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.

In vitro culture of Parascaris equorum larvae and initial investigation of parasite excretory-secretory products.

Currently, diagnosis of Parascaris equorum infection in equids is limited to patent infections. The goals of this study were to culture P. equorum larvae in vitro and identify excretory-secretory (ES) products for prepatent diagnostic testing. Parascaris equorum L2/L3 larvae were hatched and cultured for up to 3 weeks for ES product collection. Fifth stage (L5) P. equorum were also cultured for ES product collection. Examination of ES fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain revealed L2/L3 products ranging from 12-94 kDa and L5 products ranging from 12-189 kDa. Western blot analyses were conducted using polyclonal antibodies produced against P. equorum or Baylisascaris procyonis L2/L3 ES products, sera from rabbits inoculated with B. procyonis or Toxocara canis eggs, and sera from animals naturally infected with P. equorum or T. canis. Western blot results indicated parasite antigens migrating at 19 and 34 kDa may be useful for specifically detecting P. equorum infections.

Contracaecum bioccai n. sp. from the brown pelican Pelecanus occidentalis (L.) in Colombia (Nematoda: Anisakidae): morphology, molecular evidence and its genetic relationship with congeners from fish-eating birds.

Contracaecum bioccai n. sp. is described from the brown pelican Pelecanus occidentalis (L.) in northern Colombia (Totumo Marsh) based on 20 enzyme loci studied using multilocus allozyme electrophoresis. Moreover, genetic relationships between the new taxon and related congeners are presented based on allozyme data-sets and sequence analyses (519 bp) of the mtDNA-cox2 gene. Fixed allele differences were found at some of the allozyme loci analysed in comparison with other Contracaecum spp. from pelicans and cormorants [i.e. the sibling species of the C. rudolphii Hartwich, 1964 complex, C. septentrionale Kreis, 1955, C. micropapillatum (Stossich, 1890), C. microcephalum (Rudolphi, 1809) and C. pelagicum Johnston & Mawson, 1942]. The genetic distance, at the allozyme level, between C. bioccai n. sp. and its congeners ranged from D ( Nei ) = 0.80 versus C. septentrionale to D ( Nei ) = 1.40 versus C. micropapillatum. The genetic distance at the mtDNA cox-2 level ranged, on average, from K-2P = 0.12 versus the C. rudolphii species complex to K-2P = 0.15 versus C. micropapillatum. An overall concordant tree topology, obtained from UPGMA and NJ tree analyses inferred from allozyme data, as well as from MP, UPGMA and NJ inferred from mtDNA-cox2 sequence analysis, showed C. bioccai n. sp. as a separated lineage to the other Contracaecum spp. A concordant result was also obtained by PCA analysis based on both the allozyme and mtDNA cox-2 data-sets. All of the tree topologies, derived from the phylogenetic analysis inferred from both allozymes and mtDNA data-sets, were in substantial agreement and depicted C. bioccai as closely related to the sibling species of the C. rudolphii complex (C. rudolphii A and C. rudolphii B) and C. septentrionale. Morphological analysis and a differential diagnosis based on male specimens of C. bioccai, which had been genetically characterised by both allozyme markers and mtDNA sequences analysis with respect to morphologically related congeners, enabled the detection of differences in a numbers of characters, including spicule length, the morphology of the distal end of the spicule and the distribution patterns of the distal caudal papillae.

Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: ascaridoidea) defined by polymerase-chain-reaction-based restriction fragment length polymorphism.

Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction endonucleases used (HinfI and TaqI), between A. simplex sensu stricto and A. simplex C with one endonuclease (HhaI), and between A. simplex C and Aniskis pegreffii with three endonucleases (HhaI, HinfI and TaqI), while no variation in patterns was detected among individuals within each species. The species A. physeteris, A. schupakovi, A. typica and A. ziphidarum were found to be different from each other and different from the three sibling species of the A. simplex complex by distinct fragments using 10-12 of the endonucleases tested. The polymorphisms obtained by restriction fragment length polymorphisms have provided a new set of genetic markers for the accurate identification of sibling species and morphospecies.

Distribution of cobalt in parasitic helminths.

The distribution of cobalt in parasitic helminths belonging to the trematodes, cestodes or nematodes was determined by the use of an atomic absorption spectrophotometer. The results of these analyses have demonstrated that growing trematodes (smaller forms) with active oogenesis and spermatogenesis contained more cobalt that older forms (large or very old adults) with empty uteri and large lobulated testes. In cestodes the neck region of cysticerci and immature proglottids of adults showed more cobalt than the cyst portion of cysticerci and hydatid or mature and gravid proglottids of worms. Similarly, the youngest endogenous daughter cysts of Echinococcus granulosus showed more cobalt in their walls than those of larger forms. The element was found more concentrated in nematode eggs than in adult females, irrespective of species of host.