PhD

2. Monoclonal fltl~ibodies (Mabs) were raised against the vaccine strain
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of OPY and three strains of OPY Viz, Vaccine (oPY - Y), IYRl (OPY -I) and
AlIeppy strain (OPY -A) were used to raise polyclonal serum in the present
investigation.
DPY - Y was revived in 11 day old chicken embryo and the embryo
death was recorded fout to five days PI with congestion all over the body
.
and spleen and necrotic foci in liver. The cytopathy in CEF cell culture
observed was rounding ami clumping of the cells, syncytium formation and
bridge fortnation with extensive vacuolation in the Cytoplasm. The
detachment of the cells was observed at 120 h PI.
DPY -I a virulent strain was inoculated in the ducklings, death was
tecorded in all the inoculated birds with extensive hemorrhages on Serous
membranes, muscles and visceral organs. Necrotic foci on liver,'
enlargement and congestiol1 of liver and spleen, and white hecrotic foci in
the gizzard were evident. The virus was further passaged ill DOE and


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cultivated in bulk in DEF cell culture.


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The DPY - Y and DPY -J\ were titrated in CEF cell culture and the
TCID 50 was 4.7 X 10 ~ per ml of the inoculum for DPY-Y and 3.2 X 104 for
DPV -J\. DPV -I cultivated in DEF cell culture had a TCIDso of 1 XI 0 5 'per ml
of the inoculum
All the strains were partially purified at 100000 g for 4.5 hat 4" C in
Beckman ultra centrifuge and the protein concentration of the virus was
estimated by biuret method and was found to be 11 mg, 8 mg and 7 mg for
DPV-Y, J\ and I respectively.
All the three strains of DPV were inoculated in rmce to raise
polyclonal serum. Four mice out of five inoculated with DPV-Y showed '
ELISJ\ titres more than l : 12800, one mouse showed a titre of I :6400. The
mice inoculated with DPY -A showed a titre of more than 1: 12800 and those
inoculated with DPV-I, 1 :~/WO
ELISA 'Was' used to test the sera samples of the mice inoculated with
DPV strains. The test was found to be highly sensitive, easy to perform
and less time consuming. The test therefore can be recommended for routine
diagnosis of DPY


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Polyclonal serum was used to study the cross reactivity of the three
strains of DPY with ELlSA. DPY -V polyclonal serum reacted with the
homologous virus with a titre of 1: 12800. It also showed similar titer with
other two heterologous strains. Polyclonal serum raised against DPY -A had a
titre of I: 12800 with homologous and heterologous strains of DPV: DPV-l
reacted with homologous strain at a titre of 1 ;1,)400. Similar titres were
observed with hcrtologous strains.
Immuno peroxidase test was used for the detection of the tissue
antigens in DPV infected CEF monolayers and in liver and spleen sections.
Polyclonnl scrum raised against DPV - V detected homologous virus in the
CEF monolayers and hetrologous virus in the liver and spleen sections. The



staining


reaction was observed us dark brown deposits at the VIruS



localization. However background tissue was also stained brown to faint
yellow in the stained preparation .
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The virus neutralization test was used to study the cross
neutralization by employing polyclonal serum raised against the three. strains
ofOPV. Polyclonal ~rUm raised against DPV-V showed a YNT 01'64 with a
VNI of 1.8 with homologous virus and a VNT 45 with VNI 1.65 with other
two strains of DPV. Polyclonal serum raised against DPY -A neutralized the
homologous virus at a titre of 32 with a VNI of 1.5. It also neutralized the

vaccine strain of IJPV with same VNT and VNI, However it neutralized
DPV-I \ ith VNT 24 and VNI 1.35. DPV-I polyclonal scrum neutralized the
homologous virus at a titre of 45 with a VNI of I.G5 . The same neutralized
DPV-V and DPV-A with a VNT 32 and VNI 1.5.
Monocloual Antibody Production
The plates were seeded with the feeder cells by collecting the spleen
cells from a normal healthy mouse. The same plates were used for seeding
the fused cells.
Fusion of the myeloma cells and the splenocytes from the donor
mice primed with the vaccine strain of DrV was under aken in presence of
poly ethylene glyco] ,1)~U 4000). Small colonies of the hybrids were
observed in the wells and the growth of these was monitored. As soon as the
hybrids attained n satisfactory growth i.e. ~n :-,e! ,_,:1 t coverage of the well the
hybrid supornatant was tested for antibody production with the help of
indirect ELlSA and the positive hybrids were selected. Cloning of the
positive hybrids was done by limiting dilution method to ascertain 1110110-
clonality.
A tnln! er seven clones producing desired antibodies against UPV
vaccine strains were grown and Mabs were used for testing the cross

reactivity of the three strains of DPV. Out of 7 Mabs tested Mab I reacted
with homologous virus with a titre of 32. It also reacted with other two
heterologous strains with the similar titre.
Mab 3, 4 and 6 had a titre of 16 with homologous as well as
heterologous virus strains of Dry and Mab 2, 5 and 7 showed a titre of 8
with all the strains ofDPY. No differences were recorded in the- EUSJ\ titres
of the 7 Mabs with homologous as well as heterologous strains of DPV. This
suggested that there may be a close antigenic and serologic relationship
within the three strains ofDPV under study.
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Immuno peroxidase test was used to detect the viral antigens in the
DPY infected CEF and in the liver and spleen tissue sections from the birds
infected with virulent Alleppy and IYRI viruses. All the seven Mabs detected
homologous as well as heterologous virus in the infected CEF an~ in the
liver and spleen sections. The reaction was very clear and background
staining was almost negligible. The viral antigen.deposits could be detected
as dark red brown deposits in the cytoplasm and nucleus of the cells.
All the seven Mabs were tested for cross neutralization and the results
indicated that, Mab 1 neutralized the homologous virus and had a VNT of 8
with YNI 0.9. It also neutralized the heterologous strains with similar YNT


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and VNI. Mab 2, 4 and 6 had a VNT of 6 with homologous virus and a VNI
0.79. These neutralized the other two virus strains with a VNT 4 and VNI
0.75.
Mab 3 had a VNT Of 2.4 with VNI 0.39 with homologous virus and a
VNT of 2 with VNI of 0.35 with the heterologous strains. However, Mab 5
and 7 did not neutralize the homologous as well as hetrologous viruses and
may be a non neutralizing.
The survivabiIity and longevity trial was conducted to ascertain the
appropriate time of collection of supernatant along with the usual parameters
like change in the pI-I and 80 per cent coverage of the well. The criterion
designated for the collection of the hybridoma supernatant like change of pH
and 80 per cent coverage of the well seems to ,be appropriate for deciding
the medium change and collection of the supernatant.
The conclusions from the present study are, in comparison with the
'polyclonal serum Mabs are highly specific in detection of the viral antigens
in the tissue sections infected with the virus and the background staining
reaction was completely reduced, thereby refining the test and making the
diagnosis very easy. The test can be recommended for the diagnosis ofDPV.
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As regards the ELISA test it has bee: I observed that the test is highly
sensitive , easy to perform and less time consunung and should be
recommended as routine diaFlloslic test for DPV.Y
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YNT is highly specific with polyelonal sera us well as with Mab. The
specificity of the test is increased fairly by using Mabs.
As regards the possible strain variation of the DPY it can be
concluded that there may not be any strain variation in the strains tested.
The probable reason for the break down of immunity and vaccination failure
maybe because of low titered vaccine used in the field for vaccination of
the birds or thc virus may be acquiring virulence by sub clinical passage in
the ducks in the field which might result in the breakdown of immunity and
failure of the vaccine.
A potent tissue culture adapted vaccine may be useful in solving the
problem.