IBDV Vaccine Research Articles

Adjuvanting a subunit novel variant IBDV vaccine to induce protective immunity

Adjuvanting a subunit novel variant IBDV vaccine to induce protective immunity

Development of egg yolk-based polyclonal antibodies and immunoprophylactic potential of antigen-antibody complex against infectious bursal disease

A study conducted in the Faisalabad district sampled 50 cases across five IBD outbreaks, revealing an alarming 80 % infection rate among poultry. Our research focused on developing an immune complex (Antigen-antibody complex) with potential immunoprophylactic benefits to counter this formidable threat. Our study was based on producing egg yolk-derived polyclonal antibodies (IgY) targeting IBDV. Commercial layer birds were immunized with inactivated IBDV, yielding IgY antibodies extracted from their eggs exhibited substantially higher and more enduring antibody titers, with a geometric mean titer of 104. Further research involved the creation of an immune complex (ICx) where antigen was extracted from infected bursae tissues. The immunogenic response of ICx was assessed in poultry birds after a challenge with a virulent strain of IBD virus and compared to a conventional IBDV vaccine in poultry. Results revealed significantly higher and more enduring antibody titers induced by the ICx, offering enhanced protective immunity against the IBDV challenge, as evidenced by lower Bursa to bodyweight ratios in vaccinated birds.

Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals.

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7–100.0) and 94.17% specificity (95% CI: 88.4–97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

Alteration of immunological parameters in infectious bronchitis vaccinated–specific pathogen-free broilers after the use of different infectious bursal disease vaccines

The vaccines currently available to control infectious bursal disease (IBD) include live-attenuated and inactivated vaccines, immune-complex vaccines, and vaccines consisting of viral constructs of herpesvirus of turkeys genetically engineered to express VP2 surface protein. To evaluate the impact of vaccines on the chicken immune system, 2 animal trials were performed in specific pathogen-free broiler chickens. In trial 1, birds were either vaccinated when they are one-day old with a dual recombinant herpes virus of turkey construct vaccine, expressing VP2 protein of (IBDV) and F protein of Newcastle disease virus, or an immune-complex IBDV vaccine or birds were not vaccinated. At 14, 28, and 35 D, the bursa of Fabricius was collected for bursa:body weight (B:BW) ratio calculation. In trial 2, birds were vaccinated when they were 1-day old according to the same protocol as trial 1, but at day 14, all groups also received a live infectious bronchitis (IB) vaccine. At 0, 7, 14, 21, and 28 days after IB vaccination, birds were tested by ELISA for IB serology and, soon after the last blood sampling, they were euthanized for collection of Harderian glands, trachea, and spleen and testing by flow cytometry for characterization of mononuclear cells. The immune-complex vaccine groups showed significantly lower B:BW ratio, lower IBV antibody titers, and higher mean percentage of CD8+ T cells in the spleen, trachea, and Harderian glands than those in the other experimental groups. The results of the in vivo trials coupled with a depth analysis of the repertoire of parameters involved in the immune response to IBD and IB vaccinations show one vaccine may influence the immune response of other vaccines included in the vaccination program.

Algal beta-(1,3)-glucan and its effect on infectious bursal disease vaccination in poultry

Vaccination is used as a control and prevention tool for infectious bursal disease (IBDV) in poultry. A new strategy for improving vaccination efficiency is the use of in-feed immune modulating ingredients. The aim of this study was to test if the use of beta-(1,3)-glucans in feed can enhance IBDV vaccination. The trial was conducted in ROSS 308 broilers. In the study, a negative control group (not vaccinated, not supplemented), a positive control group (vaccinated, not supplemented) and a treated group (vaccinated, supplemented with beta-(1,3)-glucan) were included. All broilers, except the negative control, were orally vaccinated at 18 days of age with a live IBDV vaccine. Blood samples were taken at day 18 and 35 to measure antibody titers against IBDV. Serological analysis showed the presence of maternal derived antibodies at time of vaccination in some birds. The beta-(1,3)-glucan supplemented birds showed decreased CV% (coefficient of variation) and significantly increased average antibody titers compared to non-supplemented vaccinated birds. Additionally, the beta-(1,3)-glucan group showed increased number of birds with antibody titers above the titer threshold for protective immunity. The results obtained in the current trial clearly indicate that beta-(1,3)-glucan can increase seroconversion and serological response to IBDV vaccination.

Molecular characterization of field isolates of infectious bursal disease virus from three decades, 1987-2018, reveals a distinct genotypic subgroup in Vietnam.

The complete nucleotide sequence of the viral protein 2 (VP2) ORF was determined for 26 Vietnamese infectious bursal disease isolates collected from clinical outbreaks in vaccinated flocks from 1987 to 2018 and two commercial vaccine specimens. These sequences were compared for molecular classification with 42 reference strains representing all four main classes of serotype 1, including very virulent (vvIBDV), classical (cvIBDV), antigenic variant (avIBDV) and attenuated (atIBDV) strains, and serotype 2 strains. Amino acids at nine key positions in the VP2-HVR in 20 Vietnamese isolates, A222, I242, Q253, I256, D279, A284, I294, S299, A329, which are typical of the vvIBDV class, were found to be identical in all of the isolates. Eighteen of these isolates had a unique change at residue 212 (D212N) located in the PAB loop. Phylogenetic analysis revealed a distinct lineage/subclade with strong nodal support (96%) that included recent Chinese IBDV strains that were distinct from typical vvIBDVs. Six isolates contained the amino acid substitutions P222, V242, Q253, V256, D279, A284, I294, N299, A329, which are present in two vaccine strains derived from strain 2512 and these isolates were also closely related to the classical virulent STC strain. Data from this study show that there is considerable genetic diversity among vvIBDVs, which vary according to geographic region. Antigenic drift and differences in genetic characteristics between virulent strains and IBDV vaccine strains may be the cause of vaccine failure. Better antigenic matching of vaccines to the strains circulating in Vietnam is therefore required.

Clinicopathological Assessment of Broiler Chickens Challenged with Field Infectious Bursa Disease Virus after Oral Vaccination Using Phytogenic Mucoadhesive Gum

The rising socio‐economic loss incurred from mortalities and disharmonized vaccine control programs in poultry production has necessitated the search of adjuvants especially of natural origin. More so, productivity levels of poultry in many African countries fall far below desirable levels because of the economic losses incurred from chick mortality due to infectious bursa disease (IBD). This study determine the level of immune response conferred by Infectious bursa disease vaccine using a mucoadhesive delivery system from a phytogenic origin using the clinic‐pathological response of broilers vaccinated oraly with IBD vaccine using Khaya senegalensis and Cedrela odorata as phytogenic adjuvants and later challenged with the IBD virus. A total of 260 chicks were successfully brooded and raised from day old. The birds were distributed into four groups A to D with an average of 60 birds per group in a complete randomized block design, as A (Gum + IBD Vaccine oral + Virus challenge), B (IBD Vaccine alone oral + Virus challenge), C (Gum alone oral + Virus challenge) and D (No‐Vaccine‐No‐Gum + Virus challenge). Three birds were euthanised per Group for Serum, tracheal and intestinal washings and lymphoid tissue harvest including haderian gland, thymus, lungs, spleen, ileum, cecal tonsils and bursa of Fabricius pre and post vaccinations and post infection during the 7 weeks experiment. Data was analysed using Graph pad prism 21 at α=0.05. All the birds were apparently healthy pre and post vaccinations. The birds exhibited signs of picking and soiling of the vents and feathers with projectile yellowish diarrhoea few birds post infection (B, D). The feathers were ruffled and mortality varied from 0 to 5 in the non‐vaccinated and gum alone vaccinated birds. Pathologically, there was oedematous, hyperplastic to haemorrhagic changes in the bursa, congestive to hyperplastic changes in the spleen with severity in group D and haemorrhages of breast and pectoral muscles in some of the birds that died post infection. No recumbency was recorded in the birds given gum+IBD vaccine and challenged with live virus. Prophylactic application of IBDV vaccines and the Khaya senegalensis gum as phytogenic adjuvants enhanced the chicks immune response and provided protection against IBD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation and comparison of immune responsiveness to sheep red blood cells, PHA-P and IBDV vaccine in divergent stocks of chicken

A total of 90 birds comprising two native breeds viz. Aseel and Kadaknath and one synthetic broiler stock i.e. IBL-80 were utilized to evaluate and compare antibody response to Sheep Red Blood Cells by haemagglutination test at 0, 5 and 10 days post primary inoculation, to study in vivo cell mediated immune response to mitogen Phytohaemagglutinin (PHA-P) and to evaluate immune responsiveness to IBDV vaccine. The presence of natural antibodies against SRBC was evident in all the genetic groups. All groups showed an increase in HA titre upto 10 days post immunization. The HA titre on 10 day was significantly higher in Aseel (1.88±0.10) followed by IBL-80 (1.13±0.05) and Kadaknath (1.09±0.06). However, the differences among Kadaknath and IBL-80 at day 10 PPI were failed to attain statistical significance. The in vivo cell mediated response to mitogen was highest in Aseel (0.68 mm) followed by IBL-80 (0.59 mm) and Kadaknath (0.43 mm).There was significant difference between the layer breeds for response to phytoheamagglutinin but IBL-80 was not significantly differ from both Aseel and Kadaknath. The titre values for IBDV were lowest before immunization and got increased during 7, 14, 21 DPI. At 14 DPI the titre value were significantly different in all the breeds in which Aseel exhibited the highest titre value (2.96±0.04) followed by IBL-80 (2.77±0.09) and Kadaknath (2.64±0.06). It was found that at 21 DPI antibody response was highest in all the breeds, however differences in titre value at 21 DPI in different breeds were not significantly different.

Immune-responsiveness and performance of broiler chickens fed black cumin (Nigella Sativa L.) powder

The main objective of this study was to determine the effects of various levels of dietary black cumin seed (BCS) on immune-responsiveness, broiler performance and lymphoid organs’ ratio scores against NDV, IBV and IBDV vaccines. One hundred and sixty-one day-old broiler chicks (Rose 308) were randomly assigned into five groups with three replicates of seven birds each (21 chicks per group) and fed diets supplemented with 0.7%, 1.4%, 2.1% or 2.8% black cumin. All chickens were bled weekly for 35days post-treatment. Body weights, thymus, bursa and spleen ratio scores were determined at 21 and 35days while body weight was determined weekly. The results revealed non-significant differences in body weight between all groups compared to the control group. Antibody titers against NDV also exhibited non-significant differences at third, fifth and sixth weeks of the experiment between treated groups while they were significantly different from the control group. At the fourth week, group C showed significantly different antibody titers from other treated groups. However, antibody titers against IBV were significantly different in the fifth and sixth weeks against the control group. Similar results were recorded with IBDV vaccine compared to the control group while group C had significant differences in titers at 4, 5 and 6weeks post-vaccination. Lymphoid organs, namely thymus, bursa and spleen showed non-significant differences between groups. This study showed that dietary supplement of black cumin seed at the level of 1% or 1.4% would enhance immune responsiveness in broiler chickens.

Effects Of Mixed Vaccinations Against Newcastle Disease And Infectious Bursal Disease On Immune Response, Feed Consumption And Weight Gain In Broilers.

The effect of mixed vaccinations against Newcastle disease (ND) and Infectious bursal disease (IBD) on immune response, feed consumption and weight gain were studied in 21 days old broilers. The trial was carried out to ascertain the safety of this practice which is intended to reduce labour, cost and stress associated with chasing and catching during individual bird vaccinations in large flocks. The two diseases are among the common viral diseases found in Nigeria; a country with an expanding poultry population that is creating major impact in its meat industry. Their prevention is largely by vaccination and vaccinations with live vaccines can precipitate reactions. A total of 120 broilers were assigned into 4 groups (G1, G2, G3 and G4) of 30 chickens each at 21 days of age. Chicken in G1 were vaccinated with ND vaccine, La Sota which was given singly at 21 days of age and repeated at 35 days of age. Chickens in G2 were vaccinated with both ND vaccine, La Sota and IBD vaccine, which were given simultaneously at 21 days of age and repeated at 35 days of age. Chickens in G3 were vaccinated with both vaccines but the vaccines were given singly with ND vaccine given at 21 days of age and repeated at 35 days of age while IBDV vaccine was given at 28 days of age and repeated at 42 days of age. Chickens in G4 were vaccinated with IBDV vaccine only which was given at 21 days of age and repeated at 35 days of age. Serum samples were collected from 10 chickens in each group for serology. The chickens in the groups were monitored for feed consumption and weight gain. Results showed that mixed vaccination of the chicken was well tolerated. Mixed vaccination did not affect immune response of the chickens. There was no significant difference (p > 0.05) in both feed consumption and weight gain among chickens in the different groups.

A single amino acid in the C-terminus of VP3 protein influences the replication of attenuated infectious bursal disease virus in vitro and in vivo

The very virulent infectious bursal disease virus (vvIBDV) Gx strain causes over 60% mortality in chickens but cannot replicate in CEF cultures. The attenuated Gt strain, however, is not virulent in chickens and replicates well in CEF cultures. The two strains display differences in 6 amino acids in VP4 and 4 amino acids in VP3. To determine whether VP4 and VP3 are involved in the virulence and replication of IBDV, three chimeric viruses, in which the VP4/VP3/3′UTR, VP3/3′UTR or VP4 region of Gt were replaced by the corresponding region of Gx, were constructed and characterized in vitro and in vivo. The substituted regions in VP4 or VP3 did not affect virulence of Gt. While the substituted region in VP4 had no effect on viral replication of Gt in CEF cultures, substitution of the VP3/3′UTR region did reduce the replicative capacity of the virus. Through site-directed mutagenesis, three rescued recombinant viruses with a single amino acid substitution in the C-terminus of VP3 of the Gt strain (L981P, A990V and T1005A) were characterized in a similar manner. Amino acid substitution at position 990 reduced viral replication of Gt and reduced its efficacy of protection against vvIBDV Gx challenge in vivo. This study provides important information for the design and development of more effective IBDV vaccines using reverse genetics.

BAFF enhances B-cell-mediated immune response and vaccine-protection against a very virulent IBDV in chickens

Infectious bursal disease (IBD) is an acute, highly infectious and immunosuppressive disease caused by IBDV, which specifically targets destruction of B cells in the bursa of Fabricius. B-cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important factor for B-cell proliferation and survival. Here we demonstrate that human soluble BAFF (hsBAFF) may enhance humoral immune response by elevating B lymphocyte activity of secretion of immunoglobulin (Ig) such as IgA, IgM and IgG in chickens immunized or unimmunized with an inactivated IBDV vaccine from a very virulent strain. Of importance, we found that hsBAFF, as a co-immunostimulant for vaccination, may play a vital role in amplifying the specific protective immune response, thereby potently preventing very virulent IBDV challenge. This is supported by serological evidence that hsBAFF may effectively enhance higher specific IgG activity and titre in serum of immunized chickens. The findings strongly suggest that BAFF may be exploited in combination with specific vaccination for prevention of IBD.

Field efficacy trial of a novel HVT-IBD vector vaccine for 1-day-old broilers

Two vaccination programmes for infectious bursal disease (IBD) were compared in broiler chickens with maternal immunity, placed on two farms. A turkey herpes virus (HVT)-IBD vector vaccine was administered by the subcutaneous route, at the hatchery, into the chicks of farm A at the age of 1 day. On farm B, an attenuated intermediate live IBD vaccine was given orally at the ages of 17 and 24 days. The vaccine uptake was monitored via serology and bursa/body weight ratio evolution, as well as PCR-based viral IBDV detection in the bursa of Fabricius at various time points. It was also verified by an experimental very virulent IBDV challenge performed at the age of 30 days in birds transferred from the farms with appropriate control groups in a laboratory. An immunity gap was observed in birds from farm B between the decay of the passive and the rise of the active immunity based upon serological data. The level of protection against challenge is not possible to establish in this farm as the reduction of the bursa/body weight ratio observed could be due to the residual pathogenicity of the vaccine strain or the challenge as well. This immunity gap was not present on farm A showing higher serological titres at the ages of 26 and 45 days via a suitable ELISA test and 93% protection against the very virulent challenge at the age of 30 days was observed. The maternal immunity interfering with the live IBDV vaccine replication had no detectable effect on the vector vaccine take.

Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity

Three classic IBDV strains were previously isolated from commercial layer chicken flocks and shown to be phylogenetically related to vaccine strains but pathogenic in susceptible chickens. In this study, their viral genomes were sequenced and compared to sequences of vaccines being used in those flocks. The vaccine strains examined were sequenced directly from the manufacturer and had identical genome segment B sequences. Compared to these vaccines, the GA-1, H-30 and CS-2-35 isolates each had one silent mutation in the gene that encodes VP1. Compared to the two vaccines used at the time CS-2-35 was isolated, the segment A sequence of CS-2-35 contained numerous nucleotide and amino acid mutations suggesting the CS-2-35 virus was not closely related to these vaccines. This virus however did have amino acid mutations in VP2 that are reported to be necessary for replication in cell culture and lacked two of the three amino acid mutations previously shown to be necessary for virulence. These data suggest that CS-2-35 was a descendant from an attenuated strain of IBDV. When the segment A genomic sequences of the GA-1 and H-30 viruses were compared to the vaccines being used in those flocks they were most closely related to the attenuated D78 vaccine strain. In genome segment A, three nucleotide mutations in GA-1 and four in H-30 were observed compared to the D78 classic vaccine. These nucleotide mutations caused one amino acid (H253N) change in the GA-1 virus and two amino acids (H253Q and G259D) were different in the H-30 virus. In addition, both the GA-1 and H-30 viruses had the amino acid G76 in VP2 that appears to be unique to the vaccine D78. The data suggest that GA-1 and H-30 are genetically related and have a common ancestor even though they were isolated from geographically distant flocks. The evidence also suggests that GA-1, H-30 and CS-2-35 could be reversions from attenuated vaccine viruses or by coincidence genetically resemble classic IBDV vaccines. It should be noted that some of the classic virus vaccines were not being used according to the manufacturer's recommendations at the time the GA-1, H-30 and CS-2-35 strains were isolated. Together, the molecular and pathogenicity data indicate that a single amino acid mutation from Histidine (H) to Glutamine (Q) or Asparagine (N) at position 253 in VP2 will markedly increase the virulence of an attenuated IBDV.

DNA vaccination with VP2 gene of very virulent infectious bursal disease virus (vvIBDV) delivered by transgenic E. coli DH5α given orally confers protective immune responses in chickens

The efficacy of different doses of oral DNA vaccines carrying VP2 gene of vvIBDV delivered by E. coli DH5α was studied and compared with purified VP2 recombinant expression plasmid DNA vaccine injected intradermally and whole virus vaccine either from homologous virus or from commercial source. The recombinant plasmid pRc-VP2 was transformed in a non-pathogenic strain of E. coli, the DH5α and designated as EC/pRC-VP2. Oral immunization of maternal antibody free broiler chickens at 7 and 14-day-old with different dosages of EC/pRc-VP2 elicited specific humoral immune response as measured by ELISA. Protection in different groups was calculated through clinical signs, gross and histopathological lesions, bursa of Fabricius to body weight ratio, humoral and cellular immune responses and mortality in the chickens. Vaccination with EC/pRc-VP2 at the dose rate of 10 9 CFU per chicken conferred 95.4% protection of the chickens against the challenge with homologous virulent field strain of vvIBDV. Protection afforded by attenuated vero cell adapted UAF-06 strain of vvIBDV was comparable (94%) to that by EC/pRc-VP2 and pRc-VP2 vaccines, which was significantly higher ( P < 0.05) than the protection provided by a commercial attenuated IBDV stain D-78 vaccine (D-78 vaccine was used as positive control due to its frequent use in the field for vaccination of poultry chickens) and other control groups in the study. The results revealed that DNA vaccines against IBDV may be successfully done by adopting bacterial-vectored oral delivery system and vaccination with homologous vvIBDV (UAF-06) conferred significantly higher protection as compared with imported non-homologous commercial IBDV vaccine.

Protection capability of recombinant plasmid DNA vaccine containing VP2 gene of very virulent infectious bursal disease virus in chickens adjuvanted with CpG oligodeoxynucleotide

In the present study the efficacy of recombinant plasmids DNA vaccine encoding VP2 gene of very virulent strain of infectious bursal disease virus (vvIBDV) isolated from Pakistan was investigated with or without coadministration of cytocine-phosphate-guanine oligodeoxynucleotide (CpG ODN) to protect the chickens against the disease. VP2 gene of vvIBDV was successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR) and was cloned into eukaryotic expression plasmid vector, which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenopartite leader sequences and SV-40 polyadenylation signal, and this was designated as pRc-VP2. Seven-day-old maternal antibodies free chickens were intramuscularly injected with 500 μg of pRc-VP2 with or without CpG ODN twice at 1-week interval. At the age of 21 days the broiler chickens were challenged with 10 5 EID 50 of homologous strain of IBDV and observed for 14 days post-challenge. Immunization with pRc-VP2 plus CpG ODN conferred protection in 93% of the chickens as evidenced by the absence of clinical signs, atrophy of bursa of Fabricius (BF) and mortality followed by the group vaccinated with attenuated IBD vaccine and boosted with killed oil based IBDV vaccine, which conferred 90% protection. The protection of chickens injected with pRc-VP2 alone was 67% where as only 20% of the chickens in the negative control group were protected. However, enzyme-linked immunosorbent assay (ELISA) antibody titre in the group vaccinated with pRc-VP2 plus CpG ODN were significantly higher ( P < 0.05) than the group vaccinated with pRc-VP2 alone as well as the group vaccinated with commercial attenuated IBDV vaccine boosted with commercial oil adjuvanted killed IBDV vaccine. Responsiveness to a mitogenic lectin, phytoheamagglutinin-P was significantly reduced in group immunized with conventional vaccines (live boosted with killed) as compared to all the other groups ( P < 0.05). The results revealed that co-administration of recombinant plasmids with CpG ODN could protect chickens efficiently from IBDV challenge.

Detection of Serum Antibody Levels Against Infectious Bursal Disease (IBD) Virus Using Indirect Hemagglutination (IHA) Test in Commercial Broilers

A total of 336 Serum samples were collected from different commercial broiler farms and slaughter shops from in and around Faisalabad. These samples were divided in to three groups according to the age of the birds i.e. 0-3 weeks (86 samples), 3-6 weeks (68 samples) and 6-8 weeks (182 samples). Indirect hemagglutination (IHA) test was used to titrate the serum antibodies against infectious bursal disease virus. Hyper immune serum was raised in laboratory rabbits against IBDV vaccine (D-78). It was used to identify and confirm the IBD virus from bursal tissues isolated from the IBD affected flocks using the qualitative tests i.e. agar gel precipitation and modified counter immuno-electrophoresis. The viral antigen was sonified and used to sensitize the Human O erythrocytes without any coupling agent. 1% sensitized erythrocytes suspension was prepared and used to determine the antibody titer. Results indicated that out of 336 test serum samples 221 (65.77%) were positive. The maximum positive samples belonged to 6-8 weeks of age (78.57%) followed by 3-6 weeks (60.29%) and 0-3 weeks (43.02%), respectively. Calculated geometric mean titers for groups 0-3, 3-6 and 6-8 wks of age, were found to be 6.37, 10.84 and 16.14, respectively.

Development of a vaccine for immunization against classical as well as variant strains of infectious bursal disease virus using reverse genetics

Available IBDV vaccines based on classical IBDV strains do not induce full protection in the presence of maternally derived antibodies (MDA) against variant strains. The aim of this study was the generation of an IBDV chimera between both classical and variant strains. Characterization of the generated chimeric IBDV (D78-Chim) was performed in both cell culture and susceptible chickens using the classical vaccine strain D78 for comparison. Growth kinetics on cell culture showed that the D78-Chim replicates comparable to the live vaccine strain D78. Administration of the D78-Chim to SPF chickens resulted in equally high virus neutralizing antibodies titres against both, classical and variant IBDV strains. In vivo experiments in commercial chickens revealed that D78-Chim and D78 induced comparable bursal damage in presence of maternally derived antibodies. Both D78-Chim and D78 viruses were able to breakthrough a similar level of MDA.

Susceptibility of cell lines to avian viruses

The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain) was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA. CPE and viral RNA were detected in CER and BHK-21 cells after reovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV.