A South African Babesia sp. of cattle which is as yet unclassified, was continuously cultivated in micro-aerophilous stationary-phase culture. The parasites were resuscitated from a blood stabilate stored in
liquid nitrogen. A modified HL-1 medium supplemented with either horse or bovine serum was used.
Cultures were initiated in a humidified atmosphere containing 2% 0₂ , 5% C0₂ and 93% N₂ at 37°C.
Parasites were detected on Giemsa-stained smears after 2 d in culture. On day 4, the cultures were
split at a ratio of 1:2 (v/v) and transferred into a humidified atmosphere of 5% C0₂ in air. Starting from
day 6, subcultures were made daily at a ratio of 1:4 (v/v). The percentage of parasitized erythrocytes
ranged from 2-5%. Addition of purine bases (hypoxanthine, adenine, adenosine or guanosine) was
essential for the continuous propagation of the parasites when bovine, but not horse serum, was used
for medium supplementation.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

De Waal, D.T.

Just, M.C.

Van Niekerk, C.

Zweygarth, Erich

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.A South African Babesia sp. of cattle which is as yet unclassified, was continuously cultivated in micro-aerophilous stationary-phase culture. The parasites were resuscitated from a blood stabilate stored in
liquid nitrogen. A modified HL-1 medium supplemented with either horse or bovine serum was used.
Cultures were initiated in a humidified atmosphere containing 2% 0₂ , 5% C0₂ and 93% N₂ at 37°C.
Parasites were detected on Giemsa-stained smears after 2 d in culture. On day 4, the cultures were
split at a ratio of 1:2 (v/v) and transferred into a humidified atmosphere of 5% C0₂ in air. Starting from
day 6, subcultures were made daily at a ratio of 1:4 (v/v). The percentage of parasitized erythrocytes
ranged from 2-5%. Addition of purine bases (hypoxanthine, adenine, adenosine or guanosine) was
essential for the continuous propagation of the parasites when bovine, but not horse serum, was used
for medium supplementation.mn201

UPSpace at the University of Pretoria

Onderstepoort Journal of Veterinary Research, 62:139-142 ( 1995) RESEARCH COMMUNICATION In vitro cultivation of a Babesia sp. from cattle in South Africa E. ZWEYGARTH, C. VAN NIEKERK, M.G. JUST and D.T. DE WAAL Protozoology Division, Onderstepoort Veterinary Institute Private Bag XS, Onderstepoort, 0110 South Africa ABSTRACT ZWEYGARTH, E., VAN NIEKERK, C. , JUST, M.C. & DE WAAL, D.T. 1995.1n vitro cultivation of a Babe-sia sp. from cattle. Onderstepoort Journal of Veterinary Research, 62:139-142 A South African Babesiasp. of cattle which is as yet unclassified, was continuously cultivated in micro-aerophilous stationary-phase culture. The parasites were resuscitated from a blood stabilate stored in liquid nitrogen. A modified HL-1 medium supplemented with either horse or bovine serum was used. Cultures were initiated in a humidified atmosphere containing 2 % 0 2 , 5% C02 and 93 % N2 at 37 °C. Parasites were detected on Giemsa-stained smears after 2 din culture. On day 4, the cultures were split at a ratio of 1 :2 (v/v) and transferred into a humidified atmosphere of 5% C02 in air. Starting from day 6, subcultures were made daily at a ratio of 1 :4 (v/v). The percentage of parasitized erythrocytes ranged from 2-5%. Addition of purine bases (hypoxanthine, adenine, adenosine or guanosine) was essential for the continuous propagation of the parasites when bovine, but not horse serum, was used for medium supplementation. Keywords: In vitro, cultivation, Babesia, cattle, bovine, South Africa INTRODUCTION Babesiosis is a major tick-transmitted protozoal dis-ease of cattle. In South Africa, B. bigemina and B. bovis are the two economically important agents of bovine babesiosis. Both species are transmitted by Boophilus microplus; B. bigemina is also transmit-ted by Boophilus decoloratus (Potgieter 1977). Gray & DeVos (1981) described another Babesia of cat-tle which they isolated in South Africa. This parasite, named B. occultans, causes a mild disease and is transmitted transovarially by Hyalomma marginatum rufipes, a tick species which transmits neither B. bovis nor B. bigemina. The fourth bovine Babesia de-scribed in South Africa is as yet unclassified. It was not transmissible by B. microplus and H. m. rufipes, but by H. truncatum (De Waal , Potgieter, Combrink & Mason 1990). Accepted for publication 21 June 1995-Editor The present paper describes the culture initiation and continuous in vitro propagation of Babesia sp. MATERIALS AND METHODS Babesia sp. isolate The parasite was isolated at Kaalplaas Farm (28° 08' E, 25°38'S), Onderstepoort Veterinary Institute (South Africa) (De Waal et at. 1990). The stabi late from which the cultures were initiated was cryopre-served according to the method of DeVos, Combrink 139 In vitro cultivation of a Babesia sp. from cattle & Bessenger (1982). When the stabilate was pre-pared, the percentage of parasitized erythrocytes (PPE) was 0,002%. Culture medium The medium, referred to as complete medium, con-sisted of HL-1 medium (Hycor Biomedical Inc., Port-land, Maine, USA) with either 20% horse or bovine serum, buffered with 15 mM HEPES supplemented with 2 mM !-glutamine, 0,2 mM hypoxanthine, 200 IU penicillin/mQ and 200 IJg streptomycin/mt After 5 d, the concentration of the antibiotics in the medium was halved. Some media compositions were altered by omitting hypoxanthine or by replacing it with 0,2 mM adenine, adenosine or guanosine. Culture initiation and maintenance A vial of stabilate was thawed in a water bath at 37 oc and the contents were diluted into 20 mQ of a modi-fied Vega y Martinez phosphate-buffered saline so-lution (mVYM) (Zweygarth, Just & De Waal 1995). After centrifugation (2 000 X g, 10 min, 4 °C) , the pellet was resuspended in 4 mQ of complete culture medium with 10% (v/v) fresh, unparasitized bovine erythrocytes and distributed equally in four wells of a 24-well culture plate. The plate was incubated in a modular incubator chamber at 3JCC in an atmos-phere of 2% 0 , 5% C02 and 93% N . Medium was changed daily by the replacement of 700 IJQ of me-dium overlying the erythrocytes in each well. When the PPE reached about 0,2%, culture plates were transferred in a 5%-C02-in-air atmosphere at 3JCC. Erythrocytes Blood cells were washed five times by centrifugation (650 x g, 10 min, 4 °C} and resuspension in mVYM. After each wash the leucocytes were removed from the interphase of the supernatant and bovine-red-blood-cell (BRBC) suspension. After the last wash, the BRBCs were suspended in mVYM solution at a concentration of 50% (v/v) and stored at 4 oc until use, but not longer than 2 weeks. Subcultures Erythrocytes in the culture wells were resuspended and 0,5 mQ (1 :2, v/v) or 0,25 mQ (1 :4, v/v) was trans-ferred into each of two and four new wells, respec-tively. To each of the wells was added a 10% sus-pension of uninfected BRBCs in complete medium to make up a final volume of 1 mt Estimation of parasite growth Cultures of Babesia sp. changed colour from bright red to almost black while they were growing continu-ously. This is similar to what was described for B. 140 bovis-infected cultures (Levy and Ristic 1980). To de-termine the PPE, culture samples were smeared on microscopic slides, f ixed with methanol, Giemsa-stained (1 0% v/v; 45 min), and 1 000 cells were counted. RESULTS Two days after initiation, parasites were detected in smears prepared from cultured material. On day 4, the cultures were split at a ratio of 1 :2 (v/v) into a new plate. The original plate was left as safety back-up in the gas mixture, whereas the new plate was trans-ferred into a humidified atmosphere of 5% C02 in air. Both approaches were equally good, so that on day 5 the initiation plate was also moved into the regular C02 incubator. Starting from day 6, cultures were split daily at a ratio of 1 :4 (v/v) . Bovine and equine serum-supplemented media supported the initiation and the propagation of the parasites equally well. The average PPE obtained in culture ranged from 2- 5%. When the culture media supplemented with bovine serum did not receive additional purine bases, the PPE declined. After 10 d, the PPE was about 0,4% compared to control cultures with an average PPE of 3-4%. This first became apparent in the lighter colour of the cultures on day 4 after they had chang-ed to the extra purine-deprived medium. However, when the purine-supplemented medium was re-applied, at which stage only a few parasites were de-tected, the growth rate returned to levels of the con-trol wells. Microphotographs of cultured Babesia sp. are shown in Fig. 1. Most of the culture-derived parasites were piriform and paired (Fig. 1 a, 1 b). Multiple parasites within a cell were seen occasionally (Fig. 1 c). A troph-ozoite is shown in Fig. 1 d. DISCUSSION The unclassified Babesia sp. originally isolated from an ox (De Waal eta/. 1990} was adapted to in vitro conditions by the use of methods described for the bovine Babesia spp. , B. bovis (Rodriguez, Buening, Green & Carson 1983) and B. bigemina (Vega, Bue-ning, Green & Carson 1985). The medium used con-sisted of HL-1, a medium originally designed for the serum-free cultivation of hybridoma ce lls. HL-1-based media were previously used for the isolation and cultivation of several Babesia species, namely B. caballi (Holman, Frerichs, Chieves & Wagner 1993), B. equi (Holman, Chieves, Frerichs, Olson & Wagner 1994), a Babesia sp. from a North Ameri-can elk (Holman, Craig, Doan Crider, Petrini, Rhyan & Wagner 1994), and another species from an Ameri-can woodland caribou (Holman, Petrini , Rhyan & Wagner 1994). Cultures were initiated from stabilates FIG. 1 Giemsa-stained smears of the Babesia sp. from an ox cul-tured in the 23rd passage. A, B. Paired piriform parasites C. Multiple parasites (arrow) within a cell. D. Trophozoite Bar represents 1 0 ~m with an initial PPE ((before cryopreservation) as low as 0,002 %. Compared to the use of a splenecto-mized animal to generate parasites for culture initia-tion , this new approach saved time and money. The parasites multiplied promplty and cultures could be subcultured as early as 4 d after initiation, giving rise to continuous cultures, irrespective of whether equine- or bovine-serum-supplemented media were used. Similar results were obtained by Van Niekerk and Zweygarth (manuscript in preparation) for the initiation and propagation of B. occultans in medium supplemented with bovine or equine serum. Adapta-tion of B. bovis to equine serum needed a gradual substitution of bovine serum by the former (Yunker, Kuttler & Johnson 1987) whereas Fish, Pipano, Shkap & Frank (1993) were able to change from bovine- to equine-serum-supplemented medium without an adaptation period. When cultures were fed with bovine-serum-based medium without additional purines, the PPE declined rapidly, and the usual subculture intervals could not be maintained. This process was, however, still re-versible after 10 d of cultivation, and parasites re-sumed normal growth when complete medium was re-supplied. This negative influence on parasite growth was found only with bovine-serum-supplemented media, whereas medium containing horse serum ap-parently contained enough purine bases to sustain a normal growth. Babesia spp. depend on the sal-vage of preformed purines (Sherman 1984), therefore they readily incorporate hypoxanthine (and other E. ZWEYGARTH eta/. purines) into their nucleic acid (Irvin, Young and Pur-nell 1978; Irvin and Young 1979; Conrad 1986). In the complete medium used here, the purine concen-tration obviously became a limiting factor for the growth of Babesia sp. since either hypoxanthine, adenosine, guanosine or adenine supplementation reversed the decline in parasite growth. These re-sults were in striking contrast to those of Konrad, Phipps, Canning and Donnelly (1984) who found that medium supplemented with hypoxanthine was even deleterious to B. divergens cultures. Culture-derived parasites will be used to isolate small subunit ribosomal RNA which will be amplified by means of the polymerase chain reaction, cloned and sequenced to clarify the taxonomic position of this Babesia species. ACKNOWLEDGEMENTS We express our appreciation to Mrs E. Horn, Dr S.W. Vogel and Messrs M.P. Combrink, W.M. Maluleka, J.S. Komape and S.F. Shirinda for their valuable help and support. REFERENCES CONRAD, P.A. 1986. Uptake of tritiated nucleic acid precursors by Babesia bovis in vitro. International Journal of Parasitology, 16:263-268. DEVOS, A.J., COMBRINK, M.P. & BESSENGER, R. 1982. Babe-sia bigemina vaccine: comparison of the efficacy and safety of Australian and South African strains under experimental condi-t ions in South Africa. Onderstepoort Journal of Veterinary Re-search, 49:155-158. DE WAAL, D.T., POTGIETER, F.T. , COMBRINK, M.P. & MASON, T.E. 1990. The isolation and transmission of an unidentified Babesia sp. to cattle by Hyalomma truncatum Koch, 1844. Onderstepoort Journal of Veterinary Research, 57:229- 232. FISH, L. , PIPANO, E. , SHKAP, V. & FRANK, M. 1993. Cultivation of Babesia bovis and Babesia bigemina, in medium supple-mented with equine serum. Israel Journal of Veterinary Medi-cine, 48:11 7-119. GRAY, J .S. & DEVOS, A.J. 1981. Studies on a bovine Babesia transmitted by Hyalomma marginatum rufipes Koch, 1844. On-derstepoort Journal of Veterinary Research, 48:215-223. HOLMAN, P.J., FRERICHS, W.M., CHIEVES, L. & WAGNER, G.G. 1993. Culture confirmation of the carrier status of Babe-sia caba//i-infected horses. Journal of Clinical Microbiology, 31: 698- 701 . HOLMAN, P.J., CHIEVES, L., FRERICHS, W.M., OLSON, D. & WAGNER, G.G. 1994. Babesia equi ery1hrocy1ic stage continu-ously cultured in an enriched medium. Journal of Parasitology, 80:232-236. HOLMAN, P.J ., CRAIG, T.M., DOAN CRIDER, D. , PETRINI, K.R., RHYAN, J. & WAGNER, G.G. 1994. Culture isolation and par-tial characterization of a Babesia sp. from North American elk (Cervus elaphus) . Journal of Wildlife Diseases, 30:460-465. HOLMAN, P.J., PETRINI, K., RHYAN, J. & WAGNER, G.G. 1994. In vitro isolation and cultivation of a Babesia from an American woodland caribou ( Rangifer tarandus caribou). Journal of Wild-life Diseases, 30:195- 200. 141 In vitro cultivation of a Babesia sp. from cattle IRVIN, A .D., YOUNG, E.R. & PURNELL, R.E. 1978. The in vitro uptake of tritiated nuclear acid precursors by Babesia spp. of cattle and mice. lnternational Journal of Parasitology, 8:19- 24. IRVIN, A.D. & YOUNG, E.R. 1979. Further studies on the uptake of tritiated nuclear acid precursors by Babesia spp. of cattle and mice. International Journal of Parasitology, 9:1 09-114. KONRAD, J. , PHIPPS, L.P., CANNING, E.U. & DONNELLY, J . 1984. Long term in vitro maintenance of Babesia divergens in a stationary phase culture. Parasitology, 89:1xxvi [abstract]. LEVY, M.G. & RISTIC, M. 1980. Babesia bovis: continuous cultiva-tion in a microaerophilous stationary phase culture. Science, 207:1218- 1220. POTGIETER, F.T. 1977. The life cycle of Babesia bovis and Ba-besia bigemina in ticks and in cattle in South Africa. Ph.D. the-sis, Rand Afrikaans University. 142 RODRIGUEZ, S.D. , BUENING, G.M. , GREEN, T.J. & CARSON, C.A. 1983. Cloning of Babesia bovis by in vitro cultivation. Infec-tion and Immunity, 42:15-18. SHERMAN, I.W. 1984. Metabolism, in Antimalarial drugs. Hand-book of experimental pharmacology, edited by W. Peters & W.H.G. Richards. Springer: Berlin, Heidelberg, New York: 31-81. VEGA, C.A. , BUENING, G.M., GREEN, T.J . & CARSON, C.A. 1985. In vitro cultivation of Babesia bigemina. American Jour-nal of Veterinary Research, 46:416-420. YUNKER, C. E. , KUTTLER, K.L. & JOUNSON, L.W. 1987. Attenu-ation of Babesia bovis in in vitro cultivation. Veterinary Parasi-tology, 24:7- 13. ZWEYGARTH, E., JUST, M.C. & DE WAAL, D.T. 1995. Continu-ous in vitro cultivation of erythrocytic stages of Babesia equi. Parasitology Research, 81 :355-358. l l I I J I I 

In vitro cultivation of a Babesia sp. from cattle in South Africa

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In vitro cultivation of a Babesia sp. from cattle in South Africa

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