| The outer membrane of Actinobacillus (Haemophilus) pleuropneumoniae (HPP) serotype 1 (Shope 4074) was separated using the EDTA, lithium chloride-lithium acetate and sarkosyl extraction methods. The sarkosyl method gave the highest yield of outer membrane protein (OMP) while the EDTA method gave the least. Six prominent OMPs were consistently observed in all sarkosyl extracts. Their nomenclature and corresponding molecular weight (approximate) were as follows: A - 71kD, B - 47kD, C - 39kD, D - 36.5kD, E - 29.5kD and F - 17kD. OMPs C, D and E were heat modifiable and A, D and F were resistant to trypsin digestion. Western blots using HPP serotype 1 hyperimmune rabbit and swine sera revealed intense reactivity to major bands corresponding to OMPs A, B, C and D. The {dollar}sp{lcub}125{rcub}{dollar}I radioimmuno-precipitation method extrinsically identified a 39kD (OMP C) and a 17 kD (OMP F) OMP. Intrinsic labeling with {dollar}sp{lcub}35{rcub}{dollar}S methionine identified OMPs with the following molecular weight: 100kD, 94kD, 91kD, 67kD and 39kD (OMP C). All three procedures identified OMP C to have antibody reactivity. The OMPs of HPP serotypes 1 to 7 had similar profiles except at the 35 to 50kD region (variable region). Despite difference in OMP patterns, Western blots probed with heterologous antiserum revealed cross reactivity amongst the OMP antigens of the seven A.(H.) pleuropneumoniae serotypes. Serotype specificity of high molecular weight non-protein antigens were demonstrated for all. Immunologic cross-reactivities were also demonstrated between A. (H.) pleuropneumoniae serotype 1 (App 1), Haemophilus minor strain 202 (202), H. parasuis (Hps), Pasteurella multocida serotype A (PmA) and serotype D (PmD) antigens using both the ELISA and Western blot methods. The cross reaction studies had strengthened Pohl et al's (1981) clarification of relationships among members of Haemophilus, Pasterurella, and Actinobacillus. This relatedness was most observed especially when B. bronchiseptica OMPs were rarely recognized by antisera against the other bacterial respiratory pathogens. |
