• Aucun résultat trouvé

Identification and differentiation of canine isolates by 16S-23S rDNA PCR-RFLP


Academic year: 2021

Partager "Identification and differentiation of canine isolates by 16S-23S rDNA PCR-RFLP"


Texte intégral


HAL Id: hal-00532259


Submitted on 4 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.

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

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

Identification and differentiation of canine Mycoplasma isolates by 16S-23S 1


J. Spergser*, R. Rosengarten 3


Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, 5

University of Veterinary Medicine Vienna, Veterinaerplatz 1, A-1210 Vienna, Austria 6


* 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


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


48 49 50

Keywords: canine mycoplasmas, molecular identification, 16S-23S rDNA PCR- 51



Accepted Manuscript

1. Introduction 53


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.


feliminutum, M. felis, M. gateae, M. haemocanis, M. maculosum, M. molare, M.


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.


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


Accepted Manuscript

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.


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;


Rosendal and Vinther, 1977, Rosendal, 1978; Rosendal, 1982).


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


2.1. Mycoplasma strains, isolates, and antisera 93


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.


bovigenitalium PG11T, M. cynos H831T, M. edwardii PG24T, M. feliminutum BenT, M.


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.



Accepted Manuscript

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.


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


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


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).


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.


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%


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


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


Accepted Manuscript

3. Results 157


3.1 Length of amplicons and RFLP analysis of type strains 159


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.


edwardii, M. felis) to 380 bp (M. bovigenitalium, M. maculosum, M. opalescens) (Fig.


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


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).


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


Accepted Manuscript

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


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


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.


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.


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


Accepted Manuscript

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.


Although 11 isolates that produced unknown RFLP were identified as M.


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.


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


230 231

Acknowledgements 232

The authors wish to thank Barbara Iser and Silvia W ildmann for their excellent 233

technical assistance.



Accepted Manuscript


References 236


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.


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.


(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.


North Am. Small Anim. Pract. 33, 1453-1465.



Accepted Manuscript


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.


Am. Vet. Med. Assoc. 180, 1212-1214.

276 277

Rosendal, S., Vinther, O., 1977. Experimental mycoplasmal pneumonia in dogs:


electron microscopy of infected tissue. Acta Pathol. Microbiol. Scand. (B) 85, 462- 279


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.


Microbiol. Methods 39, 121-126.

284 285 286


Accepted Manuscript

Table 1: Identification of 262 isolates recovered from the canine genital tract by 287

serology and PCR-RFLP.


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


aNine M. edwardii and 15 M. canis isolates showed cross reaction with the respective two antisera but were


clearly identified by PCR-RFLP.


bIsolates were identified as M. bovigenitalium by serology and as M. sp. HRC689 by 16S rDNA sequence



293 294 295 296 297 298 299 300 301 302


Accepted Manuscript

Figure legends 303


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.


molare H542T; lane 5 M. cynos H831T; lane 6 M. bovigenitalium PG11T; lane 7 M.


opalescens; lane 8 M. maculosum PG15T; lane 9 M. sp 1624 (isolate); lane 10 M.


gateae CST; lane 11 M. spumans PG13T; lane 12 M. arginini G230T (NCTC10129);


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.


cynos H831T; lane 6 M. bovigenitalium PG11T; lane 7 M. opalescens; lane 8 M.


maculosum PG15T; lane 9 M. sp 1624 (isolate) with unknown RFLP-type; lane 10 M.


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


321 . 322 323


Accepted Manuscript

Figure 1


Accepted Manuscript

Figure 2


Documents relatifs

Determination of the geographical origin of fruits by using 26S rDNA fingerprinting of yeast communities by PCR-DGGE: An application to Shea tree fruits3. Aly Farag El Sheikha 1,2,

Determination of citrus fruit origin by using 16S rDNA fingerprinting of bacterial communities by PCR- DGGE: an application to clementine from Morocco and Spain.. Abstract

The thermo-mechanical properties of the continuous and dispersed phase, toughening mechanisms and tough- ness optimisation in hyperbranched polymer epoxy blends were thus

Plutôt que d’être appréhendé comme une menace, le recours à des opérateurs privés peut être vu comme une opportunité pour l’opérateur public de partager certaines de

In our study, we observed a significant alteration in BMD and bone microstructure among premenopausal HIV+ve women, with a 14.1 % lower tibial trabecular bone density compared

Validity, sensitivity and resolution limit of the PCR-RFLP analysis of the rrs (16S rRNA gene) as a tool to identify soil-borne and plant-associated

Le personnel des lignes téléphoniques pro- vinciales ou territoriales pour les personnes âgées victimes de mauvais traitements ou pour les victimes de violence familiale

Technological treatments applied to French fish sample did not have a destructive effect on bacterial communities because the main bands present on non treated samples were