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An optimised enzyme-linked immunosorbent assay (ELISA) for the detection of Dictyocaulus viviparous-specific antibodies was developed and evaluated following the testing of various microtitration plates and anti-bovine Ig-conjugates. Based on recombinant major sperm protein (MSP) expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli, sera collected from 112 cattle experimentally infected with D. viviparus, from 129 helminth-naïve calves, 8 calves experimentally infected with Ostertagia ostertagi, and 2 calves infected with Cooperia oncophora were tested. ELISA results showed a calculated specificity and sensitivity as well as positive and negative predictive values of >99%. No cross-reactions with sera from calves infected with O. ostertagi or C. oncophora were observed. Lungworm-specific immunoglobulins were first detected from 28 to 35 days post-infection onwards. To differentiate between antibody-binding to the MSP-part or the GST-part of the fusion protein, additional ELISAs were performed using pure recombinant MSP or GST. Optical densities obtained from the ELISAs with the MSP showed a similar pattern to optical densities measured in the ELISAs with the fusion protein, whereas GST gave only a low background. By testing serum samples from naturally infected calves, it was found that the MSP-ELISA is positive even for sera from calves showing very low faecal larval counts. Thus, we conclude that the ELISA using the recombinant MSP-fusion protein appears to be a suitable method for routine diagnosis and epidemiological studies of cattle lungworm.