Cytotechnology 4: 285-290, 1990. 9 1990KluwerAcademic Publishers. Printed in the Netherlands. Technical report
Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants B. Byrom and C.E. Yunker University of Florida/U.S. Agency for International Development~Zimbabwe Heartwater Research Project, P.O. Box 8101, Causeway, Zimbabwe Received 17 May 1990; accepted in revised form 28 June 1990
Key words: heartwater, cell culture, Cowdria ruminantium, bovine vascular endothelial cells Abstract The causal agent of heartwater disease of domestic ruminants, Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0 =- 6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.
Introduction Heartwater (Cowdria ruminantium infection), one of the most important livestock diseases in Africa (Mare, 1984), causes high mortality in susceptible animals of enzootic areas. Its establishment and subsequent spread in certain Caribbean islands (Burridge, 1985) and the demonstration of a carrier state in wild ruminants that might be transported or imported into non-enzootic areas (Andrew and Norval, in press) portends possible spread to domestic and wild ruminants of North and South America, where potential tick vectors (Amblyomma spp.) already exist. The disease lacks a practical diagnostic test and riskfree vaccine, basically because of unavailability of adequate amounts of the causal organism. Recent demonstrations that C. ruminantium can be grown in vitro (Bezuidenhout et al., 1985; Bezuidenhout, 1987) offer the means to resolve these inadequacies, but the culture conditions
required (irradiative or chemical treatment of monolayers) are restrictive and damaging to the cells. Subsequently, we observed that the heartwater organism could, in some instances, be grown in untreated monolayers of bovine vascular endothelial cells (Yunker et al., 1988; Byrom et al., submitted for publication). More recently, we refined the conditions for growth of C. ruminantium in these monolayers and present our procedures here.
Materials and methods Bovine endothelial cell cultures These were prepared from vascular tissues obtained from abattoir-slaughtered steers. A modification of the method of Hirumi and Hirumi (1984) was used. Short segments of aorta and pulmonary artery near the heart and proximal to
286 vessel branches were removed and immersed i n phosphate-buffered saline (pH 7.4; lacking Ca ++ and Mg ++) (PBS) containing the antibiotics penicillin-G (200 units/ml; Caps, Harare, Zimbabwe), streptomycin sulfate (200 ~g/rnl; Caps) and neomycin sulfate (80 ~tg/ml; No. N1876, Sigma, St. Louis, MO) and Fungizone; (5.0 I.tg/ml; Squibb, Isando, SA) (PBS/A). After transport to the laboratory, generally within 90 min after collection, the segments were washed 3 times with PBS/A and outer layers of connective tissue and fat were aseptically removed. The inner layers, kept wet with the solution, were cut into 1 cm 2 pieces, each of which was placed lumen side down on a drop of collagenase (No. C1764 Sigma), 1 mg/ml in PBS/A, in a sterile 90 mm petri dish. Dishes were incubated for 30 min at 37~ the squares were then overturned, and detaching cells and cell clumps were collected by gentle pipetting o f collagenase solution onto the central portion of the square. Suspensions of aortic and pulmonary artery cells were transferred to separate sterile 15 ml conical centrifuge tubes and centrifuged at 1000 rpm/5 min. The supematants were removed by aspiration and pellets were suspended in 12 ml of the Glasgow modification of Eagle's minimal essential medium (GMEM, No. 041-2100, Gibco Laboratories, Grand Island, NY). Medium contained the above antibiotics but lacked foetal bovine serum. Sodium bicarbonate, 7.5% solution was added (4.7 ml/litre), and the medium was gassed with CO2 to bring the pH to 6.0-6.5. The cells were again pelleted and resuspended in 12 ml of complete medium. Complete medium was GMEM to which was added 10% tryptose phosphate broth (TPB) solution (No. CM283, Oxoid Ltd, Basingstoke, UK), 20% heatinactivated foetal bovine serum (FBS, Flow Labs., Irvine, Ayrshire Scotland), 292 ~tg/ml Lglutamine (No. 810-1051 Gibco) and 300 [.tg/ml endothelial cell growth supplement (ECGS, No. E2759 Sigma). The two suspensions were distributed in 0.5 ml aliquots among the wells of 2-24 well plastic tissue culture clusters (No. 3524, Costar, Cambridge, MA) and an additional 0.5 ml of complete medium was added. The plates were incubated at 37~ week in a humidified cham-
ber containing 5-10% CO2. At this time, 1 ml of complete medium was added to the wells. When a number of wells showed confluent monolayers, all wells showing endothelial cell growth were washed with PBS, and were incubated at 37~ min with trypsin-EDTA solution [0.05% trypsin 1:250 (No. 840-7072, Gibco) plus 0.02% EDTA (No. E6511, Sigma) in PBS]. Loosened cells were gently aspirated with a Pasteur pipette and pooled with 5 ml per well of complete GMEM containing only 150 ~tg/ml ECGS, one-half the above-mentioned concentrations of antibiotics and 25% filtered conditioned medium from the 24 well plate. The suspension was distributed in 5 ml amounts in small (25 cm 2) plastic T-flasks (No. 3055; Costar) and incubated at 37~ for 1-2 weeks. At confluency, each culture was harvested by enzymatic treatment and passed to a large (75 cm 2) T-flask (No. 3275; Costar). At this point EGGS was omitted from the medium and foetal calf serum reduced to 10%. Again 25% conditioned medium was added. The volume of medium in each flask was made up to 25 ml and flasks were incubated as above. When monolayers were confluent, stocks of the cell lines were stored in liquid N2. Further passages were carried out in complete GMEM containing 10% FBS, 10% TPB solution, 292 I.tg/ml L-glutamine and one-half concentrations of the above-mentioned antibiotics. Stock cultures in large T-flasks of 2 such cell lines (BA987 from aorta, and BPA689 from pulmonary artery) were used in this study at various passage levels from 3-25. Assay cultures were prepared by washing one stock culture twice with PBS and enzymatically disaggregating the cells with trypsin-EDTA. Ceils were resuspended in passage-medium at a concentration of 50,000-100,000 per ml and the suspension was distributed to small (5 ml) or large (25 ml) T-flasks as required. After incubation for 3-7 days, medium was removed and replaced with one of three different maintenance media to be tested: GMEM, Leibovitz L-15, (No. 430-1300; Gibco) and Leibovitz L-15 + 0.45% glucose (L-15G). All contained 5% FBS, 10% TPB solution, 292 ~tg/ml L-glutamine, 100 ~tg/ml penicillin and 2.5 I.tg/ml fungizone. The pH was
287 adjusted to 6.0-6.5 either b y gassing with C O 2 (GMEM) or by addition of N1 HC1 (L-15 and L-15G). Cultures were inoculated with Cowdriainfected materials at 3-33 days post seeding. Maintenance medium was changed weekly on uninfected flasks.
Rickettsial Stocks and inoculation of cultures Six stocks of Cowdria ruminantium, prepared as frozen stabilates in blood from infected sheep, were used in this study. Four were from Zimbabwe (Crystal Springs, Palm River, Highway and Lemco-T3) and two were from S. Africa [Welgevonden (du Plessis, 1985) and Ball-3 (Haig, 1952)]. These stocks were inoculated into Merino sheep, and blood was collected on the 2nd to 5th day of fever when rectal temperatures were greater than 41.0~ Blood samples were heparinised, formed elements were removed, and the plasma was used as inoculum for cell cultures (Byrom et al., submitted for publication). AlternatiVely, heparinised whole blood (0.2 ml i.v.) was used to infect laboratory mice of the DBA2 strain (Stewart, 1989). Samples of infected mouse organs (kidney, heart, spleen, liver and blood) were pooled and homogenised in one of the three above mentioned maintenance media, centrifuged at 1000 rpm/5 rains, and the supernate was made up to 5 ml per mouse in maintenance medium. Mouse organ homogenate (MOH) was used at a dilution of 1:10 in maintenance medium, either as freshly prepared inoculum or after addition of 10% dimethylsulfoxide (No. D8779; Sigma) and storage in liquid nitrogen. Monolayers were infected after decanting medium. For plasma samples, 1-4 ml of inoculum were introduced into 25 cm 2 flasks and these were incubated on a slowly rocking platform at 37~ hr. Inocula were then removed, 4 ml of maintenance medium added and the cultures reincubated on the rocking platform. For MOH 1 ml of inoculum per 25 cm 2 flask was used and the adsorption period was reduced to one hour. Flasks were washed twice with maintenance medium or PBS after the adsorption period and
before adding fresh maintenance medium. Cultures were monitored for cytopathic effect (CPE) from the 7th post-inoculation day and those exhibiting CPE were sampled for microscopic examination. Samples were obtained by removing a small portion of the monolayer by means of a sterile 21 ga needle with a bent tip. These were transferred to microscope slides and smeared slightly to dissociate cells. The smears were air-dried, methanol-fixed, stained with Leukostat (Fischer Scientific, Orangeburg, NY) and examined for cytoplasmic inclusions typical of Cowdria rurninantiurn, w h e n cultures became heavily infected they were harvested for subcultivation by dislodging the monolayer into the medium with a plastic cell scraper or sterile swab and drawing the suspension into a 5 ml syringe fitted with a 21 ga needle. Cells were dispersed by expressing the suspension into the flask 3 times, taking care not to create a froth. The suspension was either distributed between two 25 cm 2 flasks or one 75 cm 2 flask, or used as a source of organisms for biochemical extractions or serological tests.
Results Thirty-three Cowdria-infected plasma samples and 12 mouse organ homogenates from infected animals were inoculated into 333 flasks containing one of three maintenance media (Table 1). w h e n GMEM was used, isolations were made from less than one-half (45.5%) of the samples tested and only 26.7% of the cultures inoculated became infected. A marked improvement in rates of isolation (78.6%) and infected cultures (43.0%) occurred when L-15 medium was used. w h e n inoculated cultures were incubated in the presence of L-15 with glucose added, all samples tested and 94.7% of the cultures inoculated proved positive. Cell culture isolates of C. ruminantium were multiply passaged into a total of 1,036 subcultures containing one of the three maintenance media (Table 2). In GMEM, the percentage of positive subcultures was low (avg. 36.9). Use of
288 Table 1. Isolations, from infected animals, of Cowdria ruminantium in bovine vascular endothelial cells cultured in three different media 1
GMEM Inoculum 2
L-15
Samples
Crystal Springs (MOH) (Plasma) Highway (MOH)
Flasks
L-15G
Samples
Flasks
Samples
Flasks
0/2
0/8
2/3
4/20
1/1
11/12
nt
nt
1/1
3/3
1/1
12/12
nt
nt
1/1
4/8
1/1
12/12
(Plasma)
4/6
17/28
3/3
15122
1/1
7/12
Welgevonden (MOH)
1/2
2/17
0/1
0/8
1/1
12112
(Plasma)
nt
nt
nt
nt
1/1
10/10
Ball 3 (Plasma)
nt
nt
nt
nt
2/2
16/16
Lemco T3 (Plasma)
1/2
1/17
nt
nt
1/1
8/8
Palm River (Plasma)
4/10
19/76
4/5
14/32
nt
nt
Total (%)
10/22 (45.5)
39/146 (26.7)
11/14 (78.6)
40/93 (43.0)
9/9 (100)
89/94 (94.7)
1 No. of positives/No, tested; nt = not tested; 2 MOH = Mouse organ homogenate; GMEM = Glasgow modification of Eagle's minimal essential medium; L-15 = Leibovitz's L-15 medium; L-15G = L-15 medium + 0.45% glucose. Table 2. Subcultivation of Cowdria ruminantium in bovine vascular endothelial cells cultured in three different media 1,2
GMEM
L-15
Stock
Flasks
Days 3
Ball-3
nt
Crystal Springs
16/37 (43.2%)
13.8
Passage 4
-
-
Flasks nt
8
25/59 (42.3%)
L-15G Days
Passage
-
-
13.7
11
176/180 (97.8%) 8.1
15+
135/141 (95.7%) 8.9
17+
42/89 (47.2%)
10.5
6
51/103 (49.5%)
8.8
7
2/5 (40.0%)
5.0
2
1/3 (33.3%)
14.0
2
Palm River
4/36 (11.1%)
23.5
4
4/41 (9.8%)
17.0
2
Welgevonden 7/25 (28.0%)
13.1
3
1/5 (20.0%)
16.0
4
Total
12.1
1,2 See footnotes for Table 1; 3 Average No. days between passages; 4 Highest passage level achieved.
82/211 (38.9%) 10.8
Passage 12+
Highway
72/74 (97.3%)
Days 9.8
Lemco T3
71/192(36.9%)
Flasks
87/90 (96.7%)
6.6
13+
112/120 (93.3%) 11.1
15+
19/28 (67.9%)
7.5
601/633 (95.0%) 8.8
5+
289 L-15 as a maintenance m e d i u m improved the success rate only slightly (avg. 38.9%). However, when infected cells were passaged in L-15 with added glucose, a marked increase in percent of successful subcultures (avg. 95.0%) was seen for all isolates. In addition, the average time between passages was reduced from 12.1 to 8.8 days.
Discussion For many years, all efforts to propagate C. ruminantium in vitro failed (Uilenberg, 1983). The first successful culture method for the organism was reported by South African workers (Bezuidenhout et al., 1985), which provided basic information necessary for refinement of techniques and a supply o f organisms necessary for molecular and serological studies (Wilkens and Ambrosio, 1989; Yunker et al., 1988). However, the special treatments o f the cultured cells (irradiative or chemical retardation of cell growth), which were d e e m e d necessary to establish initial infection, are detrimental to the cells and often cause loss o f the culture. In addition, cultures so treated do not ensure routine isolation or continuous growth of the rickettsia (Bezuidenhout, 1987; Yunker et al., 1988). These earlier studies emp l o y e d G M E M as the growth m e d i u m for Cowdria-infected cells. Because this medium is a modified basal medium, we attempted to improve the percentage o f positive cultures by substituting a more nutritious medium, Leibovitz's L-15, or, by adding to the latter an equivalent amount o f glucose (0.45%) to that contained in G M E M . Leibovitz's L-15 medium, which has high levels of amino acids was originally developed in order to increase virus yields in tissue and cell cultures (Leibovitz, 1963). The composition of this medium was based on evidence of competition between virus and cell for certain essential amino acids. The addition o f glucose to this medium makes it more suited to the acidic requirements of C. ruminantium during its extracellular phase (Yunker et al., 1988). Our results show that the use of L-15 m e d i u m with 0.45% glucose added greatly stimulates the growth of the heartwater
organism. As a result, C. ruminantium m a y now be routinely isolated and passaged in untreated bovine vascular endothelial cells.
ACknowledgements The excellent technical assistance of Miss P. Donovan and Mrs. G. Smith is gratefully acknowledged. W e thank Drs J.L. du Plessis and J.D. Bezuidenhout, Veterinary Research Institute, Onderstepoort, S.A., for providing the Welgevonden stock of Cowdria ruminantium. Dr. C.G. Stewart, Medical University of Southern Africa, Medunsa, S.A. kindly provided D B A 2 mice. This work was supported by the U.S. A g e n c y for International Development (Contract No. AFR-0435-A-009084-00; M.J. Burridge, Principal Investigator) and the Government o f Zimbabwe.
References 1. Wilkens SC, Ambrosio RE, (1989) The isolation of nucleic acid sequences specific for Cowdria ruminantium. Onderstepoort J. Vet. Res. 56: 127-129. 2. AndrewHR, Norval RAI (1989) The carrier status of sheep, cattle and African buffalo recovered from heartwater. Vet. Parasitol. 34: 261-266. 3. BezuidenhoutJD, Patterson CL, Bamard JH (1985) In vitro culture of Cowdria ruminantium. Onderstepoort J. Vet. Res. 52: 113-120. 4. Bezuidenhout JD (1987) The present state of Cowdria ruminantium cultivationin cell lines. OnderstepoortJ. Vet. Res. 54: 205-210. 5. ByromB, Yunker CE, DonovanP, Smith G (1989) In vitro isolation of Cowdria ruminantium from plasma of heartwater-infectedruminants (submitted for publication). 6. Burridge MJ (1985) Heartwater invades the Caribbean. Parasitol. Today h 175-177. 7. Du Plessis JL (1985) A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice injected with tick homogenates. Onderstepoort J. Vet. Res. 52: 55-61. 8. Haig DA (1952) Note on the use of the white mouse for the transport of strains of heartwater. S. African Vet. Med. Assoc. 23: 167-170. 9. Hirumi H, Hirumi K (1984) Continuous cultivation of animal-infective bloodstream forms of an East African Trypanosoma congolense stock. Ann. Trop. Med. Parasitol. 78: 327-330.
290 10. Leibovitz A (1963) The growth and maintenance of tissuecell cultures in free gas exchange with the atmosphere. Am. J. Hyg. 78: 173-180. 11. Mare CJ (1984) Heartwater in Foreign Animal Diseases, their Prevention, Diagnosis and Control. Committee on Foreign Animal Diseases (ed), U.S. Animal Health Assoc., Richmond, VA, pp. 186-194. 12. Stewart CG (1989) Cowdria ruminantium stocks: how significant are they? Zimbabwe Vet. J. 20: 149-153.
13. Uilenberg G (1983) Heartwater (Cowdria ruminantium infection). Current status. Adv. Vet. Sci. Comp. Med. 27: 427-480. 14. Yunker CE, Byrom B, Semu S (1988) Cultivation of Cowdria ruminantium in bovine vascular endothelial ceils. Kenya Veterinarian: 12: 12-16.
Address for offprints: B. Byrom, University of Florida/U.S. Agency for International Development/Zimbabwe Heartwater Research Project, P.O. Box 8101, Causeway, Zimbabwe
Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants B. Byrom and C.E. Yunker University of Florida/U.S. Agency for International Development~Zimbabwe Heartwater Research Project, P.O. Box 8101, Causeway, Zimbabwe Received 17 May 1990; accepted in revised form 28 June 1990
Key words: heartwater, cell culture, Cowdria ruminantium, bovine vascular endothelial cells Abstract The causal agent of heartwater disease of domestic ruminants, Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0 =- 6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.
Introduction Heartwater (Cowdria ruminantium infection), one of the most important livestock diseases in Africa (Mare, 1984), causes high mortality in susceptible animals of enzootic areas. Its establishment and subsequent spread in certain Caribbean islands (Burridge, 1985) and the demonstration of a carrier state in wild ruminants that might be transported or imported into non-enzootic areas (Andrew and Norval, in press) portends possible spread to domestic and wild ruminants of North and South America, where potential tick vectors (Amblyomma spp.) already exist. The disease lacks a practical diagnostic test and riskfree vaccine, basically because of unavailability of adequate amounts of the causal organism. Recent demonstrations that C. ruminantium can be grown in vitro (Bezuidenhout et al., 1985; Bezuidenhout, 1987) offer the means to resolve these inadequacies, but the culture conditions
required (irradiative or chemical treatment of monolayers) are restrictive and damaging to the cells. Subsequently, we observed that the heartwater organism could, in some instances, be grown in untreated monolayers of bovine vascular endothelial cells (Yunker et al., 1988; Byrom et al., submitted for publication). More recently, we refined the conditions for growth of C. ruminantium in these monolayers and present our procedures here.
Materials and methods Bovine endothelial cell cultures These were prepared from vascular tissues obtained from abattoir-slaughtered steers. A modification of the method of Hirumi and Hirumi (1984) was used. Short segments of aorta and pulmonary artery near the heart and proximal to
286 vessel branches were removed and immersed i n phosphate-buffered saline (pH 7.4; lacking Ca ++ and Mg ++) (PBS) containing the antibiotics penicillin-G (200 units/ml; Caps, Harare, Zimbabwe), streptomycin sulfate (200 ~g/rnl; Caps) and neomycin sulfate (80 ~tg/ml; No. N1876, Sigma, St. Louis, MO) and Fungizone; (5.0 I.tg/ml; Squibb, Isando, SA) (PBS/A). After transport to the laboratory, generally within 90 min after collection, the segments were washed 3 times with PBS/A and outer layers of connective tissue and fat were aseptically removed. The inner layers, kept wet with the solution, were cut into 1 cm 2 pieces, each of which was placed lumen side down on a drop of collagenase (No. C1764 Sigma), 1 mg/ml in PBS/A, in a sterile 90 mm petri dish. Dishes were incubated for 30 min at 37~ the squares were then overturned, and detaching cells and cell clumps were collected by gentle pipetting o f collagenase solution onto the central portion of the square. Suspensions of aortic and pulmonary artery cells were transferred to separate sterile 15 ml conical centrifuge tubes and centrifuged at 1000 rpm/5 min. The supematants were removed by aspiration and pellets were suspended in 12 ml of the Glasgow modification of Eagle's minimal essential medium (GMEM, No. 041-2100, Gibco Laboratories, Grand Island, NY). Medium contained the above antibiotics but lacked foetal bovine serum. Sodium bicarbonate, 7.5% solution was added (4.7 ml/litre), and the medium was gassed with CO2 to bring the pH to 6.0-6.5. The cells were again pelleted and resuspended in 12 ml of complete medium. Complete medium was GMEM to which was added 10% tryptose phosphate broth (TPB) solution (No. CM283, Oxoid Ltd, Basingstoke, UK), 20% heatinactivated foetal bovine serum (FBS, Flow Labs., Irvine, Ayrshire Scotland), 292 ~tg/ml Lglutamine (No. 810-1051 Gibco) and 300 [.tg/ml endothelial cell growth supplement (ECGS, No. E2759 Sigma). The two suspensions were distributed in 0.5 ml aliquots among the wells of 2-24 well plastic tissue culture clusters (No. 3524, Costar, Cambridge, MA) and an additional 0.5 ml of complete medium was added. The plates were incubated at 37~ week in a humidified cham-
ber containing 5-10% CO2. At this time, 1 ml of complete medium was added to the wells. When a number of wells showed confluent monolayers, all wells showing endothelial cell growth were washed with PBS, and were incubated at 37~ min with trypsin-EDTA solution [0.05% trypsin 1:250 (No. 840-7072, Gibco) plus 0.02% EDTA (No. E6511, Sigma) in PBS]. Loosened cells were gently aspirated with a Pasteur pipette and pooled with 5 ml per well of complete GMEM containing only 150 ~tg/ml ECGS, one-half the above-mentioned concentrations of antibiotics and 25% filtered conditioned medium from the 24 well plate. The suspension was distributed in 5 ml amounts in small (25 cm 2) plastic T-flasks (No. 3055; Costar) and incubated at 37~ for 1-2 weeks. At confluency, each culture was harvested by enzymatic treatment and passed to a large (75 cm 2) T-flask (No. 3275; Costar). At this point EGGS was omitted from the medium and foetal calf serum reduced to 10%. Again 25% conditioned medium was added. The volume of medium in each flask was made up to 25 ml and flasks were incubated as above. When monolayers were confluent, stocks of the cell lines were stored in liquid N2. Further passages were carried out in complete GMEM containing 10% FBS, 10% TPB solution, 292 I.tg/ml L-glutamine and one-half concentrations of the above-mentioned antibiotics. Stock cultures in large T-flasks of 2 such cell lines (BA987 from aorta, and BPA689 from pulmonary artery) were used in this study at various passage levels from 3-25. Assay cultures were prepared by washing one stock culture twice with PBS and enzymatically disaggregating the cells with trypsin-EDTA. Ceils were resuspended in passage-medium at a concentration of 50,000-100,000 per ml and the suspension was distributed to small (5 ml) or large (25 ml) T-flasks as required. After incubation for 3-7 days, medium was removed and replaced with one of three different maintenance media to be tested: GMEM, Leibovitz L-15, (No. 430-1300; Gibco) and Leibovitz L-15 + 0.45% glucose (L-15G). All contained 5% FBS, 10% TPB solution, 292 ~tg/ml L-glutamine, 100 ~tg/ml penicillin and 2.5 I.tg/ml fungizone. The pH was
287 adjusted to 6.0-6.5 either b y gassing with C O 2 (GMEM) or by addition of N1 HC1 (L-15 and L-15G). Cultures were inoculated with Cowdriainfected materials at 3-33 days post seeding. Maintenance medium was changed weekly on uninfected flasks.
Rickettsial Stocks and inoculation of cultures Six stocks of Cowdria ruminantium, prepared as frozen stabilates in blood from infected sheep, were used in this study. Four were from Zimbabwe (Crystal Springs, Palm River, Highway and Lemco-T3) and two were from S. Africa [Welgevonden (du Plessis, 1985) and Ball-3 (Haig, 1952)]. These stocks were inoculated into Merino sheep, and blood was collected on the 2nd to 5th day of fever when rectal temperatures were greater than 41.0~ Blood samples were heparinised, formed elements were removed, and the plasma was used as inoculum for cell cultures (Byrom et al., submitted for publication). AlternatiVely, heparinised whole blood (0.2 ml i.v.) was used to infect laboratory mice of the DBA2 strain (Stewart, 1989). Samples of infected mouse organs (kidney, heart, spleen, liver and blood) were pooled and homogenised in one of the three above mentioned maintenance media, centrifuged at 1000 rpm/5 rains, and the supernate was made up to 5 ml per mouse in maintenance medium. Mouse organ homogenate (MOH) was used at a dilution of 1:10 in maintenance medium, either as freshly prepared inoculum or after addition of 10% dimethylsulfoxide (No. D8779; Sigma) and storage in liquid nitrogen. Monolayers were infected after decanting medium. For plasma samples, 1-4 ml of inoculum were introduced into 25 cm 2 flasks and these were incubated on a slowly rocking platform at 37~ hr. Inocula were then removed, 4 ml of maintenance medium added and the cultures reincubated on the rocking platform. For MOH 1 ml of inoculum per 25 cm 2 flask was used and the adsorption period was reduced to one hour. Flasks were washed twice with maintenance medium or PBS after the adsorption period and
before adding fresh maintenance medium. Cultures were monitored for cytopathic effect (CPE) from the 7th post-inoculation day and those exhibiting CPE were sampled for microscopic examination. Samples were obtained by removing a small portion of the monolayer by means of a sterile 21 ga needle with a bent tip. These were transferred to microscope slides and smeared slightly to dissociate cells. The smears were air-dried, methanol-fixed, stained with Leukostat (Fischer Scientific, Orangeburg, NY) and examined for cytoplasmic inclusions typical of Cowdria rurninantiurn, w h e n cultures became heavily infected they were harvested for subcultivation by dislodging the monolayer into the medium with a plastic cell scraper or sterile swab and drawing the suspension into a 5 ml syringe fitted with a 21 ga needle. Cells were dispersed by expressing the suspension into the flask 3 times, taking care not to create a froth. The suspension was either distributed between two 25 cm 2 flasks or one 75 cm 2 flask, or used as a source of organisms for biochemical extractions or serological tests.
Results Thirty-three Cowdria-infected plasma samples and 12 mouse organ homogenates from infected animals were inoculated into 333 flasks containing one of three maintenance media (Table 1). w h e n GMEM was used, isolations were made from less than one-half (45.5%) of the samples tested and only 26.7% of the cultures inoculated became infected. A marked improvement in rates of isolation (78.6%) and infected cultures (43.0%) occurred when L-15 medium was used. w h e n inoculated cultures were incubated in the presence of L-15 with glucose added, all samples tested and 94.7% of the cultures inoculated proved positive. Cell culture isolates of C. ruminantium were multiply passaged into a total of 1,036 subcultures containing one of the three maintenance media (Table 2). In GMEM, the percentage of positive subcultures was low (avg. 36.9). Use of
288 Table 1. Isolations, from infected animals, of Cowdria ruminantium in bovine vascular endothelial cells cultured in three different media 1
GMEM Inoculum 2
L-15
Samples
Crystal Springs (MOH) (Plasma) Highway (MOH)
Flasks
L-15G
Samples
Flasks
Samples
Flasks
0/2
0/8
2/3
4/20
1/1
11/12
nt
nt
1/1
3/3
1/1
12/12
nt
nt
1/1
4/8
1/1
12/12
(Plasma)
4/6
17/28
3/3
15122
1/1
7/12
Welgevonden (MOH)
1/2
2/17
0/1
0/8
1/1
12112
(Plasma)
nt
nt
nt
nt
1/1
10/10
Ball 3 (Plasma)
nt
nt
nt
nt
2/2
16/16
Lemco T3 (Plasma)
1/2
1/17
nt
nt
1/1
8/8
Palm River (Plasma)
4/10
19/76
4/5
14/32
nt
nt
Total (%)
10/22 (45.5)
39/146 (26.7)
11/14 (78.6)
40/93 (43.0)
9/9 (100)
89/94 (94.7)
1 No. of positives/No, tested; nt = not tested; 2 MOH = Mouse organ homogenate; GMEM = Glasgow modification of Eagle's minimal essential medium; L-15 = Leibovitz's L-15 medium; L-15G = L-15 medium + 0.45% glucose. Table 2. Subcultivation of Cowdria ruminantium in bovine vascular endothelial cells cultured in three different media 1,2
GMEM
L-15
Stock
Flasks
Days 3
Ball-3
nt
Crystal Springs
16/37 (43.2%)
13.8
Passage 4
-
-
Flasks nt
8
25/59 (42.3%)
L-15G Days
Passage
-
-
13.7
11
176/180 (97.8%) 8.1
15+
135/141 (95.7%) 8.9
17+
42/89 (47.2%)
10.5
6
51/103 (49.5%)
8.8
7
2/5 (40.0%)
5.0
2
1/3 (33.3%)
14.0
2
Palm River
4/36 (11.1%)
23.5
4
4/41 (9.8%)
17.0
2
Welgevonden 7/25 (28.0%)
13.1
3
1/5 (20.0%)
16.0
4
Total
12.1
1,2 See footnotes for Table 1; 3 Average No. days between passages; 4 Highest passage level achieved.
82/211 (38.9%) 10.8
Passage 12+
Highway
72/74 (97.3%)
Days 9.8
Lemco T3
71/192(36.9%)
Flasks
87/90 (96.7%)
6.6
13+
112/120 (93.3%) 11.1
15+
19/28 (67.9%)
7.5
601/633 (95.0%) 8.8
5+
289 L-15 as a maintenance m e d i u m improved the success rate only slightly (avg. 38.9%). However, when infected cells were passaged in L-15 with added glucose, a marked increase in percent of successful subcultures (avg. 95.0%) was seen for all isolates. In addition, the average time between passages was reduced from 12.1 to 8.8 days.
Discussion For many years, all efforts to propagate C. ruminantium in vitro failed (Uilenberg, 1983). The first successful culture method for the organism was reported by South African workers (Bezuidenhout et al., 1985), which provided basic information necessary for refinement of techniques and a supply o f organisms necessary for molecular and serological studies (Wilkens and Ambrosio, 1989; Yunker et al., 1988). However, the special treatments o f the cultured cells (irradiative or chemical retardation of cell growth), which were d e e m e d necessary to establish initial infection, are detrimental to the cells and often cause loss o f the culture. In addition, cultures so treated do not ensure routine isolation or continuous growth of the rickettsia (Bezuidenhout, 1987; Yunker et al., 1988). These earlier studies emp l o y e d G M E M as the growth m e d i u m for Cowdria-infected cells. Because this medium is a modified basal medium, we attempted to improve the percentage o f positive cultures by substituting a more nutritious medium, Leibovitz's L-15, or, by adding to the latter an equivalent amount o f glucose (0.45%) to that contained in G M E M . Leibovitz's L-15 medium, which has high levels of amino acids was originally developed in order to increase virus yields in tissue and cell cultures (Leibovitz, 1963). The composition of this medium was based on evidence of competition between virus and cell for certain essential amino acids. The addition o f glucose to this medium makes it more suited to the acidic requirements of C. ruminantium during its extracellular phase (Yunker et al., 1988). Our results show that the use of L-15 m e d i u m with 0.45% glucose added greatly stimulates the growth of the heartwater
organism. As a result, C. ruminantium m a y now be routinely isolated and passaged in untreated bovine vascular endothelial cells.
ACknowledgements The excellent technical assistance of Miss P. Donovan and Mrs. G. Smith is gratefully acknowledged. W e thank Drs J.L. du Plessis and J.D. Bezuidenhout, Veterinary Research Institute, Onderstepoort, S.A., for providing the Welgevonden stock of Cowdria ruminantium. Dr. C.G. Stewart, Medical University of Southern Africa, Medunsa, S.A. kindly provided D B A 2 mice. This work was supported by the U.S. A g e n c y for International Development (Contract No. AFR-0435-A-009084-00; M.J. Burridge, Principal Investigator) and the Government o f Zimbabwe.
References 1. Wilkens SC, Ambrosio RE, (1989) The isolation of nucleic acid sequences specific for Cowdria ruminantium. Onderstepoort J. Vet. Res. 56: 127-129. 2. AndrewHR, Norval RAI (1989) The carrier status of sheep, cattle and African buffalo recovered from heartwater. Vet. Parasitol. 34: 261-266. 3. BezuidenhoutJD, Patterson CL, Bamard JH (1985) In vitro culture of Cowdria ruminantium. Onderstepoort J. Vet. Res. 52: 113-120. 4. Bezuidenhout JD (1987) The present state of Cowdria ruminantium cultivationin cell lines. OnderstepoortJ. Vet. Res. 54: 205-210. 5. ByromB, Yunker CE, DonovanP, Smith G (1989) In vitro isolation of Cowdria ruminantium from plasma of heartwater-infectedruminants (submitted for publication). 6. Burridge MJ (1985) Heartwater invades the Caribbean. Parasitol. Today h 175-177. 7. Du Plessis JL (1985) A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice injected with tick homogenates. Onderstepoort J. Vet. Res. 52: 55-61. 8. Haig DA (1952) Note on the use of the white mouse for the transport of strains of heartwater. S. African Vet. Med. Assoc. 23: 167-170. 9. Hirumi H, Hirumi K (1984) Continuous cultivation of animal-infective bloodstream forms of an East African Trypanosoma congolense stock. Ann. Trop. Med. Parasitol. 78: 327-330.
290 10. Leibovitz A (1963) The growth and maintenance of tissuecell cultures in free gas exchange with the atmosphere. Am. J. Hyg. 78: 173-180. 11. Mare CJ (1984) Heartwater in Foreign Animal Diseases, their Prevention, Diagnosis and Control. Committee on Foreign Animal Diseases (ed), U.S. Animal Health Assoc., Richmond, VA, pp. 186-194. 12. Stewart CG (1989) Cowdria ruminantium stocks: how significant are they? Zimbabwe Vet. J. 20: 149-153.
13. Uilenberg G (1983) Heartwater (Cowdria ruminantium infection). Current status. Adv. Vet. Sci. Comp. Med. 27: 427-480. 14. Yunker CE, Byrom B, Semu S (1988) Cultivation of Cowdria ruminantium in bovine vascular endothelial ceils. Kenya Veterinarian: 12: 12-16.
Address for offprints: B. Byrom, University of Florida/U.S. Agency for International Development/Zimbabwe Heartwater Research Project, P.O. Box 8101, Causeway, Zimbabwe
