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SERUM ANTIBODY LEVELS AGAINST T MYCOPLASMAS IN TWO NORTH AMERICAN INDIAN POPULATIONS

PREDISPOSED TO SPONDYLITIS DENYS K . FORD and ELIZABETH HENDERSON

Serum antibody levels against T mycoplasmas (Ureaplasma urealyticum) were determined by the meta- bolic inhibition method in several populations. A higher prevalence o f antibody was found in Haida Indians and Bella Coola Indians than in blood donors, patients with rheumatoid arthritis, and patients attending a VD clinic. Antibody levels did not correlate with the presence of spondylitis or the histocompatibility antigen HLA-B27, although both these Indian populations have a high preva- lence of spondylitis and HLA-B27.

The metabolic inhibition technique (M IT) of Purcell et a1 (1 ) was the first practical method for detect- ing antigenic differences between strains of T myco- plasmas. Initial studies showed multiple antigenic se- rotypes (2,3), but no satisfactory evidence was found of significant serum antibody elevations in response to T mycoplasma “infection” (4). Subsequent reports have defined eight antigenically distinct subtypes of T myco- plasmas ( 5 ) , one being Shepard’s type organism (6) and

From the Division of Kheumatology. Department of Medi- cine, University of British Columbia, Vancouver, British Columbia, Canada.

Supported by the Canadian Arthritis and Rheumatism Society and by Federal Health Grant 610-1055-28.

Denys K . Ford, M.D.: Professor of Medicine: Elizabeth Hen- derson. B.Sc.: Research Assistant.

Address reprint requests to Denys K . Ford. M.D.. Division of Rheumatology. Department of Medicine. 700 West 10th Avenue, Vancouver, British Columbia, Canada V5Z I M9.

Submitted for publication December 17. 1975; accepted May 20. 1976.

the other seven being serotypes initially characterized in the present authors’ laboratory and previously reported (3). Recently the test of Purcell was modified to prevent the inactivation of complement by NH: ions produced from the hydrolysis of urea (73) . In this laboratory, this modification was evaluated but was not found to alter significantly the sensitivity of the test: the original test of Purcell and the modification yielded equivalent data. Because the Purcell method was simpler to perform, it was chosen for the following study.

This investigation arose because a pilot survey of stored frozen sera revealed antibody in a sample from a Haida Indian woman with spondylitis. Immediate fol- low-up of this observation showed that her son, also a spondylitic, had a significant serum antibody titer, but 5 other members of the family without spondylitis had no demonstrable antibody. This family had been the source of a special study in 1963 (9) when polyarthritis was observed as a sequel to a flu-like illness. Serum samples from over 400 Haida Indians were available for further investigation, having been collected in 1962 during an epidemiologic study of the Haidas in the Queen Char- lotte Islands of British Columbia. At that time it was found that the prevalence of rheumatoid arthritis among the Haidas was not distinctive, but that the prevalence of spondylitis was unexpectedly high (10). A subsequent epidemiologic study of Bella Coola Indians also showed an increased prevalence of spondylitis ( I 1).

In 1973 a pilot study of a random sample of these stored sera indicated that the Haida Indian samples had

Arthritis and Rheumatism, Vol. 19, No. 6 (November-December 1976)

1329

a higher prevalence of T mycoplasma antibody than did samples from Red Cross donors. Consequently, when visits were made to Bella Coola in 1974 and the Queen Charlotte Islands in 1975 for H LA histocompatibility typing of the Bella Coola and Haida Indians respectively (12), serum samples were taken for further T myco- plasma antibody studies and the following investigation was subsequently completed.

MATERIALS AND METHODS Serum Samples. Haida Indian Serum samples were

collected at the time of HLA typing in the spring of 1975. Bella Coola Indian serum samples were gathered at the time of HLA typing in May 1974. The Bella Coola Indians live about 200 miles north of Vancouver, whereas the Haidas live about 400 miles north of Vancouver. Both serum collections were de- signed to yield a random sample of each Indian population, as described in the report of the HLA-B27 study (12).

Serum samples were also taken from Red Cross do- nors attending Blood Donor Clinics in Vancouver and from patients attending the VD Control Clinic with a variety of diagnoses. Patients with rheumatoid arthritis, ankylosing spondylitis, and Reiter's syndrome were under the care of the rheumatologists of the Division of Rheumatology of the Uni- versity of British Columbia Medical School at the Arthritis Centre in Vancouver. Sera from patients with Reiter's syn- drome and also from the VD Clinic patients were collected when the patients were not on antimycoplasmal antibiotics. All the sera were stored at -20°C before testing.

Purification of IgG Antibody. The IgG fraction of serum was prepared by the DEAE-Sephadex method of Webb (13). The purity of the fractions was confirmed by immuno- electrophoresis, which demonstrated single immunoprecipitin IgG lines in each of the fractions. Quantitative radial immuno- diffusion determined the IgG concentrations in the fractions, and the concentration of IgG was adjusted to approximate that in the original serum.

HLA Histocompatibility Testing. Tests for HLA typing were performed by the Vancouver General Hospital tissue- typing laboratory. The Terasaki microlymphocytotoxicity procedure was employed, with antisera provided by Dr. Tera- saki.

T Mycoplasma Serotypes and Media. All of the eight serotypes of Black ( 5 ) were employed in the pilot phases of the study. Seven of these were originally isolated in this labora- tory, and the remaining strain was the type organism isolated by Shepard et a1 (6). I n the final testing, serotype V was selected because this organism had been the main organism studied in this laboratory during the past 14 years. The culture medium consisted of Difco PPLO medium, supplemented with 10% unheated horse serum, I% Oxoid yeast. urea at a concen- tration o f 0.05% for growth and I % for MIT, 0.002% phenol red, and 1000 units/ml of penicillin.

Metabolic Inhibition Test. Antibody levels were mea- sured by the Microtiter method of Purcell, with 50 color change units (CCU) of organisms per well. Each well finally contained 0.05 ml of serum serially diluted twofold in 1% urea myco- plasma medium, 0.05 ml of T mycoplasma culture containing

50 CCU in the same medium, and 0.05 ml of I:20 guinea pig complement (Microbiological Associates), also in the same medium. The serial serum dilutions and pipetting were per- formed in the Cooke Minipipetter and Minidiluter. All tests were done in duplicate.

The disposable Linbro plates were sealed with Micro- titer Plate Sealers and incubated overnight at 32°C and at 37°C on the following day, until the control wells showed a pH rise to 7.5 by comparison with standards. At tha t time the titers were read as the highest dilution of serum added to the test wells that showed an inhibition of pH rise.

A modified Lin and Kass procedure (7,8) was devel- oped for comparison with the Purcell method. The T myco- plasmas, in a urea-free medium containing dialyzed horse serum, were exposed to human serum and 1:20 guinea pig complement for 1 hour at 37°C before the addition of urea- containing medium. In this laboratory the 10 CCU inoculum suggested by Lin and Kass was found to give erratic results, and therefore a 50 CCU inoculum was used.

RESULTS Preliminary studies were required to define the

MIT before proceeding to the main investigation. As noted previously, an initial comparison was made be- tween a modified Lin and Kass method and that of Purcell, but no significant difference in sensitivity was found between the two techniques in this laboratory. Table I shows the comparison when 3 human sera and 3 hyperimmune rabbit antisera were tested against 3 se- rotypes. This table demonstrates the low levels of inhibi- tion given by human sera in contrast to hyperimmune rabbit antisera; it also shows the greater serotype speci- ficity of the rabbit antisera in contrast to a broader cross-reactivity of the inhibition by human serum.

In order t o establish that the metabolic inhibition test with human serum was a measure of antibody and not a manifestation of nonspecific inhibition, the IgG fraction of serum was prepared by the Webb procedure (13) from the sera of 9 subjects, selected because they had high, moderate, or negligible inhibitory titers. The comparison of titers between the original serum and the IgG fraction, at a concentration equivalent t o that in the serum, is shown in Table 2. N o loss of inhibition re- sulted from the IgG fractionation procedure.

Table 2 also confirms the observation noted in Table 1 that the inhibitory factor in human serum was broadly reactibe against the T mycoplasma serotypes. Because of this broad reactivity of human antibody as contrasted to rabbit antibody, and because the pilot study had shown no more significant antibody levels against any other serotype, it was decided to test all sera against a single serotype, V.

In the MIT tests on serum samples from the

I330 FORD AND HENDERSON

Table 1. Comparison of Purcell's Standard MIT Method with a Modification of the Lin and Kass Method

Inhibitory Titers (Reciprocal) Against Specific Serotypes

Modified Lin and Kass Method Purcell Method

T-Ill T-V T-VII T-111 T-V T-VII

Sera Human LL 2 16 4 4 32 4

HL 4 16 32 8 64 16 DT 8 32 8 64 64 8

Antisera Rabbit 111 10.240 40 20 10,240 20 20

V 80 5,120 20 20 10,240 20 VII 160 . 160 2,560 80 80 5,120

population groups shown in Table 3, about 20 serum samples were tested at one time and sera from several population groups were included in each testing. The sera were unselected except for two factors. First, indi- viduals in the control groups were selected to achieve a male predominance similar to the spondylitic and Rei- ter's groups. Second, the individuals i n the con,trol groups were selected to achieve an average age similar to the Haida, spondylitic, and Reiter's groups. Columns 3 and 4 of Table 3 demonstrate the similarity of the groups in regard to sex and age. Red Cross donors in Vancouver were chosen as controls to compare to the

Indian population, patients attending the Vancouver VD Control Clinic were selected for comparison with the patients having Reiter's syndrome, and the rheuma- toid arthritics were chosen for comparison with the non- Haida spondylitics. In view of the resulting low titers, Table 3 shows the percentage of sera that gave inhibition at each of the twofold serum dilutions up to 1:16. The data in columns 6-9 of Table 3 show uniformity of the comparisons between the population categories, which- ever inhibitory titer is considered, with the single ex- ception that the number of 1:16 titers in the rheumatoid arthritic groups is unexpectedly high.

Table 2. Comparison of MIT Titers Between Serum and Derived IgG Fractions Againsr Seven Serotypes

Inhibitory Titers (Reciprocal) Against Specific Serotypes Patient Sample

Diagnosis Tested IgG (mg%) T-I T-I1 T-111 T-IV T-V T-VI T-VII

RA

RA

RS

RA

RA

RA

RS

HS

HS

I360 I080 1 I30 750

1360 I I70 1240 900

I890 I360 1 I70 I090 I550 1961 2320 4520 I700 1250

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 0 0 0 0 0 0 0 0 0 0 1 1 0 4 0 I 0 0 2 0 4 0 1 0 0 0 16 0 0 0 0 0 0 8 0 0 0 4 8 4 8 8 2 8 I 0 8 2 4 0 2 8 8 8 16 32 8 8 2 2 I 4 32 4 4

32 4 16 16 16 4 32 16 8 16 32 16 4 64 32 16 4 16 32 8 16 32 16 8 32 128 4 32

N D N D 32 8 64 16 16 N D N D 32 8 64 64 16

RA: rheumatoid arthritis: RS: Reiter's syndrome: HS: Haida spondylitic; N D : not done.

SERUM ANTIBODY LEVELS 1331

Table 3. Serum Antibody Titers to T Mycoplasma Serotype V ~

Percent Percent Positiveat Titer Total Percent Average with

Population No. Male Age 0 Titer I : 2 1:4 1:8 1.16

Nonspondylitic

Spondylitic Haidas Bella Coola Indians Reiter’s syndrome

patients Non-Haida

spondylitics Rheumatoid

arthritics Red Cross donors VD ClinicControls

Haidas

<Age 3 I >Age31 Total

I08

22 106 40

32

50

108

78 59

137

88 42 41.6 58.4 46.4 30.5 10.2

86 48 18.0 86.4 59.1 41.0 13.6 58.5 44 34.0 66.0 48.0 35.0 11.6

100 36 25.0 75.0 45.0 25.0 15.0

87.5 43 34.0 66.0 37.5 28.1 15.6

I00 53 69.4 30.0 20.4 18.0 10.0

68.5 37 65.8 34.2 21.2 13.8 4.6

I00 25 59.0 41.2 29.5 14.2 2.6 I00 39 59.3 40.7 30.5 15.5 5. I I00 31 59.0 41.0 29.9 14.6 3.7

Table 4 presents the statistical analysis of the 2 1.4 serum dilution data from which column 7 of Table 3 was derived. The Haida and Bella Coola Indians are significantly difyerent from the Red Cross donors, the VD Control Clinic patients, and the group with rheuma- toid arthritis. The group with Reiter’s syndrome is sig- nificantly different from the Red Cross donors and the rheumatoid arthritics, but not from the VD Clinic con- trols. The non-Haida spondylitics are only different from the Red Cross donors at a significance level of P < 0.05.

Because Haida Indians have eight to ten times the prevalence of ankylosing spondylitis as compared to populations studied elsewhere (12), and also because they have eight to ten times the prevalence of the histo- compatibility antigen HLA-B27 (12), it was of interest to determine if there was a relationship between T mycoplasma antibody titers and the presence of HLA- B27. Table 5 shows that n o correlation existed. It also includes data on 23 patients with Reiter’s syndrome in

whom HLA-B27 status was determined, and again there was no definite correlation.

DISCUSSION An unexpected finding of T mycoplasma an-

tibody in a Haida mother and her son initiated this study, which was performed to discover i f etiologic clues to the cause of spondylitis would emerge. The data derived from the investigation indicate that the Haida and Bella Coola Indian populations have a high preva- lence of low levels of antibody against T mycoplasmas, when compared to Vancouver Red Cross donors, patients attending the Vancouver VD Control Clinic, or patients with rheumatoid arthritis. There is, unfortu- nately, no suggestion that this observation is related to the high prevalence of spondylitis in these Indian popu- lations. Moreover, the findings show that antibody lev- els were not correlated with HLA-B27 status. Con- sequently, these observations do not now have clinical

Table 4. Statistical Analysis of Data in Column 7 of Table 3 (Titer 2 1:4/

Bella Coola Nonspondylitic Spondylitic Reiter’s Non-Haida Indians Haidas Haidas Syndrome Pts Spondylitics

X 2 p x2 p x 2 p x2 P x 2 P

RedCrossdonors 17.0 <0.01 15.0 <0.01 10.4 <0.01 8.1 <0.01 3.5 0.05 VDcliniccontrols 8.5 <0.01 6.8 <0.01 5.2 0.02 3.2 0.08 0.74 0.4 R heumatoid 11.3 <O.Ol 10.1 <0.01 8.6 <0.01 6.5 0.01 3.0 0.08

arthritics

1332 F O R D A N D HENDERSON

Table 5. HLA-27 Status of Individuals Compared lo T Mycoplasnia Serorype V Antibody Levels

HLA-27 Status

106 Haidas 23 Reiter’s Cases

Antibody Titer Pos Neg Pos Neg

2 1.4 23 21 10 I < 1:4 29 29 8 4

or immunologic relevance, and it is impossible to hy- pothesize profitably on the basis of the observations.

I n this laboratory a modified Lin and Kass method was not found superior to the Purcell technique for measuring T mycoplasma antibody, and thus emphasis on complement lysis of organisms does not seem important in the practical determination of anti- body levels in human serum.

Previous studies employing hyperimmune rabbit serum have differentiated distinct serotypes of T mycoplasmas, and there is currently no explanation of the broader cross-reactivity of human serum or pure IgG fractions of human serum.

The significance of T mycoplasma antibody in patients with Reiter’s syndrome is not clear at this time. The difficulties in adequate follow-up of patients with nongonococcal urethritis have prevented a satisfactory study of antibody responses to urethritis associated with T mycoplasmas, but pilot studies have indicated that antibody responses are unpredictable and often not de- monstrable. Currently, T mycoplasma serology does not add significant support to the possibility of an etiologic role for these organisms in Reiter’s syndrome, although the gats provide no evidence against such an hypothesis.

ACKNOWLEDGMENTS The authors are grateful to Ms. Judy Smith and Ms.

Beverley Wort for expert technical assistance in the initial phases of the study. They qlso acknowledge the help of Dr. J.

P. Gofton in collecting the Haida and Bella Coola sera, and of the Canadian Red Cross and Vancouver VD Control Clinic in securing the control sera.

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Shepard MC, Lunceford CD, Ford DK, et al: Ureaplasma urealyticum gen., sp.nov.: proposed nomenclature for the human T (T-strain) mycoplasmas. Int J Syst Bacteriol

Lin JS, Kass EM: Immune inactivation of T-strain myco- plasmas. J Infect Dis 122:93-95, 1970 Lin JS, Kendrick MI, Kass EH: Serologic typing of h u - man genital T-mycoplasmas by a complement-dependent mycoplasmacidal test. J Infect Dis 126:658-663, 1972 Price GE, Ford DK, Gofton JP, et al: An outbreak of “infectious” polyarthritis in a Haida Indian family. Ar- thritis Rheum 6:633-638, 1963 Robinson HS, Gofton JP, Price GE: A study of rheumatic disease in a Canadian Indian population. Ann Rheum Dis

Gofton JP, Bennet PH, Smythe HA, et al: Sacroiliitis and ankylosing spondylitis i n North American Indians. Ann Rheum Dis 31:474-481, 1972 Gofton JP, Chalrners A, Price GE, et al: HLA-B27 and ankylosing spondylitis in B.C. Indians. J Rheumatol

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