Bulk-freezing of Theileria parva stabilates for vaccine production

InfernafionaI

Journal/or

Pergamon 0020-7519(95)00102-6

Bulk-Freezing

Ponuirology,

Vol. 26, No. 1, pp. 67-70, 1996 Australian Society for Parasitology Elsevier Science Ltd Printed in Great Britain 002&7519/96 SlS.Nl + 0.00

of Theileria parva Stabilates Vaccine Production L. M. NJUGUNA

for

and F. L. MUSISI

Tick-borne Diseases Vaccine Production Centre, C/o FAO Representative, P.O. Box 30750, Lilongwe 3, Malawi (Received

9 January

1995; accepted

25 August

1995)

Abstract-Njuguna L. M, & Musisi F. L. 1996. Bulkfreezing of Theileria purvlrstabilntes for vaccine production. Znternatiomd kwalfir Parusitology 26: 67-70. The freezing and thawing characteristics of different volume samples of Treezing medium and Theileria purva stabilates were studied in order to identify suitable conditions for freezing large volumes of stabilatea for use in the preparation of vaccines. Conventionally, 1.8 ml aliquots are used but are cumbersome to handle in the field preparation of trivalent vaccines. In this study, 75 ml aliquots of the stabilates were found to exhibit freezing and thawing characteristics similar to those of the 1.8 ml aliquots. Bullc freezing of 2 stabilatea yielded sporozoites which remained infective for cattle upon thawing, induced a range of clinical reactions in vaccinated cattle and stimulated the development of inunune reactions. Key words:

bulk-freezing;

freezing

medium;

stabilates;

Theileria

parva;

vaccine

production.

infective (Musisi, personal observations). On a large scale, however, several hours would be required to thaw thousands of 2-ml vials in which individual stabilate component stocks are conventionally cryopreserved. The prolonged period of thawing, pooling each component and then mixing and dispensing the components would result in serious loss of viability of the pooled trivalent stabilate, thus rendering the technique of thawing and refreezing useless. To overcome this problem, preliminary investigations were conducted to assess the feasibility of freezing components of the T. parva trivalent stabilate in large volumes. This paper describes and discusses preliminary results obtained with freezing and thawing stabilates in aliquots ranging from 1.8 to 150 ml.

INTRODUCTION

East Coast Fever (ECF) (Theileria parva infection of cattle) is a major constraint to the improvement of the cattle industry in Eastern and Central Africa. A method of immunizing cattle against ECF using 3 Theileria parvn stocks with simultaneous administration of long-acting oxytetracyclines was developed at the former East African Veterinary Research Organization (EAVRO), Muguga, Kenya (Radley, 1981). The method requires harvesting and freezing sporozoites of 3 separate T. parva stocks into stabilates (Lumsden Kc Hardy, 1965) with subsequent thawing and mixing in appropriate predetermined ratios at the time of immunization. The procedure, however, requires extra equipment in the field (such as pipettes and beakers) and mistakes could be made in measurements during mixing of the different components, and contamination of the stabilates with dust is possible. On a small scale, it was demonstrated at the Vaccine Production Centre, Lilongwe, that T. parva stabilates could be thawed and refrozen but remain

MATERIALS AND METHODS Determination of optimalvolume to freeze. Eagle’s Minimum Essential Medium (MEM) with 3.5% bovine plasma albumin (BPA) and 7.5% glycerol constituted the freezing medium. Aliquots (1.8 ml) of the medium were dispensed into 32x 2 ml Bio-freeze vials (Costar Corporation, 67

68

L. M. Njuguna Table

Expt

l-Response

of cattle

Container

to T. parva Animal number

stabilate

and F. L. Musisi

inocula

derived

Stabilate number

from

Days

2 ml vials and 250 ml transfusion

of detection

Schizonts 1

Plastic

vials

Transfusion 2

Plastic

vials

Transfusion *Sera

collected

bags

bags

on day 21 before

of

Treatment (day)

baas Day 28 serology

Fever

153 154

77 77

755 757

77 77

9-21 8-10

7, l&21

826 829

79 79

lo-18 11-17

12-15 12-16

14/16 14116

640

832 835

79 19

11-17 l&l8

12-19 12-16

14/16 14/16

640 640

death

was positive

l&l 1 67

for IFAT

Cambridge, MA 012140, U.S.A.). The vials were arrangedin a single row inside a cardboard container. A hole was punched into a screw cap of one of the vials and a probe of a digital platinum resistance thermometer, KM200 (KaneMay Measuring Instruments) was secured tightly with adhesive tape. The vial having the probe was placed in the middle of the row of the 31 vials. The initial temperature was recorded and the entire batch of vials placed into a - 70°C

deep freezer; thereafter the temperature was recorded every 10 min for up to 90 min. This procedure was repeated using 50 and 100 ml volumes of medium in 250 ml anticoagulantfree transfusion bags, Blood Bag CPD-A250 (Helm Pharmaceuticals GmbH) and 75, 120 and 150 ml aliquots in 600 ml anticoagulant-free transfusion bags, Transfer Pack (Traveno1 Laboratories Ltd). The drop in temperature in the bags was monitored over a period of 3 h. Twenty-four hours later, the contents of the vials and bags were thawed rapidly in a waterbath at 40°C and the rise in temperature was monitored. A volume in the transfusion bags with a cooling curve closest to that of the conventional aliquots in the vials was selected for subsequent experiments involving freezing of T. parva stabilates. The experiment was repeated using T. parva stabilate at 1.8 ml aliquots in vials and 75 ml aliquots in 250 ml transfusion bags. The ability of the 2 T. parva parasite stocks in the stabilates to infect cattle was assessed. Parasites, ticks, rabbits and stabilate preparation. Two T. parve stocks, derived from cattle “Muguga” (see Brocklesby, Barnett & Scott, 1961) and “Kiambu 5” (see Irvin et al., 1974), were used to assess the freezing techniques. Rhipicephalus appendiculatus were obtained from EAVRO where a T. parva-free colony had been established by the method of Bailey (1960) and kept as a closed colony for more than 30 years. Nymphs were fed on infected cattle to become infected with T. parva. Following moulting, the ticks were fed on rabbits (American black rabbits crossed with local Malawi types) for 4 days, then detached with forceps and the salivary glands dissected out (Pumell & Joyner, 1968). Prior to preparation of the stabilates, the infection rates of T. parva in the salivary glands were estimated according to the technique of Blewett & Branagan (1973). T. parva (Muguga) and T. parva (Kiambu 5) stabilates were prepared and dispensed as 1.8 ml aliquots in 2 ml vials and 75 ml aliquots in 250 ml transfusion bags. They were frozen in a - 70°C deep freezer overnight.

-

640 640 * 640

antibodies.

Cattle and in vivo testing of stabilates. The cattle used to evaluate viability of the stabilates were Malawi ZebuFriesian crosses. They were obtained from the Southern Region of Malawi (where ECF does not occur) from farms exercising strict tick control regimes. Cattle were sprayed twice a week with chlorofenvinphos except when ticks were being fed. They were kept on concrete floors and fed concentrates and silage to avoid infestation with ticks. Viability of the stabilates was assessed in these cattle. The contents of 3 vials were pooled, allowed to equilibrate for 40 min then 1 ml was withdrawn and inoculated subcutaneously under the ear of each animal. Similarly, the thawed contents of 2 bags were pooled, equilibrated and 1 ml withdrawn for inoculation. The rectal temperatures of cattle (Table 1) were monitored daily early in the morning. Peripheral blood smears and biopsy smears from parotid and prescapular lymph nodes were collected daily starting from day 10 and upon detection of lymph node enlargement, respectively. The smears were fixed in methanol, stained with Giemsa and examined by light microscopy for Theileria

schizonts and intraerythrocytic

piroplasms. The develop-

ment of antibodies to T. parva was monitored using indirect immunofluorescent antibody test (Burridge Kimber, 1972) on days 0,21 and 28.

the &

RESULTS

changes recorded for the different aliquots in the vials or the transfusion bags are depicted in Figs 14. During freezing, the 1.8 ml aliquots of freezing medium cooled rapidly and passed through the eutectic point fastest; these aliquots cooled to -60°C within 90 min (Fig. 1). The next fastest to freeze were the 50 and 75 ml aliquots of freezing medium in the 250 and 600 ml transfusion bags; these reached -60°C within 150 min. The slowest to freeze were the 100 ml aliquots in the 250 ml transfusion bag (Fig. 1). Interestingly, the 1.8 ml aliquots took the longest time, whereas the 50 and 150 ml aliquots thawed faster. The freezing and thawing patterns of the 1.8 and 75 ml aliquots of the T. parva stabilate (Figs 3 and 4) were similar to those observed with the freezing medium alone (Figs 1 and 2). The

temperature

Bulk-freezing of Theileriu stabilates

0 -60 ’ 0

20

40

60

60

100

Time

(mln)

120

140

160

10

20

69

30

40

50

160

Fig. 1. Freezing curves for T. purva freezing medium in vials (ES, 1.8 ml) and transfusion bags (+, 50 ml; 0, 75 ml; O, 100 ml; x, 120 ml; l , 150 ml).

80

70

Time

10

20

30

40

50

60 Time

0

20

40

60

60

100

Time

(ret)

120

140

160

160

Fig. 2. Thawing curves for T. parve freezing medium in vials (ra , 1.8 ml) and transfusion bags (+, 50 ml; *, 150 ml). A total of 2600 R. appendiculatus infected with T. parva (Muguga) with 62.5% infection and a mean acini infection rate of 17.2 were subsequently used to prepare stabilate No. 77. The 260 ml of stabilate was dispensed in 1.8 and 75 ml volumes in vials and transfusion bags, respectively, and frozen at - 70°C. Stabilate No. 79 was produced from 2200 R. appendiculatus ticks infected with T. puma (Kiambu 5) with a 52.5% infection and a mean acini infection rate of 54.6. This stabilate was similarly dispensed into vials and transfusion bags and frozen to - 70°C. Both stabilates were thawed and used to infect a total of 8 cattle which were monitored for the development of fever, Theileria schizonts, piroplasms and antibody responses (Table 1). In the first experiment, stabilate frozen in the vials caused a fever for 2 days in each of the animals but no parasites were detected. In contrast, one of the 2 animals receiving the same stabilate frozen in transfusion bags developed a fever for 13 days and schizonts were detected in both

90

1001101201301

Fig. 3. Freezing curves for T. parve stabilates in vials (n , 1.8 ml) and transfusion bags (+, 75 ml).

0

-wu

80

(mln)

70

80

90

100110120130

(WC)

Fig. 4. Thawing curves for T. parva stabilates in vials (a, 1.8 ml) and transfusion bags (+, 75 ml). animals for 3 and 13 days. Piroplasms were only detected in animal No. 755 which died on day 21 despite its good physical appearance. All 4 animals had developed antibody titres 3 1640 by day 28 postinoculation. In the second experiment, all animals inoculated with stabilate from the vials and transfusion bags developed fever and schizonts. Those receiving stabilate from the vials developed piroplasms while those receiving stabilate from transfusion bags did not. The clinical reactions in both groups were severe (Table 1) and all were treated with antitheilerial drugs. None the less, one animal (No. 829) died on day 27 despite treatment. All surviving animals seroconverted to a titre > 1:640 by day 28. DISCUSSION

Predictably, the temperature gradient recorded during freezing the medium and the stabilate (Figs 1 and 3) demonstrated that large volumes froze more slowly than smaller volumes. Volumes exceeding 75 ml took more than 3 h to freeze to -60°C. The surface area:volume ratio also appeared to be important in determining the freezing rate of different

70

L. M. Njuguna and F. L. Musisi

aliquots; the greater the surface area:volume ratio, the faster the freezing rate tended to be. Slow freezing or prolonged holding of parasites between -20 and -50°C is known to be detrimental to cells and microorganisms since this exposes lipoprotein membranes to increased solute concentrations resulting in significant membrane damage (Farrant, 1970). Freezing of small volumes (1.8 ml) in vials is known to yield viable parasites as it is conventionally used to freeze T. parva sporozoites. The cooling curves of 50 and 75 ml volumes were similar to those of the smaller volumes, whereas the slower cooling curves of greater volumes, particularly between -20 and - 50°C would cause greater destruction of parasites (Farrant, 1970; Levine et al., 1958). Contrary to expectation, however, the 1.8 ml aliquots took longer to thaw at 0°C than the larger aliquots. The reasons for this are unknown although it is thought that this result may be erroneous due to either the thermometer probe being slightly off centre in the bag or the contents of the vial being insulated by an air gap. It was noted that the total thickness of each bag with its contents was thinner than that of the vial with contents, therefore it is possible that the rate of thawing was influenced by the larger surface area : volume ratio of the bags in comparison to that of the vials. Despite the similar freezing and thawing characteristics of the 1.8 and 75 ml volumes, treatment may have adversely affected the parasites contained in larger volumes so cattle were inoculated with the stabilates to determine their viability. Cattle inoculated with both stabilates became infected with detection of schizonts and piroplasms, development of fever and formation of specific antibodies. This result confirmed that T. parva sporozoite stabilates can be cryopreserved in 75 ml volumes in 250 ml transfusion bags without apparent loss of viability. Differences observed in the degree of reactions of cattle inoculated with different stabilates may be related to parasite strain differences or to quantitative differences in the stabilates (stabilate 77 with mean acini infection rate of 17.2 compared to 54.6 for stabilate 79). The demonstration of a successful bulk freezing technique for T. purvu stabilates should greatly simplify the preparation, freezing and rapid thawing of individual components for trivalent stabilate vaccines. Rather than processing thousands of small vials which take several hours to thaw, mix and dispense during preparation of the vaccine, the use of large stabilate volumes would take considerably less time and effort, and would economize on freezer storage space. Bulk freezing would also be useful in the preparation of monovalent T. parva stabilate

vaccines, since frozen samples of known concentration could be subsequently diluted to provide multiple standardized doses irrespective of the vaccine batch. AcknowledgementsWe acknowledge financial support provided by both the UNDP and Malawi Government to Project RAF/92/010 under which the work was carried out. The project was executed by FAO. We are indebted to our colleagues on the project with whom we carried out some of the experiments, especially Messrs K. E. Chamambala and M. V. Kamtotole for valuable technical assistance.

REFERENCES Bailey K. P. 1960. Notes on the rearing of Rhipicephalus appendiculatus and their infection with Theileriaparva for experimental transmission. Bulletin of Epizootic Diseases of Africa 8: 3343. Blewett D. A. & Branagan D. 1973. The demonstration of Theileria parva infection in intact Rhipicephalus appendiculatus salivary glands. Tropical Animal Health and Production 5: 27-34. Brocklesby D. W., Barnett S. F. & Scott G. R. 1961. Morbidity and mortality rates in East Coast Fever (Theileria parva infection) and their application in drug screening. British Veterinary Journal 117: 101-103. Burridge M. J. & Kimber C. D. 1972. The indirect fluorescent antibody test for experimental East Coast Fever (Theileria parva infection of cattle): evaluation of cell culture antigen. Research in Veterinary Science 13: 451455. Cunningham M. P., Brown C. G. D., Burridge M. J., Joyner L. P. & Pumell R. E. 1973. Cryopreservation of infective particles of Theileria parva. International Journal for Parasitology 3: 89-95. Farrant J. 1970. Mechanisms of injury and protection in living cells and tissues at low temperatures. In: Current Trends in Cryobiology. (Edited by Smith A. U.), pp. 139153. Plenum Press, New York. Irvin A. D., Pumell R. E., Brown C. G. D., Cunningham M. P., Ledger M. A. &Payne R. C. 1974. The application of an indirect method of infecting ticks with piroplasms for use in the isolation of infections. British Veterinary Journal 130: 28&288. Levine N. D., Miezlle M. & Houlahan D. A. 1958. Factors affecting the protective action of glycerol on Trichomonas foetus at freezing temperature. Experimental Parasitology 7: 236248. Lumsden W. H. R. &Hardy G. J. C. 1965. Nomenclature of living parasite material. Nature, London 205: 1032. Purnell R. E. & Joyner L. P. 1968. The development of Theileria parva in the salivary glands of the tick, Rhipicephalus appendiculatus. Parasitology 58: 725-732. Radley D. E. 1981. Infection and treatment method of immunization against theileriosis. In: Advances in the Control of Theileriosis: Proceedings of an International Conference Held at the International Laboratory for Research on Animal Diseases, Nairobi, 9-13 February 1981. (Edited by Irvin A. D., Cunningham M. P. & Young A. S.), pp. 227-237. Martinus Nijhoff, The Hague.