Parasitol Res (1992) 78:423-426
Parasitology Research
9 Springer-Verlag1992
Growth of Babesia bigemina parasites in suspension cultures for vaccine production W.K. Jorgensen, S.J. Waldron, J. Me Grath, R.J. Roman, A.J. de Vos, and K.E. Williams Tick Fever Research Centre, Queensland Department of Primary Industries, 280 Grindle Road, WACOL QLD 4076, Australia Accepted March 1, 1992
Abstract. An Australian Babesia bigemina vaccine strain was maintained in suspension culture for 40 days. Parasite growth was compared using two tissue-culture flask sizes (25 and 75 cm2), four gas mixes (2%, 2.5%, 3% and 3.5% 02 ; 5% CO2 ; and the balance N2) and four packed blood cell (PCV) volumes (7%, 9%, 13% and 18%). The best continuous parasite yields were obtained from suspension cultures in 75-cm 2 flasks at a PCV of 13% and gas mixtures of 2 % - 3 % 0 2 , 5% CO2 and the balance N2. Parasite yields per millilitre of culture medium were 3 times those obtained in microaerophilous stationary-phase cultures. The method has thus far been used for 6 months to produce the Australian requirements for live B. bigemina vaccine.
Babesia bigemina is an economically important haemoprotozoan parasite that can cause acute and often fatal infections in susceptible cattle. A living vaccine against the disease is produced in Australia and has proved to be effective in several tropical and sub-tropical countries in which the major tick vectors, Boophilus spp., are endemic (see reviews by Dalgliesh et al. 1990; de Vos and Jorgensen 1992). Because the vaccine is derived from the blood of calves acutely infected with a laboratory strain of B. bigemina, there is the potential for contamination with pathogenic organisms, and strict testing and quarantine procedures must be undertaken (Rogers et al. 1988; Dalgliesh et al. 1990). In vitro culture can be used us a source of parasites to minimise the risk o f contamination associated with using donor animals in vaccine production (Jorgensen et al. 1989). Growth o f B. bigemina in vitro was first described by Vega et al. (1985 a, b), who used the microaerophilous stationary-phase (MASP) culture techniques of Levy and Ristic (1980). Jorgensen et al. (1989) demonstrated that the Australian vaccine strain of B. bigemina could be maintained in culture for 3 months without either Correspondence to: W.K. Jorgensen
a loss of protective immunogenicity or an increase in virulence. Vega et al. (1986) and Figueroa et al. (1990) successfully scaled up B. bigemina MASP cultures into 75-cm 2 tissue-culture flasks but showed no improvement in parasite yields per millilitre of culture material. These previous culture methods did not produce sufficient numbers of parasites per mitlilitre o f culture medium for economic vaccine production (Jorgensen et al. 1989; Timms and Stewart 1989). At the Tick Fever Research Centre (Australia), an average of 6000 doses of B. bigemina vaccine were used per month in 1990, and the demand is currently increasing. To produce these quantities o f vaccine, large-scale suspension cultures are required, such as those described for growing B. boris (Erp et al. 1980; Timms and Stewart 1989). B. bigemina is more difficult than B. boris to culture successfully (Montenegro-James et al. 1989), and suitable methods for large-scale production have not been described. Our aim, therefore, was to develop an economic, robust method of producing B. bigemina vaccine in vitro. We describe methods of producing B. bigemina for vaccine in suspension cultures using sealed tissue-culture flasks to prevent contamination. Static cultures (MASP and modified MASP) were also prepared for comparative purposes.
Materials and methods
Babesia bigernina strain A strain of B. bigemina (designated the G strain) was used. The strain is currently used as a vaccine strain in Australia (Jorgensen et al. 1989) and South Africa (de Vos et al. 1982).
Me~um The methods used for medium formulation and for the collection and storage of red blood cells (RBC) were similar to the procedures previously described by Vega et al. (1985a, b) and Figueroa et al. (1990). The serum and RBC used were obtained from one donor
424 cow that was maintained in arthropod-free quarantine accommodation for all culture experiments.
Determination o f culture yields A direct counting technique (Parker 1973) was used to quantify the numbers of B. bigemina-parasitised RBC per millilitre of culture. Samples were taken on sub-culture days of each of the different culture methods, and parasite growth rates were compared using this technique.
flat at 37 ~ C. During incubation, the RBC settled and spent medium could be changed by decanting approximately 10 ml and replacing this quantity with fresh medium every day. The parasites were sub-cultured every 2nd day, with half of the original parasitised RBC being replaced by fresh RBC. After being opened, the flasks were gassed by slow bubbling of a gas mix of 4 % 0 2 , 5% COz and 91% N2 (Commonwealth Industrial Gases, Brisbane) into the medium through a 1-ml disposable pipette connected to the gas cylinder. When the foamed medium had reached the flask opening, the pipette was rapidly withdrawn and the flask was sealed and returned to the incubator. M M A S P cultures were used to initiate suspension cultures.
Static cultures Suspension cultures MASP cultures. Primary, microaerophilous stationary-phase (MASP) cultures (Levy and Ristic 1980) were initiated from heparinised blood collected from a splenectomised donor calf infected with G strain B. bigemina. The parasites were maintained in continuous culture using the methods of Vega et al. (1985a, b). One MASP culture of 1.2 ml from a 24-well tissue-culture plate was used to initiate modified MASP cultures (see below).
Modified MASP cultures. Modified MASP (MMASP) cultures were maintained in sealed 25-cm 2 tissue-culture flasks (Corning, New York) containing 15 ml medium plus RBC to a packed cell volume (PCV) of 8%. Flasks containing cultures were incubated
Suspension cultures were maintained in 25- or 75-cm 2 sealed tissueculture flasks (Coming, New York). The 25-cm 2 flasks contained 15 ml medium and RBC to a PCV of 9%. The 75-cm 2 flasks contained 50 ml medium plus RBC to a PCV of 7%, 9%, 13% or 18%, depending on the experiment (Table 1). As the facilities did not enable all cultures to be maintained concurrently, each PCV was tested in succession. Medium was changed daily by centrifuging the flask contents at 192 g for 4 (15 ml) or 10 min (50 ml) at 20 ~ C in a Sorvall RC5C device equipped with a HS4 swing-out rotor. After centrifugation, 10 or 30 ml supernatant was removed, depending on the culture
Table 1. Direct parasite counts used to quantify Babesia bigemina in suspension cultures in 75-cm z flasks at four gas mixes and four different PCVs and in 25-cm 2 flasks at four gas mixes and a 9% PCV Flask size 75 cm 2
25 cm 2
7% PCV % O2ingasmix"
9% PCV % O2ingasmix a
13% PCV % O2ingasmix"
18% PCV % O2ingasmix"
9% PCV % O2ingasmix"
Dayof subculture b
2
2.5
3
3.5
2
2.5
3
3.5
2
2.5
3
3.5
2
2.5
3
3.5
2
2.5
3
3.5
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
7 14 19 9 -
5 10 23 3 -
4 5 7 1
4 11 28 12
7 39 39 18 25 14 15 19 19 14 34 -
7 25 40 24 18 22 13 18 18 7 22 -
7 43 43 32 26 14 16 19 20 9 20 -
7 30 28 23 22 22 10 14 24 12 25
8 33 51 49 42 35 34 38 44 34 69 36 51 .
13 31 26 39 39 42 31 55 42 40 51 52 59 .
8 40 36 45 48 35 40 37 37 33 46 34 46 .
9 33 35 51 29 31 35 49 27 39 47 23 53 .
13 24 38 45 57 33 44 37 49 30 32 42 29
13 26 39 52 51 38 31 30 35 34 28 44 37
13 23 29 55 43 52 47 33 41 49 19 26 31
13 27 19 48 49 31 40 38 27 45 27 22 31
7 13 18 22 40 15 33 31 47 29 32 48 56 22 56 44 49 41 59 57 30
7 23 28 13 25 16 27 28 46 44 50 65 39 36 29 38 47 33 65 40 44
7 17 30 16 25 7 27 35 35 33 42 46 40 42 44 54 60 39 60 46 48
7 17 26 8 17 9 26 28 30 43 34 38 33 40 57 44 37 36 65 46 49
.
.
Data represent numbers of parasites x 106/ml culture. PCV, Packed cell volume; -, culture discontinued a Gas mix contains x % oxygen + 5 % CO2 + balance nitrogen b Direct parasitaemia count ( x 106/ml) at the initiation of culture and thereafter on each day of sub-culture
425 volume, and replaced with fresh medium. The entire culture was returned to the appropriate size flask and gassed as described for the MMASP culture method. Suspension cultures were maintained on a platform rocker (Bioline, Australia) operating at 0.4 Hz in an incubator at 37~ C. The cultures were sub-cultured every 2nd day by removing half of the pellet and supernatant after centrifugation and replacing this with fresh RBC and medium to return the flask culture volume and PCV to the original values. Parasites were grown in cultures at each PCV using 2%, 2.5%, 3% and 3.5% 0 2 with 5% COz plus the balance N2 to assess the effects of changing the percentage of oxygen in the gas mix and altering the PCV on parasite growth (Table I). One series of four replicate 25-cmz flasks was also maintained using only 2% O2, 5% COz and 93% N2 to assess between-flask variability (Table 2). Cultures were maintained for up to 40 days before being discontinued (Table 1).
Results
Static cultures Both M A S P a n d M M A S P cultures were m a i n t a i n e d c o n t i n u o u s l y for 6 m o n t h s . M e a n direct p a r a s i t a e m i a c o u n t s f r o m the latter p a r t o f this period are c o m p a r e d with the results o b t a i n e d for s u s p e n s i o n cultures in Table 3.
Suspension cultures T h e results o f direct p a r a s i t a e m i a c o u n t s for each P C V tested are displayed in Table 1. A n analysis o f v a r i a n c e of the c o u n t s i n d i c a t e d t h a t there was n o significant difference b e t w e e n the four gas mixes, regardless o f w h e t h e r the d a t a for each P C V tested were a n a l y s e d separately or pooled. U s i n g this a s s u m p t i o n , a linear regression analysis was d o n e o n the four c o m b i n e d results o b t a i n e d for each P C V g r o u p i n g f r o m s u b - c u l t u r e day 2 to culture t e r m i n a t i o n . O n l y the c o u n t s d e t e r m i n e d in the 25-cm 2 flasks at a 9 % P C V a n d in the 75-cm 2 flasks at a 13% P C V d e m o n s t r a t e d a linear rise with time. T h e rem a i n d e r d e m o n s t r a t e d a q u a d r a t i c r e l a t i o n s h i p t h a t declined, i m p l y i n g t h a t these cultures c o u l d n o t be m a i n tained indefinitely. The second set o f f o u r s u s p e n s i o n cultures initiated in 25-cm 2 flasks at a n initial p a r a s i t a e m i a o f 12 x 106 p a r a s i t e s / m l a n d m a i n t a i n e d at 9 % P C V u s i n g a gas mix of 2 % 0 2 , 5 % COz a n d 9 3 % N2 were m a i n t a i n e d for 22 days. Table 2 displays the v a r i a t i o n b e t w e e n the four replicates. The s t a n d a r d d e v i a t i o n o f the m e a n s varied between 1.12 a n d 10.17 x 106 p a r a s i t e s / m l over the sample p e r i o d a n d did n o t decrease with the age o f the cultures.
Table 2. Direct parasitaemia counts used to quantify Babesia bigemina in a set of four identical suspension cultures in 25-cm2 sealed tissue-culture flasks Day a
25-cm 2 flask; PCV 9% Replicate number
0 2 4 6 8 I0 12 14 16 18 20 22
i
2
3
4
12 50 41 43 41 42 50 43 45 43 20 33
12 40 45 44 51 37 61 35 36 59 35 23
12 35 39 45 44 42 64 45 30 39 23 22
12 48 35 42 42 50 49 41 57 42 25 16
2
SD
12 43 40 43 44 43 56 41 42 46 26 24
0 6.06 3.61 1.12 3.91 4.66 6.60 3.74 10.17 7.79 5.63 6.10
Data represent numbers of parasites x 106/ml culture. -, Culture discontinued
Comparison of mean yields of MASP, M M A S P and suspension cultures
a Initial parasite count ( x 106/ml) and those obtained on the indicated days of sub-culture
Parasite c o u n t s per millilitre o f c u l t u r e m e d i u m from M A S P , M M A S P a n d s u s p e n s i o n c u l t u r e were c o m p a r e d
Table 3. Mean direct parasitaemia counts obtained for Babesia bigemina using different culture techniques Culture vessel type 1 well in a 24-well plate 25-cm2 STCF 25-cm2 STCF 75-cm2 STCF 75-cm2 STCF 75-cm2 STCF
Culture method
MASP MMASP Suspensionc Suspensiond Suspensione Suspensionf
Culture volume (ml)
1.2 15 15 50 50 50
Culture PCV (%)
Number of samples
Mean DPC x 106/ml (SD)
Vaccine doses/ml b
8
5
5
16.5 (5.02)
1.6
8 9 9 13 18
4 2 2 2 2
9 11 10 12 12
18.0 (6.05) 29.8 (12.04) 23.6 (9.57) 43.0 (10.17) 38.3 (9.06)
1.8 2.9 2.3 4.3 3.8
DPC, Direct parasitaemia count; STCF, sealed tissue-culture flask a Gas m i x = % 0 2 + 5 % CO2+balance N2 b 1 dose= 107 parasites ~ Days 2-22, Table 1 d Days 2-20, Table 1 ~ Days 2-24, Table ] f Days 2-24, Table 1
% 02 in gas mix a
426
(Table 3). Only data for suspension cultures maintained at 2% 02 gas mixes are shown in Table 3, as no significant difference in parasite number was found between the four gas mixes tested (Table 1). An analysis of variance showed that 75-cm 2 flasks at 13% and 18% PCV produced significantly higher yields than did those of the other methods (P
Parasitology Research
9 Springer-Verlag1992
Growth of Babesia bigemina parasites in suspension cultures for vaccine production W.K. Jorgensen, S.J. Waldron, J. Me Grath, R.J. Roman, A.J. de Vos, and K.E. Williams Tick Fever Research Centre, Queensland Department of Primary Industries, 280 Grindle Road, WACOL QLD 4076, Australia Accepted March 1, 1992
Abstract. An Australian Babesia bigemina vaccine strain was maintained in suspension culture for 40 days. Parasite growth was compared using two tissue-culture flask sizes (25 and 75 cm2), four gas mixes (2%, 2.5%, 3% and 3.5% 02 ; 5% CO2 ; and the balance N2) and four packed blood cell (PCV) volumes (7%, 9%, 13% and 18%). The best continuous parasite yields were obtained from suspension cultures in 75-cm 2 flasks at a PCV of 13% and gas mixtures of 2 % - 3 % 0 2 , 5% CO2 and the balance N2. Parasite yields per millilitre of culture medium were 3 times those obtained in microaerophilous stationary-phase cultures. The method has thus far been used for 6 months to produce the Australian requirements for live B. bigemina vaccine.
Babesia bigemina is an economically important haemoprotozoan parasite that can cause acute and often fatal infections in susceptible cattle. A living vaccine against the disease is produced in Australia and has proved to be effective in several tropical and sub-tropical countries in which the major tick vectors, Boophilus spp., are endemic (see reviews by Dalgliesh et al. 1990; de Vos and Jorgensen 1992). Because the vaccine is derived from the blood of calves acutely infected with a laboratory strain of B. bigemina, there is the potential for contamination with pathogenic organisms, and strict testing and quarantine procedures must be undertaken (Rogers et al. 1988; Dalgliesh et al. 1990). In vitro culture can be used us a source of parasites to minimise the risk o f contamination associated with using donor animals in vaccine production (Jorgensen et al. 1989). Growth o f B. bigemina in vitro was first described by Vega et al. (1985 a, b), who used the microaerophilous stationary-phase (MASP) culture techniques of Levy and Ristic (1980). Jorgensen et al. (1989) demonstrated that the Australian vaccine strain of B. bigemina could be maintained in culture for 3 months without either Correspondence to: W.K. Jorgensen
a loss of protective immunogenicity or an increase in virulence. Vega et al. (1986) and Figueroa et al. (1990) successfully scaled up B. bigemina MASP cultures into 75-cm 2 tissue-culture flasks but showed no improvement in parasite yields per millilitre of culture material. These previous culture methods did not produce sufficient numbers of parasites per mitlilitre o f culture medium for economic vaccine production (Jorgensen et al. 1989; Timms and Stewart 1989). At the Tick Fever Research Centre (Australia), an average of 6000 doses of B. bigemina vaccine were used per month in 1990, and the demand is currently increasing. To produce these quantities o f vaccine, large-scale suspension cultures are required, such as those described for growing B. boris (Erp et al. 1980; Timms and Stewart 1989). B. bigemina is more difficult than B. boris to culture successfully (Montenegro-James et al. 1989), and suitable methods for large-scale production have not been described. Our aim, therefore, was to develop an economic, robust method of producing B. bigemina vaccine in vitro. We describe methods of producing B. bigemina for vaccine in suspension cultures using sealed tissue-culture flasks to prevent contamination. Static cultures (MASP and modified MASP) were also prepared for comparative purposes.
Materials and methods
Babesia bigernina strain A strain of B. bigemina (designated the G strain) was used. The strain is currently used as a vaccine strain in Australia (Jorgensen et al. 1989) and South Africa (de Vos et al. 1982).
Me~um The methods used for medium formulation and for the collection and storage of red blood cells (RBC) were similar to the procedures previously described by Vega et al. (1985a, b) and Figueroa et al. (1990). The serum and RBC used were obtained from one donor
424 cow that was maintained in arthropod-free quarantine accommodation for all culture experiments.
Determination o f culture yields A direct counting technique (Parker 1973) was used to quantify the numbers of B. bigemina-parasitised RBC per millilitre of culture. Samples were taken on sub-culture days of each of the different culture methods, and parasite growth rates were compared using this technique.
flat at 37 ~ C. During incubation, the RBC settled and spent medium could be changed by decanting approximately 10 ml and replacing this quantity with fresh medium every day. The parasites were sub-cultured every 2nd day, with half of the original parasitised RBC being replaced by fresh RBC. After being opened, the flasks were gassed by slow bubbling of a gas mix of 4 % 0 2 , 5% COz and 91% N2 (Commonwealth Industrial Gases, Brisbane) into the medium through a 1-ml disposable pipette connected to the gas cylinder. When the foamed medium had reached the flask opening, the pipette was rapidly withdrawn and the flask was sealed and returned to the incubator. M M A S P cultures were used to initiate suspension cultures.
Static cultures Suspension cultures MASP cultures. Primary, microaerophilous stationary-phase (MASP) cultures (Levy and Ristic 1980) were initiated from heparinised blood collected from a splenectomised donor calf infected with G strain B. bigemina. The parasites were maintained in continuous culture using the methods of Vega et al. (1985a, b). One MASP culture of 1.2 ml from a 24-well tissue-culture plate was used to initiate modified MASP cultures (see below).
Modified MASP cultures. Modified MASP (MMASP) cultures were maintained in sealed 25-cm 2 tissue-culture flasks (Corning, New York) containing 15 ml medium plus RBC to a packed cell volume (PCV) of 8%. Flasks containing cultures were incubated
Suspension cultures were maintained in 25- or 75-cm 2 sealed tissueculture flasks (Coming, New York). The 25-cm 2 flasks contained 15 ml medium and RBC to a PCV of 9%. The 75-cm 2 flasks contained 50 ml medium plus RBC to a PCV of 7%, 9%, 13% or 18%, depending on the experiment (Table 1). As the facilities did not enable all cultures to be maintained concurrently, each PCV was tested in succession. Medium was changed daily by centrifuging the flask contents at 192 g for 4 (15 ml) or 10 min (50 ml) at 20 ~ C in a Sorvall RC5C device equipped with a HS4 swing-out rotor. After centrifugation, 10 or 30 ml supernatant was removed, depending on the culture
Table 1. Direct parasite counts used to quantify Babesia bigemina in suspension cultures in 75-cm z flasks at four gas mixes and four different PCVs and in 25-cm 2 flasks at four gas mixes and a 9% PCV Flask size 75 cm 2
25 cm 2
7% PCV % O2ingasmix"
9% PCV % O2ingasmix a
13% PCV % O2ingasmix"
18% PCV % O2ingasmix"
9% PCV % O2ingasmix"
Dayof subculture b
2
2.5
3
3.5
2
2.5
3
3.5
2
2.5
3
3.5
2
2.5
3
3.5
2
2.5
3
3.5
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
7 14 19 9 -
5 10 23 3 -
4 5 7 1
4 11 28 12
7 39 39 18 25 14 15 19 19 14 34 -
7 25 40 24 18 22 13 18 18 7 22 -
7 43 43 32 26 14 16 19 20 9 20 -
7 30 28 23 22 22 10 14 24 12 25
8 33 51 49 42 35 34 38 44 34 69 36 51 .
13 31 26 39 39 42 31 55 42 40 51 52 59 .
8 40 36 45 48 35 40 37 37 33 46 34 46 .
9 33 35 51 29 31 35 49 27 39 47 23 53 .
13 24 38 45 57 33 44 37 49 30 32 42 29
13 26 39 52 51 38 31 30 35 34 28 44 37
13 23 29 55 43 52 47 33 41 49 19 26 31
13 27 19 48 49 31 40 38 27 45 27 22 31
7 13 18 22 40 15 33 31 47 29 32 48 56 22 56 44 49 41 59 57 30
7 23 28 13 25 16 27 28 46 44 50 65 39 36 29 38 47 33 65 40 44
7 17 30 16 25 7 27 35 35 33 42 46 40 42 44 54 60 39 60 46 48
7 17 26 8 17 9 26 28 30 43 34 38 33 40 57 44 37 36 65 46 49
.
.
Data represent numbers of parasites x 106/ml culture. PCV, Packed cell volume; -, culture discontinued a Gas mix contains x % oxygen + 5 % CO2 + balance nitrogen b Direct parasitaemia count ( x 106/ml) at the initiation of culture and thereafter on each day of sub-culture
425 volume, and replaced with fresh medium. The entire culture was returned to the appropriate size flask and gassed as described for the MMASP culture method. Suspension cultures were maintained on a platform rocker (Bioline, Australia) operating at 0.4 Hz in an incubator at 37~ C. The cultures were sub-cultured every 2nd day by removing half of the pellet and supernatant after centrifugation and replacing this with fresh RBC and medium to return the flask culture volume and PCV to the original values. Parasites were grown in cultures at each PCV using 2%, 2.5%, 3% and 3.5% 0 2 with 5% COz plus the balance N2 to assess the effects of changing the percentage of oxygen in the gas mix and altering the PCV on parasite growth (Table I). One series of four replicate 25-cmz flasks was also maintained using only 2% O2, 5% COz and 93% N2 to assess between-flask variability (Table 2). Cultures were maintained for up to 40 days before being discontinued (Table 1).
Results
Static cultures Both M A S P a n d M M A S P cultures were m a i n t a i n e d c o n t i n u o u s l y for 6 m o n t h s . M e a n direct p a r a s i t a e m i a c o u n t s f r o m the latter p a r t o f this period are c o m p a r e d with the results o b t a i n e d for s u s p e n s i o n cultures in Table 3.
Suspension cultures T h e results o f direct p a r a s i t a e m i a c o u n t s for each P C V tested are displayed in Table 1. A n analysis o f v a r i a n c e of the c o u n t s i n d i c a t e d t h a t there was n o significant difference b e t w e e n the four gas mixes, regardless o f w h e t h e r the d a t a for each P C V tested were a n a l y s e d separately or pooled. U s i n g this a s s u m p t i o n , a linear regression analysis was d o n e o n the four c o m b i n e d results o b t a i n e d for each P C V g r o u p i n g f r o m s u b - c u l t u r e day 2 to culture t e r m i n a t i o n . O n l y the c o u n t s d e t e r m i n e d in the 25-cm 2 flasks at a 9 % P C V a n d in the 75-cm 2 flasks at a 13% P C V d e m o n s t r a t e d a linear rise with time. T h e rem a i n d e r d e m o n s t r a t e d a q u a d r a t i c r e l a t i o n s h i p t h a t declined, i m p l y i n g t h a t these cultures c o u l d n o t be m a i n tained indefinitely. The second set o f f o u r s u s p e n s i o n cultures initiated in 25-cm 2 flasks at a n initial p a r a s i t a e m i a o f 12 x 106 p a r a s i t e s / m l a n d m a i n t a i n e d at 9 % P C V u s i n g a gas mix of 2 % 0 2 , 5 % COz a n d 9 3 % N2 were m a i n t a i n e d for 22 days. Table 2 displays the v a r i a t i o n b e t w e e n the four replicates. The s t a n d a r d d e v i a t i o n o f the m e a n s varied between 1.12 a n d 10.17 x 106 p a r a s i t e s / m l over the sample p e r i o d a n d did n o t decrease with the age o f the cultures.
Table 2. Direct parasitaemia counts used to quantify Babesia bigemina in a set of four identical suspension cultures in 25-cm2 sealed tissue-culture flasks Day a
25-cm 2 flask; PCV 9% Replicate number
0 2 4 6 8 I0 12 14 16 18 20 22
i
2
3
4
12 50 41 43 41 42 50 43 45 43 20 33
12 40 45 44 51 37 61 35 36 59 35 23
12 35 39 45 44 42 64 45 30 39 23 22
12 48 35 42 42 50 49 41 57 42 25 16
2
SD
12 43 40 43 44 43 56 41 42 46 26 24
0 6.06 3.61 1.12 3.91 4.66 6.60 3.74 10.17 7.79 5.63 6.10
Data represent numbers of parasites x 106/ml culture. -, Culture discontinued
Comparison of mean yields of MASP, M M A S P and suspension cultures
a Initial parasite count ( x 106/ml) and those obtained on the indicated days of sub-culture
Parasite c o u n t s per millilitre o f c u l t u r e m e d i u m from M A S P , M M A S P a n d s u s p e n s i o n c u l t u r e were c o m p a r e d
Table 3. Mean direct parasitaemia counts obtained for Babesia bigemina using different culture techniques Culture vessel type 1 well in a 24-well plate 25-cm2 STCF 25-cm2 STCF 75-cm2 STCF 75-cm2 STCF 75-cm2 STCF
Culture method
MASP MMASP Suspensionc Suspensiond Suspensione Suspensionf
Culture volume (ml)
1.2 15 15 50 50 50
Culture PCV (%)
Number of samples
Mean DPC x 106/ml (SD)
Vaccine doses/ml b
8
5
5
16.5 (5.02)
1.6
8 9 9 13 18
4 2 2 2 2
9 11 10 12 12
18.0 (6.05) 29.8 (12.04) 23.6 (9.57) 43.0 (10.17) 38.3 (9.06)
1.8 2.9 2.3 4.3 3.8
DPC, Direct parasitaemia count; STCF, sealed tissue-culture flask a Gas m i x = % 0 2 + 5 % CO2+balance N2 b 1 dose= 107 parasites ~ Days 2-22, Table 1 d Days 2-20, Table 1 ~ Days 2-24, Table ] f Days 2-24, Table 1
% 02 in gas mix a
426
(Table 3). Only data for suspension cultures maintained at 2% 02 gas mixes are shown in Table 3, as no significant difference in parasite number was found between the four gas mixes tested (Table 1). An analysis of variance showed that 75-cm 2 flasks at 13% and 18% PCV produced significantly higher yields than did those of the other methods (P
