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Biomedical subjects

J Nicolet

Publications and source records attributed to J Nicolet.

At least 127 records · Page 7Linked to original sources
PubMed1992

Influence of the transport on the outcome of the bacteriological analysis of dog urine comparison of three transport tubes.

A sterile plastic tube, a boric acid-glycerol-sodium formate tube and a dip-slide tube were compared for transport of dog urine for bacteriological examination under practice conditions, at ambient temperature. In a first part, 50 dog urine samples were cultured on agar plates and on a dip-slide kit within 2 h after collection and after storage at 20 degrees C in a sterile tube and in a boric acid tube for 24 h and 48 h. Culture of the samples stored in the boric acid tube and culture on the dip-slide yielded results which correlated very well with those of the culture of fresh urine. However, culture of the samples stored in the sterile tube yielded up to 65% false positive results. In a second part, a comparison was drawn with culture results of 100 dog urine specimens collected by different practitioners and simultaneously mailed to our laboratory in a sterile tube, in a boric acid tube and in a dip-slide kit. Samples sent in the boric acid and in the dip-slide tube showed comparable culture results. Culture of the samples sent in the plastic tube yielded 53% false positive results in comparison with those of the samples preserved in boric acid.

Animals↗
PubMed1992

Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli.

Hemolysin genes of the reference strains of Actinobacillus pleuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia coli by using polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of the corresponding appA gene from A. pleuropneumoniae serotype 5. The three genes from serotypes 1, 2, and 5 have identical restriction maps and appear to encode a hemolysin which was previously identified in serotype 2 and designated HlyII. Gene appA is different from hlyIA encoding the major hemolysin type I (HlyI) which was identified earlier in serotype 1. Polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of hlyIA of serotype 1 showed that the gene encoding HlyI is present in serotype 1 but not in serotype 2, in contrast to the gene encoding HlyII that was present in both serotypes. This was confirmed by Western blot (immunoblot) experiments using monoclonal antibodies specific for either recombinant HlyI or recombinant HlyII, which showed that A. pleuropneumoniae serotype 1 strain 4074 produces both HlyI and HlyII, whereas serotype 2 strain S1536 produces only HlyII. The expression of both hemolysins was investigated in all serotypes by the use of monoclonal antibodies. HlyI was shown to be expressed by the reference strains of serotypes 1, 5a, 5b, 9, 10, and 11, whereas HlyII was shown to be expressed by the reference strains of all 12 serotypes tested except serotype 10. A. pleuropneumoniae serotype 1 strain 4074 is the first bacterium which has been shown to contain two different actively expressed RTX toxin genes. Comparison of our data with those from other groups shows that the originally described strongly hemolytic hemolysin type I (HlyI) corresponds to cytolysin I (ClyI) which was recently described by others, while the weakly hemolytic hemolysin type II (HlyII) seems to be identical to ClyII and AppA.

Actinobacillus pleuropneumoniae↗
PubMed1992

Functional analysis of the Ca(2+)-regulated hemolysin I operon of Actinobacillus pleuropneumoniae serotype 1.

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlyI; gene designation, hlyI) by Actinobacillus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4. A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hlyD of the Escherichia coli hemolysin operon, and expression of the A. pleuropneumoniae genes in E. coli revealed that they have the same functions as their E. coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyIA) to active hemolysin I, while hlyIB and hlyID are necessary for HlyIA secretion. Northern (RNA) hybridization of A. pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyICA, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD. The level of hlyI mRNA was substantially higher in A. pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression of the hlyI determinant is Ca2+ regulated. Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hlyI determinant.

Actinobacillus pleuropneumoniae↗
PubMed1992

Detection of Campylobacter upsaliensis in diarrheic dogs and cats, using a selective medium with cefoperazone.

Using a newly formulated selective medium containing cefoperazone, we isolated 72 Campylobacter strains in fecal samples from 397 diarrheic dogs and cats. Of these, 39 were thermophilic catalase-negative Campylobacter species. We identified these Campylobacter strains by DNA:DNA hybridization, using digoxigenin-labeled total genomic DNA of 4 Campylobacter reference strains (C jejuni, C coli, C lari, and C upsaliensis) as a probe. The labeling was done with a commercially available kit. We could identify 66 of the 72 Campylobacter isolates to the species level with this method; identification with probes always agreed with conventional test results. Of the 66 identified strains, 33 were C upsaliensis and 33 were C jejuni. Six isolates could not be assigned to a known species with probes or conventional tests. On the basis of our findings, C upsaliensis is more resistant to cefoperazone than to cephalothin, thereby explaining the unexpected recovery of these campylobacters on cephalosporin-containing media.

Animals↗
PubMed1992

[Detection with nonradioactively labeled probes of the toxin genes of different E. coli pathotypes from swine].

We tested hemolytic E. coli from 86 pigs with edema disease or colidiarrhea. They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes. By slide-agglutination we detected 38 cases with E. coli O149:K88, 28 with E. coli O139:82B and 20 with E. coli O141. E. coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes. Edema disease E. coli O139:82B reacted with the SLTII probe. E. coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns. All the E. coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII. No strains isolated from pigs with edema disease possessed any of these enterotoxin genes. Gene probe technique confirmed the serological method as useful tool for diagnosing E. coli O149:K88 and O139:82B as ETEC or VTEC, respectively. On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E. coli of serogroup O141.

Animals↗
PubMed1991

Identification and partial characterization of the hemolysin (HlyII) of Actinobacillus pleuropneumoniae serotype 2.

The secreted hemolytic activity produced by Actinobacillus pleuropneumoniae serotype 2 reference strain is thermolabile, inactivated by proteinase K and requires Ca2+ as cofactor for its hemolytic activity. Purification of the hemolytic activity resulted in a fraction containing two proteins, one of 105 kDa and one of 125 kDa. These two proteins could be further separated by preparative SDS polyacrylamide gel electrophoresis. This purification step, resulted in loss of the hemolytic activity. Polyclonal antibodies were made against each of these proteins in rabbits. Neutralization experiments showed that antibodies made against the 105 kDa protein could neutralize the hemolytic activity produced by A. pleuropneumoniae serotype 2, while antibodies made against the 125 kDa protein were unable to neutralize the hemolytic activity. The 105 kDa protein therefore, is the hemolysin of A. pleuropneumoniae serotype 2, known as HlyII. This protein is closely related immunologically to the hemolysin I (HlyI) from A. pleuropneumoniae serotype 1. DNA::DNA hybridization experiments performed by the Southern blot method using the cloned structural gene of HlyI from A. pleuropneumoniae serotype 1 demonstrate that the structural genes of the two hemolysins (hlyIA and hlyIIA) are different and show at least 30% heterology. This confirms that HlyI and HlyII are two different proteins, although they have a very similar molecular weight and show strong immunological cross reactions.

Actinobacillus↗
PubMed1991

Immunological properties of Actinobacillus pleuropneumoniae hemolysin I.

The 105 kDa hemolysin I protein from Actinobacillus pleuropneumoniae serotype I type strain 4074 (HlyI) was shown by immunoblot analysis to be the predominant immunogenic protein if convalescent field sera or sera from pigs experimentally infected with A. pleuropneumoniae serotype 1 were used. SDS gel- and immunoblot-analysis using total culture, washed cells or culture supernatant showed that HlyI is essentially secreted and is not found attached to the bacteria. Proteins in the 105 kDa range that react strongly with anti-HlyI antibody, are produced by all serotypes and are presumed to be their hemolysins. Sera from pigs experimentally infected with each of the 12 serotypes strongly reacted with HlyI. In addition, some sera from pigs that were confirmed to be negative for A. pleuropneumoniae, also reacted with HlyI as well as with related proteins from Actinobacillus rossii and Actinobacillus suis. These two species produce proteins in the 105 kDa range which cross-react strongly with HlyI. They could be the source of the immunological reactions of the A. pleuropneumoniae-negative sera with HlyI. However, no cross-reactions could be found between HlyI and the Pasteurella haemolytica leukotoxin, the Escherichia coli alpha-hemolysin or related proteins from various hemolytic E. coli strains isolated from pigs. The immunological cross-reactions of HlyI with related proteins from A. rossii, A. suis and possibly from other bacterial species may create uncertainty in interpretation if HlyI is used as the antigen in serodiagnosis of A. pleuropneumoniae.

Actinobacillus↗
PubMed1991

Use of antibodies against the P36 protein of Mycoplasma hyopneumoniae for the identification of M. hyopneumoniae strains.

Mycoplasma hyopneumoniae, the principal aetiological agent of porcine enzootic pneumonia, synthesizes a 36 kDa protein (P36) which is an early and strong immunogenic factor in experimentally and naturally infected swine. Polyclonal antibodies were made against the recombinant P36 protein in rabbits and used for the identification of M. hyopneumoniae by the immunoblot technique. The proteins from the M. hyopneumoniae reference strains and from 13 M. hyopneumoniae field strains isolated from naturally infected pigs in Switzerland, Hungary, France and Canada were analysed by the immunoblot technique using anti-P36 antibodies. All 13 field strains and the three reference J strains of M. hyopneumoniae, received from different collections and laboratories, exhibited a strong reaction with a protein of 36 kDa indicating that the P36 protein is a common M. hyopneumoniae antigen. None of the different porcine Mycoplasma species including M. flocculare, M. hyorhinis, M. hyosynoviae, A. axanthum, A. laidlawii and A. granularum showed any reaction on the immunoblot with the anti-P36 antibodies. In addition, we have found no reaction with anti-P36 antibodies using 47 different Mycoplasma or Acholeplasma species isolated from human, mice, rat, poultry, ruminant, dog and cat. In conclusion we have shown that P36 is a protein that is a common antigen of M. hyopneumoniae strains and is not found in other Mycoplasma or Acholeplasma species tested. Because of its high specificity, P36 protein, or antibodies made against this protein can be used for the identification of M. hyopneumoniae strains.

Acholeplasma↗
PubMed1991

Cloning and expression of a species-specific early immunogenic 36-kilodalton protein of Mycoplasma hyopneumoniae in Escherichia coli.

Mycoplasma hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, synthesizes a 36-kDa protein which is an early and strong immunogenic factor in experimentally and naturally infected swine. The gene encoding this protein was cloned by screening a gene library of M. hyopneumoniae DNA with rabbit hyperimmune serum made against whole M. hyopneumoniae cells and convalescent-phase swine serum. Analysis of the recombinant protein expressed in Escherichia coli by immunoblot techniques showed that the protein is expressed in E. coli in its full length and does not cross-react with proteins from M. flocculare or M. hyorhinis. Genetic analysis showed that the gene was expressed from the lac promoter of the vector and seems to be translationally initiated from its own ribosome binding site. Subcloning in a transcriptional fusion vector to optimize expression resulted in production of the 36-kDa protein in E. coli at levels up to 30% of total protein.

Animals↗
PubMed1991

Nucleotide sequence of the hemolysin I gene from Actinobacillus pleuropneumoniae.

The DNA sequence of the gene encoding the structural protein of hemolysin I (HlyI) of Actinobacillus pleuropneumoniae serotype 1 strain 4074 was analyzed. The nucleotide sequence shows a 3,072-bp reading frame encoding a protein of 1,023 amino acids with a calculated molecular size of 110.1 kDa. This corresponds to the HlyI protein, which has an apparent molecular size on sodium dodecyl sulfate gels of 105 kDa. The structure of the protein derived from the DNA sequence shows three hydrophobic regions in the N-terminal part of the protein, 13 glycine-rich domains in the second half of the protein, and a hydrophilic C-terminal area, all of which are typical of the cytotoxins of the RTX (repeats in the structural toxin) toxin family. The derived amino acid sequence of HlyI shows 42% homology with the hemolysin of A. pleuropneumoniae serotype 5, 41% homology with the leukotoxin of Pasteurella haemolytica, and 56% homology with the Escherichia coli alpha-hemolysin. The 13 glycine-rich repeats and three hydrophobic areas of the HlyI sequence show more similarity to the E. coli alpha-hemolysin than to either the A. pleuropneumoniae serotype 5 hemolysin or the leukotoxin (while the last two are more similar to each other). Two types of RTX hemolysins therefore seem to be present in A. pleuropneumoniae, one (HlyI) resembling the alpha-hemolysin and a second more closely related to the leukotoxin. Ca(2+)-binding experiments using HlyI and recombinant A. pleuropneumoniae prohemolysin (HlyIA) that was produced in E. coli shows that HlyI binds 45Ca2+, probably because of the 13 glycine-rich repeated domains. Activation of the prohemolysin is not required for Ca2+ binding.

Actinobacillus↗
PubMed1990

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli.

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.

Actinobacillus↗
PubMed1990

Hemolysin patterns of Actinobacillus pleuropneumoniae.

The secreted hemolytic activities produced by the reference strains and field isolates of the 12 serotypes and 2 subtypes of Actinobacillus pleuropneumoniae were analyzed. Serotype 1 produced a Ca2(+)-inducible hemolysin, which was previously characterized as a 105-kilodalton protein and was named hemolysin I (HlyI). Serotypes 2, 4, 6, 7, and 8 produced a different hemolytic activity that was not inducible by Ca2+ but required this ion for its activity. The hemolytic activity produced by these serotypes was much weaker than that found in serotype 1 and was not neutralized by rabbit antibodies against HlyI. It was, however, neutralized by serum from pigs that were experimentally infected with a serotype 2 strain and was called hemolysin II (HlyII). Serotypes 5a, 5b, 9, 10, and 11 produced both HlyI and HlyII. In these strains, HlyI was the major contributor to the hemolytic activity. The remaining serotypes, 3 and 12, produced a very weak hemolytic activity, which was not further analyzed. Immunoblot analysis of the culture supernatants from all 12 serotypes with rabbit polyclonal antibodies directed against HlyI revealed reactions with a protein in the 105-kilodalton size range for all serotypes, indicating that HlyI and HlyII might be serologically related. Strains producing active HlyI seem to belong to serotypes that are generally considered to be virulent types and that are frequently isolated from pigs in severe pleuropneumonia outbreaks.

Actinobacillus↗
PubMed1990

Overview of the virulence attributes of the HAP-group of bacteria.

The pathogenicity requirements of the HAP bacteria include colonization of mucous surfaces, invasion of the host tissues, survival and multiplication in the host, interference with the defences of the host, and damage to the host. For these purposes the bacteria possess adhesion structures, capsular polysaccharides, surface structures such as outer membrane proteins, and lipopolysaccharides. They also secrete extracellular products, including exotoxins. The information available in this context for the major pathogens of the HAP group is reviewed.

Actinobacillus Infections↗
PubMed1990

Use of monoclonal antibodies for classifying Actinobacillus (Haemophilus) pleuropneumoniae.

The serological typing (by enzyme-linked immunosorbent assay) of 119 isolates of Actinobacillus (Haemophilus) pleuropneumoniae (representing in varying numbers the 12 serovars of this taxon) by monoclonal antibodies derived from the reference strains of serovars 1 to 5 in general correlated reasonably with the serotype previously established for these strains by conventional procedures employing polyclonal antisera. However, where there were reasonable numbers of isolates representing a given serovar to provide a decision, there was no instance where the correlation between the monoclonal and the polyclonal antibody was in complete accord. In addition, some of the differences between monoclonal and polyclonal antibody binding with some isolates suggest that the distribution of the serotype-specific antigens within the taxon may be even more complex than has previously been supposed.

Actinobacillus↗
PubMed1989

Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae.

The genetic determinant of the cohemolysin which is responsible for the CAMP phenomenon, a cohemolysis, of Actinobacillus pleuropneumoniae was cloned in Escherichia coli. Total DNA from the A. pleuropneumoniae serotype 1 type strain 4074 was used to construct a gene library in plasmid pUC18 in E. coli JM83. A total of 10,500 clones containing recombinant plasmids have been screened for hemolysis on blood plates. Fifty-five clones which showed a weak hemolytic response after 24 to 48 h of incubation were screened for the CAMP reaction with Staphylococcus aureus. This led to the identification of one clone which showed a positive CAMP reaction. Immunoblot analysis revealed that the recombinant strain expressed a protein with a molecular mass of 27,000 daltons, similar in size to the CAMP protein of the group B streptococci. Rabbit antibodies against the CAMP+ clone neutralized the CAMP reaction mediated by the E. coli strain containing the cloned CAMP gene as well as that of A. pleuropneumoniae. Antibodies raised against the cloned CAMP cohemolysin cross-reacted with Streptococcus agalactiae protein B. We designate the 27,000-dalton molecule CAMP factor protein and name its corresponding gene cfp.

Actinobacillus↗
PubMed1988

Regulation of hemolysin expression in Actinobacillus pleuropneumoniae serotype 1 by Ca2+.

Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumonia, secretes a hemolytic activity which is thought to be a factor involved in the pathogenesis of the disease. The biosynthesis of hemolysin by serotype 1 strain 4074 was strongly dependent on the activity of free Ca2+ in the growth medium. At activities of free Ca2+ below 50 microM, very low hemolytic activities could be detected in the growth medium and in cell extracts. Maximal hemolytic activities of up to 400 hemolytic units per ml could be measured in growth medium containing free Ca2+ activities above 3 mM. Other bivalent cations did not stimulate the production of hemolysin. Neither the growth rate nor the secretion of hemolysin was affected by increasing Ca2+ concentrations in the medium. The hemolysin of serotype 1 did not require Ca2+ as a cofactor for the lysis of erythrocytes. Ca2+ induced the expression of a 105-kilodalton protein, which was secreted. This protein comigrated with purified hemolysin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited hemolysin activity upon purification. Inhibition experiments with rifampin suggest that the hemolysin of A. pleuropneumoniae is regulated by Ca2+ at the transcriptional level. The threshold of hemolysin induction was around 700 microM free Ca2+, a concentration which is similar to that found in blood serum. The Ca2+-inducible hemolysin represents a novel type of positively regulated bacterial gene expression.

Actinobacillus↗
PubMed1988

Enzyme-linked immunosorbent assay for diagnosis of chronic Q fever.

From 1982 through 1987 we diagnosed 13 chronic Q fever cases. Clinically these patients presented a culture-negative endocarditis, and all but two had high complement-fixing antibody titers to Coxiella burnetii phase I (reciprocal titer above 200). With the enzyme-linked immunosorbent assay (ELISA), titers of immunoglobulin G (IgG) to phases I and II of C. burnetii averaged 158,000 and 69,900, respectively, whereas they reached 300 and 3,200 in acute Q fever cases. Similarly, IgA to both phases of C. burnetii and IgM to phase I were consistently higher during chronic than acute Q fever. The serological follow-up of one patient with chronic Q fever over a 4-year period showed a good correlation between the titers of IgG and IgM antibody titers detected by ELISA and indirect fluorescent-antibody test (IFA) to both phases of C. burnetii. Few discrepancies appeared with IgA. Shortly after initiation of antibiotic treatment, a slow and steady decrease of the antibody titers to C. burnetii phases I and II was observed. The complement fixation, IFA, and ELISA tests showed the same type of antibody response. The ELISA proved to be an excellent diagnostic test for chronic Q fever. It distinguished negative from positive reactions clearly, and results were highly reproducible. The reading is objective, and the test is simple to perform and more sensitive than the IFA and complement fixation tests. The ELISA is recommended for serologic evaluation of patients with chronic Q fever.

Antibodies, Bacterial↗