Browsing by Subject "chicken"
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Item Differential gene expression in innate immunity between commercial broilers and layers(2009-06-02) Shen, ShixueTremendous improvements have been achieved in growth rates and feed efficiency in commercial broiler birds. However, fast growth broilers generally show weak immune competence and disease resistance. Innate immunity is the first line of defense providing immediate killing effects to a broad range of infectious pathogens and limiting infections to a minimum at an early stage before the activation of more specific adaptive immunity. Acute phase proteins (APPs), defensins and Toll-like receptors (TLRs) are all important innate immune molecules functioning from recognition to killing the foreign microbes. Tibial dyschondroplasia (TD) is one chicken disease associated with rapid growth in broilers. The objective of this research was to study the differential expression of innate immune related genes in liver and spleen tissues between commercial broilers and layers with the stimulation of lipopolysaccharide (LPS). Also, this study investigated mechanisms involved in the pathogenesis of TD at molecular levels. This study first identified and annotated nineteen new chicken APPs genes from the chicken genome draft with bioinformatics tools. Using a relative quantitative real-time RT-PCR method, the expression levels of all thirty-one APPs, thirteen defensins and eight TLRs genes were systemically investigated at the transcriptional level at three time points (0-, 3-, 8-hour) with the challenge of LPS. This study showed that broiler birds generally expressed significantly lower levels of all three families of innate immune related genes than layers and the inductive extent of these genes are generally smaller in broilers too. Close investigation of some important signaling transcription factors (NF-kB and IRF-3) and cytokine (IL-6) also reached the same conclusion. This study revealed that the inadequate expression of deiodinase type 2 (DIO2) contributed to the pathogenesis of TD in rapid growth broilers. All of the experimental results solidly validate the hypothesis that a compromised innate immune response or weak disease resistence is associated with fast growth broiler birds.Item Evaluation of Sindbis-M2e Virus Vector as a Universal Influenza A Vaccine(2012-10-19) Vuong, ChristineAlthough avian influenza virus (AIV) infections in domestic poultry are uncommon, transmission of avian influenza from wild waterfowl reservoirs does occur. Depopulation of the infected flock is the typical response to AIV outbreaks in domestic chicken production, causing a loss in profits and accumulation of unexpected expenses. Because it is impossible to know which of many virus subtypes will cause an outbreak, it is not feasible for the U.S. to stockpile vaccines against all possible avian influenza threats. Currently, the U.S. does not routinely vaccinate chickens against influenza due to the inability to differentiate infected from vaccinated animals (DIVA), which would place limitations on its trade markets. A Sindbis virus vector expressing the PR8 influenza strain's M2e peptide was developed as a potential universal DIVA vaccine. M2e is a conserved peptide amongst influenza A viruses; M2e-specific antibodies induce antibody-dependent cytotoxicity or phagocytosis of infected cells, reducing production and shedding of AIV during infection. In this study, chickens were vaccinated at one-month-of-age with parental (E2S1) or recombinant Sindbis viruses expressing the PR8 M2e peptide (E2S1-M2e) by subcutaneous or intranasal routes at high (106 pfu) or low (103 pfu) dosages. Chickens were boosted at 2-weeks post-initial vaccination using the same virus, route, and dosage, then challenged with low pathogenic H5N3 AIV at 0.2 mL of 106/mL EID50 2-weeks post-boost. Serum samples were collected at 1-week and 2-weeks post-vaccination, 2-weeks post-boost, and 2-weeks post-challenge and screened for PR8 M2e-specific IgY antibody production by ELISA. Both high and low dose subcutaneously, as well as high dose intranasally vaccinated E2S1-M2e groups produced significantly higher levels of PR8 M2e-specific IgY antibodies as early as 1-week post-vaccination, while the uninoculated control and E2S1 groups remained negative for all pre-challenge time points. M2e-specific IgY antibodies capable of binding the challenge H5N3 M2e peptide were detected in groups with existing vaccine-induced M2e-specific antibodies pre-challenge, suggesting antibody M2e cross-reactivity. After challenge, all groups developed M2e-specific IgY antibodies and high HI titers, verifying successful AIV infection during challenge and production of hemagglutinin-specific antibodies. Viral shedding titers 4-days post-challenge were used to measure vaccine efficacy and were similar amongst all groups. Microneutralization assay results confirmed that post-boost serum samples, containing only M2e-specific antibodies, were unable to neutralize AIV in vitro. Although the E2S1-M2e vaccine was capable of producing high levels of M2e-specific IgY antibodies when inoculated subcutaneously, these antibodies were not able to reduce viral shedding and therefore did not protect chickens from AIV.Item Genomic Approaches to Study Innate Immune Response to Salmonella Enteritidis Infection in Chickens(2010-01-14) Chiang, Hsin-ISalmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis. Contamination of consumed poultry products continues to be a global threat to public health. Genetic resistance using genomic approach provides a promising solution to controlling SE infection in poultry. The mechanism of SE resistance in chickens remains elusive. Three different approaches, microarray techology, gene silencing, and computational gene analysis, have been utilized to study SE-induced transcriptional changes of host immune response in the chicken. A whole genome chicken 44K microarray was used to analyze the transcriptome of heterophils from SE-resistant (line A) and SE-susceptible chickens (line B) with/without in vitro SE stimulation. Many differentially expressed immune-related genes were found in the SE-infected to non-infected comparison, where more immune-related genes were down-regulated in line B than line A. These results suggested a similar Toll-like receptor (TLR) regulatory network might exist in heterophils of both lines, and provided strong candidates for further investigating SE resistance and susceptibility in chickens. In the gene silencing study, small interfering RNAs (siRNA) were used to specifically inhibit the expression of NFkB1 in the chicken HD11 macrophage cell line with SE challenge. Genes related to the NF-kB signaling pathway were selected to examine the effect of NFkB1 inhibition on TLR pathway. With 36% inhibition of NFkB1 expression, the results showed an increased expression of TLR4 and interleukin (IL)-6 following SE challenge and suggested a likely inhibitory regulation of NFkB1 on TLR signal pathway. Finally, two novel chicken C-type lectin-like receptors were identified and annotated to chicken CD69 and CD94/NKG2-like with multiple evidences generated by computational (in-silico) sequence analysis. Both genes located in a region on chicken chromosome 1 that is syntenic to mammalian Nature Killer Receptor Complex (NKC) region, which may have existed before the divergence between mammals and aves. While siRNA lays the foundation of using loss-of-function approach on testifying gene-gene interactions, in-silico analysis aids in gathering information of unknown genes of great interest. Both approaches provide great potential to use for down-stream analysis following microarray study.Item Identification of Significantly Regulated Genes in the Estrogen Induced Gallus gallus Liver Over a 24-Hour Time Course(2012-02-14) Trojacek, EricaIn birds, estrogen is a strong stimulator of gene programs that regulate the formation of very low density lipoproteins (VLDL). Apolipoprotein-B (ApoB) is an integral part of very low density lipoproteins. In mammals, the rate of ApoB synthesis is controlled by post-translational means. In contrast, estrogen treated birds show changes in ApoB transcript level; in a natural setting, the bird?s metabolism and transcription are in great flux due to yolk formation. Besides the ApoB gene, the entire complement of genes that is necessary to form a VLDL is not known. To determine the genes that play a role in the formation of VLDL 7-10d old chicks were injected with estrogen at several time points over a 24hr period. Following exsanguinations by cardiac puncture, livers were removed and RNA was extracted. The RNA was quantified and hybridized to microarrays using a dual-dye system. Slides were scanned and analyzed, and features were extracted. To qualify microarray results, quantitative real time PCR (q-RTPCR) was done on a selection of genes. Previous studies had shown that approximately 200 genes are upregulated by the treatment of hormone naive chickens with estrogen. As a result of our liver transcriptional profiling, we identified 1,528 genes at 1.5hrs, 1,931 genes at 3hrs, 2,398 genes at 6hrs, 2,356 at 12hrs, and 1,713 genes at 24hrs following estrogen exposure. We determined that these regulated genes include those responsible for the transcription of RNA used to create the gene products that serve as components of VLDL itself or that act in VLDL assembly. These include genes encoding structural proteins, like ApoB, and genes encoding assembly-related proteins. Of the differentially expressed genes as compared to time 0, there were approximately 30% which were unannotated with regards to function limiting conclusions. We hope to determine the function of these genes and to annotate them based on this information.Item Roles for extra-hypothalamic oscillators in the avian clock(2009-05-15) Karaganis, Stephen PaulAvian circadian clocks are composed of a distributed network of neural and peripheral oscillators. Three neural pacemakers, located in the pineal, the eyes, and the hypothalamus, control circadian rhythms of many biological processes through complex interactions with slave oscillators located throughout the body. This system, an astonishing reflection of the life history of this diverse class of vertebrates, allows birds to coordinate biochemical and physiological processes and harmonize them with a dynamic environment. Much work has been done to understand what roles these pacemakers have in avian biology, how they function, and how they interact to generate overt circadian rhythms. The experimental work presented in this dissertation uses the domestic chicken, Gallus domesticus, as a model to address these questions and carry forward current understanding about circadian biology in this species. To do so, we utilized a custom DNA microarray to investigate rhythmic transcription in cultured chick pineal cells. We then sought to identify genes which might be a component of the pineal clock by screening for rhythmic transcripts that are sensitive to a phase-shifting light stimulus. Finally, we surgically removed the eyes or pineal from chickens to examine the roles of these extra-SCN pacemakers in regulating central and peripheral rhythms in metabolism and clock gene expression. Using these methods, we show that the oscillating transcriptome is diminished in the chick pineal ex vivo, while the functional clustering of clock controlled genes is similar. This distribution reveals multiple conserved circadian regulated pathways, and supports an endogenous role for the pineal as an immune organ. Moreover, the robustness of rhythmic melatonin biosysnthesis is maintained in vitro, demonstrating that a functional circadian clock is preserved in the reduced subset of the rhythmic pineal transcriptome. In addition, our genomic screen has yielded a list of 28 genes that are candidates for functional screening. These should be evaluated to determine any potential role they may have as a component of the pineal circadian clock. Finally, we report that the eyes and pineal similarly function to reinforce rhythms in brain and peripheral tissue, but that metabolism and clock gene expression are differentially regulated in chick.Item Sequential application of epsilon-polylysine, lauric arginate and acidic calcium sulfate for inactivation of pathogens on raw chicken and beef(2009-05-15) Benli, HakanSalmonella and Escherichia coli O157:H7 (EC) contamination continues to be one of the major concerns for the microbiological safety of raw poultry and beef products. Application of more than one decontamination agent as a multi-hurdle intervention to carcasses in a processing line might produce greater reductions than one treatment alone due to different modes of action of individual antimicrobials. In this study, sequential spray applications of e-polylysine (EPL), lauric arginate and acidic calcium sulfate (ACS) solutions were evaluated against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) on artificially inoculated broiler carcasses and against ST and EC on beef rounds and ground beef derived from the rounds. All possible 2-way combinations and individual applications of 20 % ACS (ACS20), 300 mg/liter EPL (EPL300) and 200 mg/liter LAE (LAE200) were evaluated using a sterile membrane filter model system. The combinations that provided higher Salmonella reductions were further evaluated on inoculated chicken carcasses using either response surface methodology (RSM) or in various concentrations applied in a sequential manner. Sequential spray applications of EPL300 - ACS 30 % (ACS30) or LAE200-ACS30 produced the highest Salmonella reductions on inoculated chicken carcasses. In a subsequent experiment, treatment of Salmonella inoculated carcasses with EPL300-ACS30 or LAE200-ACS30 combinations were found effective for reducing initial Salmonella counts by 1.5 and 1.8 log CFU/ml, respectively, immediately after treatment and by 1.2 and 1.8 log CFU/ml, respectively, following 6 days of storage at 4.4 ?C. Evaluation of the resident microflora including aerobic plate counts (APC), E. coli, coliforms and psychrotrophs on uninoculated chicken carcasses after treatment with EPL300-ACS30 or LAE200-ACS30 and during storage indicated that these treatments have the potential to increase the shelf-life of poultry carcasses. Furthermore, application of warm (55 ?C) EPL300-ACS30 or LAE200-ACS30 onto inoculated beef rounds reduced both ST and EC counts over 6 days of storage at 4.4 ?C by 4.5 and 4.3 log CFU/cm2, respectively. Ground beef manufactured with EPL300-ACS30 or LAE200- ACS30 treated rounds had lower ST and EC counts initially and stayed lower over 4 days of storage at 4.4 ?C when compared to control.