International Journal of Poultry Science 4 (4): 239-243, 2005
ISSN 1682-8356
© Asian Network for Scientific Information, 2005
Detection of Infectious Bursal Disease Virus in Field Outbreaks in Broiler
Chickens by Reverse Transcription-Polymerase Chain Reaction
D. Mittal, N. Jindal*, S.L. Gupta, R.S. Kataria1 and A.K. Tiwari2
Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary Sciences,
CCS Haryana Agricultural University, Hisar-125 004, India
1
National Bureau of Animal Genetics Resources, Karnal, India
2
National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar, India
*E-mail: nareshjindal1@rediffmail.com
Abstract: During the period from July 2002 to June 2003, infectious bursal disease (IBD) was suspected
in 101 commercial flocks of broiler chickens on the basis of clinical and post mortem findings in some
districts of the Haryana state, India. Bursal samples were collected randomly from 20 flocks for the detection
of infectious bursal disease virus (IBDV) by reverse transcription- polymerase chain reaction (RT-PCR) and
nested PCR assay. IBDV could be detected in 17 samples as evidenced by amplification of 643 bp fragment
of the very variable region of VP2 gene of virus by agarose gel electrophoresis. The authenticity of the
amplicons was further confirmed by nested PCR generating amplicons of 552 bp using internal primers. The
results of the present study indicated that RT-PCR followed by nested PCR can be used to diagnose field
outbreaks of IBD in poultry because of its rapidity, accuracy and sensitivity.
Key words: Infectious bursal disease, diagnosis, RT-PCR, broiler chickens
antibodies (Eterradossi et al., 1997), where as, VP3 is
considered as a group specific antigen as it is
recognized by monoclonal antibodies directed against
VP3 from strains of both serotypes 1 and 2 (Becht et al.,
1988). VP4 is involved in the processing of 110 kDa
precursor polyprotein (Jagadish et al., 1988).
Conventional diagnosis of IBD relies upon clinical
findings, pathological changes, and virological and
serological methods such as agar gel precipitation test,
electron microscopy, fluorescent antibody test, counterimmunoelectrophoresis,
indirect
ELISA,
serum
neutralization test etc. The application of molecular
techniques like reverse transcription- polymerase chain
reaction (RT- PCR) as a tool for the diagnosis of IBDV
infection has been reported in the past (Lee et al., 1994;
Jackwood and Jackwood, 1997; Banda and Villegas,
2004). The present work aims at detection of IBDV from
field outbreaks in broiler chickens in Haryana state, a
North Western state of India.
Introduction
Infectious bursal disease (IBD) also known as Gumboro
disease, is an acute, highly contagious viral disease of
young chickens. The causative agent is infectious bursal
disease virus (IBDV) which belongs to the genus
Avibirnavirus with in the family Birnaviridae (Dobos et al.,
1979). It is a nonenveloped icosahedral, bisegmented,
double stranded RNA virus with a diameter of about 5560 nm (Ismail and Saif, 1990). This virus can be
differentiated into two serotypes by virus neutralization
test (McFerran et al., 1980). Serotype 1 contains the
pathogenic strains where as serotype 2 strains are not
pathogenic to chickens (Ismail and Saif, 1990).
Pathogenic serotype 1 IBDV field strains can be grouped
into classical, antigenic variant and very virulent (vv)
strains (Brown et al., 1994).
The genome of IBDV consists of two segments of
dsRNA. The larger segment A (3.2 kb) encodes for viral
proteins VP2, VP3 and VP4, which are produced by auto
proteolysis of a 110 kDa precursor polyprotein from a
single large open reading frame (ORF) (Hudson et al.,
1986). Segment A also encodes for putative VP5, a 17
kDa protein, encoded from a small ORF, partly
overlapping the polyprotein ORF has recently been
reported to play role in cell lysis and release of virus
(Lombardo et al., 2000). Genome segment B (2.9 kb)
encodes for a 90 kDa RNA dependent RNA polymerase
protein, VP1 (Azad et al., 1985). VP2 and VP3 are the
major structural proteins of the virion. VP2 is major host
protective antigen of IBDV and contains the antigenic
region responsible for the induction of neutralizing
Materials and Methods
Collection of samples: During the period from July 2002
to June 2003, IBD was suspected in 101 commercial
broiler flocks on the basis of clinical signs and
postmortem findings. Bursal tissues from 20 affected
flocks were collected from Hisar, Fatehabad, Sirsa,
Bhiwani and Jind districts of Haryana State, in 50%
buffered glycerine for diagnosis by RT-PCR. One
sample consisted of two to five bursa pooled from the
affected birds in a flock. The samples after collection
were kept at -20oC till use. Four IBD vaccines (Georgia,
239
Mittal et al.: Detection of Infectious Bursal Disease Virus
MB, Intermediate and Intermediate Plus) currently used
in this region were included as positive control. Bursa
samples collected from apparently healthy unvaccinated
birds served as negative control.
amplicons (643 bp) was carried out using forward
primer5’-CGCTATAGCGCTTGACCCAAAAA-3’
(nucleotide position 651- 673) and reverse primer- 5’CTCACCCCAGCGACCGTAACGACG-3’
(nucleotide
position 1179-1202) amplifying 552bp internal
sequence of the very variable region of VP2 gene of IBDV
as described by Kataria et al. (1998). Primary amplicons
(1µl) of 643 bp diluted 1:10 in nuclease free water were
used for nested PCR. Other reagents and cycling
conditions were same as for primary PCR except the
annealing temperature was 62oC.
Total RNA extraction: Total RNA was extracted directly
from the bursal tissues and the vaccines using TRIzol
reagent (Life Technologies, USA) according to the
manufacturer’s protocol. The volume of TRIzol used for
the vaccine strains corresponded to that of the distilled
water required for vaccine reconstitution. Briefly, 500µl of
TRIzol reagent was added to approximately 100mg
bursal tissue and homogenized using pestle and
mortar. The homogenate was centrifuged at 12,000rpm
for 15 minutes. The supernatant was collected and
mixed with 200µl of chloroform. The aqueous phase
was separated by centrifugation at 12,000rpm for 15 min
at 4oC. The RNA in the aqueous phase was precipitated
with 500µl of isopropanol and collected by centrifugation
at 12,000rpm for 10 min. After washing with 70%
ethanol, the pellet was dissolved in nuclease free water
and stored at -20oC until used.
Results and Discussion
Clinical findings and post mortem changes: In most of
the affected flocks, the birds revealed the signs and
symptoms of dullness, depression, anorexia, ruffled
feathers, inability to move followed by death. There was
yellowish white or greenish yellow diarrhoea in most of
the affected birds. Mortality due to the disease increased
with the progression of disease and peaked at third and
fourth day and then started declining.
In almost all the flocks, the postmortem lesions were
observed in bursa of Fabricius. The changes in bursa in
acute form of the disease included edematous and
swollen bursa, presence of gelatinous exudate around
bursa, with hemorrhages (Fig. 1). In addition, severe
hemorrhages on thigh and pectoral muscles were also
recorded (Fig. 1). In the chronic form of the disease, the
bursal changes comprised of atrophy and/or presence
of cheesy core inside the bursa. The hemorrhages on
muscles were of milder degree in sub-acute form of
disease and were mild or absent in chronic form of
disease. In some of the flocks, hemorrhages at the
junction of proventriculus and gizzard were also
recorded. Besides these, swollen kidneys and
enlargement of liver were also noticed during
postmortem examination of the broiler chicks. More or
less similar types of signs and symptoms and/or post
mortem findings have been reported earlier (Mohanty et
al., 1971; Lukert and Hitchner, 1984; Jindal et al., 2004).
Reverse Transcription- Polymerase Chain Reaction
(RT-PCR): The total RNA extracted was subjected to
reverse transcription using 100ng random hexamer
primers, 50ng heat denatured viral RNA, 50units
RNAase inhibitor, 2µl of 0.1M DTT, 1µl of 10mM dNTPs
mix, 4µl of 5X RT buffer and 200 units Superscript II
reverse transcriptase (Life Technologies, USA). The 20
µl reaction mixture was incubated at 25oC for 10min and
then at 42oC for 50min. Reverse transcriptase was
inactivated by heating at 70oC for 15min. The
oligonucleotide
primers
forward5’TCACCGTCCTCAGCTTAC-3’ (nucleotide position 587604) and reverse- 5’-TCAGGATTTGGGATCAGC- 3’
(nucleotide position 1212-1229) described by Liu et al.,
(1994) were used for the amplification of 643 bp
amplicons corresponding to very variable region of the
VP2 gene of IBDV. For the amplification, 6µl of cDNA was
incubated in total volume of 50µl reaction mix containing
5µl 10X PCR buffer, 20pmol each of the forward and
reverse primers, 1µl of 10mM dNTPs mix, 3U of Taq
DNA polymerase (Bangalore Genei, India). The
incubation temperature and duration of each cycle of the
PCR were 1min at 94oC for denaturation, 1min at 52oC
for annealing and 1min at 72oC for extension. The
amplification was carried out for 35 cycles with final
extension at 72oC for 10 min.
Diagnosis by RT-PCR: Infectious bursal disease virus
could be detected in 17 of the 20 field samples and in all
the four vaccine strains by RT-PCR. The primary PCR
amplicons yielded a single specific band of 643 bp on
ethidium bromide stained 1.5% agarose gel without any
amplification in apparently healthy unvaccinated bursal
sample (Fig. 2a and 2b). Further confirmation by nested
PCR amplification resulted in expected 552 bp product
in all the primary PCR positive field samples and the
vaccine strains. However, there was no amplification in
the negative control after nested PCR (Fig. 3a and 3b).
Infectious bursal disease is one of the major problems
faced by poultry industry in India and is responsible for
considerable economic losses. In Haryana state, the
disease has been recorded regularly in broiler chickens
Confirmation of PCR Products: The PCR products (5µl
aliquots) were separated on a 1.5% agarose gel stained
with ethidium bromide. For determining the DNA
segments size, the 100bp DNA marker (Bangalore
Genei, India) ranging from 100 to 1000 bp was used. For
further confirmation, the nested PCR of the primary
240
Mittal et al.: Detection of Infectious Bursal Disease Virus
inspite of regular vaccination. During the period from July
1994 to June 2003, the disease affected 8.89% (795)
flocks in Hisar and adjoining areas in the state and was
recorded both in the vaccinated and unvaccinated flocks.
The cumulative mortality and case fatality rates during
the nine year period due to this disease ranged from
2.47- 4.40% and 48.60- 70.55%, respectively (Jindal et
al., 2004).
The conventional methods routinely used for diagnosis
of IBDV infection are laborious, time consuming and
less sensitive. Hindrance for virus isolation is that, most
of very virulent field isolates do not replicate in common
tissue culture (van den Berg et al., 1991), where as for
virus neutralization test, the field strains need to be
adapted to grow in vitro. Furthermore, there is always a
risk of modification of antigenic and pathologic
characteristics of the virus during adaptation procedure
(Lukert and Saif, 1997).
Knowledge of the molecular make up has led to the
development of more sensitive and specific tests. In the
present study, efforts were made to assess the
diagnostic potential of highly sensitive technique of
reverse transcription- polymerase chain reaction (RTPCR) on clinical field samples. Seventeen of the 20
bursal samples were found positive for IBDV by RT-PCR
using very variable region of VP2 gene confirming the
presence of IBDV.
Total RNA extracted from infected bursal tissue
homogenates by TRIzol reagent yielded sufficiently pure
RNA for RT-PCR. Random hexanucleotide primers
instead of specific primers used for reverse transcription
of viral RNA had an advantage of producing random
cDNA fragments which could be amplified by any set of
the primers. Use of random primers increases the
sensitivity of PCR. Complete denaturation of double
stranded RNA to convert it into single stranded prior to cDNA synthesis is critical during RT-PCR. Many workers
have used denaturants like dimethyl sulphoxide (DMSO)
or methyl mercuric hydroxide (MMH), instead of heat
denaturation alone in order to increase the sensitivity of
the test. Vakharia et al. (1992) used MMH to denature
double stranded RNA prior to cDNA synthesis. Qian and
Kibenge (1994) found heat denaturation at 65oC for 90
min along with DMSO to be superior to heat denaturation
alone at 65oC for 10 min. In this study, heat denaturation
of the double standard RNA (dsRNA) in boiling water
bath at 95oC for 5 min followed by snap chilling in ice
prior to reverse transcription without using highly toxic
denaturants like methyl mercuric hydroxide (MMH) or
dimethyl sulphoxide (DMSO) was found to be sufficient
for VP2 gene amplification. More or less similar
observations were also made by Kataria et al. (1998).
Also the heat denaturation of template cDNA in the
reaction mixture prior to addition of Taq DNA polymerase
insured improved efficiency and stability of the enzyme
over longer cycling period. The problem of non specific
Fig. 1: Hemorrhagic bursa and hemorrhages on thigh
muscles in a bird suffering from infectious
bursal disease.
2a
643bp
2b
643bp
Fig. 2 a, b: Agarose gel electrophoresis of primary PCR
products of field samples (1-20) and four
vaccine strains (Intermediate Plus, I+;
Intermediate, IM; Georgia, G and MB)
showing amplicons of 643bp.
Lane M : 100 bp DNA molecular size marker
Lane-ve : negative control
241
Mittal et al.: Detection of Infectious Bursal Disease Virus
3a
apparently healthy bursal tissue taken as negative
control. The specificity of RT-PCR for diagnosis of IBDV
was further confirmed by nested PCR. Nested PCR has
been considered to be more sensitive for IBDV than
conventional RT-PCR (Liu et al., 2002). The RT- PCR
followed by nested PCR can be used as a tool for the
diagnosis of IBDV infection (Jackwood and Jackwood,
1994; Lee et al., 1994; Tham et al., 1995) because of the
existing rapidity, accuracy and sensitivity of these
methods. These nucleic acid based techniques are
useful tool for detecting IBDVs as the virus can be
detected and typed without isolation and propagation in
cell cultures or embryonated eggs, even when the virus
is present in very minute quantity and has lost its
infectivity. The application of these techniques on more
numbers of samples followed by further studies using
restriction enzyme analysis and sequencing will be
helpful in generating epidemiological information in
order to formulate a vaccination strategy for effective
control of the disease.
552bp
3b
References
Azad, A.A., S.A. Barett and K.J. Fahey, 1985.
Characterization and molecular cloning of the
double stranded genome of an Australian strain of
infectious bursal disease virus. Virol., 143: 35-44.
Banda, A. and P. Villegas, 2004. Genetic characterization
of very virulent infectious bursal disease viruses
from Latin America. Avian Dis., 48: 540-549.
Becht, H., H. Muller and H.K. Muller, 1988. Comparative
study on structural and antigenic properties of two
serotypes of infectious bursal disease virus. J. Gen.
Virol., 69: 631-640.
Brown, M.D., P. Green and M.A. Skinner, 1994. VP2
sequences of recent European 'very virulent'
isolates of infectious bursal disease virus are
closely related to each other but are distinct from
those of 'classical' strains. J. Gen. Virol., 75: 675-80.
Dobos, P., B.J. Hill, R. Hallet, D.T.C. Kells, H. Becht and
D. Teninges, 1979. Biophysical and biochemical
characterization of five animal viruses with
bisegmented double-stranded RNA genomes. J.
Virol., 32: 593-605.
Eterradossi, N., D. Toquin, G. Rivallan and M. Guittet,
1997. Modified activity of a VP2-located neutralizing
epitope on various vaccine, pathogenic and
hypervirulent strains of infectious bursal disease
virus. Arch. Virol., 142: 255-270.
Hudson, P.J., N.M. McKeru, B.E. Power and A.A. Azad,
1986. Genomic structure of the large segment of
infectious bursal disease virus. Nucleic Acid Res.,
14: 5001-5012.
Ismail, N.M. and Y.M. Saif, 1990. Differentiation between
antibodies to serotype 1 and 2 infectious bursal
disease viruses in chicken sera. Avian Dis., 34:
1002-1004.
552b
Fig. 3:a, b: Agarose gel electrophoresis of nested PCR
products of field samples (1-17) and four
vaccines
(Intermediate
Plus,
I+;
Intermediate, IM; Georgia, G and MB)
showing amplicons of 552 bp
Lane M : 100 bp DNA molecular size marker
Lane-ve : negative control
amplification observed initially was over come by setting
the annealing temperature to 52oC for primer pair #1.
The magnesium ion (Mg++) concentration of 1.5mM was
found most suitable with the primer set. However,
Jackwood and Sommer (1998) reported the requirement
of 2-4 mM Mg++ concentration for IBDV PCR.
The RT-PCR technique thus standardized could easily
be used for the amplification of very variable region of
VP2 gene of IBDV field isolates. The authenticity of PCR
products by the size of the amplicons has been verified
by other workers too (Lin et al., 1993; Liu et al., 1994;
Kataria et al., 1998). The specificity of RT-PCR was
confirmed by absence of amplification in unvaccinated
242
Mittal et al.: Detection of Infectious Bursal Disease Virus
Jackwood, D.J. and R.J. Jackwood, 1994. Infectious
bursal disease viruses: molecular differentiation of
antigenic subtypes among serotype 1 viruses. Avian
Dis., 38: 531-537.
Jackwood, D.J. and R.J. Jackwood, 1997. Molecular
identification of infectious bursal disease virus
strains. Avian Dis., 41: 97-104.
Jackwood, D.J. and S.E. Sommer, 1998. Genetic
heterogeneity in the VP2 gene of infectious bursal
disease viruses detected in commercially reared
chickens. Avian Dis., 42: 321-339.
Jagadish, M.N., V.J. Staton, P.J. Hudson and A.A. Azad,
1988. Birnavirus precuson polyprotein is processed
in Escherichia coli by its own virus encoded
polypeptide. J. Virol., 62: 1084-1087.
Jindal, N., N.K. Mahajan, D. Mittal, S.L. Gupta and R.S.
Khokhar, 2004. Some epidemiological studies on
infectious bursal disease in broiler chickens in
parts of Haryana, India. Int. J. Poult. Sci., 3: 478-482.
Kataria, R.S., A.K. Tiwari, S.K. Bandyopadhyay, J.M.
Kataria and G. Butchaiah, 1998. Detection of
infectious bursal disease virus of poultry in clinical
samples by RT-PCR. Biochem. Mol. Biol. Int., 45:
315-322.
Lee, L.H., L.J. Tingh, J.H. Shien and H.K. Sheih, 1994.
Single tube non-interrupted reverse transcriptionPCR for the detection of infectious bursal disease
virus. J. Clin. Microbiol., 32: 1268-1272.
Lin, Z., A. Kato, Y. Otaki, T. Nakamura, E. Sasmaz and S.
Ueda, 1993. Sequence comparisons of a highly
virulent infectious bursal disease virus prevalent in
Japan. Avian Dis., 37: 315-23.
Liu, H., J.J. Giambrone and T. Dormitorio, 1994.
Detection of genetic variations in serotype 1 isolates
of infectious bursal disease virus using polymerase
chain reaction and restriction endonuclease
analysis. J. Virol. Meth., 48: 281-291.
Liu, J., J. Zhou and J. Kwang, 2002. Antigenic and
molecular characterization of recent infectious
bursal disease virus isolates in China. Virus
Genes, 24: 135-47.
Lombardo, E., A. Maravet, I. Espinosa, A. FernandezArias and J.F. Rodriguez, 2000. VP5, the nonstructural polypeptide of infectious bursal disease
virus, accumulates within the host plasma
membrane and induces cell lysis. Virol., 277: 345357.
Lukert, P.D. and S.B. Hitchner, 1984. Infectious Bursal
Disease. In: Diseases of Poultry, eds. Hofstad M. S.,
H. J. Barnes, B. W. Calnek, W. M. Reid and H.
W.Yoder Jr., 8th edn. Iowa State University Press,
Ames, USA, pp: 566-576.
Lukert, P.D. and Y.M. Saif, 1997. Infectious Bursal
Disease. In: Diseases of Poultry, eds., Calnek B.W.,
H.J. Barnes, C.W. Beard, I. R., McDougald and Y.M.
Saif , 10th edn. Ames, IA: Iowa State University
Press, pp: 721-738.
McFerran, J.B., M.S. McNulty, E.R. McKillop, T.J. Connor,
R.M. McCracken, D.S. Collins and G.M. Allan, 1980.
Isolation and serological studies with infectious
bursal disease viruses from fowl, turkeys and
ducks: demonstration of a second serotype. Avian
Pathol., 9: 395-404.
Mohanty, G.C., A.P. Pandey and B.S. Rajya, 1971.
Infectious bursal disease in chicken. Curr. Sci., 40:
181-184.
Qian, B. and F.S.B. Kibenge, 1994. Observation on
polymerase chain reaction amplification of
infectious bursal disease virus ds RNA. J. Virol.
Meth., 47: 237-242.
Tham, K.M., L.W. Young and C.D. Moon, 1995. Detection
of infectious bursal disease virus by reverse
transcription-polymerase
chain
reaction
amplification of the virus segment A gene. J. Virol.
Meth., 53: 201-212.
Vakharia, V.N., B. Ahmed and J. He, 1992. Infectious
bursal disease virus structural proteins expressed
in a baculovirus recombinant confer protection in
chickens. J. Gen. Virol., 74: 1201-1206.
van den Berg, T.P., M. Gonze and G. Meulemans, 1991.
Acute infectious bursal disease in poultry: isolation
and characterisation of a highly virulent strain. Avian
Pathol., 20: 133-143.
243