Oral immunization of broilers with chitosan nano-encapsulated extracellular and cell wall proteins of necrotic enteritis-induced Clostridium perfringens

Abstract Currently, there is no commercial vaccine to control Clostridium perfringens (CP) or necrotic enteritis – a clinically and economically devastating disease. Two chitosan-nanoparticle encapsulated CP (CNP-CP) vaccines using extracellular proteins (ECP) and cell wall proteins (CWP) were synthesized: a CNP-CP ECP + CWP vaccine (SC vaccine) and a CNP-CP ECP + CWP surface-adsorbed CWP vaccine (SCC vaccine). The experiment comprised a complete randomized design of 3 treatments replicated 5 times: SC, SCC and nonimmunized control. Broilers orally gavaged with SC or SCC vaccine were primed and boosted with 70μg dose at 3- and 14-days post-hatch (dph). SDS-PAGE analysis revealed bands at 54.7 and 84.7 kDa in the ECP and 17 bands for CWP fraction. There were no differences in body weight gain, feed conversion ratio and mortality rate between treatments. At 17dph, serum of birds in the SC and SCC groups had higher neutralizing antibodies (P<0.01) compared to the control group. At 17dph, there was an increase in bile anti-ECP IgA levels in the SC vaccinated birds and a non-significant increase in SCC vaccinated birds compared to control. At 17dph, bile specific anti-CP CWP IgA levels were greater (P<0.05) in both immunized groups compared to control. At 17 and 21dph, serum anti-ECP and anti-CP CWP IgY levels in SC and SCC birds were comparable to the control. At 21dph, CD4+/CD8+ T-cell ratio in SC and SCC vaccinated groups were elevated (P ≤ 0.01) compared to control. At 17dph, SC and SCC vaccinated birds had a significant reduction (P ≤ 0.001) in α-toxin levels in caecal contents compared to control. Caecal α-toxin levels remained reduced at 21dph (P < 0.05) in SC birds and numerically reduced in SCC vaccinated birds compared to control. Jejunal CP load in SCC birds was significantly reduced by 1.4 Log10 copy numbers of CP/g compared to control and no differences were observed in liver CP load between immunized and non-immunized birds. SC and SCC immunization did not alter TNF-α, IL-10, iNOS, IL-6 or IL-17 mRNA expression. At 17 and 21dph, SC and SCC immunized birds demonstrated greater sera bactericidal activity compared to control. CNP-SC and CNP-SCC immunization induced specific immune response against C. perfringens and reduced CP colonization via oral route of administration.