Blastomyces dermatitidis: Antigenic evaluation of yeast phase lysates

The immunodiagnosis of blastomycosis has been hindered due to the fact that other dimorphic fungal organisms like H. capsulatum cross-react. In the competitive ELISA experiment six B. dermatitidis yeast phase lysates were evaluated for their efficacy as reagents for the detection of B. dermatitidis antigens in urine specimens from 170 dogs with confirmed blastomycosis. Differences between dogs were expected. However, differences between the lysates used in the ELISA brought forth the question of strain variation which may be explained by differing antigenic profiles of the lysates and the potential to purify and separate the reactive antigens from those that cross-react with H. capsulatum.; Isoelectric focusing (IEF) was used to address this question. In the initial study a B. dermatitidis isolate was focused and fractions were evaluated with an indirect ELISA to determine if reactive fractions could be separated from fractions containing antigens cross-reactive with H. capsulatum. The results indicated that separation was possible, which led to the next study to examine the immune response produced by rabbits when immunized with the fractions.; Four B. dermatitidis isolates were evaluated in the vaccination study. The reactive fractions were used to immunize rabbits. All of the rabbits immunized with the 592 IEF fractions produced the classic primary and secondary immune response whereas the other isolates produced a variable response. Isolate 394 did not appear to be effective at eliciting an immune response.; The proteins used as immunogens along with their corresponding crude lysates were evaluated with SDS-PAGE and Western blotting. More protein bands were observed in the IEF fractions than in their respective lysates as the focusing concentrated the proteins.; The IEF fractions were not as effective as compared to the crude lysates when used to detect B. dermatitidis antibodies, except for the IEF 48938 fractions. Some of the reactive antigens which did not focus at the same isoelectric point (pI) were lost, reducing sensitivity when compared to crude lysates containing all of the antigens. The IEF 48938 fractions exhibited the most cross-reactivity when tested against the H. capsulatum indicating that proteins within this pI are still cross-reactive and further purification is warranted.