Purification and kinetics of extracellular phospholipase A of Salmonella newport.

Attempts were made to purify and study the kinetics of extracellular phospholipase A of Salmonella newport (6,8, eb; 1,2). The enzyme was purified by salt precipitation followed by gel filtration, using different grades of Sephadex. The enzymically active purified preparation was found to be a protein, having molar mass ranging between 43 and 67 kDa. The enzyme had a pH optimum at 7.5, giving 18.2 micrograms of lysophosphatidylcholine per mg protein. Its activity was enhanced by all metal ions except potassium, by solvents and surfactants except sodium dodecyl sulfate. It hydrolyzed the membrane phospholipids of red blood cells and was inhibitory to the growth of other microorganisms.

Structural and functional changes in rabbit ileum by purified extracellular phospholipase A of Salmonella newport.

As phospholipases of Salmonella species may play a role in the pathogenesis of gastrointestinal tract diseases. Salmonella newport, the causative agent of infantile diarrhoea was examined for the production of phospholipase. The enzyme was purified by gel filtration chromatography and was found to be a protein of molar mass ranging from 43 to 67 kDa. The purified enzyme alone or in combination with organisms, produced both structural and functional changes in rabbit ileum, contributing towards pathogenesis of diarrhoea due to this organism.

Prevention of malaria-induced foetal abnormalities following immunization of mice with Plasmodium berghei merozoite antigen.

Pre-pregnancy immunization of Swiss albino mice with merozoite antigen of P. berghei entrapped in multilamellar phosphatidyl choline liposomes resulted in (i) increased prepatent period, (ii) either no or low parasitaemic levels, (iii) reduced mortality, and (iv) normal foetal and placental development, upon challenge with P. berghei on 13th gestational day. The unimmunized animals which received either phosphate buffered saline or empty multilamellar phosphatidyl choline liposomes before pregnancy developed high parasitaemic and 30-40 per cent animals died before parturition while 60-70 per cent unimmunized animals revealed foetal abnormalities such as low body weight and larger spleen size. Placentae of unprotected animals had hyperplasia of trophoblastic membrane and plugging of placental sinusoids with parasitized erythrocytes and malarial pigments. The data suggest that prior immunization of animals with merozoite antigen entrapped in multilamellar phosphatidyl choline liposomes could abrogate the ill effects induced by malaria infection under the stress of pregnancy.

Effect of prior eradication of Plasmodium berghei infection on the foetal development and parasitaemic levels under the stress of pregnancy.

Pregnant mice infected on gestation day (GD) 13 with Plasmodium berghei had similar rate of parasitaemia and mortality as non pregnant controls. 50% of pregnant infected mice had normal delivery, 20% had absorbed foetuses and 30% died before parturation. However, animals infected with P. berghei, treated with drugs (sulfadiazine or chloroquine) had normal foetal development. No recrudescence occurred in either of these groups of animals even under the stress of pregnancy indicating protection. Pups born to pregnant infected mice (GD) 13 had low body weight as compared to pups born to mice which were infected and treated with drugs. Histopathological findings showed hyperplasia of trophoblast and plugging of placental sinusoids with parasitized erythrocytes in animals infected on GD 13 only. It is felt that prior eradication of malaria infection results in development of sufficient immunity which prevents the mal development of foetuses or parasitaemic levels under the stress of pregnancy.

Characterization of phospholipase A2 of mycoplasma species.

As phospholipases of mycoplasma species may play a role in the pathogenesis of respiratory tract and urogenital tract diseases Mycoplasma mycoides and Acholeplasma laidlawii were examined as to the production of phospholipase A2 (PLA) and attempts were made to purify and characterize it. Both species produced PLA. The purified enzyme was found to be heat-labile, active at alkaline pH, revealing a single band in polyacrylamide gel electrophoresis. Metal ions such as calcium and barium, increased its activity whereas solvents at high concentrations decreased it. It was resistant to surfactants.

Production and purification of Escherichia coli hemolysin.

Eight strains of Escherichia coli, isolated from patients with a urinary tract infection were investigated for production of hemolysin. Six of these produced hemolysin and one revealed maximum hemolytic activity. Three urinary and two faecal isolates were positive for mannose-resistant hemagglutination. One isolate positive for hemagglutination and giving maximum hemolytic activity was then used. Hemolysin was present in the supernatant broth and the medium of choice to obtain the optimum yield was the alkaline meat extract broth followed by brain heart infusion broth. The highest yield appeared in the exponential phase of growth. Hemolysin is a heat-labile protein, being produced optimally at pH 8. A three-stage procedure was the best method for its purification.

Influence of malarial infection on the maternal-foetal relationship in pregnant mice.

Pregnant mice infected on gestation day (GD) 6 with Plasmodium berghei showed a more rapid rate of increase in parasitaemia than mice infected later in pregnancy or non-pregnant controls. All mice infected on GD 6 were dead by the 7th post-infection day. Pregnant mice infected on GD 13, in contrast, had similar rates of parasitaemia and mortality as non-pregnant controls and 50% delivered normally, the foetuses were absorbed in 20% and 30% died before parturition. The pups born to mice infected on GD 13 were smaller. This compromised foetal development was likely to be the result of maternal anaemia, hyperplasia of placental trophoblast and plugging of placental sinusoids with parasitized red cells.

Experimental haematogenous pyelonephritis in mice with uropathogenic, enteropathogenic and enterotoxigenic Escherichia coli.

Acute haematogenous pyelonephritis was produced with known uropathogenic (075), enteropathogenic (0111) and enterotoxigenic (H10407) Escherichia coli in mice. The enterotoxigenic E. coli showed the highest bacterial counts in kidney and urine and was found to have the same potential for causing pyelonephritis as a known pyelonephritogenic strain both in untreated and corticosteroid treated mice. Enteropathogenic E. coli was found to be less pyelonephritogenic in untreated mice, but under the effect of corticosteroids it showed similar pyelonephritogenic potential.

Studies on enterotoxin of Shigella dysenteriae type 1. II. Systemic effects in rabbits of Shigella dysenteriae 1 enterotoxin.

The enterotoxin of Shigella dysenteriae type 1 is an acid and heat-labile protein. It induces a gut dilatory response and increases the levels of blood glucose, serum alkaline phosphatase and serum acid phosphatase in rabbits.

Induction of stable L-forms of Salmonella typhi and Listeria monocytogenes.

Stable L-forms of Salmonella typhi and Listeria monocytogenes were produced using penicillin (4500 units/ml) as inducer, and sucrose, normal horse serum and Mg++ as stabilizers. Stable L-forms were produced after 100 and 56 passages, then adapted to grow and multiply in a medium free of inducer and stabilizers so that they did not revert to parental forms even after 12 continuous passages.

Studies of enterotoxin of Shigella dysenteriae type 1. I. Effect of aeration on the production of enterotoxin and suitability of laboratory animals for its biological assay.

Shigella dysenteriae type 1 was used to study the influence of aeration on enterotoxin production and the suitability of laboratory animals for the toxin assay. The enterotoxin produced under aerobic shake culture conditions gave a gut dilatory response of 1.436 but that obtained in aerobic and anaerobic stationary cultures exerted no activity. The toxin fractionated on Sephadex G-150 column was more effective than that obtained by ammonium sulphate precipitation. Only Sephadex column fraction II had a toxic activity. Among the three laboratory animal models tested, the rabbit ileal loop was suitable for the assay of toxin. The skin permeability test was unsatisfactory for enterotoxicity assay.

Klebsiella pneumoniae enterotoxin. II. Physicochemical properties of enterotoxin.

Physicochemical properties and systemic effects of the enterotoxin of Klebsiella pneumoniae has been studied. The enterotoxin had a molecular weight between 10 000 to 50 000. It was protein in nature, and heat and acid stable, inducing a dilatatory response in the gut. It haemolyzed the erythrocytes of various animals including man. It had a capillary permeability activity. In addition, when administered parenterally it increased the level of blood glucose, serum cholesterol, serum alkaline phosphatase and serum acid phosphatase.

Klebsiella pneumoniae enterotoxin. III. Effect of Klebsiella pneumoniae enterotoxin on the intestinal transport and histopathological changes in rabbit ileal loop.

Klebsiella pneumoniae strain B-5-1 was used to study the effect of its enterotoxin on the intestinal transport and histopathological changes in the rabbit ileal loop. Enterotoxin increased the net flux of water and of electrolytes. Increased amounts of phospholipids and proteins were also detected together with structural and functional abnormalities in the ileum. All these suggest that K. pneumoniae enterotoxin not only induce a net flux of fluid and imbalance of electrolytes but also damages the intestinal structure.

Biological properties of L-forms and their parent bacteria.

L-forms of Staphylococcus aureus, Streptococcus faecalis, Listeria monocytogenes and Salmonella typhy and their parent bacteria were examined for biological properties and compared with their parental forms. Some L-forms differed from their parent bacteria and required a longer incubation period.

Induction of stable L-forms of Staphylococcus aureus and Streptococcus faecalis.

Stable L-forms were induced from Staphylococcus aureus and Streptococcus faecalis. These formed typical foamy L-colonies and showed large and small round bodies. They grew continuously on routine antibiotic-free nutrient broth and blood agar media for 12 passages without reversion to their parental forms. At different concentrations of penicillin various morphological forms were observed. Effect of sucrose, normal horse serum and penicillin on their adaptation and stabilization is discussed.

Serological interrelationships in avian mycoplasma serotypes.

A study of 18 standard avian mycoplasma and their antisera employing indirect haemagglutination test revealed distinct relationships among avian mycoplasma serotypes, so that they could be classified into 9 groups.