Veterinary Parasitology, 39 ( 199 1) 207-2 13 Elsevier Science Publishers B.V., Amsterdam

207

Besnoitia besnoiti: quantitative in vitro studies

Varda Shkap, Hanna Bin, B. Lebovich and E. Pipano Division of Parasitology, Kimron Veterinary Institute, Bet-Dagan, P.O. Box 12, Israel

(Accepted 28 February 199 1)

ABSTRACT

Shkap, V., Bin, H., Lebovich, B. and Pipano, E., 1991. Besnoitia besnoiti: quantitative in vitro studies. Vet. Parasitol., 39: 207-2 13.

Experimental parameters for optimization of the growth conditions of Besnoitia besnoiti endozoites in vitro are presented. A combination of Hepes-buffered McCoy and Leibovitz (ML) medium with 10% bovine serum was preferable to Eagle’s Minimum Essential Medium (MEM) based on either Hank’s or Earle’s salts. The ML medium and a CO2 balanced gas phase significantly increased the growth rate of the parasite. Infection of Vero cells at I h after subcultivation resulted in higher yields of endozoites than infection of 24- or 48-h-old cells. Increased numbers of parasites (6 x IO’ and 9 x 10’) in the infection inoculum per Roux bottle containing Vero cells produced poor yields, while infection with 2 x 10’ parasites resulted in a 250 to 300-fold increase after 6 days of cultivation with one medium change.

INTRODUCTION

Besnoitia besnoiti (Marotel, 19 12) is a cyst-forming coccidian protozoan causing bovine besnoitiosis (Fayer, 1980). The parasite can be maintained in laboratory animals and in cell cultures (Bigalke, 1962; Neuman, 1974; Shkap et al., 1987). The green monkey kidney (Vero) cell line was found to be a suitable support for propagation of Besnoitia in vitro and these cells are used for vaccine production in South Africa (Bigalke et al., 1974). However, growth conditions for the in vitro production of ample amounts of Bemoitia parasites, needed for the performance of diagnostic tests, immunochemical studies and vaccine preparation, have not been reported. In the present study, we have examined a number of parameters for obtaining higher yields of the parasite in vitro, such as the culture medium, gas phase, age of host cells and the initial inoculum size.

0304-40 17/9 l/$03.50 0 199 1 Elsevier Science Publishers B.V. All rights reserved.

208 V. SHKAP ET AL.

M A T E R I A L S A N D M E T H O D S

Parasites

Besnoitia besnoiti, originally isolated from a naturally infected bull in Is- rael, was maintained in vitro by serial passages in Vero cells (Shkap et al., 1987). Infected cells were dispersed with 0.25% trypsin supplemented with 0.02% ethylenediaminetetraacetic acid (EDTA). The culture supernatants and the dispersed cells containing Besnoitia endozoites were centrifuged at 1500 × g for 20 min and the pelleted parasites were counted in a haemocytometer (Bigalke, 1962 ).

Cell cultivation and media

Vero cells were grown as monolayers in either 25 cm 2 plastic flasks or Roux bottles at 37°C. The growth media were as follows: (a) Eagle's Minimum Essential Medium (MEM) (Glasgow modification) (MacPherson and Sto- ker, 1962 ) with Earle's salts base; (b) MEM on Hank's basal salts; (c) a com- bination of equal volumes of McCoy and Leibovitz media (ML). Media were supplemented with 10% bovine serum, 20 mM Hepes (Gibco, Grand Island, NY, USA ), 20 mM glutamine, 100 U ml - 1 penicillin and 100 mg ml- 1 strep- tomycin. The density of Vero cells at seeding was 105 cells ml- ~ and confluent monolayers were subcultured routinely every 4 days. Infected cultures were grown in closed vessels or in a humidified incubator containing 5% CO2 and 95% air. In some experiments, a gas mixture of 5% CO2, 2% 02 and 93% N2 was flushed into Roux bottles which were then closed tightly until the para- sites were harvested.

Infection of cells and evaluation of multiplication rate

Dispersed Vero cells were seeded into appropriate culture vessels and var- ious numbers of parasites, as specified below, were introduced into the vessels at I, 24 or 48 h after seeding. After 4-6 days of growth, both supernatant and infected cells were collected, centrifuged at 1500 Xg for 20 min and the para- sites counted. The mean multiplication rate (MMR) was calculated from three replicate experiments and expressed as the ratio of input to output of para- sites per vessel.

R E S U L T S

Effect of culture medium

The multiplication rates of parasites grown in Vero cells, in 25 cm 2 plastic flasks with four different culture media, are shown in Fig. 1. In all the exper-

QUANTITATIVE IN VITRO STUDIES OF B. BESNOrI7 209

cc

3°0 t 200 - -

1 0 0 - -

C l o s e d f l asks

c

.,....,..., !i!iiiii!i!i

i!iiiiiiiiii ;iiiiiiijii .......,...

iiiii!i!i i iiiiiiiii:i , . . . . , , .

iiiiiiiiiii !iiiii!i!i!i

b iiiiiiiiiiii

5 % C O z + 95%ai r

Fig. I. Mult ipl icat ion of B. besnoiti in Vero cells grown in various culture media. (a) Eagle's MEM (Earle 's s a l t s )+10% serum; (b) Eagle's MEM (Hank 's s a l t s )+10% serum; (c) M c C o y + Leibovitz ( 1 : 1 ) + 10% serum; (d) M c C o y + Leibovitz ( 1 : 1 ).

iments, the initial inoculum was 10 6 parasites and the period of cultivation 4 days. The highest yield of endozoites (210 X 106) was obtained with ML me- dium containing serum grown in 5% CO2 and 95% air. The lowest number of parasites ( 14 X 106 and 18 X 106) was obtained with MEM based on Earle's salts, irrespective of gas conditions. The ML serum-free medium gave similar rates of multiplication as the MEM (Earle's salts base) when grown in closed vessels. Higher yields of endozoites, 2.8- and 4.l-fold, were obtained with MEM based on Hank's rather than on Earle's salts, either in closed flasks or in a CO2-enriched gas phase. Subsequent subcultivation for about 20 passages over 3 months constantly showed preference for the ML medium, but not when bovine serum was omitted. Without bovine serum, the growth rate of both the host cells and parasites decreased with each subculture to a level that did not allow further subcultivation (data not shown).

Effect of host cell age

Three individual experiments were performed with ML medium plus serum in Roux bottles. Vero cells seeded in triplicate were inoculated with 5 X 106 or 107 endozoites at 1, 24 and 48 h after subculturing. Cultures were grown either in closed bottles, or in a 5% CO2 and 95% air phase, or in bottles into which a mixture of 5% CO2, 2% 02 and 93% N2 was flushed through a sterile filter for a minute before sealing them. As shown in Fig. 2, after 4 days of cultiva-

2 10 V. SHKAP ET AU

150

100-

50-

b b @

O- 1 24 48

D

a b

0 - 1 24

a b c

48

Age of cel ls when infected (hours)

Fig. 2. Multiplication ofB. besnoiti in Vero cells infected with 5 X 106 (A) or 107 (B) organisms per Roux bottle at 1, 24 and 48 h after subculturing. (a) Growth in closed bottles; (b) growth in 5% CO2, 95% air; (c) growth in 5% CO2, 2% 02, 93% N2.

tion the highest yield of endozoites was obtained from cultures infected shortly after seeding of the cells. In these cultures, increased multiplication rates of up to 113-fold were achieved with an initial infection of 107 parasites, and up to 76-fold with an initial infection of 0.5X 10 v parasites. Infection at 48 h post-seeding resulted in a 4-fold increase with an infection of 107 endozoites, irrespective of the gas conditions. Cultures infected with 5 X 106 or 107 orga- nisms at 24 h yielded 15- and 14-fold increases, respectively. A similar mul- tiplication rate was obtained 48 h post-seeding when cells were infected with 5 X 106 parasites. As in the previous experiments, there was a lower yield from cultures grown in 5% CO2, 2% O2 and 93% N2.

Effect o f initial inoculum size on the multiplication rate

Vero cells in Roux bottles were inoculated with 2 × 106-9 X 10 7 endozoites in 1 h after subculturing. Cultures were grown either in closed vessels, or in a 5% CO2 and 95% air phase, or in a mixture of 5% CO2, 2% 02 and 93% N2. Infected cultures were harvested 4 days later. Figure 3 shows that a higher yield of endozoites (128 t imes increase) was collected from cultures inocu- lated with 107 parasites grown in the 5% CO2 and 95% air atmosphere. Larger inocula of 6 × 107 and 9 X 107 endozoites per bottle resulted in about ten times more final parasite output. On the other hand, infection with 2X 107 or 0.2 × 107 parasites gave a similar 30-fold increase in closed bottles, and up to a 40- and 52-fold increase in cultures grown in an enriched CO2 atmosphere.

QUANTITATIVE IN VITRO STUDIES OF B. BESNOITI 211

100

50

b

0.2 0.5 1.0 2.0 4.0 6.0 9.0

Number of parasites in inoculum (XIO')

Fig. 3. Yield of B. besnoiti grown for 96 h in Vero cells infected in 1 h with various numbers of organisms. (a) Growth in closed bottles; (b) growth in 5% CO> 95% air; (c) growth in 5% CO2, 2% 02, 93% N2.

In the mixture of 5% CO2, 2% 02 and 93% Nz, the multiplication rate was lower in all cultures tested.

In another set of experiments, Vero cells were inoculated with 0.2 × 10 v en- dozoites at 1 h after seeding. Cells were grown in either closed bottles or a mixture of 5% COR, 2% O2 and 93% N2. Sixteen bottles were harvested each day after 4 or 6 days of cultivation. The med ium was changed on Day 4 in cultures grown for up to 6 days. Changing the media and cultivating for longer periods resulted in about a 6-fold increase in the final yield compared to that from cultures mainta ined for 4 days. As before, growth in a low 02 concentra- tion resulted in lower yields.

DISCUSSION

From the results presented, it appears that Hepes-buffered ML med ium supplemented with 10% bovine serum was preferable for the growth of Besnoitia in Eagle's MEM based on either Hank's or Earle's salts. The ML media include more amino acids and vitamins, such as L-alanine, L-aspara- gine, L-serine, glycine, B~2, biotin and ascorbic acid, which might be critical for Besnoitia growth. Moreover, Leibovitz's med ium features buffering ca- pacity by utilising free amino acids and substituting galactose and pyruvate for glucose (Griffiths, 1986). Exclusion of bovine serum from the ML media resulted in lower yields of Besnoitia parasites. These results agree with those of Shimizu ( 1963 ), showing that the addit ion of calf serum to Toxoplasma-

2 1 2 V. SHKAP ET AL.

infected cultures increased the rate of multiplication. Growth in a humid at- mosphere with 5% CO2 in either 25 cm 2 flasks or Roux bottles gave higher yields of parasites compared to growth in sealed bottles. A low 02 concentra- tion (2%) in the mixture of gases lowered the growth rate in all our experi- ments, confirming that 02 is a growth-limiting factor (Freshney, 1983 ). Con- tinuous gas exchange in the CO2 incubator was beneficial for both growth of the host cells and the parasites. Therefore, Hepes-buffered medium and a CO2 balanced gas phase significantly increased the growth rate of the parasite.

From the results obtained, it is clear that the age of the Veto cells used for infection is a critical factor for the development and multiplication of the parasite. Dvorak and Howe (1977) showed that older cells were less perme- able to Toxoplasma gondii than younger cells. Hermentin and Asp6ck ( 1987 ) obtained similar results and suggested that the trypsinisation damages the gly- cocalix of the cells, thus increasing their susceptibility to infection.

Based on the data obtained, it appears that the higher initial inoculum size hampered the op t imum growth ofB. besnoiti. According to Reduker and Speer ( 1987 ), increasing numbers of sporozoites or trophozoites of Eimeria bovis had an inhibitory effect on in vitro development of the parasite. On the other hand, few parasites in the inoculum produced poor yields. Hughes et al. (1986) and Valkoun and Cinatl (1979) showed that when the inoculum of T. gondii was too small, HeLa cells overgrew the parasites, giving low yields.

We obtained up to 130 times the initial inoculum at 4 days of infection and 280-fold MMR after 6 days, including one medium change. In analogous ex- periments on the op t imum growth conditions of T. gondii, Chang and Gabrielson (1984), Hermentin and Asp6ck (1987) and Hermentin et al. ( 1987 ) obtained a 132-150-fold increase after 4 days of cultivation.

In summary, we have examined experimental parameters for mass culti- vation ofB. besnoiti endozoites in vitro with a view to obtaining an opt imum yield for immunological studies and for vaccine development.

REFERENCES

Bigalke, R., 1962. Preliminary communication on the cultivation of Besnoitia besnoiti (Morotel, 1912) in tissue culture and embryonated eggs. J.S. Afr. Vet. Med. Assoc., 33: 523-532.

Bigalke, R., Schoeman, J. and McCully, R., 1974. Immunization against bovine besnoitiosis with a live vaccine prepared from a blue wildebeest strain ofBesnoitia besnoiti grown in cell cultures. 1. Studies on rabbits. Onderstepoort J. Vet. Res., 41: 1-6.

Chang, G. and Gabrielson, D., 1984. 7bxoplasma gondii: growth in ovine fetal kidney cell cul- tures. Exp. Parasitol., 57: 81-85.

Dvorak, J. and Howe, C., 1977. Toxoplasrna gondii-vertebrate cell interactions. I. The influ- ence of bicarbonate ion, CO2, pH and host cell culture age on the invasion of vertebrate cell in vitro. J. Protozool., 24:416-419.

Fayer, R., 1980. Epidemiology of protozoan infections: the coccidia. Vet. Parasitol., 6: 75-103.

QUANTITATIVE IN VITRO STUDIES OF B. BE,'iNOI77 213

Freshney, R., 1983. Culture of Animal Cells. A Manual of Basic Technique. A.R. Liss, New York.

Griffiths, B., 1986. Scaling-up of animal cell cultures. In: R.I. Freshney (Editor), Animal Cell Culture A Practical Approach. IRL Press, Oxford/Washington, DC, pp. 33-69.

Hermentin, K. and Asp6ck, H., 1987. Higher yields and increased purity of in vitro grown Toxoplasma gondii. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1: Orig. Reihe A, 267: 272-276.

Hermentin, K., Auer, H. and Asp6ck, H., 1987. In vitro cultivation of Toxoplasma gondii under defined, serum-free conditions. J. Parasitol., 73:1276-1277.

Hughes, H., Hudson, L. and Fleck, D., 1986. In vitro culture of Toxoplasma gondii in primary and established cell lines. Int. J. Parasitol., 16:317-322.

MacPherson, I. and Stoker, M., 1962. Polyoma transformation of hamster cell clones - - an investigation of genetic factors affecting cell competence. Virology, 16:147-151.

Neuman, M., 1974. Cultivation ofBesnoitia besnoiti Maritel, 1912, in cell culture. Tropenmed. Parasitol., 25: 243-249.

Reduker, D. and Speer, C., 1987. Effect of sporozoite inoculum size on in vitro production of merozoites ofEirneria bovis (Apicompexa). J. Parasitol., 73: 427-430.

Shimizu, K., 1963. Studies on toxoplasmosis. V. Complemental observations on the tissue cul- ture method, especially the effect of the nutrient fluid upon the invasion and multiplication of the organisms. Jpn. J. Vet. Res., 11:1-11.

Shkap, V., Pipano, E. and Greenblatt, C., 1987. Cultivation ofBesnoitia besnoiti and evaluation of susceptibility of laboratory animals to cultured parasites. Vet. Parasitol., 23:169-178.

Valkoun. A. and Cinatl, J., 1979. Propagation of Toxoplasma gondii in suspension cultures of HeLa cells. Folia Parasitol. (Prague), 26: 165-167.