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Infection and Immunity, November 2003, p. 6148-6154, Vol. 71, No. 11
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.11.6148-6154.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Departments of Microbiology and Immunology,1 Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 722052
Received 8 May 2003/ Returned for modification 24 June 2003/ Accepted 28 July 2003
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ABSTRACT |
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INTRODUCTION |
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While genital tract infection models have been developed with primates (15), mice (2), marmosets (8), rabbits (16), and guinea pigs (9), sexual transmission has been demonstrated only for guinea pigs infected with GPIC. Barron first reported that male guinea pigs could be infected intraurethrally with GPIC and that, when they were housed with female guinea pigs, they transmitted the infection sexually (10). Interestingly, pregnant female guinea pigs could also transmit the infection to their offspring during parturition, producing conjunctivitis in the newborns, analogous to the human situation (9). Although Barron was able to show sexual transmission in guinea pigs, it was not possible to determine when the animals became infected or what the infecting dose from the male was.
In this laboratory, we have always infected guinea pigs and mice with high doses in order to guarantee infection and provide a strong antigenic stimulus. Other groups have used low infection doses in mice under the assumption that this dose may be more realistic. With regard to the evaluation of vaccine candidates for the animal model, it is particularly important that a dose closely approximating the natural infecting dose be used in the studies to accurately judge the protective capacity of the vaccine. Since sexual transmission has been demonstrated for the GPIC-guinea pig model, we undertook in this study to (i) develop a reliable and predictable model of sexual transmission, (ii) attempt to define the approximate infecting dose from a male guinea pig during sexual intercourse, and (iii) determine the effect of a "natural" challenge dose on the protective capacity of an inactivated chlamydial elementary body vaccine.
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MATERIALS AND METHODS |
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Female guinea pigs were infected intravaginally with various doses of chlamydiae suspended in 25 µl of sucrose-phosphate-glutamate buffer by insertion of a micropipette tip approximately 3 cm into the vagina so the suspension was deposited against the cervix. Male guinea pigs were anesthetized with methoxyflurane, and 25 µl of a GPIC suspension containing 107 inclusion-forming units (IFU) of GPIC was placed on the tip of the urethra with the external meatus retracted. Because of the anatomy, it is impossible to know exactly how many organisms entered the urethra, but it must be assumed that a large portion did not actually come in contact with the urethral epithelium.
In the dose-response and immunization experiments, animals not infected sexually were inoculated intravaginally on the same day. Thus, the stage of their estrous cycles was random in comparison to that of sexually infected animals, which, by definition, were in estrus. We have never observed a difference in the infection kinetics associated with the time of the estrous cycle when animals were infected, although we have seen significant differences in the appearances of upper genital tract pathologies (20).
Culture and isolation of chlamydiae. The agent of GPIC, a member of the species Chlamydia psittaci, was originally obtained from the late Edward Murray and has been continuously passaged in our laboratory. For infection purposes, GPIC is maintained in McCoy cells by standard techniques and aliquots of stock are frozen at -70°C in sucrose-phosphate-glutamate buffer. Chlamydiae were cultured and quantified from swabs according to standard procedures.
For isolation of chlamydiae from female animals, a sterile Dacroswab was inserted in the vagina until it came in contact with the cervix, and the swab was rotated against the cervix, removed, and placed in sucrose-phosphate buffer (23) containing 5% fetal calf serum, 50 µg of gentamicin per ml, 100 µg of vancomycin per ml, and 2.5 µg of amphotericin B (Fungizone) per ml. The swabs were frozen at -70°C until needed. Swabs were collected from male guinea pigs anesthetized with methoxyflurane. The urethra was exposed, and a Dacroswab was inserted approximately 2 cm into the urethra, rotated, removed, and placed in sucrose-phosphate buffer.
Sexual transmission. In order to establish a model in which the time of sexual transmission could be reliably determined, we took advantage of a unique aspect of the female guinea pig reproductive system. When animals are not in estrus, a membrane which covers the vagina is produced; however, as soon as the animal enters estrus, this membrane disappears and the female is receptive to copulation. Within 3 days following the onset of estrus, the membrane regenerates. Guinea pigs have an estrous cycle of approximately 15 to 17 days and are in estrus for only about 8 h.
Therefore, all female guinea pigs were monitored daily to determine the presence or absence of the vaginal membrane and each individual cycle was recorded. Females were monitored through a least two estrous cycles or until the time that the opening of the vaginal membrane could be predicted to a 3-day period (1 day with an error margin of plus or minus 1 day). Four to six days prior to a given female entering estrus, a male guinea pig was infected intraurethrally (21) and housed with the female in a large breeder cage. A swab was collected from the male to verify infection, and all males used in this study were found to be isolation positive. Monitoring for the opening of the vaginal membrane was continued until it was observed. The date when the vaginal membrane opened was considered to be the day that copulation had taken place. Beginning 3 days later, cervical swabs were collected from the female every 3 days until the resulting infection had resolved or we were convinced by repeated cultures that the animal remained uninfected. Animals were routinely monitored for at least 30 days. The male was removed 1 week after the date of copulation, and the female was moved to a single cage. Following resolution of the infection, the female was monitored to determine whether she had become pregnant. Pregnancy and/or infection was considered confirmation that the animals had copulated.
Immunization protocol. Guinea pigs were immunized subcutaneously with 100 µg of UV-inactivated GPIC elementary bodies (18) in phosphate-buffered saline emulsified to a 1:1 dilution in Freund's complete adjuvant (Sigma, St. Louis, Mo.). Two and 4 weeks later, the animals were given a booster injection with a similar preparation emulsified in Freund's incomplete adjuvant (Sigma). This regimen has been shown to elicit a significant protective response against genital challenge (18).
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RESULTS |
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The average length of the incubation period for the sexually infected guinea pigs was 8.3 days. This was significantly different from the length for the group receiving 104 IFU (P < 0.04, one-tailed t test) but not from those for the other groups. When the infection kinetics were compared (Fig. 1) by a two-factor (days and group) ANOVA with repeated measures, the infection curves of animals infected by sexual transmission were significantly different than the curves of guinea pigs artificially inoculated with either 104 IFU (P < 0.001) or 103 IFU (P < 0.01) but not 102 IFU. Only the animals which became infected in each group were compared. In general, the peak levels of infection were lower in the 102-IFU, 101-IFU, and sexual-transmission groups than the peak levels in the 104- and 103-IFU groups. The infection kinetics, with respect to the peak level of infection for this sexual-transmission group, were similar to those seen for the 102- and 101-IFU groups but were significantly different (P < 0.05, one-tailed t test) from those of the groups infected with 103 and 104 IFU. Based on the comparison of the infection kinetics, the length of the incubation period, and the percentage of animals becoming infected upon challenge, one can conclude that the female guinea pigs were receiving approximately 102 IFU by sexual transmission at this point in the males' infections.
Of major interest was the observation that the period of time that GPIC could be isolated from the genital tract was significantly shorter in the group which acquired their infection by sexual transmission (Table 1). The average length of shedding of organisms in this group was 9.9 days, whereas the minimum mean length of time of shedding for the groups receiving the inoculant artificially was 15 days. These data were highly significant when they were assessed by a one-tailed t test.
We also determined the percentage of infected animals that became pregnant versus the percentage of those that were not infected following sexual contact that became pregnant. There was no observed difference in the pregnancy rates of the uninfected animals (57%) and the infected guinea pigs (50%), suggesting that, at least in animals infected at the time of fertilization, chlamydial infection had no effect on conception or early pregnancy.
Effect of immunization upon challenge by sexual transmission. An important potential application of a model for sexual transmission is to evaluate the protective capacity of vaccines. In order to determine whether protection against a sexual challenge could be induced with a vaccine, female guinea pigs were immunized with a preparation of UV-inactivated elementary bodies of GPIC. A group of 10 females was immunized as indicated in Materials and Methods. Beginning a minimum of 2 weeks after the last booster inoculation, infected male guinea pigs were grouped singly with individual females as described above. This experiment was being performed at the same time as the experiment characterizing the course of infection by sexual transmission described above; thus, the animals described above served as unimmunized controls. Because of the variation associated with the estrous cycles of the individual guinea pigs, immunized animals could not be challenged at exactly the same time. Thus, the range of time following the last booster when the females were challenged extended from 46 to 143 days.
For a comparison of the results of the above-described experiments to those of artificial challenge of immunized guinea pigs with known numbers of IFU, we performed an experiment in which guinea pigs were inoculated intravaginally with either 103, 102, or 101 IFU 2 weeks after the last booster injection. The experiment was repeated with just 103 and 102 IFU. All of the data from the two artificial-challenge experiments and the sexual-transmission challenge experiment are shown in Fig. 1 for comparison.
When the percentage of animals becoming infected upon artificial challenge was determined, guinea pigs receiving 102 IFU demonstrated a high level (P < 0.0001 by chi-square analysis) of sterile immunity; i.e., no infectious organisms could be isolated from cervical swabs at any of the times tested (Fig. 2). Because only two of five control animals inoculated with 101 organisms became infected, we could not determine statistical differences for that group. Although only a low level of sterilizing immunity was seen in the group challenged with 103 organisms (70% positive for infection in the immunized group versus 90% in the unimmunized group), the peak level of infection was significantly reduced by immunization (P < 0.001; two-factor [days and group] ANOVA with repeated measures) (Fig. 3). However, the length of the infection remained unchanged. The length of infection in unimmunized animals was 19.7 days, compared to 17.6 days in infected immunized animals. This difference was not statistically different. This finding was very similar to what we had seen before in animals challenged with higher doses of chlamydiae (18).
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DISCUSSION |
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The GPIC genital infection model is the only experimental animal model for any sexually transmitted disease for which sexual transmission of infection has actually been demonstrated (10). It is made even more significant because the reproductive physiology of the guinea pig, in contrast to that of true rodents, resembles very closely that of the human (26). Guinea pigs have a 15- to 17-day estrous cycle, in contrast to the 5-day cycle of rodents, and are excellent models for human reproductive physiology. As we have previously reported, the pathological response in guinea pigs to GPIC genital infection is remarkably similar to that of humans in virtually all pathological parameters (19). We have also observed that, as in humans, GPIC genital infections are exacerbated by treatment with physiologic levels of estrogen (13) and with oral contraceptives (11). While we had previously found no effect on the course of infection by treatment with progesterone when high infection doses were used (12), we have recently observed that if one challenges animals receiving medroxyprogesterone acetate (Depo-Provera) with a natural-infection dose, the infection is increased in intensity (R. G. Rank, unpublished data).
Ultimately, in order to use the guinea pig model for the assessment of potential vaccine candidates, it was critical that we knew the actual infectious dose in infections acquired by sexual contact. Because the quantification of chlamydiae from urethral swabs of the male would not give the number of IFU contained in guinea pig semen during copulation, and because direct culture of guinea pig semen was impractical, we elected to gain an approximation of the infecting dose by comparing the course of the resulting infections in females to the course of those resulting from artificial intravaginal inoculation with known amounts of chlamydiae. Fortunately, the reproductive physiology and anatomy of the guinea pig are such that one can easily map the estrous cycle of individual animals and accurately predict when the female will be in estrus, the only time at which she will mate. The infection kinetics and percentage of animals that became infected were similar to those of animals artificially infected with 102 IFU. Obviously, there is likely to be a great deal of variability, especially related to the stage of the infection in the male when copulation takes place. In general, the male guinea pigs in this study were in the early part of their infection cycle (14). If the males had been at the peak of their infections, more organisms may have been transferred to the females, or conversely, if the infection was subsiding, the dose would have been considerably lower. It is difficult to know the minimal infectious dose for female guinea pigs by sexual transmission, although the ID50 for GPIC in the genital tracts of female guinea pigs was found to be 48 IFU. The infection rate for the animals infected sexually was 63.2%, so 100 IFU would be a reasonable approximation. In any case, our data suggest that, at least for the guinea pig, to model genital disease, an infectious dose of 102 to 103 IFU should be used. Note that Cotter et al. (5) observed an ID50 for mice with the mouse pneumonitis agent of 19 to 48 IFU as well. Moreover, in studies of human male volunteers with Neisseria gonorrhoeae, the infectious dose was found to be in the 102- to 103-IFU range (24).
We considered the possibility that infection may not have been via sexual contact but may have occurred through grooming or close contact in the cage. Fifty percent of the infected animals did become pregnant, which definitely indicates sexual contact. If genital infection was via grooming, we would expect to see ocular infections in the animals as well, since GPIC is by nature an ocular pathogen. We monitored the females for ocular infection, and none of the 29 females in the study developed ocular infections; thus, it is highly unlikely that transmission occurred without sexual contact.
While 102 IFU is suggested as the infectious dose for guinea pigs, there is no way to extrapolate to humans or other animal models. There are no published studies quantifying the number of chlamydiae in semen and few studies determining the number of IFU in the male genital tract. Geisler et al. (7) did analyze the association between various signs and symptoms in the male to the number of IFU in a 100-µl well. They reported that the number of IFU was in the 101-to-102 range. With this number of organisms, even considering a larger volume transmitted to the female, it is unlikely that very high numbers of organisms are in the infectious inoculum. It is interesting that in the male guinea pig, there is inflammation in the urethral epithelium but not overt exudate as one sees in human males with urethritis (21). Moreover, if one considers that frequent urine flow provides a periodic flushing action, then any organisms transmitted must be newly released from infected cells. The majority of human males infected with Chlamydia are asymptomatic, with no apparent exudate (22), so the guinea pig data support the concept that just a few organisms can elicit an infection.
Perhaps the most important and novel finding in our study was the observation that infections acquired sexually, in contrast to infections acquired by artificial intravaginal inoculation with any dose tested, have a significantly shorter course of shedding of detectable organisms. This finding was quite consistent, even when infection courses in immunized animals challenged artificially and sexually were compared. While it is certainly not clear at this point what the reasons are for the abbreviated infection, the most likely possibility is that there are factors associated with semen that inhibit chlamydial growth. One of the antimicrobial factors which may have an effect on early chlamydial infection is the group of polypeptides known as defensins. Defensins are 3- to 6-kDa cationic peptides containing six disulfide-paired cysteines which make up 5 to 15% of the total protein content of neutrophils and have been found to have activity against a number of bacterial and viral pathogens. Defensins have been found to be present in various parts of the male reproductive tract, including the semen of rats, mice, and humans (4). Moreover, two different guinea pig defensins derived from neutrophils have been described previously (25, 27). It is possible that defensins in semen deposited in the female genital tract kill some of the chlamydiae before they can infect their target cells and/or remain in the genital tract and interfere with subsequent generations of chlamydiae. It is also conceivable that cytokines and chemokines produced in the male as a result of an ongoing infection are being passively transferred and act to enhance or hasten the inflammatory response in the female.
Whatever the mechanism, these data indicate that artificial inoculation of the genital tract in animal models may not accurately mimic all of the factors involved in natural infection and that one must take into consideration the possible role of passively transferred factors from the male. Even the transfer of factors from the male vary depending on the stage of the infection course in the male. Clearly, there is great variability in the course of chlamydial genital infection from individual to individual, and this variability may result from factors beyond those associated with the host response in the female.
Finally, a major role for animal models is in the evaluation of vaccine candidates. We have previously demonstrated that the GPIC-guinea pig model resembles human female genital infection very closely with regard to the pathological response (19). Moreover, we have reported that immunity to intravaginal-challenge infection could be elicited by immunization with an inactivated whole-organism vaccine or by purified outer membrane protein 2 (3, 18). While immunization with UV-inactivated GPIC did not prevent infection in 50% of the animals, the resultant infections were much less severe. Whether infection ascending to the upper genital tract was prevented remains to be determined in future experiments. It was apparent from guinea pigs artificially challenged with GPIC that animals with low challenge doses, i.e., 102 and 101 IFU, were more likely to have solid immunity in which no organisms could be isolated. That the infection rate in animals challenged by sexual transmission was somewhat higher suggests that they may have received actual challenge doses of 102 to 103 IFU by sexual transmission. Alternatively, it is entirely possible that the viscous nature of semen blocked the attachment of vaginal or cervical antibodies to the organism, effectively increasing the infection rate compared to that of animals inoculated with organisms suspended in sucrose-phosphate-glutamate buffer. Antibody is essential for immunity to reinfection in the guinea pig model (17). This finding demonstrates the real value of a sexual-transmission model in that there may be complicating factors associated with the natural means of infection which cannot be identified in the artificial inoculation of organisms suspended in a buffer. Of interest was the observation that the resultant infections in immunized animals infected sexually were also significantly abbreviated compared to those of animals challenged artificially with similar doses. This finding again suggests some contribution of antimicrobial or immunity factors from the male.
The ability to reliably and predictably accomplish sexual transmission in the guinea pig model is a unique opportunity to evaluate possible vaccine candidates or strategies for a natural infection, especially since there is no other animal model for chlamydial infection or, for that matter, any other sexually transmitted diseases in which sexual transmission has been demonstrated. In addition, the model provides an excellent opportunity to evaluate the effect of immunizing the male on his ability to infect an immunized or unimmunized female. We have previously reported that an inactivated whole-organism vaccine could induce a marked reduction in the level of a challenge infection in the male (14). This finding suggests that an immunized male may transmit fewer organisms and, consequently, maximize the protective capability of the vaccine in a female. With the guinea pig model, this strategy as well as other strategies can finally be tested.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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