The spacer oligonucleotide typing (spoligotyping) method was evaluated for its ability to differentiate Mycobacterium bovis strains. This method detects the presence or absence of spacers of the direct repeat locus of the M. bovis genome. The spacers in the direct repeat locus are amplified by PCR and are detected by hybridization of the biotin-labelled PCR product with a membrane containing oligonucleotides derived from spacer sequences that have previously been bound to a membrane. One hundred eighty-two M. bovis isolates from domestic animals (cattle, goat, sheep, and cats) and wild animals (deer and wild boar) were spoligotyped, and the results were compared with those obtained by IS6110 restriction fragment length polymorphism analysis. Two rather homogeneous clusters of isolates containing 20 and 4 types, respectively, were identified by spoligotyping. The first cluster included isolates from cattle, cats, and feral animals. By spoligotyping, isolates from the Spanish wild boar and deer had the same pattern as some bovine isolates, suggesting transmission between these animals and cattle and highlighting the importance of the study of these reservoirs. The second cluster included all the caprine and ovine isolates. Within each cluster, the patterns of the different strains differed only slightly, suggesting that the spoligotypes may be characteristic of strains from particular animal species. Spoligotyping proved to be useful for studying the epidemiology of bovine M. bovis isolates, especially of those isolates containing only a single copy of IS6110. In view of our results, we suggest fingerprinting all M. bovis strains by the spoligotyping method initially and then by IS6110 restriction fragment length polymorphism typing of the strains belonging to the most common spoligotypes.
Summary The involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogen Actinobacillus pleuropneumoniae in virulence was investigated using haemolysin‐deficient mutants constructed by a mini‐Tn10 mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini‐Tn10 were cloned and sequenced. In the non‐haemolytic mutant, the transposon had inserted in apxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In the apxI operon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved in A. pleuropneumoniae pathogenicity. The non‐haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.
A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed.
A total of 32 Pasteurella multocida isolates were obtained from 60 cases of swine pneumonic lungs collected in “Castilla y León” (northwestern Spain) between November 2017 and April 2018. Capsular type A isolates were isolated from 96.9% cases and capsular type D from the remaining 3.1%. All isolates were characterized for their susceptibilities to eight antimicrobial agents and the presence of eight resistance genes. The frequency of susceptibility was lower than 60% in four of the drugs, 84.4% of the isolates showed resistance to at least two compounds, and 46.9% to a combination of three drugs. The resistance patterns suggested that enrofloxacin, chloramphenicol, tetracycline and cefotaxime were the compounds most likely active to P. multocida . The usage of PCR revealed that erm C, bla ROB1 , tet B and msr E genes occurred in more than 37.0% isolates, that suggested its putative accountability in the resistance of the strains harbor them. However, most were detected in susceptible strains and only a genetic explanation for the resistance could be linked to erythromycin. Therefore, the resistances to clyndamicin, cotrimoxazol, β-lactams and tetracyclin observed by phenotypic testing remains genetically unexplained and further investigations are required.
The strong bizonal hemolysis on blood agar and the positive CAMP reaction with Rhodococcus equi denotes the production of two different cytolytic factors by Listeria ivanovii. One was characterized as a thiol-activated (SH) cytolysin of 61 kilodaltons and was termed ivanolysin O (ILO) since data suggested that it is different from listeriolysin O, the SH-cytolysin produced by Listeria monocytogenes. The other is a 27-kilodalton hemolytic sphingomyelinase C that was found to be the cytolytic factor responsible for the halo of incomplete hemolysis synergistically enhanced by R. equi exosubstances. When thiol-disulfide exchange affinity chromatography and gel filtration were applied to the purification of ILO from concentrated L. ivanovii culture supernatants, the copurification of the two cytolysins was observed. This phenomenon seems to be due to the formation of intermolecular disulfide bonds between ILO and the sphingomyelinase, since the latter was found to contain free SH groups, not essential for the activity. These SH groups could react with the single cysteine residue characteristically present in the SH-cytolysins, forming a dimeric cytolytic complex. The purification of ILO was achieved by a further gel filtration with a reducing agent (dithiothreitol) in the eluent. A method for the purification of the sphingomyelinase based on selective sequestration of ILO from the L. ivanovii concentrated culture supernatant by the SH cytolysin target molecule cholesterol and thiol-disulfide affinity chromatography is described.
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