Comparison of culture and PCR to detect and subsp. in ear swabs taken from goats

HAL Id: hal-00535912

https://hal.archives-ouvertes.fr/hal-00535912

Submitted on 14 Nov 2010

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Comparison of culture and PCR to detect and subsp. in ear swabs taken from goats

Christian de la Fe, Joaquín Amores, Juan C. Corrales, Ángel Gómez Martín, Antonio Sánchez, Antonio Contreras

To cite this version:

Christian de la Fe, Joaquín Amores, Juan C. Corrales, Ángel Gómez Martín, Antonio Sánchez, et

al.. Comparison of culture and PCR to detect and subsp. in ear swabs taken from goats. Veterinary

Microbiology, Elsevier, 2009, 140 (1-2), pp.105. �10.1016/j.vetmic.2009.06.036�. �hal-00535912�

Accepted Manuscript

Title: Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in ear swabs taken from goats

Authors: Christian de la Fe, Joaqu´ın Amores, Juan C. Corrales, Angel G´omez Mart´ın, Antonio S´anchez, Antonio Contreras ´

PII: S0378-1135(09)00320-4

DOI: doi:10.1016/j.vetmic.2009.06.036

Reference: VETMIC 4490

To appear in: VETMIC

Received date: 23-3-2009 Revised date: 18-6-2009 Accepted date: 22-6-2009

Please cite this article as: de la Fe, C., Amores, J., Corrales, J.C., Mart´ın, ´ A.G., S´anchez, A., Contreras, A., Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in ear swabs taken from goats, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2009.06.036

This is a PDF file of an unedited manuscript that has been accepted for publication.

As a service to our customers we are providing this early version of the manuscript.

The manuscript will undergo copyediting, typesetting, and review of the resulting proof

before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that

apply to the journal pertain.

Accepted Manuscript

Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma

1

mycoides subsp. capri in ear swabs taken from goats

2 3

Christian de la Fe*, Joaquín Amores, Juan C. Corrales, Ángel Gómez Martín, Antonio

4

Sánchez, Antonio Contreras

5 6

Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus

7

de Espinardo s/n. Murcia, 30100, Spain

8 9 10 11

*Corresponding author: Christian de la Fe Rodríguez

12

Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia.

13

Campus de Espinardo s/n, 30100 – Murcia, Spain

14

Tel: +34 968367259; Fax: +34 968364147

15

E-mail: cdelafe@um.es

16

17

18

19

20

21

Manuscript

Accepted Manuscript

Abstract

22

This study was designed to evaluate the validity of PCR for the direct detection of

23

Mycoplasma (M.) agalactiae and Mycoplasma mycoides subsp. capri (Mmc), as the two

24

species most frequently causing contagious agalactia (CA) in goats. The PCR method was

25

compared with the traditional culture technique to determine which method was most efficient

26

at identifying all auricular carriers present in herds. The samples analysed were 307 ear swabs

27

taken from goats reared in a CA endemic area. We assessed the validity of each technique to

28

detect each species and agreement between both methods. For each species, the result was

29

taken as true-positive when at least one of the two tests was positive. Of the swabs tested, 246

30

were scored positive by PCR (235 and 11 for Mmc and M. agalactiae, respectively) and 117

31

showed a positive culture result (113 for Mmc and 4 for M. agalactiae). 133 of the PCR

32

positive-samples (124 and 9 for Mmc and M. agalactiae, respectively) yielded negative

33

culture results and 4 culture-positive samples tested negative using PCR (2 for each species).

34

Sensitivity and negative predictive values for PCR were 84.62 and 99.32 (for M. agalactiae)

35

and 99.16 and 97.22% (for Mmc) respectively, and for culture were 30.77 and 97.03 (for M.

36

agalactiae) and 47.08 and 36.08% (for Mmc), respectively. PCR proved to be a rapid and

37

sensitive method for the detection of mycoplasmas in the external ear of asymptomatic

38

carriers. Tools such as this are needed to adopt efficient control measures against CA.

39 40 41

Keywords: contagious agalactia; auricular carriers; PCR; culture; Mycoplasma agalactiae;

42

Mycoplasma mycoides subsp. capri

43

44

45

Accepted Manuscript

1. Introduction

47

Mycoplasmas have been incriminated in the aetiology of several diseases of

48

ruminants. The species Mycoplasma agalactiae (Ma) and Mycoplasma mycoides subsp. capri

49

(Mmc) have been identified as the most frequent causes of contagious agalactia (CA) (Gil et

50

al., 1999; De la Fe et al., 2005), a syndrome that produces mastitis, arthritis,

51

keratoconjunctivitis, abortion and pneumonia. Considered among the most serious diseases

52

affecting small dairy ruminants, CA has been endemic in most Mediterranean countries since

53

the end of the 1980’s, and causes high economic losses in all affected areas (Bergonier et al.,

54

1997; Corrales et al., 2007).

55

Since 1981, several mycoplasma species have been identified in the external ear canal

56

of goats, including all species involved in the aetiology of CA syndrome (Cottew and Yeats,

57

1981, 1982; Da Massa, 1983; DaMassa and Brooks, 1991; Gil et al., 1999; De la Fe et al.,

58

2005; Mercier et al., 2007). Recent results also suggest the presence of high numbers of

59

mycoplasma carriers in herds (De la Fe et al., 2005; Mercier et al., 2007; Tardy et al., 2007).

60

Although the impact of this carrier state is unknown, the most highly pathogenic

61

mycoplasmas seem to be more frequently isolated from herds with a prior history of

62

mycoplasma infection (Mercier et al., 2007), pointing to a need for efficient diagnostic

63

methods to identify all carriers present in herds.

64

Contagious agalactia has been traditionally diagnosed by taking samples for culture in

65

several specific media, and this method is also used to detect mycoplasma carriers in goats

66

(Gil et al., 1999; De la Fe et al., 2005; Corrales et al., 2007; Mercier et al., 2007). The

67

difficulties encountered in detecting and identifying mycoplasmas with this method have led

68

researchers to explore the use of newer and faster molecular techniques such as PCR for CA

69

diagnosis (Hotzel et al.,1996; Tola et al., 1996; Peyreaud et al., 2003; Manso-Silvan et al.,

70

2007). This technique has demonstrated its validity to detect mycoplasmas in different types

71

Accepted Manuscript

of clinical sample (Tola et al., 1996; Dorigo-Zetsma et al., 1999; Spergser et al., 2002;

72

Johnson et al., 2004; Stellrecht et al., 2004). However, its suitability for detecting

73

mycoplasmas in ear swabs to support the health status description of goat herds has not yet

74

been determined. The present study was designed to evaluate the validity of PCR for the

75

direct detection of Ma and Mmc in ear swab samples taken from goats in a CA endemic area

76

with no clinical signs of the disease. Results were compared to those provided by a traditional

77

culture procedure.

78

2. Materials and methods

79

2.1. Ear swabs

80

307 ear swabs were collected and subjected to culture and PCR procedures to detect

81

the presence of mycoplasmas. Samples were collected from 8 Murciano-Granadina goat herds

82

in Murcia (E Spain), where CA is endemic. Most of the samples (n=285) were obtained from

83

herds with a previously confirmed infection by Mycoplasma spp. (Ma and Mmc), while the

84

rest (n=22) were collected from a herd with no known history of CA for use as negative

85

controls.

86

All the goats tested had been previously examined to confirm they had no clinical

87

signs of CA. Following this clinical examination, ear swabs were taken, placed in transport

88

medium (Sterile transport swab, Copan, Brescia, Italy), immediately refrigerated (4ºC) and

89

transported to the laboratory, where they were processed on the day of collection. As soon as

90

they arrived at the laboratory, the swabs were twirled and left in 1 ml of PH culture medium

91

(Kirchhoff and Rosengarten, 1984) for 30 minutes at room temperature. After discarding the

92

swabs, aliquots of the remaining fluid were taken for culture and PCR and the rest was stored

93

at -80ºC.

94

2.2. DNA extraction and PCR

95

Accepted Manuscript

DNA was extracted from samples using a previously described method (Tola et al.,

96

1997) with some modifications. Briefly, 200 µl of each sample were incubated for 10 min at

97

room temperature with 100 µl of denaturation buffer (0.5 M NaOH, 1.5 M NaCl) and then for

98

a further 10 min at room temperature with 40 µ1 of silica particles suspended in 900 µ1 of

99

lysis buffer (10.12 M guanidine isothiocyanate, 0.1 M Tris-HCl, pH 6.4, 0.11 M EDTA, pH

100

8.0, 2.6% Triton X-100). After centrifugation at 14,000 X g for 30 s, the pellet was washed

101

twice with washing buffer (10.12 M guanidine isothiocyanate, 0.1 M Tris-HCl, pH 6.4), twice

102

with 70% ethanol and once with acetone. The dried pellet was then resuspended in 100 µ1 of

103

TE buffer (10 mM Tris-HCl, pH 8.0, 1mM EDTA, pH 8.01), heated at 56°C for 10 min,

104

briefly vortexed and centrifuged at 14,000 X g for 2 min. 8 µ1 of supernatant were added to

105

the reaction mixture for amplification.

106

PCR-based detection assays for Ma and Mmc (Hotzel et al., 1996; Tola et al., 1996)

107

were performed in a total volume of 25 µl. Taq DNA polymerase (Bio Line, Barcelona,

108

Spain) was used for amplification according to the manufacturer’s instructions in an i-cycler

109

DNA thermal cycler (Bio-Rad, Hercules, CA, USA). The PCR amplification products were

110

analysed by gel electrophoresis on 1% (w/v) agarose gels and visualised after staining with

111

ethidium bromide using a UV transilluminator (Syngene, Frederick, MD, USA).

112

2.3. Mycoplasma cultures

113

Solid and liquid pH media (De la Fe et al., 2005) were inoculated with 0.2 ml of each

114

sample and incubated at 37ºC in a 5% CO

humid atmosphere. Isolates from previously

115

cloned single colonies were used for preliminary identification based on biochemical and

116

serological tests. The biochemical tests performed were: sensitivity to digitonin, glucose and

117

mannose fermentation, arginine and urea hydrolysis, tetrazolium reduction, film and spot

118

production and phosphatase activity (Poveda, 1998). The serological method (Poveda and

119

Nicholas, 1998) was a growth inhibition test using monospecific hyperimmune antiserum

120

Accepted Manuscript

obtained from rabbits against the corresponding reference strains. Final identification was

121

based on the results of a specific PCR test (Hotzel et al., 1996; Tola et al., 1996; Peyreaud et

122

al., 2003).

123

2.4. Interpretation of results and statistical analyses

124

Data were analyzed to determine the number of samples testing positive using each

125

technique. A true-positive result for each species was defined as at least one of the two tests

126

(culture or PCR) proving positive. Sensitivity and negative predictive values (NPV) for each

127

technique and mycoplasma species, and agreement between the techniques were determined

128

using the kappa test as implemented in the Win Episcope 2.0 software package (Thrusfield et

129

al., 2001). By definition, the specificity and positive predictive values (PPV) of both assays

130

were 100%.

131

3. Results

132

3.1. Clinical samples

133

Of the 307 swabs from 8 goat herds analyzed, 80.7% (248) yielded one of the

134

potentially pathogenic mycoplasmas evaluated, and only 59 samples (19.3%) scored negative

135

for both species using both diagnostic methods, including the 22 samples used as negative

136

controls. Thus, the prevalence of both species in the ear canal was high in all the herds. These

137

herds all suffered the form of the diseases typically observed in CA endemic areas, which is

138

characterized by the absence of classical symptoms with the exception of some mastitis. This

139

prevalence contrasted with the absence of the mycoplasma species detected in the ear canals

140

of goats reared in the herd used as control with no history of CA. In 2 of the samples checked

141

(0.66%), both species were detected together. Despite the fact that several mycoplasma strains

142

could be concomitantly carried in the same ear canal, the combined presence of 2 pathogenic

143

species seems to be less common.

144

Accepted Manuscript

A positive result for Mmc was obtained in 237 samples whereas Ma was detected in

145

13 of the swabs tested. Only 70 samples (22.8%) scored negative for Mmc using both

146

diagnostic methods while 294 (95.7%) scored negative for Ma. According to the techniques

147

used, 235 samples tested positive for Mmc and 11 for Ma using PCR, whereas 113 samples

148

were culture positive for Mmc and only 4 for Ma. The numbers of samples testing positive for

149

each mycoplasma species according to the test used are provided in Table 1.

150

3.2. Validity variables and agreement

151

The validity variables determined (sensitivity and NPV of the culture and PCR

152

methods to detect Ma and Mmc) are shown in Tables 2 and 3. The results obtained indicate a

153

PCR detection rate for both species of mycoplasma improved by more than 100% over the

154

culture technique (235 versus 113 true-positives for Mmc, and 11 versus 4 for Ma). The

155

kappa values obtained were 0.252 (0.154-0.351, 95% CI) for Ma and 0.28 (0.2-0.360, 95%

156

CI) for Mmc.

157

4. Discussion

158

PCR techniques developed in the past 12 years for the detection of mycoplasmas

159

causing CA have demonstrated their suitability for CA diagnosis using an array of samples

160

including milk, nasal swabs, eye swabs, vaginal swabs and carpal joint fluid (Dedieu et al.,

161

1995; Hotzel et al., 1996; Tola et al., 1996; Tola et al., 1997; Greco et al., 2001; Peyreaud et

162

al., 2003; Manso Silvan et al., 2007). The results of the present study reveal that PCR is also

163

an effective rapid tool for directly detecting Ma and Mmc in ear swabs taken from goats, as

164

the agents that most frequently cause CA. The detection of asymptomatic carriers is of special

165

interest in herds assumed CA-free. In addition, the emergence of CA in non-infected herds

166

has been almost always linked to the introduction of carriers of Mycoplasma spp., and the

167

most common infection site is the external ear canal (Corrales et al., 2007). The peculiar

168

features and risks of this carrier state determine that it may occur independently of any

169

Accepted Manuscript

clinical expression (Bergonier et al., 1997), so clinically healthy and serologically negative

170

animals can carry the causative mycoplasmas at this anatomical site (DaMassa, 1983; De la

171

Fe et al., 2005). Ear-infecting mycoplasmas have also been detected in animals clinically

172

affected during an outbreak of CA (Gil et al., 1999). Hence, the successful diagnosis of

173

carriers is essential so that appropriate control measures can be quickly installed (Corrales et

174

al., 2007).

175

The specificity of the two methods tested here was 100% by definition and in the

176

absence of false positives, our results confirm that PCR is more sensitive than the culture

177

method to detect both mycoplasmas in ear swabs. The improved sensitivity of PCR over

178

culture methods for detecting Ma in milk samples has been previously reported (Tola et al.,

179

1997). This finding has also been confirmed in studies in which PCR and culture techniques

180

were used to detect other mollicutes such as M. pneumoniae and genital mycoplasmas in

181

swabs taken from different anatomical sites in humans (Luki et al., 1998; Dorigo-Zetsma et

182

al., 1999; Spergser et al., 2002; Johnson et al., 2004; Stellrecht et al., 2004). In addition,

183

culture and PCR showed weak agreement (Thrusfield, 1995) with a kappa value of 0.252

184

obtained for Ma and 0.28 for Mmc. Compared to the traditional culture method, PCR has the

185

advantage that it detects both viable and non-viable organisms (Stellrecht et al., 2004), hence

186

its enhanced sensitivity. Four of our samples were PCR negative but yielded a positive culture

187

result for Ma (n=2) and Mmc (n=2). Several factors such as the presence of Taq polymerase

188

inhibitors or gene sequence variations lead to false negative PCR results.

189

Although our study was not designed to determine the prevalence of carriers in CA

190

infected herds or confirm the presence of carriers in herds thought to be CA-free, the number

191

of swabs scoring positive for the two species of mycoplasma was high. Thus, 80.7% of the

192

samples taken proved positive for Mmc or Ma based on PCR, culture or both techniques.

193

Accepted Manuscript

number of carriers was also observed (Gil et al., 1999; De la Fe et al., 2005; Mercier et al.,

195

2007). In view of the sensitivity shown by our culture technique, the numbers of mycoplasma

196

carriers reported in these studies could be underestimated since this was the method used in

197

all these reports. Consistent with our results, in recent studies Mmc was the mycoplasma

198

species most detected in the external ear (De la Fe et al., 2005; Mercier et al., 2007). Given

199

the need for an improved understanding of the true pathogenesis of mycoplasma strains

200

isolated from this anatomical region, a recent investigation has focused on characterizing

201

isolates from ears along with those considered responsible for CA outbreaks (Tardy et al.,

202

2007). In our opinion, there is an urgent need to determine carrier proportions in infected and

203

non-infected herds and to confirm the real existence of carriers in herds assumed CA-free.

204

These two unanswered questions regarding the epidemiology of CA are crucial for real

205

control of the disease. Thus, the presence of asymptomatic carriers means the health status of

206

a herd cannot be truly known and there is a risk of subsequent propagation of the aetiological

207

agents (Mercier et al., 2007). This occurred in Sardinia (Italy), where the disease was

208

introduced by carrier sheep from the mainland (Sanguinetti and Chiocco, 1987).

209

In conclusion, PCR is a more rapid and effective method than the standard culture

210

technique for the detection of M. agalactiae and Mmc in ear swab samples taken from goats.

211

Acknowledgements

212

This work was supported by the Fundación Seneca, Agencia de Ciencia y Tecnología

213

de la Región de Murcia, Spain (Research Project 05693/PI/07) and the Ministerio de

214

Educación y Cultura, Spain (Plan Nacional I+D. Project AGL2006-03105/GAN).

215

References

216

Bergonier, D., Berthelot, X., Poumarat, F., 1997. Contagious agalactia of small ruminants:

217

current knowledge concerning epidemiology, diagnosis and control. Rev. Sci. Tech. 16, 848–

218

873.

219

Accepted Manuscript

Corrales, J., Esnal A., De la Fe, C., Sánchez, A., Assunçao, P., Poveda, J., Contreras, A.,

220

2007. Contagious agalactia in small ruminants. Small Rumin. Res. 68, 154-166.

221

Cottew, G.S., Yeats, F.R., 1981. Occurrence of mycoplasmas in clinically normal goats. Aust.

222

Vet. J. 57, 52–53.

223

Cottew, G.S., Yeats, F.R., 1982. Mycoplasmas and mites in the ears of clinically normal

224

goats. Aust. Vet. J. 59, 77–81.

225

DaMassa A.J., 1983. Prevalence of mycoplasmas and mites in the external auditory meatus of

226

goats. Calif. Vet. 12, 10–17.

227

DaMassa, A.J., Brooks, D.L., 1991. The external ear canal of goats and other animals as a

228

mycoplasma habitat. Small Rumin. Res. 4, 85–93.

229

De la Fe, C., Assunçao, P., Rosales, R.S., Antunes, T., Poveda, J.B., 2005. Microbiological

230

study of contagious agalactia in a serological endemic area. Vet. J. 170, 257-259.

231

Dedieu, L., Mady, V., Lefevre, P.C., 1995. Development of two PCR assays for the

232

identification of mycoplasmas causing contagious agalactia. FEMS Microbiol. Lett. 129, 243-

233

249.

234

Dorigo-Zetsma, J.W., Zaat, S.A., Wertheim-van Dillen, P.M., Spanjaard, L., Rijntjes, J., van

235

Waveren, G., Jensen, J.S., Angulo, A.F., Dankert, J., 1999. Comparison of PCR, culture, and

236

serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in

237

children. J. Clin. Microbiol. 37, 14-17.

238

Gil, M.C., Hermoso de Mendoza, M., Rey, J., Alonso, J.M., Poveda, J.B., Hermoso de

239

Mendoza, J., 1999. Isolation of mycoplasmas from the external ear canal of goats affected

240

with contagious agalactia. Vet. J. 158, 152–154.

241

Greco, G., Corrente, M., Martella, V., Pratelli, A., Buonavoglia, D., 2001. A multiplex-PCR

242

for the diagnosis of contagious agalactia of sheep and goats. Mol. Cell. Probes. 15, 21-25.

243

Accepted Manuscript

Hotzel, H., Sachse, K., Pfützner, H., 1996. A PCR scheme for differentiation of organisms

244

belonging to the Mycoplasma mycoides cluster. Vet. Microbiol. 49, 31-43.

245

Johnson, L.R., Drazenovich, N.L., Foley, J.E., 2004. A comparison of routine culture with

246

polymerase chain reaction technology for the detection of Mycoplasma species in feline nasal

247

samples. J. Vet. Diagn. Invest. 16, 347-351.

248

Kirchhoff, H., Rosengarten, R., 1984. Isolation of a motile mycoplasma from fish. J. Gen.

249

Microbiol. 130, 2439-2445.

250

Luki, N., Lebel, P., Boucher, M., Doray, B., Turgeon, J., Brousseau, R., 1998. Comparison of

251

polymerase chain reaction assay with culture for detection of genital mycoplasmas in

252

perinatal infections. Eur. J. Clin. Microbiol. Infect. Dis. 17, 255-263.

253

Manso-Silván, L., Perrier, X., Thiaucourt, F., 2007. Phylogeny of the Mycoplasma mycoides

254

cluster based on analysis of five conserved protein-coding sequences and possible

255

implications for the taxonomy of the group. Int. J. Syst. Evol. Microbiol. 57, 2247-2258.

256

Mercier, P., Pellet, M., Morignat, E., Calavas, D., Poumarat, F., 2007. Prevalence of

257

mycoplasmas in external ear canal of goats: Influence of the sanitary status of the herd. Small

258

Rumin. Res. 73, 296–299.

259

Peyraud, A., Woubit, S., Poveda, JB., De la Fe, C., Mercier, P., Thiaucourt, F., 2003. A

260

specific PCR for the detection of Mycoplasma putrefaciens. Mol. Cell. Probes. 17, 289-294.

261

Poveda, J.B., 1998. Biochemical Characteristics in Mycoplasma Identification. In: Miles, R.,

262

Nicholas, R.A.J. (Eds.), Methods in Molecular Biology. Vol. 104 Mycoplasma Protocols.

263

Humana Press, Totowa, 69-78.

264

Poveda, J.B., Nicholas R., 1998. Serological identification of mycoplasmas by growth and

265

metabolic inhibition tests. In: Miles, R., Nicholas, R.A.J. (Eds.), Methods in Molecular

266

Biology. Vol. 104 Mycoplasma Protocols. Humana Press, Totowa, 105-111.

267

Accepted Manuscript

Sanguinetti, V., Chiocco D., 1987. Contagious agalactia and mycoplasma mycoides: The

268

situation in Italy. In: Jones, G.E. (Ed.), Agriculture: Contagious agalactia and other

269

mycoplasmal diseases of small ruminants. European Communities, Luxembourg, 11-21.

270

Spergser, J., Aurich, C., Aurich, J.E., Rosengarten, R., 2002. High prevalence of

271

mycoplasmas in the genital tract of asymptomatic stallions in Austria. Vet. Microbiol. 87,

272

119-129.

273

Stellrecht, K.A., Woron, A.M., Mishrik, N.G., Venezia, R.A., 2004. Comparison of multiplex

274

PCR assay with culture for detection of genital mycoplasmas. J. Clin. Microbiol. 42, 1528-

275

1533.

276

Tardy, F., Mercier, P., Solsona, M., Saras, E., Poumarat, F., 2007. Mycoplasma mycoides

277

subsp. mycoides biotype large colony isolates from healthy and diseased goats: prevalence

278

and typing, Vet. Microbiol. 121, 268-277.

279

Thrusfield M., 1995. Chapter 17. Diagnostic testing. In: Thrusfield, M. (Ed.), Veterinary

280

Epidemiology. Blackwell Science Ltd., Oxford, 266-284.

281

Thrusfield, M., Ortega, C., de Blas, I., Noordhuizen, J.P., Frankena, K., 2001. Win Episcope

282

2.0: improved epidemiological software for veterinary medicine. Vet. Rec. 148, 567-572.

283

Tola, S., Idini, G., Manunta, D., Galleri, G., Angioi, P.P., Rocchigiani, A.M., Leori, G., 1996.

284

Rapid and specific detection of Mycoplasma agalactiae by polymerase chain reaction. Vet.

285

Microbiol. 51, 77-84.

286

Tola, S., Angioi, A., Rocchigiani, A.M., Idini, G., Manunta, D., Galleri, G., Leori, G., 1997.

287

Detection of Mycoplasma agalactiae in sheep milk samples by polymerase chain reaction,

288

Vet Microbiol. 54, 17-22.

289

290

291

292

293

Accepted Manuscript

Table 1. Numbers and percentages of samples testing positive for Mycoplasma agalactiae

and Mycoplasma mycoides subsp. capri using culture versus PCR techniques

Mycoplasma

species

Number of samples

Tested Positive

result (%) C+^a PCR+^b

(%) C+^a PCR-^c

(%) C-^d PCR+^c

(%) Negative

result (%)

Ma

Mmc

a

Positive culture result

b

Positive PCR result

c

Negative PCR result

d

Negative culture result

Table 1

Accepted Manuscript

Table 2. Validity variables of the culture and PCR techniques used to detect Mycoplasma agalactiae in ear swab samples

% (95% confidence interval) Culture PCR

Sensitivity

Specificity

Positive predictive value

Negative predictive value

Youden J

a

100% by definition

Table 2

Accepted Manuscript

Table 3. Validity variables of the culture and PCR techniques used to detect Mycoplasma mycoides subsp. capri in ear swab samples

% (95% confidence interval) Culture PCR

Sensitivity

Specificity

Positive predictive value

Negative predictive value

Youden J

a

100% by definition

Table 3