- Email: [email protected]
Babesia bovis: Culture of Laboratory-Adapted Parasite Lines and Clinical Isolates in a Chemically Defined Medium
Experimental Parasitology 99, 168–174 (2001) doi:10.1006/expr.2001.4655, available online at http://www.idealibrary.com on
Babesia bovis: Culture of Laboratory-Adapted Parasite Lines and Clinical Isolates in a Chemically Defined Medium
Louise A. Jackson, Susan J. Waldron, Heidi M. Weier, Claire L. Nicoll,* and Brian M. Cooke*,1 Tick Fever Research Centre, Queensland Department of Primary Industries, 280 Grindle Road, Wacol, Queensland 4076, Australia; and *The Department of Microbiology, Monash University, P.O. Box 53, Victoria 3800, Australia
Jackson, L. A., Waldron, S. J., Weier, H. M., Nicoll, C. L., and Cooke, B. M. 2001. Babesia bovis: Culture of laboratory-adapted parasite lines and clinical isolates in a chemically defined medium. Experimental Parasitology 99, 168–174. Babesiosis caused by Babesia spp. is a disease of both veterinary and human importance. Here, we describe a method to continuously culture laboratory lines and field isolates of Babesia bovis in vitro in a chemically defined medium using ALBUMAX II as an alternative to bovine serum. Further, we have successfully cultured parasite isolates directly from cattle that failed to grow in traditional serum-containing medium. Variation of atmospheric gas composition and culture volumes to determine optimal growth conditions revealed that a 600-l culture in an atmosphere comprising 5% O2, 5% CO2, and 90% N2 achieved a significantly higher percentage of parasitized red blood cells than any other combination tested. The process could be scaled up to reliably produce large volumes of parasites. Supplementation of the culture medium with hypoxanthine further improved parasite growth. B. bovis cultured in this way could be the basis of an alternative, safer vaccine and a reliable source of parasites and exoantigens for parasitological research.
INTRODUCTION
Babesiosis, caused by pathogenic species of the intraerythrocytic protozoan Babesia, is an acute and often fatal tickborne disease of domestic and wild animals in tropical and subtropical regions throughout the world (see Homer et al. 2000 for recent review). Babesia spp. can infect humans, although these cases appear to be predominantly caused by infection with the rodent parasite Babesia microti or the bovine parasite Babesia divergens. There have, however, been reports, albeit less well documented, of fatal infections in humans believed to have been caused by Babesia bovis (Calvo de Mora et al. 1985; Suarez Hernandez et al. 1997). Babesiosis in cattle is of paramount importance to the beef and dairy industry throughout North and South America, Africa, Asia, and Australia with an estimated 500 million animals at risk of infection worldwide and a mortality rate of up to 50% (McCosker 1981). In Australia, babesiosis caused by B. bovis is the most important tickborne disease in cattle and is transmitted by the cattle tick Boophilus microplus. A live attenuated vaccine for use in cattle has been available since 1966 (Callow and Mellors 1966) and a single inoculation generally affords long-lasting protective immunity. The vaccine, however, is prepared from infected blood and its use carries the risk of accidental transmission of other, perhaps as yet unidentified, pathogenic organisms (Rogers et al. 1988). Live attenuated strains of B. bovis grown in a chemically defined medium in vitro could be the
䉷 2001 Academic Press (USA)
Index Descriptors and Abbreviations: Babesia bovis; babesiosis; in vitro culture; bovine serum-supplemented medium (BS-medium); ALBUMAX II-supplemented medium (A-medium); A-medium supplemented with hypoxanthine (AH-medium); microaerophilus stationary phase (MASP); Vega y Martinez solution (VYMS); N-Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES); N-2-hydroxyethylpiperazine-N ⬘-2-ethanesulfonic acid (Hepes); red blood cell (RBC); parasitized RBC (PRBC).
To whom correspondence should be addressed. Fax: IDD ⫹ (613) 9905 4811. E-mail: [email protected]. 1
168
0014-4894/01 $35.00 䉷 2001 Elsevier Science (USA) All rights reserved.
CULTURE OF Babesia bovis IN VITRO
basis of an alternative vaccine and a source of parasites and exoantigens for immunological and biochemical studies. Traditionally, B. bovis has been grown in a microaerophilus stationary phase (MASP) culture in medium containing 40% bovine serum (Levy and Ristic 1980). In our laboratory, however, some strains of B. bovis have been difficult to cultivate using established MASP culture techniques. Grande et al. (1997) have previously demonstrated that B. divergens could be grown in a “serum-free” medium supplemented with a lipid-enriched bovine serum albumin preparation (ALBUMAX I; Life Technologies, Grand Island, NY), and high percentages of parasitized red blood cells (PRBCs), approximating those obtained in standard serum-containing medium, could be achieved. In addition, Cranmer et al. (1997) successfully used ALBUMAX II (Life Technologies) as an alternative to human serum for culturing the related apicomplexan parasite Plasmodium falciparum in vitro. The aim of the present study was to initiate and culture B. bovis in vitro using ALBUMAX II under conditions similar to those previously described for B. divergens and P. falciparum (Grande et al. 1997; Cranmer et al. 1997). Additionally, following our new method, we have examined the effect of hypoxanthine supplementation, oxygen and carbon dioxide concentration, and culture volume on the growth of B. bovis in an attempt to maximize parasite growth. Furthermore, we show that our method is capable of producing sufficiently large volumes of parasites continuously, reliably, and economically, which are required for laboratory-based studies to address outstanding questions pertinent in modern day parasitological research.
MATERIALS AND METHODS
Parasites. The three strains of B. bovis used to infect calves and then subsequently to initiate in vitro cultures were W (Bock et al. 1992), Dixie (Bock et al. 1995), and T (Bock et al. 1992). Stocks of B. bovis Peachester strain that had been previously cryopreserved either from blood taken directly from infected cattle or from previous MASP cultures grown in medium containing bovine serum were also used to initiate fresh MASP cultures. In addition, three strains of B. bovis (W, Anderson, and K strains) undergoing continuous MASP culture in bovine serum-containing medium were also used. Media. BS-medium consisted of Medium M199 (Sigma Chemical Co., St. Louis, MO) buffered with 20 mM N-Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) (Sigma) and supplemented with 40% (v/v) normal bovine serum. A-medium comprised RPMI 1640 medium (Life Technologies) buffered with 25 mM Hepes (Sigma) and 25 mM NaHCO3 supplemented with 1% (w/v) ALBUMAX II. Both media were further supplemented with 100 IU/ml penicillin, 100 g/ ml streptomycin, and 0.25 g/ml amphotericin B. Hypoxanthine
169 (Sigma) was added to A-medium to a final concentration of 200 M where indicated. Animals. One-week-old calves (Bos taurus) were purchased from an area of southeast Queensland free of endemic babesiosis and maintained under tickfree conditions. Calves were splenectomized and monitored for evidence of hemoparasite infections twice weekly by examination of Giemsa-stained blood smears. Whole blood containing B. bovis, either fresh or cryopreserved, was inoculated intravenously into calves. Four days after inoculation, when the percentage parasitemia exceeded 1%, 50 ml of venous blood was collected into heparin anticoagulant and used to initiate in vitro cultures. Initiation of MASP cultures in A-medium. Thawed cryostabilate or infected blood was added to Vega y Martinez solution (VYMS) (Vega et al. 1985) and centrifuged at 1000g for 10 min. After centrifugation, 0.5 ml of the red blood cell (RBC) pellet was added to 4.5 ml of BS-medium and to 4.5 ml of A-medium containing 5% (v/v) fresh, uninfected bovine RBCs, and 1.2 ml was added to 4 wells of a 24well tissue culture plate. Plates were incubated at 37⬚C in an atmosphere of 2% O2, 5% CO2, and 93% N2. Medium overlying the RBCs was changed on each of the 2 days following initiation of the cultures. On the third day, wells were subcultured (1:1) using a 5% suspension of normal bovine RBCs in either BS- or A-medium. The percentage of RBCs that were parasitized was determined for each of the wells from Giemsa-stained smears by counting 1000 RBCs. Extraerythrocytic parasites or parasites inside RBC ghosts were not included in the count. Culture media was changed daily and smears were prepared approximately every 3 days when parasites were subcultured. Effect of hypoxanthine supplementation to A-medium. A-medium was prepared with and without the addition of 200 M hypoxanthine (Sigma). MASP cultures already adapted to A-medium without hypoxanthine (W, Dixie, and T strains) were randomly allocated to wells of a 24-well culture plate and an equal volume of RBCs in A-medium with or without hypoxanthine was added. The total volume of culture in each well was 1.2 ml and the percentage of PRBCs was determined as described above. Effect of atmospheric gas composition and culture volume on the growth of B. bovis. A MASP culture of B. bovis W strain, already growing in A-medium containing 200 M hypoxanthine (AHmedium), was added to randomly allocated wells of a 24-well culture plate. An equal volume of RBCs in AH-medium was added to all wells. The total volume of each well was 1.2, 0.9, or 0.6 ml and each volume was tested in quadruplicate in each plate. Four 24-well plates were set up with the different well volumes so that four different gas mixtures could be tested in combination and optimum growth conditions could be determined. The plates were simultaneously incubated in individual gassed chambers within one incubator but each plate was exposed to the different gaseous environments by flushing premixed gas mixtures through each of the chambers. The gas mixtures tested were 2% O2, 5% CO2, 93% N2; 3.5% O2, 5% CO2, 91.5% N2; 5% O2, 5% CO2, 90% N2; and 2% O2, 7.5% CO2, 90.5% N2. The percentage of RBCs that were parasitized was determined by staining parasites with Hydroethidine (Polysciences Inc., Warrington, PA) and subsequent analysis by flow cytometry (Wyatt et al. 1991). Large-scale continuous culture of B. bovis in vitro. Laboratorybased studies of the biology of Babesia often require a reliable and continuous supply of larger volumes of parasites than can easily be obtained using 24-well culture plates. Parasites (W, Anderson, and K strains) were maintained in continuous culture in 25-, 75-, or 150-cm2 tissue culture flasks in AH-medium. The total volumes of RBCs for each of the different size flasks were 0.35, 1.0, and 2.0 ml for 25-,
170
JACKSON ET AL.
75-, and 150-cm2 flasks, respectively, plus 6, 20, or 50 ml of AHmedium. Flasks were gassed for 1 min with 5% O2, 5% CO2, and 90% N2 and then immediately closed with a gas-tight cap and incubated at 37⬚C. Medium was changed daily and the percentage of PRBCs determined on Giemsa-stained smears. Uninfected RBCs were then added to dilute the number of PRBCs to approximately 50% of its value or lower to prevent the number of PRBCs from rising to a high level that may have been inhibitory for continuous log-phase growth. Statistical analysis. Data are expressed as the mean percentage of PRBCs ⫾ standard error. The mean percentage of PRBCs in cultures growing in either A-medium or BS-medium were compared using the Student’s nonpaired t test. The effect of addition of hypoxanthine, different atmospheric gas mixtures, and different well volumes were tested by analysis of variance. Where significant differences were detected, a test of least significant difference was used to compare individual groups. Significance was defined as P ⬍ 0.05.
RESULTS
Initiation of MASP cultures from infected blood in Amedium. MASP cultures were successfully initiated in both BS- and A-medium from calf blood infected with the W strain of B. bovis (Fig. 1a). During the 42-day culture period, cultures maintained average percentages of PRBCs of 3.6 and 3.4% in BS- and A-medium, respectively. In contrast, however, when placed in MASP culture in A-medium, calf blood infected with either the Dixie or the T strain of B. bovis maintained an average (over all days from 3 to 48) percentage of PRBCs of 3.9% (for Dixie; Fig. 1b) and 4.8% (for T strain; data not shown) but both failed to grow in BSmedium (Fig. 1b; Dixie strain). Cryostabilates of B. bovis Peachester strain, prepared either from infected blood or from previously cultured parasites, were used to successfully initiate MASP cultures in A-medium, and average (over all days from 3 to 42) percentages of PRBCs of 3.1 and 3.2%, respectively, were maintained during culture (Fig. 2). However, parasites from infected blood that had been cryopreserved did not grow in BS-medium. Cryostabilates prepared from the previously cultured parasites were successfully grown in BS-medium and maintained an average (over all days) percentage of PRBCs of 1.9%. Effect of the addition of hypoxanthine to A-medium. Addition of 200 M hypoxanthine to A-medium significantly improved the growth of B. bovis in vitro. Figure 3 shows the growth of a representative B. bovis strain (Dixie) in medium with and without the addition of hypoxanthine. Over a 15-day culture period, the mean percentage of PRBCs was significantly (P ⬍ 0.05) greater in the presence (3.8%) than in the absence (3.2%) of hypoxanthine.
FIG. 1. Microaerophilus stationary phase (MASP) culture of Babesia bovis (a) W strain or (b) Dixie strain initiated from blood from an infected calf in either BS-medium (●-●) or A-medium (䡩-䡩). The percentage of RBCs that were parasitized was counted in Giemsastained smears made from cultures on the days indicated and is expressed as the mean ⫾ standard error. Wells were then subcultured (1:1) using a 5% suspension of normal bovine RBCs to dilute the percentage of PRBCs to 50% of their level and the plates returned to culture.
Effect of different atmospheric gas mixtures and well volumes on B. bovis growth in A-medium. Significantly higher percentages of PRBCs were obtained when cultures were incubated in an atmosphere of 5% O2, 5% CO2, 90% N2 than in any of the other atmospheric gas mixtures tested (P ⬍ 0.05) (Fig. 4a). There were no significant differences between the percentage of PRBCs obtained in any of the other gas mixtures. A culture volume of 600 l produced a higher percentage of PRBCs than either 900 or 1200 l
CULTURE OF Babesia bovis IN VITRO
171
FIG. 2. Microaerophilus stationary phase (MASP) culture of Babesia bovis Peachester strain initiated either from a culture cryostabilate in either BS-medium (●-●) or A-medium (䡩-䡩) or from an infected blood cryostabilate in either BS-medium (䡲-䡲) or A-medium (▫-▫). The percentages of RBCs that were parasitized were counted in Giemsastained smears made from cultures on the days indicated and are expressed as the mean ⫾ standard error. Wells were subcultured as described in Fig. 1 and then returned to culture.
(P ⬍ 0.05) (Fig. 4b). The combination of 600 l culture volume incubated in an atmosphere of 5% O2, 5% CO2, 90% N2 achieved a significantly higher percentage of PRBCs than any other combination of culture volume and atmospheric gas mixture (P ⬍ 0.05). FIG. 4. Microaerophilus stationary phase (MASP) culture of Babesia bovis W strain cultured in A-medium containing 200 M hypoxanthine. Parasites were (a) exposed to different gaseous environments, 2% O2, 5% CO2, 93% N2; 3.5% O2, 5% CO2, 91.5% N2; 5% O2, 5% CO2, 90% N2; 2% O2, 7.5% CO2, 90.5% N2, or (b) cultured with different volumes in the wells. The percentage of RBCs that were parasitized was determined by staining parasites with Hydroethidine and analysis by flow cytometry. Data are expressed as the mean ⫾ standard error for quadruplicate wells at each of the different gas mixtures tested. *Significant differences with 95% confidence.
FIG. 3. Microaerophilus stationary phase (MASP) culture of Babesia bovis Dixie strain cultured in A-medium with (●-●) or without (䡩-䡩) the addition of 200 M hypoxanthine. The percentages of RBCs that were parasitized were counted in Giemsa-stained smears made from cultures on the days indicated and are expressed as the mean ⫾ standard error. Wells were subcultured as described in Fig. 1 and then returned to culture.
Large-scale continuous culture of B. bovis in vitro. Parasites (W, Anderson, and K strains) were continuously propagated in 25-, 75-, and 150-cm2 tissue culture flasks in AHmedium gassed with 5% O2, 5% CO2, 90% N2 for periods of up to 5 months. The growth curve for a typical culture of K strain parasites, over a period of 6 consecutive days, is shown in Fig. 5. The purpose of the experiment described here was only to demonstrate that parasites could be cultured continuously in AH-medium using the flask method and
172
FIG. 5. Large-scale culture of Babesia bovis (K strain) in AHmedium over a period of 6 consecutive days. The percentage of RBCs that were parasitized was counted in Giemsa-stained smears made from cultures diluted daily to approximately 50% of its level by addition of fresh bovine RBCs. Three individual growth curves are shown representing each of three identical cultures that were grown in three separate 75-cm2 tissue culture flasks simultaneously during the 6 day period.
thus culture was stopped after 6 days with a percentage of PRBCs in excess of 12%. We have, however, maintained more than three parasite strains in continuous culture for periods of up to 5 months using this method and can, if required, achieve percentages of PRBCs of 20% or greater. It is also noteworthy in Fig. 5 that, after accounting for daily dilution of the percentage of PRBCs with fresh RBCs, the percentage of PRBCs in the culture doubled every approximately 17 to 20 h. This is consistent with previous estimates of the growth rate of B. bovis cultured in traditional serum-containing medium in vitro (Erp et al. 1978; Levy et al. 1981).
DISCUSSION B. bovis was successfully cultured continuously in Amedium from infected blood, from cryostabilates of either infected blood or cultured parasites, and from continuous in vitro parasite cultures growing in “traditional” BS-medium. In contrast, we have identified a number of parasite lines, isolated directly from infected blood, that failed to grow in BS-medium but which adapted immediately to in vitro culture when A-medium was used. In fact, we have been successfully culturing many different lines of B. bovis during the past 3 years in our laboratories and have not yet found
JACKSON ET AL.
a line that will not grow in A-medium. Furthermore, for B. bovis lines W, T, K, and Anderson that have been cultured continuously in this way, we have not identified any morphological differences (by examination of Giemsa-stained smears) from the same parasite lines grown in BS-medium. Furthermore, other pathophysiologically important biological properties of these parasites, such as their ability to cytoadhere to bovine vascular endothelial cells, are also maintained (B. M. Cooke, unpublished). Although addition of hypoxanthine was not an absolute requirement for parasite growth in either BS- or A-medium, its addition significantly increased parasite growth. We, therefore, recommend the addition of hypoxanthine to Amedium to maximize parasite yield. This is in direct contrast to P. falciparum where additional exogenous hypoxanthine has been shown to be an absolute requirement for parasite growth in RPMI 1640 supplemented with ALBUMAX II (Cranmer et al. 1997). Medium 199 contains 0.1 M hypoxanthine in its standard formulation while RPMI 1640 does not contain any hypoxanthine according to the manufacturers’ specifications. Goff and Yunker (1986) have previously shown that addition of extra hypoxanthine to Medium 199 did not appear to enhance growth of B. bovis. It is interesting to note that in this study, exogenous hypoxanthine was not an absolute requirement for growth of B. bovis when cultured in RPMI1 640 containing ALBUMAX II, whereas Irvin et al. (1978) reported that exogenous hypoxanthine was critical for the growth of Babesia spp. Traditionally, Babesia spp. parasites have largely been cultured in Medium 199 buffered with TES. Here, we have used the ALBUMAX-supplemented media previously described by Cranmer et al. (1997) for growth of malaria parasites which, in contrast to traditional babesia culture media, comprises RPMI 1640 buffered with Hepes. Several lines of evidence suggest that it is unlikely that the change from Medium 199 to Hepes-buffered RPMI 1640 affected our results obtained in the present study. First, a previous comparison of the growth of B. bovis in Medium 199 and RPMI 1640 showed no significant differences between these two culture media (Erp et al. 1980), albeit in suspension rather than MASP culture. Second, the addition of 25 mM Hepes showed no detrimental affect on parasite growth (Erp et al. 1980). Third, although not systematically tested here, we have in fact noted that RPMI 1640 or Medium 199 can be used interchangeably for the growth medium without any notable differences in parasite growth. The combination of 600-l well volume in a 24-well plate incubated in an atmosphere of 5% O2, 5% CO2, 90% N2 achieved a significantly higher percentage of PRBCs than any other combination of well volume and atmospheric gas
173
CULTURE OF Babesia bovis IN VITRO
mixture. It would be of interest to determine whether gas mixtures containing higher concentrations of O2 would further increase the percentage of PRBCs obtained. A previous study has in fact shown that a gas mixture consisting of 20% O2, 5% CO2, 75% N2 gave optimal growth for B. bovis (Erp et al. 1980). However, in that study B. bovis was cultivated using bovine-serum medium in a suspension rather than a stationary culture. Levy and Ristic (1980) found that a depth of approximately 0.6 cm was optimal for B. bovis growth in traditional BS-medium while a depth of 0.16 cm could not sustain continuous culture albeit under high O2 conditions (5% CO2 in air). Our results obtained here demonstrate an inhibitory effect on parasite growth for higher volumes of medium in the wells. When culture vessels different from those described here are used, it is, therefore, valuable to optimize the volume of media above the cultures to optimize parasite yield. It is perhaps not surprising that excessively large volumes of media above the cultures, resulting in increased hydrostatic pressure or compression on the RBCs, might inhibit parasite proliferation by inhibiting merozoite release or restricting their spread to neighboring RBCs. Furthermore, previous studies have demonstrated profound effects of hydrostatic pressure on the proliferation or morphological characteristics of cells, such as vascular endothelial cells including altered release of growth factors and reorganization of the normal cell cytoskeleton (Acevedo et al. 1993; Salwen et al. 1998; Schwartz et al. 1999). The direct effect of hydrostatic pressure on merozoites and the biochemical responses transduced by such mechanical stimuli are not known, but are clearly an area worthy of further investigation. Traditionally, MASP cultures of babesia parasites are subcultured every 2–3 days. Here, using our large-scale culture flask method, parasite numbers can reach sufficiently high levels in a period of 24 h that subculture can be performed routinely on a daily basis. This maximizes overall parasite yield and is particularly important when large volumes of parasites are required for production of parasites as a source of immunogens or vaccine material. Finally, at the present time, the only vaccines available for the prevention of babesia infection in cattle are live attenuated forms produced in splenectomized calves. Although still considered relatively effective, their use is associated with varying levels of protection, and the development of a more effective and betterdefined vaccine using either in vitro cultured parasites or recombinant antigens is of utmost importance. An abundant supply of genetically and phenotypically well-characterized parasites cultured in a chemically defined medium, as described here, will be a prerequisite to achieving these goals.
Moreover, until an effective recombinant vaccine is developed, use of attenuated parasites cultured in a defined medium represent a step in the right direction toward avoidance of accidental cotransmission of known (Rogers et al. 1988) or as yet unidentified infectious agents that are a potential hazard associated with the use of the currently available vaccines.
ACKNOWLEDGMENTS The authors thank Katy Williams for technical assistance and Wayne Jorgensen and Bert de Vos for expert advice. This work was supported by the Australian Centre for International Agricultural Research (ACIAR PN AS2 9690). Dr. Cooke is supported by the National Health and Medical Research Council of Australia.
REFERENCES Acevedo, A. D., Bowser, S. S., Gerritsen, M. E., and Bizios, R. 1993. Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor. Journal of Cellular Physiology 157, 603–614. Bock, R. E., de Vos, A. J., Kingston, T. G., Shiels, I. A., and Dalgliesh, R. J. 1992. Investigations of breakdowns in protection provided by living Babesia bovis vaccine. Veterinary Parasitology 43, 45–56. Bock, R. E., de Vos, A. J., Lew, A., Kingston, T. G., and Fraser, I. R. 1995. Studies on failure of T strain live Babesia bovis vaccine. Australian Veterinary Journal 72, 296–300. Callow, L. L., and Mellors, L. T. 1966. A new vaccine for Babesia argentina infection prepared in splenectomised calves. Australian Veterinary Journal 42, 464–465. Calvo de Mora, A., Garcia Castellano, J. M., Herrera, C., and JimenezAlonso, J. 1985. Human babesiosis: Report of a case with fatal outcome. Medicina Clinica 85, 515–516. Cranmer, S. L., Magowan, C., Liang, J., Coppel, R. L., and Cooke, B. M. 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 363–365. Erp, E. E., Gravely, S. M., Smith, R. D., Ristic, M., Osorno, B. M., and Carson, C. A. 1978. Growth of Babesia bovis in bovine erythrocyte cultures. American Journal of Tropical Medicine and Hygiene 27, 1061–1064. Erp, E. E., Smith, R. D., Ristic, M., and Osorno, B. M. 1980. Optimization of the suspension culture method for in vitro cultivation of Babesia bovis. American Journal of Veterinary Research 41, 2059– 2062. Goff, W. L., and Yunker, C. E. 1986. Babesia bovis: Increased percentage of parasitized erythrocytes in cultures and assessment of growth
174 by incorporation of [3H] hypoxanthine. Experimental Parasitology 62, 202–210. Grande, N., Precigout, E., Ancelin, M. L., Moubri, K., Carcy, B., Lemesre, J. L., Vial, H., and Gorenflot, A. 1997. Continuous in vitro culture of Babesia divergens in a serum-free medium. Parasitology 115, 81–89. Homer, M. J., Aguilar-Delfin, I., Telford, S. R., III, Krause, P. J., and Persing, D. H. 2000. Babesiosis. Clinical Microbiology Reviews 3, 451–469. Irvin, A. D., Young, E. R., and Purnell, R. E. 1978. The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology 8, 19–24.
JACKSON ET AL.
Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis. Australasian Veterinary Journal 65, 285–287. Salwen, S. A., Szarowski, D. H., Turner, J. N., and Bizios, R. 1998. Three-dimensional changes of the cytoskeleton of vascular endothelial cells exposed to sustained hydrostatic pressure. Medical & Biological Engineering & Computing 36, 520–527. Schwartz, E. A., Bizios, R., Medow, M. S., and Gerritsen, M. E. 1999. Exposure of human vascular endothelial cells to sustained hydrostatic pressure stimulates proliferation: Involvement of the ␣v integrins. Circulation Research 84, 315–322.
Levy, M. G., and Ristic, M. 1980. Babesia bovis: Continuous cultivation in a microaerophilus stationary phase culture. Science 207, 1218– 1220.
Suarez Hernandez, M., Alonso Castellano, M., Pelaez Martinez, R., Sanchez Perez, B., Bravo Gonzalez, J. R., and Sanchez Sibello, A. 1997. Investigation of Babesia in farm workers and blood donors in the province of Ciego de Avila. Revista Cubana de Medicina Tropical 49, 130–135.
Levy, M. G., Erp, E., and Ristic, M. 1981. Cultivation of babesia. In “Babesiosis” (M. Ristic and J. P. Kreier, Eds.), pp. 207–223. Academic Press, New York.
Vega, C. A., Buening, G. M., Green, T. J., and Carson, C. A. 1985. In vitro cultivation of Babesia bigemina. American Journal of Veterinary Research 46, 416–420.
McCosker, P. J. 1981. The global importance of babesiosis. In “Babesiosis” (M. Ristic and J. P. Kreier, Eds.), pp. 1–24. Academic Press, New York.
Wyatt, C. R., Goff, W., and Davis, W. C. 1991. A flow cytometric method for assessing viability of intraerythrocytic hemoparasites. Journal of Immunological Methods 140, 23–30.
Rogers, R. J., Dimmock, C. K., de Vos, A. J., and Rodwell, B. J. 1988.
Received 30 May 2001; accepted with revision 17 September 2001
Babesia bovis: Culture of Laboratory-Adapted Parasite Lines and Clinical Isolates in a Chemically Defined Medium
Louise A. Jackson, Susan J. Waldron, Heidi M. Weier, Claire L. Nicoll,* and Brian M. Cooke*,1 Tick Fever Research Centre, Queensland Department of Primary Industries, 280 Grindle Road, Wacol, Queensland 4076, Australia; and *The Department of Microbiology, Monash University, P.O. Box 53, Victoria 3800, Australia
Jackson, L. A., Waldron, S. J., Weier, H. M., Nicoll, C. L., and Cooke, B. M. 2001. Babesia bovis: Culture of laboratory-adapted parasite lines and clinical isolates in a chemically defined medium. Experimental Parasitology 99, 168–174. Babesiosis caused by Babesia spp. is a disease of both veterinary and human importance. Here, we describe a method to continuously culture laboratory lines and field isolates of Babesia bovis in vitro in a chemically defined medium using ALBUMAX II as an alternative to bovine serum. Further, we have successfully cultured parasite isolates directly from cattle that failed to grow in traditional serum-containing medium. Variation of atmospheric gas composition and culture volumes to determine optimal growth conditions revealed that a 600-l culture in an atmosphere comprising 5% O2, 5% CO2, and 90% N2 achieved a significantly higher percentage of parasitized red blood cells than any other combination tested. The process could be scaled up to reliably produce large volumes of parasites. Supplementation of the culture medium with hypoxanthine further improved parasite growth. B. bovis cultured in this way could be the basis of an alternative, safer vaccine and a reliable source of parasites and exoantigens for parasitological research.
INTRODUCTION
Babesiosis, caused by pathogenic species of the intraerythrocytic protozoan Babesia, is an acute and often fatal tickborne disease of domestic and wild animals in tropical and subtropical regions throughout the world (see Homer et al. 2000 for recent review). Babesia spp. can infect humans, although these cases appear to be predominantly caused by infection with the rodent parasite Babesia microti or the bovine parasite Babesia divergens. There have, however, been reports, albeit less well documented, of fatal infections in humans believed to have been caused by Babesia bovis (Calvo de Mora et al. 1985; Suarez Hernandez et al. 1997). Babesiosis in cattle is of paramount importance to the beef and dairy industry throughout North and South America, Africa, Asia, and Australia with an estimated 500 million animals at risk of infection worldwide and a mortality rate of up to 50% (McCosker 1981). In Australia, babesiosis caused by B. bovis is the most important tickborne disease in cattle and is transmitted by the cattle tick Boophilus microplus. A live attenuated vaccine for use in cattle has been available since 1966 (Callow and Mellors 1966) and a single inoculation generally affords long-lasting protective immunity. The vaccine, however, is prepared from infected blood and its use carries the risk of accidental transmission of other, perhaps as yet unidentified, pathogenic organisms (Rogers et al. 1988). Live attenuated strains of B. bovis grown in a chemically defined medium in vitro could be the
䉷 2001 Academic Press (USA)
Index Descriptors and Abbreviations: Babesia bovis; babesiosis; in vitro culture; bovine serum-supplemented medium (BS-medium); ALBUMAX II-supplemented medium (A-medium); A-medium supplemented with hypoxanthine (AH-medium); microaerophilus stationary phase (MASP); Vega y Martinez solution (VYMS); N-Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES); N-2-hydroxyethylpiperazine-N ⬘-2-ethanesulfonic acid (Hepes); red blood cell (RBC); parasitized RBC (PRBC).
To whom correspondence should be addressed. Fax: IDD ⫹ (613) 9905 4811. E-mail: [email protected]. 1
168
0014-4894/01 $35.00 䉷 2001 Elsevier Science (USA) All rights reserved.
CULTURE OF Babesia bovis IN VITRO
basis of an alternative vaccine and a source of parasites and exoantigens for immunological and biochemical studies. Traditionally, B. bovis has been grown in a microaerophilus stationary phase (MASP) culture in medium containing 40% bovine serum (Levy and Ristic 1980). In our laboratory, however, some strains of B. bovis have been difficult to cultivate using established MASP culture techniques. Grande et al. (1997) have previously demonstrated that B. divergens could be grown in a “serum-free” medium supplemented with a lipid-enriched bovine serum albumin preparation (ALBUMAX I; Life Technologies, Grand Island, NY), and high percentages of parasitized red blood cells (PRBCs), approximating those obtained in standard serum-containing medium, could be achieved. In addition, Cranmer et al. (1997) successfully used ALBUMAX II (Life Technologies) as an alternative to human serum for culturing the related apicomplexan parasite Plasmodium falciparum in vitro. The aim of the present study was to initiate and culture B. bovis in vitro using ALBUMAX II under conditions similar to those previously described for B. divergens and P. falciparum (Grande et al. 1997; Cranmer et al. 1997). Additionally, following our new method, we have examined the effect of hypoxanthine supplementation, oxygen and carbon dioxide concentration, and culture volume on the growth of B. bovis in an attempt to maximize parasite growth. Furthermore, we show that our method is capable of producing sufficiently large volumes of parasites continuously, reliably, and economically, which are required for laboratory-based studies to address outstanding questions pertinent in modern day parasitological research.
MATERIALS AND METHODS
Parasites. The three strains of B. bovis used to infect calves and then subsequently to initiate in vitro cultures were W (Bock et al. 1992), Dixie (Bock et al. 1995), and T (Bock et al. 1992). Stocks of B. bovis Peachester strain that had been previously cryopreserved either from blood taken directly from infected cattle or from previous MASP cultures grown in medium containing bovine serum were also used to initiate fresh MASP cultures. In addition, three strains of B. bovis (W, Anderson, and K strains) undergoing continuous MASP culture in bovine serum-containing medium were also used. Media. BS-medium consisted of Medium M199 (Sigma Chemical Co., St. Louis, MO) buffered with 20 mM N-Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) (Sigma) and supplemented with 40% (v/v) normal bovine serum. A-medium comprised RPMI 1640 medium (Life Technologies) buffered with 25 mM Hepes (Sigma) and 25 mM NaHCO3 supplemented with 1% (w/v) ALBUMAX II. Both media were further supplemented with 100 IU/ml penicillin, 100 g/ ml streptomycin, and 0.25 g/ml amphotericin B. Hypoxanthine
169 (Sigma) was added to A-medium to a final concentration of 200 M where indicated. Animals. One-week-old calves (Bos taurus) were purchased from an area of southeast Queensland free of endemic babesiosis and maintained under tickfree conditions. Calves were splenectomized and monitored for evidence of hemoparasite infections twice weekly by examination of Giemsa-stained blood smears. Whole blood containing B. bovis, either fresh or cryopreserved, was inoculated intravenously into calves. Four days after inoculation, when the percentage parasitemia exceeded 1%, 50 ml of venous blood was collected into heparin anticoagulant and used to initiate in vitro cultures. Initiation of MASP cultures in A-medium. Thawed cryostabilate or infected blood was added to Vega y Martinez solution (VYMS) (Vega et al. 1985) and centrifuged at 1000g for 10 min. After centrifugation, 0.5 ml of the red blood cell (RBC) pellet was added to 4.5 ml of BS-medium and to 4.5 ml of A-medium containing 5% (v/v) fresh, uninfected bovine RBCs, and 1.2 ml was added to 4 wells of a 24well tissue culture plate. Plates were incubated at 37⬚C in an atmosphere of 2% O2, 5% CO2, and 93% N2. Medium overlying the RBCs was changed on each of the 2 days following initiation of the cultures. On the third day, wells were subcultured (1:1) using a 5% suspension of normal bovine RBCs in either BS- or A-medium. The percentage of RBCs that were parasitized was determined for each of the wells from Giemsa-stained smears by counting 1000 RBCs. Extraerythrocytic parasites or parasites inside RBC ghosts were not included in the count. Culture media was changed daily and smears were prepared approximately every 3 days when parasites were subcultured. Effect of hypoxanthine supplementation to A-medium. A-medium was prepared with and without the addition of 200 M hypoxanthine (Sigma). MASP cultures already adapted to A-medium without hypoxanthine (W, Dixie, and T strains) were randomly allocated to wells of a 24-well culture plate and an equal volume of RBCs in A-medium with or without hypoxanthine was added. The total volume of culture in each well was 1.2 ml and the percentage of PRBCs was determined as described above. Effect of atmospheric gas composition and culture volume on the growth of B. bovis. A MASP culture of B. bovis W strain, already growing in A-medium containing 200 M hypoxanthine (AHmedium), was added to randomly allocated wells of a 24-well culture plate. An equal volume of RBCs in AH-medium was added to all wells. The total volume of each well was 1.2, 0.9, or 0.6 ml and each volume was tested in quadruplicate in each plate. Four 24-well plates were set up with the different well volumes so that four different gas mixtures could be tested in combination and optimum growth conditions could be determined. The plates were simultaneously incubated in individual gassed chambers within one incubator but each plate was exposed to the different gaseous environments by flushing premixed gas mixtures through each of the chambers. The gas mixtures tested were 2% O2, 5% CO2, 93% N2; 3.5% O2, 5% CO2, 91.5% N2; 5% O2, 5% CO2, 90% N2; and 2% O2, 7.5% CO2, 90.5% N2. The percentage of RBCs that were parasitized was determined by staining parasites with Hydroethidine (Polysciences Inc., Warrington, PA) and subsequent analysis by flow cytometry (Wyatt et al. 1991). Large-scale continuous culture of B. bovis in vitro. Laboratorybased studies of the biology of Babesia often require a reliable and continuous supply of larger volumes of parasites than can easily be obtained using 24-well culture plates. Parasites (W, Anderson, and K strains) were maintained in continuous culture in 25-, 75-, or 150-cm2 tissue culture flasks in AH-medium. The total volumes of RBCs for each of the different size flasks were 0.35, 1.0, and 2.0 ml for 25-,
170
JACKSON ET AL.
75-, and 150-cm2 flasks, respectively, plus 6, 20, or 50 ml of AHmedium. Flasks were gassed for 1 min with 5% O2, 5% CO2, and 90% N2 and then immediately closed with a gas-tight cap and incubated at 37⬚C. Medium was changed daily and the percentage of PRBCs determined on Giemsa-stained smears. Uninfected RBCs were then added to dilute the number of PRBCs to approximately 50% of its value or lower to prevent the number of PRBCs from rising to a high level that may have been inhibitory for continuous log-phase growth. Statistical analysis. Data are expressed as the mean percentage of PRBCs ⫾ standard error. The mean percentage of PRBCs in cultures growing in either A-medium or BS-medium were compared using the Student’s nonpaired t test. The effect of addition of hypoxanthine, different atmospheric gas mixtures, and different well volumes were tested by analysis of variance. Where significant differences were detected, a test of least significant difference was used to compare individual groups. Significance was defined as P ⬍ 0.05.
RESULTS
Initiation of MASP cultures from infected blood in Amedium. MASP cultures were successfully initiated in both BS- and A-medium from calf blood infected with the W strain of B. bovis (Fig. 1a). During the 42-day culture period, cultures maintained average percentages of PRBCs of 3.6 and 3.4% in BS- and A-medium, respectively. In contrast, however, when placed in MASP culture in A-medium, calf blood infected with either the Dixie or the T strain of B. bovis maintained an average (over all days from 3 to 48) percentage of PRBCs of 3.9% (for Dixie; Fig. 1b) and 4.8% (for T strain; data not shown) but both failed to grow in BSmedium (Fig. 1b; Dixie strain). Cryostabilates of B. bovis Peachester strain, prepared either from infected blood or from previously cultured parasites, were used to successfully initiate MASP cultures in A-medium, and average (over all days from 3 to 42) percentages of PRBCs of 3.1 and 3.2%, respectively, were maintained during culture (Fig. 2). However, parasites from infected blood that had been cryopreserved did not grow in BS-medium. Cryostabilates prepared from the previously cultured parasites were successfully grown in BS-medium and maintained an average (over all days) percentage of PRBCs of 1.9%. Effect of the addition of hypoxanthine to A-medium. Addition of 200 M hypoxanthine to A-medium significantly improved the growth of B. bovis in vitro. Figure 3 shows the growth of a representative B. bovis strain (Dixie) in medium with and without the addition of hypoxanthine. Over a 15-day culture period, the mean percentage of PRBCs was significantly (P ⬍ 0.05) greater in the presence (3.8%) than in the absence (3.2%) of hypoxanthine.
FIG. 1. Microaerophilus stationary phase (MASP) culture of Babesia bovis (a) W strain or (b) Dixie strain initiated from blood from an infected calf in either BS-medium (●-●) or A-medium (䡩-䡩). The percentage of RBCs that were parasitized was counted in Giemsastained smears made from cultures on the days indicated and is expressed as the mean ⫾ standard error. Wells were then subcultured (1:1) using a 5% suspension of normal bovine RBCs to dilute the percentage of PRBCs to 50% of their level and the plates returned to culture.
Effect of different atmospheric gas mixtures and well volumes on B. bovis growth in A-medium. Significantly higher percentages of PRBCs were obtained when cultures were incubated in an atmosphere of 5% O2, 5% CO2, 90% N2 than in any of the other atmospheric gas mixtures tested (P ⬍ 0.05) (Fig. 4a). There were no significant differences between the percentage of PRBCs obtained in any of the other gas mixtures. A culture volume of 600 l produced a higher percentage of PRBCs than either 900 or 1200 l
CULTURE OF Babesia bovis IN VITRO
171
FIG. 2. Microaerophilus stationary phase (MASP) culture of Babesia bovis Peachester strain initiated either from a culture cryostabilate in either BS-medium (●-●) or A-medium (䡩-䡩) or from an infected blood cryostabilate in either BS-medium (䡲-䡲) or A-medium (▫-▫). The percentages of RBCs that were parasitized were counted in Giemsastained smears made from cultures on the days indicated and are expressed as the mean ⫾ standard error. Wells were subcultured as described in Fig. 1 and then returned to culture.
(P ⬍ 0.05) (Fig. 4b). The combination of 600 l culture volume incubated in an atmosphere of 5% O2, 5% CO2, 90% N2 achieved a significantly higher percentage of PRBCs than any other combination of culture volume and atmospheric gas mixture (P ⬍ 0.05). FIG. 4. Microaerophilus stationary phase (MASP) culture of Babesia bovis W strain cultured in A-medium containing 200 M hypoxanthine. Parasites were (a) exposed to different gaseous environments, 2% O2, 5% CO2, 93% N2; 3.5% O2, 5% CO2, 91.5% N2; 5% O2, 5% CO2, 90% N2; 2% O2, 7.5% CO2, 90.5% N2, or (b) cultured with different volumes in the wells. The percentage of RBCs that were parasitized was determined by staining parasites with Hydroethidine and analysis by flow cytometry. Data are expressed as the mean ⫾ standard error for quadruplicate wells at each of the different gas mixtures tested. *Significant differences with 95% confidence.
FIG. 3. Microaerophilus stationary phase (MASP) culture of Babesia bovis Dixie strain cultured in A-medium with (●-●) or without (䡩-䡩) the addition of 200 M hypoxanthine. The percentages of RBCs that were parasitized were counted in Giemsa-stained smears made from cultures on the days indicated and are expressed as the mean ⫾ standard error. Wells were subcultured as described in Fig. 1 and then returned to culture.
Large-scale continuous culture of B. bovis in vitro. Parasites (W, Anderson, and K strains) were continuously propagated in 25-, 75-, and 150-cm2 tissue culture flasks in AHmedium gassed with 5% O2, 5% CO2, 90% N2 for periods of up to 5 months. The growth curve for a typical culture of K strain parasites, over a period of 6 consecutive days, is shown in Fig. 5. The purpose of the experiment described here was only to demonstrate that parasites could be cultured continuously in AH-medium using the flask method and
172
FIG. 5. Large-scale culture of Babesia bovis (K strain) in AHmedium over a period of 6 consecutive days. The percentage of RBCs that were parasitized was counted in Giemsa-stained smears made from cultures diluted daily to approximately 50% of its level by addition of fresh bovine RBCs. Three individual growth curves are shown representing each of three identical cultures that were grown in three separate 75-cm2 tissue culture flasks simultaneously during the 6 day period.
thus culture was stopped after 6 days with a percentage of PRBCs in excess of 12%. We have, however, maintained more than three parasite strains in continuous culture for periods of up to 5 months using this method and can, if required, achieve percentages of PRBCs of 20% or greater. It is also noteworthy in Fig. 5 that, after accounting for daily dilution of the percentage of PRBCs with fresh RBCs, the percentage of PRBCs in the culture doubled every approximately 17 to 20 h. This is consistent with previous estimates of the growth rate of B. bovis cultured in traditional serum-containing medium in vitro (Erp et al. 1978; Levy et al. 1981).
DISCUSSION B. bovis was successfully cultured continuously in Amedium from infected blood, from cryostabilates of either infected blood or cultured parasites, and from continuous in vitro parasite cultures growing in “traditional” BS-medium. In contrast, we have identified a number of parasite lines, isolated directly from infected blood, that failed to grow in BS-medium but which adapted immediately to in vitro culture when A-medium was used. In fact, we have been successfully culturing many different lines of B. bovis during the past 3 years in our laboratories and have not yet found
JACKSON ET AL.
a line that will not grow in A-medium. Furthermore, for B. bovis lines W, T, K, and Anderson that have been cultured continuously in this way, we have not identified any morphological differences (by examination of Giemsa-stained smears) from the same parasite lines grown in BS-medium. Furthermore, other pathophysiologically important biological properties of these parasites, such as their ability to cytoadhere to bovine vascular endothelial cells, are also maintained (B. M. Cooke, unpublished). Although addition of hypoxanthine was not an absolute requirement for parasite growth in either BS- or A-medium, its addition significantly increased parasite growth. We, therefore, recommend the addition of hypoxanthine to Amedium to maximize parasite yield. This is in direct contrast to P. falciparum where additional exogenous hypoxanthine has been shown to be an absolute requirement for parasite growth in RPMI 1640 supplemented with ALBUMAX II (Cranmer et al. 1997). Medium 199 contains 0.1 M hypoxanthine in its standard formulation while RPMI 1640 does not contain any hypoxanthine according to the manufacturers’ specifications. Goff and Yunker (1986) have previously shown that addition of extra hypoxanthine to Medium 199 did not appear to enhance growth of B. bovis. It is interesting to note that in this study, exogenous hypoxanthine was not an absolute requirement for growth of B. bovis when cultured in RPMI1 640 containing ALBUMAX II, whereas Irvin et al. (1978) reported that exogenous hypoxanthine was critical for the growth of Babesia spp. Traditionally, Babesia spp. parasites have largely been cultured in Medium 199 buffered with TES. Here, we have used the ALBUMAX-supplemented media previously described by Cranmer et al. (1997) for growth of malaria parasites which, in contrast to traditional babesia culture media, comprises RPMI 1640 buffered with Hepes. Several lines of evidence suggest that it is unlikely that the change from Medium 199 to Hepes-buffered RPMI 1640 affected our results obtained in the present study. First, a previous comparison of the growth of B. bovis in Medium 199 and RPMI 1640 showed no significant differences between these two culture media (Erp et al. 1980), albeit in suspension rather than MASP culture. Second, the addition of 25 mM Hepes showed no detrimental affect on parasite growth (Erp et al. 1980). Third, although not systematically tested here, we have in fact noted that RPMI 1640 or Medium 199 can be used interchangeably for the growth medium without any notable differences in parasite growth. The combination of 600-l well volume in a 24-well plate incubated in an atmosphere of 5% O2, 5% CO2, 90% N2 achieved a significantly higher percentage of PRBCs than any other combination of well volume and atmospheric gas
173
CULTURE OF Babesia bovis IN VITRO
mixture. It would be of interest to determine whether gas mixtures containing higher concentrations of O2 would further increase the percentage of PRBCs obtained. A previous study has in fact shown that a gas mixture consisting of 20% O2, 5% CO2, 75% N2 gave optimal growth for B. bovis (Erp et al. 1980). However, in that study B. bovis was cultivated using bovine-serum medium in a suspension rather than a stationary culture. Levy and Ristic (1980) found that a depth of approximately 0.6 cm was optimal for B. bovis growth in traditional BS-medium while a depth of 0.16 cm could not sustain continuous culture albeit under high O2 conditions (5% CO2 in air). Our results obtained here demonstrate an inhibitory effect on parasite growth for higher volumes of medium in the wells. When culture vessels different from those described here are used, it is, therefore, valuable to optimize the volume of media above the cultures to optimize parasite yield. It is perhaps not surprising that excessively large volumes of media above the cultures, resulting in increased hydrostatic pressure or compression on the RBCs, might inhibit parasite proliferation by inhibiting merozoite release or restricting their spread to neighboring RBCs. Furthermore, previous studies have demonstrated profound effects of hydrostatic pressure on the proliferation or morphological characteristics of cells, such as vascular endothelial cells including altered release of growth factors and reorganization of the normal cell cytoskeleton (Acevedo et al. 1993; Salwen et al. 1998; Schwartz et al. 1999). The direct effect of hydrostatic pressure on merozoites and the biochemical responses transduced by such mechanical stimuli are not known, but are clearly an area worthy of further investigation. Traditionally, MASP cultures of babesia parasites are subcultured every 2–3 days. Here, using our large-scale culture flask method, parasite numbers can reach sufficiently high levels in a period of 24 h that subculture can be performed routinely on a daily basis. This maximizes overall parasite yield and is particularly important when large volumes of parasites are required for production of parasites as a source of immunogens or vaccine material. Finally, at the present time, the only vaccines available for the prevention of babesia infection in cattle are live attenuated forms produced in splenectomized calves. Although still considered relatively effective, their use is associated with varying levels of protection, and the development of a more effective and betterdefined vaccine using either in vitro cultured parasites or recombinant antigens is of utmost importance. An abundant supply of genetically and phenotypically well-characterized parasites cultured in a chemically defined medium, as described here, will be a prerequisite to achieving these goals.
Moreover, until an effective recombinant vaccine is developed, use of attenuated parasites cultured in a defined medium represent a step in the right direction toward avoidance of accidental cotransmission of known (Rogers et al. 1988) or as yet unidentified infectious agents that are a potential hazard associated with the use of the currently available vaccines.
ACKNOWLEDGMENTS The authors thank Katy Williams for technical assistance and Wayne Jorgensen and Bert de Vos for expert advice. This work was supported by the Australian Centre for International Agricultural Research (ACIAR PN AS2 9690). Dr. Cooke is supported by the National Health and Medical Research Council of Australia.
REFERENCES Acevedo, A. D., Bowser, S. S., Gerritsen, M. E., and Bizios, R. 1993. Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor. Journal of Cellular Physiology 157, 603–614. Bock, R. E., de Vos, A. J., Kingston, T. G., Shiels, I. A., and Dalgliesh, R. J. 1992. Investigations of breakdowns in protection provided by living Babesia bovis vaccine. Veterinary Parasitology 43, 45–56. Bock, R. E., de Vos, A. J., Lew, A., Kingston, T. G., and Fraser, I. R. 1995. Studies on failure of T strain live Babesia bovis vaccine. Australian Veterinary Journal 72, 296–300. Callow, L. L., and Mellors, L. T. 1966. A new vaccine for Babesia argentina infection prepared in splenectomised calves. Australian Veterinary Journal 42, 464–465. Calvo de Mora, A., Garcia Castellano, J. M., Herrera, C., and JimenezAlonso, J. 1985. Human babesiosis: Report of a case with fatal outcome. Medicina Clinica 85, 515–516. Cranmer, S. L., Magowan, C., Liang, J., Coppel, R. L., and Cooke, B. M. 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 363–365. Erp, E. E., Gravely, S. M., Smith, R. D., Ristic, M., Osorno, B. M., and Carson, C. A. 1978. Growth of Babesia bovis in bovine erythrocyte cultures. American Journal of Tropical Medicine and Hygiene 27, 1061–1064. Erp, E. E., Smith, R. D., Ristic, M., and Osorno, B. M. 1980. Optimization of the suspension culture method for in vitro cultivation of Babesia bovis. American Journal of Veterinary Research 41, 2059– 2062. Goff, W. L., and Yunker, C. E. 1986. Babesia bovis: Increased percentage of parasitized erythrocytes in cultures and assessment of growth
174 by incorporation of [3H] hypoxanthine. Experimental Parasitology 62, 202–210. Grande, N., Precigout, E., Ancelin, M. L., Moubri, K., Carcy, B., Lemesre, J. L., Vial, H., and Gorenflot, A. 1997. Continuous in vitro culture of Babesia divergens in a serum-free medium. Parasitology 115, 81–89. Homer, M. J., Aguilar-Delfin, I., Telford, S. R., III, Krause, P. J., and Persing, D. H. 2000. Babesiosis. Clinical Microbiology Reviews 3, 451–469. Irvin, A. D., Young, E. R., and Purnell, R. E. 1978. The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology 8, 19–24.
JACKSON ET AL.
Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis. Australasian Veterinary Journal 65, 285–287. Salwen, S. A., Szarowski, D. H., Turner, J. N., and Bizios, R. 1998. Three-dimensional changes of the cytoskeleton of vascular endothelial cells exposed to sustained hydrostatic pressure. Medical & Biological Engineering & Computing 36, 520–527. Schwartz, E. A., Bizios, R., Medow, M. S., and Gerritsen, M. E. 1999. Exposure of human vascular endothelial cells to sustained hydrostatic pressure stimulates proliferation: Involvement of the ␣v integrins. Circulation Research 84, 315–322.
Levy, M. G., and Ristic, M. 1980. Babesia bovis: Continuous cultivation in a microaerophilus stationary phase culture. Science 207, 1218– 1220.
Suarez Hernandez, M., Alonso Castellano, M., Pelaez Martinez, R., Sanchez Perez, B., Bravo Gonzalez, J. R., and Sanchez Sibello, A. 1997. Investigation of Babesia in farm workers and blood donors in the province of Ciego de Avila. Revista Cubana de Medicina Tropical 49, 130–135.
Levy, M. G., Erp, E., and Ristic, M. 1981. Cultivation of babesia. In “Babesiosis” (M. Ristic and J. P. Kreier, Eds.), pp. 207–223. Academic Press, New York.
Vega, C. A., Buening, G. M., Green, T. J., and Carson, C. A. 1985. In vitro cultivation of Babesia bigemina. American Journal of Veterinary Research 46, 416–420.
McCosker, P. J. 1981. The global importance of babesiosis. In “Babesiosis” (M. Ristic and J. P. Kreier, Eds.), pp. 1–24. Academic Press, New York.
Wyatt, C. R., Goff, W., and Davis, W. C. 1991. A flow cytometric method for assessing viability of intraerythrocytic hemoparasites. Journal of Immunological Methods 140, 23–30.
Rogers, R. J., Dimmock, C. K., de Vos, A. J., and Rodwell, B. J. 1988.
Received 30 May 2001; accepted with revision 17 September 2001