INFECTION AND IMMUNITY, Apr. 1978, p. 248-2570019-9567/78/0020-0248$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 20, No. 1

Printed in U.S.A.

Experimental Infection of the Genital Tract of Female GrivetMonkeys by Mycoplasma hominis

B. R. MOLLER,' E. A. FREUNDT,' F. T. BLACK,' AND P. FREDERIKSEN2

Institute ofMedical Microbiology' and Institute ofPathology,2 University ofAarhus,DK-8000 Aarhus C, Denmark

Received for publication 5 October 1977

Mycoplasma hominis, a common inhabitant of the mucosae of the genitouri-nary tract of human and nonhuman primates, was inoculated directly into theuterine tubes of five laparotomized grivet monkeys. A self-limiting acute salpin-gitis and parametritis developed within a few days in all animals. Although therewere no clinical signs of overt disease, the gross pathology was characterized bypronounced oedematous swelling and hyperaemia of the tubes and parametria.Microscopically, cellular infiltrations of lymphocytes and some polymorphonu-clear leukocytes were found in the acute phase in the subserosa and muscularisof the tubes and in the parametria. Granulation tissue and fat necrosis appearedat a later stage in the parametria. The infection was associated with a markedantibody response and a moderate rise of the erythrocyte sedimentation rate andleukocyte counts. The capability of M. hominis to produce salpingitis and para-metritis in a nonhuman primate would seem to add rather significantly to theavailable evidence suggesting an etiological role of this organism in inflammatorydiseases of the internal female genitals of humans.

The first isolation of mycoplasmas from hu-mans was made in 1937 from a Bartholin glandabscess (7). Since then, two mycoplasmal spe-cies, Mycoplasma hominis and Ureaplasmaurealyticum, have been shown to be very com-mon inhabitants of the human genitourinarymucous membranes, and many attempts havebeen made to determine the pathogenic poten-tial of these organisms in relation to inflamma-tory diseases of the genital tract of males andfemales (10-12, 27, 28). In females, the possibleetiological implication of these organisms in sal-pingitis and related disorders has attracted par-ticular interest. For obvious reasons, isolationsmade from the cervix and vagina do not provideconclusive evidence as to a causal relationshipofthe organisms to infection of the uterine tubes.Although recovery of mycoplasmas directlyfrom the tubes or from tubo-ovarian and pelvicabscesses has occasionally been reported (11,12), it has generally been difficult to obtainspecimens from the uterine tubes in the acutestage of salpingitis for cultivation attempts. Thisobject could be achieved, however, with theintroduction of laparoscopy as a direct methodof diagnosing salpingitis (19). The circumstantialevidence obtained by this means in favor of theconcept that M. hominis, and possibly U. urea-lyticum, may be the cause of some cases ofsalpingitis has gained additional support from anumber of serological studies (15, 20, 22). Never-

theless, the true significance of mycoplasmas ascausal agents of salpingitis is still the subject ofdiscussion.The purpose of this study was to determine

whether salpingitis and inflammatory lesions ofother tissues ofthe internal female genitals couldbe produced experimentally by M. hominis in anonhuman primate.

MATERIALS AND METHODSOrganism. M. hominis D1887, isolated in this lab-

oratory from the cervix of a patient with acute salpin-gitis, was used in all the experiments. It had beencloned only once after the primary isolation and wasstored at -70'C. For preparation of the inoculum, theorganism was subcultured twice in a liquid medium(B-medium) consisting of heart infusion broth (Difco)enriched with 20% (vol/vol) horse serum and 10% of a25% (wt/vol) solution of yeast extract. The secondsubculture was made in 300 ml of medium and har-vested in the late log growth phase by centrifugation(10,000 rpm/20 min) and suspension of the pellet in 5ml of phosphate-buffered saline, pH 7.2. The concen-trated suspension contained about 108 colony-formingunits per ml. Portions (1 ml) were frozen and stored at-70'C. The number of colony-forming units of theindividual lots was redetermined before each experi-ment to ensure that no significant loss of viable unitshad occurred during storage. In one experiment, theculture used for inoculation was derived from an iso-late of M. hominis that had been recovered from theuterine tube of another monkey after experimentalinfection with strain D1887.

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Animals. Six female grivet monkeys (Cercopithe-cus aethiops), weight 1.5 to 2.2 kg, were used. Theanimals had been captured in East Africa and kept inquarantine for at least 6 weeks at Statens Seruminsti-tut in Copenhagen, where they had been tested forenteric pathogens (Salmonella and Shigella spp.) andfor tuberculosis by the tuberculin test. Throughoutthe experimentation period the monkeys were cagedindividually in an isolation room. They were fed acommercial primate food and were supplied with freshfruit and water ad libitum.

Preinoculation testing. Throat, vaginal, and rec-tal swabs were obtained three times, at intervals of 2to 3 days, while the animals were anesthetized withketamine chloride (Ketalar, 5 mg/kg). Cultivationswere made to ascertain that the monkeys did notharbor M. hominis or U. urealyticum in any of thesesites. In addition, vaginal swabs were cultivated foraerobic and anaerobic bacteria and for Trichomonasvaginalis (Diamond medium [6]). A retrospect exclu-sion of Chlamydia trachomatis infection was at-tempted at a later stage by cultivation of cervicalswabs in cycloheximide-treated McCoy cells (25).Blood samples for serology, leukocyte countings, anddetermination of the erythrocyte sedimentation rate(ESR) were taken by inguinal puncture of the femoralarteria while the monkeys were under anesthesia. Therectal temperature was recorded twice daily by a ther-mister.

Experimental infection. Infection was induced byinoculation directly into the uterine tubes by laparot-omy performed while the monkeys were under anes-thesia with phenyclidine hydrochloride (Sernylan, 20mg/ml), 0.15 ml; chlorpromazin (0.25% solution), 0.5ml; and atropin (0.1% solution), 0.2 ml.

Surgery was performed under aseptic conditions.The abdomen was shaved, washed with soap, anddisinfected with a 2% iodine tincture. The skin wascovered with a sterile surgical drape (Steri-drape). Thelaparotomy was made by a paramedian incision, afterwhich 0.2 ml of the mycoplasma suspension was in-jected through the wall of the lateral part of eachuterine tube by using a 0.8-mm needle. It was endea-vored to deposit the inoculum into the lumen of thetubes. In one monkey included as a control for thewhole series of experiments, phosphate-buffered salinewas injected instead of the mycoplasma suspension.The abdominal wall was closed by a suture in threelayers, and the wound was covered with spray-plaster(Nobecutan). To minimize the risk of bacterial infec-tion, daily injections of methicillin (Lucopenin, 60mg/kg) were initiated on the day of operation andwere continued until day 5 postoperation. The surgerywas tolerated very well by the monkeys.Assessment of lesions and collection of speci-

mens. Laparotomy as described above was performed,with minor temporal variations, on days 3, 7, 12, 20,and 35 postinoculation (p.Q) to follow the developmentand regression of inflammatory lesions and to collectspecimens. Swabs for recovery of mycoplasmas weretaken from the vagina, fimbriae, uterine tubes, uterinecavity, and parametria, and swabs for bacterial culti-vation were taken from the fimbriae, uterine tubes,uterus, and the peritoneal serosa covering the intes-tines. Biopsies were taken from the tubes and para-

metria by means of 2-mm surgical ear-forceps andfrom the uterus by a 3-mm skin drill.

Culturing of specimens. For cultivation of M.hominis the swabs were inserted immediately into 1.7ml of the liquid B-medium containing penicillin G, 400IU/ml, and thallium acetate, 0.01% (wt/vol). Afterincubation at 37°C for 3 days, subcultivation was madeinto (i) liquid B-medium, (ii) liquid B-medium con-taining 0.3% of arginine, and (iii) a duplicate set ofsolid B-medium plates prepared by replacing heartinfusion broth with heart infusion agar (Difco). Again,subcultivation onto solid medium was made from thesecondary liquid cultures after 3 days of incubation.One set of agar plates was incubated at 37°C in candlejars, and the other set was incubated in an atmosphereof 95% N2 and 5% CO2. The plates were read under astereomicroscope after 3 days and, if negative, werereexamined at intervals during incubation for another7 days. Identification of mycoplasma isolates was madeby the growth inhibition (2) and indirect epi-immu-nofluorescence tests (26).

Cultivations for bacteria were made in brain heartinfusion broth (Difco) and on blood agar, lactose brom-othymol blue, and chocolate agar plates.

Histology. The tissue biopsies were fixed in 10%Formalin and processed by routine histological meth-ods, including staining with hematoxylin and eosin.

Serology. The indirect hemagglutination (IHA)test was performed by the method of Krogsgaard-Jensen, by using Formalinized sheep erythrocytes sen-sitized with the supernatant fluid after centrifugationof sonically treated antigen (14).

RESULTS

All five monkeys that were experimentallyinfected by inoculation of M. hominis into theiruterine tubes presented gross pathology as wellas histological evidence of acute inflammatorylesions of the upper genital tract. The entirecourse of infection was essentially the same inall experimental animals, including the onewhich received the monkey-passaged M. hom-inis inoculum. Clinically, none of the monkeyspresented any remarkable signs of disease, theirgeneral health condition remaining fairly unim-paired.Gross lesions. Signs of an acute inflamma-

tory reaction were apparent as early as day 3 p.i.Characteristically, the lateral part of the uterinetubes were swollen and reddened, whereas atthis stage the medial part and the parametrialooked normal. On day 7 p.i., the tubes showeda pronounced swelling and hyperemia in theirentire length, and the parametria were moder-ately to markedly oedematous. After 12 days thesigns of inflammation of the tubes were decreas-ing, but the parametria still showed pronouncedswelling. During the following 2 to 3 weeks therewas a further marked regression of the inflam-matory lesions, and the upper genital tract wasvirtually normal in appearance 4 to 5 weeks after

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inoculation of the mycoplasmas.No exudation was observed from the tubes at

any stage of infection. The uterus showed onlyslight signs of inflammation, and the ovarieswere invariably normal without oedema or red-dening. Cystic structures did not develop eitherin the tubes, ovaries, or parametria.Histology. Pronounced inflammatory

changes were found microscopically in the uter-ine tubes and parametria of all five monkeysinoculated with M. hominis (Fig. 1 and 2).The lumen of the tubes always had a normal

appearance without any exudate or inflamma-tory cells. The mucosa was likewise practicallyintact except for an occasional slight hyperemiaand lymphoid infiltration. The most notablefinding in the tubes was an intense infiltrationin the subserosa of lymphoid cells together withan increased number of polymorphonuclear leu-kocytes. In most cases, the cellular infiltrationextended into the peripheral layer of the mus-cularis.The inflammatory lesions of the parametria

were characterized by marked oedema and hy-peremia, accompanied by cellular infiltrationsconsisting mainly of great numbers of lympho-cytes, but also ofsome heterophilic granulocytes.In most cases, fat necrosis and granulation tissueappeared in the parametria about days 10 to 12p.'.Body temperature. No significant rise of the

rectal temperature was observed in relation tomycoplasma infection.Hematology. The ESR showed a moderate

rise on days 3 to 12 p.i. (Fig. 3). The number ofleukocytes exhibited a slight decrease on day 3,followed by a moderate increase on days 7 to 14p.i. (Fig. 3). Differential countings showed noconsistent changes.

Isolation of M. hominis. This could be re-covered regularly from the fimbriae, tubes, par-ametria, and the uterine cavity on days 3 and 7p.i. Swabs taken from the said tissues duringsubsequent laparotomies usually yielded nogrowth of mycoplasmas, although in one case M.hominis was isolated from the uterus on day 10,and in another case from the fimbriae on day 17p.i. From the vagina, M. hominis could be iso-lated from day 3 until about 3 weeks (four cases)and 3.5 month (one case) p.i.With one exception, no growth of bacteria was

obtained from swabs taken in connection withlaparotomy. Chlamydia could not be isolatedfrom cervical swabs.Serology. Although none of the monkeys had

detectable IHA antibodies in preinoculationsera, such antibodies developed 7 to 12 days p.i.A fourfold rise was observed in all monkeysduring the course of the infection, with maxi-mum titers of 160 to 640. This titer level per-

INFECT. IMMUN.

sisted for more than 3 months in every case (Fig.3).Control monkey. The body temperature,

ESR, and leukocyte counts of the monkey in-oculated with phosphate-buffered saline re-mained unchanged within preinoculation values.Macroscopic signs of inflammation of the inter-nal genitals were not demonstrable at any time.Microscopically, very minimal inflammatorychanges were seen confined to the tubes. M.hominis could not be isolated, and no antibodiesdeveloped against this organism, although theanimal was caged in the same room as the ex-perimentally infected animals (Fig. 3).

DISCUSSIONAlthough M. pneumoniae, the major etiolog-

ical agent of primary atypical pneumonia, is theonly mycoplasma of proven pathogenicity tohumans, the possible role of other mycoplasmalspecies in human disease has been the subject ofnumerous investigations during recent decades(12, 28). In the Introduction, mention was madebriefly of findings that strongly suggest that M.hominis, and possibly U. urealyticum, may beetiologically involved in salpingitis and otherinflammatory conditions of the female genitaltract. The studies by Mardh and Westrom (22;P.-A. Mardh, M.D. thesis, University of Lund,Lund, Sweden. 1972), who obtained samples forcultivation attempts directly from inflamed sal-pinges through the laparoscope, are particularlynoteworthy. In 4 out of 52 cases of acute salpin-gitis such specimens yielded growth in pure cul-ture of M. hominis, whereas in 2 cases U. ureal-yticum was recovered. Antibodies against M.hominis were demonstrated by IHA in 54% ofthe 52 patients, with a significant change of thetiter in 7 out of 29 patients from whom morethan one serum specimen was collected duringthe course of the disease (20). The demonstra-tion, in 34% of the total number of patients, ofmarkedly increased serum levels of immuno-globulin M (17) was in accordance with similarfindings in patients suffering from M. pneu-monia pneumoniae (1, 9). The quite high inci-dence of M. hominis antibodies recovered by MArdh and Westrom in their salpingitis material,as opposed to a low incidence in healthy con-trols, provided further support for the signifi-cance of similar observations previously madeby other authors (15, 22).

Previous attempts at proving the pathogenic-ity of M. hominis and other mycoplasmas ofhuman provenance have included, on a few oc-casions, experimental infection of human vol-unteers (3, 23, 29). For obvious reasons, it wouldbe out of the question to follow this approach asa possible means of reproducing salpingitis un-der experimental conditions. The choice in the

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M. HOMINIS INFECTION OF MONKEY GENITAL TRACT

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FIG. 1. Histopathological appearance of monkey uterine tube inoculated with 0.2 x 108 colony-formingunits of M. hominis, D1887. (A) Normal tube at day of inoculation. (B) Day 3 p.i., oedema and slightinflammation in mucosal folds, pronounced inflammation in muscular coat and serosal covering. (C) Day 12p.i., inflammatory changes regressing. (D) Day 21 p.i., inflammatory lesions subsided except for moderatechanges in the serosal coat. Stained with hematoxylin and eosin, x52.

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present study of a nonhuman primate as an related to humans, but also by the fact that thealternative experimental model was motivated, normal mycoplasmal flora appears to be shared,not only by the wish to use an experimental to a wide extent, by human and nonhumananimal that is phylogenetically relatively closely primates. Thus, M. hominis has been isolated

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from the oropharynx and the lower genital tract The production by inoculation of M. hominisof a variety of primate species in captivity (4, 5, in grivet monkeys of a self-limiting acute salpin-16, 21). gitis and parametritis associated with a marked

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antibody response, together with a moderate riseof the ESR and leukocyte counts, would seem,

in our opinion, to add rather significantly to theassumption that M. hominis is also capable ofproducing salpingitis in humans under naturalconditions.On the basis of gross pathology and histology,

non-tuberculous salpingitis in humans may bedivided into two main groups (24). One type,which is caused by Neisseria gonorrhoeae, ischaracterized by a moderate swelling and red-dening of the uterine tubes, and the parametriausually look practically, normal. The lumen ofthe tubes is distended with a purulent exudate,which may also escape the abdominal ostia andproduce pelvic peritonitis and abscesses. Themucous membrane of the tubes is swollen andhyperaemic. Microscopically the epitheliumshows cloudy swelling of the cells and patchydestruction. The subepithelial tissue is infil-

trated with leukocytes, chiefly of the polymor-phonuclear variety. The pathogenesis ofthe gon-orrheal salpingitis is generally regarded to de-pend on ascending infection from a primary le-sion of the lower genital tract, the gonococcireaching the endosalpinx via the uterine mucosa.

In non-gonorrheal salpingitis, the inflamma-tory swelling, which also involves the parame-tria, is usually much more pronounced than inthe pyogenic infection. The swelling of the tubesis due to an enormous oedematous thickening ofthe tube wall, whereas there is no exudate in thelumen. The histology is characterized by a nor-

mal epithelium and marked oedema and otheracute inflammatory changes of the subserosaand muscularis of the tubes. The parametria,likewise, show intense oedema, hyperemia, andinfiltration with leukocytes. This type of salpin-gitis and parametritis may occur as a postpartumor postabortive complication, or result from

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256 M0LLER ET AL.

surgical procedures such as cauterization of thecervix or uterine curettage. In both sets of con-ditions, microorganisms are believed to gain en-trance to the tissues through lesions of the cer-vical or endometrial epithelium and to spread tothe tubes, parametria, and broad ligaments viablood vessels and lymphatics.

It will be seen from that described above thatthe pathology of the experimentally induced M.hominis genital tract infection of monkeysclosely resembles the latter type of salpingitisand parametritis described in humans. Otherpossible causes of this type of inflammation in-clude staphylococci and streptococci. The par-ticular importance of C. trachomatis as anotheretiological agent of acute salpingitis is borne outby recent investigations (8, 13, 18). Altogether,gonococci would seem to be a relatively lessfrequent cause of salpingitis and related disor-M.D. thesis, University of Lund, Lund, Sweden,1976).The production ofM. hominis infection of the

genital tract by direct inoculation into the uter-ine tubes is admittedly highly artificial. How-ever, further experiments are in progress in thislaboratory with the purpose of reproducing thedisease under conditions simulating, moreclosely, the natural infection.

ACKNOWLEDGMENTS

We thank P.-A. Mhrdh and T. Ripa, Department of MedicalMicrobiology, University of Lund, Sweden, for testing themonkeys for Chlamydia infection, and Birthe Hestbech, De-partment of Bacteriology of this Institute, who aided in theisolation and identification of bacteria.

This work was supported by grant 512-5183 from the Dan-ish Medical Research Council.

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