Thesis (M.S.)--University of Akron, Dept. of Chemical and Biomolecular Engineering, 2006. / "May, 2006." Title from electronic thesis title page (viewed 01/15/2008) Advisor, Lu-Kwang Ju; Committee members, Bi-min Zhang Newby, Donald Ott; Department Chair, Lu-Kwang Ju; Dean of the College, George K. Haritos; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

The reduction in cost and increase in throughput of whole genome sequencing (WGS) technologies, and the advent of benchtop WGS instruments such as the Illumina MiSeq, means that WGS is no longer restricted to large genome centres and consortia. The number of microbial genomes in public repositories is ever increasing due to the availability of WGS technologies to research groups, with individual genera having their own dedicated genome databases. Pseudomonas aeruginosa is an opportunistic pathogen and a major cause of healthcare associated infection in immunocompromised individuals and cystic fibrosis (CF) patients. The first complete genome sequence of a P. aeruginosa strain was that of the most commonly studied laboratory strain P. aeruginosa PAO1, sequenced in 2000. It was found in a study, published in 2010, that there were differences between the chromosomal sequences of two isolates of P. aeruginosa PAO1 originating from two different public strain collections, as well as differences compared to the reference sequence. It was therefore proposed that P. aeruginosa PAO1 exists as variable sublines in strain collections, whose differences included a 2.2~Mb chromosomal inversion and a prophage insertion compared to the reference sequence, as well as single nucleotide polymorphisms (SNPs) and short insertions and deletions (INDELs) which are unique to individual sublines. Since the current genomic reference sequence is based on one of these variable sublines, this study aims to deduce the sequence of the original P. aeruginosa Strain 1 (PAO) from which all the PAO1 sublines are derived, using sequence information from several laboratory sublines and from P. aeruginosa strains PAO2 and PAO3, which were directly derived from the PAO progenitor strain. This could be used as a more universal reference which is representative of a range of PAO1 sublines, to which they are all more closely related. Although most genetic studies of P. aeruginosa are carried out in PAO1, this strain is in no way the archetype for this diverse species. As whole genomes of other P. aeruginosa strains became available, comparative genomics revealed that PAO1 shared only 80% of its genome with other strains. P. aeruginosa is a highly ubiquitous organism which is able to adapt to a variety of environmental niches, as well as to human and animal hosts. This is thought to be related to the large genome size and genetic complexity of the organism, with 10% of its genes devoted to regulatory functions. Mutations in quorum sensing (QS) genes have been reported in multiple studies of host adapted P. aeruginosa isolates. QS is the mechanism by which a bacterium adapts from the lifestyle of an individual cell to a multicellular community via the regulation of specific target genes, and has been shown to regulate key virulence factors. A study of 49 clinical isolates, collected from various wound sites from distinct inpatients and outpatients at the Queen's Medical Centre in Nottingham within a few days of one another, revealed that this set of isolates displayed a diverse range of QS phenotypes. Two of these isolates were identified as producing high levels of N-acylhomoserine lactone (AHL) QS signal molecules (QSSMs) and low levels of alkyl quinolone (AQ) QSSMs, which had not previously been found in clinical isolates of P. aeruginosa, at the time of the study. WGS of these 49 clinical isolates was carried out in this study, where the aim was to; (1) determine the genomic diversity of these isolates, and relate this to previously sequenced P. aeruginosa strains, and (2) analyse the key QS genotypes of these isolates, and attempt to relate these to the phenotypes observed, in particular for the two isolates which were AHL-proficient and AQ-deficient.

Avian mycobacteriosis is a chronic infectious disease of poultry caused by different mycobacteria belonging to the M. avium-intracellular complex but primarily subspecies avium. Data on the interaction between M. avium and avian cells is very limited. This study describes the invasion and intracellular survival of M. avium isolates from both chicken and calf sources infecting THP-1-human-like macrophages and HD11 avian macrophage-like cells. There was strain to strain variation in initial invasion between the isolates tested but this variation was independent of the host-source of isolate. Invasion of the host cells was increased in the presence of host specific serum, both calf and poultry serum enhanced M. avium invasion of avian cells not seen with human cells and human serum increased the bacterial invasion of human cells, but not seen with avian cells. Both mannose and scavenger receptors were the dominant receptor for uptake of bacteria in human (THP-1) and avian (HD11) cells respectively. Simultaneous blocking mannose-, scavenger- and complement receptors did not result in complete inhibition of bacterial invasion in both cell lines suggesting that the phagocytic receptors are not working independently and may cooperate to interact with diverse ligands on the bacterial surface simultaneously for optimal binding and internalization. Invasion was both actin and tubulin dependent. The inhibitory effects of combinatory blocking of receptors and cell cytoskeleton are synergistic on the bacterial invasion of both THP-1 and HD11 cells. Post-invasion, nitric oxide and reactive oxygen species played different roles in the intracellular bacterial survival. Nitric oxide production was correlated with a reduction in intracellular survival of M. avium. In contrast, induction of reactive oxygen species enhanced M. avium survival and inhibition of reactive oxygen species using antioxidants led to a significant reduction in bacterial survival. Entry via specific receptors could have significant effect on the bacterial survival within the host cells. M. avium infection of HD11 can lead to disruption of tubulin and subsequent inhibition of both the phagosomal acidification and lysosome fusion so enhancing intracellular survival of the bacterial strains within the infected cells. In conclusion, the differences observed between bacterial isolates and host cells being infected suggest subtle differences in initial invasion and survival mechanisms between different isolates and hosts cells some of which are strain dependent and some are host cell dependent.

Pseudomonas aeruginosa (PA), a Gram-negative opportunistic rod with ubiquitous presence in a panoply of different environments, secretes a wide array of virulence determinants that have established it as one of the leading nosocomial pathogens. Many of these virulence factors are regulated by the quorum sensing (QS) system that responds to environmental cell density variations. PA can ultimately trigger the onset of severe acute and chronic infections, especially in immunosuppressed subjects. The QS network in PA is comprised of at least four multi-layered interconnected subsystems with hierarchical organisation. From these, three (las, rhl and pqs) play a pivotal role in the production of virulence factors (e.g., lectins and pyocyanin) with relevant participation in the development and maintenance of biofilm matrices. The QS network is divided in two major signaling pathways, the one driven by N-acylhomoserine lactone signals and the one driven by 2-alkyl-4-quinolone molecules. Two alkyl quinolones of core importance exist in PA, the 2-heptyl-3-hydroxy-4(1H)quinolone, typically recognised as the “Pseudomonas quinolone signal” (PQS) and its precursor 2-heptyl-4(1H)-quinolone (HHQ). In addition to the regulatory involvement of the las and rhl quorum sensing systems, the biosynthesis of PQS production is also positively regulated by PqsR-dependent transcription of the pqsABCDE operon (a multivirulence factor regulator also known as MvfR). For that reason, the alkyl quinolone (AQ) signalling pathway, and more specifically its major regulator PqsR, are widely seen as promising targets for novel antimicrobial approaches. Because of the growing presence of multidrug resistant PA in the clinical setting, representing both an immediate menace to immunocompromised patients and a heavy burden on hospital budgets, the development of more rapid and affordable screening strategies for detection of the pathogen are required. Thus, new screening strategies adapted to clinical samples and successful novel synthetic small PqsR antagonists can lead the way to a new Era in the battle against hyper-virulent/-resistant PA strains. Primarily focusing on the AQ system, this research project investigated three inter-related areas, namely: (a) the highthroughput screening of novel synthetic small molecule antagonists of the PqsR protein, designed for suppression of virulence-associated phenotypes, (b) the development of a luminescent PQS-based screening bioreporter to be applied in the clinical setting, and finally (c) the optimisation of a methodology combining liquid extraction surface analysis (LESA) and mass spectrometry (MS) for screening PQS-related AQs from in vivo bacterial extracts, and also intended for future screening of a variety of clinical samples (e.g., blood plasma, urine and saliva). A large number of synthetic small molecules with putative PqsR antagonism were obtained from our French partner GreenPharma, and studied for their capacity to interfere with expression of the key player of the pqs system, PqsR. By applying a rational selection strategy, based on the inhibitory effects on pqsA expression, assessment of metabolical exertion, and assay studies on the ultimate repression of key virulence-associated phenotypes (lectins LecA and LecB, pyocyanin, PQS-associated AQs) and impact on biolfilm formation, a final selection comprised of the four best antagonists was obtained. Compounds GPZ002966, GPZ004927, GPZ824390 and GPZ273902 had their cytotoxicity subsequently studied keeping in mind their applicability in pre-clinical studies. Overall, these PqsR antagonists promoted very strong inhibition of pqsA and lecA expression, strongly reduced production of pyocyanin and PQS-related AQs (HHQ, HQNO and C7-PQS itself), showed a strong degree of biofilm inhibition, with IC50 scores sitting at the nanomolar level, and no signs of metabolical arrest was reported by the test strains used. Some explanations, focusing on the functional and structural organisation/composition of these compounds are also offered based on a comparative analysis against a number of the most prolific PqsR antagonists recently developed. The bioluminescent PQS-based biosensor for the detection of PA was engineered to respond to the presence of exogenous PQS that forms a complex with the regulatory PqsR protein, ultimately stimulating the expression of a luxCDABE-fused pqsA promoter. The biosensor was subsequently inserted in a non-pathogenic E. coli recipient by means of chromosomal integration, devoid of the sdiA LuxR homolog that could potentially interfere with the recognition of PqsR. A silent reporter was observed when in E. coli, but further assessments to its genetic integrity did not reveal any single nucleotide polymorphisms (SNP). In addition, after further testings to its activity in different established PA mutants, devoid of genes that constituted the bioreporting system or to it directly associated, a fully functional bioreporter was confirmed. Finally, a few possible explanations as to what might be in the origin of a defective bioreporter in E. coli are discussed. Lastly, a new LESA-MS protocol based on the surface sampling of dried bacterial extracts, envisaging its potentialities as a rapid and cheap screening method for detection of AQs, was designed and optimised. Even though the method has been widely used in a variety of research scenarios, this is the first time LESA-MS is applied as a screening methodology in the context of bacterial extract screening. Overall, the optimisation process showed that LESA-MS is an approach with numerous potentialities and immediate advantages, where one emphasises sampling simplicity, fast delivering of results, sensitivity to AQs at the nanomolar level (especially for C7-PQS and the precursor HHQ). But simultaneously, this methodology also revealed limitations inherent to its setting up that constrain an effective screening. The most emphatic ones being the volatility of the preparations to intra-sampling variability, and to a certain degree, an unexpected insensitivity to important QS N-acyl homoserine lactones (AHLs), namely C4-HSL and 3-oxo-C12-HSL. Nevertheless, such limitations do not present themselves as an insurmountable barrier, and based on results from available studies making use of the LESA-MS a number of possibilities to work around these are also presented.

The bacterial pathogen Campylobacter jejuni is a leading cause of foodborne gastroenteritis worldwide. Consumption of contaminated poultry meat is considered a major source of infection. Changes in cell morphology were demonstrated to occur spontaneously on entry in to stationary phase, with development of filamentous cells amongst short spiral morphotypes. The aim of this study was to investigate differences between the morphotypes of C. jejuni and C. coli and gain insights into their development. Using a minimal culture medium filament formation was observed to be wide spread amongst C. jejuni strains tested but was not universal in C. coli strains. Filamentation did not appear to arise due to the release of diffusible molecules or the accumulation of either toxic metabolites or oxidative stressors in the medium. Separated filaments exhibited greater intracellular ATP contents compared to spiral cells, and were able to survive longer in water at 4 and 37 C. C. jejuni 12661 was identified as producing long filaments but genome sequence analysis provided no clear explanation for the enhanced filament formation. Using RNA-Seq, transcriptome differences were examined between cells growing in exponential phase and separated cell morphotypes recovered from stationary phase cultures of the C. jejuni strains 12661 and PT14. These studies identified profound transcriptional differences between the cell morphotypes present at stationary phase, and highlighted problems of interpreting such data without separation of these sub-populations. Stationary phase cells of either morphotype were impaired in motility, which likely resulted from down-regulation of rpoN encoding sigma factor 54, and several key motility associated genes. The spoT gene of C. jejuni mediates the synthesis of ppp(G)pp as part of the stringent response to stress. The spoT gene was differentially regulated in the stationary phase morphotypes recovered in this study, as were the genes encoding the phosphohydrolases PPX1/PPX2 and polyphosphate kinases PPK1/PPK2 that control cellular (p) pp(G)pp pools. Prominent heat-shock and oxidative stress responses were evident in stationary phase cells compared to cells in exponential growth phase but transcription of the ribosomal proteins was not down-shifted. The transcript levels of several cell division associated genes were down-regulated in stationary phase spiral and filamentous cells. The formation of long filamentous cell morphotypes by C. jejuni 12661 corresponds with reduced expression of maf (inhibitor of septum formation), mreB (actin-like rod-shape determining protein), mreC (rod-shape determining protein), parA, parB (chromosome partitioning proteins), ftsA (actin-like function in cytokinesis), ftsH (ATP-dependent zinc metallopeptidase), ftsW (lipid II-linked peptidoglycan transporter) and ftsZ (tubulin-like z-ring formation) that collectively function in septum formation between daughter cells.

The bacterial genus Clostridium is composed of Gram-positive, spore-forming rods with widespread biotechnological applications. This study focused mainly on Clostridium pasteurianum DSM 525, a saccharolytic species which is able to convert glycerol, the by-product of the biodiesel industry, into the valuable chemical commodities n-butanol, ethanol and 1,3-propanediol. The aim was to formulate reproducible methods for the creation of mutants, both directed and random, and use the tools developed to investigate genes, and their products, important in solvent production. A prerequisite for the deployment of the envisaged genetic tools was a reproducible means for their introduction into the cell. Following the observation of low frequencies of plasmid transfer by electroporation it was hypothesised that the low level of transformants observed were a consequence of the presence of rare hypertransformable variants within the population. Accordingly, successfully transformed clonal populations were cured of their acquired plasmid and retransformed. In a number of instances the cured cell lines proved hypertransformable, with plasmid transformation frequencies obtained that were 5 orders of magnitude higher than those obtained with the progenitor strain. All of the hypertransformable strains isolated were shown by whole genome sequence to contain single nucleotide polymorphisms (SNPs) in one or more genes. In one instance, the single SNP present was shown to be directly responsible for the increased transformation frequency by its deliberate restoration to wild type using the allelic exchange procedures subsequently developed. Having established reproducible, high frequencies of plasmid transformation reverse genetics was employed to establish gene function. Accordingly, allelic exchange gene knock-out procedures were used to target genes coding for enzymes of the central energy metabolism in C. pasteurianum and the phenotypes of the mutants obtained were analysed in laboratory scale fermentations. Strains in which the genes encoding the redox response regulator (rex) and a hydrogenase (hyd) were deleted showed increased n-butanol titres, representing first steps towards utilisation of C. pasteurianum as a chassis for this important chemical. With the inactivation of the dhaBCE gene, encoding glycerol dehydratase, production of 1,3-propanediol was entirely eliminated, demonstrating the importance of the reductive pathway for growth and redox homeostasis of this organism when grown on glycerol. In order to allow forward genetic approaches, a mariner-transposon system previously exemplified in Clostridium difficile was adapted for use in alternative clostridial hosts. In the absence of an efficient transformation system for C. pasteurianum, the initial exemplification of the system was undertaken in Clostridium acetobutylicum and Clostridium sporogenes. Successful transposon delivery was demonstrated through the use of a plasmid conditional for replication and through the insertion of a gene encoding an alternate sigma-factor, TcdR, into their genomes. Transposition was shown to be entirely random and the libraries obtained of sufficient size to allow the isolation of both auxotrophic and sporulation/germination deficient mutants. Steps were taken to develop the same system in C. pasteurianum which was successful by using a suicide delivery plasmid, which was only possible with the high transformation efficiency achieved as part of this study. This study presents an essential forward genetics procedure for industrially important Clostridium species and a comprehensive genetic engineering approach for the important biofuel producer C. pasteurianum.

Orientador: Viviane Moschini-Carlos / Resumo: Em geral, é comum que os reservatórios no Brasil apresentem características propensas a eutrofização, constituindo ecossistemas favoráveis para a expansão das florações de algas e de cianobactérias potencialmente tóxicas. O reservatório de Guarapiranga é responsável pelo fornecimento de água para grande parte da região metropolitana de São Paulo (RMSP), porém, nos últimos anos, tem sido observada uma aceleração do processo de eutrofização mediante interferência humana. Com a alteração das características físico-químicas da água, florações tornaram-se cada vez mais frequentes. Assim, este estudo objetivou investigar a interação e resposta da comunidade fitoplanctônica às variáveis físico-químicas da água do reservatório, bem como, analisar a variação espacial e temporal da comunidade fitoplanctônica, buscando conhecer a dinâmica das cianobactérias e a presença de microcistina. Para isso, foram coletadas amostras de água no reservatório no período chuvoso e seco na região à montante, intermediária (tributário Parelheiros) e barragem. Em laboratório, foram analisadas as características físico-químicas, quantificação de microcistina, triagem e identificação taxonômica dos organismos fitoplanctônicos. Por meio da análise descritiva e estatística foi possível constatar que composição da comunidade fitoplanctônica é diferente ao longo do reservatório e varia em resposta às características físico-químicas de cada local. Em geral, o período de seca representou um fator agravante do pr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In general, it is common that the reservoirs in Brazil have the characteristics prone to eutrophication, constituting favorable ecosystem for the expansion of algal blooms and potentially toxic cyanobacteria. The Guarapiranga reservoir is responsible for providing water for a large proportion of the metropolitan area of São Paulo (MRSP), but in recent years it has been observed an accelerated eutrophication process by human interference. By changing the physical and chemical characteristics of water, algal blooms have become increasingly frequent. This study aimed to investigate the composition and biomass of the phytoplankton community as well as the effects of main environmental variables on the phytoplankton community structure, additionally, to analyze the dynamics of cyanobacteria and the quantification of microcystin. Wather samples for phisical, chemical and byologic analysis were collected in the rainy and the dry season in the regions of the reservoir: upstream, intermediate (Parelheiros affluent) and dam. The organisms were analyzed in the microscope, identified and measured for the calculation of the biomass. Statistical analyses indicated that the composition of the phytoplankton community is different along the reservoir and varies in response to the physicochemical characteristics of each site. In general, the dry period represented an aggravating factor in the eutrophication process in the reservoir, mainly in the dam area, where it was observed a observed a si... (Complete abstract click electronic access below) / Mestre

Bdellovibrio bacteriovorus is a naturally soil-dwelling, Gram-negative predatory bacterium that attaches to, invades, and replicates within a wide range of other Gram-negative bacterial species, killing such prey in the process. A small number of previous studies testing the effect of B. bacteriovorus against known Gram-negative plant pathogens have suggested that B. bacteriovorus has potential as a ‘food security agent’ against Gram-negative bacterial infections in crop plants. My project built on this knowledge by screening a range of known Gram-negative bacterial plant pathogens and Plant Growth-Promoting Rhizobacteria (PGPRs) for susceptibility to Bdellovibrio predation in vitro; testing predation-susceptible strains in a simple, semi-sterile in vivo system on the surface of Agaricus bisporus mushrooms; and finally testing the effect of Bdellovibrio addition in a more complex, natural Triticum aestivum (wheat) soil rhizosphere mesocosm. An in vitro prey strain growth assay showed that susceptibility to B. bacteriovorus predation varied between a range of 20 Gram-negative (mostly Pseudomonas) bacterial pathogen/PGPR species, isolated from a range of different host crops or soil environments. Four of these species (Pseudomonas avellanae 48, P. syringae pv. phaseolicola, P. tolaasii 2192T and P. agarici 2289) were highly susceptible to predation, and three species (B. vietnamiensis G4, P. marginalis 667, and Pectobacterium atrosepticum SCRI1143) showed apparent resistance to predation. P. tolaasii 2192T, causes dark, pathogenic lesions on post-harvest mushroom host crops; In vivo co-inoculation tests on the surface of A. bisporus mushrooms showed that lesions were significantly reduced with B. bacteriovorus treatment, which was due to B. bacteriovorus predatory killing and reduction of prey cell numbers, preventing symptoms. B. bacteriovorus also preyed upon and killed a putative pathogenic Pseudomonas species isolated from a grey lesion on an organic, garden mushroom, but some likely commensal species isolated from mushroom tissue showed resistance to predation. These data together suggest that B. bacteriovorus could be used commercially to prolong the shelf life of mushrooms, reducing crop losses through spoilage, with minimal negative effects on mushroom PGPR species. Finally, inoculating B. bacteriovorus into the soil around young winter wheat plants in a natural pot soil mesocosm was found to increase plant growth and grain yield at harvest; this was contrary to my initial hypothesis that B. bacteriovorus would reduce wheat plant growth, by preying upon and killing PGPR species such as P. fluorescens that reduce wheat plant infection with Gaeumannomyces graminis var. tritici, the yield-reducing take-all fungal pathogen of wheat. The soil was found to be low in nitrogen; thus B. bacteriovorus inoculation could have increased wheat growth due to B. bacteriovorus death in the soil and subsequent release of nutrients including nitrogen. However, some B. bacteriovorus cells survived in the soil where they could prey upon some Gram-negative bacterial species, reducing their numbers. Some of the wheat growth and yield-producing effects of B. bacteriovorus may be due to the predation of species that are associated with late flowering, and therefore grain development, in wheat, allowing time for more grain to develop. Alternatively, it could be due to processes performed by B. bacteriovorus in the soil that are not related to predation, such as production of the plant hormone IAA, or B. bacteriovorus colonisation of the roots and predation of root-associated pathogenic bacterial species. Further studies are required to identify the mechanisms behind these unexpected crop yield-promoting effects, and the extent of any nutrient ‘boost’ effect due to death of B. bacteriovorus in the wheat soil, to determine whether B. bacteriovorus could be used as a pre-harvest growth and yield-promoting agent. Although most studies of B. bacteriovorus so far have focussed on its predatory activity, it likely performs other functions in its natural soil habitat, which may underlie some of the growth and yield-promoting effects shown here. However, these data show that B. bacteriovorus could be used commercially as a ‘food security agent’ when used as a post-harvest treatment to prevent crop spoilage and loss (as for A. bisporus mushrooms).

An examination of an organic extract of the cyanobacterium Lyngbya majuscula collected from Wasini Island off the southern Kenyan coast led to the isolation of the known cyclodepsipeptide antanapeptin A (7), recently isolated from a Madagascan collection of L. majuscula, and a new bioactive cyclodepsipeptide, homodolastatin 16 (42). Although L. majuscula is a common, pantropical cyanobacterium this study represents the first investigation of the natural product chemistry of a Kenyan population of L. majuscula. The structures of the two cyclodepsipeptides were determined from 2D NMR and mass spectrometry data. The L- stereochemistry of the proline, valine, and N-methylphenylalanine amino acids in 7 and the L – proline configuration in 42, was confirmed by Marfey’s HPLC method. Chiral GC was used to determine the absolute stereochemistry of the hydroxyisovaleric acid moiety in 7 and 42, the lactate residue in 42 and tentatively propose an L-stereochemistry for the Nmethylisoleucine amino acid in 42. Homodolastatin 16, a higher homologue of the potential anti-cancer agent, dolastatin 16, exhibited moderate activity against two oesophageal cancer cell lines.

MAP is the causative agent of a wasting disease in ruminants and other animals called Johne’s disease. Culture of the organism can take months and in the case of some sheep strains of MAP, culture can take up to a year. It can take several years for an animal infected with MAP to show clinical symptoms of disease. During this subclinical stage of infection, MAP can be shed into the environment contaminating their surroundings and infecting other animals. As well as this Johne’s disease is particularly difficult to diagnose during the subclinical stage of infection. Culture is very difficult and takes too long to be a viable method to diagnose Johne’s disease. Microscopic methods can be used on histological samples to detect MAP, however common acid-fast stains used are not specific for MAP and other mycobacteria and acid-fast organisms can be detected. Molecular methods, such as PCR, exist to rapidly detect the signature DNA sequences of these organisms, however they have the disadvantage of not being able to distinguish between live and dead organisms. Other immunological methods, such as ELISA tests, exist and are routinely used to diagnose Johne’s disease, however their sensitivity is very poor especially during the subclinical stage of disease. The aim of these studies was to develop novel rapid methods of detecting MAP to act as an alternative to methods already available. Sample processing using magnetic separation was carried out to allow good capture of MAP cells and to allow efficient phage infection. Using the phage assay, a specific, sensitive phage based method was developed that could detect approximately 10 cells per ml of blood within 24 h in the laboratory with a sensitive, specific plaque-PCR. This optimised detection method was then used to determine whether MAP cells could be detected in clinical blood samples of cattle suffering from Johne’s disease. The results suggest that animals experimentally and naturally infected with MAP harboured cells in their blood during subclinical and clinical stages of infection. A novel high-throughput method of detecting mycobacteria was also developed. Using phage D29 as a novel mycobacterial DNA extraction tool, viable MAP cells were detected within 8 h and the format of the assay means that it can be adapted to be used in a high-throughput capacity. Factors affecting phage infection and phage-host interactions were investigated to make sure the phage based methods of detection were as efficient as possible. It was found that periods of recovery were often necessary to not only make sure the phage were not inhibited but to also allow the host cells to be metabolically active as it was found that phage D29 can only infect mycobacteria cells that are metabolically active. A fluorescent fusion-peptide capable of specifically labelling MAP cells was also developed to be used as an alternative to acid-fast staining. Peptides that were found to specifically bind to MAP cells were fused with green fluorescent protein and cells mounted on slides were specifically labelled with the fluorescent fusion protein. This resulted in a good alternative to the generic acid-fast staining methods. The blood phage assay has shown that viable MAP cells can be found in the blood of animals suffering from Johne’s disease within 24 h and this can be confirmed using a MAP specific plaque-PCR protocol. A novel faster method to detect MAP was also developed, to cut down the time to detection of viable MAP cells to 8 h, which can be formatted to be used in a high-throughput capacity. The phage assay was used as a tool to determine different metabolic states of mycobacteria, and helped investigate optimal detection conditions when using the phage assay. Finally a novel fluorescent label was developed to detect MAP as an alternative to insensitive acid-fast staining. The development of these novel methods to rapidly, specifically and sensitively detect MAP will push further the understanding of Johne’s disease and help control it.