- Volume 30, Issue 2, 1989
Volume 30, Issue 2, 1989
- Review Article
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Improved ELISA with immunoabsorbent-purified mycobacterial antigen for serodiagnosis of tuberculosis
More LessSUMMARYAn antigen was purified from Mycobacterium tuberculosis H37Ra culture filtrate by immunoabsorbent affinity chromatography with CNBr-activated sepharose 4B column coupled with pooled γ-globulin fraction from patients with active tuberculosis. The column was washed extensively with PBS and eluted with 3M sodium thiocyanate. Peak fractions were pooled and used as coating antigen in an ELISA. Sera from 86 normal subjects and 54 patients with active tuberculosis were tested against the immunoabsorbed antigen by ELISA with biotin-conjugated anti-human globulin and avidin-peroxidase reagents. At a selected ‘cut-off’ dilution of 320, 49 (91%) of 54 sera from active cases and 8 (9.3%) of 86 sera from normal subjects gave positive test results—a sensitivity and specificity each of 91%, compared with our previous results of sensitivity 75% and specificity 83% with PPD as antigen.
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The virulence of Enterobacter cloacae and Serratia marcescens in experimental bladder infection in diabetic mice
Y. Obana and T. NishinoSUMMARYDiabetic mice were significantly more susceptible than normal mice to bladder infection with Enterobacter cloacae or Serratia marcescens but not to intraperitoneal infection. Normal urine inhibited the growth of E. cloacae and S. marcescens, whereas urine from diabetic mice permitted multiplication. The addition of urea to urine from diabetic mice restored its antibacterial properties for E. cloacae and S. marcescens. We consider that the decreased urea content of the urine of diabetic mice was responsible for their increased susceptibility to bacterial infection of the bladder.
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Haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii
More LessSUMMARYOf 120 laboratory-maintained strains of Listeria monocytogenes and two of L. ivanovii examined for haemolytic and lipolytic activity, 62 exhibited haemolytic activity alone, 20 of these showed haemolytic and lipolytic activity and 40 had neither activity. The L. ivanovii strains showed both activities. The results indicated a relationship between haemolysin production and lipolytic activity which was not explained by the serotype of the organism. In addition, the following hydrolytic activities were detected in the cell-free growth media of strains L. monocytogenes Boldy and L. ivanovii (formerly L. monocytogenes) Type 5 (substrates acted upon are given in parentheses): acid phosphatase (4-nitrophenylphosphate, naphthyl phosphate, glycerophosphate, phosphorylcholine and GTP); neutral phosphatase (4-nitrophenylphosphate, naphthyl phosphate, phosphorycholine, NADP and UDPG); phosphodiesterase (bis-4-nitrophenylphosphate, ATP and NADP); NADase (NAD); phospholipase C (4-nitrophenylphosphoryl-choline, phosphatidyl choline and ethanolamine, and sphingomyelin); and lipase and esterase (triacetin, tributyrin, triolein, naphthyl-laurate, -myristate, -caprylate, -palmitate and -oleate, 4-nitrophenyl-acetate -laurate and Tween 80). The preparations also showed weak catalase activity. No evidence was found for the presence of RNAase, DNAase, peptidase/amidase, phosphoamidase, α-amylase, glucosidase, galactosidase, pyranosidase or glucose aminidase.
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Separation and properties of the haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii
More LessSUMMARYDesalted ammonium-sulphate (0–65%) precipitates from the cell-free supernates of 16–24-h cultures of Listeria monocytogenes Boldy and L. ivanovii (previously L. monocytogenes) Type 5 were eluted through Sephadex G-200. The enzyme activities gave rise to two main peaks. The first peak (approximate mol. wt of protein 150 000) contained only phosphatase activity (assayed by hydrolysis of 4-nitrophenylphosphate at pH 5.0 and 7.0). The second peak (approximate mol. wts of proteins 40 000–60 000) contained the haemolysin activity and the following hydrolytic activities (assay substrates are given in parentheses): phospholipase C (phosphatidyl choline and 4-nitrophenyl-phosphoryl-choline); phosphodiesterase (bis-4-nitrophenyl-phosphate); acid phosphatase (4-nitrophenylphosphatase); and esterases and lipases (4-nitrophenyl acetate, naphthyl-acetate and -oleate, triacetin and triolein). DEAE-Sephadex chromatography of appropriate fractions from the Sephadex G-200 purification step separated the first peak into two phosphatases and resolved the second peak into its constituent activities. Polyacrylamide gel electrophoresis showed that the individual fractions from the DEAE-Sephadex step consisted of mixtures of protein. The effects of pH and potential activators and inhibitors on the active proteins purified by DEAE-Sephadex chromatography were examined.
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Characterisation of monoclonal antibodies for detection of Mobiluncus spp. in genital specimens
More LessSUMMARYMonoclonal antibodies were raised to the anaerobic curved rods, Mobiluncus curtisii subsp. curtisii NCTC 11656, M. curtisii subsp. holmesii NCTC 11657 and M. mulieris NCTC 11658. Three antibodies reacted with the two subspecies of M. curtisii and, when used in combination against clinical isolates, showed 100% sensitivity and specificity in immunofluorescence studies. Immunoblotting showed that two of these antibodies reacted with an epitope on a protein which had an electrophoretic mobility corresponding to a Mr of 75 Kda in the absence of a reducing agent and 82 Kda in its presence in both type strains and in clinical isolates. The third antibody reacted with an epitope in type strains which had a mobility corresponding to 75 Kda and was unaffected by a reducing agent. However, in clinical isolates the epitope was present on a protein of 75 Kda or 71 Kda, or on both. A fourth antibody showed reactivity with M. mulieris NCTC 11658 alone, but only 6 (24%) of 25 clinical isolates gave positive results by immunofluorescence. The epitope is believed to be present on a protein of < 90 Kda. All four antibodies were shown by immunogold staining to be directed against epitopes exposed on the cell surface.
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Mutation induced by vibriophage PS166 infection changes biotype and phage type of Vibrio cholerae
More LessSUMMARYThe effect of a newly isolated vibriophage, PS166, on Vibrio cholerae (El Tor) MAK757 was investigated. Two PS166-resistant mutants of strain MAK757 were isolated. These had undergone transition to the classical biotype with concomitant acquisition of new phage sensitivity. However, the parental Ogawa serotype remained unchanged. These mutant strains also showed a unique temperate phage-sensitivity profile, distinct from that of strain MAK757. The possible target of phage PS166 interaction is discussed.
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Selection of a Brucella vaccine strain of low residual virulence by chemical mutagenesis
More LessSUMMARYFollowing incubation of the oral vaccine Brucella suis strain 2 with diethyl sulphate (DES), a mutant designated strain S105 was selected by screening surviving bacteria for reduced virulence for mice. Strain 105 also showed low residual virulence for guinea-pigs and, unlike the parent strain, did not initiate abortion in pregnant sheep and goats after parenteral administration. Nevertheless, it was as effective as the parent strain in stimulating protective immunity to Brucella melitensis when given as an oral vaccine to sheep under both experimental and field conditions.
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Quantification of the leucocyte influx into rabbit ileal loops induced by strains of Salmonella typhimurium of different virulence
SUMMARYLeucocyte influx into rabbit ileal loops, induced by strains of Salmonella typhimurium of different virulence, was assessed with 111Indium-labelled leucocytes. Strains fell into two groups on the basis of their leucotactic potential: ‘virulent’ strains (which induced fluid secretion) caused a dose-dependent leucocyte influx; strains which did not induce fluid secretion failed to induce a significant leucocyte influx. Fluid secretion was never observed in the absence of leucocyte influx, but leucocyte influx per se did not induce fluid secretion. The phenotype of the challenge inoculum influenced fluid secretion; young log-phase organisms induced fluid secretion with a higher frequency than overnight cultures. These findings support earlier evidence implicating leucocytes in an interactive but not exclusive role in the genesis of salmonella-induced fluid secretion. They suggest, though do not prove, that interaction of leucocytes with the appropriate phenotype of organisms results in the release of a host-derived or bacterial secretagogue, or both. The bacterial factor may or may not be the antigen related to cholera toxin, described previously.
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