Pirovano, Andrea
(2019)
A study of the practical challenges associated with developing a new test for bovine tuberculosis.
MRes thesis, University of Nottingham.
Abstract
Bovine tuberculosis, a chronic disease caused by Mycobacterium bovis, is a major economic problem affecting farmers both in the UK and worldwide. In order to prevent and control bovine tuberculosis it is very important to be able to promptly and definitively diagnose infected animals. However, the current diagnostic tools show some limitations which can contribute to cause economic losses and to the transmission of the disease to other animals in a herd, to wildlife and potentially to humans. Therefore, there is an urgent need to have available new diagnostic methods that allow the definitive detection of bovine tuberculosis. In this work, the practical issues associated with the cost-effective production of two diagnostic kits that employ mycobacteriophage (ActiphageTM and ActiphageTM Rapid) were investigated. This technology exploits the ability of bacteriophage to infect live mycobacterial species in a strain specific manner. This technique was originally developed as a tool for the diagnosis of human tuberculosis (FastPlaqueTBTM, BIOTEC) and has now been refined to offer transformational tools with significant applications in veterinary, agriculture and - potentially – the human sector. These kits are manufactured and distributed by PBD Biotech Ltd. (Thurston, Suffolk, UK) and allow the detection of the presence of M. bovis and M. avium subsp. paratuberculosis in diverse sample types, such as blood and milk. The assays can therefore be used for infection control and as a part of a quality assurance programme to confirm the absence of these organisms in non-pasteurized dairy products. Furthermore, the methods can be adapted to detect other types of mycobacteria and applied to detect infection not only in cattle but also in other animal hosts including sheep, goats, deer, horses, alpacas, llamas as well as companion animals and exotic species.
In order to develop the simplest method that gives a reproducible titre of at least 1010 pfu ml-1, three different ways of manufacturing the freeze-dried bacteriophage supplied with the kits were explored. With all of the three methods, there was no difference in the titre of the phage culture before and after freeze drying. This suggests that the process does not have a negative impact on the viability of the phage. With the first two methods employed, the titre obtained was normally 109 pfu ml-1. Only one time, using method 2, the titre obtained was 1010 pfu ml-1. This titre seems to have been routinely achieved modifying method 2 in order to ensure an optimal elution of the bacteriophage from the agar to the medium. Shelf life studies on the freeze dried phage are being performed and, so far, they revealed an average stability of about 3 months if stored at +5 / +8°C. The practical issues related to manufacturing of the virucide tablets supplied with the ActiphageTM Core kit were also explored and a softer formula led to jamming of the tablet press and broken tablets but also produced a tablet that was easier to dissolve. Once the production issues were solved by adjusting the machine settings, a procedure for checking the weights of the tablets was developed. Finally, work was carried out to identify the best source of the other kits components and to design and test packaging materials, and finally an evaluation of the cost of competitor tests was performed to better understand the market price that could be charged for ActiphageTM tests.
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