INFECTION AND IMMUNITY, June 1972, p. 921-926Copyright ( 1972 American Society for Microbiology

Vol. 5, No. 6Prinited in U.S.A.

Infection of Genital Tract and Transmission ofOcular Infection to Newborns by the Agent of

Guinea Pig Inclusion ConjunctivitisDAVID T. MOUNT, PIERLUIGI E. BIGAZZI, AND ALMEN L. BARRON

Department of Microbiology, School of Medicine, State University of New York, Biffalo, New York 14214

Received for publication 24 January 1972

Female guinea pigs were inoculated intravaginally with the agent of guinea piginclusion conjunctivitis (Gp-ic). Evidence for infection was obtained by demon-stration of Gp-ic inclusions in epithelial cells of genital tract smears, histopathology,recovery of Gp-ic during infection, and antibody response. Infection of the genitaltract was produced with low doses (20-200 median egg lethal doses) of the agent.The infection lasted about 3 weeks, and there was no marked clinical response.Ocular infection that could be detected as early as 3 days after birth in offspringof infected mothers was also demonstrated.

It has long been recognized that infants acquireinclusion blennorrhea (conjunctivitis) from in-fected mothers at birth. Study of the oculo-genital relationship in this disease has beenimpeded by the lack of a suitable experimentalhost. Attempts to achieve genital transmission ofinclusion conjunctivitis in monkeys from in-fected mothers to newborns have not been suc-cessful (1).

In 1964, Murray (6) reported a naturally oc-curring conjunctivitis of guinea pigs whichclinically was a mild, self-limiting disease ofyoung animals. Examination of stained smearsobtained from conjunctival scrapings revealed thepresence of inclusions rather similar to thoseobserved in the human disease. The causativeagent of guinea pig inclusion conjunctivitis wasfound to be a member of the Chlamydia group ofagents and was designated Gp-ic. Although thebiological characteristics of Gp-ic place it in thespecies C. psittaci rather than C. trachomatis,the similarities of the infections in guinea pigsand humans stimulated interest in the guinea pigdisease as a model for the study of chlamydialoculo-genital infections. Gp-ic has been found tobe endemic in several herds, but the natural modeof spread has not been defined. Consideration hasbeen given to the possibility of genital tracttransmission. The present paper is concerned withthe demonstration of infection by Gp-ic in thevsenital tract of female guinea pigs and transmis-*;ion of ocular infection to newborns.

MATERIALS AND METHODSChlamydia. Gp-ic agent was obtained from E. S.

Murray, Harvard School of Public Health, Boston,

Mass. Stock suspensions of 20%,G yolk sacs, stored at-70 C, were used at passage levels 5 and 6 of thislaboratory. Infectivity titrations were performed byyolk sac inoculation of 0.25 ml into 7-day-old chickembryos. Those embryos which died between 3 and 12days were considered positive in the calculation of the50% egg lethal dose (ELD5o) by the method of Reedand Muench (8).

Diluent. Sucrose potassium glutamate (SPG) (3),containing 2.5 mg of streptomycin (Squibb andSons, New York, N.Y.) per ml and 0.5 mg of vanco-mycin (Lilly Research Laboratories, Indianapolis,Ind.) per ml, was used for yolk sac suspensions, in-fectivity titrations, and collection of specimens.

Guinea pigs. Mature Hartley strain female guineapigs were obtained from Simonson Laboratories, Inc.,Gilroy, Calif. This stock is apparently free of naturalGp-ic infection.

Inoculation and sampling. Gp-ic yolk sac suspensionwas centrifuged at 130 X g for 10 min at 5 C. and thesupernatant fluid was centrifuged at 27,000 X g for30 min at 5 C. The organisms were resuspended to theoriginal volume in SPG. Guinea pigs were inoculatedintravaginally with 0.05 ml by insertion of a thinvinyl tubing (no. 6106, Becton-Dickinson, Rutherford,N.J.) attached to a 23-gauge needle using a 0.25-mlsyringe. After inoculation, the animals were cagedindividually.

Vaginal scrapings were obtained with a bacterio-logical loop and conjunctival specimens were col-lected as described by Murray (6). Smears were fixedin methanol and stained with Giemsa. Specimens forisolation of Gp-ic were collected in 1.0 ml of SPGand stored at -70 C.

Evaluation of smears. Vaginal smears were gradedon a scale from 0 to 4 based on the number of epithelialcells which contained cytoplasmic inclusions (1 =<1%; 2 = 1%-9%; 3 = 10%-39%; 4 = >.40%).

Histological preparations. Genital tract tissues were

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MOUNT, BIGAZZI, AND BARRON

fixed in I10o neutral Formalin, sectioned at 5 ,m, andstained with hematoxylin and eosin.

Detection of antibodies. Sera collected from guineapigs were tested for antibodies by indirect immuno-fluorescence. Gp-ic agent was adapted to grow (A. L.Barron, unpublished data) in the BGM line of AfricanGreen monkey cells (2). The cells were grown oncover slips in Leighton tubes, and, when the majoritywere infected, the cover slips were fixed in acetone andstored at -70 C. The conjugate was a rabbit antiserumto guinea pig gamma globulin (Hyland Laboratories,Inc., Los Angeles, Calif.) and was used at a dilutionof 1:20. Cover slips were examined under a fluorescentmicroscope (American Optical Fluorolume) withbarrier filter EK no. 2A in conjunction with a Corning5840 exciter filter. The end point for antibody titra-tions was the highest dilution of serum yielding apositive reaction characterized by yellowish-greenfluorescent inclusions in the cytoplasm of infected cells.

RESULTS

Demonstration of genital tract infection. Eightguinea pigs were inoculated with approximately2 x 106 ELD5o each. Vaginal smears were ex-amined for inclusions. On the first day afterinoculation, smears collected from all of theanimals were negative for inclusions. On thesecond day, smears from four of the eight animalshad inclusions, and by day 4 inclusions wereobserved for all animals. Smears revealed thepresence of epithelial cells containing Gp-icinclusions and many polymorphonuclear leuko-cytes (Fig. 1). In two of the animals a vaginaldischarge accompanied the first detection ofinclusions in smears. Rectal temperatures were

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_y'

recorded for the first 9 days after inoculation andno elevation of temperature was found.The average inclusion scores of the eight

animals are shown in Fig. 2. Peak infection, asmeasured by inclusion scores, occurred aboutday 6 and declined gradually thereafter. Ingeneral, inclusions could be readily detectedfrom day 4 until day 20. In one animal, inclusionswere detected 30 days after inoculation.During the course of this experiment, con-

junctival smears were also examined. Inclusionswere observed in smears collected from two of theeight animals on day 16. Neither of these animalsmanifested an obvious clinical conjunctivitis.The results obtained for isolation of Gp-ic in

chick embryos during the course of the aboveexperiment are summarized in Table 1. Recoveryof the agent on day 1 could be attributed topresence of inoculum. Gp-ic was readily isolatedfrom the genital tract during the period of peakinfection described above, and after 20 days theincidence declined. In general, recovery of Gp-iccould be correlated with the presence of inclu-sions in smears.

Serology. The antibody responses of the abovegroup of animals as determined by immuno-fluorescence are shown in Table 2. Antibodieswere not detected in any of the pre-inoculationsera, or those collected on day 7. At this time, theaverage inclusion score was high. By day 16, whenthe average inclusion score had declined, anti-bodies were detected in sera of five of the sevenanimals tested. Antibody titers ranging from 40 to

__ I

_

k

FIG. 1. Vaginal smear 3 days postiinoculationi with Gp-ic. Epithelial cells with cytoplasmic inclutsions are presentwith manay polymorphoniclear leakoc) tes. Giemsa staini. Magniificationl X 1,000.

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GUINEA PIG INCLUSION CONJUNCTIVITIS

Inclusion S47

3

FIG. 2.from eightGp-ic. One

TABLE 1. AI

Days afinoculati

1361220273034

TABLE 2.

Day

0 <107 <1016 <1024 8034 160

a Reciptive resul

>320 werwere still:

Effect cpigs wereof Gp-icELDN wimnatory canimals 1only twodemonstr

core spectively. However, the intensity of the infectionas measured by inclusion scores, with the excep-tion of one animal, was equivalent to that ob-served with the higher levels.

Histopathology. A group of guinea pigs wasinoculated intravaginally with approximately2 x 106 ELD1, of Gp-ic. Pairs of animals weresacrificed at intervals through 16 days andgenital tract tissues were removed for histologicalexamination. Vaginal smears collected at the

l _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ | ,time of sacrifice were all positive for inclusions.1O 20 30 Both cervix and vagina demonstrated similar

DAYS changes in histological appearance. The out-Average inclusion scores of vaginal smears standing features of the early stages of infectionguinea pigs infected in the genital tract with (day 3 and 6) were large numbers of polymorpho-animal died on day 21. nuclear leukocytes in the lumen of the cervix,

desquamation of secretory cells, presence ofIsolation of Gp-ic in chick embryos from inclusions in the outer transitional cells, andiaginal specimens ofguinea pigs lymphocyte infiltration of the subepithelial tissues.injected in the genital tract When secretory cells were present, chlamydial

inclusions could be observed within them (Fig. 4).ter,ion -No. positive No. negative In the later stages of infection (day 9-16), the

number of polymorphonuclear leukocytes in the3 2 cervical lumen decreased, the secretory cells were

8 0 absent, and the inclusions were less numerous.4 0 The lymphocyte infiltration increased so that6 0 several animals showed marked accumulation of3 2 them in the subepithelial tissues of the cervix1 5 (Fig. 5). Well developed cornification was not

!1 5 observed in any of the animals even thoughr 0 1 several were considered to be in estrus as de-

termined by the histological appearance of theFluorescetnt antibody titers of sera from uterus. Genital tract tissues from infected animalsfemale guiniea pigs genitally which were sacrificed on day 33 were essentially

infected with Gp-ic aW normal. The uterus and urethra of all infectedanimals did not show any pathological changes.

Guinea pig no. Transmission to newborns. An experiment wasconducted to determine whether infected mothers

2 3 4 5 6 8 could transmit Gp-ic to newborns as an ocularinfection. Three of five pregnant females were

<10 <10 <10 <10 <10 <10 successfully infected in the genital tract (Table 3).<10 <10 <10 <10 <10 <10 Female no. 102, not infected with the yolk sac

80" >20 40 80 <10 4080 40 80 >320 >320 320 inoculum, was infected after inoculation with160 40 80 80 320 320 vaginal material from no. 105. Guinea pig no. 102

_____ - ___ - ----- - __ also became positive for inclusions in conjunctivalrocal of highest dilution yielding posi- scraping 7 days after delivery. The mode ofts. transmission to the eye was obscured in this

instance because both of the offspring were in-re found in all sera by day 24 and titers fected in the eyes at the time and could have beenhigh at day 34. the source of the infection.)f varying doses. Groups of four guinea The results observed in the newborn guineainoculated with varying concentrations pigs are summarized in Table 4. Conjunctival(Fig. 3). When 2 X 106 or 2 X 104 smears from the eight offspring of infectedere inoculated, inclusions and inflam- mothers were negative for at least 2 days afterells were observed in smears from all birth. When the second specimen was taken, as)y day 4. At doses of 200 or 20 ELDw , shown in the table, all of the eight newborns wereof the four animals in each group positive for inclusions. In six of the offspring,

*ated inclusions at 8 and 11 days, re- inclusions were present in smears from both

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MOUNT, BIGAZZI, AND BARRON

ELD50 Inclusion Score

2x10 21 IHiIHW HIH2IHoL

2X14 rhTW77~nLx1 __

32 x10

L

2 F2 x 10 L

DAYS2 4 6 8 11 13 15 18 22FIG. 3. Inclusion scores of vaginal smears ofgroups offour guinea pigs which received varying doses of Gp-ic.

M,~~~~~~~~~~~~~~~~~~~~1

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FIG. 4. Endocervical epitheliuni 6 days postinzocuilationi. Sup2rficial secretory cells are being shed ii the abselnceof cornification. Three secretory cells with inicllusions are present ( H & E staini. Magnification X 400.

eyes, and in none were conjunctival inclusionsfound more than 14 days after birth. Gp-ic wasisolated from conjunctival scrapings collected 7days after birth in all five of the animals tested.Three of the eight newborns also developedclinical conjunctivitis with considerable dischargeand closure of the eyelids, which in no. 115 wasbilateral. The clinical conjunctivitis was resolvedrather quickly with a return to apparent normalcy

in 2 to 3 days. Two of the newborns with clinicalconjunctivitis were markedly smaller than all theothers, and no. 115 died on day 6.The offspring of females no. 103 and no. 104,

which had never demonstrated vaginal inclusions,were uniformly negative for inclusions in con-junctival smears and gross clinical conjunctivitisduring the period of observation of 41 and 34days, respectively.

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GUINEA PIG INCLUSION CONJUNCTIVITIS

-- y W- -_rUMV

FIG. 5. Endocervix 9 days postinoculation. A focal accumulation of lymphocytes is present in the deeper tissue( T T ). An1 inclusioni may be seen at the epithelial surface ( T ). Secretory cells are absent and the epithelium isgreatly disorganized. H & E stain. Magnification X 250.

TABLE 3. Infection of pregnant guinea pigs in genital tract with Gp-ic agent

Day inclusions Inclusions Da inclusions Inclusions in Dag inclusionsGuinea pig first observed in Day delivered in vaginal o served in conjunctival o served in

no. vaginal scraping postinoculation scraping day vaginal scrapings scrapings day conjunctival scrapingspostinoculation of delivery postdelivery of delivery postdelivery

100 2 12 +a 4 - (Day 29)102 2 18 + 2 - 7105 4 25 + 2 - - (Day 16)103 - (Day 21) 10 _ - (Day 11) - - (Day 18)104 - (Day 21) 7 _ - (Day 14) - - (Day 34)

a Symbols: +, positive;-, negative.

TABLE 4. Transmission of Gp-ic infection to newborns by infected mothers

Day inclusions Day clinical Gp-ic isolated fromfirst observed conjunctivitis conjunctival scrapings

Mother Infected at Newborn in conjunctiva recognized 7 days postdeliverydelivery ____________________________ ________________

Right eye Left eye Right eye Left eye Right eye Left eye

100 + 110 4 6 _ la + +111 - 4 _ _ _ +

102 + 116b 3 NDC - - ND ND117 5 5 - 5 Cd Cd

105 + 112 5 5 - - + +113 5 5 - _ + +114 5 5 - - + +115e 5 5 2 5 ND ND

103 109 - - - ND ND104 - 106 - -- ND ND

107 - _ _ - ND ND108 - - _ - ND ND

a Many bacteria present.b Died day 3.c Not done.d Contaminated.e Died day 6.

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MOUNT, BIGAZZI, AND BARRON

DISCUSSION

The purpose of this investigation was to learn ifguinea pigs could be infected experimentally in thegenital tract with Gp-ic agent and whether theinfection could be transmitted to newbornanimals at delivery. Infection was demonstratedby detection of Gp-ic inclusions in vaginalsmears and histologic sections, recovery of theagent, and rise in antibody titer to Gp-ic. Therewas no marked clinical response to the genitaltract infection although occasional animals had aslight vaginal discharge. The mild clinical responsenoted in the genital tract has been described forthe natural conjunctivitis also. Murray (6) ob-served that many animals would not even havebeen detected without cytological examination.The time course of the genital tract infection of

approximately 21 days appeared to be compatibleto that described by others for conjunctivitisproduced experimentally (5, 6). Murray (6)reported a period of approximately 28 days fornaturally occurring disease. In the genital tract,the number of inclusions detected in smears de-clined as the infection subsided. A close correla-tion between clinical signs and the finding ofinclusion bodies in smears was reported byKazdan et al. (5) for conjunctivitis.Murray (6) described the earliest evidence of

infection in conjunctival smears as being thepresence of polymorphonuclear leukocytes.Kazdan et al. (5) described the earliest changes tobe a cellular response which was predominatelylymphocytic followed by an intense heterophilicresponse. In our experiments, large numbers ofmononuclear cells were never observed in vaginalsmears.

Interpretation of the histopathology of genitaltract infection was complicated by the stage of theestrus cycle. The animals were inoculated withoutknowledge of the stage of the cycle and infectioncould have occurred at any stage. The finding ofinclusions in secretory cells showed that such cellscould support the multiplication of Gp-ic as wellas the transitional cells. Since the secretory cellswere shed very quickly, it is reasonable to con-clude that the infection was mainly supported bythe transitional cells of the stratified squamousepithelium. In sections of genital tract, manypolymorphonuclear leukocytes were observed inthe lumen, especially in the early stages of infec-tion, followed, after a few days, by lymphocyteinfiltration of the subepithelial tissue. This patternwas also noted by Kazdan et al. (5) for sections ofconjunctiva. The presence of lymphocytic in-filtration in subepithelial tissues, observed particu-

larly in the latter stages of infection, may indicatea role of the cellular immune system.Murray (6) was unable to detect antibodies in

sera from 50 guinea pigs with primary experi-mental ocular infections by using the complementfixation test. In another study, Murray and Rad-cliffe (7) reported inconsistent antibody titerswhen sera were tested between 15 and 30 dayspost-experimental infection. The differences inantibody response obtained in our studies may bea reflection of the route of infection or themethods of detection.

In his original paper Murray (6) considered thatinfection of young guinea pigs probably occurredat 2 to 3 months of age. Kazdan et al. (5) ob-tained evidence for natural conjunctivitis asearly as 15 days after birth. In our experimentsnewborn animals were infected in the eye, ap-parently during delivery, because the infection wasdetected as early as 3 days.The results obtained in this investigation have

encouraged us to use the Gp-ic model system forthe study of oculo-genital chlamydial infectionssuch as inclusion conjunctivitis in man. Inparticular, problems concerning tissue specificity,mode of transmission, and immunity are cur-rently being investigated.

ACKNOWLEDGMENTS

This investigation was supported by Public Health ServiceGeneral Research support grant FR-05400 from the Division ofResearch Facilities and Resources to D.T.M., and grant EY00079from the National Eye Institute to A.L.B.

P. E. Bigazzi is a recipient of a Henry C. Buswell and BerthaH. Buswell Fellowship.

LITERATURE CITED

1. Alexander, E. F., and W. T. Chang. 1967. Infection of preg-nant monkeys and their offspring with TRIC agents. Amer.J. Ophthalmol. 63:1145-1153.

2. Barron, A. L., C. Olshevsky, and M. M. Cohen. 1970. Charac-teristics of the BGM line of cells from African Green mon-key kidney. Arch. Gesamte Virusforsch. 32:389-392.

3. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1950. Theinfluence of certain salts, amino acids, sugars, and proteinon the stability of rickettsiae. J. Bacteriol. 59:509-522.

4. Gordon, F. B., E. Weiss, A. L. Quan, and H. R. Dressler.1966. Observations on guinea pig inclusion conjunctivitisagent. J. Infect. Dis. 116:203-207.

5. Kazdan, J. J., J. Schachter, and M. Okumoto. 1967. Inclusionconjunctivitis in the guinea pig. Amer. J. Ophthalmol.61:116-124.

6. Murray, E. S. 1964. Guinea pig inclusion conjunctivitis virus.I. Isolation and identification as a member of the psittacosis-lymphogranuloma-trachoma group. J. Infect. Dis. 114:1-12.

7. Murray, E. S., and F. T. Radcliffe. 1967. Immunologic studiesin guinea pigs with guinea pig inclusion conjunctivitis(Gp-ic) Bedsonia. Amer. J. Ophthalmol. 63:1263-1269.

8. Reed, L. J., and H. Muench. 1938. A simple mlethod of es-timating fifty per cent endpoints. Amer. J. Hyg. 27:493-497.

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