Current status of in vitro cultivation of Cowdria ruminantium

veterinary parasitology ELSEVIER

Veterinary Parasitology 57 (1995) 205-211

Current status of in vitro cultivation of Cowdria ruminantium C.E. Yunker Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort, South Africa

Abstract Heartwater caused by Cowdria ruminantium infection is the most important tick-borne disease of ruminants in southern Africa. The in vitro culture system for this rickettsia, developed less than a decade ago, is responsible for the great majority of research accomplishments currently being reported in the areas of epidemiology, diagnosis and control of heartwater. Despite this progress, cultivation of C. ruminantium remains more of an art than a science, inasmuch as significant discrepancies exist, both among and within laboratories, in the ability to successfully and repeatedly produce cultured organisms. The current status of the in vitro system and its contributions are reviewed, and ongoing research in these areas by the Onderstepoort Veterinary Laboratory and collaborating institutions is discussed. Keywords: Cowdria ruminantium; In vitro culture

Heartwater is the most important tick-borne disease of ruminants in southern Africa, and second in importance only to East Coast Fever elsewhere in the Afrotropical region. It is known or presumed to be present in all countries south of the Sahara except Lesotho (Camus and BarrY, 1988; Yunker and AUsopp, 1994), and exists as well on offshore African islands (Sao Tom~, Zanzibar) plus some in the Indian Ocean (Comores, Madagascar Reunion, Mauritius). Its established presence in the Caribbean Islands of Antigua, Guadeloupe and Marie Galante, and possibly elsewhere in the Antilles (Muller Kobold et al., 1992), serves to threaten the livestock industry and wildlife of North, Central and South America. The heartwater agent, Cowdria ruminantium, is a rickettsial organism--an intracellular parasite dependent on a living host for survival and growth. Until re0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10304-4017 (94)03121-5

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cently, a means for producing quantities of C. ruminantium was lacking. All attempts, over many decades, to grow the organism outside of the host animal had either failed or resulted in only short-term growth (reviewed by Logan et al., 1987) and researchers requiring the organism for their studies were forced to try to obtain small quantities from infected host tissues. In 1985, however, scientists at the Veterinary Research Institute, Onderstepoort, RSA, reported a culture system for the heartwater organism which entirely changed the nature of research on the disease. That was the growth in vitro of C. ruminantium in a serially propagating line of bovine umbilical cells, the E-5 line, of endothelial origin (Bezuidenhout et al., 1985 ). With this long-awaited technology, laboratories in Africa, Europe and America renewed research on this important disease. For the first time, large quantities of relatively pure organisms became available, surface proteins could be identified and monoclonal antibody produced. Serological tests employing cell-generated antigens, as well as DNA probes were developed and an attenuated live vaccine was developed. Regrettably, each of these have limitations and additional developmental work is required. Probably the most important benefit was the ability to prepare genomic libraries of the heartwater organism, which has implications for eventual control by recombinant or synthetic vaccines. Significantly, at a recent international symposium on heartwater disease held in Guadeloupe, West Indies, in 1993, 75% of the research findings reported were based on cultured organisms or their products. Today I would like to point out problem areas in Cowdria cultivation in need of resolution, and indicate other areas where in vitro studies should be able to answer questions in both basic and applied research on heartwater disease. Despite the potential that the culture system affords, workers in different laboratories, and even different workers in a single laboratory, commonly experience difficulties in consistently growing the heartwater organism--and I include the Onderstepoort laboratory, where the technique originated. Reasons for these discrepancies are difficult to identify, and could range from occasional use of poorly rinsed glassware to an inherent inadequacy of the culture medium. Because the nutritional requirements and metabolic processes of the heartwater rickettsia are poorly known, media used for culture have been arbitrarily chosen and there is no agreement as to which will produce the most consistent and repeatable results. Each laboratory has its own favorite medium and, invariably, its share of culture failures. There is, therefore, a need to improve and refine the culture system to the point where any laboratory with a cell culture facility can easily and reliably produce quantities of the organism as needed. This can be facilitated by gaining an understanding of the metabolic processes of C. ruminantium. To improve the culture system, we have employed both an empirical and an analytical approach. Extrinsic unknowns were avoided through use of plasticware, by introducing reagent grade water, sterility tests, heat-inactivation of serum, etc. The problem of adequacy of the culture medium is more complex and different

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medium formulations must be tested in combination with sera from various sources. The medium originally prescribed for growth of Cowdria was the Glasgow modification of Eagle's Minimal Essential Medium (GMEM; MacPherson and Stoker, 1961 ). GMEM, a modification of Eagle's Basal Medium (BME), contains twice the BME concentrations of amino acids and vitamins plus tryptose phosphate broth. It is, itself, not a highly nutritious medium, and may represent a minimal medium for growth of Cowdria. Liebovitz's L- 15, which is a highly enriched medium that contains megadoses of essential and non-essential amino acids, has been used in Harare since 1990 for Cowdria cultivation (Byrom and Yunker, 1990). However, it has not supported good growth of the rickettsia in South Africa, the difference presumably being in the source of supply. However, overwhelming the system with excess nutrients will not provide answers to questions concerning Cowdria metabolism. Instead, a basal growth medium should be identified and its components either sequentially omitted or augmented until effects on growth are seen. This is essentially what we are currently doing. Using GMEM as a base, we are attempting to learn something about the metabolic needs of the rickettsia, with the aim of identifying deficiencies of the prescribed culture medium. We are currently testing the result of adding various nutrients or metabolites to the basic medium, and analyzing depleted medium. Additives were selected on the basis of experiences with other rickettsias (Table 1 ). For example, these four sugars, glucose, galactose, glucosamine and galactosamine, are involved in rickettsial UDP production and cell wall synthesis, and the last three are important in peptidoglycan synthesis. (We have sometimes seen that when Cowdria fails to complete development in cultured cells, the organisms are swollen and have an indistinct outline, which implies that the outer cell wall and membranes might be deficient or lacking altogether. ) Glucose is already known to be necessary for growth of Cowdria (Byrom and Yunker, 1990), but why this should be is not known. It is probably not an energy substrate. Glycerol and palmitic acid are important metabolites in fatty acid synthesis of rickettsias. Iron and iron-saturated transferrin might improve the culture system. Iron is, of course, vital to the synthesis of heme-proteins, especially cytochrome and cytochrome oxidases, which rickettsias require. Extra glutamine Table 1 Metabolites, nutrients and other factors possibly beneficial to Cowdria ruminantium grown in vitro

Glucose Galactose Glucosamine Galactosamine n-acetylglucosamine n-acetylgalactosamine Inositol Glycerol Palmiticacid

Transferrin Ferric citrate Glutamate Pyruvate fl-mercaptoethanol VitaminE Other non-essentialaminoacids Other fatty acids Cholesterol

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and pyruvate added to the medium should also be tested, as should the addition of free radical scavengers, beta-mercaptoethanol and vitamin E. The former scavenger also reduces sulfhydryl groups released as by-products of amino acid catabolism, and aids in swelling the phosphorylated sugar pools, which provide intermediates and nucleotides for rickettsias. Changes of amino acids and proteins in cell culture medium from both infected and uninfected cultures were studied in collaboration with Professor A.W.H. Neitz of the Biochemistry Department, University of Pretoria. Samples of culture medium were taken daily and analyzed for free amino acids and major proteins. The former were analyzed by the Pico-Tag reversed phase high performance liquid chromatography pre-column derivatization method, and proteins determined by capillary electrophoresis using a 57 cm × 75/tm fused silica open tube at high pH. Preliminary results indicate that, in general, over a 5 day period during which the organism completes a cycle of growth, the concentrations of amino acids remain fairly constant in uninfected cultures, but increase in Cowdria-infected ones. This is especially apparent in the cases ofglutamic acid, alanine, proline, valine, isoleucine, leucine and lysine, indicating that the rickettsia is capable of producing these entities, either through de novo synthesis or proteolysis. This would imply that these particular amino acids are superfluous and can be omitted from the culture medium. Proteolysis by the rickettsia was, in fact, seen. Five major proteins, which increased in uninfected cultures, decreased in infected ones throughout the course of infection. Depletion of two amino acids, glutamine and arginine, was also observed (Table 2 ); in the case ofglutamine, significantly more was lost than in the uninfected controls. Although the metabolic processes of Cowdria are unknown, glutamine is known to be the major energy source of its nearest relative, Ehrlichia. Analyses are being repeated to confirm this. If confirmed, it will mean that the medium originally recommended for culture of the rickettsia contains far too little of these acids. Effects on Cowdria growth after extra arginine and glutamine are added to GMEM are now being assessed. Obviously, if initial concentrations of either amino acid are too low, the host-cell and parasite will be in competition for them. Depending on the variable amounts of free amino acids contributed by the particular serum that is added to the medium, Cowdria may or may not complete its Table 2 Depletion of essential amino acids (mg 1 ~) in Glasgow MEM after exposure to Cowdria ruminantium in sheep endothelial cell cultures Day 0

t-arginine t-glutamine a

42.0 292.0

Day 2

2.2 85.0

Day 5

1.8 38.0

Percent loss Infected

Control

95.7 87.0

96.0 71.0

aL-glutamine held in cell culture medium at 35 °C normally degrades at the rate of 5% day- i.

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cycle before the medium is renewed. Significantly, perhaps, use of Dulbecco's MEM, which contains a double dose of GMEM's amino acids, has given promising results in preliminary growth trials. Studies along these lines could reveal the biochemical stratagem of the heartwater organism, which will not only facilitate construction of a specific culture medium, but possibly also lead to an understanding of the pathogenesis of the infection, about which very little is known. In order to judge the effect of various culture conditions on the growth of a parasite, it is necessary to have some means to assess its growth. A knowledge of the infectious potential of a given stock or preparation is vital to all work with living pathogens, including vaccine development. There is presently no means to conveniently titrate stocks of C. ruminantium other than by animal inoculation or infection of a large series of culture flasks. Animal inoculation is practical only with mouse-infective stocks of Cowdria; the use of ruminants to titrate other stocks is prohibitively expensive. The most precise titration method, plaque production, which is useful in studies of Rickettsia spp., could not be made to work with Cowdria, despite the fact that a cytopathic effect is produced in infected monolayers (C. Yunker, unpublished information, 1992) nor have we been successful in attempts to use the metabolic-inhibition test with this rickettsia. However, we have used the cytopathic effect to estimate titres by the infectious dose-50 (IDso) method. Sheep brain endothelial cells (SBE- 189; Bezuidenhout and Brett, 1992) were grown in 24-well tissue culture plates and infected by highspeed centrifugation with serial ten-fold dilutions of infectious elementary bodies of C. ruminantium of a murinotrophic stock, Kwanyanga. Infection, judged by visual inspection was verified by microscopic examination of monolayers stained with Diff-Quik or Hoechst (bis-benzamide). Tissue culture IDso values based on CPE were comparable to LDso values obtained by mouse inoculation. However, when wells beyond CPE endpoint were sampled and stained, organisms could be identified in some, possibly indicating that the tissue cuRure system is more sensitive than mice. Alternatively, it could mean that the rickettsias not inducing CPE were non-viable. Further investigations are in progress. Not long ago, before the advent of a culture system for heartwater, it was repeatedly proclaimed that when Cowdria ruminantium was finally cultured, DNA libraries could be produced and, with these, all manner of diagnostics and vaccines would evolve. Then, we could forget about cultures. (But then, we were also confident that any animal immunized to a stock of heartwater would be immune to all stocks, and that a new generation of serological diagnostics based on culture-produced antigen would resolve many problems in diagnosis.) However, as more is accomplished, more questions arise. We now know that cross-protection among certain stocks of C. ruminantium is limited at best, and that the new diagnostics are seriously flawed by false positive reactions with various species of Ehrlichia. If a live vaccine prepared from whole organisms cannot provide protection, can we expect a subunit or synthetic vaccine to do more? No doubt biotechnology will one day provide an affirmative answer; but meanwhile, we must make do

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with the existing vaccine, despite its obvious defects. This vaccine, which has been with us for half a century, consists of live, virulent organisms in sheep blood. However, there is a definite need to replace the existing vehicle for production of the vaccine with tissue cultures. This would reduce or eliminate the potential for other blood diseases to be carried with the vaccine, preclude erythrocytic reactions, and be significantly less expensive to produce. It has been estimated that one roller bottle culture of 800 cm 2 surface area will provide up to 20 000 sheep doses of vaccine (Bezuidenhout and Brett, 1992 ). Other areas where Cowdria cultures can be applied are in the development of inactivated or attenuated vaccines from locally prevalent genotypes. This is being done in Guadeloupe (Martinez et al., 1993 ), Zimbabwe (H.A. Andrew, personal communication, 1992 ) and Senegal (Jongejan et al., 1993 ). The in vitro culture system especially lends itself to modification of virulence. The heartwater organism has spontaneously attenuated on serial passage without loss of immunogenicity (Jongejan, 1991 ), and it is well established that manipulation of the culture parameters or exposure to mutagenic chemicals will serve the same purpose with other pathogens. Also, studies of the immune-response to heartwater are facilitated through use of in vitro systems. Expression and action of various cytokines are readily studied in in vitro systems, and autologous cell-lines (i.e. lines of parasite-supporting cells derived from the same experimental animals to be used as experimental subjects) are particularly valuable in studies of cell-mediated immunity. Finally, there is an urgent need to reduce the confusing genotypic heterogeneity that exists among different isolates of C. ruminantium, as reported elsewhere in this program (Allsopp and Visser, 1993 ). The existence of at least four species of Cowdria, plus numerous other unidentified organisms related to protobacteria, including Ehrlichia and Rickettsia, is suggested on the basis of phylogenetic analysis of sequences of parts of the srRNA gene of several South African isolates obtained from heartwater cases. There is evidence that cultured endothelial cells may serve to drastically reduce the heterogeneity that may exist in a given stock of C. ruminantium. Obviously, much basic and applied research remains to be done in the area of cultivation of the agent or agents associated with heartwater disease.

Acknowledgments I thank Dr. Jim C. Williams, Food and Drug Administration, Bethesda, MD, for suggestions regarding approaches leading to an understanding of metabolic processes of C. ruminantium, and Professor A.W.H. Neitz, Biochemistry Department, University of Pretoria, for allowing me to discuss portions of our work currently in progress. E. Horn and S. Brett provided technical assistance.

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