Recovery of velogenic Newcastle disease virus from dead and healthy free-roaming birds in Nigeria.

Fourteen Newcastle disease virus isolates were recovered, 10 from dead birds (nine chickens and one guinea fowl) and four from apparently healthy, free-roaming birds (one chicken, two ducks and one pigeon) in Nigeria. The pathogenicity indices showed all the isolates to be velogenic.

Incidence of cytotoxin producing isolates of Clostridium difficile in faeces of neonates and children in Nigeria.

One hundred and fifty six (156) confirmed isolates of Clostridium difficile from faeces of neonates and children in parts of Anambra State, Nigeria were screened and assayed for cytotoxin production by the tissue culture technique and the frequency of occurrence estimated. Twenty three out of 156 isolates were found to be cytotoxin positive isolates representing a frequency of 14.8%. There was no difference between the frequency of occurrence of cytotoxin positive isolates in neonates and children from rural and urban areas. Infants in the age group of one dy to 1 yr showed 16.7% frequency of occurrence of cytotoxin positive isolates, with toxin titers between 5 to 1280, 10% for children of 1-2 yrs age group, with titers between 5 to 40 and 8.3% for children of 2 to 3 yrs age group with titers of 5 and 10. No cytotoxin positive isolate was detected from children of the 3 to 5 yrs age group. Children fed by formula foods alone showed a 50% frequency of occurrence of cytotoxin positive isolates, children fed by breast milk plus formula supplementation, 19.23% and breast milk alone 17.5%. There was no significant difference in the frequency of occurrence of cytotoxin positive isolates in faeces from diarrhoeal and non diarrhoeal cases. It appears therefore, that age and mode of feeding are important factors that influence intestinal colonization of cytotoxin producing isolates of C. difficile in neonates and children.

Incidence of Clostridium difficile in infants in rural and urban areas of Nigeria.

Stool specimens collected from 320 infants under 5 years of age resident in both rural and urban centres of Nsukka and Enugu towns in Anambra State, Nigeria were examined for the presence of Clostridium difficile and the frequency of occurrence determined. Clostridium difficile was isolated from 156 out of 320 samples (48.8%). The frequency was higher in infants resident in rural areas (52.5-48.3%) than in their urban counterparts (47.8-42%). A high frequency rate (77%) was obtained in diarrheal cases as against 42% in non diarrheal cases. A frequency of 57.7% was obtained in infants in the age group of 1 day - 1 yr, which decline with age to the lowest 5.6% in the age group of 4.5 years. The frequency of 66.1% was obtained in infants breast-fed plus formula supplementation, 41.7% in infants exclusively breast-fed and 33% in infants fed exclusively with formula foods. The findings suggest that the type of living environments, together with age and mode of feeding may contribute to the high intestinal carriage of Clostridium difficile in infants in parts of Nigeria.

Isolation and some properties of beta-hemolysin produced by Nocardia asteroides.

An intracellular beta-hemolysin capable of lysing human, sheep and cow erythrocytes but not cells from some other animals was isolated from the cell walls of the three developmental cell-forms of Nocardia asteroides and characterised. The spherical cell-forms contained the highest amounts of the hemolysin (100 h.u./mg protein) and the least LD50 for mice suggesting that this may be the cell-form most pathogenic to susceptible animals. The hemolysin has the properties of a protein, was pH stable and sensitive to both catabolite repression and temperature. The activity of the hemolysin was enhanced by Ca++ and Na+ ions. The hemolysin was immunogenic in rabbits.

Antibacterial substance from Carica papaya fruit extract.

Ripe and unripe Carica papaya fruits (epicarp, endocarp, seeds and leaves) were extracted separately and purified. All the extracts except that of leaves produced very significant antibacterial activity on Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Shigella flexneri. The MIC of the substance was small (0.2-0.3 mg/ml) for gram-positive bacteria and large (1.5-4 mg/ml) for gram-negative bacteria. The substance was bactericidal and showed properties of a protein. Other proteins previously found in C. papaya did not show antibacterial activity.

Alkaline phosphatase from Nocardia asteroides.

Intracellular alkaline phosphatase (Apase) produced during growth of Nocardia asteroides in dephosphated nutrient broth supplemented with 1% glucose and some properties of the purified enzyme were studied. Filamentous cells of N. asteroides synthetized the Apase in high yields (7.0 x 10(3) units/mg cell protein) during exponential growth phase. The intracellular level dropped drastically and the extracellular amounts increased during stationary phase, probably due to the loss of the enzyme in the medium because of the fragmentation of the filamentous cells into rod-like and spherical forms and/or due to cell lysis. The purified enzyme showed typical Apase activity with optimal pH at 10 and optimal temperature at 37 degrees C. The Km of the enzyme for p-nitrophenyl phosphate was close to the value reported for Apase from other sources. Other properties of the enzyme which resembled those from other species included respectively the uncompetitive, non-competitive and competitive inhibition of L-phenylalanine, urea and inorganic phosphate. The effect of these compounds on the enzyme may be useful in the regulation of Apase activity in vivo and hence may play a vitalrole during morpho genesis of N. asteroides.

Isolation and metabolism of glycogen and poly-betahydroxybutyrate in Nocardia asteroides at different developmental stages.

Sudanophilic and iodine-staining cytoplasmic granules were isolated at various developmental stages during growth of Nocardia asteroides (strain 55) and identified as poly-beta-hydroxybutyrate (PHB) and glycogen granules, respectively. During growth in nutrient broth containing 1% glucose, maximal accumulation of PHB and glycogen granules, up to 10 and 20% respectively, of its dry-weight was obtained in the filamentous cells at 16 h just prior to the onset of cell fragmentation during the stationary phase. The decrease of the cytoplasmic granules was concomitant with fragmentation of the cells to rod-like and spherical cells, suggesting that the polymers may serve as carbon and energy source during morphogenesis. Both granules were detected in the three cell-forms. At higher concentrations of glucose (4%) more glycogen granules (9 times) accumulated than PHB (4 times) and glycogen hydrolysis was also faster than that of PHB, suggesting preferences of glycogen over PHB as energy and carbon source under this growth condition. Growth and biosynthesis of granules were significantly reduced by very high glucose concentration (10%) and the usual pleomorphic developmental stages were reduced to a dimorphic life cycle. Thus, biosynthesis of both granules and morphogenesis are under catabolite repression. Both polymers were also found in N. brasiliensis and N. otitidis-caviarum, indicating that cytoplasmic accumulation of multiple granules is common in the genus.

Immunological analysis of cells of Nocardia at various developmental stages during morphogenesis.

High antibody titres and a single precipitin line of identity were obtained with hyperimmune rabbit serum prepared against filamentous cells of Nocardia asteroides strain 55 and the cells of three other strains of Nocardia, indicating the presence of a group antigen between the strains. High titres were also obtained with the hyperimmune serum and the three different cell forms (filamentous, rod-like and spherical cells) of the homologous strain, indicating that each developmental stage was highly antigenic. The antibodies were completely removed by absorption with the various cell forms, suggesting a similarity of the antigens. A single common antigen was demonstrated between the three developmental stages by immunodiffusion, indicating that there was no antigenic alteration occuring during morphogenesis of the genus Nocardia.

Activity of some enzymes during growth and sporulation of Clostridium botulinum type E.

The activities of alkaline and acid phosphatases, glucose dehydrogenase and NADH oxidase were assayed in cell-free extracts of sporogenic and asporogenic mutants of Clostridium botulinum. During growth of both mutants, the activities of alkaline and acid phosphatases were relatively constant, but during sporulation of the sporogenic mutant, the alkaline phosphatase activity rose to a maximum of 70 mumol/min x mg protein whereas the acid phosphatase decreased rapidly before it increased, indicating a possible role in sporogenesis. Glucose dehydrogenase activity was detected only in cell-free extracts of the sporogenic mutant and reached a maximum of 7 mumol/min x mg protein during the endospore maturation stage. The NADH oxidase activity was detected in both mutants. The NADH oxidase seems to stimulate glucose oxidation in both mutants during growth and the dehydrogenation processes of the butyric type of fermentation during spore formation in the sporogenic mutant. The findings suggest that increased glucose dehydrogenase activity in C. botulinum, as in Bacillus species, may serve as a spore event marker and that alkaline and acid phosphatases may play a regulatory role in anaerobic sporulation metabolism.

Effect of cyclic AMP on catabolite repressed bacterial sporogenesis of an anaerobe.

The sporulation of a high frequency sporogenic mutant of Clostridium botulinum was reduced to less than 30% in a medium containing 270 mM glucose. The repression was reversed from 30 to greater than 80% sporulation by the addition of 10(-5) or 10(-4) M cyclic 3',5'-adenosine monophosphate (cAMP) or monobutyrul cyclic AMP (B-cAMP). No difference was observed in amount of growth with the addition of either the cAMP or B-cAMP. Glucose consumption was enhanced by the addition of either of the cyclic nucleotides and corresponding changes in pH were observed. The catabolite repression by glucose was reversed by ATP or ADP. Except for GTP, guanine nucleotides were not effective. The intracellular cyclic AMP levels were high in vegetative, sporulating and derepressed cells, but low in glucose-repressed cells. The findings suggest that the sporulation of the anaerobe was sensitive to catabolite repression which was specifically reversed by cyclic AMP.

Comparison of butyric type of fermentation in sporogenic and asporogenic mutants of Clostridium botulinum.

Glucose-adapted cells of a sporogenic mutant. MSp(+), and an asporogenic mutant, RSpoIIIa, of Clostridium botulinum type E rapidly fermented glucose, fructose, maltose, and sucrose, resulting in cytoplasmic granulation, heavy growth, a pH of <6.0, and sporulation of the MSp(+) mutant ranging from 60 to 80%. In Trypticase peptone glucose broth, the MSp(+) mutant formed >80% refractile endospores in 25 h, whereas the RSpoIIIa mutant which was blocked at early forespore stage had commenced to lyse. Both mutants accumulated acetate and intracellular granules, reaching maximal levels at early stationary phase of growth. In MSp(+), as the levels of acetokinase, phosphotransacetylase, and butyryl-coenzyme A dehydrogenase reached a maximum, butyrate accumulation continued concurrently with an increase of endospore formation, whereas the levels of poly-beta-hydroxybutyrate decreased simultaneously with its precursor, acetate. Butyrate biosynthesis was blocked in the asporogenic mutant. As shown by isotopic assays, butyrate and acetate serve as precursors of spore lipids. beta-Phenethyl alcohol, fluoroacetic acid, and 2-picolinic acid inhibited anaerobic sporogenesis almost completely, butyrate biosynthesis by >87%, and acetate accumulation by 50 to 62%, showing a direct relationship between butyric type of fermentation and anaerobic sporulation.

Poly-beta-hydroxybutyrate metabolism during growth and sporulation of Clostridium botulinum.

The granules observed in the cytoplasm of cells of sporogenic and asporogenic strains of Clostridium botulinum type E were isolated at various developmental stages of growth and sporulation. Electron microscopy of thin sections showed that most of the granules were dispersed throughout the cytoplasm. Chemical analysis and electron microscopy showed that the granules were poly-beta-hydroxybutyrate (PHB). The polymer began to accumulate after 8 h of growth, reaching 9 and 13% of the cell dry weight in the sporogenic and asporogenic strains, respectively, during early stationary phase. (14)C-acetate was readily incorporated into PHB. The rate of assimilation paralleled the production and utilization of PHB, indicating that the acetate served as its precursor. (14)C-butyric acid was not utilized to any significant extent. Most of the PHB which had accumulated in the sporogenic strain was catabolized during the development of the spore, but in the asporogenic mutant it remained essentially unchanged. The findings suggest that the PHB provides endogenous carbon and energy for the synthesis of spore-specific components required for spore maturation.

Structural changes associated with extraction of group E spore antigen of Clostridium botulinum.

Spores of type E Beluga strain of Clostridium botulinum, which were hydrolyzed with HCl to remove the spore-specific antigen, showed a spore coat which was thinner and projected into the exosporium with multiple protuberances. The changes observed suggest that the antigen is a component of the spore coat connected with the maintenance of rigidity.