Transcriptome profiling of infected chickens with newly emerged genotype VI I Newcastle disease virus strains

Since it was reported in Indonesia in 1926, Newcastle disease (ND) is an endemic disease in Indonesia resulting in devastating economic losses in poultry. Despite heavy vaccination programs, NDV infection still occurs among commercial chicken farms, including vaccinated birds in Southeast Asia, especially in Indonesia. Therefore, the first objective was to detect and perform full genome sequencing of highly virulent circulating NDVs in vaccinated chicken flocks to identify the most adequate NDV strains as vaccine candidates. Our full genome sequencing analysis on two selected isolates from vaccinated birds in Indonesia has shown that both of them belong to highly virulent NDV-GVII.2 strains, while only 0.4% of the vaccine strains used in this country are genotype VII. Moreover, sequencing analysis of the existing genotype VII vaccine revealed that it has significant differences from GI vaccine strain, which is not sufficiently representative of genotype VII viruses. This finding illustrates that virulent NDV-GVII.2 strains are mainly responsible for the high morbidity and mortality of recent ND outbreaks in poultry in Indonesia. Virulent NDV-GVII strains were previously characterised as immunopathological phenotypes triggering severe tissue damage probably through apoptosis and necrosis of lymphoid tissues. However, the underlying molecular mechanism of pathogenesis of ND remains to be fully understood. Hence, the second objective was to identify and characterise the molecular mechanism of lymphotropic behaviour of NDV-GVII by focusing on detecting key biomarkers and cellular immune response signalling pathways that contribute to severe lymphocyte destruction in infected birds using RNA sequencing, bioinformatics tools and PPI network analysis. Transcriptomic profiling indicates that virulent NDV-GVII significantly downregulates immunologically regulated genes and innate immune regulating pathways including fMLP signalling in neutrophils, Fcy receptor-mediated phagocytosis in macrophages and monocytes, and leukocyte extravasation signalling and NF-kB activation by viruses. These transcriptional changes may lead to widespread immunosuppression and enhanced replication of the virus. As a result, the host’s immune response may be diminished, delayed, incomplete or display overly strong induction that can cause severe tissue damage. This finding also implies that virulent NDV-GVII strains appear to possess the capability to inhibit the induction of immune responses by targeting lymphocytes and destroying these cells, which may be one indispensable factor of the pathogenesis of NDV-GVII. Moreover, PPI network analysis revealed that the top three significantly enriched gene modules were phagocytosis, immune response-related terms, and glutamate receptor signalling pathway. We identified novel genes EGF, LPAR5, AGT, AGTR1, RAC2, CD4, CD3D, IL7R, NPY, GRM3, and GRAP2 as potential biomarkers. This study provides valuable information to help understand novel immune evading mechanisms of highly pathogenic NDV-GVII from a host-pathogen interaction point of view. Interactions between the virus and the host determine the success of the viral infection. Hence, the third objective was to identify any particular genes of the virus that may have an important role in virulence and pathogenicity by profiling the transcription of virus in infected birds. This study revealed the transcriptional gradient of these highly pathogenic NDV-GVII genes: NP:P:M:F:HN:L, in which there were a slight attenuations at the NP:P and HN:L gene boundaries. Our result also provides a fully comprehensive qRT-PCR protocol for measuring viral transcript abundance that may be more convenient for laboratories where accessing HTS is not feasible.Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 202

Biosecurity reference : CFR-listed agent and toxin summaries.

This reference document provides summary information on the animal, plant, zoonotic, and human pathogens and toxins regulated and categorized by 9 CFR 331 and 7 CFR 121, 'Agricultural Bioterrorism Protection Act of 2002; Possession, Use and Transfer of Biological Agents and Toxins,' and 42 CFR 73, 'Possession, Use, and Transfer of Select Agents and Toxins.' Summary information includes, at a minimum, a description of the agent and its associated symptoms; often additional information is provided on the diagnosis, treatment, geographic distribution, transmission, control and eradication, and impacts on public health

Epidemiological studies on avian influenza and other respiratory viruses in backyard poultry in Oman

This thesis describes studies on the epidemiology of the avian influenza virus (AIV) and other respiratory viruses such as Newcastle disease (NDV), infectious bronchitis (IBV) and avian metapneumovirus (aMPV) in backyard poultry in Oman. Also, I utilized backyard birds as sentinels to detect the presence of West Nile virus (WNV) in Oman backyard poultry. Additionally, I tried to investigate the risk factors contributing to the spatial distribution of AIV and NDV diseases in backyard poultry farms. Management biosecurity and health programmes in commercial broiler poultry farms were also examined. Chapter 3 reviews the epidemiology of the viral respiratory diseases affecting poultry in the Middle East (ME) in relation to diseases reported in Oman. The review was undertaken to identify knowledge gaps. The review focused more on the highly pathogenic avian influenza H5N1 outbreak in the ME since most of the published poultry articles were on this virus. There was a clear gap in the knowledge on the epidemiology of respiratory viral pathogens except for H5N1. Chapter 4 describe a sero-surveillance study on backyard poultry flocks in Oman. A snapshot two-stage cluster sampling was done during the summer of 2012 on 2350 backyard poultry birds from 238 farms from all of the Oman’s regions and governorates. The dominant poultry species in the visited farms was the chickens; however, ducks, turkeys, geese and guinea fowls were present and sampled. The overall seroprevalence of the avian influenza and Newcastle disease viruses were 37.5% and 42.1%, respectively, and the flock’s positive level was 84% and 90.2%, respectively. The mean within-flock seroprevalences were 37.6% and 43.4%. All the PCR results were negative for NDV and AIV. In conclusion, both disease viruses are endemic in the backyard poultry in Oman. Chapter 5 studies the risk factors associated with the intensity of the infection of both avian influenza and Newcastle disease in Omani backyard farms (serological results from chapter 4). A number of previous studies have investigated and identified a number of risk factors for both diseases, especially the highly pathogenic avian influenza. I obtained the risk factors that are present in Oman from those previously identified and modelled their association with the intensity of the AIV and NDV infection in Omani backyard flocks using general linear models (GLM). There was a regional effect on the level of exposure to both viruses; however there was no North-South pattern. Also, there was a highly significant association between the presence of AIV and NDV infection which may be attributed to the level of biosecurity applied in the farms. Furthermore; there was a negative association between the farm altitude and the AIV intensity of infection. The flock size was marginally negatively associated with the NDV infection rate. Chapter 6 describes a study on the prevalence of IBV and aMPV using molecular methods including RT-PCR, sequencing and phylogenetic analysis from the same sampled backyard flocks. Five flocks from the northern regions were positive for aMPV subtype B and 39 flocks were positive for 5 different genotypes of the IBV virus. The 793B like was the most prominent genotype. However, genotypes M41, IS885, IS94 and D274 were also identified. The presence of the IBV viral genome in the FTA card collected from ducks and turkeys raises the question of their role in IBV epidemiology. The study concluded that both viruses are endemic in Oman backyard poultry. Chapter 7 describes a study of the serological prevalence of the West Nile virus using backyard birds as sentinels. The sera from backyard birds were used for the detection of antibodies against WNV. The total flock prevalence was 45% and the total bird prevalence was 21%. All the tested bird species showed positive ELISA results. Later a snapshot mosquito sampling was done in 16 of the previously identified WNV positive farms. The mosquito species recorded on WN positive farms were Culex quinquefasciatus, a known vector of WNV, and Anopheles stephensii, a malaria vector. The study concluded that WNV is endemic in backyard poultry in Oman. Management, biosecurity and health practices are the core of poultry farming success and the main defence against the introduction of diseases to the farm, as well as dissemination between farms. Chapter 8 describes the management and health practices applied in commercial poultry production farms in Oman. A questionnaire designed to investigate the management, biosecurity and health practices in broiler production poultry farms was filled in by Ministry of Agriculture and Fisheries animal production engineers for 69 broiler poultry farms. These 69 broiler farms produce around 95% of the poultry meat produced in Oman. The Southern region Dhofar governorate, has the highest reported number of birds, nearly 20 million, which makes up almost (82%) of the responding broiler production farms. The majority of the farms use closed houses with evaporating/cooling fans and cooling beds (46/69, 66.7%). All farms vaccinate against Newcastle disease. infectious bursal disease (IBD), avian influenza virus H9N2, avian infectious bronchitis and aMPV were also been reported in vaccination programs with different percentages. The bigger farms apply better biosecurity measures and their managers have bette