Amblyomma hebraeum is the predominant tick species on goats in the Mnisi Community Area of Mpumalanga Province South Africa and is co-infected with Ehrlichia ruminantium and Rickettsia africae.

BACKGROUND: In sub-Saharan Africa, Amblyomma ticks are vectors of heartwater disease in domestic ruminants, caused by the rickettsial pathogen Ehrlichia ruminantium. Immature tick stages often bite humans, whereby they act as vectors of tick-bite fever caused by Rickettsia africae. Moreover, Amblyomma ticks cause damage to livestock due to their feeding behaviour. In South Africa, we studied the abundance of Amblyomma hebraeum ticks on goats of emerging farmers in Mpumalanga Province. A selected number of A. hebraeum nymphs and adult ticks was tested for co-infection with E. ruminantium and R. africae. METHODS: A total of 630 indigenous goats, belonging to farmers in the Mnisi Community area, were examined for ticks in 2013 and 2014. All ticks were identified, and a selected number was tested by PCR with reverse line blot hybridisation. RESULTS: In total, 13,132 ticks were collected from goats distributed over 17 different households. Amblyomma hebraeum was the predominant species, followed by R. microplus. Rhipicephalus appendiculatus, R. simus and R. zambeziensis were also identified. Amblyomma hebraeum was present throughout the year, with peak activity of adults in summer (November) and nymphs in winter (July). The ratio between adults and nymphs ranged from 1:2.7 in summer to 1:55.1 in winter. The mean prevalence of infection for E. ruminantium by PCR/RLB in adult ticks was 17.4% (31/178), whereas 15.7% (28/178) were infected with R. africae. In pooled nymphs, 28.4% were infected with E. ruminantium and 38.8% carried R. africae infection. Co-infections of E. ruminantium and R. africae in adult and pooled nymphal ticks were 3.9% (7/178) and 10% (14.9), respectively. Lameness of goats due to predilection of ticks for the interdigital space of their feet was observed in 89% of the households. CONCLUSIONS: Goats act as important alternative hosts for cattle ticks, which underscored the necessity to include goats in control programs. It is suggested to use acaricide-impregnated leg-bands as a sustainable method to kill ticks and prevent lameness in goats. The challenge of goats by considerable numbers of E. ruminantium-infected ticks is a major obstacle for upgrading the indigenous goat breeds. Humans may be at risk to contract tick-bite fever in this area.

Comparative Transcriptome Profiling of Virulent and Attenuated Ehrlichia ruminantium Strains Highlighted Strong Regulation of map1- and Metabolism Related Genes.

The obligate intracellular pathogenic bacterium, Ehrlichia ruminantium, is the causal agent of heartwater, a fatal disease in ruminants transmitted by Amblyomma ticks. So far, three strains have been attenuated by successive passages in mammalian cells. The attenuated strains have improved capacity for growth in vitro, whereas they induced limited clinical signs in vivo and conferred strong protection against homologous challenge. However, the mechanisms of pathogenesis and attenuation remain unknown. In order to improve knowledge of E. ruminantium pathogenesis, we performed a comparative transcriptomic analysis of two distant strains of E. ruminantium, Gardel and Senegal, and their corresponding attenuated strains. Overall, our results showed an upregulation of gene expression encoding for the metabolism pathway in the attenuated strains compared to the virulent strains, which can probably be associated with higher in vitro replicative activity and a better fitness to the host cells. We also observed a significant differential expression of membrane protein-encoding genes between the virulent and attenuated strains. A major downregulation of map1-related genes was observed for the two attenuated strains, whereas upregulation of genes encoding for hypothetical membrane proteins was observed for the four strains. Moreover, CDS_05140, which encodes for a putative porin, displays the highest gene expression in both attenuated strains. For the attenuated strains, the significant downregulation of map1-related gene expression and upregulation of genes encoding other membrane proteins could be important in the implementation of efficient immune responses after vaccination with attenuated vaccines. Moreover, this study revealed an upregulation of gene expression for 8 genes encoding components of Type IV secretion system and 3 potential effectors, mainly in the virulent Gardel strain. Our transcriptomic study, supported by previous proteomic studies, provides and also confirms new information regarding the characterization of genes involved in E. ruminantium virulence and attenuation mechanisms.

Detection and molecular characterization of tick-borne pathogens infecting sheep and goats in Blue Nile and West Kordofan states in Sudan.

Tick-borne pathogens (TBPs) are common in livestock of sub-Saharan Africa. However, information regarding TBPs in sheep and goats in Sudan is limited. In this study, 178 blood samples of sheep and goats in Blue Nile and West Kordofan states were investigated for TBPs using PCR. Overall, 110 (61.8%) samples were found to be infected with at least one of the following pathogens: Anaplasma ovis, Theileria ovis, and Ehrlichia ruminantium. Babesia ovis and T. lestoquardi were not identified. A. ovis was the most prevalent pathogen (n = 107, 60.1%), followed by T. ovis (n = 23, 12.9%) and E. ruminantium (n = 1, 0.6%). The prevalence rates of A. ovis and T. ovis were significantly higher in sheep than in goats. Phylogenetic analysis of T. ovis 18S rRNA and A. ovis msp4, groEL, and 16S rRNA, revealed that the pathogens identified in this study are clustered together, indicating similar molecular characteristics. Additionally, phylogenetic analysis of E. ruminantium pCS20 revealed that E. ruminantium in this study belong to the West Africa group, and different to E. ruminantium previously identified in ticks from Sudan. We concluded that TBPs are highly prevalent in the study area and continuous monitoring of TBPs in sheep and goats in Sudan is highly required.

Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin.

Babesiosis, theileriosis, anaplasmosis, and heartwater are tick-borne diseases (TBD) that threaten livestock production in sub-Saharan Africa including Benin. This country has been faced with an invasion of Rhipicephalus microplus, a major vector for babesiosis, theileriosis, and anaplasmosis over the last decade. Yet, data on TBD and the impact of the invasive ticks are lacking, making risk level evaluation and disease control arduous. In this study, epidemiological features of Babesia bovis, B. bigemina, Theileria spp., Anaplasma marginale and Ehrlichia ruminantium infections in Benin cattle were investigated in R. microplus-invaded and non-invaded areas. Detection of pathogens was based on species-specific PCR assays and resulting data were used to identify risk factors. Genetic diversity and phylogenies were then evaluated using several markers. Out of 207 samples examined, 170 (82.1%), 109 (52.7%), 42 (20.3%) 24 (11.6%) and 1 (0.5%) were positive for T. mutans, A. marginale, B. bigemina, B. bovis and E. ruminantium, respectively. Animal gender (for B. bovis), exposure to R. microplus (for B. bigemina and A. marginale), animal age (for B. bigemina and A. marginale) and cattle breed and/or antiprotozoal treatment (for T. mutants) significantly modulated pathogen occurrence. In addition, R. microplus exposure was significantly related to co-infection patterns and cases of clinical theileriosis and/or anaplasmosis were recorded among cattle highly exposed to the tick. In the genetic characterization, Theileria spp. and E. ruminantium sequences were conserved. Babesia spp. and A. marginale, however, showed high sequence polymorphisms that indicate the presence of several strains and may be linked to R. microplus invasion. Taken together, these results ascertain the endemicity of tick-borne infections in Benin and suggest that the characteristics of Babesia spp. and A. marginale infections in R. microplus-invaded and non-invaded areas are different.

Ticks and Tick-Borne Pathogens of the Caribbean: Current Understanding and Future Directions for More Comprehensive Surveillance.

Ticks are obligate hematophagous arthropods of significant importance to human and veterinary medicine. They transmit a vast array of pathogens, including bacteria, viruses, protozoa, and helminths. Most epidemiological data on ticks and tick-borne pathogens (TBPs) in the West Indies are limited to common livestock pathogens such as Ehrlichia ruminantium, Babesia spp. (i.e., B. bovis and B. bigemina), and Anaplasma marginale, and less information is available on companion animal pathogens. Of note, human tick-borne diseases (TBDs) remain almost completely uncharacterized in the West Indies. Information on TBP presence in wildlife is also missing. Herein, we provide a comprehensive review of the ticks and TBPs affecting human and animal health in the Caribbean, and introduce the challenges associated with understanding TBD epidemiology and implementing successful TBD management in this region. In particular, we stress the need for innovative and versatile surveillance tools using high-throughput pathogen detection (e.g., high-throughput real-time microfluidic PCR). The use of such tools in large epidemiological surveys will likely improve TBD prevention and control programs in the Caribbean.

Recombination Is a Major Driving Force of Genetic Diversity in the Anaplasmataceae Ehrlichia ruminantium.

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago.

Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium.

Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria.

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.

Opportunities in diagnostic and vaccine approaches to mitigate potential heartwater spreading and impact on the American mainland.

Heartwater, caused by the Rickettsiales Ehrlichia ruminantium (ER), is a tropical tick-borne disease of wild and domestic ruminants, transmitted by Amblyomma ticks. It causes significant economic losses due to high mortality and the high cost of antibiotic treatment of affected animals, limiting herd productivity. It is present in sub-Saharan Africa, islands in the Indian Ocean and two Caribbean islands (Guadeloupe and Antigua) from where it threatens the American mainland due to risk of the spread of infected A. variegatum by migratory birds or by uncontrolled movement of animals. If an accidental introduction of a tick-free ER carrier animal occurs, autochthonous A. maculatum has proven to be a good experimental vector for heartwater. Modeling A. variegatum population dynamics has been developed, but further work is needed to predict favourable habitats and allow targeted surveillance. We overview here the advances in diagnostics, vaccines and epidemiology of heartwater and analyze the research gaps and needs to mitigate potential ER introduction and spread on the American mainland. Effective serologic ELISA tests allow prevalence studies, and several PCR-based diagnostic tests are currently available to detect ER in sick animals. However, the development of rapid assays, including multi-pathogen tests, would enhance the efficacy and cost-effectiveness of heartwater diagnosis. Several experimental vaccines (inactivated, attenuated and recombinant) are under development. Attenuated and inactivated vaccines are effective against homologous strains but their efficacy in the field is decreased due to broad antigenic diversity of ER. New molecular typing assays are now being used to study the genetic structure of ER populations worldwide, but the linking of genotyping to cross-protection is still not straightforward. Currently an inactivated vaccine would be the most appropriate vaccine for the American mainland due to its safety, the availability of a fully controlled bioprocess allowing ER mass production and the possibility to design "regional cocktail vaccines". This would require the selection and isolation of Caribbean ER strains supported by data of molecular epidemiology studies in this region. Development of an universal recombinant vaccine requires increased knowledge of ERbiology, including virulence mechanisms. Comparison of virulent and attenuated strains using"omic approaches" is on-going and will be crucial to understand these mechanisms and to develop improved vaccines.

Trends in the control of heartwater.

Heartwater is an economically serious tick-borne disease of ruminants caused by the intracellular bacterium Ehrlichia ruminantium. The disease has traditionally been controlled by four different approaches: controlling the tick vector by dipping, establishing endemic stability, performing immunisation by infection and treatment, and preventing the disease by regular administration of prophylactic antibiotics. The first three of these methods are subject to failure for various epidemiological reasons, and serious disease outbreaks can occur. Prophylaxis is effective, but very expensive, and the logistics are daunting when large herds of animals are involved. The development of a safe, cheap and effective vaccine is the only likely way in which heartwater can be economically controlled, and over the past 15 years three new types of experimental vaccine have been developed: inactivated, attenuated, and recombinant vaccines. These new vaccines have shown varying degrees of promise, but none is as yet sufficiently successful to be marketable. We describe the experimental products, and the various technical and biological difficulties which are being encountered, and report on ways in which new technologies are being used to improve vaccine effectiveness.

Heartwater: an abridged historical account.

Heartwater was first recognised and recorded in South Africa as early as 1838. Since then the disease has been described from almost all the countries in Africa south of the Sahara, from certain islands around Africa and from a number of islands in the Caribbean. Most of the research on the disease, at least until fairly recently, was conducted in South Africa. Progress in research on the disease has been slow but a few important findings are highlighted in this paper.

Ehrlichia ruminantium variants which do not cause heartwater found in South Africa.

In 1994 a batch of apparently healthy goats was selected for intended export to the USA from a heartwater-free and vector tick-free region of South Africa. The animals were tested serologically for heartwater, using either or both an IFA and an ELISA test, and 52% were found to be serologically positive. A PCR assay based on Ehrlichia ruminantium 16S gene sequences gave positive results for 54% of the animals, suggesting that apparently non-pathogenic E. ruminantium variants existed in this heartwater-free area. To identify and characterise the agents responsible for the positive serological and PCR results, ticks and animal blood samples were collected from two of the three farms involved in the original survey during two successive seasons of expected peak tick activity. Ticks were kept alive for a minimum of 3 weeks to allow digestion of any blood meal before being processed. Over the two seasons, 28% of the livestock and 15% of the ticks sampled were found to be carrying E. ruminantium. E. ruminantium 16S and pCS20 sequences were detected in all of the four tick species collected from the livestock (Rhipicephalus evertsi evertsi, Rhipicephalus evertsi mimeticus, Hyalomma truncatum, Hyalomma marginatum rufipes), suggesting that some of the species may act as vectors. Animals generally carried multiple E. ruminantium 16S genotypes, whereas ticks rarely carried more than one. Infection levels in both animals and ticks were too low to generate a marked response when a blood stabilate was sub-passaged in a clean sheep, preventing the subsequent establishment of any of the organisms in culture.

Emerging tick-borne disease in African vipers caused by a Cowdria-like organism.

Heartwater is a tick-borne infectious disease caused by the rickettsial organism Cowdria ruminantium, currently Ehrlichia ruminantium. It poses an imminent threat to the Western Hemisphere, where it could cause mortality in cattle and other ruminant livestock in excess of 70%. It has been reported in the Caribbean; and its vector, Amblyomma sparsum, has been found on imported African spurred tortoises (Geochelone sulcata) and leopard tortoises (Geochelone pardalis) in southern Florida in the United States, leading to an importation ban on these reptiles. Symptoms have not been previously reported in reptiles. Here, we report peracute and acute deaths in African vipers imported from Africa through Florida. Signs included vomiting mucoid fluid, diarrhea, emaciation, convulsions, and death. Postmortem showed few gross lesions. The most consistent peracute and acute lesions were the pulmonary lesions and pericarditis with considerable bloody fluid in the pericardial sac (hydropericardium). These lesions strongly resembled the lesions of heartwater and a coccobacillus of less than 1-micron diameter was isolated in viper cell culture. The outbreak was brought to a halt by tick control and treatment of all exposed snakes with tetracycline. This isolation, tetracycline sensitivity, clinical signs, preliminary results with polymerase chain reaction of pCS20 ORF, and the viper preference of the disease may indicate a Cowdria-related attenuated species that has adapted to infect reptiles or an emerging new form of this group of microbes.

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.

An attenuated Ehrlichia ruminantium (Welgevonden stock) vaccine protects small ruminants against virulent heartwater challenge.

Heartwater is a tick-borne disease of ruminants caused by the intracellular rickettsia Ehrlichia ruminantium. The only commercially available immunization procedure involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by treatment with tetracyclines when fever develops. The virulent Welgevonden stock of E. ruminantium was attenuated by continuous propagation of the organisms in a canine macrophage-monocyte cell line (DH82), followed by re-adaptation to grow in a bovine endothelial cell line (BA 886). The material used for the present experiments consisted of the attenuated stock between passages 43 and 64 after re-adaptation. When inoculated into sheep or goats the attenuated organisms did not produce disease, and the only symptom observed was a rise in body temperature in most, but not all, animals. All sheep injected with 2 ml of culture suspension were subsequently found to be fully protected against a lethal needle challenge with the virulent homologous stock or with one of four different heterologous stocks (Ball 3, Gardel, Mara 87/7, Blaauwkrans). Titrations of elementary body suspensions showed that 2ml of a 1:10,000 dilution of culture suspension injected into sheep or goats was still sufficient to trigger an immune response which resisted a lethal needle challenge with the virulent Welgevonden stock. Adult Amblyomma hebraeum ticks, fed as nymphs on sheep immunized with DH82-derived organisms of passage 111, were able to transmit the attenuated stock to a naive sheep, which was found to be protected against a subsequent lethal homologous needle challenge.

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater (Ehrlichia ruminantium).

Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heartwater) were demonstrated in brain smears and formalin-fixed brain sections derived from a buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility that the tick-free environment of a quarantine boma may have affected the calf's immunity, is discussed. These findings suggest that monitoring heartwater in wild ruminants and making brain smears as a routine during post mortem evaluations of wild ruminants, should be encouraged.

Ehrlichia ruminantium grows in cell lines from four ixodid tick genera.

Continuous cell lines from the ticks Amblyomma variegatum, Boophilus decoloratus, Boophilus microplus, Hyalomma anatolicum anatolicum, Ixodes scapularis, Ixodes ricinus and Rhipicephalus appendiculatus were tested for ability to support growth of the rickettsial pathogen Ehrlichia (previously Cowdria) ruminantium. Five E.ruminantium isolates, from West Africa, South Africa and the French West Indies, were used. Twelve tick cell lines were inoculated with E.ruminantium derived either from cultures of a bovine endothelial cell strain designated BPC or from other tick cell lines. Successful infection resulted in either continuous growth (in which the pathogen/cell line system could be perpetuated through regular subculture on fresh, uninfected cells for many months or years) or finite growth (in which the pathogen disappeared after one or a few subcultures). Infection with E.ruminantium from BPC was established in I.scapularis, I.ricinus and A.variegatum cell lines; E.ruminantium was transferred from these infected cell lines to B.decoloratus, B.microplus and R. appendiculatus cell lines. H.a.anatolicum cells could not be infected with E.ruminantium by any procedure. All five E.ruminantium isolates grew continuously in at least one tick cell line at temperatures between 28 degrees C and 37 degrees C; three of the isolates were successfully re-established in BPC following prolonged maintenance in tick cells. This study demonstrates that E.ruminantium is not intrinsically restricted to growth in cells from ticks of the natural vector genus Amblyomma.

Development of improved attenuated and nucleic acid vaccines for heartwater.

Heartwater, an economically important tickborne disease of wild and domestic ruminants, is caused by the intracellular rickettsia Ehrlichia (formerly Cowdria) ruminantium. The only commercially available immunization procedure is more than 50 years old and uses an infection and treatment regimen using a preparation of virulent organisms in cryopreserved sheep blood. Much research has been conducted into the development of attenuated, inactivated, and nucleic acid vaccines over the last half-century, with relatively little success until recently. We describe here the development of two new experimental vaccines, a live attenuated vaccine and a nucleic acid vaccine. The attenuation of virulent E. ruminantium was achieved by growing the organisms in a continuous canine macrophage-monocyte cell line. After more than 125 passages the cultures produced no disease when inoculated into mice or sheep, and the animals were completely protected against a subsequent lethal homologous needle challenge. The nucleic acid vaccine consists of a cocktail of four E. ruminantium genes, from a genetic locus involved in nutrient transport, cloned in a DNA vaccine vector. Sheep immunized with this cocktail were completely protected against a subsequent lethal needle challenge, either with the homologous isolate or with any one of five different virulent heterologous isolates. Protection against a field challenge in a heartwater endemic area, however, was relatively poor. Genetic characterization of the E. ruminantium genotypes in the challenge area did not identify any having major differences from those used in the heterologous needle challenge experiments, so lack of cross-immunity between the vaccine genotype and those in the field was unlikely to be the main reason for the lack of protection. We believe that a needle challenge is far less severe than a tick challenge, and that the immunity engendered by the DNA vaccine alone was not sufficient to protect against the natural route of infection. Boosting with live organisms after DNA vaccination results in much higher levels of protection against tick challenge than DNA vaccination alone, suggesting that improved methods of boosting could lead to more effective immunization.

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.