Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Abstract

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.

FIG. 1.

Short-term growth over 16 days of M. avium subsp. paratuberculosis bovine strain K5 and human strain SN6 in A. polyphaga trophozoites. MAP, M. avium subsp. paratuberculosis; qRT-PCR, quantitative real-time PCR.

FIG. 2.

Long-term growth over 24 weeks of M. avium subsp. paratuberculosis bovine strain K10 and human strain SN6 in dynamic long-term cultures of A. polyphaga undergoing encystment and reactivation to trophozoite forms. MAP, M. avium subsp. paratuberculosis; qRT-PCR, quantitative real-time PCR.

FIG. 3.

IS900 PCR analysis of DNA extracts from amoebic cultures showing amplification of the predicted 298-bp product, which was also verified by amplicon sequencing. The amoebic cultures had been fed extracts of human gut tissues and then incubated at room temperature for between 35 and 47 months with long periods of encystment. Lane R, reagent control; lane +, positive control; lane L, size ladder.

FIG. 4.

Fluorescent in situ hybridization using an IS900 probe (see text), showing clumps of M. avium subsp. paratuberculosis cells within the cytoplasm of A. polyphaga in cultures maintained for 24 months after feeding with extracts of human gut tissue. (A) Culture fed to maintain the amoebae in the trophozoite form. (B) Culture undergoing long periods of encystment of the amoebae, followed by trophozoite reactivation.

FIG. 5.

Auramine-rhodamine staining of amoebic cultures previously shown to be positive for M. avium subsp. paratuberculosis by PCR and in situ hybridization, showing fluorescent mycobacteria 24 months after feeding with extracts of human gut tissues. (A) Mycobacteria dispersed in the cytoplasm of a trophozoite. (B) Peripheral distribution of mycobacteria in an encysted A. polyphaga cell.