Infection of Genital Tract and Transmission of Ocular Infection to Newborns by the Agent of Guinea Pig Inclusion Conjunctivitis

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1 INFECTION AND IMMUNITY, June 1972, p Copyright ( 1972 American Society for Microbiology Vol. 5, No. 6 Prinited in U.S.A. Infection of Genital Tract and Transmission of Ocular Infection to Newborns by the Agent of Guinea Pig Inclusion Conjunctivitis DAVID T. MOUNT, PIERLUIGI E. BIGAZZI, AND ALMEN L. BARRON Department of Microbiology, School of Medicine, State University of New York, Biffalo, New York Received for publication 24 January 1972 Female guinea pigs were inoculated intravaginally with the agent of guinea pig inclusion conjunctivitis (Gp-ic). Evidence for infection was obtained by demonstration of Gp-ic inclusions in epithelial cells of genital tract smears, histopathology, recovery of Gp-ic during infection, and antibody response. Infection of the genital tract was produced with low doses ( 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 offspring of infected mothers was also demonstrated. It has long been recognized that infants acquire inclusion blennorrhea (conjunctivitis) from infected mothers at birth. Study of the oculogenital relationship in this disease has been impeded by the lack of a suitable experimental host. Attempts to achieve genital transmission of inclusion conjunctivitis in monkeys from infected mothers to newborns have not been successful (1). In 1964, Murray (6) reported a naturally occurring conjunctivitis of guinea pigs which clinically was a mild, self-limiting disease of young animals. Examination of stained smears obtained from conjunctival scrapings revealed the presence of inclusions rather similar to those observed in the human disease. The causative agent of guinea pig inclusion conjunctivitis was found to be a member of the Chlamydia group of agents and was designated Gp-ic. Although the biological characteristics of Gp-ic place it in the species C. psittaci rather than C. trachomatis, the similarities of the infections in guinea pigs and humans stimulated interest in the guinea pig disease as a model for the study of chlamydial oculo-genital infections. Gp-ic has been found to be endemic in several herds, but the natural mode of spread has not been defined. Consideration has been given to the possibility of genital tract transmission. The present paper is concerned with the demonstration of infection by Gp-ic in the vsenital tract of female guinea pigs and transmis- *;ion of ocular infection to newborns. MATERIALS AND METHODS Chlamydia. 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 this laboratory. Infectivity titrations were performed by yolk sac inoculation of 0.25 ml into 7-day-old chick embryos. Those embryos which died between 3 and 12 days were considered positive in the calculation of the 50% egg lethal dose (ELD5o) by the method of Reed and Muench (8). Diluent. Sucrose potassium glutamate (SPG) (3), containing 2.5 mg of streptomycin (Squibb and Sons, New York, N.Y.) per ml and 0.5 mg of vancomycin (Lilly Research Laboratories, Indianapolis, Ind.) per ml, was used for yolk sac suspensions, infectivity titrations, and collection of specimens. Guinea pigs. Mature Hartley strain female guinea pigs were obtained from Simonson Laboratories, Inc., Gilroy, Calif. This stock is apparently free of natural Gp-ic infection. Inoculation and sampling. Gp-ic yolk sac suspension was centrifuged at 130 X g for 10 min at 5 C. and the supernatant fluid was centrifuged at 27,000 X g for 30 min at 5 C. The organisms were resuspended to the original volume in SPG. Guinea pigs were inoculated intravaginally with 0.05 ml by insertion of a thin vinyl tubing (no. 6106, Becton-Dickinson, Rutherford, N.J.) attached to a 23-gauge needle using a 0.25-ml syringe. After inoculation, the animals were caged individually. Vaginal scrapings were obtained with a bacteriological loop and conjunctival specimens were collected as described by Murray (6). Smears were fixed in methanol and stained with Giemsa. Specimens for isolation of Gp-ic were collected in 1.0 ml of SPG and stored at -70 C. Evaluation of smears. Vaginal smears were graded on a scale from 0 to 4 based on the number of epithelial cells which contained cytoplasmic inclusions (1 = <1%; 2 = 1%-9%; 3 = 10%-39%; 4 = >.40%). Histological preparations. Genital tract tissues were 921

2 922 MOUNT, BIGAZZI, AND BARRON INFECT. IMMUNITY fixed in I10o neutral Formalin, sectioned at 5,m, and stained with hematoxylin and eosin. Detection of antibodies. Sera collected from guinea pigs were tested for antibodies by indirect immunofluorescence. Gp-ic agent was adapted to grow (A. L. Barron, unpublished data) in the BGM line of African Green monkey cells (2). The cells were grown on cover slips in Leighton tubes, and, when the majority were infected, the cover slips were fixed in acetone and stored at -70 C. The conjugate was a rabbit antiserum to guinea pig gamma globulin (Hyland Laboratories, Inc., Los Angeles, Calif.) and was used at a dilution of 1:20. Cover slips were examined under a fluorescent microscope (American Optical Fluorolume) with barrier filter EK no. 2A in conjunction with a Corning 5840 exciter filter. The end point for antibody titrations was the highest dilution of serum yielding a positive reaction characterized by yellowish-green fluorescent inclusions in the cytoplasm of infected cells. RESULTS Demonstration of genital tract infection. Eight guinea pigs were inoculated with approximately 2 x 106 ELD5o each. Vaginal smears were examined for inclusions. On the first day after inoculation, smears collected from all of the animals were negative for inclusions. On the second day, smears from four of the eight animals had inclusions, and by day 4 inclusions were observed for all animals. Smears revealed the presence of epithelial cells containing Gp-ic inclusions and many polymorphonuclear leukocytes (Fig. 1). In two of the animals a vaginal discharge accompanied the first detection of inclusions in smears. Rectal temperatures were IJ _y' recorded for the first 9 days after inoculation and no elevation of temperature was found. The average inclusion scores of the eight animals are shown in Fig. 2. Peak infection, as measured by inclusion scores, occurred about day 6 and declined gradually thereafter. In general, inclusions could be readily detected from day 4 until day 20. In one animal, inclusions were detected 30 days after inoculation. During the course of this experiment, conjunctival smears were also examined. Inclusions were observed in smears collected from two of the eight animals on day 16. Neither of these animals manifested an obvious clinical conjunctivitis. The results obtained for isolation of Gp-ic in chick embryos during the course of the above experiment are summarized in Table 1. Recovery of the agent on day 1 could be attributed to presence of inoculum. Gp-ic was readily isolated from the genital tract during the period of peak infection described above, and after 20 days the incidence declined. In general, recovery of Gp-ic could be correlated with the presence of inclusions in smears. Serology. The antibody responses of the above group of animals as determined by immunofluorescence are shown in Table 2. Antibodies were not detected in any of the pre-inoculation sera, or those collected on day 7. At this time, the average inclusion score was high. By day 16, when the average inclusion score had declined, antibodies were detected in sera of five of the seven animals tested. Antibody titers ranging from 40 to k I FIG. 1. Vaginal smear 3 days postiinoculationi with Gp-ic. Epithelial cells with cytoplasmic inclutsions are present with manay polymorphoniclear leakoc) tes. Giemsa staini. Magniificationl X 1,000.

3 VOL. 5,1972 Inclusion S core 47 3 A I Days af ter inoculati TABLE 2. Day GUINEA PIG INCLUSION CONJUNCTIVITIS spectively. However, the intensity of the infection as measured by inclusion scores, with the exception of one animal, was equivalent to that observed with the higher levels. Histopathology. A group of guinea pigs was inoculated intravaginally with approximately 2 x 106 ELD1, of Gp-ic. Pairs of animals were sacrificed at intervals through 16 days and genital tract tissues were removed for histological examination. Vaginal smears collected at the l time of_ sacrifice were, all positive for inclusions. 1O Both cervix and vagina demonstrated similar DAYS changes in histological appearance. The out- FIG. 2. Average inclusion scores of vaginal smears standing features of the early stages of infection from eight guinea pigs infected in the genital tract with (day 3 and 6) were large numbers of polymorphoanimal died on day 21. nuclear leukocytes in the lumen of the cervix, Gp-ic. One desquamation of secretory cells, presence of TABLE 1. Isolation of Gp-ic in chick embryos from inclusions in the outer transitional cells, and iaginal specimens of guinea 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).,ion -No. positive No. negative In the later stages of infection (day 9-16), the number of polymorphonuclear leukocytes in the 3 2 cervical lumen decreased, the secretory cells were 8 0 absent, and the inclusions were less numerous. 4 0 The lymphocyte infiltration increased so that 6 0 several animals showed marked accumulation of 3 2 them in the subepithelial tissues of the cervix 1 5 (Fig. 5). Well developed cornification was not!1 5 observed in any of the animals even though r 0 1 several were considered to be in estrus as determined by the histological appearance of the Fluorescetnt antibody titers of sera from uterus. Genital tract tissues from infected animals female guiniea pigs genitally which were sacrificed on day 33 were essentially infected with Gp-ic aw normal. The uterus and urethra of all infected animals did not show any pathological changes. Guinea pig no. Transmission to newborns. An experiment was conducted to determine whether infected mothers could transmit Gp-ic to newborns as an ocular infection. Three of five pregnant females were 0 <10 <10 <10 <10 <10 <10 <10 successfully infected in the genital tract (Table 3). 7 <10 <10 <10 <10 <10 <10 <10 Female no. 102, not infected with the yolk sac 16 <10 80" > < >320 > inoculum, was infected after inoculation with vaginal material from no Guinea pig no also became positive for inclusions in conjunctival a Reciprocal of highest dilution yielding posi- scraping 7 days after delivery. The mode of tive resul ts. transmission to the eye was obscured in this instance because both of the offspring were in- found in all sera by day 24 and titers fected in the eyes at the time and could have been >320 werre were still: high at day 34. the source of the infection. Effect c)f varying doses. Groups of four guinea The results observed in the newborn guinea pigs were inoculated with varying concentrations pigs are summarized in Table 4. Conjunctival of Gp-ic (Fig. 3). When 2 X 106 or 2 X 104 smears from the eight offspring of infected ELDN wiere inoculated, inclusions and inflam- mothers were negative for at least 2 days after mnatory cells were observed in smears from all birth. When the second specimen was taken, as animals 1 )y day 4. At doses of 200 or 20 ELDw, shown in the table, all of the eight newborns were only two of the four animals in each group positive for inclusions. In six of the offspring, demonstr*ated inclusions at 8 and 11 days, re- inclusions were present in smears from both 923

4 924 MOUNT, BIGAZZI, AND BARRON INFECT. IMMUNITY ELD50 Inclusion Score 2x10 2IH 21 IHiIHW HIH ol 2X14 Lx1 rhtw77~n 3 2 x 10 L 2 F 2 x 10 L DAYS FIG. 3. Inclusion scores of vaginal smears ofgroups offour guinea pigs which received varying doses of Gp-ic. M,~~~~~~~~~~~~~~~~~~~~1 F =7 H, :.: 1. FIG. 4. Endocervical epitheliuni 6 days postinzocuilationi. Sup2rficial secretory cells are being shed ii the abselnce of cornification. Three secretory cells with inicllusions are present ( H & E staini. Magnification X 400. eyes, and in none were conjunctival inclusions found more than 14 days after birth. Gp-ic was isolated from conjunctival scrapings collected 7 days after birth in all five of the animals tested. Three of the eight newborns also developed clinical conjunctivitis with considerable discharge and closure of the eyelids, which in no. 115 was bilateral. The clinical conjunctivitis was resolved rather quickly with a return to apparent normalcy in 2 to 3 days. Two of the newborns with clinical conjunctivitis were markedly smaller than all the others, 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 conjunctival smears and gross clinical conjunctivitis during the period of observation of 41 and 34 days, respectively.

5 VOL. 5, 1972 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 is greatly 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 inclusions Guinea 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 scrapings postinoculation of delivery postdelivery of delivery postdelivery a a 4 - (Day 29) (Day 16) (Day 21) 10 _ - (Day 11) - - (Day 18) (Day 21) 7 _ - (Day 14) - - (Day 34) Symbols: +, positive;-, negative. TABLE 4. Transmission of Gp-ic infection to newborns by infected mothers Day inclusions Day clinical Gp-ic isolated from first observed conjunctivitis conjunctival scrapings Mother Infected at Newborn in conjunctiva recognized 7 days postdelivery delivery Right eye Left eye Right eye Left eye Right eye Left eye _ la _ b 3 NDC - - ND ND Cd Cd _ e ND ND ND ND ND ND ND ND _ - ND ND a Many bacteria present. b Died day 3. c Not done. d Contaminated. e Died day 6.

6 926 MOUNT, BIGAZZI, AND BARRON INFECT. IMMUNITY DISCUSSION The purpose of this investigation was to learn if guinea pigs could be infected experimentally in the genital tract with Gp-ic agent and whether the infection could be transmitted to newborn animals at delivery. Infection was demonstrated by detection of Gp-ic inclusions in vaginal smears and histologic sections, recovery of the agent, and rise in antibody titer to Gp-ic. There was no marked clinical response to the genital tract infection although occasional animals had a slight vaginal discharge. The mild clinical response noted in the genital tract has been described for the natural conjunctivitis also. Murray (6) observed that many animals would not even have been detected without cytological examination. The time course of the genital tract infection of approximately 21 days appeared to be compatible to that described by others for conjunctivitis produced experimentally (5, 6). Murray (6) reported a period of approximately 28 days for naturally occurring disease. In the genital tract, the number of inclusions detected in smears declined as the infection subsided. A close correlation between clinical signs and the finding of inclusion bodies in smears was reported by Kazdan et al. (5) for conjunctivitis. Murray (6) described the earliest evidence of infection in conjunctival smears as being the presence of polymorphonuclear leukocytes. Kazdan et al. (5) described the earliest changes to be a cellular response which was predominately lymphocytic followed by an intense heterophilic response. In our experiments, large numbers of mononuclear cells were never observed in vaginal smears. Interpretation of the histopathology of genital tract infection was complicated by the stage of the estrus cycle. The animals were inoculated without knowledge of the stage of the cycle and infection could have occurred at any stage. The finding of inclusions in secretory cells showed that such cells could support the multiplication of Gp-ic as well as the transitional cells. Since the secretory cells were shed very quickly, it is reasonable to conclude that the infection was mainly supported by the transitional cells of the stratified squamous epithelium. In sections of genital tract, many polymorphonuclear leukocytes were observed in the lumen, especially in the early stages of infection, followed, after a few days, by lymphocyte infiltration of the subepithelial tissue. This pattern was also noted by Kazdan et al. (5) for sections of conjunctiva. The presence of lymphocytic infiltration in subepithelial tissues, observed particularly in the latter stages of infection, may indicate a role of the cellular immune system. Murray (6) was unable to detect antibodies in sera from 50 guinea pigs with primary experimental ocular infections by using the complement fixation test. In another study, Murray and Radcliffe (7) reported inconsistent antibody titers when sera were tested between 15 and 30 days post-experimental infection. The differences in antibody response obtained in our studies may be a reflection of the route of infection or the methods of detection. In his original paper Murray (6) considered that infection of young guinea pigs probably occurred at 2 to 3 months of age. Kazdan et al. (5) obtained evidence for natural conjunctivitis as early as 15 days after birth. In our experiments newborn animals were infected in the eye, apparently during delivery, because the infection was detected as early as 3 days. The results obtained in this investigation have encouraged us to use the Gp-ic model system for the study of oculo-genital chlamydial infections such as inclusion conjunctivitis in man. In particular, problems concerning tissue specificity, mode of transmission, and immunity are currently being investigated. ACKNOWLEDGMENTS This investigation was supported by Public Health Service General Research support grant FR from the Division of Research Facilities and Resources to D.T.M., and grant EY00079 from the National Eye Institute to A.L.B. P. E. Bigazzi is a recipient of a Henry C. Buswell and Bertha H. Buswell Fellowship. LITERATURE CITED 1. Alexander, E. F., and W. T. Chang Infection of pregnant monkeys and their offspring with TRIC agents. Amer. J. Ophthalmol. 63: Barron, A. L., C. Olshevsky, and M. M. Cohen Characteristics of the BGM line of cells from African Green monkey kidney. Arch. Gesamte Virusforsch. 32: Bovarnick, M. R., J. C. Miller, and J. C. Snyder The influence of certain salts, amino acids, sugars, and protein on the stability of rickettsiae. J. Bacteriol. 59: Gordon, F. B., E. Weiss, A. L. Quan, and H. R. Dressler Observations on guinea pig inclusion conjunctivitis agent. J. Infect. Dis. 116: Kazdan, J. J., J. Schachter, and M. Okumoto Inclusion conjunctivitis in the guinea pig. Amer. J. Ophthalmol. 61: Murray, E. S Guinea pig inclusion conjunctivitis virus. I. Isolation and identification as a member of the psittacosislymphogranuloma-trachoma group. J. Infect. Dis. 114: Murray, E. S., and F. T. Radcliffe Immunologic studies in guinea pigs with guinea pig inclusion conjunctivitis (Gp-ic) Bedsonia. Amer. J. Ophthalmol. 63: Reed, L. J., and H. Muench A simple mlethod of estimating fifty per cent endpoints. Amer. J. Hyg. 27: