bovisBCG are two very closely related species (8), proteins homologous to theM

bovisBCG are two very closely related species (8), proteins homologous to theM. assay (ELISA) for KHK-IN-1 hydrochloride paratuberculosis, and such a test was developed (10). Unexpectedly, the rabbit polyclonal antibodies raised against a362 by Coetsier and colleagues also react with some components ofM. bovisBCG (particularly with a 38-kDa protein), but none of their monoclonal antibodies react withM. bovisBCG (2). Whereas the possibility of cross-reactivity between peptide a362 ofM. aviumsubsp.paratuberculosisand someM. boviscomponents could not be rigorously excluded, this unexpected reaction most probably derives from the inadequate immunization protocol. Indeed, the polyclonal antibodies directed against a362 that Coetsier et al. used for a specific histopathological diagnostic test for paratuberculosis were raised in rabbits following two inoculations of a362 emulsified in complete Freunds adjuvant (CFA). CFA is an emulsion of mycobacteria in oil (1). Thus, it is KHK-IN-1 hydrochloride not surprising that antibodies to mycobacterial antigens develop in response to CFA, and this has already been demonstrated in animals Artn immunized with CFA alone (9). Preabsorption of the above-mentioned anti-a362 polyclonal serum with the a362 polypeptide and the disappearance of immunostaining in theM. bovisBCG Western blot would have provided support for the stated cross-reactivity between a362 andM. bovisBCG components. Recently, the complete genome ofM. tuberculosisH37 Rv was sequenced (3). AsM. tuberculosisandM. bovisBCG are two very closely related species (8), proteins homologous to theM. aviumsubsp.paratuberculosis34-kDa protein were searched among those encoded by theM. tuberculosisgenome. As KHK-IN-1 hydrochloride expected from our previous experiments (5), a BLAST search in protein data banks identified one protein ofM. tuberculosisH37 Rv (a 30,225-Da protein under reference Rv 0954 in the classification of Cole et al. [3]) which is usually highly similar to the 34-kDa protein ofM. aviumsubsp.paratuberculosis. Apart from this 30,225-Da protein ofM. tuberculosis, no significant homology was found with any 38-kDa protein such as the one identified inM. bovisBCG by Coetsier and colleagues. Nevertheless, anM. lepraeDNA sequence encoding a protein homologous to the 34-kDa protein ofM. aviumsubsp.paratuberculosiswas recently deposited in GenBank under accession no. U8211. The alignment of the protein identified inM. tuberculosiswith the 34-kDa protein ofM. aviumsubsp.paratuberculosisshows that regions of identical amino acids (Fig.1) are interspersed between regions containing dissimilar amino acids. The carboxyl ends of the two proteins (from 188 to 303) nevertheless seem to be more dissimilar than their amino-terminal parts, as 16 gaps were necessary for their correct alignment (Fig.1). The B-cell epitope(s) specific toM. aviumsubsp.paratuberculosisexpressed in peptide a362 must be present in the regions of dissimilarity (Fig.1). Their exact locations could easily be tested with synthetic peptides corresponding to these regions and the monoclonal antibodies produced by Coetsier and colleagues. == FIG. 1. == Alignment of the amino acid sequences of the 34-kDa protein ofM. aviumsubsp.paratuberculosis(M.para) and of the Rv0954 protein ofM. tuberculosisH37Rv (M.tube). The two homologous proteins were aligned by using the Align program (http://vega.igh.cnrs.fr/bin/align-guess.cgi). Black background indicates regions containing identical amino acids. Arrows indicate locations of potential transmembrane helices as predicted by the TMpred program (http://www.isrec.isb-sib.ch/software/TMPRED_form.html). The sequence corresponding to the a362 peptide is usually boxed. In conclusion, although the work of Coetsier and colleagues proves again that this 34-kDa protein ofM. aviumsubsp.paratuberculosiscontains species-specific B-cell epitopes, and albeit it KHK-IN-1 hydrochloride is highly probable that a similar protein does exist inM. bovisBCG, the fact that B-cell epitope(s) cross-reacting withM. bovisBCG are present in the carboxyl end of the 34-kDa protein ofM. aviumsubsp.paratuberculosis(peptide a362) needs further examination. == Recommendations ==.