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For the diagnosis and the identification of C. oncophora, D. viviparus and F. hepatica, coproscopical, molecular and serological techniques are available. For standard diagnosis, mainly coproscopical and serological methods are currently used. The sensitivity and specificity of the different techniques are extremely variable. Serological assays have the advantage of their implementation as herd health monitoring tools. All currently available assays are only capable of diagnosing Abs against one parasite and particularly serological assays are often limited in their specificity due to the use of complex crude or ES Ags.
A high-throughput multiplex fluorescence immunoassay was successfully developed for the simultaneous detection of Abs produced against C. oncophora, D. viviparus and F. hepatica in bovine serum and milk (individual and BTM) samples. It is characterised by low costs and time, high reproducibility and more importantly, by high sensitivity and specificity, due to the use of recombinant Ags. The platform was furthermore characterised by absence of cross-reactivity with other important GI nematodes of livestock (e.g. H. contortus, O. ostertagi and T. colubriformis) when examined with positive sera from experimentally infected calves. Future examination of positive control serum samples from animals specifically mono-infected with other parasite species, such as Taenia saginata, Paramphistomum cervi etc. is envisaged.
The conduction of one assay (in a 96-well format) can be performed in three hours, which is less or similar than those times usually needed for the performance of a classical ELISA. This multi-plex, multi-well format allows performance of large-scale epidemiological studies. Validation of the newly developed platform for both kinds of samples was performed using established, commercially available ELISAs that have been widely applied in the field. Correlation between the assays was good, especially when the same recombinant Ags were used.
Since this platform is modular, it on one hand enables inclusion of other parasite species e.g. O. ostertagi, H. contortus, important biomarkers and herd health parameters in the future or on the other hand exclusion of individual target species for farms where these species are irrelevant in order to limit the costs. In particular, inclusion of O. ostertagi should be a major aim for the future, though recombinant Ag suitable for diagnosis are currently not available.
Finally, the newly developed serum triplex Luminex assays was used to analyse field serum samples collected in Denmark, Switzerland and Poland. The results obtained show no or low rates of lungworm infection in all three countries. C. oncophora appears in higher rates in Poland in comparison to Denmark and Switzerland. Additionally in Poland, increased levels of liver fluke were detected.
The first use of this technique in a field survey following the collection of BTM samples from four European countries (Belgium, Germany, Ireland and Poland) revealed generally high exposure to C. oncophora and considerably less exposure to D. viviparus. For F. hepatica the situation was slightly different with two countries (Ireland and Poland) showing high exposure rates and the other two more moderate Further optimisation of the assay (e.g. determination of positive cut-off values), ones. the inclusion of more paired samples from endemic and non-endemic areas is envisaged in the future.