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Protection of rams against epididymitis by a Brucella ovis -vitamin E adjuvant vaccine
Veterinary Immunology and lmmunopathology, 7 (1984) 293--304 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
293
PROTECTION OF RAMS AGAINST EPIDIDYMITIS BY A BRUCELLA OVIS-VITAMIN E ADJUVANT VACCINE M. AFZAL I, R. P. TENGERDY I", R. P. ELLIS 1, C. V. KIMBERLING 2 and C. J. MORRIS I I Dept. of Microbiology and 2Dept. of Clinical Sciences, Colorado State University, Fort Collins, CO 80523 (U.S.A.) * Reprint requests: Dr. R. P. Tengerdy , Department of Microbiology, Colorado State University, Fort Collins, CO 80523, U.S.A. Tel. 303-491-6163 Supported by a grant from Hoffmann LaRoche, Inc. and also by grants from the USDA (Animal Health, Colorado State University Experiment Station Program). (Accepted 19 March 1984)
ABSTRACT Afzal, M., Tengerdy, R.P., Ellis, R.P., Kimberling, C.V. and Morris, C.J., 1984. Protection of rams against epidldymitis by a Brucella ovis-vitamln E adjuvant vaccine. Vet. Immunol. Immunopathol., 7: 293-304. Rams vaccinated at 7 and 8 months of age with a B. ovis-vitamin E adjuvant vaccine had increased antibody titers compared with Freund's incomplete adjuvant or commercial bacterin vaccinated rams. The percent overall infectivity in an experimental infection of B. ovis-vitamin E adjuvant vaccinated rams was 22~ compared to 44~ for B. ovis-Freund's incomplete adjuvant or bacterin vaccinated rams and 67~ for control. INTRODUCTION Ram epididymitis is considered to be a major disease in sheep. Incidence varies from 15 to 40 percent in the Western States of the U.S.A.
(Kimberling, 1980).
Major economic losses result from reduced
fertility, shortened breeding life, reduced marketability, the need for inspection and lower lambing rates in ewes (Jensen, 1974).
The major
causative agent for the disease in this region is Brucella ovis (Kimberling,
1980; Swift, et al., 1983).
Hajdu (1962) reported a preva-
lance of 30-54 percent B. ovis infection in Tsigai and Valacian rams and 14 percent in Merino rams in Australia. The efficacy of presently available vaccines varies.
A vaccine
from live B. melitensis, called Rev. I, is used extensively and with
0165-2427/84/$03.00
© 1984 Elsevier Science Publishers B.V.
294
satisfactory results in S. Africa and Peru (Jensen, is not available in the U.S.A.
1974).
This vaccine
A B. ovis vaccine, Ramedal®, produced by
Cutter Laboratories is reportedly potentiated by combination with B. abortus, Strain 19 (Buddle, Harrold, 1979).
1958; Swift and Maki, 1968; McGowen and
In McGowen's study on vaccine efficacy, the combination
of Ramedal + Strain 19 protected 80% of the rams while Ramedal alone protected only 56%. 19.
The results suggested an adjuvant action by Strain
Strain 19, however, is disapproved by USDA for use in sheep, there-
fore, other enhancers or adjuvants should he considered. Colorado Serum Company currently produces the only USDA approved B. ovis bacterin.
Our research group has contributed to the development
and testing of this bacterin (Kimberling, 1980).
Our experience and the
success of using vitamin E as an immunoenhancer in other immunization processes stimulated our interest in the development of the B. ovisvitamin E adjuvant vaccine, as a water in oil adjuvant emulsion. In previous work we found that vitamin E is a potent immunoenhancer as a dietary supplement or an immunoadjuvant in a number of animal species against different infectious diseases (Tengerdy, Tengerdy et al., 1981; Tengerdy et al., 1983).
1980;
Vitamin E, as an anti-
oxidant and cell membrane stabilizer, enhances humoral antibody production and phagocytosis, probably by regulating prostaglandin and cyclic nucleotide biosynthesis (Tengerdy et al., 1981).
Recently we reported
that vitamin E is a very good immunoadjuvant for increasing humoral antibody titer of sheep vaccinated against Clostridium perfringens toxoid D (Tengerdy et al., 1983). In the current study the efficacy of a B. ovis-vitamin E adjuvant vaccine was tested against the Colorado Serum Co. bacterin and a B. ovis-Freund's incomplete adjuvant vaccine for controlling an experimental infection with a field isolate of B. ovis.
MATERIALS AND METHODS Animals Fifty-four Collins, trate,
rams a t 6 m o n t h s o f age w e r e p l a c e d
s h o r n and f e d a g r o w i n g and f a t t e n i n g minerals
and s a l t s .
o f n i n e e a c h and v a c c i n a t e d at
8 months of age.
in a dry lot
ration
of alfalfa
The rams w e r e r a n d o m l y d i v i d e d at 7 months of age,
and b o o s t e r
into
in Fort concensix groups
vaccinated
295 Vaccines I.
Commercial bacterin suspension
is an aluminum hydroxide precipitated
of 109 B. ovis cells per ml, kindly supplied by
Colorado Serum Co., Denver, 2.
B. ovis-Vitamin
CO.
E adjuvant vaccine:
1.0 ml of 2.0 x 109 B.
ovis suspension + 0.85 ml [DL]-~-tocopheryl Arlacel® emulsified 3.
B. ovis-Freund's
incomplete
above except mineral
Placebos
adjuvant
(FIA) vaccine:
were made with 13.3% aluminum hydroxide
that was used for making the bacterin. into rams subcutaneously.
2.0 ml of each preparation was
The B. ovis strain used in the vaccines
from Rifle,
suspension
A booster of 2.0 ml bacterin was
given to all groups except the control and placebo later.
Same as
oil replaces vitamin E.
of vitamin E and FIA were prepared without B. ovis and con-
trol (sham) injections
injected
acetate + 0.15 ml
to stable emulsion.
CO., maintained by serial passage
groups one month
is a field isolate
in blood agar.
Challenge Eighty-six
days following
first vaccination
each ram was challenged
with 5 x 10 9 cells from a fresh field isolate of B. ovis suspended 5 ml of sterile normal saline preputial
.
Bacteria were inoculated
sheath and the sheath was tied with gauze to help avoid leakage.
Animals were made to run and the gauze was removed 5 minutes
Micro complement
later.
fixation test
The micro complement
fixation test was performed (1982).
in polyvinyl
microtiter plates
following Burgess and Norris
sera were diluted
1:10 in modified veronal buffer and inactivated
60°C for 1 hour.
To 0.025 ml inactivated
sheep serum,
two-fold were added 0.025 ml B. ovis test antigen Veterinary
in
into the
Services Diagnostic
Laboratory,
0.025 ml 1:35 diluted guinea pig complement
Ames,
Briefly,
sheep at
serially diluted
(kindly supplied by Iowa) diluted 1:64, and
containing
5 CHs0 units.
The plates were incubated overnight
in a refrigerator
ml sensitized
(3% v/v SRBC + 2 units of hemolysin
sheep red blood cells
in equal volumes)
(4°C), then 0.025
was added and the plates were shaken on a reciprocal
296
._. I 0 0
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o CO
80
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60
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40
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0 I
I0
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!
15
I
I
I
20 40 45 TUBE NUMBER
I
I
50
55
Separation of B. ovis antigens on Sephadex G-200 column.
shaker at 150 rpm for 40 min at 37°C.
The titer is the serum dilution
giving 50~ or less hemolysis by visual observation.
Enzyme linked immunosorbent assay (ELISA) Antigen used for ELISA was prepared as follows:
a heavy B. ovis
suspension in physiological buffered saline (PBS) (pH 7.2) was heated at 80°C for 2 hours and centrifuged at 10,000 x G at 4°C for 20 minutes. The supernate was concentrated with carbowax (polyethylene glycol, Fisher Scientific Co., Pittsburg, PA).
Concentrated antigen was separated on
Sephadex G-200 column with 0.15 M Phosphate buffer (pH 7.0); the protein peaks were monitored by absorption at 280 nm.
Fraction I eluted from the
column (Fig. 1) was concentrated by ultrafiltration through PM-10 Amicon membrane filter 10,000 M.W. cut off (Amicon Corp., Danvers, HA) and dialyzed overnight against PBS (pH 7.2). antigen in ELISA.
This fraction was used as
Fraction II did not give a specific response in ELISA.
297
ELISA was carried out in Dynatech microtiter flexvinyl plates (Dynatech Labs, Inc., Alexandria, VA).
Wells were sensitized with 50 pl
of Fraction I antigen diluted in carbonate coating buffer (pH 9.6) to 2.0 pg/ml protein concentration for 2 hours at 37°C.
Plates were washed
3x with PBS-Tween 20 and 50 pl 1:200 diluted sera were added to the wells. Plates were incubated for 30 minutes at 37°C and then washed with PBSTween 20.
Then 50 ~i of rabbit anti-sheep IgG-peroxidase conjugate
(Cappel Labs., Malvern, PA), diluted 1:500 was added and incubated for 2 hours at 37°C.
Plates were washed 3x, and 50 pl of substrate
(O-phenylenediamine HCI+H202) was added to each well.
The reaction was
stopped with 50 pl 2 N sulphuric acid after 5 minutes.
The absorption
of the samples at 488 nm was read directly in the plates in a Dynatech Microelisa Reader.
Titers were expressed as difference in absorbance
values between treatment and control groups at a constant 1:200 serum dilution.
The absorbance values of the standard reference negative and
positive sera from the Veterinary Services Diagnostic Laboratory, Ames, Iowa, were 0.05 and 0.22 respectively.
The CF titer of these reference
sera were 0 and 1:20 respectively.
Semen evaluation Semen was collected with electroejaculation one day before and 7, 17, 40, 65, 85 and 105 days post challenge. white blood cells were examined.
Motility and presence of
Semen morphology was studied after
staining with Hancock's stain (Society of Theriogenology, Hastings, NE). Each semen sample was cultured on 5~ serum tryptose agar and vancomycin chocolate agar. atmosphere.
Plates were incubated at 37°C for 3-5 days in 10~ CO 2
Isolated colonies were further examined for positive identi-
fication of B. ovis.
Histopathology Testes and epididymes were excised at slaughter, 114 days postchallenge,
and transported to the laboratory in ice.
Tissues from the
center of testes and the tail of epididymes were cut and fixed in 10~ buffered formalin.
Paraffin sections 5-7 pm thick were cut and stained
with haemotoxylin and eosin.
298 RESULTS
The time course of immunization and antibody titers by microcomplement fixation (CF) and ELISA are shown in Fig. 2a and 2b.
It is evident
that the B. ovis-vitamin E adjuvant vaccine enhanced humoral immunity more than the other vaccines, although the differences were not significant statistically.
The sharp increase and decrease in the CF titer was
probably due to the shift from IgM to IgG antibody, observed earlier as one of the major effects of vitamin E on humoral immunity (Tengerdy, et al., 1973).
CF titers are about I00 times greater for IgM than IgG
antibodies.
Such sharp rise and fall in the titers is not observed in
ELISA, which measures predominantly IgG antibodies.
B. ovis-FIA vaccina-
tion resulted in a delayed and prolonged anamnestic response at the time of challenge.
Some of the non-immunized rams (control and placebo
groups) had measurable CF titers and ELISA titers before challenge, perhaps indicating natural exposure to B. ovis or related organisms. Peak antibody titers by the CF and ELISA tests one week after booster vaccination roughly coincided with protection, but a statistically significant correlation between enhanced humoral immunity and increased protection could not be demonstrated. Protection was judged from the percentage of overall infection, based on three criteria:
]) isolation of B. ovis from semen, 2) acute
inflammatory lesions in epididymis, and 3) presence of leukocytes and more than 30 percent separated sperm heads in semen.
By these criteria
the state of infection in the treatment groups can be seen in Table I. Semen motility did not change significantly as a result of experimental infection.
The isolation of B. ovis from semen, however, was usually
associated with increase in white blood cells and higher occurrence of separated heads.
Histopathologic observations revealed lesions primarily
restricted to the epidydimis.
Only 2 rams that had lesions in the
epididymis also had lesions in the testes. one epididymis (5 rams) or in both (7 rams).
Lesions were either only in There was neutrophilic
infiltration of the thickened or hyperplastic tubular epithelium (Fig. 3). Microabscesses or cysts having cellular debris and neutrophils were formed in the epithelium. tubular lumens. observed.
Neutrophils were mixed with sperm in most
In the interstitium, lymphoid cell aggregation was
299
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o
200-
nhi 150II,, I 0 0 o
o
BACTERIN
×
x
B OVIS
+
FIA
n
n
B.OVIS
÷
VITAMIN E
"1-
50-
O
'
0
-50
l
50
I00
vaccinotion booster
chal lenge
1'
t
150
2 0 0 DAYS
t
Fig. 2a. M i c r o c o m p l e m e n t f i x a t i o n titer of rams i m m u n i z e d a g a i n s t B r u c e l l a ovis. T i t e r s are e x p r e s s e d as the m e a n serum d i l u t i o n ± s t a n d a r d e r r o r (n=9).
0.3-
E C
0.2
GO (Z) v
LU
0.1
Z rF 0
O.
nm
-0.1
i
-50
' ' ' I ....
0
I ....
50
vaccination booster
I ....
I00
I
150
'
'
'
i!
2 0 0 DAYS
chal lenge
Fig, 2b. E L I S A titers of rams i m m u n i z e d a g a i n s t B r u c e l l a ovis. Titers are e x p r e s s e d as d i f f e r e n c e in a b s o r b a n c e at 1:200 serum d i l u t i o n b e t w e e n t r e a t m e n t and control groups.
300
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301
Fig. 3 Light micrograph of ram epididymis 114 days after challenge with a field isolate of B. ovis. Note the infiltration of the neutrophils (n) in the thickened epithelium and formation of cysts with cellular debris (c). Neutrophils mixed with sperm can also be seen in the lumen (I) of tubule. Magnification = 400x. By these criteria, the vitamin E adjuvant vaccine of B. ovis afforded a significantly increased protection against ram epididymitis, as shown in Table I.
The overall infectivity dropped from 67~ in the control group
to 22~ in the vitamin E adjuvant group.
It is interesting to note that
the vitamin E placebo (emulsion without B. ovis) also had non-specific protective effect.
DISCUSSION The significant finding of this study is the strong protective effect of a vitamin E adjuvant vaccine of B. ovis against ram epididymitis. Similar protection was found in other animal species against other bacterial infections, when vitamin E was administered as a dietary supplement (Tengerdy et al., 1981).
Vitamin E as an adjuvant had a
302 g r e a t e r enhancement on antibody production than d i e t a r y supplementation in immunizing sheep a g a i n s t Clostridium p e r f r i n g e n s , type D (Tengerdy et a l . , 1983). In the present study, the increased p r o t e c t i o n could not be p o s i t i v e l y correlated with increased antibody titers, as it was the case in earlier studies (Tengerdy,
et al., 1981).
The mechanism of the observed protection
by the vitamin E adjuvant vaccine is not known, but it is possible that cellular and local immunity may play a more important role in this disease than humoral immunity.
It is known that vitamin E is involved
in T and B lymphocyte and macrophage cooperation in enhanced humoral response.
Since adjuvants generally also promote cell cooperation,
it is possible that the mechanism of the vitamin E adjuvant action is a synergistic enhancement of cell cooperation (Campbell et al., 1974, Tanaka et al., 1979, C o ~ i n and Shloss, 1980). The experimental infection procedure used in this study gave 67~ infectivity in the control rams and probably differs significantly from a natural infection process.
Producing an experimental infection in
rams depends on age, virulence of organism and route of infection.
Most
investigators reported an infection rate of 45 to 87 percent in experimental infection (Ris, 1964; Claxton, 1968; Swift and Maki, 1968). Bulgin and Anderson (1983) reported isolation of B. ovis only from mature breeding rams and not from rams under 1 year of age infected with B. ovis.
If this finding is true, our rams were challenged a bit early
(9 months of age) to have an optimal experimental infection.
Neverthe-
less, we isolated B. ovis from semen and correlated it with other criteria for infection (inflammatory lesions, semen quality) to arrive at the overall infectivity figures reported in Table I.
The superior protec-
tion by the vitamin E adjuvant vaccine is evident under the conditions of this experimental infection.
Part of the effect may be non specific,
as evidenced by the protective effect of the vitamin E placebo. In natural infection several microorganisms beside B. ovls may be present, some of which may be contributing to the disease process.
It
remains to be seen how much protection the new vaccine will give under field conditions.
It also remains to be seen which antigen(s) of B.
ovis contributes most to immune protection, which antibody class (IgM, IgG, or IgA) or cell mediated immune mechanism is most protective, and
303
what is the local mechanism of pathogenesis and immune protection.
These
questions will be approached in future research.
ACKNOWLEDGEMENTS The authors are thankful to Dr. A. E. McChesney of the Department of Pathology, Colorado State University, Fort Collins, CO for his help in the histopathological
studies.
REFERENCES Buddle, M.B., 1958. Vaccination in the control of Brucella ovis infection in sheep. New Zeal. Vet. Jour. 6:36-41. Bulgin, M. S. and Anderson, B. C., 1983. Association of sexual experience with isolation of various bacteria in cases of ovine epididymitis. J.A.V.M.A., 182:372-374. Burgess, G. W. and Morris, M. J., 1982. Evaluation of cold complement fixation test for diagnosis of ovine brucellosis. Aust. Vet. J., 59:23-25. Campbell, P. A., Cooper, H. R., Heinzerling, R. H., and Tengerdy, R. P., 1974. Vitamin E enhances in vitro immune responses by normal and non adherent spleen cells. Proc. Soc. Exp. Biol. Hed., 146:465-469. Claxton, P. D., 1968. Brucella ovis vaccination of rams. A comparison of two commercial vaccines and two methods of vaccination. Aust. Vet. J., 44:48-54. Corwin, L. M. and Shloss, J., 1980. Role of antioxidants in the stimulation of the mitogenic response. J. Nutr., 110:2497-2505. Hajdu, S., 1962. Ergebnisse der serologischen Untersuchung von Schafen auf Brucelloidose. Arch. Exp. Vet. Med., 16:19-28. Jensen, R., 1974. Diseases of Sheep. Lea & Febiger, Philadelphia. Kimberling, C. V., 1980. Ram epididymitis. CSU Extension Bulletin 8-016, Fort Collins, Colorado. Colorado State University. HcGowan, B. and Harrold, R. D., 1979. Epididymitis in rams. Studies on vaccine efficacy. Cornell Vet., 69:73-76. Ris~ D. R., 1964. An indirect haemagglutination test for the detection of Brucella ovis antibodies. 2. Comparison of the indirect haemagglutination test with other diagnostic methods. New Zeal. Vet. J., 12:72-75. Swift, B. and Maki, L., 1968. Immunologic studies on three ram epidimymitis bacterins. The Cornell Vet. 58:659-665. Swift, B. L., Dahlgren, R. R. and Maki, L. R., 1983. Ram epididymitis survey. WRCC-46 Ram Epididymitis Second Annual Meeting. Tanaka, T., Fujiwara, H. and Torisu, M., 1979. Vitamin E and immunity I. Enhancement of helper T cell activity. Immunology. 38:727-731. Tengerdy, R. P., Heinzerling, R. H., Brown, G. L. and Mathias~ M. M., 1973. Enhancement of humoral immune response by vitamin E. Intern. Arch. Allergy, 44:211-222. Tengerdy, R. P., 1980. Effect of vitamin E on immune responses, in L. J. Hachlin (ed.) Vitamin E, A Comprehensive Treatise. Marcel Dekker, Inc., pp. 42-443.
304
Tengerdy, R. P., Mathias, M. M. and Nockels, C. F., 1981. Vitamin E, immunity and disease resistance, i_nn M. Phillips and A. Baetz (Eds.) Diet and Resistance to Disease. Plenum Press, N.Y., pp. 27-42. Tengerdy, R. P., Meyer, D. L., Lauerman, L. H., Lueker, D. C. and Nockels, C. F., 1983. Vitamin E enhances humorsl antibody response to Clostridium perfringens, type D, in sheep. Brit. Vet. J., 139:147-151.
293
PROTECTION OF RAMS AGAINST EPIDIDYMITIS BY A BRUCELLA OVIS-VITAMIN E ADJUVANT VACCINE M. AFZAL I, R. P. TENGERDY I", R. P. ELLIS 1, C. V. KIMBERLING 2 and C. J. MORRIS I I Dept. of Microbiology and 2Dept. of Clinical Sciences, Colorado State University, Fort Collins, CO 80523 (U.S.A.) * Reprint requests: Dr. R. P. Tengerdy , Department of Microbiology, Colorado State University, Fort Collins, CO 80523, U.S.A. Tel. 303-491-6163 Supported by a grant from Hoffmann LaRoche, Inc. and also by grants from the USDA (Animal Health, Colorado State University Experiment Station Program). (Accepted 19 March 1984)
ABSTRACT Afzal, M., Tengerdy, R.P., Ellis, R.P., Kimberling, C.V. and Morris, C.J., 1984. Protection of rams against epidldymitis by a Brucella ovis-vitamln E adjuvant vaccine. Vet. Immunol. Immunopathol., 7: 293-304. Rams vaccinated at 7 and 8 months of age with a B. ovis-vitamin E adjuvant vaccine had increased antibody titers compared with Freund's incomplete adjuvant or commercial bacterin vaccinated rams. The percent overall infectivity in an experimental infection of B. ovis-vitamin E adjuvant vaccinated rams was 22~ compared to 44~ for B. ovis-Freund's incomplete adjuvant or bacterin vaccinated rams and 67~ for control. INTRODUCTION Ram epididymitis is considered to be a major disease in sheep. Incidence varies from 15 to 40 percent in the Western States of the U.S.A.
(Kimberling, 1980).
Major economic losses result from reduced
fertility, shortened breeding life, reduced marketability, the need for inspection and lower lambing rates in ewes (Jensen, 1974).
The major
causative agent for the disease in this region is Brucella ovis (Kimberling,
1980; Swift, et al., 1983).
Hajdu (1962) reported a preva-
lance of 30-54 percent B. ovis infection in Tsigai and Valacian rams and 14 percent in Merino rams in Australia. The efficacy of presently available vaccines varies.
A vaccine
from live B. melitensis, called Rev. I, is used extensively and with
0165-2427/84/$03.00
© 1984 Elsevier Science Publishers B.V.
294
satisfactory results in S. Africa and Peru (Jensen, is not available in the U.S.A.
1974).
This vaccine
A B. ovis vaccine, Ramedal®, produced by
Cutter Laboratories is reportedly potentiated by combination with B. abortus, Strain 19 (Buddle, Harrold, 1979).
1958; Swift and Maki, 1968; McGowen and
In McGowen's study on vaccine efficacy, the combination
of Ramedal + Strain 19 protected 80% of the rams while Ramedal alone protected only 56%. 19.
The results suggested an adjuvant action by Strain
Strain 19, however, is disapproved by USDA for use in sheep, there-
fore, other enhancers or adjuvants should he considered. Colorado Serum Company currently produces the only USDA approved B. ovis bacterin.
Our research group has contributed to the development
and testing of this bacterin (Kimberling, 1980).
Our experience and the
success of using vitamin E as an immunoenhancer in other immunization processes stimulated our interest in the development of the B. ovisvitamin E adjuvant vaccine, as a water in oil adjuvant emulsion. In previous work we found that vitamin E is a potent immunoenhancer as a dietary supplement or an immunoadjuvant in a number of animal species against different infectious diseases (Tengerdy, Tengerdy et al., 1981; Tengerdy et al., 1983).
1980;
Vitamin E, as an anti-
oxidant and cell membrane stabilizer, enhances humoral antibody production and phagocytosis, probably by regulating prostaglandin and cyclic nucleotide biosynthesis (Tengerdy et al., 1981).
Recently we reported
that vitamin E is a very good immunoadjuvant for increasing humoral antibody titer of sheep vaccinated against Clostridium perfringens toxoid D (Tengerdy et al., 1983). In the current study the efficacy of a B. ovis-vitamin E adjuvant vaccine was tested against the Colorado Serum Co. bacterin and a B. ovis-Freund's incomplete adjuvant vaccine for controlling an experimental infection with a field isolate of B. ovis.
MATERIALS AND METHODS Animals Fifty-four Collins, trate,
rams a t 6 m o n t h s o f age w e r e p l a c e d
s h o r n and f e d a g r o w i n g and f a t t e n i n g minerals
and s a l t s .
o f n i n e e a c h and v a c c i n a t e d at
8 months of age.
in a dry lot
ration
of alfalfa
The rams w e r e r a n d o m l y d i v i d e d at 7 months of age,
and b o o s t e r
into
in Fort concensix groups
vaccinated
295 Vaccines I.
Commercial bacterin suspension
is an aluminum hydroxide precipitated
of 109 B. ovis cells per ml, kindly supplied by
Colorado Serum Co., Denver, 2.
B. ovis-Vitamin
CO.
E adjuvant vaccine:
1.0 ml of 2.0 x 109 B.
ovis suspension + 0.85 ml [DL]-~-tocopheryl Arlacel® emulsified 3.
B. ovis-Freund's
incomplete
above except mineral
Placebos
adjuvant
(FIA) vaccine:
were made with 13.3% aluminum hydroxide
that was used for making the bacterin. into rams subcutaneously.
2.0 ml of each preparation was
The B. ovis strain used in the vaccines
from Rifle,
suspension
A booster of 2.0 ml bacterin was
given to all groups except the control and placebo later.
Same as
oil replaces vitamin E.
of vitamin E and FIA were prepared without B. ovis and con-
trol (sham) injections
injected
acetate + 0.15 ml
to stable emulsion.
CO., maintained by serial passage
groups one month
is a field isolate
in blood agar.
Challenge Eighty-six
days following
first vaccination
each ram was challenged
with 5 x 10 9 cells from a fresh field isolate of B. ovis suspended 5 ml of sterile normal saline preputial
.
Bacteria were inoculated
sheath and the sheath was tied with gauze to help avoid leakage.
Animals were made to run and the gauze was removed 5 minutes
Micro complement
later.
fixation test
The micro complement
fixation test was performed (1982).
in polyvinyl
microtiter plates
following Burgess and Norris
sera were diluted
1:10 in modified veronal buffer and inactivated
60°C for 1 hour.
To 0.025 ml inactivated
sheep serum,
two-fold were added 0.025 ml B. ovis test antigen Veterinary
in
into the
Services Diagnostic
Laboratory,
0.025 ml 1:35 diluted guinea pig complement
Ames,
Briefly,
sheep at
serially diluted
(kindly supplied by Iowa) diluted 1:64, and
containing
5 CHs0 units.
The plates were incubated overnight
in a refrigerator
ml sensitized
(3% v/v SRBC + 2 units of hemolysin
sheep red blood cells
in equal volumes)
(4°C), then 0.025
was added and the plates were shaken on a reciprocal
296
._. I 0 0
i
o CO
80
1.1.1 0 Z
60
m n," 0 (/) gO
40
0,,I
20 /,,-
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Separation of B. ovis antigens on Sephadex G-200 column.
shaker at 150 rpm for 40 min at 37°C.
The titer is the serum dilution
giving 50~ or less hemolysis by visual observation.
Enzyme linked immunosorbent assay (ELISA) Antigen used for ELISA was prepared as follows:
a heavy B. ovis
suspension in physiological buffered saline (PBS) (pH 7.2) was heated at 80°C for 2 hours and centrifuged at 10,000 x G at 4°C for 20 minutes. The supernate was concentrated with carbowax (polyethylene glycol, Fisher Scientific Co., Pittsburg, PA).
Concentrated antigen was separated on
Sephadex G-200 column with 0.15 M Phosphate buffer (pH 7.0); the protein peaks were monitored by absorption at 280 nm.
Fraction I eluted from the
column (Fig. 1) was concentrated by ultrafiltration through PM-10 Amicon membrane filter 10,000 M.W. cut off (Amicon Corp., Danvers, HA) and dialyzed overnight against PBS (pH 7.2). antigen in ELISA.
This fraction was used as
Fraction II did not give a specific response in ELISA.
297
ELISA was carried out in Dynatech microtiter flexvinyl plates (Dynatech Labs, Inc., Alexandria, VA).
Wells were sensitized with 50 pl
of Fraction I antigen diluted in carbonate coating buffer (pH 9.6) to 2.0 pg/ml protein concentration for 2 hours at 37°C.
Plates were washed
3x with PBS-Tween 20 and 50 pl 1:200 diluted sera were added to the wells. Plates were incubated for 30 minutes at 37°C and then washed with PBSTween 20.
Then 50 ~i of rabbit anti-sheep IgG-peroxidase conjugate
(Cappel Labs., Malvern, PA), diluted 1:500 was added and incubated for 2 hours at 37°C.
Plates were washed 3x, and 50 pl of substrate
(O-phenylenediamine HCI+H202) was added to each well.
The reaction was
stopped with 50 pl 2 N sulphuric acid after 5 minutes.
The absorption
of the samples at 488 nm was read directly in the plates in a Dynatech Microelisa Reader.
Titers were expressed as difference in absorbance
values between treatment and control groups at a constant 1:200 serum dilution.
The absorbance values of the standard reference negative and
positive sera from the Veterinary Services Diagnostic Laboratory, Ames, Iowa, were 0.05 and 0.22 respectively.
The CF titer of these reference
sera were 0 and 1:20 respectively.
Semen evaluation Semen was collected with electroejaculation one day before and 7, 17, 40, 65, 85 and 105 days post challenge. white blood cells were examined.
Motility and presence of
Semen morphology was studied after
staining with Hancock's stain (Society of Theriogenology, Hastings, NE). Each semen sample was cultured on 5~ serum tryptose agar and vancomycin chocolate agar. atmosphere.
Plates were incubated at 37°C for 3-5 days in 10~ CO 2
Isolated colonies were further examined for positive identi-
fication of B. ovis.
Histopathology Testes and epididymes were excised at slaughter, 114 days postchallenge,
and transported to the laboratory in ice.
Tissues from the
center of testes and the tail of epididymes were cut and fixed in 10~ buffered formalin.
Paraffin sections 5-7 pm thick were cut and stained
with haemotoxylin and eosin.
298 RESULTS
The time course of immunization and antibody titers by microcomplement fixation (CF) and ELISA are shown in Fig. 2a and 2b.
It is evident
that the B. ovis-vitamin E adjuvant vaccine enhanced humoral immunity more than the other vaccines, although the differences were not significant statistically.
The sharp increase and decrease in the CF titer was
probably due to the shift from IgM to IgG antibody, observed earlier as one of the major effects of vitamin E on humoral immunity (Tengerdy, et al., 1973).
CF titers are about I00 times greater for IgM than IgG
antibodies.
Such sharp rise and fall in the titers is not observed in
ELISA, which measures predominantly IgG antibodies.
B. ovis-FIA vaccina-
tion resulted in a delayed and prolonged anamnestic response at the time of challenge.
Some of the non-immunized rams (control and placebo
groups) had measurable CF titers and ELISA titers before challenge, perhaps indicating natural exposure to B. ovis or related organisms. Peak antibody titers by the CF and ELISA tests one week after booster vaccination roughly coincided with protection, but a statistically significant correlation between enhanced humoral immunity and increased protection could not be demonstrated. Protection was judged from the percentage of overall infection, based on three criteria:
]) isolation of B. ovis from semen, 2) acute
inflammatory lesions in epididymis, and 3) presence of leukocytes and more than 30 percent separated sperm heads in semen.
By these criteria
the state of infection in the treatment groups can be seen in Table I. Semen motility did not change significantly as a result of experimental infection.
The isolation of B. ovis from semen, however, was usually
associated with increase in white blood cells and higher occurrence of separated heads.
Histopathologic observations revealed lesions primarily
restricted to the epidydimis.
Only 2 rams that had lesions in the
epididymis also had lesions in the testes. one epididymis (5 rams) or in both (7 rams).
Lesions were either only in There was neutrophilic
infiltration of the thickened or hyperplastic tubular epithelium (Fig. 3). Microabscesses or cysts having cellular debris and neutrophils were formed in the epithelium. tubular lumens. observed.
Neutrophils were mixed with sperm in most
In the interstitium, lymphoid cell aggregation was
299
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Fig. 2a. M i c r o c o m p l e m e n t f i x a t i o n titer of rams i m m u n i z e d a g a i n s t B r u c e l l a ovis. T i t e r s are e x p r e s s e d as the m e a n serum d i l u t i o n ± s t a n d a r d e r r o r (n=9).
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Fig, 2b. E L I S A titers of rams i m m u n i z e d a g a i n s t B r u c e l l a ovis. Titers are e x p r e s s e d as d i f f e r e n c e in a b s o r b a n c e at 1:200 serum d i l u t i o n b e t w e e n t r e a t m e n t and control groups.
300
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301
Fig. 3 Light micrograph of ram epididymis 114 days after challenge with a field isolate of B. ovis. Note the infiltration of the neutrophils (n) in the thickened epithelium and formation of cysts with cellular debris (c). Neutrophils mixed with sperm can also be seen in the lumen (I) of tubule. Magnification = 400x. By these criteria, the vitamin E adjuvant vaccine of B. ovis afforded a significantly increased protection against ram epididymitis, as shown in Table I.
The overall infectivity dropped from 67~ in the control group
to 22~ in the vitamin E adjuvant group.
It is interesting to note that
the vitamin E placebo (emulsion without B. ovis) also had non-specific protective effect.
DISCUSSION The significant finding of this study is the strong protective effect of a vitamin E adjuvant vaccine of B. ovis against ram epididymitis. Similar protection was found in other animal species against other bacterial infections, when vitamin E was administered as a dietary supplement (Tengerdy et al., 1981).
Vitamin E as an adjuvant had a
302 g r e a t e r enhancement on antibody production than d i e t a r y supplementation in immunizing sheep a g a i n s t Clostridium p e r f r i n g e n s , type D (Tengerdy et a l . , 1983). In the present study, the increased p r o t e c t i o n could not be p o s i t i v e l y correlated with increased antibody titers, as it was the case in earlier studies (Tengerdy,
et al., 1981).
The mechanism of the observed protection
by the vitamin E adjuvant vaccine is not known, but it is possible that cellular and local immunity may play a more important role in this disease than humoral immunity.
It is known that vitamin E is involved
in T and B lymphocyte and macrophage cooperation in enhanced humoral response.
Since adjuvants generally also promote cell cooperation,
it is possible that the mechanism of the vitamin E adjuvant action is a synergistic enhancement of cell cooperation (Campbell et al., 1974, Tanaka et al., 1979, C o ~ i n and Shloss, 1980). The experimental infection procedure used in this study gave 67~ infectivity in the control rams and probably differs significantly from a natural infection process.
Producing an experimental infection in
rams depends on age, virulence of organism and route of infection.
Most
investigators reported an infection rate of 45 to 87 percent in experimental infection (Ris, 1964; Claxton, 1968; Swift and Maki, 1968). Bulgin and Anderson (1983) reported isolation of B. ovis only from mature breeding rams and not from rams under 1 year of age infected with B. ovis.
If this finding is true, our rams were challenged a bit early
(9 months of age) to have an optimal experimental infection.
Neverthe-
less, we isolated B. ovis from semen and correlated it with other criteria for infection (inflammatory lesions, semen quality) to arrive at the overall infectivity figures reported in Table I.
The superior protec-
tion by the vitamin E adjuvant vaccine is evident under the conditions of this experimental infection.
Part of the effect may be non specific,
as evidenced by the protective effect of the vitamin E placebo. In natural infection several microorganisms beside B. ovls may be present, some of which may be contributing to the disease process.
It
remains to be seen how much protection the new vaccine will give under field conditions.
It also remains to be seen which antigen(s) of B.
ovis contributes most to immune protection, which antibody class (IgM, IgG, or IgA) or cell mediated immune mechanism is most protective, and
303
what is the local mechanism of pathogenesis and immune protection.
These
questions will be approached in future research.
ACKNOWLEDGEMENTS The authors are thankful to Dr. A. E. McChesney of the Department of Pathology, Colorado State University, Fort Collins, CO for his help in the histopathological
studies.
REFERENCES Buddle, M.B., 1958. Vaccination in the control of Brucella ovis infection in sheep. New Zeal. Vet. Jour. 6:36-41. Bulgin, M. S. and Anderson, B. C., 1983. Association of sexual experience with isolation of various bacteria in cases of ovine epididymitis. J.A.V.M.A., 182:372-374. Burgess, G. W. and Morris, M. J., 1982. Evaluation of cold complement fixation test for diagnosis of ovine brucellosis. Aust. Vet. J., 59:23-25. Campbell, P. A., Cooper, H. R., Heinzerling, R. H., and Tengerdy, R. P., 1974. Vitamin E enhances in vitro immune responses by normal and non adherent spleen cells. Proc. Soc. Exp. Biol. Hed., 146:465-469. Claxton, P. D., 1968. Brucella ovis vaccination of rams. A comparison of two commercial vaccines and two methods of vaccination. Aust. Vet. J., 44:48-54. Corwin, L. M. and Shloss, J., 1980. Role of antioxidants in the stimulation of the mitogenic response. J. Nutr., 110:2497-2505. Hajdu, S., 1962. Ergebnisse der serologischen Untersuchung von Schafen auf Brucelloidose. Arch. Exp. Vet. Med., 16:19-28. Jensen, R., 1974. Diseases of Sheep. Lea & Febiger, Philadelphia. Kimberling, C. V., 1980. Ram epididymitis. CSU Extension Bulletin 8-016, Fort Collins, Colorado. Colorado State University. HcGowan, B. and Harrold, R. D., 1979. Epididymitis in rams. Studies on vaccine efficacy. Cornell Vet., 69:73-76. Ris~ D. R., 1964. An indirect haemagglutination test for the detection of Brucella ovis antibodies. 2. Comparison of the indirect haemagglutination test with other diagnostic methods. New Zeal. Vet. J., 12:72-75. Swift, B. and Maki, L., 1968. Immunologic studies on three ram epidimymitis bacterins. The Cornell Vet. 58:659-665. Swift, B. L., Dahlgren, R. R. and Maki, L. R., 1983. Ram epididymitis survey. WRCC-46 Ram Epididymitis Second Annual Meeting. Tanaka, T., Fujiwara, H. and Torisu, M., 1979. Vitamin E and immunity I. Enhancement of helper T cell activity. Immunology. 38:727-731. Tengerdy, R. P., Heinzerling, R. H., Brown, G. L. and Mathias~ M. M., 1973. Enhancement of humoral immune response by vitamin E. Intern. Arch. Allergy, 44:211-222. Tengerdy, R. P., 1980. Effect of vitamin E on immune responses, in L. J. Hachlin (ed.) Vitamin E, A Comprehensive Treatise. Marcel Dekker, Inc., pp. 42-443.
304
Tengerdy, R. P., Mathias, M. M. and Nockels, C. F., 1981. Vitamin E, immunity and disease resistance, i_nn M. Phillips and A. Baetz (Eds.) Diet and Resistance to Disease. Plenum Press, N.Y., pp. 27-42. Tengerdy, R. P., Meyer, D. L., Lauerman, L. H., Lueker, D. C. and Nockels, C. F., 1983. Vitamin E enhances humorsl antibody response to Clostridium perfringens, type D, in sheep. Brit. Vet. J., 139:147-151.