In the present study T. pyogenes isolated from milk of mastitic dairy cattle (n=57, n=1), from genital tract of metritic and apparently healthy dairy cattle (n=14) and from fatal infections of three grey slender lorises (n=3) together with 12 reference strains representing ten species of genera Trueperella and Arcanobacterium could be identified and characterized individually by using phenotypical methods, by MALDI-TOF MS fingerprinting, by FT-IR spectroscopy and by various genotypic techniques investigating the molecular targets 16S rRNA, ISR, sodA and gap. The genotypic techniques also included the amplification of the putative virulence factor encoding genes plo, cbpA, nanH, nanP, fimA, fimC, fimE and tet(W). A collection of the investigated T. pyogenes from milk of mastitic dairy cattle were subjected to epidemiological studies using MLSA and the T. pyogenes from fatal infections of three grey slender lorises using rep-PCRs , RAPD-PCR and MLSA. The T. pyogenes of mastitic origin were isolated in a period of 3 years and were mainly isolated together with various other bacteria from milk of mastitic dairy cattle with varying clinical symptoms. However, T. pyogenes seemed to be the major causative agent. The phenotypic properties, also including MALDI-TOF MS analysis and the newly described FT-IR spectroscopy and the genotypic methods, allowed a reliable identification and further characterization of the bacteria of this origin. The T. pyogenes of mastitis origin possessed several putative virulence factor encoding genes in varying combinations. These results together with the genomic fingerprinting with a newly established MLSA revealed that bovine mastitis in farms caused by T. pyogenes is mainly caused by individual bacterial clones without relation to each other. The 14 T. pyogenes isolated from genital tract of metritic and apparently healthy dairy cattle and from the three grey slender lorises could also be identified phenotypically and genotypically. However, the distribution of virulence factor encoding genes of the T. pyogenes isolated from bovine genital tract revealed no significant differences between diseased and apparently healthy animals indicating that additional criteria might be responsible for onset and etiopathology of bovine metritis. In contrast to the T. pyogenes from mastitis origin and from bovine genital tract the three T. pyogenes isolated from grey slender lorises displayed identical phenotypical and genotypical properties. The latter could also be demonstrated by genomic fingerprinting using three different rep-PCRs, by RAPD-PCR and by MLSA. These results showed that the fatal infection of the three grey slender lorises were caused by a cross infection of a single T. pyogenes clone. However, the route of infection of the three grey slender lorises at Frankfurt Zoo remains unclear.In the present study T. pyogenes of bovine and grey slender lorises origin could reliably be identified by several phenotypic and genotypic methods and further characterized by determination of putative virulence factor encoding genes and by novel DNA fingerprinting procedures. All these techniques might help to improve a future identification and characterization of T. pyogenes and might help to determine epidemiological relationships of these bacteria in animal or human infections.
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