Sustainable food production capable of feeding a growing human population is a significant global challenge, and is a priority encompassed within the United Nations Millennium Development Goal to ‘eradicate extreme poverty and hunger’. Infectious diseases reduce the productivity of farm animals, and the globalised trade of animals and their products increases the threat of disease incursion. Accurate and rapid diagnostic tests are an essential component of contingency plans to detect, control and eradicate such diseases. Diagnosis involves a ‘pipeline’ that normally starts with clinical suspicion, followed by collecting samples, transporting specimens to a centralised laboratory setting (e.g. national/international Reference Laboratories), analysing these samples using a range of diagnostic tests and reporting the results. However, the transport of specimens from the field to the laboratory can be a lengthy process that can delay critical decision-making and severely affect the quality of the samples.
This important limitation of centralised diagnostic testing has motivated the development of tools for the rapid, simple detection of livestock pathogens. Recent advances in the development of technologies for personalised human medicine have motivated the development of prototype diagnostic tests for a wide selection of diseases of livestock. However, many of these tests are not yet routinely used or commercially available. This paper critically reviews the most promising examples of such assays, and highlights the challenges that remain to transition these tests from applied research and development into routine use.

Technological advances in veterinary diagnostics: opportunities to deploy rapid decentralised tests to detect pathogens affecting livestock.

The requirements for prompt diagnosis of foot-and-mouth disease (FMD) during outbreaks, and the need to establish robust laboratory testing capacity within FMD-endemic countries, have motivated the development of point-of-care tests (POCTs) to support current diagnostic strategies. Despite numerous publications detailing the design of platforms and assays for this purpose, the majority have only been evaluated in laboratory settings, using protocols incompatible for use in challenging environments. To address this gap, this thesis describes the development of an end-to-end molecular toolbox for the detection and characterisation of FMD virus (FMDV) RNA in decentralised settings. A critical review and multiway comparison of seven assay formats and 11 sample preparation methods revealed that reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time reverse transcription PCR (rRT-PCR) POCT-formats exhibited comparable analytical and diagnostic sensitivity to their laboratory-based equivalents. Additionally, reagent lyophilisation provided a solution for cold chain and storage considerations, whilst not compromising assay performance. Both assays were compatible with simple sample preparation methods, removing the requirement for nucleic acid extraction. For example, dilution of samples in nuclease-free water enabled FMDV RNA to be detected in multiple sample types (epithelial tissue suspensions, serum, oesophageal-pharyngeal fluid and lesion swabs), from as early as one day post infection. Notably, when the robust field-ready protocols were deployed into challenging low-resource laboratory and field-settings within East Africa, POCT results (rRT-PCR = 144; RT-LAMP = 145) were consistent with clinical observations and a reference rRT-PCR, with FMDV detected from acutely infected as well as convalescent cattle. Furthermore, transitioning of East Africa-specific FMDV-typing rRT-PCR assays (for serotypes O, A, Southern African Territories [SAT] 1 and SAT 2) into a multiplex POCT-format enabled rapid identification of FMDV serotype in situ, confirming active outbreaks of both O and A. This thesis also describes the development of GoPrime, a novel real-time PCR (rPCR) primer/probe validation tool. By parameterising GoPrime with experimental data, collected to investigate the effects of primer/probe-template mismatches on cycle threshold and limit of detection, it was possible to quantitatively predict the performance of rPCR assays in silico. The work of this thesis supports the deployment of molecular POCTs into non-specialised, resource-limited and challenging settings for simple, highly sensitive and rapid detection and/or characterisation of FMDV.

The development and application of molecular tools for the diagnosis of foot-and-mouth disease in field and low-resource laboratory settings

Ministry of Livestock and Fisheries Development (MLFD) and Chief Executive Office (CEO) of Tanzania Veterinary Laboratory Agency (TVLA)

Investigating the perfomance of lateral flow devices in diagnosis and genetic characterization of foot and mouth disease virus in Tanzania

A DISSERTATION SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES IN PARTIAL FULFILLMENT FOR THE REQUIREMENT OF THE AWARD OF THE DEGREE OF MASTERS OF SCIENCE IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY OF MAKERERE UNIVERSITY.

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Serotypes of foot and mouth disease virus in african buffaloes and cattle in queen elizabeth national park

Foot - and - mouth - disease is severe, highly contagious disease of cloven - hoofed animals that affects large animal livestock species and various wildlife species. Different countries has a different FMD status which require a disparate approach defining the diagnostic and control strategy. A variety of new diagnostic tests and procedures was developed to improve FMD laboratory diagnosis. The aim of this study is to evaluate the contemporary diagnostic tools and the ability of our laboratory to detect FMD virus or viral genome in field samples and cell culture fluids using an Ag ELISA, TaqMan real-time RT-PCR and Virus isolation combined with chromatographic - LFD (lateral flow devises) tests.

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I. Chenchev

Papers

Improved diagnostic strategy for foot-and-mouth disease in Bulgaria