<li> To evaluate and compare the expression of endogenous anti- and pro-inflammatory mediators in caprine and bovine neutrophils (PMN). Neutrophil genes and proteins regulated in response to exposure to E. coli endotoxin (LPS) and phorbol myristate acetate (PMA) will be identified. Common and divergent genes and proteins in goats and cattle may serve as molecular markers and therapeutic targets against parasites of ruminants. <li> To evaluate the effect of host immune factors on global parasite gene expression in E. coli and C. elegans. Novel genes regulated in response to Colostrinin in E. coli and C.elegans will be identified. <li> To compare NRAMP1 mRNA expression using reverse transcriptase polymerase chain reaction (RT-PCR) methods in coccidia-resistant and -susceptible goats. Genes that may serve as molecular markers and therapeutic targets against coccidia in goats will be identified. <li> To incorporate these findings for the development of a farm-site parasite control demonstration program for small farmers. </ol>
NON-TECHNICAL SUMMARY: Bacteria induced mastitis and nematode parasites cause losses of 2-8 billion dollars to the US animal industry per year. The over all goal of this effort is to promote the enhancement of agricultural producers' economic opportunities through the provision of science based knowledge towards the utilization of genomic diversity for protection of animals from inflammatory and parasitic diseases and the identification of alternative therapeutics to ensure food safety, quality and adequate nutrition and health.
APPROACH: Cell, molecular and genome level evaluations using flow cytometry, protein assays, RT PCR, microarray and Bioinformatics approaches will be conducted on bovine and caprine PMN.
PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: Objective 1: A comparative analysis of anti and pro-inflammatory gene expression was conducted at the RNA and protein level. Cell surface expression of TLR4 was conducted on a bioanalyzer. In isolated bovine PMN, gene expression did not show an effect of E.coli endotoxin (LPS) type. At the level of transcription LPS stimulation activated IL-8, IL-10 and TNF alpha genes. The cytokines IL-8 and TNF- alpha were detected by ELISA following LPS activation, IL-1 beta was not detected. Natural resistance-associated macrophage protein (NRAMP-1) gene was constitutively expressed in bovine PMN and detected in genomic DNA of caprine leukocytes. In bovine PMN TLR4 was expressed following LPS stimulation. <BR><BR>Objective 2: Commercial colostrum high in Proline rich poly peptide complex a neutraceutical grade was used. Initial studies show that this colostrum promotes growth reproduction and chemotaxis of C.elegans. An earlier response (day 1) was observed in colostrum treated nematodes. High numbers of migrated nematodes were observed within colostrum treatment. Most nematodes migrated to E. coli and reproduced following treatment with colostrum more than was observed with dried milk or in controls. An evaluation of global gene expression in E.coli following exposure to colostrum has been conducted using microarray analysis with dye swap. Initial results have been used to identify five up-regulated and five down regulated genes. The mannose transporter of the bacterial phosphotransferase system was highly elevated. Data acquired using Jaguar analysis software and analyzed using the open source software Magic tool were analyzed using SAS Analysis in related experiments to validate this approach. <BR><BR>Objective 3: Studies thus far have been optimized for the detection of NRAMP-1 in Genomic DNA of caprine leukocytes. To avoid the need for large volumes of blood for PMN isolation, we are exploring alternative techniques for RNA isolation from small numbers of cells. Natural resistance-associated macrophage protein (NRAMP-1) gene was detected in genomic DNA from goats. Initial on farm screening included collection of weights, blood samples and evaluation of the levels of coccidia and assessments of the packed cell volume. The levels of coccdia in the herd were low. We have identified a farm facing coccidia problems. <BR><BR>Objective 4: Based on our results and developments in parasite management control programs, we have conducted discussions with collaborators and farm personnel on the need for and establishment of an on farm genetics based performance evaluation system. Outreach material identified as useful for management of the herd was shared (goat management calendars and 4H material). We have conducted a workshop on use of the FAMACHA for parasite management and a TV interview on Channel 14 news on marketing as part of the annual cattle and goat field day at the NC A&T farm. <BR>PARTICIPANTS: PIs, research collaborators, research assistants and students. <BR>TARGET AUDIENCES: Animal scientists, livestock producers, academicians, stakeholders, <BR>USDA PROJECT MODIFICATIONS: The evaluation of gene expression in caprine PMN may need to be modified to include all leukocytes in light of difficulties with isolation of sufficient quantities of RNA. Due to the low incidence of coccidia problems the University farm we are looking at local farms that may have coccidia problem herds.
IMPACT: 2007/01 TO 2007/12<BR>
We have identified primers that amplify the NRAMP-1 gene in bovine PMN and can identify NRAMP-1 in goat genomic DNA. The pro-inflammatory response to LPS is not influenced by the type of E.coli endotoxin used. We have established techniques and approaches that allow for the evaluation of the impact of genomic diversity on the immune response in ruminants in the lab. We have preliminary evidence for a subset of E.coli heat shock genes that are highly responsive (up and down regulated) to treatment with colostrum. Bovine colstrum may be a superior medium to milk for use in supplementation of C.elegans growth media in axenic cultures. These studies show that C.elegans is responsive to colostrum components. Microarray analysis of the effect of colostrum on gene expression in E.coli has helped identify genes for further evaluation, and established the procedures for repeat experiments. SAS analysis of microarry data complements the use of magic tool. The mannose transporter of the bacterial phosphotransferase system was highly elevated in response to colostrum in E.coli. This transporter mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. Bactericidal activity of both secreted and non-secreted microcin E492 requires the mannose permease. Fecal egg count screening has been used to evaluate the herd for the incidence of parasites. The level of coccidia was very low and reflects good management practices. Collaborative efforts with the farm personnel have resulted in successful outreach efforts. Summer research apprentice students and undergraduate students have participated in this effort. Three master's students have conducted thesis research. Two have completed the MS program. One was a USDA National Needs Fellowship recipient.