The effect of mycoplasmas and acholeplasmas of equine origin on organ cultures of chicken-embryo trachea.

Chicken-embryo tracheal organ cultures were inoculated with equine strains of Mycoplasma arginini, M. equigenitalium, 2 strains of M. subdolum, Acholeplasma laidlawii and 3 strains of A. oculi. All strains established and multiplied in the explant cultures, but only M. subdolum and A. oculi produced a cytopathic effect on ciliated epithelial cells, causing sloughing of cells and cilia after 6 days. There was a correlation between ciliostasis and increase in titre of both M. subdolum and A. oculi and this relationship was not observed with M. equigenitalium and A. laidlawii. All the strains of acholeplasma multiplied to some extent in organ culture media, but reached higher titres in the presence of explants. Cells infected with the M. subdolum strain showed sloughing of cilia, vacuolization, and increase in size of mitochondria, followed by disorganization of epithelium and marked destruction of subcellular organelles. Mycoplasmas were closely attached to the epithelial surface of the tracheal explant 8 days after infection.

Pathogenicity of mollicutes for insects: possible use in biological control.

Acholeplasmas, spiroplasmas and other non-helical sterol-requiring mycoplasmas of unknown phylogenetic affinity inhabit insects. Of these, only spiroplasmas are known to be pathogenic. Group I-2 spiroplasmas, or Spiroplasma apis, especially in combination with other organisms, reduce honey-bee longevity. Plant pathogenic mycoplasma-like organisms are often found intracellularly in insects. Spiroplasmas are found predominantly in the gut lumen or haemolymph (or both) of their insect hosts. Pathogenicity of mycoplasmas is usually altered by extended passage in unusual hosts, in only one of two alternate hosts, or in culture media. Enhancement of experimental pathogenicity may occur with extended cultural passages, but maintenance of natural pathogenicity must be accomplished by continuous exposure to the usual host. Recent data provide new information on the ecology of pathogenicity. Spiroplasmas from unique habitats also tend to be unique. Spiroplasmas isolated from flowers appear to be adapted to insect species that frequent floral surfaces. Group IV spiroplasmas have been isolated from members of 4 holometabolous insect orders (including Lepidoptera), all of which visit flowers. Social or predatory insects, or insects with an "aggregation" phase in their life histories, also appear to be prone to spiroplasma infection. Some insect species which harbor spiroplasmas also carry infections of other mollicutes, some of which involve the haemolymph. Appearance of spiroplasmas in adult insects in nature is strongly affected by seasonality. Extensive tests of the host ranges of the new insect mollicutes will be required before their suitability for biological control can be evaluated.

Isolation and pathogenicity of mycoplasmas from geese.

Several mycoplasma isolation trials were performed on infertile goose eggs and goose embryos which died during incubation, as well as on geese of different ages. A total of 43 out of 110 goose eggs proved to be contaminated by mycoplasmas. Upon autopsy of birds which laid positive eggs, lesions were observed in the airsacs. Mycoplasmas could be isolated from their air sacs and oviduct. Four out of 15 strains examined biochemically and serologically with antisera prepared against all known avian mycoplasma species were identified as Acholeplasma laidlawii and A. axanthum, respectively. Two strains proved to be glucose-positive and arginine-negative and 9 were glucose-negative but arginine-positive. Some strains caused 50-80% mortality among embryos inoculated intra-yolk-sac at 12 days. In goslings inoculated at the age of 3 days with these strains, we observed fibrinous airsacculitis and peritonitis. By inoculating laying geese with one of the strains, we demonstrated decreasing egg production, increasing early-embryo mortality and egg transmission of mycoplasmas.

Insect cell cultures in the study of attachment and pathogenicity of spiroplasmas and mycoplasmas.

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.

Isolation and characterization of Mycoplasma and Acholeplasma from apparently healthy and diseased (infectious sinusitis) turkeys.

Investigation of 136 turkeys (24 manifesting infra-orbital sinusitis, 112 apparently healthy) resulted in isolation of 79 strains of Mycoplasma and 4 of Acholeplasma. By the disc growth inhibition test with 16 reference antisera of avian serogroups, 55 strains were identified serologically and 28 remained unidentified. Thirteen strains of Mycoplasma gallisepticum, 1 of M. meleagridis, and 2 of Acholeplasma laidlawii were isolated from turkey sinusitis whereas serogroups C (2), D (19), F (8), M. meleagridis (4), M. anatis (4), A. laidlawii (2), and 28 unidentified strains were isolated from apparently healthy turkeys. Three patterns were recognized on the basis of glucose, maltose, and sucrose, fermentation. The most frequent, pattern I, included 13 M. gallisepticum strains whereas 5 M. meleagridis strains belonged to fermentation pattern III. Isolates were also studied for reduction of tetrazolium, methylene blue, potassium tellurite, resistance to methylene blue and sodium taurocholate, and production of arginine deiminase and "film and sports." Inoculation of selected isolates into developing chick embryos revealed that 2 A. laidlawii strains were nonpathogenic and 13 M. gallisepticum, 1 serogroup D and 2 serogroup F strains were pathogenic, causing 50--100% mortality. In vitro antibiotic disc sensitivity tests indicated that rovamycin (solubilized spiramycin) may be recommended for turkey mycoplasmosis. Isolation of 2 A. laidlawii strains from turkey sinusitis and 4 M. anatis strains from apparently healthy turkeys appears interesting.

Pathogenicity of some Mycoplasma and Acholeplasma species in the lungs of gnotobiotic calves.

Cloned cultures of 16 strains, representing nine different species of Mycoplasma and Acholeplasma, were inoculated intratracheally into gnotobiotic calves. Strains of M bovirhinis, M canadense, M verecundum, A axanthum and A modicum did not produce visible pneumonic lesions and were not reisolated from the lungs. Strains of M alkalescens and M arginini colonised the lower respiratory tract but failed to produce visible pneumonia. M bovigenitalium (strain M991/70) and M dispar (strain Gri226) both colonised the respiratory tract and induced pneumonic lesions estimated to involve up to 8 per cent (M bovigenitalium) and 17 per cent (M dispar) of the lung. Histologically M bovigenitalium produced a cuffing pneumonia and M dispar produced a interstitial alveolitis.