The Welgevonden stock of  Cowdria ruminantium, aetiologic agent of
   heartwater, was continuously propagated in DH82 cells, a continuous
   canine macrophage-monocyte cell line. Cultures of DH82 cells were
   readily infected provided that the culture medium was supplemented with
   cycloheximide. Cultures were split at regular 3-day intervals and
   infection rates ranged between 60% and 95%. Cultures were continuously
   propagated through more than 125 passages over a period of more than one
   year.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 and the
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.The Welgevonden stock of  Cowdria ruminantium, aetiologic agent of
   heartwater, was continuously propagated in DH82 cells, a continuous
   canine macrophage-monocyte cell line. Cultures of DH82 cells were
   readily infected provided that the culture medium was supplemented with
   cycloheximide. Cultures were split at regular 3-day intervals and
   infection rates ranged between 60% and 95%. Cultures were continuously
   propagated through more than 125 passages over a period of more than one
   year.Agricultural Research Council of South Africa and the
European Union (Cowdriosis Network) Grant no.
IC18-CT95-0008 (DG12-SNRD).mn201

UPSpace at the University of Pretoria

Onderstepoort Journal of Veterinary Research, 68: 155-157 (2001) RESEARCH COMMUNICATION Continuous in vitro propagation of Cowdria ruminantium (Welgevonden stock) in a canine macrophage-monocyte cell line E. ZWEYGARTH and ANTOINETTE I. JOSEMANS Onderstepoort Veterinary Institute, Parasitology Division Private Bag X05, Onderstepoort, 0110 South Africa ABSTRACT ZWEYGARTH, E. & JOSEMANS, A.1. 2001. Continuous in vitro propagation of Cowdria ruminantium (Welgevonden stock) in a canine macrophage-monocyte cell line. Onderstepoort Journal of Veteri-nary Research, 68:155-157 The Welgevonden stock of Cowdria ruminantium, aetiologic agent of heartwater, was continuously propagated in DH82 cells, a continuous canine macrophage-monocyte cell line. Cultures of DH82 cells were readily infected provided that the culture medium was supplemented with cycloheximide. Cultures were split at regular 3-day intervals and infection rates ranged between 60 % and 95·%. Cultures were continuously propagated through more than 125 passages over a period of more than one year. Keywords: Canine macrophage-monocyte cell line , Cowdria ruminantium, cycloheximide , DH82, heartwater, in vitro cultivation INTRODUCTION Heartwater is an infectious, non-contagious disease of domestic ruminants caused by the rickettsia Cow-dria ruminantium. It is transmitted by ticks of the genus Amblyomma (Lounsbury 1900). The preva-lence of the disease throughout sub-Saharan Africa, the islands of Madagascar, La Reunion, Mauritius and Sao Tome coincides with the distribution of the vector ticks (U ilenberg 1983). The disease also oc-curs on three Caribbean islands (Perreau, Morel , Barre & Durand 1980; Birnie, Burridge, Camus & Barre 1984). Cowdry (1926) was the first to describe, from a his-tological study, the multiplication of C. ruminantium (then known as Rickettsia ruminantium) within the endothelial cells of infected animals. It is therefore Accepted for publication 28 February 2001-Editor not surprising that the first successful in vitro propa-gation of Cowdria was achieved using bovine umbili-cal cord endothelial cells (Bezuidenhout, Paterson & Barnard 1985), although other cell types have also been used. Logan, Whyard, Quintero & Mebus (1987) devised a primary neutrophil culture system suitable for the production of Cowdria to be used in serologi-cal tests. This system was, however, of short dura-tion and unsuitable for continuous in vitro propaga-tion of the organism. Macrophages are also infected by Cowdria (Du Plessis 1975) but these cells are un-suitable for the continuous cultivation of the organ-ism, because normal mature macrophages do not proliferate. However, Jongejan & Bekker (1999) tested several cell lines, which were in use for the cultivation of other ehrlichial species (P388D1 ; MDH-SP; DH82) and were able to grow Cowdria in mono-cyte-macrophage cell lines from mice and dogs. Infec-tion rates remained low, however, and no persistent infections were established. We report here the first continuous propagation of the Welgevonden stock of C. ruminantium in DH82 cells 155 In vitro propagation of Cowdria ruminantium (Welgevonden stock) through more than 125 passages over a period of more than one year. MATERIALS AND METHODS Stock of C. ruminantium The Welgevonden stock was isolated by injecting into a mouse a tick homogenate prepared from a single tick. The tick, a male Amblyomma hebraeum, was collected on the farm Welgevonden in the Northern Province of South Africa (Du Plessis 1985). Cell cultures DH82 cells originally derived from a dog suffering from malignant histiocytosis (Wellman, Krakowka, Jacobs & Kociba 1988) were grown in Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12)(Sigma, St Louis, MO, USA; D 0547) containing 15 mM HEPES and 1.2 g/Q sodium bicar-bonate. It was further supplemented with 10% (v/v) heat-inactivated foetal bovine serum, 2 mM L-gluta-mine, 100 IU/mQ penicillin and 100 Ilg/mQ streptomy-cin. This medium was used for infected and unin-fected cell cultures. Infection of DH82 cells The propagation of C. ruminantium in endothelial cells was carried out as described in detail elsewhere (Zweygarth, Vogel, Josemans & Horn 1997). In brief, endothelial cell cultures heavily infected with C. ruminantium were harvested by scraping off the cell monolayer into the medium. The cell suspension was centrifuged (800 x 9 for 10 min at room temperature) and 2.5 mQ of the supernatant, containing mainly el-ementary bodies, was distributed into culture flasks containing DH82 cells. Attempts were made to initi-ate two different cultures, one using the medium described above and one using the same medium supplemented with 1 Ilg/mQcycioheximide. Cyclohex-imide is a specific inhibitor of protein synthesis in eucaryotic cells (Ennis & Lubin 1964) and has been used to promote growth of chlamydial organisms in cell cultures (Hobson, Johnson & Byng 1977). The cultures were incubated at 37 cC. In each case the initial medium was removed after 24 h and replaced with 5 mQ of the same medium. Monitoring of cultures C. ruminantium infections were detected by light microscopy. Small samples of adherent cells were removed from the cultures, using a sterile 21 gauge needle with a bent tip, and smears were prepared. These were air-dried, methanol-fixed, and quick-stained (RapiDiff; Clinical Sciences Diagnostics, Booysens, South Africa). Occasionally cytospin prep-arations of infected cells floating in the culture super-natant were also monitored. 156 RESULTS Infection rates with C. ruminantium in DH82 cell cul-tures in the absence of cycloheximide were 3.6 % in adherent cells, and less than 1 % in cells floating in the supernatant, and because of these low infection rates the cultures were terminated after two pas-sages. The experiments were repeated twice with similar results (data not shown). In contrast, the cultures of DH82 cells which con-tained cycloheximide were readily infected by the Welgevonden stock of C. ruminantium, with approxi-mately 35 % of adherent cells and 70 % of detached cells showing infection before the first subculture. Cultures were split at regular intervals of 3 days at a ratio of 2. Occasionally many of the adherent cells were found to have detached from the surface of the flask and were subsequently lost when the medium was changed 24 h after passage. In such cases the cultures were passaged onto fresh host cells with-out being split. Infection rates were high, i.e. usually between 60 % and 95 %. Continuous cultures of Cowdria could be maintained only when cycloheximide was added to the medium, and this continued to be true even after 116 pas-sages. If cycloheximide was omitted from cultures having an infection rate close to 100 % the rate be-gan to decline after 3 days and was close to 0 % after only four passages. DISCUSSION Cowdria ruminantium is particularly closely related to those Ehrlichia species in the phylogenetic clade known as Genogroup III (Allsopp, Visser, Du Plessis, Vogel & Allsopp 1997) with somewhat more distant relatives in Genogroups II and I. Several of these species have been successfully propagated in DH82 cells: from Genogroup III Ehrlichia canis (Dawson, Rikihisa, Ewing & Fishbein 1991) and Ehrlichia chaf-fensis (Dawson, Anderson, Fishbein, Sanchez, Gold-smith, Wilson & Duntley 1991) and from Genogroup I Ehrlichia risticii (Van Heeckeren, Rikihisa, Park & Fertel 1993) and Neorickettsia helminthoeca (Riki-hisa, Stills & Zimmerman 1991). The results of our study demonstrate that C. ruminantium too can read-ily be propagated in DH82 cells, a permanent canine macrophage-monocyte cell line, but only if cyclohex-imide is present in the culture medium. Split ratios of two and subculture intervals of 3 days were regu-larly observed, although in rare instances a split ra-tio of one had to be used. Jongejan & Bekker (1999) failed to establish persist-ent infections of Cowdria in the DH82 cell line, and we speculate that this was because they did not in-corporate cycloheximide in the medium. It should be noted that Bezuidenhout (1987) and Yunker, Byrom & Semu (1988) found that the presence of cyclohex-imide in the culture did not improve the propagation of Cowdria in bovine endothelial cells. This might have been due to the fact bovine endothelial cells usually show contact inhibition after confluency, with a concomitant reduction in their rate of metabolism. DH82 cells do not show this characteristic, and we propose that the crucial factor in our experiments was the suppression of protein synthesis in the DH82 cells by cycloheximide (Ennis & Lubin 1964) thus reducing their high metabolic turnover. It has been shown that Chlamydia psittaci and L cells compete for the amino acid isoleucine (Hatch 1975) and it is likely that competition between Cowdria and DH82 cells for certain amino acids would have been sup-pressed by cycloheximide. Such competition could have been the factor limiting the propagation of Cow-dria in the absence of cycloheximide. In conclusion , it has been shown that the Welgevon-den stock of C. ruminantium can be propagated in DH82 cells, provided suitable culture conditions pre-vail. ACKNOWLEDGEMENTS We thank Dr B.A. Allsopp for helpful comments on the manuscript. This 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 ALLSOPP, M.T.E.P., VISSER, E.S., DU PLESSIS, J.L., VOGEL, S.W & ALLSOPP, BA 1997. Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences. Veterinary Parasitology, 71 :283- 300. 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. COWDRY, E.v. 1926. Studies on the aetiology of heartwater: III. The multiplication of Rickettsia ruminantium within the en-dothelial cells of infected animals and their discharge into the circulation. Journal of Experimental Medicine, 44:803- 814 DAWSON, J.E. , ANDERSON , B.E., FISHBEIN , 0.8., SANCHEZ, J.L., GOLDSMITH, C.S., WILSON , K.H. & DUNTLEY, C.W 1991. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. Journal of Clinical Microbiology, 29:2741-2745. E. ZWEYGARTH & A.I. JOSEMANS DAWSON, J.E. , RIKIHISA, Y , EWING, SA & FISHBEIN , D.B. 1991 . Serologic diagnosis of human ehrlichiosis using two Ehrlichia canis isolates. Journal of Infectious Diseases, 163: 564-567. DU PLESSIS, J.L. 1975. Electron microscopy of Cowdria rumi-nantium infected reticulo-endothelial cells of the mammalian host. Onderstepoort Journal of Veterinary Research, 42: 1- 14. 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. ENNIS, H.L. & LUBIN, M. 1964. Cycloheximide: aspects of inhi-bition of protein synthesis in mammalian cells . Science, 146: 1474-1476. HATCH, T.P. 1975. Competition between Chlamydia psittaci and L cells for host isoleucine pools: a limiting factor in chlamydial multipl ication. Infection and Immunity, 12:211-220. HOBSON, D. , JOHNSON, F.WA. & BYNG , R.E. 1977. The growth of the ewe abortion chlamydial agent in McCoy cell cultures . Journal of Comparative Pathology, 87: 155-159. JONGEJAN, F. & BEKKER, C.P.J. 1999. Cowdria ruminantium: recent developments in diagnostic methods, molecular char-acterization and vaccines, in Rickettsia and rickettsial diseases at the turn of the third millenium, edited by D. Raoult & P. Brou-qui. Paris: Elsevier. LOGAN , L.L. , WHYARD, T.C., QUINTERO, J.C. & ME BUS, CA. 1987. The development of Cowdria ruminantium in neutrophils. Onderstepoort Journal of Veterinary Research, 54: 197-204. LOUNSBURY, C.P. 1900. Tick-heartwater experiment. Agricul-tural Journal of the Cape of Good Hope, 16:682-687. PERREAU, P., MOREL, P.C., 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 ile Maurice) . Revue de I' Elevage et de Medecine Veterinaire des Pays Tropicaux, 33: 21-22. RIKIHISA, Y , STILLS, H. & ZIMMERMAN, G. 1991. Isolation and continuous culture of Neorickettsia helminthoeca in a macro-phage cell line. Journal of Clinical Microbiology, 29: 1928-1933. UILENBERG, G. 1983. Heartwater (Cowdria ruminantium infec-tion) : current status. Advances in Veterinary Science and Comparative Medicine, 27:427-480 . VAN HEECKEREN, A.M., RIKIHISA, Y , PARK, J. & FERTEL, R. 1993. Tumor necrosis factor alpha, interleukin-1 a, interleukin-6, and prostaglandin E2 production in murine peritoneal macro-phages infected with Ehrlichia risticii. Infection and Immunity, 61 :4333-4337. WELLMAN, M.L., KRAKOWKA, S. , JACOBS, R.M. & KOCIBA, G.J. 1988. A macrophage-monocyte cell line from a dog with malignant histiocytosis. In Vitro Cellular and Developmental Biology, 24:223-229. YUNKER , C.E., BYROM , B. & SEMU , S. 1988. Cultivation of Cowdria ruminantium in bovine vascular endothelial cells. The Kenya Veterinarian, 12: 12-16. ZWEYGARTH, E. , VOGEL, S.W , JOSEMANS, A. & HORN, E. 1997. In vitro isolation and cultivation of Cowdria ruminantium under serum-free culture conditions. Research in Veterinary Science, 63:161-164. 157 

Continuous in vitro propagation of Cowdria ruminantium (Welgevonden stock) in a canine macrophage-monocyte cell line

https://repository.up.ac.za/bitstream/2263/18399/1/24zweygarth2001.pdf

Continuous in vitro propagation of Cowdria ruminantium (Welgevonden stock) in a canine macrophage-monocyte cell line

Abstract

Similar works

Full text

Available Versions

UPSpace at the University of Pretoria