Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that mediate host-responses to pathogens. In mammals, TLR ligands promote cellular activation and the production of cytokines. Several TLR ligands have been employed prophylactically for the control of bacterial or viral diseases in the mouse model. However, the TLR-mediated responses in chickens have not been well described. Importantly, the utility of TLR agonists for the control of viral pathogens, such as avian influenza virus (AIV), has not been fully explored in chickens. To this end, the studies described in this thesis characterized the kinetics of in vivo responses in chickens to the TLR4 ligand lipopolysaccharide (LPS) and the TLR21 ligand CpG ODN. It was demonstrated that both of these ligands induced the up-regulation of several immune system genes in the spleen, including those associated with pro-inflammatory and antiviral responses, as well antigen presentation. By harnessing the immunostimulatory properties of TLR ligands, it was also demonstrated that the prophylactic administration of either poly I:C (a TLR3 ligand), LPS or CpG ODN may confer immunity to a low pathogenic avian influenza virus, as determined by a reduction in both oropharyngeal and cloacal virus shedding in infected birds. Furthermore, transcriptional analysis of genes in the spleen and lungs identified interleukin (IL)-8, interferon (IFN)-α and IFN-γ as correlates of immunity. In conclusion, TLR ligands may modulate several aspects of the chicken immune system to induce an anti-viral state, thereby conferring immunity to AIV.

Many countries, including Nepal, have been affected with highly pathogenic avian influenza (HPAI) outbreaks. There have been human mortalities in some countries and large numbers of poultry either died or were culled due to HPAI. The overall objective of this thesis was to improve our understanding of the epidemiology and economics of avian influenza (AI), and particularly HPAI, in Nepal. We determined the seroprevalence of and risk factors for AI virus antibodies presence in ducks in Kathmandu, Nepal. The estimated true prevalence of AI viruses (AIV) antibodies was 27.2% [95% Confidence Interval (CI): 24.6- 29.5]. Age of the ducks was identified as the only risk factor for AIV seropositivity. Ducks older than one year were more likely to be seropositive compared to ducks less than six months of age [Odds Ratio= 2.17 (95% CI: 1.07- 4.39)]. This study provided baseline information about seroprevalence of AIVs in Kathmandu that will benefit further research to differentiate the subtypes of AIVs circulating in Kathmandu. We also evaluated alternatives to the current control program (CCP) for HPAI in Nepal. The considered alternatives were: (i) absence of control measures (ACM) and (ii) vaccinating 60% of the domestic poultry flock twice per year. Cost-benefit analysis approach was used to evaluate the economic feasibility of the programs. In terms of the benefit-cost ratio, our findings indicated that there is a return of 1.96 dollars for every dollar spent in the CCP compared to ACM. The net present value of the CCP versus ACM was US$ 989,918. The vaccination program yielded a return of 2.41 dollars for every dollar spent when compared to the CCP. The net present value of vaccination versus implementing the CCP was US$ 13,745,454. These results support a continued investment into the CCP rather than ceasing to implement government regulated control measures and suggest that vaccination may be an even better control alternative. In summary, our studies have highlighted the value of epidemiologic and economic analysis in research of AI. Our results are expected to lead to an improved understanding and awareness of AI in Nepal and to formulation of better control strategies.

Those who are interested in the public mood, including politicians and economists, comment that the public are becoming ever more sceptical about many things, but health risk information should not be one of them. If health risk information is perceived by the public as ‘just another scary story’, or ‘more of the same we heard last month’, then the ability of risk messages to convey urgency and recommend action could be greatly diminished; the ‘cry wolf’ scenario becomes more real every time a threat appears in the media but fails to materialise. This thesis explores how avian influenza, (H₅N₁), as a health risk category, has been reported and represented in the New Zealand media. By analysing avian influenza-related items in four New Zealand newspapers over a six-year period, 2002-2008, and by comparing results with those found in a U.S. study by Dudo, Dahlstrom & Brossard (2007), this thesis explores the dominant themes and discourses the media drew upon when reporting the health threat of avian influenza. In addition, data from four focus groups sessions was analysed for the purpose of exploring public perceptions of health risk messages and the influence of the media on those perceptions. This thesis was situated within a constructionist epistemology, and employed a mixed-methods methodology with content, thematic and textual analyses. Risk communication theories and models, media conventions of agenda-setting and framing, and sociological concepts informed how the topic of health risk communication was operationalised. The analysis of the focus group data explored how the participants discussed the threat of H₅N₁; how they constructed concepts of personal and community risk, what role, if any, they attributed to the media in their construction and how they positioned themselves in regards to illness and contagion. The focus group analysis revealed that three dominant themes - risk, media and ‘othering’ – represented how the focus group participants talked about the risk of avian influenza. These and several sub-dominant themes shared similarities to those found in the newspaper analysis. Whilst initial discussions seemed to indicate a nonchalant attitude towards the risk of avian influenza, the many topics and themes that characterised the way the participants discussed the risk between them, showed that they had thought about the personal consequences of a possible health risk, and had formed strong opinions about many facets of that risk. Results from the newspaper analysis largely mirrored those of the above U.S. study, and showed that the New Zealand media favoured episodic over thematic framing; sensationalising the reporting of avian influenza, whilst providing little in the way of scientific and contextual information. Moreover, the analysis showed that, when reporting health risks, media templates are well established. The analysis of the focus group data revealed that the participants wanted media health risk messages to be clear, concrete and factual. However, this desire for messages that communicate certainty about risk, which is, by definition inherently uncertain, raises questions about the very nature of risk communication. Findings of this thesis suggest that future risk communication research should focus, not on how the media are reporting health risks, but how the public conceptualise risk, construct it in times of crisis and evaluate their ability to control it.

No description available.

Surveillance of domestic poultry flocks for antibodies against avian influenza and Newcastle disease to detect and differentiate between these diseases is very important. The ability to determine if the detected influenza virus antibodies belong to one of the reportable H5 or H7 subtypes is imperative. These two major viruses are continually responsible for economic loss in poultry industries all over the world. Current serological methods of detection are an effective means of detecting antibody responses to these viruses, however continually investigating improved methods of surveillance is important. Development of a serological assay using Luminex technology which involves the use of recombinantly generated influenza A nucleoprotein, hemagglutinin H5, hemagglutinin H7, and Newcastle disease nucleocapsid proteins bound to Magplex beads allowed for the simultaneous detection of antibodies against these proteins that matches the efficiency of past methods while maintaining high levels of specificity and overall accuracy. Assay development took the form of two connected projects beginning with construction of an assay that operated in duplex, detecting antibodies against influenza nucleoprotein (AIV-NP) and Newcastle disease nucleocapsid protein (APMV-1-NC). Once optimized, the second half of development involved expansion of the assay to include detection of H5 (AIV-H5) and H7 (AIV-H7) subtypes, as well as the addition of internal assay quality controls to monitor assay performance over time. Assay thresholds and overall performance of both of these functional assays were evaluated using large quantities of field and experimental sera from chickens and turkeys to maximize specificity and overall accuracy.

Genetic immunization, also known as DNA or polynucleotide immunisation, is well documented to induce broad-based immunity in various animal models of infectious and non-infectious diseases. However, the low potency of DNA vaccines has to date precluded the development of commercial vaccines. The aim of this study was to systematically investigate a number of parameters to improve the potency of DNA vaccines for use in chickens, using a low pathogenic avian influenza (LPAI) virus as a proof-of-concept for their ability to produce a humoral immune response. The index virus used in the study was avian influenza virus A/coot/WA/2727/79 (H6N2), isolated from an apparently healthy Eurasian coot in 1979. Prior to any DNA experiments the virus was rigorously characterized. The virus strain was shown to be an H6 subtype by haemaglutination inhibition (HI) testing and as an N2 subtype by gene sequence analysis. The isolate was shown to be able to grow on MDCK cells in the absence of exogenous trypsin. It was further biologically characterized as LPAI with an intravenous pathogenicity index (IVPI) of 0.15 and a motif of 321PQAETRG328 at the cleavage site of the haemagglutinin (HA) protein. It was capable of infecting domestic chickens under experimental conditions with a low level of virus excretion via the cloaca and oropharynx following intravenous or oral and oculonasal inoculation. The full-length HA and nucleoprotein (NP) genes of this H6N2 virus were subsequently cloned into the eukaryotic expression vector VR1012 to generate VR-HA and VR-NP constructs. Six-week-old Hy-Line chickens were intramuscularly injected with either the VR-HA or VR-NP vaccine at different dose rates, with or without lipofectin as adjuvant. Minimal or no detectable antibody was produced, as measured by HI, ELISA and Western blotting-based assay, but high titres of H6-specific HI antibodies appeared 10 days after homologous virus challenge. In contrast to the empty vector controls, there was a significant difference in HI antibody titre between pre- and post-challenge in vaccinated birds, indicating some evidence for the priming effect of the DNA vaccines. Using the frequency of virus shedding as an indicator of protection, lower doses (50 or 100 ¦Ìg per chicken) of either adjuvanted VR-HA or VR-NP vaccine significantly reduced virus shedding in oropharyngeal and cloacal swabs compared to higher doses (300 or 500 ¦Ìg per chicken ) or empty vector control chickens. Although two vaccinations with naked VR-HA alone were not sufficient to induce an effective immune response against a homologous virus challenge, further repeat vaccinations and incorporation of adjuvant did lead to the generation of low to moderate HI antibody titres in some chickens and resulted in no or reduced virus shedding after challenge. Next, to examine the effect of expression vector, three different DNA vectors, pCI, pCI-neo and pVAX1 were used to clone the same HA gene and generate three DNA vaccine constructs. Once again, direct intramuscular injection of the three DNA constructs did not elicit measurable H6-specific HA antibody response in Hy-Line chickens but the 100 µg pCI-HA lipofectin adjuvanted vaccine group showed a significant increase in post-challenge HI titres from the naive control group, indicating that an anamnestic antibody response had been induced by the pCI-HA DNA vaccination. Compared with the controls, the three DNA constructs showed significantly reduced virus shedding in cloacal swabs post virus challenge, suggesting that the three DNA vaccines induced some level of immune response in vaccinated chickens. As with the VR-HA construct, the lower dose groups for each vaccine (50 or 100 g) were more effective at reducing virus shedding from the cloaca than the higher dose group (300 g). To further investigate why the DNA vaccines did not elicit a measurable antibody response, the HA gene incorporating a Kozak enhancer sequence was cloned into an alternative expression vector, pCAGGS, to produce the pCAG-HAk construct. Three-week-old SPF chickens were immunized with this construct either by the intramuscular route (IM) or electroporation (EP). H6 HI antibodies were present in some chickens by 3 weeks after the first IM vaccination and 75% of the chickens vaccinated with 10, 100 or 300 µg pCAG-HAk were antibody positive by 2 weeks after the second IM vaccination. For EP immunization, 87.5% of vaccinated birds seroconverted after the first vaccination and 100% seroconverted after the second vaccination and the H6 HI antibody titres were significantly higher than for chickens vaccinated by IM inoculation. Another group was given a single dose IM vaccination with 100 µg of the pCAG-HAk construct and showed a maximum sero-conversion rate of 53.3% with a peak H6 HI titre of 27 at 5 weeks post-vaccination. This demonstrated that optimization of the expression vector and insertion of a Kozak sequence could synergistically enhance expression of the H6 HA gene and result in a measurable H6 antibody response in SPF chickens. EP was also compared with IM inoculation with the 100 g pCI-HA construct in SPF chickens, resulting in a 50% sero-conversion rate and mean HI titre of 21.3 at 2 weeks after the second vaccination by EP. By comparison, only 25% chickens had trace HI titres by IM inoculation. This indicated that EP was more efficient than IM delivery for both constructs. A codon-optimized complete HA gene from A/coot/WA/2727/79 (H6N2) was then chemically synthesized and cloned into a pCAGGS vector to generate the pCAG-optiHAk construct. SPF chickens immunized twice with either 10 µg or 100 µg of pCAG-optHA showed 37.5% and 87.5% sero-conversion rates respectively, with a mean H6 HI tire of 21.4 and 22.6 at 3 weeks after the second immunization, but the differences were not statistically significant. There were also no significant differences in either the sero-conversion rate or the H6 HI titre between the pCAG-HAk and pCAG-optiHAk groups, suggesting that a codon-optimized HA DNA vaccine did not achieve significantly better immunogenicity than the pCAG-HAk vaccine. In vitro expression of the developed DNA constructs in chicken-, hamster-, monkey- and human-origin cells, as measured by Western blotting and immunofluorescence testing (IFT), showed the strength of H6 HA expression in the following descending order - pCAG-optiHAk/pCAG-HAk, pCI-HAk, VR-HA, pCI-HA, pCIneo-HA and pVAX-HA. The in vivo chicken vaccinations also showed that the pCI-HA construct was more effective than the pCI-neo-HA, and that the pCAG-optiHA or pCAG-HAk constructs were better than pCI-HAk in term of reduction in virus shedding after H6N2 virus challenge. Thus, in vitro HA gene expression directly correlated with the generation of immune responses in vivo, indicating that in vitro studies can be used for pre-selection of expression plasmids prior to development of avian influenza DNA vaccines. Lipofectin as a chemical adjuvant was shown to enhance the DNA-induced immune response but is prohibitively expensive for routine use in poultry vaccines. Thus, an experimental adjuvant for poultry DNA vaccines (Essai) and a new nanoparticle (Phema) adjuvant used for the first time in poultry were compared with conventional aluminum salts (alum) adjuvant in the present study. No HI antibody was detected in any adjuvant-vaccinated Hy-Line chickens following two immunizations. However, in comparison with the naive control group, the alum- and Phema adjuvanted pCAG-HAk groups significantly reduced the frequency of virus shedding in oropharyngeal swabs, but Essai adjuvant was not effective in augmenting the pCAG-HAk vaccine efficacy. This pilot study also emphasised that the traditional aluminum hydroxide adjuvant, either DNA binding or non-binding, may be useful as an adjuvant for enhancing DNA-induced immune responses in chickens owing to its low price and safety record. Overall, DNA immunization with various HA-expressing constructs was shown to be variably effective in inducing immune responses in chickens. The efficacy of DNA vaccines could be synergistically improved by taking appropriate approaches. With continuing research DNA vaccines have the potential to become an important tool for disease prevention and control.

The poultry industry occupies an important sector in Thai agriculture. The "Bird Flu" or "Avian Influenza (AI)" crisis, which first commenced in Thailand in 2004, has caused many problems. It has devastated the Thai chicken industry, the worlds fourth largest, with exports worth $A1.93 billion annually and employing hundreds of thousands of people. The European Union, the number two buyer of Thai chicken, and Japan, Thailands biggest customer, has banned imports of Thai chicken. / Locally, the marketing of chicken meat has posed a major economic problem because of a substantial fall in consumption. Many Thais, who learned about bird flu from the newspapers, have avoided buying and eating poultry as a simple precaution. Other Thais, who have continued to buy and consume fresh chicken meat, are doing so with caution. / This study attempted to examine perception factors and risk reduction strategies influencing consumers decisions to buy fresh chicken meat during a food risk situation in Thailand. In addition, an evaluation is made as to whether there is a difference between consumers perceptions and their purchasing decisions under specified demographic factors. / The current study focused on two key variables; namely, consumer perception factors and risk reduction strategies and their influence on Thai consumers purchasing decisions for fresh chicken meat. The four perception factors were 1) price, 2) product quality, 3) place, and 4) risk, based on studies by Schiffman and Kanuk (1997); Glitsch (2000), and Yeung and Morris (2001a). The risk reduction strategies, employed in this study, taken from previous literature (Glitsch, 2000); and Yeung and Morris, 2001b), include:1) purchasing reputable brands, 2) government control or guarantee labels, 3) purchasing from reliable outlets, 4) traceability, 5) avoiding “cheap prices”, and 6) personal controls. There is a long tradition for using demographic variables to explain the observed differences in consumption and food intake surveys. In this study, the demographic variables examined were age, gender, marital status, education, occupation, family size, and income. / Because the recognition of situational effects is an important guide to understanding and predicting consumer behaviour, the current study evaluated purchasing decisions in three situations: 1) in the current situation, do Thai consumers buy fresh chicken meat for consumption? 2) during the first bird flu crisis when there was a lot of adverse press on the effects of bird flu, did Thai consumers buy chicken meat for consumption? 3) in the future, if there is another bird flu crisis of similar severity, will Thai consumers be likely to buy chicken meat for consumption? / The research design was descriptive with the survey method used to gather data from the target respondents. Data was collected in six of Bangkoks largest markets, each located in a district with a population of approximately one million people. A total of 70 questionnaires were distributed in each of the six markets and 400 valid questionnaires were used to analyse the data. / The results showed that all four of the consumer perception factors, namely: price, product quality, place of purchase, and risk perception, influence purchasing decisions in the three different situations, in different ways. From the four perception factors, product quality had the highest influence on consumers purchasing decision, followed by risk and price perception. Place of purchase did not really influence their purchasing decision in the current situation. / Thesis (DBA(DoctorateofBusinessAdministration))--University of South Australia, 2007.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 194-237) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 194-237) Also available online.