1a. Objectives (from AD-416):
Objective 1: Develop immune reagents to detect host effector molecules controlling immune responses and determine the role of host effector molecules in disease resistance to discover biological determinants associated with disease resistance to infectious diseases of poultry. Sub-objective 1a: Develop immune reagents to detect host effector molecules controlling immune responses to NE. Sub-objective 1b: Determine the role of host effector molecular in NE disease resistance. Objective 2: Discover effective immune intervention strategies to prevent and control infectious diseases of poultry through use of nutrients as immune modulators to enhance gut health and develop strategies for their use in increasing production efficiency. Identify effector molecules of innate immunity and develop strategies for their use in reducing economic losses associated with enteric diseases of poultry. Sub-objective 2a: Use nutrients as immune modulators to enhance gut health and develop strategies for their use in increasing production efficiency. Sub-objective 2b: Identify effector molecules of innate immunity and develop strategies for their use in reducing economic losses associated with enteric diseases of poultry.
1b. Approach (from AD-416):
Develop immune reagents to detect host effector molecules controlling immune responses. Determine the role of the host effector molecules in disease resistance. Discover biological determinants associated with disease resistance to infectious diseases of poultry.
3. Progress Report:
For Objective 1, progress was made in developing new poultry immune reagents that detect cell surface glycoproteins of major immune cells which enable the isolation and biological characterization of two major cell populations of the poultry immune system for detailed immunological research. Two groups of mouse hybridomas that secrete mouse monoclonal antibodies specific for dendritic cells (CD80) and regulatory T cells (CD25) of the poultry immune system were developed and characterized. The CD80 marker successfully isolated chicken dendritic cells, while progress was made with the immunological characterization of CD25. Since these are the first group of immune reagents which detect chicken dendritic cells and regulatory T cells, these reagents will be useful for basic and applied research in both diseased and normal chickens. Progress in assessing gene expression of avian innate immune molecules, such as avian beta-defensin messenger RNA, was made using a necrotic enteritis (NE) disease model in two genetically disparate commercial broiler chicken lines, designated as R and C. Although the exact nature of interactions between defensins and cytokines in determining the outcome of host innate immune responses to the pathogens of NE remains to be investigated, the observation that differences in gene expression levels of beta-defensins and pro-inflammatory cytokines are associated with NE disease susceptibility and resistance could lead to genetic selection strategies to improve NE disease resistance in broilers. For Objective 2, alternative strategies to mitigate the use of antibiotics have been developed and these include dietary and recombinant vaccination strategies. Cinnamaldehyde (CINN) is a constituent of cinnamon that has been traditionally used to treat human diseases, and possesses antifungal, antipyretic, antioxidant, antimicrobial, and larvicidal activities. Our previous study showed that dietary feeding of CINN along with carvacrol and capsicum to day-old chickens improved gut immunity against coccidiosis and significantly altered the level of gene expression in intestinal intraepithelial lymphocytes. In vivo trials demonstrated that CINN-fed chickens showed significant improvement in body weight gains following challenge infection with live parasites of Eimeria, and improvement in various aspects of host immune parameters, including cytokine levels. Progress has been made in developing and improving the vaccine efficacy of recombinant proteins against coccidiosis and NE. In collaboration with a private company, the efficacy of aqueous nanoparticle-based mucosal delivery adjuvant in combination with a parasite subunit protein was demonstrated against avian coccidiosis. Progress was made in the identification of macrophage migration inhibitory factor, a proinflammatory cytokine that plays an important role in host defense against a variety of microorganisms including protozoan parasites.
1. Differential gene expression profiles of avian beta-defensins (AvBD) in commercial broiler chickens afflicted with necrotic enteritis (NE). Globally, the economic loss due to NE is estimated to cost the U.S. $2 billion annually, largely due to medical treatments and impaired growth performance. Recently, NE has re-emerged as a significant problem as a result of restricted use of in-feed antibiotics, high-density housing conditions, and re-use of litter. There is an urgent need to develop alternative disease control strategies not only to control, but also to prevent NE. Better understanding of host-pathogen, as well as pathogen-pathogen (Clostridium-Eimeria) interactions in NE will be required to realize these goals. In order to better characterize innate immune molecules which are involved in host response to C. perfringens, a causative pathogen of NE, changes in the expression levels of avian defensin messenger RNAs were evaluated in two genetically disparate commercial broiler chicken lines, R and C. Among the 14 avian defensin types examined, there was a tissue-specific expression of AvBD transcripts. The broiler chicken lines showed differential gene expression patterns of AvBD transcripts with R line chickens showing higher expression levels than C line. Both chicken lines showed enhanced gene expression levels of pro-inflammatory cytokines in the intestine whereas interleukin-17A was significantly increased only in the intestine of R line chickens following NE infection. Although the exact nature of interactions between defensins and cytokines in determining the outcome of host innate immune responses to the pathogens of NE requires further investigation, the differences in gene expression levels of beta-defensins and pro-inflammatory cytokines in the intestine, crop and spleen could explain the genetically determined disease resistance and susceptibility to NE in two commercial broiler chickens.
2. Identification of dendritic cell-associated antigen and dendritic cell presentation of parasite peptide. Avian coccidiosis is a complex intestinal disease of major economic importance in chickens that is caused by multiple species of the protozoan, Eimeria. Conventional disease control methods have relied on prophylactic administration of drugs with anticoccidial activity, or on vaccination with live or attenuated parasites. There is an urgent need to develop a field vaccine against avian coccidiosis that is safe and effective against all relevant parasites. One novel approach to vaccination against coccidiosis is to exploit dendritic cells (DCs) as an initial step in the development of a second-generation coccidiosis vaccine. Previous work demonstrated that a potent T cell-dependent protective immune response against E. tenella infection in chickens was generated by in vivo administration of antigen-loaded DC exosomes. These DC exsosomes, when used to vaccinate young birds against coccidiosis, also were effective against live parasite infection. Immune tissues of exosome-immunized and infected chickens had increased numbers of cells secreting interleukin (IL)-2-, IL-16, and interferon gamma, as well as greater antigen-stimulated proliferative responses, and higher numbers of antigen-reactive immunoglobulin-producing cells following in vitro stimulation with E. tenella, E. maxima, or E. acervulina antigens. By contrast, the numbers of cells secreting the cytokines IL-4 and IL-10 were diminished in immunized and infected chickens. Chickens immunized with antigen-loaded DC exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the non-immunized/infected controls. These results suggest that successful field vaccination may be possible against avian coccidiosis using non-viable, DC-derived exosomes.
3. Comparative gene expression analysis of intestinal lymphocytes to compare innate immunity induced by three major Eimeria species. Avian coccidiosis is caused by seven species of Eimeria protozoa (E. acervulina, E. maxima, E. tenella, E. mitis, E. necatrix, E. praecox, and E. brunetti) that differ in pathogenicity and immunogenicity. Although prophylactic chemotherapy has been traditionally used for disease control, the emergence of drug-resistant parasites and legislative bans on the use of in-feed antibiotic growth promoters encourages the development of alternative coccidiosis control strategies. Functional genomics and bioinformatics technologies were used to study host-pathogen interactions and to compare the global gene transcripts of the three species of coccidia that most commonly infect commercial poultry. The results of bioinformatic analysis of gene expression data showed that relative expression levels of immune- and non-immune-related messenger RNAs in chicken intestinal intraepithelial lymphocytes experimentally infected with E. acervulina, E. maxima, or E. tenella, were relatively similar at 1, 2, and 3 days post-primary infection. By contrast, E. tenella elicited the greatest number of altered transcripts at 4, 5, and 6 days post-primary infection, and at all time points following secondary infection. With all three coccidia, biological pathway analysis identified the altered transcripts as mainly belonging to the categories of "Disease and Disorder" and "Physiological System Development and Function." Sixteen intracellular signaling pathways were identified from the differentially expressed transcripts following infection, with the greatest significance observed following E. acervulina infection. This new information will expand our understanding of host-pathogen interactions in avian coccidiosis and contribute to the development of novel disease control strategies.
4. Identification, molecular cloning, and protein expression of two immunogenic proteins of Clostridium perfringens. Necrotic enteritis (NE) and gangrenous dermatitis (GD) are among the most important Clostridial poultry diseases in the U.S. NE is caused by C. perfringens type A, a Gram-positive, anaerobic, spore-forming, rod-shaped bacterium, whereas GD is primarily associated with infection by C. perfringens type A or C. septicum with underlying predisposing factors. To develop recombinant vaccine against C. perfringens, we used immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections to identify two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO). These two genes were also expressed in Escherichia coli and their corresponding recombinant proteins were purified for use in immunoassays to detect C. perfringens-specific antibodies. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry. Ongoing studies are directed at evaluating whether these, or other, purified clostridial proteins, either alone or in combination with novel adjuvants, can serve as potential subunit vaccines for NE and/or GD.
5. Development of alternative strategy to enhance gut immunity and to mitigate the use of antibiotics using dietary phytonutrients. Although widespread use of antibiotic-based growth promoters has improved the efficiency of worldwide poultry production, there is an increasing interest in developing alternative strategies to antibiotics to control infectious diseases in livestock and poultry due to the emergence of drug-resistant pathogens. We investigated using dietary phytogenics to enhance poultry immunity using avian coccidiosis and necrotic enteritis as disease models. Phytogenics are a group of natural growth promoters derived from herbs, spices or other plants, and many medicinal foods and herbal products are highly effective in enhancing host defense against microbial infections. Previous studies from our laboratory showed that phytogenics augment host immunity against infectious agents through their ability to alter gene expression. Cinnamaldehyde (CINN) is a constituent of cinnamon that is widely used as a flavoring compound and has been traditionally used to treat human diseases, including inflammatory diseases. CINN has been reported to possess antioxidant, and antimicrobial activities, as well as being able to modulate T cell differentiation. In chickens fed a diet supplemented with CINN, the levels of interleukin (IL)-1 beta, IL-6, IL-15 and interferon-gamma transcripts in intestinal lymphocytes were 2- to 47-fold higher compared with chickens given a non-supplemented diet. Furthermore, dietary CINN attenuated E. acervulina and E. maxima-induced bodyweight loss, decreased E. acervulina oocyst shedding, and increased E. tenella-specific antibody responses compared with the non-supplemented control diet.
6. Dietary phytonutrients enhance protective vaccinal immunity against Eimeria tenella infection. Eimeria recombinant protein vaccines, while less efficacious than coccidiostats and live vaccines, offer an alternative control strategy against avian coccidiosis. For example, Eimeria profilin which we previously identified in the merozoites of the parasite increased antigen-specific proliferation of chicken spleen lymphocytes and augmented cytokine production by splenic and intestinal lymphocytes. Furthermore, subcutaneous immunization of chickens with recombinant profilin expressed in E. coli increased protective immunity to subsequent challenge infection by virulent E. acervulina. However, the effectiveness of profilin immunization was dose-dependent, such that only partial protection against challenge infection was generated at low vaccine doses (micrograms). Therefore, we investigated using dietary phytonutrients with proven immune enhancing property to improve the effectiveness of recombinant protein vaccines by co-administration of the vaccine with adjuvants or recombinant chicken cytokines, or by feeding plant-based phytonutrients. Results showed that profilin-immunized chickens fed the supplemented diets and infected with E. tenella had increased body weight gains, greater anti-profilin antibody levels, heightened profilin-induced lymphocyte proliferation, altered intestinal cytokine transcript levels, and augmented percentages of PBL subpopulations, compared with immunized and infected animals fed with a non-supplemented diet. Although the underlying mechanisms that are responsible for the dietary immune enhancing effects remain to be determined, these results provide the foundation for further studies to identify a safe and effective alternative to prophylactic medication for control of avian coccidiosis in commercial poultry production facilities.
7. Mechanism of adaptive immunity mediated by intracellular parasites has been identified. Egression, which describes the mechanism by which intracellular parasites like Eimeria employs to exit from parasitophorous vacuoles and host cells, plays a very important role in the parasite life cycle and is central to Eimeria propagation and pathogenesis. We found that premature egression of sporozoites from E. tenella-infected primary chicken kidney cells, or from chicken peripheral blood mononuclear cells, occurred when the cells were co-cultured in vitro with spleen lymphocytes from E. tenella-infected chickens, but not when co-cultured with splenocytes from uninfected chickens. Furthermore, Eimeria-specific antibodies and cytokines derived from E. tenella-primed B and T lymphocytes, respectively, also were capable of promoting premature egress of sporozoite from infected host cells. Both egressed host cells and parasites were viable, although the latter had ability to some extent. Our proposed immune-mediated mechanism of premature parasite egression represents a novel process that is relevant to a highly immunogenic pathogen such as Eimeria. These results suggest a novel, immune-mediated mechanism that the host exploits to interrupt the normal Eimeria life cycle in vivo, and thereby blocks the release of mature parasites into the environment. Future studies to further characterize host and parasite factors that signal the egress will lead to the development of novel therapeutic approaches to interrupt the normal Eimeria life cycle in vivo.
8. Probiotic-mediated immunomodulation of poultry innate immunity to enteric pathogens. Enteric diseases in commercial poultry cause significant losses in productivity, increased mortality, and contamination of food products for human consumption. Direct-fed microbials (DFMs), also known as probiotics, have been successfully used to improve the balance of gut microflora. In particular, certain strains of Bacillus subtilis have been selected as candidate DFMs on the basis of their in vitro inhibitory effect on avian pathogenic bacteria. Dietary supplementation with a Bacillus-based DFM was shown to improve feed conversion in poultry and to beneficially alter the gastrointestinal microflora to reduce colonization by avian pathogens Escherichia coli and Clostridium perfringens type A. In this reporting period, the effect of dietary Bacillus-based DFMs (eight single strain- and one multiple-strain product [AVICORR™]) on growth performance, intestinal lesions, and innate and acquired immunities was evaluated in broiler chickens following Eimeria maxima (EM) infection. EM-induced reduction of body weight gain and intestinal lesions were significantly decreased by addition of two DFMs (15AP4 or Bs27) into broiler diets compared with EM-infected control birds. Finally, all experimental diets increased splenocyte mitogenesis in infected broilers compared with the non-supplemented and infected controls. In summary, dietary B. subtilis-based DFMs reduced the clinical signs of experimental avian coccidiosis and increased various parameters of immunity in broiler chickens in a DFM strain dependent manner.
9. Development of recombinant vaccines as antibiotics-free alternative strategies to mitigate the gut damages caused by avian intestinal pathogens. Studies using recombinant antigens common to multiple coccidia species to stimulate broad-spectrum immunity have shown limited success, mainly because of their low antigenicity, inadequate stimulation of protective host immunity, and/or restricted expression during the parasite life-cycle. Therefore, we have investigated two different adjuvant systems to increase immunogenicity of recombinant vaccine. In collaboration with a private company, a series of water-in-oil emulsion adjuvants were evaluated for their effectiveness in promoting protective immunity against avian coccidiosis following vaccination with profilin, a protein that is expressed by multiple Eimeria species at all asexual stages. The results of these studies indicated that vaccination with the E. acervulina profilin subunit vaccine in combination with MontanideTM adjuvants enhances protective immunity against avian coccidiosis. In another study, which was carried out with a private company, the effects of a novel adjuvant composed of Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC) on protective immunity against avian coccidiosis following immunization with an Eimeria recombinant protein was investigated. Chickens immunized with profilin plus QCDC showed increased body weight gains and decreased intestinal lesion scores compared with the profilin only, but no differences were found in fecal oocyst shedding among the three groups. In summary, this study provided scientific evidence to document the immunoenhancing activities of novel adjuvants in poultry.Lee, S.H., Lillehoj, H.S., Jang, S., Lee, K., Lillehoj, E., Yancy, R.J., Dominowski, P., Kim, D. 2010. Evaluation of novel adjuvant Eimeria profilin complex on intestinal host immune responses against live E. acervulina challenge infection. Avian Diseases. 28(39):6498-6504.