Veterinary Parasitology, 45 ( 1992 ) 153-156
153
Elsevier Science Publishers B.V., Amsterdam
Short Communication Human serum for in vitro cultivation of Babesia
bovis A.K. Mishra a, G. Clabaughb and I. Kakomab aDivision of Parasitology, Indian Veterinary Research Institute, Izatnagar-2 4 312 2, India bDepartment of Veterinary Pathobiology, College of Veterinary Medicine, 2001 South Lincoln Avenue, Urbana, IL 61801, USA (Accepted 10 April 1992)
ABSTRACT Mishra, A.K., Clabaugh, G. and Kakoma, I., 1992. Human serum for in vitro cultivation of Babesia boris. Vet. Parasitol., 45: 153-156. Various combinations of human serum (from blood of groups A and Rhesus positive) with bovine serum, i.e. 20%+20% (Medium I), 30%+ 10% (II), 40%+0% (III) and 0%+40% (IV) and Medium- 199 (60%) were used in the propagation of Babesia boris. Babesia boris stabilate revived by inoculation in a bovine calf was used at a level of 6% parasitized erythrocytes (PPE). The medium was replenished every 24 h. The medium changed from bright red to dark-coffee color every 24 h. The increase in PPE was maximal between 24 and 48 h. It was also observed that the increase in PPE was significantly higher in a 1 : 2 dilution compared with a 1 : 1 dilution. The increase in PPE was highest in Medium I with a 50% replacement of bovine with human serum. However, the parasites could not be subcultured and maintained continuously. Fifty percent replacement was thus optimal for the human-bovine serum combination in a microaerophilous stationary phase (MASP) system. The results indicate that B. bovis can also multiply in a human/bovine serum MASP culture system at least for a period of 48 h, and this is consistent with the zoonotic nature of Babesia species.
INTRODUCTION
Recently, Babesia bovis has been reported to be of zoonotic importance. The first case of B. bovis infection in a young splenectomized Yugoslavian was reported by Skrabalo and Deanovic (1957). Thereafter, the zoonotic importance has been confirmed by many cases of human babesiosis involving B. bovis, B. divergens, B. microti and B. equi from Northern Ireland, France, the former USSR, Scotland, and Spain (Healy and Ristic, 1989). Babesia Correspondence to: I. Kakoma, Department of Veterinary Pathobiology, College of Veterinary Medicine, 2001 South Lincoln Avenue, Urbana, IL 61801, USA.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
154
A.K. MISHRA ETAL.
bovis was propagated in a continuous microaerophilous stationary phase (MASP) culture system for the first time by Levy and Ristic (1980) using 40% bovine serum. Babesia bovis has also been successfully cultivated in a MASP culture system using 20% bovine and 20% goat serum (Mishra et al., 1991 ). In the present study, attempts have been made to cultivate B. bovis in a MASP culture system while replacing bovine serum with human serum at various levels in order to determine whether B. bovis can multiply in human serum which would be consistent with its zoonotic potential. MATERIALS AND METHODS
Parasites Babesia bovis (Mexican isolate) maintained as a stabilate in liquid nitrogen was revived in a bovine calf. At a level of 6% parasitized erythrocytes (PPE), the blood was collected and cultured following the MASP culture technique of Levy and Ristic (1980). The parasites were used in the present study after being continuously maintained in culture for 4 months. Humanserum Commercially available human serum from donors of blood groups A and Rhesus positive was used. Four combinations of human and bovine serum were used. Medium 1 contained 20% human serum + 20% bovine serum (50% replacement); Medium 2 contained 30% human serum and 10% bovine serum (75% replacement); Medium 3 contained 40% human serum without bovine serum ( 100% replacement); Medium 4 contained 40% bovine serum without human serum (0% replacement). Me~um A 1 1 packet of powdered Medium- 199 (GIBCO, Gathersburg, MD) with Hanks salt L-glutamine and without sodium bicarbonate was dissolved in 1 1 of distilled water and supplemented with 0.35 g sodium bicarbonate, 5.96 g HEPES (Sigma, St. Louis, MO), 220 400 IU penicillin and 0.215 g streptomycin. The pH of the medium was adjusted to 6.95-7.05. Culture procedure Cultures were set up in 24-well flat-bottomed plates using different combinations of human and bovine serum following the MASP culture system of Levy and Ristic (1980). Spent medium in each plate was replaced with an equal volume of fresh medium without disturbing the cells and the PPE was
155
HUMANSERUMFORCULTURINGBABESIA BOVIS TABLE 1 Average PPE of B. boris in a human/bovine serum MASP culture system Human/bovine Dilution 1: 1 serum Oh 24h
48h
72h
0h
24h
48h
72h
20/20 30/10 40/00 00/40
6.2(142) 5.2 (100) 4.5 (73) 6.4 (80)
4.0(53) 3.9 (50) 2.4 ( - 7 ) 7.3 (104)
1.7 1.7 1.5 2.4
5.2(205) 3.3 (94) 2.7 (80) 4.4 (81)
4.5(164) 3.6 (111) 2.7 (90) 6.1 (153)
2.9(70) 3.5 (105) 2.0 (33) 7.4 (207)
2.6 2.6 2.6 3.6
6.1(134) 4.3 (65) 3.0 (15) 4.8 (36)
Dilution 1:2
Values in parentheses represent percentage increases in PPE of B. boris compared with 0 h.
determined at 24-h intervals. Two dilutions of infected cultures with noninfected cultures, 1 : 1 and 1:2, were maintained and the kinetics of growth of B. boris were monitored. RESULTS
The spent medium in both dilutions was dark-coffee colored at every 24-h interval when the medium was changed. The increase in the PPE was maximum between 24 and 48 h, declining thereafter except in medium with only bovine serum (Table 1 ). The percentage increase in PPE in Dilution 1:2 compared with PPE at 0 h (i.e. initial PPE) was much higher than the percentage increase in Dilution 1 : 1. Further, it was observed that 50% replacement of bovine serum with human serum gave better parasite multiplication than 75% or 100% replacement of bovine serum with human serum. DISCUSSION
The change in the spent medium from bright red to dark-coffee color in all the culture plates coincided with the multiplication of the parasites and the release of exoantigens, consistent with the results of Levy and Ristic (1980). The percentage increase in the PPE of B. bovis was significantly higher ( P < 0.05 ) in Dilution 1 : 2 compared with Dilution 1 : 1. These observations are similar to those of Mishra et al. (1990), who reported that higher dilutions gave a better percentage increase in the PPE. The parasite multiplication was optimum between 24 and 48 h. Babesia boris can be maintained in MASP culture by subculturing for a desired period of time (Levy and Ristic, 1980) and also in medium containing 20% bovine and 20% goat serum (Mishra et al., 1991 ), but the cultures in media with different proportions of human and bovine serum cannot be subcultured and maintained continuously. After 48 h, all the cultures showed a declining trend in their multiplication rate. On subculture, the parasites failed to invade fresh healthy eryth-
156
A.K. MISHRA ET AL.
rocytes and finally disappeared. Mishra et al. ( 1989 ) were able to m a i n t a i n B. bovis without subculture in standard MASP cultures for a period of 10 days. The parasites remained viable as evidenced by their multiplication in subsequent subculture. In the present study, B. bovis parasites multiplied in the m e d i u m containing h u m a n serum only for a short period o f 24-48 h. Since the multiplication o f the parasite in h u m a n serum has been observed, it is apparent that h u m a n serum provides a favorable m e d i u m for B. boris in a MASP culture system at least for a period o f 48 h. It is tempting to speculate that the parasite m a y find the h u m a n body suitable for initial establishment at least for the first 2 days following inoculation. This appears to be the first report on the multiplication o f B. bovis in in vitro culture in a mixture of h u m a n and bovine serum. The system facilitates short-term culture o f the agent and provides a model for its transient survival in h u m a n serum consistent with its zoonotic potential.
REFERENCES Healy, G. and Ristic, M., 1989. Human babesiosis. In: M. Ristic (Editor), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, FL, pp. 209-225. Levy, M.G. and Ristic, M., 1980. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science, 207:1218-1220. Mishra, A.K., Clabaugh, G., Kakoma, I. and Ristic, M., 1989. Studies on the maintenance of Babesia bovis (Mexican isolant) without subculture using microaerophilousstationary phase (MASP) culture technique. Proc. 38th Annual Meeting, American Societyof Tropical Medicine and Hygiene, Honolulu, Hawaii, 10-14 Dec., 1989, ASTMH Press, Lawrence, KS, p. 165. Mishra, A.K., Clabaugh,G., Kakoma, I. and Ristic, M., 1990. Kinetics of the growth of Babesia boris in microaerophilous stationary phase (MASP) system. Proc. First Asian Congress of Veterinary Parasitology, 26-28 Nov. 1990, New Delhi, India, All India Society for Parasitology, New Delhi, p. 75. Mishra, A.K., Clabaugh, G., Kakoma, I. and Ristic, M., 1991. Goat serum: a substitute of bovine serum in cultivation of Babesia boris. Acta Vet. Hung., 39:1-2 and 25-28. Skrabalo, Z. and Deanovic, Z., 1957. Piroplasmosisin man. Report on a case. DOC. Med. Geogr. Trop., 9:11.
153
Elsevier Science Publishers B.V., Amsterdam
Short Communication Human serum for in vitro cultivation of Babesia
bovis A.K. Mishra a, G. Clabaughb and I. Kakomab aDivision of Parasitology, Indian Veterinary Research Institute, Izatnagar-2 4 312 2, India bDepartment of Veterinary Pathobiology, College of Veterinary Medicine, 2001 South Lincoln Avenue, Urbana, IL 61801, USA (Accepted 10 April 1992)
ABSTRACT Mishra, A.K., Clabaugh, G. and Kakoma, I., 1992. Human serum for in vitro cultivation of Babesia boris. Vet. Parasitol., 45: 153-156. Various combinations of human serum (from blood of groups A and Rhesus positive) with bovine serum, i.e. 20%+20% (Medium I), 30%+ 10% (II), 40%+0% (III) and 0%+40% (IV) and Medium- 199 (60%) were used in the propagation of Babesia boris. Babesia boris stabilate revived by inoculation in a bovine calf was used at a level of 6% parasitized erythrocytes (PPE). The medium was replenished every 24 h. The medium changed from bright red to dark-coffee color every 24 h. The increase in PPE was maximal between 24 and 48 h. It was also observed that the increase in PPE was significantly higher in a 1 : 2 dilution compared with a 1 : 1 dilution. The increase in PPE was highest in Medium I with a 50% replacement of bovine with human serum. However, the parasites could not be subcultured and maintained continuously. Fifty percent replacement was thus optimal for the human-bovine serum combination in a microaerophilous stationary phase (MASP) system. The results indicate that B. bovis can also multiply in a human/bovine serum MASP culture system at least for a period of 48 h, and this is consistent with the zoonotic nature of Babesia species.
INTRODUCTION
Recently, Babesia bovis has been reported to be of zoonotic importance. The first case of B. bovis infection in a young splenectomized Yugoslavian was reported by Skrabalo and Deanovic (1957). Thereafter, the zoonotic importance has been confirmed by many cases of human babesiosis involving B. bovis, B. divergens, B. microti and B. equi from Northern Ireland, France, the former USSR, Scotland, and Spain (Healy and Ristic, 1989). Babesia Correspondence to: I. Kakoma, Department of Veterinary Pathobiology, College of Veterinary Medicine, 2001 South Lincoln Avenue, Urbana, IL 61801, USA.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
154
A.K. MISHRA ETAL.
bovis was propagated in a continuous microaerophilous stationary phase (MASP) culture system for the first time by Levy and Ristic (1980) using 40% bovine serum. Babesia bovis has also been successfully cultivated in a MASP culture system using 20% bovine and 20% goat serum (Mishra et al., 1991 ). In the present study, attempts have been made to cultivate B. bovis in a MASP culture system while replacing bovine serum with human serum at various levels in order to determine whether B. bovis can multiply in human serum which would be consistent with its zoonotic potential. MATERIALS AND METHODS
Parasites Babesia bovis (Mexican isolate) maintained as a stabilate in liquid nitrogen was revived in a bovine calf. At a level of 6% parasitized erythrocytes (PPE), the blood was collected and cultured following the MASP culture technique of Levy and Ristic (1980). The parasites were used in the present study after being continuously maintained in culture for 4 months. Humanserum Commercially available human serum from donors of blood groups A and Rhesus positive was used. Four combinations of human and bovine serum were used. Medium 1 contained 20% human serum + 20% bovine serum (50% replacement); Medium 2 contained 30% human serum and 10% bovine serum (75% replacement); Medium 3 contained 40% human serum without bovine serum ( 100% replacement); Medium 4 contained 40% bovine serum without human serum (0% replacement). Me~um A 1 1 packet of powdered Medium- 199 (GIBCO, Gathersburg, MD) with Hanks salt L-glutamine and without sodium bicarbonate was dissolved in 1 1 of distilled water and supplemented with 0.35 g sodium bicarbonate, 5.96 g HEPES (Sigma, St. Louis, MO), 220 400 IU penicillin and 0.215 g streptomycin. The pH of the medium was adjusted to 6.95-7.05. Culture procedure Cultures were set up in 24-well flat-bottomed plates using different combinations of human and bovine serum following the MASP culture system of Levy and Ristic (1980). Spent medium in each plate was replaced with an equal volume of fresh medium without disturbing the cells and the PPE was
155
HUMANSERUMFORCULTURINGBABESIA BOVIS TABLE 1 Average PPE of B. boris in a human/bovine serum MASP culture system Human/bovine Dilution 1: 1 serum Oh 24h
48h
72h
0h
24h
48h
72h
20/20 30/10 40/00 00/40
6.2(142) 5.2 (100) 4.5 (73) 6.4 (80)
4.0(53) 3.9 (50) 2.4 ( - 7 ) 7.3 (104)
1.7 1.7 1.5 2.4
5.2(205) 3.3 (94) 2.7 (80) 4.4 (81)
4.5(164) 3.6 (111) 2.7 (90) 6.1 (153)
2.9(70) 3.5 (105) 2.0 (33) 7.4 (207)
2.6 2.6 2.6 3.6
6.1(134) 4.3 (65) 3.0 (15) 4.8 (36)
Dilution 1:2
Values in parentheses represent percentage increases in PPE of B. boris compared with 0 h.
determined at 24-h intervals. Two dilutions of infected cultures with noninfected cultures, 1 : 1 and 1:2, were maintained and the kinetics of growth of B. boris were monitored. RESULTS
The spent medium in both dilutions was dark-coffee colored at every 24-h interval when the medium was changed. The increase in the PPE was maximum between 24 and 48 h, declining thereafter except in medium with only bovine serum (Table 1 ). The percentage increase in PPE in Dilution 1:2 compared with PPE at 0 h (i.e. initial PPE) was much higher than the percentage increase in Dilution 1 : 1. Further, it was observed that 50% replacement of bovine serum with human serum gave better parasite multiplication than 75% or 100% replacement of bovine serum with human serum. DISCUSSION
The change in the spent medium from bright red to dark-coffee color in all the culture plates coincided with the multiplication of the parasites and the release of exoantigens, consistent with the results of Levy and Ristic (1980). The percentage increase in the PPE of B. bovis was significantly higher ( P < 0.05 ) in Dilution 1 : 2 compared with Dilution 1 : 1. These observations are similar to those of Mishra et al. (1990), who reported that higher dilutions gave a better percentage increase in the PPE. The parasite multiplication was optimum between 24 and 48 h. Babesia boris can be maintained in MASP culture by subculturing for a desired period of time (Levy and Ristic, 1980) and also in medium containing 20% bovine and 20% goat serum (Mishra et al., 1991 ), but the cultures in media with different proportions of human and bovine serum cannot be subcultured and maintained continuously. After 48 h, all the cultures showed a declining trend in their multiplication rate. On subculture, the parasites failed to invade fresh healthy eryth-
156
A.K. MISHRA ET AL.
rocytes and finally disappeared. Mishra et al. ( 1989 ) were able to m a i n t a i n B. bovis without subculture in standard MASP cultures for a period of 10 days. The parasites remained viable as evidenced by their multiplication in subsequent subculture. In the present study, B. bovis parasites multiplied in the m e d i u m containing h u m a n serum only for a short period o f 24-48 h. Since the multiplication o f the parasite in h u m a n serum has been observed, it is apparent that h u m a n serum provides a favorable m e d i u m for B. boris in a MASP culture system at least for a period o f 48 h. It is tempting to speculate that the parasite m a y find the h u m a n body suitable for initial establishment at least for the first 2 days following inoculation. This appears to be the first report on the multiplication o f B. bovis in in vitro culture in a mixture of h u m a n and bovine serum. The system facilitates short-term culture o f the agent and provides a model for its transient survival in h u m a n serum consistent with its zoonotic potential.
REFERENCES Healy, G. and Ristic, M., 1989. Human babesiosis. In: M. Ristic (Editor), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, FL, pp. 209-225. Levy, M.G. and Ristic, M., 1980. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science, 207:1218-1220. Mishra, A.K., Clabaugh, G., Kakoma, I. and Ristic, M., 1989. Studies on the maintenance of Babesia bovis (Mexican isolant) without subculture using microaerophilousstationary phase (MASP) culture technique. Proc. 38th Annual Meeting, American Societyof Tropical Medicine and Hygiene, Honolulu, Hawaii, 10-14 Dec., 1989, ASTMH Press, Lawrence, KS, p. 165. Mishra, A.K., Clabaugh,G., Kakoma, I. and Ristic, M., 1990. Kinetics of the growth of Babesia boris in microaerophilous stationary phase (MASP) system. Proc. First Asian Congress of Veterinary Parasitology, 26-28 Nov. 1990, New Delhi, India, All India Society for Parasitology, New Delhi, p. 75. Mishra, A.K., Clabaugh, G., Kakoma, I. and Ristic, M., 1991. Goat serum: a substitute of bovine serum in cultivation of Babesia boris. Acta Vet. Hung., 39:1-2 and 25-28. Skrabalo, Z. and Deanovic, Z., 1957. Piroplasmosisin man. Report on a case. DOC. Med. Geogr. Trop., 9:11.
