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Kubala, Anton (2024) Development and Evaluation of Novel Methods to Improve Phage-Based Detection Methods. PhD thesis, University of Nottingham.

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Abstract

Mycobacterial pathogens, such as M. tuberculosis, M. bovis and Mycobacterium avium ssp. paratuberculosis (MAP), pose significant and emerging global health risks for humans while mycobacterial disease in domesticated animals result in billions of dollars in losses to the agrarian industries, especially the dairy industry. Mycobacterial diseases are seen as endemic and persistent, with a lack of diagnostic tools able to detect mycobacterial infections quickly and with high sensitivity being the main hurdle to combating endemic mycobacterial infections. Due to the typically slow growth of mycobacteria such as MAP and M. bovis, current culture-based methods are too slow and classic microscopy is neither sensitive enough to detect low numbers of cells in clinical samples nor does it allow specific identification of the mycobacterial species encountered. PCR-based detection assays for both MAP and M. bovis exist and are able to produce a conclusive result in a few hours but they have limited sensitivity and cannot differentiate between live and dead organisms. The most common tool for the diagnosis of mycobacterial pathogens in animals are ELISA assays which are rapid, low cost and are reasonably specific but have low sensitivity, especially in the early stages of infection. To address all these issues, the Actiphage® assay was developed which utilises bacteriophage D29 to specifically infect and lyse mycobacterial cells recovered from a clinical blood sample and then PCR is used to identify species-specific signature sequences in the released mycobacterial DNA. This approach allows highly specific and sensitivity identification of the mycobacterial cells present in a blood sample within 24 h.

This project was sponsored by PBD Biotech Ltd which was set up to commercialise The Actiphage® assay and the main aim was to develop the methods required to produce the kit reagents in a stable format and to further refine the methods used to process the blood samples. The first approach that was used to stabilise the bacteriophage was to freeze them while still embedded in the agar used to grow them in the laboratory. This proved to be successful and was found to be a method that could be generically used to create stable phage samples that could be stored at –20 °C. This work also provided good information about the characteristics of the excipients required to protect the phage from freeze-thaw damage, but ultimately was not practical for large scale manufacture of kits. Thus it was decided to try and freeze dry high titre liquid lysates of the phage.

To that end research was carried that allowed fast and inexpensive production of large volumes (10-1000 ml) of high titre (> 1 x 1010 pfu ml-1) phage suspension as well as methods to allow this to be freeze dried in appropriate volumes with good shelf stability at room temperature. This provided a framework of knowledge to then develop a method for the production of a stable form of both MAP and M. bovis culture to be included as a positive control in the commercial kits. While it was possible to produce stable reagents, it was found that, unlike the phage samples, these needed to be stored at low temperature to maintain functionality. This led to the discovery that while the mycobacteria would survive freeze drying in high numbers, cells transitioned into a state that was not detectable by phage D29, possibly because they entered a stationary growth phase and were thus viable but nondetectable. However it was found that this process could be reversed by incubating the resuscitated cells for 2-3 h at 37 °C. Additional work was carried out on investigating phage host cell interaction in the presence of excipients and at low temperatures for the purpose of optimizing the efficiency and sensitivity of the phage based detection assay for the freeze dried reagents.

A second area of research focussed on optimizing the blood separation method employed for concentrating mycobacterial cells in animal blood samples. A method was developed to separate peripheral blood mononucleocytes (PBMCs) using ACK lysis buffer to specifically lyse the erythrocytes in the blood allowing the PBMCs to be isolated and concentrated by centrifugation. Replacing the standard density gradient centrifugation with ACK lysis made assay procedure cheaper, more reliable and faster thus increasing usability and sample throughput. It was then shown that this method could then be used for testing commercial cervid (farmed deer) blood samples and that the Actiphage® assay could successfully be used as an epidemiological tool for the diagnosis and monitoring of MAP in farmed deer, confirming that this assay has great potential for the control of mycobacterial diseases.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Rees, Catherine Swift, Benjamin
Keywords:	mycobacterial pathogens, mycobacterial infections, bacteriophage
Subjects:	Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria S Agriculture > SF Animal culture
Faculties/Schools:	UK Campuses > Faculty of Science > School of Biosciences
Item ID:	78341
Depositing User:	Kubala, Anton
Date Deposited:	02 Jun 2025 10:27
Last Modified:	02 Jun 2025 10:27
URI:	https://eprints.nottingham.ac.uk/id/eprint/78341

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