Anaerobic biodegradation of pentachlorophenol in a liquor obtained after extraction of contaminated chips and wood powder.

Recovery of 97.5% of the pentachlorophenol (PCP) in contaminated wood powder was obtained after extraction with 0.1% KOH solution at 60 degrees C for 75 min. Extraction with NaOH and Na2CO3 was less effective than KOH. The neutralized extract was treated using a methanogenic consortium in an upflow anaerobic fixed-film reactor. The reactor was operated at 29 degrees C for over 600 d. The best performance of the reactor was observed when the PCP liquor was supplemented with glucose and formate. Complete dechlorination of PCP and phenol removal was obtained for a PCP loading rate of 13.3-18.0 mg l(-1) of reactor volume d(-1) with recirculation of the effluent and a hydraulic retention time (HRT) of 0.5-0.6 d.

Isolation and characterization of Desulfitobacterium frappieri sp. nov., an anaerobic bacterium which reductively dechlorinates pentachlorophenol to 3-chlorophenol.

An anaerobic bacterium, strain PCP-1T (T = type strain), which dechlorinates pentachlorophenol (PCP) to 3-chlorophenol, was isolated from a methanogenic consortium. This organism is a spore-forming rod-shaped bacterium that is nonmotile, asaccharolytic, and Gram stain negative but Gram type positive as determined by electron microscopic observations. Inorganic electron acceptors, such as sulfite, thiosulfate, and nitrate (but not sulfate), stimulate growth in the presence of pyruvate and yeast extract. The optimum pH and optimum temperature for growth are 7.5 and 38 degrees C, respectively. The dechlorination pathway is: PCP-->2,3,4,5-tetrachlorophenol -->3,4,5-trichlorophenol-->3,5-dichlorophenol-->3-chlorophenol. This bacterium dechlorinates several different chlorophenols at ortho, meta, and para positions; exceptions to this are 2,3-dichlorophenol, 2,5-dichlorophenol, 3,4-dichlorophenol, and the monochlorophenols. The time course of PCP dechlorination suggests that two enzyme systems are involved in dehalogenation in strain PCP-1T. One system is inducible for ortho dechlorination, and the second system is inducible for meta and para dechlorinations. A 16S rRNA analysis revealed that strain PCP-1T exhibits 95% homology with Desulfitobacterium dehalogenans JW/IU-DC1, an anaerobic bacterium which can dehalogenate chlorophenols only in ortho positions. These results suggest that strain PCP-1T is a member of a new species and belongs to the recently proposed genus Desulfitobacterium. Strain PCP-1T differs from D. dehalogenans JW/IU-DC1 by its broader range of chlorophenol dechlorination. Strain PCP-1 is the type strain of the new species, Desulfitobacterium frappieri.

Study of the reductive dechlorination of pentachlorophenol by a methanogenic consortium.

Pentachlorophenol (PCP) dechlorination by a methanogenic consortium was observed when glucose, formate, lactate, or yeast extract was present in the mineral medium as a secondary carbon source. Acetate was not a good substrate to sustain dechlorination. The consortium was able to dechlorinate the different monochlorophenols, although the chlorine in position ortho and meta was removed more readily than in para position. Dechlorination was most efficient at 37 degrees C. At 45 degrees C, the first PCP dechlorination steps were very rapid, but 3,5-dichlorophenol (3,5-DCP) was not further dechlorinated. At 15 and 4 degrees C, dechlorination was very slow. The dechlorination of PCP to 3-chlorophenol (3-CP) was still observed after the consortium had been subjected to heat treatment (80 degrees C, 60 min), suggesting that spore-forming bacteria were responsible. The dechlorinating activity of the consortium was significantly reduced by the presence of hydrogen, 2-bromoethanosulfonic acid (BESA), or sulfate but not of nitrate. The dechlorination of 3-CP was completely inhibited by heat treatment or the presence of BESA, suggesting that a syntrophic microorganism would be involved. Vigorous agitation of the consortium stopped the dechlorination, but the presence of DEAE-Sephacel acting as a support was very efficient in restoring the activity, suggesting that association between certain members of the consortium was important.

Protection of mice and swine against infection with Actinobacillus pleuropneumoniae by vaccination.

CaCl2 and LiCl cell extracts and a crude hemolysin preparation were isolated from Actinobacillus pleuropneumoniae serotype 1 strain 4074 and tested for protection against A. pleuropneumoniae serotype 1 and 5 in mice. The LiCl cell extract adsorbed on AlPO4 and the crude hemolysin preparation adsorbed on Al(OH)3 showed a highly significant protection (P < 0.01) against both serotypes. Different vaccine preparations were used to immunize pigs by intra-muscular injection at days 0 and 14; the pigs were then challenged at day 21 by intra-tracheal inoculation of 1 x 10(8) colony forming units (CFU) of a serotype 1 strain 4074. A vaccine which combined the LiCl extract and the crude hemolysin preparation adsorbed on Al(OH)3 gave the best protection with no mortality and no sign of morbidity in the vaccinated pigs. In the other experimental groups which included a group immunized with a commercial bacterin, mortality, respiratory disease and extensive pulmonary lesions were noted. This mixture shows good potential as a vaccine against pleuropneumonia in pigs.

Production and purification of a low molecular weight haemolysin produced by Actinobacillus pleuropneumoniae serotype 1.

An unstable haemolytic activity produced by a strain of serotype 1 of Actinobacillus pleuropneumoniae was isolated when 1 per cent bovine serum albumin (BSA) was added to RPMI 1640 medium. BSA acts as a carrier molecule and stabilises activity. This haemolysin (BSA-haemolysin) was precipitated with ammonium sulphate, dialysed and lyophilised. Of the species tested, bovine erythrocytes were the most susceptible to the BSA-haemolysin while mouse and rabbit erythrocytes were the least susceptible. The haemolytic activity was dependent on the incubation temperature, no activity being observed at or below 24 degrees C. The haemolytic activity was also partly stabilised by 100 micrograms ml-1 dithiothreitol (DTT). The DTT-haemolysin was purified to homogeneity by ultrafiltration and high pressure liquid chromatography on a Protein Pak DEAE-5PW column. The molecular weight was estimated at 16 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and at 23 kDa by molecular gel filtration from the elution position of the haemolytic activity. The DTT-haemolysin activity was completely destroyed by pronase treatment suggesting that this substance could be a polypeptide. The addition of BSA to DTT-haemolysin increased its activity and stability to lyophilisation. The addition of 10 mM calcium chloride in the titration assay increased the activity of DTT-haemolysin from 220 to 476 haemolytic units ml-1. The BSA-haemolysin activity was only slightly affected by the addition of calcium chloride.

Detection and purification of a potential precursor protein or a prohaemolysin produced by Staphylococcus haemolyticus.

The haemolytic substance H7 produced by Staphylococcus haemolyticus is composed of three peptides out of 44 amino acid residues each having a structure resembling a signal peptide. The cytoplasmic fraction for the presence of a protein precursor containing this signal sequence was investigated. Specific rabbit IgGs to substance H7 were isolated by affinity chromatography on Sepharose-4B-H7. These anti-H7 IgGs recognized mainly a 51 kD protein in the cytoplasmic fraction of S. haemolyticus from 2, 4, 6, 8, 10 and 23 h cultures. These results support the idea that the 51 kD protein could be either a prohaemolysin or else the precursor of a protein of unknown function with a signal sequence showing homology with the haemolytic peptides. After affinity chromatography on Sepharose-4B-anti-H7, the 51 kD protein was shown associated with an RNA-protein complex composed of four or five proteins and an RNA estimated at 300 nt. This complex could be associated with the machinery of protein secretion. The 51 kD protein was finally purified to homogeneity by HPLC on a Protein Pak DEAE-5PW column in the presence of 5 M urea.

A potential polyvalent Neisseria meningitidis vaccine based on a mixture of CaCl2 cell extracts from groups A, B and Y.

Outer membrane proteins (OMP) from Neisseria meningitidis cells of groups A, B, C and Y were extracted with CaCl2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of each extract showed a multibanded pattern characterized by the presence of three to four major proteins. Immunization of mice with individual extracts induced homologous bactericidal reactions. In addition, the extracts from groups B and C induced heterologous bactericidal reactions (B-C, C-B). The sera of mice immunized with a mixture of group A, B, and Y extracts showed a high bactericidal titre against the three N. meningitidis groups forming the mixture. Moreover, this bactericidal activity had a large spectrum against different strains from groups A, B, Y and also C. After immunization of mice with the individual extracts, each of the sera was shown by immunoblotting to contain antibodies reacting with some of the corresponding proteins. Mice immunized with the mixture showed a significant reduction of bacteraemia following challenge with either of N. meningitidis groups A, B or C. These results suggest that a mixture of OMP from different N. meningitidis groups may have the potential for a meningococcal polyvalent vaccine.

Mouse immune response to meningococcal antigens.

The mouse immune response against Neisseria meningitidis was studied by using an extract from group Y (Slaterus) known to contain protein antigens common to other meningococci. By using a solid-phase radioimmunoassay, high titers of specific IgM and IgG class antibodies were measured which lasted over 2 months after immunization. These antibodies cross-reacted with similar extracts from other meningococci groups. Bactericidal antibodies directed against protein antigens were also elicited after immunization and they belonged to IgM, IgG2a, and IgG2b isotypes. Cellular immunity, expressed as delayed type hypersensitivity under the conditions tested, could be detected neither in homologous nor heterologous reactions.

Evaluation of the immunogenicity of a CaCl2 extract from Neisseria meningitidis group Y.

Cross-protection has already been demonstrated in mice after vaccination with a CaCl2 extract from the Neisseria meningitidis group Y Slaterus strain. The immunogenicity of such extracts from group Y cells, cultivated in a fermenter in Neisseria chemically defined medium, against virulent groups A, B, and C meningococci has been evaluated by two different animal models and a microbactericidal procedure. The mouse challenge system has revealed that the active cross-production observed 7 days after a single immunization with the extract was probably nonspecific, since bacillus Calmette-Guérin gave similar results. However, after three vaccinations, active cross-protection was observed, mainly against the strains of groups B and C, for at least 35 days after the last injection. In the mouse bacteremia model, the extract had a protective effect mainly against the homologous group Y strain but in a few experiments a significant protection was also obtained against the strains of groups A and B. The microbactericidal test revealed that even after three injections of mice, guinea pigs, or humans with the extract only the homologous bactericidal activity was induced. Since there was no close correlation between the results obtained with the two animal models and also with the microbactericidal procedure, no definitive conclusion can be drawn on the protective potential of our extract.