Comparative analysis of excretory-secretory antigens of Anisakis simplex, Pseudoterranova decipiens and Contracaecum osculatum regarding their applicability for specific serodiagnosis of human anisakidosis based on IgG-ELISA.

Serodiagnosis of human anisakidosis is presently hampered by the current lack of standardised serological assays that allow sensitive and specific detection of Anisakidae-specific antibodies in human patients. In the present study, we comparatively evaluated the diagnostic value (by IgG-ELISA) of excretory-secretory antigens (ESAgs) of Anisakis simplex, Pseudoterranova decipiens and Contracaecum osculatum, representing the most frequently found genera responsible for human infection. In addition, we tested also a mix of the three ES preparations (Mix-ESAgs) as well as two recombinant allergens of A. simplex, rAni s 1 and rAni s 7. ES antigen from C. osculatum yielded the best diagnostic performance in IgG-ELISA-based serodiagnosis of the Spanish anisakidosis patients investigated in this study (relative serodiagnostic sensitivity 100%; specificity 89%) as compared to A. simplex ES-antigen (93% versus 57%) and P. decipiens (67% versus 93%) or a mix of the three ES antigens (100% versus 44%), respectively. Cross-reactions of C. osculatum ES antigen with serum-antibodies from patients suffering from other helminth infections were rare and were exclusively found with few sera from toxocariasis, ascariasis, and filariasis patients. The two recombinant allergens rAni s 1 and rAni s 7 did not prove sufficiently sensitive and specific in order to justify a further evaluation of these antigens regarding their suitability in IgG-ELISA-based serodiagnosis of human anisakidosis. In conclusion, the C. osculatum-ESAg-ELISA remains as key candidate to be further assessed for the serodiagnosis of symptomatic anisakidosis in different endemic regions.

Baylisascaris procyonis Roundworm Seroprevalence among Wildlife Rehabilitators, United States and Canada, 2012-2015.

Baylisascaris procyonis roundworms can cause potentially fatal neural larva migrans in many species, including humans. However, the clinical spectrum of baylisascariasis is not completely understood. We tested 347 asymptomatic adult wildlife rehabilitators for B. procyonis antibodies; 24 were positive, suggesting that subclinical baylisascariasis is occurring among this population.

Potential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice.

The intestinal nematode Baylisascaris schroederi is an important cause of death for wild and captive giant pandas. Inorganic pyrophosphatases (PPases) are critical for development and molting in nematode parasites and represent potential targets for vaccination. Here, a new PPase homologue, Bsc-PYP-1, from B. schroederi was identified and characterized, and its potential as a vaccine candidate was evaluated in a mouse challenge model. Sequence alignment of PPases from nematode parasites and other organisms show that Bsc-PYP-1 is a nematode-specific member of the family I soluble PPases. Immunohistochemistry revealed strong localization of native Bsc-PYP-1 to the body wall, gut epithelium, ovary and uterus of adult female worms. Additionally, Bsc-PYP-1 homologues were found in roundworms infecting humans (Ascaris lumbricoides), swine (Ascaris suum) and dogs (Toxocara canis). In two vaccine trials, recombinant Bsc-PYP-1 (rBsc-PYP-1) formulated with Freund complete adjuvant induced significantly high antigen-specific immunoglobulin (Ig)G but no IgE or IgM responses. Analysis of IgG-subclass profiles revealed a greater increase of IgG1 than IgG2a. Splenocytes from rBsc-PYP-1/FCA-immunized mice secreted low levels of T helper (Th)1-type cytokines, interferon-γ and interleukin (IL)-2, while producing significantly high levels of IL-10 and significantly elevated levels of IL-4 (Th2 cytokines) after stimulation with rBsc-PYP-1 in vitro. Finally, vaccinated mice had 69.02-71.15% reductions (in 2 experiments) in larval recovery 7 days post-challenge (dpc) and 80% survival at 80 dpc. These results suggest that Th2-mediated immunity elicited by rBsc-PYP-1 provides protection against B. schroederi, and the findings should contribute to further development of Bsc-PYP-1 as a candidate vaccine against baylisascariasis.

Proteomic Profiling Reveals New Insights into the Allergomes of Anisakis simplex, Pseudoterranova decipiens, and Contracaecum osculatum.

Anisakis simplex, Pseudoterranova decipiens, and Contracaecum osculatum third-stage larvae (L3) are fish-borne nematodes that can cause human anisakidosis. Although A. simplex is a known source of allergens, knowledge about the allergic potential of P. decipiens and C. osculatum is limited. Therefore, we performed comparative proteomic profiling of A. simplex, P. decipiens, and C. osculatum L3 larvae using liquid chromatography-tandem mass spectrometry. In total, 645, 397, and 261 proteins were detected in A. simplex, P. decipiens, and C. osculatum L3 larvae, respectively. Western blot analysis confirmed the cross-reactivity of anti-A. simplex immunoglobulin (Ig)G antibodies with protein extracts from P. decipiens and C. osculatum L3 larvae. The identified proteins of the Anisakidae proteomes were characterized by label-free quantification and functional analysis, and proteins involved in many essential biological mechanisms, such as parasite survival, were identified. In the proteome of A. simplex 14, the following allergens were identified: Ani s 1, Ani s 2 (2 isomers), Ani s 3 (2 isomers), Ani s 4, Ani s 8, Ani s 9, Ani s 10, Ani s 11-like, Ani s 13, Ani s fructose 1,6-bisphosphatase, Ani s phosphatidylethanolamine-binding protein (PEPB), and Thu a 3.0101. The following 8 allergens were detected in P. decipiens: Ani s 2, Ani s 3 (2 isomers), Ani s 5, Ani s 8, Ani s 9, Ani s PEPB, and Ani s troponin. In C. osculatum 4, the following allergens were identified: Ani s 2, Ani s 5, Ani s 13, and Asc l 3. Furthermore, 28 probable allergens were predicted in A. simplex and P. decipiens, whereas in C. osculatum, 25 possible allergens were identified. Among the putative allergens, heat shock proteins were most frequently detected, followed by paramyosin, peptidyl-prolyl cis-trans isomerase, enolase, and tropomyosin. We provide a new proteomic data set that could be beneficial for the discovery of biomarkers or drug target candidates. Furthermore, our findings showed that in addition to A. simplex, P. decipiens and C. osculatum should also be considered as potential sources of allergens that could lead to IgE-mediated hypersensitivity.

Antigen detection: Insights into Toxocara and other ascarid infections in dogs and cats.

Detection of ascarid excreted or secreted (E/S) molecules is an alternative approach to the identification of infection by egg flotation. E/S molecules serve as direct markers for the ascarid nematode commonly found in cats and dogs (Toxocara spp., Toxascaris leonina and Baylisascaris procyonis). The nematode derived E/S material mixes with the intestinal contents of the host animal and is available for detection as a coproantigen in the host's faeces. Antigen capture immunological techniques allow sensitive coproantigen detection. Different patterns of antigen to egg agreement are demonstrated in an experimental Toxocara canis infection throughout the prepatent, patent, and post-treatment phases. Examination of faecal samples from a large field population of dogs and cats tested for both egg shedding and antigen indicates that more infections were identified by antigen. Host age influences the agreement of antigen and Toxocara egg results. Older dogs and cats were less likely to have a patent infection (egg positive and antigen positive) result pattern. An egg observation in the absence of antigen detection may indicate a spurious egg. The impact of spurious eggs was further examined by comparisons of cohorts of dogs separated by presence or absence of a pseudoparasite observation or by egg semi-quantification bin. Lastly, the antigen to egg agreement was calculated for other ascarid species.

Treatment of larva migrans syndrome with long-term administration of albendazole.

BACKGROUND: Larva migrans syndrome is a food-borne parasitic disease in humans, caused by accidental ingestion of eggs or larvae of ascarid nematodes, namely, Toxocara canis, Toxocara cati, or Ascaris suum, the roundworms commonly found in the intestines of dogs, cats and pigs respectively. When a patient is diagnosed as having larva migrans syndrome, oral-administration of albendazole is recommended, however, the regimen remains controversial worldwide. In Japan, the duration of albendazole administration is longer than those of European and North American countries. The purpose of this study was to assess the efficacy and safety of long-term administration treatment of albendazole for larva migrans syndrome. METHODS: From 2004 to 2014, our laboratory was involved in the diagnosis of 758 larva migrans syndrome cases, of which 299 cases could be followed up after the treatment. We analyzed these 299 follow-up cases on the ELISA results before and after the treatment as well as on anthelmintic used, dose and duration of medication, clinical findings, and side effects, recorded on a consultation sheet provided by the attending physicians. We have 288 cases as the subjects of this study. RESULTS: Albendazole represented a 78.0% efficacy rate. The side effects represented 15.0% in using albendazole alone cases; however, the side effects were mild to moderate and there were no severe cases reported. CONCLUSIONS: The long-term administration treatment of albendazole is safe and effective for larva migrans syndrome.

Baylisascariasis: A young boy with neural larva migrans due to the emerging raccoon round worm.

A 17-month-old boy from Vancouver, Canada, presented with a 5-day history of progressive somnolence, ataxia, and torticollis. Additional investigations revealed eosinophilic encephalitis with deep white matter changes on MR imaging. On day 13, serology came back positive for Baylisascaris procyonis antibodies. While prophylaxis after ingestion of soil or materials potentially contaminated with raccoon feces can prevent baylisascariasis, timely treatment can sometimes alter a disastrous outcome. Populations of infected raccoons are propagating globally, but cases of Baylisascaris neural larva migrans have so far only been reported from North America.

Lagochilascaris minor: experimental infection of C57BL/6 and BALB/c isogenic mice reveals the presence of adult worms.

The nematode Lagochilascaris minor is the causative agent of lagochilascariosis, a human disease that affects the neck region causing exudative abscesses with eggs, larvae, and adult parasites. Mice are currently considered intermediate hosts for the parasite. To determine the pattern of infection and the possibility of mice as definitive hosts for L. minor, experimental lagochilascariosis was studied in two distinct isogenic mouse strains: BALB/c and C57BL/6. Our results indicate that BALB/c mice are more resistant to L. minor infection than C57BL/6, having less intense lesions in the lungs, a lower number of nodules with encysted larvae and fewer adult worms, and displaying a higher serum level of IFN gamma. Both mouse strains had low levels of serum IL-10. We also observed adult parasites in both mouse strains, raising the possibility that mice are definitive hosts of L. minor. This is the first description of adult parasite development of L. minor in mice.

Detection and Evaluation of Antibody Response to a Baylisascaris-Specific Antigen in Rodent Hosts with the Use of Western Blotting and Elisa.

Diagnosis of parasitic diseases that involve tissue-stage larvae is challenging, and serology remains the most effective antemortem test for detecting these infections. Baylisascaris procyonis, the raccoon roundworm, is a zoonotic ascarid. Raccoons are the usual definitive host, and humans may be infected as accidental hosts. More than 150 species of birds and mammals may act as paratenic hosts, and rodents play an important role in the transmission and maintenance of this parasite in nature. Migratory larvae in paratenic host tissues can produce ocular disease and severe to fatal neurologic disease, but not all infected hosts develop signs. A sensitive and specific Western blot (WB) assay based on a recombinant Baylisascaris-specific antigen (rBpRAG-1) has been developed for use in humans. We evaluated the use of this antigen to detect Baylisascaris spp. infections in rodent paratenic hosts. With the use of 4 species of Peromyscus mice ( Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, Peromyscus polionotus) from a previous infection trial, we developed species-adapted WB and ELISA assays and evaluated performance compared to detection of larvae in tissue samples. These assays revealed species-level differences in seroconversion and terminal antibody concentrations, with P. leucopus developing significantly greater antibody concentrations than P. californicus and P. polionotus at all dose levels, and P. maniculatus at the low dose. Some P. californicus and P. polionotus failed to seroconvert despite the recovery of larvae from their tissues. WB and ELISA results were correlated; however, the WB demonstrated higher sensitivity than the ELISA overall (72.2% versus 63.9%, respectively). With the use of experimental samples, specificity was 100% for WB and 94.1% for ELISA. A WB was also used to test Mus and Rattus samples, and although numbers were too limited to evaluate sensitivity and specificity, all animals known to be infected by tissue digestion were WB positive, and all uninfected animals were negative. Finally, the Peromyscus-adapted WB and ELISA were used to test a set of serum samples from wild-trapped P. maniculatus and Rattus rattus. Both assays were generally sensitive, but specificity was equivocal. This emphasizes the challenge of using serology for investigation of wildlife diseases, in which hosts have unknown exposure histories. Nevertheless, serologic methods have utility in the study of Baylisascaris spp. in paratenic hosts, either wild or captive, and have advantageous attributes (non-lethal, high-throughput), but results should be interpreted carefully.

Human antibody recognition of Anisakidae and Trichinella spp. in Greenland.

High levels of total IgE are observed among children in Greenland. To evaluate the extent to which Anisakidae and Trichinella spp. contribute to the high total IgE level, an ELISA and a western blot were developed for the detection of IgG antibodies to Anisakidae, based on excretory/secretory antigens from Anisakidae larvae. Western blots with Anisakidae and Trichinella antigens discriminated between Anisakidae and Trichinella infections, enabling cross-reactivity between the two parasite infections to be eliminated. Serum samples from 1012 children in Greenland were analysed for specific antibodies to Anisakidae and Trichinella. Eleven children were IgG-positive for Trichinella and nine were IgG-positive for Anisakidae, indicating a relatively low prevalence of both infections among children in Greenland. Faecal samples from 320 children were also examined for other intestinal parasites. Enterobius vermicularis was found in one sample and Blastocystis hominis in 32 samples, but no other intestinal parasites were identified. In total, 304 children had elevated total IgE levels. There was a significant association between Trichinella seropositivity and high levels of total IgE, but not between Anisakidae seropositivity and total IgE. The data indicate that parasitic infections alone do not explain the high level of total IgE observed among children in Greenland.

Allergenic activity of Pseudoterranova decipiens (Nematoda: Anisakidae) in BALB/c mice.

BACKGROUND: Anisakis simplex is the only fishery-product associated parasite causing clinical allergic responses in humans so far. However, other anisakids, due to the presence of shared or own allergens, could also lead to allergic reactions after sensitization. The aim of this study was to determine if Pseudoterranova decipiens belonging to the family Anisakidae has allergenic activity and is able to induce sensitization after oral administration in a murine (BALB/c mice) model. RESULTS: The ingestion of A. pegreffii proteins by BALB/c mice, which had been previously sensitized by intraperitoneal inoculation with the corresponding live L3 larvae, triggers signs of allergy within 60 min, whereas P. decipiens did to a lesser extent. Beside symptoms, allergic reactions were furtherly supported by the presence of histamine in sera of sensitized mice. Specific IgG1 and IgE responses were detected in sera of all sensitized mice from week four. Specific IgG2a response was detected in sera from mice sensitized to P. decipiens. After polyclonal or specific activation with anti-CD3/anti-CD28 or antigens, respectively, splenocytes from mice infected i.p. with A. pegreffii or P. decipiens larvae showed significantly higher production of IL-10 than naïve mice. After stimulation with specific antigens, significantly higher IL-5 and IL-13 amounts were produced by specific antigen stimulated splenocytes than by the naïve cells; only P. decipiens proteins induced IFN-É£. CONCLUSIONS: The overall results suggest that infection with P. decipiens can sensitize mice to react to subsequent oral challenge with anisakid proteins, as described for A. simplex (sensu stricto) and A. pegreffii infections. The results show that anisakid proteins induce a dominant Th2 response, although P. decipiens could also induce a mixed type 1/type 2 pattern.

Immunomodulatory effects of excretory/secretory compounds from Contracaecum osculatum larvae in a zebrafish inflammation model.

Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode Contracaecum osculatum parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins, of which some had enzymatic activity, were injected solely or with LPS. ES proteins alone induced up-regulation of a number of immune-related genes, but generally to a lower degree compared to LPS. When co-injected with LPS, the worm products exacerbated merely expression of five genes affecting Th1, Th2, Th17 and innate responses compared to the LPS-injected group. However, the level of overexpression decreased in an inverse dose-dependent manner. The immune regulating action of C. osculatum ES products is interpreted as an important evolutionary ability of larval parasites in the transport host which makes it less susceptible to host immune responses whereby the probability of reaching the final host is increased.

Equine antibody response to larval Parascaris equorum excretory-secretory products.

Parascaris equorum is an intestinal nematode of foals and young horses that can produce mild to severe pathology. Current diagnosis is limited to detection of patent infections, when parasite eggs are identified during fecal examinations. This study examined the use of larval P. equorum excretory-secretory (ES) products in a western blot test for diagnosis of prepatent equine P. equorum infection. Sera from adult mares negative for patent P. equorum infections, foals prior to consuming colostrum, and P. equorum infected foals were used as controls in this study. Study samples included sera from 18 broodmares prior to parturition and sera from their foals throughout the process of natural infection. Sera from study horses were examined for IgG(T) antibody recognition of ES products. Foals naturally infected with P. equorum possessed IgG(T) antibodies against 19kDa, 22kDa, 26kDa, and 34kDa ES products. However, passive transfer of colostral antibodies from mares was shown to preclude the use of the crude larval ES product-based western blot test for diagnosis of prepatent P. equorum infections in foals.

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

Eye findings of diffuse unilateral subacute neuroretinitis and multiple choroidal infiltrates associated with neural larva migrans due to Bbaylisascaris procyonis.

PURPOSE: To report childhood infection with Baylisascaris procyonis (raccoon round worm) manifesting as diffuse unilateral subacute neuroretinitis (DUSN) and choroidal infiltrates in association with neurologic disease (neural larva migrans). METHOD: Observational case series, one with eye manifestations of DUSN, the other with choroidal infiltrates, both with severe neurologic degeneration. RESULTS: Indirect immunofluorescence assays on serum and cerebrospinal fluid were positive for B. procyonis in one and serially positive and increasing in the other. Both children had a history of pica and raccoon exposure. CONCLUSIONS: Baylisascaris procyonis infection is associated with two cases of severe neurologic degeneration with ocular lesions: DUSN and choroidal infiltrates. Although B. procyonis is known to cause DUSN, these cases indicate that concomitant ocular migration may accompany neural larva migrans. These are the third and forth cases in the US literature of neural larva migrans due to B. procyonis with eye findings.

Determination of IgE in the serum of patients with allergic reactions to four species of fish-parasite anisakids.

This study investigates cross-reactions between somatic and metabolic antigens of various anisakids in the serum of patients with allergic processes. Twenty patients with allergic reactions after eating fish were studied using the skin-prick test for sensitivity to four species of anisakids. IgE was also determined, by blotting, in the serum of these patients when confronted with somatic and metabolic antigens of Anisakis simplex s.l. and Hysterothylacium aduncum, and somatic antigens of A. physeteris and H. fabri. The results obtained with both techniques basically agree, the following facts being of particular note: four patients presented specific IgE for A. simplex s.l. and 1 for Hysterothylacium sp. only, eight patients presented cosensitivity for A. simplex s.l. and A. physeteris, two for A. simplex s.l. and Hysterothylacium sp., one for A. physeteris and Hysterothylacium sp., and two for A. simplex s.l., A. physeteris and Hysterothylacium sp. Given the results obtained, it should be considered that other species of fish-parasite anisakids, apart from A. simplex s.l., may be involved in the allergic reactions presented by a large number of patients.

Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens.

We investigated the occurrence of antibodies against protein antigens of the nematode parasite Pseudoterranova decipiens in the plasma and bile of the Antarctic teleost Trematomus bernacchii. Three different P. decipiens protein solutions were prepared: excreted/secreted proteins from live larvae (ESP); surface-associated proteins obtained by mild extraction of larval bodies (SAP); and cuticular soluble proteins recovered by extraction in strong reducing conditions (CSP). Using different immunoassays, these 3 preparations were tested for their ability to bind fish antibody. As determined by ELISA, the specific antibody binding activity was higher in SAP than in CSP. As determined by dot-blot immunoassay, the specific antigen binding activity versus SAP was higher in bile than in plasma antibodies. A different number of antigenic components of SAP and ESP were identified by immunoblotting performed with plasma or bile antibodies. These results led to the conclusion that T. bernacchii parasitism by nematodes involves plasma and bile anti-parasite antibodies. Furthermore bile antibodies were found to be more reactive and more heterogeneous than plasma.

Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice.

Raccoon roundworms (Baylisascaris procyonis) and other Baylisascaris species cause patent or latent larva migrans (LM) in a variety of mammals and birds, including humans. It is not clear whether LM by Baylisascaris transfuga, roundworms of bears, is associated with clinical neurological disorders. To clarify this issue, ICR and BALB/c mice as well as Mongolian jirds (Meriones unguiculatus) were orally inoculated with 2,000-5,000 embryonated eggs of B. transfuga. In mice, the ascarid caused symptomatic LM of limited extent and duration, whereas the infection was fatal in jirds; i.e., they exhibited general signs such as severe depression and emaciation on days 8-11 postinfection (PI) and died, or they developed progressive and fatal neurological disorders after day 14 PI. Histological examination showed B. transfuga larvae in the brain of all mice and jirds examined, and the larvae collected from them developed to a size comparable with that of B. procyonis. There existed, however, critical differences in host reactions against larvae localized in the brain of mice and jirds; B. transfuga larvae found in mice were surrounded by granulomatous reactions and immobilized, whereas larvae found in jirds were free from any host reaction and mobile, causing extensive malacia.

The development of immunity to Parascaris equorum infection in the foal.

Following infection with 8000 Parascaris equorum eggs in two- to four-week-old foals reared under worm-free conditions a high percentage of the infective dose completed its tissue migration and returned to the small intestine. Patent infections were establisehd between 81 and 104 days after infection and high faecal egg counts were recorded. A group of six- to 12-month-old foals, which had been either reared under worm-free conditions or exposed to natural ascarid and strongyle infections on pasture, received a similar infection of 8000 P equorum eggs. Compared with the younger foals there was a marked reduction in the number of larvae reaching the small intestine. Also, patent infections were established less frequently (50 per cent of cases) and, when present, the faecal egg counts remained low.

Comparison of counterimmunoelectrophoresis with indirect haemagglutination test in the detection of antibodies in rabbits experimentally infected with various species of ascarids.

Counterimmunoelectrophoresis (CIEP) and indirect haemagglutination (IHA) were used for the detection of antibodies in 437 sera of rabbits experimentally infected with Ascaris suum, Toxocara canis and Toxascaris leonina. CIEP showed 44.0-52.3% positivity in A. suum, 25.5% in T. canis and 19.7-29.2% in T. leonina infection, whereas the positivity detected by IHA was 59.8-90.2% in A. suum, 44.8% in T. canis and 59.6% in T. leonina infection. A comparison of the two tests reveals that CIEP is a suitable, rapid and simple method for orientation examinations in ascarid infections.