Electrophoretic Patterns of Strains of Mycoplasma pulmonis

Abstract

Strains of Mycoplasma pulmonis, examined by polyacrylamide-gel electrophoresis of proteins extracted in a phenol-acetic acid-water solvent, gave patterns with reproducible differences. Some strains could be placed into groups, but a number could not be associated with any group. In all but one case a 'basic M. pulmonis pattern ' was easily recognizable. This exception suggests that occasionally one might fail to recognize a strain of M.pulmonis by relying solely on the electrophoretic technique. I N T R O D U C T I O N Fowler, Coble, Kramer & Brown (1963) suggested that the electrophoretic separation of proteins of mycoplasmas be used as a new method of identifying members of this species. Their observations were extended by Rottem & Razin (1967). The patterns obtained by these workers were distinct for the species of mycoplasmas examined. Sometimes there were differences between the electrophoretic patterns of strains within a species; for example, Myco-plasma yulmonis (Razin & Rottem, I 967 ; Razin, I 968). In the present investigation 19 strains of Mycoplasma pulmonis were examined by poly-acrylamide-gel electrophoresis to find out to what extent the electrophoretic patterns of their proteins differed and to see if a typing scheme could be formed on the basis of these patterns. M E T H O D S Organisms. The strains of mycoplasmas and the sources from which they were obtained are given in Table I. The strains included reference strains and recent isolates. Preliminary identification of recently isolated strains was made by the growth inhibition test (Clyde, 1964) employing at least two antisera to Mycoplasma pulmonis plus antiserum to the other rodent mycoplasmas M. neurolyticum and M. arthritidis. Strains which were inhibited were regarded as members of the species M. pulmonis. Clones. All strains were cloned at least three times. Where an original culture showed several colonial forms, representative colonies were cloned six times. Media and growth conditions. The media used for the cultivation of the mycoplasmas were modified from that described by Chanock et al. (1962). Thallous acetate was omitted and ampicillin at a final concentration of 0-1 yo was used in place of penicillin (Fallon, 1969). Medium which contained I % (w/v) glucose and 0'002 % phenol red was inoculated from a logarithmic phase culture and incubated aerobically at 37 "C until a colour change in the medium showed a drop of 0.5 pH unit, which took approximately 48 h. For solid medium I yo (wlv) Ionagar no. 2 (Oxoid) was added.

The comparison and characterisation of glycolytic mycoplasmas isolated from the respiratory tract of sheep

“…Where the same tests were performed, the results agreed with those of Carmichael et a[. (1972) andFurlong andCottew (1973). Furthermore, the appearance of colonies of the Scottish strains and the appearance of organisms from broth cultures stained with MacNeal's stain corresponded with the descriptions given by these authors.…”

“…Phenol-acetic acid-water ( 2 : 1 : 0-5 v/v/v) extraction of the sedimented organisms and PAGE of the extracted proteins was essentially as described by Forshaw (1972). The gels, which contained 35 % (v/v) acetic acid, 5h1 urea and 7.5 % (w/v) acrylamide, were stained with 1 % amido black.…”

Serological Heterogeneity of Mycoplasma pulmonis

“…Serological analysis produced some groups of strains which were not always distinct from one another, because some strains shared antigens with members of more than one group. With the exception that Ash and 63 fell into the same group, both by the electrophoretic and metabolic inhibition technique, the groups of strains with similar electrophoretic patterns (Forshaw, 1972) were not the same as those formed from the results of the serological studies. This is not unexpected, since in the serological studies, antigens are being analysed, whereas in the electrophoretic studies, extracted proteins are being analysed .…”

“…In the present study both serological and electrophoretic (Forshaw, 1972) methods were used. As detailed in Results, complement fixation and growth-inhibition tests showed no intraspecies groupings, but the metabolic inhibition test indicated possible relationships between the few strains.…”

Isoelectric focusing of mycoplasma proteins

“…The use of polyacrylamide, as a disc gel (1, 3, 6, 7, 9, 11, 14-17, 19, 20, 22), or direct comparison, as a flat gel (24,25), requires solubilization of the cells, either in a phenolacetic acid-water mixture (14) or in sodium dodecyl sulfate (6). It has been difficult to distinguish species-strain-specific antigens in such preparations (9). Polyacrylamide gel isoelectric focusing (PAGIF) enables high-resolution separation of proteins as first demonstrated by Awdeh et al (2), Leaback and Rutter (13), and Dale and Latner (5) with immunoglobulins, hemoglobins, and serum proteins, respectively.…”