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.     

Mycoplasma infection of geese. 1. Incidence of mycoplasmas and acholeplasmas in geese

This paper presents data about the isolation of members of the order Mycoplasmatales from material of goose origin. Acholeplasma laidlawii strains were isolated from 2 to 8 day old goslings with heavy fibrinous airsacculitis, peritonitis and perihepatitis. Losses reached 30% of the flock by the end of the 8th week of age. Acholeplasma axanthum strains were detected in goose-embryos that died on the 13th day of incubation. A significant loss (up to 60%) of embryos was observed in the flock and some layers died showing fibrinous peritonitis, salpingitis and abdominal airsacculitis. Mycoplasma gallinarum also was isolated from goose-embryo fibroblast tissue cultures. All strains except A. laidlawii caused cytoplasmic vacuolization and intracytoplasmic inclusion bodies in goose-embryo fibroblast tissue cultures. The alteration observed in chicken-embryo fibroblast cell cultures were similar; in addition, the A. laidlawii caused a marked pycnosis of the cells.     

The influence of mycoplasmas on the cytopathic effect of varicella virus

Summary The development of varicella virus microplaques in human embryo lung fibroblasts and human thyroid cells was completely or almost completely inhibited by an arginine-metabolizing mycoplasma,M. arginini. On the other hand, the cytopathic effect of a rhinovirus was unaffected, and varicella virus microplaque formation was uninfluenced by two glucose-fermenting mycoplasmas. These facts, together with the observation that varicella virus microplaque formation was dependent on the presence of arginine in the cell cultures, suggested that depletion of arginine was responsible for the inhibition of microplaque development. However, the addition of arginine to both varicella virus- and herpes simplex virus-infected cultures only partially reversed the inhibitory effect of the mycoplasma. This indicates that other factors, in addition to arginine depletion, may be involved. The results clearly show that only those cultures which are free from arginine-metabolizing mycoplasmas are likely to be useful for isolating and studying varicella virus.     

Possible teratogenic effect of mycoplasmas on the human fetus]

The viscera and brains of 18 stillborns and dying newborn babies infected with mycoplasmas were examined. The etiological diagnosis was done by direct Coons method using antisera against M. hominis and M. pneumoniae. A high percentage (50%) of congenital malformations in mycoplasma-infected babies was observed. Histological and histochemical examinations of the brains and internal organs revealed marked circulatory disorders and degenerative changes indicative of a pathological process which had occurred. Mycoplasmal infection contracted by the fetus in the uterus or during the delivery may be assumed to be pathogenic. Based on the data from the literature and the own observations it is suggested that urogenital mycoplasmas may exert a teratogenic effect on the human fetus.     

Adjuvant effect of lysolecithin analogues on the development of contact sensitivity in mice.

The influence of two lysolecithin analogues on the development of contact sensitivity (CS) to picryl chloride in mice was studied. Both substances were shown to have an adjuvant effect on the primary CS response, but depending on dosage of adjuvant and antigen, the timing of adjuvant injection, and the strain of mice employed, the relative activities of the two substances varied considerably. Large amounts of one of the adjuvants were less stimulatory than small amounts. Both substances had a suppressive effect on the development of CS following repeated administration of picryl chloride. The results are discussed in relation to hypotheses on the cellular targets for, and mode of action of, immunological adjuvants.     

Effect of lysolecithin analogues on plant viruses

Newly synthesized lysolecithin analogues 2-0-hexadecyl-glycero-3-phosphocholine (1) and its methyl (2), ethyl (3) and benzyl (4) derivatives were tested with regard to the anti- phytoviral effect on potato virus X ( PVX ), red clover mottle virus ( RCMV ) and tobacco mosaic virus (TMV). The first two compounds (1, 2) reduced markedly the content of PVX and RCMV in systemically infected host plants as evidenced by precipitin test and bio-assay. Whereas compounds (1), (2) and (3) did not significantly influence the local lesion formation caused by TMV, compound (4) increased the number of necrotic lesions. In the presence of all four lysolecithin analogues, especially of (1), (2) and (3) the infectivity of virus particles was reduced.     

The prolonged persistence of mycoplasmas in culture

Mycoplasma fermentans organisms in medium containing 20% horse serum multiplied to 10(7)-10(10) ccu/ml within 5 days at 37 degrees C and were dead usually after 9 days. There was no growth in medium without serum, nor in such medium with the addition of cholesterol or palmitic acid or both, but in some experiments addition of bovine plasma albumin (BPA) increased the number of organisms by up to 1000-fold and some remained viable for up to 84 days. BPA and cholesterol or BPA, palmitic acid and cholesterol more often enhanced growth, in terms of the maximum number of organisms and their survival, than did the addition of BPA alone. The maximum number of organisms in such supplemented serum-free media was usually at least ten-fold less than in medium with horse serum, but some organisms remained viable for up to 131 days. Survival of Ureaplasma urealyticum was also longer in the supplemented serum-free medium than in standard horse-serum medium. The possible factors affecting persistence of mycoplasmas in culture are discussed in relation to these observations.     

Guanylate cyclase from bovine adrenal medulla: Subcellular distribution and studies on the effect of lysolecithin on enzyme activity

The localization of guanylate cyclase in bovine adrenal medulla was explored by preparation of subcellular organelles. Forty per cent of the activity was recovered in the soluble fraction (cytosol), and 46% in the low-speed (800g) pellet as particulate. In order to determine the exact localization of the particulate enzyme, continuous sucrose density gradient centrifugation was used. The results indicate that guanylate cyclase activity sedimented together with acetylcholinesterase and adenylate cyclase, two plasma membrane markers. Enrichment of specific activity over that present in the crude chromaffin granule fraction was identical for these three enzymes (from 4.4 to 6.2-fold). Particulate guanylate cyclase appears therefore to be a constituent of plasma membranes.The non-ionic detergent Triton X-100 and the natural phospholipid, lysolecithin, stimulated particulate guanylate cyclase activity 20-fold, but that of the soluble enzyme only 2- to 3-fold. The kinetic behaviour of particulate guanylate cyclase was not altered by treatment with lysolecithin: the Hill n coefficient for guanosine 5′-triphosphate remained close to 1.35 and the S_0.5 value to 320 μM. Among lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce activation of particulate guanylate cyclase. Incubation of membranes with phospholipase A₂ led to a 5-fold stimulation of particulate guanylate cyclase activity, while the soluble enzyme activity was not affected. These results suggest a possible role for lysolecithin in the regulation of the intracellular levels of cyclic guanosine monophosphate during or following the excitation-secretion coupling process in the medullary cell.     

Effect of lymecycline on pathogenic mycoplasmas

Results of sensitivity to antibiotic in vitro and results of antibiotic treatment in vivo seem to recognize so far lymecycline as the tetracycline to prescribe in infections by mycoplasmas.     

The differential effect of cooling on responses of cerebellar cortex.

1. Responses of the cerebellar cortex in anaesthetized cats were evoked by mossy fibre and/or climbing fibre inputs, and the effects of graded cooling of the cerebellar cortex were investigated. Cooling was applied either globally by flooding the exposed cortex with cooled Ringer Locke, or in later experiments locally be passing cooled fluid through a silver tube in contact with the cerebellar cortex. The cortical temperature was continuously monitored by a thermistor inserted to a depth of 0.5 mm close to the recording site. 2. In the granular layer the cooling caused a large increase in the diphasic P1N1 wave generated by the afferent mossy fibre volley. The waves generated by synaptic excitation and discharge of granule cells, N2P2, were not diminished until the temperature fell towards 20 degrees C. In contrast the N3 wave of the molecular layer was largest with cooling in the range of 35 to 25 degrees C, often several times larger than at 38 to 40 degrees C. Associated with the enhanced N3 wave there was an enhanced N4 wave, which indicates an increased discharge by Purkyn? cells. 3. Climbing fibre inputs generate a negative field potential in the molecular layer due to the powerful excitation of Purkyn? cells. In contrast to the N3 potential this climbing fibre wave was largest at the higher temperatures 35-40 degrees C and declined progressively with cooling, being usually suppressed at moderate coolings of 31-27 degrees C. Intracellular recording revealed that the diminution was due both to the elimination of all but the first impulses of the normal burst discharge of the climbing fibre impulses and to the diminution of the synaptic excitation of a single climbing fibre impulse. 4. It is shown that the negative potentials produced in the molecular layer by combinations of mossy fibre and climbing fibre inputs can be very effectively distinguished by this differential effect of cooling. 5. The effects of cooling even to a severe level are immediately recoverable on warming. Repeated cooling has no untoward effects and there is no sign of the hysteresis reported for the cuneate nucleus. 6. There is a discussion of the factors that could cause cooling to differentiate between the actions of the mossy fibre and climbing fibre impulses on Purkyn? cells.     

Glycosidase activities of mycoplasmas

The activities of alpha- and beta-glucosidase, beta-galactosidase and beta-N acetylglucosaminidase were assessed at acidic pH by fluorimetry using the appropriate 4-methylumbelliferyl substrate in four Mycoplasma species (M. pneumoniae, M. gallisepticum, M. hominis and M. capricolum) and in Acholeplasma laidlawii. The glycosidase activities were in a low range (0.1-4.2 nmole per h per mg protein) with the exception of higher activities of beta-N-acetylglucosaminidase in A. laidlawii. The enzyme levels of a virulent and a nonvirulent strain of M. pneumoniae were comparable. Despite the very sensitive assay, neuraminidase activity was not detected in M. pneumoniae and M. gallisepticum. No induction of alpha-glucosidase could be demonstrated for M. pneumoniae or A. laidlawii. At least part of the glycosidase activities was localized in the membrane fraction of all mycoplasmas studied. This may support the hypothesis that pathogenic mycoplasmas, being membrane parasites, may modify, by their glycosidases, some host cell glycoconjugates. However, our study did not distinguish the pathogenic mycoplasmas to possess a characteristic glycosidase profile.     

The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation

This study aims to explore the differential effect of scaffold composition and architecture on chondrogenic response to dynamic strain stimulation using encapsulating PEG-based hydrogels and primary bovine chondrocytes. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic materials to be used as scaffolds. Four different compositions were tested, including: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only. Primary articular chondrocytes were encapsulated in the hydrogel scaffolds and subjected to 15% dynamic compressive strain stimulation at 1-Hz frequency for 28 days. Stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the unconfined true compressive modulus by 32%, 45.4%, 33.6%, and 28.2%, compared to their static controls. The PF showed a significantly larger relative increase in the modulus in comparison to all other scaffolds tested. These results support the hypothesis that mechanical stimulation and material bioactivity have a significant effect on the reported chondrocyte response. Similar trends were observed with the swelling ratio of the constructs. These findings indicate that while stimulation causes metabolic changes in chondrocytes seeded in PEG hydrogels, the matrix bioactivity has a significant role in enhancing chondrocyte mechanotransduction in encapsulating scaffolds subjected to physical deformations.     

Effects of Baytril, Tylosin and Tiamulin on avian mycoplasmas

The minimum inhibitory concentration (MIC) of Baytril, Tylosin and Tiamulin for strains of Mycoplasma gallisepticum (MG) M. synoviae (MS), M. meleagridis (MM) and M. iowae (MI) and serovars were compared. In general the lowest MIC for MG, MS and MI was obtained with Baytril, while for MM both Baytril and Tiamulin gave the lowest MICs. Protection against mortality was best attained with Baytril for broiler chicks and poults but against prevention of growth depression Baytril was best for broiler chicks whilst Baytril and Tylosin were equally effective in poults. MG was recovered from fewer birds following treatment with Tiamulin and then Baytril in that order. Baytril also had a suppressive effect on the natural infection of MI in poults. Fewer poults showed rapid serum agglutination reactions to MG antigen following Baytril treatment than with the other antimicrobials. The tolerance of turkey embryos for Baytril was between 1 and 2 mg/ egg. Baytril reduced MG infection of turkey embryos.     

Haemagglutinins of pathogenic avian mycoplasmas

The pathogenic avian mycoplasmas, Mycoplasma gallisepticum , Mycoplasma synoviae , Mycoplasma meleagridis , Mycoplasma iowae and Mycoplasma imitans , synthesize haemagglutinins that are immunogenic, variably expressed, surface proteins. The haemagglutinins of M. gallisepticum (pMGA), M. synoviae (VlhA) and M. imitans are lipoproteins, encoded by related multigene families that appear to have arisen by horizontal gene transfer. M. gallisepticum also has genes encoding cytadhesins in its genome but these are present as a single copies, while the pMGA gene family contains 30 to 70 genes. The switch in expression of distinct pMGA genes (e.g. pMGA1.1 to pMGA1.9) generates antigenic variation, which is thought to be important in immune evasion but also has significance in the preparation of M. gallisepticum antigens for serological diagnosis. In the majority of M. synoviae strains, post-translational cleavage of the VlhA protein generates an amino-terminal part (the lipoprotein MSPB) and a carboxyl-terminal part (MSPA), which mediates binding to erythrocytes. The 5' vlhA gene region, which encodes proline-rich repeats in the amino-terminal part of MSPB, is highly polymorphic among M. synoviae strains. Insertions or deletions in the part of vlhA encoding the proline-rich repeats cause MSPB length variation in different M. synoviae strains. Recombination between the 5' vlhA gene and pseudogenes in the genome generates changes in antigenic determinants in the carboxyl two-thirds of the MSPB molecule, and in MSPA, resulting in changes in the domains involved in the binding of M. synoviae to erythrocytes. Variant haemagglutinins of M. gallisepticum (pMGA1.7) and M. synoviae (diverse VlhA forms) share sequences that may be responsible for antigenic cross-reactions between M. gallisepticum and M. synoviae . Shared epitopes have been demonstrated using specific antibodies against MSPB that also recognize proteins of M. gallisepticum and of M. iowae (serotype N). Size and antigenic variants have also been reported for M. meleagridis and M. iowae proteins, but it is not known if these are their haemagglutinins. Advances in the molecular characterization of M. gallisepticum (pMGA, pvpA ) and M. synoviae ( vlhA ) genes and their sequencing in numerous strains is likely to enable significantly improved epidemiological studies and improved tracing of M. gallisepticum and M. synoviae strains in different flocks.     

In vitro activity of nitroxoline on urogenital mycoplasmas

The product nitroxoline was studied in vitro for its activity towards Ureaplasma urealyticum and Mycoplasma hominis. In view of the low MIC values obtained, it seems nitroxoline could be used in the treatment of urinary infections. It is bactericidal, and should not produce resistant strains.