Veterinary Science Collected Works - Theses

Permanent URI for this collection

Search Results

Now showing 1 - 4 of 4
  • Item
    Thumbnail Image
    Novel platforms for full genomic characterisation of avian pathogens directly from clinical samples
    Asif, Kinza ( 2021)
    Identification of the microbial strains involved in infectious disease is imperative for epidemiological investigation and to implement control strategies using vaccination. The conventional techniques used for strain identification including microneutralisation assays and polymerase chain reaction (PCR)-based assays such as PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-High resolution melt (PCR-HRM) curve analysis are not only cumbersome but also their results are difficult to interpret. Analysis of the whole genomes of the pathogens is considered the gold standard for the characterisation of strains. However, whole-genome sequencing (WGS) requires the in vitro isolation of the pathogens which can potentially introduce variations in the genome. In addition, some pathogens are not cultivable in vitro. Therefore, WGS directly from clinical tissues would be the most suitable option. In this study, four viral pathogens namely Fowl adenovirus (FAdV), avian hepatitis E virus (aHEV), fowlpox virus (FPV), and infectious laryngotracheitis virus (ITLV) affecting the hepatic, cutaneous, and respiratory tissues were targeted in four independent studies to assess the suitability of WGS directly from clinical tissues using Illumina and Nanopore sequencing platforms. To extract sequencing grade viral DNA/RNA directly from hepatic tissues, the liver homogenate was treated with 5% kaolin hydrated aluminium silicate to remove excess lipid tissue present in the liver before proceeding to the phenol-chloroform extraction. FAdV DNA extracted from treated tissue, resulted in the complete genome assembly of FAdV using both Illumina and Nanopore sequencing platforms. A similar extraction technique was used to extract aHEV RNA directly from liver tissues followed by long range RT-PCR and sequencing. . Analysis of the resultant WGS from clinical samples revealed that Australian aHEV isolates had emerged as a result of recombination between the US and European strains possibly following the importation of poultry into Australia and dissemination through vertical transmission. To evaluate the suitability of WGS directly from cutaneous tissues affected with FPV, homogenate of the affected comb tissue was subjected to DNA extraction and sequencing. The sequencing results were compared with the same FPV isolate grown in chorioallantoic membranes (CAMs). Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar, but not identical to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of SNPs in the two sequences when compared to the reference genome, essentially classifying the two sequences as the same strain, but also highlighting the impact of in vitro passage on WGS of viral pathogens. Sequencing the whole genome of ILTV was attempted directly from tracheal scrapings of experimentally infected birds to circumvent in vitro culturing. Despite the high number of quality reads obtained from sequencing, assembling the genome was not possible due to poor overlapping sequences and the presence of multiple gaps. A concatenated sequence covering 91% of the ILTV genome was obtained after excluding the regions with low coverage. Further analysis performed on the concatenated genome classified the ILTV isolate as the same class used for infection of the birds (class 9) but revealed the presence of 50 single nucleotide polymorphisms (SNPs) between the two. The results of this study suggest that despite the failure to assemble ILTV genome directly from clinical tissues, the technique has a potential to replace the current PCR-HRM technique used for ILTV typing since it provides far more detailed information about the genome of the ILTV. Thus, the studies reported in this thesis have served as a proof of concept and have contributed to the evaluation of the suitability of WGS as a tool for accurate strain identification directly from clinical specimens of these pathogens. Also, this thesis provided insights into the origin of aHEV isolates currently circulating in Australia.
  • Item
    Thumbnail Image
    Genomic recombination of infectious laryngotracheitis virus (ILTV) vaccines
    Fakhri, Omid ( 2020)
    Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens. This disease causes economic loss in poultry industries worldwide and is a major concern for animal health and welfare. Although, ILTV vaccines are in use to control disease, biosecurity breaches and continuous evolution of the ILTV genome make outbreak prevention difficult. Genomic recombination plays a role in diversification of herpesvirus genomes and has been suggested to be an important alternative evolutionary mechanism in herpesviruses such as ILTV. In previous studies of ILTV, two new genotypes of virulent field strains were shown to be independent recombinants derived from distinct attenuated commercial ILTV vaccines. These new strains became the dominant field viruses responsible for widespread and severe disease outbreaks in Australian poultry flocks. This project aimed to determine the frequency of these recombination events and the conditions that can facilitate recombination using in vitro, in ovo and in vivo studies. In these series of studies, a high-throughput and cost-effective method for detection of recombinant ILTVs was developed and was used to analyse the viruses generated under multiple infection conditions. The results from these analyses indicated that genomic recombination between vaccine strains of ILTV is a frequent event and takes place under a broad range of conditions. De novo mutation was detected in addition to genomic recombination. The capacity of ILTV vaccines to allow secondary infection by another ILTV vaccine (i.e. superinfection) was investigated at both cellular and natural host levels. The result of this investigation showed that superinfection can occur after administration of ILTV vaccines, providing the virus with ample opportunity for genomic recombination. The outcomes of these studies include advances in fundamental understanding about the factors that lead to ILTV recombination.
  • Item
    Thumbnail Image
    Tissue tropism and latency of infectious laryngotracheitis virus: A study in the natural host
    Thilakarathne, Dulari Samanthika ( 2019)
    Herpesviruses are evolutionarily successful pathogens that infect a large number of animal species. This success is partly attributed to their capacity to establish latency in the host. The avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) causes an acute upper respiratory tract infection in chickens that has considerable impacts on world poultry economy and welfare. The disease caused by ILTV, infectious laryngotracheitis (ILT), is currently controlled by vaccination principally with live attenuated vaccines. Limitations associated with live attenuated vaccines, including the ability to establish latency, may provide avenues for the emergence of novel, more virulent, recombinant strains of ILTV, further complicating the epidemiology of ILTV. In this project, a sensitive nested polymerase chain reaction (NPCR) protocol was developed and the sensitivity of this assay was compared with that of other commonly used PCRs in ILTV research. A trigeminal ganglia (TG) co-culture system was established and optimised and a tracheal co-culture system was reproduced to study in vitro reactivation of latent ILTV. A genotyping system based on allelic variations in multiple genomic regions of ILTV to discriminate ILTV strains prevalent in Victoria, Australia, is also described. These methodologies revealed that a large proportion of the ILTV-vaccinated birds in a commercial layer flock, close to the end of their productive laying period, were shedding multiple vaccine strains of ILTV in the upper respiratory tract, presumably due to reactivation of latent infection. Further, co-culture systems showed in vitro reactivation of latent ILTV in TG and trachea of these birds. The capacity of four vaccine strains of ILTV (SA2, A20, Serva and a glycoprotein G deleted mutant vaccine candidate) to establish latency in specific pathogen free chicken (SPF) following eye-drop vaccination was investigated in vivo. This study revealed ILTV vaccines differed in their capacities to establish latency in TG, and also showed that nearly half of the population had detectable ILTV in their upper respiratory tract (URT), 21 days post vaccination, possibly due to reactivation of infection. A second in vivo experiment was performed to study latency characteristics and late systemic lymphocyte responses in SPF chickens following intratracheal inoculation with a vaccine ILTV (SA2) or a virulent field ILTV (class 9; CL9) strain. Results from this study indicated that latency characteristics did not significantly differ between these strains at 21 days post inoculation (dpi) or at 35 dpi, and suggested that the trachea may be a more significant site of latency and reactivation than the TG. Moreover, regardless of the ILTV strain inoculated, SPF birds showed lymphocytosis during the latent stage of infection. Additionally, tissue tropism of two newly emerged recombinant strains of ILTV (CL9 and class 10; CL10) was investigated using commercial broiler and SPF chickens. The possibility of using feathers as a diagnostic sample was explored. This study revealed that both CL9 and CL10 ILTV strains caused severe disease in both types of birds, distributed to visceral organs and persisted for up to 14 dpi in URT. The NPCR developed in this project detected ILTV DNA in feathers of infected broiler and SPF birds at 14 dpi. Taken together, these studies have shown that tissue tropism and latency is a complex area of research and to a large extent these properties are strain dependent. The studies reported in this thesis have enriched the literature on tissue tropism and latency of ILTV. The results will be valuable for future latency studies and for selection of vaccines to control ILTV.
  • Item
    Thumbnail Image
    Chicken immune responses to infectious laryngotracheitis virus (ILTV) glycoproteins
    Sabir, Ahmad Jawad ( 2019)
    Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease, primarily infecting the upper respiratory tract and conjunctiva. The disease results in significant economic losses to the poultry industry worldwide. Currently, the immune status of the vaccinated flocks or individuals is assessed by the presence of systemic antibodies using commercially available whole virus ELISAs. These ELISAs are good indicators of exposure to either field or vaccine virus, however, the assays are poor in predictors of the level of protective immunity. In this study, individual ILTV glycoproteins (gC, gD, gE, gG, gI and gJ) were assessed for their potential to predict the levels of protective antibody and/or cell-mediated immunity as well as for their capacity to induce neutralising antibodies. To examine whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, individual glycoproteins expressed in mammalian cells were used in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation between systemic antibodies to individual glycoproteins and protection. The possibility that the protective epitopes of glycoproteins may not be readily detectable in ELISA was investigated by evaluation of the antibodies to each glycoprotein in an in-vitro virus neutralisation assay. Monospecific polyclonal antibodies to individual ILTV glycoproteins gC, gD, gE, gI and gJ were generated in rats and examined for their capacity to neutralise the virus. Neutralising antibodies were detected to gC, gD and gJ individually or in combination. Polyclonal antibodies to gE and gI failed to neutralise the virus when tested individually or combined. In-vitro splenocyte stimulation assays using individual glycoprotein stimulating antigen were conducted to examine the transcription profiles of selected cytokines from nonvaccinated-nonchallenged (NVXNCh), vaccinated-challenged (VXCh) and nonvaccinated-challenged (NVXCh) groups of chickens. The transcription profiles of cytokines referencing Th1, Th2 and Th17 responses were then examined against tracheal pathology results to identify correlation between responses against viral glycoprotein(s) and protective immune response. Expression of IFN-gamma was significantly upregulated in VXCh and NVXCh groups of birds in response to stimulation with gD, with expression levels moderately correlated with protection. Stimulation with gE and gI resulted in significantly higher levels of IL-6 in VXCh birds and response to gE was moderately correlated with protection. Expression of IL-17a was significantly elevated in response to stimulation with gD in NVXCh birds and results were moderately linked to disease severity. The results of this study suggest that IFN-gamma and IL-6 cytokine responses to gD and gE, respectively, may be correlated with protective cell-mediated immunity, whereas IL-17a cytokine responses to gD may be linked to disease. Finally, whole-genome analysis of an unusually virulent “vaccine-like” isolate revealed a new class of ILTV, identified here as class 7b, emerged as a result of recombination probably between another recombinant strain and a vaccine strain. Further analysis of the genome detected recombination hotspots within the unique long (UL) region comprising of genes encoding gB, gC and gM and assembly proteins UL28 and ICP18.5. Boot-scanning analysis of concatenated glycoprotein sequence alignments detected recombination breakpoints within glycoproteins C and H, but not within glycoproteins located in the unique short (US) region. Further studies would be needed to explore the possible role of these glycoproteins in virulence. Thus, the studies reported in this thesis have contributed to understanding (1) the relationship between antibodies to ILTV glycoproteins and protective immunity, (2) virus neutralisation potential of specific antibodies to ILTV glycoproteins, (3) the potential role of ILTV glycoproteins to induce cell-mediated protective immunity, (4) and laid foundation to explore inter-strain genetic diversity within ILTV glycoproteins.