1Four acholeplasma, 11 animal mycoplasma (mostly bovine) and 7 human mycoplasma types were examined using the indirect immunoperoxidase (IP), indirect immunofluorescence (IF) and growth inhibition (GI) tests to compare the sensitivity and specificity of these tests for the identification of Mycoplasmatales. The IP and IF tests were applied to unfixed colonies on agar blocks. Colonies of Acholeplasma laidlawii, Acholeplasma modicum, Mycoplasma agalactiae and Mycoplasma bovis showed a bright yellow autofluorescence in the IF test making it inapplicable in these cases. The IP test was as specific as the GI test and more specific than the IF test. In the IP test, cross-reactions were only found between M . agalactiae and M . bovis strains, two types originally considered to be related to each other. Crossreactions between these organisms were not seen in the GI test. With one exception, the IP test was also found to be more sensitive than the GI test and as sensitive as the IF test; thus for two Acholeplasma axanthum antisera the IP test was inferior to the IF and GI tests with A. axanthum strains. A close relationship was found in the IP and IF tests between Mycoplasma mycoides strain Gladysdale and the unclassified bovine group 7 strain ~2 9 . The results of the IP test were much easier to read than those of the IF tests. Thus colonies treated with homologous antiserum became dark brown and, in mixed cultures, could be readily distinguished from heterologous colonies using a light microscope. All controls were negative and strains isolated from cattle, cell cultures, humans and monkeys were easily identified.
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.
SUMMARY Two hundred and thirty seven semen samples from 10 institutes for artifical 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. Two hundred and thirty seven semen samples frozen at -1 96°C, from ten AID institutes in the Netherlands and Belgium were supplied in 0 25 ml or 0 5 ml straws. The semen had been given voluntarily by donors, who received a small sum for expenses. The samples were all diluted 2:1 or 1:1 with a cryoprotective liquid containing glycerol and sometimes also egg yolk or bovine serum albumin. Fifty samples contained ampicillin 380 mg/l and 36 contained kanamycin 5 mg/l. Forty samples came from an institution that used only fresh semen. The semen was diluted 1:1 with Eagle's modified minimum essential medium (EMEM) with 15% fetal calf serum, and was frozen to -196°C. Volumes of 120-200 ,l were diluted with 2 ml of EMEM for culture for Ngonorhoeae, C trachomatis, HSV, and CMV. Volumes of 60-200 ,l were suspended in 2 ml of U-9 medium' for culture for M hominis and U urealyticum. Materials and methods CULTURE METHODSCulture for N gonorrhoeae Semen diluted with EMEM was inoculated with a loop on to a medium consisting of GC medium base (Difco), haemoglobin (Difco), and IsoVitalex (BBL), and was incubated for 48 hours.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.