INCO-DC ACTION 97
Concerted Action EC-CHINA(Contract no: IC18-CT98-0330)

ACUTE INFECTIOUS BURSAL DISEASE IN POULTRY :
EPIDEMIOLOGY OF THE DISEASE,
BASIS OF VIRULENCE AND
IMPROVEMENT OF VACCINATION

Poultry production is one of the fundamental industry of Chinese animal production. Although the total number and production of poultry are the first in the world, the per capita data is still low and the efficiency of the production is not high. According to the national statistical data the mortality rate of birds in China is 20-30%. The most important cause of death is infectious diseases, resulting in 60-90% of the loss, and especially those caused by viruses. Infectious Bursal Disease Virus (IBDV) is a major cause of infectious disease losses in Chinese chicken flocks due to high morbitity and mortality during acute infection and/or immunosuppression during a chronic course of infection. Immunosuppression enhances the susceptibility of chickens to other infections and interferes with vaccination against other disease.

The objectives of this project are to increase knowledge of the epidemiology of IBD by establishing common systems of diagnosis and epidemiosurveillance in order to reveal the incidence and prevalence of the acute and immunosuppressive forms of the disease in Asia and Europe. The use of generated tools in the ongoing research program should allow a better understanding of the pathogenesis and, therefore, a better control of the disease. The prevention and control of poultry infectious disease in China will help to catch up with the great development in intensive poultry production and will improve the supply of sufficient healthy meat and eggs for people.

The goals of the C.A. will be the following:

1. Selection and characterization of Chinese and European very virulent (vv) IBDV isolates following their pathogenicity (specific pathogen free (SPF) chicken), their antigenicity (monoclonal antibodies (Mabs)) and their genotype (RT-PCR and restriction enzyme (RE) analysis).
2. Development and adaption of the diagnostic tools (antigen capture (AC) ELISA; RT-PCR and RE analysis) based on the isolation and characterization procedure by selecting a reference panel of monoclonal antibodies (competition studies, epitope mapping), primers and restriction enzymes.
3. Analysis of the molecular epidemiology of vvIBDV based on Mabs, sequences and phylogenetic analysis. Comparison of the sequences with serotype I (classical, attenuated, variant, very virulent) and serotype II strains. Harmonize the nomenclature of IBDV strains based on molecular criteria.
4. Reveal markers for virulence (molecular markers) and pathogenesis (cell tropism, molecular basis of attenuation, cell mediated immune response, cytopathic effect) important for the development of effective vaccination procedures and diagnostic tools.
5. Constitution of an IBDV genomic cDNA bank and generation of relevant full length cDNAs that could be used in the reverse genetics system to generate genetically engineered recombinant infectious viral clones.
6. Application of the generated knowledge to diagnostic tools and vaccination procedures.

This C.A. will be open to other groups working in the field of IBDV, without having claim on financial support.

To achieve the objectives and goals, the project has been divided into five task groups, each divided into sub-tasks following the respective ongoing of the work. A flow chart illustrates the interactions between different tasks and participants.
 

 Task 1: Selection and characterization of reference material

1.1. Selection of vv IBDV strains isolated in Europe and in China
1.2. Classification following their
a. antigenicity (AC-ELISA with a panel of Mabs from different laboratories)
b. genotype (RT-PCR on VP2 gene followed by restriction enzyme analysis)
c. pathogenicity (cloning on embryonated SPF eggs; determination of EID5O; inoculation into
 SPF chicken).
Comparison of the obtained results with regards to pathogenicity, antigenicity and genotype with those from standard strains.
1.3. Development and adaption of the diagnostic system that will allow the determination of the incidence and prevalence of the acute and immunosuppressive forms of IBDV in China and to distinguish between classical virulent and very virulent strains.
1.4. Preparation of reference material for the distribution to each partner.

Task 2: Generation of a genomic data bank

2.1. Standardization and optimization of cloning and sequencing procedures with the help of well defined reference strains. The selection of primers, restriction sites and cloning vectors will be undertaken together in order to warrant suitable constructs for expression systems and the assembly of full length infectious cDNA clones.
2.2. Sequence analysis of the selected isolates with special attention on the 5' and 3' ends of both segments of the viral genome and on the VP5 gene. The Chinese partners will focus on segment A while the European partners will focus on segment B. The amplimers will be cloned in cloning vectors.
2.3. Generation of an IBDV data bank. Analysis of sequences and comparison with serotype I (classical, attenuated, variant, very virulent) and sertoype II strains.

Task 3: Studies on pathogenesis with respect to the development of diagnostic tools and vaccination procedures

3.1. Virulence markers: Search for specific markers for vvIBDV (antigenic (VP2, VP3) or pathotypic (rest of genome)) by alignements of the sequences with an appropriate (and standardized) analysis software.
3.2. Virus cell interactions: Identification of the virus-encoded and cellular domains responsible for the differences in cell tropism.
3.3. Role of macrophages: generated tools will be tested in vitro for the activation of macrophages and the ability to induce secretion of particular cytokines.
 3.4. Role of cellular immune response.
3.5. Cytopathic effects: analysis of the cytopathic effects (necrosis, apoptosis) induced by selected viruses.
3.6. Attenuation: vvIBDV will be attenuated by passages in SPF-eggs or cell culture. Attenuated strains will be sequenced and tested in the different in vitro systems.

Task 4: Generation of recombinant viruses by a reverse genetics system

4.1. Construction of full length cDNA clones.
4.2. Generation of recombinant viruses (reassortants, recombinants, mutants) on the basis of results obtained in tasks 1 - 3.
4.3. Evaluation of the recombinant viruses in vitro and in vivo according to methodology described in tasks 1 - 3 in order to identify sequences potentially responsible for antigenicity, virulence, attenuation and cell tropism.

Task 5: Application of the generated tools for the development of diagnostic tools and vaccination procedures

5.1. Evaluation of the protection induced after vaccination with viruses of different pathogenicity generated by reverse genetics.
5.2. Testing of plasmids encoding for different IBDV proteins in vivo for their protective potential.
5.3. In ovo vaccination and antigenicity of vaccines: the vaccination potential of different immune-
  complexes will be tested in embryonated eggs.

Progress reports will be made to the Commission after each meeting, with a final report to be delivered within four month after completion of the project. All participants will meet at the beginning of the project and subsequently each 9 (regionally) or 18 months (internationally).
 

Report of the First International Meeting (Jan99)

Report of the First  Regional Meeting (Europe Oct 99)

The virus particles

Symptoms of the disease

Structure of IBDV

Classification of IBDV

Sequence Data

Related Websites

Symptoms of the disease

The incubation period of IBDV in chicken is about 2 to 4 days. The first symptom in infected chicken is an acute onset of depression. Birds are disinclined to move and peck at their vents (photo). Affected chickens suffer from whitish or watery diarrhoea, anorexia, depression, ruffled feathers, trembling and severe prostration (Cosgrove, 1962). Feed intake is depressed but water consumption is elevated and they also become dehydrated and have a subnormal temperature. Affected flocks show depression for 5-7 days during which mortality rises rapidly for the first two days and then declines sharply as clinical normality returns (Parkhurst, 1964). Bursa, the primary target organ of the virus, is the first internal organ that shows lesions within 24 hours after infection (Helmboldt et al., 1964). The infected bursa has a gelatinous yellowish transudate covering the serosal surface as shown in photo. The transudate disappears as the bursa returns to its normal size and becomes grey in colour during the period of atrophy. At day 3, the bursa increases in size and weight because of edema and hyperemia. At day 4, the size of the bursa has usually doubled  its normal weight and then it begins to recede in size. At day 8, the bursa is usually one-third of its original weight. The infected bursa always shows necrotic foci and petechial haemorrhages on the mucosal surface. The spleen is slightly enlarged and has small grey foci which disperses uniformly on the surface (Lukert et al., 1991). As shown in photo, birds become dehydrated with darkened dislocation of pectoral muscles due to impairment of clotting mechanism. The infected kidney shows an  enlargement and pallor with accumulation of crystalline urate in tubules (Cosgrove, 1962).
 

Structure of IBDV

     The double stranded RNA genome of IBDV has two segments. The smaller (2.8 kbp) RNA segment (segment B) encodes VP1 (90 kDa), which is the viral RNA polymerase. The (3.1 kbp) larger genomic segment (segment A) encodes a 115 kDa VP2-VP4-VP3 precursor protein which is autocleaved into three polypeptides of molecular weights 41 kDa (VP2), 32 kDa (VP3) and 28 kDa (VP4). VP2 is the most abundant viral protein, making up 51% of serotype I IBDV protein. It is the major host-protective immunogen of IBDV which contains the antigenic sites responsible for the induction of neutralizing antibodies (Azad et al., 1987). The structural protein VP3 has a minor neutralization site (Jagadish and Azad, 1991). VP4 which represents 6% of the viral proteins is a protease responsible for the cleavage of the polyprotein into VP2, VP4 and VP3 (Duncan et al., 1987). Recently, a second open reading frame (ORF), preceding and partially overlapping the polyprotein gene was discovered. This ORF encodes VP5, a 17 kDa polypeptide shown to be present in IBDV-infected cells (Mundt et al., 1995).

Classification of IBDV

     Two serotypes of IBDV are recognised based on virus-neutralisation test. Serotype I viruses are pathogenic to chickens and vary in their virulence, whereas serotype II viruses isolated from turkey are apathogenic for both turkey and chicken (Lukert et al., 1991). Neutralizing antibodies to a serotype II strain (strain OH) cannot protect chicks from the challenge with a virulent serotype I (strain STC) strain (Lasher et al., 1994). Serotype II IBDV produces mild histological lesions in the bursa, spleen and Harderian gland of 1-day old SPF chicks (Sivanandan et al., 1986). The serotype I viruses are antigenically heterogeneous and can be divided into four groups: classical virulent strains, attenuated strains, antigenic variant strains and 'very virulent' strains.

     Classical virulent strains cause severe lymphoid necrosis and bursal inflammation in infected chicken and lead to immunodeficiency and moderate mortality. Mortality peaks in the third day of post-infection but death may still occur over the next 5 to 7 days giving a total flock mortality of up to 20%-30% in SPF chickens. Strains 52-70, STC and 002-73 belong to the classical virulent strains.

      Attenuated strains have been adapted to primary cell culture, such as chicken bursal lymphoid cells, chicken embryo kidney (CEK) cells and CEF cell (Lukert et al., 1991). Cell culture adapted IBDV strains have their virulence reduced and are used as attenuated live virus vaccines. However, attenuated IBDV strains may not induce immunity in chicken in the presence of high level of maternal antibodies because the IBDV strains are neutralized by maternal antibodies.

      In 1984-85, appearance of antigenic variants of IBDV were reported on the Delmarva peninsula (Rosenberger et al., 1986; Saif  1984). Antigenic variant strains are recognised by their abilities to escape cross-neutralization from the antiserum against classical strains (McFerran et al., 1980).  The strains are highly cytolytic and cause rapid bursal atrophy (within 72 d..p.i.) with minimal inflammatory response. There is no IBD symptom in 3-4 weeks old SPF leghorns (Rosenberger & Cloud, 1986; Rosenberger et al., 1987). A variety of field infections, especially those of the respiratory system, may arise from immunosuppression caused by the variant strains (Faragher et al., 1974). Some companies incorporated both classic and variant IBDV vaccine strains into commercial vaccine to enhance antigenic spectrum.

       The 'very virulent' strains, such as UK 661, spread rapidly throughout Europe, including U.K in the late eighties. In summer 1990, outbreaks of an acute infectious bursal disease with a high mortality rate occurred in broiler chicken flocks in western Japan. The disease spread dramatically throughout the country within the following 6 months and resulted in heavy economic losses in Japan. The initial outbreaks were characterized by high morbidity (80%) and correspondingly significant mortality, attaining 25% broilers and 60% pullets over a 7 day period (Chettle et al., 1985; van den Berg et al., 1991b; Nunoya et al., 1992). The European very virulent strains were regarded as classical serotype I strains because  antibody against classical strain could neutralize the very virulent strain in virus neutralization test. The new strains caused typical lesions of IBDV and were antigenically similar to the classical European strains, which had been prevalent for some decades. The infection caused completed loss of follicular architecture. The strains could establish infection in the presence of high levels of maternal antibody that were protective against 'classical' strains (Chettle et al., 1989) and cause up to 60-100% mortality (van den Berg et al., 1991).

International Cooperation (INCO)
 
 
 
 

Related Websites

All the Virology on the WWW
Office International des Epizooties (OIE)
National Institute of Animal Health(Japan)
THE "VIRTUAL" VETERINARY CENTER
World's Poultry Science Association (WPSA)
American Association of Avian Pathologists (AAAP)
Poultry Science Virtual Library