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United States Department of Agriculture

Agricultural Research Service

Research Project: AVIAN GENOMIC AND IMMUNOLOGIC APPROACHES FOR CONTROLLING MUCOSAL PATHOGENS
2009 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
High-throughput gene expression analysis was developed using avian intestinal epithelial lymphocyte-specific cDNA microarray (AVIELA) for host-pathogen immunobiology research. AVIELA was successfully applied to unravel complex host-pathogen interactions and to investigate local host immune responses to economically important poultry mucosal pathogens including Eimeria, avian influenza and Salmonella. Although much has been reported on the immunopathology caused by these pathogens in the susceptible hosts, there is very limited information on host-pathogen interactions in poultry due to the lack of adequate immune reagents for poultry. In all these host-pathogen interactions, there are serious knowledge gaps that hinder the development logical and effective control and prevention strategies. Towards this goal, the following major progress has been made in this reporting period:.
1)Identification of significantly altered gene transcripts that are critically involved in local immune response against Eimeria, avian influenza and Salmonella;.
2)development of bioassays and immune reagents (recombinant molecules and mouse monoclonal antibodies) that can identify Th17 and Treg cells, and poultry dendritic cells;.
3)development of a novel control strategy against coccidiosis using hyperimmune egg yolk IgY antibodies;.
4)identification of new polymorphic genetic DNA markers associated with disease resistance to avian coccidiosis;.
5)development and proof-of-concept for dietary enhancement of protective immunity against coccidiosis using plant-derived phytonutrients;.
6)development of nutrigenomics to identify metabolic and immune pathways associated with nutrition-mediated immunity, and.
7)expression of a novel anti-microbial peptide, NK-lysin, in an insect vector and demonstration of its efficacy against invasive stage of Eimeria. Major progress also includes the technology transfer of new knowledge on host-pathogen immunobiology, gut immunity and host genomics to commercial companies to develop practical disease prevention strategies against many economically important infectious diseases via formal agreements.


4.Accomplishments
1. Identify immunological mechanisms associated with genetically determined disease resistance in Fayoumi MHC-congenic chickens. Genetic selection for disease resistance and enhanced immune responses to pathogens can lead to a reduction of drug use and decrease the risks associated with vaccination in commercial poultry production. The present study was conducted to compare local gene expression profiles between two congenic chicken lines, using our avian intestinal intraepithelial lymphocyte microarray (AVIELA). Comparison of the local gene expression levels between M5.1 and M15.2 chickens following E. maxima infection revealed alterations in 32 (10 up, 22 down), 98 (43 up, 55 down), and 92 (33 up, 59 down) mRNAs at 3 time points. Functional analysis using Gene Ontology (GO) categorized the genes exhibiting the different expression patterns into several GO terms including immunity and defense. In summary, transcriptional profiles showed that more gene expression changes occurred with E. maxima infection in the M15.2 than the M5.1 line. The most gene expression differences between the two chicken lines were exhibited at days 4 and 5 following E. maxima infection. These results demonstrate that differential gene expression patterns associated with the host genetic difference in coccidiosis resistance provides deeper insights into the host protective immune mechanisms which are under the host genetic control.

2. Molecular cloning and functional characterization of IL-17 gene familes in chicken and duck. The IL-17 cytokine family contains pro-inflammatory cytokines (IL-17A through IL-17F in mammalian species) that play a critical role in immunoregulation at mucosal surfaces. To extend our knowledge on the evolution of IL-17 cytokines in birds, duck IL-17 cDNA and genomic DNA were cloned from mitogen (ConA)-activated splenic lymphocytes. Duck IL-17 shared 84% amino acid sequence identity to the previously described chicken IL-17 and 36-47% to mammalian homologues and the open reading frame 13 of Herpesvirus saimiri (HSV 13). The genomic structure of duck IL-17 was remarkably similar to those of chicken and mammalian conterparts. Transcripts of duck IL-17 could be strongly detected in ConA-activated splenic lymphocytes while a variety of normal tissues did not express detectable levels by RT-PCR. Cross-reactivity of two mAbs against chicken and duck IL-17 with IL-17 was detected by indirect ELISA and Western blot analyses. These findings indicate that the structure of IL-17 is highly conserved among poultry, and two mAbs can be used for molecular and immunological studies of IL-17 in birds. This new information about the duck IL-17 gene will be useful in the development of immunological reagents for basic and applied disease research.

3. High-throughput gene expression analysis to investigate local innate immunity to Highly Pathogenic Avian Influenza Virus (HPAIV) in immunosuppressed host. Comprehensive understanding of host-pathogen interaction at the local infection site is critical to develop a logical immunological control strategy against avian influenza infection. Most commercial chickens are infected with Infectious bursal disease virus (IBDV) early in life, but little is known about the potential effects of pre-exposure to IBDV and the subsequent susceptibility to AIV infection and/or associated clinical signs, lesions, and virus shedding. In a collaborative study with the School of Veterinary College at the University of Maryland, tissue-specific cDNA microarray was used to study local gene expression of lung tissues to identify host immune mechanisms involved in AIV evolution of virulence in an immunocompromised host model. The results indicated that LPAIV, after adaptation in the immunosuppressed host becomes more virulent in both immunosuppressed and immunocompetent chickens highlighting the potential of prior IBDV infections as portal of entry of AIV in chickens. This study provided important new knowledge for IBDV-AIV interactions in the field.

4. Morphological and functional characterizations of chicken interdigitating dendritic cells (IDC) and follicular dendritic cells (FDC) in the intestine. Dendritic cells (DCs) are highly efficient antigen processing cells (APCs) found in tissues that are in contact with the external environment, as well as in the bone marrow. Despite the important roles that FDCs and IDCs play in the immune response, there is a lack of phenotypic and functional studies compared with mammalian systems due to the absence of isolation protocols for avian DCs. In order to explore various antigen delivery strategies targeting APCs, gut DCs were isolated following Eimeria infection. Since E. tenella primarily infects the intestinal caeca where DCs reside, chickens were orally infected with E. tenella sporozoites based on our previous studies which showed enhanced numbers of DCs in the cecal tonsils compared with other lymphoid organs. Using a combination of methods involving antigen-specific antibodies against E. tenella and CD45, it was possible for the first time to obtain a highly enriched IDCs and FDCs which are functionally active in chickens. This novel method will enable the detailed biochemical and immunological characterizations of avian DCs and is expected to facilitate the investigation of the role of DCs in initiating immune response in normal and disease states.

5. Expression of chicken NK-lysin in a baculovirus vector-driven insect protein expression platform and validation of its biological activity against Eimeria. We identified a new anti-microbial chicken gene from a chicken intestinal cDNA library whose sequence was homologous to human NK-lysin. A full-length chicken NK-lysin cDNA was cloned, and its sequence showed relatively low amino acid sequence homology to mammalian NK-lysins or granulysin (< 20%). Recombinant chicken NK-lysin expressed in COS7 cells was cytotoxic for E. acervulina and E. maxima parasites indicating its important role in innate immune responses to avian coccidiosis. Future studies indicated that recombinant chicken NK-lysin has target specificity against other apicomplexan parasites including Neospora and Cryptosporidia. Because of their exquisite specificity against apicomplexa parasites, chicken NK-lysin is a potential candidate as an anti-parasitic immunotherapeutic that may be useful for pharmaceutical and agricultural applications in the animal health industry.

6. Identification of single nucleotide polymorphic (SNP) DNA markers associated with disease resistance phenotype in coccidiosis in meat birds. Conventional disease control methods which include chemoprophylaxis with coccidiostatic drugs or vaccination with live and attenuated parasites suffer from serious drawbacks and there is an increasing need to develop alternative approaches for disease control in poultry. Genetic selection of disease resistant chickens using molecular genetics and functional genomics tools provides the potential to address this need. To better define genetic markers near the coccidiosis quantitative trait loci (QTL), single nucleotide polymorphisms (SNPs), were identified using SNP genotyping of commercial broiler chickens segregating for coccidiosis resistance. The current study focused on candidate genes which are located between the LEI0071 and LEI0101 QTLs that may play a role in the host response to coccidia infection and, if present, to determine whether or not these polymorphisms were associated with resistance to experimental E. maxima infection. Three selected genes, zyxin, CD4, and tumor necrosis factor receptor super family 1A (TNFRSF1A), were chosen based upon their genomic location, immunological function, and previous results suggesting involvement in protective immunity to coccidiosis. SNPs were genotyped in 24 F1 generation and 290 F2 generation animals. No SNPs were identified in the TNFRSF1A gene while 10 were located in the zyxin gene and 4 in the CD4 gene. None of the CD4 SNPs were correlated with disease resistance. However, by single marker association, several of the zyxin SNPs were significantly associated with elevated carotenoid or NO2- + NO3- concentrations. These results suggest that zyxin is the first candidate gene potentially associated with increased resistance to E. maxima in meat-type chickens.

7. Induction of immediate protective immunity against coccidiosis using dietary feeding with hyperimmune IgY egg yolk antibodies. Due to increasing concerns with prophylactic drug use, much interest has been devoted toward the development of drug-independent control strategies against coccidiosis. An alternative control strategy potentially applicable to intestinal diseases such as coccidiosis involves passive immunization using hyperimmune, pathogen-specific, parasite-neutralizing antibodies. The current study was conducted to evaluate the effect of dietary supplementation with a purified IgY fraction (Supracox, or SC) derived from egg yolk of hens hyperimmunized with Eimeria oocysts on protection against E. tenella and E. maxima infection. Dietary supplementation with SC significantly improved body weight gain of birds infected with E. tenella at 0.02% and 0.05% of diet, and body weight gain of birds infected with E. maxima at 0.05% of diet, compared with chicken given the unsupplemented control diet. Among the birds infected with E. tenella, oocyst shedding was reduced in birds fed 0.05% SC compared with controls. Lesion scores were significantly lower only in birds given 0.5% SC among the birds infected with E. maxima compared with the unsupplemented diet. We conclude that passive immunization of chickens with hyperimmune IgY antibodies provides protection against coccidiosis by reducing body weight loss, oocysts production, or intestinal lesions.

8. Development of dietary strategies to enhance local immunity to mucosal pathogens using plant-derived phytonutrients and investigation of immune mechanisms. Recent interest in the nutrition-based enhancement of host immunity in humans and animals has spawned new interest in discovering and exploring the potential immune-stimulating properties of naturally occurring dietary substances. For example, the seed oil of safflower (Carthamus tinctorius) is known to inhibit the production of pro-inflammatory cytokines by endotoxin (LPS)-stimulated human monocytes and safflower petals contain polysaccharides that activate macrophages in vitro. In the Asian countries, safflower seeds have been traditionally used as an herbal medicine to enhance resistance to infectious diseases and treat cancers. However, in spite of the well-known medicinal effects of safflower, few studies have been reported describing the effects of safflower on the immune system. Because of recent industry interest in using natural plant-derived products to enhance host defense against microbial infections in domestic animals and poultry, new studies were initiated to evaluate the 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 Oriental plum activated 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-¿, interleukin-8 (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.

9. Gene expression analysis to study local host-pathogen interaction in Salmonellosis. Salmonella enterica serovar Enteritidis (SE) is the principal etiologic agent of non-typhoidal salmonellosis, accounting for 18.6% of all cases in the U.S. SE outbreaks are associated with the consumption of contaminated foods such as poultry meats and egg products. Little information regarding the response of avian macrophages to Salmonella infection is known. In order to better define molecular and cellular pathways associated with host response to SE, our newly developed avian macrophage-specific cDNA microarray (AMM) was used to monitor the expression of thousands of gene transcripts in infected chicken macrophages. The significant alterations in macrophage gene expression following SE infection revealed SE-induced changes in genes associated with many functions that are potentially unique to avian macrophages as well as those conserved among vertebrate hosts. Out of the total 4,906 genes on the AMM, 269 different transcripts exhibited significant expression changes (> 2-fold, P < 0.001) with 86 being up-regulated and 183 down-regulated. Most of the transcriptional changes occurred at 5 hours post inoculation with more genes being repressed than activated. SE infection strongly induced the expression of pro-inflammatory immune mediators, pro-survival molecules, and translation factors. In contrast, SE infection suppressed the classical apoptosis pathways, cytoskeleton biogenesis, cell adhesion and migration, cell differentiation, and the initiation of cell-mediated immunity. Furthermore, SE infection triggered the expression of genes encoding the components of the endocytic pathway and repressed those coding for protein and ion transporters. These results clearly indicate that the expression of macrophage genes belonging to diverse functional classes is modulated by SE.


6.Technology Transfer

Number of Invention Disclosures Submitted1

Review Publications
Kim, D., Hong, Y.H., Park, M.S., Lamont, S.J., Lillehoj, H.S. 2008. Differntial immune-related gene expression in two genetically disparate chicken lines during infection by Eimeria maxima. Developmental Biology. 132:131-140.

Zhang, S., Lillehoj, H.S., Kim, C., Keeler, C.L., Babu, U., Zhang, M. 2008. Transcriptional response of chicken macrophages to salmonella enterica serovar enteritidis infection. Developmental Biology. 132:153-160.

Kim, C.H., Lillehoj, H.S., Keeler, C.L. 2008. Comparison of transcriptional responses from avian gut tissues after E. acervulina and E. maxima infections using cdna microarray technology. Developmental Biology. 132:131-140.

Lee, S., Lillehoj, H.S., Park, D., Hong, Y.H., Cho, S., Chun, H., Park, H., Lillehoj, E.P. 2008. Immunomodulatory properties of dietary plum on coccidiosis. Clinical Immunology. 31:389-402.

Lee, S., Lillehoj, H.S., Dalloul, R., Park, D., Hong, Y. 2008. Influence of pediococcus-based probiotic on coccidiosis in broiler chickens. Poultry Science. 86:63-66.

Cheeseman, J.H., Levy, N.A., Kaiser, P., Lillehoj, H.S., Lamont, S.J. 2008. Salmonella enteritidis-induced alteration of inflammatory CXCL chemokine messenger-RNA expression and histologic changes in the ceca of infected chicks. Avian Diseases. 52:229-34.

Hong, Y.H., Lillehoj, H.S., Park, D., Lee, S., Han, J., Shin, J., Park, M., Kim, J. 2008. Cloning and functional characterization of chicken interleukin-17D. Veterinary Immunology and Immunopathology. 126(1-2):1-8.

Cheeseman, J.H., Lillehoj, H.S., Lamont, S.J. 2008. Reduced nitric oxide production and INOS mrna expression in ifn-g stimulated chicken macrophages transfected with inos sirnas. Veterinary Immunology and Immunopathology. 125:375-80.

Lillehoj, H.S., Hong, Y.H., Kim, C. 2008. Quantitative genetic and functional genomics approaches to investigating parasite disease resistance and protective immune mechanisms in avian coccidiosis. Developmental Biology. 132:67-76.

Kim, Chul, H., Lillehoj, H.S., Bliss, T.W., Keeler, C.L., Hong, Y.H., Park, Dong, W., Yamage, M., Lillehoj, E.P., Min, W. 2008. Construction and Application of an Avian Intestinal Intraepithelial Lymphocyte cDNA Microarray for Gene Expression Profiling during Eimeria maxima Infection. Molecular Immunology. 124:341-354.

Hong, Y.H., Kim, E., Lillehoj, H.S., Lillehoj, E.P., Song, K. 2008. Association of resistance to avian coccidiosis with single nucliotide polymorphisms in the zyxin gene. Poultry Science. 88:562-466.

Li, S., Zhang, Z., Pace, L., Zhang, S., Lillehoj, H.S. 2008. Functions exerted by the virulence-associated type 3 secretion systems during Salmonella enterica serovar Enteritidis infection of chicken oviduct epithelial cells and macrophages. Avian Pathology. 38:97-106.

Lee, S., Lillehoj, H.S., Cho, S., Chun, H., Park, H., Lim, C., Lillehoj, E.P. 2009. Immunostimulatory effects of oriental plum (Prunus salicina Lindl). Comparative Immunology Microbiology and Infectious Diseases. 32(5):407-17.

Lillehoj, H.S., Kim, D., Kim, C., Lamont, S.J. 2008. Immune-related gene expression in two B-complex disparate, genetically inbred Fayoumi chicken lines that differ in susceptibility following Eimeria maxima infection. Animal Genetics. 87:433-443.

Del Cacho, E., Gallego, M., Lillehoj, H.S., Sanchez-Acedo, C. 2009. Avian Follicular and Interdigitating Dendritic Cells: Isolation and morphologic, phenotypic, and functional analyses. Veterinary Immunology and Immunopathology. 129:66-75.

Last Modified: 12/19/2014
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