In vitro propagation and genome sequencing of three 'atypical' Ehrlichia ruminantium isolates.

Three isolates of Ehrlichia ruminantium (Kümm 2, Omatjenne and Riverside), the causative agent of heartwater in domestic ruminants, were isolated in Ixodes scapularis (IDE8) tick cell cultures using the leukocyte fraction of infected sheep blood. All stocks were successfully propagated in IDE8 cells, whereas initiation attempts using endothelial cell cultures were unsuccessful. Therefore, the new technique should be included in any attempt to isolate field strains of E. ruminantium to enhance the probability of getting E. ruminantium isolates which might not be initiated in endothelial cells. Draft genome sequences of all three isolates were generated and compared with published genomes. The data confirmed previous phylogenetic studies that these three isolates are genetically very close to each other, but distinct from previously characterised E. ruminantium isolates. Genome comparisons indicated that the gene content and genomic synteny were highly conserved, with the exception of the membrane protein families. These findings expand our understanding of the genetic diversity of E. ruminantium and confirm the distinct phenotypic and genetic characteristics shared by these three isolates.

Comparative Proteomic Profiling of Ehrlichia ruminantium Pathogenic Strain and Its High-Passaged Attenuated Strain Reveals Virulence and Attenuation-Associated Proteins.

The obligate intracellular bacterium Ehrlichia ruminantium (ER) causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir) and its high-passaged attenuated strain (ERGatt). Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation) are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments) involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt), and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

Global gene expression profiling of Ehrlichia ruminantium at different stages of development.

Ehrlichia ruminantium (ER), the causative agent of heartwater on ruminants, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma. Previous studies have shown that early stages of development may be critical for Ehrlichia pathogenicity. To gain insights into the biology of intracellular ER, we determined the genome-wide transcriptional profile of ER replicating inside bovine aortic endothelial cells using DNA microarrays. At intermediate and late stages of infection (reticulate and elementary bodies, respectively), a total of 54 genes were differentially expressed. Among them, we measured by q-RTPCR the overexpression of 11 of 14 genes. A number of genes involved in metabolism, nutrient exchange, and defense mechanisms, including those involved in resistance to oxidative stress, were significantly induced in ER reticulate bodies. This is consistent with the oxidative stress condition and nutrient starvation that seem to occur in Ehrlichia-containing vacuoles. During the lysis stage of development, when ER is infectious, we showed the overexpression of a transcription factor, dksA, which is also known to induce virulence in other pathogens such as Salmonella typhimurium. Our results suggest a possible role of these genes in promoting ER development and pathogenicity.

In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures.

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.

Process development for the mass production of Ehrlichia ruminantium.

This work describes the optimization of a cost-effective process for the production of an inactivated bacterial vaccine against heartwater and the first attempt to produce the causative agent of this disease, the rickettsia Ehrlichia ruminantium (ER), using stirred tanks. In vitro, it is possible to produce ER using cultures of ruminant endothelial cells. Herein, mass production of these cells was optimized for stirring conditions. The effect of inoculum size, microcarrier type, concentration of serum at inoculation time and agitation rate upon maximum cell concentration were evaluated. Several strategies for the scale-up of cell inoculum were also tested. Afterwards, using the optimized parameters for cell growth, ER production in stirred tanks was validated for two ER strains (Gardel and Welgevonden). Critical parameters related with the infection strategy such as serum concentration at infection time, multiplicity and time of infection, and medium refeed strategy were analyzed. The results indicate that it is possible to produce ER in stirred tank bioreactors, under serum-free culture conditions, reaching a 6.5-fold increase in ER production yields. The suitability of this process was validated up to a 2-l scale and a preliminary cost estimation has shown that the stirred tanks are the least expensive culture method. Overall, these results are crucial to define a scaleable and fully controlled process for the production of a heartwater vaccine and open "new avenues" for the production of vaccines against other ehrlichial species, with emerging impact in human and animal health.

Characterization of Ehrlichia ruminantium replication and release kinetics in endothelial cell cultures.

Ehrlichia ruminantium is the causative agent of Heartwater, a fatal tick-borne disease affecting ruminants in African countries and West Indies and can be used as an inactivated vaccine for wild and domestic animals. In order to improve E. ruminantium production yields we characterize E. ruminantium growth kinetics in terms of duplication time, maximum production yield, and peak of infectivity. After a 24 h period for E. ruminantium attachment/internalization and a lag phase of 12 h, the exponential growth occurred within 36-108 h post-infection (hpi) with a net increase of up to 2.2 orders of magnitude. Maximum E. ruminantium infectivity was observed at 120 hpi and was defined as the best time of harvesting (TOH) for propagation of E. ruminantium cultures. This study showed that considering the quality constraint of the final product (E. ruminantium vaccine), the E. ruminantium suspension should be harvested at 113 hpi. Overall, the characterization of E. ruminantium progression through the average infection cycle, not only can contribute to the maximization of E. ruminantium production yield, with important consequences for the large scale production and utilization of an inactivated Heartwater vaccine, but also to elucidate growth mechanisms of some of the other ehrlichial species, with emerging impact in human and animal health.

Quantification of Ehrlichia ruminantium by real time PCR.

Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.

Ehrlichia ruminantium seroprevalence in domestic ruminants in Ghana; I. Longitudinal survey in the Greater Accra Region.

Serum samples collected monthly over a 34-month period from cattle, sheep and goats in the Greater Accra Region of Ghana were tested for antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, by polyclonal competitive ELISA (PC-ELISA). Maternal antibodies, detected in about half of animals followed from under 1 month old, declined to negative levels within 2-4 months. Amblyomma variegatum tick vectors were present on livestock in rural areas throughout the year, and first seroconversion occurred at any age, although the majority of calves seroconverted between 1 and 10 months old, sheep by 11 months, and goats by 7 months. All the cattle in the study became seropositive by 20 months of age, except one animal which subsequently died of heartwater. Following seroconversion, 25% of bovine sera tested negative in the PC-ELISA. Just over half the sheep in the survey seroconverted before or during the study period; following seroconversion, less than 3% of ovine sera became PC-ELISA negative. About a quarter of the goats seroconverted, and 34% of their post-seroconversion sera tested negative in the PC-ELISA. Overall, the serology indicated that virtually all cattle on the survey farms were exposed to E. ruminantium without suffering disease, but that a substantial proportion of sheep and goats escaped exposure and thus formed a susceptible population. E. ruminantium was detected in brains of 14, 36 and 4% of cattle, sheep and goats submitted for post mortem at the Accra Veterinary Laboratory, indicating that sheep were most at risk from heartwater disease.

In vitro infection by Ehrlichia ruminantium of baby hamster kidney (BHK), Chinese hamster ovary (CHO-K1) and Madin Darby bovine kidney (MDBK) cells.

The Welgevonden stock of Ehrlichia ruminantium, aetiological agent of heartwater, was propagated in baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO-K1) cells and Madin Darby bovine kidney (MDBK) cells. The cultures required supplementation of the medium with cycloheximide for reliable growth of E. ruminantium. Growth of the Welgevonden stock in BHK and CHO-K1 cells could lead to the development of suspension cultures suitable for the mass production of E. ruminantium for an inactivated elementary body vaccine.

In vitro infection of nonendothelial cells by Ehrlichia ruminantium.

The Welgevonden stock of Ehrlichia ruminantium was propagated in eight nonendothelial cell cultures derived from different animal species, both ruminants and nonruminants. The origins of the cells were: bovine fetal testis (BFT), cat ovary (COC), donkey fibroblasts (DFC), sheep fibroblasts (E(2)), horse testis (HTC), lamb fetal testis (LFT), mouse connective tissue (L), and African green monkey kidney (Vero). Four cell culture types (BFT, E(2), LFT and Vero) required supplementation of the medium with cycloheximide for suitable growth of E. ruminantium, whereas the other four (COC, DFC, HTC, and L) did not. Three other stocks of E. ruminantium, Senegal, Ball 3, and Gardel, were also propagated, either in LFT cultures only or in both E(2) and LFT cell cultures. The Welgevonden stock was successfully initiated using E(2) and LFT cell cultures.

Amino acid content of cell cultures infected with Cowdria ruminantium propagated in a protein-free medium.

The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985. Culture media were usually supplemented with serum and tryptose phosphate broth, both undefined components, contributing to great variability. Recently, we reported about the propagation of stocks of C. ruminantium in a protein-free culture medium referred to as SFMC-23, which is chemically fully defined. To clarify whether the amino acid composition in SFMC-23 is adequate for the in vitro propagation of Cowdria, the Welgevonden stock was propagated in SFMC-23 medium. After a 3-day culture period, samples were taken from uninfected and infected bovine endothelial cell cultures. They were analyzed for free amino acids by the Pico Taq reversed-phase HPLC precolumn derivatization method. Eighteen different amino acids were examined. A considerable decrease in concentration was observed with proline (29%) and glutamine (62%). Further dramatic changes were observed with amino acids which accumulated in the culture medium: aspartic acid, serine, asparagine, tryptophane, glycine, and alanine. The concentration of alanine increased by approximately 660%. The concentrations of all other amino acids analyzed remained within a 25% range, either increasing or decreasing. These results suggest that only glutamine may run short during in vitro cultivation. It seems more likely that accumulation of various amino acids may impact negatively on long-term Cowdria propagation.

The Kümm isolate of Ehrlichia ruminantium: in vitro isolation, propagation and characterization.

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.

Kinetics of experimental infection of sheep with Ehrlichia ruminantium cultivated in tick and mammalian cell lines.

Inoculation of sheep with Ehrlichia (previously Cowdria) ruminantium which has been cultivated in mammalian endothelial cell cultures is almost always followed by a severe clinical reaction, whereas inoculation of the agent cultivated in tick cell lines usually does not provoke a clinical response, but may result in seroconversion and/or protection against subsequent challenge with virulent stabilates. A quantitative, real-time PCR assay was developed to determine the kinetics of infection (rickettsaemia) in sheep inoculated with tick cell- and mammalian cell-derived E. ruminantium (Gardel isolate). The method and initial results are described, and the significance of the findings is discussed in relation to the clinical responses of the sheep.

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

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.

A chemically defined medium for the growth of Cowdria ruminantium.

Chemically defined media, termed SFMC-23 and SFMC-36, were devised for the in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants. Both media were based on Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12) containing various supplements. Medium SFMC-23 and SFMC-36 supported the long-term growth of the Welgevonden stock of C. ruminantium for a total of 55 and 28 passages, respectively, with regular passage intervals of 3 days. Using SFMC-23, split ratios varied from 5-10, depending on which host cell line was used. Other stocks of C. ruminantium (Sankat, Blaauwkrantz, Senegal) were successfully propagated for a test period of ten passages.

Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line.

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells.

Electron microscopy of Cowdria-infected macrophages suggests that in the absence of binary fission a mosaic of organisms develops from an amorphous electron dense matrix.

Electron microscopy of mouse peritoneal macrophages infected with the Kümm stock of Cowdria ruminantium suggests that in the final stage of intracellular growth, a mosaic of organisms develops from an amorphous matrix of varying electron density by a process in which double unit membranes portion off the Cowdria particles. This stage is preceded by inclusions consisting of a network of aggregated electron dense granules and these in turn by homogeneous dense bodies. The study failed to show how these dense bodies develop from internalized Cowdria particles introduced in the infective inoculum. The replication of the heartwater agent in macrophages differs from that in vascular endothelial cells in two important respects. First, at no stage during the course of development in macrophages is binary fission in evidence and second, in the absence of a limiting membrane the inclusions and colonies of organisms throughout the cycle of development in macrophages are in intimate contact with the host cell cytoplasm.

Serum-free media for the in vitro cultivation of Cowdria ruminantium.

The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985; since then, most groups working with this culture system have used media which were supplemented with serum and, in addition, most of them contained tryptose phosphate broth. These undefined products vary from batch to batch and often fail to support the growth of C. ruminantium. We are therefore working towards the development of a completely chemically defined medium for Cowdria culture. We attempted the propagation of the Welgevonden stock of C. ruminantium in bovine endothelial cell cultures in a variety of serum-free culture media. Four synthetic media gave unsatisfactory results, these were: SFRE-199, Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's medium, and Leibovitz L-15. These media were all supplemented with a proprietary solution A (components solution A of the HL-1 medium kit, containing transferrin, testosterone, sodium selenite, ethanolamine, saturated and unsaturated fatty acids, and stabilizing proteins). Three other serum-free media did support the growth of C. ruminantium: a modified HL-1 medium, Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12), and RPMI 1640. The chemical composition of DME/F-12 and RPMI 1640 are published, but not that of the HL-1 medium. Each of these media was supplemented with proprietary solution A. Various supplements were investigated as alternative to the incompletely specified solution A; bovine lipoproteins and bovine transferrin were identified as essential supplements which effectively replaced compound solution A. C. ruminantium was propagated in the three growth-supportive media for at least 10 passages.

Growth of Cowdria ruminantium in tissue culture endothelial cell lines from wild African mammals.

Endothelial cell cultures were established from several wild African mammalian species. Long-term cultures were established from three ruminants, stable antelope (Hippotragus niger), buffalo (Syncerus caffer), and eland (Tragelaphus oryx), and from an omnivore, the bushpig (Potamochoerus porcus). Cowdria ruminanntium was isolated from plasma of clinically affected animals in these four cell lines and in bovine endothelial cells used routinely for C. ruminantium propagation. Nineteen different strains of C. ruminantium from Africa and the Caribbean region were grown and maintained in these cell lines and their growth was comparable with growth in the bovine endothelial cells. The role of sable antelope, eland, and bushpigs in the epidemiology of heartwater is unknown. However, these results extend the number of cell lines that can be used to isolate and grow C. ruminantium.

Development of an in vitro cloning method for Cowdria ruminantium.

Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium.