foxp3+IL-10+ and CD8+CD25+Foxp3+IL-10+. IFN-γ expression raised in BALB/c CD4+CD25+ and CD4+CD25- for CE and ES, respectively. ES-stimulated-DCs increased CD4+CD25+ Foxp3+ and CD8+CD25- Foxp3+ expression in T cells. The association of ES or CE with LPS produced the increase in splenocyte activity in C57BL/6J. The association of CE with CpG decreased the proliferation caused by CpG in C57BL/6J. MAIN CONCLUSIONS A. simplex increase the frequency of Tregs, which in turn produce IL-10 and IFN-γ. The host genetic base is essential in the development of anti-Anisakis immune responses (Th2, Th1, Treg).

Induction of tolerogenic properties by Anisakis larval antigens on murine dendritic cells.

AIMS: The objective of this work is to investigate whether Anisakis simplex larval antigens present immunomodulatory properties by the induction of tolerogenic dendritic cells (DCs) from two strains of mice (BALB/c and C57BL/6J). METHODS AND RESULTS: We used mouse bone marrow-derived DCs. We determined their antigen-presenting ability by expression of membrane markers (MHC I and MHC II, CD80, CD86) and intracellular expression levels of IL-10 and IL-12 cytokines. We also analysed whether stimulation with A simplex larval antigens is enhanced by the co-administration of the TLR4 and TLR9 agonists [LPS E coli 026B6 and CpG (ODN1826), respectively]. Two differential types of responses were found in the two mouse strains studied: the BALB/c strain showed an acute and inflammatory response, whereas the C57BL/6J mice developed a more discrete and resistant response. This suggests the coexistence of two opposing responses generated by A simplex larval antigens and confirms that the host genetic basis plays a role in the development of a Th2 or Treg response. CONCLUSION: The study of the mechanisms by which Anisakis manipulates the immune response through anti-inflammatory molecules is of interest not only for the direct application on the development of anthelmintic strategies, but also for the development of new anti-inflammatory products.

New insights into the allergenicity of tropomyosin: a bioinformatics approach.

The invertebrate panallergen tropomyosin is a protein with an extremely simple folding. This makes it a perfect target for investigating structural differences between invertebrate and vertebrate tropomyosins, which are not considered allergenic. Phylogenetic and sequence analyses were conducted in order to explore the differences in primary structure between several tropomyosins and to promote an experimental development in the field of food allergy, based on the study of tropomyosin. The phylogenetic analyses showed that tropomyosin is a useful evolutionary marker. The phylogenetic trees obtained with tropomyosin were not always phylogenetically correct, but they might be useful for allergen avoidance by tropomyosin allergic individuals. Sequence analyses revealed that the probability of alpha helix folding in invertebrate tropomyosins was lower than in all the studied vertebrate ones, except for the Atlantic bluefin tuna Thunnus thynnus tropomyosin. This suggested that the lack of alpha helix folding may be involved in the immunogenicity of tropomyosins. More specifically, the regions adjacent to the positions 133-135 and 201 of the invertebrate tropomyosins, presented lower probability of alpha helix folding than those of vertebrates and are candidates to be responsible for their allergenicity.

Avidity Studies in Anisakis simplex-Associated Allergic Diseases.

Gastroallergic anisakiasis (GAA) and Anisakis-sensitization-associated chronic urticaria (CU+) differ with respect to specific IgE levels. We hypothesised different immunoglobulin avidities in both entities as well as their dependence on TI and fish consumption. 16 patients with GAA and 17 patients with CU+ were included, and immunoglobulin levels were analysed by CAP (Phadia). IgE and IgG avidity indexes (AvIgE and AvIgG, resp.) were also determined. IgG avidity was higher in GAA than in CU+ (P = 0.035), whereas there was a tendency to lower IgE avidity in GAA (P = 0.095). When analysing all patients, AvIgG was positively correlated with specific IgE, IgG, and IgG4 as well as total IgE (Rho between 0.66 and 0.71; P < 0.002), but AvIgE was negatively correlated with specific IgE (Rho -0.57; P < 0.001), specific IgG4 (Rho -0.38; P < 0.05), and total IgE (Rho 0.66; P < 0.001). In GAA, weekly fish consumption was positively associated with AvIgE (Rho 0.51; P = 0.05). A multivariate regression showed that time interval was the main explaining factor for AvIgE in GAA. We could show a differential behaviour of immunoglobulin isotype avidities in both entities and their dependence on fish-eating habits as well as on the time elapsed to the last parasitic episode.

Different fish-eating habits and cytokine production in chronic urticaria with and without sensitization against the fish-parasite Anisakis simplex.

BACKGROUND: Anisakis simplex sensitization has been associated with acute, but also with chronic urticaria. The objective of this study is to characterize chronic urticaria with (CU+) and without sensitization (CU-) against the ubiquitous fish parasite A. simplex in a transversal and longitudinal evaluation. METHODS: 16 CU+ and 22 CU- patients were included and assessed for Urticaria activity score (UAS), fish-eating habits by standardized questionnaire and cytokine production (assessed by flow cytometric bead-based array) of peripheral blood mononuclear cells after stimulation with A. simplex extract or Concanavalin A (Con A). Patients were randomly put on a fish-free diet for three months and UAS, as well as cytokine production were again assessed. A difference of ≥1 in UAS was defined as improvement. RESULTS: There was no difference in UAS in both groups. Anisakis induced IL-2, IL-4 and IFN-γ production was higher in CU+. Con A induced IL-6 and IL-10 production was higher in CU+. CU+ was associated with higher total fish intake, whereas CU- was associated with oily fish intake. The correlation of UAS was positive with oily fish, but negative with total fish intake. There was a better UAS-based prognosis in CU+ without diet. Improvement was associated with higher Con A induced IL-10/IFN-γ as well as IL-10/IL-6 ratios. Further, previous higher oily fish intake was associated with improvement. CONCLUSIONS: Our data confirm the different clinical and immunological phenotype of CU+. Our results show a complex relationship between fish-eating habits, cytokine production and prognosis, which could have important consequences in dietary advice in patients with CU. When encountering A. simplex sensitization, patients should not be automatically put on a diet without fish in order to reduce contact with A. simplex products.

The Anisakis allergy debate: does an evolutionary approach help?

Allergic phenomena share common pathways with the immune response against helminth parasites. The definitions regarding allergens and their related concepts have their roots in the area of allergy research. The experience with the fish parasite Anisakis simplex-associated allergic features still nurtures an open debate on the necessity of larvae being alive to induce allergic reactions such as urticaria or anaphylaxis. Conceptual definitions of allergen, major allergen, as well as putatively crossreacting antibodies, as are used in food allergy, depend on the clinical relevance of specific IgE and deserve careful interpretation in the various forms of A. simplex-associated allergic features. Conversely, an evolutionary based interpretation of the presence of specific IgE depends on the viability of A. simplex.

Chronic urticaria is associated with a differential helminth-arthropod-related atopy phenotype.

The relationship between atopic sensitization and chronic urticaria is still controversial. In this study, we aimed to compare the prevalence of aeroallergen sensitization in chronic urticaria patients with (CU/As+) and without (CU/As-) sensitization against Anisakis simplex. Forty-nine CU/As+ and 80 CU/As- patients were studied and skin prick tests (SPT) were performed against aeroallergens. We assessed sensitization in a subgroup of patients with allergic rhinoconjunctivitis and/or bronchial asthma (RCBA) and compared the prevalence with a control group of 522 non-urticaria patients with RCBA. Forty-five percent of CU/As- and 60.4% of CU/As+ patients displayed positive SPT to at least one aeroallergen. CU/As+ patients had a higher prevalence of sensitization against pollen, mould or dander (PMD) (52.2% vs 29.1%, P < 0.01), whereas the prevalence of house dust mite (HDM) sensitization was not statistically different (26.3% in CU/As- and 36.7% in CU/As+). However, in chronic urticaria patients with RCBA, 53.8% of CU/As- and 57.9% of CU/As+ patients differed in the prevalence of HDM sensitization compared to the control group (33.5%, P = 0.03), whereas no difference could be stated for PMD sensitization. Compared to RCBA patients, both CU/As+ and CU/As- patients have a higher clinically relevant sensitization rate against HDM, thus displaying a differential atopy phenotype.

Encephalitozoon cuniculi: implementation of a new fluorimetric method for the detection of anti-microsporidia antibodies.

A new fluorimetric method for the diagnosis of microsporidia was compared to the indirect immunofluorescence (IIF) method. Plates were coated with Encephalitozoon cuniculi spores, sera were incubated and an anti-human IgG FITC-conjugate was added. Finally, the plates were read using a fluorimeter. The results obtained were compared using the IIF technique confirming the positive sera with Fluorescence Index (FI) values of 3.75 and 5.24 in the fluorimetric method. Sera with FI values of 2.03 and 2.35 had borderline results when the IIF technique was used. The present results confirm the usefulness of fluorimetric methods in the diagnosis of human microsporidia, both in cases of the absence of immunodeficiency as well as in epidemiological studies.

Prophylaxis with ketotifen in rats with portal hypertension: involvement of mast cell and eicosanoids.

BACKGROUND: Since we have previously shown an increase of mast cells in the small bowel and in the mesenteric lymph nodes in the rats with prehepatic portal hypertension, it can be hypothesized that this essential inflammatory cell would be involved in the pathogeny of the splanchnic changes related to portal hypertension. METHODS: To verify this hypothesis, we first studied mast cell infiltration in the ileum and in the mesenteric lymph nodes in sham-operated male Wistar rats (n=12) and in short-term prehepatic portal hypertensive rats (n=12), and the serum levels of rat mast cell protease II (RMCP-II) by ELISA. In a second set of experiments ketotifen, a mast cell stabilizer drug, was administered to sham-operated (n=10) and portal hypertensive (n=12) rats 24 hours before the intervention and prostanoids (PGE2, PGI2, TXB2) and leukotrienes (LTC4, LTB4) were assayed by RIA, mast cell infiltration in the ileum and in the mesenteric lymph nodes and the serum levels of RMCP-II were also studied, to show its effectiveness to prevent the mesenteric alterations produced by the inflammatory mediators released by the mast cell. RESULTS: Forty-eight hours after the intervention RMCP-II (P<0.05), PGE2 (P<0.001) and LTC4 serum levels decreased and mast cell number and RMCP-II levels increased in mesenteric lymph nodes in portal hypertensive rats. Prophylactic administration of ketotifen reduced portal pressure (P<0.001), serum levels of PGE2 (P<0.001) and RMCP-II (P<0.001) in mesenteric lymph nodes. CONCLUSIONS: In acute portal hypertension in the rat, the mast cell translocation from intestinal mucosa to mesenteric lymph nodes, where they are activated and degranulates, would represent a defence mechanism to avoid the activation of an acute and massive inflammatory response in this location. Prophylactic administration of ketotifen is able to reduce the splanchnic inflammatory changes related to acute portal hypertension in the rat.

Mast cell inhibition by ketotifen reduces splanchnic inflammatory response in a portal hypertension model in rats.

Experimental early prehepatic portal hypertension induces an inflammatory exudative response, including an increased infiltration of the intestinal mucosa and the mesenteric lymph nodes by mast cells and a dilation and tortuosity of the branches of the superior mesenteric vein. The aim of this study is to verify that the prophylactic administration of Ketotifen, a stabilizing drug for mast cells, reduces the consequence of splanchnic inflammatory response in prehepatic portal hypertension. Male Wistar rats were used: Sham-operated and with Triple Partial Portal Vein Ligation, which were subcutaneously administered poly(lactide-co-glycolide) acid microspheres with vehicle 24h before the intervention and SO and rats with Triple Partial Portal Vein Ligation, which were administered Ketotifen-loaded microspheres. Around 48h after surgery, the portal pressure was measured; the levels of chymase (Rat Mast Cell Protease-II) were assayed in the superior mesenteric lymph complex and granulated and degranulated mast cells in the ileum and cecum were quantified. Prophylactic administration of Ketotifen reduced portal pressure, the incidence of dilation and tortuosity of the superior mesenteric vein branches, the amount of Rat Mast Cell Protease-II in the superior mesenteric lymph complex and the number of activated mast cells in the cecum of rats with portal hypertension. In summary, the administration of Ketotifen reduces early splanchnic inflammatory reaction in the rat with prehepatic portal hypertension.

Skin prick test of Kudoa sp. antigens in patients with gastrointestinal and/or allergic symptoms related to fish ingestion.

A majority of Kudoa spp. infects the somatic muscle of fish establishing cysts. Previously, elevated humoral responses were detected in BALB/c mice immunised with Kudoa sp. pseudocyst extracts and in BALB/c mice orally inoculated with Kudoa sp. pseudocysts, as well as the presence of anti-Kudoa sp. antibodies in human sera by enzyme-linked immunosorbent assay. The objective of this work was to test Kudoa sp. pseudocyst extracts by the skin prick test. Fifteen patients with gastroallergic and/or allergic symptoms related to fish ingestion were examined. Kudoa sp. pseudocyst extracts were administered (1 mg/ml) on the volar forearm skin. Four of the 15 selected patients were positive to Kudoa sp. extracts. The saline solution negative control did not induce any reaction.

Anisakis simplex: the high prevalence in Madrid (Spain) and its relation with fish consumption.

Anisakiosis is a nematodosis with high prevalence in Spain. In this work we (a) investigated whether a recently introduced ELISA of Anisakis simplex-specific IgE in serum suffers from cross-reactivity with other common allergens; (b) used this assay to obtain an estimate of the prevalence of A. simplex-specific IgE in the population of Madrid; and (c) related positivity to fish consumption habits. No evidence of cross-reactivity between the ELISA and other allergens was found. The prevalence of positivity was 12.4% (11.7% among healthy subjects and 16% among patients with non-digestive non-allergic pathologies). All interviewed subjects reported consumption of uncooked fish (known to be the most likely source of infection); in addition, positivity was more prevalent among subjects who habitually consumed fresh and possibly undercooked fish than among those who generally consumed frozen fish or boiled or baked fish. These results are discussed in relation to the much lower prevalence observed in Galicia (N.W. Spain).

Seroprevalence of anti-Kudoa sp. (Myxosporea: Multivalvulida) antibodies in a Spanish population.

A majority of Kudoa species infect the somatic muscle of fish establishing cysts. Because there is no effective method to detect infected fish without destroying them, these parasitised fish reach the consumer. The elevated humoral responses detected previously by us in BALB/c mice immunised with Kudoa sp. pseudocyst extracts and the high IgG1 and IgE levels induced by the oral administration of Kudoa pseudocysts to BALB/c mice showed the possible immunopathological effects in man from the ingestion of Kudoa-infected fish. In this work, we investigated the seroprevalence of anti-Kudoa sp. antibodies in a Spanish healthy population and the possible association between the manifestation of allergic reactions after fish consumption and the humoral responses to Kudoa sp. antigens. Specific anti-Kudoa sp. antibody levels in sera of patients diagnosed with several digestive pathologies were also determined, studying their possible association with the alteration of analytic parameters in these patients.

ELISA antibody determination in patients with anisakiosis or toxocariosis using affinity chromatography purified antigen.

One of the fundamental aspects of a parasitic infection diagnosis is the use of adequate antigens to develop specific and sensitive immunoassays. This fact is especially complicated in nematode infection cases because of the high cross-reactivity among different parasites in this group. We performed an evaluation of Anisakis simplex antigens purified by affinity chromatography. We used sera from 38 patients diagnosed with Anisakis sensitization and sera from 35 patients with clinical suspicion of visceral larva migrans (VLM). These sera were assayed by the ELISA method against the crude extracts (CEs) and the purified antigens. When the sera from patients diagnosed with Anisakis sensitization were tested against the A. simplex CE, the IgG was the most abundant immunoglobulin. When the A. simplex larval antigens were purified using a column of IgG anti-A. simplex (PAK) or a column of IgG anti-Ascaris suum (PAS) were tested, we observed a higher diminution in the IgG levels, which coincides with the augmentation of the mean values against the "eluted of Ascaris" (EAS antigen). When the IgE was detected, only 18.4% of the sera reacted with the PAS antigen. We have observed that in the purification process of A. simplex antigen by affinity chromatography, the majority of the proteins that produced cross-reactivity against A. suum and Toxocara canis were eliminated.