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Experimental immunization of calves against Anaplasma marginale infection: observations on the use of living A. centrale and A. marginale
Veterinary Parasitology, 7 (1980) 305--311 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
305
EXPERIMENTAL IMMUNIZATION OF CALVES AGAINST ANAPLASMA MARGINALE INFECTION: OBSERVATIONS ON THE USE OF LIVING A. CENTRALE AND A. MARGINALE
A.J. W I L S O N ' , R. P A R K E R and K.F. TRUEMAN
Queensland Department of Primary Industries, Oonoonba Veterinary Laboratory, Townsville, Queensland 4810 (Australia) (Accepted for publication 9 June 1980)
ABSTRACT Wilson, A.J., Parker, R. and Trueman, K.F., 1980. Experimental immunization of calves against Anaplasma marginale infection: observations on the use o f living A. centrale and A. marginale. Vet. Parasitol., 7: 305--311. Groups of Bos indicus cross calves aged 6 months were immunized with Anaplasma centrale o r A . marginalc. All animals were challenged with 101° A. marginale 7 months later. Groups immunized with A. marginale were refractory to challenge, whereas only a portion of the animals vaccinated with A. centrale were immune. Following immunization, animals that had experienced a good primary antibody response as measured by a complement fixation test, a marked reduction in packed cell volume, and a high parasitaemia, resisted a subsequent challenge with A. marginale. Resistance was characterised by a weak secondary antibody response and the absence of A. marginale in blood films.
INTRODUCTION
Bovine anaplasmosis caused by Anaplasma marginale is widespread throughout the tropics and causes great economic loss (McCallon, 1973). A. centrale has been used extensively in Africa and Australia as a living vaccine to immunize cattle against the more pathogenic A. marginale (Malherbe, 1963; Callow, 1976). The number of vaccine doses used in Australia annually has increased dramatically in recent years from 6722 in 1968 to a peak of 434 124 in 1976. Vaccination is recommended at weaning, which is normally at 6--9 months of age. The protection afforded to cattle by A. centrale against A. marginale has been known for many years (Theiler, 1911; Legg, 1936; Kuttler, 1967b). However, this immunity is not absolute and in some instances may not be fully effective (Rogers and Shiels, 1979). The mechanism of this protection 1Present address: C/- Department of Pathology, Veterinary Faculty. P.O. Box 29053, Nairobi, Kenya.
0304-4017/80/0000--0000/$ 02.50 © 1980 Elsevier Scientific Publishing Company
306 is n o t clear. It is assumed t h a t both organisms have antigens in c o m m o n (Kuttler, 1967a). Schindler et al. (1966) considered that each organism possessed at least one antigen c o m p o n e n t n o t shared by the other parasite. A study was undertaken to examine the i m m u n i t y to A. marginale infection in groups of calves following immunization with A. centrale or A.
marginale. MATERIALS AND METHODS
AnapIasma organisms Two isolates of A. marginale, obtained from infected cattle in areas about 700 kilometres apart, were each inoculated into a splenectomised calf. Stabilates of the organisms were prepared at peak parasitaemia using the m e t h o d of Dalgliesh and Mellors (1974) and designated Oonoonba-1 and Oonoonba-136, respectively. Derivatives of the two stabilates, designated Strain A and B in individual splenectomised calves, were used. The source of A. centrale organisms was a commercial living vaccine prepared at the Tick Fever Research Centre, Wacol 4076, Australia.
Experimental animals Bos indicus cross animals, aged 6 months, were obtained from a property in north-west Queensland which was free of the cattle tick (Boophilus microplus). All animals were negative for anaplasmosis by serological and microscopical examination, and were maintained in a tick-free area similar to t h a t described by Johnston and Tammemagi (1969). Experimental design Fifty-four calves of mixed sexes, aged 6 months, were divided into 6 groups of 9 by stratified randomisation based on packed cell volume (PCV) and body weight (Groups 1--6). Groups 2 and 5 were inoculated intravenously with a standard vaccine dose (107 organisms) of A. centrale. Groups 3 and 4 received approximately 107 A. marginale (Strain A). Groups 1 and 6 were uninoculated controls. Jugular blood was examined three times weekly for parasites and PCV from the seventh to the fortysecond day after inoculation. Body weight was measured and serum collected weekly until day 42, and thence m o n t h l y until day 216, after inoculation. Groups 1--3 were challenged on day 216 with approximately 10 '° A. marginale organisms (Strain A). Groups 4--6 were similarly challenged with approximately 10 l° of Strain B. Jugular blood from all animals was examined three times weekly for parasites and PCV from the seventh to the twenty-eighth day post-challenge. The cattle were weighed and serum collected weekly until the forty-second day post-challenge.
307 A f t e r v a c c i n a t i o n a n d challenge, G r o u p s 2 a n d 5 were sub-divided i n t o 10 animals w h i c h failed t o resist challenge ( G r o u p 7) a n d 8 animals w h i c h resisted challenge ( G r o u p 8), based o n degree o f PCV depression and parasitaemia. T h e responses o f G r o u p s 7 and 8 were c o m p a r e d with t h o s e o f G r o u p s 1 a n d 6, a n d 3 and 4, respectively. Antibody
response
S e r u m a n t i b o d y levels t o A . m a r g i n a l e antigen were d e t e r m i n e d b y a microc o m p l e m e n t f i x a t i o n test (CFT) described b y Martin a n d Ritchie ( 1 9 7 3 ) , with s o m e m o d i f i c a t i o n s (Parker et al., 1 9 7 8 ) . Statistical analysis
T h e d a t a were e x a m i n e d b y analysis o f variance based on a r a n d o m i s e d b l o c k design. Where significant differences were d e t e c t e d b e t w e e n groups, pair-wise c o m p a r i s o n s were m a d e b y " t " test. PCV a n d b o d y weight were c o r r e c t e d f o r initial values b y analysis o f covariance. The C F T titres were t r a n s f o r m e d ( l o g ( X + l ) t r a n s f o r m a t i o n ) prior t o analysis. RESULTS T h e m e a n m a x i m u m parasitaemia and m e a n m a x i m u m % depressions o f PCV a f t e r i m m u n i z a t i o n a n d challenge o f G r o u p s 1--8 are s h o w n in Table I. TABLE I Mean maximum parasitaemia and mean % fall in PCV in the animals of Groups 1--8 after immunisation and challenge. Figures in brackets are days post-inoculation Group number
Age (months)
Immunisation
Challenge I
Organism used
Mean maximum parasitaemia
Mean % fall in PCV
Mean maximum parasitaemia
Mean % fall in PCV
0 1.1 4.6 3.0 1.3 0 0.3 2.1 0 3.5
0 20.0 39.4 33.3 18.4 0 8.5 25.9 0 36.3
3.2 2.5 0 0 1.3 5.2 3.4 0 4.2 0
33.2 21.7 0 0 19.4 42.8 36.5 0 37.3 0
1
6
n.d.
2 3 4 5
6 6 6 6
A. A. A. A.
6
6
n.d.
7 8
6 6
A. centrale A. centrale
1 and 6 3 and 4
6
n.d.
6
A. marginale
centrale marginale marginale centrale
(28) (11) (11) (28) (28) (28) (12)
Challenge performed 217 days after immunisation. n.d. = not done.
(33) (13) (13) (33) (33) (33) (13)
(12) (12) (12) (12) (12) (12)
(12) (14) (17) (14) (14) (14)
308
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group 3 & 4 group I & 6 group 7
e--e
group
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40'
8
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30'
20
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:._., 5" .o
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POST
~o IMMUNIZATION
[
2"0 DAYS
POST
CHALLENOE
Fig. 1. The results of challenge with Anaplasma marginale following immunisation with either A. marginale or A. centrale, as measured by parasitaemia, % reduction in packed cell volume and complement fixing antibody titre. Groups 3 and 4 were immunised with A. marginale and resisted challenge. Groups 1 and 6 were not immunised and were susceptible to challenge. Group 7 had a mild reaction to vaccination with A. centrale and was more susceptible to challenge than Group 8, that had a strong reaction to vaccination with A. centrale.
T h e m e a n p a r a s i t a e m i a s , m e a n % d e p r e s s i o n s o f P C V a n d t h e m e a n antib o d y r e s p o n s e s i n G r o u p s 7 a n d 8, a n d t h e c o m b i n e d d a t a f r o m G r o u p s 1 a n d 6, a n d 3 a n d 4 a f t e r i m m u n i z a t i o n a n d c h a l l e n g e are s h o w n i n Fig. 1.
309 Parasitaemia Anaplasms were observed in all immunized animals (Groups 2--5; Table I). Parasitaemias were low grade, prolonged and occasionally with poorly defined peaks. All animals showed their maximum parasitaemia within a day of the group mean peak. The animals of Groups 3 and 4 infected with A. marginale showed a peak, mean parasitaemia of 4.6 and 3.0%, respectively, 11 days post-inoculation. The animals of groups 2 and 5 vaccinated with A. centrale showed a peak, mean parasitaemia of 1.1 and 1.3%, respectively, 28 days post-inoculation. No anaplasms were observed in any animals of Groups 3 and 4 after challenge (Table I). These results were significantly different from those in control Groups 1 and 6 which showed peak, mean parasitaemias of 3.2 and 5.2%, respectively, after challenge (P<0.01), and from those in Groups 2 and 5 which showed peak, mean parasitaemias of 2.5 and 1.3%, respectively, after challenge (P<0.01). The mean parasitaemia in Group 8 was significantly higher than in Group 7, 28--33 days after vaccination (P<0.01; Table I). Following challenge, no anaplasms were observed in Group 8 animals, whereas Group 7 animals had a mean maximum parasitaemia of 3.4%. Packed cell volume A depression of the PCV was seen in all immunised animals, maximum depressions occuring within a day of the group mean maximum. Groups 3 and 4, which received A. marginale, showed mean maximum depressions of 33.3 and 39.4%, respectively, 13 days after inoculation. This was significantly greater than the depressions of 18.4 and 20.0% shown by Groups 2 and 5, respectively, 33 days after receiving A. centrale (P<0.01; Table I). Following challenge, the animals that had been immunised with A. marginale (Groups 3 and 4) showed no depression of PCV. This was significantly less than the depressions of 21.7 and 19.4% shown by Groups 3 and 4, 14 and 17 days post-challenge and the depressions of 33.2 and 42.8% shown by the non-immunised control Groups 1 and 6, 12 and 14 days post-challenge (P< 0.01; Table I). Antibody response Group 8 showed a significantly greater primary antibody response than Group 7, 28--35 days after vaccination, with mean CFT titres of 1:130 and 1:30, respectively (P<0.05; Fig. 1). The primary antibody response in Groups 3 and 4 reached a peak o f 1:219 at day 21 and was higher than Group 8 at any time (P<0.01; Fig. 1). Following challenge, the maximum response of control Groups 1 and 6 (1:247) was significantly greater than that of the other groups (P<0.01; Fig. 1). Group 7 had a greater secondary antibody response (maximum 1:71} than groups 3 and 4 (1:37) and 8 (1:12), 7--4 days post-challenge.
310 Body weight No differences in mean b o d y weight changes were detected between Groups 1--6 during the experiment. Mean weights increased from 141.5 to 198.8 kg, an increase of 40.5%. However, Group 7 lost significantly more weight than Group 8 during the period 0--21 days post-challenge (0.5 and 0.04 kg/day, respectively) (P<0.05). DISCUSSION
This study showed that vaccination with A. centrale m a y not provide adequate protection against anaplasmosis in all animals. Increased susceptibility to A. marginale with increasing age is known to occur in Bos taurus (Jones et al., 1968) and Bos indicus cattle (Wilson et al., 1979). A similar lack of susceptibility of y o u n g animals to A. centrale m a y have interfered with effective immunisation. However, under Australian conditions there has been no field evidence to suggest that vaccination of y o u n g animals with A. centrale is not effective. It is well known that A. centrale has c o m m o n antigenic components with A. marginale (Kuttler, 1967a, b). Infection with A. centrale does n o t always result in subsequent immunity to A. marginale (see Group 7). Resistance to A. marginale challenge was related to good primary antibody response, as observed in all A. marginale and some A. centrale (Group 8) immunized animals. A characteristic of this resistance was a very weak response at challenge. Antibody response could thus be used as an indicator of the level of immunity achieved by vaccination. Because calfhood vaccination with A. centrale left 10 out of 18 animals without adequate immunity, a second A. centrale vaccination m a y be indicated when they are older. Alternatively, A. centrale vaccination could b'e followed by A. marginale, as has been suggested for cattle going to regions of severe challenge (Callow, 1976). Further experimental work appears desirable to evaluate these procedures. A primary inoculation with A. marginale, combined with chemotherapy (Todorovic and TeUez, 1975), could be considered for valuable animals. It is generally accepted that field strains of A. marginale are immunologically similar or identical, although Uilenberg (1970) indicated that this may n o t be the case in Madagascar. Callow et ai. (1976) observed that a Bolivian strain of A. marginale was serologically similar to an Australian strain. Cross-immunity testing in the present work suggested that Strains A and B of A. marginale, isolated 700 kilometres apart, were immunologically identical.
311 REFERENCES Callow, L.L., 1976. Tick-borne livestock diseases and their vectors. 3. Australian methods of vaccination against anaplasmosis and babesiosis. World Anim. Rev., 18: 9--15. 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. Dalgliesh, R.J. and Mellors, L.T., 1974. Survival of the parasitic protozoan Babesia bigemina in blood cooled at widely different rates to --196 ° C. Int. J. Parasitol., 4: 169--172. 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 bigemina. Aust. Vet. J., 45: 445--449. Jones, E.W., Kliewer, I.O., Norman, B.B. and Brock, W.E., 1968. Anaplasma marginale infection in young and aged cattle. Am. J. Vet. Res., 29: 535--544. Kuttler, K.L., 1967 a. Serologic relationships of A naplasma marginale and A naplasma centrale as measured by the complement fixation and capillary tube agglutination tests. Res. Vet. Sci., 8: 207--211. Kuttler, K.L., 1967b. A study of the immunological relationship of Anaplasma marginale andAnaplasma centrale. Res. Vet. Sci., 8: 467--471. Legg, J., 1936. Cross immunity tests between Anaplasma centrale (South Africa) and Anaplasma marginale (Australia). Aust. Vet. J., 12: 230--233. Malherbe, W.D., 1963. Some observations on anaplasmosis. Cornell Vet., 53: 71--77. Martin, W.H. and Ritchie, W.H., 1973. A microtitre technique for the complement fixation test for anaplasmosis. Proc. U.S. Anim. Health Assoc., 77: 582--592. McCallon, B.R., 1973. Prevalence and economic aspects of anaplasmosis. Proc. 6th Natl. Anaplasmosis Conf., pp. 1--3. Parker, R., Parker, M.L. and Wilson, A.J., 1978. Liquid nitrogen storage of Anaplasma marginale complement fixation antigen by a multiple small aliquot technique. Res. Vet. Sci., 25: 401--402. Rogers, R.J. and Shiels, I.A., 1979. Epidemiology and control of anaplasmosis in Australia. J.S. Afr. Vet. Assoc., 50: 363--366. Schindler, Von K., Ristic, M. and Wokatsch, R., 1966. Vergleichende untersuchungen mit Anaplasma marginale und A. centrale. Z. Tropenmed. Parasitol., 17: 337--360. Theiler, G., 1911. First report of Director Vet. Res., Union of South Africa, pp. 7--46. Todorovic, R.A. and Tellez, C.H., 1975. The premunition of adult cattle against babesiosis and anaplasmosis in Columbia, South America. Trop. Anim. Health Prod., 7: 125--131. Uilenberg, G., 1970. Notes sur les babesioses et l'anaplasmose des bovins ~ Madagascar. Rev. Elev. Med. Vet. Pays Trop., 23: 439--454. Wilson, A.J., Parker, R. and Trueman, K.F., 1979. Anaplasmosis in Bos indicus type cattle. Aust. Adv. Vet. Sci., p. 76.
305
EXPERIMENTAL IMMUNIZATION OF CALVES AGAINST ANAPLASMA MARGINALE INFECTION: OBSERVATIONS ON THE USE OF LIVING A. CENTRALE AND A. MARGINALE
A.J. W I L S O N ' , R. P A R K E R and K.F. TRUEMAN
Queensland Department of Primary Industries, Oonoonba Veterinary Laboratory, Townsville, Queensland 4810 (Australia) (Accepted for publication 9 June 1980)
ABSTRACT Wilson, A.J., Parker, R. and Trueman, K.F., 1980. Experimental immunization of calves against Anaplasma marginale infection: observations on the use o f living A. centrale and A. marginale. Vet. Parasitol., 7: 305--311. Groups of Bos indicus cross calves aged 6 months were immunized with Anaplasma centrale o r A . marginalc. All animals were challenged with 101° A. marginale 7 months later. Groups immunized with A. marginale were refractory to challenge, whereas only a portion of the animals vaccinated with A. centrale were immune. Following immunization, animals that had experienced a good primary antibody response as measured by a complement fixation test, a marked reduction in packed cell volume, and a high parasitaemia, resisted a subsequent challenge with A. marginale. Resistance was characterised by a weak secondary antibody response and the absence of A. marginale in blood films.
INTRODUCTION
Bovine anaplasmosis caused by Anaplasma marginale is widespread throughout the tropics and causes great economic loss (McCallon, 1973). A. centrale has been used extensively in Africa and Australia as a living vaccine to immunize cattle against the more pathogenic A. marginale (Malherbe, 1963; Callow, 1976). The number of vaccine doses used in Australia annually has increased dramatically in recent years from 6722 in 1968 to a peak of 434 124 in 1976. Vaccination is recommended at weaning, which is normally at 6--9 months of age. The protection afforded to cattle by A. centrale against A. marginale has been known for many years (Theiler, 1911; Legg, 1936; Kuttler, 1967b). However, this immunity is not absolute and in some instances may not be fully effective (Rogers and Shiels, 1979). The mechanism of this protection 1Present address: C/- Department of Pathology, Veterinary Faculty. P.O. Box 29053, Nairobi, Kenya.
0304-4017/80/0000--0000/$ 02.50 © 1980 Elsevier Scientific Publishing Company
306 is n o t clear. It is assumed t h a t both organisms have antigens in c o m m o n (Kuttler, 1967a). Schindler et al. (1966) considered that each organism possessed at least one antigen c o m p o n e n t n o t shared by the other parasite. A study was undertaken to examine the i m m u n i t y to A. marginale infection in groups of calves following immunization with A. centrale or A.
marginale. MATERIALS AND METHODS
AnapIasma organisms Two isolates of A. marginale, obtained from infected cattle in areas about 700 kilometres apart, were each inoculated into a splenectomised calf. Stabilates of the organisms were prepared at peak parasitaemia using the m e t h o d of Dalgliesh and Mellors (1974) and designated Oonoonba-1 and Oonoonba-136, respectively. Derivatives of the two stabilates, designated Strain A and B in individual splenectomised calves, were used. The source of A. centrale organisms was a commercial living vaccine prepared at the Tick Fever Research Centre, Wacol 4076, Australia.
Experimental animals Bos indicus cross animals, aged 6 months, were obtained from a property in north-west Queensland which was free of the cattle tick (Boophilus microplus). All animals were negative for anaplasmosis by serological and microscopical examination, and were maintained in a tick-free area similar to t h a t described by Johnston and Tammemagi (1969). Experimental design Fifty-four calves of mixed sexes, aged 6 months, were divided into 6 groups of 9 by stratified randomisation based on packed cell volume (PCV) and body weight (Groups 1--6). Groups 2 and 5 were inoculated intravenously with a standard vaccine dose (107 organisms) of A. centrale. Groups 3 and 4 received approximately 107 A. marginale (Strain A). Groups 1 and 6 were uninoculated controls. Jugular blood was examined three times weekly for parasites and PCV from the seventh to the fortysecond day after inoculation. Body weight was measured and serum collected weekly until day 42, and thence m o n t h l y until day 216, after inoculation. Groups 1--3 were challenged on day 216 with approximately 10 '° A. marginale organisms (Strain A). Groups 4--6 were similarly challenged with approximately 10 l° of Strain B. Jugular blood from all animals was examined three times weekly for parasites and PCV from the seventh to the twenty-eighth day post-challenge. The cattle were weighed and serum collected weekly until the forty-second day post-challenge.
307 A f t e r v a c c i n a t i o n a n d challenge, G r o u p s 2 a n d 5 were sub-divided i n t o 10 animals w h i c h failed t o resist challenge ( G r o u p 7) a n d 8 animals w h i c h resisted challenge ( G r o u p 8), based o n degree o f PCV depression and parasitaemia. T h e responses o f G r o u p s 7 and 8 were c o m p a r e d with t h o s e o f G r o u p s 1 a n d 6, a n d 3 and 4, respectively. Antibody
response
S e r u m a n t i b o d y levels t o A . m a r g i n a l e antigen were d e t e r m i n e d b y a microc o m p l e m e n t f i x a t i o n test (CFT) described b y Martin a n d Ritchie ( 1 9 7 3 ) , with s o m e m o d i f i c a t i o n s (Parker et al., 1 9 7 8 ) . Statistical analysis
T h e d a t a were e x a m i n e d b y analysis o f variance based on a r a n d o m i s e d b l o c k design. Where significant differences were d e t e c t e d b e t w e e n groups, pair-wise c o m p a r i s o n s were m a d e b y " t " test. PCV a n d b o d y weight were c o r r e c t e d f o r initial values b y analysis o f covariance. The C F T titres were t r a n s f o r m e d ( l o g ( X + l ) t r a n s f o r m a t i o n ) prior t o analysis. RESULTS T h e m e a n m a x i m u m parasitaemia and m e a n m a x i m u m % depressions o f PCV a f t e r i m m u n i z a t i o n a n d challenge o f G r o u p s 1--8 are s h o w n in Table I. TABLE I Mean maximum parasitaemia and mean % fall in PCV in the animals of Groups 1--8 after immunisation and challenge. Figures in brackets are days post-inoculation Group number
Age (months)
Immunisation
Challenge I
Organism used
Mean maximum parasitaemia
Mean % fall in PCV
Mean maximum parasitaemia
Mean % fall in PCV
0 1.1 4.6 3.0 1.3 0 0.3 2.1 0 3.5
0 20.0 39.4 33.3 18.4 0 8.5 25.9 0 36.3
3.2 2.5 0 0 1.3 5.2 3.4 0 4.2 0
33.2 21.7 0 0 19.4 42.8 36.5 0 37.3 0
1
6
n.d.
2 3 4 5
6 6 6 6
A. A. A. A.
6
6
n.d.
7 8
6 6
A. centrale A. centrale
1 and 6 3 and 4
6
n.d.
6
A. marginale
centrale marginale marginale centrale
(28) (11) (11) (28) (28) (28) (12)
Challenge performed 217 days after immunisation. n.d. = not done.
(33) (13) (13) (33) (33) (33) (13)
(12) (12) (12) (12) (12) (12)
(12) (14) (17) (14) (14) (14)
308
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~. 1: 2 0 0 ' LL. U 1:1OO'
50,
o--o i--m
group 3 & 4 group I & 6 group 7
e--e
group
&--A
40'
8
> u a. c
30'
20
I0'
:._., 5" .o
io DAYS
POST
~o IMMUNIZATION
[
2"0 DAYS
POST
CHALLENOE
Fig. 1. The results of challenge with Anaplasma marginale following immunisation with either A. marginale or A. centrale, as measured by parasitaemia, % reduction in packed cell volume and complement fixing antibody titre. Groups 3 and 4 were immunised with A. marginale and resisted challenge. Groups 1 and 6 were not immunised and were susceptible to challenge. Group 7 had a mild reaction to vaccination with A. centrale and was more susceptible to challenge than Group 8, that had a strong reaction to vaccination with A. centrale.
T h e m e a n p a r a s i t a e m i a s , m e a n % d e p r e s s i o n s o f P C V a n d t h e m e a n antib o d y r e s p o n s e s i n G r o u p s 7 a n d 8, a n d t h e c o m b i n e d d a t a f r o m G r o u p s 1 a n d 6, a n d 3 a n d 4 a f t e r i m m u n i z a t i o n a n d c h a l l e n g e are s h o w n i n Fig. 1.
309 Parasitaemia Anaplasms were observed in all immunized animals (Groups 2--5; Table I). Parasitaemias were low grade, prolonged and occasionally with poorly defined peaks. All animals showed their maximum parasitaemia within a day of the group mean peak. The animals of Groups 3 and 4 infected with A. marginale showed a peak, mean parasitaemia of 4.6 and 3.0%, respectively, 11 days post-inoculation. The animals of groups 2 and 5 vaccinated with A. centrale showed a peak, mean parasitaemia of 1.1 and 1.3%, respectively, 28 days post-inoculation. No anaplasms were observed in any animals of Groups 3 and 4 after challenge (Table I). These results were significantly different from those in control Groups 1 and 6 which showed peak, mean parasitaemias of 3.2 and 5.2%, respectively, after challenge (P<0.01), and from those in Groups 2 and 5 which showed peak, mean parasitaemias of 2.5 and 1.3%, respectively, after challenge (P<0.01). The mean parasitaemia in Group 8 was significantly higher than in Group 7, 28--33 days after vaccination (P<0.01; Table I). Following challenge, no anaplasms were observed in Group 8 animals, whereas Group 7 animals had a mean maximum parasitaemia of 3.4%. Packed cell volume A depression of the PCV was seen in all immunised animals, maximum depressions occuring within a day of the group mean maximum. Groups 3 and 4, which received A. marginale, showed mean maximum depressions of 33.3 and 39.4%, respectively, 13 days after inoculation. This was significantly greater than the depressions of 18.4 and 20.0% shown by Groups 2 and 5, respectively, 33 days after receiving A. centrale (P<0.01; Table I). Following challenge, the animals that had been immunised with A. marginale (Groups 3 and 4) showed no depression of PCV. This was significantly less than the depressions of 21.7 and 19.4% shown by Groups 3 and 4, 14 and 17 days post-challenge and the depressions of 33.2 and 42.8% shown by the non-immunised control Groups 1 and 6, 12 and 14 days post-challenge (P< 0.01; Table I). Antibody response Group 8 showed a significantly greater primary antibody response than Group 7, 28--35 days after vaccination, with mean CFT titres of 1:130 and 1:30, respectively (P<0.05; Fig. 1). The primary antibody response in Groups 3 and 4 reached a peak o f 1:219 at day 21 and was higher than Group 8 at any time (P<0.01; Fig. 1). Following challenge, the maximum response of control Groups 1 and 6 (1:247) was significantly greater than that of the other groups (P<0.01; Fig. 1). Group 7 had a greater secondary antibody response (maximum 1:71} than groups 3 and 4 (1:37) and 8 (1:12), 7--4 days post-challenge.
310 Body weight No differences in mean b o d y weight changes were detected between Groups 1--6 during the experiment. Mean weights increased from 141.5 to 198.8 kg, an increase of 40.5%. However, Group 7 lost significantly more weight than Group 8 during the period 0--21 days post-challenge (0.5 and 0.04 kg/day, respectively) (P<0.05). DISCUSSION
This study showed that vaccination with A. centrale m a y not provide adequate protection against anaplasmosis in all animals. Increased susceptibility to A. marginale with increasing age is known to occur in Bos taurus (Jones et al., 1968) and Bos indicus cattle (Wilson et al., 1979). A similar lack of susceptibility of y o u n g animals to A. centrale m a y have interfered with effective immunisation. However, under Australian conditions there has been no field evidence to suggest that vaccination of y o u n g animals with A. centrale is not effective. It is well known that A. centrale has c o m m o n antigenic components with A. marginale (Kuttler, 1967a, b). Infection with A. centrale does n o t always result in subsequent immunity to A. marginale (see Group 7). Resistance to A. marginale challenge was related to good primary antibody response, as observed in all A. marginale and some A. centrale (Group 8) immunized animals. A characteristic of this resistance was a very weak response at challenge. Antibody response could thus be used as an indicator of the level of immunity achieved by vaccination. Because calfhood vaccination with A. centrale left 10 out of 18 animals without adequate immunity, a second A. centrale vaccination m a y be indicated when they are older. Alternatively, A. centrale vaccination could b'e followed by A. marginale, as has been suggested for cattle going to regions of severe challenge (Callow, 1976). Further experimental work appears desirable to evaluate these procedures. A primary inoculation with A. marginale, combined with chemotherapy (Todorovic and TeUez, 1975), could be considered for valuable animals. It is generally accepted that field strains of A. marginale are immunologically similar or identical, although Uilenberg (1970) indicated that this may n o t be the case in Madagascar. Callow et ai. (1976) observed that a Bolivian strain of A. marginale was serologically similar to an Australian strain. Cross-immunity testing in the present work suggested that Strains A and B of A. marginale, isolated 700 kilometres apart, were immunologically identical.
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