Analysis by one-dimensional (1-D) SDS-PAGE/Western blotting of whole mite extract (larval and adult stages) with sheep sera taken 0-84 days after infection with the sheep scab mite, Psoroptes ovis revealed a progressive IgE antibody response, with a dominant high molecular weight allergen (MW 180 kDa) detected early during infection. Further analysis by two-dimensional (2-D) SDS-PAGE/Western blotting with post-infection sera, revealed a more complex picture with numerous (> 20) IgE reactive spots. Trypsin digest and Maldi-ToF analyses of these spots identified two house dust mite allergen homologues, namely tropomyosin (Der p 10) and paramyosin (Der p 11), and analysis of a third spot indicated an apolipoprotein-like IgE reactive protein (Der p 14). Further 1-D and 2-D SDS/Western blotting, with specific antibodies to the house dust mite allergens Der p 10, 11, and to the IgE reactive peptide of the high molecular weight house dust mite allergen, Der p 14 (vitellogenin/apolipophorin), provided firm evidence for the presence of these three allergens in extracts of the Psoroptes mite. These studies show for the first time that homologues of the house dust mite 10, 11 and 14 group allergens are represented as immunodominant allergens of the sheep scab mite, and may represent important vaccine candidates.
Infestation of sheep with the ectoparasitic mite Psoroptes ovis, results in a severe allergic dermatitis. Currently, little is known about the allergens/antigens that stimulate the allergic response. We have isolated an 836-bp cDNA from a P. ovis cDNA library which displays strong homology to cysteine proteases and, in particular, to the group I house dust mite allergens Der p 1, Der f 1 and Eur m 1. The cDNA was expressed in Escherchia coli, fused to a hexahistidine tag and the recombinant protein (Pso o 1) purified using a nickel-affinity column. The recombinant Pso o 1 was tested for recognition by immunoglobulin (Ig)G and IgE in serum from P. ovis naïve and P. ovis infested sheep. Using Western blots, both classes of antibody to Pso o 1 were detected in postinfestation serum. In enzyme-linked immunosorbent assays, a pronounced IgG-antibody response to Pso o 1 was detected in five of five sheep 3 weeks postinfestation. The IgE-antibody response to whole mite extract was poor in four of five animals. However, a marked IgE response occurred in the fifth animal, and IgE anti Pso o 1 was detected in the serum.
Five tests for antibodies against chlamydial (enzootic) abortion of ewes were compared using 255 sera from experimentally (group 1) or naturally (group 2) infected animals, flocks free of the disease (group 3) and individual animals testing positively by the complement fixation test but from flocks with no evidence of chlamydial abortion (group 4). Sera from five specific pathogen-free lambs vaccinated with two different subtypes of Chlamydia pecorum were also included (group 5). All tests used some form of processed culture of C psitiaci as antigen. Specificities, established with group 3 and 4 sera, ranged between 96 per cent (ELISA using lipopolysaccharide antigen) and 59 per cent (Immunocomb). Reactions with group 5 sera suggested that the cause of false positive results in the field might be cross-reactive antibodies against the arthritogenic subtype of C pecorum. Sensitivities, established with groups 1 and 2 sera, ranged between 81 per cent (Immunocomb) and 51 per cent (ELISA using solubilised protein antigen). The minimum sample sizes required to be 95 per cent certain of detecting at least five seropositives in two infected flocks (combined data) were 15 to 48, dependent on the test applied. The Western blot test, applied to a proportion of samples, yielded no false positives with group 3 sera but 31.7 per cent with group 4 sera. Thus, none of the tests in this comparison emerged as sufficiently satisfactory in all respects, suggesting that further improvements in chlamydial serology must come through the use of non-native antigens or in the form of a competitive ELISA.
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