Studies on the immunopathogenesis, diagnosis and control of infectious bronchitis and avian metapneumoviruses in chicken

This thesis describes field and experimental investigations on various aspects of the immunopathogenesis, diagnosis and vaccination of infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) in the chicken. The immunopathogenesis of an economically important variant IBV (IS/885/00 like) seen in the Middle East and North Africa was examined in one day old specific pathogen free (SPF) and commercial broiler chicks (Chapter 3). The virus caused respiratory distress, depression and diarrhoea in both SPF and broiler chicks but the severity was milder in the latter. Mild head swelling was observed in one infected broiler chick at 15 days post infection (dpi) and virus with 100% nucleotide level similarity to the inoculum was detected by reverse transcription polymerase chain reaction (RT-PCR) and virus isolation (VI). In the IS/885/00-like infected SPF chicks, cystic oviducts were found in two female chicks. IBV with 99% part S1 sequence similarity to the initial inoculum was isolated from the cystic fluid. The protection provided by current commercial vaccines against variant IBV IS/885/00 like and IS/1494/00 like was investigated in day old commercial broiler chicks (Chapter 4). Protection was evaluated based on the clinical signs, gross lesions, tracheal ciliary scores and virus detection by RT PCR. It was found that administering combined live H120 and CR88 vaccines simultaneously at day old, followed by CR88 vaccine at 14 days old gave more than 80% ciliary protection against both of the Middle East isolates. Cellular and local immune responses in the trachea following vaccination of day old broiler chicks with different live IBV vaccines were evaluated (Chapter 5). In addition, protection conferred against virulent IBV was also examined. All vaccination programmes were able to induce measurable levels of CD4+, CD8+ and IgA bearing B cells in the trachea following vaccination when compared to unvaccinated birds. Expression levels of CD4+ and CD8+ cells varied between the vaccinated groups. Vaccines containing Mass2 combined with 793B2 produced good protection against challenge with virulent IBV QX compared to vaccines containing Mass (Mass1 or Mass2) alone or Mass1 with D274 or CR88. All vaccination programmes produced more than 80% protection against homologous (M41 and 793B) challenge. In Chapter 6, IBVs with high nucleotide level similarity to IS/885/00 like and IS/1494/06 like strains were detected by RT PCR in a broiler flock exhibiting high mortality and respiratory distress in Libya. For the first time, these findings have highlighted the circulation of variant IBVs in the Eastern part of Libya. Humoral and cellular immune responses and protection studies in SPF chicks that received live Newcastle disease virus (NDV), aMPV and IBV vaccines in single, dual or triple combinations were examined (Chapter 7). Protection against virulent IBV or aMPV was not affected when the vaccines were given either singly or in combination. There were no significant differences in the mean antibody titres of the NDV-vaccinated groups and they remained above the protective titre. The mean titres of antibodies against aMPV were suppressed when aMPV vaccine was given with other live vaccines but the aMPV-vaccinated groups were fully protected when challenged with virulent aMPV. The mean titres of antibodies were similar in the IBV-vaccinated groups and all IBV-vaccinated groups gave almost 100% protection against M41 challenge. Between the vaccinated groups, there were no significant differences in the mean numbers of CD4+, CD8+ and IgA-bearing B cells, reflecting similar levels of tracheal cellular and IgA responses irrespective of single, dual or triple vaccine applications. Despite the aMPV humoral antibody suppression, the efficacy of the live vaccines was not compromised when they were given simultaneously to young SPF chicks. Comparative studies in day old SPF chicks using both aMPV subtype A or B, separately or in combination, were evaluated (Chapter 8). There were significant differences in the degree of the clinical signs induced by the single subtypes A, B or A+B given together, with most severe signs observed in the latter two groups. By RT-PCR or VI, subtype B virus persisted longer than subtype A. Even though similar titres of the viruses were used, birds given subtype B alone or in combination showed a greater increase in antibody titres than those given A. These findings demonstrate that for the two strains used, subtype B was more pathogenic than subtype A and was excreted and persisted in the tissues for longer. The use of Flinders Technology Associates (FTA) cards for detection of several avian pathogens has been previously reported. To date, no information has been published on the use of FTA cards for detection of aMPV. In Chapter 9, the feasibility of using FTA cards for the molecular detection of aMPV subtype A and B by RT-PCR was investigated. Findings showed that FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization

Evaluation of Flinders Technology Associates cards for storage and molecular detection of avian metapneumoviruses

The feasibility of using Flinders Technology Associates (FTA) cards for the molecular detection of avian metapneumovirus (aMPV) by reverse transcriptase-polymerase chain reaction (RT-PCR) was investigated. Findings showed that no virus isolation was possible from aMPV-inoculated FTA cards, confirming viral inactivation upon contact with the cards. The detection limits of aMPV from the FTA card and tracheal organ culture medium were 101.5 median ciliostatic doses/ml and 100.75 median ciliostatic doses/ml respectively. It was possible to perform molecular characterization of both subtypes A and B aMPV using inoculated FTA cards stored for up to 60 days at 4 to 6°C. Tissues of the turbinate, trachea and lung of aMPV-infected chicks sampled either by direct impression smears or by inoculation of the tissue homogenate supernatants onto the FTA cards were positive by RT-PCR. However, the latter yielded more detections. FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization

Protection conferred by live infectious bronchitis vaccine viruses against variant Middle East IS/885/00-like and IS/1494/06-like isolates in commercial broiler chicks.

The ability of the infectious bronchitis H120 (a Massachusetts strain) and CR88 (a 793B strain) live attenuated vaccine viruses to protect from two Middle East infectious bronchitis virus isolates, IS/885/00-like (IS/885) and IS/1494/06-like (IS/1494) in broiler chicks was investigated. Day-old chicks were separated into three groups, (I) vaccinated with H120 at day-old followed by CR88 at 14 days-old, (II) vaccinated with H120 and CR88 simultaneously at day-old and again with CR88 at 14 days-old, (III) control unvaccinated. At 30 days-old, each of the groups was challenged with virulent IS/885 or IS/1494. Protection was evaluated based on the clinical signs, tracheal and kidney gross lesions and tracheal ciliostasis. Results showed that administering combined live H120 and CR88 vaccines simultaneously at day-old followed by CR88 vaccine at 14 days-old gave more than 80 per cent tracheal ciliary protection from both of the Middle East isolates. In addition, this programme conferred 100 per cent protection from clinical signs and tracheal or kidney lesions. The other vaccination programme, H120 at day-old followed by CR88 at 14 days-old, the tracheal ciliary protection conferred were 60 per cent and 80 per cent from IS/885/00-like and IS/1494/06-like, respectively

Detection of variant infectious bronchitis viruses in broiler flocks in Libya

A number of broiler flocks with respiratory disease and high mortality in five broiler farms in Libya were sampled for detection of infectious bronchitis virus (IBV). Twelve IBV strains from these farms were detected by reverse transcription polymerase chain reaction (RT-PCR) and differentiated by nucleotide sequencing of the hypervariable region of the S1 gene. A pair-wise comparison of the sequences showed two distinctive patterns. Those from farms 1, 2, 4 and 5, formed a separate cluster with 94–99% relatedness to the Egyptian IBV strains CK/Eg/BSU-2/2011, CK/Eg/BSU-3/2011 and Eg/1212B. Sequences from the farm 3 formed another cluster with 100% relatedness to Eg/CLEVB-2/IBV/012 and IS/1494/06. This appears to be the first report on the co-circulation these variant IBVs in Libya

Mucosal, Cellular, and Humoral Immune Responses Induced by Different Live Infectious Bronchitis Virus Vaccination Regimes and Protection Conferred against Infectious Bronchitis Virus Q1 Strain

The objectives of the present study were to assess the mucosal, cellular, and humoral immune responses induced by two different infectious bronchitis virus (IBV) vaccination regimes and their efficacy against challenge by a variant IBV Q1. One-day-old broiler chicks were vaccinated with live H120 alone (group I) or in combination with CR88 (group II). The two groups were again vaccinated with CR88 at 14 days of age (doa). One group was kept as the control (group III). A significant increase in lachrymal IgA levels was observed at 4 doa and then peaked at 14 doa in the vaccinated groups. The IgA levels in group II were significantly higher than those in group I from 14 doa. Using immunohistochemistry to examine changes in the number of CD4(+) and CD8(+) cells in the trachea, it was found that overall patterns of CD8(+) cells were dominant compared to those of CD4(+) cells in the two vaccinated groups. CD8(+) cells were significantly higher in group II than those in group I at 21 and 28 doa. All groups were challenged oculonasally with a virulent Q1 strain at 28 doa, and their protection was assessed. The two vaccinated groups gave excellent ciliary protection against Q1, although group II's histopathology lesion scores and viral RNA loads in the trachea and kidney showed greater levels of protection than those in group I. These results suggest that greater protection is achieved from the combined vaccination of H120 and CR88 of 1-day-old chicks, followed by CR88 at 14 doa

Heterologous live infectious bronchitis virus vaccination in day-old commercial broiler chicks: clinical signs, ciliary health, immune responses and protection against variant infectious bronchitis viruses

Groups of one-day-old broiler chicks were vaccinated via the oculo-nasal route with different live infectious bronchitis virus (IBV) vaccines: Massachusetts (Mass), 793B, D274 or Arkansas (Ark). Clinical signs and gross lesions were evaluated. Five chicks from each group were humanely killed at intervals and their tracheas collected for ciliary activity assessment and for the detection of CD4+, CD8+ and IgA-bearing B cells by immunohistochemistry (IHC). Blood samples were collected at intervals for the detection of anti-IBV antibodies. At 21 days post-vaccination (dpv), protection conferred by different vaccination regimes against virulent M41, QX and 793B was assessed. All vaccination programmes were able to induce high levels of CD4+, CD8+ and IgA-bearing B cells in the trachea. Significantly higher levels of CD4+ and CD8+ expression were observed in the Mass2 + 793B2-vaccinated group compared to the other groups (subscripts indicate different manufacturers). Protection studies showed that the group of chicks vaccinated with Mass2 + 793B2 produced 92% ciliary protection against QX challenge; compared to 53%, 68% and 73% ciliary protection against the same challenge virus by Mass1 + D274, Mass1 + 793B1 and Mass3 + Ark, respectively. All vaccination programmes produced more than 85% ciliary protection against M41 and 793B challenges. It appears that the variable levels of protection provided by different heterologous live IBV vaccinations are dependent on the levels of local tracheal immunity induced by the respective vaccine combination. The Mass2 + 793B2 group showed the worst clinical signs, higher mortality and severe lesions following vaccination, but had the highest tracheal immune responses and demonstrated the best protection against all three challenge viruses