Download Identification of Biuetongue Virus-specific Immunoglobulin E in Cattle

J. gen. Virol. (1987), 68, 2509-2514.
Printed in Great Britain
2509
Key words: bluetongue virus/IgE/type I hypersensitivity
Identification of Biuetongue Virus-specific Immunoglobulin E in Cattle
By G A R Y A. A N D E R S O N , 1. J E F F R E Y L. S T O T T , 2
L A U R E L J. G E R S H W l N 2 AND B E N N I E I. O S B U R N 2
l Department of Veterinary Science, IANR, University of Nebraska-Lincoln, Lincoln, Nebraska
68583 and 2School of Veterinary Medicine, University of California-Davis, Davis, California 95616,
U.S.A.
(Accepted 28 May 1987)
SUMMARY
A modified passive cutaneous anaphylaxis test and an ELISA were used to identify
IgE in calves vaccinated (sensitized) with chlorine dioxide-inactivated bluetongue
virus (BTV) and in calves inoculated with infectious BTV. The levels of IgE were
greatest in the vaccinated calves after challenge with infectious virus, which correlated
with development of clinically apparent dermatitis and stomatitis. These findings
suggest that some aspects of clinical bluetongue disease in cattle may have an
immunopathological mechanism mediated by IgE (type I hypersensitivity).
Various immunological and immunopathological mechanisms may be initiated during the
course of a viral infection. Although much is known about humoral and cellular immunity in
many viral infections, evidence of the importance of immediate (type I) hypersensitivity
reactions in viral diseases is scanty. High concentrations of IgE have been correlated with the
development of allergic disease in man. Frick et al. (1979) determined that viruses could initiate
immunological pathways to induce allergic sensitizations to viruses and pollens in children,
probably by altering populations of T and B lymphocytes. Additionally, Ida et al. (1977) found
an enhancement of IgE-mediated histamine release from human basophils by viruses, in
conjunction with interferon.
Homocytotropic antibodies were demonstrated in cattle (Wells & Eyre, 1970) and bovine IgE
has been characterized as a distinct Ig class analogous to human IgE (Hammer et al., 1971).
Gershwin & Dygert (1983) developed an ELISA to measure relative serum concentrations of
total IgE in cattle. The 72 h passive cutaneous anaphylaxis (PCA) test has been utilized for
identification and/or quantification of antigen-specific bovine IgE. Ovalbumin, horse serum,
rabbit serum albumin, methyl cellulose, Fasciola hepatica, and Micropolysporafaeni are antigens
that have been shown to invoke IgE responsiveness in cattle (Black et al., 1975; Doyle, 1973;
Gershwin, 1981 ; Gershwin & Olsen, 1984; Hammer et al., 1971 ; Wells & Eyre, 1970; Wilkie et
al., 1978). In this report, we describe the identification of virus-specific IgE in calves vaccinated
(sensitized) with inactivated bluetongue virus (BTV) serotype 17 and challenged with infectious
virus and in calves inoculated with infectious BTV serotype 17 without prior vaccination/sensitization. Our results indicate that a type I hypersensitivity may be important in the dermatitis
and stomatitis associated with bluetongue disease in cattle.
The calves used in this study were housed in insect-secure facilities and determined to be free
of BTV by the agar gel immunodiffusion test and failure to isolate the virus. Twelve calves were
divided into four groups as follows: vaccinated/sensitized (six calves), adjuvant control (two
calves), challenge control (two calves) and animal control (two calves). The vaccinated/sensitized group was divided further into three groups: two calves received vaccine with levamisole
phosphate at 3.3 mg/kg body wt, two calves received vaccine with cimetidine hydrochloride at
3.3 mg/kg body wt, and two calves received only vaccine (vaccinated controls). The vaccine
consisted of chlorine dioxide-inactivated BTV emulsified in an aluminium hydroxide adjuvant
(obtained from Dr T. L. Barber, USDA/ARS, Denver, Co. U.S.A.). Vaccinated and adjuvant
control calves were given two inoculations of the appropriate preparation 5 weeks apart. In
0000-7729 © 1987 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 12:05:11
2510
Short communication
addition, immunomodulators (levamisole or cimetidine) were given to four of the six vaccinated
but not to the vaccinated control or adjuvant control calves.
All calves, except the animal controls, were challenged with virulent BTV serotype 17 5 weeks
after the second vaccination/sensitization. Each calf was inoculated subcutaneously with blood
from an experimentally infected sheep, and a total of 2-5 x 105 5 0 ~ embryo lethal doses
(ELDs0) were given to each animal. All calves inoculated with virus became viraemic.
Heparinized blood samples were collected by jugular venipuncture weekly, prepared as
previously described (Foster & Luedke, 1968) and inoculated into embryonating chicken eggs
for BTV isolation. Challenge controls were viraemic by 7 days post-challenge (p.c.) and
remained viraemic for an average of 29 + 10 days p.c. Sensitized calves were also viraemic at 7
days p.c. and remained viraemic for an average of 48 + 14 days p.c. Clinical disease was
apparent in five of the six vaccinated/sensitized calves after challenge with infectious BTV;
furthermore, four of the five clinically affected animals had received immunomodulators. The
clinical signs apparent on day 11 p.c. were characterized as an exudative dermatitis and an
ulcerative stomatitis. Histologically, the lesions were infiltrated by mononuclear inflammatory
cells, with eosinophils being prominent in the dermatitis. Challenge control calves and one calf
that had been vaccinated/sensizited but not immunomodulated also exhibited inflammation
within the skin and oral mucosa, but the inflammation was only apparent microscopically.
Weekly serum samples from each calf were stored at - 20 °C until tested. BTV-neutralizing
antibodies were quantitatively determined by plaque reduction (Stott et al., 1978), and nonneutralizing, precipitating antibodies were detected by agar gel immunodiffusion. BTV groupspecific precipitating antibodies (IgG) were detected in all sensitized calves 7 to 14 days
following the second vaccination/sensitization with inactivated BTV. Minimal virus neutralization titres (titres of 1:10 to 1:40) were detected in some vaccinated/sensitized animals, and
similar low level titres were present in some control calves. Following live virus challenge, virusneutralizing antibody titres developed in all vaccinated/sensitized and challenge control calves.
Vaccinated/sensitized calves tended to have lower neutralizing antibody titres than the
challenge controls. Peak titres (28 to 42 days p.c.) in vaccinated/sensitized calves ranged from
2.5 x 10 3 to 4.0 x 104, as compared to peak titres of 1.0 x 104 to 1.6 × 105 in challenge controls.
All vaccinated/sensitized animals continued to have group-specific antibodies, and the
challenge controls developed these antibodies 7 to 21 days p.c.
BTV-specific IgE was determined by modified PCA tests (Gershwin, 1981; Wilkie et al.,
1978) and an indirect ELISA (Gershwin & Dygert, 1983; Hfibschle et al., t981 ; Poli et al., 1982).
BTV serotype 17 (62-45-S strain obtained from Dr T. L. Barber) was plaque-picked three times
from agar-overlaid Vero cells. Virus was purified as previously described, except for
centrifugation through sucrose density gradients (Huismans et al., 1979; Verwoerd, 1969). Cell
culture virus was extracted three times in fluorocarbon and Sephadex G-200, and the combined
water phases were extracted in a mixture of Tween 80 and ether. The virus was pelleted through
4 0 ~ sucrose, resuspended in 0.002 M-Tris buffer pH 8.6 and stored at 4 °C. For use in the PCA
and ELISA tests, the protein concentration of the virus was adjusted to 50 ~tg/ml and 5 ~tg/ml,
respectively (Bradford, 1976).
The modified PCA tests were performed in calves as Prausnitz-Kfistner tests with the
addition of intravascular dye injection. Briefly, the hair was clipped on three recipient calves
and intradermal injections of 0.2 ml of pre-inoculation sera, selected test sera and heatinactivated (4 h at 56 °C) test sera, respectively, were made in the skin. After 72 h, Evans' blue
dye (20 ml of a 2.5 ~ solution in phosphate-buffered saline, PBS) was injected intravenously and
0.2 ml of partially purified BTV was injected at each serum-inoculated site. In addition, selected
serum-inoculated sites were injected with 0.2 ml of a control antigen (50 ~tg/ml) prepared
similarly to the viral antigen but from uninfected Vero cells. Histamine at 100 ~tg/ml, BTV
antigen, Vero cells, 0.002 M-Tris buffer pH 8.6 and PBS pH 7.4 were injected (0-2 ml) into skin
sites not inoculated previously with test sera. The oedema and blueing were recorded 30 min
after challenge, and the reactions were evaluated 0 to 5 +. Weals scored as 1 + measured at least
0.5 cm in diameter, and 5 + weals were 2.5 to 3.0 cm in diameter.
The results of PCA tests with pre-inoculation sera and sera collected at selected intervals
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 12:05:11
Short communication
2511
Table 1. BTV-specific PCA reactivity in calves
Vaccinated/sensitized
Controls
¢
Vaccination/
sensitization
•
+ Levamisole
+ Cimetidine
Calf
1
Calf
5
Calf
2
Calf
6
Controls
Calf
9
I
Adjuvant
Calf
10
Calf
I1
Calf
12
Challenge
Calf
13
Calf
14
ND
0*
0
0
0
0
0
0
0
0
ND$
14
35.~
0
0
0
0
0
0
0
0
0
ND
ND
49
4+
0
0
0
5+
0
3+
0
ND
0
ND
0
0
0
0
ND
ND
ND
ND
56
Post-challenge
0§
7
14
21
28
35
42
3+
0
4+
2+
0
2+
ND
ND
ND
ND
0
4+
0
4+
0
3+
0
2+
0
0
2+
3+
0
0
ND
ND
0
0
0
0
4+
4+
5+
2+
5+
4+
3+
3+
3+
2+
2+
2+
4+
2+
3+
2+
0
0
0
0
0
2+
0
0
0
0
0
2+
ND
3+
2+
2+
ND
1+
0
2+
ND
0
0
ND
ND
0
ND
3+
3+
ND
0
0
ND
Nt~
* First day of vaccination/sensitization.
t ND, Not determined.
:~Day of second vaccination/sensitization.
§ Day of challenge with virulent BTV.
during the experiments are shown in Table 1. Pre-inoculation sera from all these calves failed to
sensitize the skin of recipient calves, and heat inactivation (56 °C for 4 h) of the PCA-positive
sera eliminated the ability to elicit a P C A reaction. Positive reactions were commonly observed
at sites injected with sera from the vaccinated/sensitized group, particularly the immunomodulated calves. Three of six and four of six sera were positive 14 and 21 days after the second
vaccination/sensitization, respectively. The sera from three o f the four immunomodulated
calves and one of the two vaccinated controls were positive at 7 days p.c. and remained positive
to day 42. The greatest responses were consistently observed with sera collected 7, 14 and 21 days
p.c. One calf (no. 6) receiving cimetidine failed to elicit a positive reaction after day 14 p.c.,
whereas one vaccinated control (no. 9) did not elicit a positive reaction until 28 days p.c. A
moderate positive P C A reaction was observed with sera from one adjuvant control at day 28 p.c.
( 2 + ) and with one challenge control at days 28 and 35 p.c. ( 3 + ) . Selected sera that elicited a
positive reaction with the BTV antigen were also tested using the B H K control antigen in the
P C A test. Of the sera tested with B H K antigen, calf 5 showed a 1 + reaction with serum
collected at day 14 p.c. ; this same serum elicited a 4 + reaction with the BTV antigen. Tris
buffer, PBS and BTV and B H K antigen each failed to elicit a reaction when injected into
unsensitized skin sites, but the response to injections of histamine varied between 3 + and 5 + .
BTV-specific IgE was also measured by modification of the E L I S A for BTV antigen
described by Hfibschle et al. (1981) and Poll et al. (1982) and for IgE by Gershwin & Dygert
(1983). The virus was processed as indicated above, and 100 ~tl ali quots of the BTV preparations
were fixed to the bottoms of wells of P V C m i c r o E L I S A plates for 12 to 16 h (4 °C) following
dilution in carbonate buffer p H 9.6. R a b b i t serum albumin (100 txl of 1 ~ solution in carbonate
buffer) was added and incubated at 37 °C for 2 h, to block any remaining binding sites in the
well. The plates were washed six times with a solution consisting of PBS and 0 . 0 5 ~ Tween 20.
Aliquots (100 ~tl) of undiluted test sera, positive and negative control sera and PBS, respectively,
were added in duplicate wells. A serum sample that was strongly positive with the P C A test and
had a high absorbance in the E L I S A (calf 1 at 21 days p.c.) was used as the positive control, and
the negative controls consisted of PBS and a pool of serum collected from two healthy 6 month
old calves. The plates were incubated at 37 °C for 30 min, soaked in P B S - T w e e n 20 for 20 min
and washed six times with P B S - T w e e n 20. R a b b i t anti-bovine IgE, pj'oduced as previously
described (Gershwin & Dygert, 1983), was diluted 1:150 and a d d e d to the wells in 100 ~tl
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 12:05:11
2512
Short communication
0-6 -(a) I
I
I
I
I
J
0.5
-(b) l
I
I
I
I
1
I
28
42
56
70
14
28
\
0.4
0,3
0.2~
0.11
0. °
(c)
0-5
0-4
0-3
0-2
0-1
0. °
-~)Ji"~l
'"'l"~"'(
J'~ ~j .''i'+'''l "
-'
0-5--
14
1st
0.4-
2nd
42
Virulent BTV-17
Time (days)
0.30.20.1
0 N
1st
14
28
42
2nd
56
70
14
28
42
Virulent BTV-17
Time (days)
Fig. 1. A,05readings of ELISA for anti-BTV IgE in calves vaccinated/sensitized with chlorine dioxideinactivated BTV and subsequently challenged with infectious virus. One group received levamisole (a)
and one group received cimetidine (b) during sensitization/vaccination. There were control groups
consisting of calves vaccinated/sensitized (vaccinated control) without immunomodulators (c),
inoculated with only the alum adjuvant (adjuvant control) during sensitization/vaccination (d) and not
vaccinated/sensitized but challenged with infectious virus (challenge control) (e). The absorbance for
the negative control serum pool and PBS fell within the shaded background (not shown).
volumes; incubation at 37 °C for 30 m i n was followed by six washes with P B S - T w e e n 20.
Alkaline phosphatase-goat anti-rabbit conjugate was added to each well at a 1:750 dilution in
100 tll volumes, and the plates were incubated 30 m i n at 37 °C. Prior to the addition of substrate,
plates were washed eight times with PBS-Tween 20. Finally, 200 btl ofp-nitrophenylphosphate
in diethanolamine buffer was added, and the A4os were read at 5 m i n with an automated
microplate reader. Plate-to-plate variation of the data was corrected by using the previously
described method (Volter et al., 1980).
The development of anti-BTV IgE in the sera of the calves in the experiment is shown in Fig.
1. The sera of three of the four vaccinated/sensitized and immunomodulated calves had elevated
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 12:05:11
Short communication
2513
absorbance readings, after the second vaccination/sensitization and after challenge, which
peaked 21 days p.c. There was little difference in the absorbance among sera collected from one
of the immunomodulated animals (no. 6), one vaccinated control calf (no. 9), the adjuvant
control calves and one challenge control calf (no. 14). The other vaccinated and challenge
control calves had the highest absorbance readings at 42 and 28 days, respectively.
In these experiments IgE anti-virus antibodies developed in cattle given chlorine dioxideinactivated BTV, infectious BTV, or both. Reaginic antibodies were detected after the second
vaccination/sensitization, reaching peak levels after challenge with infectious virus, and this
peak correlated with the occurrence of clinical disease. The route of immunization, antigen
concentrations and adjuvants are considered to be important in specific IgE production
(Siraganian, 1981). Injections were subcutaneous in all our animals, using chlorine dioxideinactivated BTV suspended in alum. Additionally, the effect of levamisole and cimetidine in
these experiments is unknown. Although the specific immunomodulating mechanism of
levamisole and cimetidine are incompletely understood, it is known that levamisole can
potentiate the immune response in some species by modification of macrophage and
lymphocyte function (Brunner & Muscoplat, 1980; Renoux, 1978), and it has been hypothesized
that cimetidine may potentiate the immune response in humans by blocking the action of
histamine on H-2 receptors located on suppressor T lymphocytes (Jorizzo et al., 1980; Mavligit
et al., 1981). In our studies, the IgE anti-BTV response was greatest in three of the
immunomodulated calves, but a challenge control calf and a vaccinated control calf also
developed BTV-specific IgE. The IgE levels in the control calves were lower and they peaked 7
and 21 days later (days 28 and 42 p.c.) than the immunomodulated animals.
The 'allergic breakthrough phenomenon' proposed by Katz (1978) is a possible mechanism
for a viral effect on IgE production. Chiorazzi et al. (1977) suggested that treatments with low
dose irradiation, cyclophosphamide or anti-lymphocyte serum may deplete highly sensitive
suppressor cell populations and therefore allow escape of helper T lymphocytes to stimulate IgEproducing B lymphocytes. Viral infections also may selectively deplete suppressor T
lymphocytes and allow an 'allergic breakthrough' (Frick & Brooks, 1983).
Although specific mechanisms important in causing high levels of IgE anti-virus antibodies
are poorly understood, it appears that BTV-specific IgE can be present during BTV infections
and that the level of IgE may be important for development of clinical bluetongue disease. The
present experiments suggest that direct interaction of BTV proteins with virus-specific IgE
located on basophils or mast cells may modulate mediator release. It is well known that the
triggering of these cells results in the release of histamine, leukotrienes, chemotactic and other
inflammatory factors. Some of these factors, i.e. prostaglandins, thromboxane A2 and
histamine, were shown to be elevated in the vaccinated/sensitized calves in our studies (Emau et
al., 1984), which also could be important in the dermatitis and stomatitis observed in natural
bluetongue disease. The capacity of BTV to induce specific IgE and the role of these antibodies
in immunological defence and injury merit further investigation. Furthermore, the implications
for vaccine development are obvious.
We thank Craig Olsen for technical assistance during the enzyme-linked immunosorbent assays. This work was
funded in part by funds provided by the United States Department of Agriculture under the Animal Health Act,
public law 95-113, and contract 58-9AHZ-9-464 and published with the approval of the Director as paper 85-7714,
Nebraska Agricultural Research Division.
REFERENCES
BLACK, L., MENARD, F., BEADLE, G. & PAY, T. (1975). Hypersensitivity in cattle after foot-and-mouth disease
vaccination: response to hydroxypropylmethylcellulose. Journal of Hygiene 75, 79-86.
BRADFORD, i . i . (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.
BRUNNER, C. J. & MUSCOPLAT,C. C. (1980). Immunomodulatory effects of levamisole. Journal of the American
Veterinary Medical Association 176, 1159-1162.
CHIORAZZI,N., FOX,D. A. & KATZ,D. H. (1977). Hapten-specific IgE antibody responses in mice. VII. Conversion of
IgE "nonresponder" strains to IgE "responder" by elimination of suppressor cell activity. Journal of
Immunology 118, 48-54.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 12:05:11
2514
Short communication
DOYLE, J. J. (1973). Homocytotropic antibodies induced in calves by infection with Fasciola hepatica. International
Archives of Allergy 45, 744-751.
EMAU,P., GIRl, S. N., ANDERSON,G. A., STOTT, J. L. & OSBURN,B. I. (1984). Function of prostaglandins, thromboxane
A2, and histamine in hypersensitivity reaction to experimental bluetongue disease in calves. American Journal
of Veterinary Research 45, 1852-1857.
FOSTER, N. M. & LUEDKE, A. J. (1968). Direct assay for bluetongue virus by intravascular inoculation of
embryonating chicken eggs. American Journal of Veterinary Research 29, 749-753.
FRICK, O. L. & BROOKS,D. L. (1983). Immunoglobulin E antibodies to pollens augmented in dogs by virus vaccines.
American Journal of Veterinary Research 44, 440-445.
FRICK, O. L., 6ERMAN, D. F. & MILLS, J. (1979). Development of allergy in children. I. Associations with virus
infections. Journal of Allergy and Clinical Immunology 63, 228-241.
GERSHWIN, L. J. (1981). Heterologous passive cutaneous anaphylaxis with bovine immunoglobulin E. American
Journal of Veterinary Research 42, 1184-1187.
GERSHWlN, L. J. & DYGERT, B. S. (1983). Development of a semiautomated microassay for bovine immunoglobulin
E: definition and standardization. American Journal of Veterinary Research 44, 891-895.
GERSHWlN, L. J. & OLSEN, C. L. (1984). Humoral response of cattle to aerosolized Micropolysporafaeni. American
Journal of Veterinary Research 45, 2135-2142.
HAMMER, D. K., KIRCKnOFEN, B. & SCm,IID, T. (1971). Detection of homocytotropic antibody associated with a
unique immunoglobulin class in the bovine species. Journal oflmmunology 1, 249-257.
.OaSCHLE, O. J. B., LORENZ, R. J. & MATHEKA,X. D. (1981). Enzyme-linked immunosorbent assay for detection of
bluetongue virus antibodies. American Journal of Veterinary Research 42, 61-65.
HUISMANS,H., nREMER, B. C. W. & BARBER,T. L. (1979). The nucleic acid and proteins of epizootic hemorrhagic
disease virus. Onderstepoort Journal of Veterinary Research 46, 95-104.
IDA, S., nOOKS, J. J., SIRAGANIAN,t . P. & ~OTKINS, X. L. (1977). Enhancement of IgE-mediated histamine release
from human basophils by viruses: role of interferon. Journal of Experimental Medicine 145, 892-895.
JORIZZO, J. L., SAMS, W. M., JEGASOTHY, B. V. & OLANSKY, A. J. (1980). Cimetidine as an immunomodulator:
mucocutaneous candidiasis as a model. Annals of Internal Medicine 92, 192-195.
KATZ, D. (1978). The allergic phenotype. Manifestation of "allergic break-through" and imbalance in normal
"damping" of IgE antibody production. Immunological Reviews 41, 77-108.
MAVLIGIT,G. M., CALVO,D. B., PATT, Y. Z. & HERSH, E. M. (1981). Immune restoration and/or augmentation of local
xenogeneic graft versus host reaction by cimetidine in vitro. Journal of Immunology 126, 2272-2274.
POLl, G., STOTT, J., LIN, Y. S. & MANNING, 1. S. (1982). Bluetongue virus: comparative evaluation of enzyme-linked
immunosorbent assay, immunodiffusion and serum neutralization for detection of viral antigens. Journal of
Clinical Microbiology 15, 159-162.
RENOUX, G. (1978). Modulation of immunity by levamisole. Pharmacology and Therapeutics [A] 2, 97-123.
SlRAGANIAN,R. P. (1981). Immediate hypersensitivity reactions. Cellular Function in Immunity and Inflammation,
pp. 323-354. Amsterdam: Elsevier/North-Holland.
s r o r r , J. L., BARSER,T. L. & OSBURN,B. I. (1978). Serotyping bluetongue virus: a comparison of plaque inhibition
(disc) and plaque neutralization methods. Proceedings of the 21st Annual Meeting of the American Association of
Veterinary Laboratory Diagnosticians, pp. 399--410.
VERWOERD, D. W. (1969). Purification and characterization of bluetongue virus. Virology 38, 203-212.
VOLLER, A., BIDWELL, D. & BARTLETT, A. (1980). Enzyme-linked immunosorbent assay. In Manual of Clinical
Immunology, pp. 359-371. Edited by N. R. Rose & H. Friedman. Washington, D.C. : American Society for
Microbiology.
WELLS, P. W. & EYRE, 1'. (1970). Homocytotropic antibodies demonstrated by passive cutaneous anaphylaxis in
calves. Veterinary Record 87, 173-175.
WlLKIE, ~. N., NIELSEN, K. n. & LrrrLE, J. (1978). Experimental hypersensitivity pneumonitis: serum
immunoglobulins G1, G2, and E in Micropolysporafaeni sensitized and desensitized calves. International
Archives of Allergy and Applied Immunology 51, 441-450.
(Received 5 March 1987)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 12:05:11