In vitro cultivation of Anaplasma marginale in bovine erythrocytes co-cultured with endothelial cells

veterinary parasitology ELSEVIER

Veterinary Parasitology 73 (1997) 43-52

In vitro cultivation of Anaplasma marginale in bovine erythrocytes co-cultured with endothelial cells S.D. Waghela a,*, D. Cruz a, R.E. Droleskey b, J.R. DeLoach b, G.G. Wagner a a Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843, USA b USDA/ARS Food Animal Protection Research Laboratory College Station, TX 77845, USA Received 8 November 1996; accepted 10 March 1997

Abstract Primary cultures of Anaplasma marginale infected erythrocytes were used to determine conditions for in vitro cultivation of the rickettsia. The infected erythrocytes that were maintained by regular addition of Glasgow's MEM with fetal calf serum and uninfected erythrocytes showed a 1-5% increase in percent infected erythrocytes on the evaluation of Giemsa stained smears. This increase in parasitemia resulted in up to 70% change in the number of infected erythrocytes. Co-culture of the infected erythrocytes with endothelial cell monolayers allowed for longer maintenance with the parasitemia ranging from 5 - 1 3 % through four passages over 16 weeks. Examination of cultures using transmission electron microscopy showed initial bodies within the erythrocytes at 10 days after the initial passage of the primary culture. The endothelial cell monolayers in the co-cultures contained multiple initial bodies. We have demonstrated that A. marginale can be grown for a limited number of passages in the co-culture system, which will facilitate the development of a continuous culture of the organism. © 1997 Elsevier Science B.V. Keywords: Anaplasma marginale; In vitro culture; Erythrocytes; Endothelial cells

I. Introduction B o v i n e anaplasmosis, a v e c t o r - b o r n e disease, c a u s e d by the rickettsia A n a p l a s m a marginale occurs w o r l d w i d e with an e s t i m a t e d one half-billion cattle at risk (Ristic and

* Corresponding author. Tel.: + 1 409 8455176; fax: + 1 409 8622344; e-maih [email protected] 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0304-401 7(97)00045-9

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Kreier, 1974; Dallwitz et al., 1987). The lack of significant improvements in either vaccines or drugs and increase in the disease distribution area (Kocan, 1994) suggest that the annual losses in the USA are as severe as estimated in the early 1970s (McCallon, 1973). The loss in production, combined with the increased risk of improper use of various chemicals and drugs, and the social pressures to examine these issues, make research on an antigen defined vaccine for anaplasmosis a priority (NRC, 1982). Vaccination against anaplasmosis in many countries includes preimmunization with a virulent strain of A. marginale, or with the less virulent A. centrale, followed by treatment with tetracyclines as needed. Several killed A. marginale products are available in the USA and a few other countries. Both of these vaccines are derived from blood of artificially infected cattle and problems associated with their use include standardization, storage and delivery. Other factors, such as the risk of transmission of other diseases via erythrocytes and of isoerythrolysis have promoted the search for an alternate immunogen. Since initial body membrane fractions of A. marginale may induce protection against anaplasmosis (Tebele et al., 1991), in vitro cultures would provide a defined source of initial bodies and for preparation of sub-unit or defined antigens. A. marginale has reportedly been grown in vitro in various cell types including bovine erythrocytes, bovine lymph node cells, bovine embryonic kidney cells, bovine endothelial cells, bovine turbinate cells, bovine and rabbit bone marrow cells, and tick cell lines (Hidalgo, 1973; Marble and Hanks, 1973; Hidalgo, 1975; Kessler et al., 1979; Kessler and Ristic, 1979; Baradji, 1986; Samish et al., 1988; Hidalgo et al., 1989; Blouin, 1991; Blouin et al., 1992; Blouin et al., 1993). Nevertheless, continuous in vitro culture of A. marginale as a more efficacious and economical source of antigen for vaccine production has not been achieved (Blouin, 1991). Recently, Munderloh et al. (1996) were able to maintain A. marginale continuously in a tick cell line for 14 serial passages over a period of 16 months. We report in vitro culture of A. marginale in bovine erythrocytes co-cultured with endothelial cells as a prelude to eventual development of a continuous culture system.

2. Materials and methods

2.1. Infection of cattle as a source of A. marginale Calves (6-12 months old) were splenectomized under mild sedation and local anesthesia, then monitored for Eperythrozoon sp., Anaplasma sp. and other hemoparasites for 21 days. The calves were infected with approx. 10 9 A. marginale (Texas isolate, cryopreserved at 10% PCV with an estimated 1.125 X 10 9 infected erythrocytes/ml). Blood for culture inoculum, antigen preparation and additional stabilate was collected in sodium heparin (140 USP units/10 ml blood) from the calves at peak parasitemia, usually about 25 days after inoculation.

2.2. Culture media The following culture media were tested: RPMI 1640 (JRH Biosciences) containing 25 mM HEPES (4-[2-hydroxyethyl]-l-piperzine-ethanesulfonic acid) and 0.02%

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NaHCO3; Glasgow's MEM (Gibco-BRL) containing 0.02% NanCO3; Leibovitz L-15 (Gibco-BRL) containing 20 mM TES (2-[2-hydroxy-l,l-bis(hydroxymethyl)-ethyl] aminoethanesulfonic acid), 2 mM MgC12, 25 mM glucose, 5 mM adenosine and 5 mM inosine. All media also contained 10% ( v / v ) tryptose phosphate broth (Gibco-BRL), 2 mM L-glutamine, 100 U penicillin, 100 Ixg streptomycin, 25 txg fungizone and were supplemented with 20% fetal calf serum. 2.3. Donor erythrocytes Defibrinated blood collected from an adult bovine steer was centrifuged at 1000 X g for 20 min at 4°C and the buffy coat cells carefully removed. The remaining erythrocyte pellet was resuspended in an equal volume of Puck's saline G (Gibco-BRL) solution containing added glucose (2% w / v ) , 100 U penicillin, 100 p~g streptomycin, and 25 Ixg fungizone/ml and stored at 4°C. The donor animal was free of A. marginale infection as indicated by negative blood smear examination and negative hybridization of whole blood DNA with an A. marginale specific DNA probe (Goff et al., 1988). The serum from this animal was negative for antibody activity to A. marginale by card agglutination and indirect fluorescent antibody (IFA) tests, and by Western blot analysis. 2.4. Source of bovine endothelial cells Primary endothelial cells were grown from bovine pulmonary artery and characterized as previously described (Hirumi and Hirumi, 1984; Goetze et al., 1985). After the 10th passage, the endothelial cells grown to confluence were used for co-culture of A. marginale infected bovine erythrocytes. 2.5. Culture of A. marginale infected erythrocytes Blood from an A. marginale infected splenectomized calf was collected when the percent infected erythrocytes (PIE) was 24%. The blood was centrifuged at 1000 X g for 20 rain and the plasma and buffy coat discarded. The erythrocyte pellet was washed twice with Hank's balanced salt solution and resuspended in each medium with the packed cell volume (PCV) of 12.5%. The erythrocyte suspension in the three different media was dispensed in triplicate wells (1.25 ml volume per well) of a 24-well culture plate with or without a monolayer of endothelial cells. Culture plates were incubated at 37°C in a humidified atmosphere of 5% CO 2 in air. One ml of the spent medium in the primary cultures was removed and replaced daily with fresh medium. Prior to adding fresh medium, 1 I~1 of the sedimented erythrocytes was taken and smeared on glass slides. The smears were stained with Giemsa and the number of A. marginale infected erythrocytes in a total of 1000 cells determined. Once a week, 25 Ixl of uninfected erythrocytes at 50% PCV prepared from the blood of the donor steer was added to each of the wells. Subculture was accomplished with resuspended material transferred to a new well containing uninfected erythrocytes to obtain a PIE of two with 12.5% PCV in 1.25 ml of the medium/well. Controls included similar cultures of erythrocytes from an uninfected splenectomized calf. Periodically, 5 pA aliquots of sedimented erythrocytes

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were washed twice with Dulbecco's phosphate-buffered saline (DPBS) and resuspended in 1.0% glutaraldehyde in 0.01 M phosphate, 0.15 M NaCI, pH 7.4 in distilled water for electron microscopy (DeLoach et al., 1991). A 100-~1 sample containing about 1.7 X 108 erythrocytes was periodically removed from a replicate well of the culture or subcultures, centrifuged at 2000 x g for 10 min, washed twice with DPBS and the pellet stored at - 7 0 ° C prior to processing for hybridization with the specific A. marginale DNA probe. Blood for a second series of experiments was collected when the PIE was 21 and co-cultured at erythrocyte concentrations of either 1.56 X 109 cells (PCV 6.25%) or 3.12 X 10 9 cells (PCV 12.5%) suspended in 1.25 ml of Glasgow's MEM per well. The change in percent of numbers of infected erythrocytes was determined by estimating the increase in numbers of infected erythrocytes/well following the removal or addition of erythrocytes at the previous sampling. A third series of experiments was initiated with blood collected at peak parasitemia (23%), washed and resuspended in Glasgow's MEM medium at 12.5% PCV and plated onto endothelial cell monolayers. One ml medium from each well was removed daily, 1 ~1 of the erythrocytes used to make smears and the erythrocytes resuspended in 1 ml of fresh Glasgow's MEM. The suspension was shaken for 1 h at 250 rpm in a 37°C orbital shaker prior to returning to a well for incubation. Also, A. marginale infected erythrocytes were co-cultured with endothelial cells in Glasgow's MEM at 3.12 x 10 9 cells in 1.25 ml/well (PCV 12.5%) to study the effect of early versus late subculture. Cultures were incubated in a humidified atmosphere of 5% CO 2 in air at 37°C as in the first experiment. Subcultures were prepared by diluting with donor uninfected erythrocytes at a ratio of 1:3 during either the first ('early') or sixth ('late') week of incubation. Further passages of the subcultures were done at the same ratio of infected erythrocytes to uninfected erythrocytes. Samples for electron microscopy and DNA hybridization were collected periodically.

2.6. Electron microscopy Cell samples were post-fixed in 1% osmium tetroxide, post-stained in 0.5% uranyl acetate, dehydrated and embedded as a pellet in epoxy resin for ultrathin sectioning and examination.

2. 7. Dot blot hybridization The DNA from the erythrocyte pellets was prepared and applied to nylon membranes as described previously (Shompole et al., 1989; Waghela et al., 1991). The blotted DNA was evaluated for hybridization to a 2 kb DNA fragment, removed by SstI digestion from a 3.9 kb A. marginale insert in the recombinant plasmid, pAm97, (Barbet et al., 1987). The 2-kb fragment was labeled with biotin-14-d-CTP by random priming using the BioPrime TM DNA labeling system (Gibco-BRL). The labeled probe was hybridized as previously described (Goff et al., 1988) and chemiluminescence (Photogene TM nucleic acid detection system, Gibco-BRL) detected by exposure of the blot to Kodak X-Omat AR film for 15 min.

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3. Results

3.1. A. marginale cultures Fig. l shows the percentage of A. marginale infected erythrocytes in three different media. The A. marginale PIE during the first week of culture in Glasgow's MEM was 1% to 10% higher than in either Leibovitz L-15 or RPMI 1640. At 2.5 weeks of culture, the PIE in all media was similar. The PIE in Glasgow's MEM remained approximately l0 for at least 6 weeks. After an initial rise (within 48 h), the PIE of A. marginale cultured in Glasgow's MEM without endothelial cells decreased to 17 within 1 week (Fig. 2A). The PIE in endothelial cell co-cultures increased initially to about 35, dropped to 25 by the third week, then increased to approximately 30 before subculture. The PIE increased 3 - 5 fold (Fig. 213) following the initial two passages, with or without endothelial cell co-cultures. However, this increase was not maintained in the subsequent subcultures, and the PIE remained lower than 5.

3.2. Effects of erythrocyte concentration, shaking and time of subculture Decreasing the PCV in the primary cultures from 12.5% to 6.25% resulted in an initial increase in the total number of infected erythrocytes but did not affect the PIE in subsequent passages. Hemolysis was observed earlier in the cultures which were shaken and the PIE decreased faster. No PIE increase was observed in subsequent subcultures. The primary culture in the third series of the experiments was monitored for a total of 16 weeks. Although fresh erythrocytes were added regularly, the culture showed a declining PIE which stabilized at 2 - 3 for the last 2 weeks. The PIE increased within 4 days in both early and late first passages. The PIE began to decline and stabilized at 2 - 3 following the third subculture of the early, but not late, passage material.

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Fig. 2. Percent infected erythrocytes in primary cultures and subcultures. (A) Average percent of infected erytbrocytes in three replicate primary cultures of A. marginale initiated by mixing erythrocytesfrom infected steer with erythrocytes from an uninfected donor. 0, erythrocytes only; a, erythrocytes co-cultured with endothelial cell monolayers. (B) Replicate subcultures made 7 weeks after initiation of primary co-cultures. Endothelial cells periodically sampled from co-cultures and stained with Giemsa contained A. marginale infected cells, usually 1 - 3 c e l l s / 5 fields (1000 × magnification). When passaged in the absence of erythrocytes, the infection in the endothelial cells diminished.

3.3. Electron microscopy Electron micrographs of samples taken from the cultures at various times showed multiple initial bodies within each marginal body in erythrocytes cultured with endothe-

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A

Fig. 3. Electron micrographs showing initial bodies of Anaplasma marginale: (1) in erythrocytes derived from the first passage of subculture at 7 weeks post-initiation (A) with endothelial cells; (B) without endothelial cells; and (2) in bovine endothelial cells (C) taken 30 days post-inoculation of primary co-culture in erythrocytes. Bar = 0.5 tzm.

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lial cells compared to degenerating initial bodies observed in the absence of endothelial cells (Fig. 3A and B). Electron microscopy of endothelial cells taken at 30 days from a primary A. marginale co-culture showed initial bodies bounded by a cell membrane (Fig. 3C).

3.4. Detection of A. marginale in infected erythrocytes by hybridization with a specific DNA probe Hybridization of the A. marginale specific probe to the DNA samples prepared from erythrocytes collected from the co-cultures at various times indicated the presence of the rickettsial DNA as long as 63 days post-inoculation.

4. Discussion Our objective was to evaluate conditions for A. marginale growth in bovine erythrocytes co-cultured with endothelial cell monolayers using the short-term in vitro procedure. Previous studies have shown the difficulty in obtaining sustained growth of A. marginale in vitro for long periods (Hidalgo, 1973; Blouin, 1991). We were able to maintain A. marginale infections of bovine erythrocytes up to 16 weeks in primary co-cultures or subcultures, longer than previously reported (Kessler et al., 1979). Presence of endothelial cells appeared to be critical for anaplasma growth over an extended period. The trend toward decreasing PIE with passage was not altered by shaking, changing the erythrocyte concentration or by passaging during the early or late phase of primary culture. The presence of A. marginale in the cultures was confirmed by electron microscopy, antigenic analysis and hybridization with a specific DNA probe. The A. marginale was also detected by IFA test with monoclonal antibody, by immunoprecipitation and in Western blot analysis (Waghela et al., 1997). Blouin et al. (1993) noted no increase in the numbers of inclusions in endothelial cells inoculated with tick salivary gland stage of A. marginale. Since the numbers of the rickettsia decreased on further passages of the endothelial cells, the presence of initial bodies may be due to the phagocytic capacity of these cells. This study indicates that A. marginale can be grown in continuous culture for a limited number of passages. Further studies may help define the specific culture conditions required to establish a reproducible in vitro source of A. marginale for antigen and vaccine studies. Since this study, Munderloh et al. (1996) have propagated A. marginale in tick cell cultures, and the continuously in vitro grown rickettsia proved infective for calves and the ticks.

Acknowledgements This work was supported by research grant Research Development Funding, RD-7-91; and USDA Animal Health Formula Fund, AH-8117. We thank Dr. T.C. McGuire, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, for supplying the recombinant plasmid pAm 97.

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