Molecular Diagnostic Methods for Mycobacterium avium subsp. paratuberculosis
1 Mycobacterium avium subsp. paratuberculosis persistence in a controlled 1 dairy cattle farm
Transcript of 1 Mycobacterium avium subsp. paratuberculosis persistence in a controlled 1 dairy cattle farm
1
Mycobacterium avium subsp. paratuberculosis persistence in a controlled 1
dairy cattle farm environment studied by culture and quantitative IS900 real-2
time PCR 3
4
5
M. Moravkova, V. Babak, A. Kralova, I. Pavlik, I. Slana* 6
Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic 7
8
*Corresponding author: 9
Tel.: +420 5 33331618 10
fax: +420 5 41211229 11
E-mail address: [email protected] (I. Slana). 12
13
The aim of this study was to monitor the persistence of Mycobacterium avium subsp. 14
paratuberculosis (MAP) in environmental samples taken from a Holstein farm with a 15
long history of clinical paratuberculosis. A herd of 606 heads was eradicated and 16
mechanical cleaning and disinfection with Chloramin B with ammonium (4%) was 17
carried out on the farm; in its surroundings (on the field and field midden) lime was 18
applied. Environmental samples were collected before and over a period of 24 19
months after destocking. Only one sample out of 48 (2%) examined on the farm 20
(originating from a waste pit and collected before destocking) was positive for MAP 21
by cultivation on solid medium (HEYM). The results using real-time PCR (qPCR) 22
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01264-12 AEM Accepts, published online ahead of print on 6 July 2012
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
2
showed that a total of 81% of environmental samples with an average mean MAP cell 23
number of 3.09x103 were positive for MAP before destocking compared to 43% with 24
an average mean MAP cell number of 5.86x102 after 24 months. MAP-positive 25
samples were detected in the cattle barn as well as in the calf barn and surrounding 26
areas. MAP was detected from different matrices: floor and instrument scraping, 27
sediment or scraping from watering troughs, waste pit and cobwebs. MAP DNA was 28
also detected in soil and plants collected on the field midden and field 24 months 29
after destocking. Although the proportion of positive samples decreased from 64% to 30
23% over time, the number of MAP cells was comparable. 31
32
MAP is the causal agent of a chronic inflammatory intestinal infection known 33
as paratuberculosis or Johne´s disease. Its worldwide spread in ruminants means 34
that it bears a high financial impact, especially in dairy cattle. Losses attribute to 35
clinical infection include decreases in milk production, increasing calving intervals, 36
reductions in slaughter weight, infertility and increased incidence of mastitis (12, 24). 37
Additionally, the potential role of MAP in triggering Crohn´s disease has been 38
discussed for many years (22, 38, 42). Furthermore, new studies describing a 39
significant association of MAP with diabetes mellitus type I and multiples sclerosis 40
patients have appeared (4, 5). 41
Due to the long incubation period that is necessary for the development of 42
clinical signs as well as variable immunological responses and irregular shedding of 43
MAP in faeces, it is difficult to diagnose paratuberculosis and eliminate this pathogen 44
from the farm and pastures before it spreads to other susceptible animals. It has 45
been documented that cows with clinical signs can shed pathogens in average 46
amounts of 106 MAP cells per gram (6). Therefore, the natural excretion of MAP in 47
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
3
the faeces of infected animals as well as the land application of their fresh manure 48
onto fields and pastures contributes to the spread of the pathogen in the environment 49
and its persistence. Interspecies transmission between cattle and wild animals 50
sharing the same habitats was documented by some authors (9, 17). 51
Although MAP is generally unable to replicate outside the host, it is able to 52
survive for long periods in different environments (44, 46). This is attributed to the 53
pathogen´s thick waxy cell wall that possesses high amounts of lipids responsible for 54
properties such as acid fastness, hydrophobicity, biofilm formation, increased 55
resistance to chemicals and physical processes (47). Furthermore, a dormant state 56
as well as spore-like forms are thought to occur in this pathogen (20, 46). 57
Livestock manure provides nutrients for vegetables and helps build and 58
maintain soil fertility; hence, it is routinely applied on agricultural lands. However, 59
before application on land proper maturing processes need to be followed in order to 60
eliminate potential pathogens excreted in faeces. For that reason, the storing of 61
manure (fermentation) for 6 months before spreading on land is generally 62
recommended. Unfortunately, MAP is more resistant than other faecal bacteria and 63
the survival of the pathogen depends on the treatment method used, which include 64
anaerobic and aerobic lagoons, composting system as static piles, aerated static 65
piles, turned and aerated turned windrows. Grewal et al. (10) reported that with 66
anaerobic liquid lagoon storage treatment MAP can survive for 56 days and other 67
bacteria such as Escherichia coli, Salmonella sp. and Listeria sp. for 28 days. This 68
was in comparison to thermophilic composting at 55 °C when numbers of all bacteria 69
were reduced over three days. Nevertheless, MAP DNA was detectable for up to 175 70
days in an anaerobic liquid lagoon and for up to 56 days in response to other 71
treatments. 72
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
4
Recently, the number of biogas plants, which produce biogas but also 73
fermented mass that is used as land fertilizer has increased. During such treatment 74
MAP was isolated from the fermentors for up to two months and MAP DNA was 75
detected 16 months after manure introduction (40). 76
Due to the economical impact of paratuberculosis, control programs for this 77
disease in cattle, sheep and goats have been introduced in many countries. 78
Programs are based on early detection and elimination of infected animals. The most 79
important steps are considered to be the culling of heavy shedders and separation of 80
young animals from adults (potentially infected animals). Regardless, complete 81
information about MAP transmission, survival and pathogenesis are also still missing 82
and therefore these control programs are not as successful as was expected. 83
The first aim of our study was to monitor the distribution and persistence of 84
viable MAP cells and MAP DNA on one dairy cattle farm before and after destocking 85
of all animals and following the application of recommended measures (disinfectant 86
and lime application). The second aim of this study was to determine environmental 87
contamination with MAP outside the farm and to study the persistence of MAP cells 88
or DNA over time. 89
90
MATERIALS AND METHODS 91
92
Farm history. Clinical and subclinical infection of MAP was determined in one 93
dairy cattle herd containing 606 heads. The prevalence of MAP (based on qPCR 94
examination of faecal samples older than 15 months) reached 75.6% with a 95
maximum shedding of around 108 MAP cells per gram. An outbreak of MAP infection 96
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
5
was declared and due to the unsuccessful control program it was instructed that the 97
herd be eradicated. Therefore, measures were taken in accordance with the 98
declaration of sources of infection. A hot pressure cleaner with steam was used for 99
mechanical cleaning and activated Chloramin B with ammonium (4%) was applied for 100
subsequent disinfection in stables and operational areas around the farm. 101
Chloramin B was applied outside the stables to concrete and asphalt, while lime was 102
applied to soil and green areas. Lime was also used for decontamination of the field 103
and field midden in doses of 1000 kg/ha. 104
105
Sample collection. To study the effect of herd elimination on MAP 106
persistence on and outside the dairy farm, a total of 84 environmental samples were 107
analyzed (Table 1 and 2). Environmental samples were collected into sterile plastic 108
bags or vials. Sampling sites were located inside and outside the stables and the 109
building for storing animal feed (scrapings from floor, spider web, silage, plant and 110
liquid lagoon storage). These samples were collected before and 12, 18 and 24 111
months after the removing of all animals (Table 1). The samples from outside the 112
farm (field midden with the remains of the manure from an infected cow, a field with a 113
history of intensive fertilization with the manure of infected cows and a pond that 114
caught the water from the fertilized field) were collected 12, 18 and 24 months after 115
the removing of all animals from the farm (Table 2). After transportation to the 116
laboratory, samples were stored at 4 °C in the refrigerator until the next day or at -117
70 °C in the freezer for up to four weeks prior to laboratory examination. 118
119
Sample preparation and identification of isolates. All environmental 120
samples were submitted for MAP culture and qPCR. Methods for processing, 121
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
6
decontamination and inoculation of environmental samples on solid media were 122
published previously (16, 30), and were carried out as follows. Fifty millilitres of liquid 123
samples were centrifuged at 4,500 x g for 45 min, the sediment was mixed with 25 ml 124
of sterile distilled water and used for the following procedure. Three grams of each 125
sample were mixed with 25 ml of sterile distilled water and shaken for 30 min, 126
followed by sedimentation for 30 min at room temperature. After that, 5 ml of the 127
supernatant were added to 20 ml 0.9% hexadecylpyridinium chloride (HPC, Merck, 128
Darmstadt, Germany) to reach a final concentration of 0.72% HPC; samples were 129
then put on a shaker for 30 min. After 72 hours and in a dark room at room 130
temperature, 100 µl of sediment were inoculated on each of tree vials containing 131
Herrold´s Egg Yolk Medium (HEYM) with Mycobactin J (Allied Monitor, Fayette, MO, 132
USA). The incubation was at 37 °C for three to four months. The presence of acid-133
fast bacilli was determined by Ziehl-Neelsen (ZN) staining and ZN-positive cultures 134
were examined for MAP by conventional multiplex PCR detecting IS900 described 135
previously (25). 136
137
DNA isolation and qPCR. Fifty millilitres of water or 0.25 g of another type of 138
sample (e.g., soil, scraping, cobweb and plant) were employed for DNA isolation. 139
Before the isolation, water samples were concentrated by centrifugation at 4,500 x g 140
for 45 min. For DNA isolation the resulting sediment was used. The roots of plants 141
were washed in PBS buffer at the beginning of the processing. DNA from the upper 142
part of the plants was isolated using the PowerFood Microbial DNA isolation kit 143
(MoBio, Carlsbad, CA, USA) followed the manufacturer’s instructions. DNA from 144
roots and other environmental samples was isolated using the MoBio PowerSoil DNA 145
isolation kit (MoBio) with slight modifications (14). 146
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
7
The conditions for qPCR using the primers for IS900 and an internal 147
amplification control as well as the absolute number of MAP cells per g/ml of 148
environmental sample calculation was described previously (39). qPCR was 149
performed on the LightCycler 480 Instrument (Roche Molecular Diagnostic, 150
Mannheim, Germany) and subsequent analysis was carried out using the “Fit point 151
analysis” option of the LightCycler 480 software (Roche Molecular Diagnostic). Each 152
sample was analyzed in duplicate using qPCR assays. 153
154
Statistical analysis. Data analysis was performed using the statistical software 155
Statistica 9 (StatSoft, Inc., Tulsa, OK, USA) and GraphPad Prism 5 (GraphPad 156
Software, Inc., San Diego, CA, USA). Proportions of MAP DNA-positive samples 157
were evaluated by Fisher´s exact test and Chi-Squared test for trend. Logarithmically 158
transformed values of MAP cells were compared by Unpaired t-test and Kruskal-159
Wallis test. 160
RESULTS 161
Distribution of MAP on the farm studied by culture and qPCR. A total of 48 162
environmental samples were collected inside and outside the building on the studied 163
dairy farm with only one (2%) culturing positive for MAP. This sample originated from 164
a waste pit (water lagoon) and was collected at time 0 (when the infected herd was 165
present). Applying the qPCR method, 28 environmental samples (58%) were positive 166
for MAP DNA during the studied periods. Significant differences in MAP positivity 167
were shown before (0 months) and in the period after the removal of the infected 168
herd (months 12, 18 and 24), respectively 81% and 47% (P-value for Fisher's exact 169
test is <0.05). The proportion of MAP DNA-positive samples decreased over time 170
from 81% at time 0 to 43% at time 24 months after the removal of the infected herd 171
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
8
(Table 1). This trend was statistically significant (P-value for Chi-Squared test for 172
trend is <0.05). 173
A total of 82% of MAP DNA-positive samples were detected in the cattle barn 174
environment with heavily shedding animals, compared to other locations with low 175
expected levels of MAP DNA detection (58%; calf barn and mow and feed 176
preparation room) but the difference was not statistically significant (P-value for 177
Fisher's exact test is >0.05; Table 1). 178
MAP DNA was isolated from organic substrate (85%; scraping of floor, wall, 179
instrument and sediment from watering trough) as well as from cobwebs (53%) found 180
usually above animal level (Table 1). Regardless, statistically significant differences 181
were not found (P-value for Fisher's exact test is >0.05). 182
All examined samples from the water lagoon (waste pit) were MAP DNA-183
positive at each studied time. The number of MAP cells (1.96x104) at time 18 months 184
after destocking is noteworthy. One out of six silage samples was positive with a low 185
number of MAP cells. Two faecal samples of roe deer (Capreolus capreolus) 186
collected on the farm were negative (Table 1). 187
The average number (geometric mean) of MAP cells in positive samples at the 188
beginning (0 months) and after destocking (average of months 12, 18 and 24) 189
decreased, respectively 3.09x103 and 3.02x102 (P-value for Unpaired t-test <0.01). 190
The geometric mean of MAP cells in positive samples collected in the cattle barn 191
(8.81x102) and calf barn (9.8x102) is not significantly different (P-value for Unpaired t-192
test is >0.05). No differences in the geometric mean of MAP cells in positive organic 193
(3.61x102) and cobweb (9.36x102) samples was seen (P-value for Unpaired t-test is 194
>0.05; Table 3). 195
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
9
Distribution of MAP outside the farm studied by culture and qPCR. A total 196
of 36 environmental samples from outside the farm, e.g., from field midden, field and 197
pond were collected during the study period, 16 (44%) of which were positive for 198
MAP DNA. However, no sample was positive by culture. MAP DNA was isolated from 199
the soil, remains of manure, vegetables originating from the field or field midden but 200
also from the pond (Table 2). A trend toward a decrease in proportions of positive 201
samples over time was observed (P-value for Chi-Squared test for trend is <0.05) but 202
differences between the separate time periods were not statistically significant (P-203
values for Fisher's exact tests are >0.05). The geometric means of MAP cells at 204
individual times (12, 18 and 24 months) were 5.03x102, 8.76x102, 5.43x102, 205
respectively; differences between time periods were not statistically significant (P-206
value for Kruskal-Wallis test is >0.05; Table 2). 207
208
DISCUSSION 209
Although MAP infection has been studied for many decades, no adequate 210
information about the survival of MAP and persistence of MAP DNA in different 211
environments after the destocking of a dairy cattle herd exists. In the present study, 212
we focused on monitoring MAP contamination in a farm environment before and after 213
the removal of an infected herd and subsequent high pressure cleaning and 214
decontamination with Chloramin B and ammonium and lime application in adjacent 215
places around barns. We examined areas with adult cows that were the source of 216
infection on the farm but also the places thought to harbour low levels of MAP such 217
as the calf barn or mow and feeding room. We were able to detect MAP by culture 218
only in one sample from the waste pit before destocking of the infected herd although 219
the herd prevalence at this time was estimated to be approximately 75.6%. The 220
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
10
environmental positivity as determined by qPCR on the farm at this time was found to 221
be high and reached 81% (Table 1). A similar situation was observed by Berghaus et 222
al. (2), who determined that the prevalence of paratuberculosis among cows was 223
proportional to the environmental contamination by MAP. Since MAP excretion into 224
the environment via the faeces of infected cows is common, environmental sampling 225
was also proposed as a convenient method for estimating herd prevalence (2, 29, 226
32). 227
Early diagnosis of MAP infection is crucial for preventing MAP transmission in 228
a herd. It is recommended to remove massive shedders as well as low shedders 229
(21). For this, it is necessary to have a sensitive and rapid method for MAP detection. 230
qPCR assays have been shown, in comparison with liquid and solid cultures, to be 231
suitable and sensitive for faecal as well as environmental samples (1, 3, 15). 232
Moreover, the qPCR method applied in this study has been previously compared to 233
culture on solid media HEYM (18), and a predictive model for MAP detection that 234
demonstrates the probability of culture-positive samples according to the number of 235
cells per gram of faeces was designed. In this model, when the number of cells per 236
gram is 104, there is only a 40% probability of MAP isolation by culture on HEYM 237
medium. It was documented that cultivation in liquid media compared to solid media 238
had better results; unfortunately, this method is not up and running in our laboratory. 239
The lower sensitivity of cultivation may relate to the effects of the decontamination 240
method as well as the absorption of the pathogen to some particles and consequent 241
discarding during processing. Further, loss of MAP viability may be associated with 242
storing of the sample. Aly et al. (1) demonstrated a loss of viability due to freezing 243
and thawing; on the other hand, minimal loss of DNA was observed. 244
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
11
The existence of false positive reaction in qPCR may be also considered but 245
due to the negative results of isolation control and tested specify of qPCR in our 246
previous study (39) we eliminated this possibility. 247
Although MAP contamination of the environment found by cultivation was very 248
low and we were not able to demonstrate a reduction in live MAP cells at different 249
time periods after the destocking and decontamination of the farm, we were able to 250
observe a significant decrease in MAP DNA before and during the time after 251
destocking when the qPCR method was applied. Although a declining trend of 252
amounts of MAP DNA was noted over time, MAP DNA was detected in cobweb and 253
organic substrate (floor scraping) localized in building 24 months after destocking and 254
decontamination of the farm (Table 1). The high sensitivity of qPCR and a failure to 255
fully eliminate MAP DNA after destocking and high-pressure cleaning was also 256
documented by Eisenberg et al. (7). They demonstrated that more than 50% of 257
samples from a barn (after destocking and high-pressure cleaning) were positive 258
using a qPCR method. 259
A crucial preventative measure on farms is the separation of calves from the 260
adults and their faeces to minimize MAP transmission. It has been demonstrated that 261
calves are most susceptible to a MAP infection but MAP shedding and clinical signs 262
appear in adult animals. That is why the most commonly contaminated areas on 263
farms are cow alleyways and sites of manure storage (29, 41). We detected MAP 264
DNA in 82% of the samples from the cattle barn during the studied period (Table 1). 265
In contrast to our expectations, we also found a high number of positive samples in 266
the calf barn area. The difference in proportions of MAP DNA-positive samples and 267
average number of MAP cells between cattle barn and calf barn was not statistically 268
significant, respectively 82% with an average number of cells of 8.81x102 and 55% 269
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
12
with an average number of cells of 9.80x102. MAP DNA was also found at sites 270
without any animals such as the mow and feed preparation room. The MAP DNA-271
positive samples originated from cobweb as well as organic substrate (Table 1). In 272
contrast to these results, other authors described the isolation of live MAP from calf 273
area, feed and water; however, compared to flooring and manure storage this 274
detection was minor only (29, 41). 275
Due to the presence of high shedding animals in the cow barn, we assume 276
that distribution to the calf barn was through contaminated boots, instruments or dust. 277
Our results also indicate the importance of MAP spreading by aerosol (presence of 278
MAP in cobweb) within the farm (Table 1). This is supported by the studies of 279
Eisenberg et al. (7), who described the spreading of MAP through bio-aerosols after 280
the introduction of an infected shedding cow. They were able to detect live MAP in 281
the floor and settled dust in the barn and later also in the floor dust outside the barn. 282
As the faeces of the calf were not tested, contamination caused by MAP shedding by 283
the calf cannot be ruled out (32, 43). 284
Composting is generally viewed as a way to decrease pathogen 285
concentrations; however, most studies of pathogen dynamics in compost did not 286
report complete elimination of all pathogens. Wastewater lagoons were estimated as 287
one of the most common areas to be contaminated with MAP and have been 288
described to be more contaminated than manure or alleyways (2, 32). Our study also 289
confirmed a high range of contamination of the waste pit; MAP DNA was found in 290
each examined sample from the waste pit at each time up to 547 days, when 291
1.96x104 (cells/g) were detected (Table 1). Grewal et al. (10) were able, using qPCR, 292
to detect MAP DNA in liquid manure up to day 175 at 20 to 25 °C but MAP cells were 293
isolated by culture during the studied period only for up to 56 days. Jorgensen (13) 294
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
13
observed that a drastic reduction in MAP survival in cattle slurry occurred over the 295
first 7 days but that MAP cells were isolated by culture for up to 252 days at 5 °C. 296
Therefore the practice of manure spreading on fields and open access to middens 297
may contribute to the possible transmission of different pathogens including MAP 298
(40). 299
In this study, we analyzed samples from the midden (remains of filed manure) 300
as well as from a field that was previously fertilized with contaminated manure (soil, 301
upper part of plants and its roots) and subsequently limed. Further, we examined 302
samples from an adjacent pond. We did not detect culturable MAP in any of these 303
samples during the studied period. In contrast, Whittington et al. (44, 46) observed 304
the survival of MAP in soil and water sediment for approximately one year and 305
Salgado et al. (36) detected viable MAP in the upper part of soil 21 months after 306
manure application on the top of the 90-cm soil columns. Differences in physical, 307
chemical, and other conditions, e.g., nutrient availability and competition with other 308
microorganisms present in the soil may interact and affect pathogen persistence. 309
Whittington et al. (45) reported a 99% decrease in MAP isolation in soil samples 310
mixed with contaminated faeces. This was explained by the adsorption of bacteria to 311
soil particles and the later removal of these particles by sedimentation during sample 312
processing. This reduction was two orders of magnitude less than that from faeces. 313
On the other hand, using qPCR we detected MAP DNA in 44% of the 314
examined samples from the field, field midden as well as from the pond. In spite of 315
the decline in MAP DNA detection over time, the qPCR method revealed IS900 gene 316
copies 24 months after destocking of infected herd from the farm and subsequent 317
manure application on field and manure storage. MAP DNA has not only been 318
revealed in soil but also in upper parts of plants and in its roots. In our previous study 319
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
14
we detected MAP DNA in the roots of plants and soil samples (20 cm below the soil´s 320
surface) 15 weeks after natural exposure to muflon faeces on one muflon farm (30). 321
This is in an agreement with other studies, in which slow MAP movement in soil and 322
adsorption to soil particles under in vitro conditions was demonstrated (31, 36). 323
Manure application together with rainfall may also pose a risk in terms of 324
surface runoff and in this way the pathogen may be transmitted to water 325
environments. Pickup et al. (28) found that more that 60% of river water samples 326
from an area with infected cows tested positive for IS900 genes indicating the 327
presence of MAP. Similarly, in the present study, we also detected MAP DNA in three 328
out of nine samples from a pond placed close to a fertilized field. MAP survival in this 329
environment may be supported by persistence in protists as was documented by 330
Mura et al. (26) who proved the presence of MAP in Acanthamoeba polyphaga for up 331
to four years. 332
qPCR positivity may be caused by the presence of viable culturable MAP, 333
non-viable MAP, viable but non-culturable MAP cells or by residual DNA. It has been 334
documented that short DNA fragments can persist in soil and other environments for 335
extended periods of time despite the presence of DNA-degrading enzymes. These 336
DNA fragments can be partially protected against enzymatic degradation due to the 337
DNA adsorption to soil particles which depends mainly on clay proportion, pH and the 338
length of the fragments (27, 33, 34). Romanowski et al. (35) demonstrated the 339
persistence of plasmid DNA in soil for weeks and even for months after its release 340
from the cell. England et al. (8) detected a Pseudomonas aureofaciens gene in soil 341
four weeks after inoculation of the cell lysates. However, in contrast to these reports, 342
Young et al. (48) showed that PCR amplification of a specific gene is directly 343
correlated to the presence of viable cells. DNA from dead M. bovis cells was not 344
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
15
detectable in soil for more than 10 days and when free DNA was introduced into soil 345
the detection time was only approximately three days. 346
Viable but non-culturable bacteria or dormant bacterial states were observed 347
in many bacterial species including mycobacteria (19, 23, 37). This dormant state is 348
characterized by drastically decreased metabolic activity and enhanced resistance to 349
physical and chemical factors. Some studies suggest that a dormant state also exists 350
in MAP. This suspicion is supported by the observations that MAP produces a large 351
array of specific proteins in response to starvation stress which are linked with 352
dormancy state in other bacteria (11, 46). Recently, Lamont et al. (20) described 353
spore-like structures in one-year old broth cultures of MAP. These spore-like 354
structures survived exposure to heat, lysozyme and proteinase K and upon 355
germination in a well-established bovine macrophage model displayed enhanced 356
infectivity. 357
In conclusion, although we did not isolate MAP by culture after the destocking 358
of an infected herd and following application of decontamination methods, we cannot 359
be sure if MAP DNA detected for up 24 months after destocking of the infected herd 360
came from viable but non-culturable MAP cells, from dead cells, or was simply 361
residual DNA. The low probability of MAP isolation by culture compared to qPCR was 362
described in our previous study (18). Our present study illustrates the difficulty with 363
interpretation of qPCR results and highlights the possibility of underestimation of 364
culture results from environment. Since the infection doses for different animal 365
species are unknown and it is not clear if repeated small amounts of MAP can cause 366
infection, it is necessary to take into account qPCR results that are usually more 367
sensitive than culture methods. Furthermore, MAP aerosol transmission should also 368
be considered when prevention measures are applied on a farm. From these results, 369
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
16
the question also arises of how long after the removal of an infected herd is it 370
possible to stock new animals and if it is safe to apply the manure of infected cows 371
on a field. 372
373
ACKNOWLEDGEMENTS 374
The authors wish to thank Lenka Palasova, and RNDr Iva Bartejsova for providing 375
technical assistance. Neysan Donnelly is thanked for the grammatical correction of 376
the manuscript. This work was supported by the Ministry of Agriculture, Czech 377
Republic (Grants Nos. MZe0002716202 and QH81065) and project ADMIREVET 378
(No. CZ 1.05/2.1.00/01.0006/ED0006/01/01). 379
380
REFERENCES 381
1. Aly SS, Mangold BL, Whitlock RH, Sweeney RW, Anderson RJ, Jiang JM, 382
Schukken YH, Hovingh E, Wolfgang D, Van Kessel JAS, Karns JS, 383
Lombard JE, Smith JM, Gardner IA. 2010. Correlation between 384
Herrold egg yolk medium culture and real-time quantitative polymerase 385
chain reaction results for Mycobacterium avium subspecies 386
paratuberculosis in pooled fecal and environmental samples. J. Vet. 387
Diagn. Invest. 22:677-683. 388
2. Berghaus RD, Farver TB, Anderson RJ, Jaravata CC, Gardner IA. 2006. 389
Environmental sampling for detection of Mycobacterium avium ssp. 390
paratuberculosis on large California dairies. J. Dairy Sci. 89:963-970. 391
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
17
3. Cook KL, Britt JS. 2007. Optimization of methods for detecting Mycobacterium 392
avium subsp. paratuberculosis in environmental samples using 393
quantitative, real-time PCR. J. Microbiol. Methods. 69:154-160. 394
4. Cossu A, Rosu V, Paccagnini D, Cossu D, Pacifico A, Sechi LA. 2011. 395
MAP3738c and MptD are specific tags of Mycobacterium avium subsp. 396
paratuberculosis infection in type I diabetes mellitus. Clin. Immunol. 397
141:49-57. 398
5. Cossu D, Cocco E, Paccagnini D, Masala S, Ahmed N, Frau J, Marrosu MG, 399
Sechi LA. 2011. Association of Mycobacterium avium subsp. 400
paratuberculosis with Multiple Sclerosis in Sardinian Patients. PloS 401
One. 6 (4):e18482. 402
6. Eamens GJ, Whittington RJ, Turner MJ, Austin SL, Fell SA, Marsh IB. 2007. 403
Evaluation of radiometric faecal culture and direct PCR on pooled 404
faeces for detection of Mycobacterium avium subsp. paratuberculosis in 405
cattle. Vet. Microbiol.125:22-35. 406
7. Eisenberg SWF, Nielen M, Santema W, Houwers DJ, Heederik D, Koets AP. 407
2010. Detection of spatial and temporal spread of Mycobacterium 408
avium subsp. paratuberculosis in the environment of a cattle farm 409
through bio-aerosols. Vet. Microbiol. 143:284-292. 410
8. England LS, Lee H, Trevors JT. 1997. Persistence of Pseudomonas 411
aureofaciens strains and DNA in soil. Soil. Biol. Biochem. 29:1521-412
1527. 413
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
18
9. Fritsch I, Luyven G, Kohler H, Lutz W, Mobius P. 2012. Suspicion of 414
Mycobacterium avium subsp. paratuberculosis transmission between 415
cattle and wild-living red deer (Cervus elaphus) by multitarget 416
genotyping. Appl. Environ. Microbiol. 78:1132-1139. 417
10. Grewal SK, Rajeev S, Sreevatsan S, Michel FC, Jr. 2006. Persistence of 418
Mycobacterium avium subsp. paratuberculosis and other zoonotic 419
pathogens during simulated composting, manure packing, and liquid 420
storage of dairy manure. Appl. Environ. Microbiol. 72:565-574. 421
11. Gumber S, Taylor DL, Marsh IB, Whittington RJ. 2009. Growth pattern and 422
partial proteome of Mycobacterium avium subsp. paratuberculosis 423
during the stress response to hypoxia and nutrient starvation. Vet. 424
Microbiol. 133:344-357. 425
12. Hasonova L, Pavlik I. 2006. Economic impact of paratuberculosis in dairy cattle 426
herds: a review. Vet. Med. 51:193-211. 427
13. Jorgensen JB. 1977. Survival of Mycobacterium paratuberculosis in slurry. Nord. 428
Vet. Med. 29:267-270. 429
14. Kaevska M, Slana I, Kralik P, Reischl U, Orosova J, Holcikova A, Pavlik I. 430
2011. "Mycobacterium avium subsp. hominissuis" in neck lymph nodes 431
of children and their environment examined by culture and triplex 432
quantitative real-time PCR. J. Clin. Microbiol. 49:167-172. 433
15. Kawaji S, Begg DJ, Plain KM, Whittington RJ. 2011. A longitudinal study to 434
evaluate the diagnostic potential of a direct faecal quantitative PCR test 435
for Johne's disease in sheep. Vet. Microbiol. 148:35-44. 436
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
19
16. Kopecna M, Parmova I, Dvorska-Bartosova L, Moravkova M, Babak V, Pavlik 437
I. 2008. Distribution and transmission of Mycobacterium avium 438
subspecies paratuberculosis in farmed red deer (Cervus elaphus) 439
studied by faecal culture, serology and IS900 RFLP examinations. Vet. 440
Med. 53:510-523. 441
17. Kopecna M, Trcka I, Lamka J, Moravkova M, Koubek P, Heroldova M, Mrlik 442
V, Kralova A, Pavlik I. 2008. The wildlife hosts of Mycobacterium 443
avium subsp. paratuberculosis in the Czech Republic during the years 444
2002-2007. Vet. Med. 53:420-426. 445
18. Kralik P, Slana I, Kralova A, Babak V, Whitlock RH, Pavlik I. 2011. 446
Development of a predictive model for detection of Mycobacterium 447
avium subsp. paratuberculosis in faeces by quantitative real time PCR. 448
Vet. Microbiol. 149:133-138. 449
19. Kudykina YK, Shleeva MO, Artsabanov VY, Suzina NE, Kaprelyants AS. 450
2011. Generation of dormant forms by Mycobacterium smegmatis in the 451
poststationary phase during gradual acidification of the medium. 452
Microbiology 80:638-649. 453
20. Lamont EA, Bannantine JP, Armien A, Ariyakumar DS, Sreevatsan S. 2012. 454
Identification and characterization of a spore-like morphotype in 455
chronically starved Mycobacterium avium subsp. paratuberculosis 456
cultures. PloS One 7:e30648. 457
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
20
21. Lu Z, Mitchell RM, Smith RL, Van Kessel JS, Chapagain PP, Schukken YH, 458
Grohn YT. 2008. The importance of culling in Johne's disease control. 459
J. Theor. Biol. 254:135-146. 460
22. Man SM, Kaakoush NO, Mitchell HM. 2011. The role of bacteria and pattern-461
recognition receptors in Crohn's disease. Nat. Rev. Gastroenterol. 462
Hepatol. 8:152-168. 463
23. Marsh P, Morris NZ, Wellington EMH. 1998. Quantitative molecular detection of 464
Salmonella typhimurium in soil and demonstration of persistence of an 465
active but non-culturable population. FEMS Microbiol. Ecol. 27:351-363. 466
24. McKenna SL, Keefe GP, Tiwari A, VanLeeuwen J, Barkema HW. 2006. 467
Johne's disease in Canada part II: disease impacts, risk factors, and 468
control programs for dairy producers. Can. Vet. J. 47:1089-1099. 469
25. Moravkova M, Hlozek P, Beran V, Pavlik I, Preziuso S, Cuteri V, Bartos M. 470
2008. Strategy for the detection and differentiation of Mycobacterium 471
avium species in isolates and heavily infected tissues. Res. Vet. Sci. 472
85:257-264. 473
26. Mura M, Bull TJ, Evans H, Sidi-Boumedine K, McMinn L, Rhodes G, Pickup 474
R, Hermon-Taylor J. 2006. Replication and long-term persistence of 475
bovine and human strains of Mycobacterium avium subsp. 476
paratuberculosis within Acanthamoeba polyphaga. Appl. Environ. 477
Microbiol. 72:854-859. 478
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
21
27. Ogram AV, Mathot ML, Harsh JB, Boyle J, Pettigrew CA. 1994. Effects of Dna 479
polymer length on its adsorption to soils. Appl. Environ. Microbiol. 480
60:393-396. 481
28. Pickup RW, Rhodes G, Bull TJ, Arnott S, Sidi-Boumedine K, Hurley M, 482
Hermon-Taylor J. 2006. Mycobacterium avium subsp. 483
paratuberculosis in lake catchments, in river water abstracted for 484
domestic use, and in effluent from domestic sewage treatment works: 485
Diverse opportunities for environmental cycling and human exposure. 486
Appl. Environ. Microbiol. 72:4067-4077. 487
29. Pillars RB, Grooms DL, Kaneene JB. 2009. Longitudinal study of the 488
distribution of Mycobacterium avium subsp. paratuberculosis in the 489
environment of dairy herds in the Michigan Johne's disease control 490
demonstration herd project. Can. Vet. J. 50:1039-1046. 491
30. Pribylova R, Slana I, Kaevska M, Lamka J, Babak V, Jandak J, Pavlik I. 2011. 492
Soil and plant contamination with Mycobacterium avium subsp. 493
paratuberculosis after exposure to naturally contaminated mouflon 494
feces. Curr. Microbiol. 62:1405-1410. 495
31. Raizman EA, Habteselassie MY, Wu CC, Lin TL, Negron M, Turco RF. 2011. 496
Leaching of Mycobacterium avium subsp paratuberculosis in soil under 497
in vitro conditions. Vet. Med. Int. 2011:506239. 498
32. Raizman EA, Wells SJ, Godden SM, Bey RF, Oakes MJ, Bentley DC, Olsen 499
KE. 2004. The distribution of Mycobacterium avium ssp. 500
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
22
paratuberculosis in the environment surrounding Minnesota dairy farms. 501
J. Dairy Sci. 87:2959-2966. 502
33. Romanowski G, Lorenz MG, Wackernagel W. 1991. Adsorption of plasmid Dna 503
to mineral surfaces and protection against Dnase-I. Appl. Environ. 504
Microbiol. 57:1057-1061. 505
34. Romanowski G, Lorenz MG, Wackernagel W. 1993. Plasmid Dna in a 506
groundwater aquifer microcosm-adsorption, Dnase resistance and 507
natural genetic-transformation of Bacillus subtilis. Mol. Ecol. 2:171-181. 508
35. Romanowski G, Lorenz MG, Wackernagel W. 1993. Use of polymerase chain-509
reaction and electroporation of Escherichia coli to monitor the 510
persistence of extracellular plasmid Dna introduced into natural soils. 511
Appl. Environ. Microbiol. 59:3438-3446. 512
36. Salgado M, Collins MT, Salazar F, Kruze J, Bolske G, Soderlund R, Juste R, 513
Sevilla IA, Biet F, Troncoso F, Alfaro M. 2011. Fate of 514
Mycobacterium avium subsp. paratuberculosis after application of 515
contaminated dairy cattle manure to agricultural soils. Appl. Environ. 516
Microbiol. 77:2122-2129. 517
37. Shleeva MO, Bagramyan K, Telkov MV, Mukamolova GV, Young M, Kell DB, 518
Kaprelyants AS. 2002. Formation and resuscitation of 'non-culturable' 519
cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in 520
prolonged stationary phase. Microbiology 148:1581-1591. 521
38. Singh AV, Singh SV, Singh PK, Sohal JS, Singh MK. 2011. High prevalence of 522
Mycobacterium avium subspecies paratuberculosis ('Indian bison type') 523
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
23
in animal attendants suffering from gastrointestinal complaints who 524
work with goat herds endemic for Johne's disease in India. Int. J. Infect. 525
Dis. 15:E677-E683. 526
39. Slana I, Kralik P, Kralova A, Pavlik I. 2008. On-farm spread of Mycobacterium 527
avium subsp. paratuberculosis in raw milk studied by IS900 and F57 528
competitive real time quantitative PCR and culture examination. Int. J. 529
Food Microbiol. 128:250-257. 530
40. Slana I, Pribylova R, Kralova A, Pavlik I. 2011. Persistence of Mycobacterium 531
avium subsp. paratuberculosis at a farm-scale biogas plant supplied 532
with manure from paratuberculosis-affected dairy cattle. Appl. Environ. 533
Microbiol. 77:3115-3119. 534
41. Smith RL, Schukken YH, Pradhan AK, Smith JM, Whitlock RH, Van Kessel 535
JS, Wolfgang DR, Grohn YT. 2011. Environmental contamination with 536
Mycobacterium avium subsp. paratuberculosis in endemically infected 537
dairy herds. Prev. Vet Med. 102:1-9. 538
42. Tuci A, Tonon F, Castellani L, Sartini A, Roda G, Marocchi M, Caponi A, 539
Munarini A, Rosati G, Ugolini G, Fuccio L, Scagliarini M, Bazzoli F, 540
Belluzzi A. 2011. Fecal Detection of Mycobacterium avium 541
paratuberculosis using the IS900 DNA sequence in Crohn's Disease 542
and ulcerative colitis patients and healthy subjects. Dig. Dis. Sci. 543
56:2957-2962. 544
43. van Roermund HJW, Bakker D, Willernsen PTJ, de Jong MCM. 2007. 545
Horizontal transmission of Mycobacterium avium subsp. 546
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
24
paratuberculosis in cattle in an experimental setting: Calves can 547
transmit the infection to other calves. Vet. Microbiol. 122:270-279. 548
44. Whittington RJ, Marsh IB, Reddacliff LA. 2005. Survival of Mycobacterium 549
avium subsp. paratuberculosis in dam water and sediment. Appl. 550
Environ. Microbiol. 71:5304-5308. 551
45. Whittington RJ, Marsh IB, Taylor PJ, Marshall DJ, Taragel C, Reddacliff LA. 552
2003. Isolation of Mycobacterium avium subsp. paratuberculosis from 553
environmental samples collected from farms before and after 554
destocking sheep with paratuberculosis. Aust. Vet. J. 81:559-563. 555
46. Whittington RJ, Marshall DJ, Nicholls PJ, Marsh AB, Reddacliff LA. 2004. 556
Survival and dormancy of Mycobacterium avium subsp. 557
paratuberculosis in the environment. Appl. Environ. Microbiol. 70:2989-558
3004. 559
47. Wu CW, Schmoller SK, Bannantine JP, Eckstein TM, Inamine JM, Livesey M, 560
Albrecht R, Talaat AM. 2009. A novel cell wall lipopeptide is important 561
for biofilm formation and pathogenicity of Mycobacterium avium 562
subspecies paratuberculosis. Microb. Pathog. 46:222-230. 563
48. Young JS, Gormley E, Wellington EMH. 2005. Molecular detection of 564
Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil. 565
Appl. Environ. Microbiol. 71:1946-1952. 566
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
25
Table 1 Environmental samples from the farm collected before (Spring 0 months) and after removal of the infected herd (12, 18 and 567 24 months) studied by qPCR and cultivation 568
569
Spring (0 months)# Spring (12 months)## Autumn (18 months)## Spring (24 months)## Total (%) Localisation Matrix exam/
posit qPCR$ C exam/ posit qPCR$ C
exam/ posit qPCR$ C
exam/ posit qPCR$ C
Shed cattle cobweb 3/3 102-105 0 1/0 0 0 1/0 0 0 1/1 102 0 organic
substrate* 1/1 102 0 3/3 102 0 1/1 102 0 0
Subtotal 4/4 4/3 2/1 1/1 11/9 (82%) Shed calf cobweb 4/2 103 0 0 1/0 0 0 0
organic substrate
3/2 102-103 0 1/0 0 0 0 2/2 102-103 0
Subtotal 7/4 1/0 1/0 2/2 11/6 (55%) Mow and feed. prepar. room
cobweb 0 3/2 102 0 2/1 100 0 1/0 0 0
organic substrate
0 0 2/2 101- 102 0 0
Subtotal 0 3/2 4/3 1/0 8/5 (63%) Outside environment
waste pit 5/5 103-104 1 1/1 102 0 1/1 104 0 0
silage 0 2/0 0 0 2/1 101 0 2/0 0 0
faecal** 0 1/0 0 0 1/0 0 0 0 others*** 0 0 2/0 0 0 1/0 0 0 Subtotal 5/5 4/1 6/2 3/0 18/8 (44%) Total 16/13 1 12/6 0 13/6 0 7/3 0 48/28 (58%)(%) (81%) (50%) (46%) (43%) Geom. mean& 3.09x103 2.98x102 2.20x102 5.86x102
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
26
exam/posit – number of samples examined/positive 570
C – cultivation 571
# - examination was done before removal of animals from the farm 572
## - examination was done 12, 18 and 24 months after removal of animals from the farm 573
$ - qPCR was enumerated in number of MAP cells per g/ml 574
* floor scraping, instrument, sediment or scraping from watering troughs 575
** faecal samples from wild animals (roe deer; Capreolus capreolus) 576
*** vegetables and flushing 577
& - geometric mean calculated according absolute values 578
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
27
Table 2 Environmental samples originating from close to the farm housing the infected cattle studied by qPCR 579
580
581
exam/posit – number of samples examined/positive 582
Spring (12 months)* Autumn (18 months)* Spring (24 months)* Total (%)
Locality Matrix exam/ posit
qPCR ¥ exam/ posit
qPCR¥ exam/ posit qPCR¥
Field midden Soil 2/2 102-103 1/1 102 1/0 0 Vegetable 1/1 102 # 4/2 102 #$ 2/0 0 Biofilm 1/1 100 NA NA NA NA Subtotal 4/4 5/3 3/0 12/7 (58%) Ditch at field midden
Soil nd nd 1/1 103 1/1 103 Water sediment 1/1 103 1/1 103 NA NA
Vegetable nd nd nd nd 2/1 103 $ Subtotal 1/1 2/2 3/2 6/5 (83%) Field Soil 2/0 0 1/0 0 1/0 0 Vegetable 1/1 102 $ 2/0 0 2/0 0 Subtotal 3/1 3/0 3/0 9/1 (11%) Pond Water sediment 2/1 102 1/0 0 1/1 101 Biofilm 1/0 0 1/1 102 nd nd Vegetable nd nd nd nd 3/0 0 Subtotal 3/1 2/1 4/1 9/3 (33%) Total 11/7 12/6 13/3 36/16 (44%)(%) (64%) (50%) (23%) Geom. mean& 5.03x102 8.76x102 5.43x102
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
28
C - cultivation 583
*12, 18 and 24 months after removal of the infected cattle herd 584
¥ - qPCR was enumerated in number of MAP cells per g/ml 585
# - upper part of the vegetable 586
$ - roots of the vegetable 587
nd – not done 588
NA – not avialable 589
& - geometric mean calculated according to absolute values 590
591
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from
29
Table 3 Comparison of MAP DNA presence before and after destocking of animals, on cattle barn and calf barn, in cobwebs and organic 592 substrate 593
594
Time (0)#
Time (after destocking)* Cattle barn Calf barn Cobweb Organic
substrate
3.09x103 3.02x102 8.81x102 9.8x102 9.36x102 3.61x102
595
# time before destocking of animals 596
* geometric mean from month 12, 18 and 24 597
598
on January 2, 2019 by guesthttp://aem
.asm.org/
Dow
nloaded from