Background:The mechanisms involved in heme handling in trematodes are poorly understood. Results: The biochemical and functional characteristics of a new family of small proteins (MF6p/FhHDM-1) secreted by Fasciola and other trematodes are reported. Conclusion:The Fasciola MF6p/FhHDM-1 major antigen is a heme-binding protein. Significance:Our results provide new insights into the biology of hematophagous trematodes.

ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1–10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis.

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BackgroundHuman fasciolosis is a re-emerging disease worldwide and is caused by species of the genus Fasciola (F. hepatica and F. gigantica). Human fasciolosis can be diagnosed by classical coprological techniques, such as the Kato-Katz test, to reveal parasite eggs in faeces. However, although 100% specific, these methods are generally not adequate for detection of acute infections, ectopic infections, or infections with low number of parasites. In such cases immunological methods may be a good alternative and are recommended for use in major hospitals where trained personnel are available, although they are not usually implemented for individual testing.Methodology/Principal FindingsWe have developed a new lateral flow test (SeroFluke) for the serodiagnosis of human fasciolosis. The new test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO) the SeroFluke test showed maximal specificity and sensitivity and can be used with serum or whole blood samples.Conclusions/SignificanceThe new test can be used in major hospitals in hypoendemic countries as well as in endemic/hyperendemic regions where point-of-care testing is required.

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein ( apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as and, also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme.

BackgroundThe main objective of the study was to determine the degree of sensitization to Anisakis spp. antigens in healthy coastal population of Dalmatia given the high thermally unprocessed fish intake rate present in this area, suggested as a significant risk factor for anisakiasis. We performed a monocenter, cross-sectional pilot study stratified by geographic area of residence, conducted at the County secondary healthcare provider Medicine-biochemical Laboratory in Split (Croatia), from November 2010 till December 2011, on 500 unpaid volunteer subjects undergoing routine blood analysis and belonging to the south coast of the Adriatic Sea.Methodology/Principal FindingsWe studied the IgE seroprevalence to Anisakis spp. Ani s l and Ani s 7 allergens by indirect ELISA in healthy subjects, which were selected at random in the region of Dalmatia (Southern Croatia), among islands, coastal urban and inland rural populations. In order to detect possible cross-reactivity to other human helminthes, serum samples were tested also for the presence of IgG antibodies to Ascaris lumbricoides and Toxocara canis. The overall and coastal Anisakis seroprevalences for the sampled population were 2% and 2.5%, respectively. The logistic univariate regression analysis confirmed that regarding anti-Anisakis IgE seroprevalence, raw fish intake, daily fish intake, homemade origin of fish dish and occupational contact (professional, artisanal or hobby contact with fishery or fish industry) were risk factors associated to Anisakis spp. sensitization, but neither of the variables was exclusive for a particular seropositive population. Also, a significant difference was observed between seropositive and seronegative subjects that had stated allergy or symptoms associated with allergy (atopic dermatitis, asthma or rhinitis) in their previous history.Conclusions/SignificanceBeing the first in Croatia, our study underlines the necessity of incorporating Anisakis spp. allergens in routine hypersensitivity testing of coastal population.

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