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Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babesia bovis immunogen
Veterinary Parasitology, 18 (1985) 321--337 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
321
HETEROLOGOUS STRAIN IMMUNITY IN BOVINE BABESIOSIS USING A CULTURE-DERIVED SOLUBLE BABESIA BO VIS IMMUNOGEN
SONIA MONTENEGRO-JAMES and MIODRAG RISTIC
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL (U.S.A.) MANUEL TORO BENITEZ, EDGAR LEON and ROGER LOPEZ
Instituto de Investigaciones Veterinarias, Fondo Nacional de Investigaciones Agropecuarias, Maracay (Venezuela) (Accepted for publication 25 February 1985)
ABSTRACT Montenegro-James, S., Toro Benitez, M., Leon, E., Lopez, R. and Ristic, M., 1985. Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babesia bovis immunogen. Vet. Parasitol., 18: 321--337. The cross-protective capacity of culture-derived soluble immunogens against heterologous Babesia bovis strains from different geographical locations of Latin America was examined. Susceptible yearling cattle were either immunized with immunogens derived from Venezuelan or Mexican strains, or were administered a multi-component immunogen containing antigens of the Australian, Mexican and Venezuelan strains. Cattle were challenged with virulent B. boris organisms of the Argentinian, Colombian, Ecuadorean, Mexican and Venezuelan strains. The major parameters used to evaluate cross-protection were the following: presence, level and duration of parasitemia; maximal PCV reduction; level and duration of fever; determination of fibrinogen and cryofibrinogen; homologous and heterologous antibody levels; and net gains in body weight. Results showed good protection with a Venezuelan B. bovis immunogen after homologous and heterologous challenge exposures. A low degree of cross-immunity was observed when cattle vaccinated with the Mexican immunogen were challenged with each of the heterologous strains.
INTRODUCTION
Bovine babesiosis is caused by the intra-erythrocytic protozoan Babesia bovis, and represents a major problem for the livestock industry in tropical and sub-tropical areas of the world. Inter-strain antigenic differences exist within the B. bovis (syn. argentina) species, and have been well documented (Callow, 1964, 1968; Curnow, 1968, 1973). Strains maintain their antigenic identity, reversing to a common antigenic type even after tick transmission (Curnow, 1973). Callow et al. {1979) have shown that the virulence of B. bovis organisms may be re-
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© 1985 Elsevier Science Publishers B.V.
322
duced with their rapid passage in splenectomized calves. Attenuated and avirulent parasites are apparently able to induce protective strain immunity to heterologous challenge (Callow et al., 1979; De Vos et al., 1982). Furthermore, there is evidence that avirulent B. bovis strains may better express antigens involved in the induction of protective immune responses (Kahl et al., 1982). Although live avirulent parasites have been effectively used for vaccination against bovine babesiosis in Australia (Callow et al., 1979), inherent disadvantages such as variable infectivity, severe post-vaccination clinical reactions (Callow and Dalgliesh, 1982) and a short shelf life (Mahoney et al., 1981) have proved to be problematic for the widespread use of these live vaccines. Therefore, the potential for a safe and effective non-living vaccine would appear limitless. Since the development of cell culture systems for the continuous propagation of B. bovis parasites (Erp et al., 1980; Levy and Ristic, 1980), several studies (Smith et al., 1981; Kuttler et al., 1982, 1983) have confirmed the efficacy of soluble parasite antigens (James et al., 1981) in providing homologous protection against challenge exposure with virulent B. bovis organisms. The logistic of vaccine development and its subsequent application would probably require heterologous immunity to geographically isolated B. bovis strains. Consequently, culture-derived soluble immunogens should also induce a desirable degree of heterologous strain protection. Recently, Timms et al. (1983) reported partial protection of cattle when vaccinated twice with a culture-derived soluble B. bovis (Australian origin) immunogen, and challenge-exposed with a virulent heterologous strain. The present study was designed to determine the cross-protective effect among B. bovis isolates from various regions (Venezuela, Mexico, Argentina, Ecuador and Colombia) of Latin America, and to assess the efficacy and potential of a culture-derived multi-strain immunogen produced from less virulent B. bovis isolates. MATERIALS AND METHODS
Location
Vaccination trials were performed at the Veterinary Research Institute (VRI) in Maracay, Venezuela. Experimental animals
Animals used in the study were susceptible (negative according to the indirect fluorescent antibody (IFA) test) cross-bred Holstein (Bos taurus) cattle, approximately 18 months of age. They were obtained from a highaltitude tick-free region of Colombia and were maintained in isolation facili-
323 ties at the laboratory. These adult animals were utilized in t w o immunization trials. Additionally, 3--4-month-old calves were splenectomized for use in reactivation of B. bovis strains.
Strains Strains of B. bovis isolated from Venezuela, Mexico, Argentina, Ecuador, Colombia and Australia were kindly provided as stabilates by veterinary research institutes located in those countries. The Australian and Colombian strains, originally isolated during outbreaks of babesiosis, had been attenuated b y rapid passage in splenectomized calves (Callow et al., 1979; Gonzalez et al., 1979) and utilized as live vaccines. All the strains had been maintained b y transmission in Boophilus microplus under laboratory conditions and/or b y syringe-passage in splenectomized animals.
Reactivation o f B. bovis strains Reactivation of each of the six strains was established separately by inoculation of blood stabilates into splenectomized calves. Infections were monitored by examining thin smears of peripheral blood, and by determining b o d y temperatures and packed cell volumes (PCV). When a certain preestablished parasitemia was reached, infected blood of each strain was aseptically collected to initiate in vitro cultivation (1% parasitemia) of the parasites according to the MASP method of Levy and Ristic (1980), to produce stabilates (2--3% parasitemia) by cryopreservation in liquid nitrogen and to prepare infected erythrocytic (at least 5% parasitemia) antigen for IFA tests.
Production o f immunogens Soluble immunogens were prepared from supernatant fluids of Venezuelan, Mexican and Australian B. bovis cultures. Supernatant antigen was collected daffy for 3 days from cultures with parasitemias averaging 6.0-8.0%. Immunogens were prepared as previously described (Kuttler et al., 1982) and mixed with 1 mg Quil-A saponin (Superfos, Denmark) adjuvant per 1 ml vaccinal dose. Each dose consisted of 10X -concentrated supernatant fluid (10 ml-equivalent). In experiment 1, Venezuelan and Mexican immunogens were administered separately, but in Experiment 2 a multi-strain combination of Venezuelan, Mexican and Australian immunogens was formulated. Vaccinal dosages in the latter experiment, consisting of 10 ml of supernatant fluid from each strain, were concentrated 10-fold to 3 ml and then mixed with saponin as above.
Experimental design Two successive experiments were conducted as detailed below.
324
Experiment 1 This experiment was designed to assess heterologous protective i m m u n i t y among B. bovis isolates from various regions of Latin America. Eighteen experimental cattle were divided into two groups and each group was vaccinated with either the Venezuelan (n = 10) or Mexican (n = 8) immunogens. Ten non-vaccinated animals were used as negative controls. The vaccine was administered subcutaneously in two 1-ml doses on Days 0 and 21. All vaccinated and control animals were divided into sub-groups (n = 2) and each challenged 3 months following the second vaccination with 6 × 108 virulent B. bovis organisms of either the Venezuelan, Mexican, Argentinian, Ecuadorean or Colombian strains. No homologous challenge was conducted with the Mexican strain. Experiment 2 The purpose of this subsequent experiment was to examine the protective effect among various B. bovis strains when culture-derived antigens from selected, less virulent isolates were used together as components of a soluble B. bovis immunogen. Thirty-two susceptible yearling cross-bred Holstein cattle from a tick-free zone in Colombia were used in this experiment. Sixteen animals were vaccinated with soluble antigen derived in vitro from high-passage Venezuelan, Mexican and Australian strains. Another 16 animals were retained as nonvaccinated controls. The vaccine was administered subcutaneously in two 3ml doses on Days 0 and 21. The vaccinated and control animals were divided into 4 sub-groups (n = 4) and challenged 1 m o n t h after the second vaccination with 1 × 109 virulent B. bovis organisms of either the Venezuelan, Mexican, Argentinian or Ecuadorean strains. Collection and analysis o f data Body temperature, PCV and platelet levels, parasitemia (thin blood film), IFA titers and selected erythrophilic proteins were monitored throughout the experiments. Plasma fibrinogen and cryofibrinogen levels were determined by the radial immunodiffusion test and by cold precipitation, respectively, according to methods described by Goodger and Wright (1977). Homologous and heterologous IFA titers were measured from the sera of vaccinated animals. Net gains in b o d y weight post-challenge were also calculated. The statistical significance of the difference between means was established by the Student's t-test. RESULTS
Vaccination with Venezuelan B. bovis immunogen In this first experiment, protection was assessed on the basis of differences
Vac.
Vac.
1 G r o u p m e a n s ± s t a n d a r d error. Presence (+) o f p l a s m a c r y o f i b r i n o g e n a t 4 ° C. * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l values, P < 0.05.
45.5±2.5"12.5+-3.5
79.0±5.0 . 46.5+-2.5"14.0+-5.0
79.0±5.0 +
17.5±2.5
Vac.
Mexico
13.0±7.0
14.0±1.0
73.5±5.0 +
8.0±1.0 10.0±2.0 4.0+-2.0 5.0±1.0 3.5+-0.5 6.0±1.0 34.0±2.0 50.0±5.0
Cont.
62.0+-6.0 8 0 . 0 ± 5 . 0 _ +
59.0±3.0 _
Vac.
71.0±4.0 --
Cont.
Colombia
13.0+-0" 10.0+-2.0 1 2 . 0 ± 0 " 2.0 +- 1.0 1 . 0 ± 0 3.0+-0 1.5±1.5 3.0+-1.5 4.0+3.0 25.0+-1.0"38.0±1.0 35.0±7.0
Vac.
Argentina
1 5 . 0 ± 3 . 0 13.0+-1.0 2.0±0* 5.0+2.0 0.5±0.5 5.0+-0 22.0±1.0"35.0±2.0
Cont.
Ecuador
Venezuela Cont.
Heterologous
Homologous
Type of challenge
P r e p a t e n t p e r i o d (days) 0±0" 14.0±0 D u r a t i o n of p a r a s i t e m i a (days) 0 ± 0 " 2.0±0 D u r a t i o n of fever (days) 0.5±0.5 3.5±0 Maximum%PCVreduction 20.0+-1.0"37.0±5.0 Maximum % platelet reduction 51.0±3.0 79.0±3.0 Cryofibrinogen 2 -+ Weight gain (kg) 6 w e e k s post-challenge 37.5±0.5"13.5±4.5
Measurements
12.0±3.0
83.0±3.0 +
7.0+-0 8.0+-0 6:0-+0 51.0±0
Cont.
Clinical a n d p a t h o p h y s i o l o g i c a l r e s p o n s e s ' o f v a c c i n a t e d ( V e n e z u e l a n B. bovis i m m u n o g e n ) and c o n t r o l a n i m a l s t o c h a l l e n g e e x p o s u r e w i t h h o m o l o g o u s a n d h e t e r o l o g o u s B. bovis strains
TABLE I
b~
326
in the duration of prepatent period (established as the time required to visualize parasites in thin blood films), duration of parasitemia, reduction in PCV, presence of erythrophilic cryofibrinogen and net weight gains between immunized and control groups. The data are presented in Table I. Vaccinated animals were fully protected against challenge with the homologous strain, as their clinical condition remained unaltered during the challenge period. No parasitemia was detected and only a slight decrease in PCV was seen, with a maximal reduction of 20%. Additionally, weight gains in vaccinated cattle were 37.5 kg as compared to 13.5 kg in controlanirrials (P <
0.05). The Venezuelan immunogen also demonstrated the ability to induce heterologous strain immunity to challenge with the Ecuadorean and Argentinian strains. Most notably, vaccinated cattle showed maximum PCV reductions of 22 of 25% following the Ecuadorean and Argentinian challenges, respectively, whereas control cattle demonstrated respective decreases of 35 and 38%. Good protection was further evident when net weight gains were analyzed. Significant increases in weight were observed in vaccinated animals 6 weeks after challenge exposure with the Ecuadorean and Argentinian strains (45.5 and 46.5 kg, respectively), although control animals demon-
5120
H H []
[]
z~
A
O
O
Venezuela (Homol) Ecuador Argentina Colombia Australia Mexico
2560
,,i ÷1
1280
I$$ o
2
640
u a}
320
160
//
I
I
I
I
28
35
42
49
Days
Fig. 1. Mean homologous and heterologous anti-B, bouis IFA titers following vaccination with the Venezuelan immunogen.
327
strated limited gains of 12.5 and 14.0 kg, respectively (P < 0.05). However, animals vaccinated with the Venezuelan immunogen failed to protect against challenge with the Mexican and Colombian strains according to most clinical parameters. Following challenge with the Mexican strain, both vaccinated and non-vaccinated cattle were equally affected as PCV reductions (vaccinated = 50%, controls = 51%), presence of erythrophilic plasma cryofibrinogen and duration of fever were similar in all animals. A comparison of peak h o m o l o g o u s and heterologous antibody titers postvaccination is shown in Fig. 1. Maximal h o m o l o g o u s antibody responses were reached 1 week after the second vaccination (mean IFA titers = 1 : 2 2 2 9 ) with high antibody levels sustained for a 3-week period. Heterolog o u s antibody levels were lower for all strains, with the Mexican strain demonstrating the greatest difference during the vaccination period. The kinetics of IFA responses observed throughout the vaccination and challenge periods are shown in Fig. 2. Homologous antibody levels declined slowly postvaccination to a pre-challenge titer of 1:160 on Day 109. A strong anamnestic response began soon afterwards, with titers peaking at 1:327 650 approximately 4 weeks post-challenge (peak antibody titer of control animals = 1:9801). The increase in the heterologous antibody response was more gradual, as a 10-fold difference between h o m o l o g o u s and heterologous IFA
327680 Vaccinates Controls . . . . .
163840
~
Venezuela ~ Ecuador H Argentina I3
81920 40960
_
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.o--~-~-------(3 ~ " ' ~ / ~'~tg~ ~/i /// ~ "ll
20480 10240
/
5120 2560
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./C.i~<~,
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e
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14 28 42 56 70 84 98112/0
t
il°
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Challenge
7
I
I
I
14
21
28
Days
Fig. 2. Mean homologous and heterologous anti-B, boris IFA titers following vaccination with the Venezuelan immunogen and challenge with virulent paxasit~s of the Venezuelan, Ecuadorean, Argentinian, Colombian and Mexican strains, respectively.
14.0-+0 2.0± 0 3.5-+ 1.0 37.0±5.0 79.0-+5.0 + 13.5_+4.5
10.0±3.0 4.0+ 2.0 4.5-+ 1.0 34.0±6.0 77.0±5.0 +
13.5-+1.5
12.5±3.5
+
+
11.0+6.0
13.0±1.0 5.0-+2.0 5.0±0 35.0-+2.0 79.0-+4.0
Cont.
9.0+-1.0 5.0±2.0 3.5-+1.0 37.0-+3.0 74.0±4.0
Vac.
Vac.
Cont.
Ecuador
Venezuela
T y p e of c h a l l e n g e
1 G r o u p m e a n s ± s t a n d a r d error. 2 Presence (+) of p l a s m a c r y o f i b r i n o g e n at 4 ° C. *Significantly d i f f e r e n t f r o m c o n t r o l values, P < 0.05.
P r e p a t e n t p e r i o d (days) D u r a t i o n o f p a r a s i t e m i a (days) D u r a t i o n o f fever (days) Maximum % PCV reduction Maximum % platelet reduction Cyrofibrinogen 2 Weight gain (kg) 6 weeks post-challenge
Measurements
18.5±1.5
4-
10.0+-2.0 4.0_+2.0 2.5-+1.5 29.0±8.0 64.0±4.0
Vac.
Argentina
14.0+-5.0
+
10.0±2.0 1.0-+0 3.0-+1.5 36.0±1.0 77.0-+5.0
Cont.
14.0±1.0
--
10.0-+3.0 3.0-+1.0 1.5±0" 25.0-+2.0 54.0±6.0
Vac.
Colombia
13.0±7.0
4-
8.0-+1.0 4.0-+1.0 3.5_+0.5 34.0±2.0 80.0-+5.0
Cont.
Clinical a n d p a t h o p h y s i o l o g i c a l r e s p o n s e s I o f v a c c i n a t e d ( M e x i c a n B. bovis i m m u n o g e n ) a n d c o n t r o l a n i m a l s to challenge e x p o s u r e w i t h h e t e r o l o g o u s B. bovis strains
T A B L E II
e.O t~ QO
329
titers was observed 10 days post-challenge. Nevertheless, high levels of antib o d y to all B. bovis strains were eventually achieved. Vaccination with Mexican B. bovis immunogen Animals administered with the Mexican B. bovis immunogen responded poorly to heterologous challenge exposure with all the B. bovis strains utilized, as vaccinated cattle showed clinical reactions similar to those found in control animals (Table II). Homologous and heterologous IFA titers produced in response to vaccination are depicted in Fig. 3. Maximal homologous titers (1:5583) occurred 2 weeks after the second vaccinal dose, and were followed by a transient decline in antibody levels until challenge. Heterologous responses were once again consistently lower, especially those specific to the Venezuelan strain. In the absence of clinical protection, high antibody titers to all strains (average maximal titer = 1:106, 211) were obtained postchallenge. 10240
5120
ui ui
2560
÷1
1280 o .o n
"G ID
64O
320 -
Antigens O O II----II O [] • • z~----~
160 -
Mexico (Homol) Ecuador Argentina Colombia Australia Venezuela-
I
I
I
I
28
35
42
49
Days Fig. 3. Mean homologous and heterologous anti-B, boris IFA titers following vaccination with the Mexican immunogen.
330
Vaccination with multi-strain B. bovis immunogen Homologous antibody levels produced in response to vaccination with the single-strain and multi-strain immunogens are shown in Fig. 4. Pre-challenge IFA titers to the multi-strain B. bovis immunogen (Experiment 2) were significantly lower, with a more rapid diminution to baseline levels, than those of either the single-strain Venezuelan or Mexican immunogens (Experiment 1). Maximal post-vaccinal titers corresponded to those induced by the Australian strain (1:1000), followed by those of the Venezuelan (1:766) and Mexican (1:448) strains, respectively. The degree of protection was also assessed by comparing relevant clinical and pathophysiological parameters with those observed in control animals. Data collected following heterologous challenge are summarized in Table III. Results were similar to those obtained in the first experiment, as vaccinated 10240 Immunogen --
5120
---
Venezuelan Mexican M u l t i - strain
2560
1280
$ •-'I-
640
~ rr"
320
160
80
40
< 40
~ io
L 14
I 21
I 28
I 35
I 42
I 49
t Days II °
Fig. 4. Mean homologous anti-B, bovis IFA titers following vaccination with either the Mexican, Venezuelan or multi-strain immunogens.
331 TABLE III Clinical and pathophysiological responses' of vaccinated (multi-strain' B. boris immunogen) and Control animals to challenge exposure with heterologous B. bovis strains Measurements
Type of challenge Argentina
Prepatent period (days) Duration of parasitemia (days) Duration of fever (days) Duration of anemia (days) Maximum PCV reduction (%) Maximum platelet reduction (%) Maximum fibrinogen increase (%) Duration of elevated fibrinogen levels (days) Weight gain (kg) 3 weeks post-challenge
Ecuador
Vac.
Cont.
Vac.
Cont.
3.3-+0.3 2.8-+0.8* 1.0-+0"* 1.0-+0.4"* 37.5-+0.9**
3.3-+0.3 7.3-+0.9 4.0-+0.7 9.7-+0.9 54.3-+5.5
4.5-+0.9 1.8-+0.5" 1.5-+0.3"* 2.8-+ 1.1"* 41.5-+1.5"*
3.7-+0.3 5.0-+0.6 4.7-+0.9 11.3-+3.2 54.7-+6.2
75.9_+6.7
83.9_+4.6
72.0-+3.0"
82.6-+6.6
54.2_+10.1
81.4-+13.9
67.7-+10.5" 103.5-+16.1 4.5-+2.1"
13.3-+1.7
13.5_+2.2"
4.5-+1.2"
4.7_+2.9
10.3-+1.5
12.3-+2.8"
3.1_+2.3
' Group mean + standard error. : B. bovis from Venezuela, Mexico, and Australia (high-passage).
*Significantly different from control values, P < 0.05. **Significantly different from control values, P < 0.01. 163840 81920 Argentinian Antigen : : Vaccinated
40960u~ 20480 --
Q----O Controls
f
Lr~ 10240 -+I 5120
-
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/
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.~ 2580t..1280 .O o_ •~
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160
-
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I I I 14 21 28
t
II°
I 35
I y/ 42 49 0
t
Challenge
Fig. 5. M e a n h e t e r o l o g o u s anti-B,
~fl
I
I
1
7
14
21
28
Days
boris I F A titers f o l l o w i n g v a c c i n a t i o n w i t h the m u l t i -
strain immunogen and challenge with virulent organisms of the Argentinian strain.
332
animals were protected against heterologous challenge with B. bovis strains from Argentina and Ecuador. Only mild, transient clinical changes were observed among vaccinates, whereas all control cattle manifested severe clinical disease characterized b y ataxia, labored respiration, profuse salivation, anorexia and jaundice. Some significant clinical differences were: duration of parasitemia (vaccinates = 2.8 days, controls = 7.3 days after the Argentinian challenge; P < 0.05); duration of anemia (vaccinates = 2.8 days, controls = 11.3 days after the Ecuadorean challenge; P < 0.01); duration of elevated fibrinogen (vaccinates of both groups = 4.5 days, controls = 13.3 days and 10.3 days after Argentinian and Ecuadorean challenges, respectively, P
0.05). A typical IFA response after heterologous challenge is illustrated in Fig. 5. An early anamestic response, as observed following the Argentinian challenge, was characteristic with antibody titers (1:40 960 for vaccinated animals and 1:5120 for controls) peaking 2 weeks post-challenge. Data pertaining to homologous challenge with the Venezuelan and Mexican strains are detailed in Table IV. Cattle vaccinated with the multi-strain B. bovis immunogen demonstrated solid immunity to acute babesiosis subsequent to challenge with the virulent homologous strains. However, nonvaccinated control animals showed significantly greater differences with respect to most pathophysiological parameters (P ~ 0.01). T A B L E IV Clinical a n d p a t h o p h y s i o l o g i c a l r e s p o n s e s ~ o f v a c c i n a t e d ( m u l t i - s t r a i n ~ B. bovis i m m u n o g e n ) a n d c o n t r o l a n i m a l s t o challenge e x p o s u r e w i t h h o m o l o g o u s B. bovis strains Measurements
T y p e o f challenge Venezuela
P r e p a t e n t p e r i o d (days) D u r a t i o n of p a r a s i t e m i a (days) D u r a t i o n of fever (days) D u r a t i o n of a n e m i a (days) M a x i m u m P C V r e d u c t i o n (%) Maximum platelet reduction
(%)
Mexico
Vac.
Cont.
Vac.
Cont.
5.0+_0.6 2.3-+0.5* 0.8-+0.5* 0-+ 0 " * 31.5-+1.7"*
5.7+_0.9 3.7+_0.9 3.7+_0.7 5.2-+2.1 45.3-+2.3
4.0-+0.4* 3.8-+0.5** 3.8-+0.5** 5.5-+0.5** 43.0-+1.2"*
2.6-+0.3 8.0+_1.0 8.7-+0.3 14.3-+2.2 62.7_+4.5
52.6-+3.3*
85.0-+2.9
82.0-+6.0
89.0-+2.7
61.7-+6.1"
84.2+-4.2
59.6-+9.6
58.1+_11.9
7.5+_2.1
7.3+_1.7
7.5+_0.5
8.0_+1.2
14.0+_2.8"
5.0_+2.1
8,8_+3.1
2.1+_1.8
M a x i m u m f i b r i n o g e n increase
(%)
D u r a t i o n of e l e v a t e d fibrinogen levels (days) Weight gain (kg) 3 weeks post-challenge
G r o u p m e a n +- s t a n d a r d error. B. boris f r o m V e n e z u e l a , Mexico, a n d A u s t r a l i a ( h i g h passage). * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l values, P < 0.05. * * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l values, P < 0.01.
333 DISCUSSION Solid homologous protection against challenge exposure with virulent Venezuelan B. bovis organisms was demonstrated in cattle vaccinated with a soluble immunogen derived from cultures of the same B. bovis strain. This finding confirms previous reports where strong homologous immunity was induced with culture-derived immunogens of the Mexican strain (Smith et al., 1981; Kuttler et al., 1982, 1983). ' An important consideration for the potential use of soluble immunogens, however, is their cross-protective capacity between different isolates (strains) of B. bovis, particularly since strain-specific antigenic differences are known to exist (Curnow, 1973). The multiplicity of strain-specific antigens among various Latin American isolates was evident in the present study, as heterologous IFA titers were invariably lower than those obtained in homologous tests. A good degree of cross-protection was shown when cattle vaccinated with the Venezuelan strain were challenged with virulent strains from Ecuador and Argentina, respectively. This indicates that c o m m o n protective antigens exist among strains from various geographical regions. There was little or no protection of cattle vaccinated with the Venezuelan strain and subsequently challenged with Colombian or Mexican strains. Similarly, there was a low degree of cross-protection when animals vaccinated with the Mexican strain were infected with the heterologous strains specified above. An interesting finding in this study was the difference in cross-immunity between the Mexican isolate and all the other strains utilized. One possible explanation for this uniqueness may be the long-standing prohibition of cattle imports into Mexico for the purpose of controlling foot-and-mouth disease. An immunogen containing culture
334 identity was shown for the Australian and Bolivian B. bovis strains (Callow et al., 1976). Based on these findings, an Australian strain was incorporated for use in the present study. Homologous pre-challenge IFA titers to the multi-strain B. bovis immunogen were significantly lower than those produced in response to either the single-strain Venezuelan or Mexican immunogens (Fig. 4). Possibly, excess antigen (3-fold increase in dosage, i.e., one dosage-equivalent per strain) provoked a state of antigenic competition or immunodepression due to an antigen overload at the lymphocyte level. Maximal pre-challenge titers corresponded to those specific for antigens of the Australian strain. Callow et al. (1976) previously found the Australian antigen to be slightly mol'e reactive than the Bolivian antigen in the IFA test. Antigen(s) of the Australian strain may be displaying a unique or structurally dominant epitope which could enhance immunogenicity in vivo. Non-vaccinated control animals in this study showed acute clinical disease following challenge infection, whereas vaccinated cattle showed no marked signs of illness and gained weight throughout the experimental period. Any transient pathophysiological reactions in the latter group were reversed by the fifth day post-challenge. Likewise, parasitemias were rapidly cleared. These observations are similar to those reported during cross-immunity experiments on different Australian isolates (Callow, 1968; Johnston and Tammemagi, 1969; Mahoney et al., 1979b). An assessment of protection may also be based on the prevention of the hypotensive shock syndrome characteristic of acute B. bovis infections (Goodger et al., 1981a). Much of the pathophysiology in bovine babesiosis is associated with changes in coagulation parameters. Indeed, thrombocytopenia, hyperfibrinogenemia and cryofibrinogenic complexes are often observed in non-immune animals suffering from acute infection (Goodger and Wright, 1977; Goodger et al., 1981b). The strong heterologous strain immunity induced by culture-derived soluble immunogens in the present study was able to prevent or ameliorate any damage caused by these pathophysiological reactions. During B. bovis infections, babesial antigen may bind with fibrinogen to form immunogenic complexes (Goodger, 1976). Parasite antigens found within the erythrocytic stroma of infected cells are thought to be fibrinogenassociated (Goodger et al., 1980; Montenegro-James et al., 1983). In addition to the formation of antigen--fibrinogen complexes, fibrinogen may be altered by Babesia parasites, making it antigenic for the host. Thus, autoantibodies to fibrinogen-related products and erythrocytic stroma are detected in Babesia infections (Mahoney and Goodger, 1972; Thoongsuwan et al., 1979). Therefore, it seems conceivable that the elevated plasma fibrinogen concentrations observed in vaccinated and control animals post-challenge could lead to the stimulation of high amounts of non-protective antibody (IFA-reactive) directed toward parasite antigens and/or host autoantigens. Vaccination with culture-derived soluble immunogens has shown that ade-
335
quate protection can be provided against acute infection with heterologous B. bovis strains. Immunogens derived from the Venezuelan and Australian strains would appear to have the best heterologous protective capabilities. Recently, Timms et al. (1983) reported partial heterologous protection of cattle vaccinated with a culture-derived soluble immunogen (Australian), and subsequently challenged with another Australian strain. Mahoney et al. (1979a) have suggested that protective immune responses are mediated by strain-specific antibody. Variant-specific immune mechanisms are obviously initiated by protective antigens having unique antigenic epitopes. However, there is now increasing evidence that cell-mediated immune responses may also play a role in the regulation of protective immunity to babesiosis (Allison, 1984). Cell-mediated immunity to Babesia parasites could act with, or independently from, humoral mechanisms in vaccinated animals. In addition to inducing the strong antibody responses observed in the present study, culture-derived soluble antigens are also potent stimulators of T lymphocyte (Timms et al., 1984) and macrophage (Montealegre et al., 1983) function. One is hopeful that with careful selection and optimization of an adjuvant, both humoral and cell-mediated immune mechanisms can be synergized to provide long-lasting protection against acute babesiosis. ACKNOWLEDGEMENTS
This study was conducted as part of a ,cooperative research project between the Venezuelan Institute for Veterinary Research and the University of Illinois, and was supported by funds from the United States--Israel (Binational) Agricultural Research and Development Fund (BARD) and the Rockefeller Foundation.
REFERENCES Allison, A.C., 1984. Cellular immunity to malaria and Babesia parasites: a personal viewpoint. Contemp. Top. Immunobiol., 12: 463--490. Callow, L.L., 1964. Strain immunity in babesiosis. Nature (London), 204: 1213. Callow, L.L., 1968. A note on homologous strain immunity in Babesia argentina infections. Aust. Vet. J., 44: 268--269. Callow, L.L. and Dalgliesh, R.J., 1982. I m m u n i t y and immunopathology in babesiosis. In: S. Cohen and K.S. Warren (Editors), Immunology of Parasitic Infections. Blackwell Scientific, Oxford, 2nd edn., pp. 475--520. Callow, L.L., Quiroga, Q.C. and McCosker, P.J., 1976. Serological comparison of Australian and South American strains of Babesia argentina and Anaplasma marginale. Int. J. Parasitol., 6 : 307--310. Callow, L.L., Mellors, L.T. and McGregor, W., 1979. Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle. Int. J. Parasitol., 9: 333--338. Curnow, J.A., 1968. In vitro agglutination of bovine erythrocytes infected with Babesia argentina. Nature (London), 217 : 267--268. Curnow, J.A., 1973. Studies on antigenic changes and strain differences in Babesia argentina infections. Aust. Vet. J., 49: 279--283.
336 De Vos, A.J., 1978. Immunogenicity and pathogenicity of three South African strains of Babesia boris in Bos indicus cattle. Onderstepoort J. Vet. Res., 45 : 119--124. De Vos, A.J., Bessenger, R. and Fourie, C.G., 1982. Virulence and heterologous strain immunity of South African and Australian Babesia boris strains with reduced pathogenicity. Onderstepoort J. Vet. Res., 49: 133--136. Emmerson, F.R., Knott, S.G. and Callow, L.L., 1976. Vaccination with Babesia argentina in 5 beef herds in South Eastern Queensland. Aust. Vet. J., 52: 451--454. Erp, E.E., Smith, R.D., Ristic, M. and Osorno, B.M., 1980. Continuous in vitro cultivation of Babesia boris. Am. J. Vet. Res., 41: 1141--1142. Gonzalez, E.R., Todorovic, R.A., Lopez, G. and Garcia, O.A., 1979. Attenuation of a Babesia boris (syn. argentina) strain isolated in Colombia. Rev. Inst. Colomb. Agropecu., 14: 33--39. 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. and Wright, I.G., 1977. Babesia boris (argentina): observations of coagulation parameters, fibrinogen catabolism and fibrinolysis in intact and splenectomized cattle. Z. Parasitenkd., 54: 9--27. Goodger, B.V., Wright, I.G., Mahoney, D.F. and McKenna, R.V., 1980. Babesia boris (argentina): studies on the composition and location of antigen associated with infected erythrocytes. Int. J. Parasitol., 10: 33--36. Goodger, B.V., Wright, I.G. and Mahoney, D.F., 1981a. The use of pathophysiological reactions to assess the efficacy of the immune response to Babesia boris in cattle. Z. Parasitenkd., 66: 41--48. Goodger, B.V., Wright, I.G. and Mahoney, D.F., 1981b. Initial characterization of cryoprecipitates in cattle recovering from acute Babesia boris (argentina) infection. Aust. J. Exp. Biol. Med. Sci., 59: 521--529. James, M.A., Levy, M.G. and Ristic, M., 1981. Isolation and partial characterization of culture-derived soluble Babesia boris antigens. Infect. Immun., 31 : 358--361. Johnston, L.A.Y. and Tammemagi, L., 1969. Bovine babesiosis: duration of latent infection and i m m u n i t y to Babesia argentina. Aust. Vet. J., 45: 445--449. Kahl, L.P., Anders, R.F., Rodwell, B.J., Timms, P. and Mitchell, G.F., 1982. Variable and c o m m o n antigens of Babesia boris parasites differing in strain and virulence. J. Immunol., 129: 1700--1705. Kuttler, K.L., Levy, M.G., James, M.A. and Ristic, M., 1982. Efficacy of a nonviable culture-derived Babesia boris vaccine. Am. J. Vet. Res., 43: 281--284. Kuttler, K.L., Levy, M.G. and Ristic, M., 1983. Cell culture-derived Babesia boris vaccine: sequential challenge exposure of protective i m m u n i t y during a 6-month postvaccination period. Am. J. Vet. Res., 44: 1456--1459. Levy, M.G. and Ristic, M., 1980. Babesia boris: continuous cultivation in a microaerophilous stationary phase culture. Science, 2 0 7 : 1 2 1 8 - - 1 2 2 0 . Mahoney, D.F. and Goodger, B.V., 1972. Babesia argentina: immunogenicity of plasma from infected animals. Exp. Parasitol., 32: 71--85. Mahoney, D.F., Kerr, J.D., Goodger, B.V. and Wright, I.G., 1979a. The immune response of cattle to Babesia boris (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., 1979b. I m m u n i t y in cattle to Babesia boris after single infections with parsites of various origin. Aust. Vet. J., 55: 10--12. Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1981. Bovine babesiosis: the immunization of cattle with fractions of erythrocytes infected with Babesia boris (syn. B. argentina). Vet. Immunol. Immunopathol., 2 : 145--156. McCosker, P.J., 1975. Control of Piroplasmosis and Anaplasmosis in Cattle. A Practical Manual. F.A.O., Santa Cruz, Bolivia.
337 Montealegre, F., Levy, M.G. and Ristic, M., 1983. In vitro secretory activity of bovine blood mononuclear phagocytes and its effect on the growth of Babesia bovis. In: Proc. Second Int. Conf. Malaria and Babesiosis, 19--22 September 1983, Annecy, France, pp. 0--2 (abstract). Montenegro-James, S., James, M.A. and Ristic, M., 1983. Localization of culture-derived soluble Babesia bovis antigens in the infected erythrocyte. Vet. Parasitol., 13: 311-316. Smith, R.D., James, M.A., Ristic, M., Aikawa, M. and Vega, C.A., 1981. Bovine babesiosis: protection of cattle with culture-derived soluble Babesia bovis antigen. Science, 212: 335--338. Thoongsuwan, S., Cox, H.W. and Rickman, W.J., 1979. A n t i b o d y to fibrinogen/fibrin products (anti-F) in malaria, babesiosis, and trypanosomiasis of rodents. J. Parasitol., 65 : 426--429. Timms, P., Dalgliesh, R.J., Barry, D.N., Dimmock, C.K. and Rodwell, B.J., 1983. Babesia bovis: comparison of culture-derived parasites, nonliving antigen and conventional vaccine in the protection of cattle against heterologous challenge. Aust. Vet. J., 60: 75-77. Timms, P., Stewart, N.P., Rodwell, B.J. and Barry, D.N., 1984. Immune responses of cattle following vaccination with living and non-living Babesia bovis antigens. Vet. Parasitol., 16 : 243--251.
321
HETEROLOGOUS STRAIN IMMUNITY IN BOVINE BABESIOSIS USING A CULTURE-DERIVED SOLUBLE BABESIA BO VIS IMMUNOGEN
SONIA MONTENEGRO-JAMES and MIODRAG RISTIC
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL (U.S.A.) MANUEL TORO BENITEZ, EDGAR LEON and ROGER LOPEZ
Instituto de Investigaciones Veterinarias, Fondo Nacional de Investigaciones Agropecuarias, Maracay (Venezuela) (Accepted for publication 25 February 1985)
ABSTRACT Montenegro-James, S., Toro Benitez, M., Leon, E., Lopez, R. and Ristic, M., 1985. Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babesia bovis immunogen. Vet. Parasitol., 18: 321--337. The cross-protective capacity of culture-derived soluble immunogens against heterologous Babesia bovis strains from different geographical locations of Latin America was examined. Susceptible yearling cattle were either immunized with immunogens derived from Venezuelan or Mexican strains, or were administered a multi-component immunogen containing antigens of the Australian, Mexican and Venezuelan strains. Cattle were challenged with virulent B. boris organisms of the Argentinian, Colombian, Ecuadorean, Mexican and Venezuelan strains. The major parameters used to evaluate cross-protection were the following: presence, level and duration of parasitemia; maximal PCV reduction; level and duration of fever; determination of fibrinogen and cryofibrinogen; homologous and heterologous antibody levels; and net gains in body weight. Results showed good protection with a Venezuelan B. bovis immunogen after homologous and heterologous challenge exposures. A low degree of cross-immunity was observed when cattle vaccinated with the Mexican immunogen were challenged with each of the heterologous strains.
INTRODUCTION
Bovine babesiosis is caused by the intra-erythrocytic protozoan Babesia bovis, and represents a major problem for the livestock industry in tropical and sub-tropical areas of the world. Inter-strain antigenic differences exist within the B. bovis (syn. argentina) species, and have been well documented (Callow, 1964, 1968; Curnow, 1968, 1973). Strains maintain their antigenic identity, reversing to a common antigenic type even after tick transmission (Curnow, 1973). Callow et al. {1979) have shown that the virulence of B. bovis organisms may be re-
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322
duced with their rapid passage in splenectomized calves. Attenuated and avirulent parasites are apparently able to induce protective strain immunity to heterologous challenge (Callow et al., 1979; De Vos et al., 1982). Furthermore, there is evidence that avirulent B. bovis strains may better express antigens involved in the induction of protective immune responses (Kahl et al., 1982). Although live avirulent parasites have been effectively used for vaccination against bovine babesiosis in Australia (Callow et al., 1979), inherent disadvantages such as variable infectivity, severe post-vaccination clinical reactions (Callow and Dalgliesh, 1982) and a short shelf life (Mahoney et al., 1981) have proved to be problematic for the widespread use of these live vaccines. Therefore, the potential for a safe and effective non-living vaccine would appear limitless. Since the development of cell culture systems for the continuous propagation of B. bovis parasites (Erp et al., 1980; Levy and Ristic, 1980), several studies (Smith et al., 1981; Kuttler et al., 1982, 1983) have confirmed the efficacy of soluble parasite antigens (James et al., 1981) in providing homologous protection against challenge exposure with virulent B. bovis organisms. The logistic of vaccine development and its subsequent application would probably require heterologous immunity to geographically isolated B. bovis strains. Consequently, culture-derived soluble immunogens should also induce a desirable degree of heterologous strain protection. Recently, Timms et al. (1983) reported partial protection of cattle when vaccinated twice with a culture-derived soluble B. bovis (Australian origin) immunogen, and challenge-exposed with a virulent heterologous strain. The present study was designed to determine the cross-protective effect among B. bovis isolates from various regions (Venezuela, Mexico, Argentina, Ecuador and Colombia) of Latin America, and to assess the efficacy and potential of a culture-derived multi-strain immunogen produced from less virulent B. bovis isolates. MATERIALS AND METHODS
Location
Vaccination trials were performed at the Veterinary Research Institute (VRI) in Maracay, Venezuela. Experimental animals
Animals used in the study were susceptible (negative according to the indirect fluorescent antibody (IFA) test) cross-bred Holstein (Bos taurus) cattle, approximately 18 months of age. They were obtained from a highaltitude tick-free region of Colombia and were maintained in isolation facili-
323 ties at the laboratory. These adult animals were utilized in t w o immunization trials. Additionally, 3--4-month-old calves were splenectomized for use in reactivation of B. bovis strains.
Strains Strains of B. bovis isolated from Venezuela, Mexico, Argentina, Ecuador, Colombia and Australia were kindly provided as stabilates by veterinary research institutes located in those countries. The Australian and Colombian strains, originally isolated during outbreaks of babesiosis, had been attenuated b y rapid passage in splenectomized calves (Callow et al., 1979; Gonzalez et al., 1979) and utilized as live vaccines. All the strains had been maintained b y transmission in Boophilus microplus under laboratory conditions and/or b y syringe-passage in splenectomized animals.
Reactivation o f B. bovis strains Reactivation of each of the six strains was established separately by inoculation of blood stabilates into splenectomized calves. Infections were monitored by examining thin smears of peripheral blood, and by determining b o d y temperatures and packed cell volumes (PCV). When a certain preestablished parasitemia was reached, infected blood of each strain was aseptically collected to initiate in vitro cultivation (1% parasitemia) of the parasites according to the MASP method of Levy and Ristic (1980), to produce stabilates (2--3% parasitemia) by cryopreservation in liquid nitrogen and to prepare infected erythrocytic (at least 5% parasitemia) antigen for IFA tests.
Production o f immunogens Soluble immunogens were prepared from supernatant fluids of Venezuelan, Mexican and Australian B. bovis cultures. Supernatant antigen was collected daffy for 3 days from cultures with parasitemias averaging 6.0-8.0%. Immunogens were prepared as previously described (Kuttler et al., 1982) and mixed with 1 mg Quil-A saponin (Superfos, Denmark) adjuvant per 1 ml vaccinal dose. Each dose consisted of 10X -concentrated supernatant fluid (10 ml-equivalent). In experiment 1, Venezuelan and Mexican immunogens were administered separately, but in Experiment 2 a multi-strain combination of Venezuelan, Mexican and Australian immunogens was formulated. Vaccinal dosages in the latter experiment, consisting of 10 ml of supernatant fluid from each strain, were concentrated 10-fold to 3 ml and then mixed with saponin as above.
Experimental design Two successive experiments were conducted as detailed below.
324
Experiment 1 This experiment was designed to assess heterologous protective i m m u n i t y among B. bovis isolates from various regions of Latin America. Eighteen experimental cattle were divided into two groups and each group was vaccinated with either the Venezuelan (n = 10) or Mexican (n = 8) immunogens. Ten non-vaccinated animals were used as negative controls. The vaccine was administered subcutaneously in two 1-ml doses on Days 0 and 21. All vaccinated and control animals were divided into sub-groups (n = 2) and each challenged 3 months following the second vaccination with 6 × 108 virulent B. bovis organisms of either the Venezuelan, Mexican, Argentinian, Ecuadorean or Colombian strains. No homologous challenge was conducted with the Mexican strain. Experiment 2 The purpose of this subsequent experiment was to examine the protective effect among various B. bovis strains when culture-derived antigens from selected, less virulent isolates were used together as components of a soluble B. bovis immunogen. Thirty-two susceptible yearling cross-bred Holstein cattle from a tick-free zone in Colombia were used in this experiment. Sixteen animals were vaccinated with soluble antigen derived in vitro from high-passage Venezuelan, Mexican and Australian strains. Another 16 animals were retained as nonvaccinated controls. The vaccine was administered subcutaneously in two 3ml doses on Days 0 and 21. The vaccinated and control animals were divided into 4 sub-groups (n = 4) and challenged 1 m o n t h after the second vaccination with 1 × 109 virulent B. bovis organisms of either the Venezuelan, Mexican, Argentinian or Ecuadorean strains. Collection and analysis o f data Body temperature, PCV and platelet levels, parasitemia (thin blood film), IFA titers and selected erythrophilic proteins were monitored throughout the experiments. Plasma fibrinogen and cryofibrinogen levels were determined by the radial immunodiffusion test and by cold precipitation, respectively, according to methods described by Goodger and Wright (1977). Homologous and heterologous IFA titers were measured from the sera of vaccinated animals. Net gains in b o d y weight post-challenge were also calculated. The statistical significance of the difference between means was established by the Student's t-test. RESULTS
Vaccination with Venezuelan B. bovis immunogen In this first experiment, protection was assessed on the basis of differences
Vac.
Vac.
1 G r o u p m e a n s ± s t a n d a r d error. Presence (+) o f p l a s m a c r y o f i b r i n o g e n a t 4 ° C. * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l values, P < 0.05.
45.5±2.5"12.5+-3.5
79.0±5.0 . 46.5+-2.5"14.0+-5.0
79.0±5.0 +
17.5±2.5
Vac.
Mexico
13.0±7.0
14.0±1.0
73.5±5.0 +
8.0±1.0 10.0±2.0 4.0+-2.0 5.0±1.0 3.5+-0.5 6.0±1.0 34.0±2.0 50.0±5.0
Cont.
62.0+-6.0 8 0 . 0 ± 5 . 0 _ +
59.0±3.0 _
Vac.
71.0±4.0 --
Cont.
Colombia
13.0+-0" 10.0+-2.0 1 2 . 0 ± 0 " 2.0 +- 1.0 1 . 0 ± 0 3.0+-0 1.5±1.5 3.0+-1.5 4.0+3.0 25.0+-1.0"38.0±1.0 35.0±7.0
Vac.
Argentina
1 5 . 0 ± 3 . 0 13.0+-1.0 2.0±0* 5.0+2.0 0.5±0.5 5.0+-0 22.0±1.0"35.0±2.0
Cont.
Ecuador
Venezuela Cont.
Heterologous
Homologous
Type of challenge
P r e p a t e n t p e r i o d (days) 0±0" 14.0±0 D u r a t i o n of p a r a s i t e m i a (days) 0 ± 0 " 2.0±0 D u r a t i o n of fever (days) 0.5±0.5 3.5±0 Maximum%PCVreduction 20.0+-1.0"37.0±5.0 Maximum % platelet reduction 51.0±3.0 79.0±3.0 Cryofibrinogen 2 -+ Weight gain (kg) 6 w e e k s post-challenge 37.5±0.5"13.5±4.5
Measurements
12.0±3.0
83.0±3.0 +
7.0+-0 8.0+-0 6:0-+0 51.0±0
Cont.
Clinical a n d p a t h o p h y s i o l o g i c a l r e s p o n s e s ' o f v a c c i n a t e d ( V e n e z u e l a n B. bovis i m m u n o g e n ) and c o n t r o l a n i m a l s t o c h a l l e n g e e x p o s u r e w i t h h o m o l o g o u s a n d h e t e r o l o g o u s B. bovis strains
TABLE I
b~
326
in the duration of prepatent period (established as the time required to visualize parasites in thin blood films), duration of parasitemia, reduction in PCV, presence of erythrophilic cryofibrinogen and net weight gains between immunized and control groups. The data are presented in Table I. Vaccinated animals were fully protected against challenge with the homologous strain, as their clinical condition remained unaltered during the challenge period. No parasitemia was detected and only a slight decrease in PCV was seen, with a maximal reduction of 20%. Additionally, weight gains in vaccinated cattle were 37.5 kg as compared to 13.5 kg in controlanirrials (P <
0.05). The Venezuelan immunogen also demonstrated the ability to induce heterologous strain immunity to challenge with the Ecuadorean and Argentinian strains. Most notably, vaccinated cattle showed maximum PCV reductions of 22 of 25% following the Ecuadorean and Argentinian challenges, respectively, whereas control cattle demonstrated respective decreases of 35 and 38%. Good protection was further evident when net weight gains were analyzed. Significant increases in weight were observed in vaccinated animals 6 weeks after challenge exposure with the Ecuadorean and Argentinian strains (45.5 and 46.5 kg, respectively), although control animals demon-
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327
strated limited gains of 12.5 and 14.0 kg, respectively (P < 0.05). However, animals vaccinated with the Venezuelan immunogen failed to protect against challenge with the Mexican and Colombian strains according to most clinical parameters. Following challenge with the Mexican strain, both vaccinated and non-vaccinated cattle were equally affected as PCV reductions (vaccinated = 50%, controls = 51%), presence of erythrophilic plasma cryofibrinogen and duration of fever were similar in all animals. A comparison of peak h o m o l o g o u s and heterologous antibody titers postvaccination is shown in Fig. 1. Maximal h o m o l o g o u s antibody responses were reached 1 week after the second vaccination (mean IFA titers = 1 : 2 2 2 9 ) with high antibody levels sustained for a 3-week period. Heterolog o u s antibody levels were lower for all strains, with the Mexican strain demonstrating the greatest difference during the vaccination period. The kinetics of IFA responses observed throughout the vaccination and challenge periods are shown in Fig. 2. Homologous antibody levels declined slowly postvaccination to a pre-challenge titer of 1:160 on Day 109. A strong anamnestic response began soon afterwards, with titers peaking at 1:327 650 approximately 4 weeks post-challenge (peak antibody titer of control animals = 1:9801). The increase in the heterologous antibody response was more gradual, as a 10-fold difference between h o m o l o g o u s and heterologous IFA
327680 Vaccinates Controls . . . . .
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14.0-+0 2.0± 0 3.5-+ 1.0 37.0±5.0 79.0-+5.0 + 13.5_+4.5
10.0±3.0 4.0+ 2.0 4.5-+ 1.0 34.0±6.0 77.0±5.0 +
13.5-+1.5
12.5±3.5
+
+
11.0+6.0
13.0±1.0 5.0-+2.0 5.0±0 35.0-+2.0 79.0-+4.0
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9.0+-1.0 5.0±2.0 3.5-+1.0 37.0-+3.0 74.0±4.0
Vac.
Vac.
Cont.
Ecuador
Venezuela
T y p e of c h a l l e n g e
1 G r o u p m e a n s ± s t a n d a r d error. 2 Presence (+) of p l a s m a c r y o f i b r i n o g e n at 4 ° C. *Significantly d i f f e r e n t f r o m c o n t r o l values, P < 0.05.
P r e p a t e n t p e r i o d (days) D u r a t i o n o f p a r a s i t e m i a (days) D u r a t i o n o f fever (days) Maximum % PCV reduction Maximum % platelet reduction Cyrofibrinogen 2 Weight gain (kg) 6 weeks post-challenge
Measurements
18.5±1.5
4-
10.0+-2.0 4.0_+2.0 2.5-+1.5 29.0±8.0 64.0±4.0
Vac.
Argentina
14.0+-5.0
+
10.0±2.0 1.0-+0 3.0-+1.5 36.0±1.0 77.0-+5.0
Cont.
14.0±1.0
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10.0-+3.0 3.0-+1.0 1.5±0" 25.0-+2.0 54.0±6.0
Vac.
Colombia
13.0±7.0
4-
8.0-+1.0 4.0-+1.0 3.5_+0.5 34.0±2.0 80.0-+5.0
Cont.
Clinical a n d p a t h o p h y s i o l o g i c a l r e s p o n s e s I o f v a c c i n a t e d ( M e x i c a n B. bovis i m m u n o g e n ) a n d c o n t r o l a n i m a l s to challenge e x p o s u r e w i t h h e t e r o l o g o u s B. bovis strains
T A B L E II
e.O t~ QO
329
titers was observed 10 days post-challenge. Nevertheless, high levels of antib o d y to all B. bovis strains were eventually achieved. Vaccination with Mexican B. bovis immunogen Animals administered with the Mexican B. bovis immunogen responded poorly to heterologous challenge exposure with all the B. bovis strains utilized, as vaccinated cattle showed clinical reactions similar to those found in control animals (Table II). Homologous and heterologous IFA titers produced in response to vaccination are depicted in Fig. 3. Maximal homologous titers (1:5583) occurred 2 weeks after the second vaccinal dose, and were followed by a transient decline in antibody levels until challenge. Heterologous responses were once again consistently lower, especially those specific to the Venezuelan strain. In the absence of clinical protection, high antibody titers to all strains (average maximal titer = 1:106, 211) were obtained postchallenge. 10240
5120
ui ui
2560
÷1
1280 o .o n
"G ID
64O
320 -
Antigens O O II----II O [] • • z~----~
160 -
Mexico (Homol) Ecuador Argentina Colombia Australia Venezuela-
I
I
I
I
28
35
42
49
Days Fig. 3. Mean homologous and heterologous anti-B, boris IFA titers following vaccination with the Mexican immunogen.
330
Vaccination with multi-strain B. bovis immunogen Homologous antibody levels produced in response to vaccination with the single-strain and multi-strain immunogens are shown in Fig. 4. Pre-challenge IFA titers to the multi-strain B. bovis immunogen (Experiment 2) were significantly lower, with a more rapid diminution to baseline levels, than those of either the single-strain Venezuelan or Mexican immunogens (Experiment 1). Maximal post-vaccinal titers corresponded to those induced by the Australian strain (1:1000), followed by those of the Venezuelan (1:766) and Mexican (1:448) strains, respectively. The degree of protection was also assessed by comparing relevant clinical and pathophysiological parameters with those observed in control animals. Data collected following heterologous challenge are summarized in Table III. Results were similar to those obtained in the first experiment, as vaccinated 10240 Immunogen --
5120
---
Venezuelan Mexican M u l t i - strain
2560
1280
$ •-'I-
640
~ rr"
320
160
80
40
< 40
~ io
L 14
I 21
I 28
I 35
I 42
I 49
t Days II °
Fig. 4. Mean homologous anti-B, bovis IFA titers following vaccination with either the Mexican, Venezuelan or multi-strain immunogens.
331 TABLE III Clinical and pathophysiological responses' of vaccinated (multi-strain' B. boris immunogen) and Control animals to challenge exposure with heterologous B. bovis strains Measurements
Type of challenge Argentina
Prepatent period (days) Duration of parasitemia (days) Duration of fever (days) Duration of anemia (days) Maximum PCV reduction (%) Maximum platelet reduction (%) Maximum fibrinogen increase (%) Duration of elevated fibrinogen levels (days) Weight gain (kg) 3 weeks post-challenge
Ecuador
Vac.
Cont.
Vac.
Cont.
3.3-+0.3 2.8-+0.8* 1.0-+0"* 1.0-+0.4"* 37.5-+0.9**
3.3-+0.3 7.3-+0.9 4.0-+0.7 9.7-+0.9 54.3-+5.5
4.5-+0.9 1.8-+0.5" 1.5-+0.3"* 2.8-+ 1.1"* 41.5-+1.5"*
3.7-+0.3 5.0-+0.6 4.7-+0.9 11.3-+3.2 54.7-+6.2
75.9_+6.7
83.9_+4.6
72.0-+3.0"
82.6-+6.6
54.2_+10.1
81.4-+13.9
67.7-+10.5" 103.5-+16.1 4.5-+2.1"
13.3-+1.7
13.5_+2.2"
4.5-+1.2"
4.7_+2.9
10.3-+1.5
12.3-+2.8"
3.1_+2.3
' Group mean + standard error. : B. bovis from Venezuela, Mexico, and Australia (high-passage).
*Significantly different from control values, P < 0.05. **Significantly different from control values, P < 0.01. 163840 81920 Argentinian Antigen : : Vaccinated
40960u~ 20480 --
Q----O Controls
f
Lr~ 10240 -+I 5120
-
/
/
±
~
~
~
-
.~ 2580t..1280 .O o_ •~
320
¢r
160
-
-
640 --
8O 4O <40~. I°
_ 7
I I I 14 21 28
t
II°
I 35
I y/ 42 49 0
t
Challenge
Fig. 5. M e a n h e t e r o l o g o u s anti-B,
~fl
I
I
1
7
14
21
28
Days
boris I F A titers f o l l o w i n g v a c c i n a t i o n w i t h the m u l t i -
strain immunogen and challenge with virulent organisms of the Argentinian strain.
332
animals were protected against heterologous challenge with B. bovis strains from Argentina and Ecuador. Only mild, transient clinical changes were observed among vaccinates, whereas all control cattle manifested severe clinical disease characterized b y ataxia, labored respiration, profuse salivation, anorexia and jaundice. Some significant clinical differences were: duration of parasitemia (vaccinates = 2.8 days, controls = 7.3 days after the Argentinian challenge; P < 0.05); duration of anemia (vaccinates = 2.8 days, controls = 11.3 days after the Ecuadorean challenge; P < 0.01); duration of elevated fibrinogen (vaccinates of both groups = 4.5 days, controls = 13.3 days and 10.3 days after Argentinian and Ecuadorean challenges, respectively, P
0.05). A typical IFA response after heterologous challenge is illustrated in Fig. 5. An early anamestic response, as observed following the Argentinian challenge, was characteristic with antibody titers (1:40 960 for vaccinated animals and 1:5120 for controls) peaking 2 weeks post-challenge. Data pertaining to homologous challenge with the Venezuelan and Mexican strains are detailed in Table IV. Cattle vaccinated with the multi-strain B. bovis immunogen demonstrated solid immunity to acute babesiosis subsequent to challenge with the virulent homologous strains. However, nonvaccinated control animals showed significantly greater differences with respect to most pathophysiological parameters (P ~ 0.01). T A B L E IV Clinical a n d p a t h o p h y s i o l o g i c a l r e s p o n s e s ~ o f v a c c i n a t e d ( m u l t i - s t r a i n ~ B. bovis i m m u n o g e n ) a n d c o n t r o l a n i m a l s t o challenge e x p o s u r e w i t h h o m o l o g o u s B. bovis strains Measurements
T y p e o f challenge Venezuela
P r e p a t e n t p e r i o d (days) D u r a t i o n of p a r a s i t e m i a (days) D u r a t i o n of fever (days) D u r a t i o n of a n e m i a (days) M a x i m u m P C V r e d u c t i o n (%) Maximum platelet reduction
(%)
Mexico
Vac.
Cont.
Vac.
Cont.
5.0+_0.6 2.3-+0.5* 0.8-+0.5* 0-+ 0 " * 31.5-+1.7"*
5.7+_0.9 3.7+_0.9 3.7+_0.7 5.2-+2.1 45.3-+2.3
4.0-+0.4* 3.8-+0.5** 3.8-+0.5** 5.5-+0.5** 43.0-+1.2"*
2.6-+0.3 8.0+_1.0 8.7-+0.3 14.3-+2.2 62.7_+4.5
52.6-+3.3*
85.0-+2.9
82.0-+6.0
89.0-+2.7
61.7-+6.1"
84.2+-4.2
59.6-+9.6
58.1+_11.9
7.5+_2.1
7.3+_1.7
7.5+_0.5
8.0_+1.2
14.0+_2.8"
5.0_+2.1
8,8_+3.1
2.1+_1.8
M a x i m u m f i b r i n o g e n increase
(%)
D u r a t i o n of e l e v a t e d fibrinogen levels (days) Weight gain (kg) 3 weeks post-challenge
G r o u p m e a n +- s t a n d a r d error. B. boris f r o m V e n e z u e l a , Mexico, a n d A u s t r a l i a ( h i g h passage). * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l values, P < 0.05. * * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l values, P < 0.01.
333 DISCUSSION Solid homologous protection against challenge exposure with virulent Venezuelan B. bovis organisms was demonstrated in cattle vaccinated with a soluble immunogen derived from cultures of the same B. bovis strain. This finding confirms previous reports where strong homologous immunity was induced with culture-derived immunogens of the Mexican strain (Smith et al., 1981; Kuttler et al., 1982, 1983). ' An important consideration for the potential use of soluble immunogens, however, is their cross-protective capacity between different isolates (strains) of B. bovis, particularly since strain-specific antigenic differences are known to exist (Curnow, 1973). The multiplicity of strain-specific antigens among various Latin American isolates was evident in the present study, as heterologous IFA titers were invariably lower than those obtained in homologous tests. A good degree of cross-protection was shown when cattle vaccinated with the Venezuelan strain were challenged with virulent strains from Ecuador and Argentina, respectively. This indicates that c o m m o n protective antigens exist among strains from various geographical regions. There was little or no protection of cattle vaccinated with the Venezuelan strain and subsequently challenged with Colombian or Mexican strains. Similarly, there was a low degree of cross-protection when animals vaccinated with the Mexican strain were infected with the heterologous strains specified above. An interesting finding in this study was the difference in cross-immunity between the Mexican isolate and all the other strains utilized. One possible explanation for this uniqueness may be the long-standing prohibition of cattle imports into Mexico for the purpose of controlling foot-and-mouth disease. An immunogen containing culture
334 identity was shown for the Australian and Bolivian B. bovis strains (Callow et al., 1976). Based on these findings, an Australian strain was incorporated for use in the present study. Homologous pre-challenge IFA titers to the multi-strain B. bovis immunogen were significantly lower than those produced in response to either the single-strain Venezuelan or Mexican immunogens (Fig. 4). Possibly, excess antigen (3-fold increase in dosage, i.e., one dosage-equivalent per strain) provoked a state of antigenic competition or immunodepression due to an antigen overload at the lymphocyte level. Maximal pre-challenge titers corresponded to those specific for antigens of the Australian strain. Callow et al. (1976) previously found the Australian antigen to be slightly mol'e reactive than the Bolivian antigen in the IFA test. Antigen(s) of the Australian strain may be displaying a unique or structurally dominant epitope which could enhance immunogenicity in vivo. Non-vaccinated control animals in this study showed acute clinical disease following challenge infection, whereas vaccinated cattle showed no marked signs of illness and gained weight throughout the experimental period. Any transient pathophysiological reactions in the latter group were reversed by the fifth day post-challenge. Likewise, parasitemias were rapidly cleared. These observations are similar to those reported during cross-immunity experiments on different Australian isolates (Callow, 1968; Johnston and Tammemagi, 1969; Mahoney et al., 1979b). An assessment of protection may also be based on the prevention of the hypotensive shock syndrome characteristic of acute B. bovis infections (Goodger et al., 1981a). Much of the pathophysiology in bovine babesiosis is associated with changes in coagulation parameters. Indeed, thrombocytopenia, hyperfibrinogenemia and cryofibrinogenic complexes are often observed in non-immune animals suffering from acute infection (Goodger and Wright, 1977; Goodger et al., 1981b). The strong heterologous strain immunity induced by culture-derived soluble immunogens in the present study was able to prevent or ameliorate any damage caused by these pathophysiological reactions. During B. bovis infections, babesial antigen may bind with fibrinogen to form immunogenic complexes (Goodger, 1976). Parasite antigens found within the erythrocytic stroma of infected cells are thought to be fibrinogenassociated (Goodger et al., 1980; Montenegro-James et al., 1983). In addition to the formation of antigen--fibrinogen complexes, fibrinogen may be altered by Babesia parasites, making it antigenic for the host. Thus, autoantibodies to fibrinogen-related products and erythrocytic stroma are detected in Babesia infections (Mahoney and Goodger, 1972; Thoongsuwan et al., 1979). Therefore, it seems conceivable that the elevated plasma fibrinogen concentrations observed in vaccinated and control animals post-challenge could lead to the stimulation of high amounts of non-protective antibody (IFA-reactive) directed toward parasite antigens and/or host autoantigens. Vaccination with culture-derived soluble immunogens has shown that ade-
335
quate protection can be provided against acute infection with heterologous B. bovis strains. Immunogens derived from the Venezuelan and Australian strains would appear to have the best heterologous protective capabilities. Recently, Timms et al. (1983) reported partial heterologous protection of cattle vaccinated with a culture-derived soluble immunogen (Australian), and subsequently challenged with another Australian strain. Mahoney et al. (1979a) have suggested that protective immune responses are mediated by strain-specific antibody. Variant-specific immune mechanisms are obviously initiated by protective antigens having unique antigenic epitopes. However, there is now increasing evidence that cell-mediated immune responses may also play a role in the regulation of protective immunity to babesiosis (Allison, 1984). Cell-mediated immunity to Babesia parasites could act with, or independently from, humoral mechanisms in vaccinated animals. In addition to inducing the strong antibody responses observed in the present study, culture-derived soluble antigens are also potent stimulators of T lymphocyte (Timms et al., 1984) and macrophage (Montealegre et al., 1983) function. One is hopeful that with careful selection and optimization of an adjuvant, both humoral and cell-mediated immune mechanisms can be synergized to provide long-lasting protection against acute babesiosis. ACKNOWLEDGEMENTS
This study was conducted as part of a ,cooperative research project between the Venezuelan Institute for Veterinary Research and the University of Illinois, and was supported by funds from the United States--Israel (Binational) Agricultural Research and Development Fund (BARD) and the Rockefeller Foundation.
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337 Montealegre, F., Levy, M.G. and Ristic, M., 1983. In vitro secretory activity of bovine blood mononuclear phagocytes and its effect on the growth of Babesia bovis. In: Proc. Second Int. Conf. Malaria and Babesiosis, 19--22 September 1983, Annecy, France, pp. 0--2 (abstract). Montenegro-James, S., James, M.A. and Ristic, M., 1983. Localization of culture-derived soluble Babesia bovis antigens in the infected erythrocyte. Vet. Parasitol., 13: 311-316. Smith, R.D., James, M.A., Ristic, M., Aikawa, M. and Vega, C.A., 1981. Bovine babesiosis: protection of cattle with culture-derived soluble Babesia bovis antigen. Science, 212: 335--338. Thoongsuwan, S., Cox, H.W. and Rickman, W.J., 1979. A n t i b o d y to fibrinogen/fibrin products (anti-F) in malaria, babesiosis, and trypanosomiasis of rodents. J. Parasitol., 65 : 426--429. Timms, P., Dalgliesh, R.J., Barry, D.N., Dimmock, C.K. and Rodwell, B.J., 1983. Babesia bovis: comparison of culture-derived parasites, nonliving antigen and conventional vaccine in the protection of cattle against heterologous challenge. Aust. Vet. J., 60: 75-77. Timms, P., Stewart, N.P., Rodwell, B.J. and Barry, D.N., 1984. Immune responses of cattle following vaccination with living and non-living Babesia bovis antigens. Vet. Parasitol., 16 : 243--251.