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United States Department of Agriculture

Agricultural Research Service

Research Project: AVIAN GENOMIC AND IMMUNOLOGIC APPROACHES FOR CONTROLLING MUCOSAL PATHOGENS
2008 Annual Report


1a.Objectives (from AD-416)
* Identify host nucleic acid and protein markers and functional genetic variations associated with disease susceptibility and resistance to mucosal pathogens of economic importance. * Discover effective immune interventions strategies to prevent and control mucosal pathogens of poultry. * Determine the host-pathogen interactions that result in immune evasion or protective immunity to avian mucosal pathogens.


1b.Approach (from AD-416)
High throughput genomic approaches will be interfaced with disease modeling studies to decipher genetic and biological determinants of disease susceptibility. This approach will lead to the discovery of innovative tools to prevent and control avian mucosal pathogens such as avian coccidiosis, avian influenza, infectious bronchitis, and other important mucosal pathogens of poultry.


3.Progress Report
An integrative approach toward the development of novel control strategies against poultry mucosal pathogens using genomics, molecular biology and immunology is being developed. Comprehensive understanding of the protective host immunity and the identification of various effector molecules against various mucosal pathogens need to be better investigated before successful prevention and disease control strategies can be developed. Using information available from poultry genome sequencing and rapidly developing functional genomics technology, much progress is being made to unravel the nature of host-pathogen interactions in coccidiosis, necrotic enteritis (NE) and avian influenza infections. Many new poultry genes involved in host innate and adaptive immune responses to mucosal pathogens have been identified, fully sequenced and immunological reagents to detect them are being developed. Nature of host immune response which is involved in immune protection is being investigated using new functional genomics tools. Based on the new information gathered from these studies, new vaccination strategies are being developed in collaboration with poultry industry scientists under formal ARS trust agreements. Furthermore, several immunologically-based strategies such as recombinant vaccines, hyperimmune antibodies and probiotics are also being developed and applied to the control of field diseases. New knowledge on host-pathogen immunobiology, gut immunity and disease genomics is being actively applied in commercial settings to develop practical novel disease prevention strategies against many poultry diseases via formal agreements with private companies. Necrotic enteritis (NE) is an emerging and devastating poultry disease. However, lack of a reproducible NE disease model has been a major obstacle in understanding the basic parameters of immunity in NE. Successful development of NE disease model was accomplished for the first time using E. maxima and Clostridium perfringens dual infection. Using this experimental model, expression of a large repertoire of genes involved in innate immunity was monitored using a real-time RT-PCR. Gene expression profiles associated with avian influenza (AI) virus infection were monitored and genes associated with local innate immune response to AIV were identified in a collaborative study which was carried out with scientists at the Southeastern Poultry Research laboratory (SEPRL) in GA. This research relates to avian genomic and immunologic approaches for controlling mucosal pathogens that aligns with NP103 objectives under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry.


4.Accomplishments
1. Identification of polymorphic gene markers for coccidiosis resistance in broiler chickens. An alternative to anti-coccidial drugs or live parasite vaccines for controlling avian coccidiosis is to select for breeds of chickens that have increased natural resistance to Eimeria parasites. Using two commercial, pure broiler lines with different levels of resistance to coccidiosis, a new QTL located near the LEI0101 on chromosome 1 was found to have a significant association with resistance to Eimeria infection. Additionally, new SNP markers which are located near the coccidiosis QTLs are being identified and their biological roles are being investigated. This new information may be used to improve lines of broilers for natural resistance to avian coccidiosis and to understand genetic basis for coccidiosis control. This research relates to avian genomic and immunologic approaches for controlling mucosal pathogens that aligns with NP103 objectives under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry.

2. Molecular cloning and functional characterization of chicken genes controlling host innate immunity. Limited information on host genes involved in innate immunity to mucosal pathogens hinders progress in understanding immune mechanism of protection and subsequent development of control strategies against many economically important diseases of poultry. A new chicken cytokine gene, LPS-induced TNF-a factor, LITAF, which is important in host inflammatory response to parasites, bacteria, and viruses has been cloned and its biological function characterized. TNF-a is one of the most pleiotropic cytokines in mammals, but has yet to be identified in avian species. Recently, we isolated a full-length cDNA encoding the chicken homologue of LPS-induced TNF-a factor (LITAF), transcription factor, with an open reading frame of 148 amino acids and a predicted molecular mass of 16.0 kDa. Quantitative RT-PCR analysis showed that chicken LITAF mRNA is predominantly expressed in spleen and intestinal intraepithelial lymphocytes (IELs). LITAF mRNA levels were up-regulated following in vitro stimulation of macrophages with E. coli or S. typhimurium endotoxin, and 18-48 hr after treatment with E. acervulina, E. maxima, or E. tenella. Recombinant LITAF protein exhibited cytotoxic activity against chicken tumor cell lines in vitro. This new information about chicken LITAF will be useful in the development of immunological reagents for poultry immunology and disease research. This research relates to avian genomic and immunologic approaches for controlling mucosal pathogens that aligns with NP103 objectives under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry.

3. Development of necrotic enteritis (NE) model and investigation of host immunity to NE. Necrotic enteritis is an emerging disease of poultry resulting from increasing outbreaks of coccidiosis which predispose chickens to infection by Clostridium perfringens (CP). Lack of scientific data concerning local host immune responses to pathogens causing NE hinders progress in the development of effective control strategies against NE. In this report, local host-pathogen interaction leading to immunopathology associated with NE was investigated using a real-time RT-PCR. Following CP infection, there were significant increases in the expression of several important immune mediators in the gut, namely IFN-a, IFN-¿, IL-1ß, IL-2, IL-12, IL-13, IL-17 and TGF-ß4. On the other hand, TGF-ß4, which is considered as an anti-inflammatory cytokine, was repressed during EM/CP coinfection. The results of this study indicated for the first time molecular evidence that pre-exposure to E. maxima infection represses the ability of CP to induce an effective inflammatory cytokine response, which may account for the exacerbated pathological findings and increased CP colonization seen during experimental NE. Furthermore, these results established that a synergistic relationship exists between EM and CP during the course of experimental NE resulting in an intestinal disease phenotype that is qualitatively and quantitatively different from that produced by infection by either enteric microorganism alone. The results provide the foundation for future development of novel control strategies against NE. This research relates to avian genomic and immunologic approaches for controlling mucosal pathogens that aligns with NP103 objectives under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry.

4. Identification of a new anti-infective protein secreted by activated lymphocytes Due to increasing concerns over the use of drugs to control coccidiosis, there is a need to develop non-drug methods to control many poultry pathogens including coccidia. Using a high-throughput sequencing strategy, a new gene whose sequence is homologous to human NK-lysin was identified, and a full-length chicken NK-lysin cDNA was cloned. Although chicken NK-lysin showed relatively low amino acid sequence similarity to mammalian NK-lysins or granulysin (< 20%), it possessed the characteristically conserved 6 cysteine and 1 proline residues, hallmarks of the saposin protein family required to form 3 disulfide bonds needed for antimicrobial activity. Although our preliminary studies did demonstrate an anti-tumor effect of chicken NK-lysin, it lacked antibacterial activity. Chicken recombinant NK-lysin, was cytotoxic for E. acervulina and E. maxima parasites indicating its important role in innate immune response to avian coccidiosis. Future studies using synthetic peptides derived from NK-lysin may be useful for pharmaceutical and agricultural uses in the food animal industry. This is the first isolation of an anti-infective protein from intestinal IEL. TThis research relates to avian genomic and immunologic approaches for controlling mucosal pathogens that aligns with NP103 objectives under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry.

5. Identified and evaluated immunomodulatory properties of plant-derived phytonutrients. Coccidiosis is caused by several species of Eimeria and is an important disease in poultry production. Avian coccidiosis has traditionally been controlled by chemoprophylaxis using anticoccidial synthetic products or antibiotic ionophores. However, with increasing concerns over the emergence of drug-resistant Eimeria strains, alternative control methods are needed. Because of recent increases in industry’s interest in using natural plant-derived products to enhance host defense against microbial infections in domestic animals and poultry, we initiated studies to evaluate immunomodulatory properties of several plant-derived phytonutrients. The results of these studies showed for the first time that crude plant extracts from safflower leaf and plum activate macrophage function and enhance protective immunity against avian coccidiosis. For example, following E. acervulina infection, chickens fed a 0.1% safflower-supplemented diet exhibited decreased fecal oocyst shedding, elevated splenic lymphocytes proliferation and CD4+ splenic lymphocytes, and higher transcripts for IFN-¿, IL-8, IL-15, and IL-17, compared with chickens fed a nonsupplemented diet. These results clearly indicate that safflower leaf possesses immune enhancing properties and improves protective immunity against experimental coccidiosis when given as a dietary supplement. Thus, the addition of safflower leaf to chicken feed may provide an alternative method against coccidiosis if and when future restrictions are placed on the use of anti-coccidial drugs in commercial production. This research relates to avian genomic and immunologic approaches for controlling mucosal pathogens that aligns with NP103 objectives under Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Mucosal Diseases of Livestock and Poultry.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of New CRADAS2
Number of Active CRADAs6
Number of Non-Peer Reviewed Presentations and Proceedings4
Number of Newspaper Articles and Other Presentations for Non-Science Audiences2

Review Publications
Lillehoj, H.S. 2007. Host innate immunity against intestinal parasites. Journal of Korean Poultry Science. 34:77-83.

Scott, T.R., Lillehoj, H.S. 2007. Monoclonal antibodies against chicken interleukin-6. Veterinary Immunology and Immunopathology 114:173-177.

Hong, Y.H., Lillehoj, H.S., Lee, S.H., Park, D.W., Lillehoj, E.P. 2007. Molecular Cloning and Characterization of Chicken Lipopolysaccharide-Induced TNF-alpha Factor (LITAF). Developmental and Comparative Immunology 30:919-929.

Lee, S., Lillehoj, H.S., Park, D.W., Hong, Y.H., Lin, J.J. 2007. Effects of Pediococcus and Saccharomyces-based Probiotic (MitoMax) on Coccidiosis in Broiler Chickens. Comparative Immunology Microbiology and Infectious Diseases 30:261-268.

Gay, C.G., Zuerner, R., Bannantine, J.P., Lillehoj, H.S., Zhu, J., Green, R.D., Pastoret, P.P. 2007. Genomics and vaccine development. OIE Rev. Sci Tech. 26:49-67.

Park, S.S., Lillehoj, H.S., Hong, Y.H., Lee, S. 2007. Functional Characterization of Tumor Necrosis Factor Superfamily 15(TNFSF15) Induced by Lipopolysaccharides and Eimeria Infection. Developmental and Comparative Immunology 31:934-944.

Jang, S.I., Jun, M., Lillehoj, H.S., Dalloul, R.A., Kong, I.K., Kim, S., Min, W. 2007. Anticoccidial effect of green tea-based diets against Eimeria maxima. Veterinary Parasitology 144:172-175.

Shini, J.H., Kim, H., Lim, D., Jeon, M., Hani, B.K., Park, T.S., Kim, J.K., Lillehoj, H.S., Cho, B.W., Han, J.Y. 2006. Analysis of chicken embryonic gonad ests. Animal Genetics 37:85-86.

Dalloul, R.A., Bliss, T.W., Hong, Y.H., Ben Chouikha, I., Park, D., Keeler Jr, C.L., Lillehoj, H.S. 2007. Unique Responses of the Avian Macrophage to Different Species of Eimeria. Molecular Immunology 44:558-566.

Del Cacho, E., Gallego, M., Sanchez-Acedo, C., Lillehoj, H.S. 2008. Identification of flotillin-1 on eimeria tenella sporozoites and its role in host cell invasion. Journal of Parasitology. 93:328-332.

Yoo, J., Chang, H.H., Bae, Y., Seoung, C., Lillehoj, H.S., Min, W. 2008. Monoclonal antibodies reactive with chicken interleukin-17. Veterinary Immunology and Immunopathology. 121:359-363.

Park, S.S., Allen, P., Lillehoj, H.S., Park, D., Fitzcoy, S., Bautista, D.A., Lillehoj, E.P. 2008. Immunopathology and Cytokine Responses in Broiler Chickens Coinfected with Eimeria maxima and Clostridium perfringens Using an Animal Model of Necrotic Enteritis. Avian Diseases. 52:14-22.

Hong, Y.H., Lillehoj, H.S., Siragusa, G.R., Bannerman, D.D., Lillehoj, E.P. 2008. Antimicrobial activity of chicken NK-lysin against Eimeria sporozoites. Avian Diseases. 52:302-305.

Lee, S., Lillehoj, H.S., Heckert, R.A., Chun, H., Cho, S., Tuo, W. 2008. Immunomodulatory Effects of Safflower Leaf (Carthamus tinctorius)on Chicken lymphocytes and macrophages. Journal of Poultry Science. 45:147-151

Kim, D., Lillehoj, H.S., Hong, Y.H., Park, D., Lamont, S.J., Han, J., Lillehoj, E.P. 2008. Immune-related gene expression in two genetically disparate fayoumi chicken lines following eimeria maxima infection. International Journal of Poultry Science. 87:433-443.

Lillehoj, H.S., Kim, C., Keeler, C.L., Zhang, S. 2008. Immunogenomic approaches to study host immunity to enteric pathogens. Poultry Science. 86:1491-1500.

Lee, S., Lillehoj, H.S., Heckert, R.A., Cho, S., Hong, Y.H., Park, D., Lillehoj, E.P., Chun, H., Park, H. 2007. Immunomodulatory effect of dietary Safflower leaf on coccidiosis. 18:715-724.

Lillehoj, H.S., Del Cacho, E., Gallego, M., Lopez-Bernard, F., Sanchez-Acedo, C. 2008. Isolation of chicken follicular dendritic cells. Journal of Immunological Methods. 334:59-69.

Last Modified: 12/22/2014
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