Babesia occultans, the causative agent of a benign form of cattle babesiosis in South Africa, was continuously
 cultivated in microaerophilous stationary-phase culture. A modified medium, 199, supplemented
 with either 40% (v/v) bovine or 40% (v/v) horse serum, was used. Cultures were initiated in a humidified
 atmosphere containing 2% 0₂, 5% C0₂, and 93% N₂. The highest percentage of parasitized erythrocytes
 (PPE) reached 4,5% in horse-serum- and 2,4% in bovine-serum-supplemented medium. Parasite
 suspensions were cryopreserved and successfully resuscitated.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Van Niekerk, C.J.

Zweygarth, Erich

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Babesia occultans, the causative agent of a benign form of cattle babesiosis in South Africa, was continuously
 cultivated in microaerophilous stationary-phase culture. A modified medium, 199, supplemented
 with either 40% (v/v) bovine or 40% (v/v) horse serum, was used. Cultures were initiated in a humidified
 atmosphere containing 2% 0₂, 5% C0₂, and 93% N₂. The highest percentage of parasitized erythrocytes
 (PPE) reached 4,5% in horse-serum- and 2,4% in bovine-serum-supplemented medium. Parasite
 suspensions were cryopreserved and successfully resuscitated.mn201

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Onderstepoort Journal of Veterinary Research, 63:259-261 (1996) In vitro C.J.VAN N and E. ZWEYGARTH OnderstefXtrJtt Veterinary Institute lnd~ersl~eD£>ort. ()~10 Soath Africa ABSTRACT VAN N IEKERK, C.J. & ZWEYGARTH, E. 1996. In vitro cultivation of Babesia occultans. Onderstepoort Journal of Veterinary Research, 63:259-261 Babesia occultans, the causative agent of a benign form of cattle babesiosis in South Africa was con-tinuously cultivated in microaerophilous stationary-phase culture. A modified medium, 199, supplemented w1th e1ther 40% (v/v) bov1ne or 40% (v/v) horse serum, was used. Cultures were initiated in a humidi-fied atmosphere containing 2% 0 2 , 5% C02, and 93% N2. The highest percentage of parasitized eryth-rocytes (PPE) reached 4,5% 1n horse-serum- and 2,4% in bovine-serum-supplemented medium. Para-Site suspens1ons were cryopreserved and successfully resuscitated. Keywords: In vitro cultivation, Babesia occultans, bovine babesiosis, South Africa INTRODUCTION In a study by Thomas & Mason (1981 ), "large merozoi-tes" of a Babesia sp. were found in the haemolymph of engorged adult female Hyalomma marginatum ru-fipes ticks. This tick species was not previously known to be a vector of any Babesia sp. The same authors showed that the piroplasm stages and the kinetes of the parasite ranged in size between those of B. bovis and B. bigemina. It was subsequently shown that this new parasite, B. occultans (Gray & DeVos 1981 ), was highly infective for H. m. rufipes, but not for Boophi-lus microplus, the vector for B. bovisand B. bigemina. The present study describes the establishment of B. occultans in microaerophilous stationary-phase cul-ture (Levy & Ristic 1980). Accepted for publication 25 June 1996- Editor MATERIALS AND METHODS Babesia occultans isolate The isolation of B. occultans has been described in detail elsewhere (Gray & deVos 1981 ;Thomas & Ma-son 1981 ). Briefly, ticks were collected on a farm near Ellisras in the Northern Transvaal (South Africa). The progeny of these ticks were used to transmit the par-asite to splenectomized cattle. Blood stabilates were prepared with 8% (v/v) dimethylsulphoxide (DMSO) as a cryoprotectant, and stored in the gas phase of a liquid-nitrogen container. At the time of stabilate preparation, the percentage of parasitized erythro-cytes (PPE) was approximately 0 ,2%. Experimental animal A 1-year-old splenectomized ox, raised under tick-free conditions and free from blood protozoa, was in-jected intravenously with 4,5 mQ of a B. occultans blood stabilate (Gray & DeVos 1981 ). Blood smears were prepared daily and examined for the presence 259 In vitro cultivation of Babesia occultans of babesia. In addition, the packed-cell volume (PCV) and rectal temperature of the steer were determined daily. Starting 2 d before stabilate inoculation, the ani-mal was injected on alternate days with prednisolone 1 mg kg- 1 (Deltacortril, Pfizer), until the parasitaemia started to drop. The dose was then halved each day to prevent an iatrogenic adrenocortical insufficiency by the sudden withdrawal of the drug. The animal was kept under tick-free conditions during the whole ex-periment. Culture medium The medium used, consisted of medium 199 (with Hanks' salts) with either 40% bovine serum or 40% horse serum, buffered with 20 mM ofTAPSO (3-[N-tris-{ hyd roxymethyl} methyl-ami no ]-2-hyd roxypropane-sulfonic acid; Sigma, St. Louis, Mo. , USA; Goff & Yun-ker 1988) and supplemented with 100 IU/mQ of penicillin and 1 00 IJg/mQ of streptomycin. Culture initiation and maintenance For initiation of cultures, blood from the infected ox was collected by venipuncture into sterile vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant. The blood cells were washed three times by centrifugation (1 000 x g, 10 min, room tem-perature) and resuspended in modified Vega y Mar-tinez (mVYM) phosphate-buffered saline solution (Zweygarth, Just & De Waal 1995). After each cen-trifugation, the white-blood cells were removed from the bovine red-blood cells (BRBC) by the use of an Eppendorf pipette. After the last wash, 100 IJQ of the packed RBC suspension and 900 IJQ of medium were pipetted into a 24-well culture plate. The plates were initially incubated in an atmosphere of 2% 0 2; 5% C02 and 93% N2 at 3rC. The culture plates were transferred to a 5-%-C02-in-air atmosphere when the PPE was between 0,5 and 1 %. In another experiment, cultures were initiated directly in a 5-%-C02-in-air atmosphere. Medium was replaced daily, 20 IJQ of fresh , uninfected BRBC was added once a week until the cultures were subcultured. The washing procedure of BRBC from an uninfected do-nor animal, was the same as that described above. Estimation of parasite growth The PPE of cultures was determined by counting five fields of approximately 500 BRBC, each on a thin blood film stained with either 10% Giemsa for 35 min or with a quick-staining kit (RapiD iff, Clinical Sciences Diagnostics, Booysens, South Africa). Preparation and retrieval of stabilates For cryopreservation, erythrocytes from cultures were collected and centrifuged (1 000 x g, 10 min, room temperature) and a 40% (v/v) suspension of 260 erythrocytes in fresh complete culture medium was prepared. Equal volumes of cell suspension and pre-cooled 4% (v/v) DMSO in complete medium were mixed, and 800 IJQ aliquots of the mixture were then transferred into 1-mQ cryotubes and kept at -70 °C overnight in a polystyrene container. The following day, they were transferred for long-term storage into the gas phase of a liquid-nitrogen container. Resuscitation was done by thawing two stabilate vi-als in a water bath at 3?DC and the contents were di-luted in 40 mQ of mVYM solution. After centrifugation (3,000 x g, 1 0 min, 4 °C) the pellet was resuspended in 2 mQ of complete culture medium with 1 0% (v/v) fresh , unparasitized erythrocytes; this was divided into two wells of a 24-well culture plate. The plate was then incubated at 3rC in the oxygen-reduced gas mixture as described above. RESULTS Parasites were detected in Giemsa-stained thin blood smears of the experimental animal between days 5 and 14, after stabilate injection. The parasitaemia was generally low, and a PPE of more than 1 ,8% was observed only on days 10 and 11 (19% and 16%, respectively). On days 12 and 13, the parasitaemia dropped to 0,8% and< 0,01 %, respectively. After day 14, parasites were no longer detected. The rectal temperature of the steer ranged between 40,0 and 41 ,3°C on days 9, 10 and 11. The PCV dropped from 35% pre-infection to 19%, on day 11 post infection. The infection of the steer with B. occultans did not produce severe clinical symptoms. The course of in-fection was very mild, similar to that described by Thomas & Mason (1981). After 14 d, parasites dis-appeared rapidly from the blood, and therefore treat-ment was not necessary. This is in contrast to obser-vations made of other bovine Babesia species where splenectomy exacerbated clinical reactions in the host animal (Riek 1968). Blood collected on day 6 was adjusted to a PPE of 0,6% before being used for culture initiation. Incu-bation was done with horse-serum-supplemented medium in the oxygen-reduced gas mixture. After an initial increase in the PPE, there was a steady de-crease, until they were only rarely detected in stained smears. From day 14 onwards, the PPE increased slowly and subcultures were first made on day 19, when a PPE of 0,8 % was recorded. Subsequently, subcultures were made every other day. On day 35, the PPE reached 4% in subcultures. The highest PPE obtained at any stage, was 4,5%. The parasites were propagated for ten weeks before they were cryo-preserved. In parallel experiments, bovine-serum-supplemented medium did not support culture initia-tion. When cultures were initiated in an atmosphere of 5 % C02 in air, prior to the reduction of the parasite concentration, the PPE in culture initially increased from 1 ,8% to 2,4% (from 1 ,8% to 3,2% in horse-serum-supplemented medium), but when cultures were split at a ratio of 1 :2 (v/v) , parasites ceased to grow. Using another batch of bovine serum, however, and the oxygen-reduced gas mixture, it was possi-ble to initiate cultures from blood obtained on day 13 of infection, when the PPE was below 0,01 %. The parasites remained viable and resumed growth after cryopreservation. They were detected in blood smears on day 4 after resuscitation. Subcultures were done on day 7, when the PPE reached approxi-mately 4%. CONCLUSIONS The experiments showed that B. occultans can be initiated and propagated in vitro under conditions similar to those described for B. bovis (Rodriguez, Buening, Green & Carson 1983) and B. bigemina (Vega, Buening, Green & Carson 1985). Bovine and horse serum are both suitable for the cultivation of B. occultans, provided a good serum batch is cho-sen. Similar results were described for B. bovisand B. bigemina cultures (Fish, Pipano, Shkap & Frank 1993) where bovine serum could be replaced by horse serum without negatively influencing parasite growth. In our experiments, higher PPEs were pro-duced in horse serum than in bovine serum. Research is currently under way, which aims to in-crease the PPE in the culture system. To date, cul-ture-derived parasites have been successfully used by us as antigen for the indirect immunofluorescence antibody test. C.J. VAN NIEKERK & E. ZWEYGARTH ACKNOWLEDGEMENTS We express our appreciation to Mrs E. Horn, Dr S.W. Vogel, Ms M.C Just and Mr M.C. Combrink for their valuable help and support. REFERENCES 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, 48:117-119. GOFF, W.L. & YUNKER, C.E. 1988. Effects of pH, buffers and medium-storage on the growth of Babesia bovis in vitro. Inter-national Journal of Parasitology, 18:775-778. GRAY, J.S. & DE VOS, A.J. 1981 . Studies on a bovine Babesia transmitted by Hyalomma marginatum rufipes Koch, 1844. On-derstepoort Journal of Veterinary Research, 48:215- 223. LEVY, M.G. & RISTIC, M. 1980. Babesia bovis: continuous culti-vation in a microaerophilous stationary phase culture. Science, 207:1218-1220. RIEK, R.F. 1968. Babesiosis, in Infectious blood diseases of man and animals, II , edited by D. Weiman & M. Ristic. New York and London: Academic Press: 219-268. RODRIGUEZ, S.D., BUENING, G.M., GREEN, T.J. & CARSON, C.A. 1983. Cloning of Babesia bovis by in vitro cultivation. In-fection and Immunity, 42:15- 18. THOMAS, S.E. & MASON, T.E. 1981.1solation and transmission of an unidentified Babesia sp. infective for cattle. Onderstepoort Journal of Veterinary Research, 48:155-158. 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. ZWEYGARTH, E. , JUST, M.G. & DE WAAL, D.T. 1995. Continu-ous in vitro cultivation of erythrocytic stages of Babesia equi. Parasitology Research, 81 :355-358. 261 

In vitro cultivation of Babesia occultans

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In vitro cultivation of Babesia occultans

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