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Identification and differentiation of canine isolates by 16S-23S rDNA PCR-RFLP
J. Spergser, R. Rosengarten
To cite this version:
J. Spergser, R. Rosengarten. Identification and differentiation of canine isolates by 16S-23S rDNA PCR-RFLP. Veterinary Microbiology, Elsevier, 2007, 125 (1-2), pp.170.
�10.1016/j.vetmic.2007.04.045�. �hal-00532259�
Accepted Manuscript
Title: Identification and differentiation of canineMycoplasma isolates by 16S-23S rDNA PCR-RFLP
Authors: J. Spergser, R. Rosengarten
PII: S0378-1135(07)00226-X
DOI: doi:10.1016/j.vetmic.2007.04.045
Reference: VETMIC 3686
To appear in: VETMIC Received date: 12-2-2007 Revised date: 24-4-2007 Accepted date: 26-4-2007
Please cite this article as: Spergser, J., Rosengarten, R., Identification and differentiation of canineMycoplasmaisolates by 16S-23S rDNA PCR-RFLP,Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2007.04.045
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Accepted Manuscript
Identification and differentiation of canine Mycoplasma isolates by 16S-23S 1
rDNA PCR-RFLP 2
J. Spergser*, R. Rosengarten 3
4
Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, 5
University of Veterinary Medicine Vienna, Veterinaerplatz 1, A-1210 Vienna, Austria 6
7
* Corresponding author at: Institute of Bacteriology, Mycology and Hygiene, 8
Department of Pathobiology, University of Veterinary Medicine Vienna, 9
Veterinaerplatz 1, A-1210 Vienna, Austria. Tel.: +43 1 25077 2127, Fax: +43 1 25077 10
2190, 11
E-mail address: joachim.spergser@vu-wien.ac.at 12
13 14 15 16 17 18 19 20 21 22 23 24 25 26 Manuscript_revised
Accepted Manuscript
Abstract 27
28
Conventional serological methods for the identification of canine mycoplasma 29
isolates depend on the availability of a panel of species-specific diagnostic antisera 30
and are not always reliable in terms of specificity. To enable simultaneous 31
identification of field isolates, PCR-RFLP analysis of the 16S-23S rRNA intergenic 32
spacer region was used to characterize the type strains of the 12 currently described 33
canine mycoplasmas of the Genus Mycoplasma which represent the “classic” non- 34
hemotropic species. The use of 16S-23S rDNA PCR in the first step of this analysis 35
revealed specific size differences of amplicons which allowed to classify these 12 36
canine Mycoplasma species into three groups. Depending on the length of the 37
amplicon, subsequent RFLP analysis of PCR products using two restriction 38
endonucleases in a single digest (ApoI/DdeI or TaqI/VspI) generated unique banding 39
patterns. For further evaluation of the 16S-23S rDNA PCR-RFLP assay system as 40
identification and differentiation tool, a total of 262 field isolates collected from the 41
canine genital tract were tested. PCR-RFLP results for 251 field isolates correlated 42
with traditional serological test results.The remaining 11 isolates had an RFLP 43
pattern distinct from the type strains included in this study and were identified by 16S 44
rDNA sequencing as closely related to M. sp. HRC689. The PCR-RFLP assay 45
established in this study enabled a rapid, accurate and easily performed identification 46
and differentiation of all 12 currently described non-hemotropic canine Mycoplasma 47
species.
48 49 50
Keywords: canine mycoplasmas, molecular identification, 16S-23S rDNA PCR- 51
RFLP 52
Accepted Manuscript
1. Introduction 53
54
So far, fifteen mycoplasma species of the genera Acholeplasma (1), Mycoplasma 55
(13) and Ureaplasma (1) and two not yet fully described species of the genus 56
Mycoplasma have been isolated from or detected in dogs: Acholeplasma laidlawii, 57
Mycoplasma (M.) arginini, M. bovigenitalium, M. canis, M. cynos, M. edwardii, M.
58
feliminutum, M. felis, M. gateae, M. haemocanis, M. maculosum, M. molare, M.
59
opalescens, M. sp. HRC689, M. sp. VJC358, M. spumans and Ureaplasma 60
canigenitalium (Chalker, 2005). W ith the exception of the former Haemobartonella 61
species M. haemocanis that is not cultivable on standard mycoplasma growth media, 62
most of these mycoplasmas grow rapidly on modified Hayflick’s medium under 63
aerobic conditions at 37°C (Rosendal, 1975a). Cultivation is therefore still the most 64
commonly used current method for their detection in canine clinical samples.
65
However, only few laboratories routinely culture for mycoplasmas, as the required 66
mycoplasma media are complex and expensive, and identification is traditionally 67
achieved by serological methods that are dependent on specific antisera to each 68
individual species. Such diagnostic antisera are not readily available in most 69
diagnostic laboratories, and in addition, cross-reactions may occur with some other 70
species (Rosendal, 1975b). Biochemical tests only allow grouping but not 71
identification of canine mycoplasma isolates (Chalker, 2005). Recently, PCR assays 72
have been developed to identify canine mycoplasmas by amplification of species- 73
specific sequences of the 16S-23S rRNA intergenic spacer region appropriate to 74
clinical laboratories focussing on specific species. However, these assays are not 75
ideally suited for diagnostic laboratories testing for several canine mycoplasma 76
species because they require multiple PCR (Chalker et al., 2004). Because of the 77
difficulties in identifying canine mycoplasma isolates, the majority of studies 78
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focussing on canine mycoplasmas neglected to identify the particular species 79
present. Thus little is known about specific infections caused by canine mycoplasma 80
species, although certain species have been associated with canine anaemia (M.
81
haemocanis), respiratory disease (M. cynos) and urogenital tract infections (M. canis) 82
(Chalker et al., 2004; L’Abee-Lund et al., 2003; Messick et al., 2003; Rosendal, 1972;
83
Rosendal and Vinther, 1977, Rosendal, 1978; Rosendal, 1982).
84
The present study describes the identification of the non-hemotropic canine 85
Mycoplasma species using a single PCR of the 16S-23S rRNA intergenic spacer 86
region (IGS), followed by a single digestion reaction incorporating two restriction 87
endonucleases based on the length of the amplicon. The assay is sufficiently rapid 88
and at low cost, making it applicable as standard assay in routine diagnostics.
89 90
2. Materials and methods 91
92
2.1. Mycoplasma strains, isolates, and antisera 93
94
Mycoplasma type strains were purchased from the National Collection of Type 95
Cultures (NCTC; Colindale, UK) (M. arginini G230T NCTC10129, M. canis PG14T 96
NCTC10146), or were obtained from the Friedrich-Loeffler-Institute (Federal 97
Research Center for Animal Health, Jena, Germany), the former ‘Bundesinstitut für 98
gesundheitlichen Verbraucherschutz und Veterinaermedizin’ (BgVV) (M.
99
bovigenitalium PG11T, M. cynos H831T, M. edwardii PG24T, M. feliminutum BenT, M.
100
felis COT, M. gateae CST, M. maculosum PG15T, M. molare H542T, M. opalescens 101
MH5408DT, M. spumans PG13T). In addtion, species-specific antisera against 102
mycoplasma type strains listed above were obtained from the Friedrich-Loeffler- 103
Institute, Jena, Germany.
104
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A total of 262 field isolates of canine mycoplasmas were recovered from vaginal (n = 105
98) and semen (n = 102) samples of dogs without (female n = 39; male n = 48) and 106
with (female n = 59; male n = 54) symptoms of genital disorder such as genital 107
lesions and inflammation, semen abnormalities and/or infertility. Statistical 108
comparisons were made with the statistics package SPSS for Windows (SPSS Inc., 109
Chicago, Illinois, USA). Distribution patterns between groups were analysed by 110
ANOVA and χ2-test.
111
Samples were cultured at 37°C in 5% CO2 and 95% air in/on modified Hayflick’s 112
medium/agar. Isolates belonging to the genera Acholeplasma and Ureaplasma were 113
excluded by digitonin testing and testing for urease production in Ureaplasma 114
medium, respectively. Mycoplasma isolates were identified to species level using 115
immunofluorescence as conventional serological test. In those cases where 116
serological identification revealed mixed cultures, five single colonies were picked 117
and incubated in modified Hayflick’s medium. After propagation cultures were tested 118
for purity by serology. For further analysis, cultures were stored at -80°C until use in 119
PCR-RFLP assays.
120 121
2.2. PCR-RFLP analysis 122
123
DNA was extracted from 250 µl of culture using GenElute DNA extraction kit (Sigma, 124
Vienna, Austria). A 16S-23S rRNA IGS sequence was amplified using primer pair 125
F2A (5’-GTG GGG ATG GAT CAC CTC CT-3’) and R2 (5’-GCA TCC ACC AAA AAC 126
TCT T-3’) as described previously (Tang et al., 2000). In vitro amplification was 127
carried out in a reaction mixture of 50 µl containing 1 mM MgCl2, 0.1 mM of each 128
dNTP, 5 µl of 10 x PCR buffer, 1 U of Taq DNA polymerase (Promega, Mannheim, 129
Germany), 20 pmol of each primer (F2A/R2) and 5 µl of template. PCR was 130
Accepted Manuscript
performed in a thermal cycler (Gene Amp PCR System 2400-thermal cycler, Perkin 131
Elmer Applied Biosystems, Branchburg, New Jersey, USA). The reaction mixture was 132
incubated at 94°C for 30 s, followed by 30 cycles of 94°C for 30 s, 55°C for 120 s and 133
72°C for 120 s. The cycling was followed by a final incubation at 72°C for 300 s. A 134
total of 10 µl of the PCR amplification products were analysed by 1.5% agarose gel 135
electrophoresis and ethidium bromide staining (Sigma, Vienna, Austria).
136
Amplified DNA was digested using two restriction endonucleases depending on the 137
length of the amplicon: TaqI and VspI (Fermentas, Leon-Rot, Germany) were used 138
for products ranging from 340 to 380 bp and ApoI (Fermentans, Leon-Rot, Germany) 139
and DdeI (Segenetic, Borken, Germany) for amplicons at 250 bp.
140
Of the PCR products, 8 µl were digested with 4 U of each enzyme, 100 µg/ml of BSA 141
and 2 µl 10 x buffers for 5 h at 37°C. Digests were electrophoresed in a 2.5%
142
agarose gel and stained in ethidium bromide. Restriction fragment sizes were 143
determined by comparison to GeneRuler™ 100 bp DNA ladder (Fermentans, Leon- 144
Rot, Germany).
145 146
2.3 Sequencing of the 16S rRNA gene to identify mycoplasmas with unknown RFLP 147
pattern 148
149
Mycoplasma isolates producing an RFLP pattern distinct from the type strains were 150
analysed by partial DNA sequencing of the 16S rRNA gene using the eubacteria 151
primers 27f and 1492r (Lane, 1991). The identity of the compiled DNA sequences 152
was determined by comparison to DNA sequences available in the EMBL database 153
using FASTA (Pearson and Lipman, 1988).
154 155 156
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3. Results 157
158
3.1 Length of amplicons and RFLP analysis of type strains 159
160
Amplified products ranged in size from 210 bp (M. feliminutum), 250 bp (M. arginini, 161
M. gateae, M. spumans), 340 bp (M. cynos, M. molare), 360 bp (M. canis, M.
162
edwardii, M. felis) to 380 bp (M. bovigenitalium, M. maculosum, M. opalescens) (Fig.
163
1). M. feliminutum was clearly distinguishable from the other 11 species by its IGS 164
length and was therefore not included in RFLP analysis. By combining two enzymes 165
(TaqI/VspI for 340-380 bp products, ApoI/DdeI for amplicons at 250 bp) in a single 166
digest, unique RFLP patterns were produced for all the 12 canine Mycoplasma 167
species included in this study (Fig. 2A, 2B).
168 169
3.2. PCR-RFLP analysis of field isolates 170
171
PCR and restriction enzyme combinations used for the above canine Mycoplasma 172
reference strains were selected to test 262 Mycoplasma field isolates of canine 173
origin. For most isolates, conventional serological identification matched with the 174
identification based on RFLP patterns (Tab. 1).
175
There were, however, 9 M. edwardii and 15 M. canis isolates primarily categorised as 176
mixed cultures by serology and, after propagating of single colonies, constantly 177
showing cross reaction with the respective two antisera. Those isolates were clearly 178
identified to the species level by PCR-RFLP. Overlapping RFLP patterns indicating 179
the presence of mixed cultures were not observed. A further 11 isolates identified as 180
M. bovigenitalium using serological tests, produced RFLP patterns different from all 181
of the reference strains tested (Fig. 2A, Lane 9). Mixed cultures containing two or 182
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more canine mycoplasma species have been found in 11% of vaginal samples and 183
50% of semen samples, respectively. Statistical analysis revealed no correlation 184
between particular Mycoplasma species and clinical status.
185 186
3.3 Identification of Mycoplasma isolates with unknown RFLP pattern 187
188
The 11 isolates with an RFLP pattern distinct to the reference strains were further 189
analysed using 16S rDNA sequencing. All of these isolates were identified as most 190
closely related to M. sp. HRC689 (99% sequence identity).
191 192
4. Discussion 193
194
This is the first report of incorporating two restriction enzymes in a single digest of 195
amplified 16S–23S rRNA IGS to provide an identification system to the species level 196
of canine Mycoplasma isolates. The 16S–23S rRNA IGS was targeted for the 197
development of a PCR–RFLP, as the IGS exhibit a high degree of heterogeneity 198
varying in length and composition (Chalker and Brownlie, 2004). Two restriction 199
enzymes were incorporated in one tube, as single restriction enzyme digests did not 200
produce characteristic patterns for each of the Mycoplasma species examined.
201
Evaluation of the 16S–23S rRNA IGS RFLP was conducted using TaqI/VspI and 202
ApoI/DdeI digestion of genital field isolates from healthy and diseased individuals.
203
Nine M. edwardii and 15 M. canis isolates representing pure cultures and clearly 204
identified to the species level by PCR–RFLP were constantly crossreacting with the 205
respective two antisera indicating the existence of closely related strains within the 206
two species probably sharing surface epitopes. As appropriate dilutions of antisera 207
for immunfluorescence have been determined using type strains cross-reactive 208
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results within field isolates may occur. Serological crossreactions have been shown 209
previously (Rosendal, 1975b) but studies on strain heterogeneity within canine 210
mycoplasma species resulting in variable serological results are still missing.
211
Although 11 isolates that produced unknown RFLP were identified as M.
212
bovigenitalium by serology, DNA sequencing of the 16S rRNA gene identified these 213
isolates as closely related to M. sp. HRC689. As M. sp. HRC689, representing a 214
single unclassified strain, is a yet not validly described mycoplasma species, a 215
species-specific antiserum for serological identification was not available and 216
therefore not included in our study. Serological misidentification of those isolates 217
might be explaned by their close phylogenetic relationship to M. bovigenitalium.
218
Previous reports describing the isolation and serologically based identification of the 219
bovine Mycoplasma species M. bovigenitalium from the canine host should therefore 220
be interpreted with caution (Buchim et al., 1978; Rosendal, 1978). The PCR–RFLP of 221
the 16S–23S rRNA IGS described in this study offers a more accurate and rapid 222
identification scheme than identification by serology. As molecular technology is 223
available in more laboratories than specific canine mycoplasma antisera, this assay 224
may improve identification of Mycoplasma isolates leading to an increase in 225
diagnosis and identification of specific infection in the canine host. The PCR–RFLP 226
assay system developed here can be reliably applied to subcultivated Mycoplasma 227
isolates as demonstrated in this study. Its application to primary cultures from 228
randomly collected samples, however, requires further validation of its sensitivity and 229
specificity.
230 231
Acknowledgements 232
The authors wish to thank Barbara Iser and Silvia W ildmann for their excellent 233
technical assistance.
234
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235
References 236
237
Bruchim, A., Lutsky, I., Rosendal, S., 1978. Isolation of mycoplasmas from the canine 238
genital tract: a survey of 108 healthy dogs. Res. Vet. Sci. 25, 243-245.
239 240
Chalker, V.J., Brownlie, J., 2004. Taxonomy of the canine Mollicutes by 16S rRNA 241
gene and 16S/23S rRNA intergenic spacer region sequence comparison. Int. J. Syst.
242
Evol. Microbiol. 54, 537-542.
243 244
Chalker, V.J., Owen, W .M.A., Paterson, C., Barker, E., Brooks, H., Rycroft, A.N., 245
Brownlie, J., 2004. Mycoplasmas associated with canine infectious respiratory 246
disease. Microbiology 150, 3491-3497.
247 248
Chalker, V.J., 2005. Canine mycoplasmas. Res. Vet. Scie. 79, 1-8.
249 250
L’Abee-Lund, T.M., Heiene, R., Friis, N.F., Ahrens, P., Sorum, H., 2003. Mycoplasma 251
canis and urogenital disease in dogs in Norway. Vet. Rec. 153, 231-235.
252 253
Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M.
254
(Eds.), Nucleic Acid Techniques in Bacterial Systematics. John W iley & Sons Ltd., 255
Chichester, UK, pp. 115-175.
256 257
Messick, J.B., 2003. New perspectives about hemotropic mycoplasma (formerly, 258
Haemobartonella and Eperythrozoon species) infections in dogs and cats. Vet. Clin.
259
North Am. Small Anim. Pract. 33, 1453-1465.
260
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261
Rosendal, S., 1972. Mycoplasmas as a possible cause of enzootic pneumonia in 262
dogs. Acta Vet. Scand. 13, 137-139.
263 264
Rosendal, S., 1975a. Canine mycoplasmas: cultural and biochemical studies of type 265
and reference strains. Acta Pathol. Microbiol. Scand. (B) 83, 457-462.
266 267
Rosendal., S., 1975b. Canine mycoplasmas: serological studies of type and 268
reference strains, with a proposal for the new species, Mycoplasma opalescens. Acta 269
Pathol. Microbiol. Scand. (B) 83, 463-470.
270 271
Rosendal, S., 1978. Canine mycoplasmas: pathogenicity of mycoplasmas associated 272
with distemper pneumonia. J. Infect. Dis. 138, 203-210.
273 274
Rosendal, S., 1982. Canine mycoplasmas: their etiologic niche and role in disease. J.
275
Am. Vet. Med. Assoc. 180, 1212-1214.
276 277
Rosendal, S., Vinther, O., 1977. Experimental mycoplasmal pneumonia in dogs:
278
electron microscopy of infected tissue. Acta Pathol. Microbiol. Scand. (B) 85, 462- 279
465.
280 281
Tang, J., Hu, M., Lee, S., Roblin, R., 2000. A polymerase chain reaction based 282
method for detecting Mycoplasma/Acholeplasma contaminants in cell culture. J.
283
Microbiol. Methods 39, 121-126.
284 285 286
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Table 1: Identification of 262 isolates recovered from the canine genital tract by 287
serology and PCR-RFLP.
288
Mycoplasma species Vaginal samples (n = 98) Semen samples (n = 102)
M. canis a 61 66
M. edwardii a 10 21
M. felis 0 0
M. cynos 2 8
M. molare 1 0
M. bovigenitalium/M. sp. HRC b 5 6
M. opalescens 0 1
M. maculosum 12 18
M. arginini 4 9
M. spumans 14 24
M. gateae 0 0
M. feliminutum 0 0
289
aNine M. edwardii and 15 M. canis isolates showed cross reaction with the respective two antisera but were
290
clearly identified by PCR-RFLP.
291
bIsolates were identified as M. bovigenitalium by serology and as M. sp. HRC689 by 16S rDNA sequence
292
analysis.
293 294 295 296 297 298 299 300 301 302
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Figure legends 303
304
Fig. 1. PCR of the 16S-23S rRNA intergenic spacer region (IGS). Lane 1 M. canis 305
PG14T (NCTC10146), lane 2 M. edwardii PG24T; lane 3 M. felis COT; lane 4 M.
306
molare H542T; lane 5 M. cynos H831T; lane 6 M. bovigenitalium PG11T; lane 7 M.
307
opalescens; lane 8 M. maculosum PG15T; lane 9 M. sp 1624 (isolate); lane 10 M.
308
gateae CST; lane 11 M. spumans PG13T; lane 12 M. arginini G230T (NCTC10129);
309
lane 13 M. feliminutum BenT; M molecular weight marker (GeneRuler™ 100 bp DNA 310
ladder (Fermentans, Leon-Rot, Germany).
311 . 312 313
Fig. 2. Restriction endonuclease digestion of the IGS. (A) Lanes 1-9 TaqI and VspI 314
digest; (B) Lanes 10-12 ApoI and DdeI digest. Lane 1 M. canis PG14T (NCTC10146), 315
lane 2 M. edwardii PG24T; lane 3 M. felis COT; lane 4 M. molare H542T; lane 5 M.
316
cynos H831T; lane 6 M. bovigenitalium PG11T; lane 7 M. opalescens; lane 8 M.
317
maculosum PG15T; lane 9 M. sp 1624 (isolate) with unknown RFLP-type; lane 10 M.
318
gateae CST; lane 11 M. spumans PG13T; lane 12 M. arginini G230T (NCTC10129); M 319
molecular weight marker (GeneRuler™ 100 bp DNA ladder (Fermentans, Leon-Rot, 320
Germany).
321 . 322 323
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Figure 1
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Figure 2
