Search

The insect cell lines Dm-1 (Drosophila melanogaster), AS-2 (Aceratagallia sanguinolenta) and AC-20 (Agallia constricta) were infected with spiroplasmas, mycoplasmas and Acholeplasma laidlawii. In Dm-1 cultures maintained at 25 degrees C in M1A medium, all strains multiplied except M. hyorhinis and the uncultivable sex-ratio organism. Spiroplasma citri R8A2, S. floricola BNR-1 and OBMG, S. apis PPS-1 and the strains BC-3, corn stunt spiroplasma (CSS) and 277F produced cytopathogenic effects (CPE), whereas S. mirum SMCA, M. orale, M. arginini and A. laidlawii did not. Cytadsorption was found with the cultivable spiroplasmas and A. laidlawii. At 30 degrees C SMCA, M. orale, M. arginini and A. laidlawii killed the Dm-1 cultures. M. hyorhinis grew without any CPE. In AS-2 and AC-20 cultures grown at 28 degrees C in LB medium, R8A2, B88, 277F, BNR-1 and PPS-1 multiplied and reached titres of 2 X 10(8) to 4 X 10(9) CFU/ml. They produced CPE leading to culture death. CSS did not grow. R8A2 reached higher titres in AS-2 cultures than in fresh LB medium. This stimulating factor was studied by means of conditioned medium. All 6 spiroplasmas cytadsorbed to AS-2 and AC-20 cells. B88 and 277F adsorbed heavily, while the other 4 strains adsorbed only slightly. Fluorescent DNA staining with "Hoechst 33258" revealed the presence of non-helical forms inside the cells.

Greening disease of citrus is characterized by the presence of procaryotic organisms in the sieve tubes of infected plants. These procaryotes have often been called mycoplasma-like. We have previously shown that the envelope of the organism was composed of two membranes, each with a triple-layered structure: an inner membrane (cytoplasmic membrane) and an outer membrane. Penicillin treatment of greening-affected plants results in remission of symptoms, suggesting the presence of a peptidoglycan (PG) layer in the envelope of the organism. However, when observed by conventional electron microscopy, no PG layer could be detected in the envelope of the greening organism (GO). Recently, we were able to transmit the GO from citrus to periwinkles by dodder. In periwinkles, GO multiply to high titres and, therefore, characterization studies can be carried out directly on the organisms in situ. Using papain treatment of GO in greening-infected periwinkles, we were able to visualize a PG-like layer in the envelope of the GO. This layer was removed by lysozyme treatment. In these respects, the structure of the GO envelope was nearly identical to that of E. coli, a Gram-negative bacterium, but was different from that of Staphylococcus aureus (a Gram-positive bacterium) treated in the same way. From the presence of a membranous PG-containing cell wall, the GO appears to be a true bacterium of the Gram-negative type, and not a mycoplasma.

Two spiroplasma strains (B31 and B39) recovered from diseased honey-bees (Apis mellifera) in southwestern France were similar in biochemical, serological and pathological properties. The organisms grew at 30 degrees C, required cholesterol for growth, fermented glucose, catabolized arginine and produced a film and spot reaction. The two spiroplasmas were serologically indistinguishable but were related to serogroup IV spiroplasmas, which had been previously isolated from flower surfaces and from insects. The isolates were distinct from the three previously established species of Spiroplasma and from other presently known serogroups. The G + C content of the DNA from strain B31 was 30 +/- 1 mol %. Both B31 and B39 strains were associated with a lethal infection ("May disease") of the honey-bee. On the basis of the characterization presented here, it is proposed that these spiroplasmal pathogens of bees and allied strains be classified as a new species, Spiroplasma apis, the type strain of which is B31 (ATCC 33834).