Development and validation of a field deployable test for the diagnosis of high-priority infectious animal diseases in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatu, Palmerston North, New Zealand

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Massey University
Figure 2-1 is reused with permission.
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In the event of infectious disease incursions, rapid and accurate diagnosis is essential for ensuring appropriate and prompt control measures are put in place to minimise further transmission. Foot and mouth disease (FMD) is one example of an exotic disease that could severely affect New Zealand’s livestock industries if introduced to this country. Pen-side testing can help by providing a rapid confirmation of a provisional diagnosis without the delays and risks associated with sending samples to a diagnostic laboratory. The aim of the work presented in this thesis was to develop and validate a field deployable diagnostic test system for prompt and accurate detection of FMD virus (FMDV). In addition, the test can be used to simultaneously detect two other viruses that would be expected to be on the differential diagnosis list: bovine viral diarrhoea type 1 (BVDV-1) and type 2 (BVDV-2). Chapter 1 comprises a brief literature review of FMDV infections in susceptible species, followed by a review of the current and emerging trends in field deployable diagnostics as applicable to animal diseases. In Chapter 2, a multi-criteria scoring and ranking model for identifying the best test platform for development of the deployable field test is presented. The general flow of the method consisted of defining the requirements for the ideal test platform, identifying, and shortlisting potential candidate systems, describing the criteria for evaluation, and scoring the candidate platforms against the criteria by a panel of recruited experts. This participatory and collective opinion provided a basis for selecting T-COR 8™ (Tetracore®) as the best overall fit-for-purpose. In Chapter 3, several easy techniques for processing clinical samples compatible with the selected test platform were examined. These protocols were applied to test panels comprising serial dilutions of BVDV-1 or equine rhinitis A virus (ERAV) in serum or oral swab samples. The latter was used as a proxy for FMDV. The protocols were compared to a reference extraction method based on the observed detection limit, as judged by quantification cycle (Cq) values generated in virus-specific reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays. The complexity of sample manipulation and time required were also considered. Dilution of the sample with phosphate-buffered saline (PBS), with or without a pre-heating step, was chosen as the most suitable method for integration in the pen-side PCR testing. Development of the field assay’s controls is described in Chapter 4. These included a synthetic positive control transcript (R3+) that could be safely used with assays aimed at the detection of several pathogens associated with development of vesicular disease in cattle. The universal control transcript also incorporated an exogenous internal control (IC) target, which was designed to be used with a phage based (Qβ) internal control (IC) system. Optimization of a Qβ IC assay for use in the pen-side multiplex RT-qPCR (mRT-qPCR) is also included in this Chapter. In Chapter 5, development, and optimisation of mRT-qPCR for the differential detection of FMDV, BVDV-1 and BVDV-2, including detection of a Qβ as exogenous IC, is presented. The optimised mRT-qPCR showed linearity over five 10-fold dilutions of R3+ transcript, good efficiency, and low intra-and inter-assay variability. The mRT-qPCR was highly specific for the detection of representative FMDV serotypes and was also able to simultaneously detect BVDV-1 and BVDV-2 isolates. The assay did not react with other viruses that can produce vesicular lesions, nor did it react with unrelated bovine pathogens endemic in New Zealand. Multiplexing the four primer- and probe sets did not affect the performance and analytical sensitivity of the assay for the detection of individual components when compared to the respective singleplex assays. The diagnostic performance of the optimised mRT-qPCR for detecting FMDV, BVDV-1 and BVDV-2 is presented in Chapters 6 and 7. Diagnostic specificity was evaluated using sera and oral swabs from New Zealand cattle. Diagnostic sensitivity for FMDV detection was assessed using mock oral swabs from outbreak samples in two endemic countries (Lao PDR and Myanmar). The robustness of the field PCR was evaluated at three field locations with varied environmental conditions (New Zealand, Lao PDR, and Myanmar). Overall, the diagnostic specificity (DSp) of the field mRT-qPCR for three target viruses (FMDV, BVDV-1 and BVDV-2) was close to 100%, which was similar to the performance of respective reference PCRs. Although the diagnostic sensitivity (DSe) of the FMDV component was comparable to that obtained with the reference method, care must be taken in interpreting the result since FMD positive samples used for evaluation of the sensitivity of the mRT-qPCR were not sourced from New Zealand cattle. The mRT-qPCR also had high DSe for detecting BVDV-1 infected cattle when the BVDV RNA levels expected to be present in clinical samples from either persistently infected (PI) or transiently infected animals were considered. Pre-heating of samples increased the sensitivity of the BVDV-1 component of the assay. Further validation using additional FMDV-positive and negative clinical specimens should be attempted in the future. Overall, the work presented in this thesis resulted in the development of a simple, extraction-free pen-side PCR test that can be deployed around New Zealand for rapid and reliable detection of FMDV in the event of a suspected incursion. Future work to enhance its use would involve exploration of other methods of preparing samples so that the test can be utilised in screening sub-clinical FMDV infections during post-outbreak surveillance.
Cattle, Virus diseases, Testing, Diagnostic reagents and test kits, Design, Foot-and-mouth disease, Bovine viral diarrhea, Diagnosis, Veterinary virology, field deployable test, FMD, foot and mouth disease, pen-side test validation