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Bovine babesiosis: The passive immunization of calves against Babesia argentina with special reference to the role of complement fixing antibodies
EXPERIMENTAL PARASITOLOGY 20, 119-124 (1967)
Bovine
Babesiosis:
against the
The Passive
Babesia
argentina
Role
Complement
of
D. Division
of Animal Laboratory,
Immunization
with
Special Fixing
of Calves
Reference
to
Antibodies’
F. Mahoney
Health, C.S.I.R.O. Veterinary Parasitology Yeerongpilly, Brisbane, Australia
(Submitted for publication, 23 November 1966) MAHONEY, D. F. 1967. Bovine babesiosis: the passive immunization of calves against Babesia argentina with special reference to the role of complement fixing antibodies. Experimental Parasitology 20, 119-124. The presence of protective antibodies to Babesia argentina was demonstrated by the passive transfer of immunity with serum from carriers of the organism to highly susceptible splenectomized calves. Serum, obtained from donors after one infection, apparently contained a low concentration of protective antibodies, but higher concentrations were evident in serum from donors after four or more superinfections. Serum from naturally infected donors was less effective in conferring protection than serum from donors infected in the laboratory. As probably only the laboratory infections were homologous for the challenge strain, this anomaly might have been due to antigenic variation among strains of B. argentina. No positive correlation between protective capacity and complement fixation (CF) titer of serum could be demonstrated. It was concluded that CF antibodies were unlikely to be concerned with immunological resistance to B. argentina.
In studies of the significance of the complement fixation (CF) reaction in bovine babesiosis (Mahoney, 1964)) evidence was obtained suggesting that functional immunity was not dependent upon the presence of CF antibodies. When carriers of either Babesia argentina (Lignikes) of B. bigemina (Smith and Kilborne) were reinfected with the homologous parasite, different degrees of resistance were observed in individual animals as indicated by the levels of parasitaemia observed after -challenge. These differences, however, could not be correlated with titers of CF antibodies in the serum. Because of the importance of the CF test in epidemiological * In partial fulfilment of the requirements of the Ph.D. degree in the Department of Parasitology, University of Queensland.
work, further studies were undertaken to examine the relationship of CF antibodies to the state of acquired immunity. Experiments on the passive transfer of immunity to B. argentina were undertaken to determine whether serum from infected animals contained protective antibodies and, if so, to examine the association between protection and CF titers. MATERIALS
Experimental
AND
METHODS
Animals
Forty 4-month-old calves of mixed beef breeds were purchased, as required, in a Babesia-free area and subsequently maintained free from babesial infection at the laboratory. The calves weighed between 180 and 250 lb and were splenectomized
119
120
MAHONEY
1-2 weeks before being used in the experiments.
was by either the intravenous (i/v) traperitoneal (i/p) route.
or in-
Seven animals of mixed beef breeds, obtained from a Babes&z-freearea, were pre- Assessment of Protection The splenectomized calves were infected pared as donors of serum. No. 086 and of 10c parasitized MPS, purchased at 4-8 months of age and by injection (i/v) maintained at the laboratory under condi- erythrocytes and were then subjected to tions that excluded extraneous infestation various regimes of treatment with serum with ticks (Boophilus microplus), were from either infected or noninfected animals. subjected to infection by controlled tick The details are given in the description of infestation at intervals of 5-10 months. each experiment that appears with the reAnother group consisted of three yearling sults. The degree of passive protection afheifers (No. T4, T13, T15), first infected forded by the injected serum from infected at the laboratory by controlled tick in- animals was assessedon differences in surfestation and then exposed to further in- vival, in the course and severity of the fection while depastured in a naturally tick- clinical response, and in parasitemia beinfested paddock for 2 years. Two other tween experimental and control groups. donors, (No. 216, 218) were given an Daily observations included rectal temperinfection by controlled tick infestation at ature readings, packed red cell volume, the laboratory at 2 years of age and bled period of survival after infection, and the 2 weeks after clinical recovery; they pro- number of parasites per cubic millimeter vided sera with high concentrations of CF of jugular blood. antibodies. RESULTS
Strain of B. argentina
One strain of B. argentina, previously described (Mahoney, 1964), was used at the laboratory for infecting the experimental calves and the serum donors. However, those donors subsequently exposed to infection in the field, T4, T13, and T15, probably experienced infection with other strains of B. argentina and possibly infection with B. bigemina as well.
Immunity Calves
Tests with
Splenectomized
This experiment was conducted to determine whether there was any evidence of passive transfer of immunity by serum from infected animals, and also to find the dose required and the most effective method of administration. Serum was obtained from a donor after eight successive laboratory infestations with Collection, Storage, and Administration of infected ticks, the last infestation at 5 Serum months prior to bleeding. Ten splenectoUp to 9 liters of blood were drawn from mized calves were infected with B. argena donor at any one time. After the blood tina and five of them were then given a had remained for l-2 hours at room tem- large dose of serum on the same day; two perature, the clots were broken up and received 2 ml/lb body weight, two received kept overnight at 4°C. The serum was then 4 ml/lb, and one 8 ml/lb. In 3 other decanted, clarified by centrifugation, and calves, serum treatment was not comstored in 400-ml quantities at -20°C. menced until the fourth day after infection, Before use, it was thawed rapidly in a water- when parasites were first detected in their bath at 40°C. Administration of the serum blood, and was then given daily at a rate
PASSIVE IMMUNIZATION
TABLE Clinical
Reactions and Survival
Treatment and serum donor No.
Calf No. P. P. 1’. P.
121
AGAINST Babesia argentina I
of Treated and Control Calves in Passive Protection Experiments
Parasite density (parasites/ cu mm)
Fall in P.C.V. (% of preinfection level)
Rectal temperature
Death (days after infection)
To Survival
72 77 108 111 157 180 261 262
N 198,000 135,000 205,000 260,000 73,500 N N
47 13 31 33 55 32 61 51
106.4 105.8 107.0 106.6 107.0 106.8 106.4 106.4
13 10 10 10 11 12 S 12
Serum from uninfected donors
72 76 118 124
51,000 92,000 148,000 137.000
73 75 46 40
107.4 107.0 107.0 106.4
11 12 9 10
Convalescent serum with high CF titer. Donor No. 216, 218, MP5 Serum from donor No. MP5 after 2nd infection
122 123 250 257 119 125
19) 100 20,000 N 31,500 11,400 12,300
79 82 72 74 51 62
106.4 106.8 106.4 107.0 106.6 106.8
15 13 13 13 S 10
69 75 P. 76 P. 107 P. 110 249 251
7 10 3 6 10 24 90
12 19 7 12 15 18 12
104.0 104 8 104.4 104.0 104.6 103.2 104.8
S S s S S 8 S
Serum from naturally infected donors (No. T4, T13, T15).
66 71 74
12,000 205 500
55 60 62
106.0 107.0 106.2
S S S
100
Serum from donor 5 mor after last infection. Donor No. 086. Single dose of serum at time of infection. Donor No. 086.
175 176 178
550 32,400 46,400
26 27 46
103.8 105.6 105.6
S 12 S
66
158 160 149 156 159
53,000 35,100 500,000 4,350 21) 000
47 40 33 56 31
106.0 106.0 106.2 106.2 106.2
10 11 10 S 14
20
Untreated
Serum from donor No. 086, MP5 after 4 or more infections. Low CF titer
Note. P.C.V.
P.
= Packed red cell volume;
N = observation
8.3
0 50
100
not made; S = survived.
of 1 ml/lb. The two remaining calves were that received a single dose of 2 ml/lb, and untreated controls and received no serum. one given a single dose of 4 ml/lb, died The two control calves, the two calves lo-12 days after infection. The calf given
122
MAHONEY
a single dose of 8 ml/lb died on the 14th day. The other calf given 4 ml/lb survived. The most striking effect of the administration of serum in a single dose immediately after infection was a delay in the appearance of parasites in the peripheral blood, and this period increased in parallel with the amount of serum given. Parasites were first detected in control calves at 4 days, in the group that received 2 ml/lb at 5 days, in the group that received 4 ml/lb at 6-7 days, and in the calf that received 8 ml/lb at 8 days. Of the three calves that received 8 ml/lb in small daily doses, one died on the 1lth day without showing any benefit from the serum injection but the remaining two survived. The latter showed low parasitemias and no appreciable diminution in packed red cell volumes. Serum treatment was discontinued after 11 days and, over the next 5 days, parasitemias increased but then declined; and by 21 days no clinical signs of infection were apparent. As this preliminary trial suggested that some protection had been conferred by daily injections of serum commencing after parasites were first detected in the peripheral blood, this method of administration was further explored. Fourteen splenectomized calves were used. The control group comprised six calves of which two received injections of pooled serum from noninfected calves, and four were untreated. The test group comprised eight calves of which three were injected with serum from the naturally infected donors, one was injected with serum obtained from one laboratory donor after seven successive infections, and four were injected with pooled serum obtained from two laboratory donors after ten and five successive infections, respectively. The total quantity of serum given to each calf was 8 ml/lb in daily doses of approximately 1 ml/lb. All six control animals died 10-l 3 days
after infection and parasite densities of 51 ,OOO-205,000 organisms/cu mm of blood were recorded. All calves injected with the serum from infected animals survived. Three that were given serum from naturally infected donors suffered clinical reactions with parasite densities as high as 12,OOO/cu mm and 55-62% reduction in packed red cell volume. The five calves given serum from laboratory infected donors did not develop clinical reactions; parasite densities did not exceed 10 organisms/cu mm, reductions in packed red cell volume were only 7-19%) and only slight rises in rectal temperature were recorded. Relationship between Protective Activity and CF Titer Twelve splenectomized calves were used in treatment regimes designed to correlate protection with CF titer of serum. Four were control animals two of which received serum from uninfected animals, and two were untreated. Four calves were injected with serum obtained from three animals after a single infection that showed high CF titers (l/80-1/640). Two further calves were injected with serum from a donor after its first reinfection, and finally, two were injected with pooled serum from laboratory donors after 4-9 reinfections. These latter sera had CF titers of only l/10. One of the control calves survived, but the other three died 9-12 days after infection. Maximum parasite densities in this group were 137,000-148,000 organisms/ cu mm of blood. All four calves that received serum with a high CF titer died 13-15 days after infection and showed parasite densities of 19,100-3 1,500 organisms/cu mm. Of the two calves given serum (CF titer l/10) from the donor after its first reinfection, one survived and the other died 10 days after infection; these calves had maximum parasite densities of
PASSIVE IMMUNIZATION AGAINST Babesia argentina
123
11,400-12,300 organisms/cu mm, with single dose was 8 days after a challenge reductions in packed red cell volume of dose of 10” organisms. From experience 51-62%. The two calves given pooled with a particular B. argentina strain in use serum from laboratory donors with a low at this laboratory, an incubation period of CF titer survived without showing obvious 8 days is usually associated with the injecclinical reactions. Maximum parasite densi- tion of less than 10,000 parasites, and ties were 24-90 organisms/cu mm, and therefore it was concluded that the transreductions in packed red cell volume were ferred serum caused some mortality or re12-18%. tardation in multiplication of the organisms. The data from these experiments are It is possible that protective antibodies were summarized in the accompanying table. not maintained in sufficient concentration The results have been grouped according to sustain this effect or the parasites may to the type of serum used. The clinical be particularly vulnerable to antibody atreactions of the calves and their periods of tack during the phase of active multiplicasurvival are stated. tion, as, for example, when passing from one red cell to another. If this is so, the timing of the injection of serum could be DISCUSSION important in conferring a high degree of In other experiments not previously re- protection. These observations disagree ported, 2 year-old nonspenectomized cattle with those that have shown a naturally were used in passive immunization trials. acquired passive immunity to B. argentina However, under the conditions of the ex- persisting in calves for 30-40 days after periment these cattle were not highly sus- ingesting colostrum from immune dams ceptible to B. argentina, and only small (Hall, 1960, 1963). This greater period differences in the severity of infection in of protection could be due to a higher treated and control groups were observed. concentration of protective antibodies in Because of their greater and more con- colostrum than in the serum used in the sistent susceptibility splenectomized calves present experiments; the colostral antiwere used in the present experiments. Such bodies might also be eliminated more calves rarely survived more than lo-13 slowly than those present in serum. days after infection with an estimated lo6 The results of the second experiment organisms, and any procedure prolonging demonstrated that the serum from carrier their survival beyond this period could be animals that had been subjected to reregarded as indicating some acquired re- peated infection, contained protective antisistance. bodies that conferred a high degree of The daily injection of donor serum into immunity on splenectomized calves. The an infected animal immediately after the calves treated with serum from naturally detection of parasites in its blood, was infected animals, however, were more more effective in changing the course of severely affected than those that received the disease and conferring some protec- serum from laboratory donors. This result tion than the administration of a single may have been associated simply with a large dose of serum at the time of infection. difference in protective antibody titer, or Injection of a single large dose of serum possibly, as has been demonstrated for at the time of infection did, however, delay B. bigemina by Callow (1964), with the the appearance of parasitemia; the incuba- existence of antigenically different strains tion period in the calf given the largest of B. argentina. The laboratory serum was
124
MAHONEY
homologous for the challenge strain, but the naturally infected donors may have encountered other strains during their 2 years on pasture. The protection of splenectomized calves with serum from infected cattle demonstrates the importance of humoral factors in the mechanism of acquired immunity. Babesiosis is a disease in which immunity almost certainly depends on the presence of infection. Along with malaria it has been placed in an immunological category in which antibodies are assigned little or no functional importance (Sergent and Sergent, 1956). The immune response of the host is presumably mediated through the agency of cells maintained in a state of readiness by the stimulus of latent infection. The findings reported in the present paper are not completely compatible with this concept, as they suggest that the mechanism of immunity in babesiosis is dependent, at least in part, upon the presence of protective antibodies, and may not be fundamentally different from that in other infectious diseases. McGregor (1964) reached a similar conclusion in relation to malaria. In the third experiment, protection was obtained only with serum with a low level of CF antibody collected from animals that had been infected 4-9 times. The importance of frequent superinfection to promote adequate protective antibody levels is suggested by the slight protection demonstrated with serum taken from a donor after only two infections. The failure of convalescent serum which contained high ‘Fresh infection of a host while the previous one is still present (Anon, 1963).
levels of CF antibody to give protection, suggests that these antibodies are not important in the immune reaction. In earlier work (Mahoney, 1964)) it was shown that the CF antibody response attained a peak shortly after the first infection, and then declined to a titer of l/10-1/20, which could be maintained by regular superinfection with infected ticks. In contrast, the concentration of protective antibodies increased to high levels only after four or more superinfections.
I wish to thank Professor J. F. A. Sprent, Department of Parasitology, University of Queensland, for advice during the course of the work, and Mr. G. B. Mirre for technical assistance. REFERENCES
1963. Terminology HEALTH ORGANIZATION, of malaria and of malaria eradication. Report of a drafting committee, p. 120. CALLOW, L. L. 1964. Strain immunity in babesiosis. Nature, London 204, 1213-1214. HALL, W. T. K. 1960. The immunity of calves to Babesia argenrina infection. Australian Veterinary Journal 36, 361-366. HALL, W. T. K. 1963. The immunity of calves to tick transmitted Babesia argentina infection, WORLD
Australian
Veterinary
Journal
39, 386-389.
D. F. 1964. Bovine babesiosis: An assessment of the significance of complement fixing antibody based upon experimental infection. Australian Veterinary Journal 40, 369-375. MCGREGOR, I. A. 1964. The passive transfer of human malarial immunity. American Journal of Tropical Medicine and Hygiene 13, (supplement part 2), 237-239. SERGENT, ED., AND SERGENT, ET. 1956. Historique du concept de l’immunite relative ou “permunition” correlative d’une infection latente. MAHONEY,
Archives
52-89.
de L’lnstitut
Pasteur
d’Al.qerie
34,
Bovine
Babesiosis:
against the
The Passive
Babesia
argentina
Role
Complement
of
D. Division
of Animal Laboratory,
Immunization
with
Special Fixing
of Calves
Reference
to
Antibodies’
F. Mahoney
Health, C.S.I.R.O. Veterinary Parasitology Yeerongpilly, Brisbane, Australia
(Submitted for publication, 23 November 1966) MAHONEY, D. F. 1967. Bovine babesiosis: the passive immunization of calves against Babesia argentina with special reference to the role of complement fixing antibodies. Experimental Parasitology 20, 119-124. The presence of protective antibodies to Babesia argentina was demonstrated by the passive transfer of immunity with serum from carriers of the organism to highly susceptible splenectomized calves. Serum, obtained from donors after one infection, apparently contained a low concentration of protective antibodies, but higher concentrations were evident in serum from donors after four or more superinfections. Serum from naturally infected donors was less effective in conferring protection than serum from donors infected in the laboratory. As probably only the laboratory infections were homologous for the challenge strain, this anomaly might have been due to antigenic variation among strains of B. argentina. No positive correlation between protective capacity and complement fixation (CF) titer of serum could be demonstrated. It was concluded that CF antibodies were unlikely to be concerned with immunological resistance to B. argentina.
In studies of the significance of the complement fixation (CF) reaction in bovine babesiosis (Mahoney, 1964)) evidence was obtained suggesting that functional immunity was not dependent upon the presence of CF antibodies. When carriers of either Babesia argentina (Lignikes) of B. bigemina (Smith and Kilborne) were reinfected with the homologous parasite, different degrees of resistance were observed in individual animals as indicated by the levels of parasitaemia observed after -challenge. These differences, however, could not be correlated with titers of CF antibodies in the serum. Because of the importance of the CF test in epidemiological * In partial fulfilment of the requirements of the Ph.D. degree in the Department of Parasitology, University of Queensland.
work, further studies were undertaken to examine the relationship of CF antibodies to the state of acquired immunity. Experiments on the passive transfer of immunity to B. argentina were undertaken to determine whether serum from infected animals contained protective antibodies and, if so, to examine the association between protection and CF titers. MATERIALS
Experimental
AND
METHODS
Animals
Forty 4-month-old calves of mixed beef breeds were purchased, as required, in a Babesia-free area and subsequently maintained free from babesial infection at the laboratory. The calves weighed between 180 and 250 lb and were splenectomized
119
120
MAHONEY
1-2 weeks before being used in the experiments.
was by either the intravenous (i/v) traperitoneal (i/p) route.
or in-
Seven animals of mixed beef breeds, obtained from a Babes&z-freearea, were pre- Assessment of Protection The splenectomized calves were infected pared as donors of serum. No. 086 and of 10c parasitized MPS, purchased at 4-8 months of age and by injection (i/v) maintained at the laboratory under condi- erythrocytes and were then subjected to tions that excluded extraneous infestation various regimes of treatment with serum with ticks (Boophilus microplus), were from either infected or noninfected animals. subjected to infection by controlled tick The details are given in the description of infestation at intervals of 5-10 months. each experiment that appears with the reAnother group consisted of three yearling sults. The degree of passive protection afheifers (No. T4, T13, T15), first infected forded by the injected serum from infected at the laboratory by controlled tick in- animals was assessedon differences in surfestation and then exposed to further in- vival, in the course and severity of the fection while depastured in a naturally tick- clinical response, and in parasitemia beinfested paddock for 2 years. Two other tween experimental and control groups. donors, (No. 216, 218) were given an Daily observations included rectal temperinfection by controlled tick infestation at ature readings, packed red cell volume, the laboratory at 2 years of age and bled period of survival after infection, and the 2 weeks after clinical recovery; they pro- number of parasites per cubic millimeter vided sera with high concentrations of CF of jugular blood. antibodies. RESULTS
Strain of B. argentina
One strain of B. argentina, previously described (Mahoney, 1964), was used at the laboratory for infecting the experimental calves and the serum donors. However, those donors subsequently exposed to infection in the field, T4, T13, and T15, probably experienced infection with other strains of B. argentina and possibly infection with B. bigemina as well.
Immunity Calves
Tests with
Splenectomized
This experiment was conducted to determine whether there was any evidence of passive transfer of immunity by serum from infected animals, and also to find the dose required and the most effective method of administration. Serum was obtained from a donor after eight successive laboratory infestations with Collection, Storage, and Administration of infected ticks, the last infestation at 5 Serum months prior to bleeding. Ten splenectoUp to 9 liters of blood were drawn from mized calves were infected with B. argena donor at any one time. After the blood tina and five of them were then given a had remained for l-2 hours at room tem- large dose of serum on the same day; two perature, the clots were broken up and received 2 ml/lb body weight, two received kept overnight at 4°C. The serum was then 4 ml/lb, and one 8 ml/lb. In 3 other decanted, clarified by centrifugation, and calves, serum treatment was not comstored in 400-ml quantities at -20°C. menced until the fourth day after infection, Before use, it was thawed rapidly in a water- when parasites were first detected in their bath at 40°C. Administration of the serum blood, and was then given daily at a rate
PASSIVE IMMUNIZATION
TABLE Clinical
Reactions and Survival
Treatment and serum donor No.
Calf No. P. P. 1’. P.
121
AGAINST Babesia argentina I
of Treated and Control Calves in Passive Protection Experiments
Parasite density (parasites/ cu mm)
Fall in P.C.V. (% of preinfection level)
Rectal temperature
Death (days after infection)
To Survival
72 77 108 111 157 180 261 262
N 198,000 135,000 205,000 260,000 73,500 N N
47 13 31 33 55 32 61 51
106.4 105.8 107.0 106.6 107.0 106.8 106.4 106.4
13 10 10 10 11 12 S 12
Serum from uninfected donors
72 76 118 124
51,000 92,000 148,000 137.000
73 75 46 40
107.4 107.0 107.0 106.4
11 12 9 10
Convalescent serum with high CF titer. Donor No. 216, 218, MP5 Serum from donor No. MP5 after 2nd infection
122 123 250 257 119 125
19) 100 20,000 N 31,500 11,400 12,300
79 82 72 74 51 62
106.4 106.8 106.4 107.0 106.6 106.8
15 13 13 13 S 10
69 75 P. 76 P. 107 P. 110 249 251
7 10 3 6 10 24 90
12 19 7 12 15 18 12
104.0 104 8 104.4 104.0 104.6 103.2 104.8
S S s S S 8 S
Serum from naturally infected donors (No. T4, T13, T15).
66 71 74
12,000 205 500
55 60 62
106.0 107.0 106.2
S S S
100
Serum from donor 5 mor after last infection. Donor No. 086. Single dose of serum at time of infection. Donor No. 086.
175 176 178
550 32,400 46,400
26 27 46
103.8 105.6 105.6
S 12 S
66
158 160 149 156 159
53,000 35,100 500,000 4,350 21) 000
47 40 33 56 31
106.0 106.0 106.2 106.2 106.2
10 11 10 S 14
20
Untreated
Serum from donor No. 086, MP5 after 4 or more infections. Low CF titer
Note. P.C.V.
P.
= Packed red cell volume;
N = observation
8.3
0 50
100
not made; S = survived.
of 1 ml/lb. The two remaining calves were that received a single dose of 2 ml/lb, and untreated controls and received no serum. one given a single dose of 4 ml/lb, died The two control calves, the two calves lo-12 days after infection. The calf given
122
MAHONEY
a single dose of 8 ml/lb died on the 14th day. The other calf given 4 ml/lb survived. The most striking effect of the administration of serum in a single dose immediately after infection was a delay in the appearance of parasites in the peripheral blood, and this period increased in parallel with the amount of serum given. Parasites were first detected in control calves at 4 days, in the group that received 2 ml/lb at 5 days, in the group that received 4 ml/lb at 6-7 days, and in the calf that received 8 ml/lb at 8 days. Of the three calves that received 8 ml/lb in small daily doses, one died on the 1lth day without showing any benefit from the serum injection but the remaining two survived. The latter showed low parasitemias and no appreciable diminution in packed red cell volumes. Serum treatment was discontinued after 11 days and, over the next 5 days, parasitemias increased but then declined; and by 21 days no clinical signs of infection were apparent. As this preliminary trial suggested that some protection had been conferred by daily injections of serum commencing after parasites were first detected in the peripheral blood, this method of administration was further explored. Fourteen splenectomized calves were used. The control group comprised six calves of which two received injections of pooled serum from noninfected calves, and four were untreated. The test group comprised eight calves of which three were injected with serum from the naturally infected donors, one was injected with serum obtained from one laboratory donor after seven successive infections, and four were injected with pooled serum obtained from two laboratory donors after ten and five successive infections, respectively. The total quantity of serum given to each calf was 8 ml/lb in daily doses of approximately 1 ml/lb. All six control animals died 10-l 3 days
after infection and parasite densities of 51 ,OOO-205,000 organisms/cu mm of blood were recorded. All calves injected with the serum from infected animals survived. Three that were given serum from naturally infected donors suffered clinical reactions with parasite densities as high as 12,OOO/cu mm and 55-62% reduction in packed red cell volume. The five calves given serum from laboratory infected donors did not develop clinical reactions; parasite densities did not exceed 10 organisms/cu mm, reductions in packed red cell volume were only 7-19%) and only slight rises in rectal temperature were recorded. Relationship between Protective Activity and CF Titer Twelve splenectomized calves were used in treatment regimes designed to correlate protection with CF titer of serum. Four were control animals two of which received serum from uninfected animals, and two were untreated. Four calves were injected with serum obtained from three animals after a single infection that showed high CF titers (l/80-1/640). Two further calves were injected with serum from a donor after its first reinfection, and finally, two were injected with pooled serum from laboratory donors after 4-9 reinfections. These latter sera had CF titers of only l/10. One of the control calves survived, but the other three died 9-12 days after infection. Maximum parasite densities in this group were 137,000-148,000 organisms/ cu mm of blood. All four calves that received serum with a high CF titer died 13-15 days after infection and showed parasite densities of 19,100-3 1,500 organisms/cu mm. Of the two calves given serum (CF titer l/10) from the donor after its first reinfection, one survived and the other died 10 days after infection; these calves had maximum parasite densities of
PASSIVE IMMUNIZATION AGAINST Babesia argentina
123
11,400-12,300 organisms/cu mm, with single dose was 8 days after a challenge reductions in packed red cell volume of dose of 10” organisms. From experience 51-62%. The two calves given pooled with a particular B. argentina strain in use serum from laboratory donors with a low at this laboratory, an incubation period of CF titer survived without showing obvious 8 days is usually associated with the injecclinical reactions. Maximum parasite densi- tion of less than 10,000 parasites, and ties were 24-90 organisms/cu mm, and therefore it was concluded that the transreductions in packed red cell volume were ferred serum caused some mortality or re12-18%. tardation in multiplication of the organisms. The data from these experiments are It is possible that protective antibodies were summarized in the accompanying table. not maintained in sufficient concentration The results have been grouped according to sustain this effect or the parasites may to the type of serum used. The clinical be particularly vulnerable to antibody atreactions of the calves and their periods of tack during the phase of active multiplicasurvival are stated. tion, as, for example, when passing from one red cell to another. If this is so, the timing of the injection of serum could be DISCUSSION important in conferring a high degree of In other experiments not previously re- protection. These observations disagree ported, 2 year-old nonspenectomized cattle with those that have shown a naturally were used in passive immunization trials. acquired passive immunity to B. argentina However, under the conditions of the ex- persisting in calves for 30-40 days after periment these cattle were not highly sus- ingesting colostrum from immune dams ceptible to B. argentina, and only small (Hall, 1960, 1963). This greater period differences in the severity of infection in of protection could be due to a higher treated and control groups were observed. concentration of protective antibodies in Because of their greater and more con- colostrum than in the serum used in the sistent susceptibility splenectomized calves present experiments; the colostral antiwere used in the present experiments. Such bodies might also be eliminated more calves rarely survived more than lo-13 slowly than those present in serum. days after infection with an estimated lo6 The results of the second experiment organisms, and any procedure prolonging demonstrated that the serum from carrier their survival beyond this period could be animals that had been subjected to reregarded as indicating some acquired re- peated infection, contained protective antisistance. bodies that conferred a high degree of The daily injection of donor serum into immunity on splenectomized calves. The an infected animal immediately after the calves treated with serum from naturally detection of parasites in its blood, was infected animals, however, were more more effective in changing the course of severely affected than those that received the disease and conferring some protec- serum from laboratory donors. This result tion than the administration of a single may have been associated simply with a large dose of serum at the time of infection. difference in protective antibody titer, or Injection of a single large dose of serum possibly, as has been demonstrated for at the time of infection did, however, delay B. bigemina by Callow (1964), with the the appearance of parasitemia; the incuba- existence of antigenically different strains tion period in the calf given the largest of B. argentina. The laboratory serum was
124
MAHONEY
homologous for the challenge strain, but the naturally infected donors may have encountered other strains during their 2 years on pasture. The protection of splenectomized calves with serum from infected cattle demonstrates the importance of humoral factors in the mechanism of acquired immunity. Babesiosis is a disease in which immunity almost certainly depends on the presence of infection. Along with malaria it has been placed in an immunological category in which antibodies are assigned little or no functional importance (Sergent and Sergent, 1956). The immune response of the host is presumably mediated through the agency of cells maintained in a state of readiness by the stimulus of latent infection. The findings reported in the present paper are not completely compatible with this concept, as they suggest that the mechanism of immunity in babesiosis is dependent, at least in part, upon the presence of protective antibodies, and may not be fundamentally different from that in other infectious diseases. McGregor (1964) reached a similar conclusion in relation to malaria. In the third experiment, protection was obtained only with serum with a low level of CF antibody collected from animals that had been infected 4-9 times. The importance of frequent superinfection to promote adequate protective antibody levels is suggested by the slight protection demonstrated with serum taken from a donor after only two infections. The failure of convalescent serum which contained high ‘Fresh infection of a host while the previous one is still present (Anon, 1963).
levels of CF antibody to give protection, suggests that these antibodies are not important in the immune reaction. In earlier work (Mahoney, 1964)) it was shown that the CF antibody response attained a peak shortly after the first infection, and then declined to a titer of l/10-1/20, which could be maintained by regular superinfection with infected ticks. In contrast, the concentration of protective antibodies increased to high levels only after four or more superinfections.
I wish to thank Professor J. F. A. Sprent, Department of Parasitology, University of Queensland, for advice during the course of the work, and Mr. G. B. Mirre for technical assistance. REFERENCES
1963. Terminology HEALTH ORGANIZATION, of malaria and of malaria eradication. Report of a drafting committee, p. 120. CALLOW, L. L. 1964. Strain immunity in babesiosis. Nature, London 204, 1213-1214. HALL, W. T. K. 1960. The immunity of calves to Babesia argenrina infection. Australian Veterinary Journal 36, 361-366. HALL, W. T. K. 1963. The immunity of calves to tick transmitted Babesia argentina infection, WORLD
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D. F. 1964. Bovine babesiosis: An assessment of the significance of complement fixing antibody based upon experimental infection. Australian Veterinary Journal 40, 369-375. MCGREGOR, I. A. 1964. The passive transfer of human malarial immunity. American Journal of Tropical Medicine and Hygiene 13, (supplement part 2), 237-239. SERGENT, ED., AND SERGENT, ET. 1956. Historique du concept de l’immunite relative ou “permunition” correlative d’une infection latente. MAHONEY,
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