Differentiation of virulent and biological control Paenibacillus larvae strains associated with American foulbrood in bee hives

The detection and control of American Foulbrood (AFB) is made more challenging due to a lack of evidence regarding the virulence mechanisms employed by this honeybee pathogen. Whilst incidence of this pathogen within the UK has recently declined, ~100 colonies were identified as infected with AFB in 2011 (to end of September), so AFB should still be considered a serious threat to honeybee health. It is known that within the species many phenotypes exist, and the infection caused by the phenotypes differs greatly. This PhD thesis presents several advances towards a greater understanding of the intra-specific differences occurring within the species. Chapter 2 evaluates the use of 16S rRNA sequencing as a method of Paenibacillus larvae identification, as well as exploring the use of this ribosomal subunit for differentiation of the species. The sequencing of two housekeeping (purH and PyrE) genes assesses the potential of a Multi Locus Sequence Typing (MLST) method, as a means of subspecies differentiation. Chapter 3 assesses what can be inferred from use of Enterobacterial Repetitive Intergenic Consensus (ERIC) sequence fingerprinting, with regards to prior knowledge genetic differences. Lateral Flow Devices (LFDs), a commonly used diagnostic tool, are tested to ensure P. larvae isolates representing all 4 ERIC types are detected. In Chapter 4 an in-vitro honeybee rearing method is employed to observe the correlation between proteolytic activity of isolates and in-vitro virulence. The method is applied to a wider range of reference isolates, to observe the intra-species differences existing. The ability to produce large numbers of viable spores is explored as a potential difference existing between ERIC types I and IV. Whole genome shotgun sequencing is used in chapter 5 to perform comparative genomic studies on 4 P. larvae isolates also utilising 646 contigs from a previous sequencing project. The possible presence of plasmid DNA is explored, through GC content analysis. The genetic basis of a sporulation phenotypic difference is examined by BLAST analysis of orthologous genes. In Chapter 6 the findings of this thesis are discussed in more detail, and potential areas of further study are identified.