Download Virus enhancement following infection with antibody-coated

Indian Journal of Experimental Biology
Vol. 39, December 2001, pp. 1314-1317
Virus enhancement following infection with antibody-coated
infectious bursal disease virus (IBDV) in chickens
Rajesh Kumar & Shiv Charan*
Deptt. of Veterinary Microbiology, College of Veterinary Sciences,
Chaudhary Charan Singh Haryana Agricultural University, Hisar 125 004, India
Received /6 March 2001; revised 13 August 2001
A novel concept of vaccination, employing virus-antibody complex has been reported for the control of infectious bursal disease in chickens. A comparison of virus replicati on, serum neutralizing antibody response and pathogenicity in chickens inoc ulated with the antibody coated virus, prepared by mixing virus and antibody in different ratios (1:1, 1:0.1, 1: 0.01)
and virus alone without antibody, has been made. Antibody coated virus (when mixed in certain crucial ratios) replicated to
a hi gher magnitude in the target organ, caused more severe pathogenesis but induced a primary serum neutrali zing antibody
response almost comparable. The results may have important implications in understanding of pathogenesis and development of co ntrol strategies against infectious bursal disease vi rus, specially employ ing immune complex vaccine.
Infectious bursal disease virus (IBDV) mainly infects
the B-cells of bursa of Fabricius. However, other
lymphoid organs e.g. caecal tonsils, thymus, spleen
and Harderian glands are also affected 1• The virus is
known to produce severe combined B- and T -cell
immuno-suppression 2 .3 .
To control IBD, both killed and live vaccines have
been used 45 . Recently, usc of virus-antibody complex
has been suggested as prophylactic measure6-8. Although , the exact mechanism of induction of protection by the antibody complex vaccine has not been
fully understood, it has been suggested that better
immune response with antibody complex vaccine
over the conventional vaccines is associated with delayed release of virus, lesser degeneration of lymphoid organs and higher localization of IBDV antigen
on bursal and splenic follicular dendritic cells 9 . In
these studies we have investigated the replication kinetics of antibody coated virus vs virus alone and we
report that the antibody-coated IBDV may lead to enhancement of the virus replication in vivo under certain conditions. This is probably the first report in
IBDV on antibody mediated enhancement of virus
replication in vivo. Therefore, these studies may have
important implications in the field applications of
immune-complex vaccines.
Chicks-Broiler chicks (18 days old; 80) from a
farm with the hi story of no outbreak were procured.
*Correspondent author: Phone: +91 1662 44460;
Fax: +91 1662 34952; E. mail: [email protected]
The chicks were tested for anti-IBDV antibody status.
For raisi ng polyclonal anti-IBDV antibodies 7-9
weeks old chickens were used.
Pre-immune status of chicks for IBD antibodyAlthough the chicks were obtained from a farm with
the history of no outbreak of IBD, the chicks were
tested for anti-IBDV antibody possibly for any
maternal antibody. The sera were collected randomly
from 5-6 chicks and pooled for the assessment of
antibody employing SN test and the fter was found to
be I: 160.
Virus-Georgia strain (intermediate) of IBDV was
procured from M/s Indovax Ltd, Hisar (Haryana) and
was grown and titrated in chickens embryo fibroblast
(CEF).
Titration of virus-Micro-titration method was
used for titration of the virus. Various dilutions of the
virus ranging from 10"1 to 10"8 were made in the
growth medium-199. 100 Jll of each dilution of the
virus was added to the well starting from highest dilution. This was followed by 100 Jll of cell suspension
(l x 106 cells/ml) to all the wells. Proper cell controls
were also set up simultaneously. The plates were observed daily for CPE. After 4-5 days supernatant was
discarded and the cells were stained with crystal violet (1 %). The virus titer was calculated by the method
of Reed and Munch (10). The titer of stock virus was
found to be 5x I 0 6 TCID 5ofml.
Production and titration of hyper-immune serumAntiserum against the Georgia strain of IBDV, propa-
NOTES
gated in CEF, was raised in 7-9 weeks old 6 chickens
by inoculating three doses of lx106 TCID 50 (approx.)
virus at 15 day interval by ocular-oral route. The
chickens were bled 10 days after the last injection.
Hyper-immune serum raised in White Leghorn chickens against Georgia strain grown in CEF was titrated
by serum neutralizing test and titer was found to be 1 :
5000 (approx.), whereas the serum from control
chickens had a titer of less than I :5. The antibody titer
of the hyper-immune serum against IBDV was further
confirmed in a commercial ELISA and was found to
be 5300.
Preparation of Virus antibody complex-For preparing virus-antibody complex for immunization, appropriate dilutions of anti-serum were mixed in equal
volume of virus 1x 105 TCID 50 . This virus antibody
was kept at room temperature (23°-25°C) for I hr
with frequent gentle mixing. Various preparations
were prepared so as to give l x l05 TCID 50 of IBDV
and unit activity of IBDV antibody so that the final
ratio of I : I; 1:0.1 ; 1:0.01 was achieved between
IBDV and unit activity of IBDV antibody (unit activity of antiserum has been defined as reciprocal of serum neutralizing antibody titer per milliliter of antiserum, ref 8).
Experimental design-Chicks (80) were divided
into 5 groups of 16 birds each. These groups were
inoculated with different ratio of virus and antibody
i.e. Group I with I: I , Group II with I : 0.1, Group III
with I: 0.0 I, Group IV with virus with normal chickens serum, while Group V with phosphate buffered
saline (PBS) alone. After 6 weeks these groups were
re-inoculated with the virus via ocular-oral route.
Various parameters i.e. clinical signs, mortality, gross
lesions, bursa/ body weight ratio (b:b ratio) including
microscopic examination of organs (bursa, thymus,
spleen) were recorded. In addition, virus isolation
from bursa specimen was al so done. Blood samples
were collected at 0, 3, 6, I 0, 15, 21 and 28 days after
primary infection as well as 5 and 10 days after second infection for serum neutralization test.
Bursal preparation for virus titration-Bursa\
samples from 2-3 birds at various time intervals from
Group I to V were pooled, grounded in Dounce's homogenizer in Hank's balanced salt solution (HBSS) to
make 10% (w/v ) homogeneous suspension and subjected to antibiotic treatment for I hr at 37°C. The
suspension was then subjected to three cycles of
freezing and th awing. Finally, it was centrifuged at
2000g for 15 min . The supernatant was passed
1315
through 0.22 Jl Millipore membrane. The titer was
determined as described earlier and expressed as per g
of bursal tissue.
Neutralization test (SN)-Constant virus varying
serum method was followed for performing SN test in
96-well micro tissue culture plates (Nunc, Denmark).
Sera were inactivated at 56°C for 30 min. in water
bath. Test was run in triplicate. Two-fold dilutions of
each serum was made starting from 1:10 to 1:5120 in
50 )li volume using medium M-199 . IBDV was diluted to give titer of 100 TCID 5o/50f.ll and added to
each well in 50 111 amount, except in wells marked for
cell control. In the wells marked for cell control , 100
111 of growth medium (M-199 supplemented with 10%
bovine serum, !-glutamine, sodium bicarbonate and
antibiotics) was added. The plates were incubated at
37°C for 1 hr to allow serum to neutralize the virus.
CEF cells were diluted in growth medium (M-199) to
give a concentration of 1x 106 cells/ml and added in
I 00 f.ll amounts in each well. The plates were incubated at 37"C in 5% C0 2 in air. After 5 days, the supernatant was discarded and cells were stained with
crystal violet and examined .
Microscopic examination of bursa- Samples of
bursal tissues were collected in 10% formal saline
from each group at various time post inoculation and
the tissue sections were examined after Haematoxi lin
and Eosin staining for comparison of di sease severity.
Virus titration in the bursa- The results of virus
titration in bursa (pooled from 2-3 birds) of chickens
inoculated with IBDV antibody complex or virus
alone at various time intervals have been shown in
Fig 1. The maximum virus replication was found in
Gp II inoculated with virus antibody ratio (1 :0.1). The
degree of virus replication was minimum in Gp I
which had maximum amount of antibody. The virus
replication in chickens inoculated with virus along
with normal chickens serum (Gp IV) or with virus
antibody ratio of 1:0.01 (Gp III) were almost similar.
A direct correlation between concentration of antibodies and inhibition of virus replication in bursa was
not seen in these studies, virus titers in bursae of Gp II
chickens reached highest peak with virus titre 7
log 1o/g (approx.) of tissue and was detectable till day
28. Thus, in Gp II ( 1:0.1) maximum titre of virus in
bursa suggests that probably thi s ratio of virus and
antibody was very crucial and may support enhanced
viral replication.
Serum neutralization (SN) antibody responseAfter primary immunization, SN antibodies appeared
INDIAN J EXP BIOL, DECEMBER 2001
1316
-o--Group I
:;"'"'
-ft-- Group Ill [v+Ab)1:0.01))
.0
~ Group
0
E -
g>
IV [v+N) Normal serum)
- * -Group V [PB Scontrol]
C)~
Q;
(v+Ab) ~ : 1)J
-<>-Group II [v+Ab)1:0.1)]
4
Q._J
~-
.,.
2
>
Days post inoculation
Fig. 1- Effect of coating infec ti ons bursal di sease virus with antibodies on in vivo replication of virus [Groups of chickens were
inoc ulated with 1x I 0 5 TCID 50 of virus coated with the antibodies
in different ratios (Group f, If, Ill) of virus alone in (Group IV) or
no virus (Group V) and in vivo replication of the virus was assessed at various time points between day 3 and day 28]
between day 10 and 15 post-inoculation and declined
to basal level between day 28 and 35 in all the virus
inoculated groups (I-IV). After secondary immunization, there was rapid increase in the level of SN antibody titers (2560) in I, III and IV groups, whereas in
Gp II where virus antibody ratio was (1 :0.1), SN antibody titers remained low (20) at day 5 and 10 days
post secondary immunization. This was the same
group where there was maximal replication of virus.
Gross and microscopic lesions-The group inoculated with IBDV alone showed mild lesions characterized by petecheal haemorrhages in thigh muscles.
However, the lesions were more severe in the groups
inoculated with virus antibody complex. The highest
degree of the haemorrhages in thigh and pectoral
mu scle and splenomegaly was found in Gp II at 15
day post infection . This was the same group in which
virus replication was maximal and SN antibody titers
were lowest after secondary immunization.
Similarly, histopathological lesions in the group of
chickens inoculated with virus alone, showed milder
reaction of heterophil infiltration and depletion of
lymphocytes in follicles of bursa of Fabricius between
day 3 and 6, as compared to the groups inoculated
with viru s antibody complex which showed moderate
to severe lesions characterized by atrophy of follicles
and necrosis of lymphocytes, thickening of interfollicular space due to connective ti ssue proliferation
and infiltration of reticular cells and leucocytes . The
max imum lesions were also found on day 15 as was
the virus titers in bursa. With the virulent strain of
IBDV, generally the maximum lesions and virus replication in the bursae have been reported around
day 4. This delayed reaction in the present studies
may be because of the use of vaccine virus and due to
the effect of coating with antibodies in these experiments .
In the present study, the virus replication, SN antibody responses and pathogenesis of the li ve IBDV
alone (as in case of a conventional live vaccine) and
after mixing with the antibodies (as in case of an immune-complex vaccine) were compared. It was observed that in chickens (Gp II) inoculated with the
virus coated with the antibody, there was about 100fold more virus replication and the severity of the lesions was markedly higher than in chickens inoculated with the virus alone. Primary SN antibody responses, however, were almost comparable in the
groups of chickens whether the virus was gi ven alone
or together with the antibodies. Interestingly, in the
chickens showing higher virus replication there was a
remarkably suppressed secondary SN antibody response. Such a remarkably lower secondary antibody
response may be attributable to degeneration of bursal
cells after primary inoculation since the target cells
for the replication of IBDV are bursal B-cell leading
to poor priming of B and/or T cells during immunization. The immunosuppressive feature of IBDV has
been well documented with respect to B-cells resulting into lower antibody responses against a number of
infectious agents and vaccines in chickens which have
been previously exposed to IBDV 2' 11.1 2 . Suppression
ofT cells responses as a result of IBDV infection has
been reported more recently and is of tran sient nature3.
Possibility of such phenomenon was recognized
si nce the findings that the sera from human immune
deficiency virus (HJV) infected individual can enhance HIV infection in vitro as well as in vivo where
antibody dependent enhancement could be demonstrated at high dilutions (up to I :65000) while SN antibody activity can rarely be demonstrated at dilution s
higher than 1: 1000 13' 14 • Similarly, in studies with feline immunodeficiency virus (FIV) enhancement of
FIV infection has been reported. The cats vaccinated
with FIV-env showed accelerated acute infection ,
early appearance of viraemia and greater vi ral load
15 16
after challenge than the control cats ' • In our studies, an enhanced virus replication in the group of
chickens inoculated with virus antibody ratio of I :0. 1
may reflect a similar phenomenon. To the best of our
knowledge this appears to be the first report with respect to IBDV .
NOTES
Thus, the present studies highlight the importance
of routine monitoring the level of IBDV antibodies in
commerci al flocks since lower levels of SN antibodies may rather support replication of IBDV . Therefore, acceleration or enhancement of IBDV infection
by antibodies during field vaccination employing live
vaccines and more particularly when immunecomplex vaccine are used could be very crucial. The
results of these studies will therefore provide a novel
bas is for understanding pathogenesis and formulating
effi cient vaccination program against IBDV critically.
The authors wish to thankfully acknowledge the
valuable help of Dr R P Gupta, Deptt. of Veterinary
Pathology for the interpretation of histopathological
slides. RK was a recipient of a scholarship from
Chaudhary Charan Singh Haryana Agricultural
Uni versity, Hisar.
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