In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis ( IDE 8 ) cell cultures

ZWEYGARTH, E., JOSEMANS, A.I. & STEYN, H. 2008. In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures. Onderstepoort Journal of Veterinary Research, 75:121–126 Four stocks of Ehrlichia ruminantium (Welgevonden, Ball3, Nonile and Blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into Ixodes scapularis (IDE8) tick cells using the leukocyte fraction of the blood of infected sheep. Organisms of two of the E. ruminantium stocks (Welgevonden and Blaauwkrans) propagated in IDE8 cells were also successfully used to infect bovine endothelial cells. All stocks were successfully propagated in IDE8 cells using Dulbecco’s modified Eagle’s medium nutrient mixture Ham F-12 containing 10 % foetal bovine serum (FBS). The technique should be included in any attempt to isolate uncharacterized E. ruminantium stocks.


INTRODUCTION
Heartwater or cowdriosis is an infectious, non-contagious, tick-borne disease caused by the intra cel lu lar rickettsial agent Ehrlichia ruminantium.The dis ease affects cattle, sheep, goats and also some wild ruminants.It is transmitted by ticks of the genus Amblyomma and has been reported from almost all African countries south of the Sahara, from the adjacent islands in the Indian and Atlantic Oceans (Uilen berg 1983) and from some Caribbean islands (Perreau, Morel, Barré & Durand 1980;Birnie, Burridge, Camus & Barré 1984).Heartwater is usually an acute disease and may be fatal within hours or days after the onset of clinical signs.
The mammalian cell culture system first described by Bezuidenhout, Paterson & Barnard (1985) is the method of choice for in vitro isolation and propagation of E. ruminantium, whereas the successful prop agation of E. ruminantium in tick cells was only reported recently (Bell-Sakyi, Paxton, Munderloh & Sumption 2000;Bekker, Bell-Sakyi, Paxton, Martinez, Bensaid & Jongejan 2002;Bell-Sakyi 2004).Initiation of infection in tick cell cultures was achieved using elementary bodies derived from mammalian cell cultures (Bell-Sakyi et al. 2000) or from other tick cell lines previously infected with mammalian cell culture forms (Bell-Sakyi 2004) but not directly from infected animals.Attempts to infect tick cells with blood from sheep undergoing clinical responses following experimental E. ruminantium infection were unsuccessful (Bell-Sakyi 2004).
The present experiments describe the first successful establishment of infection in Ixodes scapularis (IDE8) tick cell cultures directly from the blood of four sheep, each infected with a different South African stock of E. ruminantium, and the subsequent infection of endothelial cells from infected IDE8 cells.

Stocks of E. ruminantium
Four stocks of E. ruminantium isolated in South Africa were used: the Welgevonden stock was originally isolated by injecting a tick homogenate into a mouse; the tick had been collected on the farm Welgevonden in the Northern Transvaal (Du Plessis 1985); the Ball3 stock was isolated from a nonspecified host in the Northern Province (Haig 1952); the Blaauwkrans stock was isolated from an eland near Port Elizabeth in 1996 (Zweygarth & Josemans 2001); and the Nonile stock from a sheep in Kwa-Zulu-Natal (MacKenzie & McHardy 1984).

Cell cultures
The tick cell line IDE8, derived from I. scapularis embryos (Munderloh, Liu, Wang, Chen & Kurtti 1994) was used throughout these experiments.IDE8 cultures, infected and uninfected, were maintained at 32 °C in complete L-15B medium, unless otherwise stated.IDE8 cell cultures were used at passage levels between 54 and 62.Three fifths of the medium was replaced weekly.Propagation of infected and un infected bovine aorta endothelial (BA 886) cell cultures was carried out as described previously (Zweygarth, Vogel, Josemans & Horn 1997).

Sheep-derived infective culture inoculum
Each of the four E. ruminantium stocks-Ball3, Blaauwkrans, Nonile or Welgevonden-was used to infect a Merino sheep by intravenous injection of a 5 mℓ blood stabilate.The body temperature of each sheep was monitored daily and a blood sample was drawn when it had risen to more than 41.5 °C, unless stated otherwise.Blood was collected by venipuncture into sterile Vac-u-test® tubes containing heparin (lithium heparin, 14.3 USP per mℓ blood) as anticoagulant and put on ice.The cooled blood was centrifuged (800 x g; 10 min; 4 °C) and the buffy coat was collected and washed with cold phosphate-buffered saline (PBS).The buffy coat was again collected, and the red blood cells were lysed for approximately 30 s in 20 mℓ sterile distilled water followed by the addition of 5 mℓ of a 5 x concentrated physiological NaCl solution (4.5 % w/v).The leukocytes were centrifuged and the resulting cell pellet was re-suspended in 5 mℓ of DF-12 or complete L-15B medium.The leukocyte suspensions were then inoculated into 25 cm² culture flasks containing IDE8 cells.The cultures were incubated at 32 °C.
Attempts to infect tick cell cultures with the Wel gevonden stock of E. ruminantium were also carried out according to the method described by Byrom, Yunker, Donovan & Smith (1991) with minor modifications.Briefly, heparinized blood (lithium heparin, 14.3 USP per mℓ blood) was centrifuged (800 x g; 10 min; room temperature) and 3 mℓ of plasma were inoculated into each of two 25 cm² culture flasks containing a layer of IDE8 cells.The cultures were put on a rocking platform for 3 h at three cycles per min at 25 °C, after which the plasma was decanted and the cell monolayer was rinsed three times with 5 mℓ PBS.Finally DF-12 medium was added.

In vitro infection of bovine endothelial cells by E. ruminantium organisms derived from IDE8 cell cultures
IDE8 cells infected with the Welgevonden and Blaauwkrans stocks of E. ruminantium were used to demonstrate infectivity of IDE8-derived organisms for BA 886 cells.Aliquots of 2.5 mℓ of infected IDE8 cell suspension were distributed into culture flasks containing BA 886 cells.Fresh DF-12 medium was added to give a final volume of 5 mℓ.The cultures were then incubated at 37 °C and the medium was replaced every 3 days.IDE8 and BA 886 cultures were monitored for infection by microscopic examinations.Small samples from the cell layer were removed and smears were prepared.Cytospin smears were made from cultures where some of the cells were in suspension.Smears were allowed to dry before being fixed with methanol and stained with eosin-methylene blue.

Molecular characterization
Extraction and amplification of DNA DNA was extracted from the four stocks of E. ruminantium with the QIAamp DNA kit extraction kit (Qiagen).The primers for the pCS20 PCR diagnostic test (Van Heerden, Steyn, Allsopp, Zweygarth, Josemans & Allsopp 2004) specific for E. ruminantium was used to sequence of E. ruminantium in the blood and in tick cell cultures.Briefly, the PCRs were performed with 2 μℓ of genomic DNA extracted from cell culture as template in a 50 μℓ reaction with 0.5 mM of each of the primers HH1F and HH2R, 2.5 mM dNTP; 25 mM MgCl 2 , 10 x PCR reaction buffer and 0.5U of TaKaRa EX Taq enzyme (Takara Shuzo Co., Ltd Japan).PCR conditions were: incubation of 25 s at 94 °C, 35 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 45 s, elongation at 72 °C for 30 s, final elongation at 72 °C for 10 min and hold at 4 °C (Gene Amp PCR System 9700, Applied Biosystems).Each set of PCRs included a positive control containing 1 μℓ purified genomic DNA of E. ruminantium (Welgevonden) and a negative control containing 5 μℓ distilled water.Ten microliter of each sample was separated on a 1 % agarose gel with molecular mass marker PHiX74.

Sequencing
The PCR amplicons from samples showing bands of the expected size were purified with a High Pure PCR product purification kit (Roche) and sequenced using an ABI Prism 3100 Automatic DNA sequencer (BigDye terminator cycler sequencing kit, Perkin Elmer Applied Biosystems) with primers HH1F, HH2R (Van Heerden et al. 2004), and the data was assembled in gap 4 (Staden, Beal & Bonfield 2000) and analysed using ClustalX (Thompson, Gibson, Plewniak, Jeanmougin & Higgins 1997).

Sheep-derived infective culture inoculum
Leukocytes isolated from the blood of infected sheep were used as infective inoculum.All four South African E. ruminantium stocks were established successfully in IDE8 cell cultures by this method.The Welgevonden stock was detected in stained smears 9 days after initiation, when leukocytes harvested 14 days after infection were used.In contrast, all attempts to initiate the Welgevonden stock in the "conventional" way (Byrom et al. 1991), i.e. by incubating plasma from the infected animal together with host cells (IDE8), failed (data not shown).During prolonged incubation of the latter IDE8 cultures some cytotoxic effects induced by the ovine plasma became manifest as the pH of the medium failed to fall in the same way as it did in untreated controls.DF-12 medium was used for these experiments.
Infection with the Blaauwkrans and Ball3 stocks of E. ruminantium was detected in stained smears in IDE8 cell cultures after 21-29 and 18-25 days, respectively.Both stocks were initiated using DF-12 medium.Leukocytes contained morula-like inclusions, presumably E. ruminantium.These were only demonstrated with the Blaauwkrans stock before culture initiation.The results of successful culture initiations are shown in Table 1.
Attempts to infect IDE8 cell cultures using the Nonile stock were carried out on two successive occasions.The first attempt was carried out on Day 13 post infection of the donor sheep when its body temperature was 41.3 °C.A Giemsa-stained cytocentrifuge smear prepared from the leukocyte inoculum revealed that more than 99 % of the cells were mononuclear.Cultures initiated on this occasion remained negative throughout an observation period of 60 days.In contrast, the initiation experiment carried out the next day revealed a switch to a granulocyte cell distribution pattern, with mononuclear cells being a minor contaminating population only.Thirteen days after initiation, infected tick cells were detected in Giemsa-stained cytocentrifuge smears prepared from culture supernatant.Complete L-15B medium was used for the initiation, but was replaced by DF-12 medium 39 days after culture initiation due to unsatisfactory growth of the E. ruminantium organisms.The first subculture was carried out 100 days after culture initiation.These results are summarized in Table 2.

In vitro infection of BA 886 cells by E. ruminantium organisms derived from IDE8 tick cell cultures
IDE8 cultures infected with the Welgevonden stock for 149 days and the Blaauwkrans stock for 165 days were used successfully to infect BA 886 cell cultures.When the Welgevonden stock was used, both BA 886 cultures were positive 3 days after inoculation as determined by stained smears, whereas the Blaauwkrans stock was detected in the two infected cultures on Days 9 and 15, respectively.BA 886 cultures infected with both stocks were then subcultured after a further 15 days.The results are summarized in Table 3.

Molecular characterization
Sequence analysis using the pCS20 specific primers confirmed that the E. ruminantium stocks recovered from the tick cell cultures were the same as those which were injected into the donor sheep, showing that the organisms used to infect the sheep were also of the stocks isolated in the respective cultures.

DISCUSSION
The first continuous propagation of E. ruminantium in a tick cell line was achieved by Bell-Sakyi et al. (2000), who cultivated the Gardel stock in the I. scapularis-derived cell line IDE8 (Munderloh et al. 1994).Elementary bodies derived from bovine endothelial cell cultures of several stocks of E. ruminantium were used to establish continuous, infected tick cell cultures (Bell-Sakyi 2004).However, attempts to infect three different tick cell lines, AVL/CTVM13, IDE8 and RAN/CTVM3, with blood from sheep undergoing clinical responses following experimental E. ruminantium infection were unsuccessful (Bell-Sakyi 2004).Similar unsuccessful results were obtained by us when we attempted to infect IDE8 cultures using heparinized plasma from infected sheep.However, infective organisms are not only found free-floating in the blood or plasma of an infected animal but also in circulating leukocytes, in which the organisms are able to proliferate.Logan, Why ard, Quintero & Mebus (1987) observed E. ruminantium colonies in up to 35 % of neutrophils maintained in vitro for between 18 h and 5 days.Further more, leukocytes from infected animals were able to transmit E. ruminantium to naive animals (Ilemobade & Blotkamp 1978).The experiments in the present study show that leukocytes may also be used to initiate in vitro cultures.In fact, IDE8 tick cell cultures can be infected with E. ruminantium directly from the blood of infected sheep, provided leukocytes are used as the inoculum.Cultures of all four stocks used in the experiments-Ball3, Blaauwkrans, Nonile and Welge vonden-were successfully initiated in IDE8 cells using this technique.Similar approaches were carried out with Ehrlichia canis (Ewing, Munderloh, Blouin, Kocan, Kurtti 1995) and with the other former Ehrlichia species: Ehrlichia equi (Munderloh, Madigan, Dumler, Goodman, Hayes, Barlough, Nelson & Kurtti 1996), the human granulocytic ehrlichiosis (HGE) agent (Munderloh, Jauron, Fingerle, Leitritz, Hayes, Hautman, Nelson, Huberty, Kurtti, Ahlstrand, Greig, Mellencamp & Goodman 1999), and Ehrlichia phagocytophila (Woldehiwet, Horrocks, Scaifer,  3) -( 2) 100 (1) Remained negative for 60 days (2) Not applicable (3) Detected in a cytocentrifuge smear of pooled samples from the three culture flasks Ross, Munderloh, Brown, Edwards & Hart 2002) (all now reclassified as Anaplasma phagocytophilum [Dumler, Barbet, Bekker, Dasch, Palmer, Ray, Rikihisa & Rurangirwa 2001]) which have been successfully isolated from infected blood into I. scapularis cell cultures using leukocytes as infective inoculum.
Besides neutrophils, in vitro cultured macrophages also revealed the presence of inclusion bodies of E. ruminantium (Sahu 1986).Interestingly, infection of IDE8 cells with the Nonile stock using a leukocyte fraction containing almost exclusively mononuclear cells failed, although monocytes/macrophages have been described as containing the organism.In contrast, the Nonile stock could be established in IDE8 cells using granulocytes from the same infected sheep.
Ehrlichia ruminantium is regarded as an obligatory parasite of endothelial cells (Martinez, Sheikboudou, Couraud & Bensaid 1993) but it has been shown recently that not all E. ruminantium isolates can be initiated in endothelial cells.Different types of host cell were required to isolate these organisms which have previously resisted all attempts at isolation in the conventional way (Zweygarth, Josemans, Van Strijp, Van Heerden, Allsopp & Allsopp 2002).Consequently, isolating E. ruminantium using only endothelial cells limits the probability of success.Here we show that IDE8 cells can be used as an alternative to conventional methods.Tick cell cultures may thus increase the chances of isolating organisms which do not generally grow in endothelial cells as the primary culture.
Both the Welgevonden and Blaauwkrans stocks in IDE8 cells, which were used to inoculate BA 886 cells, not only infected them but also gave rise to continuous infected mammalian cell culture lines.Similar results were reported by Bell-Sakyi et al. (2000).However, their IDE8 cultures were not consistently infective for bovine pulmonary artery endothelial cells.The fact that endothelial cells can be infected with IDE8-derived organisms makes the system very valuable, especially when cultures cannot be directly initiated in endothelial cells.Infected IDE8 cells can thus be used as a stable source of inoculum to identify suitable mammalian cell lines that support growth of the organisms.It has been reported recently that the Kümm isolate, which resisted all attempts at conventional in vitro culture, was isolated in culture in non-endothelial cells (Zweygarth et al. 2002).
Unlike others (Bell-Sakyi et al. 2000;Bekker et al. 2002;Bell-Sakyi 2004), who only used a L-15B-based medium for the propagation of E. ruminantium in IDE8 cultures, we also used DF-12 medium, which was originally devised for the propagation of E. ruminantium in mammalian cell cultures at 37 °C (Zweygarth et al. 1997).DF-12 medium was a suitable alternative for the propagation of E. ruminantium in IDE8 cells and it also supported the growth of uninfected IDE8 cells over a three-month period with several subcultures (data not shown).
In conclusion, it has been shown that the technique of isolating E. ruminantium from infected animals using IDE8 tick cell cultures is a valuable method which should be included in any attempt to isolate uncharacterized E. ruminantium stocks.

TABLE 1
Infection of IDE8 cell cultures using leukocytes isolated from sheep infected with three E. ruminantium stocks

TABLE 3
In vitro infection of BA 886 cells by E. ruminantium organisms derived from IDE8 tick cell cultures