Effect of Animal Passage on Arthritogenic and Biological

INFECTION AND ImmuNrry, June 1970, p. 538-545Copyright @ 1970 American Society for Microbiology

Vol. 1, No. 6Printed in U.S.A.

Effect of Animal Passage on Arthritogenic andBiological Properties of Mycoplasma arthritidis'

L. GOLIGHTLY-ROWLAND, B. C. COLE, J. R. WARD, AND B. B. WILEY

Division of Arthritis, Department of Internal Medicine, and Department of Microbiology, University of UtahCollege of Medicine, Salt Lake City, Utah 84112

Received for publication 12 January 1970

Determinations of the ED50 of cultures of Mycoplasma arthritidis disclosed theexistence of great diversity in the arthritogenic properties of the various strains. Inmost cases, the ED50 values were lowered after passaging in rats, and a more severearthritis with a shorter period of onset was observed. Heavy suspensions of arthrito-genic M. arthritidis did not appear to induce any toxic symptoms. Prior injections ofendotoxin did not enhance the toxicity of M. arthritidis suspensions. The more ar-thritogenic strains grew more slowly in the basal medium and remained viable forlonger periods of time than those with lower arthritogenic properties. All strainswere identical on the basis of complement-fixation tests. Minor differences observedbetween strains during gel-diffusion studies could not be correlated with arthrito-genic properties. Arthritogenic strains appeared to be less susceptible to the inhibit-ing action of rabbit antisera than were the nonarthritogenic strains.

Rat arthritis induced by Mycoplasma arth-ritidis has been studied extensively in relation toclinical and histological characteristics (10, 24,27, 34). The immunological responses of thehost have been described (5, 15, 17), and it hasrecently been suggested that a heterogeneticantigen common to M. arthritidis and rat tissuemay play a role in the pathogenesis of this disease(5). In spite of these studies, many questionsremain unanswered, such as the mechanism ofimmunity to reinfection and the pathogenesisof the spontaneous and experimental disease.Continued passage of M. arthritidis on arti-

ficial media has been observed to decrease thevirulence of the mycoplasma for the rat (13, 14,32). (In this paper the term "virulence" is usedto indicate the ability of M. arthritidis to inducearthritis.) Although quantitative data are notavailable, passage of the agent in rats appearsto increase the virulence of the organism (8, 13,24, 27, 35). A recent study by Cole, Miller, andWard (7) demonstrated that significant differ-ences existed between the ability of fresh isolatesand laboratory-maintained strains to inducearthritis. It was also shown that if sufficientnumbers of colony-forming units (CFU) wereinjected into rats even the less virulent strainswould induce arthritis. Other workers resortedto the use of agar as an enhancer of virulence in

Most of this study was submitted in thesis form to the Uni-versity of Utah by Laura Golightly-Rowland in partial fulfillmentof the requirements for the M.S. degree.

order to produce arthritis (9). Because of thelarge numbers of organisms required to inducearthritis experimentally, it is likely that ancillaryfactors such as stress, trauma, or concurrent in-fections are required for the disease to developnaturally.

Except for a few reports, differences in thebiological and serological properties of virulentand avirulent strains of M. arthritidis have notbeen adequately described. No conclusive evi-dence for the existence of a toxin has been pre-sented in spite of a brief report by Thomas (31).However, differences in colonial morphologybetween a virulent and an avirulent strain of M.arthritidis were reported by Howell and Jones(12). Warren (35) also reported a positive corre-lation of virulence with the rate of reduction ofmethylene blue.

It is essential for the study of host-parasiterelationships that the disease can be consistentlyreproduced experimentally. Thus, the presentstudy was designed to quantitate the arthritogenicproperties of a variety of M. arthritidis strains.This property was measured by determiningthat dose of organisms which caused arthritis in50% of the animals (ED50). The feasibility of in-creasing the virulence of laboratory-maintainedstrains by passaging in rats was also explored.Finally, attempts to differentiate virulent andavirulent strains of M. arthritidis on the basis ofbiological and serological properties will be re-ported.

538

ARTHRITOGENIC PROPERTIES OF M. ARTHRITIDIS

MATERLILS AND METHODSStrains used. The sources of M. arthritidis strains

14152, 158, 14124, El, and PN have been describedelsewhere (5, 7). In addition, strains R6 and DL wereisolated by the authors from a rat lung and an in-flamed rat joint, respectively. M. arthritidis strainsPG6 and H606 were kindly supplied by M. Davidsonand L. Thomas (New York University), who origi-nally obtained them from D. G. FF. Edward and R.Wittler, respectively. Strains El, R6, and DL havebeen passaged 10 to 20 times on agar and are repre-sentative of fresh isolates. Strain PN has been pas-saged on agar approximately 30 times, and the re-maining strains have been transferred in artificialmedia for several years.

Medium. Difco PPLO agar or broth supplementedwith 10% (v/v) sterile horse serum, 5% (v/v) sterileyeast extract (3, 11), and 1,000 units of penicillinG per ml was used as the base medium. L-Argininehydrochloride (0.2%, w/v) was occasionally addedto enhance growth (29).

Passaging techniques. Stock strains of mycoplasmawere grown for 2 to 3 days in 250-ml amounts of thebasal broth. Cultures were concentrated by centrifu-gation at 27,000 X g for 15 min and were resuspendedin 2.5 ml of broth. Albino male rats (Holtzman Co.,Madison, Wis.) weighing 120 to 200 g were injectedeither intravenously (iv) via the caudal vein or sub-cutaneously (sc) into the lumbo-spinal region with0.7-ml amounts of the suspensions. After 3 to 5days, the rats were sacrificed and the pus was trans-ferred from the arthritic joints or abcesses to freshserum broth. After 2 to 3 days of incubation, thecultures were concentrated and reinjected into ad-ditional rats. When no purulent material was avail-able, inflamed joint or muscle tissue was used asinoculum for the next culture. M. arthritidis strains14124, 14152, and 158 were passaged at least 10times by sc injection. M. arthritidis strain 14152was also passaged 10 times by iv injection. Strains14124 and 158 could not be regularly passed iv bythis technique because of occasional failure to inducearthritis. Unpassaged cultures were suffixed Po;passaged cultures were designated by the suffixesPIO sc, P13 sc, or P10 iv.

After passaging, all strains were grown in largequantities and concentrated; samples were stored at-20 C.

Production and scoring of arthritis and ED5o de-terminations. Samples of each frozen culture werethawed approximately 5 days prior to injection andwere counted for CFU by a modification of themethod of Miles and Misra (20). Additional tubeswere thawed immediately prior to injection, andappropriate dilutions were prepared according to theresults of the previous plate count. As a final checkon the inoculum, the cultures were recounted at thetime of injection: in most instances, the two countsdid not vary by more than 50% provided that thesuspensions were stored at -20 C for no longer than3 weeks. Male albino rats (Holtzman) weighing 120to 150 g were injected iv in groups of 10 with 5- or10-fold dilutions of the mycoplasma suspensions.The animals were examined and scored for arthritis

and paralysis at 3, 4, 5, 8, 10, and 14 days after injec-tion. The severity of arthritis was recorded by scoringeach of the limb joints on a 0 to 4 scale with 1 repre-senting minimum arthritis and 4 representing maxi-mum arthritis. Total scores for each animal of 1 orgreater which persisted for at least 2 consecutivedays were considered positive cases of arthritis. Theaverage day of onset, maximum, and decline ofarthritis was calculated, as were the mean number oflimbs involved, the mean scores for each group,and the mean scores of arthritic animals in eachgroup.

Estimation of the dose of organisms causingvisible arthritis in 50% of the injected animals (ED50)was calculated on two-cycle semilogarithmic graphpaper as described by Miller and Taintor (21). Thestandard error was also determined.

Serological studies. Rabbit hyperimmune serawere prepared by use of organisms grown in rabbitinfusion broth (30) and were injected accordingto the immunization schedule of Morton and Roberts(22).

Complement-fixation titers were determined by theone-fifth quantitative test of Kolmer and Boemer(16). Antigens were prepared as previously described(6), and a stock dilution in modified barbital buffer(2) was made up to give an optical density readingof 0.16 at a wavelength of 535 nm on a model DBspectrophotometer (Beckman Instruments Inc.,Fullerton, Calif.).Growth inhibition tests were carried out by a

modification of the method of Clyde (4). Antisera in0.1-ml amounts were placed in wells cut into theinoculated plates. After 3 days of incubation, thesizes of the zones of inhibition were recorded andgraded on a scale of 0 to 4.

Metabolic inhibition tests were performed asdescribed by Cole et al. (5) by means of a modificationof the technique of Purcell et al. (28).

Generation time determinations. Test tubes con-taining 5-ml amounts of the serum basal PPLO brothsupplemented with 0.05% (w/v) L-arginine hydro-chloride were inoculated with 103 CFU of the strainsto be tested and were incubated at 37 C. Viablecounts were made (20) at zero time, at 6, 12, 24, 36,48 hr, and at 3, 4, 5, 6, and 8 days. Three tubes ofbroth culture were combined for each count. Thelogio of the number of viable organisms was plottedon graph paper against time. The steepest slope ofthe curve, i.e., that corresponding to the log phase ofgrowth, was used for generation time determination(23).The experiment was repeated with rat muscle

infusion broth substituted for PPLO broth. Theformer was prepared by homogenizing 100-g amountsof cleaned and skinned rat tissue in 300 ml of distilledwater. After being heated to boiling and standing for1 hr, the infusion was filtered through gauze andWhatman no. 1 filter paper. Proteose peptone (1%,w/v), sodium chloride (0.5%, w/v), and L-argininehydrochloride (0.05%, w/v) were added, and themixture was adjusted to pH 7.8. After autoclaving,10% (v/v) sterile rat serum and 5% (v/v) fresh yeastextract were added.

VOL. 1, 1970 539

GOLIGHTLY-ROWLAND ET AL.

RESULTS

Development of arthritis and ED58 determina-tions. The range of doses given and the ED50 esti-mations of the various M. arthritidis strains are re-

corded in Table 1. With the exception of strainsPG6 and H606, all strains of M. arthritidis were

capable of inducing arthritis. The ED50 valuesranged between 4 X 107 and 5.6 X 109 CFU. Therecent isolates El, DL, and PN were noticeablymore virulent than the laboratory-maintainedstrains. The rat-passaged cultures, PIO iv and P13sc of strain 14152 were at least 26 and 94 timesmore virulent, respectively, than the original un-passaged culture. The latter values were calculatedon the assumption that maximum standard error

had occurred. The EDao of the sc passage of strain158 was only slightly lower than that of the un-

passaged culture. However, strain 158 PlO sc

produced a more severe arthritis with a shorteronset than the unpassaged culture (Table 2).Two values are given for the unpassaged 14124

strain. The first virulence test was performed be-fore most of this work was undertaken (7). Theresults were characteristic of a strain of very lowvirulence (designated 14124 in Table 1). Thesecond test indicated that the unpassaged culturewas more virulent than the rat-passaged culture.Subsequent investigations indicated the presenceof a gram-negative bacillus in the suspension ofthis culture (designated 14124 Po Con in Tables 1

TABLE 1. Arthritis ED8o determinations for M. arthritidis strains

Strains ~No. of animal ..No. ofStrains Nopassages Range of CFU injected per rat animals ED5so SE

14152a P13 sc 3.5 X 106-3.5 X 109 40 4.0 X 107 - 8.0 X 106PN 4 X 107-4 X 108 20 4.0 X 107 4 8.2 X 106DL 1 X 107-4 X 101 60 5.0 X 107 4 7.5 X 105El 4 X 107-2 X I08 30 6.2 X 107 i 7.1 X 10614124 Po Con 4.6 X 108-1.5 X 10'0 40 1.0 X 108 i 2.6 X 10l158 PIO sc 6 X 100-6 X 109 40 1.3 X 108 ± 2.1 X 10714152 P1O iv 1 X 107-8 X 108 30 1.6 X 108 ± 1.1 X 107R6 2.5 X 107-9 X 108 30 1.8 X 108 + 2.0 X 107158 Po 1.2 X 107-2 X 10'0 70 2.0 X 108 4 1.4 X 10714124 PlO sc 1.5 X 107-1.5 X 1010 40 3.1 X 108 4 6.1 X 10714124b 2 X 109-1.5 X 101' 20 >2 X 109; < 1.5 X 101014152 Po 1.4 X 109-5 X 109 40 5.6 X 109 4 9.0 X 108H6060 4.2 X 108-2 X 10" 20 >2.0 X 10"PG6c 5 X 108-3 X I10" 40 >3.0 X 10"

a Strains listed in order of decreasing virulence (ED50).Results taken from Cole et al. (7).Arthritis could not be demonstrated with these strains.

TABLE 2. Severity and persistence of arthritis in2duced in rats by M. arthritidis strains

Mean no. of Av-g d y of

Straina Dose given per rat |~Mean score Mean score limbs in-Strain' Dose given per of alas of arthri- volved perofllras tic rats arthritic Onset of Maximum Decline ofrat arthritis arthritis arthritis

14152 P13 sc 3.5 X 1081 5.5 6.3 1.9 3.0 8.7 14.0PN 4 X 108 9 11 1.8 3.7 8.0 14.0DL 4 X 108 8.3 12 2.4 3.6 9.0 13.9El 2 X 108 6.3 7 1.5 4.8 10.0 14.014124 Po Con 4.6 X 108 3.5 5.0 1.3 3.4 7.1 9.7158 PlOsc 6 X 108 6.3 7.0 1.8 3.7 8.0 13.814152 PlO iv 8 X 108 8.5 8.5 2.0 4.5 8.1 14.0R6 9 X 108 11.0 11.0 2.5 4.1 8.0 13.0158 Po 7.7 X 108 2.7 3.4 1.2 5.5 8.5 12.414124 PlOsc 1.5 X 109 5.9 6.6 1.6 5.8 9.5 13.014152 Po 1 X 109 1.7 4.2 1.0 5.5 8.5 12.5

a Each strain was injected into a group of 10 rats. Strains are listed in order of decreasing virulenceas measured by ED,o determination.

540 I NFEC. IMMUN.

ARTHRITOGENIC. PROPERTIES OF M. ARTHRITIDIS

and 2). Although no further work was conductedwith this bacillus, it appeared to lower the ED50of the mycoplasma.The severity and course of arthritis obtained

with various mycoplasma strains when inocula be-tween 2 X 108 and 1.5 X 109 CFU were used aresummarized in Table 2. The arthritis scores werelarger with the more virulent strains, and morelimbs were involved. The day of onset of arthritiswas delayed in the case of the less virulent strains.No significant differences between virulent andless virulent strains could be detected either in thetime required for maximum arthritis to develop or

for the arthritis to decline. Maximum arthritis wasusually obtained after 8 to 10 days, whereas thedecline of arthritis occurred 12 to 14 days afterinjection.

Toxicity of mycoplasma suspensions. Duringthe ED50 determinations, no evidence of death dueto the toxic effects of the mycoplasma suspen-sions was encountered. To determine the effectsof massive doses of virulent M. arthritidis in rats,strain PN was injected iv into groups of six rats(120 to 150 g) in the following amounts: 2 X 109,1010, 5 X 101°, and 2.5 X 1011 CFU per rat. Theseverity of the resulting arthritis is recorded inTable 3.With inocula of 5 X 1010 to 2.5 X 1011 CFU

per rat, the earliest symptoms, consisting ofminimal arthritis, appeared at 2 to 3 days. In noinstance did death occur before 8 days, the timeat which arthritis was maximal. As was observedduring the ED50 tests, the severity of arthritis andnumber of limbs involved gradually increased,and the day of onset decreased with increasingdoses of organisms.

In an attempt to increase the chances of detect-ing toxic products, the following experiments were

set up. Groups of 10 rats each were injected with3 X 1010 CFU of (i) M. arthritidis strain 158PIO, (ii) M. arthritidis strain H606, (iii) M. sali-varium, and (iv) M. hominis. Another set of ani-mals was injected with the above mycoplasmasand 100 ,tg of endotoxin (Escherichia coli; Difco)

TABLE 3. Effect of increasing doses ofM. arthritidisstrain PN on the development of arthritis

Mean ofMean Mea no. of Mea

Dose gvniv Incidence soecreanf limbseanof of al or dayvolof

(CFU) arthritis of all art onset ofrats rats per arthritis

arthriticrat

2 X 109 5/6 12 15 2.0 3.81 X 1010 5/6 15 18 2.6 2.65 X 1010 6/6 27 32 3.7 2.0

2.5 X 1011 6/6 37 37 3.8 2.2

per rat. Animals injected with sterile broth con-taining endotoxin served as controls. The animalswhich received mycoplasma suspensions aloneshowed no evidence of toxic effects for the 36 hrduring which they were observed. The highestincidence of death occurred in animals receivingendotoxin together with the avirulent M. arthri-tidis (H606) and with M. hominis. In these groups8 of 10 animals were dead in 4 hr and 10 of 10 in22 hr. In the M. salivarium, virulent M. arthritidis(158 P10), and control groups, 2 of 10 animalswere dead after 4 hr and 3 of 10 animals weredead after 22 hr. Thus, the virulent strain of M.arthritidis appeared to be no more toxic than theother mycoplasmas which were tested.Growth characteristics. Studies were carried

out to determine whether growth characteristicscould be correlated with virulence. Minor differ-ences in the colonial morphology of the variousstrains were observed. The size of the peripheralsurface growth of the colonies varied, and somestrains were noticeably more granular than others.However, these differences could not be corre-lated with virulence.The generation times and rates of decline of

viable organisms grown in the PPLO broth arerecorded in Table 4. In general, the more virulentstrains had longer generation times than the lessvirulent or avirulent strains. Similarly the rat-passaged strains grew more slowly than unpas-saged cultures. The growth characteristics ofstrain 14124 Po were typical of an avirulentstrain, thus supporting the contention that abacterial contaminant present in the culture usedfor ED50 determinations contributed to increased

TABLE 4. Growth ofM. arthritidis strains in PPLObroth

aGeneration Rate of decline ofStraina time viable organisms(hr)

14152 P13 sc 2.6 ModeratebPN 1.5 RapidDL 2.7 SlowEl 3.4 Slow158 PlO sc 2.1 Moderate14152 PIO iv 2.4 Moderate158 Po 1.9 Rapid14124 PIO sc 1.8 Rapid14124 Po 1.3 Rapid14152 Po 1.9 RapidH606 1.5 RapidPG6 1.7 Rapid

a Strains listed in order of decreasing virulence(ED50).bThese designations are based upon the angle

of the growth curve slopes during the declinephase.

VOL. 1, 1970 541


virulence (see above). Strain PN, although ofhigh virulence, exhibited rapid growth in PPLObroth. It should be pointed out that this strainmaintained virulence in spite of some 30 trans-fers in artificial media.

Similar experiments were set up with somestrains in which rat muscle infusion broth wasused as the medium. The differences between thegeneration times were less pronounced than be-fore, although the less virulent strains still grewsomewhat faster than the others. It was also ob-served that the generation times of the less virulentand avirulent strains were longer in rat infusionbroth than in PPLO broth.The rates of decline of viability (designated

slow, moderate, and rapid in Table 4) after maxi-mum growth had been reached were much slowerin the case of the more virulent strains than withthe less virulent or avirulent strains.

Serological investigations. All strains of M.arthritidis were inhibited to some extent by thevarious homologous and heterologous seraagainst which they were tested (Table 5). Byscoring the zones of inhibition on a scale of 0 to 4,it was possible to estimate the average amount ofinhibition of each strain by the various antisera.In general, the less virulent strains were inhibitedto a much greater extent with both homologousand heterologous antisera than were the morevirulent strains. This was particularly noticeablein the size of the inhibition zone with the non-arthritogenic strains H606 and PG6. Strain DLwas the least inhibited of all, and evidence ofinhibition was sometimes noticeable only by areduction in the size of the colonies. Further ex-amination of some strains by the metabolic inhibi-tion test (Table 6) indicated considerable hetero-geneity. Whereas all strains were inhibited to someextent, strain DL appeared to be the most distinctin these tests. The avirulent strain H606 was themost susceptible to the inhibiting action of allantisera. The rat-passaged and unpassaged cul-tures of strain 14152 could not be differentiatedon the basis of the metabolic inhibition test.Comparisons between avirulent and virulent,

passaged and unpassaged strains by the comple-ment-fixation test failed to reveal any significantdifferences. The titers obtained never varied morethan fourfold between virulent and avirulent orpassaged and unpassaged strains.Gel diffusion tests were set up to determine

whether the presence or absence of any antigeniccomponents could be correlated with virulenceor lack of virulence. No such relationship wasfound to exist, although minor differences be-tween strains were detected. Some antigen prep-arations reacted poorly with all sera, although

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TABLE 6. Relationship of M. arthritidis strains by the metabolic inhibition test

Reciprocal of metabolic inhibition antibody titer with indicated sera

Strain14152 Po 14152 14152 PN DL R6 H606 PG6P13 sc Plo iv PN DR6 H0 PG

14152 Po 5,120 5,120 2,560 NTa NT NT NT NT14152 P13 sc 1,280 5,120 2,560 NT NT NT NT NT14152 P10 iv 2,560 2,560 2,560 NT NT NT NT NTPN NT NT NT 320 20 320 640 320DL NT NT NT 40 I,280 40 640 320R6 NT NT NT 80 40 1,280 1,280 640H606 NT NT NT 5,120 320 2,560 20,480 5,120

a Not tested.

harvesting younger cultures resulted in greaterserological reactivity. Prolonged storage of the an-tigens often resulted in a decrease in reactivity.

DISCUSSIONThe present study indicates that great variation

in virulence exists between different strains of M.arthritidis. Some laboratory-maintained strains(H606 and PG6) failed to induce arthritis at dosesof 3 X 1011 CFU, whereas others induced minimalarthritis with an inoculum of 2 x 107 CFU.

Several investigators have reported that whenM. arthritidis is passaged in rats a more severe in-fection can be induced (8, 13, 24, 27, 35). Pre-viously, little attempt was made to quantitatethese observations. The present study indicatesthat passaging in rats lowers the ED50 values andresults in more severe arthritis. This was particu-larly apparent with the two passaged cultures ofstrain 14152 in which the virulence was at least26 and 94 times greater, respectively, than thatof the unpassaged culture (assuming that maxi-mum error had occurred in the ED50 determina-tions). Although the passaged culture of strain158 was found to have only a slightly lower ED50value, the arthritis scores were higher, more limbswere involved, and the day of onset occurredearlier than with the unpassaged organisms.The observation that the unpassaged culture of

strain 14124 was more virulent than the rat-pas-saged culture was explicable on the basis of bac-terial contamination (see above).Although significant differences in the virulence

of passaged and unpassaged organisms were ob-served in this study, it is likely that many sub-sidiary factors contributed to experimental errorin the EDEo determinations. Viable counts forCFU are based upon the assumption that, in com-paring different cultures and strains, each colonyis derived from either a single organism or a simi-lar number of organisms. Mycoplasmas may existsingly or in chains, and the proportion of these

may vary from strain to strain or with the age ofthe culture. Additional factors such as clumpingof the suspensions, differences in the growth phaseof the organisms at the time of harvesting, leakagefrom injection sites, and variability in the re-sistance to infection of individual animals mayalso contribute to experimental error. Further-more, since unknown numbers of nonviable or-ganisms may also be present in the inoculum, it ispossible that virulence factors contained in thesecells could contribute to the severity of the dis-ease. Despite these potential errors in EDw de-terminations, a fivefold variation in inoculum wasfound to result in significant differences in thedegree of arthritis.

It was recently reported in a brief statement byThomas (31) that M. arthritidis induced toxicdeath in rats 1 hr after the injection of 1010 to 1011organisms. The strain, size of the inoculum, andthe weight of the rats were not specified. No evi-dence of rapid toxic deaths due to M. arthritidiswas encountered in the present study. Even whenmassive doses of virulent organisms were used, noclinical symptoms were visible for 2 days. Animalsgiven high doses occasionally died between 6 and8 days, owing to a massive infection and result-ing interference with the ability to carry outnatural functions. Attempts to increase thechances of detecting M. arthritidis toxins by em-ploying a preliminary injection of endotoxinfailed. However, we have observed that massivedoses of virulent M. arthritidis can induce deathin mice within 24 hr (unpublished data).

It was reported by Howell and Jones (12)that the colonies of a virulent strain of M. arthriti-dis which they studied had a rougher appearancethan those of an avirulent strain. Differences incolonial morphology were detected in the presentstudy, but they could not be correlated with viru-lence. However, generation time studies revealedthat the less virulent strains grew mucb faster inMycoplasma broth (Difco) than did the virulent

VOL. 1, 1970 543


strains. Thus, in a mixed population of virulentand avirulent variants, selection pressure could re-sult in the emergence of an avirulent variant underconditions of artificial cultivation. Although thevirulent strains survived for longer periods oftimein broth cultures, this could be explained by thefact that these strains produced less growth andhence metabolized smaller amounts of arginine,resulting in a lower pH as compared with the lessvirulent and avirulent strains. Lipman, Cl de, andDenny (19) detected differences in growth ratesfor various strains of M. pneumoniae, but thesedifferences could not be correlated with virulence.

Serological studies by means of complement-fixation and gel-diffusion tests failed to reveal anydifferences between strains which could be corre-lated with virulence. Lipman et al. (19) failed todifferentiate virulent and avirulent strains of M.pneumoniae by means of complement-fixation,metabolic inhibition, and immunodiffusion tech-niques. In our study, all of the complement-fixa-tion titers were virtually identical in the ho-mologous and heterologous systems. Using geldiffusion techniques, Lemcke (18) detected anextra precipitin band in the M. hominis type 2antiserum. In the present study, M. arthritidisstrains could not be differentiated from those pre-viously classified as M. hominis type 2. Antigensprepared from the slower-growing (virulent)strains often produced fewer bands of precipita-tion than the avirulent or less virulent strains.However, more bands could invariably be pro-duced by harvesting younger cultures. It wasalso found to be important to employ fresh anti-gens, as storage frequently reduced the numberof precipitin bands which developed. Factors in-fluencing reactivity of antigens have been referredto by other investigators (25, 26, 30).

Although all strains were inhibited in thegrowth inhibition test, the zones varied greatly insize. The slow-growing, more virulent strainswere noticeably less susceptible to inhibition thanthe avirulent or less virulent strains by eitherhomologous or heterologous sera. In the meta-bolic inhibition test, although the homologoussystems usually produced the highest titers, theavirulent strains were inhibited to a greater extentby all sera, in spite of the fact that they grew moreluxuriantly. If virulent strains of M. arthritidisare less susceptible to the action of antibody,then this property could play a role in the diseaseprocess. It may be that the greater resistance ofvirulent M. arthritidis strains to growth-inhibit-ing antibody is sufficient to overcome the negli-gible or very low levels of metabolic inhibitingantibody produced by rats in response to infec-tion with M. arthritidis (5).The growth characteristics and inhibition by

antiserum of the unpassaged culture of strain14124 were typical of an avirulent strain. Previouswork with this organism (7) indicated that theED50 for rats was between 2 X 109 and 1.5 X1010 CFU. Thus, the lower ED50 observed inthe present study may have been due to a bac-terial contaminant.

Since large numbers of mycoplasmas arerequired to induce experimental arthritis, it isdifficult to determine how the disease occursnaturally. A wide variety of stress factors, in-cluding mixed infections with other microor-ganisms, may be involved (9, 15, 32). It hasrecently been shown that Pasteurella pneumo-tropica may contribute to pneumonia of micecaused by M. pulmonis (1). Surprisingly, littlecontrolled work has been conducted on theconditions necessary for spontaneous rat ar-thritis, and further experimentation in this areais greatly needed. The present study has pro-vided a basis for this future work in defining thedegree of reproducibility of the disease and inquantitating the dose-response effects of a widevariety of M. arthritidis strains.The passaging of M. arthritidis in rats has been

observed to increase the virulence of the or-ganisms. Apart from some differences in growthcharacteristics and susceptibility to the inhibitingaction of hyperimmune serum, no other prop-erties could be correlated with virulence of theorganisms.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantsAM-02255 and AM5016 from the National Institute of Arthritisand Metabolic Diseases.

LITERATURE CITED

1. Brennan, P. C., T. E. Fritz, and R. J. Flynn. 1969. Role ofPasteurella pneumotropica and Mycoplasma pulmonis inmurine pneumonia. J. Bacteriol. 97:337-349.

2. Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H.Sussdorf. 1963. Methods in immunology, p. 246-247. W. A.Benjamin, Inc., New York.

3. Chanock, R. M., L. Hayflick, and M. F. Barile. 1962. Growthon artificial medium of an agent associated with atypicalpneumonia and its identification as a PPLO. Proc. Nat.Acad. Sci. U.S.A. 48:41-49.

4. Clyde, W. A. 1964. Mycoplasma species identification basedupon growth inhibition by specific antisera. J. Immunol.92:958-965.

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Effect of Animal Passage on Arthritogenic and Biological