Screening for immune biomarkers associated with infection or protection against Ehrlichia ruminantium by RNA-sequencing analysis.

Heartwater is one of the most economically important tick-borne fatal diseases of livestock. The disease is caused by the bacteria Ehrlichia ruminantium transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma controls E. ruminantium growth and that cellular immune responses are protective, an effective recombinant vaccine for this disease is lacking. Analyses of markers associated with infection as well as protection will lead to a better understanding of the E. ruminantium immune response and corresponding pathways induced in sheep peripheral blood mononuclear cells (PBMC) will assist in development of such a vaccine. In this study, Biomarkers of infection (BMI) were identified as uniquely expressed genes during primary infection and biomarkers of protection (BMP) associated with immune to heartwater were identified post challenge. Sheep were experimentally infected and challenged with E. ruminantium infected ticks. The immune phenotypic and transcriptome profile of their PBMC were compared to their own naïve PBMC collected before infection. The study revealed 305 differentially expressed genes (DEGs) as BMI, of these 17 were upregulated at all three time-points investigated. These DEGs, form part of the bacterial invasion of epithelial cells Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway, and others detected from day 1 post infection and are considered predictive markers for early heartwater infection in ruminants. Similarly, a total of 332 DEGs were identified as BMP, of these 100 were upregulated and 75 were downregulated at all three time-points investigated. However, at D1PC most DEGs were downregulated (n = 1312) that correlated with a reduction in the % CD4 and CD8 T cells detected with flow cytometry. KEGG pathway analyses showed complete down regulation of T cell specific pathways possibly due to homing of immune cells to the site of infection after acquired immunity developed. At D4PC, expression levels of most of these downregulated genes increased and by D6PC they were upregulated. This indicates that the sampling time-point for biomarker analyses is important when results for acquired immune responses are inferred. This data identified DEGs that could be considered as biomarkers of protective immunity that can be used for identification of vaccine antigens and provides a strong foundation to further development of heartwater recombinant vaccines.

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium.

Cowdria polymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in an Escherichia coli host expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominant rproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived from rprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510 rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.Contribution: This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

The economic impact of heartwater on the South African livestock industry and the need for a new vaccine.

Heartwater is a dangerous tick-borne disease for livestock farmers in South Africa and results in annual economic losses for the livestock industry. This study determined the total economic impact of heartwater in South Africa by calculating this disease's direct and indirect costs. Survey data from 272 livestock farmers from six provinces of South Africa was used for this study. Available heartwater research in South Africa only focused on the prevention and control of and vaccination against heartwater, with no research having undertaken an in-depth study of the total economic impact of this disease. The highest cost due to heartwater at provincial level was calculated for the Eastern Cape Province, followed by the North West and Mpumalanga Provinces. Heartwater has an enormous impact on the cattle industry, with an approximate R1 059 million losses experienced each year. The sheep industry experiences a total cost loss of approximately R168 million per year, with the goat industry experiencing about R39 million per year. The total economic impact of heartwater on the South African livestock industry is estimated at R1 266 million per annum. The direct cost contributed 66.47%, and indirect costs contributed 33.57% to the total cost of heartwater. The annual cost of heartwater vaccines contributed the least to the total cost of heartwater, with only 10% of the participants administering the heartwater vaccine. A total of 84.29% of the respondents indicated a need for a new and improved heartwater vaccine. This study emphasises the importance of heartwater in South Africa and farmers' high costs because of this disease. If an improved heartwater vaccine can be developed, the direct cost of heartwater will reduce, which is the bulk of its economic impact. This research can serve as a basis for future research on heartwater, where cost-benefit analyses could be conducted on heartwater's different prevention and control methods.

Development of inactivated heartwater (Ehrlichia ruminantium) vaccine in South Africa.

Heartwater, Ehrlichia ruminantium infection in cattle, sheep, goats, and some wild ruminants, is an economically important disease in Africa characterized by high mortality rates in susceptible populations. In South Africa, the current commercial heartwater vaccine is an infection and treatment type of immunization using virulent live E. ruminantium organisms generated from blood of infected sheep with subsequent treatment of the animals with antibiotics at specific times during the course of infection. This vaccine has several inherent problems preventing its wide use as the vaccine must be administered intravenously and it does not protect against all the South African field isolates. A vaccine based on inactivation of Zimbabwean E. ruminantium Mbizi strain organisms produced in endothelial cell cultures can be a sustainable option because it will not require antibiotic treatment and will be safe as there is no potential for reversion to virulence. Previous data generated in laboratory trials and under natural field setting provides support for this vaccine approach. Four inactivated vaccine formulations using the E. ruminantium Mbizi strain were tested for their efficacy in Merino sheep compared to an unvaccinated control group (11 sheep per group). Two vaccines were prepared by beta-propiolactone (BPL) inactivation, and two were inactivated with binary ethylenimine (BEI) while purification was done with both percoll and polyethylene glycol (PEG). The four vaccine preparations were formulated with Montanide ISA 50V2 adjuvant and administered twice subcutaneously (2 ml per dose) at an interval of 4 weeks. All groups were challenged with a virulent homologous cell-cultured E. ruminantium inoculated via the intra-venous route on day 56. The primary variable of efficacy was measured by the percentage survival rate or mortality between the Controls and Vaccine Groups. Three vaccine formulations (BEI/Percoll (Group 3), BEI/PEG (Group 4), BPL/Percoll, (Group 1) had a significantly higher percent of animal surviving challenge compared to the unvaccinated control (p-values 0.001, 0.035, 0.030, respectively). The highest number of survivors was obtained in Group 3 BEI/Percoll; 10/11 (91%). Groups 4 (BEI/PEG) and Group 1 (BPL/Percoll) produced similar percentage of survivals of 64%. In contrast, the lowest survival rate of 50% was observed in Group 2 (BPL/PEG) which was numerically different but not significantly different from the unvaccinated control which had an 18% survival rate (2/11). The inactivated vaccine using BEI or BPL as inactivating agents blended with ISA 50 adjuvant induced protective immunity against challenge. The BEI/Percoll (Group 3) vaccination regimen was most efficacious against a lethal heartwater challenge as it significantly protected sheep against mortality which is the most important aspect of heartwater infections. Future work should be directed towards improvement of this vaccine formulation especially from the down-stream processing point of view as the percoll method is not scalable for commercialization purposes.

Safety and efficacy of an attenuated heartwater (Ehrlichia ruminantium) vaccine administered by the intramuscular route in cattle, sheep and Angora goats.

Heartwater is an economically important tick-borne disease of ruminants in Africa. The current commercial vaccine uses live Ehrlichia ruminantium from blood of infected sheep, requires antibiotic treatment during infection, needs to be administered intravenously and does not protect against all South African isolates. An attenuated tissue culture vaccine not requiring antibiotic treatment and effective against different field strains in small groups of goats and sheep was reported previously. The objective of the present study was to test safety and efficacy of this vaccine administered by intramuscular (i.m.) inoculation in larger groups of sheep, Angora goats and cattle. Animals were vaccinated via intravenous (i.v.) and i.m. routes and received E. ruminantium homologous challenge by feeding of infected ticks or by i.v. inoculation of infected blood. For vaccine titration in sheep and goats, the optimum safe and efficacious dose was determined using 2 ml equivalent of 102-105 culture-derived live elementary bodies (EBs). Similarly, the vaccine was titrated in cattle using 5 ml containing 105-107 EBs. Seventy percent of i.v. vaccinated and 9.7% of i.m. vaccinated Angora goats receiving 105 EBs, developed severe reactions to vaccination and were treated. These treated animals and the remaining 90.3% of i.m.- vaccinated goats showed 100% protection against i.v. or tick challenge. Sheep and Angora goats vaccinated i.m. with 104 EBs had no vaccination reactions and were fully protected against i.v. or tick challenge. Similarly, vaccinated cattle (dose 106 EBs) did not react to vaccine inoculation and were fully protected against i.v. or tick homologous challenge. Control non-vaccinated animals reacted severely to challenge and required oxytetracycline treatment. This successfully demonstrated that Angora goats, sheep and cattle can be safely vaccinated with the attenuated E. ruminantium Welgevonden vaccine via the i.m. route, with no clinical reactions to vaccination and 100% protection against virulent i.v. and homologous tick challenge.

A multi-epitope DNA vaccine co-administered with monophosphoryl lipid A adjuvant provides protection against tick transmitted Ehrlichia ruminantium in sheep.

Previously, a heartwater experimental DNA vaccine provided 100% protection following laboratory challenge with Ehrlichia ruminantium administered by needle but not against an E. ruminantium tick challenge in the field. A multi-epitope DNA vaccine incorporating both CD4+ and CD8+ cytotoxic T lymphocytes epitopes could provide a better alternative. In this study, we investigated the use of multi-epitope DNA vaccines against an E. ruminantium experimental tick challenge in sheep. The multi-epitope DNA vaccines were delivered via the intramuscular route and intradermal route using the gene gun in the presence of monophosphoryl lipid A (MPL) adjuvant, which was either applied topically to the gene gun inoculation site or co-administered with the vaccine via the intramuscular route. Initially two constructs namely, pSignal plus and pLamp were tested with MPL applied topically only and no protection was obtained in this formulation. However, when pLamp was co-administered with MPL via the intramuscular route in addition to topical application, its protective efficiency improved to protect 60% of the sheep against tick challenge. In this formulation, the vaccine induced enhanced activation of memory T cell responses both before and after challenge with variations amongst the different sheep possibly due to their different genetic backgrounds. In conclusion, this study showed that a heartwater multi-epitope DNA vaccine, co-administered with MPL adjuvant can protect sheep following a laboratory E. ruminantium tick challenge.

Ehrlichia ruminantium antigens and peptides induce cytotoxic T cell responses in vitro.

Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.

Cellular immune responses induced in vitro by Ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides.

Secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (IFN)-γ. In this study three open reading frames (ORFs) (Erum8060, Erum7760, Erum5000) encoding secreted proteins were selected from the Ehrlichia ruminantium (Welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. The whole recombinant protein of the three ORFs as well as four adjacent fragments of the Erum5000 protein (Erum5000A, Erum5000B, Erum5000C, Erum5000D) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-His antibodies and sheep sera. These recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (PBMCs), spleen and lymph node (LN) cells to determine whether they induce recall cellular immune responses in vitro. Significant proliferative responses and IFN-γ production were evident for all recombinant proteins, especially Erum5000A, in both ruminant species tested. Thus overlapping peptides spanning Erum5000A were synthesised and peptides that induce proliferation of memory CD4+ and CD8+ T cells and production of IFN-γ were identified. These results illustrate that a Th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine.

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.

Heartwater--Ehrlichia ruminantium infection.

Summary Heartwater is a notifiable disease that is listed by the World Organisation for Animal Health. It is caused by Ehrlichia ruminantium, an obligately intracellular Gram-negative bacterium in the order Rickettsiales and the family Anaplasmataceae. The disease is borne byticks in the genus Amblyomma and causes heartwater, or cowdriosis, in wild and domestic ruminants, primarily in Africa, but also in parts of the Caribbean. The disease was recognised in South Africa in the 19th Century and determined to be tick borne in 1900, while the organism was identified in 1925 and first cultured in vitro in 1985. This latter achievement boosted research into the disease at a time when biology was moving into the molecular genetic age. Over the last 20 years, there have been significant improvements in our understanding of E. ruminantium, yielding major advances in diagnosis, epidemiology, genetic characterisation, phylogeny, immunology, and vaccine development. The organism is genetically highly variable; this has important implications for future control measures, and is making it difficult to develop an effective vaccine for protection against tick challenge. Research is continuing into three different types of vaccine, inactivated, attenuated, and recombinant, and the current state of development of each is discussed.

A user-friendly and scalable process to prepare a ready-to-use inactivated vaccine: the example of heartwater in ruminants under tropical conditions.

The use of cheap and thermoresistant vaccines in poor tropical countries for the control of animal diseases is a key issue. Our work aimed at designing and validating a process for the large-scale production of a ready-to-use inactivated vaccine for ruminants. Our model was heartwater caused by the obligate intracellular bacterium Ehrlichia ruminantium (ER). The conventional inactivated vaccine against heartwater (based on whole bacteria inactivated with sodium azide) is prepared immediately before injection, using a syringe-extrusion method with Montanide ISA50. This is a fastidious time-consuming process and it limits the number of vaccine doses available. To overcome these issues, we tested three different techniques (syringe, vortex and homogenizer) and three Montanide ISA adjuvants (50, 70 and 70M). High-speed homogenizer was the optimal method to emulsify ER antigens with both ISA70 and 70M adjuvants. The emulsions displayed a good homogeneity (particle size below 1 µm and low phase separation), conductivity below 10 µS/cm and low antigen degradation at 4 °C for up to 1 year. The efficacy of the different formulations was then evaluated during vaccination trials on goats. The inactivated ER antigens emulsified with ISA70 and ISA70M in a homogenizer resulted in 80% and 100% survival rates, respectively. A cold-chain rupture assay using ISA70M+ER was performed to mimic possible field conditions exposing the vaccine at 37 °C for 4 days before delivery. Surprisingly, the animal survival rate was still high (80%). We also observed that the MAP-1B antibody response was very similar between animals vaccinated with ISA70+ER and ISA70M+ER emulsions, suggesting a more homogenous antigen distribution and presentation in these emulsions. Our work demonstrated that the combination of ISA70 or ISA70M and homogenizer is optimal for the production of an effective ready-to-use inactivated vaccine against heartwater, which could easily be produced on an industrial scale.

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.

Ethnoknowledge of Bukusu community on livestock tick prevention and control in Bungoma district, western Kenya.

ETHNOPHARMACOLOGICAL RELEVANCE: To date, nomadic communities in Africa have been the primary focus of ethnoveterinary research. The Bukusu of western Kenya have an interesting history, with nomadic lifestyle in the past before settling down to either arable or mixed arable/pastoral farming systems. Their collective and accumulative ethnoveterinary knowledge is likely to be just as rich and worth documenting. AIM OF THE STUDY: The aim of the present study was to document indigenous knowledge of the Bukusu on the effect of livestock ticks and ethnopractices associated with their management. It was envisaged that this would provide a basis for further research on the efficacy of these practices that could also lead to the discovery of useful tick-control agents. MATERIALS AND METHODS: Non-alienating, dialogic, participatory action research (PAR) and participatory rural appraisal (PRA) approaches involving 272 women and men aged between 18 and 118 years from the Bukusu community were used. RESULTS: Ticks are traditionally classified and identified by colour, size, host range, on-host feeding sites, and habitat preference. Tick-associated problems recognised include kamabumba (local reference to East Coast fever, Anaplasmosis or Heartwater diseases transmitted by different species of livestock ticks) and general poor performance of livestock. Traditional methods of controlling ticks include handpicking, on-host use of ethnobotanical suspensions (prepared from one or more of over 150 documented plants) to kill the ticks and prevent re-infestation, fumigation of infested cattle with smoke derived from burning ethnobotanical products, burning pastures, rotational grazing ethnopractices, and livestock quarantine. CONCLUSIONS: The study confirms that the Bukusu have preserved rich ethnoveterinary knowledge and practices. It provides some groundwork for elucidating the efficacy of some of these ethnopractices in protecting livestock from tick disease vectors, particularly those involving the use of ethnobotanicals, which may lead to the discovery of useful ant-tick agents.

Treatment and control of chlamydial and rickettsial infections in sheep and goats.

Small ruminants are susceptible to several chlamydial and rickettsial infections. Some of them, such as Ehrlichia ruminantium, have a great impact on the sheep and goat industry while others, such as Coxiella burnetii, are important zoonotic agents. This review focuses on measures of treatment and control for the following organisms: Chlamydophila abortus (formerly Chlamydia psittaci immunotype 1), Coxiella burnetii, Anaplasma ovis, Anaplasma phagocytophilum, and Ehrlichia ruminantium.

Efficiency of inactivated vaccines against heartwater in Burkina Faso: impact of Ehrlichia ruminantium genetic diversity.

In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N=153, p=0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N=173, p<0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.

Studies of a polymorphic Ehrlichia ruminantium gene for use as a component of a recombinant vaccine against heartwater.

A previously identified polymorphic Ehrlichia ruminantium gene, Erum2510, was investigated to determine its ability to induce protective immunity in ruminants following two different DNA immunisation strategies; DNA-only and a DNA prime/recombinant protein (rprotein) boost immunisation. The DNA-only vaccine was also compared to a cocktail of three polymorphic E. ruminantium (Welgevonden) open reading frames (ORFs) adjacent to Erum2510 in the genome. Weak protection was observed in animals immunised with the pCMViUBs_Erum2510 construct alone, while none of the animals immunised with the DNA cocktail were protected. In contrast, all five animals immunised using a DNA prime/rprotein boost strategy survived challenge, thereby indicating that Erum2510 is a good candidate for inclusion in a recombinant vaccine against heartwater. One drawback of using polymorphic genes is a possible lack of cross-protection between genotypes, therefore the genetic diversity of Erum2510 was investigated to establish the degree of polymorphism among different E. ruminantium stocks. Three distinct genotypes were identified indicating that if this gene is used as a vaccine (prime/boost strategy) the vaccine should include a representative Erum2510 gene from each genotype.

Preparation and in vitro characterisation of Ehrlichia ruminantium plasmid DNA and proteins encapsulated into and DNA adsorbed onto biodegradable microparticles.

Four E. ruminantium 1H12 open reading frames and their proteins known to protect sheep against heartwater needle challenge were encapsulated into, or adsorbed onto poly(d,l-lactide-co-glycolide) microparticles. Microspheres with smooth surface and smaller than 5 µm diameters were produced, with high adsorption and encapsulation efficiencies. Gel electrophoresis showed that neither encapsulation nor adsorption affected the stability of the DNA or proteins. Cationic microparticles released ∼40% of plasmid DNA on day 1 while PLGA 50:50-COOH microparticles co-encapsulating plasmid DNA and polyvinyl alcohol only started to release from days 12-28. Recombinant proteins were released from PLGA 85:15 and homopolymer R 203 S microparticles in a biphasic manner with a high initial burst release (∼45-80%). In contrast, PLGA 50:50 microparticles had low (15-65%) initial burst release followed by (25-80%) release by days (days 28-42). A cocktail of these microparticles could therefore be used as single-dose auto-booster vaccine.

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.

A heterologous prime/boost immunisation strategy protects against virulent E. ruminantium Welgevonden needle challenge but not against tick challenge.

Heterologous prime/boost immunisation strategies using the Ehrlichia ruminantium 1H12 pCMViUBs_ORFs [Pretorius A, Collins NE, Steyn HC, Van Strijp F, Van Kleef M, Allsopp BA. Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames. Vaccine 2007;25(12):2316-24] were investigated in this study. All the animals immunised twice with a recombinant (r) DNA cocktail of four 1H12 pCMViUBs_ORFs followed by a r1H12 protein and those immunised 3x with 1H12 plasmid rDNA showed 100% protection against a virulent E. ruminantium Welgevonden needle challenge. In addition, 90% of the sheep immunised twice with rDNA and boosted with r1H12 lumpy skin disease virus (LSDV) survived. Only the lymphocytes isolated from the r1H12 protein boost group showed specific proliferation and increased interferon (IFN)-gamma expression. In contrast, only 20% protection was obtained in animals immunised with the rDNA prime/r1H12 protein boost when subjected to natural tick challenge in the field. Thus this heterologous prime/boost immunisation strategy had not conferred any significant protection against a field challenge.