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Experiences with multispecies polymerase chain reaction and specific oligonucleotide probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae H. Salisch , M. Ryll , K.-H. Hinz & U. Neumann Published online: 17 Jun 2010.

To cite this article: H. Salisch , M. Ryll , K.-H. Hinz & U. Neumann (1999) Experiences with multispecies polymerase chain reaction and specific oligonucleotide probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae, Avian Pathology, 28:4, 337-344, DOI: 10.1080/03079459994588 To link to this article: http://dx.doi.org/10.1080/03079459994588

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Avian Pathology (1999) 28, 337±344

Experiences with multispecies polymerase chain reaction and speci® c oligonucleotide probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae H. Salisch, M. Ryll, K.-H. Hinz & U. Neumann

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Hannover School of Veterinary Medicine, Clinic for Poultry, BuÈnteweg 17, D-30559 Hannover, Germany

Ampli® ed fragments of the rDNA coding for 16S rRNA of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) were blotted on nylon membranes, followed by dot-blot detection with two species-speci® c digoxigenin-(DIG)-labeled oligonucleotide probes. The sensitivity and speci® ty of the tests were determined in titration studies with puri® ed homologous and heterologous DNA. With the detection protocol used, the MSYV8/31 probe showed 100% speci® ty for MS, while both MG and the related species Mycoplasma imitans were recognized by the MGAV8/31 probe. Both DIG-labeled oligonucleotides gave positive results in the colorimetric assay with 10 to 100 ng homologous non-ampli® ed DNA and polymerase chain reaction (PCR) ampli® cates of 100 fg homologous template DNA. There was no reaction with heterologous strains when ampli® cates starting with a 106-fold amount of template DNA (100 ng) were tested in dot-blots. The suitability for ® eld samples was demonstrated with tracheal swabs from turkeys and chickens, and the results were compared with mycoplasma growth in cultures of the same swabs. Both tests had an accuracy of over 95%, a high sensitivity and speci® city, and high predictive values of positive or negative results. There was no signi® cant difference between the results obtained by the two methods. PCR in combination with dot-blotting is a relatively simple method for the detection of mycoplasma infections, and a valuable extension of current diagnostic tools.

Introduction Mycoplasmas are small wall-less prokaryotic bacteria which colonise mucosal surfaces. Mycoplasma gallisepticum (MG) is commonly involved in chronic respiratory disease in chickens and infectious sinusitis in turkeys, and infections are often complicated by Escherichia coli and/or respiratory viruses. Mycoplasma synoviae (MS) may cause subclinical or clinical infections. Some strains cause airsacculitis on their own or in mixed infections with bacteria or respiratory viruses. Exudative synovitis of joints and tendon sheaths followed by lameness and poor growth may occur as a result of systemic infection with MS. Neither species is fully controlled by antibiotic treatment. Therefore, monitoring of the health status and eradication of infected chicken and turkey breeder

¯ ocks is the control measure of choice (Stipkovits & Kempf, 1996; Kleven, 1997; Ley & Yoder, 1997). Although mycoplasma detection is still carried out routinely by serology and culture, efforts have been made in recent years to establish polymerase chain reaction (PCR)-based tests which allow more rapid diagnosis than culture but have comparable sensitivity and speci® ty (Kempf, 1997, 1998). Primers which amplify sequences of the genome coding for the 16S rRNA have been selected to recognize one or more organisms such as a particular mycoplasma species (Lauerman et al., 1993), three pathogenic avian mycoplasma species (Garcia et al., 1995), 14 avian mycoplasma species (Fan et al., 1995a) or numerous bacteria including mycoplasmas (Weisburg et al., 1991). In this

Received 18 September 1998. Accepted 20 December 1998. ISSN 0307-945 7 (print)/ISSN 1465-3338 (online)/99/040337-0 8

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1999 Houghton Trust Ltd

338

H. Salisch et al. Table 1. Species and strains of avian mycoplasmas Species

Strain

Source

Origin

Mycloplasma gallisepticum

S6 Conn F R A5969 IPDH 162±77 36±90 10±93 WVU 1853 76±93 91±93 152±93 9/1±95 PG9 85±91 DD PG16 WR1 486 4229 1/2 F-95 R171 17529 CKK

G.C. Snell (UK) E. Vielitz (GER) S.H. Kleven (USA) S.H. Kleven (USA) K.-H. Hinz (GER) K.-H. Hinz (GER) K.-H. Hinz (GER) ATCC type strain 25204 K.-H. Hinz (GER) K.-H. Hinz (GER K.-H. Hinz (GER) K.-H. Hinz (GER) F.T.W. Jordan (UK) K.-H. Hinz (GER) F.T.W. Jordan (UK) F.T.W. Jordan (UK) F.T.W. Jordan (UK) M. Runge (GER) J.M. Bradbury (UK) K.-H. Hinz (GER) M. Runge (GER) R. Yamamoto (USA) F.T.W. Jordan (UK)

Turkey brain Chicken trachea Chicken trachea Chicken trachea Chicken lung Broiler breeder trachea Turkey trachea Chicken hock joint Broiler breeder trachea Turkey sinus Broiler breeder trachea Turkey trachea Sewage Goose cloaca Chicken trachea Chicken respiratory tract Turkey air sac Chicken oviduct Duck turbinate Turkey poult yolk sac Chicken sinus Turkey sinus Chicken trachea

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M. synoviae

Acholeplasma laidlawii M. cloacale M. gallinaceum M. gallinarum M. gallopavonis M. glycophilum M. imitans M. iowae M. lipofaciens M. meleagridis M. pullorum

Materials and Methods

or better was used. Preparations containing 100 ng DNA in 3 m l TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), the volume added to one PCR mix, were made up, and tenfold dilutions from 10 ng to 100 ag DNA per 3 m l of MG A5969 and MS WVU 1853 were prepared with highly puri® ed water (AmpuwaÔ , Fresenius, Bad Homburg, Germany) for titration studies or with TE buffer for positive control DNA (1 pg/3 m l). DNA solutions were stored at 2 21 and 2 70 °C until use.

Mycoplasma strains

Collection and processin g of clinical specimens

paper, we describe a comparison of culture and PCR-based DNA probe tests similar to those of Garcia et al. (1996) to detect MG and MS in clinical specimens.

The mycoplasm a strains used for DNA preparation are shown in Table 1. Strains from clinical material were puri® ed by subcultur e of well-separated colonies picked with a sterile loop from mycoplasm a agar plates. Strains were propagate d in broth medium, and the identity and purity of all cultures except Acholeplasma laidlawii, Mycoplasma cloacale, M. imitans and M. lipofaciens were con® rmed by indirect immunoperoxidas e tests (Imada et al., 1987).

Culture and serology Mycoplasma media and culture from tracheal swabs have been described previousl y (Salisch et al., 1998). Sera were examined by rapid serum agglutination (RSA) tests on the day of blood sampling using stained MG, MS or Mycoplasma meleagridi s (MM) antigen (Intervet International, Boxmeer, Netherlands). Occasionally , RSA results were con® rmed by haemagglutinatio n inhibition tests with antigen prepared in this laboratory from strains MG A5969 and MS WVU 1853, using the microtest method (Hinz, 1977) or using the appropriate commercial enzyme-linked immunosorben t assay (ELISA) kits for MG, MM or MS (KPL Gaithersburg, MD, USA).

Turkeys and chickens with serological results and/or pathologica l ® ndings suggestive of mycoplasm a infection were swabbed for culture and PCR-based DNA probe tests. They were also bled for con® rmation of serological results. The history of the samples and number s of swabs taken from each ¯ ock are presented in Table 2. DNA extraction from sinus, tracheal, air sac, lungs or tendon sheath swabs was carried out as described earlier (Salisch et al., 1998). Brie¯ y, after swabbing the tissues with a dry rayon swab (Greiner, Frickenhausen , Germany), the swabs were placed in a second set of tubes ® lled with 3 ml mycoplasm a broth medium for MS to keep the tips wet during transport. In the laboratory, after swabbing of the agar culture plates with the tip, they were replaced into the transpor t tubes, then brie¯ y put on a vortex mixer for a better release of the material from the tip. They were removed with sterile foreceps, and a 1 ml sample was taken for processing . After centrifugation, the sediment was washed twice with sample wash (SW) buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.05 v/v% Tween 20), and the DNA was released from the sedimented cells by boiling in 25 m l SW buffer for 10 min. After chilling on ice and centrifugation (13 880 3 g, 2 min), the supernatant s were collected in fresh tubes. The crude DNA samples were used fresh for PCR or stored at 2 21°C.

Preparation of genomi c DNA

Ampli® cation

Cultures grown to the logarithmic phase of developmen t were harvested at the colour-changin g point. DNA was extracted and puri® ed as described by Wilson (1988) and Maass & Dalhoff (1994). Concentration and purity were checked on a Beckman model 25 photometer (Beckman, Fullerton, CA). Only DNA with an OD260/280 ratio of 1.7

A protocol similar to that of Garcia et al. (1995) was used for PCR. One reaction mix (50 m l) contained 42.8 m l AmpuwaÔ , 5 m l 10 3 reaction buffer (160 mM (NH4)2 SO4 , 670 mM Tris-HCl (pH 8.8 at 25°C), 15 mM MgCl2 , 0.1% Tween 20), 1 m ml dNTP mix (10 mM each in AmpuwaÔ ), 0.5 m l of each primer (MGPCR-39 , 59 -GTC TCG

PCR for M. gallisepticum and M. synoviae

339

Table 2. Details of ¯ ocks and samples Flock no. 1 2

3 4

5

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6 7 8 9 10 to 13

14 to 16 a

Type of bird (age Serological status (number positive/number in weeks, if known) examined), ¯ ock history or pathological ® ndingsa

Organs sampled (number of swabs)

Turkey (17)

Trachea Trachea Trachea Trachea

MS RSA 2/12; HI 4/12; ELISA 5/12; MG and MM negative; some airsacculitis Turkey MS RSA 70/98, ELISA 22/98; MG RSA 7/98, ELISA negative; MM 7/98, ELISA 1/98; no pathological ® ndings Turkey MS RSA positive on routine screening Turkey (15) MG RSA 60/60; MS and MM RSA 0/60; increased mortality, sinusitis, retarded growth, leg weakness Turkey (8) MG, MS, and MM RSA 1/1; sinusitis, (tracheitis, airsacculitis, bronchopneumonia ) Broiler chicken (5) Dead after showing respiratory signs; airsacculitis Turkey (8) Dead after showing respiratory signs; airsacculitis Turkey MG RSA and ELISA 1/40 on routine screening Turkey (10) MG RSA 1/60; MM RSA 14/60; MG infection in previous crops; clinically normal Broiler breeder (36) MS RSA 64/64; MG RSA 42/64; sinusitis, sporadic raà les or leg weakness, sinusitis, tracheitis, airsacculitis, tendosynovitis Layer chicken (27) MS RSA 75/75; MG 0/75; clinically normal

(12) on farm (2), airsac (4) at necropsy (40) on farm (3), air sac (3), lung (3) at necropsy

Trachea (9) on farm Trachea (60) on farm

Trachea (1), air sac (1), lung (1) at necropsy Trachea Trachea Trachea Trachea

(1), air sac (1), lung (1) at necropsy (2), air sac (2), lung (2) at necropsy (40) on farm (60) on farm

Trachea (16/¯ ock of 5000) on farm; Trachea (3), sinus (3), hock joint tendon sheath (3) at necropsy Trachea (25/¯ ock of 1500) on farm

HI, haemagglutination inhibition.

TTA GAT AAA GT-39 (Garcia et al., 1995) and MSY16PCR59 , 59 -AGG CAG CAG TAG GGA AT-39 (Fan et al., 1995b) and 0.2 m l (1 U) BioThermÔ polymerase. The primers were synthesize d and high performance liquid chromatograph y (HPLC) puri® ed by Biometra (GoÈttingen, Germany) and supplied as 100 m l solutions. The 10 3 buffer, 100 mM dNTP solutions and polymerase came from GeneCraft (MuÈnster, Germany). Puri® ed DNA in 3 m l TE buffer or clinical specimens in 3 m l sample wash buffer were added to 50 m l reaction mix just before PCR. A protocol similar to that of Garcia et al. (1995) was chosen for ampli® cation: 94 °C for 10 min, 15 cycles at 94 °C for 1 min 20 s, 45 °C for 1 min and 74°C for 1 min, followed by 20 cycles at 94 °C for 1 min 20 s, 45°C for 1 min and 74°C for 1 min 1 5 s/cycle. At the end of 35 cycles, the samples were held at 4°C in the PTC-100±96V hot bonnet cycler (MJ Research, Watertown, MA, USA) until removal for further analysis.

Ampli® cate detection Speci® c DNA was detected in dot-blots with digoxigeni n (DIG)labeled probes on 0.45 m m pore size positively charged nylon membranes (Boehringer, Mannheim, Germany). After capillary transfer of 3 m l ampli® cate from puri® ed DNA solutions, swab preparations or buffers used for DNA extraction, the DNA was denatured with 0.4 M NaOH in 1.5 M NaCl for 15 min., followed by a 10 min neutralization of the nylon strips with 1 M Tris-HCl pH 7.4, 1.5 M NaCl to allow the re-use of the prehybridizatio n solution in the hybridization step. After backing (30 min at 120°C), the membranes were prehybridized at 56°C for 60 min in 5 3 sodium citrate buffer (SSC) (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0), pH 7.0, with 2 w/v% Boehringer blocking reagent (BBR) (Boehringer, Mannheim, Germany), 0.2% N-lauroylsarcosin e and 0.08% sodium lauryl sulfate (SDS) and 50% deionized formamide (Sigma-Aldrich, Deisenhofen, Germany). The buffer was adjusted to pH 7.5 before use. For hybridization, 10 pM/ml of the appropriate DIG-labeled DNA probe (Fernandez et al., 1993) was added to the prehybridization solution. The probe was either the original MGAV8/31-DIG probe (59 -DIG-ACT GCA GCA CCG AAG TAT TCG CTC CGA CAC T-39 ) or a MSYV8/31-DIG probe modi® ed in three positions (5© -DIG-CGT GCG TCG ATG GTT TCT ATC AAC TAG TCA T-39 )

for a perfect match with the published 16S rRNA sequenc e of MS WVU 1853 (X52083, EMBL bases, Hinxton, Cambridge, UK). Both probes were prepared and HPLC puri® ed by Biometra (GoÈttingen, Germany) and supplied as 100 pM/m l solutions. Membranes were hybridized at 56°C for 60 min, then washed twice with 2 3 SSC, 0.1 w/v% SDS at 56 °C for 5 min and twice with 0.5 3 SSC, 0.1% SDS at 56°C for 5 min, and were equilibrated in Tris buffer (100 mM TrisHCl pH 7.5, 150 mM NaCl). After reblocking with the same buffer with 2% BBR at 37 °C for 30 min, anti-digoxigenin-alkalin e phosphatase-labele d Fab fragments (0.75 U/m l, Boehringer) were added to the reblocking buffer at a concentratio n of 1 m l/10 ml (1:15 000 or 50 mU/ml). The membranes were incubated for 30 min at 37°C, washed three times for 5 min each with sodium maleic acid tween buffer (Engler-Blum et al., 1993; SMT, 3 M NaCl, 0.1 M maleic acid, 0.3% Tween 20, pH 8.0) at 37°C, and were equilibrated again for 5 min in substrate buffer (100mM Tris-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl 2 ). Substrates 5-bromo-4-chloro-3-indoly l phosphat e and nitro blue tetrazolium (165 m g BCIP and 330 m g NBT/ml substrate buffer) were used for the colorimetric detection of ampli® cates. Membranes were incubated for 2 h at 37°C for optimum sensitivity.

Statistical evalution For comparison of culture and PCR-based DNA probe tests, McNemar’ s tests (2 3 2 tables) were done with SigmaStat 2.0 software (Jandel, Erkrath, Germany). Sensitivity, speci® ty, predictive values of positive and negative results, and accuracy were calculated according to Hopert et al. (1993) from 294 and 236 clinical specimens.

Results In preliminary studies with dot-blots, 10 and 100 ng homologous non-ampli® ed DNA were detected by both probes. The sensitivity after ampli® cation was 100 fg DNA for MG A5969 and MS WVU 1853. Both probes detected all homologous strains

340

H. Salisch et al. Table 3. Detection of M. gallisepticum (MG) by culture and a PCR-based DNA probe test Flock suspicious for MG infection by

Flock number

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2a 4 5 6 7 8 9 10 to 13 Total Sensitivity of the DNA probe test Speci® ty of the DNA probe test Predictive value of a positive result Predictive value of a negative result Accuracy of the DNA probe test

Serology

Clinical signs

1

2 1

1 NDb ND 1 1 1

1 1 1 1 2 2 1

90.2% 96.8% 82.2% 98.4%

Number of swabs positive for MG Number of swabs examined

cultures

MGAV8/31 probe test

49 60 3 3 6 40 60 73 294

0 38 3 0 0 0 0 0 41

0 43 2 0 0 0 0 0 45

True positivesc True negativesc False positivesd False negativese

41

37

253

245

0

8

0

4

95.8%

a

Flocks not listed here were all negative. ND, not done. c Corresponding results between culture and PCR-based DNA probe tests. d Positive results not con® rmed by culture (likely to be true positives from non-viable MG, and cultures `negative’ due to bacterial overgrowth). e Culture positives not detected in the PCR-based DNA probe test. b

and did not react with heterologous strains when a 106-fold amount of DNA (100 ng) was added to the PCR mix. The only exception was Mycoplasma imitans (MIM), which could not be distinguished from MG. The OD260/280 ratio of the DNA preparations was at least 1.70, with the average above 1.8 for all those strains which grew well. The results of ® eld specimens examined for MG and MS are summarised in Tables 3 and 4, respectively. The turkeys from ¯ ock 1 were negative by culture and the MG and MS DNA probe test. A few colonies were cultured from the tracheal swabs from the turkeys of ¯ ock 2 but PCR was negative. The cultures were identi® ed as MS by indirect immunoperoxidase tests and by ampli® cation of culture material and the MS DNA probe test. The remaining 47 swabs were negative for MG, MM and MS by culture, and neither MG nor MS could be detected with the DNA probe tests. One MSpositive swab was detected by both culture and the PCR-based DNA probe test for the turkeys from ¯ ock 3, with only few colonies seen on the culture plate of that swab. In ¯ ock 4, which had clinical mycoplasmosis, all turkeys were positive for MG and negative for MS in RSA tests, and both culture and PCR were equally suitable for diagnosis. There were 35

matching positive results among the 43 swabs positive by the PCR-based MG DNA probe test. One of the remaining eight swabs was culture negative, and seven were unsuitable for mycoplasma culture due to bacterial overgrowth. Eight matching results were seen for the 17 swabs negative by PCR. The sensitivity of the PCR-based DNA probe test in this series was 92.1%. MS could not be detected by culture or with the MS DNA probe test con® rming the negative MS serology seen for this ¯ ock. Because MS infection was not expected, this result was not used for calculation of the operational characteristics of the MS test. All three swabs from the turkeys of ¯ ock 5 were positive for MG and negative for MS by culture. With the DNA probe test, only the air sac and lung sample were positive for MG, but not the suspension from the tracheal swab. All three swabs were negative for MS. In a second PCR, 100 pg internal control DNA of MG were added, and the resulting blots were still negative for the tracheal swab, which indicates the presence of inhibitors in the sample. The nine swabs from one broiler and two turkeys from ¯ ocks 6 and 7 with ® brinous airsacculitis were negative for MS and MG by both

PCR for M. gallisepticum and M. synoviae

341

Table 4. Detection of M. synoviae (MS) by culture and a PCR-based DNA probe test Flock suspicious for MG infection by

Flock number

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1a 2 3 5 6 7 10 to 13 14 to 16 Total Sensitivity of the DNA probe test Speci® ty of the DNA probe test Predictive value of a positive result Predictive value of a negative result Accuracy of the DNA probe test

Serology

Clinical signs

1

1 1

2

1 1 NDb ND 1 1

2 1 1 1 1 2

93.4% 98.2% 98.3% 93.3%

Number of swabs positive for MG Number of swabs examined

cultures

MGAV8/31 probe test

18 49 9 3 3 6 73 75 236

0 2 1 0 0 0 59 60 122

0 0 1 0 0 0 60 55 116

True positivesc Truec negativesc False positivesd False negativese

122

114

114

112

0

2

0

8

95.8%

a

Flocks not listed here were all negative. ND, not done. c Corresponding results between culture and PCR-based DNA probe tests. d Positive results not con® rmed by culture (likely to be true positives from non-viable MG, and cultures `negative’ due to bacterial overgrowth). e Culture positives not detected in the PCR-based DNA probe test. b

detection methods. The 40 turkeys from ¯ ock 8 were negative by mycoplasma culture and DNA probe tests. Also, mycoplasmas could not be detected by culture or by either DNA probe test in the 60 tracheal swabs taken from the turkeys of ¯ ock 9, although one serum positive for MG and 14 positive for MM were found in RSA tests. These turkeys were unvaccinated and unmedicated. Infection with MS was found in the broiler breeder chickens of ¯ ocks 10 to 13, and also in the layer chickens of ¯ ocks 14 to 16, which were clinically normal. There was no signi® cant difference between the results of culture and the DNA probe test using the MGAV8/31-DIG probe (P 5 0.386) or MSYV8/31-DIG probe (P 5 0.114). Both DNA probe tests had an accuracy and speci® ty of over 95%. Discussion The multispecies PCR described by Garcia et al. (1995, 1996) is a cost-effective alternative to the commercial tests available (Idexx, Portland, ME, USA). Ampli® cates produced with the primers provided in the kits are not recognized by the probes of Fernandez et al. (1993), and the commercial probes do not recognize ampli® cates

obtained with the primers described by Fan et al. (1995b) and Garcia et al. (1995). Therefore, both tests are independent. In combination with culture and serology, such tests can be used for the detection of MG and MS in control programmes and for con® rmation of dubious results obtained by serology or culture. About 10 to 100 mycoplasma cells contain suf® cient amounts of 16S rDNA for successful detection in dot-blots after PCR, whereas DNA probe tests without a prior PCR, as carried out in the 1980s, are much less sensitive and are unsuitable for diagnosis. At ® rst sight, it might be disappointing that this 16S rRNA-based test was unable to distinguish MG from MIM, but with the exception of three bases (out of over 1500 bases), the 16S rDNA sequences of these two species are identical (Boyle & Morrow, 1994). Amplicon digestion with VspI might be used for further differentiation (Boyle, 1993; Boyle & Morrow, 1994), but appears unnecessary for samples originating from chickens and turkeys, since so far MIM has been found only in ducks, geese, and partridges (Bradbury et al., 1993). Due to the close phylogenetic relationship, we were not able to differentiate MG and MIM by culture followed by immunoperoxidase assay, nor by serology with commercially available RSA

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342

H. Salisch et al.

antigens or MG ELISAs. Although differentiation is not easy, it can be done by immuno¯ uorescence (Bradbury et al., 1993). There is approximately 40% DNA:DNA homology between MG and MIM (Dupiellet et al., 1990). The commercial PCRbased DNA probe test (Campbell et al., 1993), with a different target and protocol to the test described in this paper, selectively detects MG and not MIM (Bradbury et al., 1993; Salisch et al., 1998), and might therefore be used to distinguish both species in samples from waterfowl or partridges. It is of special importance that diagnostic tests do not give false positive results, while some false negative results might be tolerated and be compensated for by increasing the number of samples taken. For the MG DNA probe test, there were eight results which were classi® ed as `false positives’ , all in samples from a ¯ ock with clinical MG infection con® rmed by pathology, serology and culture. Bacterial overgrowth of the culture plates leading to the classi® cation, `negative’ was observed for seven swabs from ¯ ock 4, and the last swab may have picked up non-viable MG cells. All eight `false positive’ results seen with the MG tests are therefore more likely to be true positives. As well as samples with numerous bacteria, there are also some with a low number of mycoplasma cells, and it is possible that culture is favoured by an uneven distribution of cells on the swab or in the suspension. In one of the four false negative swabs, the presence of PCR inhibitors was detected by use of internal control DNA. In general, there were no obvious problems with inhibitors when tracheal swabs were taken for mycoplasma detection, and the routine use of a second PCR reaction with control DNA therefore seems unnecessary, although more samples containing inhibitors might be found if each sample is checked. While use of internal controls is common for individual samples to avoid false negatives, ¯ ock screening is usually done with at least 10 samples, and simply doubling the number of swabs instead of screening for inhibitors might be more useful for avoiding false negative ¯ ock results. With the MS DNA probe test, two `false positive’ specimens were found, both coming from ¯ ocks shown to be MS positive by serology and culture, and therefore likely to be true positives. The false culture negatives might be partially due to bacterial overgrowth of cultures and low numbers of cells on the swabs, as discussed earlier. Garcia et al. (1996) have described a similar test using 40mer DIG-labeled probes for the detection of MG and MS in three broiler breeder ¯ ocks (A, B, C). Flock A was found to be MG positive both by culture and DNA probe tests, with eight out of 24 swabs being positive by each. The other two ¯ ocks were culture negative, but PCR and dot-blot positive for MS or MG. In our study, eight ¯ ocks gave nearly identical

results with culture and DNA probe tests, and the number of samples examined allowed the calculation of the operational characteristics of the tests when applied to ® eld samples. With the exception of the low predictive value for a positive result with the MG probe (82.2%) (possibly explained by culture `negatives’ due to bacterial overgrowth), both PCR-based DNA probe tests gave 90% or better results for all operational characteristics measured, including an accuracy of more than 95%. Serological results or pathological ® ndings can be misleading in mycoplasma diagnosis. For example, despite the fact that 42 serum samples from ¯ ocks 10 to 13 were positive for MG and that vaccination could be ruled out, MG was not detected by culture or the MG DNA probe test. With the exception of three ¯ ocks where only one or two birds were examined, representative sample sizes were taken for the evaluation. Only the two ¯ ocks also MG positive by culture where found to be positive when the MG DNA probe test was used, and all nine ¯ ocks MS culture positive were also positive with the MS test. All the remaining ¯ ocks considered suspicious for mycoplasma infection either by serology or clinical signs and pathology were negative by culture and PCR. No signi® cant differences were observed between plate culture and DNA probe tests. Broth culture is thought to to be more sensitive than plate culture, but our experience is that this advantage vanishes when samples contain very low numbers of mycoplasmas. The few cells present might be lost when a part of the primary culture is taken for subculture, and broth culture favors the growth of some other bacteria present in the specimens as well, resulting in more cultures unsuitable for mycoplasma detection (K.-H. Hinz, personal communication, 1994). However, broth and/or plate culture cannot be completely replaced by DNA probe tests. Other mycoplasma species would be overlooked by these tests, and it is important to know whether viable or non-viable mycoplasmas have been picked up with the swabs, e.g. after chemotherapy in meat ¯ ocks. On the other hand, PCR techniques give earlier results, usually within 1 or 2 days, and can be less laborious than culture, depending on the number of samples and the method used for the characterization of the ampli® cates. Multiplex PCR has been recently shown to be useful for simultaneous ampli® cation of MG, MS, MM amd M. iowae in pure and mixed culture samples (Wang et al., 1997). In this method, four primer pairs in each reaction tube produce ampli® cates of different size, which are then identi® ed by agarose electrophoresis. A multiplex PCR with two primer pairs based on 16S rRNA sequences of M. gallinarum and M. gallinaceum has been examined by Lauerman & van Santen (1996). This kind of test might become an alterna-

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PCR for M. gallisepticum and M. synoviae

tive, especially for small numbers of samples, but sensitivity studies with ® eld samples and nontarget DNA from other sources remain to be done. When care is taken to avoid false positives due to contamination, multispecies PCR for MG and MS meets the demands of diagnosis with respect to sensitivity, speci® ty and accuracy, and will ease and improve diagnosis in epidemiological studies with large numbers of samples. It will also suit other applications such as species identi® cation in cultures or detection of mycoplasmas in medicated ¯ ocks or tissues taken at necropsy. Thus, PCR is the method of choice for rapid detection of pathogenic mycoplasmas but should be done in parallel with culture. Isolation of the organisms allows for collection of strains from the full spectrum of cultivable mycoplasmas and allows quantitation of growth. It also permits drug sensitivity tests to be carried out and it provides con® rmation of PCR results. This is especially important in cases where infected ¯ ocks face slaughter. Acknowledgements We wish to thank the veterinarians and technicians of the turkey and broiler integrations and veterinary poultry practices, and J. M. Bradbury, F. T. W. Jordan, S. H. Kleven, M. Runge, G. C. Snell, E. Vielitz and R. Yamamoto for strains given to our laboratory, and Rita Leise and Myriam Heise for excellent technical assistance.

References Boyle, J.S. (1993). Phylogeny and diagnosi s of several avian mycoplasm a species. B.Sc. Thesis. Melbourne University, 86 pp. Boyle, J.S. & Morrow, C.J. (1994). Phylogeny of avian mycoplasmas : implications for mollicute taxonomy. Internationa l Organisation for Mycoplasmolog y Letters, 3, 589±590. Bradbury, J.M., Abdul-Wahab , O.M.S., Yavari, C.A., Dupiellet, J.-P. & Bove , J.M. (1993) . Mycoplasma imitans sp. nov. is related to Mycoplasma gallisepticum and found in birds. International Journal of Systematic Bacteriology, 43, 721±728. Campbell, G., van Dam, B. & Tyrrell, P. (1993). Commercial DNA probe test kits for Mycoplasma gallisepticum and Mycoplasma synoviae: a ® eld report. Proceeding s of the 42nd Western Poultry Disease Conference, Sacramento, CA, 28 February±2 March 1993, pp. 80±81. Dupiellet, J.P., Vuillaume, A., Rousselot, D., Bove , J.M. & Bradbury, J.M. (1990). Serological and molecular studies on Mycoplasma gallisepticum strains. Zentralblatt fuÈr Bakteriologie, Suppl. 20, 859±864. Engler-Blum, G., Meier, M., Frank, J. & MuÈller, G.A. (1993) . Reduction of backgroun d problems in nonradioactiv e northern and southern blot analyses enables higher sensitivity than 32P-based hybridizations. Analytical Biochemistry, 210, 235±244. Fan, H.H., Kleven, S.H., Jackwood , M.W., Johansson, K.-E., Pettersson, B. & Levisohn, S. (1995a). Species identi® cation of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism analysis. Avian Diseases, 39, 398±407. Fan, H.H., Kleven, S.H. & Jackwood , M.W. (1995b) . Studies of intraspecies heterogenicit y of Mycoplasma synoviae, M. meleagridis, and M. iowae with arbitrarily primed polymerase chain reaction. Avian Diseases, 39, 766±777. Fernandez, C., Mattsson, J.G., Bolske, G., Levisohn, S. & Johansson , K.E. (1993). Species-speci ® c oligonucleotide probes complemen tary to 16S rRNA of Mycoplasma gallisepticum and Mycoplasma synoviae. Research in Veterinary Science, 55, 130±136. Garcia, M., Jackwood, M.W., Levisohn, S. & Kleven, S.H. (1995) .

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Detection of Mycoplasma gallisepticum, M. synoviae, and M. iowae by multiple-species polymerase chain reaction and restriction fragment length polymorphism . Avian Diseases, 39, 606±616. Garcia, M., Jackwood , M.W., Head, M., Levisohn, S. &. Kleven, S.H. (1996). Use of species-speci ® c oligonucleotid e probes to detect Mycoplasma gallisepticum, M. synoviae, and M. iowae PCR ampli® cation products. Journal of Veterinary Diagnostic Investigation, 8, 56±63. Hinz, K.-H. (1977). Untersuchunge n zur Spezi® taÈt des HAH-Tests an experimentel l mit Mycoplasma gallisepticum oder Mycoplasma synoviae in® zierten HuÈhnerkuÈken. Deutsche tieraÈrztliche Wochenschrift, 84, 475±479. Hopert, A., Uphoff, C. C., Wirth, M., Hauser, H. & Drexler, H. G. (1993). Sensitivity and speci® ty of polymerase chain reaction in comparison with other methods for the detection of mycoplasm a contamination in cell lines. Journal of Immunologica l Methods, 164, 91±100. Imada, Y., Uchida, I. & Hashimoto, K. (1987). Rapid identi® cation of mycoplasmas by indirect immunoperoxidas e test using small square ® lter paper. Journal of Clinical Microbiology, 25, 17±21. Kempf, I. (1997). DNA ampli® cation methods for diagnosis and epidemiologica l investigations of avian mycoplasmosis . Acta Veterinaria Hungarica, 45, 373±386. Kempf, I. (1998). DNA ampli® cation methods for diagnosis and epidemiologica l investigation of avian mycoplasmosis . Avian Patholology, 27, 7±14. Kleven, S.H. (1997). Mycoplasma synoviae infection. In B.W. Calnek, H.J. Barnes, C.W. Beard, L.R. McDougald & Y.M. Saif (Eds.), Diseases of Poultry 10th edn (pp. 220±228). London: MosbyWolfe. Lauerman, L.H. & van Santen, V.L. (1996). Multiplex PCR for Mycoplasma gallinarum and M. gallinaceum identi® cation. Journal of the American Veterinary Medical Association, 209, 388. Lauerman, L.H., Hoerr, F.J., Sharpton, A.R., Shah, S.M. &. van Santen, V.L. (1993). Development and application of a polymerase chain reaction assay for Mycoplasma synoviae. Avian Diseases, 37, 829±834. Ley, D.H. & Yoder H.W. Jr. (1997). Mycoplasma gallisepticum infection. In B.W. Calnek, H.J. Barnes, C.W. Beard, L.R. McDougald & Y.M. Saif, (Eds.), Diseases of Poultry 10th edn (pp. 194±207). London: Mosby-Wolfe. Maass, M. & Dalhoff, K. (1994). Comparison of sample preparation methods for detection of Chlamydia pneumoniae in broncheoalve olar lavage ¯ uid by PCR. Journal of Clinical Microbiology, 32, 2616±2619. Salisch, H., Hinz, K.-H., Graack, H.-D. & Ryll, M. (1998). A comparison of a commercial PCR-based test to culture methods for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in concurrently infected chickens. Avian Patholology, 27, 142±147. Stipkovits, L. & Kempf, I. (1996). Mycoplasmoses in poultry. International Of® ce of Epizootics Scienti® c and Technical Review, 15, 1495±1525. Wang, H., Fadl, A.A. &. Khan, M.I. (1997). Multiplex PCR for avian pathogeni c mycoplasmas. Molecular and Cellular Probes, 11, 211± 216. Weisburg, W.G., Barns, S.M., Pelletier, D.A. & Lane, D.J. (1991). 16S ribosomal DNA ampli® cation for phylogeneti c study. Journal of Bacteriology, 173, 697±703. Wilson, K. (1988). Preparation of genomi c DNA from bacteria. In F.M., Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J.G. Seidman & K. Struhl (Eds.), Current Protocols in Molecular Microbiology (pp. 2.4.1±2.4.5). New York: Greene Publishing and Wiley Interscience

REÂSUME DeÂtection de Mycoplasma gallisepticum et Mycoplasma synoviae aÁ l’aide d’un test PCR multi-espeÁce et de sondes oligonucleÂotidiques speÂci® ques Des fragments ampli® e s d’ ADNr codant pour l’ ARNr 16S de Mycoplasma gallisepticum (MG) et M. synoviae (MS) ont e teÂde pose s sur des membranes de nylon, ensuite une de tection par dot-blot a e teÂ

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re alise e aÁ l’ aide de deux sondes d’ oligonucle otides marque es aÁ la digoxige nine (DIG) spe ci® ques d’ espeÁce. La sensibilite et la spe ci® citeÂdes tests ont e teÂde termine es par des titrages de l’ ADN puri® eÂhomologue et he te rologue. Dans les conditions du protocole, la sonde MSYV8/31 a montreÂ100% de spe ci® citeÂvis-aÁ-vis de MS, alors que la sonde MGAV8/31 a reconnu les deux souches MG et M. imitans. Les deux oligonucl e otides marque s DIG ont donne des re sultats positifs par colorime trie avec des quantite s d’ ADN homologue non ampli® eÂallant de 10 aÁ 100 ng et des produits issus de la matrice d’ ADN homologue ampli® eÂpar PCR de 100 fg. Il n’ a pas e te observeÂde re action avec des souches he te rologues quand les produits d’ ampli® cation de la matrice d’ ADN ont e teÂutilise s aÁ partir de la dilution 106 pour re aliser les dot-blots. L’ applicabiliteÂde ces techniques aÁ des e couvillonnage s trache aux de dinde et de poulet e leve s conventionnellemen t a e teÂde montre e et les re sultats ont e teÂcompar e s aÁ ceux obtenus apreÁs la mise en culture des meà mes e couvillonnages . Les deux tests ont une ® de liteÂsupe rieure aÁ 95 %, sont treÁs sensibles et spe ci® ques et pre sentent des valeurs pre dictives e leve es des re sultats positifs ou ne gatifs. Il n’ y a pas e teÂobserveÂde diffe rence signi® cative entre les re sultats obtenus par les deux me thodes. La PCR associe e au dot-blot est une me thode relativement simple pour la de tection des infections mycoplasmique s et peut eà tre facilement utilise comme me thode de diagnostic. ZUSAMMENFASSUNG Erfahrungen mit der Multispezies-PCR und spezi® schen Oligonukleotid-Sonden zum Nachweis von Mycoplasma gallisepticum und M. synoviae Ampli® zierte Fragmente der rDNA, die fuÈr die 16S-rRNA von Mycoplasma gallisepticum (MG) und M. synoviae (MS) kodieren, wurden auf Nylon-Membrane n geblottet und anschliessen d im DotBlot-Nachweis mit zwei speziesspezi ® schen Digoxigenin (DIG)markierten Oligonukleotid-Sonde n untersucht . Die Emp® ndlichkeit und Spezi® taÈt der Tests wurden in Titrationsstudien mit gereinigter homologe r und heterologer DNA ermittelt. Mit dem verwendeten Nachweisverfahre n wies die Sonde MSYV8/31 eine 100%ige Spezi® taÈt fuÈr MS auf, waÈhrend durch die Sonde MGAV8/31 sowohl MG als auch die verwandte Spezies M. imitans erkannt wurden. Beide DIG-markierten Oligonukleotid e lieferten im kolorimetrischen Test mit 10 bis 100 ng homologer nicht-ampli® zierter DNA und PCRAmpli® katen von 100 fg homologer Template-DNA positive Ergebnisse. Es gab keine Reaktion mit heterologen StaÈmmen, wenn die

Ampli® kate beginnen d mit einer 10 6fachen Menge Template-DNA (100 ng) in Dot-Blots untersucht wurden. Die Eignung fuÈr Feldproben wurde mit Trachea-Abstrichen von Puten und HuÈhnern nachgewiesen , und die Ergebnisse wurden mit dem Mykoplasma-Wachstu m in Kulturen derselben Tupferproben verglichen. Beide Tests hatten eine Genauigkei t von uÈber 95%, eine hohe Emp® ndlichkeit und Spezi® taÈt und hohe Vorhersagewert e positiver oder negativer Resultate. Es gab keinen signi® kanten Unterschied zwischen den mit den zwei Methoden erzielten Ergebnissen. Die PCR in Verbindun g mit dem Dot-Blotting ist eine relativ einfache Methode fuÈr den Nachweis von Mykoplasma-Infektione n und eine wertvolle ErgaÈnzung der gegenwaÈrtigen diagnostische n Hilfsmittel.

RESUMEN Experimentos con pcr multiespeci® co y sondas de oligonucleoÂtidos especi® cas para la deteccioÂn de mycoplasma gallisepticun y mycoplasma synoviae Se depositaron fragmentos ampli® cados del rRNA codi® cante para el 16S rRNA de Mycoplasma gallisepticum (MG) y Mycoplasma synoviae (MS), realiza ndose a continuacio n te cnicas de dot-blot con dos sondas de oligonucleo tidos especie-espec õ  ® cos y marcados con digoxigenina (DIG). La sensibilidad y especi® cidad de los tests se determinoÂmediante titulacio n con DNA homo logo y hetero logo puri® cado. Con el protocol o de deteccio n utilizado, la sonda MSYV8/31 presento un 100% de especi ® cidad para MS, mientras que MG y M. imitans fueron identi® cados por la misma sonda MGAV8/31. Ambos oligonucleo tidos marcados con DIG, dieron resultados positivos en la te cnica colorime trica con 10±100ng de DNA homo logo no ampli® cado y con 100 fg de DNA homo logo ampli® cado. No se detectoÂreaccio n con cepas hetero logas mediante te cnicas de dot-blot cuando se realizaban las ampli® caciones a partir del DNA (100 ng) a una dilucio n de 106 . Se demostroÂla utilidad en muestras de campo con hisopos traqueales de pavos y pollos, y los resultados se compararon con el crecimiento de micoplasma en cultivos a partir de los mismos hisopos. Ambos tests presentaron una exactitud de ma s del 95%, una elevada sensibilidad y especi® cidad y elevados valores predictivos de resultados positivos o negativos. No hubo una diferencia signi® cativa entre los resultados obtenidos mediante los dos me todos. La te cnica de la PCR en combinacio n con el dot-blotting es un me todo relativament e sencillo para la deteccio n de infecciones por micoplasmas y constituye una ampliacio n de las te cnicas habituales de diagno stico.