Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: Marek's disease virus (MDV) causes lymphomas of T lymphocytes in chickens. The pathogenesis of infection is complex and many MDV genes play a role in the establishment of infection. We concentrated on the importance of splice variants of the pp38 gene, which is an early gene expressed during virus replication. Previously we had shown that full length pp38 affects the mitochondrial pathway in contrast with splice variants A and B. To determine the importance of these findings for the pathogenesis of infection we generated three new recombinant viruses using the MDV RB-1B BAC clone. In RB-1Bmut the splice acceptor sites were mutated so that only full length pp38 is expressed, RB-1BspA and RB-1BspB expressed only splice variant A and splice variant B, respectively. The wild-type RB-1B (RB-1BWT), RB-1Bmut, RB1BspA and RB1BspB were rescued in cell culture and the replication of these viruses was determined in chick kidney cells. In addition, Marek's disease (MD) susceptible P2a chickens were infected at 7 days of age to determine the importance of the splice variants for virus replication in vivo. P2a chickens were also inoculated to determine the importance for the development of tumors. The results of these experiments have been reported at the annual meeting of the American Association of Avian Pathologists in 2009 and a manuscript is being prepared for submission to a peer-reviewed journal. In addition the results have been reported at the annual meetings of the NE-1034 project and disclosed to the major breeders in the poultry industry. Patents or disclosures have not been filed. The most significant outputs have been the increased understanding about the role of pp38 in the pathogenesis of MD. For the first time it has been shown that MDV can affect the mitochondrial oxidative phosphorylation pathway and that the expression of full length pp38 is linked to rapid induction to tumor formation probably by increased virus replication compared to expression of only the splice variants. PARTICIPANTS: K. A. Schat, DVM, PhD, PI M.S. Piepenbrink, PhD, Postdoctoral Associate Priscilla H. OConnell, Research Support Specialist K.W. Jarosinski, PhD, Senior Research Associate TARGET AUDIENCES: Poultry veterinarians, poultry researchers and poultry producers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The following new findings are pertinent for the understanding of the role of pp38 in the pathogenesis of MD. RB-1BspA and RB-1BspB replicated at significant lower levels in chick kidney cell cultures then RB-1BWT and RB-1Bmut. The plaque sizes were also significantly smaller for the two splice variants than for RB-1BWT and RB-1Bmut. In vivo experiments also showed that the splice variants replicated to significantly lower levels in spleen cells than RB-1B WT and RB-1Bmut. The latter two viruses caused MD tumors in all birds within 4 to 5 weeks post infection. In contrast, chickens infected withRB-1BspA survived significantly longer although 60% of the birds developed tumors between 5 and 11 weeks post-infection. Chickens inoculated with RB-1BspB survived significantly longer than chickens infected with RB-1BsplA and had significantly fewer birds with tumors. These results demonstrated that expression of full length pp38 is important for early virus replication leading to an increase in virus-infected lymphocytes with therefore an increased likelihood that some of the infected lymphocytes transform. The funds provided by the grant provided parts of the salaries of Dr Piepenbrink and Mrs. Priscilla O'Connell which was essential for the research to proceed.
Publications
- Piepenbrink M.S., X. Li, P.H. OConnell, and K.A. Schat. Marek's disease virus phosphorylated polypeptide pp38 alters transcription rates of mitochondrial electron transport and oxidative phosphorylation genes. Virus Genes 39:102-112, 2009.
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Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: The results of this project have been disseminated through three broad avenues 1) National Scientific Meetings. Results were presented at the annual meetings of the American Association of Avian Pathologists (AAAP). Participants include scientist from the poultry industry in addition to scientists from research institutes and universities. In addition, every year a report was presented at the annual NE1016 meeting which includes representatives from the poultry industry. In 2005 some of the results were also presented at the National Breeders Round Table Conference. 2) Specialized meetings. Results were presented at 3 specialist Marek's disease conferences in 2004 (Oxford, UK), 2006 (Delaware, USA) and 2008 (Townsville, Australia). These meetings not only attract the top scientists in the field but also representatives from the poultry and vaccine industry. 3) General International Meetings. Information on Marek's disease and chicken infection anemia virus were presented at the World Veterinary Poultry Congress in Istanbul, Turkey (1985), several Congresses in Latin America, China, Thailand and meetings with industry representatives in India. It has to be emphasized that the poultry industry is a truly global industry and that information generated by this project benefits the global poultry industry. In addition collaborations were developed with several NE-1016 participants. The information on the control of chicken infectious anemia virus was also provided to producers of specific pathogen-free chickens. The impact of the research will benefit the poultry breeders by providing insight in the mechanisms of resistance to Marek's disease, which may lead to changes in the selection process of the primary chicken lines. The producers of specific-pathogen-free chickens have a better understanding of the problems associated with the control of chicken infectious anemia virus. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The outcomes for the NE-1016 project are divided into the parts: A) Marek's disease virus (MDV) and B) chicken infectious anemia virus (CAV). Specific-pathogen-free (SPF), major histocompatibility complex (MHC)-defined chickens resistant to MDV (N2a-line, MHC B21B21) and susceptible to MDV (P2a-line, MHC: B19B19) were used for both projects. A) Several MDV glycoproteins were identified that are recognized by vaccine-induced cytotoxic T lymphocytes (CTL) in N2a but not in P2a chickens. Similar data were obtained for natural killer (NK) cell responses, demonstrating that genetic resistance to MD is, at least in part, linked to innate and acquired cell-mediated immune responses. However, infection of N2a chickens with very virulent plus (vv+) strains of MDV resulted in a strong proinflammatory cytokine and nitric oxide response leading to similar levels of neurological lesions as in P2a chickens. It is therefore important to obtain a balance in genetic selection between strong cell-mediated immune responses and cytokine responses. We examined the importance of three genes for the pathogenesis of MDV using bacterial artificial chromosomes (BAC) to generate mutant strains. It was learned that RLORF4 of MDV contributes significantly to the pathogenicity of MDV. Deletion of both copies of RLORF4 resulted in increased plaque sizes and replication in cell culture which is typically associated with attenuation of MDV. In vivo experiments showed that the early pathogenesis and pathology of the deletion mutant was comparable to that of tissue culture attenuated MDV. The deletion mutant caused tumors in only a small percentage of chickens at 13 weeks of age, in contrast all chickens infected with the wild-type virus developed MD lesions between 4 and 6 weeks post-infection. We also showed that deletion of the first intron of the vIL8 gene resulted in significant attenuation. The function of the pp38 gene of MDV is controversial. We found that this gene produces two splice variants which are expressed in vivo, but after the initial virus replication phase. We found that expression of the full length pp38 protein, but not the spliced products interferes with the mitochondrial oxidative phosphorylation pathway in DF1 cells and QT35 cell lines. BAC clones have been generated expressing the spice variants or the mutated full length pp38 genes lacking the splice acceptor sites. BACs expressing the splice variants replicate poorly in cell culture. In vivo experiments are in progress to determine the contributions of the different pp38-derived proteins to the pathogenesis of MD. B) We demonstrated that co-infection of chickens with CAV and reticuloendotheliosis virus (REV) resulted in the absence of significant decrease in REV-specific CTL at 7 days post infection. This was the first evidence linking CAV infection directly to decreased pathogen-specific CTL responses. We also showed that the promoter/enhancer of CAV is positively controlled by estrogen, but negatively by COUP-TF1 (chicken ovalbumin upstream promoter transcription factor 1) and a protein binding to an E-box like element at the transcription initiation site of CAV.
Publications
- Miller, M. M., Jarosinski, K.W. and Schat, K.A. 2008. Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding {delta}EF1. J. Gen. Virol. 89:2998-3003.
- Schat, K.A. 2009. Chicken anemia virus. Curr. Top. Microbiol. Immunol. 331:151-184.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The phosphorylated polypeptide (pp)38 of oncogenic Marek's disease (MD) herpesvirus (MDV) is expressed during lytic infections in vivo and in vitro, but the functions have not been fully elucidated. The quail cell line QT-35, latently infected with MDV, was used to generate QTP32 in which stably transfected pp38 gene is expressed under control of a tetracycline (Dox) controlled promoter to examine possible functions of pp38. Previously, we found two new pp38 splice variants (A and B) in tetracycline-induced QTP32 cells and MDV-infected chickens (Li et al., 2006). To determine if the pp38 splice variants play a role in the metabolism of QTP32 cells, we developed additional inducible QT35 lines transfected with pp38 spliceA, splice B or pp38 in which the splice acceptor sites were mutated (MM). Expression of the protein was verified for the 3 cell lines. The metabolic activity of the cells transfected with splice variant A or B was not altered after induction with Dox, but
cells transfected with pp38MM had a significant increase in mitochondrial dehydrogenase activity without a corresponding increase in cell numbers. Because of the difference in the activity of mitochondrial succinate dehydrogenase an up-regulation in the number of transcripts of key mitochondrial enzymes in the oxidative phosphorylation pathway was expected after Dox-induction of QTP32 and QTPMM cell lines. To further investigate the potential changes, we developed real-time RT-PCR (qRT-PCR) assays for Japanese quail Cytochrome C oxidase-1 (Cox-1), Cytochrome B, and ATP synthase A chain. qRT-PCR revealed that Cox-I and ATP synthase A-chain were significantly down regulated in Dox-induced QTP32 and QTPMM cells. Transcription of cytochrome B was more variable but similar for all cells. Transcription was similar for Dox-induced and control cells expressing pp38 splice variants. To determine if enzyme activity was also influenced in a similar manner to transcription of mitochondrial genes,
ATP was measured in isolated mitochondria from Dox induced and control QTP32 cells. Significant differences in ATP were detected with decreased activity in Dox-induced cells. Vaccination to protect young chickens against CIAV-induced immunosuppression is an important aspect of modulating environmental factors that may influence immune responses. We examined the possibility of using immune complex vaccines to protect against subsequent challenge. This approach may mitigate some of the potential negative effects on the immune responses of replicating live CIAV vaccines and may lead to development of protective immunity in maternal antibody positive chicks. In several experiments we examined the optimal ratio of virus to antibody based on delayed virus replication and the absence of anemia. The results indicate that immune complexes may be useful to protect against challenge with field strains. We are currently testing the protective efficacy of this type of approach.
PARTICIPANTS: Marsh and Dietert no longer participate in the project. Micheal Piepenbrink worked on the project as a postdoctoral fellow under the direction of Schat. He was assisted by Pricilla O'Connell, research support specialist with Schat. Results of the studies were reported at the annual meeting of NE1016 and made available to the poultry industry. Funding for the vaccine study with the chicken anemia virus was also provided by US Egg and Poultry and they received appropriate information for distribution to the poultry industry. Schat chaired the annual meeting and was responsible for submission of the overall report of the NE1016 group for the year 2006-2007.
Impacts
Change in knowledge: The development of inducible cell lines allowed the functional study of Marek's disease proteins using in vitro approaches. The results indicate that pp38 alters cell metabolism. The work with chicken infectious anemia virus (CIAV) may lead to better vaccines to protect chickens against CIAV-induced immunossuppression.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
By comparing the immunotoxic outcomes from a variety of embryonic exposures involving environmental chemicals, drugs, and infectious agents, it has been possible to construct a sequence of seven critical windows though which disruption of immune development most commonly occurs (Dietert and Piepenbrink, 2006). Additionally, it is becoming clear that a predominant phenotype for embryonic-induced immunotoxicity exists and needs to be considered when desiring strategies for effective immune management (Dietert and Dietert, submitted). The most critical issues involve protection of normal thymic developmental processes while facilitating macrophage and dendritic cell maturation surrounding hatching. Understanding the pathogenesis of Marek's disease (MD) is important for the development of the next generation of vaccines, which probably will be needed in the next 5 to 10 year. The phosphorylated polypeptide (pp)38 of oncogenic MD herpesvirus (MDV) is expressed during lytic
infections in vivo and in vitro, but its functions remain unknown. The quail cell line QT-35, latently infected with MDV, was used to generate QTP32 in which pp38 is expressed under control of a tetracycline controlled promoter to examine possible functions of pp38. Induction of pp38 did not influence late MDV genes expression, induce apoptosis, or prevent apoptosis, but it enhanced mitochondrial dehydrogenase activity significantly. Two new pp38 splice variants were found in induced QTP32 cells, in additional in vitro systems and MDV-infected chickens. Polypeptides of 40 KD and 20 KD were detected by western blot using monoclonal antibody H19. These polypeptides were also produced in DF-1 cells transfected with a pp38 construct in which the splice acceptor sites had been mutated (Li and Schat, 2006). Transfection of QT-35 cells with full length pp38 in which the splice acceptor sites were mutated indicated that the full length pp38 gene is responsible for increased mitochondrial
activity (Piepenbrink et al, 2006). The development of lymphomas in MDV-infected chickens is a complex process in which many genes play a role. Last year we reported that deletion of both copies of RLORF4, located in the repeat regions flanking the unique long region of the MDV genome, significantly attenuated MDV. During these studies it became clear that this region, which also includes the genes for vIL-8 (essential for early virus replication), Meq and RLORF5a, produces multiple alternative spliced messages. Several new spliced messages were detected Analysis of expression in MDV-infected chickens showed that the RLORF5a/vIL8 and 3 of 4 RLORF4/vIL-8 transcripts were only expressed at 4 days post-infection. A number of these transcripts encoded vIL-8 exons II and III, suggesting that exon I may not be essential for vIL-8 functions. However, deletion of vIL-8 exon I decreased early viral replication and reduced tumor incidence similar to deletion mutants lacking the complete vIL-8
gene (Jarosinski and Schat 2007).
Impacts
It is critical for post-hatch health of the chicken that appropriate immune balance be achieved at hatching to meet infectious agent challenges and to mount desired vaccine responses. The present summary findings address both the risks of in ovo low-dose immunotoxicity as well as the opportunities to use in ovo adjuvants to improve the poultry health. Marek's disease (MD) is one of the target areas of the USDA-NRI competitive research grant program. The studies on the pathogenesis of MD indicate that the molecular basis for the pathogenicity remains only partially elucidated. Our studies on the role of several genes in the pathogenesis add to our understanding of the disease process and may ultimately lead to the development of improved vaccines.
Publications
- Jarosinski, K. W. and K.A. Schat. 2007. Multiple alternative splicing to exons II and III of viral interleukin 8 (vIL-8) in the Mareks disease virus genome: the importance of vIL-8 exon I. Virus Genes 34:9-22. 2007.
- Li, X., K.W. Jarosinski, and K.A. Schat. 2006. Expression of Mareks disease virus phosphorylated polypeptide pp38 produces splice variants and enhances metabolic activity. Vet. Microbiol. 117:154-168.
- Luebke, R.W., D.H. Chen, R. Dietert, Y. Yang, and M.I. Luster. 2006. Immune system maturity and sensitivity to chemical exposure. J. Toxicol. Environ. Health Part A 69:811-825.
- Piepenbrink, M., X. Li, P.H. O'Connell, and K.A. Schat. 2006. The role of oncogenic Mareks disease virus pp38 splice variants in the up-regulation of cellular metabolic activity. In: Proceedings 4th International Workshop of the MolecularPpathogenesis of Mareks disease virus.University of Delaware, newark, DE. p 3 (Abstract).
- Dietert, R.R. and M. J. McCabe, Jr. 2006. Lead immunotoxicity. In Luebke, R. and R. House (Eds) Immunotoxicology and Immunopharmacology. 3rd edition. CRC Press LLC. Boca Raton, FL. pp207-223.
- Dietert, R.R. and M.S. Piepenbrink. 2006a. Perinatal immunotoxicity: Why adult exposure- assessment fails to predict risk. Environ. Health Perspect. 114:477-483.
- Dietert, R.R. and M.S. Piepenbrink. 2006b. Lead and immune function. Crit. Rev. Toxicol. 36:359-385. 2006b.
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Progress 01/01/05 to 12/31/05
Outputs
Chicken T cell lines transformed by Mareks disease virus (MDV) were compared for relative sensitivity to lead nitrite-induced toxicity. Among the lines sampled, those double negative for the T cell markers CD4 and CD8 and carrying T cell receptor 2 (TCR2) were more sensitivity to lead than single positives (CD4+CD8- or CD4-CD8+) also with TCR2. The two single positives did not differ from each other in relative cell line sensitivity to lead. Genetic strain source for derivation of the MDV-transformed cell lines did not appear to be important in differential sensitivity T cells although these comparisons were limited. The work to better understand the linkage that exists between thymulin and selenium in their mediation of very similar effects on avian NK cells. This has led to a more careful examination of the control and regulation of each of these mediators. It has been clearly established that both of these mediators are closely linked to thyroid function and it is
the hypothesis of the current work that Se status can directly effect thymic function and thymulin production through the regulation of thyroidal activity. Dietary Se deficiencies result in significantly decreased plasma T3 levels, while specific vitamin E deficiencies produced no effect. Conversely, regardless of whether dietary vitamin E was adequate or not, plasma T4 levels were elevated in those animals fed the Se-deficient diets. This resulted in a highly significant positive correlation between Se status and plasma T3 levels and a significant negative correlation between Se status and T4 levels. To assess the role of RLORF4 in MDV pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The transcripts for MDV RLORF4 and RLORF5a were not longer present in the reconstitued viruses, while the adjacent vIL-8 transcript was unchanged. Reconstituted virus
from pRB-1BdeltaRLORF5a (rRB-1BdeltaRLORF5a) produced similar plaque sizes when compared to parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1BdeltaRLORF4 (rRB-1BdeltaRLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was increased when compared to rRB-1B or rRB 1BdeltaRLORF5a using quantitative (q)PCR assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days post-inoculation (pi), as determined by virus isolation and qPCR assays. At 14 days pi, virus isolations from chickens infected with rRB 1BdeltaRLORF4 were comparable to highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. rRB-1BdeltaRLORF5a produced tumors that were similar in number and kinetics to rRB-1B in P2a chickens. In stark contrast, none of the chickens inoculated with rRB-1BdeltaRLORF4 died up to 13 weeks pi; however, two chickens had tumors at termination of the
experiment. Additional experiments using deletion mutants in which only one copy of RLORF4 was deleted showed that both copies need to be deleted to attenuate MDV.
Impacts
Since the chicken must achieve immune balance at hatching to meet disease challenges and mount desired vaccine responses, the current results on lead toxicity are an important step in understanding the risks and optimizing the in ovo environment to improve the poultry health. The results of the studies on thymulin and selenium demonstrated that innate immunity may be enhanced by dietary deficiencies and that these treatments may affect immune development and function. The work on the characterization of Mareks disease virus genes adds important information to the understanding of the genes involved in pathogenesis of infection and tumor development. The deletion of RLORF4 reduced the pathogenicity significantly, but unfortunately not completely.
Publications
- Dietert, R.R. 2005. New developments in the assessment of developmental immunotoxicology. J. Immunotoxicology 2 (4): 185-189.
- Dietert, R.R. and Piepenbrink, M.S.. 2005. Environment-immune interactions: biomarkers for breeding healthier chickens. Proceeding of the 54th Annual National Breeders Roundtable. pp. 15-35.
- Dietert, R.R. and Piepenbrink, M.S. 2005. The managed immune system: Protecting the womb to delay the tomb. BELLE Newsletter 13 (1): 7-11.
- Miller, M.M., Jarosinski, K.W., and Schat, K.A. 2005. Positive and negative regulation of chicken anemia virus transcription. J. Virol. 79:2859-2868.
- Jarosinski, K.W., Osterrieder, N., Nair, V.K., and Schat, K.A. 2005. Attenuation of Mareks disease virus (MDV) by deletion of open reading frame RLORF4 but not RLORF5a. J Virol 79, 11647-11659.
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Progress 01/01/04 to 12/31/04
Outputs
Mareks disease (MD) virus (MDV) strains are divided into pathotypes based on their ability to induce disease in genetically defined, vaccinated chickens. We found that very virulent plus (vv+) strains cause a strong proinflammatory response in spleens and in brain tissues of genetically susceptible and resistant chicken lines. Using quantitative real-time PCR and RT-PCR assays, we were able to differentiate between virulent and vv+ MDV strains during the first 10 days post infection. This approach offers a major advantage over previous pathotyping methods, because the read-out does not rely on the induction of tumors. Isolation of MDV remains problematic especially because many investigators use inappropriate cell cultures. We revisited this issue and confirmed that chicken kidney cells are superior for virus isolation compared to chicken embryo fibroblasts and chicken embryo kidney cells. We used two quail cell lines for infection with MDV. One of the cell lines,
CU447, was susceptible to persistent infection, in which cells produced late viral proteins, e.g., glycoprotein B, without inducing cytopathic effects. The heavy metal, lead, induces different early-life immune alterations depending upon gender. We examined the potential role of embryonic hormone balance on lead-induced developmental immunotoxicity following sequential in ovo exposure of chickens to testosterone (on embryonic day 8) and lead (on embryonic day 12). Developing thymocytes from embryonic day 20 females were assessed using cell surface markers analyzed by flow cytometry (CD3, CD4, CD8). Combined exposure to testosterone and lead significantly altered thymocyte subpopulations whereas exposure to either testosterone or lead alone at the doses employed produced no detectable alteration among the thymocytes. In complementary in vitro studies, lead-induced toxicity of chicken T lymphocyte cell lines suggests that a differential susceptibility may exist among the lines. We have
examined the effect of dietary supplementation with selenium-containing amino acids (Se-AA) on natural killer (NK) cell function. This was assessed both in splenocyte NK preparations and in NK preparations of cells harvested from the respiratory tract of infectious bronchitis virus (IBV)-infected chickens. The NK response of splenocytes was assayed and these treatments were compared to the NK cells from animals fed an unsupplemented diet. NK activity was significantly increased by Se-AA administered over 4 wks and these treatments also increased responsiveness of NK cells to interferon-gamma. The effect of treatment on NK cells harvested from the lungs of IBV-infected chickens was examined. Almost a 3-fold increase in NK activity was observed in supplemented chicks and responsiveness to interferon-gamma was enhanced by Se-AA supplementation. The protective effects of this supplementation were also apparent from the growth data.
Impacts
Improved methods to isolate Mareks disease virus (MDV), to pathotype and characterize lesion development of new strains of MDV are important to further improve protection against this disease.Hormone balance impacts the effect of lead on developing thymocyte populations and may explain, in part, differential gender susceptibility. The results of supplementing diets with selenium-containing amino acids resulted in enhanced innate immunity, which may increase resistance to viral infections.
Publications
- Chang, W.-P., Combs, G.F., Jr., Scanes, C.G. and Marsh, J.A. 2005. The effects of dietary vitamin E and selenium deficiencies on plasma thyroid and thymic hormone concentrations in the chicken. Devel. Comp. Immunol. 29:265-274.
- Dietert, R.R., Lee, J-E., Hussain I., and Piepenbrink, M. 2004. Developmental immunotoxicology of lead. Toxicol. Appl. Pharmacol. 198:86-94.
- Hussain, I., Piepenbrink, M.S. and. Dietert, R.R. 2004. Impact of in ovo-administered lead and testosterone on developing female thymocytes. J. Toxicol. Env. Health (Part A). in press.
- Hussain, I., Piepenbrink, M. and Dietert, R. 2004. Effect of testosterone and lead on T cell maturation in the developing thymus. Poultry Sci. 83 (Suppl. 1):148. (Abstract).
- Jarosinski, K.W., Njaa, B.L., OConnell, P.H. and Schat, K.A. 2005. Pro-inflammatory responses in chicken spleen and brain tissues after infection with very virulent Mareks disease virus. Viral Immunol. 18: In press.
- Li, X. and Schat, K.A. 2004. Quail cell lines supporting replication of Mareks disease virus serotype 1 and 2 and herpesvirus of turkeys. Avian Dis. 48:803-812.
- Oliver, M.A. and Marsh, J.A. 2003. In vitro thymulin treatments enhance avian lung natural killer cell cytotoxicity in response to infectious bronchitis virus. Interntl. Immunopharmacol. 3:107-113.
- Schat, K.A. 2005. Isolation of Mareks disease virus: revisited. Avian Pathol. 34. In press.
- Yunis, R., Jarosinski, K.W. and Schat, K.A. 2004. Association between rate of viral genome replication and virulence of Mareks disease herpesvirus strains. Virology 328:142-150.
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Progress 01/01/03 to 12/31/03
Outputs
The effect of thymulin injections(10 ng/100 BWt)on virus clearance from the respiratory tract of birds infected with IBV was examined in an extension of our studies on the enhancement of innate immunity in response to viral infection. These treatments significantly reduced the harvest of viable virus from the lungs. This supports the conclusion that the enhanced NK activity observed following thymulin supplementation translates into enhanced viral resistance within the respiratory tract. Our most recent studies have tested the hypothesis that supplementation with the trace nutrient, selenium (Se) may produce similar effects. The effect of thymulin, selenocysteine, or the vehicle control on chicken NK cell activity was examined. Se, given in the form of a selenocysteined was as effective as thymulin in enhancing the cytotoxic activity of NK cells. One of the primary effects of this treatment appears to be to make NK cells more responsive to chicken IFN-gamma. Changes
in cell population following in ovo exposure to lead were examined. Doses employed were sufficient to produced depressed cell mediated immunity post hatching. An examination of thymus cell populations for CD markers (4, 8, T cell receptors) did not detect overt changes shortly following the exposure at Embryonic Day 12. The study will continue as additional markers are utilized.The studies on Marek's disease virus (MDV) were directed toward the recognition of proteins by cytotoxic T cells (CTL) and natural killer cells (NK cells) and to understanding the virulence factors of MDV.Several glycoproteins are recognized by CTL from genetically resistant (N2a) and susceptible (P2a) lines.Glycoprotein (g)B and gI are recognized by CTL from both lines, while CTL from the resistant line recognizes also gC, gK and to a lesser extend gH, gL, and gM.These glycoproteins are not recognized by CTL from the P2a line, but P2a CTL do lyse cells expressing gE. We tested the hypothesis that nitric oxide
(NO) induction after infection with very virulent (vv)+ strains of MDV would be decreased, because earlier studies using the virulent (v) JM-16 strain had indicated that the induction of NO reduced viremia levels. It was shown that infection with vv+ strains caused a very high level of NO production, which may be responsible for the induction of neurological lesions. The studies with chicken anemia virus (CAV)indicated that co-infection of CAV with other pathogens, MDV and reticuloendotheliosis virus (REV), causes the absence of CTL to MDV or REV 7 days post infection.If chickens have maternal antibodies to CAV, CAV will nor replicate and CTL to MDV or REV can be detected.
Impacts
These data demonstrate that either hormonal or dietary approaches have the potential for enhancing innate immunity to a virus-mediated infection such as IBV. This has significance for the possibilities of modulating or enhancing the activity of those cells that serve as the first line of defense. The information demonstrates that significant immune response capabilities arise following low level in ovo exposure to the heavy metal, lead. To date the nature of the developmental changes at the level of the thymic cell population has not been identified using the most standard markers but cellular characterization continues. The results with chicken anemia virus clearly indicate that infection with this pathogen can have a major impact on infectious diseases that require cytotoxic T cells for recovery.
Publications
- Lee, J-E., and Dietert, R.R. 2002. Developmental immunotoxicology of lead: impact on thymic function. Birth Defects Res. (Part A Clinical and Molecular Teratology. 67: 861-867.
- Lee,J-E., Naqi, S.A., Kao, E., and Dietert R.R. 2002. Embryonic exposure to lead: comparison of immune and cellular changes in unchallenged and virally-stressed chickens. Arch. Toxicol. 75: 717-734.
- Orringer, D.A., Staeheli, P., and Marsh, J.A. 2002. The effects of thymulin on macrophage responsiveness to interferon-(.Develop. Comp. Immunol.26:95-102.
- Merlino, P., and Marsh, J.A. 2002. The enhancement of avian NK cell cytotoxicity by thymulin is not mediated by the regulation IFN-(production. Develop. Comp. Immunol. 26:103-110.
- Oliver, M.A., and Marsh, J.A. 2003. In vitro thymulin treatments enhance avian lung natural killer cell cytotoxicity in response to infectious bronchitis virus. Interntl. Immunopharacol. 3:107-113.
- Dietert, R.R., Lee, J-E. and Bunn T.L. 2002. Developmental immunotoxicology: emerging issues. Hum. Exp. Toxicol. 21: 479-485.
- Dietert, R.R. and Lee, J-E. 2003. Toxicity of lead to the developing immune system. In (Ed.) Holladay, S.D. Developmental munotoxicology. Taylor and Francis, NY. in press.
- Dietert, R.R., Lee, J-E., Hussain, I. and Piepenbrink, M. 2003. Developmental imunotoxicology of lead. Toxicol. Appl. Pharmacol. in press.
- Heggen-Peay, C. L., Qureshi, M.A., Edens, F.W., Sherry, B., Wakenell, P.S.,O'Connell, P.H., and Schat, K.A. 2002. Isolation of a reovirus from poult enteritis and mortality syndrome (PEMS) and its pathogenicity in turkey poults. Avian Dis 46:32-47.
- Heggen-Peay, C. L., Cheema, M. A., Ali, R. A., Schat, K. A., Qureshi, M.A. 2002. Interactions of Poult Enteritis and Mortality Syndrome-associated reovirus with various cell types in vitro Poultry Sci 81:1661-1667.
- Jarosinski, K.W., Yunis, R.,O'Connell, P. H., Markowski-Grimsrud, C.J., and Schat, K.A. 2002. Increased nitric oxide in chickens resistant to Marek's disease, but not susceptible chickens infected with Marek's disease virus (MDV), and in chickens infected with very virulent + MDV. Avian Dis 46:636-649.
- Markowski-Grimsrud, C.J., and Schat, K.A. 2002. Cytotoxic T lymphocyte responses to Marek's disease herpesvirus-encoded glycoproteins. Vet Immunol Immunopathol 90:133-144.
- Markowski-Grimsrud, C.J., Miller, M.M., and Schat, K.A. 2002. Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus. J Virol Meth 101:135-147.
- Jarosinski, K.W., O'Connell, P.H., and Schat, K.A. 2003. Impact of deletions within the Bam HI-L fragment of attenuated Marek's disease virus on vIL-8 expression and the newly identified transcript of open reading frame LORF4.Virus Genes 26:255-269.
- Markowski-Grimsrud, C.J., and K.A. Schat 2003. Infection with chicken anemia virus impairs the generation of pathogen-specific cytotoxic T lymphocytes.Immunology 109:283-294.
- Garcia Camacho, L., Schat, K.A., Brooks, R., Jr.,Bounous, D.I. 2003. Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens. Vet Immunol Immunopathol 95:145-153.
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