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.
- SourceAvailable from: Annette Summers Engel[Show abstract] [Hide abstract]
ABSTRACT: Terrestrial sulfidic springs support diverse microbial communities by serving as stable conduits for geochemically diverse and nutrient-rich subsurface waters. Microorganisms that colonize terrestrial springs likely originate from groundwater, but may also be sourced from the surface. As such, the biogeographic distribution of microbial communities inhabiting sulfidic springs should be controlled by a combination of spring geochemistry and surface and subsurface transport mechanisms, and not necessarily geographic proximity to other springs. We examined the bacterial diversity of seven springs to test the hypothesis that occurrence of taxonomically similar microbes, important to the sulfur cycle, at each spring is controlled by geochemistry. Complementary Sanger sequencing and 454 pyrosequencing of 16S rRNA genes retrieved five proteobacterial classes, and Bacteroidetes, Chlorobi, Chloroflexi, and Firmicutes phyla from all springs, which suggested the potential for a core sulfidic spring microbiome. Among the putative sulfide-oxidizing groups (Epsilonproteobacteria and Gammaproteobacteria), up to 83% of the sequences from geochemically similar springs clustered together. Abundant populations of Hydrogenimonas-like or Sulfurovum-like spp. (Epsilonproteobacteria) occurred with abundant Thiothrix and Thiofaba spp. (Gammaproteobacteria), but Arcobacter-like and Sulfurimonas spp. (Epsilonproteobacteria) occurred with less abundant gammaproteobacterial populations. These distribution patterns confirmed that geochemistry rather than biogeography regulates bacterial dominance at each spring. Potential biogeographic controls were related to paleogeologic sedimentation patterns that could control long-term microbial transport mechanisms that link surface and subsurface environments. Knowing the composition of a core sulfidic spring microbial community could provide a way to monitor diversity changes if a system is threatened by anthropogenic processes or climate change.Frontiers in Microbiology 09/2014; 5:473. · 3.94 Impact Factor
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ABSTRACT: Slurry can harbor multiple microbial pathogens among which Mycobacterium avium subsp. paratuberculosis (MAP).•We evaluated the persistence of MAP in soil and infection of soil Acanthamoeba•Infection of amoeba by MAP provides a protected niche for the persistence•As others have suggested, MAP-infected amoeba may act like a “Trojan horse”Veterinary Microbiology 10/2014; · 2.73 Impact Factor
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ABSTRACT: Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay.PLoS ONE 09/2014; 9(9). · 3.53 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2004, p. 2989–3004
0099-2240/04/$08.00?0 DOI: 10.1128/AEM.70.5.2989–3004.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 5
Survival and Dormancy of Mycobacterium avium subsp. paratuberculosis
in the Environment
Richard J. Whittington,1* D. Jeff Marshall,2Paul J. Nicholls,3Ian B. Marsh,3
and Leslie A. Reddacliff3
Faculty of Veterinary Science, The University of Sydney, Sydney,1Orange Agricultural Institute, Orange,2and
Elizabeth Macarthur Agricultural Institute, Camden,3New South Wales, Australia
Received 17 November 2003/Accepted 3 February 2004
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 corre-
lated 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 dor-
mancy. However, survival of M. avium subsp. paratuberculosis in the environment is finite, consistent with its
taxonomic description as an obligate parasite of animals.
Paratuberculosis, or Johne’s disease, occurs worldwide and
is a chronic, enteric infection of animals caused by Mycobac-
terium avium subsp. paratuberculosis. It is transmitted insidi-
ously from adults to juveniles mainly by the fecal-oral route,
and grazing animals are most commonly affected. The organ-
ism can cause significant mortality rates when large doses are
acquired by young animals. The infection is difficult and ex-
pensive to diagnose, and it is very costly to eliminate from a
farm (7, 47). As M. avium subsp. paratuberculosis may also be
associated with Crohn’s disease in humans (1, 17), pressure is
building to reduce the prevalence of infection in farmed live-
Programs for M. avium subsp. paratuberculosis control in
livestock are being developed and promoted in many devel-
oped countries (4, 24). They are based either on testing and
culling of individual animals from within infected populations
to reduce prevalence or on depopulation of entire infected
herds and flocks to eliminate the infection. Central to these
efforts is an assumption about the causative organism. M.
avium subsp. paratuberculosis is described taxonomically as an
obligate pathogen and parasite of animals (36), so in theory it
can be eradicated by removal of all infected animals. However,
this organism can survive for long periods outside the host,
enabling it to persist and spread in the grassland environment
and to withstand a periodic lack of suitable hosts. The trans-
mission of the organism in animal feces was recognized early in
the last century, and the question of how long pastures remain
infective was raised as early as 1912 (30).
In Australia, where livestock production accounts for 13
billion Australian dollars in export earnings per annum, re-
search is being conducted on the feasibility of eradication of
ovine paratuberculosis by whole-flock depopulation, resting of
pasture, and restocking with healthy sheep. However, the time
that is required to eradicate the organism from the environ-
ment is unknown. It has been suggested that at least 6 months
to a year is required to render pastures safe after grazing by
infected cattle (7, 26). Data on the resistance of the organism
were reported in 1944 (26) when feces from a cow with para-
tuberculosis was placed in an open bowl in an exposed place in
London, United Kingdom, and cultured at intervals. The or-
ganism survived for about 9 months.
There are some other published data on the survival of M.
avium subsp. paratuberculosis (Table 1), with a trend toward
prolonged environmental viability, except in situations such as
animal house slurry in which urine is also present, or in silage
with low pH or high ammonia levels (22, 23). These data come
from the northern hemisphere, where livestock are commonly
housed indoors during winter on straw bedding, and where
climates tend to be milder than in the temperate grazing re-
gions of Australia. Furthermore, the data come from experi-
mental models without field validation and pertain to the C or
cattle strain of the organism, which is distinct from the impor-
tant S or sheep strain that is prevalent in Australian sheep
flocks (42). The S strain also occurs commonly in sheep in New
Zealand (8) and some European countries such as Iceland (49)
and Spain (R. J. Whittington and R. Juste, unpublished data).
In Australia, the S strain is found mostly in sheep, but it may
also infect goats and less commonly cattle (42, 48), and in New
Zealand it is also found in goats and red deer (11). The S strain
has cultural requirements different from those of the C strain
(8, 43), but little else is known about it microbiologically.
* Corresponding author. Mailing address: Faculty of Veterinary Sci-
ence, The University of Sydney, Private Bag 3, Camden, NSW 2570,
Australia. Phone: 61 2 93511611. Fax: 61 2 93511618. E-mail:
The apparent duration of survival of M. avium subsp. para-
tuberculosis strain S can be inferred from observations on farms
in southeastern Australia, where it was recovered from approx-
imately 20% of 163 soil-pasture, water, and sediment samples
from six farms with clinically affected sheep or goats (45).
When the same sites were sampled again about 5 months later,
after removal of affected animals, only one was culture posi-
tive, and none were culture positive ?12 months later.
The aim of this study was to determine the duration of
environmental survival of the S strain of M. avium subsp. para-
tuberculosis under Australian conditions and to investigate the
effects of a number of factors, including solar radiation and soil
pH and moisture, that might influence survival.
MATERIALS AND METHODS
Natural pasture plots and boxes of soil containing sown grasses were contam-
inated with infected sheep feces. Fecal, soil, and pasture samples were collected
at intervals for up to 117 weeks and cultured to detect viable M. avium subsp.
paratuberculosis. There were four experiments (Table 2). Experiment 1 began in
January 1998 and was undertaken at two locations with seven plots at each to
TABLE 1. Summary of reported duration of survival of M. avium subsp. paratuberculosis C strain in natural substrates exposed to conditions
mimicking the natural environment and in various laboratory models
SubstrateSource of bacilli
concn or no. of
Temp (°C); light Duration of survivalReference
In dip fluids, slurry, feces, and urine
Amitraz cattle dip fluid (pH 12.4)
Anaerobic bovine slurry (mixture
of feces, urine, straw, and water)
Anaerobic bovine slurry
Anaerobic bovine slurry
Anaerobic bovine slurry
Bovine feces and strains
Naturally infected feces
?2, ?3 wk
?252, ?287 days
Naturally infected feces,
Naturally infected feces
Naturally infected feces
Naturally infected feces
Naturally infected feces
?98, ?112 days
?21, ?28 days
Bovine feces (liquid) in open bowl
Bovine feces in open bowl
Bovine feces in open bowl
Caprine feces in open bowl
Bovine urine and feces
Ambient, ?3 to 23; exposed
Ambient, ?2 to 23; exposed
Ambient, ?2 to 23; exposed
Ambient, ?2 to 23; exposed
?246, ?284 days
?208, ?236 days
Tap water (pH 7)
Tap water (pH 5 or 8.5)
Distilled water, sterile (pH 6.4–
6.8), in sealed bottle
Tap water, sterile (pH 7.1–8.0), in
Pond water plus mud, sterile (pH
5.3–5.9), in sealed bottle
River water in bottle
Ambient, 9 to 26
?17, ?19 mo
?14, ?17 mo
?9, ?13 mo
Ambient, 9 to 26
?9, ?13 mo26
Ambient, 9 to 26
?9, ?13 mo26
UncertainAmbient, January to May in
Ambient, ?7 to 18; shade
?113, ?141 days26
River water in open bowlBovine intestinal
?135, ?163 days26
River water in open bowlUncertainAmbient, ?7 to 18; sun
?163, ?218 days 26
Uncertain455 days; D value;
69 to 92 days
In laboratory models
Silage model (pH 4.4)
Silage model NH3?1%
Spotted on paper
Spotted on paper
30 to 37
?14 for 5 mo, then 4 for 5
mo, then 38 for 8 mo
?14 for 12 mo, then 4 for 5
Up to 44
?17, ?19 mo
Desiccated cultureCultured bacilli1010
Naturally infected feces
Naturally infected feces
Naturally infected feces
?65, ?100 h
aBased on authors’ raw data (colony count) or, if no other data given, assuming 1 mg (wet weight) of bacilli ? 107organisms and 1 mg (dry weight) of bacilli ? 108
bStrain type uncertain.
2990WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.
evaluate the effects of shade, irrigation, and lime application on survival. Exper-
iment 2, a pilot study, began in November 1998 with plots at the same locations
and boxes containing soil and pasture at a third site to evaluate the effect of
shade and potential differences between plots and boxes. Experiment 3 was
undertaken at two locations with plots and boxes, was started in November 1999,
and was repeated as experiment 4 in January 2000, to examine the seasonal
effects of solar radiation and shade on the survival of the organism.
Field sites. Field sites were established in areas of endemicity for ovine para-
tuberculosis at Borenore and Carcoar (altitude, 1,000 m above sea level; 33°S
latitude) in the Central Tablelands district and at Camden (70 m above sea level;
34°S) in the Sydney district of New South Wales. At Camden there was an
exposed unshaded site, as well as a protected partially shaded site on the veranda
of a building. An intentionally shaded site (70%) was constructed on the veranda
with knitted polypropylene cloth, and a second site on the veranda was totally
shaded (100%). Shaded enclosures (70%), fully covered with knitted polypro-
pylene cloth and measuring 10 by 6 by 2.4 m (length by breadth by height,
respectively) were constructed in open paddocks on farms at Borenore and
Carcoar. Each was surrounded by a secure perimeter fence to exclude livestock
and an earth mound to prevent surface water runoff. With a handheld radiometer
it was confirmed that 70% of incident solar radiation was absorbed by the woven
cloth at each site and that there was little or no UV radiation in the 100% shade
treatment at Camden. The shaded sites at Camden were completely protected
from natural rainfall.
At Borenore and Carcoar, marker pegs and string lines were used to create
pasture plots and square subplots either 1.5 by 1.5 m in triplicate (experiments 1
and 2) or 1.1 by 1.1 m in quadruplicate (experiments 3 and 4), within the shade
enclosures and also in unshaded locations on the northern side of each enclosure.
Microirrigation sprayers were installed in plots 3, 4, 5, and 6 to provide water for
15 min each night to ensure constantly moist soil conditions. To increase soil pH,
fine agricultural lime was applied to plots 4 and 5 at rates of 50 and 250 g/m2(0.5
and 2.5 tonnes/ha), respectively, immediately prior to application of fecal mate-
rial. Very little pasture was present at the start of experiment 1, and fecal
material was applied to bare soil, but pasture was allowed to grow during this
experiment, and this created shade at soil level. By 5 months there was a dense
cover of grasses, broadleaf weeds, and clover, particularly inside the shaded
enclosures. For experiments 2 to 4, pasture was kept ?10 to 15 cm high by
regular manual cutting and removal to simulate grazing by sheep. The vegetation
was grass dominant with broadleaf weeds and clover and covered between 40 and
85% of the soil surface in shaded plots and 50 to 95% in unshaded plots.
Soil boxes composed of expanded polystyrene (58 by 38 by 23 cm) were filled
to a depth of 20 cm with soil. A commercial grass seed mixture (couch, 20%;
chewing fescue, 10%; perennial ryegrass, 70%) was sown with a light dressing (10
g/box) of fertilizer (4.8% nitrogen as ammonium, 5.7% phosphorus, 5.9% po-
tassium chloride, 12.6% sulfur, and 12.4% calcium) 7 days before application of
infected feces so that the grasses would germinate after contamination of the
boxes. Boxes were lightly watered to maintain the viability of the grasses, gen-
erally at a rate of ?0.5 liter per box per week. The boxes generally had an even
cover of grass shoots to 75 mm high by 1 week after contamination with feces. At
Camden in experiments 3 and 4, rainfall in unshaded boxes supported grass
growth whereas grass was not watered after 3 months and allowed to brown off
in the shaded boxes. A drainage tube was fitted to the base of one box in
experiment 2 to enable collection of runoff water.
Weather data. Automatic weather data loggers (Easydata Mk4; Environdata
Australia Pty. Ltd., Warwick, Queensland, Australia) were installed at the Bore-
nore and Carcoar sites (experiments 1 to 3) and also at Camden (experiments 3
and 4). These recorded dry bulb air temperature, soil temperature at 1-cm depth,
UV radiation (290 to 400 nm), solar radiation (500 to 1,000 nm with correction
to encompass 400 to 3,000 nm), and rainfall. Daily maximum, minimum, and
average dry bulb air temperature, soil temperature, rainfall, solar radiation, and
UV radiation were recorded or derived from these measurements. For experi-
ment 2 at Camden, only the daily maximum and minimum dry bulb air temper-
atures in the immediate environment of the boxes were recorded.
Source and preparation of naturally infected feces. Feces containing M. avium
subsp. paratuberculosis were collected from groups of sheep on three separate
occasions, namely, just prior to experiments 1, 2, and 3. The feces used in
experiment 4 were from the same sheep as those used in experiment 3 but were
stored at ?80°C for about 2 months. The sheep were infected with M. avium
subsp. paratuberculosis strain BstEII type S1, IS1311 type S (42). Sheep were
TABLE 2. Experimental design, starting levels of contamination, and maximum observed period of survival
Expt no.Starting datea
Unit (no. of
Viable counts of M. avium
Per g of
1Jan 1998Plot 1 (3)c
Plot 2 (3)c
Plot 3 (3)c
Plot 4 (3)c
Plot 5 (3)c
Plot 6 (3)
Plot 7 (3)
Plot 8 (3)
Plot 9 (4)
Plot 10 (4)
Plot 11 (4)
Plot 12 (4)
5.00 ? 106
7.11 ? 105
7.11 ? 105
7.11 ? 105
7.11 ? 105
7.11 ? 105
3.34 ? 104
3.34 ? 104
1.07 ? 105
1.63 ? 105
3.16 ? 104
3.16 ? 104
2.10 ? 104
2.10 ? 104
2.10 ? 104
2.10 ? 104
2.10 ? 104
3.16 ? 104
3.16 ? 104
2.10 ? 104
2.10 ? 104
2.10 ? 104
2.10 ? 104
2.10 ? 104
5.00 ? 105
2Nov 19981.20 ? 106
3Nov 19991.58 ? 105
4Jan 20001.58 ? 105
aJan, January; Nov, November.
bVegetative cover provided significant shade if not removed.
cThese plots received slurry mix. All other treatments received pellet mix.
VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2991
individually identified; purchased from a farm at Goulburn, New South Wales;
housed in a secure animal house; and fed lucerne pellets, lucerne hay, chaff, and
oats, and feces were collected as described elsewhere (46). The feces from each
animal from each day were collected into plastic bags and held at 4°C, or at
?80°C if not required for use within a few days. The quantities of feces collected
in the first year were representative of those in later years and are reported
separately (46). Feces from animals with soft-formed stools were premixed with
chaff (4 liters/kg of feces) to obtain discrete masses of feces which were added to
the other feces with some water and additional chaff to obtain a dry, flaky,
free-falling pelleted mixture (85% feces by weight), or they were mixed for longer
and pellets were broken down by hand to form a slurry mix. The pooled feces
were thoroughly mixed in a large mechanically rotated drum and then divided
into portions and stored overnight in sealed plastic bags at 4°C prior to contam-
ination of sites. Subsamples were retained at ?80°C for later enumeration of M.
avium subsp. paratuberculosis cells (see below). The fecal mixtures contained 105
to 106viable organisms per g (Table 2). The organisms in both fecal mixtures
used in experiments 3 and 4 were enumerated in April 2000, and counts were the
Contamination of plots and boxes. Plots were contaminated evenly by hand
with pellet mix at a rate of 0.9 to 1.7 kg/m2, being a level of fecal contamination
consistent with usual sheep stocking rates and equivalent visually to a pellet upon
every few square centimeters of soil surface, or with slurry mix at a rate of 0.7
kg/m2. Similarly each box was contaminated evenly with 300 g of fecal pellet mix.
The contamination rates applied to soil were in the range of 104to 106viable
organisms per square centimeter (Table 2). The boxes were contaminated in situ,
except for experiment 3, where the fecal mixture was applied at Camden and the
contaminated boxes were then transported for 3 h by vehicle to Borenore.
Movement during transport caused surface pooling of water and coating of fecal
material in some boxes with mud and adversely affected grass seed germination.
Sampling from plots and boxes. In each experiment all the subplots of each of
the field plots were sampled at each time. A galvanized steel wire grid, 1 m2, with
1,600 numbered cells each of 2.5 cm2was placed on the ground and aligned
carefully with a fixed marker peg at the corner of each subplot, and random
numbers were used to select cells for the collection of samples. A fecal pellet was
collected from the cell containing a pellet that was nearest to the selected cell.
Vegetation was parted carefully, and after removal of the pellet, a core 1 cm in
diameter by 2 cm deep was taken from the soil beneath the pellet by using a
sterile 10-ml syringe barrel from which the tip had been cut. Soil cores from plots
contaminated with fecal slurry mixture included the slurry mixture on the surface
of the soil, and separate collection of fecal material was not attempted. Soil cores
included surface litter, soil, and plant roots to a depth of about 2 cm as well as
some aerial parts of plants where these could not be avoided.
Boxes in experiment 2 were marked out into two equal segments and used for
two consecutive collections of pellets and soil beneath them. About 50 ml of
runoff water was collected weekly from a drainage tube in the base of box 10 after
overwatering this box. In experiment 3, each box was marked out into three equal
segments (A, B, and C). One segment of each of two (Borenore) or three
(Camden) boxes was sampled at each time.
At each sampling, two pools of 10 pellets and subjacent soil cores were taken
at random from each subplot or box segment for culture. Pellets and soil cores
were pooled in separate containers. At the final sampling of boxes in each
experiment, between 4 and 16 times the usual number of samples were collected
to increase the probability of isolating low numbers of organisms. Culture results
for the pooled samples of pellets and soil were paired to determine whether
viable M. avium subsp. paratuberculosis organisms were present in that subplot or
box segment (culture site) at each sampling time. After primary culture, all
samples were stored at ?80°C to enable enumeration of the organisms in se-
lected culture-positive samples. Precontamination samples consisting of two
pools of 10 soil cores were collected from representative subplots as negative
controls for soil inside and outside the shade enclosures, and negative-control
soil samples were taken from boxes. These samples were all culture negative.
Immediate-postcontamination samples were collected from all subplots and
boxes to confirm uniform contamination and effective sampling. These samples
were all culture positive. Sampling of pellets was continued for as long as they
were recognizable as discrete objects. Grass samples were collected with scissors,
with careful cutting so as to avoid contamination with feces or soil.
Culture methods. Samples were thoroughly mixed prior to subsamples of 2 g
being taken for culture. Initially mixing was undertaken by hand with a mortar
and pestle and scissors to break up plant material, but in most cases a high-speed
electric blender with metal cutting blades was used (41). Cultures were per-
formed using a double incubation and centrifugation method to decontaminate
samples and modified BACTEC 12B radiometric medium (Becton Dickinson) as
previously described (43, 44). Vials were incubated at 37°C for 20 weeks to detect
low numbers of the target organism (45). Identification of M. avium subsp.
paratuberculosis was achieved by detection of IS900 by PCR directly from the
BACTEC culture medium, with restriction endonuclease analysis of PCR prod-
uct to ensure specificity (10). Grass samples were placed in resealable plastic
bags, and 250 to 500 ml of saline with 0.1% (vol/vol) Tween 80 was added so that
the grass was completely covered. The bag was placed on a rocking platform for
1 h at room temperature and turned over every 15 min to ensure thorough
washing of the grass. The washing water was collected and centrifuged at 11,000
? g for 20 min. The pellet was then added to a tube containing 10 ml of saline
to sediment debris, and the remainder of the procedure was identical to that used
for culture of feces. Water samples from box 10 in experiment 2 were centrifuged
at 11,000 ? g for 20 min, and the pellet was added to saline and cultured as
Enumeration of M. avium subsp. paratuberculosis. Unless otherwise stated, five
replicate cultures, each of 2 g, were undertaken for each sample, and the organ-
ism was enumerated by endpoint titration in radiometric culture medium (46).
Dilutions were made in phosphate-buffered saline. Rates of contamination of M.
avium subsp. paratuberculosis per unit surface area of soil were calculated based
on the results of enumeration of the organism in the fecal mixture and the
amount of mixture applied per unit area.
Direct PCR analysis of fecal pellets. DNA was extracted from fecal pellets by
boiling, purified over a silica column, and examined for IS900 exactly as de-
scribed elsewhere (27).
Soil analysis. The soil used in boxes was well mixed, and 1-kg samples were
submitted for analysis. Standard soil samples were collected from plots in Sep-
tember 1999, 20 months after liming plots for experiment 1, with the use of a
corer 2 cm in diameter by 10 cm in depth. Twelve cores were collected in a grid
pattern from each subplot in plots 2, 3, 4, 5, and 6 (36 cores per plot). Samples
were well mixed before analysis. Surface samples were also collected from the
upper 50 mm of selected plots. Soil analyses were performed by Analysis Sys-
tems, Incitec Ltd., Port Kembla, New South Wales, Australia, by standard meth-
ods: color and texture by observation; pH meter; conductivity meter; colorimetry
for organic carbon, nitrate nitrogen, sulfur (also measured turbidimetrically),
phosphorus, and chloride; and atomic absorption spectroscopy for potassium,
calcium, magnesium, sodium, aluminum, and iron.
In silico analysis of dormancy-associated genes. The Dps protein (DNA bind-
ing protein from starved cells) and the relA gene product (GTP pyrophosphoki-
nase) are active in survival and dormancy responses of bacteria under starvation
conditions, with homologues known in mycobacteria (2, 15). The DNA se-
quences for Mycobacterium smegmatis dps (GenBank accession no. AY065628)
and Mycobacterium tuberculosis relA (relA gene accession no. Rv2583c, Tuber-
culist Web Server, http://genolist.pasteur.fr/TubercuList/) were submitted to the
M. avium subsp. paratuberculosis genome database (http://www.ncbi.nlm.nih.gov,
accession no. NC 002944). Matching sequences from M. avium subsp. paratuber-
culosis were then analyzed in each reading frame for amino acid sequences
similar to those of Dps and RelA. Alignments were done in GAP with the
BLOSUM62 amino acid substitution table (16, 28) through the Bionavigator
facility, Australian National Genomic Information Service, University of Sydney.
Statistical analysis. (i) Assessment of treatment and time effects on the pro-
portion of culture-positive samples. For experiment 1, totals of the culture-
positive sites for each treatment in weeks 5 to 9 and weeks 14 to 18 were
expressed as proportions of the corresponding total number of cultured sites. For
experiments 3 and 4 combined, proportions of culture-positive sites for each
treatment in weeks 2 to 6 and weeks 8 to 16, and also weeks 20 to 36 for the
shaded treatments at Camden, were similarly determined. Mixed-model logistic
regression analyses of the proportions were used to assess the fixed effects of
periods and treatments and their two-factor interactions, with the effects of
locations and the location-period and location-treatment interactions taken as
random. The fixed effects in experiment 1 were source of contamination (slurry
mix or pellet mix), period (5 to 9 and 14 to 18 weeks), shade (nil and 70%), and
slurry treatment (control, lime rate, and irrigation), and those in experiments 3
and 4 were month of contamination (November or January), period (2 to 6 and
8 to 16 weeks, and 20 to 36 weeks for the shaded treatments at Camden), shade
(nil and 70% at both sites; 70 and 100% at Camden), and plot type (field, and box
at Borenore). For the latter experiments, the interactions with location involved
only the periods and treatments common to the locations. The analyses were
performed using ASReml statistical software (14). All tests were conducted at
the 5% level of significance (P ? 0.05).
(ii) Rates of decay of the number of viable organisms. Counts of the number
of viable organisms [log10(counts/gram)] over time were plotted (Prism; Graph-
pad Software Incorporated). Linear regressions of log10(counts/gram) on weeks
after contamination were performed for experiments 1 and 2, excluding the data
in later weeks, which were statistical outliers. For experiment 4, a linear mixed
2992WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.
model which comprised a fixed linear term and a random nonlinear term, fitted
as a cubic smoothing spline (37), was first fitted. This model showed that there
were two distinct phases of decline, so separate linear regressions were fitted for
each phase. For each regression relation, 95% confidence limits for the predicted
mean values were calculated.
Duration and patterns of survival. Over the first 12 to 18
weeks in the experiments, there were generally marked de-
clines in the mean proportions of culture-positive sites to low
or zero values (Fig. 1 to 4). No positive results occurred be-
tween 18 and 24 weeks in any experiment. However, in exper-
iments 1, 3, and 4, culture-positive results occurred at and after
24 weeks for some treatments and on earlier occasions follow-
ing one or more negative samplings, although the mean pro-
portions were usually low (Fig. 1, 3, and 4).
In experiment 1, the organism was recovered from plots at
both sites to 32 weeks after contamination. No significant ef-
fects of shade, source of contamination, lime treatment, or
irrigation on the proportion of culture-positive sites were de-
tected (P ? 0.05) (Fig. 1).
In experiment 2, which was a pilot study using boxes for the
first time, the duration of survival of the organism in feces and
soil on unshaded plots was up to 5 weeks, and up to 10 weeks
in soil boxes at the partially shaded location. The rate of
isolation from the shaded location appeared to be greater than
that from the unshaded location (Fig. 2). Grass samples from
the boxes were culture positive each week up to and including
week 4. The time for grass samples to reach peak growth index
(5 to 8 weeks) was similar to that of fecal pellets, implying
similar viable counts of M. avium subsp. paratuberculosis. Run-
off water collected from box 10 was culture positive to week 3,
and this represented water that had moved through the soil
profile and between the soil and the inside surfaces of the box.
As the duration of survival of the organism was considerably
longer in experiment 1, which started in January, than in ex-
periment 2, which started in November, and was shorter in
unshaded vegetated plots than in partially shaded boxes in
experiment 2, it was hypothesized that differences in the
amounts of solar radiation due to season, vegetation, and di-
rect shading may have been important. Therefore, experiment
3 was started in early November, shade was included as a
treatment at two levels (0 and 70%) for plots and boxes at
Borenore and three levels (0, 70, and 100%) for boxes at
Camden, and this design was repeated as experiment 4, which
started about 3 months later, at the end of January (Fig. 3 and
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.
VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2993
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.
2994 WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.
4). In the latter experiment M. avium subsp. paratuberculosis
survived for up to 55 weeks in fecal pellets in the shade but for
much shorter periods in unshaded locations.
In experiments 3 and 4, there was a significant interaction
between month of contamination and period: the mean pro-
portions of culture-positive sites in November and January
were 68.3 and 29.3%, respectively, for weeks 2 to 6 compared
with 10.2 and 14.2%, respectively, for weeks 8 to 16. Over
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.
VOL. 70, 2004 SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2995
weeks 2 to 16 the mean proportion of positive sites for 70%
shade (56.1%) was significantly higher than that for nil shade
(9.3%). At Camden, over weeks 2 to 36 there was a significant
increase of 17.2% in the mean proportion of positive sites
between the 70 and 100% shade treatments. At Borenore, over
weeks 2 to 16 the mean proportion of positive sites for boxes
(38.2%) was significantly higher than that for plots (20.3%).
In experiment 3, grass samples from boxes in 70% shade at
Camden were culture positive for 4 weeks while those in 100%
shade were positive for 10 weeks. The corresponding values for
experiment 4 were 9 and 24 weeks. The organism was not
recovered from grass from unshaded boxes at Camden in ei-
ther experiment. There were few positive cultures from grass
from boxes at Borenore, but survival was found after 9 weeks
in 70% shade in experiment 3.
A feature of the results for experiments 1, 3, and 4 was the
reappearance of culture-positive results after one or more time
points at which all samples were culture negative (Fig. 1, 3, and
4). To provide additional information on this phenomenon,
samples from experiment 3 (boxes, 100% shade, Camden)
were examined using direct PCR. M. avium subsp. paratuber-
culosis DNA was demonstrated in six of six culture-positive
samples from time zero, six of six samples taken at 10 weeks
(only three of which had been culture positive), four of five
culture-negative samples taken at 12 weeks, and six of six
culture-negative samples taken at 32 weeks. Thus, M. avium
subsp. paratuberculosis cells were present in pellets in most
samples even though the organism was not cultivable. In each
experiment the incubation time required for cultures to reach
peak growth index increased over time, consistent with a de-
cline in the number of viable organisms. However, in some of
the cases where the organism was cultured after a previous
culture-negative time point, growth occurred more quickly at
the later time point, suggesting an increase in the viable count
or recruitment of viable cells from a dormant state.
Retrospective enumeration of M. avium subsp. paratubercu-
losis in selected culture-positive samples from experiments 1, 2,
and 4 was undertaken and confirmed these observations. There
was an initial phase of rapid decline in viable count lasting
several weeks to a few months, but thereafter the pattern was
variable (Fig. 5). In experiment 1 counts were low or 0 from 9
to 32 weeks, while in experiment 2 the count was 0 at weeks 7
and 8 but rose to 75 at week 9. For experiment 4 there was a
significant spline trend in the mean count over weeks after
contamination, with a local minimum estimated near week 8
and a local maximum near week 18. The estimated increase in
mean count between the sampled weeks 6 and 16 was 0.97 ?
0.37 logs, which was significant (P ? 0.05) and indicated that
there were two decline phases (Fig. 5). This increase in viable
count coincided with a reduction in time to peak growth index
from 10 to 6 weeks when these samples were cultured origi-
nally. There was a small rise in the viable count in experiment
2 between weeks 3 and 4 coinciding with a reduction in time to
peak growth index from 8 to 6 weeks.
Rates of decay of the number of viable organisms in the
decline phases of experiments 1, 2, and 4 (with the week 16
data included as part of the second decline phase) were esti-
mated by linear regression, and estimates ranged from 0.55 to
0.10 logs/week (Fig. 5). When grouped according to the dura-
tion of the decline phase, there was an inverse relation (Table
Weather data. Representative weather data for a 12-month
period at Camden are shown in Fig. 6. Rainfall was evenly
FIG. 5. Log10counts 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 col-
lected 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.
2996WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.
distributed at each site, with periodic heavy falls of up to 100
mm/week associated with storms. Carcoar and Borenore re-
ceived about 700 mm rainfall annually compared to 500 mm at
Camden, which was warmer than the other sites. Maximum dry
bulb air temperatures approached 40°C at Carcoar and Bore-
nore and 45°C at Camden, and minima were below 0°C at each
site. The main factors varying between shade treatments were
the degree of solar radiation and soil temperature. In un-
shaded locations total weekly solar radiation levels exceeded
200 MJ/m2in summer and were as low as 25 MJ/m2in winter,
while total weekly UV levels were 5 to 7 W/m2in summer and
0.5 to 1 W/m2in winter. In unshaded plots or boxes soil tem-
perature at the 1-cm depth ranged from about 50°C in summer
to just above 0°C in winter at Carcoar and Borenore and
approached 60°C in summer at Camden. In 70% shaded plots
and boxes the maximum soil temperature recorded was about
40°C whereas in 100% shade it was about 30°C. The diurnal
range of soil temperatures was much less for shaded than for
unshaded locations (Fig. 6).
Analyses of soils. The soil used in boxes was a dark yellow-
brown, light, sandy loam with low organic matter content, a pH
of 5.8 to 6.1, and iron levels of 12 to 30 mg/kg (Table 4). The
soil present in pasture plots at Borenore and Carcoar was a
brown clay loam, had a higher organic matter content than that
in the boxes, was slightly acidic (pH 5.7 to 6.7 across plots), and
had iron levels of up to 130 mg/kg. The application of lime
resulted in an increase in pH of about 0.4 U for low lime and
1.0 U for high lime at Borenore and 0.2 U for low lime and 0.7
U for high lime at Carcoar. The high-lime plots at both Bore-
nore and Carcoar had a pH of 7.4 in surface samples.
In silico analysis of dormancy-associated genes. Regions
highly similar to dps of M. smegmatis and relA of M. tuberculosis
were identified in the M. avium subsp. paratuberculosis genome
sequence. The 552-bp DNA sequence (GenBank accession no.
AY065628) that codes for the 184-amino-acid Dps protein
from M. smegmatis was used to locate the corresponding region
in the M. avium subsp. paratuberculosis genome database
through a Blast search. The predicted amino acid sequences
had 82.5% similarity and 75.6% identity, including a perfect
match for each of the amino acids thought to be involved in the
DNA binding signature of the active protein in M. smegmatis
(see Fig. 8) (15).
There was a homologue of M. tuberculosis relA in the M.
avium subsp. paratuberculosis genome sequence (88% similar-
ity over 2,373 bp). The predicted amino acid sequence of M.
avium subsp. paratuberculosis RelA excluded amino acids aris-
ing from a 6-bp deletion corresponding to bp 49 to 54 in M
tuberculosis but had 96% similarity and 93.4% identity (see Fig.
The results of this study support those from trials in the
northern hemisphere with the cattle strain of M. avium subsp.
paratuberculosis and confirm that this taxon can be extremely
persistent in nature, with survival for more than 1 year. Unlike
earlier trials where contaminated material was placed in small
containers, survival was studied on farms where Johne’s dis-
ease is prevalent, in natural pasture plots and in boxes con-
taining soil and grass. The presence of soil and pasture pro-
TABLE 3. Decay rates of M. avium subsp. paratuberculosis in
shaded locations estimated by linear regression of actual counts
Pellets and soil from plots
Pellets from boxes
Pellets from boxes
Pellets from boxes
aShaded veranda boxes.
TABLE 4. Soil analysis
Expt 2 Expt 3
Low lime High limeNo limea
Low limeHigh lime
pH (water), superficial soil
Organic carbon (% C)
Sulfate sulfur (KCl40) (mg/kg)
Phosphorus, Colwell (mg/kg)
Phosphorus, Bray (mg/kg)
Potassium (meq/100 g)
Calcium (meq/100 g)
Magnesium (meq/100 g)
Aluminum (meq/100 g)
Sodium (meq/100 g)
Electrical conductivity (dS/m)
Nitrogen Kjeldahl (%)
aMean value for plots 2, 3, and 6.
bNT, not tested.
cDTPA, diethylenetriamine pentaacetic acid.
VOL. 70, 2004 SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2997
vided a more realistic substrate than what could be achieved in
a laboratory environment.
When M. avium subsp. paratuberculosis in feces becomes
mixed with soil, there is a reduction of 90 to 99% in the
apparent viable count of the organism. This is probably caused
by binding of bacteria to soil particles, which are excluded from
culture by sedimentation during sample preparation (45). At-
tachment to soil also occurs with other nontuberculous myco-
bacteria (5). The culture method used, in particular the use of
antibiotics and disinfectants during sample preparation, fur-
ther reduces the analytical sensitivity of in vitro culture by
killing more than 2 log10M. avium subsp. paratuberculosis cells
(32). Thus, estimates of viable count or duration of survival of
M. avium subsp. paratuberculosis based on culture from soil are
likely to be underestimates. The duration of survival assessed
in boxes containing soil and grass was comparable to that
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; I, mean; Œ, minimum.
2998 WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.
observed in pasture plots, although there were some differ-
ences, generally favoring recovery from soil in boxes. This was
probably explained by the use in boxes of soil with low organic
matter content. It is easier to isolate the organism from such
soils than from soils of higher organic matter content (45).
Boxes were a useful substitute for plots and may be used to
advantage in future studies because they are simple to set up
and maintain, soil type can be chosen, and contamination can
In addition to recoverability from samples and losses during
culture preparation, and assuming log-linear decay, the ob-
served duration of survival of microbes also depends on the
starting level of contamination, so we attempted to standardize
this between trials. However, the measurement of decay rates
was also important, because these may be able to be extrapo-
lated to situations with different starting levels of contamina-
The survival of the organism in fecal material applied to soil
was greatest (55 weeks) in a fully shaded environment and was
least where fecal material and soil were fully exposed to the
weather and where vegetation was also removed. Vegetation
provides shade at the soil surface, and in experiment 1 this
explained the observation of survival for 32 weeks in plots that
were not otherwise shaded. In experiment 3 the duration of
survival was only 2 weeks in unshaded plots from which vege-
tation was removed to simulate grazing by sheep. Moderate
degrees of shade were significantly protective when organisms
were most numerous soon after contamination, but over a
longer period a higher level of shade was required for signifi-
cant protection. Factors such as moisture and soil pH did not
appear to influence the duration of survival. Soil pH level has
been suggested as a risk factor for Johne’s diseases, through
mechanisms related to iron availability (19). Iron levels in soils
in plots (32 to 129 mg/kg) were higher than those in soils in
boxes (12.5 to 23 mg/kg), but survival of M. avium subsp.
paratuberculosis was greater in boxes than in plots. This result
may be due to confounding with soil organic matter content,
which was higher in plots than in boxes.
Natural rainfall was at times extremely heavy and conceptu-
ally may have caused leaching of bacteria from fecal material in
all plots and the exposed boxes. However, we were unable to
significantly reduce the contamination levels in fecal material
in a laboratory trial in which a rainfall event of 400 mm over 4
days was simulated by repeatedly soaking pellets in water (data
not shown). Therefore it is unlikely that the organism was
eluted completely from fecal material in exposed plots and
M. avium subsp. paratuberculosis was isolated for up to 24
weeks from the aerial parts of grasses in this study. Following
seed germination, grass shoots penetrated the surface litter
and feces and presumably became contaminated with the or-
ganism in this way. The organism may then have been washed
VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS 2999
from grass shoots by rainfall. The shaded boxes at Camden
were not exposed to natural rainfall and were watered very
carefully by hand, which might explain the higher rate and
longer duration of recovery of the organism from grass at
Camden than of that from grass at Borenore.
What factors could explain the principal observation from
this study that survival of M. avium subsp. paratuberculosis was
favored by shade? Moisture was not a factor promoting sur-
vival. Factors apart from moisture that differed dramatically
between shaded and unshaded treatments included solar radi-
ation, soil temperature, and the diurnal range or flux of soil
temperature. In a recent study of the effect of UV light on the
cattle strain of M. avium subsp. paratuberculosis, the organism
was irradiated while suspended in distilled water and appeared
to be no more resistant than many other bacterial species (9).
The following principles need to be considered: UV radiation
cannot penetrate fecal pellets, and therefore it can cause only
surface disinfection and cannot affect the shaded underside of
pellets; pellets, being dark objects, absorb radiant energy and
in turn radiate heat; heat would be conducted to deeper re-
gions of the pellet; temperature ranges in pellets on the soil
surface would be greater than those measured in soil at a depth
of 1 cm; and evaporation may cool fresh fecal pellets but not
dry pellets. Temperature flux stands out as an obvious factor
correlated with “shade” that could affect survival of M. avium
Experiments 3 and 4 began with contamination of plots and
boxes in early November (presummer) or late January (end of
summer), respectively. For the first 6 weeks after contamina-
tion, the survival rate of M. avium subsp. paratuberculosis in
experiment 3 was more than double the rate in experiment 4.
Over the same period, air and soil temperatures in experiment
3 were lower and had narrower ranges than those in experi-
ment 4 (Fig. 6) but the differences in cumulative solar radiation
were negligible (Fig. 7). These results strongly suggest that
temperature flux influences survival more than solar radiation
does and support our interpretation that the effect of shade is
primarily through a reduction in temperature flux.
The decay rates reported here were estimated from counts
from fully shaded or partially shaded treatments, as these had
a reasonable time series of culture-positive samples. These
decay rates are therefore assumed to be the worst-case sce-
nario. Although first-order kinetics for log-linear survival
curves is commonly assumed for microbial inactivation, there
are examples where this is not the case, and tailing of microbial
survival is sometimes reported (6). Decay rates estimated from
the linear regressions in this study ranged from 2.2 to 0.4
logs/month and were inversely related to the period of obser-
vation (Table 3). Differences in environment, climate, and
other factors may impact decay rates such that the decline
might occur in a different pattern from that seen in this study.
Decay rates for unshaded locations are likely to be higher than
those for shaded sites. They were inferred from starting counts
of M. avium subsp. paratuberculosis in feces, and the observed
durations of survival in feces consequently were highly vari-
able, ranging from 1.1 to 7 logs/month. However, when the
effect of dormancy (see below), which led to culture-positive
outliers, was removed, the decay rates were more consistent
FIG. 7. Cumulative solar radiation for experiments 3 and 4 measured at Camden and aligned by week after contamination. I, experiment 3,
commencing 8 November 1999; Œ, experiment 4, commencing 31 January 2000.
TABLE 5. Decay rates of M. avium subsp. paratuberculosis in
pellets in unshaded locations where pasture was either light or was
removed to simulate grazing, inferred from starting concentrations
of the organism and the observed duration of survival, which was
assumed to be the closest week after the last culture-positive
1.2 ? 106
1.2 ? 106
1.6 ? 105
1.6 ? 105
1.6 ? 105
1.6 ? 105
1.6 ? 105
1.6 ? 105
aFigures in parentheses ignore isolation of low numbers of organisms follow-
ing the occurrence of dormancy.
3000WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.
(range of 3 to 7 logs/month) (Table 5) and greater than those
measured for shaded locations. Inclusion of observations of the
small numbers of viable organisms present following a period
of dormancy is relevant when considering eradication of the
organism from the environment but less relevant when consid-
ering control of the infection in livestock. The reliability of
these inferred estimates for unshaded sites is unclear.
In this study M. avium subsp. paratuberculosis was cultured
from all fecal-soil samples collected soon after contamination,
and afterwards there were a progressive reduction in the num-
ber of culture-positive samples and an increase in the incuba-
tion period required to reach peak growth index. This is con-
sistent with a gradual decline in the viability of the organism.
However, the time required to reach peak growth index tended
to stabilize, often at around 9 weeks of incubation, with sub-
sequent cultures being negative. For soils and feces, incubation
periods to peak growth index greater than about 6 weeks are
consistent with there being only one or several viable organ-
isms in the sample (31). Growth index reaching a peak after
this interval is suggestive of M. avium subsp. paratuberculosis
cells requiring a resuscitation phase of several weeks in the
culture medium prior to commencement of replication. After
one or more time points at which all samples were culture
negative, the organism was again recovered from soil and fecal
pellets, sometimes with a reduction in the time required for
cultures to reach peak growth index compared to that for
earlier time points, and in some cases with a sudden increase in
the proportion of culture-positive samples, coinciding with an
increase in viable counts. There are four possible reasons for
these observations: uneven distribution of organisms, system-
atic laboratory error, changes in properties of binding of the
organism to feces or soil, and bacterial dormancy.
Firstly, consider uneven distribution of fecal material and a
sampling effect, such that the organism was not included in all
samples. This is unlikely because well-mixed feces were evenly
spread by hand, all postcontamination control samples from all
subplots and boxes were culture positive, the sampling method
was random and was replicated, and M. avium subsp. paratu-
berculosis DNA was demonstrated in numerous samples of
culture-negative fecal pellets. We infer the continuing pres-
ence of intact bacterial cells in these pellet samples, as extra-
cellular DNA would have been degraded by the ubiquitous
DNases from other organisms present in feces.
Secondly, systematic laboratory error influencing the sensi-
tivity of culture (medium or operator effect) was unlikely be-
cause medium controls were used, there was little or no tem-
poral overlap in testing batches of samples across the four
experiments, and both positive- and negative-culture outcomes
were obtained at common test times.
Thirdly, a physicochemical effect that causes the organism to
change its binding properties with fecal material or soil com-
ponents so that its availability in the culture system changes
over time was unlikely within fecal pellets or soil.
The fourth explanation is dormancy of M. avium subsp.
paratuberculosis cells. The data presented in this study are
consistent with M. avium subsp. paratuberculosis being able to
enter a dormant or viable-noncultivable state and later revert-
ing to a vegetative form. This phenotypic property has not been
reported before for M. avium subsp. paratuberculosis. Dor-
mancy is defined as the state permitting survival of a non-
spore-forming bacterial cell without requiring replication. It is
genetically programmed, reversible, and induced by an unfa-
vorable environment, classically when an essential nutrient re-
quired for growth becomes limiting. Evidence for dormancy is
inability to culture the organism until the environment again
becomes favorable and cells regain the ability to divide and
thus become detectable (21).
In rapidly growing bacterial species dormancy is associated
with expression of specific genes, at least some of which are
known in mycobacteria. Oxygen depletion of cultures of M.
smegmatis (12), Mycobacterium bovis (18), and M. tuberculosis
(39) leads to dormancy and increased resistance to antibiotics
(40). In M. tuberculoisis prolonged in vitro culture with reduced
growth rate is associated with expression of heat shock proteins
in the stationary phase of culture (50). Recently, Dps-like
protein, which confers protection by binding to DNA during
nutritional and oxidative stress in other bacteria, was identified
in M. smegmatis and a homologue was found in the M. avium
genome (15). An in silico investigation identified a putative
sequence in M. avium subsp. paratuberculosis which contained
each of the amino acid residues that form the DNA binding
signature in the M smegmatis protein (Fig. 8). A second gene,
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.
VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS 3001
relA, which is active during the stringent response of M. tuber-
culosis to amino acid or carbon source depletion (2), is also
present in M. avium subsp. paratuberculosis (Fig. 9). These
findings add weight to the proposition that M. avium subsp.
paratuberculosis is capable of dormancy. However, the stimulus
for dormancy in the present study is unclear apart from sepa-
ration of this obligate parasite from its host with consequences
inferred for access to nutrients. Similarly, there must have been
an environment favorable for reversion to the vegetative state,
which might have occurred in nature or might have occurred
once dormant cells were added to culture media. However, the
culture media alone, which were constant throughout the
study, were not sufficient to resuscitate dormant cells, as there
were time points in the longitudinal study at which all samples
were culture negative and later time points at which some
samples were culture positive.
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.
3002 WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.
Sporadic environmental replication of M. avium subsp. para-
tuberculosis is another explanation for some of the observa-
tions in this study but is less likely than dormancy. Environ-
mental replication has not been reported for M. avium subsp.
paratuberculosis and is precluded by the current taxonomic
definition of the taxon (36). Further experiments, some of
which may now be conducted in silico by evaluation of the M.
avium subsp. paratuberculosis genome (3) for dormancy-asso-
ciated genes, are indicated to evaluate dormancy in M. avium
In conclusion, M. avium subsp. paratuberculosis is capable of
prolonged survival in the environment in Australia. However,
under the conditions of the present study, survival was finite.
Significant degrees of pasture decontamination can be
achieved in a relatively short period, and this will have benefits
for disease reduction in a flock or herd because of the likely
beneficial effects that lower doses of M. avium subsp. paratu-
berculosis would have on incubation period and disease out-
come (47). Eradication of the organism from pasture and soil
requires very prolonged decontamination intervals. The pro-
tective effect of shade has important practical implications for
control and eradication of paratuberculosis, even under the
harsh environmental conditions in Australia. Pasture manage-
ment, such as selective grazing with nonsusceptible hosts or
mechanical slashing, may be used to maintain a relatively low
level of shade at the soil surface to hasten decontamination.
Dormancy of the organism appears to be a feature in the
Australian environment, and this is supported by the presence
of dormancy-related genes in the M. avium subsp. paratuber-
culosis genome. This may also have implications in vivo where
survival in the intracellular environment is required.
This study was funded by Meat and Livestock Australia and NSW
Skilled technical assistance was provided by Elissa Choy, Scott
McAllister, Vanessa Saunders, Aparna Vadali, Brian Maddaford,
Christine Kearns, and Phil Slattery. Terry and Cecily Hayes, Hillwood,
Goulburn; Bess Vickers, Barrawinga, Carcoar; and Australian Na-
tional Field Days, Borenore, assisted us with hospitality, supply of
sheep, and access to their land.
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