Progress 09/15/00 to 09/14/04
Outputs
Overall, the results of this grant showed that oral vaccination with live attenuated SE vaccine enhanced cell mediated immunity with increased SE clearance. However, killed vaccine enhanced the humoral immune response with no impact on the SE clearance. CpG oligonucleotides were shown to be efficacious in poultry and can be used to modulate the immune response. When given with killed vaccines CpG oligoneucleotides were shown to increase the resistance to infection if given parenterally or in ovo. These results suggest that vaccination with attenuated live Salmonella could be an effective method for controlling Salmonella enteritidis infection in young chickens.
Impacts
Better methods of affecting Salmonella infection or persistence of infection in laying hens may lead to decreased on-farm Salmonella prevalence and therefore potentially less human food borne illness.
Publications
- Dalloul, Rami, A., Hyun S. Lillehoj, Xicheng Ding, Wongi Min, Robert A. Heckert and Dennis M. Klinman. 2004. CpG oligodeoxynucleotides as vaccine adjuvants to Eimeria in chickens. American Association of Avian Pathologists, American Veterinary Medical Association, p21.
- Okamura, M., H. S. Lillehoj, R. B. Raybourne, U. S. Babu and R. A. Heckert 2004. Cell-mediated Immune Responses to a Killed Salmonella enteritidis Vaccine: Comp. Immunology, Microbiology and Infectious Diseases. 27:255-272.
- Babu, U., Dalloul, R.A., Okamura, M., Lillehoj, H.S., Xie, H., Raybourne, R., Gaines, I., and Heckert, R. 2004. Salmonella enteritidis Clearance and Immune Responses in Chickens Following Salmonella Vaccination and Challenge. Veterinary Immunol Immunopathol. 101:251-257.
- Dalloul, R. A., Hyun S. Lillehoj, Masashi Okamura, Hang Xie, Wongi Min, Xicheng Ding, and Robert A. Heckert. 2004. In Vivo Effects of CpG Oligodeoxynucleotide on Eimeria Infection in Chickens. Avian Diseases. Avian Diseases: 48: p 733-740
- Okamura, M, H. S. LillehojAD, R.B. RaybourneB, U.BabuB, and R. HeckertC 2003. Antigen-specific Lymphocyte Proliferation, Interleukin-6 (IL-6) and IL-2 Production in Chickens Immunized with Outer Membrane Protein of Salmonella enteritidis. In Proceeding of Congress of the World Veterinary Poultry Association. P 163.
- Babu, US, Okamura M, Raybourne RB, Dalloul RA, Xie H, Gaines D, Lillehoj HS, and Heckert R. A. 2003. Cellular and Humoral Immune Responses and Salmonella enteritidis Clearance in Young Broiler Chickens Following Salmonella Vaccination and Challenge. Proceeding of the Conference of research workers in animal diseases, P 12.
- Dalloul, R., Lillehoj, H.S., Babu, U., Raybourne, R., Klinman, D. 2003. In ovo immunomodulatory effects of CpG-containing oligodeoxynucleotides in Eimeria-infected chickens. Proceeding of the 92nd Annual Poultry Science meeting. p8.
- Dalloul, Rami, A., Hyun S. Lillehoj,Uma S. Babu, Richard B. Raybourne,,Robert A. Heckert. 2004. CpG-containing Oligodeoxynucleotides Protect Chickens Against Eimeria Infections. Proceeding of American Association of Immunologists meeting, p120.
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Progress 01/01/03 to 12/31/03
Outputs
Cytokine profiling following Salmonella vaccination was carried out. The effect of Salmonella vaccination on cell-mediated immune responses to Salmonella enteritidis (SE) was assessed using ELISAs specific for IFN-gamma and IL-2. Chickens were given two intramuscular injections with SE vaccine 2 weeks apart, and at various times after vaccination, the levels of serum IFN-gamma and IL-2 were assessed. In general, SE-vaccinated chickens showed significantly higher levels of IL-2 and IFN-gamma following immunizations with the Salmonella vaccine compared with untreated controls. In another study, phagocytosis, apoptosis and nitric oxide (NO) production induced by Salmonella enterica serovars enteritidis and typhimurium in the chicken peripheral blood monocyte-derived macrophages were evaluated. The number of cells engulfing SE was similar to ST. At 2 h post infection (PI), the SE-infected cells produced significantly higher NO than ST-infected cells. However, NO
production was significantly higher in ST-infected cells than SE-infected cells at 24 h PI. At 24 h PI, the cells undergoing apoptosis increased upon ST but not SE infection. The infection showed little effect on interleukin IL-1 and IL-6 production in vitro. When NO production was assessed following in vitro stimulation with different Salmonella antigens, SE-flagella induced a significantly higher amount of NO than ST-flagella. The treatment of macrophages with recombinant chicken interferon-? significantly enhanced NO production in response to LPS derived from ST but not SE. These results suggest that these 2 serovars of Salmonella induce different types of immune responses in chicken macrophages. In another study, lymphocyte proliferation and interleukin IL-2 and IL-6 levels in serum were measured as indicators of cell-mediated immunity after immunization of chickens with a commercial killed SE vaccine or experimental subunit vaccines of crude protein (CP) extract or outer membrane
protein (OMP). Significantly increased proliferative responses to SE flagella, but not lipopolysaccharide, porin, CP, or OMP, were observed at 1 wk post immunization (PI). Use of the killed SE vaccine appeared most effective because it induced higher flagella-stimulated lymphocyte proliferation at 1 and 2 wk PI compared with the CP- and OMP-vaccinated groups. We conclude that cell-mediated immunity to SE after vaccination with the killed bacterial vaccine or subunit vaccines is transient and mainly limited to flagella. Short oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) have been recently shown to be effective immunoprotective agents in domestic animals. In ovo and in vivo studies were conducted using 18-day-old SPF embryos and birds of multiple ages, respectively. ODNs with different CpG motifs were applied at different doses followed by challenge. The data showed that in vivo CpG treatment enhanced the birds' resistance to challenge from an intestinal
infection in a normally susceptible chicken strain, as shown by reduced agent shedding and improved weight gain. Two CpG ODNs tested in ovo significantly reduced agent shedding while exhibiting a differential antibody response.
Impacts
Better methods of affecting Salmonella infection or persistence of infection in laying hens may lead to decreased on-farm Salmonella prevalence and therefore potentially less human food borne illness.
Publications
- Sheela RR, Babu US, Mu J, Elankumaran S, Bautista DA, Raybourne RB, Heckert RA, Song W. Immune responses against Salmonella enterica serovar enteritdis infection in virally immunosuppressed chickens Clin Diag Lab Immunol 10:670-679, 2003.
- Okamura M, Lillehoj HS, Raybourne RB, Babu US, Heckert RA. Antigen-specific lymphocyte proliferation and interleukin production in chickens immunized with killed Salmonella enteritidis vaccine or experimental subunit vaccines. Avian Dis. 47:1331-1338, 2003.
- Dalloul RA, Lillehoj HS, Babu US, Raybourne RB, Klinman DM, Heckert RA. In ovo immunomodulatory effects of CpG-containing oligodeoxynucleotides in Eimeria-infected chickens. 92nd Annual Meeting of the Poultry Science Association. Madison WI, July 6-9, 2003.
- Lillehoj H, Okamura M, Raybourne R, Babu U, Heckert RA. Antigen-Specific Lymphocyte Proliferation, Interleukin-6 (IL-6) And IL-2 Production In Chickens Immunized With Outer Membrane Protein Of Salmonella Enteritidis. Proc. 140th American Veterinary Medical Association Meeting, Denver, CO, July 19-23, 2003.
- Dalloul R, Lillehoj H, Heckert RA. In Vivo Effects Of CpG-ODN On Eimeria Acervulina-Infected SC and TK Chickens. Proc. 140th American Veterinary Medical Association Meeting, Denver, CO, July 19-23, 2003.
- Babu US, Scott M, Myers MJ, Okamura M, Gaines D, Yancy HF, Lillehoj H, Heckert RA, Raybourne RB. Effects of live attenuated and killed salmonella vaccine on T-lymphocyte mediated immunity in laying hens. Vet Immunol Immunopathol 91:39-44, 2003.
- Xie H, Raybourne RB, Babu US, Lillehoj HS, Heckert RA. CpG-induced immunomodulation and intracellular bacterial killing in a chicken macrophage cell line. Develop and Comp Immuno 27:823-834, 2003.
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Progress 01/01/02 to 12/31/02
Outputs
Vaccination to control Salmonella: Two studies specifically examined the immunological and inflammatory responses to various killed Salmonella enteritidis (SE) vaccines in various age birds. It wad concluded that younger chickens mounted a more robust antigen-specific immune response to the SE vaccine compared with older birds and vaccination induced not only T-cell-mediated responses but also host innate and pro-inflammatory responses. These results suggest that killed vaccine elicits cell-mediated immunity, which might be associated with protective immune response to SE. Finally, the effects of live attenuated and killed Salmonella vaccines on T-lymphocyte mediated immunity in laying hens was investigated. Overall, live vaccine was more effective in increasing the lymphocyte proliferation to Con A as well as Salmonella antigens. We concluded that this enhanced CMI may prove beneficial in protecting chickens against SE infection. Immunomodulation to control
Salmonella: In a chicken macrophage cell line (HD11) SE survived, while Salmonella typhimurium (ST) invasion resulted in immediate cell death. When compared to SE, ST quickly induced higher levels of nitric oxide and a faster/stronger apoptotic response in HD 11 cells. Both SE and ST induced apoptosis in HD 11 cells through an iNOS-dependent pathway, which lead to the intracellular bacterial killing and induction of host immunity. To affect this persistent infection, two CpG oligonucleotides (ODNs) (2006, 1826) were tested. ODN 2006 showed strong immunomodulatory effects on HD11 cells, whereas ODN 1826 had weak influences. ODN 2006 induced strong IL-6 and nitric oxide secretion by HD11 cells in both dose- and time-dependent manners. Intracellular killing of SE was also increased in 2006 activated HD11 cells. Furthermore, ODN 2006-stimulated HD11 cells had reduced proliferation and underwent apoptosis after CpG stimulation. These findings may provide a new strategy for developing more
efficient vaccine strategies for Salmonellosis in poultry.
Impacts
Better methods of affecting Salmonella infection or persistence of infection in laying hens may lead to decreased on-farm Salmonella prevalence and therefore potentially less human food borne illness.
Publications
- Babu, U., Okamura, M., Gained, D., Myers, M., Raybourne, R., Lillehoj, H. and Heckert, R. 2002. Effects of live attenuated and killed salmonella vaccines on cell-mediated immunity among young chickens. Cellular immune responses to Salmonella enteritidis vaccination. In Proceedings of 91st Annual Poultry Science meeting. Newark. DE, August 11-14, P8.
- Lillehoj, H. S., M. Okamura, R. B. Raybourne, U. S. Babu and R. A. Heckert. 2002. Cellular immune responses to Salmonella enteritidis vaccination. In Proceedings of 139th American Veterinary Medical Association Annual Convention. July 13-17, Nashville, TN. P1274.
- Okamura, M., Lillehoj, H. S., Raybourne, R. B., Babu, U. S. and R. A. Heckert. 2002. Age difference in lymphocyte proliferation, and IL-2 and IFN-gamma production following Salmonella enteritidis vaccination. In Proceedings of 91st Annual Poultry Science meeting. Newark. DE. August 11-14, 2002. P103.
- Okamura, M., Lillehoj, H.S., Raybourne, R. B., Babu, U. S. and Heckert, R.A. 2002. Age difference in lymphocyte proliferation, and IL-2 and IFN-gamma production following Salmonella enteritidis vaccination. In Proceedings of The Fifth Annual Conference on Vaccine Research. Baltimore, MD. May 6-8, P68.
- Xie, H., Raybourne, R., Babu, U., Kamura, M., Lillehoj, H., and Heckert, R. 2002. S. enteritidis and S. typhimurium have different pathogenic mechanisms in infected HD11 cells. In Proceedings of 91st Annual Poultry Science meeting. Newark. DE. August 11-14, P59.
- Xie, H., Raybourne, R. B., Babu, U. S., Lillehoj, H. S., Heckert, R.A. 2003 CpG-induced immunomodulation and intracellular bacterial killing in a chicken macrophage cell line. Developmental and Comparative Immunology (accepted for publication).
- Babu, U., Scott, M., Myers, M. J., Okamura, M., Gaines, D., Raybourne, R., Yancy, H. F., Lillehoj, H., and Heckert, R. 2002. Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens. Vet. Immunol. Immunopathol. 6729:1-6.
- Live Attenuated and Killed Salmonella Vaccines Cause an Increase in Cell-mediated Immunity among Laying Hens. 2002. Babu, U., Scott, M., Myers, M.,J., Okamura, M., Gaines, D., Yancy, H.,F., Lillehoj, H., Heckert, R.,A. and Raybourne, R. In Proceedings of 139th American Veterinary Medical Association Annual Convention. July 13-17th, Nashville, TN.
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Progress 01/01/01 to 12/31/01
Outputs
In the last 20 years there has been an increase in human food-poisoning outbreaks attributable to Salmonella enteritidis (SE) in the United States. Epidemiological studies of this increase have indicated that grade A shell eggs are an important source of SE. Despite the tremendous efforts made by the poultry industry, no effective measures for elimination of SE colonization have been developed. The humoral immune responses after infection with SE have been extensively studied for diagnostic purposes. However, the fundamental mechanism of mucosal resistance to infection and clearance of SE from the gut has received scant attention. Protection from infection by SE through humoral mechanisms alone is unlikely, due to the organism being a facultative intracellular bacterium. There is enough evidence in various animal models that cell mediated immunity plays a major role in controlling Salmonella infection. In chickens, thus far, there is lack of detailed knowledge on the
immune mechanisms involved in defense against Salmonella infection. Our studies thus far have supported earlier conclusions that natural infection produces weak and transient immune responses that are ineffective at preventing or clearing a Salmonella infection. We have found that immunization with a killed vaccine provides a better immune response than using a modified live vaccine. This immunity is better with respect to intestinal IgA (which is the main barrier to Salmonella colonization of the intestinal tract) and better in producing cellular immunity (that which is responsible for eliminating the infection). This is somewhat surprising, as traditional thinking indicated that killed vaccines were not effective at eliciting good cellular immunity. We are currently preparing several publications regarding these findings. A further goal in this research was to evaluate various methods of increasing the chick's natural resistance to infection. To this end we have been developing and
evaluating various compounds that could be administered in ovo, to increase the innate immunity of the chick at hatch. One of the first substances we evaluated was a genetic adjuvant. This is a short stretch of nucleic acids containing many C and G bases in a particular motif. These sequences are bacterial in origin and have been shown in the mammalian system to up regulate the immune system. We have synthesized several of these sequences in various motifs and have shown that some of them are also powerful stimulants in activating avian cells. We have shown the CpG oligonucleotides to increase macrophage intracellular nitric oxide, increase expression of cell surface antigens and increase expression of IL-6 in vitro. Preliminary studies have also shown it to have similar effects when delivered in ovo. We are in the process of preparing a publication reporting this novel and important finding. As further goals in this project we are continuing to explore vaccination and other methods
of increasing the immunity of the hatched chick, such that it is better able to resist Salmonella infection at hatch.
Impacts
This research should not only provide basic information on the pathogen-host cell interaction, but also give vital information on how the immune system of the chicken can be used to control or eliminate the spread of Salmonella from animals to man.
Publications
- No publications reported this period
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