1.4.4 Disease diagnosis
1.4.4.1 Clinical and histological diagnosis
Clinical diagnosis is based on disease evolution (mortality peak followed by recovery in five to seven days), observation of symptoms and examination for specifically distinctive lesions, particularly in the bursa (Lukert and Saif, 1991). Diseases such as avian coccidosis, Newcastle disease, stunting syndrome, chicken infectious anaemia, mycotoxicoses and nephropathogenic forms of infectious bronchitis can be clinically mistaken for IBD. In subclinical cases, an atrophy of the bursa may be confused with Marek's disease or infectious anaemia,and requires histological examination for differentiation(McFerran, 1993).
Histological diagnosis is based on detection of changes to the bursa as described in Section 1.4.3.1. The ability to cause lesions in lymphoid organs such as the thymus, spleen or bone marrow (Inoue et al., 1999) is considered potentially diagnostic of vvIBDV. Although labour-intensive and requiring necessary experience, the advantage of histology-based diagnosisis that acute,chronic and subclinical formscan be distinguished.
1.4.4.2 Serological diagnosis
In areas where IBDV is endemic, most broiler flocks possess anti-IBDV antibodies which may be induced by pathogenic IBDV or by attenuated vaccines. Current serological tests cannot distinguish between these antibodies and are thus of little use for diagnosis. However, the quantification of anti-IBDV antibodies is important to 1), confmn the success of vaccination in laying hens, and 2), measure the titre of passive maternal antibodies in young chickens and thereby determine the appropriate date for vaccination (DeWit, 1999). Serology is likewise essential to confmn the disease-free status of SPF flocks.
The most commonly used quantitative tests are the detection of precipitating antibodies by gel immunodiffusion (Cullen and Wyeth, 1975), enzyme-linked immunosorbent assay (ELISA) (Marquardt et al., 1980) and serum neutralisation tests in cell culture (Weisman and Hitchner, 1978). Agar gel immunodiffusion is the simplest and cheapest but least sensitive assay, and results are only obtained after a 48-h incubation. Variability in results may depend on the particular investigator, as well as the viral strain used as antigen (Wood et al., 1984). Serum neutralisation tests have the disadvantage of needing specialised equipment and five days for incubation. These assays are much more sensitive than gel immunodiffusion and have better correlation with the level of protection of subjects tested (Weisman and Hitchner, 1978). The ELISA is the most rapid and sensitive method and presents the fewest variations due to the strain of viral antigen, although considerable variability can occur with certain commercial kits (Kreider et al., 1991). Although the correlation between serum neutralisation and ELISA is high, ELISA does not detect low neutralising titres of residual maternal antibodies, which are sufficient to hamper vaccine administration. ELISAs which use a recombinant VP2 protein as the sole antigen may be better correlated with protection (Jackwood et al., 1999).
1.4.4.3 Virus-based diagnosis
Infectious bursal disease virus may be detected in the bursa of Fabricius of chicks in the acute phase of infection, ideally within the first three days after the appearance of clinical signs.
The virus may be identifiedby its proteins,or by its nucleic acids, as described below.
a). Detection of viral antigens.
IBDV antigens may be detected in thin sections of an infected bursa by direct and indirect immunofluorescence (Allan et al., 1984), or by peroxidase staining in the bursal follicles.
The specificity of these tests is enhanced by the use of mAbs. Bursal suspensions can be used in gel immunodiffusion assays which examine the reaction of the test material with a specific
antiserum or mAb (Snyder et al., 1992), and may also be probed via agglutination tests.
These may utilise latex beads or sheep red blood cells coated with anti-IBDV antibodies (Nakamura etal., 1993; Nachimutu et al., 1995).
Viral antigens in bursal suspensions may also be captured and detected by sandwich ELISA, using an anti-IBDV antibody followed by an adapted anti-species enzyme conjugate (Snyder et al., 1988). The use of po1yc1onal serum for capture enhances the sensitivity of the test, and the use of mAbs in the capture or detection stages allows for more precise antigenic characterisation of the virus. Different batteries of mAbs enable a tentative identification of the United States variants (Snyder etal., 1988) and vvIBDVs (Eterradossi et al., 1997).
b). Detection of viral genome.
DNA probes labelled with 32p (Kibenge, 1992), biotin (Jackwood et al., 1990) or digoxigenin (Hatchcock and Giambrone, 1992) have been used on blots of infected tissues to detect the multiple virus strains of IBDV serotypes 1 and 2. No genomic probe enabling differentiation between variant viruses or vvIBDVs has yet been described, probably because of the high sequence homology among serotype 1 strains.
RT-PCR allows the detection of viral RNA in homogenates of infected organs or embryos and in cell culture, irrespective of viral viability. The choice of amplified zone is dependent on the objective, hence when the aim is to detect multiple strains of the virus, primers are selected from regions where the sequence is highly preserved (Wu et al., 1997). When characterisation of the amplified fragment is to allow for identification of different IBDV strains, the central, so-called 'variable' section of VP2 is generally chosen (Liu et al., 1994).
The fragment may then be characterised by direct sequencing and analysis of the analogous amino acid sequence. The simultaneous presence of four amino acid residues (Ala222, Ile256, Ile294, Ser299) is considered indicative of vvIBDV (Brown et al., 1994;Cao et al., 1998). In the study of genomic variations as a whole, RT-PCR with nucleotide sequence analysis is usually applied. Even though this method can obtain clearer genetic information, it is laborious, relatively expensive and not suitable for some microorganisms with large genomes using current sequencing techniques (Liu etal., 1994). Davis and Boyle (1990) and Lee etal.
(1992) adapted the RT-PCR test for detection of IBDV and Wu et al. (1992) used RT-PCR and sequencing to detect differences in IBDV isolates. Lin et al. (1993) were able to show differences between highly virulent and less virulent Japanese IBDV isolates using PCR and sequencing of the cDNA fragments.
The electrophoretic profile of the amplified fragment may also be examined after digestion with different restriction endonucleases (REs), which can be compared to detect any RFLPs (Jackwood and Nielsen, 1997; Liu et al., 1994). The absence of restriction sites for enzymes BstN I and Sty I, located respectively at codons 222 and 253 of the VP2 gene, has been correlated with an atypical antigenicity, such as that found in the variant virus strains from the United States (Jackwood and Jackwood, 1994; Jackwood and Nielsen, 1997). Liu et al.
(1994) have also showed that there are genetic variations among most IBDV isolates by RT- PCR-RFLP analysis. Determination of genomic variation is more sensitive and reproducible than the use of serum neutralisation tests for classification of viruses, hence RT-PCR followed by RFLP analysis offers additional information for classification of IBDV strains that are closely related (Liu et al., 1994), or indeed that are widely dissimilar (Jackwood and Sommers, 1997). Locally relevant RT-PCR and RFLP tests continue to be developed for detection and differentiation of IBDV in clinical samples (Katariaet al., 1998).
Virus isolation, electron nucroscopy, immunofluorescence, immunodiffusion, VIrUS neutralisation, ELISA and monoclonal antibody assays have all been used for the diagnosis of IBD (Lukert and Saif, 1991). These techniques suffer several disadvantages; they are time- consuming, labour-intensive, expensive, non-specific or insensitive. And importantly, these techniques do not have the ability to detect low levels of IBDV antigens in tissues or in matter such as fowl litter. Conversely, PCR is a most suitable identifying technique which is highly sensitive and specific, and has been successfully used to detect a vast number of significant viruses, including human immunodeficiency virus (HIV) type 1 (Guatelli et al., 1989), hepatitis B virus (Ulrichet al., 1989) and numerous agricultural pathogens.
1.4.5 Disease prevention and control 1.4.5.1 Exclusion and eradication
The extreme resistance of IBDV to physical and chemical agents (Benton et al., 1967) accounts for its persistence in the external environment despite disinfection, and as such, eradication seems unrealistic. Currently, prevention of IBD is a matter of strict hygiene standards and effective vaccination (Lasher and Shane, 1994). Sanitary precautions include 'all inlall out' farming methods, cleaning and disinfection of all equipment and premises, efficient fumigation and pest removal, and a rest period between depopulation and restocking. All bedding and dung must be eliminated and composted and under no circumstances should feed remaining from previous flocks be re-used (Lukert and Saif, 1991;McFerran, 1993).