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Title: Studies on immunopathogenesis and diagnosis of infectious bronchitis virus in chicken
Author: Manswr, B. M.
ISNI:       0000 0004 7658 6829
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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An RT-PCR assay was developed to amplify and sequence the full S1 gene of classical and variant infectious bronchitis virus (IBVs) enriched in allantoic fluid (AF) or the same AF inoculated onto Flinders Technology Association (FTA) cards. Seven IBV strains (M41, D274, 793B, IS/885/00, IS/1494/06, Q1 and QX) were grown in SPF eggs and RNA was extracted from AF. Full S1 gene amplification was achieved by using two primers and products were sequenced using primers; A, SX3, 1050+ and 1380+ to achieve full S1 gene coverage. Following serial dilutions of AF, it was found that detection limits of the partial assay were higher than those of the full S1 gene. Partial S1 sequences exhibited higher than average nucleotide similarity percentages (79%; 352bp) compared to full S1 sequences (77%; 1,756bp), suggesting that the full S1 protocol has greater strain differentiation efficiency. For IBV detection from AF inoculated FTA cards, four serotypes were incubated and stored for up to 21 days at three temperatures; 4°C, room temperature (24°C) and 40°C. RNA extracted and tested with partial and full S1 protocols. Through partial sequencing, all IBVs were successfully detected at all sampling points and storage temperatures. In contrast, for full S1 sequencing, was not able to amplify the gene beyond 14 days of storage or when stored at 40°C. Full S1 sequencing appears to be suited for detection of IBVs enriched in AF, and has limited application for samples directly embedded onto FTA cards. Following Q1-like infection of specific pathogen free (SPF) or two different breeds of broiler chicks, the live body weight of all three types of chicks were significantly reduced. Also, respiratory clinical signs were found in all types of chick. For the infected SPF chicks, all swabs were RT-PCR positive, with the IBV viral load peaking in the trachea and kidneys at 9 dpi, whereas the proventriculus peaked later at 14 dpi. Significant up-regulation in the expression of several genes (IFNα, TLR3, MDA5, LITAF, IL-1β and IL-6) were seen in the trachea and kidneys at 3, 7 and 9 dpi. In the broiler chicks, the immunopathogenesis of Q1 was cross-compared between fast and slow growing broiler birds. For the fast-growing line (Line-A), swabs from the infected group were RT-PCR positive at all sampling days, whereas the slow growing line (Line-B) were positive until 14 dpi. At 7-9 dpi, higher viral loads were found in the trachea, proventriculus and kidney of fast growers compared to slow growers. Mean IBV ELISA antibody titres in the Line-B were higher than Line-A. Tracheal innate immune response showed IFN-α up-regulation only in Line-A but IFN-β was up- regulated in both lines. For TLR3, an up-regulation was seen in Line-A up to 7 dpi, and for all sampling days in Line-B. MDA5 was up-regulated in Line-A and down-regulated in Line-B at 1 dpi. In the kidneys, for Line-A birds, IFN-α and IFN-β were up-regulated at 1 and 1-3 dpi respectively. There was up-regulation in TLR3 in Line-B throughout the study period but not for Line-A. MDA5 was up-regulated in both lines at 7 and 9 dpi. It appears that the immunopathogenesis of IBV Q1 infection in slow growing (Line-B) chicks was milder in terms of the proinflammatory cytokines levels when compared to the fast growers (Line-A), which could be associated with the genetic differences between these breeds. In an attempt to establish an in vitro infectious model to cross-compare the virulence of IBV live vaccines, eight vaccines and three virulent strains of IBVs were assessed in tracheal organ cultures (TOCs). At 24 and 72 hours post infection (hpi), TOC media and tracheal rings were collected and used for RT-PCR, qRT-PCR, measurement of innate immune responses and examination of total apoptotic cells. Differences in virulence were noted between the strains as certain vaccine strains resulted in cilia destruction comparable with the virulent strain. Average cilia motility readings showed that Mass1 and VirMass reached complete ciliostasis at 96 and 72 hpi respectively, whereas, in the 793B and QX groups complete ciliostasis was reached for all strains by 120 hpi. The qRT-PCR analysis revealed decreased viral presence in the media at 24 and 72 hpi. Differences were found between the total apoptotic cell counts in the tracheal rings among virulent and vaccine strains. Down-regulation in mRNA expression of IFN-α at 24 and 72 hpi occurred in all virulent and vaccine infected TOCs. At 24 hpi, there was up-regulation in IFN-β which was down regulated by 72 hpi in virulent infected TOCs. At 24 and 72 hpi, there was up-regulation in the mRNA expression of TLR3 in all vaccine and variant strains. An up-regulation of MDA5 was seen at 24 hpi in Mass serotype strains, vir793B, 793B1 and QX strains. This study demonstrates successful use of the in vitro TOC model for distinguishing differences in virulence and replication rates among the vaccine IBV strains. Four of the vaccine strains used above were included in an in vivo experiment to validate previous data using a chicken host model. Following inoculation of different vaccine strains in SPF chicks, the immunopathogenesis was evaluated. IBV vaccines (Mass1, Mass2, 793B1 and 793B3) were administered by the oculo-nasal route and chicks were monitored daily for clinical signs. At 1, 3, 5, 7, 9 and 14 dpi, OP and CL swabs were collected for virus detection, and blood was collected for antibody assay. Necropsy was carried out on 1, 3, 5, 7, 9 and 14 dpi, with gross lesions observed and tracheal tissues collected for qRT-PCR, immunohistochemistry (CD4+ and CD8+), histopathology and host innate immune gene expressions. Respiratory signs started from 3 dpi. Viral load was lower in the 793B3 group at 5-7 dpi, compared to other groups. Higher expression of IFN-β was seen at 3 dpi in 793B groups, whereas793B1 showed a lower expression of TLR3 at 5-7 dpi compared to other groups. Down-regulation of IL-6 was seen at 7-9 dpi in all inoculated groups except for Mass2. There was higher up-regulation of MYD88 in the tracheal tissue in all inoculated groups. There was higher up-regulation of IFN-γ at 7-9 dpi in Mass1 and Mass2 compared to the control and 793B1 and 793B3. From lachrymal fluid, tracheal washes and tracheal tissue there was higher up-regulation in IgG expression in the all inoculated groups at 9-14 dpi. In Mass2 and 793B3, there was higher up-regulation of IgA in the tracheal tissue at 7-9 dpi. The 793B1 inoculated groups demonstrated higher cell mediated immune responses, represented by higher CD8β gene expression in the period of 5-9 dpi, and higher cell counts of CD4+ and CD8+ at 14 dpi. Results demonstrated that the in vivo were generally comparable to in vitro findings. This demonstrates that large number of IBV live vaccines could be screened for virulence and early immune responses using TOCs, instead of birds.
Supervisor: Chantrey, Julian ; Ganapathy, Kannan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral