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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF RESPIRATORY DISEASE SUSCEPTIBILITY Title: Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages

Authors
item Mcvey, David -
item Chitko Mckown, Carol
item Kuszak, Jennifer -
item Barletta, Raul -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 7, 2010
Publication Date: December 1, 2010
Citation: McVey, D.S., Chitko-Mckown, C.G., Kuszak, J.A., Barletta, R.G. 2010. Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages [abstract]. Annual Conference of Research Workers in Animal Diseases. Abstract No. 113 [on-line].

Technical Abstract: Johne’s disease is a significant problem in many North American cattle herds. The efficacy of currently available vaccines is questionable. There is a need to develop efficacious vaccines and strains of Mycobacterium avium subsp. paratuberculosis (MAPTB) that could serve as potential candidates for live attenuated vaccines. The confirmation of attenuation requires reliable methods to assess reductions in virulence. In these studies, we characterized the ability of wild-type and transposon insertion mutants of MAPTB to invade and survive in bovine monocyte-derived macrophages. Blood was obtained from feedlot animals and mononuclear cells were separated by density-gradient centrifugation. Adherent cells were retained in 24-well tissue culture plates with RPMI medium. Macrophage cultures were inoculated with either wild-type MAPTB (K-10) or transposon insertion mutant strain 4H2 (derived from K-10) at an MOI of 10. Cell culture supernatant medium or cell lysates were diluted and cultured on Middlebrook agar, with mycobactin supplement to enumerate surviving bacteria at 2, 24 and 72 hours. There was little difference in surviving bacteria at 2 hours, but the numbers of surviving 4H2 bacteria were reduced by approximately 15% and 45% at 24 and 72 hours, respectively. A second assay was developed using the monocyte-derived macrophages in 48-well plates. The adherent macrophages were inoculated with descending MOI ratios of 10**2 through 10**-**4 in replicates of three or four wells. After a 4-hour inoculation, the macrophage cultures were incubated for an additional 24 hours. The macrophages were lysed and plated to detect the presence of viable bacteria. The number of bacteria required to infect 50% of the macrophages (MID50) was over 10-fold higher for the 4H2 strain of MAPTB. The results indicated that the 4H2 strain was not phagocytosed and/or did not survive as well as the K-10 strain in macrophages. Continued development of macrophage attenuation assays (e.g., the MID50 method) may be necessary to compare possible vaccine candidates. Final confirmation of attenuation must be achieved in immunologically competent host animals.

Last Modified: 7/25/2014
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