Chemically defined media, termed SFMC-23 and SFMC-36, were devised for
   the  in vitro culture of  Cowdria ruminantium, the causative agent of
   heartwater in domestic ruminants. Both media were based on Dulbecco's
   modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12) containing
   various supplements. Medium SFMC-23 and SFMC-36 supported the long-term
   growth of the Welgevonden stock of  C. ruminantium for a total of 55 and
   28 passages, respectively, with regular passage intervals of 3 days.
   Using SFMC-23, split ratios varied from 5-10, depending on which host
   cell line was used. Other stocks of  C. ruminantium (Sankat,
   Blaauwkrantz, Senegal) were successfully propagated for a test period of
   ten passages.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Agricultural Research Council of South Africa. 
 European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD).mn201

Josemans, A.I.

Zweygarth, Erich

Onderstepoort Journal of Veterinary Research

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Chemically defined media, termed SFMC-23 and SFMC-36, were devised for
   the  in vitro culture of  Cowdria ruminantium, the causative agent of
   heartwater in domestic ruminants. Both media were based on Dulbecco's
   modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12) containing
   various supplements. Medium SFMC-23 and SFMC-36 supported the long-term
   growth of the Welgevonden stock of  C. ruminantium for a total of 55 and
   28 passages, respectively, with regular passage intervals of 3 days.
   Using SFMC-23, split ratios varied from 5-10, depending on which host
   cell line was used. Other stocks of  C. ruminantium (Sankat,
   Blaauwkrantz, Senegal) were successfully propagated for a test period of
   ten passages.Agricultural Research Council of South Africa. 
 European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD).mn201

UPSpace at the University of Pretoria

Onderstepoort Journal of Veterinary Research, 68:37--40 (2001) A chemically defined medium for the growth of Cowdria ruminantium E. ZWEYGARTH and ANTOINETTE I. JOSEMANS Parasitology Division, Onderstepoort Veterinary Institute, Private Bag X05 Onderstepoort 0110, South Africa ABSTRACT ZWEYGARTH, E. & JOSEMANS, A.1. 2001 . A chemically defined medium for the growth of Cowdria ruminantium. Onderstepoort Journal of Veterinary Research, 68:37--40 Chemically defined media, termed SFMC-23 and SFMC-36, were devised for the in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants. Both media were based on Ou lbecco's modified Eagle's medium nutrient mixture Ham F-12 (OME/F-12) containing var ious supplements . Medium SFMC-23 and SFMC-36 supported the long-term growth of the Welgevonden stock of C. ruminantium for a tota l of 55 and 28 passages, respectively, with regular passage intervals of 3 days. Using SFMC-23, spl it ratios varied from 5- 10, depending on which host cell line was used. Other stocks of C. ruminantium (Sankat, Blaauwkrantz, Senegal) were success-fully propagated for a test period of ten passages. Keywords: Chemically defined medium, Cowdria ruminantium, in vitro culture INTRODUCTION Heartwater is a tick-borne rickettsial disease of rumi-nants caused by Cowdria ruminantium. The disease is prevalent in sub-Saharan Africa and surrounding islands (Ui lenberg 1983), and in the Caribbean (Per-reau, Morel, Barre & Durand 1980; Birnie, Burridge, Camus & Barre 1984). The first vector of the disease to be identified was Amblyomma hebraeum in South Africa (Lounsbury 1900). Currently, twelve species of Amblyomma ticks are known to be capable of transmitting heartwater (Walker & Olwage 1987). The in vitro culture of C. ruminantium was f irst achieved by Bezuidenhout, Paterson & Barnard (1985). They used calf endothelial cells as host cells and Eagle's medium containing 10% bovine serum. Subsequently, Glasgow minimal essential medium Accepted for publication 15 November 200D-Editor (GMEM) (Bezuidenhout 1987), Leibovitz L-15 (By-rom & Yunker 1990) and Dulbecco's minimal essen-tial medium (DMEM) (Martinez, Sheikboudou, Cou-raud & Bensaid 1993) supplemented with various amounts of foetal bovine serum and tryptose phos-phate broth (TPB) were used for Cowdria culture . However, the culture of C. ruminantium in the pres-ence of undefined components such as serum and TPB has some serious disadvantages. Batch-to-batch variation of components, lack of standardiza-tion of experimental protocols, and the difficulty of identifying specific growth requirements are a few of the factors involved. Recently, the propagation of C. ruminantium in se-rum-free media supplemented with bovine lipopro-teins and transferrin was achieved (Zweygarth , Jose-mans & Horn 1998). In this report it is shown that C. ruminantium can be cultured in a protein-free, chemi-cally defined medium , by replacing bovine lip?-proteins by chemically defined lipids and transfernn by protein-free iron complexes. 37 Chemically defined medium for growth of Cowdria ruminantium MATERIALS AND METHODS Stocks of C. ruminantium Four different stocks of C. ruminantium were used the origins of which are described below. The We~ Igevonden stock was isolated by injecting a homoge-nate from a tick collected on the farm Welgevonden (Northern Transvaal, South Africa) into a mouse (Du Plessis 1985). The Senegal stock was isolated in 1981 from cattle in Senegal by subinoculation into sheep (Jongejan, Uilenberg, Franssen, Gueye & Nieuwenhuijs 1988). Both the Senegal and the Wel-gevonden stock express a conserved immuno-dominant 32 kDa protein which is currently used for serodiagnosis of the disease (Van Vliet, Jongejan, Van Kleef & Van der Zeijst 1994). The Pokoase stock was isolated in Ghana by Dr Bell-Sakyi , Centre for Tropical Veterinary Medicine, Edinburgh, Scotland. The Blaauwkrantz stock was originally isolated from an eland on the farm Blaauwkrantz (Eastern Cape, South Africa) in 1996 by Dr J.L. du Plessis, formerly of the Onderstepoort Veterinary Institute. Endothelial cell lines The E5 calf endothelial (Bezuidenhout et al. 1985) and the bovine aorta BA 886 (Yunker, Byrom & Semu 1988), cell lines were used. Uninfected cells were propagated at 37 °C as monolayers in medium con-sisting of Dulbecco's modified Eagle's medium (High-veld Biological, Kelvin, South Africa) with 10% (v/v) heat-inactivated foetal bovine serum (FBS; Delta Bioproducts, Kempton Park, South Africa). The me-dium was buffered with 10 mM HEPES (N-[2-hydrox-yethyl]piperazine-N'-[2-ethanesulfonic acid]) (Sigma, St. Louis, MO, USA), and 10 mM sodium bicarbo-nate, and supplemented with 2 mM L-glutamine, 100 IU/m~ penicillin and 1 00 I-lg/m ~ streptomycin. Before being used for C. ruminantium cultivation , each mo-nolayer was rinsed three times with 5 m~ of phos-phate-buffered saline to reduce serum contamina-tion. Endothelial cell lines were used at passage 128-177 (E5) and 33 to 65 (BA 886). Media for the propagation of C. ruminantium Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham (Sigma) containing 15 mM HEPES and 1.2 giL sodium bicarbonate was used. This was supple-mented with 2 mM L-glutamine, 1 00 IU/m~ penicillin, 1 00 I-lg/m ~ streptomycin, and is referred to as serum-free medium for Cowdria no. 36 (SFMC-36). SFMC-23 was prepared by the addition of a mixture con-taining 1 0 I-l~ of 40 mM ferric ch loride hexahydrate (Sigma) in 10 mM HCI and 1 m~ of 0.5 M glycylglycine (Sigma) to 1 00 m~ SFMC-36 supplemented with 0.1 % (v/v) chemically defined lipids (CDL Life Tech-nologies, Paisley, Scotland) . Propagation of cultures Propagation of cultures was carried out as described previously (Zweygarth, Vogel, Josemans & Horn 1997) with minor modifications. Briefly, infected cul-tures were harvested for subcultivation by scraping off the cell monolayer into the medium using a Pas-teur pipette bent at the tip to form a small hook. Cells were dispersed by pipetting the suspension up and down. The cell suspension was centrifuged (800 x 9 for 10 min at room temperature) and the appropri-ate amount (0.5 m~ or 1 m~) of supernatant was dis-tributed into each of two culture flasks containing the uninfected replacement endothelial cell line. Fresh medium was then added to 2.5 m~. The culture flasks were placed onto a rocker platform (three cycles per min) for 30 min, after which a further 2.5 mQ aliquot of fresh medium was added. After 24 h all the me-dium was discarded and replaced with 5 m~ fresh medium. Smears were prepared for microscopic ex-amination by removal of small samples from the monolayer using a sterile 21 -gauge needle with a bent tip, in order to determine the presence of C. rumi-nantium in the endothelial cells. The smears were air-dried, methanol-fixed and stained with RapiDiff (Clini-cal Sciences Diagnostics, Booysens, South Africa). RESULTS SFMC-23 and SFMC-36 were successfully used to propagate the C. ruminantium (Welgevonden) stock. The growth characteristics obtained with SFMC-23 and SFMC-36 were almost identical with an average passage interval of 3 days. After transfer of the Wel-gevonden stock into SFMC-36, a 4-day passage interval was initially observed, followed by 3-day TABLE 1 Cultivation of C. ruminantium stocks in medium SFMC-23 or SFMC-36 Stock of Total number of Number of passages Passage intervals C. ruminantium passages at termination in serum-free medium (days) Average Range Welgevonden 157 55 3.0 3 Welgevonden (SFMC-36) 157 28 3.0 3-4 Blaauwkrantz 25 10 3.3 3-5 Sankat 20 10 4.6 3-12 Senegal 26 10 4.4 3-6 38 intervals. The split ratio used was five when BA cells were used and ten when E5 cells were used as host cells. The cultures were terminated after 55 and 28 passages, respectively, due to a Mycoplasma con-tamination of the host cells. The Blaauwkrantz, San-kat and Senegal stocks were propagated in SFMC-23 over an observation period of ten passages with average passage intervals of 3.3, 4.6 and 4.4 days, respectively. A split ratio of two for the latter three stocks was obseNed throughout the cultivation pe-riod. The results are summarized in Table 1. DISCUSSION Two protein-free, chemically defined media, referred to as SFMC-23 and SFMC-36, one of which supports the growth of at least four different stocks of C. ruminantium are described in this study. The media are, however, only chemically defined to the extent of the purity of culture ingredients themselves. The in vitro cultivation of C. ruminantium under serum-free culture conditions has been reported previously (Zweygarth et al. 1997). Three C. ruminantium stocks were propagated in a serum-free medium based on a modified HL-1 medium, a DME/F-12-based me-dium supplemented with the HL-1 proprietary solu-tion A, consisting of transferrin, testosteron, sodium selenite, ethanolamine, saturated and unsaturated fatty acids, and stabilising proteins. In further experi-ments, two components, namely bovine transferrin and bovine lipoproteins that have a growth-promot-ing action on Cowdria were identified. These two components were able to replace solution A in the culture medium (Zweygarth et al. 1998). A further step towards a completely defined medium was then attempted by replacing the bovine lipoproteins and transferrin with chemically defined compounds. In-stead of bovine lipoproteins, a commercially avail-able mixture of chemically defined lipids was used. Transferrin was replaced with a complex prepared from glycylglycine and ferric chloride (Yabe, Kato, Matsuya, Yamane, lizuka, Takayoshi & Suzuki 1987). This supplemented medium, SFMC-23, supported the propagation of the Welgevonden stock in a way never obseNed in our laboratory before with respect to regularity of subculture and level of infection. Cow-dria was propagated for 55 passages at regular 3-day inteNals at split ratios, which are rarely achieved by conventional, serum-containing medium. During culture propagation it also became evident that the E5 cell line allowed a higher split ratio than the BA cell line. The reasons for this are not yet clear. Both cell lines were bovine-derived, although from different anatomical sites, the umbilical cord (E5) and the aorta (BA). Therefore, careful selection of the host cell line is necessary for optimal Cowdria growth. Surprisingly, SFMC-36, the medium which was sup-plemented with L-glutamine and antibiotics only, gave similar results to SFMC-23. This conflicted with E. ZWEYGARTH & A.I. JOSEMANS previous findings, where supplements of lipids and transferrin had to be added to maintain growth of C. ruminantium in culture (Zweygarth et al. 1998). Nev-ertheless, DME/F-12, the basis of the serum-free media, contains ferric nitrate and ferrous sulphate as a source of iron, and linoleic acid as a source of fatty acids. The results presented here demonstrate that supplementation of the Cowdria culture medium with components other than L-glutamine and antibiotics is not necessary. In addition to the Welgevonden stock, three other stocks of C. ruminantiumwere propagated in SFMC-23. However, passage intervals were slightly longer and subculture ratios usually lower than with the Welgevonden stock. This may have been due to the fact that these stocks had far fewer passages in culture than the Welgevonden stock and were there-fore less adapted to growth in vitro. Although the experiments are not completely conclu-sive as to the requirement of iron and/or lipid acids, it has been shown that Cowdria can be propagated in a protein-free, completely defined synthetiC me-dium (DMEM/F-12) which is commercially available. The media described here may be of value for ex-periments in which serum has a negative impact on results. They can certainly contribute to the elucida-tion of the amino acid requirements of cultured Cow-dria. Furthermore, these media may contribute to a more economical production of C. ruminantium or-ganisms in vitro. Whether these media can also be used for culture initiation remains to be evaluated. ACKNOWLEDGEMENTS We thank Dr M.T.E.P. Allsopp for helpful comments on the manuscript. The research was supported by the Agricultural Research Council of South Africa and the European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD). REFERENCES BEZUIDENHOUT, J.D. 1987. The present state of Cowdria rumi-nantium in cell lines. Onderstepoort Journal of Veterinary Re-search, 54:205-210. BEZUIDENHOUT, J.D., PATERSON , C.L. & BARNARD, B.J.H. 1985. In vitro cultivation of Cowdria ruminantium. Onderste-poort Journal of Veterinary Research, 52: 113-120. BIRNIE, E.F , BURRIDGE, M.J. , CAMUS, E. & BARRE, N. 1984. Heartwater in the Caribbean: isolation of Cowdria ruminantium from Antigua. The Veterinary Record, 116:121-123. BYROM, B. & YUNKER, C.E. 1990. Improved culture condition for Cowdria ruminantium (Rickettsiales) , the agent of heartwa-ter disease of domestic ruminants. Cytotechnology, 4:285-290. DU PLESSIS, J.L. 1985. A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice injected with tick homogenates. Onderstepoort Jour-nal of Veterinary Research, 52:55-61. JONGEJAN, F , UILENBERG, G., FRANSSEN, FF.J. , GUEYE, A. & NIEUWENHUIJS, J. 1988. Antigenic differences between 39 Chemically defined medium for growth of Cowdria ruminantium stocks of Cowdria ruminantium. Research in Veterinary Sci-ence, 44:186-189. LOUNSBURY, C.P 1900. Tick-heartwater experiment. Agricul-tural Journal of the Cape of Good Hope, 16:682-687. MARTINEZ, D., SHEIKBOUDOU, C., COURAUD, P.O. & BEN-SAID. A.1993. In vitro infection of bovine brain endothelial cells by Cowdria ruminantium. Research in Veterinary Science, 55: 258-260. PERREAU, P., MOREL, PC., BARRE, N. & DURAND, P. 1980. Existence de la cowdriose (heartwater) a Cowdria ruminantium chez les ruminants des Antilles fran9aises (La Guadeloupe) et des Mascareignes (La Reunion et lie Maurice) . Revue d' Elevage et de Medecine Veterinaire des Pays Tropicaux, 33: 21-22. UILENBERG, G. 1983. Heartwater (Cowdria ruminantium infec-tion): Current status. Advances in Veterinary Science and Com-parative Medicine, 27:427-480. 40 WALKER, J.B. & OLWAGE, A. 1987. The tick vectors of Cowdria ruminantium (Ixodoidea, Ixogenus Amblyomma) and their dis-tribution . Onderstepoort Journal of Veterinary Research, 54: 353-379. VAN VLIET, A., JONGEJAN, F., VAN KLEEF, M. & VAN DER ZEIJST, BAM. 1994. Molecular cloning, sequence analysis and expression of the gene encoding the major antigenic pro-tein of Cowdria ruminantium. Infection and Immunity, 62: 1451-1456. YABE, N. , KATO, M. , MATSUYA, Y , YAMANE, I. , IIZUKA, M., TAKAYOSHI , H. & SUZUKI , K. 1987. Role of iron chelators in growth-promoting effect on mouse hybridoma cells in a chemi-cally defined medium. In Vitro Cellular & Developmental Biol-ogy, 23:815-820 . YUNKER, C.E., BYROM, B. & SEMU, S. 1988. Cultivation of Cowdria ruminantium in bovine vascular endothelial cells . The Kenya Veterinarian, 12:12-16. 

A chemically defined medium for the growth of Cowdria ruminantium

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A chemically defined medium for the growth of Cowdria ruminantium

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