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Attachment of Mycoplasma pneumoniae to human WiDr cell culture monolayers was examined by using radiolabeled M. pneumoniae. The amount of attachment was proportional to the density of the WiDr cells and to the concentration of M. pneumoniae in the assay. Saturation of the monolayers was achieved with 40 micrograms of virulent strain M129 per assay, whereas binding of avirulent strain B176 was 70% less than that of strain M129. A competitive attachment inhibition assay was used to measure specific binding component activity. Attachment was inhibited when WiDr cells were pretreated with unlabeled virulent strain M129, whereas avirulent noncytadsorbing strain B176 did not inhibit attachment as well as the virulent strain. A protein-rich extract prepared from virulent, cytadsorbing strains of M. pneumoniae also inhibited attachment. The amount of inhibition was dependent on the amount of extract used, and units for binding component activity in the extract were calculated from the competitive attachment inhibition assays. The competitive attachment inhibition assay was also used to investigate the nature of the receptor site on the WiDr cells. Attachment was inhibited when the radiolabeled M. pneumoniae suspensions were pretreated with human sialoglycoproteins, such as orosomucoid and ceruloplasmin, and bovine gangliosides. These findings support the present concept that the mammalian receptor site for M. pneumoniae is a sialic acid-containing glycoprotein.

A diphtheria toxin shown to have high toxicity and avidity and to combine with antitoxin in multiple proportions was selected for the U.S. standard diphtheria toxin for the Schick test and freeze-dried. Assayed values per vial were 1.09 L f , 1.09 L r , 1,090 Schick test doses (STD), 33 LD 80 , 38 LD 50 , and 43,000 minimum skin reactive doses. One STD (L r /1000) is slightly more toxic than one unit of the international standard (L f /1,000). Experiments showed that the potency assay of Schick test toxin by guinea pig erythema toxicity, determined relative to the toxicity of the standard, was highly reproducible and significantly more reproducible than the lethal (minimum lethal dose) test and that the STD, defined as one L r /1000, was equivalent to approximately 1/50 minimum lethal dose. The erythema potency assay was prescribed in the U.S. standards for Schick test toxin effective in 1969.

Schimke, Robert T. (National Institutes of Health, Bethesda, Md.) and Michael F. Barile . Arginine metabolism in pleuropneumonia-like organisms isolated from mammalian cell culture. J. Bacteriol. 86: 195–206. 1963.—Arginine degradation is a significant metabolic process for pleuropneumonia-like organisms (PPLO; Mycoplasma ) isolated from cell culture. The conversion of arginine to ornithine in PPLO-contaminated cell culture was rapid, and occurred by the arginine dihydrolase pathway involving arginine deiminase, ornithine transcarbamylase, and carbamyl phosphokinase. In the absence of PPLO contamination, arginine conversion to ornithine was minimal and took place by an arginase activity present in the cell culture, but not in the PPLO. All five PPLO strains isolated from cell culture accomplished the conversion of arginine to ornithine, and contained the requisite enzyme of the arginine dihydrolase system, whereas PPLO-free cell cultures did not. Supplementation of PPLO culture broth with arginine increased the extent of PPLO growth. When the arginine content of the culture limited growth, arginine was completely converted to ornithine. When growth was limited in the presence of excess arginine, citrulline was the major breakdown product. It is suggested that the conversion of arginine to ornithing constitutes a significant, and possibly major, source of adenosine triphosphate for this class of organisms.

Fourteen conjunctival strains of mycoplasma were isolated from infected sheep and goats in two separate naturally occurring outbreaks of keratoconjunctivitis (pink-eye). The biological and serological properties and cell protein patterns of these strains are presented in this report. These strains were related to each other and were unrelated to 40 recognized Mycoplasma and Acholeplasma species and serotypes. We are proposing that this group of mycoplasmas be named M. conjunctivae. The role of M. conjunctivae in the etiology of keratoconjunctivitis in sheep and goats remains to be determined.