Progress 11/15/06 to 06/15/11

Outputs
OUTPUTS: This project directly supported the efforts of two graduate students who have now both successfully defended their PhD theses. Dr. Edward Kabara completed his work studying interactions between Mycobacterium avium subspecies paratuberculosis (MAP) and bovine macrophages in May 2012 and Dr. John P. Jerome completed his work on Campylobacter Jejuni in August 2012. In addition, matching funds from Michigan State University supported the work of two additional students who are nearly complete in their PhD programs. The work of Dr. Kabara focused on the effect of MAP on bovine macrophages. This work began with a series of functional genomics studies using custom made bovine microarrays. These studies demonstrated that 93 genes were differentially expressed in MAP infected macrophages relative to uninfected cells, regardless of the MAP strain used. One pathway that was consistently highlighted in these experiments was regulation of cellular apoptosis. It also became clear that MAP infections were not always 100% and that in order to properly study MAP-macrophage interactions, cells need to be sorted using flow cytometry to separate uninfected cells (bystanders) from MAP infected cells in the same cultures. Dr. Kabara then focused on the ability of MAP to affect apoptosis in host cell macrophages using electronic gating and cell sorting techniques. It was clearly demonstrated that MAP prevented host cell apoptosis. This is critical since apoptosis and the ensuing engulfment of apoptotic bodies by other cells is a key mechanism of mycobacterial clearance and important in proper immune cell activation. Dr. Jerome's PhD thesis work focused on the important food safety concern, Campylobacter Jejuni using a mouse infection model involving C57BL/6 mice lacking the IL-10 gene (C57BL/6 IL-10-/-). Variation of 22 strains with respect to colonization, development of clinical signs, and lesion scores was observed. Dr. Jerome was able to separate these 22 strains into 5 pathotypes. Using a combination of in silico virulence gene content examination and a C. jejuni genome microarray Dr. Jerome showed that virulence gene content was not necessarily associated with pathotype. In another mouse infection model, Dr. Jerome demonstrated that pathotype was more closely associated with differential expression of ninety open reading frames in the C. jejuni genome. Thus, C. jejuni virulence is more associated with gene expression levels than with basic gene content. PARTICIPANTS: Jeanne L. Burton, Assoc. Professor, Mentor Paul M. Coussens, Professor, Mentor Mike Allen, Professor, Mentor Linda Mansfield, Professor, Mentor John P. Jerome, Student Edward A. Kabara, Student TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: None

Impacts
The main impact of this project was the training of two outstanding graduate students, seeing them through completion of their PhD degrees. Both former USDA-NNGF supported students are now in successful post-doctoral programs. Dr. Kabara was recently awarded a USDA-NIFA Post-doctoral Fellowship for work in the area of inflammation with regards to mastitis and Dr. Jerome is continuing work on C. jejuni with Dr. Linda Mansfield at Michigan State University. Both are this continuing to apply their talents to topics of importance to agriculture and the USDA. During the program, Drs. Kabara and Jerome were regular presenters at the annual Conference fo research Workers in Animal Disease Conference, the Michigan Chapter of the American Society for Microbiology, and in various other international meetings. Dr. Jerome also traveled to the UK to work at the Sanger Center, where he completed work on a draft C. jejuni genome sequence. Dr. Kabara presented his work at the International Association for Paratuberculosis meeting in Japan and regularly at the Johnes Disease Integrated Project annual meetings.

Publications

• Kabara, E., Kloss, C.C., Wilson, M.R., Sreevatsen, S., Janagama, H., and P.M. Coussens. 2010. A Large-Scale Study of Differential Gene Expression in Macrophages Infected with several strains of Mycobacterium avium subspecies paratuberculosis. Briefings in Functional Genomics and Proteomics. 9(3):220-37.
• Kabara, E.A. and P.M. Coussens. 2012. Infection of primary bovine macrophages with Mycobacterium avium subspecies paratuberculosis suppresses host cell apoptosis. Frontiers in Microbiology. 3:215.
• Murphy, J.T., S. Sommer, E.A. Kabara, N.Verman, M.A. Kuelbs, P. Saama, R. Halgren, and P.M. Coussens. 2006. Gene Expression Profiling of Monocyte-Derived Macrophages Following Infection with Mycobacterium avium subspecies avium and Mycobacterium avium subspecies paratuberculosis. Physiological Genomics, 28: 67-75
• Bell, JA., Jerome, JP., Plovanich-Jones, AE., Smith, EJ., Gettings, JR., Kim, HY., Landgraf, JR., Lefebure, T., Kopper, JJ., Rathinam, VA., St. Charles, JL., Buffa, BA., Brooks, AP., Poe, SA., Eaton, KA., Stanhope, MJ., and LS Mansfield. 2012. Outcome of infection of C57BL/6 IL-10 (-/-) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up- and down-regulated in vivo. Microb. Pathog. PMID: 22960579.
• Jerome, JP, Klahn, BD, Bell, JA, Barrick, JE, Brown, CT, and LS Mansfield. 2012. Draft genome sequences of two Campylobacter jejuni clinical isolates, NW and D2600. J. Bacteriology. 194: 5707-5708.

Progress 11/15/06 to 11/14/07

Outputs
Two Students were recruited to fill the USDA NNGF program these students are Edward Kabara and John Jerome. Mr. Kabara joined the program in fall of 2007 while Mr. Jerome joined in January 2007. In addition, due to the provision of matching fellowships from Michigan State University, two additional students were recruited. These students are Jiesi Luo and Sarah Stebulis. All students have made excellent progress toward their respective programs. The following list is a brief description of theses areas John Jerome Whole genome studies of in vivo adapted Campylobacter jejuni. Ed Kabara Pathogenomics of M. paratuberculosis in primary bovine macrophages. Sarah Stebulis Propionate Regulation of Feed Intake: Interaction with Lipolytic State; Jiesi Luo Identification of genes in the cumulus cells of bovine oocytes that can be used as predictors of healthy embryos. All students have assembled guidance committees and participated in a forum where NNGF students presented their research topics and addressed questions from the greater audience. Another such forum is scheduled for spring 2009. PRODUCTS: Products of the program include a complete reannotation of the BOTL5 bovine immunity microarray for the Center for Animal Functional Genomics, the generation of two matching fellowships from MSU, Several new mutants of campylobacter jujuni have been generated, and novel methods for high efficiency cloning of bovine embryos have been developed. OUTCOMES: As a result of this program, four new graduate students have entered an area of extreme importance to the animal and veterinary sciences. These students are being well trained through coursework and research in functional genomics and bioinformatics. Collectively their continued contributions as the complete their degrees and move on to post-doctoral studies or faculty positions should advance these fields. DISSEMINATION ACTIVITIES: The two NNGF supported students and their MSU funded counterparts have been extremely active in presenting their research activities at local, national and international meetings. A lost of these presentations follows. Jiesi Luo Anti-apoptotic Effect of Sphingosine-1-Phosphate on in vitro Development of Bovine Embryo, Graduate forum of functional genomics center, MSU, 03/17/2009 Sarah Stebulis Propionate Regulation of Feed Intake: Interaction with Lipolytic State; FSC 892 Seminar; February 19, 2009; Department of Animal Science, MSU Edward Kabara Poster presentation at the Conference of Research in Animal Disease (CRWAD), Chicago, 2006 Poster presentation at the Johnes Disease Integrated Program (JDIP) conference, Texas A&M University, 2007 Poster presentation at the 8th International Veterinary Immunology Symposium (8th IVIS), Brazil, 2007 Oral presentation at Animal Science Graduate Research Forum, MSU, 2007 Oral presentation at Conference of Research in Animal Disease (CRWAD), Chicago, 2007 Poster presentation at JDIP conference, MSU, 2008 Poster presentation at 10th International Colloquium on Paratuberculosis, 2009 John Jerome Phi Zeta Research day, October 10, 2008 Determining the genetic changes contributing to increased virulence of Campylobacter jejuni during serial passage in C57BL/6 IL-10-/- mice. Gordon Research Conference: Microbial Population Biology. July 19-24, 2009. Gene expression changes and slipstrand mutation accompany increased virulence of Campylobacter jejuni isolated after passage in C57BL/6 IL-10-/- mice. Annual Meeting Food and Waterborne Diseases Integrated Research Network, National Institutes of Health. March 31, 2009. (My research presented by Dr. Linda Mansfield) Whole genome studies of in vivo adapted Campylobacter jejuni. FUTURE INITIATIVES: It is anticipated that Edward Kabara and John Jerome will complete the requirements for their PhD degrees in 2010 or early 2011, in line with a normal 4-year program. It is also anticipated that MSU supported students Jeisi Luo and Sarah Stebulis will complete their respective programs later in 2011 or 2012. These two students were recruited behind the USDA NNGF supported students. In keeping with program outlines, all students will continue to meet once per year and present results of their research programs at an open forum. As students progress through their programs, the number of research based publications and presentations will increase.

Impacts
This program has begun to produce four students trained in the application of genomics and bioinformatics to problems in veterinary and animal science. All students have been active in disseminating their research results, with the more advanced USDA NNGF students presenting commensurately more than the remaining two. The results of John Jerome's research on campylobacter virulence have provided new insights on how infection with this pathogen, an important food safety concern, leads to disease. Results of Edward Kabara's research have provided new information on how M. paratuberculosis, an organism of considerable importance to the global dairy industry, survives inside the host. Results of Jiesi Luo's research have contributed new information on methods to improve the success of cloning with bovine embryos.

Publications

• Murphy JT, Sommer S, Kabara EA, Verman N, Kuelbs MA, Saama P, Halgren R, Coussens PM. Gene expression profiling of monocyte-derived macrophages following infection with Mycobacterium avium subspecies avium and Mycobacterium avium subspecies paratuberculosis. Physiol Genomics. 2006 Dec 13;28(1):67-75