High spoligotype diversity within a population: clues to understanding the demography of the pathogen in Europe

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High spoligotype diversity within a population: clues to understanding the demography of the pathogen in

Europe

Sabrina Rodríguez, Beatriz Romero, Javier Bezos, Lucía de Juan, Julio Álvarez, Elena Castellanos, Nuria Moya, Francisco Lozano, Sergio González,

José Luis Sáez-Llorente, et al.

To cite this version:

Sabrina Rodríguez, Beatriz Romero, Javier Bezos, Lucía de Juan, Julio Álvarez, et al.. High spolig- otype diversity within a population: clues to understanding the demography of the pathogen in Europe. Veterinary Microbiology, Elsevier, 2010, 141 (1-2), pp.89. �10.1016/j.vetmic.2009.08.007�.

�hal-00560849�

Accepted Manuscript

Title: High spoligotype diversity within aMycobacterium bovispopulation: clues to understanding the demography of the pathogen in Europe

Authors: Sabrina Rodr´ıguez, Beatriz Romero, Javier Bezos, Luc´ıa de Juan, Julio ´Alvarez, Elena Castellanos, Nuria Moya, Francisco Lozano, Sergio Gonz´alez, Jos´e Luis S´aez-Llorente, Ana Mateos, Lucas Dom´ınguez, Alicia Aranaz

PII: S0378-1135(09)00365-4

DOI: doi:10.1016/j.vetmic.2009.08.007

Reference: VETMIC 4535

To appear in: VETMIC Received date: 14-5-2009 Revised date: 9-7-2009 Accepted date: 3-8-2009

Please cite this article as: Rodr´ıguez, S., Romero, B., Bezos, J., de Juan, L., ´Alvarez, J., Castellanos, E., Moya, N., Lozano, F., Gonz´alez, S., S´aez-Llorente, J.L., Mateos, A., Dom´ınguez, L., Aranaz, A., High spoligotype diversity within a Mycobacterium bovispopulation: clues to understanding the demography of the pathogen in Europe, Veterinary Microbiology(2008), doi:10.1016/j.vetmic.2009.08.007

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Accepted Manuscript

High spoligotype diversity within aMycobacterium bovis population: clues to

1

understanding the demography of the pathogen in Europe.

2

Sabrina Rodríguez

, Beatriz Romero

, Javier Bezos

, Lucía de Juan

, Julio 3

Álvarez

, Elena Castellanos

, Nuria Moya

, Francisco Lozano

, Sergio 4

González

, José Luis Sáez-Llorente

, Ana Mateos

, Lucas Domínguez

, Alicia 5

Aranaz

and the Spanish Network on Surveillance and Monitoring of Animal 6

Tuberculosis 7

a

Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad 8

Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain.

9

b

Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad 10

Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain.

11

c

Subdirección General de Sanidad Animal, Dirección General de Ganadería, 12

Ministerio de Medio Ambiente, y Medio Rural y Marino, 28071 Madrid, Spain.

13

*Corresponding author:

14

Alicia Aranaz, Departamento de Sanidad Animal, Facultad de Veterinaria, 15

Universidad Complutense de Madrid, 28040 Madrid, Spain. Phone: +34 91 16

3943721, Fax: +34 91 3943795, e-mail address: alaranaz@vet.ucm.es 17

Manuscript

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Abstract

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Mycobacterium bovis is the main causative agent of bovine tuberculosis.

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This zoonotic disease produces important economic losses and must be considered 20

a threat to endangered animal species and public health. This study was performed 21

(1) to assess the degree of diversity of the Spanish M. bovis isolates and its effect 22

on the epidemiology of the infection, and (2) to understand the connection of M.

23

bovis populations within a European context. In this report we resume the DVR- 24

spoligotyping results of 6215 M. bovis isolates collected between 1992 and 2007 25

from different hosts. The isolates clustered into 252 spoligotypes which varied 26

largely in frequency, geographical distribution and appearance in different animal 27

species. In general, the most frequent spoligotypes were found all over the country 28

and in different animal species, though some were restricted to a geographical 29

area. Among our most often isolated spoligotypes, SB0121 and SB0120 (BCG- 30

like) are a common feature between mainland European countries, however, the 31

spoligotypes differ with those found in the UK, the Republic of Ireland and 32

abroad. A comparison of spoligotypes reported from other countries reveals hints 33

for the M. bovis demography in Europe and suggests a common ancestor strain.

34

This study gives insight into the usefulness of the standardized DVR- 35

spoligotyping technique for epidemiological studies in a country with a high 36

degree of strain diversity.

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Keywords:

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1. Introduction

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Mycobacterium bovis, the main causative agent of bovine tuberculosis, 41

affects a wide range of domestic animals and wildlife. This infection entails 42

important environmental, economic and public health risks (Briones et al., 2000;

43

Corner, 2006; Michel et al., 2006).

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In Spain all regions are subjected to a national eradication programme 45

based on intradermal tuberculin testing of cattle and the slaughter of reactor 46

animals. The annual herd period prevalence rate has been reduced to 1.63% in 47

2007. However, large differences exist between geographical areas (0.19-9.51%) 48

(Spanish Ministry of Environment and Rural and Marine Affairs, 49

http://www.mapa.es/ganaderia/pags/sanidad_ganadera/zoonosis/Informe2007.pdf) 50

Nowadays we count on a set of molecular typing techniques in order to 51

investigate epidemiological backgrounds. Direct variable repeat spacer 52

oligonucleotide typing (DVR-spoligotyping) (Kamerbeek et al., 1997) detects 53

polymorphism within the genomic Direct Repeat (DR) locus. The DR locus 54

consists of multiple sequences interspersed with non-repetitive spacer sequences 55

(spacers). Strains vary in the presence and absence of these spacers and can 56

therefore be clustered in so-called spoligotypes. Since the implementation of 57

spoligotyping for M. bovis (Aranaz et al., 1998; Roring et al., 1998), the 58

technique has been considered useful as a fast and cost-effective method for first- 59

line typing (Haddad et al., 2004; Milian-Suazo et al., 2008). The applicability of 60

the technique can vary between countries or even regions, because of the

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differences between strain diversity and the discriminatory power of 62

spoligotyping. However, the reasons underlying these differences in diversity 63

remain largely unknown.

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This paper provides a survey of the M. bovis population in Spain by 65

resuming the spoligotyping results for 6215 isolates obtained during the last 15 66

years. We describe the spoligotype diversity of M. bovis and offer insight into the 67

usefulness of spoligotyping for epidemiological studies when a high degree of 68

strain diversity is present.

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2. Materials and methods

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2.1. Mycobacterial isolates and strains

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This study comprises 6215 M. bovis isolates from Spain, collected between 73

January 1992 and December 2007. Most of the isolates were cultured in the 74

laboratory VISAVET (Facultad de Veterinaria, Universidad Complutense 75

Madrid), but about 40% of the isolates were submitted by Regional Laboratories 76

within the national bovine tuberculosis eradication program. The isolates were 77

obtained from cattle (Bos taurus, n=5585), domestic goats (Capra aegagrus 78

hircus, n=33), domestic pigs (Sus scrofa domestica, n=7), wild boars (Sus scrofa, 79

n=204), red deer (Cervus elaphus, n=141), fallow deer (Dama dama, n=229), 80

Iberian lynxes (Lynx pardinus, n=6), foxes (Vulpes vulpes, n=2), chamois 81

(Rupicapra rupicapra, n=2), a badger (Meles meles, n=1), cats (Felis silvestris 82

catus, n=3), a dog (Canis lupus familiaris, n=1) and a mouflon (Ovis musimon, 83

n=1, zoo animal). The geographical distribution is represented in the 84

supplementary figure according to the number of isolates.

85

The tissue samples were decontaminated and cultured onto Coletsos and 86

0.2% (w/v) pyruvate-enriched Löwenstein-Jensen media (bioMérieux España and 87

Biomedics, Madrid, Spain) and incubated at 37

C. The DNA was prepared from 88

colonies by suspending them in 200 µl of distilled water and boiling for 10 min at 89

100ºC. The isolates were confirmed as members of the M. tuberculosis complex 90

by acid-alcohol-fast staining and PCR amplification of Mycobacterium genus- 91

specific 16S rRNA fragment and MPB70 sequences (Wilton and Cousins, 1992).

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2.2. DVR-spoligotyping

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The isolates were spoligotyped following the protocol described by 94

Kamerbeek et al. (Kamerbeek et al., 1997). The DR region was amplified using 95

the primers DRa (GGTTTTGGGTCTGACGAC, 5’ biotinylated) and DRb 96

(CCGAGAGGGGACGGAAAC) (Roche Molecular Biochemical, Berlin, 97

Germany) and the amplified product was hybridised onto a spoligotyping 98

membrane (Isogen Bioscience BV, Maarssen, The Netherlands). The DNA was 99

detected with the streptavidin-peroxidase conjugate (Boehringer-Mannheim, 100

Germany) and the ECL system (GE Healthcare, Barcelona, Spain), followed by 101

exposure of an X-ray film to the membrane. We included purified sterile water as 102

a negative control and a clinical isolate of M. tuberculosis (Aranaz et al., 1996) as 103

a positive control in every PCR-batch and hybridisation assay. Authoritative 104

names (prefix SB followed by four digits) for spoligotype patterns were obtained 105

from the Mycobacterium bovis Spoligotype Database website 106

(http://www.Mbovis.org). Part of the results have been previously published [5%

107

(Aranaz et al., 2004), 1.6% (Romero et al., 2008)].

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The spoligotyping results were enlisted in a Microsoft® Office Access 109

database along with the epidemiological data (isolation date, animal species and 110

geographical origin).

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2.3. Discriminatory power calculation

112

The index of discrimination (D) described by Hunter and Gaston (Hunter 113

and Gaston, 1988) was calculated in order to determine the discriminatory power

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of the spoligotyping technique at the national level and for provinces (Spanish 115

administrative subdivision). We used the in-silico website of the University of the 116

Basque Country (http://www.insilico.ehu.es), filling in the number of unrelated 117

strains for each spoligotype. For this purpose we only counted one spoligotype 118

when isolates of the same herd or a precise geographical area (i.e. National Parks, 119

hunting estates) shared identical patterns.

120

Additionally, we calculated the quotient of the number of unrelated strains 121

(with unknown epidemiological link) and the number of different spoligotyping 122

patterns observed for that region in order to describe the degree of diversity in 123

each province.

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3. Results

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3.1. Outline 127

The 6215 M. bovis isolates included in this study originated from sampling 128

performed during 1992-2007 that almost achieved full country coverage, 129

excluding only the Autonomous Regions Murcia, Valencia, Ceuta and Melilla 130

(supplementary Figure). The characterization by spoligotyping of the 6215 M.

131

bovis isolates resulted in 252 different patterns (supplementary Table).

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The 15 most frequent Spanish types (the ones which made up at least 1%

133

of our samples) represent 77.43% of the totality of the isolates; the corresponding 134

prevalence rates are shown in Table 1. Interestingly, 12 of the 15 most frequent 135

types appeared throughout the years covered by this study, always accounting for 136

approximately the same fraction of the total strains. The other three types 137

(SB0135, SB1232, SB1258) were associated with intensive sampling of domestic 138

and/or wildlife animals in determined areas. The most frequent spoligotype is 139

SB0121 [spb-7 in previous reports (Aranaz et al., 1996)] that clustered 27.89% of 140

the isolates, followed by SB0134 (spb-13) and SB0339 (spb-16) which accounted 141

for 11.19% and 8.05% of the isolates (Table 1).

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The 237 other spoligotyping patterns comprised 153 patterns (1319 isolates) 143

with very low frequency (28.1% of them had been found infecting only one herd 144

each), and 84 singular patterns, so-called orphans, which were only isolated once 145

(Table 1).

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Altogether we identified 148 spoligotypes that have not been previously

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listed on www.Mbovis.org. The 252 spoligotypes are attached as supplementary 148

table.

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3.2. Discriminatory power and diversity ratio 150

We calculated the discriminatory power (D) for the spoligotyping of the 151

Spanish M. bovis isolates, which means the average probability that the technique 152

will assign a different type to two isolates randomly sampled in the population of 153

M. bovis, using the equation by Hunter and Gaston (Hunter and Gaston, 1988;

154

Hunter, 1990). We achieved a discriminatory index D=0.87 at national level.

155

We also calculated the discriminatory index of spoligotyping in Spanish 156

provinces from which more than 60 isolates were obtained, and D oscillated 157

between 0.74 and 0.93. In addition, the strain/pattern ratio ranged from 1.5 to 7.1 158

(supplementary Figure). We compared these results with the respective number of 159

isolates and the annual herd period prevalence of bovine tuberculosis (MARM, 160

2007). In general, provinces with lower herd prevalence (<0.8%) showed a 161

slightly higher D value (average D=0.90) compared to national average and lower 162

strain/pattern ratio (average ratio=2.7), whereas provinces with extreme herd 163

prevalence (>4%) showed lower D values (average D=0.82) and higher 164

strain/pattern ratio (average ratio=4).

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3.3. Geographical distribution 166

The M. bovis strains were unequally distributed all over Spain 167

(supplementary Table). The predominant patterns were repeatedly found in all

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regions and in different animal species. We observed accumulation of some 169

spoligotypes in certain regions. For instance, SB0339 was isolated from animals 170

from central and northern regions, whereas SB1230 originated from isolates from 171

the South. The analysis of the geographical distribution of 137 types (excluding 172

the orphan and epidemiologically-linked isolates) showed that the majority 173

(75.9%) is disseminated while 33 types appear localized. The strains that appeared 174

in delimited areas and presented a high prevalence, especially SB0339 (Monte El 175

Pardo Nature Reserve) and SB1232 (Doñana National Park), must not be 176

overestimated, given that intensive sampling for study purposes had been carried 177

out in those areas.

178

3.4. Animal species 179

The vast majority of the types were isolated from cattle, in fact 207 of our 180

spoligotypes were found only in this species (Table 2). Six patterns were 181

exclusively isolated from wild boars (SB0868, SB1095, SB1260, SB1274, 182

SB1336, and SB1367). Another five singular patterns were obtained from samples 183

of red deer (SB1330, SB1393), a goat (SB1062), a mouflon (SB1280) and a cat 184

(SB0972). The cattle-specific spoligotypes represented 86.61% of the patterns 185

found in M. bovis from cattle. By comparison, the percentage of exclusive types 186

from wild boar and red deer was notably lower (23.08% and 9.09%, respectively).

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Interestingly, the most common spoligotypes were shared by different 188

animal species, as shown in Table 1. There are only two exceptions: SB1305, 189

isolated from cattle from the north of Spain, and SB1337 isolated from cattle from

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Catalonia. In both regions intensive sampling for typing purposes has been carried 191

out in 2006 and 2007.

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3.5. Frequency of spacers 193

We compared the frequencies of the 43 spacers throughout the 252 194

spoligotypes (Figure 1) and could confirm the absence of spacers 3, 9, 16, and 39 195

to 43. Though none of the spacers were present in all the patterns, we found a 196

rather homogenous distribution. Each spacer was present in about 79.96% (69.5- 197

89.2%) of the strains, except for spacer 21 which could be found in only 36% of 198

our strains. The patterns which lack this spacer account for 67.13% of the typed 199

isolates, but neither belonged to a determined animal species, nor geographical 200

area.

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4. Discussion

203

In this study we report the characterisation of a representative collection of 204

M. bovis isolates from the different animal host species described in Spain. This 205

collection resulted from the involvement of numerous contributors which reflects 206

the commitment at national level on the study of the epidemiology of bovine 207

tuberculosis. The DVR-spoligotyping clustered the isolates in 252 spoligotypes 208

with a discriminatory index of D=0.87. To our knowledge this study reveals the 209

highest diversity within a M. bovis population that has been described in scientific 210

literature.

211

While most spoligotypes were distributed throughout the country, a small 212

number of patterns were restricted to determined regions. Consequently, extension 213

of the analysis to Spanish regions not studied before yields patterns not previously 214

described. In general, we found a higher discriminatory index for spoligotyping in 215

the Spanish northern regions which started eradication of bovine tuberculosis first 216

and present lower annual herd period prevalence. However, some exceptions 217

exist, likely reflecting animal species, cattle breeds and herd management. This 218

fact suggests that test-and-slaughter policy has not affected the local strain 219

diversity.

220

Spoligotyping results show common traits among western continental EU 221

countries and to a limited extent also to the British Isles. As previously described 222

in other reports, a frequent type clusters a high percentage of isolates. Our most 223

often identified spoligotype, SB0121, is also the most frequent strain in Portugal,

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the second most predominant type in France after SB0120 (Haddad et al., 2001) 225

and one of the five most frequent Italian types (Boniotti et al., 2009). The 226

similarities between Spain, France, Portugal and Italy can be due to the 227

geographical nearness and trade relationship. SB0121 has also been found with a 228

very low frequency (<1%) in mainland Great Britain (Hewinson et al., 2006).

229

Surprisingly, SB0120 represents 3.95% of the isolates in Spain but is infrequent in 230

Portugal (Duarte et al., 2008). Spoligotypes SB0130 and SB0134, which make up 231

3.39 % and 11.19% of our isolates respectively, were isolated frequently in 232

Britain (Smith et al., 2006) and Ireland (only SB0130) (Costello et al., 1999;

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Skuce et al., 2005). SB0134 was also reported from French cattle and wildlife 234

(Zanella et al., 2008). Furthermore, we have repeatedly isolated SB0140, the most 235

common spoligotype of the British Isles Advanced studies using additional 236

techniques such as Variable Number Tandem Repeat analysis would be needed to 237

reveal further epidemiological relationships.

238

The analysis of diversity within each species offered insight into the 239

relative contribution of livestock and wildlife to the epidemiology of bovine 240

tuberculosis in Spain. Wildlife, especially wild boar has been suggested as a 241

reservoir (Aranaz et al., 2004; Hermoso et al., 2006; Naranjo et al., 2008). The 242

finding of 207 spoligotypes exclusive to cattle gives evidence that cattle-specific 243

spoligotypes are responsible for much of the diversity of the Spanish M. bovis 244

population. The involvement of wildlife in the epidemiology of the infection is 245

highlighted by the fact that 12 out of the 15 most frequent types are present both 246

in cattle and at least in one wild Artiodactyla species. The special farming system

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in Spain may have promoted the transmission from cattle to sympatric wildlife 248

and the broad host range of these strains reflects its success as a pathogen.

249

The results of this study also contribute to the knowledge of the 250

demography of M. bovis in Spain within the European context. A comparison of 251

the spoligotypes and D values reported in publications could explain the spread of 252

M. bovis infection. Modern genetic and archeological evidence suggests that the 253

domestication centre of European cattle (B. taurus) was the Near East at the very 254

beginning of the Neolithic period (Beja-Pereira et al., 2006; Edwards et al., 2007).

255

Recent articles of cattle phylogeny based on mitochondrial DNA showed the 256

origin of European cattle in the Fertile Crescent and the subsequent expansion 257

from this ancestral population (Götherström et al., 2005; Beja-Pereira et al., 258

2006). They also highlight the strong influence of cattle of North African origin 259

introduced by maritime routes into the Mediterranean countries. These findings 260

are coherent with the hypothesis of ancient cattle being infected with ancestral M.

261

bovis strains which have a maximum number of DVR spacers. This ancestor 262

would derive from a M. tuberculosis-like organism (Brosch et al., 2002; Mostowy 263

et al., 2005; Smith et al., 2006). We assume that original cattle populations 264

remained with human settlements, giving rise to locally adapted strains and 265

further diversification. This evolutionary scenario is congruent with those 266

described for the demography of the M. tuberculosis complex and its association 267

with the human host (Wirth et al., 2008; Hershberg et al., 2008). Cattle arrived 268

into Spain through the Pyrenees and along the Mediterranean cost during the 269

Neolithic period. Likely, the cattle population increased in the region providing an

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ecological niche for clonal expansion of the founder strains. Due to the orography 271

and history of the Iberian Peninsula, the exchange of cattle between certain 272

regions has been impeded for a long time and therefore favoured an independent 273

evolution of M. bovis strains. The high diversity in Spain may have been caused 274

by this dual entrance of cattle from land and maritime routes. Nevertheless, we 275

could not distinguish two specific spoligotyping signatures suggesting two major 276

lineages that had evolved independently.

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The evolution of the DR region is probably unidirectional, occurring by 278

single spacer deletions or loss of contiguous spacer sequences (Fang et al., 1998;

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van Embden et al., 2000). Among our strains, SB0120 (BCG-like) is the most 280

likely common ancestor, from which SB0121 may have emerged by loss of spacer 281

21. The role of spacer 21 and the possible implications of its deletion have to be 282

elucidated, as it has also been described for Portuguese isolates.

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5. Conclusion

284

DVR-spoligotyping revealed a high degree of strain diversity among 285

Spanish M. bovis isolates (D=0.87) and thus can be considered a useful tool for 286

the study of epidemiology of the infection in our country. The distribution of 287

spoligotypes was unequal in Spain both in terms of geographical presence and 288

frequency, and even in infection in the different host species. The implementation 289

of the international database (www.Mbovis.org) has simplified the comparison 290

between countries. A comparison of spoligotypes available in the scientific

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literature reveals a hypothesis about the spread of bovine tuberculosis that has 292

shaped the M. bovis population structure in Europe. Our next target is the 293

completion of a national database which integrates conventional epidemiology 294

and GIS in order to support the national eradication campaign by exploiting the 295

molecular epidemiology data.

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Acknowledgements

298

This research was funded by EU project TB-STEP (KBBE-2007-1-3-04, 299

no. 212414) and the Ministry of Environment and Rural and Marine Affairs. S.

300

Rodríguez is recipient of a PhD studentship AP2006-01630 of the Spanish 301

Ministry of Science and Innovation.

302

We would like to thank the National and Regional Animal Health 303

authorities, in especial L. Carbajo and J.L Paramio for their continuous 304

encouragement. We are grateful to T. Alende and A. Gutiérrez for technical help.

305

Contributing members to the Spanish Network on Surveillance and Monitoring of 306

Animal Tuberculosis are F. Garrido (Laboratorio Central de Sanidad Animal de 307

Santa Fé, Granada, MARM), staff of Government and Regional and Research 308

Laboratories of Autonomous Communities [J. Tellez, C. Fornell, A. Jiménez, J.M.

309

Gómez, E.J. Villalba and I. Muñoz (Andalucía), J. Gracia, I. Belanche, S.

310

Izquierdo, N. Abacens, J.M. Malo (Aragón), E. Fernandez, M.F. Copano, I.

311

Merediz, J.M. Prieto and A. Espí (Asturias), V. Vigo (Canarias), F.M. Fernández, 312

E. Sola and C. Fernández (Cantabria), P. García, M.R. Bermúdez, V. Alcaide, C.

313

Rojas, M.L. Rando, A. Sánchez, J. Alonso, F. Plaza, C. Fernández, J.A. Viñuelas, 314

J. Alia and E. Grande (Castilla La Mancha), O. Mínguez, F. Fernández, C.

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Domínguez, J.A. Anguiano, F. Moreno, I. Romero, C. Martínez, I. Burón, A.

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Grau and O. Martín (Castilla y León), J. Gou (Cataluña), J.R. Puy (Euskadi), C.

317

Sanz and E. Dorado (Extremadura), J.E. Mourelo, D. Fernández and C. Calvo 318

(Galicia), M.J. Portau, C. Aguilo and C. Vidal (Islas Baleares), J. Carpintero, E.

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Fernández, M. García, L.M. Portas, C. Delso, J.M. Cámara, E. Legaz and J.

320

Urquía (Madrid), J. Pastor and C. Rivas (Murcia), J, Eguiluz, C. Fernández and F.

321

Eslava (Navarra), F.J. Puertolas and J.F. Soldevilla (La Rioja), M. Lazaro and C.

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Caballero (Valencia)]; A. Jacoste and M. Moreno (Patrimonio Nacional);

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academic and research members from Faculties of Veterinary Sciences [A. Perea 324

(Univeridad de Córdoba), M.V. Latre (Universidad de Zaragoza), O. Quesada and 325

A. Fernández (Universidad de Las Palmas de Gran Canaria), S. Lavin and G.

326

Mentaberre (Universidad Autónoma de Barcelona), M. Pizarro, M. Castaño, F.

327

Mazzucchelli, I. Simarro and G. Santurde (Universidad Complutense de Madrid), 328

A. Contreras and J. Sánchez (Universidad de Murcia)]; colleagues from research 329

centers on Animal Health [M. Domingo, B. Soria and S. Lopez (CRESA, 330

Cataluña), and C. Sánchez and M. Galka (P.N. Doñana)]; veterinary inspectors at 331

abattoirs [J.M. Rubio, A.J. Domínguez, M. Fernandez (Ciudad Real), J.L. del 332

Pozo, M. García, F. Osuna and J. Guedeja (Madrid)]; M.D. E. Gomez-Mampaso 333

(H. Ramón y Cajal, Madrid) and R. Borrás (Facultad de Medicina, Valencia); and 334

veterinary practitioners [P. Díez de Tejada and J. M. Fernández (A.D.S. Cabra del 335

Guadarrama, Madrid), F. Moneo-López, I. Larrauri and C. Gil (Albacete), J.

336

Cermeño and D. Martín (Badajoz), J.L. García (Burgos), A. Rodríguez, E. Sainz 337

(Cáceres), P.J. Mora (Ciudad Real), O. González-Llamazares (León), J.L.

338

Cumbreño, J. Blanco, L.M. Portas, L. Sánchez, M.P. Herranz, J.M. Finat, T.

339

Yuste and J.M. Amigo (Madrid), A. Santos (Toledo), J. Fonbellida (Zamora), J.

340

Rodriguez (Laboratorios Syva), among many others, that have made this study 341

possible by submitting samples and epidemiological information. We are also

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grateful to Devin J. Morey for careful revision of the manuscript.

343

The group is a partner of the coordination action “Veterinary European 344

Network on Mycobacteria (VENoMYC)” funded by the European Union.

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References

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Accepted Manuscript

TABLE 1.

Distribution of the spoligotypes of the Spanish Mycobacterium bovis isolates according to prevalence [frequent types (>1%), infrequent types and orphans (singular types)] by animal species.

Spoligotype

No. of isolates from

Cattle Goat Pig Deer Wild boar Iberian lynx Fox Dog Cat Mouflon Badger Chamois Prevalence

SB0140 62 1 1.01

SB1305 65 1.05

SB1258 66 1 1 1 1.11

SB1337 90 1.45

SB0135 104 1 1 1.71

SB0152 106 1 1.72

SB0119 112 4 13 2.08

SB1232 37 49 54 3 1 2.32

SB0130 204 2 4 1 3.40

SB0120 241 2 3 3.96

SB0295 240 1 1 3 8 2 4.10

SB0265 377 6 2 7 6.31

SB0339 235 254 12 8.06

SB0134 686 2 9 11.21

SB0121 1653 31 16 37 27.94

Orphans

(84) 76 1 3 2 1 1 1.35

Others

(153) 1231 32 52 1 1 2 21.22

Accepted Manuscript

Table 2.

Distribution of the Mycobacterium bovis isolates according to animal species, and number of spoligotypes which were found only in a determined species.

Animal species Isolates Types Exclusive types

Cattle 5585 239 207

Wild boar 204 26 6

Red deer 141 22 2

Goat 33 3 1

Cat 3 3 1

Mouflon 1 1 1

Fallow deer 229 13 1

Pig 7 2 0

Iberian Lynx 6 3 0

Chamois 2 1 0

Fox 2 2 0

Badger 1 1 0

Dog 1 1 0

Accepted Manuscript

Figure 1.

Frequencies of the 43 spacers of the DR locus that are included in the 484

spoligotyping membrane (designed by Kamerbeek et al., 1997) detected by PCR 485

and reverse-blotting. Data expressed as percentage of spoligotypes (n = 252) in 486

which the spacer is present.

487

Accepted Manuscript

Figure 1

Click here to download high resolution image

Accepted Manuscript

Supplementary Table.The SB numbers and the binary code of the 252 spoligotypes described in this study are represented, as well as the total number of isolates and the prevalence in Spain of each spoligotype at national and regional level.

SB

NUMBER^a BINARY CODE^b T^c P^d AR^e

N^f AS^e

N^f BC^e N^f CA^e

N^f CT^e

N^f GA^e N^f LR^e

N^f NA^e N^f CL^e

C^f CM^e C^f EX^e

C^f MA^e C^f AN^e

S^f BI^e CI^e SB0121 1101111101111110111101111111111111111100000 1737 27,94 0,9 8,8 0,1 19,7 6,6 2,0 0,4 1,6 39,7 1,4 0,7 12,3 5,5 0,3 0,1 SB0134 1100011101111110111111111111111111111100000 697 11,21 3,4 1,9 53,5 7,0 1,7 0,1 3,9 15,6 0,9 0,3 2,9 8,8

SB0339 1101111101111110111101111000011111111100000 501 8,06 0,2 5,0 3,0 1,0 0,2 2,0 16,8 0,2 69,9 1,8 SB0265 1101101101111110111101111111111111111100000 392 6,31 0,3 20,7 31,4 0,3 0,8 0,5 24,0 0,8 1,3 18,4 1,8 SB0295 1101111101111110111101111111111111110100000 255 4,10 0,8 3,9 3,9 2,0 2,0 1,2 27,5 3,9 8,6 5,5 40,8 SB0120 1101111101111110111111111111111111111100000 246 3,96 0,8 13,0 20,7 11,0 7,3 1,2 1,2 35,0 0,8 2,4 3,7 2,8

SB0130 1101111101011110111111111111111111111100000 211 3,40 30,8 0,9 37,4 24,2 3,8 1,9 0,9

SB1232 1100111101110110111111111111111111111100000 144 2,32 1,4 98,6

SB0119 1101111101111100111101111111111111111100000 129 2,08 0,8 38,8 10,9 17,8 0,8 7,0 24,0

SB0152 1000000000000000000001111111111111111100000 107 1,72 2,8 15,9 2,8 2,8 59,8 1,9 1,9 5,6 6,5

SB0135 1100001101111110111101111111111111111100000 106 1,71 100,0

SB1337 1101111101111110111101111110001111111100000 90 1,45 95,6 1,1 2,2 1,1

SB1258 1101111101111110111101101000011111111100000 69 1,11 98,5 1,4

SB1305 1000111101111110111101111101111111111100000 65 1,05 32,3 67,7

SB0140 1101101000001110111111111111111111111100000 63 1,01 3,2 14,3 73,0 7,9 1,6

SB0882 1101110101111110111111111111111111011100000 50 0,80 28,0 70,0 2,0

SB1019 1100111101011110111111111111111111111100000 46 0,74 43,5 56,5

SB0867 0001111101111110111111111111111111111100000 37 0,60 2,7 62,2 35,1

SB0828 1101111101111110111111111111111110111100000 29 0,47 6,9 3,4 65,5 3,4 10,3 10,3

SB0818 1101111101111110111111111111111101111100000 29 0,47 100,0 SB1188 1101111101111100011101111111111111111100000 29 0,47 100,0

SB0875 1000011101111110111111111111111111111100000 28 0,45 50,0 39,3 10,7

SB0920 1101110101111110111111111111111111111100000 28 0,45 32,1 32,1 21,4 7,1 7,1

SB0886 1101111101111110110111111111111011111100000 27 0,43 11.1 18.5 70.4

SB1230 1101111000000110111101111111111111110100000 27 0,43 100,0

SB0294 1101111101101110111101111111111111111100000 27 0,43 22,2 3,7 7,4 55,6 11,1

SB1312 1101111101011110111111111111110111111100000 27 0,43 85,2 14,8

SB0832 1101001101111110111111111111111101111100000 26 0,42 3,8 57,7 34,6 3,8

Supplemenatry table

Accepted Manuscript

SB

NUMBER^a BINARY CODE^b T^c P^d AR^e

N^f AS^e

N^f BC^e N^f CA^e

N^f CT^e

N^f GA^e N^f LR^e

N^f NA^e N^f CL^e

C^f CM^e C^f EX^e

C^f MA^e C^f AN^e

S^f BI^e CI^e

SB1016 1101101000001110111111111111111001111100000 22 0,35 54,5 9,1 36,4

SB1167 1101111101111110111111011111111111111100000 22 0,35 100,0

SB0132 1101111101111110111101111100011111111100000 21 0,34 9,5 90,5

SB1193 1101111101101110111101111000011111111100000 21 0,34 4,8 95,2

SB1021 1101111101111110111101111111111111111000000 20 0,32 20,0 10,0 5,0 60,0 5,0

SB1174 1100111101110110111111111111111111001100000 19 0,31 5,3 36,8 42,1 15,8

SB1142 1101111101111110111101011111111111111100000 18 0,29 5,6 27,8 66,7

SB1265 1100011101111100111111111111111111111100000 18 0,29 94,4 5,6

SB1357 1101111101011110110001111111111111111100000 17 0,27 100,0

SB0329 1101111101111110111101111111111111110000000 16 0,26 25,0 43,8 31,2

SB0122 0101111101111110111101111111111111111100000 15 0,24 6,7 53,3 40,0

SB1296 1100111101111110111101111100111111111100000 15 0,24 13,3 86,7

SB0833 1101111101111110111111111111110111111100000 15 0,24 13,3 6,7 80,0

SB1345 1101111101110010111101111111111111111100000 15 0,24 100,0

SB1254 1101111101111110111101111110111111111100000 14 0,23 57,1 28,6 14,3

SB1259 1101111100000110111101111111111111111100000 14 0,23 71,4 14,3 14,3

SB1322 1100011101111110111111111000000111111100000 13 0,21 92,3 7,7

SB1275 1101111101111110111101111111101111111100000 12 0,19 91,7 8,3

SB0933 1100000101111110111111111111111111111000000 12 0,19 66,7 33,3

SB1301 0101101101111110111101111000000011111100000 12 0,19 100,0

SB1308 1101111101111110111101111111111110111100000 12 0,19 16,7 58,3 25,0

SB1303 1101111101011010111111111111111111111100000 11 0,18 100,0

SB1218 1101111101111110100001111111111111111100000 11 0,18 63,6 9,1 27,3

SB0948 0101111101111110111111111111111111111100000 10 0,16 40,0 50,0 10,0

SB0896 1101101101111110111101011111111111111100000 10 0,16 10,0 90,0

SB1325 1101111101111110100101111111111111111100000 10 0,16 10,0 90,0

SB1336 1101111101101000111101111111111111111100000 10 0,16 50,0 50,0

SB1191 1101111101100010111101111111111111111100000 10 0,16 70,0 20,0 10,0

SB1091 1101111101110110111101111111111111110100000 9 0,14 55,6 33,3 11,1

Accepted Manuscript

SB0296 1101111101111110111101111111011111111100000 7 0,11 71,4 28,6