GENERAL | RESISTANCE | SURVIVAL Survival in surface water Survival in biofilms Survival in free living amoebae Survival in feces applied to the soil surface Survival in feces injected into soils Survival in slurry pits and anaerobic digesters Survival in compost		Compensating for the fact it can seemingly only grow in animals, M. paratuberculosis has developed the ability to persist in the environment for long periods of time waiting for the chance to again infect an animal.

Survival in surface water

Lovell et al. reported in 1944 that after spiking sterilized pond water (pH 5.5) with 0.1, 1.28 or 3.4 mg wet weight of M. paratuberculosis per 100 ml of water and holding it at room temperature the organism was recovered in samples tested monthly for up to 9 months. In 1956 Larsen et al. reported on the survival of M. paratuberculosis in spiked tap water of different pHs held at 38 C in the dark. In neutral pH water (7.0) M. paratuberculosis was recovered up to 17 months (517 days) post-inoculation while for pH 5.0 and pH 8.5 water M. paratuberculosis was isolated up to 14 months post-inoculation. Consistent with these earlier reports, Sung and Collins found that when distilled water (pH 7.2) was inoculated with 10⁶ (1 million) M. paratuberculosis cells/ml and viable counts determined on a monthly basis, the time for a 1 log reduction (D-value) was 68.5 days, and viable M. paratuberculosis cells were found up to 455 days (strain Dominic). Similar findings were reported for M. avium. When suspended in tap water and held at 4C or 20C , M. avium survived beyond 485 days.

For more general references regarding mycobacteria in water the reader is directed to the review article by C.H. Collins. Subsequent to publication of this review, recognition of the global AIDS epidemic and associated opportunistic M. avium infections led to a resurgence of research interest in this opportunistic mycobacterial pathogen and, in particular, investigations concerning the source of this organism. Most investigations point to domestic water supplies as the source of human M. avium infections due in part to their abundance in the environment and their resistance to chlorination. Under natural environmental conditions, warmer waters with lower pHs have been associated with higher levels of M. avium. Similar factors could affect survival of M. paratuberculosis in water.

Survival in biofilms

No studies have been reported concerning M. paratuberculosis in biofilms (complex communities of microbes, and slime secreted by microbes, that cover surfaces of pipes and other surfaces exposed to bacteria-containing fluids). Work on other mycobacteria indicates that biofilms may be an important replication site for mycobacteria found in water. Organic substances like plastics and rubber were found to be more intensely colonized than inorganic substances such as copper and glass. The significance of mycobacteria in biofilms is that all bacteria in such locations are more resistant to chemical stress than bacteria suspended free in water.

Survival inside free-living amoeba

No published studies on M. paratuberculosis replication in amoeba have been reported. M. avium, however, has been shown to be capable of replicating inside free living amoebae such as Acanthamoeba. Moreover, the virulence of the organism was enhanced after growth inside these amoebae. Unpublished data indicates that similar relationships could exist between free living amoebae and M. paratuberculosis. If true, this would provide an ecological niche for the organism outside the infected animal and provide a location for the organism to multiply in the environment.

Survival in soil, feces and soil-feces mixtures
applied to the surface of fields/pastures

On the subject of environmental survival of M. paratuberculosis, the 1944 publication by Lovell et al. has become a classic reference. A series of studies using naturally infected bovine feces were conducted in which the infected fecal matter was exposed to a variety of natural conditions such as freezing, drying, sunlight, changes in ambient temperature, and rain, with regular attempts to reisolate M. paratuberculosis. In general they found survival of M. paratuberculosis in feces kept outdoors up to 152 to 246 days depending on specific conditions. Drying of soil appeared to shorten survival. The authors recommend considering a pasture contaminated by the organism as a potential source of infection for at least one year given the longevity of M. paratuberculosis. They mention that this supports recommendations of earlier workers (1929-1933). The commonly made statement that M. paratuberculosis survives a year on pastures, found in most literature on Johne's disease, likely originates from the work of Lovell et al. It would be interesting to repeat the study using more sensitive M. paratuberculosis detection methods than those available to Lovell at the time.

Factors that may shorten the estimated survival time of M. paratuberculosis in soil are drying, exposure to sunlight, pH above 7.0 and low iron content. Bovine urine is also hostile to M. paratuberculosis survival and increasing concentrations of bovine urine (2-10%) caused decreasing survival rates (at pH 6.3 to 6.6). A 1999 study on M. paratuberculosis survival in spiked soils in Australia found shorter survival in dry alkaline soils and no apparent effect of UV light.

Observations regarding the associations among soil pH, calcium or iron content and the incidence of paratuberculosis have a long history. The 1956 review article on Johne's disease by Doyle covers most of the early observations on association of soil type and paratuberculosis incidence and the 1997 review by Johnson-Ifearulundu complements it by covering more recent literature. The fact that this observed association (i.e. not a proven causal link), in particular concerning soil pH, has been made in England, France, The Netherlands, and the U.S. adds credibility to the idea that somehow soil composition and paratuberculosis are connected. In addition, Johnson-Ilfearulundu and Kaneene recently showed by careful epidemiologic analysis that in the state of Michigan in the U.S. the practice of application of lime to pastures (a practice that should increase soil pH) in 1993 was associated with ten-fold lower odds of a dairy herd being serologically test-positive for M. paratuberculosis infection in 1996. As an aside, it is interesting to note that in a very different study an association was demonstrated between soil acidity and frequency of skin test reactions (diagnostic tests for exposure or infection with mycobacteria) in humans.

These epidemiological observations have led to speculation concerning mechanisms by which soil pH, or its interaction with soil calcium and iron content, affect M. paratuberculosis survival. It should be noted, however, that the relationships observed are indirect, i.e. simply the association between and incidence of clinical bovine paratuberculosis and broadly characterized soil types. No laboratory studies have been done to verify if a particular soil type affects M. paratuberculosis survival or to explain the mechanism.

Survival of M. paratuberculosis in feces injected into soils

Manure management is a component of domestic agriculture enterprises. Modes of dispersal include spreading it on fields, injection into the soil and slurry pit storage for later dispersal.

No published reports on M. paratuberculosis survival after injection of contaminated feces into soil were found. Intuitively, the advantage of this practice is that it lessens opportunities for contact between M. paratuberculosis and animals or forage crops and decreases the chance for contamination of surface water by rainfall run-off. However, feces injection might also favor survival of M. paratuberculosis by placing it away from more harsh conditions of drying and sunlight. Minimal research has been conducted in this area.

Survival in feces stored in slurry pits

Jörgensen published the first comprehensive study of its kind on survival of M. paratuberculosis in slurry in Denmark in 1977. In his work he used cattle slurry (pH 8.5, dry matter 7%), swine slurry (pH 8.3, dry matter 8.3%), and a mixture of the two (pH8.4, dry matter 7.7%). After spiking each slurry preparation with 3 x 10⁷ M. paratuberculosis/ml, he bubbled a mixture of hydrogen and nitrogen gas through the slurry to secure anaerobic conditions and then stored the slurry at 5°C or 15°C. Jörgensen reported that the number of colonies of M. paratuberculosis isolated on modified Löwenstein-Jensen medium dropped drastically between sampling days 1 and day 7 but then remained relatively stable until recovery of the organism stopped indicating the limit of survival. At 5°C the survival time was 252 days in all three kinds of slurry, and at 15°C it was 182 days in swine slurry, 98 days in cattle slurry, and 168 days in mixed slurry. Comparable findings have been reported on survival of M. bovis in cattle slurry.

The second major study on M. paratuberculosis in slurry was reported by Olsen, Jörgensen and Nansen in 1985. Their study concerned conditions found during anaerobic digestion of slurry as in bio-gas plants. Slurry was spiked to yield initial counts of 3.3 x 10³ to 2.7 x 10⁴ M. paratuberculosis /gm slurry and held at mesophilic conditions (moderate temperatures; 35°C or 95°F) or thermophillic conditions (high temperatures; 53-55°C or 127-131°F). At mesophillic conditions M. paratuberculosis was re-isolated at 7, 14, and 21 but not 28 days. At thermophillic conditions viable M. paratuberculosis could not be detected in as short as 3 hours.

Survival in compost

No research on survival of M. paratuberculosis in composted animal wastes was found. However, the time-temperature profiles of properly composted animal waste suggests that such conditions would be lethal to the organism.

Key References

Thorel, M.-F, M. Krichevsky, and V. V. Levy-Frebault. 1990. Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. Nov., and Mycobacterium avium subsp. silvaticum subsp. Nov. Int. J. Syst. Bacteriol. 40:254-260.

Collins CH, Grange JM, Yates MD. Mycobacteria in water, 1984. J Appl Bacteriol 57:193-211.

Lovell R, Levi M , Francis J. Studies on the survival of Johne's bacilli, 1944. J Comp Path 54:120-129.

Doyle TM. Johne's disease, 1956. Vet Rec 68:869-878.

Johnson-Ifearulundu YJ, Kaneene JB. Relationship between soil type and Mycobacterium paratuberculosis, 1997. J Am Vet Med Assoc 210:1735-1740.

Jörgensen JB. Survival of Mycobacterium paratuberculosis in slurry, 1977. Norsk Vet. Med. 29:267-270.

Olsen JE, Jörgensen JB, Nansen P. On the reduction of Mycobacterium paratuberculosis in bovine slurry subjected to batch mesophilic or thermophilic anaerobic digestion, 1985. Agricultural Wastes 13:273-280.

Falkinham III, J.O., C.D. Norton, and M.W. LeChevallier. 2001. Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other mycobacteria in drinking water distribution systems. Appl. Env. Microbiol. 67:1225-1231.