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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 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.Item Frequency of latent equine herpesvirus 1 (EHV-1) in New Zealand horses : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies at Massey University, Palmerston North, New Zealand(Massey University, 2019) Bueno, Maria Cristina PantojaAIM: To estimate the frequency of infection with equine herpesvirus 1 among a selected population of horses from the central North Island of New Zealand, including determination of the open reading frame (ORF) 30 N/D752 genotype. METHODS: Fresh heads were collected from horses euthanised for unrelated reasons between March and November 2015. Small pieces of retropharyngeal lymph nodes (RLN) and submandibular lymph nodes (SLN) were dissected from the heads and transported to the laboratory in RNA later solution. DNA extracted from these tissues was subjected to enrichment for EHV-1 sequences by hybridisation with biotin-labelled EHV-1 specific probe, followed by recovery of EHV-1 sequences on streptavidin-coated magnetic beads. The enriched samples were tested for the presence of EHV-1 using nested PCR. The EHV-1 amplicons were sequenced to determine the ORF30 genotype of the virus. RESULTS: Overall, EHV-1 DNA was detected in RLN samples from 6/63 (9.5%) horses. Of those, three were also positive for EHV-1 DNA in SLN samples. There was no association between EHV-1 positivity and age, sex, or breed of the animals sampled. All EHV-1 positive horses harboured ORF30 N752 genotype. The D752 genotype, which has been linked to increased neurovirulence, has not been detected in any of the samples. CONCLUSION: Equine herpesvirus 1 continues to circulate among horses in New Zealand. The RLN appear to be the sample of choice for detection of EHV-1 DNA in a recently euthanised horse. The frequency of latent EHV-1 infection among sampled horses may have been higher than detected, as some of latently infected horses may have harboured EHV-1 DNA at the levels beyond the sensitivity limit of the assay or at anatomical sites not sampled in the study. Lack of detection of EHV-1 with ORF30 D752 genotype, together with detection of only one horse positive for that genotype in the previous South Island based study (Dunowska et al., 2015) suggest that infection with this genotype is not common in New Zealand. CLINICAL RELEVANCE: If live animals are tested using SLN biopsy it should be kept in mind that negative results do not rule-out the presence of latent EHV-1 at other sites inaccessible for testing. While EHV-1 with ORF30 D752 genotype was not detected in this study, the importance of this genotype should not be over-interpreted because the markers for EHV-1 neurovirulence are most likely more complex than this single amino acid substitution. Viruses with either genotype have been recovered from equine herpesvirus encephalopathy cases worldwide. The data presented provided baseline information on the frequency of EHV-1 infection among horses in New Zealand. These can provide useful information during any future outbreaks of EHV-1 associated diseases and for the development of control measures to minimise the impact of such viral disease for horses and their owners.Item The biology of avipoxvirus in New Zealand avifauna : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology at Massey University, Palmerston North, New Zealand(Massey University, 2013) Ha, Hye JeongAvipoxvirus (APV) infection is a highly contagious disease of birds which is comparable to poxvirus infections in various mammalian species, including smallpox in humans. The infection has been reported in more than 200 bird species, affecting both domesticated and free-ranging birds around the world. The disease is associated with economic loss in the poultry industry and is implicated with the decline in biodiversity in free-ranging birds, particularly in island ecosystems. This study was the first investigation into APV infection in New Zealand free-ranging birds. The initial focus of this study was the phylogenetic analysis of APV in New Zealand. Avipoxvirus antibody was then detected using enzyme-linked immunosorbent assay (ELISA) in several introduced species and an endemic passerine species in New Zealand. The pathogenicity of two major APV strains isolated from New Zealand birds was evaluated and the safety and efficacy of a commercial fowlpox (FWPV) vaccine was investigated in a model passerine species. This study confirms that various New Zealand birds including endangered species are susceptible to APV infection and that at least three different strains of APV are present in New Zealand, with overlaps in the geographic distributions between different strains. The results suggest that APV had been introduced to New Zealand through avian hosts, insect vectors or human intervention such as poultry vaccination. A high seroprevalence to APV has been observed in introduced and an endemic bird species in New Zealand, confirming that the virus is well established. A significant relationship between birds seropositive to APV and the ones positive to Plasmodium spp. has also been observed, both of which are known to be pathogens responsible for dramatic declines in island bird populations. Two major New Zealand APV strains isolated from clinical cases were pathogenic in Zebra finches (Taeniopygia guttata), which we used as a model passerine species. A commercial FWPV vaccine was safe and effective in our model species against New Zealand APV isolates and I conclude that vaccination of native passerine birds using the FWPV vaccine could be an effective tool to reduce APV mortality, particularly in endangered species.Item Equine respiratory viruses in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Veterinary Studies in Virology at Massey University, Turitea, Palmerston North, New Zealand(Massey University, 2011) McBrearty, Kaylyn AliceEquine respiratory disease has been recognised as an important cause of wastage resulting in financial loss for the equine industry worldwide. Limited studies have been conducted on equine respiratory viruses in New Zealand, particularly within the past ten years. As such, the objective of the present study was to determine 1) which respiratory viruses circulate among horses from selected New Zealand locations and 2) whether or not infection with any of the viruses identified was associated with clinical disease. A survey was conducted on 85 horses to detect the presence of viruses known to be associated with equine respiratory disease. Nasal swabs were taken from 52 horses with signs of respiratory disease and from 33 healthy horses. Horses were sampled from within the Manawatu and Hawkes Bay regions by convenience. Species specific PCR was performed directly on nasal swabs. The only viruses detected were equine herpesviruses (EHV) types 1, 2, 4 and 5. Of the 52 horses with respiratory disease, 3 tested positive for EHV-1, 14 for EHV-4, 23 for EHV-2 and 26 tested positive for EHV-5. Of the 33 healthy horses 2 tested positive for EHV-2, one of which also tested positive for EHV-5. Over all, the detection of herpesviruses was significantly associated with respiratory disease (p value <0.0001). Detection of individual virus species (EHV-2, EHV-4 or EHV-5) was also significantly associated with respiratory disease (p value 0.0002, 0.0006, <0.0001, respectively). The sample size was not large enough to evaluate the significance of EHV-1 detection and respiratory disease. Virus isolation performed on the samples from the 52 horses with respiratory disease detected EHV types 1, 2, 4 and 5. No viruses were detected from the 33 samples of healthy horses. There was poor correlation between virus isolation and PCR results, particularly with regard to EHV-4. This work gives a recent contribution to the knowledge of equine respiratory viruses in New Zealand. Although the sampling was performed by convenience, the results suggest an association between equine herpesviruses types 2, 4 and 5 and equine respiratory disease.