Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens.

In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.

Mokhtarian K ，Akhlaghi L ，Meamar AR ，Razmjou E ，Manouchehri Naeini K ，Gholami S ，Najafi Samei M ，Falak R ... -  《-》

Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens.

Current methods for the serodiagnosis of sheep fascioliasis show suboptimal sensitivity, specificity, or both. With the aim of developing an improved method, we fractionated native Fasciola hepatica excretory-secretory antigens (ESAs) by size-exclusion FPLC (fast protein liquid chromatography) on a Superdex 75 HR 10/30 column and then tested the serodiagnostic value of the antigens contained in each one of the 4 peaks obtained (peaks I-IV). Serodiagnostic value was assessed using sera from sheep naturally infected with F. hepatica (group A); sera from the individuals of a fluke-free herd (most of which also had other intestinal nematodes, lung nematodes, Moniezia spp., and/or Cysticercus tenuicollis) sera from a fluke-free herd (group B); sera from lambs experimentally infected with 10-40 F. hepatica metacercariae (group C); and sera from uninfected control lambs (group D). Enzyme-linked immunosorbent assay (ELISA) with peak I or II as target antigens (and to a lesser extent with peak III as target) showed reactivity with negative sera, so that it was not possible to establish cutoff values discriminating infected and uninfected animals. In contrast, when peak IV was used as target, a low cutoff value of 0.235 optical density units (mean + 4 SD) discriminated infected and uninfected animals, with 100% sensitivity and 100% specificity. ELISA with peak IV as a target identified infected animals (even animals that had received only 10 metacercariae) within 3-5 wk of infection and subsequently throughout the rest of the 14-wk monitoring period. In Western blotting analysis, again only the antigens contained in peak IV (range 7-40 kDa, under reducing conditions) were specific for diagnosis of infected animals. These results indicate that molecular sieving of F. hepatica ESAs by this procedure is a fast, simple, reproducible way of obtaining antigens useful for serodiagnosis of sheep fascioliasis.

Mezo M ，González-Warleta M ，Ubeira FM 《JOURNAL OF PARASITOLOGY》

Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis.

Mokhtarian K ，Meamar AR ，Khoshmirsafa M ，Razmjou E ，Masoori L ，Khanmohammadi M ，Akhlaghi L ，Falak R ... -  《-》

The use of MM3 monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis.

This study reports a new capture ELISA (MM3-SERO) for the serodiagnosis of sheep fascioliasis, based on the use of the monoclonal antibody (mAb) MM3. Like our previously reported indirect ELISA method, based on the use of a FPLC-purified fraction (fraction IV) of the Fasciola hepatica excretion/secretion antigens (ESAs), this new test was able to detect animals infected with very small numbers of metacercariae (5-40) and showed no cross-reaction with sera from sheep infected with other parasites, i.e., Moniezia spp., Cysticercus tenuicollis, and Dicrocoelium dendriticum. In contrast with these 2 methods, some sera (mainly those obtained from animals infected with D. dendriticum) showed high reactivities in indirect ELISA with whole F. hepatica ESAs used as control. Interestingly, the MM3-SERO ELISA has a better signal-to-noise ratio than the fraction-IV ELISA, thus allowing detection of seroconversion in infected sheep on average 1 wk earlier (3.2 +/- 0.4 wk postinfection [PI] for MM3-SERO ELISA vs. 4.2 +/- 0.9 wk PI for fraction IV ELISA). Moreover, the antibody response detected with MM3-SERO ELISA was more uniform, with seroconversion always occurring at 4 wk PI in sheep with 1-2 flukes and at 3 wk PI in sheep with more than 2 flukes. The MM3-SERO ELISA was also used to evaluate the kinetics of antibody response against MM3-recognized antigens in sera from sheep experimentally infected with F. hepatica and then treated with triclabendazole. Our results showed that antibody levels dropped by about 25% during the 4-wk observation period following the flukicide treatment, whereas they remained invariably high in all sheep left untreated. We conclude that the MM3-SERO ELISA is a 100% sensitive and 100% specific test for the early serodiagnosis of sheep fascioliasis. Preliminary studies in our laboratory seem to indicate that this method may also be useful for the determination of anti-F. hepatica antibodies in serum and milk of other ruminants. A commercial version of MM3-SERO is currently available from BIO X Diagnostics (La Jemelle, Belgium).

Mezo M ，González-Warleta M ，Ubeira FM 《JOURNAL OF PARASITOLOGY》

Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis.

Martínez-Sernández V ，Perteguer MJ ，Hernández-González A ，Mezo M ，González-Warleta M ，Orbegozo-Medina RA ，Romarís F ，Paniagua E ，Gárate T ，Ubeira FM ... -  《-》