Cloned genes of the aerobactin system of virulent avian Escherichia coli do not confer virulence to recombinant strains

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli

Spectrophotometric determination of total proteins in blood plasma with p-benzoquinone

In the present study we have documented the use of the reagent p-benzoquinone (PBQ) for the spectrophotometric determination of total protein in blood plasma. Since the products of reaction are stable for several hours at room temperature after the 20-min boiling step, the time at which absorbance is measured is not a critical factor. Common anticoagulants such as EDTYA, citrate, or heparin do not interfere with the PBQ method at concentrations used in clinical laboratories. The products of the reaction between PBQ and either plasma (specific absorbance 2.33 x 10-3 ñ 0.20 x 10-3 ug cm -2) or purified proteins (specific absorbance 2.61 x 10-3 ñ 0.31 x 10-3 ug cm-2) show an absorption band at 350 nm, which follows Beer's law, and therefore can be used for analytical purposes. The PBQ method has a lower limit of detection (4 ug/ml) than that of biuret method (45 yg/ml) for a final reaction mixture of 5.0 and 4.2 ml, respectively

Phagocytosis of enteropathogenic Escherichia coli and Candida albicans by lectin-like receptors

1. Ingestion of enteropathogenic Escherichia coli or Candida albicans by thioglycollate-elicited macrophages and polymorphonuclear leukocytes was investigated in vitro, 2. Goat antiserum against mannose receptors caused about 50% inhibition of E. coli phagocytosis and about 90% inhibition of C. albicans phagocytosis. 3. E. coli and C. albicans uptake was inhibited by about 60% and 98%, respectively, by plating the macrophages onto substrates coated with poly-L-lysine-mannan. Further addition of 50 mM mannose to the medium significantly increased the inhibition of phagocytosis of E. coli by macrophages from 60.7 +/- 1.5 to 79.8 +/- 13.1 and by polymorphonuclear cells from 58.9 +/- 3.7 to 88.7 +/- 4.9. 4. Preincubation of phagocytic cells with antiserum against substance A of human erythrocytes reduced E. coli ingestion by 95%, but this inhibition was not observed when the antiserum was incubated with N-acetylgalactosamine (50 mM) before being added to the phagocytes. The phagocytosis of C. albicans was not inhibited by anti-substance A antiserum. 5. The phagocytosis of E. coli was inhibited by about 25% by the addition of 7.8 micrograms/ml soluble mannan to the medium, and by about 50% by the addition of 50 mMN-acetylgalactosamine; when both substances were added to the medium, an additive inhibition of about 75% was observed. 6. These results indicate that mannose receptors on the surface of phagocytic cells mediate E. coli or Candida albicans uptake and that the binding of bacteria to N-acetylgalactosamine residues from the membrane of phagocytes is also involved in the phagocytosis of E. coli