Browsing by Author "Abdul-Careem, Mohamed Faizal"
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Item Open Access Activation of Toll-Like Receptor-Mediated Antiviral Response Against Infectious Laryngotracheitis Virus Infection(2023-09-21) Mohamed Abdul Cader, Mohamed Sarjoon; Abdul-Careem, Mohamed Faizal; van der Meer, Frank; Van Marle, Guido; Gomis, SusanthaInfectious laryngotracheitis virus (ILTV) is a highly prevalent avian respiratory virus in Canada and globally, which can cause mild to severe respiratory illnesses. Although the live attenuated ILTV vaccines are commonly used for control, they pose challenges such as establishing lifelong latent infections, reactivating and shedding latent viruses, and regaining virulence in vaccine strains. Therefore, it is essential to develop novel control measures to address the limitations of current approaches. Inducing innate antiviral responses via the activation of toll-like receptors (TLRs) is a promising strategy for reducing ILTV replication. Endosomal TLRs in chickens, such as TLR7 and TLR21, recognize viral genetic materials, while surface TLRs (e.g., TLR4) primarily recognize bacterial molecules, but may also contribute to antiviral responses by recognizing viral proteins. Synthetic TLR ligands have been shown to induce antiviral responses against some avian viruses, such as avian influenza virus (AIV), infectious bursal disease virus (IBDV), ILTV, and infectious bronchitis virus (IBV). However, the impacts of in-ovo delivery of endosomal TLR7 and TLR21 ligands and surface TLR4 ligand (single-stranded (ss) RNA, cytosine-guanosine deoxynucleotides (CpG) DNA, and lipopolysaccharide (LPS), respectively) in reducing ILTV replication in chickens post-hatch through induction of antiviral responses is unknown. This thesis aimed to study the enhanced immune response following in-ovo treatment of these TLR ligands against ILTV infection in young chickens. Our hypothesis is that in-ovo delivery of these ligands will enhance antiviral immune responses and reduce ILTV replication in chickens post-hatch. Our results confirmed the following findings: Firstly, the in-ovo administration of synthetic TLR7 ligand, resiquimod, reduces ILTV shedding post-hatch, correlating with enhanced macrophage responses. Secondly, the in-ovo delivered CpG DNA stimulates cellular immune responses in multiple organs post-hatch, potentially reducing ILTV infection. Thirdly, the in-ovo LPS treatment stimulates protective antiviral responses against ILTV infection post-hatch, correlating with the expansion of macrophage population in the lungs. Overall, the studies provide insights into the mechanisms of host responses elicited following in-ovo delivery of these three TLR ligands against ILTV in chickens. The outcomes of the current studies can be helpful in fine-tuning the currently used vaccine strategies against ILTV in chicken to achieve maximum protection.Item Open Access Antiviral Response Against Avian Influenza Virus and Avian Infectious Bronchitis Virus: Focus on MicroRNA Regulation(2024-04-29) O'Dowd, Kelsey; Abdul-Careem, Mohamed Faizal; Gagnon, Carl A.; van der Meer, Franciscus Johannes; Niu, Dongyan Xu; Niikura, Masahiro; Susta, LeonardoAvian influenza virus (AIV) and avian infectious bronchitis virus (IBV) are respiratory RNA viruses with significant economic implications due to morbidity, mortality, secondary bacterial infections, and overall decreased flock performances. Current AIV and IBV control methods have limitations and new variants continue to emerge; therefore, there is a need to develop new control strategies and gain a better understanding of host-pathogen interactions during the early stages of viral infection. Micro-ribonucleic acids (microRNAs) are small, non-coding RNA molecules that regulate gene expression by targeting complementary messenger RNA (mRNA) sequences. MicroRNAs can be packaged into extracellular vesicles (EVs), which are membrane-encapsulated vesicles released from host cells that play a role in cell-to-cell communication and regulation. This study focuses on investigating some aspects of the antiviral response against AIV and IBV infections in chickens, focusing on the role of microRNAs. The first two objectives focused on AIV H4N6 infection of chicken tracheal organ cultures (TOCs) to determine the profile of cellular and released EV microRNAs and evaluate the proteomic signature of EVs, as well as the function of EVs on chicken macrophages. The last two objectives explored infection with different strains of IBV in a chicken tracheal epithelial cell (cTEC) in vitro model and in the chicken trachea in vivo to characterize the mRNA and microRNA expression profiles. For the AIV experiments, we demonstrated for the first time that chicken tracheal cells secrete EVs. Differentially expressed (DE) microRNAs, such as miR-146a, miR-146b, miR-205a, miR-205b, and miR-449, were identified as potential targets for further functional validation studies. We determined that released EVs contain DE proteins involved in immune responses and cell signaling pathways and showed that these EVs can activate macrophages. For the IBV experiments, we showed that IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass)41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA and microRNA expression profiles that are strain-, time-and model-dependent. We identified the candidate DE microRNAs, such as gga-miR-155, gga-miR-1388a, gga-miR-7/7b and gga-miR-21-5p. Overall, these findings provide valuable insights into key expression profiles and underlying mechanisms involved in the antiviral response against AIV and IBV infections in chickens.Item Open Access Comparative innate responses induced by Toll-like receptor (TLR)7 and 21 ligands against infectious bronchitis virus infection(2019-01-26) De Silva Senapathi, Yaseshwari Upasama; Abdul-Careem, Mohamed Faizal; Czub, Markus; Van Marle, GuidoToll-like receptor (TLR)7 and 21 ligands, resiquimod and cytosine-guanosine (CpG) oligonucleotides (ODNs) respectively have been used in ovo (pre-hatch) to enhance or prime an early immune response in chickens to provide protection against microbial pathogens. Yet, their protective efficacy against an infectious bronchitis virus (IBV) infection encountered post-hatch has not been evaluated. Thus, our objectives were to investigate the efficacy of resiquimod and CpG ODNs against a post-hatch IBV infection by delivering the ligands in ovo at embryo day (ED)18 and then, to determine possible mechanisms of protection. We found upregulation of interleukin (IL)-1β and interferon (IFN)-γ mRNA levels and considerable expansions of macrophage and cluster of differentiation (CD)8α+ T cell populations in lungs of chicken as early as day one post-hatch, following pre-hatch delivery of resiquimod. When the resiquimod pre-treated day-old chickens were infected with IBV, reduction in viral shedding via oral and fecal routes was observed at 3 days post-infection (dpi). Similarly, in CpG ODN pre-treated birds at 3 dpi, we found increased recruitment of macrophages, CD8α+ and CD4+ T lymphocytes in addition to up-regulation of IFN-γ and IL-1β mRNA concentrations in chicken lungs. However, only in ovo delivered CpG ODNs significantly reduced the morbidity and mortality associated with IBV infection. Overall, these studies bring us closer to understanding mechanisms behind CpG ODNs and resiquimod induced immune responses in chickens when used as stand-alone prophylactic agents in ovo.Item Open Access Creating a Marked Mycobacterium avium subsp. paratuberculosis Vaccine Strain and Detecting Marker-Specific Immune Responses in Calves(2018-04-17) Luo, Yi Yi; De Buck, Jeroen M.; Dong, Tao G.; Abdul-Careem, Mohamed FaizalMycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a chronic, contagious granulomatous enteritis with a high global prevalence in dairy cattle. This disease causes significant economic loss in the dairy industry and has been challenging to control due to its ability to survive in harsh environmental conditions for long periods of time, its easily transmittable nature through the fecal-oral route of transmission, the difficulty to detect the pathogen, and lack of an effective treatment or cure. The difficulty with detecting the pathogen stems from current diagnostic assays lacking specificity, posing issues with mycobacterial cross-reactivity, and low sensitivity, particularly during subclinical stages of disease. Previously developed vaccines do not prevent infection and are not approved in Canada in part due to interference with bovine tuberculosis diagnostics. To remediate this issue, positive and negative immune markers were inserted into a field strain of MAP as part of a vaccine capable of differentiating infected from vaccinated animals (DIVA). Specialized transducing mycobacteriophages were used to replace a gene coding for an immunogenic protein (MAP1693c) in the MAP genome with an HA (human influenza hemagglutinin) epitope-tagged immunogenic gene (PepA) via allelic exchange. Once gene replacement was confirmed, these markers were evaluated in a calf infection trial, where 17 Holstein-Friesian dairy calves were inoculated with either 109 CFUs of the marked strain (n = 6), 109 CFUs of a wild-type (WT) field strain (n = 6), or remained uninfected controls (n = 5). Cellular and humoral immune responses were measured using an interferon gamma release assay (IGRA) and enzyme-linked immunosorbent assay (ELISA), respectively. Marker-specific IFN- release was measured by stimulating whole blood with marker peptides and proteins, while antibody response was assessed by incubating serum with the same marker peptides and proteins. Some calves inoculated with the marker strain showed increased cellular immune responses to the HA epitope positive marker. Unexpectedly, a scrambled version of the HA epitope induced a significant IFN- response in marker-infected calves compared to WT-infected (p = 0.016) and uninfected (p = 0.019) groups at 4.5 months post-inoculation. This positive marker thus holds potential as a valuable diagnostic tool as part of a DIVA vaccine for Johne’s disease. We have shown that immune markers, both immunogenic proteins and epitope tags, can be introduced into the MAP genome using a stable allelic exchange method. The specificity of the scrambled HA epitope as an antigen to test for marker-specific cellular immune responses requires further investigation.Item Open Access Investigations into Macrophage-Infectious Bronchitis Virus (IBV) Interaction and Shell-less Egg Syndrome(2018-08-29) Amarasinghe, Aruna; Abdul-Careem, Mohamed Faizal; van der Meer, Frank; Cork, S. C.; Gilch, Sabine; Gomis, Susantha MuhandiramgeInfectious bronchitis virus (IBV) is a coronavirus and infects chickens globally causing economic losses. The disease caused by IBV is known as infectious bronchitis (IB) and is prevalent in commercial broiler and layer chickens and breeder flocks in Canada. The control of IB relies on vaccination done on the day of hatch and then several times during the grower period depending on the purpose of rearing the chickens. Although the vaccine-induced immunity protects chickens from production losses induced by IBV infection, vaccine failures are frequent. Given the issues in current IB control measures, sustainable control measures developed understanding the host-IBV interaction is required. The studies conducted in the thesis focused in two major areas; 1) understanding the interaction between IBV and host immune system mainly macrophages and 2) investigating the role of IBV in a recently emerged concern of Western Canadian table-egg layer industry, shell-less egg syndrome (SES). The work described in chapter 2 of the thesis led to the finding that IBV replicates in avian macrophages in vivo and in vitro. In vitro, we showed that IBV not only targets macrophages leading to productive infection but also affects selected functions of macrophages, particularly the production of nitric oxide (NO). As shown in chapter 3, IBV infection upregulates the expression of interleukin (IL)-1β in both tracheal and lung tissues. An additional observation made was that there was a significant association between the IBV genome load and macrophage recruitment in lungs. Overall, we found that macrophages can act as a source of cytokines, which is beneficial against IBV infection. However, the ability of IBV to replicate within macrophage by decreasing selected immune functions can be detrimental to the host. Chapter 4 provides details of our work leading to the elucidation of the etiology of SES. First, molecular characterization showed that about 70% of the IBV strains isolated from layer flocks affected with SES in Western Canada were Massachusetts (Mass) genotype. Infection of layer chickens with one of the Mass IBV isolate induced shell-less eggs. The work of chapter 5 compared two Mass IBV isolates recovered from Western Canadian layer flocks for whole genome variations and documented the differences in pathogenicity, tissue distribution, and macrophage response. The knowledge generated in the thesis increased the understanding of IBV-macrophage interaction, documented the IBV genotypes observable in Western Canada layer flocks and elucidated the etiology of SES observed in layer operations in Canada.Item Embargo Microbiological and Molecular Identification and Characterization of Avian Pathogenic Escherichia coli from Poultry Farms, Alberta(2024-01) Wang, Yanqi; Niu, Dongyan; Niu, Dongyan; Abdul-Careem, Mohamed Faizal; Fairbrother, John; Zhang, KunyanAvian pathogenic Escherichia coli (APEC) is the etiological agent of avian colibacillosis, leading to significant economic losses in Canada and elsewhere. Avian fecal E. coli (AFEC) is considered to serve as a potential reservoir for APEC given shared genetic features. This study aimed to profile phenotypic and genotypic features of causative APEC in Alberta. Here, 100 APEC isolates from diseased and dead chickens (broiler and layer) and turkeys from 45 farms collected between 2021 and 2023 were subjected to serogroup typing, APEC-associated virulence gene (VG) profiling, antimicrobial susceptibility testing, multilocus sequence typing, and phylogenetic analysis. In addition, 121 AFEC isolates from five layer flocks were collected to investigate APEC-associated VG carriage in healthy birds. Furthermore, genetic similarity between 22 AFEC strains identified as potential APEC (poAPEC) and APEC strains was assessed through phylogenetic analysis. We identified 27 serogroups, 38 known and two novel sequence types (STs), and varied VG distribution across APEC isolates from chickens and turkeys. Interestingly, variations in VG among AFEC strains were found across distinct egg laying stages or housing systems. Antimicrobial resistance (AMR) genes, especially extended-spectrum beta-lactamase genes were detected in APEC and poAPEC strains across all poultry species. Moreover, phylogenetic analysis revealed close clonal relationships among APEC and poAPEC strains. Notably, we identified a poAPEC isolate that shared ST155 and VGs with an APEC strain. This study is the first to report the characteristics of APEC and poAPEC isolates that cause or potentially cause colibacillosis morbidity and mortalities in chickens and turkeys in Alberta. A variety of APEC phenotypes and genotypes can lead to colibacillosis in poultry farms and may originate from the commensal E. coli found in the gastrointestinal tract of birds.Item Open Access Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus isolates(2021-02-02) Perez Contreras, Ana Paulina; Abdul-Careem, Mohamed Faizal; Fonseca, Kevin; van der Meer, FrankThe extensive use of live-attenuated vaccines to control the upper respiratory tract viral infection in chicken known as infectious laryngotracheitis (ILT), has been associated with a surge in vaccine-related ILT outbreaks. It is documented that these ILT outbreaks are due to the regaining of virulence of the vaccine viruses due to multiple bird to bird passages following vaccination. These vaccine-originated infectious laryngotracheitis virus (ILTV) isolates are known as vaccine revertants. An additional concern is that the multiple live-attenuated vaccine ILTV and wild-type ILTV can recombine, resulting in ILTV strains with higher pathogenicity. To date, little is known about the molecular nature of the Canadian ILTV. The objectives of the present thesis work are to, 1) molecularly characterize the ILTV associated with ILT outbreaks in poultry flocks in Canada using a whole genome sequence approach and 2) study the pathogenicity of representative ILTV isolates in vivo. In achieving objective 1, It was found that most of the ILTV isolates of Canadian origin used in this study were genetically related to chicken embryo origin (CEO) live-attenuated vaccine ILTV strains. Evidence of recombination involving commonly used live-attenuated ILT vaccines was also detected in an ILTV isolate belonging to the British Columbia province. A second recombination event was found this time involving an ILTV isolate belonging to Alberta. This Alberta ILTV strain acted as a parental strain along with another live-attenuated ILT vaccine strain to give rise to an ILTV strain previously isolated in United States (US) territory. In objective 2, the pathogenicity of two wild-type and one CEO vaccine revertant ILTV isolates was compared, by infecting specific pathogen free chickens along with age-matched mock infected controls. We also used naïve contact chickens in order to determine the transmission potential of these ILTV isolates. It was found that the tested CEO vaccine revertant ILTV isolate can induce not only severe disease but also to transmit more efficiently than the wild-type ILTV isolates used for this study.Item Open Access Molecular characterization of economically important poultry viruses in western Canada(2020-12-04) Palomino-Tapia, Victor A.; Abdul-Careem, Mohamed Faizal; Sellers, Holly S.; van der Meer, Frank; Mitevski, DarkoAvian Reovirus (ARV), Chicken Astrovirus (CAstV), and Hemorrhagic Enteritis Virus (HEV) are important enteric pathogens affecting poultry production around the world. These agents are the causative agents of Viral Arthritis (VA), White Chick Syndrome (WCS), and Hemorrhagic Enteritis (HE), respectively. In meat-type chickens, pathogenic strains of ARV can replicate in the joints leading to edema, inflammatory cell infiltrate, and fibrosis, which results in rupture of tendons. Similarly, pathogenic strains of CAstV can cause transient increase in mid to late embryo mortality, reducing hatchability between 4-68%, with some hatched birds exhibiting pale plumage; these “white chicks” (WCS) usually die within the first week of life. In turkeys, HEV infection has two presentations: 1) A clinical disease consisting on gastrointestinal hemorrhages, depression and immunosuppression (Clinical HE); and 2) subclinical infection, consisting in immunosuppression and causing economical losses due to secondary infections and plant condemnations. In recent years, these diseases have gained importance in western Canada as a result of the economic losses sustained from these infections in: a) feed conversion, b) high number of culls/first week mortality, c) secondary bacterial infections, c) processing plant condemnations; and d) costly disruptions to the Canadian Supply Management system. This thesis focuses on molecular characterization of ARV, CAstV, and HEV obtained either from clinical samples (ARV, CAstV), or from cases suspected to have subclinical infection (HEV) in poultry farms located in western Canada. The biological samples from chickens (ARV, CAStV) and turkeys (HEV) were collected from cases submitted for post-mortem examination and diagnosis to Poultry Health Services (PHS), a private poultry consulting firm, located in Airdrie, Alberta, western Canada. Further studies are required to assess the virulence of these isolates for understanding their impact in the western Canada Poultry Industry and for the implementation or enhancement of vaccination practices.Item Open Access Molecular Investigation of Wildlife Herpesvirus and Parapoxvirus: Benefits and Limitations of Genetic Characterization of dsDNA Viruses from Tissues(2019-12-18) Dalton, Chimoné Stefni; van der Meer, Frank; Abdul-Careem, Mohamed Faizal; Kutz, Susan J.; Ruckstuhl, Kathreen E.Wildlife populations can be reservoirs or victims of pathogens shared with humans and/or domestic animals. Most diseases at the wildlife-livestock interface are caused by viruses. Herpesviridae and Parapoxviridae are families of important double-stranded DNA (dsDNA) viruses that have been implicated in diseases of wildlife, domestic animals, and humans resulting from spill-over or zoonotic transmission, yet still little is known about viruses circulating in wildlife. Wildlife health surveillance is a primary tool for the management of zoonotic diseases, the control of diseases of domestic animals, and the preservation of wildlife populations. Studies herein conduct molecular surveillance of herpesviruses (HV) and orf virus (a parapoxvirus) through diagnostic polymerase-chain reactions (PCR), sequencing, and phylogenetic analysis using tissues of various wildlife animal species in Canada. The viral DNA polymerase (DPOL) gene is an effective target for the detection and characterization of HV present in infected animals. Previously uncharacterized HV were characterized in marten across Canada, and Reindeer gamma-HV 1 was characterized in caribou from different herds. Phylogenetic analysis suggests HV have coevolved with their wildlife host at a species level. Detection of orf virus was most successful when targeting the viral immunodominant envelope protein gene: B2L. Orf virus was detected in muskoxen on Victoria Island in areas managed by the Northwest Territories (NT) and Nunavut (NU), and on the adjacent mainland of NU, Canada. Orf virus was present in males and females, from calf to adulthood, indicating this virus represents a disease threat for muskoxen. Next-generation sequencing was performed directly on the DNA extracted from tissues of four clinically infected, geographically distant muskoxen in our study area. Phylogenetic analysis revealed Muskox orf virus (MxOV), to be unique from known orf viruses. This thesis documents the diversity of HV circulating in wildlife, increases our awareness of limitations when using tissues for molecular surveillance, increases our understanding of orf virus infection in muskoxen, and highlight areas of much-needed research. Methodologies herein can be adapted for the surveillance of other dsDNA viruses, while the data directly contribute to the database of HV and orf virus sequences in Canadian wildlife that provide context for new or emerging pathogens.Item Open Access Nitric Oxide Mediated Antiviral Response in Avian Cells(2013-10-02) Haddadi, Siamak; Abdul-Careem, Mohamed FaizalLPS activates TLR4 signaling pathway eliciting antiviral host responses in mammals although information on such responses in avian species is scarce. Objectives of the work described in the thesis were to 1. characterize the LPS induced expression of LPS receptors and NO in two avian cell lines, LMH and MQ-NCSU, and 2. observe whether NO can elicit antiviral response against ILTV replication. We found that LPS was capable of inducing the expression of TLR4, CD14 and NO production only in MQ-NCSU cells. We also showed that TLR4 mediated NO production in MQ-NCSU cells confers antiviral response against ILTV in susceptible LMH cells. Using a selective inhibitor of inducible NO synthase and a NO donor as a source of NO, we confirmed that this effect is positively correlated with NO. Our data showed that LPS can be a potential innate immune stimulant that can be used against ILTV infection in chickens.