Generation of a Bacterial Artificial Chromosome of CVRM strain to Express VP2 gene of Infectious Bursal Disease Virus

The CVI988 strain of Marek’s disease virus (MDV) is a highly effective vaccine to protect chicken against very virulent strains of MDV. From Texas A&M University, Blanca Lupiani reported that she and her colleagues recently showed that insertion of LTR (long term repeat) sequences into the CVI988 resulted in the generation of CVRM, a virus with similar protective efficacy.The CVRM virus was cloned as a bacterial artificial chromosome, by insertion of mini-F sequences into the US2 gene by homologous recombination.

The objective of the work presented in Chicago was to evaluate CVMR-BAC as an expression vector of foreign genes. For this purpose, the VP2 gene of infectious bursal disease virus (IBDV) was cloned into CVRM genome by recombination. VP2 protein was expressed at high levels as determined by immunofluorescence of CVRM-VP2 infected chicken embryo fibroblasts.

Data on expression of VP2, as measured by antibody levels and protection against challenge, in commercial chickens was presented.

It is our expectation that this recombinant vector can provide protection against IBD and highly virulent MD challenge, concluded Dr Lupiani.

Expression of Infectious Bursal Disease VP2 in Turkey Herpesvirus Vector

The use of turkey herpesvirus (HVT) as a viral vector to express immunogenic proteins has been shown to be successful in the poultry industry.

The objective of work presented by Sanjay M. Reddy of Texas A&M University was to evaluate potential cloning sites in the HVT genome for stable expression of foreign genes.

For this purpose, the VP2 gene of infectious bursal disease virus (IBDV) was cloned into HVT genome by homologous recombination. VP2 protein was expressed at similar levels regardless of the cloning location, as determined by immunofluorescence of HVT-VP2 infected chicken embryo fibroblasts.

The recombinant HVT-VP2 viruses were stable and showed continuous expression of VP2 even after several passages in cell culture.

Data on expression of VP2, as measured by antibody levels and protection against challenge, in commercial chickens was presented.

Dr Reddy said it was his expectation that this recombinant vector can provide protection against IBD and Marek’s disease challenge.

Virulence of Canadian isolates of 'Variant' Infectious Bursal Disease Viruses in Specific Pathogen Free Chickens and Commercial Broilers

Shanika Kurukulasuriya of the Western College of Veterinary Medicine at the University of Saskatchewan in Canada explained recent studies have demonstrated that the majority of Infectious Bursal Disease Viruses (IBDVs) circulating in Canada are 'variant' strains and capable of immunosuppression in broilers.

The objective of this study was to compare pathogenesis of 'variant' IBDVs in SPF chickens and commercial broilers. Expression of INF-γ, INF-β, IL-1β, IL-2, IL-6, IL-18 in the bursa of Fabricius using a multiplex assay, percentage of bursal weight to body weight (BW: BW), bursal histopathology, and antibody titres against IBDV were compared following oral administration of 'variant' IBDV in groups of birds at day-3 or day-6 post-hatch. Cytokine analysis was conducted on days 1, 3, 5, 7, 14 and 21 following IBDV challenge.

The geometric mean of maternal antibodies against IBDV was 9,000 in commercial broilers at hatch.

The percentage BW:BW ratio was significantly lower (0.05 per cent) at day-35 post-hatch than the non-challenged control group (0.19 per cent). Also, severe bursal atrophy was noted microscopically at day-35 post-hatch.

In contrast, SPF birds had a significantly low BW:BW (0.12 per cent) and severe bursal atrophy compared to the non-challenged control group (0.31 per cent), at day 9 post-hatch (P<0.05).

Expression of INF-γ, INF-β, IL-1β, IL-2, IL-6 and IL-18 in the bursa of Fabricius was normalised with TUBB1 and ACTB.

This data demonstrated that 'variant' IBDV rapidly in fects SPF birds compared to commercial broilers however bursal atrophy in commercial broilers was similar to SPF birds day-35 post-hatch, concluded Kurukulasuriya.

Characterisation of the Immune Response and Immunosuppression in Commercial Chickens Challenged with Very Virulent Infectious Bursal Disease Virus Reassortants

Infectious bursal disease (IBD) is an acute, contagious, viral infection characterised by increased mortality in chickens typically between three and six weeks of age, and immunosuppression if infected before three weeks of age, according to Rodrigo Gallardo from the University of California Davis School of Veterinary Medicine.

In December 2008, the very virulent form of IBD virus (vvIBDV) was identified in a commercial egg layer operation in Northern California. This event is recognised as the first known case of vvIBDV in North America.

Since this first case, previously unseen vvIBDV strains have been detected in several commercial and backyard flocks. By RT-PCR and sequencing, these strains have proven to be reassortants of different serotype 1 and serotype 2 IBDV genome segments A and B.

To understand better the effects of these vvIBDV reassortants, three groups of commercial chickens, including broilers (20 per group) and layers (20 per group) were set up. Group 1 was challenged with a variant strain of IBDV, group 2 with CA-K785 vvIBDV strain (segment A vvIBDV, segment B serotype 2), and group 3 with CA-S7610 (segment A vvIBDV, segment B Australian variant). A fourth group (of 25 broilers and 25 layers) served as uninoculated control.

Cytokine and chemokine expression were evaluated at the mRNA level in the bursa and spleen at one and four days post-infection in all the experimental groups. In addition the immunosuppression induced by these viruses was evaluated using histopathology scores from bursa, thymus and spleens collected at seven and 21 days post IBDV challenge. Finally, the humoral immune response and clinical signs to an infectious bronchitis virus (IBV) challenge was characterised in all the groups by measuring IBV specific antibodies using ELISA.

The results were discussed at the Convention.

Effect of Variant Infectious Bursal Disease Virus on Avian Influenza Virus Vaccine Efficacy

Immunosuppressive viruses are known to affect vaccinal immunity, which is one reason vaccines efficacy may be over-estimated by laboratory studies, reported Erica Spackman from the USDA ARS Southeast Poultry Research Laboratory.

The impact of virally induced immunosuppression on avian influenza vaccine efficacy has not been quantified. In order to determine the effect of exposure to infectious bursal disease virus (IBDV) on vaccinal immunity to highly pathogenic avian influenza virus (AIV), vaccination-challenge studies that incorporated an evaluation of mean infectious dose were conducted.

Specific pathogen-free White Rock chickens were exposed to variant E IBDV at one week of age, then were inoculated with an inactivated, oil adjuvanted H7 AIV vaccine. Three weeks post-vaccination, the chickens were challenged with four different doses of the same highly pathogenic AIV used to prepare the vaccine.

Mortality in chickens exposed to IBDV was similar to mortality in challenge controls, regardless of vaccination status. Chickens that had been vaccinated but not exposed to IBDV were protected from mortality and morbidity.

Virus shed and antibody response to the vaccine are currently being evaluated, added Dr Spackman.

Use of a Classical ELISA to Detect Antibodies Following Vaccination with Recombinant HVT Vectored Vaccines for IBD

For many of the conventional live and inactivated IBD vaccines, ELISA guidelines have been reported. However, for the new generation of recombinant viral vector vaccines, based on HVT as a vector expressing genes that code for specific IBD VP2 antigens, new practical guidelines are needed to assist with interpretation of ELISA results, according to Bart van Leerdam of Biocheck BV.

Results have been previously reported for the recombinant (r) rHVT-IBD vaccine, Vaxxitek® HVT+IBD, using two different IBD ELISA kits. It has been suggested that the classical IBD ELISA kits, coated with the whole IBD antigen, are unable to detect sufficiently the titre levels of VP2 antibodies produced after vaccination with a recombinant HVT IBD vaccine.

Dr van Leedam's paper highlights the results observed on a classical IBD ELISA after vaccination with the recombinant r-HVT-IBD vaccine and provides guidelines to assess successful vaccination and aid in the interpretation of field results.

More specifically it aimed to answer practical questions like: "Can I use a classical IBD ELISA to monitor r-HVT-IBD vaccines?” and "How does the ELISA serology relate to VN in detecting VP2 antibodies?" and "Can I differentiate between normal vaccination response and field challenge, using a classical IBD ELISA?”

Re-emergence of GA-07 Variant IBV in Clinical Cases of Flushing

Enteric and respiratory diseases cause major economic losses in the poultry industry. During 2011-2012, reported Vijay Durairaj, 12 clinical cases were submitted to the Poultry Diagnostic and Research Center at the University of Georgia with the primary complaint of slicked over floors, flushing, airsacculitis and high mortality.

Tracheas and kidneys were collected or submitted to the lab for histopathological evaluation and virus isolation.

Among these cases, the most common histopathological findings were described as multifocal lymphocytic infiltration in the kidneys.

Embryo lesions and mortality patterns in SPF chicken embryos inoculated with the field material were consistent with infectious bronchitis virus (IBV). IBV was confirmed by RT-PCR and characterised by sequence analysis of the IBV S1.

Phylogenetic analysis of the nucleotide and deduced amino acid sequences was performed and revealed that these isolates were closely related to nephrotropic infectious bronchitis viruses, specifically GA-07.

IBV GA-07 is a variant IBV previously isolated from clinical cases of flushing in commercial broilers, added Dr Durairaj and its re-emergence determined to be the cause of flushing in the 2011-2012 case submissions.

Heterotypic Protection to Infectious Bronchitis Virus

From Auburn University, Haroldo Toro reported the development of Newcastle disease virus (NDV) LaSota (rLS) expressing a distinct spike (S) protein gene of IBV. This recombinant vaccine technology confers cross-protection among different IBV strains.

Toro demonstrated that the recombinant construct maintains the biological properties of the parental NDV and provides complete protection against challenge with velogenic NDV.

Protection Induced by IBV S1 Gene Expressed from Recombinant Adenovirus

Recombinant viruses encoding the S1 protein of IBV population C2, which becomes selected in chickens after attenuated ArkDPI vaccination were also developed by Toro at Auburn. As an expression model, they used previously described replication-defective recombinant adenovirus vectors (AdIBVS1.C2ch).

Groups of SPF chickens were primed intramuscularly at three days of age and boosted via the ocular route at 20 days of age with AdIBVS1.C2_ch. All chicken groups except the unvaccinated and unchallenged control group were challenged on day 41 after hatch via eyes and nostrils with 10^6.5 EID₅₀ per bird of an Ark serotype virulent strain.

Evaluation of protection was assessed four days after challenge by clinical signs (incidence and severity of respiratory signs), concentration of IBV viral RNA in tears, and histopathological evaluation of the trachea.

Detection and Differentiation of IBV Vaccine Serotypes Using a Multiplexed Microsphere-Based Assay

Infectious bronchitis virus (IBV) causes an upper-respiratory tract disease in chickens and has a major economic impact on the commercial chicken industry worldwide.

Ha-Jung Roh of the University of Georgia explained that vaccination with live-attenuated virus is used to control the disease but, due to multiple serotypes and variants of IBV that do not provide cross-protection, it is extremely important to choose the right vaccine type based on current virus circulating in the field.

Virus isolation followed by a virus neutralisation (VN) test in embryonated eggs is considered the gold standard procedure for IBV serotyping of field samples. Molecular diagnostic tools, such as reverse transcriptase-polymerase chain reaction (RT-PCR)and nucleotide sequencing, have also been widely used for detection and typing of virus.

In an effort to improve on those diagnostic tests, the Athens-based researchers developed a multiplexed microsphere-based assay for simultaneous detection and differentiation of the five most common IBV serotypes identified in the USA; Arkansas (Ark), Connecticut (Conn), Delaware (DE), Georgia 98 (GA98), and Massachusetts (Mass).

Serotype-specific probes were designed targeting the hypervariable region of the spike glycoprotein.

Sensitivity and specificity of each probe in single-plexed and multiplexed assays were analysed and shown to be serotype-specific by mean fluorescent intensity (MFI).

To evaluate the assay as a potential diagnostic tool, blind tests using previously identified clinical samples were performed and the results were compared to previous nucleotide sequencing of these samples.

Roh and colleagues found that the assay results were in agreement with previous sequencing and demonstrated that this microsphere-based assay could be used for rapid detection and differentiation of IBV serotypes.

October 2013