Growth and Pathogenesis of Mycoplasma mycoides Organ ... - NCBI

INFECrION AND IMMUNITy, Oct. 1970, p. 431-438

Vol. 2, No. 4 Printed In U.S.A.

Copyright © 1970 American Society for Microbiology

Growth and Pathogenesis of Mycoplasma mycoides var.

capri in Chicken Embryo Tracheal

Organ Cultures J. D. CHERRY' AND D. TAYLOR-ROBINSON Medical Research Cowicil, Clinical Research Centre, Harvard Hospital, Salisbury, Wiltshire, England

Received for publication 20 May 1970

The growth and pathogenicity of Mycoplasma mycoides var. capri were studied in chicken embryo tracheal rings in rolled tubes. In these organ cultures, M. mycoides var. capri attained titers of 107 to 101 color-changing units/ml of Eagle's medium and there was inhibition of ciliary activity. The rapidity of inhibition was directly related to the number of organisms inoculated. Growth of organisms was closely associated with the tracheal tissue because multiplication was not demonstrated in "conditioned medium," that is Eagle's medium from which the rings had been removed. Mycoplasma growth occurred when the cultures were incubated at 37 C, 33 C, and room temperature, but the cilia-stopping effect (CSE) was most rapid at 37 C and was not demonstrable at room temperature. Furthermore, there was no CSE when cultures were maintained in medium in which M. mycoides var. capri had been grown but was either filtered to remove the organisms or treated with tetracycline to stop their multiplication. This indicated that the CSE of M. mycoides var. capri was dependent upon a close association of multiplying organisms with the tracheal rings and was not due to toxic products persisting in the medium. Chicken tracheal epithelial cells did not adsorb to colonies of M. mycoides var. capri but HeLa cells did. Although their adsorption was unaffected by prior neuraminidase treatment, the addition of neuraminidase to the tracheal organ culture system delayed the CSE of M. mycoides var. capri. Peroxide production was demonstrated in M. mycoides var. capri-infected tracheal rings but not in uninfected cultures. The addition of glucose to the organ cultures delayed the CSE of M. mycoides var. capri, possibly because of the stimulation of peroxidase activity. Similarily, catalase protected the cilia from the usual damaging effect of M. mycoides var. capri. This protective effect was partially reversed by the addition of 3-amino-1 ,2,4,-triazole and it did not occur if the catalase had been inactivated by boiling. The addition of hydrogen peroxide to tracheal cultures also resulted in loss of ciliary activity. The present results show that the liberation of peroxide is an important factor in the pathogenesis of M. mycoides var. capri infection of chicken tracheal organ cultures. It is speculated that this also may be so in the natural host.

Mycoplasma mycoides var. capri causes a severe respiratory disease of goats. The

illness, contagious caprine pleuropneumonia, has been extensively investigated in natural and experimental infections in goats and the organism has been grown in artificial media and characterized (7). Although the pathology of the natural illness and the laboratory characteristics of the mycoplasma have been well described, there is relatively little known about pathogenesis. Recently, it has been shown that the organ culture system pro-

' Visiting worker from Department of Pediatrics, St. Louis University School of Medicine, St. Louis, Mo.

vides an opportunity to study the pathogenesis of mycoplasma infections (2, 3, 6). The biological effects of human mycoplasmas have been studied in hamster tracheal organ cultures (6), and M. mycoides var. capri has been shown to produce histopathological changes in human tracheal organ cultures (2). In an investigation of a chicken embryo tracheal organ culture system, it was found that M. mycoides var. capri multiplied and had a marked destructive effect on the ciliated epithelium (3). Chicken embryo tracheal organ cultures can be produced in quantities sufficient to assess ac-

431

432

CHERRY AND TAYLOR-ROBINSON

curately the effect of mycoplasmas or other agents on the ciliary epithelium and to evaluate carefully factors related to pathogenesis (3). This report describes the growth of M. mycoides var. capri in chicken embryo tracheal organ cultures and attempts to elucidate the mechanism of

INFEC. IMMUN.

from 0 to 6+. In the compilation of data for the figures and tables, the extent of ciliary activity and the extent of vigor have been combined and are recorded as percentage of ciliary activity. In all experiments, five culture tubes were used for each factor that was investigated. Cytadsorption. A 0.5% suspension of viable chicken pathogenesis. tracheal epithelial cells was prepared by scraping the cells from a trachea and suspending them in phosMATERIALS AND METHODS phate-buffered saline (PBS). HeLa cells with and without neuraminidase treatment (13) were also susOrgan cultures. The method of preparation has pended at a 0.5% concentration after washing three been described in detail elsewhere (3). In brief, rings times in PBS. A 2-ml amount of cell suspension was 1 in mm thickness were cut from the tracheas of flooded over 1- to 4-day-old colonies of M. mycoides 0.5 to chicken embryos that were 19 to 20 days old. Each var. capri, and the cultures were incubated at 37 C for ring was placed in a screw-capped tissue-culture tube 15 min. The cell suspensions were then removed, 2 ml (16 by 125 mm) to which was added 1 ml of medium. of PBS was and the colonies were observed for Tracheal rings were allowed to adhere by the surface cytadsorptionadded, under a "plate" microscope at 5OX tension of the medium to the sides of the tubes about magnification. 20 mm from the bottom. The tubes were placed in a Detection of peroxide in tracheal ring cultures. The roller drum, revolving at 15 revolutions per hr, and benzidine was a modification of that used by Cole incubated at 37 C unless otherwise stated. The rings, et al. (5).testBenzidine-blood-agar contained which were adherent to the glass during rolling, were mycoplasma agar medium (withoutplates serum or yeast) detached every 24 to 48 hr by shaking the tubes and with guinea pig erythrocytes, 0.1% benzidine, and were then allowed to adhere again. This was done to 1,0004% units of penicillin per ml. Wells 3 mm in diameremove cellular debris from the center of the rings and ter and depth were cut into the agar. Tracheal rings to prevent the outgrowth of cells. cultures infected with M. mycoides var. capri and Media. Unless otherwise noted, serum-free medium from rings were placed in the wells and for organ cultures was Eagle's basal medium with 200 uninfected surrounded medium from their respective cultures. by units of penicillin G per ml and 0.05 M N-2-hydroxy- Plates were incubated aerobically and were examined ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) daily for black coloration of the tracheal ring or the buffer. The pH of the medium was adjusted to 7.0 with surrounding agar which indicated peroxide produc0.2 N NaOH. Occasionally, when reagents potentially tion. contaminated with bacteria were used, ampicillin (500 Reagents. Purified neuraminidase (from Vibrio pg/ml) was added. 500 units/ml and 3-amino-1 ,2,4-triazole Medium for the growth and titration of M. cholerae; (AT) were purchased from Koch Light Laboratories mycoides var. capri has been described in detail pre- Ltd., England. Beef liver catalase was obviously (12). Thallium acetate was omitted from the tainedColnbrook, the from Boehringer Corp. Ltd., London. It medium used to produce organisms for organ culture supplied as a crystalline suspension in water (20 inoculation. Standard agar plates were used for colo- was mg/ml) saturated with thymol and had an activity of nial growth. approximately 39,000 units/mg; three lots were used. Mycoplasma. The strain of M. mycoides var. capri was received as colonies on agar from G. R. Smith RESULTS (Nuffield Institute, Regent's Park, London) and had Mycoplasma growth and effect on ciliary been isolated from a goat in Turkey (14). It was inhibited in a disc growth-inhibition test by antiserum activity. M. mycoides var. capri readily grew in to the PG3 strain of M. mycoides var. capri. One the chicken tracheal organ culture system. Viable colony was taken to inoculate liquid medium, and, titers of 107 to 108 CCU/ml usually occurred. after incubation at 37 C for 1 day, this culture was Such growth was about 10-fold less than that in stored at -70 C in divided samples and was used liquid mycoplasma medium. An inoculum of 0.1 throughout this study. Titration of mycoplasmas. Specimens were diluted ml which contained 10 CCU resulted in growth in in serial 10-fold steps in mycoplasma liquid medium some but not in all inoculated tubes. The ciliawhich contained 0.1% glucose and phenol red as a pH stopping effect (CSE) was directly related to the indicator. The highest dilution which produced a color inoculum size. The results of an experiment in change on incubation at 37 C for 7 days was the end which this relationship is demonstrated are point of the titration and was considered to contain 1 recorded in Table 1. An inoculum of 107 CCU color-changing unit (CCU). caused complete loss of ciliary activity in 4 days, Ciliary activity. Tubes were held in a conventional whereas an inoculum of 102 CCU required 12 days roller tube observation platform on a microscope for a similar result. The CSE coincided with that the tracheal rings adherent to the glass stage so surface were close to the objective. Ciliary activity was visible desquamation of ciliated epithelium and graded as to its extent, that observed in the entire ring was followed by more obvious dissolution of being recorded as 100% and, also as to its vigor of tissue. It is important to point out that comparimovement. The vigor was initially graded arbitrarily sons between inoculum size and the resulting

VOL. 2, 1970

MYCOPLASMA MYCOIDES VAR. CAPRI

TABLE 1. Relationship between concentration of Mycoplasma mycoides var. capri inoculated and the time taken to affect ciliary activity in chicken tracheal organ cultures Concn of inoculum

Time for a 50% reduction of

ciliary activity

Time for complete inhibition of ciliary activity

CCU

days

days

107 106 105 104 10 102 10'

2.6 3.3 4.1 5.5 8.2 7.9 12

A

E ' 10

1

02 1z

4 5 9 12 13 12 >13

effect on cilary activity could be made accurately only within a particular experiment; results varied from one experiment to another. Thus, in 10 separate experiments in which cultures were inoculated with 105 CCU of M. mycoides var. capri, the time required for 50% reduction in ciliary activity was 4.3 days i 1 day. In Fig. 1, the growth of organisms and their effect on cilia after inocula containing high and low concentrations of M. mycoides var. capri had been introduced into the cultures are compared. It would appear that the delay in CSE after inoculation of a few organisms was due to the time required for the organisms to grow to a titer of 106 to 107 CCU/ml. Mycoplasma growth in "conditioned" medium. It was possible that mycoplasma growth was dependent upon the presence of tracheal tissue or soluble products derived from it. To study this, growth of M. mycoides var. capri in tubes with tracheal ririgs was compared with growth in tubes with Eagle's meu'Hm without rings and with growth in tubes which contained rings for 7 days but which were removed before inoculation (conditioned medium). Immediately after inoculation, the tubes contained 103 CCU of M. mycoides var. capri per ml. Five days after inoculation, the organisms could not be recovered from tubes containing Eagle's medium only. Organisms could be recovered for 14 days from tubes which contained conditioned medium, but at no time was the viable titer greater than at the start of the experiment. On the other hand, growth of organisms in tubes containing tracheal rings was demonstrated from the 2nd to the 19th day, at which time the experiment was terminated. It was clear that growth of the mycoplasma was closely associated with the tracheal tissue. Effect of temperature on mycoplasma growth and CSE. Replicate groups of cultures each initially containing 105 CCU/ml of M. mycoides

433

2

4

6

8

10

12

crI -i

DAYS

FiG. 1. Growth and cilia-stopping effect of Mycoplasma mycoides var. capri in chicken tracheal organ cultures after inoculation ofhigh and low concentration.s of organisms.

var. capri were incubated at 37 C, 33 C, and room temperature (19 to 23 C). The results of this experiment are recorded in Fig. 2. The survival of control cultures at 33 C and room temperature was poor. The CSE was greater at 37 C than at 33 C even though the mycoplasma grew at least as weU at the lower temperature. Furthermore, at room temperature no CSE could be demonstrated when the infected cultures were compared with the control cultures, although the titer of viable organisms on the 6th day after inoculation was 106 CCU/ml. Attempts to demonstrate toxic metabolic products. In the first of two experiments, the medium in tubes containing tracheal rings was replaced at 2-day intervals by medium which had been harvested from M. mycoides var. capri-infected tube cultures. This replacement medium had been present in the infected cultures for 2 days and before being reused it was filtered through a 220-nm membrane filter (Millipore Corp., Bedford, Mass.). The medium in control tubes was similarly replaced with fluid from tubes which

434

u

(5) z

m 0

>

>4

4r J

DAYS

FIG. 2. Growth and cilia-stopping effect of Mycoplasma mycoides var. capri in chicken tracheal organi cultures after incubation at 37 C, 33 C, and room temperature (19 to 23 C).

contained uninfected ring cultures. The CSE in cultures inoculated with M. mycoides var. capri complete in 6 days, whereas there was no demonstrable effect over a 7-day period in cultures maintained in filtered medium. The experiment was terminated on the 7th day because the filtered medium of the 6th day unfortunately contained viable organisms. In a second experiment designed to determine whether actively metabolizing organisms were required to inhibit ciliary activity, the medium in tubes containing tracheal rings was replaced daily for 9 days by medium which had been harvested from M. mycoides var. capri-infected cultures and treated with 25 ,g of tetracycline per ml to inhibit mycoplasma growth. Control cultures had an initial concentration of 105 CCU/ml of M. mycoides var. capri, and the CSE in these cultures was complete by the 6th day. In the cultures that received the tetracycline-treated fluid, there was no significant CSE after 16 days. At the time of medium change, the removed tetracycline medium was titrated for viable organisms. On all occasions these were demonstrated, the highest titer was

INFEC. IMMUN.


being 106 CCU/ml. These studies suggest that the CSE of M. mycoides var. capri is not the result of diffusible toxic products in the medium or that the toxin is unstable. Cytadsorption. HeLa cells with and without neuraminidase treatment readily adsorbed to colonies of M. mycoides var. capri, whereas chicken tracheal epithelial cells did not adsorb. Effect of neuraminidase on mycoplasma growth and CSE. Purified neuraminidase (25 units/ml) was added to tracheal ring cultures, and the CSE and mycoplasma growth were compared with those in similar cultures without neuraminidase. As may be noted in Table 2, the addition of neuraminidase delayed slightly the CSE of M. mycoides var. capri. The maximum titers attained in the neuraminidase cultures were similar to those in the control cultures. Detection of peroxide. Twenty-four to 72 hr after the M. mycoides var. capri-infected tracheal rings were placed in the wells of the benzidineblood-agar plates, they became intensely black, whereas uninfected rings remained unstained for the 5-day observation period. Effect of glucose on mycoplasma growth and CSE. It seemed possible that peroxide produced by M. mycoides var. capri might be a cause of the loss of ciliary activity. Because glucose stimulates peroxide production (4) we added it to some cultures to determine whether this resulted in a more rapid loss of ciliary activity. Thus, the growth and CSE of M. mycoides var. capri were evaluated in cultures containing Eagle's medium with additional 0.1 o glucose (final concentration, 0.2%) and 1.0% glucose (final concentration, 1.1 %), Eagle's medium with normal glucose (0.1 %), and Eagle's medium without glucose. The results of a typical experiment are shown in Fig. 3. Contrary to expectation, 1.0% and 0.1% added glucose protected the cilia against damage even though the mycoplasma growth was similar to that in cultures with standard Eagle's medium. In this and two other experiments, the addition TABLE 2. Cilia-stopping effect of Mycoplasma mycoides var. capri in chicken tracheal organ cultures maintained in Eagle's medium compared with cultures maintained in Eagle's medium with nieuraminidase Time (days) for 50% reduction of ciliary activity Expt

1 2

Eagle's medium

Eagle's medium with neuraminidase (25 units/ml)

4.7 5.3

5.8 7.5

VOL. 2, 1970

MYCOPLASMA MYCOIDES VAR. CAPRI

435

the results of five experiments with catalase are recorded. Three different lots of catalase were used all to some extent protected the cilia from JE /,; usual cytopathology caused by M. mycoides ,04 _the / lo! var. capri infection. cn Studies with AT. In initial attempts to inhibit Z ,0- < /the protective effect of catalase, it was found that a concentration of 0.35% AT was mildly toxic to 0 lo the organ cultures, although M. mycoides var. capri readily grew in its presence and the CSE m0 was similar to that in control cultures. To determine a nontoxic dose of AT that could be used 8 12 2 4 6 10 in the organ culture system, concentrations of 0.035, 0.07, and 0.35% were comparatively ,oo- * i ^A ^ ^,studied. Unexpectedly, the 0.035% concentration protected the cilia from the usual CSE of M. mycoides var. capri (Table 4).

\\and

A

A

60-

°40

i.i%glucose

20\

0.2%1glucose

O Catalase

No glucose

A Boiled catalase * No catalase

al'glucose 2

4

6

8

10

12

DAYS

FIG. 3. Growth and cilia-stopping effect of Mycoplasma mycoides var. capri in chicken tracheal organ cultures containing different concentrations of glucose in the medium.

X

2

l

4

6

8

10

of 0.1 % glucose caused a delay of 4.1 days in the occurrence of 50% reduction of ciliary activity. If glucose had a protective effect, it seemed that its absence might enhance the CSE. This was not so because omission of glucose from the Eagle's medium resulted in a loss of ciliary activity similar to that in cultures maintained in standard Eagle's medium (0.1 % glucose). However, the mycoplasma grew 1,000-fold less readily in the absence of glucose, which may explain why the CSE was not potentiated. Effect of catalase on mycoplasma growth and CSE. In an attempt to establish whether peroxide was responsible for the CSE of M. mycoides var. capri, we added catalase to some cultures. Thus, M. mycoides var. capri was inoculated into tracheal cultures maintained in standard Eagle's medium and similar cultures to which catalase or boiled catalase had been added. The results of one DAYS experiment are shown in Fig. 4. Mycoplasma FIG. 4. Growth and cilia-stopping effect of Mycogrowth in the catalase-containing tubes was equal plasma mycoides var. capri in chicken tracheal organ to that in control cultures, but the CSE was de- cultures after treatment with catalase and boiled catalayed in the catalase-treated cultures. In Table 3, lase.

436


TABLE 3. Effect of catalase on the cilia-stopping effect of Mycoplasma mycoides var. capri in chicken tracheal organ cultures Catalase

Expt

Initial concn of my

plasma) (CCU/mlm) tube)

LotLt Concn (mg no

1 2 3 4 5

Time (days) for ciliary activity

a 50% reduction of

1 2 2 3 3

0.5 0.5 1.0 0.5 1.0

105 105 105 105 106

Eagle's Eagle's

medium

4 4.6 4.9 4.1 2.9

medium with catalase

>7 6.4 5.5 7.8 7.3

TABLE 4. Effect of 3-amino-1,2,4-triazole (AT) > on the cilia-stopping effect of Mycoplasma mycoides var. capri in chicken tracheal organ cultures Concn of AT in medium

Timeoffor a 50%activity reduction ciliary

%

days

0.35 0.07 0.035

4.4 5.8 9.2 5.1

In experiment 4 (Table 3), an additional five cultures contained catalase and 0.14% AT; cultures with only Eagle's medium had a 50% reduction of ciliary activity in 4.1 days, whereas in the catalase-AT cultures and the catalase cultures 50% reduction occurred at 6.5 and 7.8 days, respectively. This indicated that the AT partially reversed the effect of catalase. Effect of hydrogen peroxide on ciliary activity. Tracheal cultures were inoculated with a solution of hydrogen peroxide (20 volumes); the dilution of this solution in the culture medium was 0.06%. From other studies (J. D. Cherry and D. TaylorRobinson, unpublished data), we calculated that this concentration of peroxide would be about the same as that present in M. mycoides var. capri-infected cultures after there had been mycoplasma multiplication to produce at least 106 CCU/ml. There was complete cessation of ciliary activity between 2 and 3 days after inoculation of hvdrogen peroxide. This period was less than that observed for the loss of activity in M. mycoides var. capri-infected cultures, the longer period for the latter probably resulting from a more gradual accumulation of peroxide.

DISCUSSION Mycoplasmas are a cause of respiratory infections in many animal species including man. In

INFEC. IMMUN.

of the lower animals, the pathology has been extensively studied in both natural and experimental infections. Infection of man with M. pneumoniae has been evaluated from the clinical and epidemiological aspects, but little opportunity for pathological investigation has occurred. In recent years, the various mycoplasmas which cause respiratory infection syndromes have been studied in laboratory in vitro systems. These studies have suggested that several factors may be responsible for the pathogenicity of the mycoplasmas under natural in vivo conditions. In general, pulmonary infections are the result of downward invasion of microbial agents from the upper air passages to the lungs. Frequently, tracheo-bronchitis is an integral part of the pathology. In the study of respiratory disease caused by viruses, organ cultures of tracheal ciliated epithelium have been of considerable value in assessing the etiology and pathogenesis of infection (8). The organ culture system also would appear to offer many opportunities for the study of the pathogenesis of mycoplasma respiratory infections (2, 3, 6). Butler (2) showed that M. mycoides var. capri stopped the activity of cilia in human embryo tracheal organ cultures and that the effect was dose-dependent. Collier et al. (6) showed that M. pneumoniae, which was grown in hamster tracheal ring cultures that were bathed in mycoplasma medium, stopped ciliary activity and again the effect was dose-dependent. Their data suggested that the effect was more likely the result of the direct action of the mycoplasma on the cilia rather than a delayed effect due to toxic products accumulating in the medium. The results of in vitro and animal studies have suggested several factors which could have a role in the pathogenesis of mycoplasma infections. M. neurolyticum has been shown to produce a thermolabile protein exotoxin which produces rolling disease in rats and mice (19). In diffusion chamber studies, Lloyd (11) showed that M. mycoides var. mycoides produced a diffusible toxin which caused necrosis of rabbit tissues. The hemolysin produced by several mycoplasmas has been shown to be a peroxide, and it has been suggested that peroxide may be an important factor in virulence (15, 18). The in vivo studies of Brennan and Feinstein (1) support this hypothesis. They have shown that acatalasemic mice were more likely to develop pneumonia 3 days after infection with M. pulmonis than normal mice that were similarly infected. The ability of certain mycoplasmas to attach to respiratory epithelial cells may also be important in pathogenesis. In the case of M. pneumoniae, Sobeslavsky et al. (16) have suggested that cytadsorption and the liberation of many

VOL. 2, 1970

MYCOPLASMA MYCOIDES VAR. CAPR4437

peroxide directly upon the cell membrane might be important in the pathogenesis of disease. This idea is attractive but the exact mechanism may be more complex. Lipman and Clyde (9) noted that a stzain of M. pneumoniae, which was partially attenuated for the hamster lung, liberated the same amount of peroxide as the disease-producing strain from which it was derived, and, in addition, the attenuated strain still cytadsorbed. However, it may be significant that a completely attenuated strain, although producing as much peroxide as its parent virulent strain, did not cytadsorb. The present study was undertaken in an attempt to define factors concerned with the loss of ciliary activity caused by M. mycoides var. capri. It is apparent that M. mycoides var. capri grows readily in chicken tracheal organ cultures, and since the medium alone will not support growth of the organisms the infection must be associated with the tracheal ring and its ciliated epithelium. The CSE is dose-related and at least 106 organisms/ml for about 4 days was required for complete cessation of activity. The observations in this study indicate that several factors influence the effect of M. mycoides var. capri on ciliary activity and that caution should be observed in evaluating any one of them. For example, the CSE was markedly dependent upon temperature and medium composition. Differences in results from one week to another are likely to be due to minor temperature variations during incubation. The critical relationship between the concentration of glucose in the medium and the CSE is of note. Probably other adjustments of medium composition could either potentiate or decrease the CSE of M. mycoides var. capri without necessarily affecting its over all growth potential. A toxic factor could not be demonstrated in medium in which M. mycoides var. capri organisms had grown. When the medium was replaced with medium that contained high concentrations of organisms, the further multiplication of which was inhibited by tetracycline, there was no significant reduction of cihary activity after prolonged incubation. It would appear, therefore, that cytopathology is related to organisms multiplying on or close to the surface of the organ culture and is not due to the accumulation of toxic products in the medium. HeLa cells adsorbed to colonies of M. mycoides var. capri, but the adsorption was apparently not dependent upon neuraminic acid receptors because treatment of the cells with neuraminidase did not reduce their adsorption capacity. Chicken tracheal cells did not adsorb to the colonies so that it is hard to explain the slight sparing effect that neuraminidase had in the organ culture

system. It is possible that cytadsorption, utilizing neuraminic acid receptors, occurs in the tracheal ring cultures but that the in vitro adsorption test with colonies is not sensitive enough to demonstrate this. Perhaps of greatest interest in our studies was the fact that the addition of catalase to the medium delayed the cytopathic effect of M. mycoides var. capri. It has been demonstrated that M. mycoides var. capri is a vigorous producer of peroxide (5). The staining of tracheal rings placed in the wells of the benzidine-blood-agar confirms that peroxide is also produced in this organ culture system. Our results also show that peroxide is an important cause of pathogenicity in this model system. Unfortunately, concentrations of AT which may have reversed completely the effect of catalase were toxic. However, partial reversal of the catalase effect by 0.14% AT and complete reversal after boiling the catalase indicated that the effect is specific and not the result of nonspecific medium factors. The idea that peroxide is important was supported further by the knowledge that its introduction into chicken tracheal organ cultures, in the absence of M. mycoides var. capri organisms, resulted in

impaired ciliary activity. Assuming, therefore, that peroxide is the cause of virulence in the chicken tracheal system, it would seem that the medium in our toxin studies would have contained enough peroxide to affect ciliary activity. However, Somerson et al. (17) have shown that peroxide-mediated hemolysis only occurs when there is a continued production of peroxide, and Cohen and Somerson (4) have demonstrated that the amount of peroxide existing in a culture after a period of incubation is considerably less than the amount actually produced during the incubation period. The sparing effect of glucose was unexpected. Glucose was originally added to the medium in an attempt to potentiate the CSE because it was known to increase the production of peroxide by both M. pneumoniae and M. gallisepticum (4). The protective effect of glucose may be explained by the fact that, in addition to increased peroxide production, the mycoplasma may have its own peroxidase which is also stimulated. Peroxidaselike activity resulting from glucose stimulation has been demonstrated by Cohen and Somerson (4) in M. pneumoniae suspension cultures. Also unexplained was the marked protective effect that 0.035% AT had against the usual ciliary inhibition resulting from M. mycoides var. capri infection. It would seem that if either the epithelial cells or the mycoplasmas were liberating catalase then the addition of AT would potentiate the CSE; as noted the reverse occurred.

438


Perhaps the liberation of peroxide unchecked by natural catalase was more harmful to mycoplasmas than the organ cultures and therefore reduced the concentration of organisms below a crucial level for cytopathogenicity. This supposition was not borne out by the titers of viable organisms in the medium. However, the concentration of organisms in the medium may not represent the effective concentration at the ceHlular site. The present study shows that peroxide is important in the pathogenesis of M. mycoides var. capri infection of chicken tracheal organ cultures, and it seems reasonable to postulate that this is the means whereby respiratory disease is produced in the natural host. On the other hand, a similar mechanism is not necessarily involved in other mycoplasma-host model systems. For example, although M. gallisepticum produces peroxide, the CSE of this agent in chicken tracheal organ cultures is not adversely affected by the addition of catalase (J. D. Cherry and D. TaylorRobinson, unpublished data). ACKNOWLEDGMENTS We thank Pamela F. Clayton, Kathleen A. Keast, and Elizabeth M. Minton for excellent technical assistance.

LITERATURE CITED 1. Bremnan, P. C., and R. N. Feinstein. 1969. Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice. J. Bacteriol. 98:10361040. 2. Butler, M. 1969. Isolationand growth of mycoplasma inhuman embryo trachea cultures. Nature (London) 224:605-606. 3. Cherry, J. D., and D. Taylor-Robinson. 1970. Large quantity production of chicken embryo tracheal organ cultures and use in virus and mycoplasma studies. Appl. Microbiol. 19: 658-662. 4. Cohen, G., and N. L. Somerson. 1969. Glucose-dependent secretion and destruction of hydrogen peroxide by Mycoplasma pneumontiae. J. Bacteriol. 98:547-551.

INFEC. IMMUN.

5. Cole, B. C., J. R. Ward, and C. H. Martin. 1968. Hemolysin and peroxide activity of Mycoplasma species. J. Bacteriol. 95:2022-2030. 6. Collier, A. M., W. A. Clyde, Jr., and F. W. Denny. 1969. Biologic effects of Mycoplasma pneumoniae and other mycoplasmas from man on hamster tracheal organ culture. Proc. Soc. Exp. Biol. Med. 132:1153-1158. 7. Cottew, G. S., and R. H. Leach. 1969. Mycoplasmas of cattle, sheep aiid goats. In L. Hayflick (ed.), The Mycoplasmatales and the L-phase of bacteria, p. 527-570. Appleton-CenturyCrofts, New York. 8. Hoorn, B., and D. A. J. Tyrrell. 1969. Organ cultures in virology. Progr. Med. Virol. 11:408-450. 9. Lipman, R. P., and W. A. Clyde, Jr. 1969. The interrelationship of virulence, cytadsorption, and peroxide formation in Mycoplasma pnzeumontiae. Proc. Soc. Exp. Biol. Med. 131: 1163-1167. 10. Lipman, R. P., W. A. Clyde, Jr., and F. W. Denny. 1969. Characteristics of virulent, attenuated, and avirulent Mycoplasma pneumoniae strainis. J. Bacteriol. 100:1037-1043. 11. Lloyd, L. C. 1966. Tissue necrosis produced by Mycoplasma mycoides in intraperitoneal diffusion chambers. J. Pathol. Bacteriol. 92:225-229. 12. Manchee, R. J., and D. Taylor-Robinson. 1968. Haemadsorption and haemagglutination by mycoplasmas. J. Gen. Micro-

biol. 50:465-478. 13. Manchee, R. J., and D. Taylor-Robinson. 1969. Utilization of neuraminic acid receptors by inycoplasmas. J. Bacteriol. 98:914-919. 14. Smith, G. R. 1967. Experimental infection of mice with Mycoplasma mycoides var. capri. J. Comp. Pathol. 77:21-27. 15. Sobeslavsky, O., and R. M. Chanock. 1968. Peroxide formation by mycoplasmas which infect man. Proc. Soc. Exp. Biol. Med. 129:531-535. 16. Sobeslavsky, O., B. Prescott, and R. M. Chanock. 1968. Adsorption of Mycoplasma pneumoniiae to neuraminic acid receptors of various cells and possible role in virulence. J. Bacteriol. 96:695-705. 17. Somerson, N. L., R. H. Purcell, D. Taylor-Robinson, and R. M. Chanock. 1965. Hemolysin of Mycoplasma pneumoniae. J. Bacteriol. 89:813-818. 18. Somerson, N. L., B. E. Walls, and R. M. Chanock. 1965. Hemolysin of Mycoplasma pneumoniae: tentative identification as a peroxide. Science 150:226-228. 19. Thomas, L., F. Aleu, M. W. Bitensky, M. Davidson, and B. Gesner. 1966. Studies of PPLO infection. I1. The neurotoxin of Mycoplasma neurolyticum. J. Exp. Med. 124:1067-1082.