Research in Veterinary Science 1992, 52, 115-116
Lyophilised bovine serum as a substitute for frozen serum in the cultivation of Babesia bigemina and B boris L. FISH, E. PIPANO, Division of Parasitology, Kimron Veterinary Institute, Beit-Dagan 50250, Israel, P. I N D R A K A M H A N G , National Animal Health and Production Institute, Bangkok 10900, Thailand
Lyophilised serum offers significant advantages over frozen serum when it comes to shipping such material over long distances. Babesia bigemina and B boris were cultured in medium supplemented by either frozenthawed or lyophilised-rehydrated serum. There were no significant differences between the two types of medium in the growth of parasites and percentage of infected cells during subcultivation for 18 days.
BABESIA bigemina and Bbovis are tick-borne haemoprotozoan parasites of cattle of considerable economic importance in tropical, subtropical and some temperate areas. Both parasites have been successfully propagated in vitro (Levy and Ristic 1980, Vega et al 1985) in a basic synthetic culture medium supplemented with bovine serum. However, since not all babesia-free young or adult cattle provide serum that supports growth, potential donors need to be screened in ongoing cultures for growth-supporting capability. It follows that setting up cultures for the first time in babesiosis-endemic areas can be a problem unless serum known to be suitable is available for reference control purposes. Suitable reference control serum from laboratories already maintaining cultures of babesia can usually be provided, most often in the frozen state. However, the transport over long distances of frozen serum packed in dry ice is associated with practical difficulties of unpredictable delays and ambient conditions that may result in the thawing of the serum before it is delivered. On the other hand, lyophilised serum can be shipped at ambient temperature, greatly improving the likelihood of safe arrival. In the work reported here lyophilised and frozen bovine sera were compared for their suitability in cultures of B bigemina and B boris. Serum that had previously proved to be suitable for growing B bigemina and B boris in culture was obtained by defibrination of whole blood from an adult cow (about six years old). The serum was divided into two portions: one was stored at -20°C; the other was dispensed in 6-7 ml aliquots into 20 ml bottles. The latter portion was
lyophilised in the bottles in a shelf-freeze drier, stoppered under vacuum and stored at 4°C. For use, the lyophilised serum was rehydrated with 6.7 ml doubledistilled water for each bottle. M 199 medium in Earle's balanced salt solution plu s 2 per cent TES solution (574 g of 2.2-hydroxy-1, 1 bis [hydroxymethyJ] ethylaminoethane sulphonic acid in 25 ml M199) was used for initiation and maintenance of cultures. The medium was supplemented with 40 per cent frozen-thawed or lyophilised-rehydrated serum. Cultures were maintained in 24-well cluster plates according to techniques described by Levy and Ristic (1980) and Vega and others (1985) with the following minor modifications. All cultures were incubated in an atmosphere of 2 per cent oxygen, 5 per cent carbon dioxide and 93 per cent nitrogen at 37°C. Medium was replaced every 24 hours and subcultures were performed every 48 hours. For subcultivation, the cells in the wells were resuspended and the suspension was diluted 1:10 in a 10 per cent suspension of uninfected red blood cells in complete medium. One ml aliquots of the diluted suspension were distributed into each of four wells of a 24-well cluster plate. Before each passage, thin blood films were prepared and stained with Giemsa. The percentage ofparasitised erythrocytes was determined by counting 500 red blood cells per slide. Parasites used in the trials were cloned cultures of B bigemina and B boris retrieved from cryopreservation (Vega et al 1985a) and grown for about six months before use in these experiments. The cumulative increase (c0 of parasitaemia was calculated according to the following equation: cI = (re/iF) CD, where FP = final percentage of parasitemia, Ip = initial percentage of parasitemia and co = cumulative dilution (the factor by which the erythrocytes present at the start of the experiment are diluted with fresh erythrocytes during subculture). The maximal percentages of B bigemina-parasitised cells after 48 hours were 8 to 14 per cent in medium with frozen serum, compared to 8 to 12 per cent in medium with lyophilised serum (Fig 1). There was no significant difference in the cumulative increase of par115
L. Fish, E. Pipano, P. Indrakamhang
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FIG 1 : Multiplication of B bigemina in medium supplemented with frozen-thawed (- - -) or lyophilised-rehydrated ( - - ) serum
FIG 2: Multiplication of Bbovis in medium supplemented with frozenthawed (- - -) or lyophilised-rehydrated ( ) serum
asitaemia over a period of 18 days in medium sup plemented with either type of serum.
Acknowledgement
Similar results were obtained in B boris cultures (Fig 2), the percentages of infected cells varying from 8 to 14 per cent with frozen serum, and 7 to 19 per cent with lyophilised serum. The cumulative increase of parasitaemia was similar with both sera. It thus appears that lyophilised bovine serum can be substituted for frozen serum in cultures of B bigemina and B bovis with no sacrifice of the ability to support growth, but with a considerable improvement in the convenience for shipping and handling.
This study was partially supported by the us-Israeli Binational Agricultural Research and Development Fund, grant 1080-86. References
LEVY, M. & RISTIC, M. (1980) Babesia boris: continuous cultivation in a microaerophilous stationary phase culture. Science 207, 12181220 VEGA, C. A., BUENING, G. M., GREEN, T. Y. & CARSON, C. A. (1985) In vitro cultivation ofBabesia bigemina. American Journal of Veterinary Research 46, 416-420
Received April 4, 199l Accepted August 19, 1991
Lyophilised bovine serum as a substitute for frozen serum in the cultivation of Babesia bigemina and B boris L. FISH, E. PIPANO, Division of Parasitology, Kimron Veterinary Institute, Beit-Dagan 50250, Israel, P. I N D R A K A M H A N G , National Animal Health and Production Institute, Bangkok 10900, Thailand
Lyophilised serum offers significant advantages over frozen serum when it comes to shipping such material over long distances. Babesia bigemina and B boris were cultured in medium supplemented by either frozenthawed or lyophilised-rehydrated serum. There were no significant differences between the two types of medium in the growth of parasites and percentage of infected cells during subcultivation for 18 days.
BABESIA bigemina and Bbovis are tick-borne haemoprotozoan parasites of cattle of considerable economic importance in tropical, subtropical and some temperate areas. Both parasites have been successfully propagated in vitro (Levy and Ristic 1980, Vega et al 1985) in a basic synthetic culture medium supplemented with bovine serum. However, since not all babesia-free young or adult cattle provide serum that supports growth, potential donors need to be screened in ongoing cultures for growth-supporting capability. It follows that setting up cultures for the first time in babesiosis-endemic areas can be a problem unless serum known to be suitable is available for reference control purposes. Suitable reference control serum from laboratories already maintaining cultures of babesia can usually be provided, most often in the frozen state. However, the transport over long distances of frozen serum packed in dry ice is associated with practical difficulties of unpredictable delays and ambient conditions that may result in the thawing of the serum before it is delivered. On the other hand, lyophilised serum can be shipped at ambient temperature, greatly improving the likelihood of safe arrival. In the work reported here lyophilised and frozen bovine sera were compared for their suitability in cultures of B bigemina and B boris. Serum that had previously proved to be suitable for growing B bigemina and B boris in culture was obtained by defibrination of whole blood from an adult cow (about six years old). The serum was divided into two portions: one was stored at -20°C; the other was dispensed in 6-7 ml aliquots into 20 ml bottles. The latter portion was
lyophilised in the bottles in a shelf-freeze drier, stoppered under vacuum and stored at 4°C. For use, the lyophilised serum was rehydrated with 6.7 ml doubledistilled water for each bottle. M 199 medium in Earle's balanced salt solution plu s 2 per cent TES solution (574 g of 2.2-hydroxy-1, 1 bis [hydroxymethyJ] ethylaminoethane sulphonic acid in 25 ml M199) was used for initiation and maintenance of cultures. The medium was supplemented with 40 per cent frozen-thawed or lyophilised-rehydrated serum. Cultures were maintained in 24-well cluster plates according to techniques described by Levy and Ristic (1980) and Vega and others (1985) with the following minor modifications. All cultures were incubated in an atmosphere of 2 per cent oxygen, 5 per cent carbon dioxide and 93 per cent nitrogen at 37°C. Medium was replaced every 24 hours and subcultures were performed every 48 hours. For subcultivation, the cells in the wells were resuspended and the suspension was diluted 1:10 in a 10 per cent suspension of uninfected red blood cells in complete medium. One ml aliquots of the diluted suspension were distributed into each of four wells of a 24-well cluster plate. Before each passage, thin blood films were prepared and stained with Giemsa. The percentage ofparasitised erythrocytes was determined by counting 500 red blood cells per slide. Parasites used in the trials were cloned cultures of B bigemina and B boris retrieved from cryopreservation (Vega et al 1985a) and grown for about six months before use in these experiments. The cumulative increase (c0 of parasitaemia was calculated according to the following equation: cI = (re/iF) CD, where FP = final percentage of parasitemia, Ip = initial percentage of parasitemia and co = cumulative dilution (the factor by which the erythrocytes present at the start of the experiment are diluted with fresh erythrocytes during subculture). The maximal percentages of B bigemina-parasitised cells after 48 hours were 8 to 14 per cent in medium with frozen serum, compared to 8 to 12 per cent in medium with lyophilised serum (Fig 1). There was no significant difference in the cumulative increase of par115
L. Fish, E. Pipano, P. Indrakamhang
116 109,
109
108,
108
107 ,
107
106' ._=o 105.
Lz /
106
'dr/ #i
105 t
>~ 104. E
1041
103,
1031
102`
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101
1011
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i
i
i
20
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117 I1
//'
6 8 1'o 1'2 Number of days
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ll//
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4 2
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J/f J// / 2 / /
8
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FIG 1 : Multiplication of B bigemina in medium supplemented with frozen-thawed (- - -) or lyophilised-rehydrated ( - - ) serum
FIG 2: Multiplication of Bbovis in medium supplemented with frozenthawed (- - -) or lyophilised-rehydrated ( ) serum
asitaemia over a period of 18 days in medium sup plemented with either type of serum.
Acknowledgement
Similar results were obtained in B boris cultures (Fig 2), the percentages of infected cells varying from 8 to 14 per cent with frozen serum, and 7 to 19 per cent with lyophilised serum. The cumulative increase of parasitaemia was similar with both sera. It thus appears that lyophilised bovine serum can be substituted for frozen serum in cultures of B bigemina and B bovis with no sacrifice of the ability to support growth, but with a considerable improvement in the convenience for shipping and handling.
This study was partially supported by the us-Israeli Binational Agricultural Research and Development Fund, grant 1080-86. References
LEVY, M. & RISTIC, M. (1980) Babesia boris: continuous cultivation in a microaerophilous stationary phase culture. Science 207, 12181220 VEGA, C. A., BUENING, G. M., GREEN, T. Y. & CARSON, C. A. (1985) In vitro cultivation ofBabesia bigemina. American Journal of Veterinary Research 46, 416-420
Received April 4, 199l Accepted August 19, 1991
