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Claus Koch - One of the best experts on this subject based on the ideXlab platform.

  • production and characterization of mouse monoclonal antibodies against Afipia felis
    Apmis, 1997
    Co-Authors: Kraesten Engbaek, Lars Otto Uttenthal, Claus Koch
    Abstract:

    : A series of 10 monoclonal antibodies reacting with Afipia felis antigens were selected from mice immunized with live organisms of the reference strain ATCC 53690. Immunoblotting against SDS-PAGE-separated A felis sonicate allowed the antibodies to be classified into three groups: 1) 168-4, -6, -7 and -10 reacted with a 53 kDa antigen, 2) 168-1, -3 and -9 reacted with both 53 kDa and 60 kDa antigens, and 3) 168-2, -5 and -9 reacted with other antigens. Antibodies of group 1 did not cross-react with other Afipia species or 36 unrelated bacteria, whereas those of groups 2 and 3 reacted with other Afipia species and some unrelated bacteria. Immunoblots of crossed immunoelectrophoretic patterns of A. felis sonicate against rabbit antiserum showed that antibodies of groups 1 and 2 bound to the same precipitin arcs. Antibodies of group 1 reacted with a species-specific epitope on the 53 kDa antigen, while those of group 2 reacted with other epitopes shared by the 53 kDa and 60 kDa antigens. The binding of antibodies of group 1 to A. felis sonicate was inhibited by post-infection rabbit serum, whereas no inhibition was observed for antibodies of group 2. The species-specific epitope of the 53 kDa antigen and the early appearance of antibodies against this epitope after infection suggest that this antigen can be used in a serodiagnostic test for A. felis infection.

  • identification of Afipia felis antigens in culture medium reaction with human sera
    Apmis, 1997
    Co-Authors: Kraesten Engbaek, Lars Otto Uttenthal, Claus Koch
    Abstract:

    : Fourteen protein antigens were identified on SDS-PAGE of Afipia felis culture supernatant. Immunoblotting against 10 monoclonal antibodies obtained from mice infected with live A. felis showed that 4 antibodies reacted with a 56 kDa band and 3 with both 56 kDa and 62 kDa bands. Compared with A. felis sonicate, the reacting proteins in culture supernatant showed an increase in molecular mass of 2-3 kDa, suggesting that they were more glycosylated. Purified antigen obtained by affinity chromatography of culture supernatant on the seven immobilized antibodies was tested against antibodies reacting with the 56 kDa and 62 kDa bands. All eluates contained both components, suggesting that the antibodies were directed against different epitopes of a double antigen held together during the affinity chromatography but cleaved by reduction and SDS-PAGE. The molecular size of the uncleaved protein in culture supernatant was determined by size-exclusion chromatography as > 1000 kDa. Testing of pre- and post-infection rabbit sera in immunoblotting against culture supernatant demonstrated that the 56 kDa and 62 kDa components gave the most prominent specific reactions with post-infection sera. One of fifty human sera submitted for testing for cat-scratch disease and 1 of 50 sera from healthy blood donors reacted with several bands in A. felis culture supernatant, including the 56 kDa and 62 kDa bands.

  • antibody response in rabbits infected with rochalimaea henselae rochalimaea quintana and Afipia felis
    Apmis, 1994
    Co-Authors: Kraesten Engbaek, Claus Koch
    Abstract:

    : Antibody responses in three pairs of rabbits inoculated with live Rochalimaea henselae, Rochalimaea quintana and Afipia felis were studied by enzyme immunoassay with whole-cell and bacterial sonicates as antigen. No differences in measured antibody responses were found with the two types of antigen preparation. Two rabbits did not respond with antibody production. In the remaining rabbits there was a low-titred antibody response that showed no significant cross-reaction with related bacteria. After rechallenge the antibody response rose significantly and there was significant cross-reaction with related bacteria. The antigens involved in the antibody response were examined by crossed immunoelectrophoresis. After the initial inoculation 5-7 precipitin lines of the reference diagrams were deflected, including lines which cross-reacted with antigens found in related bacterial species. After reinoculation several more precipitin lines were deflected, including additional lines cross-reacting with antigens present in related bacteria and common bacterial antigens.

  • immunoelectrophoretic characterization and cross reactivity of rochalimaea henselae rochalimaea quintana and Afipia felis
    Apmis, 1994
    Co-Authors: Kraesten Engbaek, Claus Koch
    Abstract:

    : The soluble antigens of Rochalimaea henselae, Rochalimaea quintana and Afipia felis were characterized by crossed immunoelectrophoresis using bacterial sonicates as antigens against pooled hyperimmune rabbit sera. A precipitin pattern was drawn for each bacterium and shown to be reproducible and stable even when normal or preimmune rabbit serum was incorporated in the intermediate gel. By this technique 56 antigens were identified from R. henselae, 49 from R. quintana, and 39 from A. felis. The serological cross-reaction between R. henselae, R. quintana and A. felis, and between these 3 bacteria and 32 pathogenic bacteria was analysed by rocket-line immunoelectrophoresis, crossed-line immunoelectrophoresis, and tandem-crossed electrophoresis. It was concluded that (i) 4-7 antigens distinguish R. henselae, R. quintana and A. felis from each other, (ii) both Gram-positive and Gram-negative bacteria cross-react with R. henselae, R. quintana and A. felis antisera, (iii) the cross-reacting antigens of Gram-negative bacteria have both precipitating and non-precipitating specificities, whereas Gram-positive bacteria have mainly non-precipitating specificities, (iv) the cross-reacting antigens are common to several species, and (v) fewer cross-reacting antigens are found in phylogenetically disparate species than in more closely related species.

Kraesten Engbaek - One of the best experts on this subject based on the ideXlab platform.

  • production and characterization of mouse monoclonal antibodies against Afipia felis
    Apmis, 1997
    Co-Authors: Kraesten Engbaek, Lars Otto Uttenthal, Claus Koch
    Abstract:

    : A series of 10 monoclonal antibodies reacting with Afipia felis antigens were selected from mice immunized with live organisms of the reference strain ATCC 53690. Immunoblotting against SDS-PAGE-separated A felis sonicate allowed the antibodies to be classified into three groups: 1) 168-4, -6, -7 and -10 reacted with a 53 kDa antigen, 2) 168-1, -3 and -9 reacted with both 53 kDa and 60 kDa antigens, and 3) 168-2, -5 and -9 reacted with other antigens. Antibodies of group 1 did not cross-react with other Afipia species or 36 unrelated bacteria, whereas those of groups 2 and 3 reacted with other Afipia species and some unrelated bacteria. Immunoblots of crossed immunoelectrophoretic patterns of A. felis sonicate against rabbit antiserum showed that antibodies of groups 1 and 2 bound to the same precipitin arcs. Antibodies of group 1 reacted with a species-specific epitope on the 53 kDa antigen, while those of group 2 reacted with other epitopes shared by the 53 kDa and 60 kDa antigens. The binding of antibodies of group 1 to A. felis sonicate was inhibited by post-infection rabbit serum, whereas no inhibition was observed for antibodies of group 2. The species-specific epitope of the 53 kDa antigen and the early appearance of antibodies against this epitope after infection suggest that this antigen can be used in a serodiagnostic test for A. felis infection.

  • identification of Afipia felis antigens in culture medium reaction with human sera
    Apmis, 1997
    Co-Authors: Kraesten Engbaek, Lars Otto Uttenthal, Claus Koch
    Abstract:

    : Fourteen protein antigens were identified on SDS-PAGE of Afipia felis culture supernatant. Immunoblotting against 10 monoclonal antibodies obtained from mice infected with live A. felis showed that 4 antibodies reacted with a 56 kDa band and 3 with both 56 kDa and 62 kDa bands. Compared with A. felis sonicate, the reacting proteins in culture supernatant showed an increase in molecular mass of 2-3 kDa, suggesting that they were more glycosylated. Purified antigen obtained by affinity chromatography of culture supernatant on the seven immobilized antibodies was tested against antibodies reacting with the 56 kDa and 62 kDa bands. All eluates contained both components, suggesting that the antibodies were directed against different epitopes of a double antigen held together during the affinity chromatography but cleaved by reduction and SDS-PAGE. The molecular size of the uncleaved protein in culture supernatant was determined by size-exclusion chromatography as > 1000 kDa. Testing of pre- and post-infection rabbit sera in immunoblotting against culture supernatant demonstrated that the 56 kDa and 62 kDa components gave the most prominent specific reactions with post-infection sera. One of fifty human sera submitted for testing for cat-scratch disease and 1 of 50 sera from healthy blood donors reacted with several bands in A. felis culture supernatant, including the 56 kDa and 62 kDa bands.

  • antibody response in rabbits infected with rochalimaea henselae rochalimaea quintana and Afipia felis
    Apmis, 1994
    Co-Authors: Kraesten Engbaek, Claus Koch
    Abstract:

    : Antibody responses in three pairs of rabbits inoculated with live Rochalimaea henselae, Rochalimaea quintana and Afipia felis were studied by enzyme immunoassay with whole-cell and bacterial sonicates as antigen. No differences in measured antibody responses were found with the two types of antigen preparation. Two rabbits did not respond with antibody production. In the remaining rabbits there was a low-titred antibody response that showed no significant cross-reaction with related bacteria. After rechallenge the antibody response rose significantly and there was significant cross-reaction with related bacteria. The antigens involved in the antibody response were examined by crossed immunoelectrophoresis. After the initial inoculation 5-7 precipitin lines of the reference diagrams were deflected, including lines which cross-reacted with antigens found in related bacterial species. After reinoculation several more precipitin lines were deflected, including additional lines cross-reacting with antigens present in related bacteria and common bacterial antigens.

  • immunoelectrophoretic characterization and cross reactivity of rochalimaea henselae rochalimaea quintana and Afipia felis
    Apmis, 1994
    Co-Authors: Kraesten Engbaek, Claus Koch
    Abstract:

    : The soluble antigens of Rochalimaea henselae, Rochalimaea quintana and Afipia felis were characterized by crossed immunoelectrophoresis using bacterial sonicates as antigens against pooled hyperimmune rabbit sera. A precipitin pattern was drawn for each bacterium and shown to be reproducible and stable even when normal or preimmune rabbit serum was incorporated in the intermediate gel. By this technique 56 antigens were identified from R. henselae, 49 from R. quintana, and 39 from A. felis. The serological cross-reaction between R. henselae, R. quintana and A. felis, and between these 3 bacteria and 32 pathogenic bacteria was analysed by rocket-line immunoelectrophoresis, crossed-line immunoelectrophoresis, and tandem-crossed electrophoresis. It was concluded that (i) 4-7 antigens distinguish R. henselae, R. quintana and A. felis from each other, (ii) both Gram-positive and Gram-negative bacteria cross-react with R. henselae, R. quintana and A. felis antisera, (iii) the cross-reacting antigens of Gram-negative bacteria have both precipitating and non-precipitating specificities, whereas Gram-positive bacteria have mainly non-precipitating specificities, (iv) the cross-reacting antigens are common to several species, and (v) fewer cross-reacting antigens are found in phylogenetically disparate species than in more closely related species.

David F. Welch - One of the best experts on this subject based on the ideXlab platform.

  • cat scratch disease the rare role of Afipia felis
    Journal of Clinical Microbiology, 1998
    Co-Authors: Michael Giladi, D. J. Brenner, Yehudith Kletter, Suzy Abulafia, Leonard N Slater, Arnold G Steigerwalt, Anne M Whitney, Boaz Avidor, David F. Welch, Moshe Ephros
    Abstract:

    Since its isolation in 1988, Afipia felis has been associated with cat scratch disease (CSD) in only one report and its role in CSD has been questioned. We have cultured A. felis from a lymph node of a patient with CSD. 16S rRNA gene sequencing, DNA relatedness studies, fatty acid analysis, and PCR of the A. felis ferredoxin gene showed that the isolate is identical to the previously reported A. felis isolate. To determine the role of A. felis in CSD, PCR of the 16S rRNA gene followed by hybridizations with specific probes were performed with lymph node specimens from CSD patients. All 32 specimens tested positive for Bartonella henselae and negative for A. felis. We conclude that A. felis is a rare cause of CSD. Diagnostic tests not conducive to the identification of A. felis might cause the diagnosis of CSD due to A. felis to be missed.

  • Murine antibody responses distinguish Rochalimaea henselae from Rochalimaea quintana
    Journal of Clinical Microbiology, 1992
    Co-Authors: Leonard N Slater, Danese W. Coody, L K Woolridge, David F. Welch
    Abstract:

    Rochalimaea henselae causes persistent bacteremia, bacillary angiomatosis, and parenchymal bacillary peliosis. Detection of a specific antibody response to R. henselae infection may represent an alternative to cultivation as a means of diagnosis. We assessed the specificity of induced murine antibodies for antigens from R. henselae and the closely related species R. quintana. Groups of CD-1 mice were inoculated with whole organisms of six strains of R. henselae and two of R. quintana. Pre- and postinoculation blood specimens were collected. Enzyme immunosorbent assays were performed by using as antigens preparations of immunogenic proteins from one isolate of R. henselae or from the R. quintana type strain. These demonstrated high specificity of R. henselae-induced antibodies for proteins of R. henselae and of R. quintana-induced antibodies for proteins of R. quintana. Protein preparations extracted from all of the strains were separated electrophoretically. After their transfer to membranes, immunoblots were performed by using 1:1,000 dilutions of all of the pre- and postinoculation sera in combination with proteins from all of the strains. Preinoculation sera had minimal reactivity. All of the R. henselae-induced immune sera reacted with numerous proteins of all of the R. henselae strains but cross-reacted minimally with proteins of R. quintana. Immune sera from R. quintana-inoculated mice had similar species specificity. An immunofluorescence assay was developed by using antiserum to one strain of R. henselae. A 1:1,000 dilution yielded fluorescence with all strains of R. henselae but with none of R. quintana, Bartonella bacilliformis, or Afipia felis. Acinetobacter calcoaceticus subsp. anitratus was also unreactive with a dilution of 1:500. A 1:10 dilution yielded weak fluorescence with R. quintana but none with Staphylococcus epidermidis.

Bernard La Scola - One of the best experts on this subject based on the ideXlab platform.

  • genome sequence of Afipia felis strain 76713 isolated in hospital water using an amoeba co culture procedure
    Genome Announcements, 2014
    Co-Authors: Samia Benamar, Bernard La Scola, Olivier Croce
    Abstract:

    ABSTRACT Afipia felis is a Gram-negative alphaproteobacterium originally described as the agent of cat-scratch disease (CSD). We sequenced the genome from a strain of A. felis, which was recovered from a hospital water sample using an amoebal co-culture procedure. It is composed of 3,989,646 bp, with a G+C content of 61.27% and encodes 4,068 protein-coding genes and 53 RNA genes.

  • genome sequence of Afipia birgiae a rare bacterium associated with amoebae
    Journal of Bacteriology, 2012
    Co-Authors: Isabelle Pagnier, Olivier Croce, Catherine Robert, Didier Raoult, Bernard La Scola
    Abstract:

    Afipia birgiae is an alphaproteobacterium from the family Bradyrhizobiaceae, growing in amoebae, and a potential human pathogen. We sequenced the genome of type strain 34632(T). It is composed of 5,325,467 bp and contains 5,160 protein-coding genes and 53 RNA genes, including 3 rRNA genes.

  • ameba associated microorganisms and diagnosis of nosocomial pneumonia
    Emerging Infectious Diseases, 2006
    Co-Authors: Pierre Berger, Bernard La Scola, Laurent Papazian, Michel Drancourt, J P Auffray
    Abstract:

    To elucidate the role of ameba-associated microorganisms (AAMs) as etiologic agents of pneumonia, we screened for Legionella spp., Parachlamydia acanthamoeba, Afipia sp., Bosea spp., Bradyrhizobium spp., Mesorhizobium amorphae, Rasbo bacterium, Azorhizobium caulinodans, Acanthamoeba polyphaga mimivirus, and conventional microorganisms in 210 pneumonia patients in intensive-care units by using culture, polymerase chain reaction, and serologic testing. These resulted in 59 diagnoses in 40 patients. AAMs and non-AAMs were implicated in 10.5% of the patients. The infectious agents were identified in 15 patients: Acanthamoeba polyphaga mimivirus, 8; Legionella pneumophila, 3; L. anisa, 1; Parachlamydia sp., 1; Bosea massiliensis, L. worsleiensis, L. quinlivanii, and L. rubrilucens, 1; and M. amorphae and R. bacterium, 1. A. polyphaga mimivirus was the fourth most common etiologic agent, with a higher seroprevalence than noted in healthy controls. This finding suggested its clinical relevance. Therefore, AAM might cause nosocomial pneumonia and should be suspected when conventional microbiologic results are negative.

  • usefulness of rpob gene sequencing for identification of Afipia and bosea species including a strategy for choosing discriminative partial sequences
    Applied and Environmental Microbiology, 2003
    Co-Authors: Atieh Khamis, Didier Raoult, Philippe Colson, Bernard La Scola
    Abstract:

    Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

  • description of Afipia birgiae sp nov and Afipia massiliensis sp nov and recognition of Afipia felis genospecies a
    International Journal of Systematic and Evolutionary Microbiology, 2002
    Co-Authors: Bernard La Scola, Marie-noëlle Mallet
    Abstract:

    On the basis of phenotypic characterization and DNA relatedness, two novel species are proposed, Afipia birgiae sp. nov. (type strain 34632T = CIP 106344T = CCUG 43108T) and Afipia massiliensis sp. nov. (type strain 34633T = CIP 107022T = CCUG 45153T). A new genospecies is described, named Afipia felis genospecies A, closely related to Afipia felis. The complexity encountered in the taxonomy of the Bradyrhizobiaceae group within the alpha-2 subgroup of the Proteobacteria is discussed and the description of these novel species highlights the need for new tools for phylogenetic analysis in the group. The novel species herein described are fastidious bacteria isolated from a hospital water supply in co-culture with amoebae. It is hypothesized that this group of bacteria are a potential cause of nosocomial infections.

Frederick D Quinn - One of the best experts on this subject based on the ideXlab platform.

  • The Afipia toolbox and its use to isolate flagellar mutants.
    Fems Microbiology Letters, 2009
    Co-Authors: Christian Schueller, Frederick D Quinn, Albert Haas
    Abstract:

    Afipia felis, a Gram-negative alphaproteobacterium, has been implicated as one of the causative agents of cat scratch disease. To identify and begin to examine the virulence traits of this organism, we developed and tested a highly efficient transposon delivery system and a stable plasmid vector expressing green fluorescent protein. The transposome system is based on a Tn5-derived transposon and a phage restriction endonuclease type I inhibitor. Electroporation of this construct produced a library of >2600 mutants, which were screened for flagella biosynthesis mutants using a monoclonal antibody to Afipia flagellin. Insertion loci for two selected mutants were located in the genes for flagellin and flagellin biosynthesis FlhA, confirming the validity of the approach.

  • intracellular growth of Afipia felis a putative etiologic agent of cat scratch disease
    Infection and Immunity, 1992
    Co-Authors: Kristin A Birkness, V G George, E H White, David S. Stephens, Frederick D Quinn
    Abstract:

    The organism Afipia felis, which is though to be an etiologic agent of cat scratch disease, is a gram-negative rod that is clearly seen in infected tissue but is very difficult to isolate from clinical specimens; there has been only one report to date of the successful isolation and maintenance of the bacterium on artificial medium. We have found that A. felis will attach, invade via phagocytosis, and multiply intracellularly within the phagosomes of primary human monocytes and HeLa cells. Once in the cell, the bacterium appears to change morphologically, becoming longer and more pleomorphic, and loses its ability to grow on an artificial medium. Unique proteins have been identified in both the intra- and extracellular variants of A. felis. Convalescent-phase sera from patients with cat scratch disease react poorly with intracellular and extracellular bacteria, suggesting a poor humoral response. The tissue culture protocol presented has been used to isolate 14 new strains of A. felis and has for the first time permitted study of the pathogenesis of this unique organism. Images

  • proposal of Afipia gen nov with Afipia felis sp nov formerly the cat scratch disease bacillus Afipia clevelandensis sp nov formerly the cleveland clinic foundation strain Afipia broomeae sp nov and three unnamed genospecies
    Journal of Clinical Microbiology, 1991
    Co-Authors: D. J. Brenner, C. K. English, D G Hollis, William F. Bibb, C W Moss, Kristin A Birkness, J Vincent, G S Hall, Jagoda Radošević, Frederick D Quinn
    Abstract:

    On the basis of phenotypic characterization and DNA relatedness determinations, the genus Afipia gen. nov., which contains six species, is described. The type species is Afipia felis sp. nov. (the cat scratch disease bacillus). Afipia clevelandensis sp. nov., Afipia broomeae sp. nov., and three unnamed not associated with cat-borne disease. All but one strain (Afipia genospecies 3) were isolated from human wound and respiratory sources. All Afipia species are gram-negative, oxidase-positive, nonfermentative rods in the alpha-2 subgroup of the class Proteobacteria. They are motile by means of a single flagellum. They grow on buffered charcoal-yeast extract agar and nutrient broth, but rarely on MacConkey agar, at 25 and 30 degrees C. They are urease positive; but they are negative in reactions for hemolysis, indole production, H2S production (triple sugar iron agar), gelatin hydrolysis, esculin hydrolysis, and peptonization of litmus milk. They do not produce acid oxidatively from D-glucose, lactose, maltose, or sucrose. The major cell wall fatty acids are 11-methyloctadec-12-enoic (CBr19:1), cis-octadec-11-enoic (C18:1omega7c), and generally, 9,10-methylenehexadecanote and 11,12-methyleneoctadecanoate; and there are only trace amounts of hydroxy acids. The guanineplus-cytosine content is 61.5 to 69 mol%. A. felis is positive for nitrate reduction and is delayed positive for acid production from D-xylose, but it is catalase negative. A. clevelandensis is negative in all of these tests. A. broomeae is weakly positive for catalase production and acid production from D-xylose, but it is negative for nitrate reduction. Images