Acholeplasma vituli sp. nov., from bovine serum and cell cultures.

Organisms isolated from commercial foetal bovine serum and from cell culture lines containing such serum supplements were found to consist of non-helical, non-motile, pleomorphic coccoid forms. One strain (FC 097-2T) cultivated directly from foetal bovine serum was selected for characterization. In ultrastructural examination, individual round cells lacked cell wall structures and cells varied in size, with a mean diameter of about 700 nm. However, variable numbers of cells were filterable through membranes of 300 nm. Optimum growth occurred between 30 and 37 degrees C. The organism fermented glucose, fructose and mannose, but did not hydrolyse arginine. The strain was insensitive to 500 U penicillin ml(-1) and was capable of growing in the absence of serum or cholesterol. The organism was serologically distinct from all 13 currently described species in the genus Acholeplasma and from other sterol-requiring species in the genus Mycoplasma, using growth inhibition, immunoperoxidase and immunofluorescence tests. Strain FC 097-2T was found to have a DNA G+C composition between 37.6 +/- 1 mol% and 38.3 +/- 1 mol%. The genome size was determined to be 2095 kbp. The 16S rDNA sequence of strain FC 097-2T was compared to 16S rDNA sequences of other mollicutes in nucleotide databases. No deposited sequence was found to be identical; the closest relatives were several members of the genus Acholeplasma. On the basis of these findings and other similarities to acholeplasmas in morphology and growth, the absence of a sterol requirement for growth, and similar genomic characteristics, the organism was assigned to the genus Acholeplasma. Strain FC 097-2T is designated the type strain (ATCC 700667T) of a new species, Acholeplasma vituli.

Acholeplasma multilocale sp. nov., isolated from a horse and a rabbit.

Acholeplasma strains were isolated from the nasopharynx of a horse (strain PN525T [T = type strain]) and the feces of a rabbit (strain B1). One clone of strain PN525T and one clone of strain B1 were examined in detail. These clones were indistinguishable from each other and were serologically distinct from the previously described Acholeplasma and Mycoplasma spp. The strains had the following properties: guanine-plus-cytosine content of 31 mol%; sterol was not required for growth, which occurred under both aerobic and anaerobic conditions; glucose was metabolized; and arginine was hydrolyzed. Strain PN525 (= NCTC 11723) is the type strain of a new species, Acholeplasma multilocale.

Survival of Mycoplasma hyorhinis in trypsin solutions.

The survival of four strains of Mycoplasma hyorhinis in stock solutions of trypsin was tested at 22, 4 and -15 degrees C. Low (10(4)-10(5) cfu/ml) and high (10(6)-10(7) cfu/ml) initial concentrations of each strain were used, each was tested three times. A regular decrease of low and high concentrations (1 log in 10 and 20 min, respectively) was seen at 22 degrees C. At 4 degrees C the low concentrations showed a reduction of about 1 log/h, while apart from one strain high concentrations hardly decreased during the first 6 h and the survival time ranged from 24 to more than 30 h at the end of which there was a reduction of 4 logs. At -15 degrees C low concentrations survived up to 1 week in only one of the three tests, high concentrations survived for more than 12 weeks (reduction 3 logs). These latter results suggest that mycoplasmas may be present in trypsin as clumps, which deteriorate very slowly. A study was also performed to compare the sensitivity of different cultural procedures for detecting mycoplasmas.

Indicator cell lines for the detection of hidden mycoplasma contamination, using an adenosine phosphorylase screening test.

Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in all common mycoplasma species but is considered to have a low activity in mammalian cells. However, using an adoP screening test, we have observed that some cell cultures do possess an intrinsic adoP activity leading to false positive results. Moreover, as a false negative result, we encountered a variant of Mycoplasma orale (identified after cultivation on agar and immunostaining) which was not detectable with the adoP screening in cell culture supernatants and only at low levels in cell lysates. To increase the low signal/noise adoP ratio found there, we used an indicator cell line with low intrinsic activity. Indicator cells were inoculated with the test supernatant and the adoP activity of these infected cells were measured after lysis. The procedure diminished the effect of biological variation in intrinsic enzyme activity between the several cell lines tested. Furthermore, in another mycoplasma infected cell line (with M. fermentans), this infection was only reliably detected using these indicator cells. With this procedure we obtained rapid results which were concordant with those obtained using the time consuming cultivation on agar.

Clinical efficacy of ciprofloxacin versus doxycycline in the treatment of non-gonococcal urethritis in males.

In a randomised study the clinical efficacy of ciprofloxacin was compared with that of doxycycline administered in two different dosage schemes to male patients suffering from non-gonococcal urethritis. Fourteen days after completion of therapy (day 21) pyuria was absent in 30 of 100 patients in the ciprofloxacin group; Chlamydia trachomatis was isolated from five and Ureaplasma urealyticum from eight patients. In the 100 mg doxycycline group (n = 60) pyuria was absent in 36 patients (60%) and Ureaplasma urealyticum was isolated from six patients on day 21. In the 200 mg doxycycline group (n = 45) pyuria was absent in 18 patients (40%) and Ureaplasma urealyticum was isolated from two patients on day 21. Side-effects were mild and transient in all groups. It is concluded that ciprofloxacin given in a dosage of 1 g for seven days is not effective in the treatment of non-gonococcal urethritis.

Isolation of Acholeplasmatales from rabbit faeces.

Acholeplasma laidlawii was isolated from the faeces of 23.5% and 24% of groups of 51 conventional and 45 specified-pathogen-free (SPF) rabbits respectively. Isolation of the organism from individual animals could often be repeated, suggesting that infection was not merely transient. Two further acholeplasmas were isolated from two SPF rabbits. One was serologically related to Acholeplasma modicum. The other could not be identified and may be a new species.

Elimination of Mycoplasma from cell cultures by means of specific bovine antiserum.

Heifers were immunized against Mycoplasma arginini, M. fermentans, M. hyorhinis and M. orale and the antisera were applied for elimination of these species from cell cultures. From fifteen out of nineteen contaminated human and animal cell cultures the mycoplasmas could be eliminated by treating the cells with medium with 10% or 20% antiserum (eight cases) or antiserum combined with one or two antibiotics (six cases). In ten cases two treatments were sufficient, in four cases respectively four, six or eight (2 X) treatments were necessary, in one case antiserum combined with a heat treatment (42 degrees C) was successful. The efficacy of the treatment depended on the antibody titer of the serum, the contaminating mycoplasm species (M. arginini being more difficult to eliminate than the other three species) and the cells involved. The bovine sera were not cytotoxic, except for a slight toxicity for a mouse lymphoma cell line. The application of specific bovine antiserum for elimination of mycoplasmas is an easy and often successful method.

An alternative method for the decontamination of rats carrying Mycoplasma pulmonis without the use of germfree isolators.

Two strains of Lewis rat were successfully freed from Mycoplasma pulmonis infection by using a combination of oral treatment with oxytetracycline hydrochloride and obtaining young by hysterectomy. Laminar flow cabinets were used to perform hysterectomies on donor animals and for rearing hysterectomy-derived animals. After thorough microbiological examination the rats were brought to the breeding colony of the Laboratory Animal Centre. Periodic laboratory tests using both cultural and enzyme-linked immunosorbent assay methods showed that the animals have remained free from M. pulmonis for the last 3 years.

Sexually communicable micro-organisms in human semen samples to be used for artificial insemination by donor.

Two hundred and thirty seven semen samples from 10 institutes for artificial insemination by donor (AID) in Belgium and the Netherlands were tested for the presence of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, herpes simplex virus, and cytomegalovirus. The incidence of these micro-organisms in the semen samples was 0%, 6.3%, 4.6%, 35.9%, 0%, and 0.4% respectively, and 47% of all samples were infected with one or more of the micro-organisms. As the ejaculates from which the samples had been taken had already been, or would be, used for AID, the exclusion of microbiological contamination with sexually communicable micro-organisms before insemination is indicated.

Comparison of two methods for detection of mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in the Netherlands.

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.

Evaluation of roxithromycin in the treatment of non-gonococcal urethritis in males.

One-hundred and fifty-two male patients suffering from non-gonococcal urethritis were treated with an oral dosage of 300 mg roxithromycin daily for seven days. Chlamydia trachomatis was isolated from the urethra in 53 patients (35%), and Ureaplasma urealyticum in 42 patients (28%). After treatment, 49 (92%) of the 53 patients with positive Chlamydia trachomatis cultures and 34 (81%) of the 42 patients with positive Ureaplasma urealyticum cultures had negative cultures at follow-up. A clinical cure was observed in 137 patients (90%). Ten patients (7%) showed side effects consisting of nausea, sensation of distended abdomen, headache and fatigue. Seventy-eight male patients suffering from nongonococcal urethritis were treated with an oral dosage of 2 X 150mg roxithromycin daily for seven days. Chlamydia trachomatis was isolated from the urethra in 22 patients (28%), and Ureaplasma urealyticum in 30 patients (38%). After treatment, all of the 22 patients with formerly positive Chlamydia trachomatis cultures and 23 (77%) of the 30 patients with formerly positive Ureaplasma urealyticum cultures were negative at follow-up. A clinical cure was observed in 70 patients (90%). Three patients (4%) showed side-effects consisting of nausea and headache. It is concluded that roxithromycin is a good alternative to tetracycline and erythromycin in the treatment of non-gonococcal urethritis in males.

Naturally occurring genital mycoplasmosis in mice.

The first isolations of Mycoplasma pulmonis were made from inflamed ovaries of 2 C3H/F1 mice. Investigation of cultures from a further 110 apparently healthy mice revealed 14 cases of M. pulmonis localized in the ovaries and associated with oophoritis.

Prevalence of antibodies to Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma hominis in infertile women.

A total of 57 infertile women, who had been referred for in vitro fertilisation or for diagnostic laparoscopy, were tested for the presence of antibodies to Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma hominis. Four were excluded from the study. Of the remaining 53, 33 had laparoscopically obvious tubal disorders, such as adhesions, distal occlusions and strictures, and 20 did not. Antibodies to C trachomatis were found in 7/33 (21.2%) v 0/20, antibodies to N gonorrhoeae in 20/38 (60.6%) v 5/20 (25%), and antibodies to M hominis in 18/24 (75%) women with tubal disorders v 13/19 (68.4%) of those with no disorder. Antibodies to C trachomatis and N gonorrhoeae were significantly (p less than 0.05) more common in women with tubal disorders. The high prevalence of antibodies to N gonorrhoeae in infertile women without tubal disorders suggests that ciliated tubal epithelium is damaged after inflammation without this being laparoscopically visible. Our results confirm the important role of N gonorrhoeae and C trachomatis in the aetiology of infertility after tubal inflammation.

Detection of mycoplasma contamination lymphoblastoid cell lines by monoclonal antibodies.

We have produced two monoclonal antibodies, SFR1-Myco 1 and SFR1-Myco 2, that detect Mycoplasma fermentans found to contaminate lymphoblastoid cell lines (LCL). The specificity of these monoclonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoclonal antibodies with known reference strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycoplasma in these cells as determined by a standard mycoplasma detection assay. Because of the potential for widespread contamination of B lymphoblastoid cell lines transformed with Epstein-Barr virus-containing supernatants obtained from marmoset cell lines contaminated with M. fermentans, these monoclonal antibodies have value as screening reagents for this mycoplasma species in LCL.

Role of mycoplasmas in chronic prostatitis.

In 17 out of 102 patients with clinically diagnosed chronic prostatitis the disease could be attributed to known urogenital tract pathogens. Of the remaining 85 patients, Ureaplasma urealyticum was isolated from 38, Chlamydia trachomatis from five, and both organisms together from two. The results of antimicrobial treatment of the patients suggest an etiological relationship between Ureaplasma urealyticum and certain cases of chronic prostatitis. In these cases urethritis seems to be an accompanying symptom (urethro-prostatitis). No relation could be demonstrated between a favorable outcome of therapy and particular serotypes of ureaplasma. Our study could not establish any pathogenic role for Mycoplasma hominis.

Comparison of various atmospheric conditions for isolation and subcultivation of Mycoplasma hyorhinis from cell cultures.

The efficiency of aerobic incubation was compared with incubation under various oxygen and carbon dioxide conditions for the isolation and subcultivation of three strains of Mycoplasma hyorhinis from VERO-cell cultures and subcultivation of three laboratory strains. Under anaerobic conditions with a low oxidation-reduction potential (at or below -115 mV) as obtained in jars, with catalysts, containing mixtures of 5%-10% CO2 in H2, very poor or no growth of any of the six M. hyorhinis strains was observed. When traces of oxygen were present (that is, under conditions with higher oxidation-reduction potentials, e.g. when omitting the catalyst in the above gas mixtures or in 5% CO2 + 95% N2) isolation from cell cultures was successful in most tests, but subcultivation of these primary isolates was seldom possible under these semi-anaerobic conditions. However, in most cases these primary isolates could be subcultivated aerobically, although aerobic conditions were unsatisfactory for isolation in about half of the experiments. Isolation of M. hyorhinis was optimal in 5% O2 + 95% N2, under which condition the isolates could also always be subcultivated. Isolation failed occasionally when 5% O2 + 5% CO2 + 90% N2 was used, thus indicating that 5% CO2 was slightly inhibitory. 5% CO2 in air and 10% CO2 either in air, H2 or N2 were also inadequate for isolation from cell cultures. In contrast to the findings with these cell culture-adapted M. hyorhinis strains, the laboratory strains could be subcultivated easily under all conditions tested except those with an oxidation-reduction potential at or below -115 mV; 100% CO2 was inhibitory for all 6 strains. Our findings may partly explain why M. hyorhinis is often considered "non-cultivable" on artificial media once adapted to cell cultures. The findings emphasize the need to employ also a micro-aerophilic condition (5% O2 in 95% N2) in the examination of cell cultures for mycoplasma.