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

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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


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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


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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


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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


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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


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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


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(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


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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


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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


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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


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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


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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


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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


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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


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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

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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


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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


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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


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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


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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


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1 mycobacterium avium subsp. paratuberculosis persistence in a controlled 1 dairy cattle farm

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