Erysipelothrix rhusiopathiae: epidemiology, virulence factors and neuraminidase studies

Qinning Wang

Research output: ThesisDoctoral Thesis

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Abstract

Erysipelothrix rhusiopathiae, a Gram-positive bacillus, has long been an important pathogen in veterinary medicine as well as a cause of serious disease in humans. Infections caused by this organism have economic impact on animal industries, causing erysipelas in swine and morbidities in other farmed animals. Human infections are commonly erysipeloid (skin cellulitis) and occasionally septicaemia or endocarditis. Little is known of the diagnosis, epidemiology and pathogenesis of such infections in Western Australia. The aims of this thesis were to establish new diagnostic techniques for the detection and recovery of E. rhusiopathiae, to describe the epidemiology of Erysipelothrix infection in Western Australia in humans and animals, and to characterize virulence-associated characteristics, especially focusing on the neuraminidase produced by the organism. A protocol using 48 h Brain Heart Infusion enrichment followed by subculture to selective agar containing antibiotics achieved the highest recovery rate of 37% in a seafood survey. Twentyone isolates of Erysipelothrix spp., of which 19 were identified as E. rhusiopathiae, were obtained. Two published PCR assays for differentiating E. rhusiopathiae and other Erysipelothrix species were evaluated and the best PCR detection rate achieved was 67% following selective enrichment. The PCR method was 50% more sensitive than the culture method. Epidemiological surveys using the above methods showed that E. rhusiopathiae infection is present in farmed animals in Western Australia. The PCR positive frequencies (3.3-3.7%) and isolate recovery rate (2.8-3.3%) in samples from pig and sheep abattoirs and carcass washings indicate a potential threat to the economy of the farmed animal industry as well as a public health concern with the occurrence of E. rhusiopathiae in meat for consumption. Positive PCR results (1.1%) from human skin swabs of patients with cellulitis and wounds may suggest the existence of Erysipelothrix colonization in the general population. Genetic relatedness of 92 isolates of Erysipelothrix species from various sources was analyzed and a total of 64 distinct PFGE patterns identified. Isolates were further classified into 20 clonal groups based on pattern similarities, and most E. rhusiopathiae were clustered into six groups. A few patterns of other Erysipelothrix species were clustered into separate groups from E. rhusiopathiae but shared greater than 70% similarity with E. rhusiopathiae. The genetic relatedness of colonial variants was well demonstrated using this method. PFGE typing promises to be a useful tool for epidemiological and taxonomic studies of Erysipelothrix. Several virulence-associated factors were characterized in 86 isolates of Erysipelothrix spp. A rapid and sensitive peanut lectin hemagglutination assay for neuraminidase was developed and the influence of media, incubation conditions and pH on the production of the enzyme was investigated. All 61 isolates of E. rhusiopathiae produced neuraminidase in cooked meat broth with titres between 1:10 and 1:320, with no significant difference in titre among isolates from different sources. The enzyme activity was not detected in non-pathogenic Erysipelothrix spp. Capsule was produced by 78.7% of isolates of E. rhusiopathiae but not by other species, while both hyaluronidase and haemolysin were produced by non-pathogenic Erysipelothrix spp. It was concluded that neuraminidase and capsule are most likely to be virulence factors of E. rhusiopathiae. The gene encoding neuraminidase was cloned from the type strain E. rhusiopathiae ATCC 19414. The cloned fragment was a functional partial nanH gene with a mol% G+C of 39.7. The predicted amino acid sequence displayed homology with many microbial neuraminidases and contained conserved sequences found in most bacterial neuraminidases. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial genomic DNA. A neuramin
Original languageEnglish
QualificationDoctor of Philosophy
Publication statusUnpublished - 2003

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