| Necrotic enteritis is an important disease of broiler chickens caused by Clostridium perfringens, the incidence rate is up to 30% and motality is from 18% to 31%. Currently, no effective vaccine against NE is available and immunity to NE is not well characterized. Recently, it has showed the ability of certain immunogenic secreted proteins uniquely expressed by virulent C. perfringens in protecting broiler chickens against an experimental NE rather than avirulent C. perfringens when delivered intramuscularly as well as orally. The current study was carried out with two main objectives, one is to identify three highly immunogenic secreted proteins, establish their importance in NE and determine their value as immunogens; the other one is to assess the abilities to protect broiler chickens against NE and in reducing colonization and shedding of broiler chickens by different serovars of Salmonella.The first part separated and identified 3 secreted proteins apparently uniquely expressed by virulent, immunoprotective, C. perfringens additional to those previously described. Secreted proteins of virulent and avirulent C. perfringens were electrophoretically separated and reacted with serum of chickens immune to NE. Three immunoreactive protein bands unique to the virulent C. perfringens were identified by Mass Spectrometry as the toxin C. perfringens large cytotoxin (TpeL), Endo-beta-N-acetylglucosaminidase (Naglu), and Phosphoglyceromutase (Pgm). TpeL is a novel toxin of C. perfringens that was first time identified in a C. perfringens type A strain and found in a strain causing NE (CP4) by our lab, and the complete sequences of the TpeL gene has been amplified and submitted to GenBank of NCBI (Accession No. EU848493), this is a very important discovery for the study of C. perfringens type A strain.The second part focused on to establish the importance of those identified secreted proteins above in NE, and determine their value as immunogens. The genes encoding Naglu and Pgm proteins were cloned, and their gene products were purified as histidine-tagged recombinant proteins from Escherichia coli and used in immunizing chickens. Immunized and non-immunized control broiler chickens were then challenged with two different strains (CP1, CP4) of C. perfringens and assessed for the development of NE. Of the two immunogens, Pgm immunization showed significant protection of broiler chickens against experimental NE although protection reduced as challenge severity increased. However, birds immunized with Naglu were protected from challenge only with strain CP4. Birds immunized with these proteins had antigen-specific antibodies when tested in ELISA. In conclusion, this study demonstrated the partial efficacy of additional secreted proteins in immunity of broiler chickens to NE. The study also showed that there may be differences in the protective ability of immunogens depending on the infecting C. perfringens strain.The third part assessed the efficacy of oral immunization with two attenuated Salmonella Typhimurium-vectored vaccine strains expressing tHP antigen confers protection against NE in broiler chickens. Birds were immunized orally with 1×108 cfu of the attenuated S. Typhimurium vaccine strainsχ9241 orχ9352 expressing plasmid-encoded a partial recombinant Hypothetical Protein gene (tHP) of C. perfringens at days 1 and 10 of age, and then challenged at 4 weeks of age with CP4. Birds were necropsied at 5 weeks of age, the protection was evident from the gross pathological scores as well as body weight gains during the challenge period. Chickens vaccinated withχ9241-tHP andχ9352-tHP showed significant protective immune response, however, the degree of protection was less than that observed previously when these and other proteins, including tHP, were administered intramuscularly to immunize birds against NE. Meanwhile, immunized birds showed Salmonella as well as C. perfringens antigen-specific IgY and IgA antibodies in their serum and intestines detected by ELISA. Interestingly, the intestinal IgY response was higher than the IgA response for Salmonella antigens and to some extent also for C. perfringens antigens, suggested the dominance of intestinal IgY compared to IgA in NE immunity. This study described here demonstrating that an attenuated S.Typhimurium vaccine strain can successfully deliver C. perfringens antigens through the oral route to elicit partial protection efficacy against an experimental NE model in broiler chickens.The fourth part assessed the protective efficacy of oral immunization with two experimental attenuated Salmonella Typhimurium-vectored vaccine strains expressing tHP antigen in protecting chickens against intestinal and invade visceral organs colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were immunized orally with 1×108 cfu of the attenuated S.Typhimurium vaccine strainsχ9241 orχ9352 expressing plasmid-encoded a partial recombinant Hypothetical Protein gene (tHP) of C. perfringens at days 1 and 7 of age, and then challenged at 14 days of age with 106 cfu of Salmonella serovars S. Anatum, S. Enteritidis, S. Heidelberg, S. Kentucky or S. Typhimurium (representative serovars of serogroups B, C, D and E). Birds were necropsied at 4 weeks of age, and samples were collected to determine reduction in tissue and intestinal colonization. Chickens vaccinated withχ9241-tHP showed reduced colonization by S. Enteritidis (serogroup D) and by S. Heidelberg and S. Typhimurium (serogroup B) compared to control birds. No reduction in colonization was observed in chickens vaccinated withχ9352-tHP. There was association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. In conclusion, the choice of an attenuated S. Typhimurium vaccine vector for delivery of heterologous antigens in chickens should be based partly on its value in protecting against colonization with serovars within the serogroup B and S. Enteritidis (serogroup D). Such vectors would also have the additional benefit of reducing colonization of important Salmonella serovars.In summary, for the first time identified three additional immunogenic secreted proteins from virulent strain of Clostridium perfringens, it strengthened previous findings that some other secreted proteins such as Pgm and Naglu are important proteins in immunity to NE rather thanα-toxin. The recombinant attenuated Salmonella typhimurium vaccine showed partial protective fficacy against NE while reducing Salmonella colonization, reveals the importance of choosing a suitable attenuated Salmonella strain as vaccine vector in protective immunity. There are also differences in the abilities of vector plasmids to clone and express clostridial antigens, and these are possibly affected by the vehicle. The effective reducing Salmonella colonization as a guide for choosing an attenuated Salmonella vaccine vector, provided a solid basis for the development of attenuated recombinant Salmonella Typhimurium vaccine expressing C. perfringens antigens to prevent and control necrotic enteritis.
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