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Isolation of Bedsoniae from Human Arthritis and Abortion Tissues
ISOLATION O F BEDSONIAE FROM HUMAN A R T H R I T I S AND ABORTION T I S S U E S JULIUS SCHACHTER, P H . D .
The role of Bedsoniae in human abortion and human arthritis is not known. The pres ent study, to explore their possible influ ence, is still in a preliminary stage, and had its genesis in analogies with dis eases of lower animals. Mendlowski1 showed that a psittacosislike agent caused a polyarthritis in sheep. This finding was confirmed and intensively studied by Shupe, Storz and associates2'3 who showed that the infection is systemic and not limited to the joints. The infected sheep have gastrointes tinal involvement and develop conjunctivitis (Storz, personal communication). Abortions due to infection by a Bedsonia are common ly found in sheep4 and cattle.5
The initial emphasis in the studies on ar thritis was placed on Reiter's syndrome be cause of its suggestive triad of symptoms: arthritis, urethritis and conjunctivitis. The agents of trachoma and inclusion conjunc tivitis (TRIC) and LGV are capable of in fecting the conjunctiva and/or urethra. Siboulet and Galistin8 may have isolated a tra chomalike agent from the urethra of a pa tient with Reiter's syndrome and other Workers have demonstrated intracytoplasmic inclusions in the synovial tissues of such patients.9-10 Results of serologie and isola tion studies performed in this laboratory have been reported elsewhere.11
Some evidence suggests that Bedsoniae may be involved in the same diseases in hu mans. Arthritis is described as a rare com plication of lymphogranuloma venereum (LGV). e Cech,7 studying habitual aborters in Czechoslovakia, presented serologie data indicating a correlation between psittacosis infection (as measured by complementfixing antibody to a group antigen) and in creased incidence of abortion. The present study was undertaken in col laboration with the University of California Rheumatic Disease Group (under the direc tion of Dr. E. P. Engleman) and the Ob stetrics Division (Dr. A. Margolis). Ophthalmologic examination of subjects was made by Dr. C. R. Dawson at the Proctor Foundation. These investigators were re sponsible for clinical diagnosis.
MATERIALS AND METHODS CLINICAL SPECIMENS
Abortions. Expelled fetuses, in some cases pooled with material obtained by curettage, were collected within eight hours of abortion. All pregnancies appeared to have been in the first trimester and differentiation of tissue was impossible. The material was refrigerated until collected. Suspected in duced or septic abortions were excluded from the study. Impression smears were made from the specimen and stained with Giemsa. Specimens heavily contaminated with bac teria were discarded. Those selected for iso lation were cut into cubes and flamed on all sides. The tissue was then ground with car borundum, and prepared as a 50,% suspen sion in a broth containing 2.5 mg strepto mycin, 0.5 mg neomycin, 0.5 mg ristocetin and 100 units nystatin per ml. The suspension was kept at room temperature for one hour, clarified by centrifugation and inoculated into eggs. Bedsonia isolation was attempted on 22 abortions. Cervical smears were also collected from a group of 72 nor mal women appearing for a routine Papanicolaou stain.
From the George Williams Hooper Foundation and the Francis I. Proctor Foundation for Re search in Ophthalmology, University of Califor nia San Francisco Medical Center. This investi gation was supported by United States Public Health Service grants AI-04759, TW-00144 and NB-06207. Reprint requests to: Dr. Julius Schachter, G. W. Hooper Foundation, San Francisco Medical Center, School of Medicine, San Francisco, Cali Arthritis. Synovial fluid was obtained by fornia 94122. 1082/56
TRACHOMA AND ALLIED DISEASES, aspiration and used directly for isolation. Synovial membrane specimens were col lected by punch or open surgical biopsy. The tissue was ground with sterile nutrient broth for isolation studies. Conjunctival, urethral or cervical scrapings were placed in 1.0 ml of the antibiotic broth already de scribed and inoculated into eggs after one hour at room temperature. Topical anesthe sia was used for conjunctival and urethral scrapings and specimens were collected with Jones-Dunlop spatulas. Blood specimens were taken for serologie studies. Attempts to isolate Bedsonia were performed on tis sues from 59 patients with arthritis and five of their consorts. Serology. Complement fixation (CF) tests were performed as described by Meyer and Eddie.12 The antigen was a boiled phenolized suspension of the psittacosis iso late 6BC. A serum titer of 1:16 was consid ered significant. Complement fixation tests were run on all arthritis patients on whom isolation studies were done. In addition, sera from 300 patients appearing at the Ar thritic Clinic were tested. Isolation technique. The test specimens were inoculated into the yolk sacs (YS) of seven-day-old embryonated hens' eggs that had been incubated at 37°C before inocula tion and were kept at 34°C thereafter. The eggs were candled daily and the YS harvest ed from any egg dying more than three days after inoculation. Embryos still viable 13 days postinoculation were killed by chilling and the YS harvested. All yolk sacs were tested for sterility with thioglycolate broth. Impression smears were made and stained by a modified Macchiavello stain. One milliliter of a 50% YS suspension was inocula ted into each of four seven-day-old eggs for blind passage. If neither elementary bodies nor egg lethality could be seen by the end of the second blind passage, the attempt was terminated as negative. A positive isolation depended upon maintenance of regular egg lethality, the presence of characteristic ele mentary bodies in the YS smears and
1083/57
demonstrable (by CF test) Bedsonia group antigen in the YS suspensions. RESULTS
Abortion. Bedsoniae were isolated from four of the 22 abortion specimens tested. Experiments not yet reported indicate that at least two of these isolates differ from the TRIC agents by being resistant to sulfonamides and virulent for mice and do not pro duce follicular conjunctivitis in the subhu man primate. Inclusions were not seen in impression smears of the abortion specimens. The tis sues were macerated, and often few cells, other than red blood cells and polymorphonuclear elements, were found in the smears. An incidence of bizarre cellular forms compounded the difficulty of reading the slides. Of the 72 cervical smears taken from normal women, typical intracytoplasmic inclusions were found in four (5.5%). Arthritis. Of the 59 patients with arthri tis, the tissues of eight yielded Bedsoniae. Table 1 presents the laboratory results on known infected patients. Six of these eight patients had Reiter's syndrome. The other two (Patients 69 and 147) were clinically diagnosed as having rheumatoid arthritis. One of them had an atypical episodic arthri tis consistent with that seen in Reiter's syn drome, and each had a complement fixation titer of 1:16. Of the six patients with Rei ter's syndrome, whose tissues yielded Bed soniae, three had significant CF titers. Of the other patients with Reiter's syndrome, on whom isolation attempts were negative or whose serum only was available for study, two had CF titers greater than 1:16. The total of five of 15 (or 33%) patients with CF titers for the Reiter's syndrome cases compares to 12 of 300 (or 4%) positive ti ters in patients with other types of arthritis. When possible, the patients from the Ar thritis Clinic who had positive CF titers were brought back for isolation studies. This led to the positive isolation attempts on Patients 69 and 147. Isolation attempts on
AMERICAN JOURNAL OF OPHTHALMOLOGY
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MAY, 1967
TABLE 1 LABORATORY RESULTS ON PATIENTS WITH ARTHRITIS WHOSE TISSUES YIELDED PSITTACOSIS GROUP AGENTS
Isolation Results
Patient No.
Diagnosis
25 65 69 74 98 108 121 147
Reiter's syndrome Reiter's syndrome Rheumatoid arthritis Reiter's syndrome Reiter's syndrome Reiter's syndrome Reiter's syndrome Rheumatoid arthritis
CF Titer
Synovial Membrane
+
1:32 <1:2 1:16 1:4 1:128 1:128 1:2 1:16
ND ND
+ + + ND +
Synovial Fluid
Conjunctiva
ND
+
+
ND ND
+ + — + ND
ND ND
+ — + —
—
+ —
ND
Urethra
—
+ = positive; — = negative; ND = not done synovial tissue obtained from traumatized joints or autopsy specimens were uniformly negative. The isolation results on all patients are summarized in Table 2. Inclusions were not seen in smears pre pared from any of the specimens. In several instances, particles similar to elementary bodies were seen individually and in clusters. However, typical intracytoplasmic forms were not found. Sections of synovial membranes were difficult to read because of the presence of many mast cells. A consis tent finding in the synovial specimens from patients with Reiter's syndrome was the presence of many large phagocytic cells reminiscent of the Leber's cells of trachoma. Possibly degenerating material within these cells could be mistaken for microbial inclu sions.
In each instance of isolation from the synovium, the membrane (if available) was positive, but the fluid from two patients was negative. Three of the eight isolation-posi tive patients did not have significant CF titers against the psittacosis antigen. Two of them (Patients 65 and 121) did not have active arthritis at the time of examination. The main findings were urethritis and uveitis, and the isolates were achieved from the urethra and/or conjunctiva. The Bedsoniae isolated in this study differ from the previously described agents causing trachoma, inclusion conjunctivitis and lymphogranuloma venereum. Prelimi nary results indicate that the isolates are sulfonamide-resistant and do not produce iodine-staining matrices. The Bedsoniae iso lated from patients with arthritis were viru-
TABLE 2 RESULTS OF ATTEMPTS TO ISOLATE PSITTACOSIS GROUP AGENTS FROM PATIENTS WITH ARTHRITIS
Clinical Diagnosis
Isolation: Number positive/Attempts No. of Synovial ,Patients Fluid Synovial ConSkin Cervix Membrane junctiva Urethra
I. Reiter's syndrome a. Definite, early b. Definite, long standing c. Possible II. Consorts III. Rheumatoid arthritis IV. Other arthritis V. Normal or traumatici
8 5 4 5 15 18 17
*>1:16 t Includes 8 postmortem specimens
2/6
4/5
2/7
2/7
0/2
—
0/2
—
— —
0/4 0/2
1/5 0/3
0/1
-
1/8 0/11 0/1
1/8 0/6 0/17
0/2 0/4 —
0/1 0/1
— -
0/5 0/1 0/1
—
—
—
—
CF Titers*
3/8 0/5 0/4 3/5 2/15 3/18 0/9
TRACHOMA AND ALLIED DISEASES lent for mice and hamsters, but infrequently lethal for guinea pigs. However, guinea pigs, after intraperitoneal infection, irregu larly developed a transient arthritis. The only isolate tested regularly produced an acute arthritis in Macaca irus monkeys after intra-articular infection, and these animals developed flexion contractures. Attempts to produce a follicular conjunctivitis in subhu man primates were unsuccessful. DISCUSSION
The isolation of Bedsoniae from abortion specimens or patients with arthritis does not establish a causal relationship between the agent and the disease. It does demonstrate that members of the group, other than the agents of lymphogranuloma venereum, tra choma or inclusion conjunctivitis, naturally infect man and that infection by these newly isolated agents is associated with certain pathologic conditions. Isolates were achieved from the tissues of six patients with Reiter's syndrome and from two patients with rheumatoid arthritis who had positive Bedsonia complement fixa tion titers. Each patient with Reiter's syn drome, whose synovial tissue yielded a Bed sonia, was in the initial attack of the dis ease. Of the six patients with Reiter's syn drome whose tissues yielded isolates, only three had positive CF titers. This propor tion agrees generally with the results on pa tients infected with the agent of inclusion conjunctivitis. In our series of patients, 60% had CF titers greater than 1:16. Eight other patients with Reiter's syndrome were also studied and results of isolation and se rologie tests were negative. The clinical course in the two groups was essentially the same, although some of the patients whose test results were negative were in later stages or recurrences of the disease. These patients may not have been infected by a Bedsonia. However, in some cases the nega tive results could be ascribed to the stage of the disease when the specimens were col
1085/59
lected. The C F titer of Patient 102 was 1:128 when he was seen at the height of his illness, but several weeks later his titer dropped precipitously to > 1:2. If the Bed soniae agents and the disease are in a simple cause-and-effect relationship, then there may be another cause for an identical symptom complex in the isolate-negative patients. This assumes that negative results were not due to temporal effects or insensitive tech niques. The relationship between the infec tion and the disease may be more complex, however, involving immune mechanisms, double infections or predisposing factors. The epidemiology of these infections is unknown. Although sexual transmission offers an attractive hypothesis, attempts to isolate Bedsoniae from the cervix failed with five consorts, three of whom had significant C F titers. Cervical infection with the agent isolated from patients with arthritis or with the agent of inclusion conjunctivitis could ex plain the isolates obtained from abortion material. The specimens could have been contaminated during passage through the cervical canal. The handling of specimens was designed to minimize sujface contami nation but offered no certainty. The sig nificance of these isolations, be they cau sative for the abortions or cervical in origin, is therefore not assessable. The inclusions formed by different Bedsoniae appear iden tical in clinical specimens. The agents are unidentifiable, but the cytologie studies on cervical scrapings indicate a much higher prevalence (5.5%) of agent than that of clinical inclusion conjunctivitis. This is to be expected, for a chain of venereal infection precedes the ocular disease. Cervical infec tion need not result in neonatal conjunctivi tis. I have seen an instance where only the second-born of twins developed a unilateral conjunctivitis and the mother's cervix yield ed an inclusion conjunctivitis agent. The first-born child was apparently not infected. The agents isolated in this study demand
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AMERICAN JOURNAL OF OPHTHALMOLOGY
respect in the laboratory. T h e y are of high virulence for mice and probably present a potential risk to laboratory personnel. Rigid precautions must be taken during isolation procedures. A total synovectomy was per formed on Patient 147. This presented a much greater mass of tissue than is usually available. T h e eggs died in six to eight days of the first passage and the smears were rich in elementary bodies. I n this laborato ry, blind-passage material and established isolates are handled in different facilities. Before it was realized that the isolation at tempt on this patient was positive, two other groups of eggs were harvested immediately after the first specimens from Patient 147 were handled. T h e yolk sacs from these other eggs became contaminated. More often positive results became evident in the second passage and only occasionally at the end of the first. T h e potential significance of the findings in this study is obvious. N o conclusions, however, can be reached at this time. A n etiologic role for members of the Bedsonia group in certain types of human arthritis or abortion may exist but is not assumed. A proper assessment requires further research and will be made retrospectively. SUMMARY
Bedsoniae were isolated from eight pa tients with arthritis, six of whom had Reiter's syndrome. Attempts to isolate Bed soniae from human abortion specimens were positive in four of 22 instances. Inclusions were found in four of 72 cervical scrapings from a normal population.
MAY, 1967
REFERENCES
1. Mendlowski, B. and Segre, D. : Polyarthri tis in sheep: I. Description of the disease and ex perimental transmission. Am J. Vet. Res. 21:68, 1960. 2. Shupe, J. L. and Storz, J. : Pathologic study of psittacosis-lymphogranuloma polyarthritis of lambs. Am. J. Vet. Res., 25:943, 1964. 3. Storz, J., Shupe, J. L., James, L. F. and Smart, R. A. : Polyarthritis of sheep in the intermountain region caused by a psittacosis lymphogranuloma agent. Am. J. Vet. Res. 24:1201, 1963. 4. Stamp, J. T., McEwen, A. D., Watt, J. A. A. and Nisbet, D. I. : Enzootic abortion in ewes. I. Transmission of the disease. Vet. Ree. 62:251, 1950. 5. McKercher, D. G., Wada, E. M., Robinson, E. A. and Howarth, J. A.: Epizootiologic and im munologie studies of epizootic bovine abortion. Cornell Vet. 56:433, 1966. 6. Dawson, M. H., and Boots, R. L. : Arthritis associated with lymphogranuloma venereum. JAMA, 113:1162, 1939. 7. Cech, J., Drasnar, J., Strauss, J. and Skvrnovâ, K. : Die Diagnostik und Therapie latenter Ornithoseinfektionen bei infertilen Frauen. Arch. Gynaek. 194:239,1960. 8. Siboulet, A. and Galistin, P . : Arguments in favour of a virus aetiology of non-gonococcal urethritis illustrated by three cases of Reiter's dis ease. Brit. J. Ven. Dis. 38:209, 1962. 9. Levy, J.-P., Ryckewaert, A., Silvestre, D., Kahn, M.-F. and Mitrovic, D. : Etude par mi croscopie électronique des inclusions des cellules synoviales dans un cas de syndrome oculo-uré thro-synovial (Fiessinger-Leroy-Reiter). Path. & Biol. 14:216, 1966. 10. Amor, B., Coste, F. and Delbarre, F. : Sur l'origine virale possible du syndrome oculo-uré thro-synovial. Presse Med. 73:1825, 1965. 11. Schachter, J., Barnes, M. G., Jones, J. P. Jr., Engleman, E. P. and Meyer, K. F . : Isolation of. Bedsoniae from the joints of patients with Reiter's syndrome. Proc. Soc. Exper. Biol. & Med. 122:283, 1966. 12. Meyer, K. F. and Eddie, B. : In: Diag nostic Procedures for Viral and Rickettsial Dis eases (E. H. Lennette and N. J. Schmidt, eds). New York, Am. Pubi Hlth Ass. Inc., 1964, ed. 3, pp. 603-639.
The role of Bedsoniae in human abortion and human arthritis is not known. The pres ent study, to explore their possible influ ence, is still in a preliminary stage, and had its genesis in analogies with dis eases of lower animals. Mendlowski1 showed that a psittacosislike agent caused a polyarthritis in sheep. This finding was confirmed and intensively studied by Shupe, Storz and associates2'3 who showed that the infection is systemic and not limited to the joints. The infected sheep have gastrointes tinal involvement and develop conjunctivitis (Storz, personal communication). Abortions due to infection by a Bedsonia are common ly found in sheep4 and cattle.5
The initial emphasis in the studies on ar thritis was placed on Reiter's syndrome be cause of its suggestive triad of symptoms: arthritis, urethritis and conjunctivitis. The agents of trachoma and inclusion conjunc tivitis (TRIC) and LGV are capable of in fecting the conjunctiva and/or urethra. Siboulet and Galistin8 may have isolated a tra chomalike agent from the urethra of a pa tient with Reiter's syndrome and other Workers have demonstrated intracytoplasmic inclusions in the synovial tissues of such patients.9-10 Results of serologie and isola tion studies performed in this laboratory have been reported elsewhere.11
Some evidence suggests that Bedsoniae may be involved in the same diseases in hu mans. Arthritis is described as a rare com plication of lymphogranuloma venereum (LGV). e Cech,7 studying habitual aborters in Czechoslovakia, presented serologie data indicating a correlation between psittacosis infection (as measured by complementfixing antibody to a group antigen) and in creased incidence of abortion. The present study was undertaken in col laboration with the University of California Rheumatic Disease Group (under the direc tion of Dr. E. P. Engleman) and the Ob stetrics Division (Dr. A. Margolis). Ophthalmologic examination of subjects was made by Dr. C. R. Dawson at the Proctor Foundation. These investigators were re sponsible for clinical diagnosis.
MATERIALS AND METHODS CLINICAL SPECIMENS
Abortions. Expelled fetuses, in some cases pooled with material obtained by curettage, were collected within eight hours of abortion. All pregnancies appeared to have been in the first trimester and differentiation of tissue was impossible. The material was refrigerated until collected. Suspected in duced or septic abortions were excluded from the study. Impression smears were made from the specimen and stained with Giemsa. Specimens heavily contaminated with bac teria were discarded. Those selected for iso lation were cut into cubes and flamed on all sides. The tissue was then ground with car borundum, and prepared as a 50,% suspen sion in a broth containing 2.5 mg strepto mycin, 0.5 mg neomycin, 0.5 mg ristocetin and 100 units nystatin per ml. The suspension was kept at room temperature for one hour, clarified by centrifugation and inoculated into eggs. Bedsonia isolation was attempted on 22 abortions. Cervical smears were also collected from a group of 72 nor mal women appearing for a routine Papanicolaou stain.
From the George Williams Hooper Foundation and the Francis I. Proctor Foundation for Re search in Ophthalmology, University of Califor nia San Francisco Medical Center. This investi gation was supported by United States Public Health Service grants AI-04759, TW-00144 and NB-06207. Reprint requests to: Dr. Julius Schachter, G. W. Hooper Foundation, San Francisco Medical Center, School of Medicine, San Francisco, Cali Arthritis. Synovial fluid was obtained by fornia 94122. 1082/56
TRACHOMA AND ALLIED DISEASES, aspiration and used directly for isolation. Synovial membrane specimens were col lected by punch or open surgical biopsy. The tissue was ground with sterile nutrient broth for isolation studies. Conjunctival, urethral or cervical scrapings were placed in 1.0 ml of the antibiotic broth already de scribed and inoculated into eggs after one hour at room temperature. Topical anesthe sia was used for conjunctival and urethral scrapings and specimens were collected with Jones-Dunlop spatulas. Blood specimens were taken for serologie studies. Attempts to isolate Bedsonia were performed on tis sues from 59 patients with arthritis and five of their consorts. Serology. Complement fixation (CF) tests were performed as described by Meyer and Eddie.12 The antigen was a boiled phenolized suspension of the psittacosis iso late 6BC. A serum titer of 1:16 was consid ered significant. Complement fixation tests were run on all arthritis patients on whom isolation studies were done. In addition, sera from 300 patients appearing at the Ar thritic Clinic were tested. Isolation technique. The test specimens were inoculated into the yolk sacs (YS) of seven-day-old embryonated hens' eggs that had been incubated at 37°C before inocula tion and were kept at 34°C thereafter. The eggs were candled daily and the YS harvest ed from any egg dying more than three days after inoculation. Embryos still viable 13 days postinoculation were killed by chilling and the YS harvested. All yolk sacs were tested for sterility with thioglycolate broth. Impression smears were made and stained by a modified Macchiavello stain. One milliliter of a 50% YS suspension was inocula ted into each of four seven-day-old eggs for blind passage. If neither elementary bodies nor egg lethality could be seen by the end of the second blind passage, the attempt was terminated as negative. A positive isolation depended upon maintenance of regular egg lethality, the presence of characteristic ele mentary bodies in the YS smears and
1083/57
demonstrable (by CF test) Bedsonia group antigen in the YS suspensions. RESULTS
Abortion. Bedsoniae were isolated from four of the 22 abortion specimens tested. Experiments not yet reported indicate that at least two of these isolates differ from the TRIC agents by being resistant to sulfonamides and virulent for mice and do not pro duce follicular conjunctivitis in the subhu man primate. Inclusions were not seen in impression smears of the abortion specimens. The tis sues were macerated, and often few cells, other than red blood cells and polymorphonuclear elements, were found in the smears. An incidence of bizarre cellular forms compounded the difficulty of reading the slides. Of the 72 cervical smears taken from normal women, typical intracytoplasmic inclusions were found in four (5.5%). Arthritis. Of the 59 patients with arthri tis, the tissues of eight yielded Bedsoniae. Table 1 presents the laboratory results on known infected patients. Six of these eight patients had Reiter's syndrome. The other two (Patients 69 and 147) were clinically diagnosed as having rheumatoid arthritis. One of them had an atypical episodic arthri tis consistent with that seen in Reiter's syn drome, and each had a complement fixation titer of 1:16. Of the six patients with Rei ter's syndrome, whose tissues yielded Bed soniae, three had significant CF titers. Of the other patients with Reiter's syndrome, on whom isolation attempts were negative or whose serum only was available for study, two had CF titers greater than 1:16. The total of five of 15 (or 33%) patients with CF titers for the Reiter's syndrome cases compares to 12 of 300 (or 4%) positive ti ters in patients with other types of arthritis. When possible, the patients from the Ar thritis Clinic who had positive CF titers were brought back for isolation studies. This led to the positive isolation attempts on Patients 69 and 147. Isolation attempts on
AMERICAN JOURNAL OF OPHTHALMOLOGY
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MAY, 1967
TABLE 1 LABORATORY RESULTS ON PATIENTS WITH ARTHRITIS WHOSE TISSUES YIELDED PSITTACOSIS GROUP AGENTS
Isolation Results
Patient No.
Diagnosis
25 65 69 74 98 108 121 147
Reiter's syndrome Reiter's syndrome Rheumatoid arthritis Reiter's syndrome Reiter's syndrome Reiter's syndrome Reiter's syndrome Rheumatoid arthritis
CF Titer
Synovial Membrane
+
1:32 <1:2 1:16 1:4 1:128 1:128 1:2 1:16
ND ND
+ + + ND +
Synovial Fluid
Conjunctiva
ND
+
+
ND ND
+ + — + ND
ND ND
+ — + —
—
+ —
ND
Urethra
—
+ = positive; — = negative; ND = not done synovial tissue obtained from traumatized joints or autopsy specimens were uniformly negative. The isolation results on all patients are summarized in Table 2. Inclusions were not seen in smears pre pared from any of the specimens. In several instances, particles similar to elementary bodies were seen individually and in clusters. However, typical intracytoplasmic forms were not found. Sections of synovial membranes were difficult to read because of the presence of many mast cells. A consis tent finding in the synovial specimens from patients with Reiter's syndrome was the presence of many large phagocytic cells reminiscent of the Leber's cells of trachoma. Possibly degenerating material within these cells could be mistaken for microbial inclu sions.
In each instance of isolation from the synovium, the membrane (if available) was positive, but the fluid from two patients was negative. Three of the eight isolation-posi tive patients did not have significant CF titers against the psittacosis antigen. Two of them (Patients 65 and 121) did not have active arthritis at the time of examination. The main findings were urethritis and uveitis, and the isolates were achieved from the urethra and/or conjunctiva. The Bedsoniae isolated in this study differ from the previously described agents causing trachoma, inclusion conjunctivitis and lymphogranuloma venereum. Prelimi nary results indicate that the isolates are sulfonamide-resistant and do not produce iodine-staining matrices. The Bedsoniae iso lated from patients with arthritis were viru-
TABLE 2 RESULTS OF ATTEMPTS TO ISOLATE PSITTACOSIS GROUP AGENTS FROM PATIENTS WITH ARTHRITIS
Clinical Diagnosis
Isolation: Number positive/Attempts No. of Synovial ,Patients Fluid Synovial ConSkin Cervix Membrane junctiva Urethra
I. Reiter's syndrome a. Definite, early b. Definite, long standing c. Possible II. Consorts III. Rheumatoid arthritis IV. Other arthritis V. Normal or traumatici
8 5 4 5 15 18 17
*>1:16 t Includes 8 postmortem specimens
2/6
4/5
2/7
2/7
0/2
—
0/2
—
— —
0/4 0/2
1/5 0/3
0/1
-
1/8 0/11 0/1
1/8 0/6 0/17
0/2 0/4 —
0/1 0/1
— -
0/5 0/1 0/1
—
—
—
—
CF Titers*
3/8 0/5 0/4 3/5 2/15 3/18 0/9
TRACHOMA AND ALLIED DISEASES lent for mice and hamsters, but infrequently lethal for guinea pigs. However, guinea pigs, after intraperitoneal infection, irregu larly developed a transient arthritis. The only isolate tested regularly produced an acute arthritis in Macaca irus monkeys after intra-articular infection, and these animals developed flexion contractures. Attempts to produce a follicular conjunctivitis in subhu man primates were unsuccessful. DISCUSSION
The isolation of Bedsoniae from abortion specimens or patients with arthritis does not establish a causal relationship between the agent and the disease. It does demonstrate that members of the group, other than the agents of lymphogranuloma venereum, tra choma or inclusion conjunctivitis, naturally infect man and that infection by these newly isolated agents is associated with certain pathologic conditions. Isolates were achieved from the tissues of six patients with Reiter's syndrome and from two patients with rheumatoid arthritis who had positive Bedsonia complement fixa tion titers. Each patient with Reiter's syn drome, whose synovial tissue yielded a Bed sonia, was in the initial attack of the dis ease. Of the six patients with Reiter's syn drome whose tissues yielded isolates, only three had positive CF titers. This propor tion agrees generally with the results on pa tients infected with the agent of inclusion conjunctivitis. In our series of patients, 60% had CF titers greater than 1:16. Eight other patients with Reiter's syndrome were also studied and results of isolation and se rologie tests were negative. The clinical course in the two groups was essentially the same, although some of the patients whose test results were negative were in later stages or recurrences of the disease. These patients may not have been infected by a Bedsonia. However, in some cases the nega tive results could be ascribed to the stage of the disease when the specimens were col
1085/59
lected. The C F titer of Patient 102 was 1:128 when he was seen at the height of his illness, but several weeks later his titer dropped precipitously to > 1:2. If the Bed soniae agents and the disease are in a simple cause-and-effect relationship, then there may be another cause for an identical symptom complex in the isolate-negative patients. This assumes that negative results were not due to temporal effects or insensitive tech niques. The relationship between the infec tion and the disease may be more complex, however, involving immune mechanisms, double infections or predisposing factors. The epidemiology of these infections is unknown. Although sexual transmission offers an attractive hypothesis, attempts to isolate Bedsoniae from the cervix failed with five consorts, three of whom had significant C F titers. Cervical infection with the agent isolated from patients with arthritis or with the agent of inclusion conjunctivitis could ex plain the isolates obtained from abortion material. The specimens could have been contaminated during passage through the cervical canal. The handling of specimens was designed to minimize sujface contami nation but offered no certainty. The sig nificance of these isolations, be they cau sative for the abortions or cervical in origin, is therefore not assessable. The inclusions formed by different Bedsoniae appear iden tical in clinical specimens. The agents are unidentifiable, but the cytologie studies on cervical scrapings indicate a much higher prevalence (5.5%) of agent than that of clinical inclusion conjunctivitis. This is to be expected, for a chain of venereal infection precedes the ocular disease. Cervical infec tion need not result in neonatal conjunctivi tis. I have seen an instance where only the second-born of twins developed a unilateral conjunctivitis and the mother's cervix yield ed an inclusion conjunctivitis agent. The first-born child was apparently not infected. The agents isolated in this study demand
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AMERICAN JOURNAL OF OPHTHALMOLOGY
respect in the laboratory. T h e y are of high virulence for mice and probably present a potential risk to laboratory personnel. Rigid precautions must be taken during isolation procedures. A total synovectomy was per formed on Patient 147. This presented a much greater mass of tissue than is usually available. T h e eggs died in six to eight days of the first passage and the smears were rich in elementary bodies. I n this laborato ry, blind-passage material and established isolates are handled in different facilities. Before it was realized that the isolation at tempt on this patient was positive, two other groups of eggs were harvested immediately after the first specimens from Patient 147 were handled. T h e yolk sacs from these other eggs became contaminated. More often positive results became evident in the second passage and only occasionally at the end of the first. T h e potential significance of the findings in this study is obvious. N o conclusions, however, can be reached at this time. A n etiologic role for members of the Bedsonia group in certain types of human arthritis or abortion may exist but is not assumed. A proper assessment requires further research and will be made retrospectively. SUMMARY
Bedsoniae were isolated from eight pa tients with arthritis, six of whom had Reiter's syndrome. Attempts to isolate Bed soniae from human abortion specimens were positive in four of 22 instances. Inclusions were found in four of 72 cervical scrapings from a normal population.
MAY, 1967
REFERENCES
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