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In the past decade, cross-species transmission of zoonotic pathogens from wild bird reservoirs has increased the emergence of new viruses, largely as a consequence of growth in intensive poultry farming and small free range holdings. The genetic diversity of viruses has most likely evolved in parallel leading to emergence of new virus variants. Early detection of such pathogens through active monitoring of their host reservoirs would greatly assist in early detection and implementation of control strategies. Current front line investigative tools face drawbacks with their wider availability or are limited in their scope to detect novel viruses. To address these shortcomings, a low cost, user-friendly avian viral array was developed in this study. The study applied novel probe design and target labelling approaches to increase sensitivity and accommodate detection of all known avian viruses in a miniaturised array. Sensitivity and specificity of the array was also evaluated using virus isolates and known clinical samples. In addition, the array was applied successfully to detect a novel avian reovirus in diseased swans. Furthermore, attempts were made to reduce running costs of an existing pan viral array by reducing reagent usage, turnaround time and re-use of microarray slides. The array was also assessed for its sensitivity and applied to investigate several viral disease outbreaks, where it was proven to be valuable in detecting viral co-infections in diseased pig lungs. Finally, next generation sequencing (NGS), as a sequence independent detection platform was applied for the detection and characterisation of a novel swan reovirus. In conclusion, the multiplex detection platforms described here attested to be valuable as front line investigative tools. However, the use of these platforms for the aforesaid purpose should also be considered alongside traditional detection assays to maximise capture of novel viruses.

No description available.

No description available.

No description available.

The present study has shown that age resistance to R. cesticillus, manifested by worm destrobilization, occurred in male chickens. In females, age resistance developed more rapidly than in males, until the birds were about 84 days old and from this age onwards the manifestations of resistance declined. The roles of immune responses, mucus secretion and host hormone balance in the development of age resistance to R. cesticillus were subsequently investigated. Host resistance to superinfection with R. cesticillus was demonstrated for the first time and this resistance was inhibited by the immunosuppressant dexamethasone. Further evidence for the immunogenicity of the cestode was provided by histological studies and by the demonstration of immunocytes and of ring precipitin and immunofluorescent antibodies. Experimental evidence suggested that these antibodies are not protective and it is probable the protective immune response is cellular. An in vitro culture system, described by Schiller (1970), kept young adult cestodes in an active condition for more than eight days, although growth was poor. Chicken intestinal mucus extracts were shown to be lethal to R.cesticillus in vitro and mucus from old birds appeared to have a greater effect than 'young' mucus. The effects of gonadal steroids on the course of infection of R. cesticillus were investigated by caponising male birds and implanting oestradiol or testosterone pellets. This procedure reproduced to some degree the patterns of infection of R. cesticillus observed in intact, untreated, mature birds. Scolex transplantation and the correlation of worm weights down the intestine with destrobilization strongly implicated intestinal environmental factors as being responsible for the phenomenon of worm destrobilization. The possible mechanisms whereby the immune response, mucus secretions and host hormone balance affect worm viability are discussed.

The primary cereal grain used in poultry feed worldwide is maize. An understanding of how and where broiler chickens digest native starches, this being the major energy component of maize, and the influence that exogenous xylanase has on this process, is essential when optimising nutrient utilisation of a diet. In this project, both in vitro and in vivo models were used to understand broiler digestion of starches in different maize-based diets: native maize starch, maize grit and maize-soy with or without xylanase (Econase XT 25 at 100 g/t; 18,100 BXU/kg). An in vitro model to simulate the digestive tract of broilers using a ball milling technique to mimic the chicken gizzard was designed to investigate differences in the digestion of starches between the various diets. Reduced starch crystallinity and particle size of the diet was shown when samples were ball milled in HCl-pepsin, compared to roller mixed samples, leading to a greater glucose release when these samples were incubated with pancreatin. The maize-soy diet gave the greatest glucose release with pancreatin, with native maize starch giving the lowest, indicating that the processing of diets prior to digestion as well as the surrounding food matrix can impact on the digestion of starches in feed. In all in vivo trials, starch fraction measurements and SEM imaging of digesta collected from the gizzard, upper and lower small intestine, revealed the progression of starch granule digestion through the tract. Despite birds being starved prior to feeding, broilers ingested little of the native maize starch diet. Nonetheless, predominantly intact granules were evident in the gizzard, with signs of amylase attack becoming apparent in the small intestine. Following starvation, starch granules were still observed throughout the tract of birds, suggesting the presence of resistant starches. Higher digesta pH values were observed when birds were fed a maize-soy diet which gave pH 3.56, 6.70, 7.70 for the gizzard, and upper and lower small intestine, respectively, compared to pH 2.47, 6.49, 7.34 for the maize grit diet, an effect that could potentially impact on digestive processes. In feeding trials with broilers, inclusion of xylanase into the maize-soy diet and feeding on a single occasion revealed no apparent difference in the matrices surrounding the starch granules in digesta. This suggested that xylanase may be working indirectly in vivo through some physiological change in the digestion mechanism rather than direct action on the feed. To test this hypothesis, birds were fed xylanase for different lengths of time before slaughter. Peptide YY concentration in the blood was higher during the first few weeks of supplementation, with longer periods of supplementation nulling this effect, suggesting that xylanase may be acting through a prebiotic mechanism. RT-qPCR results revealed a trend towards an increase in glucose transporter (GLUT2 and SGLT1) expression at 2 and 3 weeks of xylanase supplementation, respectively. This also correlated to an increase in glucose concentration in the blood from 3 weeks of xylanase inclusion, suggesting a greater absorption capacity of birds. These results may indicate a potential mechanism of xylanase action in maize-based diets, although further work is required to confirm these findings.

Campylobacter is the leading cause of bacterial food-borne diarrhoeal disease in the developed world and a significant cause of infant morbidity and mortality in developing countries. Epidemiological studies implicate poultry as a key source of infection, with up to 80% of human cases being attributable to the avian reservoir. An effective vaccine for broilers is predicted to limit the incidence of human campylobacteriosis. Vaccination of chickens with CjaA, either in recombinant form or vectored in live-attenuated Salmonella, has been reported to significantly reduce caecal colonisation by C. jejuni, with more invasive carriers eliciting greater protection. However, protection remains modest and is slow to develop. I therefore sought to improve such vaccines, first by vectoring codon-optimised CjaA in a licensed avian pathogenic E. coli ΔaroA vaccine. In two independent trials, White Leghorn birds were vaccinated subcutaneously on the day of hatch and 14 days later then challenged with C. jejuni M1 at 28 days post-hatch. No protection was observed despite significant induction of CjaA-specific serum IgY, however, a previously described S. Typhimurium ΔaroA vaccine vectoring CjaA also failed to protect. Owing to the variability observed with live CjaA-based vaccines in these and previous studies, other candidate antigens were sought and evaluated as subunits. Twenty-one candidate C. jejuni antigens were cloned and expressed as glutathione-S-transferase (GST) fusions. Nine of these could be purified in adequate soluble quantities to be tested in vivo. The intervals of vaccination and challenge were as above, with GST alone or GSTCjaA acting as negative and positive controls, respectively. Each antigen was administered subcutaneously in TiterMax Gold® adjuvant at the molar equivalent of the doses of GSTCjaA. Repeated testing of initially promising candidates revealed that, when averaged across three independent trials, GST-SodB and GST-FliD induced statistically significant reductions in caecal colonisation of 1-2 log10 colony-forming units of C. jejuni at 48 and 56 days post-hatch compared to negative controls. Induction of antigen-specific serum IgY was measured by enzyme linked-immunosorbent assays using maltose-binding protein fusions to each antigen. This revealed significant induction of antigen specific serum IgY for the majority of the antigens tested, even when no protection was observed. In the SodB- and FliD-vaccinated groups, the peak of antigen-specific serum IgY was not coincident with the onset of protection and the fold-change in specific IgY levels in individual birds did not correlate with caecal Campylobacter numbers. Furthermore, sera from SodB-vaccinated birds failed to detect SodB in the outer membrane or surface of Campylobacter cells, indicating that SodB-specific antibodies are unlikely to be neutralising. Taken together, these studies identified two novel protective antigens that, with further optimisation, could form part of an anti-Campylobacter vaccine for broilers. However further studies are required to define the nature and consequences of immune responses required for protection.

This thesis examines the epidemiology of avian influenza viruses (AIVs) in domestic poultry in Pakistan. Major aim of the current research was: to identify risk factors associated with the spread of these viruses; to quantify their prevalence in live bird retail stalls (LBRSs) and backyard poultry in Lahore district and to genetically characterize AIVs circulating in these stalls. Four independent studies were conducted which included (i) a retrospective matched case-control study in commercial poultry farms in Pakistan to identify the risk factors (ii) an estimation of the seroprevalence of AI from a cross-sectional study of backyard poultry flocks in Lahore district (iii) a cross-sectional study of LBRSs to estimate virus prevalence and identify associated risk factors and (iv) the genetic characterization of isolates collected from LBRSs. The retrospective matched case-control study identified five risk factors for AI infection. Multivariate conditional logistic regression model showed that distance of less than 0.5 kilometer of a commercial farm from the nearest case farm (OR= 145.4; 95% CI: 13.6-1553.5), followed by “previous history of infection of flock with infectious bursal disease (OR= 3.77; 95% CI: 1.18-11.97)”, selling of birds/eggs directly to live bird retail stalls from the farm premises (OR= 9.5; 95% CI: 1.7-51.9) have significant influence on spread of AI infection amongst the commercial farms. Other significant potential risk factors are “age of flock at the time of testing (OR= 1.0; 95% CI: 1.00-1.02)” and “a truck entering the farm areas (OR= 30.74; 95% CI: 1.56-604.78)”. Complete fencing of the farm was observed to be a protective factor (OR= 0.12; 95% CI: 0.02-0.63). The cross-sectional survey of backyard poultry flocks for AI (H9, H7 and H5) showed a seroprevalence of 67% (95% CI: 56.9-77.1) for H9 and 21% (95% CI: 13.8-28.1) for H5. Co-infection with both H9 and H5 was observed in 17 villages. Seroprevalence for H9 was significantly associated with the breed of bird. No samples were positive for H7. The cross-sectional survey of LBRSs in 07 towns of Lahore district showed the prevalence of H9N2 virus to be estimated at 10% (95% CI: 6.4-13.6). Subtypes H5N1 and H7N3 were not detected in any sample. Three risk factors showed a strong association with prevalence of H9N2 which are “adding new birds to the cages that already contained birds (OR= 9.2; 95% CI: 2.4-35.1)”, “purchasing birds for sale on the stall from mixed sources (other live poultry markets, auction markets, farm/individual producers) (OR= 3.4; CI 95%: 1.3-8.8)”, and “keeping birds partially inside and outside on the stalls during the day (OR= 1.7; CI 95%: 1.0-3.0)”. Phylogenetic analysis of ten H9N2 viruses isolated from LBRSs of Lahore district showed that four genes (HA, NA, M and NP) of all viruses belonged to G1-lineage and clustered with A/Quail/Hong Kong/G1/97 reference virus while the other four genes (PB2, PB1, NP and NS) from two of the viruses analysed clustered with a group of viruses from Indian subcontinent, Persian Gulf and Middle East. One recently reported H7N3 isolate from Pakistan also clustered with these genes. All H9N2 viruses examined harboured the mutation known to alter the receptor binding profile to one that preferentially binds to human receptors. The analysis shown in this study confirmed that further gene assortment has occurred since its emergence in poultry in Pakistan and Middle East, which could evolve into new genotype. Understanding the epidemiology of avian influenza has always been considered important in formulating and implementing control policies. Results from the current studies illuminate various aspects of epidemiological features of avian influenza viruses within poultry marketing systems in Asia. The current thesis has identified different risk factors and has also reported the prevalence estimates in backyard birds and LBRSs. The presence of reassortants of H9N2 with public health importance in LBRSs has also been reported in the current thesis. These results could be considered to plan future research and appropriate control and prevention strategiess for AIV by the global community. Continued surveillance and monitoring is essential to identify the viral gene pool circulating in live bird retail stalls and backyard poultry and to better understand the public-health risk posed by these viruses.

Within the last 100 years, commercial chickens have been split into two main groups. Broiler chickens are produced for meat production while layers are produced for egg production. This has caused large phenotypic changes and the genomic signatures of selection may be detectable using statistical techniques. Genomic regions identified by these techniques may include genes associated with production traits, and is therefore of interest to animal breeders. This thesis investigates signatures of selection in a number of commercial chicken lines using several statistical techniques based on population differentiation and levels of genetic diversity. First, signatures of selection were investigated using population differentiation in nine lines of broiler chickens. Weir and Cockerham's pairwise FST was calculated for genome-wide markers between the broiler lines and averaged into overlapping sliding windows to remove stochastic effects. A chromosome bound, circular permutation method was used to generate a null distribution and determine the significance of each window. A total of 51 putative selection signatures were found shared between lines and 87 putative selection signatures were found to be unique to one line. The majority of these regions contain peak positions for broiler QTL found in previous studies and eight regions were significantly enriched for broiler QTL. One region located on chromosome 27 contained 39 broiler QTL and 114 genes, several of which were functional candidates for association with broiler traits. Secondly, areas of low diversity were investigated in three different SNP datasets. All three datasets were taken from the same broiler line at different time points and consisted of different SNP densities, including 12k, 42k and 600k. A number of zero diversity regions were found in each dataset and several were shared between the datasets. The 600k dataset was also analysed using a regression test, which investigates the patterns of diversity as the distance from the selected site increases. This method searches for signatures of selections by fitting a regression to the diversity data to test the fit of the data to the theoretical model. A total of 15 regions were found displaying significant asymptotic regression and diversity values less than 0.005. One of these regions located on chromosome 1 was also found as a fixed region in the 12k and 42k datasets and contained the gene IGF1, which encodes an important protein for growth. Finally, signatures of selection were investigated between broiler and layer datasets by investigating population differentiation and diversity based analysis. Weir and Cockerham's pairwise FST was calculated between the two lines and outliers extracted. A total of 32 regions were found displaying high differentiation. Seven regions of low diversity in the layer dataset were also investigated. Several broiler and layer QTL had been previously identified in these regions. Two genes related to hedgehog proteins were identified within selected regions, which are known to be involved in embryogenesis. Finally seven regions were found to be highly differentiated between the broiler and layer lines, and the nine broiler lines in the first chapter. This may indicate selection which occurred during breed separation. Signatures of selection were identified in four broiler and layer datasets using several statistical techniques. A number of regions were identified in multiple datasets by a number of techniques and are therefore good candidate regions for selection. Other statistical techniques could be used in future studies to further confirm these regions and identify causative genes and variants.