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Attenuation of Babesia bovis by in vitro cultivation
Veterinary Parasitology, 24 (1987) 7--13 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
7
ATTENUATION OF B A B E S I A BO VIS BY IN VITRO CULTIVATION
C.E. Y U N K E R 1, K.L. K U T T L E R ~, and L.W. JOHNSON
U.S. Department of Agriculture, Agricultural Research Service, Hemoparasitic Diseases Research Unit, Washington State University, Pullman, WA 99164-6470 (U.S.A.) (Accepted for publication 25 April 1986)
ABSTRACT Yunker, C.E., Kuttler, K.L. and Johnson, L.W., 1987. Attenuation of Babesia bovis by in vitro cultivation. Vet. Parasitol., 24: 7--13. A virulent strain of Babesia boris was adapted to grow in erythrocyte culture in the presence of equine serum and in lieu of bovine serum. F o u r splenectomized calves inoculated with the adapted strain, 429, developed hematologic signs of infection and a low grade fever, but remained free of central nervous system (CNS) signs and recovered. All of six control animals inoculated with a virulent strain reacted severely and five showed CNS signs and died. The calves injected with the attenuated strain were solidly immune when challenged with the virulent strain at 44 or 78 days after vaccination.
INTRODUCTION
The attenuation of intracellular pathogens by cultivation in vitro is a well k n o w n phenomenon; yellow fever, polio, rinderpest and other pathogenic animal viruses have been rendered avirulent or less pathogenic by prolonged growth in vertebrate tissue or cell cultures, and have subsequently been used as modified live virus vaccines. Similarly, strains of rickettsial agents of disease, such as Rickettsia prowazeki and Coxiella burneti, have also been modified in culture and employed as live vaccines. In this paper, we report that Babesia bovis, a virulent protozoan parasite of cattle, may likewise undergo a reduction in virulence under certain conditions of culture and, when the attenuated organisms are used to vaccinate cattle, protection is afforded.
1 Present address: University of Florida Heartwater Research Project, P.O. Box 8101, Causeway, Harare, Zimbabwe. 2 To whom requests for reprints should be addressed.
0304-4017/87/$03.50
© 1987 Elsevier Science Publishers B.V.
MATERIALS AND METHODS
Parasites B. bovis of Mexican origin, originally isolated by Dr. Ron Smith of the University of Illinois, was used in these experiments. The organism used in cultures was obtained from Dr. G.M. Buening, University of Missouri at Columbia, where it had undergone numerous passages in bovine e r y t h r o c y t e (Brbc) cultures. Throughout this time, this strain, designated KB, retained its virulence for cattle (G.M. Buening, personal communication, 1983). Cultures
Stock cultures of the parasite were grown in microaerophilic stationary phase (MASP) cultures of bovine rbc (Levy and Ristic, 1980). Cultures, at a 10% packed cell volume (PCV), were maintained in 25-cm 2 plastic tissue culture flasks (No. 25100, Coming Glass Works, Coming, NY), at 37°C in humidified air containing 5% CO2. They were given daily changes of medium and were subdivided at intervals of 2--3 days. Basic medium consisted of tissue culture medium 199 with Earle's salts and w i t h o u t glutamine (No. LM-191, Kansas City Biologicals, Lenexa, KS), containing the antibiotics penicillin G (100 U ml-1), streptomycin sulfate (100 #g m1-1) and amphotericin B (0.25 /~g ml-1). (This medium was later modified by the addition of 10 mg ml-1 extra glucose [W.L. G o l f and C.E. Yunker, in preparation] and the substitution of 50/~g ml- 1 gentamycin sulfate for penicillin and streptomycin.) Complete medium (CM) was prepared daffy by mixing refrigerated basic medium with adult bovine serum in a 3:2 proportion and adding 20 mM TES [n-tris ( h y d r o x y m e t h y l ) methyl-2-aminoethane sulfonic acid] (U.S. Biochemical Corp., Cleveland, OH). After adjustment to pH 7.0 with 1 N NaOH, CM was sterilized by pressure filtration and used to replace spent medium over the layer of settled rbc in culture flasks. On days when cultures were to be subdivided, existing medium was removed and a thin film of rbc was made for Giemsa-staining. Medium was replaced and parasitemias were estimated in order to determine splitratio (usually 1:1 to 1:4). Depending on this ratio, an aliquot of infected culture material was introduced into a new flask containing fresh CM and normal Brbc (e.g., if a 1:3 split was desired 5 ml of culture suspension was transferred to a flask containing 12 ml CM and 3 ml of a normal adult Brbc suspension). Normal Brbc were obtained from defibrinated whole blood of adult cattle and were washed three times in phosphate buffered saline (PBS). After each wash, cells were centrifuged at 1000 × g for 10 min, and b u f f y coats and supernatants were removed. A final wash in Puck's saline plus added glucose (PSG+; Palmer et al., 1982), was followed by centrifugation, as above, and the Brbc pellet was dispersed in PSG+ to make a 50% suspension. This was stored at 4°C for n o t longer than 2 weeks and cells were oxygenated daily by inverting the tube.
Parasites cultured in 96-well tissue culture plates (No. 3596, Costar, Cambridge, MA), were adapted to grow in the presence of equine serum by gradually substituting the latter for a portion of bovine serum in culture medium (Levy and Ristic, 1983). At intervals, part of the bovine serum was replaced with equine serum until the serum c o m p o n e n t of the medium was entirely of equine origin. Fluid levels of culture plates were adjusted to give a depth of 6 mm (200 ~1). Later, cultures were transferred to a gas-regulating incubator (Heraeus Type B-5060 EC/O2; Rupp and Bowman Co., Farmington Hills, MI), set to supply 2% 02, 5% CO2 and 93% N2, whereupon fluid depths were adjusted to 4 mm (130 pl) (Rodriguez et al., 1983). Cultures were later expanded into 25-cm 2 Coming tissue culture flasks, held in this same atmosphere, and fluid levels were adjusted to give a depth of 4 mm (15 ml). Parasites were frozen in 10% dimethyl sulfoxide or according to the m e t h o d of Palmer et al. (1982). In the latter case infected Brbc were separated from merozoites, each fraction was suspended in PSG+ containing 10% polyvinylpyrrolidone, and suspensions were frozen at a cooling rate of 20°C min-1. Experimental and donor animals
Four 6--9-month-old, splenectomized, dairy-type, steer calves were inoculated intravenously (IV) with culture material containing approximately 0.75 × 109 intraerythrocytic parasites. These four calves were similarly inoculated 44 days (3 calves) and 78 days (1 calf) after culture inoculation, with approximately 2.2 × 107 virulent B. bovis intraerythrocytic parasites of Mexican origin, obtained from frozen stabilates. Six comparable splenectomized calves were similarly inoculated with virulent B. bovis stabilate. A 1:10 dilution of the stabilate challenge material was made in PBS containing 10% normal bovine serum, kept cold, and inoculated within 30 min of thawing. Inoculated calves were bled, rectal temperatures were recorded and clinical observations were made daily. Percent parasitized erythrocytes (PPE) was estimated from Giemsa-stained smears and PCV's were determined. The donors for normal Brbc and serum were mature beef cattle of mixed breeding. Normal equine serum was obtained from Shetland and Welch cross ponies. In addition, pooled lots of horse serum from controlled herds were obtained from commercial suppliers. RESULTS Donor serum screening
Samples of horse serum were tested for their effect on B. bovis in vitro. The equine serum was used to replace one-fourth of the bovine c o m p o n e n t
10
of CM. Only one lot, a commercially available 'defined' horse serum (HyClone Laboratories, Logan, UT), supported growth of the parasite at levels seen in the equine serum-free control. This serum was used in adaptation experiments.
Adaptation o f B. bovis to growth in equine serum Parasites survived for various lengths of time after inclusion of equine serum in medium. In two trials where cultures were initiated in CM containing 40% equine serum and no bovine serum, the parasites survived, with evidence of multiplication, for 17 and 22 days, while undergoing three and four subdivisions, respectively. However, despite this, peak parasitemias gradually declined during these times and cultures were eventually lost. In cultures initiated in medium containing equine and bovine sera in various proportions (e.g., 1:3, 1:1), parasites grew w i t h o u t apparent detriment. The relative proportion of equine c o m p o n e n t could be increased at intervals of 3--4 weeks to 87.5% of total serum w i t h o u t a notable decrease in parasitemia. However, when medium containing solely equine serum was then offered, parasitemias invariably declined within days. At these times, the decline could be reversed if the percentage of bovine serum was increased. One culture initiated in medium containing equal quantities of equine and bovine serum was transferred, after 21 days and seven subdivisions, to CM containing equine and bovine sera in a 3:1 ratio. Parasites continued to multiply for the next 60 days, while cultures underwent 41 subdivisions. At the succeeding passage, parasite levels failed to rebound and parasitemias remained at low levels (< 0.1%) for 9 days. Thereafter, parasites resumed their typical growth and, on t w o occasions, 117 and 132 days after initiation, respectively, were successfully transferred into medium containing serum solely of equine origin. To date, one of these adapted strains, No. 429, has been in 40% equine serum for 307 days while undergoing 127 TABLE I Responses of splenectomized calves to infection with attenuated (429) and virulent (153) strains of Babesia boris
Group
Maximum temp. (° C)
Persistence o f fever (days)
Lowest PCV
% PCV depression
3.5 + 1.0
41.3 + 0.4
5.8 -+ 2.2
17.2 +- 5.5
49.0 + 15.0
Six calves inoc. with 153 str.
10.0 ± 0.8
41.7 -~ 0.1
5.3 ± 2.4
20.0 ± 8.0
33.0 ± 19.0
Significance
P < 0.01
P < 0.05
NS
NS
NS
Four calves inoc. with 4 2 9 str.
1st day o f fever
11
subdivisions. Peak parasitemias during this time ranged from 1.2% to 5.9% {average = 2.98 + 0.83, n = 100). Inoculation o f calves
Both the attenuated, culture-derived strain and the virulent strain of B. boris were infective for splenectomized calves, causing febrile reactions, parasitemias and lowered PCV's. Average results for these and other observations are given in Table I. Post-inoculation day of onset of fever and highest temperature attained were significantly different between vaccinated and control groups (P <: 0.01 and <~ 0.05, respectively), but persistence of fever, lowest PCV attained, percent drop in PCV and highest parasitemia attained between groups showed no significant differences. However, marked differences were seen in the clinical response of these animals to the infection. None of the four animals vaccinated with the 429 strain showed central nervous system (CNS) signs and all four survived, whereas, of six control calves inoculated with the virulent stabilate, five developed CNS signs and died. The continued virulence of the parent strain (KB) of B. boris, was confirmed by L. Chieves, of the National Animal Disease Center, Ames, IA. On June 6, 1985 a splenectomized calf was inoculated IV with approximately 1 X 109 intraerythrocytic parasites in 15 ml culture medium. It became febrile and oligocythemic, and exhibited a high percentage (22%) of parasitized erythrocytes. The animal developed CNS signs and became moribund shortly thereafter. It was killed in extremis for antigen production. The calves vaccinated with Strain 429 were later challenged with virulent B. bovis. Forty-four days after exposure to the avirulent strain, each of three calves was given an IV injection of 2 ml of a 1:10 dilution of B. bovis stabilate (Strain 153), which contained 2.2 X 107 organisms at the time of
% Maximum parasitemia
CNS signs
Deaths
Persistence of anemia
Response to challenge
<0.1
0/4
0/4
10.0 +- 6.0
0/4
< 0 . 4 +- 0.6
516
5/6
NA
NA
NS
.
.
.
.
.
12
freezing. The remaining vaccinee was given an identical dosage of the same stabilate 78 days after exposure. All survived this challenge while exhibiting no clinical or hematological or clinical evidence of infection. Parasites from Strain 429 cultures were successfully revived from frozen storage u p o n cattle inoculation. Stabilates prepared from calves which received the culture material retained their attenuated characteristics. However, these stabiliates could n o t be re-established in MASP cultures. DISCUSSION The development of cell culture systems for the propagation of Babesia bovis (Erp et al., 1978, 1980; Levy and Ristic, 1980) has greatly facilitated laboratory studies of babesiosis, including the production of experimental immunogens. The latter include soluble and corpuscular antigens (Gravely et al., 1979; James et al., 1981), and an attenuated strain of B. bovis selected by means of cell cloning (G.M. Buening, personal communication, 1983). Reduction in virulence of B. bovis had previously been demonstrated in vivo through rapid, serial syringe passage in splenectomized calves (Callow, 1971; Callow et al., 1979) and by irradiation of parasitemic blood (Mahoney et al., 1973). We have employed the e r y t h r o c y t e culture system for B. bovis to produce and maintain, for an extended period of time, an equine serum-adapted strain that is both avirulent and highly protective for splenectomized calves. Our results are similar to those of Weiss and DeGuisti (1966) who f o u n d that continued passage of Plasmodium berghei in cultures containing hamster serum yielded an attenuated strain that protected mice against exposure to a virulent strain. Babesia bovis had previously been adapted to grow in vitro in the presence of exotic blood components, yielding soluble antigens free of bovine c o m p o n e n t s (Ristic et al., 1981). The equine serum-adapted strain reported here warrants consideration as a candidate vaccine strain. Additional tests of this strain, in splenectomized as well as intact animals, are in progress, as are efforts to devise a more satisfactory system for the recovery of the parasites from frozen storage. ACKNOWLEDGEMENTS We thank Janis Woolbright and David Jones for excellent technical assistance. The help of our colleagues, G.M. Buening, T o y o h i k o Yoshihara and Lucious Chieves, in various aspects of this study is also gratefully acknowledged.
13 REFERENCES Callow, L.L., 1971. The control of babesiosis with a highly infective, attenuated vaccine. World Vet. Congr., Proc., 1 : 357--360. Callow, L.L., MeUors, 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. Erp, E., Gravely, S.M., Smith, R.D., Ristic, M., Osorno, B.M. and Carson, C.A., 1978. Growth of Babesia boris in bovine erythrocyte cultures. Am. J. Trop. Med. Hyg. 27: 1061--1064. Erp., E., Smith, R.D., Ristic, M. and Osorno, B.M., 1980. Continuous in vitro cultivation ofBabesia bovis. Am. J. Vet. Res., 41: 1141--1142. Gravely, S.M., Smith, R.D., Erp, E., Canto, G.T., Aikawa, M., Osorno, B.M. and Ristic, M., 1979. Bovine babesiosis: partial characterization of blood culture-derived Babesia boris. Int. J. Parasitol., 9: 591--598. James, M.A., Levy, M.G. and Ristic, M., 1981. Isolation and partial characterization of culture-derived soluble Babesia bovis antigens. Infect. Immun., 31: 358--361. Levy, M.G. and Ritic, M., 1980. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science, 207 : 1218--1220. Mahoney, D.F., Wright, I.G. and Keterer, P.J., 1973. Babesia argentina: the infectivity and immunogenicity of irradiated blood parasites for splenectomized calves. Int. J. Parasitol., 3 : 209--217. Palmer, D.A., Buening, G.M. and Carson, C.A., 1982. Cryopreservation of Babesia bovis for cultivation. Parasitology, 84 : 567--572. Ristic, M., Smith, R.D. and Kahoma, I., 1981. Characterization of Babesia antigens derived from cell cultures and ticks. In: M. Ristic and J.P. Kreier (Editors), Babesiosis. Academic Press, New York, pp. 337--380. Rodriguez, S.D., Buening, G.M., Green, T.J. and Carson, C.A., 1983. Cloning of Babesia bovis by in vitro cultivation. Infect. Immun., 42: 15--18. Weiss, M.L. and DeGuisti, D.L., 1966. Active immunization against Plasmodium berghei malaria in mice. Am. J. Trop. Med. Hyg., 15: 472--482.
7
ATTENUATION OF B A B E S I A BO VIS BY IN VITRO CULTIVATION
C.E. Y U N K E R 1, K.L. K U T T L E R ~, and L.W. JOHNSON
U.S. Department of Agriculture, Agricultural Research Service, Hemoparasitic Diseases Research Unit, Washington State University, Pullman, WA 99164-6470 (U.S.A.) (Accepted for publication 25 April 1986)
ABSTRACT Yunker, C.E., Kuttler, K.L. and Johnson, L.W., 1987. Attenuation of Babesia bovis by in vitro cultivation. Vet. Parasitol., 24: 7--13. A virulent strain of Babesia boris was adapted to grow in erythrocyte culture in the presence of equine serum and in lieu of bovine serum. F o u r splenectomized calves inoculated with the adapted strain, 429, developed hematologic signs of infection and a low grade fever, but remained free of central nervous system (CNS) signs and recovered. All of six control animals inoculated with a virulent strain reacted severely and five showed CNS signs and died. The calves injected with the attenuated strain were solidly immune when challenged with the virulent strain at 44 or 78 days after vaccination.
INTRODUCTION
The attenuation of intracellular pathogens by cultivation in vitro is a well k n o w n phenomenon; yellow fever, polio, rinderpest and other pathogenic animal viruses have been rendered avirulent or less pathogenic by prolonged growth in vertebrate tissue or cell cultures, and have subsequently been used as modified live virus vaccines. Similarly, strains of rickettsial agents of disease, such as Rickettsia prowazeki and Coxiella burneti, have also been modified in culture and employed as live vaccines. In this paper, we report that Babesia bovis, a virulent protozoan parasite of cattle, may likewise undergo a reduction in virulence under certain conditions of culture and, when the attenuated organisms are used to vaccinate cattle, protection is afforded.
1 Present address: University of Florida Heartwater Research Project, P.O. Box 8101, Causeway, Harare, Zimbabwe. 2 To whom requests for reprints should be addressed.
0304-4017/87/$03.50
© 1987 Elsevier Science Publishers B.V.
MATERIALS AND METHODS
Parasites B. bovis of Mexican origin, originally isolated by Dr. Ron Smith of the University of Illinois, was used in these experiments. The organism used in cultures was obtained from Dr. G.M. Buening, University of Missouri at Columbia, where it had undergone numerous passages in bovine e r y t h r o c y t e (Brbc) cultures. Throughout this time, this strain, designated KB, retained its virulence for cattle (G.M. Buening, personal communication, 1983). Cultures
Stock cultures of the parasite were grown in microaerophilic stationary phase (MASP) cultures of bovine rbc (Levy and Ristic, 1980). Cultures, at a 10% packed cell volume (PCV), were maintained in 25-cm 2 plastic tissue culture flasks (No. 25100, Coming Glass Works, Coming, NY), at 37°C in humidified air containing 5% CO2. They were given daily changes of medium and were subdivided at intervals of 2--3 days. Basic medium consisted of tissue culture medium 199 with Earle's salts and w i t h o u t glutamine (No. LM-191, Kansas City Biologicals, Lenexa, KS), containing the antibiotics penicillin G (100 U ml-1), streptomycin sulfate (100 #g m1-1) and amphotericin B (0.25 /~g ml-1). (This medium was later modified by the addition of 10 mg ml-1 extra glucose [W.L. G o l f and C.E. Yunker, in preparation] and the substitution of 50/~g ml- 1 gentamycin sulfate for penicillin and streptomycin.) Complete medium (CM) was prepared daffy by mixing refrigerated basic medium with adult bovine serum in a 3:2 proportion and adding 20 mM TES [n-tris ( h y d r o x y m e t h y l ) methyl-2-aminoethane sulfonic acid] (U.S. Biochemical Corp., Cleveland, OH). After adjustment to pH 7.0 with 1 N NaOH, CM was sterilized by pressure filtration and used to replace spent medium over the layer of settled rbc in culture flasks. On days when cultures were to be subdivided, existing medium was removed and a thin film of rbc was made for Giemsa-staining. Medium was replaced and parasitemias were estimated in order to determine splitratio (usually 1:1 to 1:4). Depending on this ratio, an aliquot of infected culture material was introduced into a new flask containing fresh CM and normal Brbc (e.g., if a 1:3 split was desired 5 ml of culture suspension was transferred to a flask containing 12 ml CM and 3 ml of a normal adult Brbc suspension). Normal Brbc were obtained from defibrinated whole blood of adult cattle and were washed three times in phosphate buffered saline (PBS). After each wash, cells were centrifuged at 1000 × g for 10 min, and b u f f y coats and supernatants were removed. A final wash in Puck's saline plus added glucose (PSG+; Palmer et al., 1982), was followed by centrifugation, as above, and the Brbc pellet was dispersed in PSG+ to make a 50% suspension. This was stored at 4°C for n o t longer than 2 weeks and cells were oxygenated daily by inverting the tube.
Parasites cultured in 96-well tissue culture plates (No. 3596, Costar, Cambridge, MA), were adapted to grow in the presence of equine serum by gradually substituting the latter for a portion of bovine serum in culture medium (Levy and Ristic, 1983). At intervals, part of the bovine serum was replaced with equine serum until the serum c o m p o n e n t of the medium was entirely of equine origin. Fluid levels of culture plates were adjusted to give a depth of 6 mm (200 ~1). Later, cultures were transferred to a gas-regulating incubator (Heraeus Type B-5060 EC/O2; Rupp and Bowman Co., Farmington Hills, MI), set to supply 2% 02, 5% CO2 and 93% N2, whereupon fluid depths were adjusted to 4 mm (130 pl) (Rodriguez et al., 1983). Cultures were later expanded into 25-cm 2 Coming tissue culture flasks, held in this same atmosphere, and fluid levels were adjusted to give a depth of 4 mm (15 ml). Parasites were frozen in 10% dimethyl sulfoxide or according to the m e t h o d of Palmer et al. (1982). In the latter case infected Brbc were separated from merozoites, each fraction was suspended in PSG+ containing 10% polyvinylpyrrolidone, and suspensions were frozen at a cooling rate of 20°C min-1. Experimental and donor animals
Four 6--9-month-old, splenectomized, dairy-type, steer calves were inoculated intravenously (IV) with culture material containing approximately 0.75 × 109 intraerythrocytic parasites. These four calves were similarly inoculated 44 days (3 calves) and 78 days (1 calf) after culture inoculation, with approximately 2.2 × 107 virulent B. bovis intraerythrocytic parasites of Mexican origin, obtained from frozen stabilates. Six comparable splenectomized calves were similarly inoculated with virulent B. bovis stabilate. A 1:10 dilution of the stabilate challenge material was made in PBS containing 10% normal bovine serum, kept cold, and inoculated within 30 min of thawing. Inoculated calves were bled, rectal temperatures were recorded and clinical observations were made daily. Percent parasitized erythrocytes (PPE) was estimated from Giemsa-stained smears and PCV's were determined. The donors for normal Brbc and serum were mature beef cattle of mixed breeding. Normal equine serum was obtained from Shetland and Welch cross ponies. In addition, pooled lots of horse serum from controlled herds were obtained from commercial suppliers. RESULTS Donor serum screening
Samples of horse serum were tested for their effect on B. bovis in vitro. The equine serum was used to replace one-fourth of the bovine c o m p o n e n t
10
of CM. Only one lot, a commercially available 'defined' horse serum (HyClone Laboratories, Logan, UT), supported growth of the parasite at levels seen in the equine serum-free control. This serum was used in adaptation experiments.
Adaptation o f B. bovis to growth in equine serum Parasites survived for various lengths of time after inclusion of equine serum in medium. In two trials where cultures were initiated in CM containing 40% equine serum and no bovine serum, the parasites survived, with evidence of multiplication, for 17 and 22 days, while undergoing three and four subdivisions, respectively. However, despite this, peak parasitemias gradually declined during these times and cultures were eventually lost. In cultures initiated in medium containing equine and bovine sera in various proportions (e.g., 1:3, 1:1), parasites grew w i t h o u t apparent detriment. The relative proportion of equine c o m p o n e n t could be increased at intervals of 3--4 weeks to 87.5% of total serum w i t h o u t a notable decrease in parasitemia. However, when medium containing solely equine serum was then offered, parasitemias invariably declined within days. At these times, the decline could be reversed if the percentage of bovine serum was increased. One culture initiated in medium containing equal quantities of equine and bovine serum was transferred, after 21 days and seven subdivisions, to CM containing equine and bovine sera in a 3:1 ratio. Parasites continued to multiply for the next 60 days, while cultures underwent 41 subdivisions. At the succeeding passage, parasite levels failed to rebound and parasitemias remained at low levels (< 0.1%) for 9 days. Thereafter, parasites resumed their typical growth and, on t w o occasions, 117 and 132 days after initiation, respectively, were successfully transferred into medium containing serum solely of equine origin. To date, one of these adapted strains, No. 429, has been in 40% equine serum for 307 days while undergoing 127 TABLE I Responses of splenectomized calves to infection with attenuated (429) and virulent (153) strains of Babesia boris
Group
Maximum temp. (° C)
Persistence o f fever (days)
Lowest PCV
% PCV depression
3.5 + 1.0
41.3 + 0.4
5.8 -+ 2.2
17.2 +- 5.5
49.0 + 15.0
Six calves inoc. with 153 str.
10.0 ± 0.8
41.7 -~ 0.1
5.3 ± 2.4
20.0 ± 8.0
33.0 ± 19.0
Significance
P < 0.01
P < 0.05
NS
NS
NS
Four calves inoc. with 4 2 9 str.
1st day o f fever
11
subdivisions. Peak parasitemias during this time ranged from 1.2% to 5.9% {average = 2.98 + 0.83, n = 100). Inoculation o f calves
Both the attenuated, culture-derived strain and the virulent strain of B. boris were infective for splenectomized calves, causing febrile reactions, parasitemias and lowered PCV's. Average results for these and other observations are given in Table I. Post-inoculation day of onset of fever and highest temperature attained were significantly different between vaccinated and control groups (P <: 0.01 and <~ 0.05, respectively), but persistence of fever, lowest PCV attained, percent drop in PCV and highest parasitemia attained between groups showed no significant differences. However, marked differences were seen in the clinical response of these animals to the infection. None of the four animals vaccinated with the 429 strain showed central nervous system (CNS) signs and all four survived, whereas, of six control calves inoculated with the virulent stabilate, five developed CNS signs and died. The continued virulence of the parent strain (KB) of B. boris, was confirmed by L. Chieves, of the National Animal Disease Center, Ames, IA. On June 6, 1985 a splenectomized calf was inoculated IV with approximately 1 X 109 intraerythrocytic parasites in 15 ml culture medium. It became febrile and oligocythemic, and exhibited a high percentage (22%) of parasitized erythrocytes. The animal developed CNS signs and became moribund shortly thereafter. It was killed in extremis for antigen production. The calves vaccinated with Strain 429 were later challenged with virulent B. bovis. Forty-four days after exposure to the avirulent strain, each of three calves was given an IV injection of 2 ml of a 1:10 dilution of B. bovis stabilate (Strain 153), which contained 2.2 X 107 organisms at the time of
% Maximum parasitemia
CNS signs
Deaths
Persistence of anemia
Response to challenge
<0.1
0/4
0/4
10.0 +- 6.0
0/4
< 0 . 4 +- 0.6
516
5/6
NA
NA
NS
.
.
.
.
.
12
freezing. The remaining vaccinee was given an identical dosage of the same stabilate 78 days after exposure. All survived this challenge while exhibiting no clinical or hematological or clinical evidence of infection. Parasites from Strain 429 cultures were successfully revived from frozen storage u p o n cattle inoculation. Stabilates prepared from calves which received the culture material retained their attenuated characteristics. However, these stabiliates could n o t be re-established in MASP cultures. DISCUSSION The development of cell culture systems for the propagation of Babesia bovis (Erp et al., 1978, 1980; Levy and Ristic, 1980) has greatly facilitated laboratory studies of babesiosis, including the production of experimental immunogens. The latter include soluble and corpuscular antigens (Gravely et al., 1979; James et al., 1981), and an attenuated strain of B. bovis selected by means of cell cloning (G.M. Buening, personal communication, 1983). Reduction in virulence of B. bovis had previously been demonstrated in vivo through rapid, serial syringe passage in splenectomized calves (Callow, 1971; Callow et al., 1979) and by irradiation of parasitemic blood (Mahoney et al., 1973). We have employed the e r y t h r o c y t e culture system for B. bovis to produce and maintain, for an extended period of time, an equine serum-adapted strain that is both avirulent and highly protective for splenectomized calves. Our results are similar to those of Weiss and DeGuisti (1966) who f o u n d that continued passage of Plasmodium berghei in cultures containing hamster serum yielded an attenuated strain that protected mice against exposure to a virulent strain. Babesia bovis had previously been adapted to grow in vitro in the presence of exotic blood components, yielding soluble antigens free of bovine c o m p o n e n t s (Ristic et al., 1981). The equine serum-adapted strain reported here warrants consideration as a candidate vaccine strain. Additional tests of this strain, in splenectomized as well as intact animals, are in progress, as are efforts to devise a more satisfactory system for the recovery of the parasites from frozen storage. ACKNOWLEDGEMENTS We thank Janis Woolbright and David Jones for excellent technical assistance. The help of our colleagues, G.M. Buening, T o y o h i k o Yoshihara and Lucious Chieves, in various aspects of this study is also gratefully acknowledged.
13 REFERENCES Callow, L.L., 1971. The control of babesiosis with a highly infective, attenuated vaccine. World Vet. Congr., Proc., 1 : 357--360. Callow, L.L., MeUors, 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. Erp, E., Gravely, S.M., Smith, R.D., Ristic, M., Osorno, B.M. and Carson, C.A., 1978. Growth of Babesia boris in bovine erythrocyte cultures. Am. J. Trop. Med. Hyg. 27: 1061--1064. Erp., E., Smith, R.D., Ristic, M. and Osorno, B.M., 1980. Continuous in vitro cultivation ofBabesia bovis. Am. J. Vet. Res., 41: 1141--1142. Gravely, S.M., Smith, R.D., Erp, E., Canto, G.T., Aikawa, M., Osorno, B.M. and Ristic, M., 1979. Bovine babesiosis: partial characterization of blood culture-derived Babesia boris. Int. J. Parasitol., 9: 591--598. James, M.A., Levy, M.G. and Ristic, M., 1981. Isolation and partial characterization of culture-derived soluble Babesia bovis antigens. Infect. Immun., 31: 358--361. Levy, M.G. and Ritic, M., 1980. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science, 207 : 1218--1220. Mahoney, D.F., Wright, I.G. and Keterer, P.J., 1973. Babesia argentina: the infectivity and immunogenicity of irradiated blood parasites for splenectomized calves. Int. J. Parasitol., 3 : 209--217. Palmer, D.A., Buening, G.M. and Carson, C.A., 1982. Cryopreservation of Babesia bovis for cultivation. Parasitology, 84 : 567--572. Ristic, M., Smith, R.D. and Kahoma, I., 1981. Characterization of Babesia antigens derived from cell cultures and ticks. In: M. Ristic and J.P. Kreier (Editors), Babesiosis. Academic Press, New York, pp. 337--380. Rodriguez, S.D., Buening, G.M., Green, T.J. and Carson, C.A., 1983. Cloning of Babesia bovis by in vitro cultivation. Infect. Immun., 42: 15--18. Weiss, M.L. and DeGuisti, D.L., 1966. Active immunization against Plasmodium berghei malaria in mice. Am. J. Trop. Med. Hyg., 15: 472--482.