DNA probes for Mycoplasma gallisepticum and Mycoplasma synoviae: Application in experimentally infected chickens

Vcterinao, Microbiology, 20 (1989) 323 337 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

323

D N A P r o b e s for Mycoplasma gallisepticum and Mycoplasma synoviae: Application in E x p e r i m e n t a l l y Infected C h i c k e n s HANA C. HYMAN I, SHARON LEVISOHN ~, DAVID YOGEV 1and SHMUEL RAZIN 1

~Department o/Membrane and Ultrastructure Research, Hadassah Medical School, The Hebrew University, Jerusalem, 91010 (Israel) :~Department of Poultry Diseases, Kimron Veterinary Institute, Bet Dagan 50250 (Israel) (Accepted for publication 16 March 1989 )

ABSTRACT Hyman, H.C., Levisohn, S., Yogev, D. and Razin, S., 1989. DNA probes h)r Mycoplasmagallisepticum and Mycoplasma synoviae: application in experimentally infected chickens. Vet. Micro biol., 20: 323-337. D NA probes specific for Mycoplasma gaUisepticum and M. synoviae were selected from genomic libraries prepared in the pUC13 vector. The probes hybridized with the DNA of a wide spectrum of strains within each homologous species, but did not react with the heterologous species or with DNA from any other avian mycoplasma or bacteria tested. Experimental infection and contact exposure of chickens to M. gallisepticum served as models to test the effectiveness of the DNA probe in diagnosis as compared with serological and culture detection methods carried out in parallel. A correlation was generally found between the level of M. gaUisepticum in tracheal swabs and the effectiveness of the probe, although a predictably reactive level of mycoplasmas was not always detected. Treatment of clinical specimens with acetylcysteine to disrupt mucus improved the detection rate. Dot-blot hybridization with probe pMG4 enabled positive identification of M. gallisepticum at an early stage of infection, prior to the development of a serological response in the infected chicken. Results are obtainable within 4 days of sampling, much more rapidly than culture, and also in clinical specimens from which mycoplasma isolation is impossible, such as carcasses. The results indicate that the use of DNA probes for the early and rapid detection of M. £,allisepticum infection is feasible; a development which can replace laborious culture techniques and less effective serological methods, and thus reduce the time required for diagnosis.

INTRODUCTION T h e lack of a rapid a n d reliable m e t h o d for diagnosis of m y c o p l a s m a infect i o n s in p o u l t r y h a m p e r s t h e e f f e c t i v e c o n t r o l o f t h e s e i n f e c t i o n s . T h e m a j o r a v i a n p a t h o g e n s M y c o p l a s m a gaUisepticum a n d M y c o p l a s m a synoviae n e e d t o be e r a d i c a t e d f r o m c o n t a m i n a t e d c h i c k e n a n d t u r k e y b r e e d e r f l o c k s in o r d e r to avoid the s p r e a d of i n f e c t i o n a n d u n n e c e s s a r y m a i n t e n a n c e costs ( J o r d a n ,

0:~78-1135/89/$03.50

© 1989 Elsevier Science Publishers B.V.

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1979). The difficult decision to slaughter a flock is currently based on periodic screening of random serum samples for IgM antibodies by a rapid, but inherently unreliable test, the rapid slide agglutination (RSA) test, followed by another, more specific serological test such as the hemagglutination inhibition (HI) test, or culture [United States Department of Agriculture (U.S.D.A.), 1985; Avakian et al., 1988]. The existence of antigens shared by M. gaUisepticure and M. synoviae and the genotypic heterogeneity characterizing these species (Yogev et al., 1988a,b, 1989; Kleven et al., 1988) further complicate accurate diagnosis. When properly carried out, confirmation of infection by culture may take from 1 to 3 weeks from the time of initial antibody detection. Development of DNA probes for rapid diagnosis of microbial infections constitut.es a new approach to solution of the problems involved in detecting fast idious organisms, such as mycoplasmas (Hyman et al., 1987; Khan et al., 1987; Razin et al., 1987; Santha et al., 1987; Zwadyk and Cooksey, 1987). DNA of the target organism is cloned into a plasmid vector which is easily maintained and produced in large quantities in an Escherichia coli host. Clones carrying DNA fragments of the target organisms are screened for size and tested for cross-reactivity with other bacteria likely to be present in the natural microbial flora (Hyman et al., 1987 ). Cross-reactivity of M. gallisepticum probes with M. .~ynoviae DNA and vice versa must be carefully tested because these mycoplasmas were shown to share genomic sequences (Yogev et al., 1989). The plasmid clone selected may then be used as a probe in hybridization tests with isolated DNA, intact bacteria, or clinical specimens deposited directly onto filters using a dot-blot system, thereby eliminating the necessity of cultivating organisms. Most importantly, a probe must be specific for the targeted organism in order to avoid false-positive responses while retaining adequate sensitivity to detect early infections, thereby speeding up the diagnostic process. In this report, we describe the development of specific DNA probes for detection of M. gallisepticum and M. synoviae and the application of the M. gaiIisepticum probe to samples taken from chickens experimentally infected with M. gallisepticum. The applicability of the DNA probe to detect M. gallisepticum in specimens from suspected field outbreaks in chicken and turkey flocks is the suhject of a separate communication (Levisohn et al., 1989). M A T E R I A L S AND M E T H O D S

Bacterial strains Strains used for probe preparation were M. gaUisepticum S-6 (isolated by R. Yamamoto, University of California, CA) and M. synoviae WVU1853 (type strain, isolated by N.O. Olson, University of West Virginia, WV). Experimental infection was carried out with M. gallisepticum R (isolated by D.G. Ritchie and obtained from S.H. Kleven, University of Georgia, GA). The origin of'

325

other strains used for testing specificity of the probes has been described elsewhere (Yogev et al., 1988a, 1989). M. synoviae strains MS-1 and MS-6 are isolates made in Israel from turkey synovitis. Mycoplasma spp. other than the above were obtained from E.A. Freundt ( F A O / W H O Collaborating Centre for Animal Mycoplasmas, University of Aarhus, D e n m a r k ) and include: M. gaUinarium PG16 and a non-numbered laboratory strain; M. pullorum CCK; M. gaUinaceum DD; M. gaUopavonis WR1; M. columbinsale 694 (serotype L); M. iowae 695 (serotype I); M. iners PG30; M. meleagridis N; A. laidlawii PGS. Escherichia coli and Alcaligenes faecalis were isolated by us from the experimental birds and identified by standard methods. Mycoplasma gaUisepticum and M. synoviae strains were grown in Frey's medium (Olson, 1984);other mycoplasmas were grown in Hayflick's mycoplasma medium (Hayflick, 1965 ) supplemented with glucose or arginine, according to the nutritional requirements of the organism.

Selection of probes Probes were prepared as described in detail elsewhere for other mycoplasma spp. ( H y m a n et al., 1987). Total D N A o f M . gaUisepticum S-6 or M. synoviae WVU1853 was digested with HindIII. Fragments were separated on a 0.7% low-gelling-temperature agarose gel, and those larger than 3 kb were ligated into plasmid pUC13 (Viero and Messing, 1982). Clones containing M. gallisepticum or M. synoviae D N A fragments were selected (Maniatis et al., 1982). Clones were screened to determine the size of the mycoplasmal D N A insert and those containing the longest inserts (about 6 kb) were chosen for further study. Preliminary screening of clones was carried out by Southern hybridization of' the M. synoviae clones to total D N A of M. gaUisepticum and of the M. gallisepticum clones with total D N A of M. synoviae. Hybridization was carried out at 56 °C and washing was performed under non-stringent conditions (Hyman et al., 1987) in order to maximize the detection of possible cross-reactions. Four clones of each mycoplasma carrying the largest inserts and showing no cross-reactions with the heterologous species were chosen for further testing.

Preparation of hybridization filters Dot-blot hybridization with purified DNAs was performed as described elsewhere ( H y m a n et al., 1987). For hybridization to D N A of bacterial cultures or with samples from experimentally infected chickens, release of D N A from the organisms was performed as follows: the air-dried nitrocellulose filter containing the samples to be tested was placed for a 5 min contact on a filter pad containing 0.5 M N a O H plus 1.5 M NaC1, followed by an additional 5-min contact with a filter pad saturated with 1.5 M NaC1 plus 0.5 M Tris-HC1 (pH

326

8.0). The nitrocellulose filters were then air-dried and washed twice for 15min periods in 2 X SSC (1 X SSC = 0.15 M NaC1 plus 0.0125 M sodium citrate). After thorough drying, the filters were baked for 2 h at 80°C under vacuum.

Sampling for tracheal infection Tracheal swabs from live experimentally infected birds were vigorously suspended in 2 ml sterile basal mycoplasma medium (BM). Aliquots were taken to determine the number of viable mycoplasmas and to test for infection by other bacteria and the rest of the suspension was frozen at - 70 ° C until tested for dot-blot hybridization. Aliquots of the tracheal lumen suspension containing mycoplasmas (0.1-0.4 ml) were usually applied directly to the nitrocellulose filters.

Disruption of mucus Acetylcysteine (AC), a reagent used in the t r e a t m e n t of h u m a n clinical specimens, was dissolved at a concentration of 0.5% in a solution consisting of 2% NaOH and 1.38% trisodium citrate-H20 (Lennette, 1985). Equal volumes of AC solution and samples to be treated were mixed vigorously on a Vortex mixer tbr 30 s and incubated at room temperature with occasional mixing for 15 min.

Hybridization of probe Hybridization of DNA immobilized on nitrocellulose filters was carried out according to procedures described by Maniatis et al. ( 1982 ). Pre-hybridization was carried out in sealed plastic bags at 56 or 60 ° C with shaking for 4 h in prehybridization buffer (0.1 ml buffer cm ~) consisting of: 6 X SSC; 5 X Denhardt's solution; 0.5% sodium dodecyl sulfate (SDS); 100 yg m1-1 sonicated and denatured salmon sperm DNA. Hybridization was carried out in the same buffer (0.05 ml cm -~) with the addition of 0.01 M EDTA, tbr 18-22 h. For hybridization, probes were labelled with :~2p using the Multiprime DNA Labelling System kit (Amersham, Gt. Britain); about 5 × 10 ~ cpm of probe per 100 em ~ filter was used. Unless otherwise specified, post-hybridization washing was carried out with shaking as follows: 2 X 10 min each with 2 × SSC at room temperature: 1 h with 0.5 X SSC plus 0.1% at 58°C; 2 X 10 min each with 0.1 × SSC at room temperature.

Titration of viable mycoplasmas The number of viable cells in mycoplasma cultures or in tracheal swab suspensions was determined by plate count (colony-forming units, CFU) or by

327 end-point dilution in liquid culture (color-changing units, CCU), using standard methods (Rodwell and Whitcomb, 1983).

Serological testing Chickens were bled at designated intervals, or at the time of necropsy. The serum samples were tested for the presence of antibodies to M. gallisepticum or M. synoviae by the RSA test using commercial stained antigen (Intervet, Holland). Positive samples in the RSA test were tested for activity in the HI test using standard methods (U.S.D.A., 1985).

Experimental birds and maintenance M. gallisepticum and M. synoviae-free chicks (strain " A n a k " ) were obtained from a commercial heavy breeder flock at 1 day of age, and maintained in a positive-pressure controlled-environment isolation cell. At time of inoculation birds were divided into experimental t r e a t m e n t groups and maintained in separate cells. A non-infected control group was maintained under the same conditions.

Experimental infection The usual method for experimental infection was by intratracheal inoculation, as described elsewhere (Levisohn and Dykstra, 1987). Infection by finespray aerosol and intra-air sac inoculation were also employed. Contact infection of chickens was carried out by placing non-infected birds together with infected birds in a Horsfal-Bauer isolation cell or by placing 1 or 2 "seeder" chickens in a M. gallisepticum-free group.

Experimental design The results of four separate experimental infection trials are reported, following the same general design. The experimental protocol in Experiments 13 called for inoculation of birds at ~ 2 weeks of age, with sampling at various time intervals post-infection (p.i.) for ~ 3 weeks (Experiments 1 and 2) or 1 m o n t h (Experiment 3). In Experiment 4, chickens were maintained in standard batteries until 1 m o n t h of age when one group was infected by air sac inoculation; other birds were infected later by contact exposure. At each time of sampling, birds were tested for serological activity, feasibility to reisolate M. gallisepticum, the number of viable mycoplasmas in the tracheal lumen suspension and reaction with the DNA probe. Whereas in Experiment 1 birds were sampled after necropsy, in later experiments, samples were also

328

taken by swabbing the trachea of the live bird. Thus, in Experiments 3 and 4, some birds were sampled repeatedly over a period of weeks and then necropsled. Other birds died during the course of the experiment; some of these were sampled for the D N A probe test at the time of post-mortem examination. Additional experimental details are given in table legends or text, where relevant to the data presented. RESULTS

Properties of the DNA probe Four probes prepared from the D N A ofM. gaUisepticum S-6 and selected on the basis of preliminary screening were tested by the dot-blot procedure against the purified D N A of 29 strains of M. gaUisepticum (10 ng per dot) and nine strains of M. synoviae (100 ng per dot). Hybridization was carried out under non-stringent conditions in order to detect any possible cross-reaction between the two Mycoplasma species. Differences were seen in the intensity of the reaction between certain probes and some of the M. gaUisepticum strains.

DNA ng

~S c~u/dot

,o 5 1

O

10 s

0.5

•

10"

0.25

"

°

I0 3

Fig. 1. Sensitivity of the M. gallisepticum DNA probe pMG4. (A) Dot-blot hybridization of the probe with purified M. gaUisepticum S-6 (Mg-S6) DNA and with DNA ( 100 ng) of M. synoviae (Ms); (B) dot-blot hybridization of the probe with organisms of various M. gallisepticum strains.

329

For the present purpose, a probe which reacted strongly with all the M. gaUlsepticum DNAs and showed no reaction with any M. synoviae D N A was selected; this was designated pMG4. Preliminary screening of the clones originating from the M. synoviae genomic library was carried out by Southern blot hybridization to total D N A of M. gaUisepticum, as described in Materials and methods. One of the clones selected was designated pSN27 and was used for testing with bacterial cultures. The sensitivity of Probe p M G 4 was estimated by reaction with purified homologous D N A (M. gaUisepticum S-6) as shown in Fig. 1A. A clear positive reaction is seen with 250 pg DNA. No reaction with 100 ng M. synoviae D N A was seen. Figure 1B shows the reaction of Probe pMG4 with M. gallisepticum organisms. The number of viable cells in the first dot-blot well was about 107 CFU; the M. gallisepticum probe detected 10 ~ CFU. Figure 2 shows the reaction of the M. synoviae probe pSN27 with the homologous type strain of M. synoviae and five other strains, including two local isolates. Positive reaction was found with 10'~-10 ~ CFU of all the M. synoviae strains tested. On testing post-log phase cultures, the reactivity with the probe did not. de-

DILUTION UNDIL.IO "t 10-2 K 1415 WVU 1853

FMT

I

I

MS1 K1938 MS6 Mg A5969 Fig. 2. R e a c t i o n o f M. synoviae D N A P r o b e p S N 2 7 in d o t - b l o t h y b r i d i z a t i o n w i t h M. synoviae o r g a n i s m s . E a c h d o t c o n t a i n e d 0.1 m l of u n d i l u t e d or 10-fold d i l u t i o n s of' l o g a r i t h m i c c u l t u r e s of v a r i o u s M. ,~ynoviae s t r a i n s . Viable c o u n t s o f M . synoviae c u l t u r e s were: ~ 10' C F U ml ~for s t r a i n M S 1; ~ 10 ~ C F U ml ~for s t r a i n s K 1 4 1 5 a n d F M T ; ~ 107 C F U m l ~for s t r a i n s W V U 1 8 5 3 , K1938, a n d M S 6 . A c u l t u r e of M. gallisepticum A5969 (10 ~ C F U m l ' ) s e r v e d as a n e g a t i v e control.

;{30

crease proportionally to the decline in number of viable cells, but was usually 10-100-fold lower than the detection level to logarithmic viable cultures. In order to test this experimentally, a log phase culture of M. gallisepticum Strain R was inactivated by heating at 56 ° C for 30 min, causing a reduction of viable cells from 5 × l0 s C F U m l - 1 to < 1 0 4 CFU ml i (the lower limit of the detection method used in this experiment). The sensitivity of detection of the heated culture by Probe pMG4 was reduced by only 10-fold, as was found also for Probe pSN27 hybridized with heat-inactivated M. synoviae Strain WVU1873. No reaction of either probe was found with cultures of other avian mycoplasmas (as listed in Materials and methods) containing at least 107 CFU per sample or with the respective purified DNAs (1000 ng). No reaction was seen with DNAs of other bacteria (E. coli, A. faecalis) found in the chicken trachea.

Experimental infection Chickens infected by intratracheal inoculation showed sporadic positive results in the RSA test when examined at 10-14 days p.i.; all birds were serologically positive when sampled at about 3 weeks p.i. Preliminary observations (Experiment 1) indicated the ability of the D N A Probe pMG4 to detect M. gallisepticum in tracheal suspensions from chickens necropsied at 9 days (3 out of 5 samples tested) and 20 days p.i. (3/5). No activity was detected in birds 3 days p.i. although all infected chickens were positive for M. gallisepticure on culture at each time of testing. In this experiment, as in those that follow, no hybridization was found with pMG4 in non-infected control birds or in any of the experimental chickens using the M. synoviae Probe pSN27. Table 1 shows the results of another experiment in which tracheal suspensions from experimentally infected birds sampled from 3 to 55 days p.i. were tested tor reaction with the probe pMG4. Two methods of infection were used, and chickens were also sampled by two methods. A positive reaction with pMG4 was found in most infected birds during the first 2 weeks p.i., but thereafter an increasing percentage of samples appeared negative on testing with the DNA probe. Levels of mycoplasmas detected by tracheal swab from live older birds in which the swab penetrates deeply into the trachea were usually equivalent to and occasionally higher than those found in the excised trachea of the same bird at necropsy. However, the data in Table 1 suggest that sampling of the excised trachea may be advantageous in young birds, at least after inoculation by aerosol. Most birds infected by intratracheal inoculation, and necropsied 4-23 days p.i. contained at least 1 0 4 CCU M. gaUisepticum in tracheal suspensions, and often much more. Birds killed later showed lower mycoplasma levels, with an increasing percentage of birds showing < 102 CCU. Nonetheless, M. gallisepticum could usually be isolated after long incubation from these chickens it' secondary infections did not interfere. In Table 2 the results of an experiment

331 TABLE 1 Hybridization of Probe p M G 4 with tracheal suspensions from live or necropsied chickens at various times after infection with M. gallisepticum Days p.i.' 3 4 7 11 14 16 19 22 28 34 44 55

Intratracheal inoculation

Aerosol infection

Live-'

Necropsied

Live

Necropsied

NT 6/8 4/6 NT NT 4/8 NT 1/4 0/5 0/6 NT

NT NT NT 7/9 NT NT 1/2 0/2 NT NT O/5

5/6 NT NT NT

2/3 NT 6/6 2/2

0/4

2/2

NT 0/3

NT NT

~p.i. = post infection. Intratracheal inoculation or aerosol inoculation was carried out at 1 m o n t h of age in M. gallisepticum-free chickens m a i n t a i n e d in a s t a n d a r d brooder battery; after infection, groups were m a i n t a i n e d in isolator cells until 22 days p.i.; birds remaining at t h a t time were transferred to cages. "Number of positive hybridization reactions per n u m b e r of samples tested; N T = n o t tested. Chickens were sampled by tracheal swab or killed and a swab taken from the excised trachea at designated intervals. T h e aerosol-inoculated group was sampled until 22 days p.i. and the intratracheal-inoculated group until 55 days p.i.

TABLE 2 Relationship between reactions with D N A Probe p M G 4 and level of M. gallisepticum in trachealswab suspensions at various times after experimental infection Days p.i. ~

7

12 19

Total birds tested

CCU in tracheal suspension ~ l0 s

10 ~

10 ~

5 / 7 :~ 3/6 5/8

1/1

1/2 1/1 -

3/4

10';

10 ~

10 :~

2/4

1/4 3/3

1/1 0/1

'p.i. = post infection. Intratracheal inoculation of chickens was carried out at 2 weeks of age. Birds were m a i n t a i n e d in isolator cells prior to a n d after infection. "CCU = color changing units of M. gallisepticum ml ~tracheal swab suspension. :'Number of positive dot blot reactions per n u m b e r of chickens tested.

332

17d PI PROBE

t

CCU/ML

lo 1o` lo

RSA

HI

13d PC 0

+

40 40 80

i

CONT

I:

i06 i0

i04

±

_+

--

10<10<,10

<102 <102

Oo

Fig. 3. Relationship between reactivity with Probe pMG4, number of M. gallisepticum organisms (CCU ml -~) and serological reactions. Samples were tracheal-swab suspensions taken at one sampling time; 17 days post-infection (d PI) from three air-sac-inoculated chickens and 13 days post-contact (d PC ) from three contact-exposed chickens. Samples from two non-infected control chickens (CONT) are also included. Aliquots of 0.1 ml of the swab suspensions were applied directly to the nitrocellulose filters for the probe test. Each suspension was also tested for number of organisms (listed below the corresponding probe reaction). Serological reactions (RSA and HI ) in serum samples obtained from the same chickens are indicated.

in w h i c h t h e a m o u n t of M. gaUisepticum in t h e t r a c h e a is r e l a t e d to r e a c t i v i t y w i t h t h e p r o b e are p r e s e n t e d . M o s t birds s h o w i n g t r a c h e a l levels of > 10 '~'C C U m l - 1Mo gallisepticum r e a c t e d w i t h P r o b e p M G 4 , a l t h o u g h at e a c h t i m e of testing o n l y a b o u t h a l f of the i n f e c t e d c h i c k e n s reacted. T r a c h e a l levels of m y c o p l a s m a in t h i s e x p e r i m e n t d e c r e a s e d m a r k e d l y 12 d a y s p.i. (relative to 7 d a y s p.i.), w h i c h c o i n c i d e d w i t h t h e clinical o b s e r v a t i o n of a c c u m u l a t i o n of large a m o u n t s of m u c u s in t h e t r a c h e a . T h e ability of t h e M. gallisepticum p r o b e to d e t e c t early stages of i n f e c t i o n was t e s t e d in a n o t h e r e x p e r i m e n t . T r a c h e a l s w a b s were t a k e n f r o m c h i c k e n s i n f e c t e d b y a i r - s a c i n o c u l a t i o n a n d f r o m c o n t a c t - i n f e c t e d chickens. Six d a y s a f t e r b e i n g p l a c e d in c o n t a c t , two c h i c k e n s t e s t e d were n e g a t i v e for R S A a n d s h o w e d no a c t i v i t y w i t h t h e probe; t r a c h e a l levels of M. gallisepticum were < 10 ~ a n d 104 C C U . At 10 d a y s a f t e r i n i t i a t i o n of c o n t a c t , four o t h e r birds were

333

3

4

5

6

7'

11 12

13

14 15

Q

Q

8

"I'AC -AC

9

10

"I-AC

16

.....

-AC

17 18 +AC

19

20

21 22 2-,3 24 O

-AC Fig. 4. Reaction of the M. gallisepticurn DNA probe pMG4 with tracheal-swab suspensions from chickensexperimentallyinfectedwith M. gaUisepticum.Samplesrepresent individualbirds at >/4 weeks p.i. + AC, acetylcysteine-treatedsamples; -AC, samples not treated with acetylcysteine. Samples 1 and 3 were taken at post-rnortem from carcasses of experimentallyinfected chickens. found to contain 10 ~ CCU M. gallisepticum ml-1 tracheal swab suspension. Although only 1/3 tested showed a positive RSA activity, three birds out of four showed positive hybridization with pMG4. Figure 3 shows further results of this experiment. The samples shown in this figure were taken at one sampling time, 17 days p.i. for air-sac-inoculated chickens and 13 days for contactexposed chickens, thereby representing different stages of infection. Chickens containing less t h a n 104 CCU in the dot (0.1 ml) on the hybridization filter did not react with pMG4 even when they showed RSA activity and diagnostically significant HI titers. In order to overcome the technical difficulties encountered in processing tracheal samples containing mucus, t r e a t m e n t of the samples with AC was attempted, as described in Materials and methods. Figure 4 shows a dot-blot filter hybridization of samples from 24 experimentally infected chickens, >~4 weeks p.i. The reagent liquified the viscous mucus, allowing a more even and rapid loading of the filters. Comparison between AC-treated and non-treated samples indicates cases in which a positive hybridization reaction was found only after AC treatment. A strong hybridization reaction was also found in tracheal-swab specimens from carcasses of M. gaUisepticum infected chickens (Fig. 4, Samples 1 and 3 ).

334 DISCUSSION

This communication describes the development of specific DNA probes for

M. gaUisepticurn and M. synoviae. An experimental infection model was used to determine the applicability of the D N A probes for testing clinical specimens. Chickens were infected with pathogenic M. gaUisepticum by various methods, and tested repeatedly for mycoplasma culture, numbers of viable mycoplasmas, serological response and reaction with the probe. As has been shown previously (Yagihashi and Tajima, 1986; Levisohn and Dykstra, 1987) tracheal levels ofM. gaUisepticurn increase, peak and decrease with time after infection, although the time course varies, probably due to differences in the severity of clinical signs. The infection model employed is useful in examining some of the questions related to the applicability of D N A probes for diagnosis and may have implications for other systems which are less accessible for experimentation, e.g. Mycoplasma pneurnoniae and Mycoplasma genitaliurn in human clinical samples ( H y m a n et al., 1987; Risi et al., 1988). The model which we used in our studies is a reasonable reproduction of the classic avian mycoplasma disease (Chronic Respiratory Disease ). Mycoplasma gallisepticum-infected chickens in our experiments usually manifested clinical signs of' disease (e.g. rales and respiratory stress). The trachea contained increased amounts of mucus and higher levels of secondary bacterial infection which, in general, increased with time after infection and exacerbated the elfect of the mycoplasmas. The changes were most marked in experiments in which birds were maintained for long periods, or in which a high level of other bacteria were present at the time of inoculation (as in Experiment 4, in which the birds were not raised in isolator cells prior to infection). Thus, in addition to a natural decrease in the number of organisms with time, there was difficulty in applying tracheal suspensions containing mucus onto the nitrocellulose filters. This factor presumably accounts for the observation that at later times af'ter infection hybridization with the probe was not effective in every infected chicken, even when a predictably detectable level of mycoplasmas was present. T r e a t m e n t of samples to disrupt mucus does indeed improve the detection rate. The experimental infection model does not specifically reproduce the field situation which is of major current economic importance in the poultry sector the detection of subclinical silent infection in chicken breeder flocks. However, our studies with contact-infected chickens indicate that the tracheal levels under these conditions are at least transiently within the range of the detection level by the probe. It is relevant to note that in this particular field situation, rapid reliable detection of M. gallisepticum on a flock (not individual) basis is critical. The applicability of the D N A probe for this purpose is borne out by our successful use of this method in field outbreaks (Levisohn et al., 1989 ).

335 The specificity of the probe is of crucial importance under field conditions. In particular, it is important to demonstrate that the probe does not crossreact with M. synoviae since both M. gallisepticum and M. synoviae may be present in the chicken trachea. Sharing of genomic sequences, as well as serological cross-reactions between these two species, has been demonstrated (Avakian et al., 1988; Yogev et al., 1989). It is not clear if specificity with respect to M. synoviae was established for the M. gallisepticum D N A probe recently reported by Santha et al. (1987). Other avian Mycoplasma spp. and bacteria found in the chicken trachea also did not react with the D N A probes. We tbund high levels of secondary infection with a variety of bacteria in the diseased trachea of the experimentally infected chickens which made mycoplasma isolation impossible in many cases, but did not interfere with the D N A probe test. Moreover, the ability of the probe to detect M. gaUisepticum in the trachea of carcasses represents another advantage of the probe, as this clinical material is not suitable for examination by any other method currently used. The effectiveness of the probe in detecting M. gaUisepticum in tracheal swabs from live or necropsied chickens was generally correlated with the levels of viable mycoplasmas found in the tracheal suspension, in spite of the inherent imprecision in quantitation of sampling in clinical specimens. The sensitivity of the probe in clinical specimens appeared to be about the same as that found in hybridization tests of M. gallisepticum organisms in culture (104 cfu), equivalent to about 100 pg D N A ( H y m a n et al., 1987). Thus, while detectable levels of' M. gaUisepticum were reached in some chickens at very early stages of infection, before a detectable humoral serological response, the hybridization method did not appear suitable for testing convalescent or carrier birds in which tracheal levels of M. gallisepticum are reduced. However, as we have shown in tests of' aged mycoplasma cultures, the probe may detect M. gallisepticum D N A when the level of viable mycoplasmas is below the nominal sensitivity of the method. Probe pMG4 also reacts strongly with atypical strains of M. gaUisepticum, which differ markedly in their genomic fingerprints and with variant strains which produce equivocal and delayed serological responses (Yogev et al., 1988a). The existence of a strain-specific F strain probe (Khan et al., 1987), together with the species-specific M. gaUisepticum probe described in the present paper open the way for epidemiological studies that were previously impossible. The ability of the D N A probe to detect M. gallisepticum before the appearance of a specific serological response (e.g. H I ) suggests that the particular advantage of the method will be in the early and rapid verification of M. gaUisepticum outbreaks in breeder flocks - a relatively narrow, but critical, window of applicability in the control of mycoplasma infections.

336 ACKNOWLEDGEMENTS

This study was supported by a grant from the United States-Israel Binational Agricultural Research and Development Fund (BARD). We thank Mordechai Wormser for technical assistance.

REFERENCES Avakian, A., Kleven, S.H. and Glisson, J.R., 1988. Evaluation of the specificity of two commercial EL1SA kits, the serum agglutination test, and the hemagglutination inhibition test for Mycoplasma gallisepticum. Avian Dis., 32: 262-272. Hayflick, L., 1965. Tissue cultures and mycoplasmas. Tex. Rep. Biol. Med. (Suppl. 1 ), 23:285 303. Hyman, H.C., Yogev, D. and Razin, S., 1987. DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium. J. Clin. Microbiol., 25: 726-728. Jordan, F.T.W., 1979. Avian mycoplasmas. In: J.G. Tully and R.F. Whitcomb (Editors), The Mycoplasmas, Vol. I. Academic Press, New York, pp. 1-48. Khan, M.I., Kirkpatrick, B.C. and Yamamoto, R., 1987. A Mycoplasma gallisepticum strain-specific DNA probe. Avian Dis., 31: 907-909. Kleven, S.H., Browning, G.F., Bulach, D.M., Ghiocas, E., Morrow, C.J. and Whithear, K.G., 1988. Examination of' Mycoplasma gallisepticum strains using restriction endonuclease DNA analysis and DNA-hybridization. Avian Pathol., 17: 559-570. Lennette, E. (Chief Editor), 1985. Manual of Clinical Microbiology. 4th edn. American Society of Microbiology, Washington, D.C., pp. 228-229. Levisohn, S. and Dykstra, M.J., 1987. A quantitative study of single and mixed infection of the chicken trachea by Mycoplasma gallisepticum. Avian Dis., 31:1 12. Levisohn, S., Hyman, H.C., Perelman, D. and Razin, S., 1989. The use of a specific DNA probe for detection of Mycoplasma gallisepticum in field outbreaks. Avian Pathol., in press. Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, NY. Olson, N.O., 1984. Mycoplasma synoviae infection. In: M.S. Hofstad, H.J. Barnes, B.W. Calnek, W.M. Reid, and H.W. Yoder (Editors), Diseases of Poultry. 8th edn. Iowa State University Press, Ames, IA, pp. 212-220. Razin, S., Hyman, H.C., Nur, I. and Yogev, D., 1987. DNA probes for detection and identification of mycoplasmas (Mollicutes). Isr. J. Med. Sci., 23: 735-741. Risi, Jr., G.F., Martin, D.H., Silberman, J.A. and Cohen, J.C., 1988. A DNA probe for detecting Mycoplasma genitalium in clinical specimens. Mol. Cell. Probes, 2: 327-335. Rodwell, A.W. and Whitcomb, R.F., 1983. Methods for direct and indirect measurement of mycoplasma growth. In: S. Razin and J.F. Tully (Editors), Methods in Mycoplasmology, Vol. 1. Academic Press, New York, pp. 185-196. Santha, M., Burg, K., Rasko, I. and Stipkovits, L., 1987. A species-specific DNA probe for the detection of Mycoplasma gallisepticum. Infect. Immun., 55: 2857-2859. United States Department of Agriculture, 1985. National poultry improvement plan and auxiliary provisions, publication VS-91-40, U.S.D.A., Beltsville, MD. Viero, 3. and Messing, J., 1982. The pUC plasmids and M13 mp7-derived system for insertion, mutagenesis and sequencing with synthetic universal primers. Gene, 19: 259-268. Yagihashi, T. and Tajima, M., 1986. Antibody responses in sera and respiratory secretions from chickens infected with Mycoplasma gaUisepticum. Avian Dis., 30: 543-550.

337 Yogev, D., Levisohn, S., Kleven, S., Halachmi, D. and Razin, S., 1988a. Ribosomal RNA gene probes detect intraspecies heterogeneity in Mycoplasma gallisepticum and Mycoplasma synoviae. Avian Dis., 32: 220-231. Yogev, D., Sela, S., Bercovier, H. and Razin, S., 1988b. Elongation factor (EF-Tu) gene probe detects polymorphism in mycoplasma strains. FEMS Microbiol. Lett., 50: 145-149. Yogev, D., Levisohn, S. and Razin, S., 1989. Genetic and antigenic relatedness between Mycoplasma gallisepticum and Mycoplasma synoviae. Vet. Microbiol., 19: 75-84. Zwadyk, Jr., P. and Cooksey, R.C., 1987. Nucleic acid probes in clinical microbiology. CRC Crit. Rev. Clin. Lab. Sci., 25: 71-101.