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Infectivity of Cryopreserved Babesia bovis, Babesia bigemina and Anaplasma centrale for cattle after thawing, dilution and incubation at 30°C
Veterinary Parasitology, 31 ( 1989 ) 243-251 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
243
I n f e c t i v i t y of C r y o p r e s e r v e d Babesia boris, Babesia bigemina and Anaplasma centrale for Cattle after T h a w i n g , D i l u t i o n and Incubation at 30°C W.K. JORGENSEN 1, A.J. DE VOS 1and R.J. DALGLIESH2
'Tick Fever Research Centre and ZAnimal Research Institute, Queensland Department o[ Primary Industries, Wacol, Qld. 4076 (Australia) {Accepted for publication 26 August 1988)
ABSTRACT Jorgensen, W.K., de Vos, A.J. and Dalgliesh, R.J., 1989. Infectivity of cryopreserved Babesia boris, Babesia bigemina and Anaplasma centrale for cattle after thawing, dilution and incubation at 30°C. Vet. Parasitol., 31: 243-251. Blood containing either Babesia boris, Babesia bigemina or Anaplasma centrale was mixed with an equal volume of 3 M glycerol in phosphate-buffered saline with or without glucose and then stored in liquid nitrogen for 2-30 days. After being thawed, the parasitized blood was subjected to various procedures, includingdilution up to 1000-fold followed by incubation at 30 or 4 ° C for 8 h, before infectivity of the parasites was tested in a total of 70 cattle. The results showed that the blood cryopreserved with glycerol remained highly infective after thawing, despite dilution and incubation for 8 h at 30 ° C. The results have practical application in the use of frozen, live vaccines against bovine babesiosis and anaplasmosis.
INTRODUCTION
Vaccination of cattle against the haemoparasites Babesia boris, Babesia bigemina and Anaplasma marginale is a worthwhile practice in many environments where the tick vectors, Boophilus spp., are endemic. Vaccine containing live attenuated strains of the babesias as well as Anaplasma centrale, an avirulent relative of A. marginale, has been used in some countries for many years (Callow, 1977; Callow and Dalgliesh, 1980; de Vos et al., 1982; Pipano et al., 1986). Chilled vaccine is the most widely used (Callow, 1977; de Vos, 1979) but its short shelf-life is a constraint to its wider application (Callow and Dalgliesh, 1980). Cryopreservation has been used to extend the period of usefulness of vaccine (Mellors et al., 1982; Pipano and Hadani, 1984), and offers a practical method for ensuring its availability in regions or countries where 0304-4017/89/$03.50
© 1989 Elsevier Science Publishers B.V.
244
continuous, local production of the chilled product is unwarranted or not costeffective. In preparing frozen vaccine, Mellors et al. (1982) and Pipano et al. (1986) used dimethylsulphoxide (DMSO) as a cryoprotective additive. A disadvantage of DMSO is its toxicity to Babesia at high environmental temperatures (Dalgliesh, 1972a). Vaccine cryopreserved with it therefore has to be used as soon as possible after thawing, preferably within 15 to 30 min (Mellors et al., 1982; Pipano et al., 1986). Glycerol, another cryoprotectant widely used for haemoparasites, is less toxic than DMSO at temperatures above 4°C (Dalgliesh, 1972a). We chose not to use glycerol previously at this laboratory because of its detrimental osmotic effect on Babesia when cattle are inoculated intravenously (Dalgliesh, 1972b). This disadvantage of glycerol is not a serious constraint in practical vaccination, for which the subcutaneous route is more convenient. We have therefore re-examined glycerol as a cryoprotective additive for frozen vaccines against bovine babesiosis and anaplasmosis, placing emphasis on testing the longevity of the parasites after thawing. This paper records the results obtained for periods of up to 8 h after thawing. MATERIALS AND METHODS
Experimental cattle Seventy Bos taurus yearling steers were used to test the infectivity of experimental inocula. The cattle were purchased from a Boophilus-free area of Queensland. At the laboratory, they were found to be serologically negative for antibodies to Babesia boris in an indirect fluorescent antibody (IFA) test (Callow et al., 1987), and to Anaplasma in a card agglutination test (Amerault and Roby, 1968). The cattle were maintained on pasture under tick-free conditions. The cattle were purchased in two consignments of 30 and 40 cattle each and used in two separate experiments approximately 3 months apart.
Parasites and collection of blood The strains of B. boris and B. bigemina, designated K and G, respectively, and the strain of A. centrale used were described by Mellors et al. (1982). All three strains are used routinely in the commercial vaccines prepared at this laboratory. Infections with each parasite are established separately by blood inoculation in splenectomized calves, these being used subsequently as vaccine donors (Callow and Dalgliesh, 1980 ). Parasitized blood was collected from vaccine donors into heparin for the present study. Samples for estimation of the parasite concentration (Parker, 1973 ) were taken from the blood immediately after collection.
245
Cryopreservation of parasitized blood The following procedures were used for each parasite. Immediately after collection, parasitized blood was gradually diluted at 37 °C with an equal volume of 3 M glycerol in phosphate buffered saline (PB S ) supplemented with 10 m M glucose. The diluted blood was held at 37 °C for 20 min to allow the glycerol to equilibrate with the erythrocytes (Dalgliesh, 1969 ). In some observations, supplementary glucose was excluded from the glycerol solution to determine whether parasite infectivity was affected. After equilibration, the diluted blood was dispensed in 1.5-ml aliquots into 12-cm lengths of plastic drinking straws ("Deeko" Australia Pty, Ltd., Melbourne, Victoria), with one end of the straws being previously heat sealed. The straws were then placed horizontally on a cardboard rack 4 cm above the surface of liquid nitrogen, approximately 5 cm in depth, within an insulating polystyrene carton. After 15 min, the straws were put into polypropylene goblets and immersed in liquid nitrogen for storage. Different batches of frozen vaccine were prepared and tested in the two different experiments. The respective parasite concentrations in each millilitre of glycerolized blood prior to freezing are shown in Table 1. However, the proportion of parasites killed by the freezing and thawing processes was unknown, thus making calculation of the number of viable organisms in the eventual inocula impossible.
Thawing and dilution of parasitized blood After 2-30 days of frozen storage, the straws were placed vertically in water at 37°C and the thawed contents pooled in 100-ml, scew-top, glass jars. The thawed blood was diluted at room temperature with PBS containing 1.5 M glycerol and, when appropriate, 5 m M glucose. TABLE 1 Parasite concentrations in glycerolized blood prior to cryopreservation and testing for infectivity in two experiments Experiment No.
Species of parasites B. bov~
B. bigemina
A. centrale
(X106)
(X106)
(X106)
1
128
205
Not done
2
145
30
212
246
Inoculation and monitoring of infection The dose of experimental inoculum was I ml when a single parasite species was being tested, and 2 ml in one experiment in which thawed blood with B. bigemina and A. centrale were mixed in equal volumes. Inoculation was by the subcutaneous route. B. boris and B. bigemina infections were monitored by preparing thick smears (Mahoney and Saal, 1961 ) of tail-tip blood daily for up to 28 days after inoculation. For A. centrale, thin smears of tail-tip blood were taken twice weekly for 10 weeks. The smears were stained with Giemsa's stain and examined under oil immersion at 800 X magnification. B. boris parasites were not detected in a proportion of inoculated cattle due to the low parasitaemias resulting from infection with the strain used in this study. In these cases, the presence of circulating B. bovis antibodies was taken as an indication of infection. To determine if seroconversion occurred, sera were collected from all cattle before and again 4 weeks after inoculation and tested for antibodies to B. bovis by the IFA test.
Experimental design In this work, we regarded cryopreserved, parasitized blood as 'frozen vaccine concentrate' suitable for dilution following thawing, thereby increasing the usable quantity. The general aim was to test the effects of dilution and exposure to a moderately high ambient temperature (30 °C) on the infectivity of parasitized blood after cryopreservation.
Experiment 1 Six groups of five cattle were used to test B. bovis vaccine, then 6 weeks later, B. bigemina vaccine. The principle of using the same animals to test the two Babesia spp. is valid, as B. boris induces no cross-protective immunity against B. bigemina (Legg, 1935; L.L. Callow, unpublished observations, 1979). In the case of B. boris, three groups were respectively inoculated with undiluted vaccine diluted 1:10 and 1:1000 within 30 min of thawing. The remaining three groups were inoculated with undiluted vaccine held at 30 ° C for 2, 4 and 8 h, respectively (see Table 2). Glucose was added to the vaccine used in all the groups. In the case ofB. bigemina, three groups were inoculated with undiluted vaccine containing added glucose, within 30 min of thawing and after incubation of the vaccine at 30 ° C for 4 and 8 h, respectively. The remaining three groups were treated in a similar fashion but with vaccine lacking supplementary glucose (see Table 3).
247
Experiment 2 Eight groups of five cattle were used to test B. bovis vaccine initially, then 4 weeks later, a combination of B. bigemina and A. centrale vaccines. Three of the groups were respectively inoculated within 30 min of thawing with undiluted vaccine, and vaccine diluted 1:10 and 1:100. A further three groups were inoculated with vaccine diluted as above and held for 8 h at 30 °C before use. In the case of B. boris, the final two groups were inoculated with vaccine cryopreserved without supplementary glucose; one of these groups was inoculated within 30 min after thawing the blood and the other after 8 h at 30°C (see Table 2). In the case of B. bigemina and A. centrale, the final two groups were used to demonstrate the infectivity of undiluted vaccine and vaccine diluted 1:100 after 8 h storage at 4°C.
RESULTS
To facilitate comparisons between results obtained from the two experiments, the results are described under headings for the three parasite species. TABLE 2 Effect of dilution, incubation a n d addition of glucose on the infectivity for cattle of frozen-thawed
Babesia bovis after subcutaneous inoculation Experiment No.
Dilution
Incubation
Glucose added l
IR 2
DP 3
1
Undiluted Diluted 1/10 Diluted 1/1000 Undiluted Undiluted Undiluted
< 30 rain < 30 min < 30 rain 2 h at 30 °C 4 h at 30 °C 8 h at 30 °C
+ + + + + +
5/5 4/5 5/5 5/5 5/5 5/5
4 4 5 4 3 5
2
Undiluted Diluted 1/10 Diluted 1/100 Undiluted Diluted 1/10 Diluted 1/100 Undiluted Undiluted
< 30 rain < 3 0 rain < 30 rain 8 h at 30°C 8 h at 30°C 8 h at 30°C < 30 m i n 8 h at 30 °C
+ + + + + + -
5/5 5/5 4/5 5/5 5/5 5/5 5/5 4/44
5 5 4 5 4 2 5 4
1Glucose added to cryoprotectant a n d diluent. 2No. of cattle infected per n u m b e r inoculated based on seroconversion. ~No. of cattle with detectable parasitaemias. 4One animal in group not inoculated.
248 TABLE3 Effect of dilution, incubation a n d addition of glucose on the infectivity for cattle of frozen-thawed
Babesia bigemina a n d Anaplasma centrale after subcutaneous inoculation Experiment No.
Dilution
Incubation
Glucose added 1
Infectivity 2
B. bigernina
A. centrale
Undiluted Undiluted Undiluted Undiluted Undiluted Undiluted
< 30 m i n < 30 m i n 4 h at 30°C 4 h at 30°C 8 h at 30 ° C 8 h at 30 °C
+ + +
5/5 5/5 5/5 5/5 5/5 5/5
ND :~ ND ND ND ND ND
Undiluted Diluted 1/10 Diluted 1/100 Undiluted Diluted 1/10 Diluted 1/100 Undiluted Diluted 1/100
< 30 m i n < 30 m i n <30 min 8 h at 30 °C 8 h at 30°C 8 h at 30°C 8 h at 4°C 8 h at 4°C
+ + + + + + + +
5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5
5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5
~Glucose added to cryoprotectant a n d diluent. 2No. of cattle infected per n u m b e r inoculated. :~Not done.
B. bovis The results are summarized in Table 2. Parasitaemias were difficult to detect in many cattle because the strain used is relatively avirulent. In fact, no parasites were seen in 10 of 69 cattle inoculated. However, all but two seroconverted, indicating that infections had occurred. Thus, neither dilution nor incubation at 30°C for 8 h, nor a combination of both, caused a pronounced decrease in infectivity; neither did omission of glucose from the diluent. B. bigemina and A. centrale All recipient cattle became infected (Table 3), indicating that none of the treatments had a pronounced effect on infectivity. The strains of B. bigemina and A. centrale used produced consistently detectable parasitaemias, making serology unnecessary to assess infectivity of the inocula. DISCUSSION
The efficacy of living vaccines against bovine babesiosis and anaplasmosis relies on infection being established in recipient cattle. In the present work,
249
cryopreserved parasites of the three species studied were consistently infective for at least 8 h after thawing without special precautions, such as storage at 4 ° C or the addition of glucose {Farlow, 1976), to maintain their viability after thawing. Moreover, parasitized blood diluted at least 100-fold after thawing provided infective inocula. We consider that the findings increase the potential of cryopreserved vaccines for use under adverse environmental conditions. An 8-h "safety margin" for cryopreserved vaccines has particular relevance to Third World countries where financial and environmental constraints to effective vaccination are greatest. In some countries, cattle at risk to tick-borne disease may be housed individually or in small groups in areas not easily reached by modern transport. Our results suggest that frozen vaccines could be thawed at a central depot, or within a suitably equipped vehicle, and transported for considerable distances without the need for refrigeration or bulky, insulated packaging. The possibility that the vaccines can be diluted following thawing, thus providing additional quantities if required, adds to the flexibility and economic attractiveness of their use in the field. However, the concept of dilutable, cryopreserved vaccines should be considered with caution. The use of blood with lower initial parasite concentrations than those used in this study may result in infectivity being lost with even a 10-fold dilution following thawing. Moreover, many factors affect the survival rate of Babesia and other haemoparasites following cryopreservation (Dalgliesh, 1972a), so that a standard procedure for post-thawing dilution may be inappropriate. Ideally, a safety margin for dilution would be established with each batch of cryopreserved vaccine before it is used in the field. In the preparation and use of frozen vaccines containing Babesia, potentially damaging osmotic effects of cryoprotective additives, particularly glycerol, are an important consideration because they may result in loss of infectivity (Dalgliesh, 1972a,b; Mellors et al., 1982). Procedures adopted in the present work to minimize these effects included the initial mixing of parasitized blood with glycerol solution at 37 ° C, thus avoiding apparent osmotic effects during equilibration of glycerol at lower temperatures (Dalgliesh, 1972a), dilution following thawing with diluent containing an equivalent concentration of glycerol and inoculation of cattle by the subcutaneous rather than intravenous route. None of the procedures is technically difficult. In fact, subcutaneous inoculation is more convenient for practical vaccination. Various types of containers have been used in the cryopreservation of Babesia and Anaplasma. We chose plastic drinking straws as containers mainly for their availability and low cost compared to proprietary cryovials. The straws used were a blend ofpolypropylene and high density polyethylene (J.S. Rosenthal, Operations Manager, "Deeko" Australia, personal communication, 1987), and were able to be autoclaved and heat sealed. We tested their toxicity for the rodent parasite Babesia microti, using methods similar to those of Timms
250 (1981), a n d f o u n d t h e m to be n o n - t o x i c ( W . K . J o r g e n s e n , u n p u b l i s h e d data, 1987). ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y f u n d s p r o v i d e d by t h e A u s t r a l i a n C e n t r e for I n t e r n a t i o n a l A g r i c u l t u r a l R e s e a r c h w i t h i n P r o j e c t N u m b e r 8321, a n d b y the Q u e e n s l a n d D e p a r t m e n t of P r i m a r y I n d u s t r i e s . W e t h a n k D. S t e v e n s o n a n d D. M c L e l l a n for t h e i r able assistance. T h e e x p e r i m e n t s p e r f o r m e d were app r o v e d b y t h e A n i m a l R e s e a r c h I n s t i t u t e A n i m a l E t h i c s Review C o m m i t t e e .
REFERENCES Amerault, T.E. and Roby, T.O., 1968. A rapid card agglutination test for bovine anaplasmosis. J. Am. Vet. Med. Assoc., 153: 1828-1834. Callow, L.L., 1977. Vaccination against bovine babesiosis. In: L.H. Miller, J.A. Pino and J.J. McKelvey, Jr. (Editors), Immunity to Blood Parasites of Animals and Man. Plenum Press, New York, pp. 121-149. Callow, L.L. and Dalgliesh, R.J., 1980. The development of effective, safe vaccination against babesiosis and anaplasmosis in Australia. In: L.A.Y. Johnston and M.G. Cooper (Editors), Ticks and Tick-borne Diseases. Proc. Symp. 56th Annu. Conf. Aust. Vet. Assoc., Townsville, 14-18 May 1979. Aust. Vet. Assoc. Sydney, pp. 4-8. Callow, L.L., Rogers, R.J. and de Vos, A.J., 1987. Standard Diagnostic Techniques for Tick-borne Diseases (Babesiosis and Anaplasmosis ) of Cattle in Australia. Australian Agricultural Coum cil, CSIRO, Melbourne, pp. 1-28. Dalgliesh, R.J., 1969. Permeation of bovine erythrocytes with glycerol and their protection during rapid freezing and thawing. Res. Vet. Sci., 10: 351-354. Dalgliesh, R.J., 1972a. Theoretical and practical aspects of freezing parasitic protozoa. Aust. Vet. J., 48: 233-239. Dalgliesh, R.J., 1972b. Effects of low temperature and route of inoculation on infectivity of Babesia bigemina in blood diluted with glycerol. Res. Vet. Sci., 13: 540-545. De Vos, A.J., 1979. Epidemiology and control of bovine babesiosis in South Africa. J. S. Afr. Vet. Assoc., 50: 357-362. De Vos, A.J., Bessenger, R. and Fourie, C.G., 1982. Virulence and heterologous strain immunity of South African and Australian Babesia boris strains with reduced pathogenicity. Onderstepoort J. Vet. Res., 49: 133-136. Farlow, G.E., 1976. Differences in the infectivity of Babesia incubated in plasma and serum and the role of glucose in prolonging viability. Int. J. Parasitol., 66: 513-516. Legg,J., 1935. The occurrence of bovine babesiellosis in Northern Australia. Council for Scientific and Industrial Research, Australia, Pamphlet No. 56, pp. 1-48. Mahoney, D.F. and Saal, J.R., 1961. Bovine babesiosis: thick blood films for the detection of parasitaemia. Aust. Vet. J., 37: 44-47. Mellors, L.T., Dalgliesh, R.J., Timms, P., Rodwell, B.J. and Callow, L.L., 1982. Preparation and laboratory testing of a frozen vaccine containing Babesia bovis, Babesia bigemina and Anaplasma centrale. Res. Vet. Sci., 32: 194-197. Parker, R., 1973. A direct counting technique for estimating high parasitaemias in infections of Babesia argentina, Babesia bigemina and Plasmodium berghei. Ann. Trop. Med. Parasitol., 67: 387-390.
251 Pipano, E. and Hadani, A., 1984. Control of bovine babesiosis. In: M. Ristic, P. Ambroise-Thomas and J.P. Kreier (Editors), Malaria and Babesiosis. Martinus Nijhoff, Dordrecht, pp. 263-281. Pipano, E., Krigel, Y., Frank, M., Markovics, A. and Mayer, E., 1986. Frozen Anaplasma centrale vaccine against anaplasmosis in cattle. Br. Vet. J., 142: 553-556. Timms, P., 1981. Inhibition ofBabesia spp. by plastics. Appl. Environ. Microbiol., 41: 17-19.
243
I n f e c t i v i t y of C r y o p r e s e r v e d Babesia boris, Babesia bigemina and Anaplasma centrale for Cattle after T h a w i n g , D i l u t i o n and Incubation at 30°C W.K. JORGENSEN 1, A.J. DE VOS 1and R.J. DALGLIESH2
'Tick Fever Research Centre and ZAnimal Research Institute, Queensland Department o[ Primary Industries, Wacol, Qld. 4076 (Australia) {Accepted for publication 26 August 1988)
ABSTRACT Jorgensen, W.K., de Vos, A.J. and Dalgliesh, R.J., 1989. Infectivity of cryopreserved Babesia boris, Babesia bigemina and Anaplasma centrale for cattle after thawing, dilution and incubation at 30°C. Vet. Parasitol., 31: 243-251. Blood containing either Babesia boris, Babesia bigemina or Anaplasma centrale was mixed with an equal volume of 3 M glycerol in phosphate-buffered saline with or without glucose and then stored in liquid nitrogen for 2-30 days. After being thawed, the parasitized blood was subjected to various procedures, includingdilution up to 1000-fold followed by incubation at 30 or 4 ° C for 8 h, before infectivity of the parasites was tested in a total of 70 cattle. The results showed that the blood cryopreserved with glycerol remained highly infective after thawing, despite dilution and incubation for 8 h at 30 ° C. The results have practical application in the use of frozen, live vaccines against bovine babesiosis and anaplasmosis.
INTRODUCTION
Vaccination of cattle against the haemoparasites Babesia boris, Babesia bigemina and Anaplasma marginale is a worthwhile practice in many environments where the tick vectors, Boophilus spp., are endemic. Vaccine containing live attenuated strains of the babesias as well as Anaplasma centrale, an avirulent relative of A. marginale, has been used in some countries for many years (Callow, 1977; Callow and Dalgliesh, 1980; de Vos et al., 1982; Pipano et al., 1986). Chilled vaccine is the most widely used (Callow, 1977; de Vos, 1979) but its short shelf-life is a constraint to its wider application (Callow and Dalgliesh, 1980). Cryopreservation has been used to extend the period of usefulness of vaccine (Mellors et al., 1982; Pipano and Hadani, 1984), and offers a practical method for ensuring its availability in regions or countries where 0304-4017/89/$03.50
© 1989 Elsevier Science Publishers B.V.
244
continuous, local production of the chilled product is unwarranted or not costeffective. In preparing frozen vaccine, Mellors et al. (1982) and Pipano et al. (1986) used dimethylsulphoxide (DMSO) as a cryoprotective additive. A disadvantage of DMSO is its toxicity to Babesia at high environmental temperatures (Dalgliesh, 1972a). Vaccine cryopreserved with it therefore has to be used as soon as possible after thawing, preferably within 15 to 30 min (Mellors et al., 1982; Pipano et al., 1986). Glycerol, another cryoprotectant widely used for haemoparasites, is less toxic than DMSO at temperatures above 4°C (Dalgliesh, 1972a). We chose not to use glycerol previously at this laboratory because of its detrimental osmotic effect on Babesia when cattle are inoculated intravenously (Dalgliesh, 1972b). This disadvantage of glycerol is not a serious constraint in practical vaccination, for which the subcutaneous route is more convenient. We have therefore re-examined glycerol as a cryoprotective additive for frozen vaccines against bovine babesiosis and anaplasmosis, placing emphasis on testing the longevity of the parasites after thawing. This paper records the results obtained for periods of up to 8 h after thawing. MATERIALS AND METHODS
Experimental cattle Seventy Bos taurus yearling steers were used to test the infectivity of experimental inocula. The cattle were purchased from a Boophilus-free area of Queensland. At the laboratory, they were found to be serologically negative for antibodies to Babesia boris in an indirect fluorescent antibody (IFA) test (Callow et al., 1987), and to Anaplasma in a card agglutination test (Amerault and Roby, 1968). The cattle were maintained on pasture under tick-free conditions. The cattle were purchased in two consignments of 30 and 40 cattle each and used in two separate experiments approximately 3 months apart.
Parasites and collection of blood The strains of B. boris and B. bigemina, designated K and G, respectively, and the strain of A. centrale used were described by Mellors et al. (1982). All three strains are used routinely in the commercial vaccines prepared at this laboratory. Infections with each parasite are established separately by blood inoculation in splenectomized calves, these being used subsequently as vaccine donors (Callow and Dalgliesh, 1980 ). Parasitized blood was collected from vaccine donors into heparin for the present study. Samples for estimation of the parasite concentration (Parker, 1973 ) were taken from the blood immediately after collection.
245
Cryopreservation of parasitized blood The following procedures were used for each parasite. Immediately after collection, parasitized blood was gradually diluted at 37 °C with an equal volume of 3 M glycerol in phosphate buffered saline (PB S ) supplemented with 10 m M glucose. The diluted blood was held at 37 °C for 20 min to allow the glycerol to equilibrate with the erythrocytes (Dalgliesh, 1969 ). In some observations, supplementary glucose was excluded from the glycerol solution to determine whether parasite infectivity was affected. After equilibration, the diluted blood was dispensed in 1.5-ml aliquots into 12-cm lengths of plastic drinking straws ("Deeko" Australia Pty, Ltd., Melbourne, Victoria), with one end of the straws being previously heat sealed. The straws were then placed horizontally on a cardboard rack 4 cm above the surface of liquid nitrogen, approximately 5 cm in depth, within an insulating polystyrene carton. After 15 min, the straws were put into polypropylene goblets and immersed in liquid nitrogen for storage. Different batches of frozen vaccine were prepared and tested in the two different experiments. The respective parasite concentrations in each millilitre of glycerolized blood prior to freezing are shown in Table 1. However, the proportion of parasites killed by the freezing and thawing processes was unknown, thus making calculation of the number of viable organisms in the eventual inocula impossible.
Thawing and dilution of parasitized blood After 2-30 days of frozen storage, the straws were placed vertically in water at 37°C and the thawed contents pooled in 100-ml, scew-top, glass jars. The thawed blood was diluted at room temperature with PBS containing 1.5 M glycerol and, when appropriate, 5 m M glucose. TABLE 1 Parasite concentrations in glycerolized blood prior to cryopreservation and testing for infectivity in two experiments Experiment No.
Species of parasites B. bov~
B. bigemina
A. centrale
(X106)
(X106)
(X106)
1
128
205
Not done
2
145
30
212
246
Inoculation and monitoring of infection The dose of experimental inoculum was I ml when a single parasite species was being tested, and 2 ml in one experiment in which thawed blood with B. bigemina and A. centrale were mixed in equal volumes. Inoculation was by the subcutaneous route. B. boris and B. bigemina infections were monitored by preparing thick smears (Mahoney and Saal, 1961 ) of tail-tip blood daily for up to 28 days after inoculation. For A. centrale, thin smears of tail-tip blood were taken twice weekly for 10 weeks. The smears were stained with Giemsa's stain and examined under oil immersion at 800 X magnification. B. boris parasites were not detected in a proportion of inoculated cattle due to the low parasitaemias resulting from infection with the strain used in this study. In these cases, the presence of circulating B. bovis antibodies was taken as an indication of infection. To determine if seroconversion occurred, sera were collected from all cattle before and again 4 weeks after inoculation and tested for antibodies to B. bovis by the IFA test.
Experimental design In this work, we regarded cryopreserved, parasitized blood as 'frozen vaccine concentrate' suitable for dilution following thawing, thereby increasing the usable quantity. The general aim was to test the effects of dilution and exposure to a moderately high ambient temperature (30 °C) on the infectivity of parasitized blood after cryopreservation.
Experiment 1 Six groups of five cattle were used to test B. bovis vaccine, then 6 weeks later, B. bigemina vaccine. The principle of using the same animals to test the two Babesia spp. is valid, as B. boris induces no cross-protective immunity against B. bigemina (Legg, 1935; L.L. Callow, unpublished observations, 1979). In the case of B. boris, three groups were respectively inoculated with undiluted vaccine diluted 1:10 and 1:1000 within 30 min of thawing. The remaining three groups were inoculated with undiluted vaccine held at 30 ° C for 2, 4 and 8 h, respectively (see Table 2). Glucose was added to the vaccine used in all the groups. In the case ofB. bigemina, three groups were inoculated with undiluted vaccine containing added glucose, within 30 min of thawing and after incubation of the vaccine at 30 ° C for 4 and 8 h, respectively. The remaining three groups were treated in a similar fashion but with vaccine lacking supplementary glucose (see Table 3).
247
Experiment 2 Eight groups of five cattle were used to test B. bovis vaccine initially, then 4 weeks later, a combination of B. bigemina and A. centrale vaccines. Three of the groups were respectively inoculated within 30 min of thawing with undiluted vaccine, and vaccine diluted 1:10 and 1:100. A further three groups were inoculated with vaccine diluted as above and held for 8 h at 30 °C before use. In the case of B. boris, the final two groups were inoculated with vaccine cryopreserved without supplementary glucose; one of these groups was inoculated within 30 min after thawing the blood and the other after 8 h at 30°C (see Table 2). In the case of B. bigemina and A. centrale, the final two groups were used to demonstrate the infectivity of undiluted vaccine and vaccine diluted 1:100 after 8 h storage at 4°C.
RESULTS
To facilitate comparisons between results obtained from the two experiments, the results are described under headings for the three parasite species. TABLE 2 Effect of dilution, incubation a n d addition of glucose on the infectivity for cattle of frozen-thawed
Babesia bovis after subcutaneous inoculation Experiment No.
Dilution
Incubation
Glucose added l
IR 2
DP 3
1
Undiluted Diluted 1/10 Diluted 1/1000 Undiluted Undiluted Undiluted
< 30 rain < 30 min < 30 rain 2 h at 30 °C 4 h at 30 °C 8 h at 30 °C
+ + + + + +
5/5 4/5 5/5 5/5 5/5 5/5
4 4 5 4 3 5
2
Undiluted Diluted 1/10 Diluted 1/100 Undiluted Diluted 1/10 Diluted 1/100 Undiluted Undiluted
< 30 rain < 3 0 rain < 30 rain 8 h at 30°C 8 h at 30°C 8 h at 30°C < 30 m i n 8 h at 30 °C
+ + + + + + -
5/5 5/5 4/5 5/5 5/5 5/5 5/5 4/44
5 5 4 5 4 2 5 4
1Glucose added to cryoprotectant a n d diluent. 2No. of cattle infected per n u m b e r inoculated based on seroconversion. ~No. of cattle with detectable parasitaemias. 4One animal in group not inoculated.
248 TABLE3 Effect of dilution, incubation a n d addition of glucose on the infectivity for cattle of frozen-thawed
Babesia bigemina a n d Anaplasma centrale after subcutaneous inoculation Experiment No.
Dilution
Incubation
Glucose added 1
Infectivity 2
B. bigernina
A. centrale
Undiluted Undiluted Undiluted Undiluted Undiluted Undiluted
< 30 m i n < 30 m i n 4 h at 30°C 4 h at 30°C 8 h at 30 ° C 8 h at 30 °C
+ + +
5/5 5/5 5/5 5/5 5/5 5/5
ND :~ ND ND ND ND ND
Undiluted Diluted 1/10 Diluted 1/100 Undiluted Diluted 1/10 Diluted 1/100 Undiluted Diluted 1/100
< 30 m i n < 30 m i n <30 min 8 h at 30 °C 8 h at 30°C 8 h at 30°C 8 h at 4°C 8 h at 4°C
+ + + + + + + +
5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5
5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5
~Glucose added to cryoprotectant a n d diluent. 2No. of cattle infected per n u m b e r inoculated. :~Not done.
B. bovis The results are summarized in Table 2. Parasitaemias were difficult to detect in many cattle because the strain used is relatively avirulent. In fact, no parasites were seen in 10 of 69 cattle inoculated. However, all but two seroconverted, indicating that infections had occurred. Thus, neither dilution nor incubation at 30°C for 8 h, nor a combination of both, caused a pronounced decrease in infectivity; neither did omission of glucose from the diluent. B. bigemina and A. centrale All recipient cattle became infected (Table 3), indicating that none of the treatments had a pronounced effect on infectivity. The strains of B. bigemina and A. centrale used produced consistently detectable parasitaemias, making serology unnecessary to assess infectivity of the inocula. DISCUSSION
The efficacy of living vaccines against bovine babesiosis and anaplasmosis relies on infection being established in recipient cattle. In the present work,
249
cryopreserved parasites of the three species studied were consistently infective for at least 8 h after thawing without special precautions, such as storage at 4 ° C or the addition of glucose {Farlow, 1976), to maintain their viability after thawing. Moreover, parasitized blood diluted at least 100-fold after thawing provided infective inocula. We consider that the findings increase the potential of cryopreserved vaccines for use under adverse environmental conditions. An 8-h "safety margin" for cryopreserved vaccines has particular relevance to Third World countries where financial and environmental constraints to effective vaccination are greatest. In some countries, cattle at risk to tick-borne disease may be housed individually or in small groups in areas not easily reached by modern transport. Our results suggest that frozen vaccines could be thawed at a central depot, or within a suitably equipped vehicle, and transported for considerable distances without the need for refrigeration or bulky, insulated packaging. The possibility that the vaccines can be diluted following thawing, thus providing additional quantities if required, adds to the flexibility and economic attractiveness of their use in the field. However, the concept of dilutable, cryopreserved vaccines should be considered with caution. The use of blood with lower initial parasite concentrations than those used in this study may result in infectivity being lost with even a 10-fold dilution following thawing. Moreover, many factors affect the survival rate of Babesia and other haemoparasites following cryopreservation (Dalgliesh, 1972a), so that a standard procedure for post-thawing dilution may be inappropriate. Ideally, a safety margin for dilution would be established with each batch of cryopreserved vaccine before it is used in the field. In the preparation and use of frozen vaccines containing Babesia, potentially damaging osmotic effects of cryoprotective additives, particularly glycerol, are an important consideration because they may result in loss of infectivity (Dalgliesh, 1972a,b; Mellors et al., 1982). Procedures adopted in the present work to minimize these effects included the initial mixing of parasitized blood with glycerol solution at 37 ° C, thus avoiding apparent osmotic effects during equilibration of glycerol at lower temperatures (Dalgliesh, 1972a), dilution following thawing with diluent containing an equivalent concentration of glycerol and inoculation of cattle by the subcutaneous rather than intravenous route. None of the procedures is technically difficult. In fact, subcutaneous inoculation is more convenient for practical vaccination. Various types of containers have been used in the cryopreservation of Babesia and Anaplasma. We chose plastic drinking straws as containers mainly for their availability and low cost compared to proprietary cryovials. The straws used were a blend ofpolypropylene and high density polyethylene (J.S. Rosenthal, Operations Manager, "Deeko" Australia, personal communication, 1987), and were able to be autoclaved and heat sealed. We tested their toxicity for the rodent parasite Babesia microti, using methods similar to those of Timms
250 (1981), a n d f o u n d t h e m to be n o n - t o x i c ( W . K . J o r g e n s e n , u n p u b l i s h e d data, 1987). ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y f u n d s p r o v i d e d by t h e A u s t r a l i a n C e n t r e for I n t e r n a t i o n a l A g r i c u l t u r a l R e s e a r c h w i t h i n P r o j e c t N u m b e r 8321, a n d b y the Q u e e n s l a n d D e p a r t m e n t of P r i m a r y I n d u s t r i e s . W e t h a n k D. S t e v e n s o n a n d D. M c L e l l a n for t h e i r able assistance. T h e e x p e r i m e n t s p e r f o r m e d were app r o v e d b y t h e A n i m a l R e s e a r c h I n s t i t u t e A n i m a l E t h i c s Review C o m m i t t e e .
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