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》

An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3).

A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.

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

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》

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 ... -  《-》

MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica.

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4-7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3-6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33-66 adults), while it was for F. gigantica infection (burden of 17-69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.

Valero MA ，Ubeira FM ，Khoubbane M ，Artigas P ，Muiño L ，Mezo M ，Pérez-Crespo I ，Periago MV ，Mas-Coma S ... -  《VETERINARY PARASITOLOGY》