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