EXPERIMENTAL EPIDIDYMITIS AND URETHRITIS IN GRIVET MONKEYS PROVOKED BY CHLAMYDIA TRACHOMA TIS

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1 FERTIIJTY AND STERlLlTY Copyright e 1980 The American Fertility Society Vol. 34, No.3, September 1980 Printed in U.SA. EXPERIMENTAL EPIDIDYMITIS AND URETHRITIS IN GRIVET MONKEYS PROVOKED BY CHLAMYDIA TRACHOMA TIS BIRGER R. M0LLER, M.D.* PER-ANDERS MARDH, M.D.t Institute of Medical Microbiology, University of Aarhus. Aarhus. Denmark. and Department of Medical Microbiology, University of Lund. Lund. Sweden Recently Chlamydia trachomatis has been indicated as a common cause of acute epididymitis in young men. In the present study, the grivet monkey was established as an experimental model for such infection. A yolk sac suspension of C. trachomatis, immunotype K, was inoculated into the left spermatic cords of two monkeys. On days 7 and 14 postinoculation (pi), the left testis of each monkey was enlarged to twice its normal size. The epididymis and the spermatic cord on the same side were swollen and reddened. All layers of spermatic cord were infiltrated with lymphocytes and polymorphonuclear leukocytes, and the lumen, which was filled with an exudate. was reduced in diameter. The luminar epithelium was destroyed. The ducts of the left epididymis were filled with an inflammatory exudate. The epithelium was flattened. but there were no lesions. The seminiferous tubules of the left testis were normal, but edema and hyperemia were observed in the interstitial tissue. The right spermatic cord was inoculated with noninfected yolk sac suspension. On the right side, no inflammatory reactions were seen in the testis, the epididymis, or the spermatic cord. The monkeys had a yellowish discharge 7 to 14 days pi. Urethral smears revealed an increased number of polymorphonuclear leukocytes 7 to 42 days pi; 60 days pi, preinoculation values were found. Fertil Steril34: Chlamydia trachomatis is a frequent cause of nongonococcal and postgonococcal urethritis in males,1 but there is no evidence that the organism is a common cause of chronic, nonbacterial prostatitis.2 Berger and co-workers 3 reported a high prevalence of nongonococcal urethritis among men with acute epididymitis. C. trachomatis was isolated from the urethra and epididymal aspirates in a high proportion of these patients. In an extended study, 4 these authors found that twothirds of "idiopathic" epididymitis in 50 men under 35 years of age was associated with C. Received January 23, 1980; revised May 28, 1980; accepted June 4, *Institute of Medical Microbiology, University of Aarhus. Aarhus. tdepartment of Medical Microbiology, University of Lund. To whom reprint requests should be addressed at Solvegatan 23, S Lund, Sweden. trachomatis infection. The infection was often accompanied by azoospermia or oligospermia. In previous studies 5, 6 we found the female grivet monkey to be a suitable model for the study of experimental genital chlamydial infections. In such monkeys chlamydiae may spread canalicularly from the cervix to the uterine tubes and produce salpingitis. 6 In the present study C. trachomatis was inoculated into the spermatic cord of two grivet monkeys and provoked epididymitis. The gross lesions and the histologic findings in these two monkeys are described. MATERIALS AND METHODS Organism. A strain (no. 5155) ofc. trachomatis, immunotype K, was used. This strain had been re-isolated from a female grivet monkey which had 275

2 276 MOLLER AND MARDH September 1980 been inoculated with a strain recovered from the uterine tube of a woman with salpingitis. 7 The strain was inoculated into embryonated hen's eggs from which a yolk sac specimen was prepared as described elsewhere.5 The suspension contained 2 x 105 inclusion-forming units of chlamydiae/ml. A yolk sac suspension from a noninfected embryonated hen's egg served as control. Animals. Two male grivet monkeys (Cercopithecus aethiops) that had been captured in East Africa were used. Before use, they had been kept in quarantine for 6 weeks. The monkeys were caged individually in an isolation room during the investigation period. They weighed 2.6 kg and 2.8 kg, respectively. Operative Technique and Experimental Infection. All operations were performed under general anesthesia, usingo.15 ml ofphenycline hydrochloride (Sernylan, 20 mg/ml), 0.5 ml of chlorpromazine (0.25% solution), and 0.3 ml of atropine (0.1 % solution). Surgery was performed under aseptic conditions. The scrotum and the lower part of the abdomen were washed with soap and disinfected with 2% iodine tincture. The skin was covered with a sterile surgical drape. In both monkeys, the right spermatic cord was isolated by an incision in the proximal part of the scrotum. The spermatic cord was opened under an operating microscope. A needle, 0.2 mm in diameter, was introduced into the lumen of the cord through which 0.3 ml of the noninfected yolk sac suspension was inoculated. The spermatic cord was closed with suture within 2 minutes after its being opened. The same operative procedure was then undertaken on the left side, where the spermatic cord received an injection of 0.3 ml ofthe yolk sac suspension infected with chlamydiae. The scrotal wall was closed by sutures in two layers. After the operation was completed, the wounds were covered with spray plaster. Seven Days after Infection. The right testis, the epididymis, and the spermatic cord from the epididymis to the external inguinal ring of monkey I were extirpated in toto. A biopsy specimen was taken from the left spermatic cord. From monkey II the left testis, the epididymis, and the spermatic cord were extirpated. A biopsy specimen was taken from the right spermatic cord. Fourteen Days after Infection. The testis, the epididymis, and the spermatic cord of monkey I were extirpated from the left side. In monkey II, the same procedures were undertaken on the right side. After extirpation of the testes, the monkeys were given 0.5 mg of testosterone daily. Preparation of Specimens for Histologic Studies. The tissue biopsies and the extirpated genital organs were fixed in 10% formalin, paraffinembedded, and stained with hematoxylin and eosin. Preparation of Urethral Smears. Urethral secretion was collected under general anesthesia on the day of infection and 7, 14, 21, 28, and 35 days postinfection (pi) with the aid of a loop which was introduced 2 cm into the urethra. The secretion was Gram-stained and the number of leukocytes per high-power field (HPF, x 800) was counted. Culture Studies. Specimens were collected by a swab from the lumen of the left and the right spermatic cord on the day of infection and on days 7 and 14 pi. On the same days and on days 21, 28, 35, and 42 pi, specimens were also collected from the urethra. All specimens were cultured for C. trachomatis,8 mycoplasmas,5 and ureaplasmas5 as described earlier. Before the experiment was begun, cultures for these organisms had also been performed in samples obtained from the throat, the urethra, and the rectum of each monkey. Specimens collected from the spermatic cord on the day of infection and on the 2 days of reoperation were also cultured for bacteria, using brainheart infusion broth (Difco), and blood, hematin, and lactulose bromthymol blue agar plates. Serologic and Hematologic Studies. Blood samples were obtained by inguinal puncture of the femoral artery. Serum antibody to C. trachoma tis was determined by a microimmunofluorescence test. 9 The erythrocyte sedimentation rate, in millimeters per hour, and the leukocyte count, in cells per cubic millimeter, were also determined. RESULTS The general conditions of the two monkeys remained unaffected during the experimental period. Neither the leukocyte count nor the erythrocyte sedimentation rate was changed in either monkey. On day 7 pi, the right testis, the epididymis, and the spermatic cord were normal in both monkeys. On the left side, the spermatic cord and the epididymis were swollen and reddened. The testis on the left side was enlarged to twice its normal size, and its surface was pale (Fig. 1). The findings were identical in the two monkeys. In monkey I, the left testis was still enlarged on day 14 pi. The epididymis and the spermatic cord were swollen and reddened. In monkey II, the right

3 Vol. 34, No.3 EPIDIDYMITIS AND URETHRITIS PROVOKED BY C. TRACHOMATIS 277 no lesions or inflammatory infiltrates. The interstitial tissue was edematous and hyperemic (Fig. 3B). The left testis showed normal seminiferous tubules, but edema and hyperemia were observed in the interstitial tissue. No inflammatory cells or cystic structures were found. In both monkeys, the testis, the epididymis, and the spermatic cord on the right side were invariably normal when extirpated after 1 and 2 weeks, respectively (Figs. 2A and 3A). Before inoculation, the urethral smears of both monkeys contained 1 or 2 polymorphonuclear leukocytes/hpf. During the course of the infection, the corresponding figure rose to a maximum of 18 to 20 leukocytes/hpf on day 7 pi. Thereafter the leukocyte count declined steadily, although it was still increased on day 42 pi, when 8 and 10 to 12 leukocytes/hpf were demonstrated in monkeys I and II, respectively. Two months after the FIG. 1. Right and left testes of monkey I, day 7 pi. The right testis is normal, the left testis is enlarged to twice the size ofthe right testis ( x 1). testis, the epididymis, and the spermatic cord were normal. A yellowish discharge from the urethra was observed in both monkeys on days 7 and 14 pi. Marked inflammatory lesions were present in the left epididymis and the spermatic cord (infected with C. trachomatis), when extirpated on days 7 and 14 pi, respectively. On the right side, where the noninfected yolk sac suspension had been injected, no inflammatory reactions were seen in the specimens extirpated after 1 and 2 weeks. In both monkeys (I and II) there was an intense infiltration of lymphocytes and of some polymorphonuclear leukocytes in all layers of the wall of the left spermatic cord. The lumen ofthe spermatic cord was reduced in diameter and filled with an exudate containing lymphocytes and desquamated cells. The luminal epithelium was injured and infiltrated with inflammatory cells. The muscularis layers of the spermatic cord were infiltrated with a great number of lymphocytes. The cellular infiltration extended to the subserosa (Fig. 28). The lumina of the ducts of the left epididymis were filled with an exudate dominated by lymphocytes and polymorphonuclear leukocytes. The epididymal epithelium was flattened, but there were FIG. 2. A, Cross-section of the right spermatic cord of monkey I, inoculated with noninfected yolk sac suspension, day 7 pi. There are no signs of inflammation. B, Cross-section of the left spermatic cord of monkey II, inoculated with Chlamydia traclwmatis, day 7 pi. There is marked infiltration of lympho cytes and of some polymorphonuclear leukocytes through all layers ofthe spermatic cord; the lumen is filled with an exudate containing lymphocytes and desquamated cells, and the epithe lium is injured (hematoxylin and eosin, original magnification x 120l.

4 278 M0LLER AND MARDH September 1980 FIG. 3. A, Section ofthe right epididymis of monkey I, inoculated with noninfected yolk sac suspension, day 7 pi. There are no signs of inflammation. B, Section of the left epididymis of monkey II, inoculated with Chlamydia trachomatis, day 7 pi. The lumina of the ducts are filled with an exudate containing mostly lymphocytes and polymorphonuclear leukocytes. The epithelium is flattened, but no lesions are present <hematoxylin and eosin, original magnification x 120). inoculation there were 1 to 3 leukocytes/hpf in both monkeys. Before the experimental infection, no chlamydiae, mycoplasmas, or ureaplasmas were isolated from the monkeys. C. trachomatis was isolated from the urethra of monkey I on day 14 pi and from the left spermatic cord of monkey II on day 7 pi, whereas mycoplasmas and ureaplasmas were never isolated. No bacteria were isolated from the spermatic cord on any occasion. The microimmunofluorescence antibody tests revealed IgG antibodies to C. trachomatis in monkey I at a titer of 16 on day 42 pi, whereas in monkey II such antibodies at the same titer were found after 35 days. The titer in monkey II was 64 on day 42 pi. By day 75 no antibodies could be detected in either monkey. DISCUSSION A variety of organisms has been implicated as causes of chronic epididymitis in man. In such cases with granuloma formation, fungal, myco- bacterial, and parasitic infections may be demonstrated. Chronic epididymitis may also occur in patients with lymphogranuloma venereum, viz., in infections caused by C. trachomatis, immunotypes LGV 1 to 3.10 The majority of cases of acute epididymitis, particularly in young adult males, has until recently been classified as "idiopathic," that is, of unknown etiology.h, 12 In elderly men with hyperplasia of the prostate, established urinary tract pathogens, such as bacteria of the family Enterobacteriaceae, often are the causative agents.4 In young men the condition often seems to be a complication of sexually transmitted infection. It has long been known that Neisseria gonorrhoeae may cause acute epididymitis. Recent studies, however, suggest that C. trachomatis is a more common cause of this condition in men under 35 year of age. In such patients chlamydiae have been isolated from epididymal aspirates and a significant rise in the titer of serum antibodies to C. trachomatis has been demonstrated.3, 4, 13 Apart from infection, it has also been suggested that reflux of sterile urine may cause "idiopathic" epididymitis,14 but this has been refuted by othersy, 12 In males with acute epididymitis, the epididymis is enlarged and tender. The spermatic cord is generally involved and may be edematous. Urethritis and a discharge may also be demonstrated. Biopsy studies indicate that the testes are also frequently involved.15 The present study has shown that C. trachomatis may provoke epididymitis in grivet monkeys when injected into the lumen ofthe spermatic cord. Seven and fourteen days after the inoculation an inflammatory reaction was found in the epididymis, the spermatic cord, and the testis. In contrast, there were no signs of inflammation on the opposite side, on which sterile yolk sac suspension was injected into the lumen of the spermatic cord. Both monkeys also had signs of urethritis which persisted for more than 6 weeks. Both monkeys studied developed an antibody response to the organisms, although the titers were low. The poor antibody response may be due to the fact that the infected organs were extirpated within 1 to 2 weeks, thereby removing the deepsited infection. After the infection C. trachomatis could be reisolated from both animals. The inability to recover the organism from some of the' specimens studied might be due to an antichlamydial activity of components produced by the accessory genital glands. 16

5 Vol. 34, No.3 EPIDIDYMITIS AND URETHRITIS PROVOKED BY C. TRACHOMATIS 279 In chlamydial epididymitis in man the infection is believed to spread canalicularly through the spermatic cord, since hematogenous spread is not known to occur in C. trachomatis infections with immunotypes D through K (TRIC agents). Experimental infections also suggest that a canalicular spread of chlamydiae may occur in the genital tracts of female grivet monkeys.6 Thus, in such monkeys infected with the same strain ofchlamydia as used in the present study, salpingitis occurred after inoculation of the organism into the cervix and the uterine cavity, but not when the tubal isthmus had previously been closed with a ligature. In both monkeys, the lumina of the epididymal tubuli on the side that have been infected with chlamydiae were filled with an inflammatory exudate containing lymphocytes and polymorphonuclear leukocytes. The epididymal epithelium was flattened, but there was no inflammatory cell reaction in the interstitial tissue. There are few histologic studies on young men with acute "idiopathic" epididymitis. However, Wolin 17 reported acute tubular destruction and some micro-abscesses in the tubules of 13 of 21 such men. In these 13 patients there was an interstitial inflammation dominated by polymorphonuclear leukocytes. In the remaining eight patients no tubular involvement was found, but there was an interstitial inflammation dominated by lymphocytes. The design of the present study, in which the extirpations were performed 1 to 2 weeks pi, did not allow the establishment of whether experimental chlamydial epididymitis may lead to the destruction of epididymal tissue. Occlusion of the proximal parts of the epididymis may also result in atrophy of the testes. Azoospermia or oligospermia is a common finding in patients with acute epididymitis 4, 18 and might be explained by total or partial occlusion of the ductus deferens. The findings in the present study suggest that chlamydial epididymitis may be accompanied by such an occlusion, since a prominent feature in the two monkeys was edema and intense infiltration oflymphocytes and some polymorphonuclear leukocytes in all layers of the wall of the spermatic cord on the side inoculated with chlamydiae. The luminal epithelium was injured, and the lumen was reduced in size. The present study also suggests that ascending chlamydial infections can provoke inflammatory changes in the spermatic cord and the epididymis which might affect fertility. REFERENCES 1. Holmes KK, Handsfield HH, Wang SP, Wentworth BB, Turck M, Anderson JB, Alexander ER: Etiology of nongonococal urethritis. 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Acta Pathol Microbiol Scand Sect B 88:107, Mardh P-A, Ripa T, Svensson L, Westrom L: Chlamydia trachomatis infection of the 'fallopian tubes in patients with acute salpingitis. N Engl J Med 296:1377, Ripa T, Mardh P-A: The culture of Chlamydia trachomatis in cycloheximide-treated McCoy cells. J Clin Microbiol 6:326, Treharne JD, Darougar S, Jones BR: Modification of the micro immunofluorescence test to provide a routine serodiagnostic test for chlamydial infection. J Clin Pathol 30:510, Borvie W: Urethritis, prostatitis, epididymitis. Paper read at WHO Scientific Group for Nongonococcal Urethritis and Other Selected Sexually Transmitted Diseases of Public Health Importance, Geneva, November WHO Working Document INTNDT/ Mittemeyer BT, Lennox KW, Borshi AA: Epididymitis: a review of 610 cases. J Urol 95:390, Ross WM, Maynard JM: Non-specific epididymitis in the military service. US Armed Forces Med J 8:841, Harnish JP, Berger RE, Alexander ER, Monda G, Holmes KK: Aetiology of acute epididymitis. Lancet 1:819, Graves RS, Engel WJ: Experimental production ofepididymitis with sterile urine: clinical implications. J Urol 64:601, Nilsson S, Obrant KD, Persson PS: Changes in the testes parenchyma caused by acute non-specific epididymitis. Fertil Steril 19:748, Mardh P-A, Colleen S, Sylwan G: Inhibitory effect on growth of Chlamydia trachomatis by seminal fluid and some of its components. Invest Urol 17:510, Wolin LH: On the etiology of epididymitis. J Urol105:531, Tozzo PJ: Semen analysis in unilateral epididymitis. NY State J Med 1:2764, 1968