2.7 Immunization and challenge procedure
We included 100 two-days-old chicks separated randomly into four groups of 25 chicks each. Three groups were vaccinated orally with seeds powder containing 15 µg recombinant TM-1 protein, same amount of WT seeds powder, and equal amount of PBS (Phosphate-buffered saline) buffer twice at day 7 and 21. The fourth group was inoculated with MG attenuated vaccine (8 x 106 CFU) via ocular method. MG virus S6 strain (5 x 106 CFU) challenge was conducted through eye route for the all groups at week 4 and 5. Clinical parameters were recorded daily after challenge with MG virus strain as described previously for two weeks [18]. Dead chickens before termination of the study were necropsied. All others were euthanized and examined two weeks after MG challenge. Briefly, the main clinical scores consisted of four levels in terms of symptomatic severity as follows: 0 - no symptom; 1 - clear nasal exudate; 2 - cloudy nasal exudate; 3 - turbid nasal exudate with foamy eyes and/or swollen infra-orbital sinuses. For air sac lesion, scores were observed and examined at 14 days after challenge as follows [19]: 0 - no lesions; 1 - mild lesions; 2 - moderate lesions; and 3 - severe lesions. Individual bodyweight was also recorded every week. All experiment groups were challenged with MG virulent strain (5 x 106 CFU) one week after second immunization by ocular delivery, followed by recording clinical parameters for additional two weeks.
2.8 Statistical analysisThe statistical analysis of this study was carried out using the one-way ANOVA analysis and indicated by P value.
Results