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The optimum conditions for the detection of human immunoglobulin G (IgG), IgM, and IgA antibodies to Ureaplasma urealyticum by an enzyme-linked immunosorbent assay (ELISA) were established by using a cell lysate antigen and commercially available alkaline phosphatase conjugates. No significant cross-reactions were observed among rabbit antisera to a variety of mycoplasmas of human origin and ureaplasma antigen, thus demonstrating the specificity of the ELISA. All human sera were assayed at a 1:200 dilution. Antigen was used at 20 mug of protein/ml and conjugates were diluted 1:500. Presence of IgG antibody to U. urealyticum was significantly associated with isolation of U. urealyticum (P < 0.001) in 110 women. Seventeen acute-phase and 19 convalescent-phase sera from male nongonococcal urethritis (NGU) patients were tested for the presence of antibody by both the metabolism inhibition assay and by ELISA, with overall agreements of 82 and 95% for acute- and convalescent-phase sera, respectively. Serum antibody responses were demonstrated to selected serotypes in the metabolism inhibition test, but the response as measured by the ELISA was independent of the serotype of the antigen used. Serum antibody levels in NGU patients were significantly higher (P < 0.002) than the normal serum standard in the IgG, IgM, and IgA classes. Additionally, the magnitude of change between acute- and convalescent-phase sera was greater for NGU patients than for normal asymptomatic ureaplasma-positive male controls. A significant change in antibody levels of one or more antibody classes was detected for 12 of 18 (67%) NGU patients by ELISA. Ten of the 12 (83%) individuals had a change in the IgM class, which is suggestive of an active infectious process. The ELISA is advantageous in that it requires only a single serotype antigen, uses one serum dilution, is class specific, and allows quantitative detection of differences between acute- and convalescent-phase sera.

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Growth of Ureaplasma urealyticum isolates from clinical exudates and urine specimens was significantly improved by supplementation of a Ureaplasma differential agar medium by putrescine. The improved medium was designated medium A7B.

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An agar growth inhibition method for serotyping Ureaplasma urealyticum is described, and the results of applying this method to serotyping 338 strains of the organism are presented. The serotyped strains consisted of cloned isolates from male patients with primary and recurrent nongonococcal urethritis (NGU), isolates from symptomatic patients with other genitourinary tract infections and disorders, and isolates from asymptomatic carriers of U. urealyticum in the genitourinary tract (controls). Among 122 male patients with NGU, serotype 4 was associated most frequently (52%) with this disease at Camp Lejeune, N.C. Seventeen percent of the isolates were type 2. The remaining isolates consisted of types 1, 3, 6, and 8 and accounted for 6 to 9% each of the serotypes isolated from the NGU group. Types 5 and 7 were not isolated. Among 91 symptomatic patients with other genitourinary tract infections and disorders, U. urealyticum type 4 also was associated most frequently (37%) with these disorders. The remaining isolates, represented by types 1, 2, 3, 6, 7, and 8, accounted for 9 to 15% each of the types isolated from this group. Type 5 was not isolated. Among 125 symptomfree carriers of U. urealyticum in the genitourinary tract, type 8 was recovered most frequently (30%), whereas type 4 was isolated next most frequently (24%). The remaining isolates consisted of types 1, 2, 3, 5, and 6 and accounted for 2 to 15% each in this asymptomatic control group. Type 7 was not isolated. Of the present eight serotypes of U. urealyticum studied in this investigation, type 4 was associated most frequently with disease (NGU) and certain other disorders of the genitourinary tract at Camp Lejeune. A previously unknown association of U. urealyticum with frequently abacteriuric, unexplained pyuria (with or without urethral pruritus and dysuria) is reported, suggesting the existence of asymptomatic Ureaplasma urethritis.

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A differential agar medium for the identification of Ureaplasma urealyticum in primary cultures of clinical specimens is described. The differential medium (no. A7) is specific for the identification of U. urealyticum and other members of the genus Ureaplasma. Large-colony, classical Mycoplasma and Acholeplasma species and Proteus L colonies are unreactive on this differential medium. The medium incorporates the biochemical principle of the direct spot test for urease in colonies of Ureaplasma and contains added urea and a sensitive indicator of ammonia, manganous sulfate. Ureaplasma colonies on this medium are identified as dark golden-brown or rich deep-brown colonies, in sharp contrast to the light background of the medium, when viewed by direct transmitted illumination under the low power of the microscope.

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The existence of unusual colonies of Ureaplasma urealyticum (T mycoplasmas) in primary agar cultures of certain urine specimens is reported and their morphology is illustrated.

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T-strain mycoplasmas were isolated from the genital tracts of 3 genera of female monkeys: the talapoins, Miopithecas talapoin, the patas, Erythrocebus patas, and the macaques, Macaca fascicularis. The talapoins have not reproduced successfully for the past 2 yr. Fertility in the patas has also been reduced, with a high incidence of stillbirths and spontaneous abortions. The macaques were not permitted to breed.

PIP: T-strain mycoplasmas have been incriminated in nongonococcal urethritis in human males and human reproductive failure in females. In this study, T-strain mycoplasmas were isolated from the genital tracts of 3 genera of female monkeys: talapoins (Miopithecas talapoin), patas (Erythrocebus patas), and macaques (Macaca fascicularis). The talapoin monkeys had a record of a high rate of reproductive failure due to infertility, spontaneous abortions, and stillbirths. The patas monkeys also had reduced fertility. The 3 genera had no contact with each other. Details of cultural methods for detecting mycoplasmas are given. T-strains isolated from the vaginas of the monkeys were similar in cultures to those isolated from humans but not closely related to human T-mycoplasma serotypes. These findings broaden the number of species which harbor T-mycoplasma and provide an animal model for experimental studies.

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A urease color test fluid medium (U-9) for the detection and identification of T (T-strain) mycoplasmas in clinical material is described which is sensitive and specific for this group of mycoplasmas. The medium was prepared from commercially available components and contained 95% half-strength, tryptic digest broth (pH 5.5), 4% unheated horse serum, 0.05% highest-purity urea, 0.001% sodium phenolsulfonphthalein, and 1,000 units of potassium penicillin G per ml. The final reaction of medium U-9 was pH 6.0. The overall agreement (positive and negative) between urease reactions in U-9 urease color test medium and culture findings in a standard agar primary culture system among 686 clinical specimens was 98.1%. The disagreement consisted of 13 false-positive urease reactions which were recognized visually as false-positive reactions due to other microorganisms. For specimens from the female genitourinary tract, the inclusion of 2.5 mug of amphotericin B (Fungizone) per ml of medium U-9 is recommended for the suppression of growth of Candida species and filamentous fungi.

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A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.

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