Survival and dormancy of Mycobacterium avium subsp. paratuberculosis in the environment

Abstract

The survival of Mycobacterium avium subsp. paratuberculosis was studied by culture of fecal material sampled at intervals for up to 117 weeks from soil and grass in pasture plots and boxes. Survival for up to 55 weeks was observed in a dry fully shaded environment, with much shorter survival times in unshaded locations. Moisture and application of lime to soil did not affect survival. UV radiation was an unlikely factor, but infrared wavelengths leading to diurnal temperature flux may be the significant detrimental component that is correlated with lack of shade. The organism survived for up to 24 weeks on grass that germinated through infected fecal material applied to the soil surface in completely shaded boxes and for up to 9 weeks on grass in 70% shade. The observed patterns of recovery in three of four experiments and changes in viable counts were indicative of dormancy, a hitherto unreported property of this taxon. A dps-like genetic element and relA, which are involved in dormancy responses in other mycobacteria, are present in the M. avium subsp. paratuberculosis genome sequence, providing indirect evidence for the existence of physiological mechanisms enabling dormancy. However, survival of M. avium subsp. paratuberculosis in the environment is finite, consistent with its taxonomic description as an obligate parasite of animals.

FIG. 1.

Percentages of culture-positive sites in experiment 1 grouped by shade treatment. Data for the plots at Carcoar and Borenore were pooled. There were no culture-positive sites for week 57, 61, 65, 69, or 72. Solid bars, no shade; striped bars, 70% shade.

FIG. 2.

Percentages of culture-positive sites in experiment 2 grouped by shade treatment. There were no culture-positive sites for week 29, 33, or 117; there were no samples for weeks 6 and 7 for the no-shade treatment. Results for grass are not shown. Solid bars, no shade, pooled results for the plots at the sites at Borenore and Carcoar; striped bars, 70% shade, results for boxes at Camden.

FIG. 3.

Percentages of culture-positive sites in experiment 3 grouped by shade treatment. (A) Plots at Borenore, fecal pellets sampled only to week 20 in 0% shade and week 16 in 70% shade; (B) boxes at Borenore, fecal pellets sampled only to week 32 in 0% shade and week 20 in 70% shade; (C) boxes at Camden, fecal pellets sampled only to week 32 in 0% shade, week 48 in 70% shade, and week 88 in 100% shade. Results for grass are not shown. Solid bars, no shade; striped bars, 70% shade; open bars, 100% shade.

FIG. 4.

Percentages of culture-positive sites in experiment 4 grouped by shade treatment. (A) Plots at Borenore, fecal pellets sampled only to week 16 in 0% shade and week 10 in 70% shade; (B) boxes at Borenore, fecal pellets sampled only to week 24 in 0% shade and week 12 in 70% shade; (C) boxes at Camden, fecal pellets sampled only to week 32 in 0% shade and week 76 in 70 and 100% shade. Results for grass are not shown. Solid bars, no shade; striped bars, 70% shade; open bars, 100% shade.

FIG. 5.

Log₁₀ counts of M. avium subsp. paratuberculosis and linear regressions on weeks after contamination. (A) Experiment 1, fecal pellet and soil samples, data from Borenore and Carcoar pooled; (B) experiment 2, fecal pellet samples collected from partially shaded pasture boxes at Camden; (C) experiment 4, fecal pellet samples collected from boxes in the 100% shade treatment at Camden. Results shown are the counts for the individual samples, the regression line with 95% confidence limits for the predicted means, and the slope of the line ± standard errors.

FIG. 6.

Weather data from Camden for a 12-month period corresponding to experiments 3 and 4. Contamination occurred on 8 November 1999 and 31 January 2000. Temperature data are weekly maxima, averages, and minima. (A) Mean weekly dry bulb air temperatures and total weekly rainfall; (B) weekly total solar radiation; (C) mean weekly soil temperature, no shade; (D) mean weekly soil temperature, 70% shade; (E) mean weekly soil temperature, 100% shade. ♦, maximum; ▪, mean; ▴, minimum.

FIG. 6.

Weather data from Camden for a 12-month period corresponding to experiments 3 and 4. Contamination occurred on 8 November 1999 and 31 January 2000. Temperature data are weekly maxima, averages, and minima. (A) Mean weekly dry bulb air temperatures and total weekly rainfall; (B) weekly total solar radiation; (C) mean weekly soil temperature, no shade; (D) mean weekly soil temperature, 70% shade; (E) mean weekly soil temperature, 100% shade. ♦, maximum; ▪, mean; ▴, minimum.

FIG. 6.

Weather data from Camden for a 12-month period corresponding to experiments 3 and 4. Contamination occurred on 8 November 1999 and 31 January 2000. Temperature data are weekly maxima, averages, and minima. (A) Mean weekly dry bulb air temperatures and total weekly rainfall; (B) weekly total solar radiation; (C) mean weekly soil temperature, no shade; (D) mean weekly soil temperature, 70% shade; (E) mean weekly soil temperature, 100% shade. ♦, maximum; ▪, mean; ▴, minimum.

FIG. 6.

Weather data from Camden for a 12-month period corresponding to experiments 3 and 4. Contamination occurred on 8 November 1999 and 31 January 2000. Temperature data are weekly maxima, averages, and minima. (A) Mean weekly dry bulb air temperatures and total weekly rainfall; (B) weekly total solar radiation; (C) mean weekly soil temperature, no shade; (D) mean weekly soil temperature, 70% shade; (E) mean weekly soil temperature, 100% shade. ♦, maximum; ▪, mean; ▴, minimum.

FIG. 6.

Weather data from Camden for a 12-month period corresponding to experiments 3 and 4. Contamination occurred on 8 November 1999 and 31 January 2000. Temperature data are weekly maxima, averages, and minima. (A) Mean weekly dry bulb air temperatures and total weekly rainfall; (B) weekly total solar radiation; (C) mean weekly soil temperature, no shade; (D) mean weekly soil temperature, 70% shade; (E) mean weekly soil temperature, 100% shade. ♦, maximum; ▪, mean; ▴, minimum.

FIG. 7.

Cumulative solar radiation for experiments 3 and 4 measured at Camden and aligned by week after contamination. ▪, experiment 3, commencing 8 November 1999; ▴, experiment 4, commencing 31 January 2000.

FIG. 8.

Alignment of the amino acid sequences for the Dps-like protein from M. avium subsp. paratuberculosis (M. ptb) and Dps from M smegmatis (M. smeg) (GenBank accession no. AY065628). Amino acid residues in boldface and underlined are reported to be involved in the DNA binding signature (15). Symbols: bar, identical; colon, highly related; period, more distantly related; no symbol, unrelated.

FIG. 9.

Alignment of the amino acid sequences for the RelA-like element from M. avium subsp. paratuberculosis (M. ptb) and RelA from M. tuberculosis (M. tb) (relA gene accession no. Rv2583c, TubercuList Web Server, http://genolist.pasteur.fr/TubercuList/). Symbols: bar, identical; colon, highly related; period, more distantly related; no symbol, unrelated.