RESUMO
The lungworm Dictyocaulus viviparus is one of the most economically important bovine parasites in temperate climate regions. Following infection, D. viviparus induces a temporary protective immunity, and a vaccine based on attenuated, infective larvae is commercially available. However, due to several disadvantages of the live vaccine, the development of a recombinant subunit vaccine is highly desirable. Therefore, the major sperm protein (MSP), which is essential for the parasite's reproduction, was tested as a recombinantly Escherichia coli-expressed glutathione-S-transferase (GST)-fused vaccine antigen in immunization trials with two different adjuvants, Quil A and Al(OH)3. Calves (N = 4 per group) were immunized on study day (SD) 0, 21 and 42 and given a challenge infection on SD 63-65. The two control groups received only the respective adjuvant. Based on geometric means (GM), a 53.64% reduction in larvae per female worm was observed in the rMSP Quil A group vs. its control group (arithmetic means (AM): 54.43%), but this difference was not statistically significant. In the rMSP Al(OH)3 group, the mean number of larvae per female worm was even higher than in the respective control group (GM: 9.24%, AM: 14.14%). Furthermore, male and female worm burdens and the absolute number of larvae did not differ significantly, while the Al(OH)3 control group harbored significantly longer worms than the vaccinated group. Vaccinated animals showed a rise in rMSP-specific antibodies, particularly IgG and its subclass IgG1, and the native protein was detected by immunoblots. Although rMSP alone did not lead to significantly reduced worm fecundity, it might still prove useful as part of a multi-component vaccine.
RESUMO
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.
