Identification and characterization of Mycobacterium paratuberculosis virulence genes expressed in vivo by negative selection
Authors: Shpigel Nahum , Barletta Raul , Rosenshine Ilan , Chaffer Marcelo 
Published: March 2022

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of a severe inflammatory bowel disease (IBD) in ruminants, known as Johne’s disease or paratuberculosis. Johne’s disease is considered to be one of the most serious diseases affecting dairy cattle both in Israel and worldwide. Heavy economic losses are incurred by dairy farmers due to the severe effect of subclinical infection on milk production, fertility, lower disease resistance and early culling. Its influence in the United States alone is staggering, causing an estimated loss of $1.5 billion to the agriculture industry every year. Isolation of MAP from intestinal tissue and blood of Crohn's patients has lead to concern that it plays a potential pathogenic role in promoting human IDB including Crohn’s disease. There is great concern following the identification of the organism in animal products and shedding of the organism to the environment by subclinically infected animals. Little is known about the molecular basis for MAP virulence. The goal of the original proposed research was to identify MAP genes that are required for the critical stage of initial infection and colonization of ruminants’ intestine by MAP. We proposed to develop and use signature tag mutagenesis (STM) screen to find MAP genes that are specifically required for survival in ruminants upon experimental infection. This research projected was approved as one-year feasibility study to prove the ability of the research team to establish the animal model for mutant screening and alternative in-vitro cell systems. In Israel, neonatal goat kids were repeatedly inoculated with either one of the following organisms; MAP K-10 strain and three transposon mutants of K-10 which were produced and screened by the US PI. Six months after the commencement of inoculation we have necropsied the goats and taken multiple tissue samples from the jejunum, ileum and mesenteric lymph nodes. Both PCR and histopathology analysis indicated on efficient MAP colonization of all the inoculated animals. We have established several systems in the Israeli PI’s laboratory; these include using IS900 PCR for the identification of MAP and using HSP65-based PCR for the differentiation between MAV and MAP. We used Southern blot analysis for the differentiation among transposon mutants of K-10. In addition the Israeli PI has set up a panel of in-vitro screening systems for MAP mutants. These include assays to test adhesion, phagocytosis and survival of MAP to/within macrophages, assays that determine the rate of MAPinduced apoptosis of macrophages and MAP-induced NO production by macrophages, and assays testing the interference with T cell ã Interferon production and T cell proliferation by MAP infected macrophages (macrophage studies were done in BoMac and RAW cell lines, mouse peritoneal macrophages and bovine peripheral blood monocytes derived macrophages, respectively). All partners involved in this project feel that we are currently on track with this novel, highly challenging and ambitious research project. We have managed to establish the above described research systems that will clearly enable us to achieve the original proposed scientific objectives. We have proven ourselves as excellent collaborative groups with very high levels of complementary expertise. The Israeli groups were very fortunate to work with the US group and in a very short time period to master numerous techniques in the field of Mycobacterium research. The Israeli group has proven its ability to run this complicated animal model. This research, if continued, may elucidate new and basic aspects related to the pathogenesis MAP. In addition the work may identify new targets for vaccine and drug development. Considering the possibility that MAP might be a cause of human Crohn’s disease, better understanding of virulence mechanisms of this organism might also be of public health interest as well.