Development and evaluation of whole cell and membrane protein vaccines against mycoplasma gallisepticum

Abstract

A study was carried out to develop and evaluate the efficacy of vaccines against M. gallisepticum infections in Gallus domesticus using whole cell and membrane proteins produced under iron sufficient and restricted conditions, incorporating the adjuvants aluminium hydroxide and saponin either alone or in combination. A total of 50 samples were collected in broth and in addition 25 tracheal swabs were streaked onto PPLO agar plates also, from birds showing respiratory ailments. Mycoplasma genus specific PCR revealed twelve positive samples and on subjecting these samples to species specific PCR, a total of four samples showed positive results. From four species specific PCR samples, two isolates could be obtained, named as MG UPF-1 and MG UPF-2, which were sub cultured and subjected to morphological, biochemical and serological characterization. The SDS-PAGE profile of whole cell proteins of the two isolates revealed similar profile, indicating their homogeneity. Whole cell and membrane proteins were isolated from cultures grown under iron sufficient and iron deficient conditions. Both the whole cell and membrane proteins revealed bands ranging from 24 kDa to 200 kDa on SDS-PAGE. The whole cell proteins had two prominent bands at 75 kDa and 35 kDa and eight faint bands, which the membrane proteins lacked. The whole cell and membrane proteins produced under iron restricted conditions had an additional band of 52 kDa, which was not seen in the respective protein groups isolated under iron sufficient condition. Most of the proteins resolved on SDS-PAGE in the four sets of proteins were found to be antigenic as found out by western blot analysis. The optimum concentration of the proteins used for vaccine preparation was 84.2 µg per dose of the vaccine. Formalin inactivated vaccines were formulated with saponin and aluminium hydroxide alone and in combination at a final concentration of 100 µg/dose and 25 per cent v/v respectively, thereby producing eight different sets of vaccines. The sterility and safety testing of the vaccines were carried before vaccination trials. The infective dose of M. gallisepticum cells for challenge studies was found to be 1.1 x 105. For vaccination studies, 240 chicks of five weeks age were divided into 10 groups and were inoculated with eight different sets of vaccines. One group was inoculated with commercial vaccine and other was kept as control. Potency testing for humoral response was conducted with HI test and CMI response with LMIT and Blastogenic calorimetry. The mean HI titre of groups vaccinated with iron sufficient and restricted saponin adjuvanated vaccines showed an increase in their titre at first week post booster vaccination followed by decline of the titre on subsequent collections on third and fifth week post booster vaccination. The mean HI titres of groups vaccinated with iron sufficient and restricted saponin-aluminium hydroxide adjuvanated vaccines maintained significantly high HI titres during the entire study period. Commercial vaccine group also showed a similar response. The control group showed significantly less titre from other vaccinated groups during the entire time period. Leukocyte migration inhibition test and Blastogenic calorimetry were carried out to assess the CMI response evoked by different sets of vaccines. A significant CMI response was shown by all vaccinated groups except commercial vaccine and control group. To assess the protective response evoked by different vaccines, two challenge studies were conducted two weeks apart. All the vaccinated birds were having significantly lower lesion score when compared to the control group. Mycoplasma gallisepticum was re-isolated from all the control birds with lesions during the first challenge study and from 70 per cent of the birds in the second challenge study. A latex agglutination test was developed using iron restricted M. gallisepticum whole cell proteins. When compared with HI test, LAT had a sensitivity of 95 per cent and specificity of 93 per cent. The test was statistically significant as indicated by a kappa value greater than 0.81. The saponin-aluminium hydroxide adjuvanated whole cell and membrane protein vaccines had elicited similar humoral immune response than vaccines adjuvanated with saponin alone. With regard to CMI response, the experimental vaccines produced a significant CMI response whereas, commercial vaccines failed to evoke a protective CMI response. In brief, saponin-aluminium hydroxide adjuvanated whole cell and membrane protein vaccines, especially those produced under iron restricted conditions, could be an effective tool to afford protection against M. gallisepticum infection, as it would stimulate both humoral and cell mediated immune responses in host Latex agglutination test was found to be an effective tool for flock screening of birds for M. gallisepticum antibodies and the test is simple, rapid, and easy to conduct, with applicability under field conditions.