Molecular genetic analysis of a large Mycobacterium avium subsp. paratuberculosis mutant bank

Authors: RATHNAIAH, G - University Of Nebraska; Bayles, Darrell; Stabel, Judith; ZINNIEL, D - University Of Nebraska; Bannantine, John; GROHN, Y - Cornell University; BARLETTA, R - University Of Nebraska

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/1/2017
Publication Date: 12/3/2017
Citation: Rathnaiah, G., Bayles, D.O., Stabel, J.R., Zinniel, D., Bannantine, J.P., Grohn, Y.T., Barletta, R.G. 2017. Molecular genetic analysis of a large Mycobacterium avium subsp. paratuberculosis mutant bank. Meeting Abstract. P010/144.

Interpretive Summary: Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterium that causes Johne’s disease in ruminants, resulting in a chronic inflammation of the small intestine. At end stages of disease, animals will present with severe diarrhea and weight loss. Animals usually become infected when they are young by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd. Current vaccines do not prevent infection but are helpful in reducing fecal shedding and the bacterial burden in the animal, thus allaying the spread of infection in the herd. In the present study, specific genes of the bacterium were deleted, followed by inoculation into calves to determine which genes were essential for pathogenesis of infection. Calves demonstrated immune responses to the inoculation after 12 months, suggesting that they were infected. Analysis of the genes responsible for pathogenesis is still underway but will be helpful in selecting vaccine candidates that may prevent infection.

Technical Abstract: Purpose: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and a potential cause of Crohn’s disease in humans. Our goal was to identify MAP genes encoding essential and non-essential functions under in vitro and in vivo conditions. Methods: MycoMarT7 transposon mutagenesis was carried out and about one million independent MAP K-10 mutants were collected. Chromosomal DNA was isolated and fragments were subjected to Illumina sequencing. The MAP mutant pool was orally inoculated into five calves by feeding milk replacer three times containing 5.0 x 105 CFU live bacilli. Feces and blood were collected on the day prior to inoculation and during the course of the 12 month infection. Animals were necropsied to obtain sections of jejunum, ileum and their associated lymph nodes, ileoceccal valve and iliac lymph nodes. Samples were processed for culture, mutant pool analysis, RT-PCR, and histopathologic and gene expression analyses. Results: To classify genes, we applied a 4-state Hidden Markov model analysis that assigns each TA site to a “state call” based on the increasing number of sequence reads: ES (essential), GD (growth defect), NE (non-essential) and GA (growth advantage). In the in vitro study, out of 4,350 genes we identified 328 ES; 1,103 GD; 2,603 NE, 258 GA and 58 unassigned. Some essential genes match functions for DNA replication (dnaE), transcription (rpoB), translation (rpmC, rpsL) and lipid biosynthesis (fabD2, fadE9). For the in vivo study, analysis of animal samples indicated that the IFN-g test was positive after 90 days up until day 270 for the mutant pool. For K-10, the test was positive at 180 days. This indicates that the animals became MAP positive. Serum antibodies remained negative throughout the entire study. Analysis of the transposon mutant pool in vivo is still in progress. Conclusions: This study defines for the first time the in vitro gene essentiality in MAP on a whole genome basis. Increase of IFN-g secretion and the lack of fecal shedding are consistent with a low infection model that is at early stages of disease progression.