- Email: [email protected]
Bovine babesiosis: The immunization of cattle with fractions of erythrocytes infected with Babesia bovis (syn B. argentina )
Veterinary Immunology and Immunopathology, 2 (1981) 145--156 Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- Printed in Belgium
145
BOVINE BABESIOSlS: THE IMMUNIZATIONOF CATTLE WITH FRACTIONS OF ERYTHROCYTES INFECTED WITH BABESIA BOVIS (SYN B. ARGENTINA) D.F. MAHONEY, I.G. WRIGHT and B.V. GOODGER CSIRO, Division of Animal Health, Long Pocket Laboratories, Private Bag No. 3, P.O., I n d o o r o o p i l l y , Queensland, AUSTRALIA 4068 (Accepted 12 January 1981) ABSTRACT Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1981. Bovine babesiosis: the immuni z a t i o n of c a t t l e with f r a c t i o n s of erythrocytes infected with Babesia bovis (syn B__ L. argentina). Vet. Immunol. Immunopathol., 2: 145-156. Soluble antigen which protected susceptible c a t t l e against challenge with Babesia bovis was extracted from B. bovis-infected erythrocytes by sonic d i s i n t e g r a t i o n and separation of the soluble from the insoluble matter by u l t r a c e n t r i f u g a t i o n . The material was then fractionated by the p r e c i p i t a t i o n of f i b r i n o g e n - l i k e proteins. The p r e c i p i t a t e contained the babesial antigens that were located on the stroma of the infected erythrocytes. Antigen o r i g i n a l l y located on the parasite remained in s o l u t i o n . Both f r a c t i o n s conferred protection on splenectomized calves against challenge with B. bovis. However, the f r a c t i o n containing the parasite antigens appeared to have more potential for development as a k i l l e d vaccine because i t was not heavily contaminated with antigenic material from bovine erythrocytes. INTRODUCTION Mahoney (1967a) showed that c a t t l e , immunized with k i l l e d suspensions of Babesia bovis prepared by the l y s i s of infected erythrocytes in d i s t i l l e d water followed by c e n t r i f u g a t i o n and freeze-drying of the sediment, were protected from c l i n i c a l disease a f t e r challenge with organisms from the same s t r a i n used to prepare the antigen. These observations were confirmed and extended by Mahoney and Wright (1976).
A
crude antigen prepared by d i s r u p t i o n of infected erythrocytes in a French pressure c e l l immunized c a t t l e against heterologous challenge with B. bovis, and the degree of immunity was s i m i l a r to that observed in a group of n a t u r a l l y infected animals. I t appeared that immunization of c a t t l e in the f i e l d against B. bovis with a k i l l e d antigen might be feasible.
Up to the present time, the only successful anti-babesial
vaccines consist of l i v i n g organisms which confer protection by producing i n f e c t i o n (Callow, 1977).
The disadvantages of such vaccines are (a) the dissemination of the
organisms in the environment, (b) the accidental spread of other host diseases, 0 1 6 5 - 2 4 2 7 / 8 1 / 0 0 0 0 - - 0 0 0 0 / $ 02.50 © 1981 Elsevier Scientific Publishing C o m p a n y
146
(c) the production of haemolytic anaemia in the new born (Dimmock, 1973) and (d) a l i m i t e d shelf l i f e .
A vaccine composed of s p e c i f i c babesial antigen appropriately
p u r i f i e d which s a t i s f i e d basic requirements regarding e f f i c i e n c y and longevity of protection would circumvent these problems.
The use of extracts of infected erythro-
cytes for the immunization of c a t t l e against B. bovis and evaluation of t h e i r potent i a l f o r f u r t h e r i n v e s t i g a t i o n as vaccines are described in this paper. MATERIALS AND METHODS Experimental Animals Cattle used in the experiments were of Bos taurus breeds and were e i t h e r 2-3 months or 2-3 years of age.
They were purchased in a Babesia-free area and were
maintained in the laboratory under conditions that precluded accidental babesial infection.
At the time of purchase, a l l were negative to the i n d i r e c t haemagglutin-
ation (IHA) test (Goodger, 1971) for B. bovis antibodies.
The 2-3 month old calves
were splenectomized 14 days before the commencement of the experiments and thick blood films were examined to detect those calves infected with other blood parasites. The Organisms The two strains of B. bovis designated "S" and "L" respectively, described by Mahoney and Wright (1976), were used in the experiments. Preparation of Extracts The "S" s t r a i n of B. bovis was used for antigen production.
Infected erythro-
cytes were concentrated from infected blood to approximately 95% supensions by d i f f e r e n t i a l l y s i s of the non-infected c e l l s in hypotonic s a l t solutions (Mahoney, 1967b).
The infected erythrocytes were then suspended in phosphate buffered saline
(PBS), pH 7.2, at a concentration of 5 x 109/ml and were disrupted in e i t h e r a French Pressure Cell at 20,000 p . s . i , or in a sonic d i s i n t e g r a t o r (Raytheon Co., Norwood Mass., U.S.A., Model DF-lOl) at maximum power f o r 4 mins.
This m a t e r i a l ,
designated infected e r y t h r o c y t i c antigen (IEA), was stored in the vapour phase of l i q u i d nitrogen. follows:
Before use, i t was frozen and thawed 3 times and fractionated as
A crude soluble extract (CSE) was the supernatant f l u i d obtained by c e n t r i -
fugation of IEA at 145,000 g for 60 mins. was the crude insoluble extract (CIE). steps.
The sediment a f t e r u l t r a c e n t r i f u g a t i o n
CSE was then passed through two f r a c t i o n a t i o n
The f i r s t was to remove haemoglobin with CM Sephadex (Dozy and Huisman, 1969)
and the second was to i s o l a t e the fibrinogen-associated antigens (FAA) of B. bovis described by Goodger (1973, 1976).
The methods used involved the p r e c i p i t a t i o n of
147 fibrinogen at 37°C by mixing CSE with either equal volumes of a solution containing O.05M CaCl2 and 20 units of thrombin per ml or with 0.1% aqueous protamine sulphate in proportion 2:1 (v/v).
The insoluble precipitates i.e. CIE and FAA were washed 3
times in PBS prior to use. The residual supernatant f l u i d after precipitation of FAA was designated the soluble parasitic extract (SPE). The fractionation scheme is shown in Figure I. INFECTED ERYTHROCYTES
I Disintegration INFECTED ERYTHROCYTEANTIGEN (IEA) Centrifuge at 145,000g x 60 mins
l
I CRUDE INSOLUBLE EXTRACT (CIE)
CRUDE SOLUBLE EXTRACT (CSE) CM
Sephadexj
HAEMOGLOBIN-FREE~ EXTRACT (HFE) ~ Precipitation of Fibrinogen
1
Precipitation of Fibrinogen
~
SOLUBLE PARASITE EXTRACT (SPE)
I
FIBRINOGENASSOCIATED ANTIGEN (FAA)
Figure I. Flow diagram of the fractionation procedure for bovine erythrocytes infected with B. bovis. Immunization of Cattle One dose of each antigen fraction for inoculation into cattle was equivalent to approximately 1.25 x lOlO organisms. This was determined as follows: IEA, 2.5 ml; CIE, the quantity of sediment obtained by ultra-centrifugation of 2.5 ml of IEA; CSE, 2.5 ml; FAA, the quantity of fibrinogen-like precipitate obtained from 5.0 ml of CSE; SPE, the quantity of supernatant f l u i d remaining after precipitation of FAA from 5.0 ml of CSE concentrated to 2.5 ml by membrane f i l t r a t i o n . Precipitates were suspended in 2.5 ml of PBS. All solutions were emulsified with an equal quantity of Freund's Complete Adjuvant (FCA) and inoculated into the animals either subcutaneously (s/c) or intramuscularly (i/m). each 2 weeks apart.
Three doses were given,
148
Assessment of Protection Two models were used to assess protection against B. bovis i n f e c t i o n . consisted of non-splenectomized adult c a t t l e , 2-3 years of age. used in each of the treatment and control groups.
The f i r s t
Four animals were
Two weeks a f t e r the t h i r d injec-
t i o n of antigen they were challenged by the intravenous ( i / v ) inoculation of 108 infected erythrocytes of the heterologous "L" s t r a i n .
Protection was assessed on
the differences in levels of parasitaemia measured by counting the number of organisms/mm3 in jugular blood, f a l l s in packed cell volume (PCV) and rises in rectal temperature between immunized and control groups.
This model was relevant to the
f i e l d s i t u a t i o n , and was used when information on the f e a s i b i l i t y of a p a r t i c u l a r f r a c t i o n f o r practical immunization was required.
The second model employed 2-3
months old splenectomized calves and 5 animals were used in each of the control and treatment groups.
They were splenectomized before the commencement of the experi-
ments in order that they should recover from the stress of the operation before immunization.
They were immunized by the same regime as that used f or the adult
c a t t l e but were challenged by the i / v i n o c u l a t i o n of 106 infected erythrocytes of the homologous "S" s t r a i n .
The results were assessed on the differences between immun-
ised and control groups in f a l l s in PCV, levels of parasitaemia, rates of parasite m u l t i p l i c a t i o n and survival of the animals.
This model was not d i r e c t l y relevant to
the f i e l d s i t u a t i o n , but was useful in screening f rac t io n s for immunogenic a c t i v i t y . Analysis of variance was used to determine the significance of the differences in changes of PCV and rectal temperature between groups.
Differences in levels of
parasiteamia shown by non-splenectomized c a t t l e were analysed by log transformation of the sum of d a i l y parasite counts of i n d i v i d u a l animals.
Differences in levels of
parasitaemia between splenectomized groups were analysed on d a i l y means for each group. Serological Tests The i n d i r e c t fluorescent antibody (IFA) test described by Goodger (1973) was used to detect s p e c i f i c babesial antibodies in the sera of c a t t l e a f t e r immunization and to determine the possible o r i g i n of the babesial antigen in the extracts. Lysins f o r normal bovine erythrocytes in the sera of immunized c a t t l e were assayed as follows:
erythrocytes from a non-infected c a l f were washed 3 times in the test
d i l u e n t ( i s o t o n i c veronal buffer containing Ca++ and Mg++, pH 7.2) and made up to a I% suspension.
Equal volumes of cell suspension, d i l u t e d c a t t l e serum and fresh
undiluted r a b b i t serum as a source of complement were mixed in the wells of standard haemagglutination trays and incubated at 37°C. The trays were shaken every I0 mins and haemolysis read at 30 mins.
Each serum was tested in d i l u t i o n s of I / 5 , I / I 0 . . .
149
. . . . 1/160 against a panel of i n d i v i d u a l c e l l suspensions from I0 normal calves.
Titres
were read as the d i l u t i o n immediately above the l a s t one showing complete haemolysis. RESULTS Extraction of Soluble Protective Antigen from Infected Erythrocytes Experiment I :
Infected erythrocytes were disintegrated in the French Pressure Cell
and soluble (CSE) and insoluble (ClE) f r a c t i o n s were prepared from IEA by u l t r a c e n t r i fugation.
Three groups of 2-3 year old steers were immunized with IEA, CSE and ClE
r e s p e c t i v e l y and the fourth control group received FCA alone.
A f t e r challenge i n f e c -
t i o n with a heterologous B. bovis s t r a i n "L", the four control animals showed severe i l l n e s s characterized by depression, anorexia, weakness, a t a x i a , and one died on the l l t h day a f t e r i n f e c t i o n . severe c l i n i c a l ment.
The animals in the three vaccinated groups did not develop
i l l n e s s , remained strong and continued to eat throughout the experi-
Differences in the percentage f a l l
munized and control groups f i r s t t i o n (PI).
in PCV from p r e i n f e c t i o n levels between im-
reached s i g n i f i c a n c e (p
Mean maximum parasitaemia in the control group was 1600 organisms/mm 3, and
mean maximum levels of 3, 12 and 308 in the IEA, ClE and CSE groups respectively were s i g n i f i c a n t l y lower (p
Rises in rectal temperature
above the p r e i n f e c t i o n level occurred in a l l groups but differences between the CIE, CSE groups and controls were not s i g n i f i c a n t .
However, mean temperature in the IEA
group was s i g n i f i c a n t l y lower than that in the controls on day 8.
There was no other
s i g n i f i c a n t difference between the immunized groups in any of the parameters measured. The data for day 8 PI are given in Table I.
The differences in f a l l s in PCV between
immunized and control groups were h i g h l y s i g n i f i c a n t (p
Mean Max. % Fall PCV*
Mean Max. % Rise Rectal Temperature*
26.5+7.4 28.378.4 31.8¥5.4 46.8¥3
1.8+2.3 3.7¥2.6 6.1¥0.2 5.9¥I.0
* Standard deviations given.
Mean M a x . Parasitaemia~ (Organisms/ m m ' ) 3 12 308 lO00
Proportion of group surviving 4/4 4/4 4/4 3/4
150
Experiment 2:
A batch of infected erythrocytes was divided into two parts.
One
part was disrupted by sonic d i s i n t e g r a t i o n and the other in the French Pressure Cell. The two samples of IEA were then fractionated into soluble and insoluble fractions by ultracentrifugation.
Four f r a c t i o n s , Crude soluble e x t r ac t French Press, CSE (FP),
Crude Insoluble e x t ra c t French Press, ClE (FP), Crude Soluble Extract sonicate CSE (SD), and Crude Insoluble e x t r a c t sonicate, ClE (SD) were used to immunize four groups of 2-3 year old steers.
A control group was inoculated with FCA alone.
After
heterologous challenge the control group showed severe c l i n i c a l i l l n e s s , and two animals died between 9 and I0 days PI. (FP) became c l i n i c a l l y i l l
One animal in the group immunized with CSE
and died on day 9.
Two others in this group developed
higher levels of parasitaemia than those animals in other immunized groups and showed depressed demeanour and anorexia.
The animals in the CIE (FP), CSE (SD) and CIE (SD)
groups showed rises in rectal temperature but no other overt signs of disease.
The
differences in f a l l s in PCV between immunized and control groups became s i g n i f i c a n t (p
There were no differences in this parameter between any of the All groups showed rises in rectal temperature and there was
no s i g n i f i c a n t difference between the control and Group CSE (FP).
The difference
between the controls and the other immunized groups was s i g n i f i c a n t (p
Levels of parasitaemia in a l l immunized groups were s i g n i f i c a n t l y d i f f e r e n t from
those in the controls (p
Group CSE (FP) was also s i g n i f i c a n t l y d i f f e r e n t from
ClE (FP), and CIE (SD) (p
Table 2 shows the data for
day 8 PI. TABLE 2 Experiment 2: The changes in PCV, rectal temperature and parasitaemia, and the survival rates in groups of 2-3 year-old steers, immunized with soluble and insoluble f r a c t i o n s of erythrocytes infected with B. bovis prepared by disruption in the French Press and sonic d i s i n t e g r a t o r . The animals were challenged with i n t r a venous i n o c u l a t i o n of 1 x 108 heterologous organisms. CSE (FP) = crude soluble e x t r a c t (French Press); CSE (SD) = crude soluble ex t r ac t (sonic d i s i n t e g r a t o r ) ; ClE (FP) = crude insoluble e x tr a c t (French Press); ClE (SD) = crude insoluble e xtract (sonic d i s i n t e g r a t o r ) . Antigen Preparation
CSE ( F P ) CSE ( S D ) CIE ( F P ) CIE ( S D ) Control
Mean Max. % Fall PCV*
Mean Max. % Rise Rectal Temperature*
30.0+9 29.0¥8.1 32.1T4.6 21.3¥3.1 54.5¥7.2
4.9+2.2 1.3¥0.5 1.4¥1.0 0.3~0.3 4.6¥0.3
* Standard deviations given.
Mean M a x . Parasitaemia3 (Organisms/ mm ) 2000 14 I0 1.5 8700
Proportion of group surviving 3/4 4/4 4/4 4/4 2/4
151
Protection of Splenectomized Calves with Crude Soluble Extract Infected erythrocytes were disrupted by sonic d i s i n t e g r a t i o n and a soluble fract i o n CSE (SD) was prepared from IEA by u l t r a c e n t r i f u g a t i o n . ectomized calves were used.
Four groups of splen-
Group 1 received 3 doses of IEA, Group 2 received 2
doses of IEA 2 weeks apart, Group 3 received 3 doses of CSE and Group 4 received FCA alone.
Two weeks a f t e r the l a s t i ~ j e c t i o n , a l l calves were challenged with 106
infected red c e l l s of the homologous s t r a i n "S".
All calves developed severe c l i n -
ical i l l n e s s between 6 and I0 days a f t e r i n f e c t i o n .
The f a l l
in PCV of the control
group was s i g n i f i c a n t l y lower (p
No s i g n i f i c a n t differences in PCV were observed between the vaccinated
groups.
There were also s i g n i f i c a n t differences between the levels of parasitaemia
and i t s rate of increase in the immunized and the control groups.
All 3 immunized
groups took s i g n i f i c a n t l y longer (p<.05) than the controls to develop the level of 103 organisms/mm3 in jugular blood, and numbers of parasites/~l were s i g n i f i c a n t l y lower (P=O.O01). Groups 1 and 3 showed lower maximum parasitaemias than Group 2 which received only 2 doses of antigen. survival of the calves.
Marked differences were observed in the
All controls died between days I0 and I I .
All but two
calves in Group 2 died between days I0 and 14 but in each of Groups 1 and 3 only one death occurred on days I0 and 14 respectively. Protection of Splenectomized Calves with Fractions of Crude Soluble Extract Three f ra c t i o n s were prepared from the CSE (SD) by (a) extracting haemoglobin on CM Sephadex (HFE) and (b) p r e c i p i t a t i o n of the fibrinogen-associated antigens with protamine sulphate (FAA).
The t h i r d f r a c t i o n was the supernatant f l u i d a f t e r removal
of the f i b r i n o g e n - l i k e p r e c i p i t a t e (SPE). Twenty splenectomized calves were a l l o c ated at random into 4 groups.
Group 1 received HFE, Group 2, FAA, Group 3, SPE, and
the fourth ( c o n t r o l ) group received FCA alone.
After challenge with the parasite
s t r a i n homologous f o r the antigen a l l the calves again developed c l i n i c a l i l l n e s s a f t e r day 6.
There was no s i g n i f i c a n t difference in f a l l s in PCV and rises in
rectal temperature between any of the groups.
Only one of the f i v e calves in the
control group survived, and a l l deaths occurred on days I0 and I I . parasitaemia f o r the group was 1.3 x 105/mm3.
Mean maximum
In the immunized calves four out of
f i v e calves survived in Groups 1 and 2 and a l l survived in Group 3.
Maximumpara-
sitaemias ranged from 5 x 104/mm3 in Groups 1 and 2 to 3.2 x 104/mm3 in Group 3. Mean levels of parasitaemia in each group, are shown in Figure 2.
The difference in
levels of parasitaemia between the immunized and control groups was s i g n i f i c a n t (p
The s p e c i f i c i t y of the antibody response of the calves in
each group to the i n o c u l a t i o n of antigen was determined by the IFA test.
Antibodies
to HFE stained both parasites and stroma of infected erythrocytes, but the antibodies
152
6.
--.-.... S'
( )
O 0
Control Fibrinogen Associated Antigen Hb-Free Extract Soluble Parasite Extract ~ Survival /
/
(~ ~) ~, _,~,--~,~k
.,.,# - ~ ' ; ' ~ " ...........
(4 s)
~'
,,
~4. ,," S ,
~E E3
%(5",)
e"" $ / o" $ ..-" t / S#
'~ 2
•
~ sf ;V"
O. 0 ..1
o
o
"~
~
~ ~ ~ Days After Infection
~
~
~
Fig. 2. Mean d a i l y parasitaemia a f t e r challenge in groups of splenectomized calves p r e v i o u s l y immunized with e x t r a c t s of erythrocytes i n f e c t e d with B. bovis. to FAA stained the e r y t h r o c y t e membranes and not the p a r a s i t e s .
Dark patches were
observed in the i n f e c t e d c e l l s which were i n t e r p r e t e d to i n d i c a t e the p o s i t i o n of the unstained p a r a s i t e .
The a n t i b o d i e s to SPE stained the parasites and not the
membranes of the i n f e c t e d red c e l l s . are demonstrated in Figure 3.
These features of the antibody responses
P r i o r absorption of the sera with normal e r y t h r o -
cyte stroma reduced the i n t e n s i t y of the s t a i n i n g of the membranes of i n f e c t e d red c e l l s by a n t i b o d i e s to FAA but did not a l t e r the s t a i n i n g of the parasites by a n t i b o d i e s to SPE. L y t i c tests on the sera using a panel of i n d i v i d u a l eryt h r o c y t e suspensions from I0 normal calves showed t h a t the groups immunized with crude haemoglobin-free e x t r a c t and the f i b r i n o g e n - a s s o c i a t e d antigens gave 97% and 76% p o s i t i v e t e s t s r e s p e c t i v e l y .
However the sera from the group t h a t was
immunized w i t h s o l u b l e p a r a s i t e a n t i g e n , gave only 16% p o s i t i v e t e s t s , a l l of which were o f low t i t r e
( I / 2 0 - 1/40) and occurred with the serum of one animal.
153
I
Fig. 3a
Fig. 3b
Fig. 3c
Fig. 3. The s t a i n i n g of B. bovis in IFA tests by antiserum from calves a f t e r 3 inoculations with (a) crude soluble e x t r a c t from B. bovis infected erythrocytes (b) the f i b r i n o g e n associated antigens precipitated ~ the extract and (c) the residual soluble material remaining a f t e r the p r e c i p i t a t i o n step. Antiserum d i l u t e d l:lO0. DISCUSSION Mahoney and Wright (1976) showed that immunization with IEA protected c a t t l e against B. bovis i n f e c t i o n as e f f e c t i v e l y as immunity associated with active i n f e c tion.
This material was a crude mixture of the p a r t i c u l a t e and soluble components
contained by infected erythrocytes and the f i r s t
stage in the p u r i f i c a t i o n of the
babesial antigen(s) was e x t r a c t i o n from t h i s mixture in aqueous s o l u t i o n .
The e a r l -
i e r studies suggested that protective antigen was associated with material insoluble in water, because Mahoney (1967a) immunized c a t t l e with washed parasite-stroma suspensions.
However, Goodger (1971) showed that sonic d i s i n t e g r a t i o n of such prepara-
tions released soluble components that reacted in the i n d i r e c t haemagglutination
154
test f o r B. bovis i n f e c t i o n .
I t was therefore decided to investigate the effects
of the mechanical destruction of infected red c e l l s on the s o l u b i l i t y of the prot e c t i v e antigen.
The soluble extracts prepared a f t e r d i s i n t e g r a t i o n of infected
erythrocytes in the French Pressure Cell gave r e l a t i v e l y poor p r o t e c t i o n , but sonic d i s i n t e g r a t i o n improved t h e i r protective a c t i v i t y to a degree equivalent to that shown by the corresponding sediment (CIE) and crude material (IEA). The reactions of splenectomized calves to challenge a f t e r immunization with IEA and the soluble extracts showed that these animals could be used experimentally to t e s t the p r o t e c t i v e a c t i v i t y of the f r a c t i o n s of infected erythrocytes.
Both of the
above preparations conferred s i g n i f i c a n t degrees of protection in this model, a l though d i f f e r e n t c r i t e r i a from those employed in evaluating results in non-splenectomized adult c a t t l e were used.
I t was found f o r example, that immunized splenectom-
ized calves developed obvious c l i n i c a l disease a f t e r i n f e c t i o n and that maximum f a l l s in PCV were often not s i g n i f i c a n t l y d i f f e r e n t from those in the controls.
However,
differences in the reaction to challenge were manifest in increased survival of the calves, and in parameters related to parasite m u l t i p l i c a t i o n such as the time to reach a specified level of parasitaemia in the blood and the maximum levels attained. The r a t i o n a l e of the f r a c t i o n a t i o n procedure was based on studies by Goodger (1971, 1973, 1976) who showed that the antigens comprising the B. bovis-infected erythrocyte contained several d i f f e r e n t s p e c i f i c i t i e s and could be c l a s s i f i e d into three groups.
The f i r s t
group was composed of two antigens associated with the
stroma of the erythrocyte.
These were responsible for the staining of the membrane
by f l u o r e s c e i n - l a b e l l e d antibodies (Ludford, 1969).
One of the antigens was located
as a dense band in or under the cell membrane and the other had a granular d i s t r i b u t i o n throughout the stroma.
Characterization of the stromal antigens showed that
they were b a s i c a l l y fibrinogen molecules, altered by conjugation with a number of peptides, some of which were of babesial and others of host o r i g i n (Goodger et a l . , 1979).
P u r i f i c a t i o n of the fibrinogen-associated antigen complexes was achieved by
methods that s p e c i f i c a l l y p r e c i p i t a t e d fibrinogen (Goodger, 1976). was composed of antigens located on the parasite. those on the stroma and membrane, but l i t t l e properties.
The second group
They d i f f e r e d in s p e c i f i c i t y from
was known of t h e i r physical and chemical
A t h i r d antigen was found in the cytoplasm of the infected erythrocyte
and extracted from the haemoglobin solution obtained a f t e r l y s i s of the erythrocytes in d i s t i l l e d water.
I t was probably not concerned with protection because protective
antigen remained in the washed parasite-stroma suspension (Mahoney, 1967a).
The
fibrinogen-associated antigens were p r e c i p i t a t e d from the crude soluble e x t r ac t and t h i s step e f f e c t i v e l y separated those antigens that were associated with the erythrocyte stroma and membrane from those located on the parasite.
The differences in
s p e c i f i c i t y of the antibodies that were produced by the calves in each group con-
155
firmed that separation of the two groups was achieved.
Antibodies from the calves
immunized with fibrinogen-associated antigen stained the stroma of infected erythrocytes and the antibodies from the group immunized with the antigen(s) l e f t in solut i o n a f t e r the removal of the p r e c i p i t a t e stained only the parasites. Both f r a c t i o n s protected splenectomized calves against challenge and the group that received the soluble parasite antigen may have shown s l i g h t l y stronger immunity than the other.
A possible i n t e r p r e t a t i o n of t h i s r e s u l t was that an antigenic com-
ponent, common to both f r a c t i o n s but not detectable by IFA test was responsible for protection. B. bovis.
However, there may be more than one mechanism for protection against For example, Mahoney et al.
(1979) showed that antibodies removed the
parasitized erythrocytes from the c i r c u l a t i o n , but t h e i r experiments, did not preclude the p o s s i b i l i t y that protective antibodies might also be directed against the merozoites of B. bovis during t h e i r passage from one c e l l to another.
The
r e s u l t s of the present work would be consistent with the operation of a dual mechanism in which the target antigens for each were of d i f f e r e n t s p e c i f i c i t y and independently capable of conferring protection on susceptible animals.
Another
important aspect of antigen preparation was the contamination of f r a c t i o n s with bovine e r y t h r o c y t i c antigens.
To be p o t e n t i a l l y useful as a vaccine, babesial
antigen must be free of such substances because the r i s k of causing neonatal haemol y t i c anaemia in calves through concurrent immunization of breeding cows against bovine blood-group antigens is unacceptable.
Assessed by t h i s c r i t e r i o n , the poten-
t i a l of the fibrinogen-associated antigens for vaccination appears to be less favourable than that of the soluble parasite antigens. former (Goodger et a l . ,
Antigenic analysis of the
1979) showed that a component of normal erythrocyte stroma
and a babesial moiety which cross-reacted with normal erythrocyte stroma were part of the FAA complex and the separation of these from Babesia-specific antigen would be d i f f i c u l t .
In contrast the low level of a c t i v i t y against bovine antigens
detected in the antiserum to the soluble parasite antigen f r a c t i o n probably resulted from simple contamination of the preparation which should be r e a d i l y eliminated by appropriate design of the f r a c t i o n a t i o n procedure. Abbreviations ClE = crude insoluble e x t r a c t ; CSE = crude soluble e x t r a c t ; FAA = fibrinogen associated antigen; FCA = Freund's Complete Adjuvant; FP = french press; HFE : haemoglobinfree e x t r a c t ; IEA = infected e r y t h r o c y ~ a n t i g e n ; SD = sonic d i s i n t e g r a t i o n , SPE = soluble parasite antigen.
156
Acknowledgments We wish to thank Mr. J.D. Kerr of CSIRO, Division of Mathematics and S t a t i s t i c s f o r assistance with analysis of results and Messrs G.B. Mirre, K. Rode-Bramanis and D. Waltisbuhl for technical assistance. The work was supported in part by funds from the Australian Meat Research Committee. REFERENCES Callow, L . L . , 1977. Vaccination against bovine babesiosis. Adv. Exp. Med. B i o l . , 93: 121-149. Dimmock, C.K., 1973, Blood group antibody production in c a t t l e by a vaccine against Babesia argentina. Res. Vet. S c i . , 15:305-309. Dozy, A.M. and Huisman, T.H.J., 1969. Chromatography of normal and abnormal hemoglobin types on CM Sephadex. J. Chrom., 40:62-70. Goodger, B.V., 1971. Preparation and preliminary assessment of p u r i f i e d antigens in the passive haemagglutination test for bovine babesiosis. Aust. Vet. J., 47: 251-256. Goodger, B.V., 1973. Babesia argentina: i n t r a e r y t h r o c y t i c location of babesial antigen extracted from parasite suspensions. I n t . J. P a r a s i t o l . , 3:387-391. Goodger, B.V., 1976. Babesia argentina: studies on the nature of an antigen associated with i n f e c t i o n . Int. J . P a r a s i t o l . , 6:213-216. Goodger, B.V., Wright, I . G . , Mahoney, D.F. and McKenna, R.V., 1979. Babesia bovis (argentina): studies on the composition and location of antigen associated with infected erythrocytes. Int. J. P a r a s i t o l . , in press. Ludford, C.G., 1969. Fluorescent antibody staining of four Babesia species. Exp. P a r a s i t . , 24:327-335. Mahoney, D.F., 1967a. Bovine babesiosis: the immunization of c a t t l e with k i l l e d Babesia argentina. Exp. P a r a s i t o l . , 20:125-129. Mahoney, D.F., 1967b. Bovine babesiosis: preparation and assessment of complement f i x i n g antigens. Exp. P a r a s i t o l . , 20:232-241. Mahoney, D.F. and Wright, I . G . , 1976. Babesia argentina: immunization of c a t t l e with a k i l l e d antigen against i n f e c t i o n with a heterologous s t r a i n . Vet. Paras i t o l . , 2:273-282. Mahoney, D.F., Kerr, J.D., Goodger, B.V. and Wright, I . G . , 1979. The immune response of c a t t l e to Babesia bovis (synonym B. argentina). Studies on the nature and s p e c i f i c i t y of protection. I n t . J. P a r a s i t o l . , 9: 297-306.
145
BOVINE BABESIOSlS: THE IMMUNIZATIONOF CATTLE WITH FRACTIONS OF ERYTHROCYTES INFECTED WITH BABESIA BOVIS (SYN B. ARGENTINA) D.F. MAHONEY, I.G. WRIGHT and B.V. GOODGER CSIRO, Division of Animal Health, Long Pocket Laboratories, Private Bag No. 3, P.O., I n d o o r o o p i l l y , Queensland, AUSTRALIA 4068 (Accepted 12 January 1981) ABSTRACT Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1981. Bovine babesiosis: the immuni z a t i o n of c a t t l e with f r a c t i o n s of erythrocytes infected with Babesia bovis (syn B__ L. argentina). Vet. Immunol. Immunopathol., 2: 145-156. Soluble antigen which protected susceptible c a t t l e against challenge with Babesia bovis was extracted from B. bovis-infected erythrocytes by sonic d i s i n t e g r a t i o n and separation of the soluble from the insoluble matter by u l t r a c e n t r i f u g a t i o n . The material was then fractionated by the p r e c i p i t a t i o n of f i b r i n o g e n - l i k e proteins. The p r e c i p i t a t e contained the babesial antigens that were located on the stroma of the infected erythrocytes. Antigen o r i g i n a l l y located on the parasite remained in s o l u t i o n . Both f r a c t i o n s conferred protection on splenectomized calves against challenge with B. bovis. However, the f r a c t i o n containing the parasite antigens appeared to have more potential for development as a k i l l e d vaccine because i t was not heavily contaminated with antigenic material from bovine erythrocytes. INTRODUCTION Mahoney (1967a) showed that c a t t l e , immunized with k i l l e d suspensions of Babesia bovis prepared by the l y s i s of infected erythrocytes in d i s t i l l e d water followed by c e n t r i f u g a t i o n and freeze-drying of the sediment, were protected from c l i n i c a l disease a f t e r challenge with organisms from the same s t r a i n used to prepare the antigen. These observations were confirmed and extended by Mahoney and Wright (1976).
A
crude antigen prepared by d i s r u p t i o n of infected erythrocytes in a French pressure c e l l immunized c a t t l e against heterologous challenge with B. bovis, and the degree of immunity was s i m i l a r to that observed in a group of n a t u r a l l y infected animals. I t appeared that immunization of c a t t l e in the f i e l d against B. bovis with a k i l l e d antigen might be feasible.
Up to the present time, the only successful anti-babesial
vaccines consist of l i v i n g organisms which confer protection by producing i n f e c t i o n (Callow, 1977).
The disadvantages of such vaccines are (a) the dissemination of the
organisms in the environment, (b) the accidental spread of other host diseases, 0 1 6 5 - 2 4 2 7 / 8 1 / 0 0 0 0 - - 0 0 0 0 / $ 02.50 © 1981 Elsevier Scientific Publishing C o m p a n y
146
(c) the production of haemolytic anaemia in the new born (Dimmock, 1973) and (d) a l i m i t e d shelf l i f e .
A vaccine composed of s p e c i f i c babesial antigen appropriately
p u r i f i e d which s a t i s f i e d basic requirements regarding e f f i c i e n c y and longevity of protection would circumvent these problems.
The use of extracts of infected erythro-
cytes for the immunization of c a t t l e against B. bovis and evaluation of t h e i r potent i a l f o r f u r t h e r i n v e s t i g a t i o n as vaccines are described in this paper. MATERIALS AND METHODS Experimental Animals Cattle used in the experiments were of Bos taurus breeds and were e i t h e r 2-3 months or 2-3 years of age.
They were purchased in a Babesia-free area and were
maintained in the laboratory under conditions that precluded accidental babesial infection.
At the time of purchase, a l l were negative to the i n d i r e c t haemagglutin-
ation (IHA) test (Goodger, 1971) for B. bovis antibodies.
The 2-3 month old calves
were splenectomized 14 days before the commencement of the experiments and thick blood films were examined to detect those calves infected with other blood parasites. The Organisms The two strains of B. bovis designated "S" and "L" respectively, described by Mahoney and Wright (1976), were used in the experiments. Preparation of Extracts The "S" s t r a i n of B. bovis was used for antigen production.
Infected erythro-
cytes were concentrated from infected blood to approximately 95% supensions by d i f f e r e n t i a l l y s i s of the non-infected c e l l s in hypotonic s a l t solutions (Mahoney, 1967b).
The infected erythrocytes were then suspended in phosphate buffered saline
(PBS), pH 7.2, at a concentration of 5 x 109/ml and were disrupted in e i t h e r a French Pressure Cell at 20,000 p . s . i , or in a sonic d i s i n t e g r a t o r (Raytheon Co., Norwood Mass., U.S.A., Model DF-lOl) at maximum power f o r 4 mins.
This m a t e r i a l ,
designated infected e r y t h r o c y t i c antigen (IEA), was stored in the vapour phase of l i q u i d nitrogen. follows:
Before use, i t was frozen and thawed 3 times and fractionated as
A crude soluble extract (CSE) was the supernatant f l u i d obtained by c e n t r i -
fugation of IEA at 145,000 g for 60 mins. was the crude insoluble extract (CIE). steps.
The sediment a f t e r u l t r a c e n t r i f u g a t i o n
CSE was then passed through two f r a c t i o n a t i o n
The f i r s t was to remove haemoglobin with CM Sephadex (Dozy and Huisman, 1969)
and the second was to i s o l a t e the fibrinogen-associated antigens (FAA) of B. bovis described by Goodger (1973, 1976).
The methods used involved the p r e c i p i t a t i o n of
147 fibrinogen at 37°C by mixing CSE with either equal volumes of a solution containing O.05M CaCl2 and 20 units of thrombin per ml or with 0.1% aqueous protamine sulphate in proportion 2:1 (v/v).
The insoluble precipitates i.e. CIE and FAA were washed 3
times in PBS prior to use. The residual supernatant f l u i d after precipitation of FAA was designated the soluble parasitic extract (SPE). The fractionation scheme is shown in Figure I. INFECTED ERYTHROCYTES
I Disintegration INFECTED ERYTHROCYTEANTIGEN (IEA) Centrifuge at 145,000g x 60 mins
l
I CRUDE INSOLUBLE EXTRACT (CIE)
CRUDE SOLUBLE EXTRACT (CSE) CM
Sephadexj
HAEMOGLOBIN-FREE~ EXTRACT (HFE) ~ Precipitation of Fibrinogen
1
Precipitation of Fibrinogen
~
SOLUBLE PARASITE EXTRACT (SPE)
I
FIBRINOGENASSOCIATED ANTIGEN (FAA)
Figure I. Flow diagram of the fractionation procedure for bovine erythrocytes infected with B. bovis. Immunization of Cattle One dose of each antigen fraction for inoculation into cattle was equivalent to approximately 1.25 x lOlO organisms. This was determined as follows: IEA, 2.5 ml; CIE, the quantity of sediment obtained by ultra-centrifugation of 2.5 ml of IEA; CSE, 2.5 ml; FAA, the quantity of fibrinogen-like precipitate obtained from 5.0 ml of CSE; SPE, the quantity of supernatant f l u i d remaining after precipitation of FAA from 5.0 ml of CSE concentrated to 2.5 ml by membrane f i l t r a t i o n . Precipitates were suspended in 2.5 ml of PBS. All solutions were emulsified with an equal quantity of Freund's Complete Adjuvant (FCA) and inoculated into the animals either subcutaneously (s/c) or intramuscularly (i/m). each 2 weeks apart.
Three doses were given,
148
Assessment of Protection Two models were used to assess protection against B. bovis i n f e c t i o n . consisted of non-splenectomized adult c a t t l e , 2-3 years of age. used in each of the treatment and control groups.
The f i r s t
Four animals were
Two weeks a f t e r the t h i r d injec-
t i o n of antigen they were challenged by the intravenous ( i / v ) inoculation of 108 infected erythrocytes of the heterologous "L" s t r a i n .
Protection was assessed on
the differences in levels of parasitaemia measured by counting the number of organisms/mm3 in jugular blood, f a l l s in packed cell volume (PCV) and rises in rectal temperature between immunized and control groups.
This model was relevant to the
f i e l d s i t u a t i o n , and was used when information on the f e a s i b i l i t y of a p a r t i c u l a r f r a c t i o n f o r practical immunization was required.
The second model employed 2-3
months old splenectomized calves and 5 animals were used in each of the control and treatment groups.
They were splenectomized before the commencement of the experi-
ments in order that they should recover from the stress of the operation before immunization.
They were immunized by the same regime as that used f or the adult
c a t t l e but were challenged by the i / v i n o c u l a t i o n of 106 infected erythrocytes of the homologous "S" s t r a i n .
The results were assessed on the differences between immun-
ised and control groups in f a l l s in PCV, levels of parasitaemia, rates of parasite m u l t i p l i c a t i o n and survival of the animals.
This model was not d i r e c t l y relevant to
the f i e l d s i t u a t i o n , but was useful in screening f rac t io n s for immunogenic a c t i v i t y . Analysis of variance was used to determine the significance of the differences in changes of PCV and rectal temperature between groups.
Differences in levels of
parasiteamia shown by non-splenectomized c a t t l e were analysed by log transformation of the sum of d a i l y parasite counts of i n d i v i d u a l animals.
Differences in levels of
parasitaemia between splenectomized groups were analysed on d a i l y means for each group. Serological Tests The i n d i r e c t fluorescent antibody (IFA) test described by Goodger (1973) was used to detect s p e c i f i c babesial antibodies in the sera of c a t t l e a f t e r immunization and to determine the possible o r i g i n of the babesial antigen in the extracts. Lysins f o r normal bovine erythrocytes in the sera of immunized c a t t l e were assayed as follows:
erythrocytes from a non-infected c a l f were washed 3 times in the test
d i l u e n t ( i s o t o n i c veronal buffer containing Ca++ and Mg++, pH 7.2) and made up to a I% suspension.
Equal volumes of cell suspension, d i l u t e d c a t t l e serum and fresh
undiluted r a b b i t serum as a source of complement were mixed in the wells of standard haemagglutination trays and incubated at 37°C. The trays were shaken every I0 mins and haemolysis read at 30 mins.
Each serum was tested in d i l u t i o n s of I / 5 , I / I 0 . . .
149
. . . . 1/160 against a panel of i n d i v i d u a l c e l l suspensions from I0 normal calves.
Titres
were read as the d i l u t i o n immediately above the l a s t one showing complete haemolysis. RESULTS Extraction of Soluble Protective Antigen from Infected Erythrocytes Experiment I :
Infected erythrocytes were disintegrated in the French Pressure Cell
and soluble (CSE) and insoluble (ClE) f r a c t i o n s were prepared from IEA by u l t r a c e n t r i fugation.
Three groups of 2-3 year old steers were immunized with IEA, CSE and ClE
r e s p e c t i v e l y and the fourth control group received FCA alone.
A f t e r challenge i n f e c -
t i o n with a heterologous B. bovis s t r a i n "L", the four control animals showed severe i l l n e s s characterized by depression, anorexia, weakness, a t a x i a , and one died on the l l t h day a f t e r i n f e c t i o n . severe c l i n i c a l ment.
The animals in the three vaccinated groups did not develop
i l l n e s s , remained strong and continued to eat throughout the experi-
Differences in the percentage f a l l
munized and control groups f i r s t t i o n (PI).
in PCV from p r e i n f e c t i o n levels between im-
reached s i g n i f i c a n c e (p
Mean maximum parasitaemia in the control group was 1600 organisms/mm 3, and
mean maximum levels of 3, 12 and 308 in the IEA, ClE and CSE groups respectively were s i g n i f i c a n t l y lower (p
Rises in rectal temperature
above the p r e i n f e c t i o n level occurred in a l l groups but differences between the CIE, CSE groups and controls were not s i g n i f i c a n t .
However, mean temperature in the IEA
group was s i g n i f i c a n t l y lower than that in the controls on day 8.
There was no other
s i g n i f i c a n t difference between the immunized groups in any of the parameters measured. The data for day 8 PI are given in Table I.
The differences in f a l l s in PCV between
immunized and control groups were h i g h l y s i g n i f i c a n t (p
Mean Max. % Fall PCV*
Mean Max. % Rise Rectal Temperature*
26.5+7.4 28.378.4 31.8¥5.4 46.8¥3
1.8+2.3 3.7¥2.6 6.1¥0.2 5.9¥I.0
* Standard deviations given.
Mean M a x . Parasitaemia~ (Organisms/ m m ' ) 3 12 308 lO00
Proportion of group surviving 4/4 4/4 4/4 3/4
150
Experiment 2:
A batch of infected erythrocytes was divided into two parts.
One
part was disrupted by sonic d i s i n t e g r a t i o n and the other in the French Pressure Cell. The two samples of IEA were then fractionated into soluble and insoluble fractions by ultracentrifugation.
Four f r a c t i o n s , Crude soluble e x t r ac t French Press, CSE (FP),
Crude Insoluble e x t ra c t French Press, ClE (FP), Crude Soluble Extract sonicate CSE (SD), and Crude Insoluble e x t r a c t sonicate, ClE (SD) were used to immunize four groups of 2-3 year old steers.
A control group was inoculated with FCA alone.
After
heterologous challenge the control group showed severe c l i n i c a l i l l n e s s , and two animals died between 9 and I0 days PI. (FP) became c l i n i c a l l y i l l
One animal in the group immunized with CSE
and died on day 9.
Two others in this group developed
higher levels of parasitaemia than those animals in other immunized groups and showed depressed demeanour and anorexia.
The animals in the CIE (FP), CSE (SD) and CIE (SD)
groups showed rises in rectal temperature but no other overt signs of disease.
The
differences in f a l l s in PCV between immunized and control groups became s i g n i f i c a n t (p
There were no differences in this parameter between any of the All groups showed rises in rectal temperature and there was
no s i g n i f i c a n t difference between the control and Group CSE (FP).
The difference
between the controls and the other immunized groups was s i g n i f i c a n t (p
Levels of parasitaemia in a l l immunized groups were s i g n i f i c a n t l y d i f f e r e n t from
those in the controls (p
Group CSE (FP) was also s i g n i f i c a n t l y d i f f e r e n t from
ClE (FP), and CIE (SD) (p
Table 2 shows the data for
day 8 PI. TABLE 2 Experiment 2: The changes in PCV, rectal temperature and parasitaemia, and the survival rates in groups of 2-3 year-old steers, immunized with soluble and insoluble f r a c t i o n s of erythrocytes infected with B. bovis prepared by disruption in the French Press and sonic d i s i n t e g r a t o r . The animals were challenged with i n t r a venous i n o c u l a t i o n of 1 x 108 heterologous organisms. CSE (FP) = crude soluble e x t r a c t (French Press); CSE (SD) = crude soluble ex t r ac t (sonic d i s i n t e g r a t o r ) ; ClE (FP) = crude insoluble e x tr a c t (French Press); ClE (SD) = crude insoluble e xtract (sonic d i s i n t e g r a t o r ) . Antigen Preparation
CSE ( F P ) CSE ( S D ) CIE ( F P ) CIE ( S D ) Control
Mean Max. % Fall PCV*
Mean Max. % Rise Rectal Temperature*
30.0+9 29.0¥8.1 32.1T4.6 21.3¥3.1 54.5¥7.2
4.9+2.2 1.3¥0.5 1.4¥1.0 0.3~0.3 4.6¥0.3
* Standard deviations given.
Mean M a x . Parasitaemia3 (Organisms/ mm ) 2000 14 I0 1.5 8700
Proportion of group surviving 3/4 4/4 4/4 4/4 2/4
151
Protection of Splenectomized Calves with Crude Soluble Extract Infected erythrocytes were disrupted by sonic d i s i n t e g r a t i o n and a soluble fract i o n CSE (SD) was prepared from IEA by u l t r a c e n t r i f u g a t i o n . ectomized calves were used.
Four groups of splen-
Group 1 received 3 doses of IEA, Group 2 received 2
doses of IEA 2 weeks apart, Group 3 received 3 doses of CSE and Group 4 received FCA alone.
Two weeks a f t e r the l a s t i ~ j e c t i o n , a l l calves were challenged with 106
infected red c e l l s of the homologous s t r a i n "S".
All calves developed severe c l i n -
ical i l l n e s s between 6 and I0 days a f t e r i n f e c t i o n .
The f a l l
in PCV of the control
group was s i g n i f i c a n t l y lower (p
No s i g n i f i c a n t differences in PCV were observed between the vaccinated
groups.
There were also s i g n i f i c a n t differences between the levels of parasitaemia
and i t s rate of increase in the immunized and the control groups.
All 3 immunized
groups took s i g n i f i c a n t l y longer (p<.05) than the controls to develop the level of 103 organisms/mm3 in jugular blood, and numbers of parasites/~l were s i g n i f i c a n t l y lower (P=O.O01). Groups 1 and 3 showed lower maximum parasitaemias than Group 2 which received only 2 doses of antigen. survival of the calves.
Marked differences were observed in the
All controls died between days I0 and I I .
All but two
calves in Group 2 died between days I0 and 14 but in each of Groups 1 and 3 only one death occurred on days I0 and 14 respectively. Protection of Splenectomized Calves with Fractions of Crude Soluble Extract Three f ra c t i o n s were prepared from the CSE (SD) by (a) extracting haemoglobin on CM Sephadex (HFE) and (b) p r e c i p i t a t i o n of the fibrinogen-associated antigens with protamine sulphate (FAA).
The t h i r d f r a c t i o n was the supernatant f l u i d a f t e r removal
of the f i b r i n o g e n - l i k e p r e c i p i t a t e (SPE). Twenty splenectomized calves were a l l o c ated at random into 4 groups.
Group 1 received HFE, Group 2, FAA, Group 3, SPE, and
the fourth ( c o n t r o l ) group received FCA alone.
After challenge with the parasite
s t r a i n homologous f o r the antigen a l l the calves again developed c l i n i c a l i l l n e s s a f t e r day 6.
There was no s i g n i f i c a n t difference in f a l l s in PCV and rises in
rectal temperature between any of the groups.
Only one of the f i v e calves in the
control group survived, and a l l deaths occurred on days I0 and I I . parasitaemia f o r the group was 1.3 x 105/mm3.
Mean maximum
In the immunized calves four out of
f i v e calves survived in Groups 1 and 2 and a l l survived in Group 3.
Maximumpara-
sitaemias ranged from 5 x 104/mm3 in Groups 1 and 2 to 3.2 x 104/mm3 in Group 3. Mean levels of parasitaemia in each group, are shown in Figure 2.
The difference in
levels of parasitaemia between the immunized and control groups was s i g n i f i c a n t (p
The s p e c i f i c i t y of the antibody response of the calves in
each group to the i n o c u l a t i o n of antigen was determined by the IFA test.
Antibodies
to HFE stained both parasites and stroma of infected erythrocytes, but the antibodies
152
6.
--.-.... S'
( )
O 0
Control Fibrinogen Associated Antigen Hb-Free Extract Soluble Parasite Extract ~ Survival /
/
(~ ~) ~, _,~,--~,~k
.,.,# - ~ ' ; ' ~ " ...........
(4 s)
~'
,,
~4. ,," S ,
~E E3
%(5",)
e"" $ / o" $ ..-" t / S#
'~ 2
•
~ sf ;V"
O. 0 ..1
o
o
"~
~
~ ~ ~ Days After Infection
~
~
~
Fig. 2. Mean d a i l y parasitaemia a f t e r challenge in groups of splenectomized calves p r e v i o u s l y immunized with e x t r a c t s of erythrocytes i n f e c t e d with B. bovis. to FAA stained the e r y t h r o c y t e membranes and not the p a r a s i t e s .
Dark patches were
observed in the i n f e c t e d c e l l s which were i n t e r p r e t e d to i n d i c a t e the p o s i t i o n of the unstained p a r a s i t e .
The a n t i b o d i e s to SPE stained the parasites and not the
membranes of the i n f e c t e d red c e l l s . are demonstrated in Figure 3.
These features of the antibody responses
P r i o r absorption of the sera with normal e r y t h r o -
cyte stroma reduced the i n t e n s i t y of the s t a i n i n g of the membranes of i n f e c t e d red c e l l s by a n t i b o d i e s to FAA but did not a l t e r the s t a i n i n g of the parasites by a n t i b o d i e s to SPE. L y t i c tests on the sera using a panel of i n d i v i d u a l eryt h r o c y t e suspensions from I0 normal calves showed t h a t the groups immunized with crude haemoglobin-free e x t r a c t and the f i b r i n o g e n - a s s o c i a t e d antigens gave 97% and 76% p o s i t i v e t e s t s r e s p e c t i v e l y .
However the sera from the group t h a t was
immunized w i t h s o l u b l e p a r a s i t e a n t i g e n , gave only 16% p o s i t i v e t e s t s , a l l of which were o f low t i t r e
( I / 2 0 - 1/40) and occurred with the serum of one animal.
153
I
Fig. 3a
Fig. 3b
Fig. 3c
Fig. 3. The s t a i n i n g of B. bovis in IFA tests by antiserum from calves a f t e r 3 inoculations with (a) crude soluble e x t r a c t from B. bovis infected erythrocytes (b) the f i b r i n o g e n associated antigens precipitated ~ the extract and (c) the residual soluble material remaining a f t e r the p r e c i p i t a t i o n step. Antiserum d i l u t e d l:lO0. DISCUSSION Mahoney and Wright (1976) showed that immunization with IEA protected c a t t l e against B. bovis i n f e c t i o n as e f f e c t i v e l y as immunity associated with active i n f e c tion.
This material was a crude mixture of the p a r t i c u l a t e and soluble components
contained by infected erythrocytes and the f i r s t
stage in the p u r i f i c a t i o n of the
babesial antigen(s) was e x t r a c t i o n from t h i s mixture in aqueous s o l u t i o n .
The e a r l -
i e r studies suggested that protective antigen was associated with material insoluble in water, because Mahoney (1967a) immunized c a t t l e with washed parasite-stroma suspensions.
However, Goodger (1971) showed that sonic d i s i n t e g r a t i o n of such prepara-
tions released soluble components that reacted in the i n d i r e c t haemagglutination
154
test f o r B. bovis i n f e c t i o n .
I t was therefore decided to investigate the effects
of the mechanical destruction of infected red c e l l s on the s o l u b i l i t y of the prot e c t i v e antigen.
The soluble extracts prepared a f t e r d i s i n t e g r a t i o n of infected
erythrocytes in the French Pressure Cell gave r e l a t i v e l y poor p r o t e c t i o n , but sonic d i s i n t e g r a t i o n improved t h e i r protective a c t i v i t y to a degree equivalent to that shown by the corresponding sediment (CIE) and crude material (IEA). The reactions of splenectomized calves to challenge a f t e r immunization with IEA and the soluble extracts showed that these animals could be used experimentally to t e s t the p r o t e c t i v e a c t i v i t y of the f r a c t i o n s of infected erythrocytes.
Both of the
above preparations conferred s i g n i f i c a n t degrees of protection in this model, a l though d i f f e r e n t c r i t e r i a from those employed in evaluating results in non-splenectomized adult c a t t l e were used.
I t was found f o r example, that immunized splenectom-
ized calves developed obvious c l i n i c a l disease a f t e r i n f e c t i o n and that maximum f a l l s in PCV were often not s i g n i f i c a n t l y d i f f e r e n t from those in the controls.
However,
differences in the reaction to challenge were manifest in increased survival of the calves, and in parameters related to parasite m u l t i p l i c a t i o n such as the time to reach a specified level of parasitaemia in the blood and the maximum levels attained. The r a t i o n a l e of the f r a c t i o n a t i o n procedure was based on studies by Goodger (1971, 1973, 1976) who showed that the antigens comprising the B. bovis-infected erythrocyte contained several d i f f e r e n t s p e c i f i c i t i e s and could be c l a s s i f i e d into three groups.
The f i r s t
group was composed of two antigens associated with the
stroma of the erythrocyte.
These were responsible for the staining of the membrane
by f l u o r e s c e i n - l a b e l l e d antibodies (Ludford, 1969).
One of the antigens was located
as a dense band in or under the cell membrane and the other had a granular d i s t r i b u t i o n throughout the stroma.
Characterization of the stromal antigens showed that
they were b a s i c a l l y fibrinogen molecules, altered by conjugation with a number of peptides, some of which were of babesial and others of host o r i g i n (Goodger et a l . , 1979).
P u r i f i c a t i o n of the fibrinogen-associated antigen complexes was achieved by
methods that s p e c i f i c a l l y p r e c i p i t a t e d fibrinogen (Goodger, 1976). was composed of antigens located on the parasite. those on the stroma and membrane, but l i t t l e properties.
The second group
They d i f f e r e d in s p e c i f i c i t y from
was known of t h e i r physical and chemical
A t h i r d antigen was found in the cytoplasm of the infected erythrocyte
and extracted from the haemoglobin solution obtained a f t e r l y s i s of the erythrocytes in d i s t i l l e d water.
I t was probably not concerned with protection because protective
antigen remained in the washed parasite-stroma suspension (Mahoney, 1967a).
The
fibrinogen-associated antigens were p r e c i p i t a t e d from the crude soluble e x t r ac t and t h i s step e f f e c t i v e l y separated those antigens that were associated with the erythrocyte stroma and membrane from those located on the parasite.
The differences in
s p e c i f i c i t y of the antibodies that were produced by the calves in each group con-
155
firmed that separation of the two groups was achieved.
Antibodies from the calves
immunized with fibrinogen-associated antigen stained the stroma of infected erythrocytes and the antibodies from the group immunized with the antigen(s) l e f t in solut i o n a f t e r the removal of the p r e c i p i t a t e stained only the parasites. Both f r a c t i o n s protected splenectomized calves against challenge and the group that received the soluble parasite antigen may have shown s l i g h t l y stronger immunity than the other.
A possible i n t e r p r e t a t i o n of t h i s r e s u l t was that an antigenic com-
ponent, common to both f r a c t i o n s but not detectable by IFA test was responsible for protection. B. bovis.
However, there may be more than one mechanism for protection against For example, Mahoney et al.
(1979) showed that antibodies removed the
parasitized erythrocytes from the c i r c u l a t i o n , but t h e i r experiments, did not preclude the p o s s i b i l i t y that protective antibodies might also be directed against the merozoites of B. bovis during t h e i r passage from one c e l l to another.
The
r e s u l t s of the present work would be consistent with the operation of a dual mechanism in which the target antigens for each were of d i f f e r e n t s p e c i f i c i t y and independently capable of conferring protection on susceptible animals.
Another
important aspect of antigen preparation was the contamination of f r a c t i o n s with bovine e r y t h r o c y t i c antigens.
To be p o t e n t i a l l y useful as a vaccine, babesial
antigen must be free of such substances because the r i s k of causing neonatal haemol y t i c anaemia in calves through concurrent immunization of breeding cows against bovine blood-group antigens is unacceptable.
Assessed by t h i s c r i t e r i o n , the poten-
t i a l of the fibrinogen-associated antigens for vaccination appears to be less favourable than that of the soluble parasite antigens. former (Goodger et a l . ,
Antigenic analysis of the
1979) showed that a component of normal erythrocyte stroma
and a babesial moiety which cross-reacted with normal erythrocyte stroma were part of the FAA complex and the separation of these from Babesia-specific antigen would be d i f f i c u l t .
In contrast the low level of a c t i v i t y against bovine antigens
detected in the antiserum to the soluble parasite antigen f r a c t i o n probably resulted from simple contamination of the preparation which should be r e a d i l y eliminated by appropriate design of the f r a c t i o n a t i o n procedure. Abbreviations ClE = crude insoluble e x t r a c t ; CSE = crude soluble e x t r a c t ; FAA = fibrinogen associated antigen; FCA = Freund's Complete Adjuvant; FP = french press; HFE : haemoglobinfree e x t r a c t ; IEA = infected e r y t h r o c y ~ a n t i g e n ; SD = sonic d i s i n t e g r a t i o n , SPE = soluble parasite antigen.
156
Acknowledgments We wish to thank Mr. J.D. Kerr of CSIRO, Division of Mathematics and S t a t i s t i c s f o r assistance with analysis of results and Messrs G.B. Mirre, K. Rode-Bramanis and D. Waltisbuhl for technical assistance. The work was supported in part by funds from the Australian Meat Research Committee. REFERENCES Callow, L . L . , 1977. Vaccination against bovine babesiosis. Adv. Exp. Med. B i o l . , 93: 121-149. Dimmock, C.K., 1973, Blood group antibody production in c a t t l e by a vaccine against Babesia argentina. Res. Vet. S c i . , 15:305-309. Dozy, A.M. and Huisman, T.H.J., 1969. Chromatography of normal and abnormal hemoglobin types on CM Sephadex. J. Chrom., 40:62-70. Goodger, B.V., 1971. Preparation and preliminary assessment of p u r i f i e d antigens in the passive haemagglutination test for bovine babesiosis. Aust. Vet. J., 47: 251-256. Goodger, B.V., 1973. Babesia argentina: i n t r a e r y t h r o c y t i c location of babesial antigen extracted from parasite suspensions. I n t . J. P a r a s i t o l . , 3:387-391. Goodger, B.V., 1976. Babesia argentina: studies on the nature of an antigen associated with i n f e c t i o n . Int. J . P a r a s i t o l . , 6:213-216. Goodger, B.V., Wright, I . G . , Mahoney, D.F. and McKenna, R.V., 1979. Babesia bovis (argentina): studies on the composition and location of antigen associated with infected erythrocytes. Int. J. P a r a s i t o l . , in press. Ludford, C.G., 1969. Fluorescent antibody staining of four Babesia species. Exp. P a r a s i t . , 24:327-335. Mahoney, D.F., 1967a. Bovine babesiosis: the immunization of c a t t l e with k i l l e d Babesia argentina. Exp. P a r a s i t o l . , 20:125-129. Mahoney, D.F., 1967b. Bovine babesiosis: preparation and assessment of complement f i x i n g antigens. Exp. P a r a s i t o l . , 20:232-241. Mahoney, D.F. and Wright, I . G . , 1976. Babesia argentina: immunization of c a t t l e with a k i l l e d antigen against i n f e c t i o n with a heterologous s t r a i n . Vet. Paras i t o l . , 2:273-282. Mahoney, D.F., Kerr, J.D., Goodger, B.V. and Wright, I . G . , 1979. The immune response of c a t t l e to Babesia bovis (synonym B. argentina). Studies on the nature and s p e c i f i c i t y of protection. I n t . J. P a r a s i t o l . , 9: 297-306.