Immunization against babesiosis: Current studies and future outlook

Preventive Veterinary Medicine, 2 (1984) 401--408

401

Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

IMMUNIZATION AGAINST BABESIOSIS: CURRENTSTUDIES AND FUTURE OUTLOOK D.F. Mahoney, I.G. Wright and B.V. Goodger CSIRO Division of Tropical Animal Science Long Pocket Laboratories, Private Bag No. 3, P.O. IndooroopiIIy, Queensland 4068, Australia

ABSTRACT

Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1984. Immunization against babesiosis: current studies and future outlook. Prev. Vet. Med., 2: 401-408. Experiments were conducted on the induction of immunity to Babesia bovis in cattle with antigen extracted from erythrocytes infected wit-B--t~-~parasite. Protection after inoculation with crude parasite-erythrocyte stroma mixtures was as strong as that induced by natural infection. B. bovis appears to contain several different protective antigens, and one o f ~ h ~ was p a r t i a l l y purified by immunoabsorption using a monoclonal antibody. It is a protein consisting of a single peptide of low molecular weight and appears to be located in or on the surface of the parasite. INTRODUCTION I t is now over a decade since the f i r s t demonstration that immunity could be induced against Babesia parasites by inoculation of their hosts with crude antigenic material prepared from infected blood (Mahoney 1967a, Phillips 1967). However, notwithstanding these developments in immunological research, vaccination with the l i v i n g organism (Callow 1977) is the only available means of preventing babesiosis, even though a number of serious disadvantages are associated with this form of immunization (Mahoney 1981).

The slow progress has

been caused by the nature of the organism and its close association with the host erythrocyte.

It is d i f f i c u l t to separate either the parasites or their

antigens from red-cell material (Mahoney and Goodger 1981) and this invariably results in the immunization of the host with erythrocyte antigens in addition to those from the parasite.

Unlike many bacterial diseases for which k i l l e d

suspensions of the causal organisms are used for immmunization, the isolation of protective antigens of Babesia spp. is a prerequisite for such development. Until recently, Babesia spp. had not been grown successfully in v i t r o .

A cul-

ture system for B. bovis is now available (Levy and Ristic 1980), but although it

increased the a v a i l a b i l i t y of infected blood, the problem of erythrocytic

contamination of antigen remained. Therefore the purification and characterization of the protective antigen(s) is relevant and the eventual aim is to clone and express such substances in a suitable organism in order to solve the dual problem of production and purification of antigen for vaccination. 0167-5877/84/$03.00

© 1984 Elsevier Science Publishers B.V.

402

EXPERIMENTAL METHODS AND RESULTS The experimental work described in t h i s

paper r e f e r s to B. bovis which is a

p a r a s i t e of c a t t l e and one of major economic s i g n i f i c a n c e throughout the w o r l d . The basic requirement f o r s t u d i e s on the antigens of t h i s species was to o b t a i n a concentrated

source

of

the

organism.

This

s e p a r a t i o n of i n f e c t e d from n o n - i n f e c t e d c e l l s

requirement

was met by the

in blood (Mahoney, 1967b).

The

technique was based on the o b s e r v a t i o n t h a t the i n f e c t e d e r y t h r o c y t e s were less susceptible

to

hypotonic

lysis

than the u n i n f e c t e d

cells.

It

was t h e r e f o r e

possible to select a concentration of salt solution that lysed all uninfected erythrocytes

leaving the infected ones intact to be recovered by d i f f e r e n t i a l

centrifugation.

The method was rapid,

preparative scale.

reproducible

and applicable on a

It was used to produce suspensions of 95-100% infected

erythrocytes from blood with parasitaemia in the range of 5 to 15%. Extracts of crude antigen were prepared from these infected cell suspensions by sonic disintegration for 2-4 minutes at maximum power of the instrument followed by centrifugation at 145,000 g for 60 minutes at 4°C. the

crude soluble antigen

and i t

The supernatant f l u i d was

was fractionated by precipitation with

protamine sulphate and by immunoabsorption (IA) using antibodies from immune cattle and monoclonal antibodies produced by the techniques established by Kohler and Milstein (1975).

A flow diagram for fractionation procedures is

shown in Figure 1.

INFECTED ERYTHROCYTES I Disintegration INFECTED ERYTHROCYTE ANTIGEN I Centrifuge at 145,000g x 60mins I

I CRUDE SOLUBLE $ EXTRACT $

CRUDE INSOLUBLE EXTRACT

I

Affinity Purification

I SUPERNATANT FRACTION (SPE)

I Antibodies

Precipitation with ~1 Protamine Sulphate

I PRECIPITATE FRACTION (FAA) I

in Calves

Antibodies in Calves

I

I

Affinity Purification

I ,

Figure 1. Flow diagram of the fractionation procedure for bovine erythrocytes infected with B. bovis.

403

Tests for the immunogenic a c t i v i t y of antigens consisted of the subcutaneous inoculation of the antigen as a water-in-oil emulsion with Freund's Complete Adjuvant (FCA) into either two-year-old steers or 6-month-old splenectomized calves.

In early work three inoculations, two weeks apart were used but in

later studies the number of inoculations were reduced to two and to one. Two to four weeks after immunization, the two-year-old steers were challenged by I/V inoculation with a different strain of B. bovis from the one used to prepare the antigen.

For splenectomized calves the same strain as that used to

prepare the antigen was used for challenge.

The immune response was then

assessed by comparing daily rectal temperatures, levels of parasitaemia and f a l l s in packed cell volume in vaccinated and control groups. The l a t t e r were sham-immunised with FCA alone. 0 ~(9 -IO.E

~. ~ o20, 0 ~ -30. m o ~ -4o >

~

(a) m" ,,o,O-

/ .. ....

.

~ ,'~-~ ~

~.;''" ! !ii!ie~a~ed

~

~

~ - 5C 0

g ~ -~0

"~'~c~:

#~..,.~-'~

.~

~bl

~ -20' ....

~ -ao

g ~ -40.

g

~ -5~' -~0-

o

i

i

A

g

YO

1"2

1~

i'~

Days

Figure 2. (a) Comparison of the changes in packed cell volume (PCV) in cattle vaccinated with crude B. bovis antigen and in cattle immunised by B. bovis infection, after c h a l ~ g ~ h virulent heterologous B. bovis organlsms. Taken from Mahoneyand Wright (1976). (b) The changes in PCV in cattle vaccinated with soluble and insoluble fractions of crude B. bovis antigen and challenged with an heterologous strain of the organism.

404

Protection by crude antigen. Mahoney and Wright (1976) showed that prepared by disruption heterologous B. bovis -

of

immunization with

infected erythrocytes

protected

crude antigen cattle

against

infection as effectively as immunity associated with

-

active infection (Figure 2a).

T h i s material was a mixture of all particulate

and soluble components contained by infected erythrocytes.

It was fractionated

into soluble and insoluble components by ultracentrifugation and both fractions induced similar protection (Figure 2b), demonstrating that protective antigen was released into solution. Fractionation of the crude soluble antigen. The f i r s t

fractionation procedure was based on studies by Goodger (1971,

1973, 1976) who showed that the antigens extracted from the B. bovis-infected erythrocyte contained several different specificities and he classified them 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 staining of the

membrane by fluorescein-labe|led antibodies (Ludford,

1967).

One of

the

antigens was located as a dense band in or under the cell membrane and the other had a granular distribution throughout the stroma. Characterization of the stromal antigens showed that they were basically fibrinogen molecules, altered by conjugation with a number of babesial and others of host origin 1980).

peptides,

some of which were of

(Goodger, Wright, Mahoney and McKenna,

Separationof the fibrinogen-associated antigen complexes was achieved

by methods that

specifically precipitated fibrinogen (Goodger, 1976).

second group was composed of antigens located on the parasite.

The

They differed

in specificity from those on the stroma and red-cell membrane, but l i t t l e was known of their physical and chemical properties.

Another antigen was found in

the cytoplasm of the infected erythrocyte and extracted from the haemoglobin solution obtained after lysis of the erythrocytes

in d i s t i l l e d water.

The

fibrinogen-associated antigens were precipitated from the crude soluble extract with protamine sulphate and this step effectively separated those antigens that were associated with the erythrocyte stroma from those located on the parasite and in the erythrocyte cytoplasm. fraction seemed to confirm

The response in calves inoculated with each

this broad separation because antibodies from those

immunized with fibrinogen-associated antigen stained the stroma of infected erythrocytes in the indirect fluorescent antibody (IFA) test and the antibodies from the group immunized with the antigen(s) l e f t in solution after the removal of the precipitate stained only the parasites (Mahoney, Wright and Goodger, 1981).

405 IA

Eluate

of S P E

4~

~

34

m ~.

2,

~ ( 3 / 5 )

.....

6 0 ._~

~'

t

(a) i

IA Eluate

._ ~

6 0 1,

o

~...--

100 ug

m,~ ( )

500ug Survival

~

(515)

--.. . .

(b)

{

FAA

~

Control

3

of

~

;,

~

(,3/5)

~

~

A

9

Days

Figure 3. Parasitaemia and survival in cattle vaccinated with different doses (100 ug, 500 ~g protein) of antigen obtained by immunoabsorption from (a) a soluble extract of B. bovis (SPE) and (b) a fibrinogen-like precipitate from the soluble e x t r a c T S , bovis (FAA), and challenged with a virulent heterologous strain of t h e ~ r ~ s m . Both fractions protected cattle against challenge.

One interpretation of

this result was that an antigenic component, common to both fractions but not distinguishable by IFA test was responsible for protection.

However, there

could be more than one target antigen involved in protection against B. bovis and these could be located on both the infected red cells and the parasites. Mahoney, Kerr,

Goodger and Wright (1979) concluded that

antibodies attacked targets at both sites.

the

protective

The immunoglobulins from the serum

of calves inoculated with each of the above fractions ( i . e . precipitate and supernatant)

were purified and coupled to

immunoabsorption.

CNBr Sepharose 4B columns for

These columns were then used to extract antigen from the

crude soluble material

(Figure 1).

The concentration of

protein

in

the

extracts was approximately 200 pg/ml and contained in addition to B. bovis-

406

specific

material,

components from normal

specific

absorption of such m a t e r i a l

bovine

erythrocytes

owing to

on the Sepharose columns.

non-

Each f r a c t i o n

contained t h r e e a n t i g e n i c components from B. b o v i s , but t h e r e were no r e a c t i o n s of i d e n t i t y tests.

or even p a r t i a l - i d e n t i t y

between the two groups in immunodiffusion

C a t t l e were immunized w i t h each f r a c t i o n

dose l e v e l s ,

100 ~g and 500 ~g of p r o t e i n ,

the supernatant f r a c t i o n (Figure

3a)

isolated

but

the

(SPE), the dose of

higher

dose

was

from crude s o l u b l e m a t e r i a l

the 500 ug dose gave more e f f e c t i v e

by a s i n g l e i n j e c t i o n .

Two

were used. With the antigens from 100 ug of p r o t e i n gave p r o t e c t i o n

less

protective.

With

the

antigens

by a n t i b o d i e s to the p r e c i p i t a t e

(FAA),

p r o t e c t i o n (Figure 3b).

The Use of Monoclonal Antibodies. Antigen

was

obtained

from

the

c r u d e soluble

extract

by

affinity

chromatography using antibodies from calves previously inoculated with the supernatant fraction

(Figure I).

T h i s material was used to immunize BALB/c

mice and their spleen cells were fused with P3-NSI-Ag4-1 mouse myeloma cells. After screening of the hybrid-cell lines for babesial antibody production by radio immuno assay (RIA) and indirect fluorescent antibody (IFA) tests, three clones were f i n a l l y obtained which showed different staining specificities for B. bovis.

Each clone was used to produce ascites f l u i d in BALB/c mice and

gamma-globulin was extracted from the f l u i d and used for immunoabsorption. The specificity of each monoclonal antibody determined by IFA analysis and the preliminary characterization of

the

fractions extracted by

IA from crude

soluble material are show in Table 1 below. TABLE I . Hybridoma Clone No.

2C3 15B1 18A5

S p e c i f i c i t y of Monoclonal Antibodies by IFA S t a i n i n g

No. of Antigens in IA Eluate (Western t r a n s f e r analysis)*

Parasites + Erythrocyte Stroma Parasites only Parasites only. Preference for blunt end

1 I i

M.W. (daltons)

Strain Specificity of Antigen

1.3X10~ 44X10~ 180X103

multiple multiple multiple

IFA = I n d i r e c t f l u o r e s c e n t a n t i b o d y . IA = A f f i n i t y chromatography. M.W. = m o l e c u l a r w e i g h t . Estimates were made on the n a t i v e p r o t e i n s . * These are p r e l i m i n a r y r e s u l t s using the bovine antiserum to each e l u a t e and a peroxidase l a b e l l e d - a n t i b o v i n e y - g l o b u l i n . Analysis of the 15B1 e l u a t e on p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s showed t h a t i t also contained a number of n o n - a n t i g e n i c contaminating p r o t e i n s , presumably of host o r i g i n . Each e x t r a c t calf

was used to

immunize groups of 4 splenectomized c a l v e s .

was given 2 doses in FCA each c o n t a i n i n g

I00 ~g p r o t e i n ,

Each

4 weeks a p a r t ,

407

and then challenged with the homologous strain of parasites, 2 weeks a f t e r the last i n o c u l a t i o n .

Only the calves immunized with the fraction isolated by 15BI

antibodies showed evidence of protection. days of

infection,

occurred

at

but

3 of

14 days.

the

The control calves died within I0

15BI group survived and the single death

Levels

of

parasitaemia

in

the

group

were

also

s i g n i f i c a n t l y lower than those in the controls from day 5 onwards. DISCUSSION An

effective

d e a d vaccine

characteristics: different

(a)

for

B. bovis

prevention of c l i n i c a l

should

h a v e the

disease;

immunological strains of the parasite;

foliowing

(b) efficacy against

(c) one or two inoculations

should induce protection; (d) protection should be induced for a minimum of 6 months;

(e) no concurrent immunisation against host blood group antigens; ( f )

a v a i l a b i l i t y in large q u a n t i t i e s ; (g) s t a b i l i t y on storage. (b) and (c) above have been f u l f i l l e d duration

of

determined.

protection However, i t

after

The c r i t e r i a

(a),

by the crude antigenic preparations.

immunization

with

antigen

has

The

not

been

has been found that immunity to r e i n f e c t i o n remained

for at least 6 months a f t e r termination of i n fec t io n with chemotherapy (Callow, McGregor, Parker and Dalgliesh, 1974) and a s i m i l a r duration of immunity would be expected to follow antigen inoculation. The p u r i f i c a t i o n of protective antigens w i l l the knowledge accumulated so far.

require some reassessment of

For example, i t

is not yet known whether a

single p u r i f i e d antigen w i l l give protection under a l l circumstances or whether several

antigens

ch a ra c t e r i s t i c s

are

needed to

give

the

artifically-induced

immunity

implied in the essential c r i t e r i a outlined above.

the

A further

complication is the possible i n t e r a c t i o n of a mixture of p u r i f i e d antigens in the host.

Under d i f f e r e n t conditions, one might e i t h e r i n h i b i t or enhance the

effect

another.

of

absorption precipitate least

three

to

(FAA) f r a c t i o n s , babesial

immunodiffusion tests. but

The experiments with

mat er ial,

antibodies from cows immunized with produced

antigens,

that

partially

two preparations, were a l l

purified

crude soluble

by

(SPE) and

each containing at

different

on analysis

with

Both preparations contained impurities of host o r i g i n

nevertheless protected c a t t l e

against

disease in

small

doses.

These

results suggested that B. bovis might contain more than one antigen capable of conferring

protection

on

the

host.

The

three

antigens

detected

by

immunodiffusion in SPE were presumably those isolated by using the monoclonal antibodies 2C3, 15BI and 18A5, but only 15BI was p r o t e c t i v e . induced in

splenectomized calves

by the

The protection

15BI antigen was s i m i l a r

to

that

produced in such animals by crude antigenic extracts of the parasite (Goodger, Wright and Mahoney, 1981).

This suggests therefore that the antigen is a major

408

common protective component of B. bovis parasites.

I t remains to be determined

which of the antigens detected in the FAA fraction are also protective, and i f each can, on its own, provide an effective protection system that w i l l f u l f i l l the

above c r i t e r i a .

If

the

confirmed, there seems l i t t l e available in

protective a c t i v i t y doubt that i t

will

of

the

15B1 protein is

soon be cloned and become

larger quantities suitable for more extensive testing.

This

should also allow work to proceed on the molecular basis of its antigenicity. REFERENCES

Callow L.L., McGregor W., Parker R.J. and Dalgliesh R.J. 1974. The immunity of cattle to Babesia argentina after drug s t e r i l i s a t i o n of infections of varying duration. Aust. Vet. J., 50: 6-11. Callow, L.L., 1977. Vaccination against bovine babesiosis. A d v . Exp. Med. B i o l . , 93, 121-149. Goodger B.V., 1971. Preparation and preliminary assessment of purified antigens in the passive haemagglutination test for bovine babesiosis. Aust. Vet. J., 47: 251-256. Goodger B.V., 1973. Babesiaargentina: intraerythrocytic location of babesial antigen extracted from parasite suspensions. Int. J. Parasitol., 3: 387-391. Goodger B.V., 1976. Babesia argentina: studies on the nature of an antigen associated with infection. Int. J. Parasitol., 6: 213-216. Goodger B.V., Wright I.G., Mahoney D.F. and McKenna R.V., 1980. Babesia bovis (argentina): studies on the composition and location of antigen associated with infected erythrocytes. Int. J. Parasitol., I0: 33-36. Goodger, B.V., Wright, I.G. and Mahoney, D.F., 1981. The use of pathophysiological reactions to assess the efficacy of the immune response to Babesia bovis in cattle. Z. Parasitenkd., 66: 41-48. KoTG.~d Milstein, C., 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London), 256: 495-497. Levy M.G. and Ristic M., 1980. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science, 20/: 1218-1220. Ludford C.G., 1967. Studies on Babesia rodhaini, its morphology, course of infection and immunity in rats; with observations on Babesia affecting cattle. Ph.D. Thesis, University of Queensland. Mahoney D.Fo, 1967a. Bovine babesiosis: the immunization of cattle with k i l l e d Babesia argentina. Exp. Parasitol., 20: 125-129. Mahoney ~ 1 9 6 7 b . B o v i n e babesiosis: preparation and assessment of complement fixing antigens. Exp. Parasitol., 20: 232-241. Mahoney D.F., 1981. Prospects for an antibabesial vaccine. In: M. Ristic and J.P. Kreier (Editors), Babesiosis. Academic Press, New York, pp. 555-562. Mahoney D.F. and Goodger, B.V., 1981. The isolation of Babesia parasites and t h e i r products from the blood. In: M. Ristic and J.P. Kreier (Editors), Babesiosis. Academic Press, New York, pp. 323-335. Mahoney D.F. and Wright I.G., 1976. Babesiaargentina: immunizationof cattle with a k i l l e d antigen against infec ~--Ct-i-6~---with a heterologous strain. Vet. Parasitol., 2: 273-282. Mahoney D.F., K e r r J.D., Goodger B.V. and Wright I.G., 1979. The immune response of cattle to Babesia bovis (syn. B. argentina). Studies on the nature and specificity of protection. Int. J. Parasitol., 9: 297-306. Mahoney D.F., Wright I.G. and Goodger B.V., 1981. Bovine babesiosis: the immunization of cattle with fractions of erythrocytes infected with Babesia bovis (syn. B. argentina). V e t . Immunol. and Immunopathol., 2: 145-156. Ph~s R.S.~--1967. Active immunization of rats against Babesia (Nuttallia) rodhaini using a k i l l e d vaccine. Parasitology, 57: i i p . -

-