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Item 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(Massey University, 2024-03-15) Bueno, RudolfoIn 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.Item Interpreting highly diverse dsRNA viral sequences from Picobirnaviridae within and among species for interspecies transmission inference : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Sciences at the School of Veterinary Science, Massey University, Manawatu, New Zealand(Massey University, 2021) Wierenga, JanelleCross-species transmission of infectious diseases has been at the forefront of our current reality considering the COVID-19 global pandemic and its repercussions. Now more than ever, we realise that the factors involved in these transmissions between species is both simple, such as the impact of increased contact between wildlife and humans, and complicated, such as the impact of biodiversity loss. Our understanding of these influences, and the complexity, has progressed considerably in the last decade though we are still far from predicting epidemics and pandemics. We do, though, understand that many of these infectious diseases cross species barriers frequently, with some types of pathogens more likely to cross between species. Picobirnaviruses (genus Picobirnavirus, family Picobirnaviridae), a genus of double-stranded RNA (dsRNA) viruses, have been identified on almost every continent, though the pathogenic potential for these viruses is still debated. Picobirnaviruses are known to have massive genetic diversity. Picobirnaviruses were first identified in rabbits and then humans and since then have been reported in many mammalian species, birds, reptiles, along with wastewater, and potentially in protozoa; prokaryotes have been implicated as potential hosts, based on conserved ribosomal-binding motifs from prokaryotic messenger RNA. Although they are highly diverse, they have often been found to have very high similarity in different host species present at the same geographic site, raising the hypothesis that cross-species transmission has occurred. The study system from Uganda described in this thesis provides a setting for the possible transmission of infectious diseases between wildlife, humans and domestic livestock. I identified multiple potential pathogens to study cross-species transmission within this system and focused on Picobirnavirus. I identified multiple picobirnaviruses in humans, wildlife and domestic animals from Uganda and New Zealand, for comparison, by using metagenomic and targeted PCR amplicon sequencing. Initial phylogenetic analyses revealed some host clustering among the picobirnaviruses studied, but host and geographical clustering was not upheld when known picobirnaviruses from global databases were included. Furthermore, the use of near-complete and two genogroup-specific viral sequences did not reveal host or geographic clustering on phylogenetic analyses. I identified multiple picobirnaviruses within the same host, detecting up to eight distinct viral sequence types in the samples. Despite the wide-ranging within-host diversity of the viruses, nearly identical virus sequences were also identified in different hosts from the study. Given the high diversity, unclear host and geographic associations, and that picobirnaviruses may infect hosts from across the natural kingdoms, including bacteria and protozoa, I sought to identify picobirnaviruses from bacterial and protozoal samples, to try to determine the host range of the viruses. I discovered picobirnavirus RNA in purified Cryptosporidium oocysts from New Zealand, suggesting a possible alternative Protist host. However, using an alternative genetic code from invertebrate mitochondria removed within putative open reading frame stop codons from some picobirnavirus sequences, supporting other studies. Further analyses of the picobirnavirus sequences revealed alternative genetic code usage or motifs in the untranslated regions that could indicate the virus has a prokaryotic rather than a eukaryotic host—which have been commonly assumed to be the definitive hosts. The discovery that prokaryotes may also be considered hosts of picobirnaviruses, as well as the presence of homologous sequence between species of this highly diverse RNA virus genera, will influence our understanding of these vexatious and under-studied viruses.Item Bovine viral diarrhoea virus in New Zealand dairy cattle : a review of diagnostic methodologies and preliminary data on circulating genotypes : a thesis presented in fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Science at Massey University, Manawatu Campus, New Zealand(Massey University, 2021) Dissanayake, Sonali DarshikaBovine viral diarrhoea (BVD) is a well-established disease in New Zealand beef and dairy cattle that causes significant economic losses for the industries each year. The pathogenesis of the disease is well understood and there is a wide range of reliable diagnostic tools that have allowed many European countries to successfully eradicate BVD through coordinated national disease control programmes. However, it is difficult to directly apply many of these frameworks in New Zealand due to the unique characteries of the pastoral farming systems that create different logistical and epidemiological challenges. There is a strong need to identify the most cost-effective means of applying existing diagnostic tests to design a feasible national control programme for New Zealand. To support this goal, this thesis has focused on filling two existing knowledge gaps: one around the performance of BVD diagnostics tests in herd and industry level disease control programmes and the other around using molecular diagnostic test methods to characterise the circulating bovine viral diarrhoea virus (BVDV) strains in the cattle populations. The last formal literature review on BVD diagnostic tests was published more than 15 years ago and there have been significant advances in the availability and performance of the tests since then. In Chapter 2, a non-systematic review was conducted on the evolution of BVD diagnostic tests over the past 60 years and how they have been applied to different herd and industry level BVD control programmes. The review includes an in-depth overview of key feature in the pathogenesis of BVD that impact test performance followed by detailed descriptions of the different diagnostic methodologies, their performance and their current applications. The discussion section highlights the remaining limitations and technical gaps in the current BVD diagnostic tests along with suggestions for future research directions. In particular, molecular epidemiology has been successfully applied in some countries as a part of their BVD control to understand suspected transmission routes but has not yet been applied in New Zealand. As a preliminary investigation, Chapter 3 was designed to understand current circulating BVDV strains in dairy cattle across New Zealand using a convenience sample virus positive serum samples that were submitted to commercial diagnostic laboratories during the study time period. Both the 5'UTR and NPro genes were sequenced from each sample to identify the strain type and phylogenetic analyses were performed to explore the genetic relatedness of each sequence. Although BVDV 1-A was found as the only subtype circulating among dairy cattle, there was a high variation among the sequences within group 1-A. The phylogenetic analysis showed a fair homogeny of New Zealand dairy isolates with overseas isolates, particularly with BVDV strains previously identified in China. There was also 100% homogeny between the New Zealand dairy isolates and those from cattle that were recently sequenced for another study, which is most likely from the sales of dairy calves into the beef industry for fattening. Several farms were also found to have multiple different phylogenetically distinct BVDV strains, which suggests that they may have been infected from multiple different sources. Molecular sequencing may provide a valuable tool for helping farmers understand the origins of BVDV outbreaks. However, there were some limitations with the sampling and detection methods used. Future research directions were proposed in the discussion including broadening the study into a national survey with adequate representative samples. Finally, Chapter 4 provides a brief discussion of how the findings from this work can be used to assist New Zealand in designing a more cost-effective national BVD control programme.Item Canine respiratory viruses in New Zealand dogs : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2019) More, Gauri DilipAcute infectious tracheobronchitis (ITB) is an important health issue in dogs worldwide. It predominantly affects kenneled dogs but has also been described in pet dogs. The aetiology of the disease is complex and likely to involve various respiratory pathogens, as well as host- and environment-related factors. Traditionally, canine parainfluenza virus (CPiV), canine adenovirus type 2 (CAdV-2), and Bordetella bronchiseptica were regarded as most commonly involved in canine ITB, and are incorporated into available vaccines. Other bacterial species as well as canine herpesvirus type 1 (CHV-1), canine reoviruses, and canine distemper virus have also been detected in the samples from diseased dogs. Within the past two decades, several novel canine respiratory viruses that may contribute to canine ITB have been discovered. These include canine influenza viruses, canine respiratory coronavirus (CRCoV), canine pneumovirus CnPnV), canine bocavirus, and canine hepacivirus. It appears that the contribution of various respiratory pathogens to canine ITB differs between different geographical locations and hence, the availability of local data is important for the implementation of the most appropriate disease prevention strategies. To date, very little data are available on the respiratory pathogens circulating among dogs in New Zealand. As such, the objective of the present study was to identify what viruses circulate among local dog populations including determination of the presence/absence of the recently identified canine respiratory viruses and to determine which of these pathogens are likely to be aetiologically involved in canine ITB. The second objective was to characterise at the molecular level selected novel viruses identified. In order to identify viruses associated with canine ITB, metagenomic shot-gun sequencing approach was used to determine what viral nucleic acids were present in the pooled samples prepared from oropharyngeal swabs collected from dogs with clinical signs of ITB (n = 50) and from healthy dogs (n = 50). Following shot-gun sequencing, assembly and mapping, sequences of CHV-1, CRCoV, CnPnV, canine picornavirus and influenza C virus were identified in the pooled sample from dogs with ITB, while none of these sequences were identified in the pooled sample from healthy dogs. This is the first molecular identification of CnPnV, CRCoV, CanPV and influenza C virus in the New Zealand dog population. Real-time PCR assays were then designed to assess the frequency of detection of five canine respiratory viruses (CPiV, CAdV-2, CHV-1, CRCoV and CnPnV) in individual oropharyngeal swab samples from dogs with signs of ITB and from healthy dogs. Infections with at least one canine respiratory virus were more commonly detected in dogs with signs of ITB (21/56 (37.50 %)) than in healthy dogs (15/60 (25.00 %)). Dogs with signs of ITB were most commonly positive for CnPnV (26.78 %) followed by CAdV-2 (8.92 %), CPiV (3.57 %), CHV-1 (3.57 %), and CRCoV (1.78 %). Only CnPnV (23.33 %) and CAdV-2 (5.00 %) were identified in samples from healthy dogs. The overall prevalence of CnPnV in the sampled population was 29/116 (25.00 %). This research revealed the first molecular evidence for the presence of CRCoV and CnPnV in dogs in New Zealand. As such, these viral sequences were further analyzed. The attachment gene (G) of three CnPnVs (CnPnV NZ-007, CnPnV NZ-048 and CnPnV NZ-049) was sequenced to characterise CnPnV circulating among dogs in New Zealand. Sequence analysis of the CnPnV G gene revealed that both group A and group B subtypes of CnPnV circulate in New Zealand. The genetic analysis of the 3‘ genomic region of CRCoV from New Zealand (CRCoV NZ-046/16) revealed closer relation to the British CRCoV 4182, Italian CRCoV 240/05 and Chinese CRCoV BJ232 than to the Korean CRCoVs (K9, K37 and K39). A deletion of one nucleotide at the region between the genes encoding the spike protein and 12.8 kDa accessory protein in CRCoV NZ-046/16 resulted in deletion of a stop codon with subsequent translation of predicted 5.9 kDa and 2.7 kDa proteins instead of two accessory proteins (4.9 kDa and 2.7 kDa) encoded at that region by most other CRCoVs. In order to gain some insight into the epidemiology of CRCoV among dogs in New Zealand, canine sera (n = 100) were randomly selected from diagnostic laboratory submissions on a monthly basis from March to December 2014, and analyzed for the presence of CRCoV antibodies using a commercial blocking ELISA with bovine coronavirus antigen. Overall, 53 % of 1015 sera tested were positive for CRCoV antibody. The present study revealed an increase in the prevalence of CRCoV antibodies with age (p = 0.014). The work presented in this thesis has contributed to our understanding of the viruses involved in canine respiratory disease in New Zealand. Although infections with CPiV, CAdV-2, and CHV-1 were detected, it appeared that recently discovered canine respiratory viruses, including CRCoV and CnPnV, may also play an important role in canine ITB in New Zealand. This may provide one explanation for the development of respiratory disease in some fully vaccinated dogs, as anecdotally reported by field veterinarians. If so, their potential role and local epidemiology should be investigated in future studies.