ABSTRACT Heartwater, a tick-borne disease caused by Ehrlichia ruminantium, is considered to be a significant cause of mortality amongst domestic and wild ruminants in South Africa. The main vector is Amblyomma hebraeum and although previous epidemiological studies have outlined endemic areas based on mortalities, these have been limited by diagnostic methods which relied mainly on positive brain smears. The indirect fluorescent antibody test (IFA) has a low specificity for heartwater organisms as it cross-reacts with some other species. Since the advent of biotechnology and genomics, molecular epidemiology has evolved using the methodology of traditional epidemiology coupled with the new molecular techniques. A new quantitative real-time polymerase chain reaction (qPCR) test has been developed for rapid and accurate diagnosis of heartwater in the live animal. This method can also be used to survey populations of A. hebraeum ticks for heartwater. Sampling whole blood and ticks for this qPCR differs from routine serumsampling, which is used for many serological tests. Veterinary field staff, particularly animal health technicians, are involved in surveillance and monitoring of controlled and other diseases of animals in South Africa. However, it was found that the sampling of whole blood was not done correctly, probably because it is a new sampling technique specific for new technology, where the heartwater organism is much more labile than the serumantibodies required for other tests. This qPCR technique is highly sensitive and can diagnose heartwater in the living animal within 2 hours, in time to treat it. Poor sampling techniques that decrease the sensitivity of the test will, however, result in a false negative diagnosis. This paper describes the development of a skills training programme for para-veterinary field staff, to facilitate research into the molecular epidemiology of heartwater in ruminants and eliminate any sampling bias due to collection errors. Humane handling techniques were also included in the training, in line with the current focus on improved livestock welfare.

Ehrlichia ruminantium is a tick-borne pathogen transmitted by ticks in the genus Amblyomma. This bacterial pathogen causes heartwater, a fatal disease affecting domestic and wild ruminants in sub-Saharan Africa. The prevalence of heartwater in western Kenya is not well documented. In this study, reverse line blot (RLB) hybridization and quantitative real-time PCR (qPCR) assays were used to detect E. ruminantium DNA in 545 blood samples collected from calves from twenty sublocations distributed across five agro-ecological zones of western Kenya. Ehrlichia ruminantium DNA was detected in 1.10% and 0.92% of the samples using RLB and qPCR, respectively. There were discrepancies in the detection of E. ruminantium by the RLB and the qPCR. Five samples were positive with the qPCR while six were positive with the RLB, but only three of the samples were positive by both tests. The occurrence of E. ruminantium in western Kenya appears to be low, but this might be attributed to the inability of the tests used to detect E. ruminantium carriers. The most prevalent haemoparasites detected by the RLB in the Ehrlichia/Anaplasma group were Anaplasma (formerly Ehrlichia) sp. Omatjenne and Anaplasma bovis at 37.98% each, while Theileria mutans (66.61%) was the most prevalent in the Theileria/Babesia group. In addition, a nested p104 PCR was used to detect Theileria parva in a subset of 86 of the samples; T. parva was detected in 32.56% (28/86) of these samples. The RLB detected T. parva in 27.91% (24/86) of the same sample subset, but only 17 were positive by both tests. The molecular tests used in this study suggest that, of the pathogenic haemoparasites known to cause disease in Kenya, T. parva occurs the most commonly in western Kenya, while E. ruminantium, A. marginale, B. bigemina and B. bovis are less frequently detected. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

In the search for a better vaccine against Ehrlichia ruminantium infection in ruminants, four E. ruminantium open reading frames (ORFs) derived from the Welgevonden isolate were tested using either DNA vaccination or DNA primemodified viral or DNA prime-recombinant protein boost strategies. Both the DNA vaccination and the DNA prime.recombinant protein boost strategy provided complete protection against E. ruminantium Welgevonden needle challenge, while the DNA prime.modified viral boost strategy only provided 90 % protection. The DNA prime.recombinant protein boost strategy also coincided with elevated cellular immunology as was evident from increased IFN-ã production. Furthermore, we could show that the 1H12 DNA vaccine could induce protection against heterologous needle challenge when animals were immunised with the Welgevonden-derived 1H12 ORFs and challenged with selected E. ruminantium stocks. Unfortunately the DNA only and the DNA prime.recombinant protein boost strategy were not protective in the field. Therefore, our results suggest that there is a vast difference between needle challenge and natural tick infestation and that E. ruminantium organisms transmitted by ticks have the ability to evade the protective immunity induced by immunization with the four 1H12 ORFs. / Thesis (PhD)--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

Four 1H12 E. ruminantium open reading frames cloned into the pCMViUBs mammalian expression vector and used as a recombinant DNA vaccine against heartwater repeatedly provided complete protection in sheep (using a cocktail or the individual ORFs) against a laboratory needle challenge while 1/5 of sheep were protected after a natural tick challenge. The lack of protection under natural field conditions could be attributed to the delivery strategy used and therefore there is a need to investigate other delivery methods. Polymeric microparticles based on PLGA polymers have been used extensively to target the delivery of vaccine to antigen presenting cells, play a role in the induction of cellular immunity and can be used as a single dose vaccine mimicking prime/boost vaccination. In this study, the four 1H12 pCMViUBs_ORFs and their respective recombinant proteins were either encapsulated into or adsorbed onto microparticles using a modified double emulsion solvent evaporation technique. The particles were formulated to release DNA on day zero and day 21 and recombinant proteins on day 42 thus mimicking a two times DNA prime/recombinant protein-boost immunization strategy. Encapsulation did not have any detrimental effects on the stability of the recombinant proteins as determined by gel electrophoresis and western blotting. The in vitro incubation of microparticles in either a Float-A-Lyzer® dialyzer or an eppendorf tube showed the potential of microparticles to be used as a vaccine because of their release profiles that mimics a heterologous prime/boost immunization strategy. Microparticles formulated using polymers with low glycolide ratios released 80% of the encapsulated proteins within the first week of in vitro incubation with most of the proteins released on day 1. Microparticles formulated using polymers with 50:50 monomer ratios released the recombinant proteins during week 1 and 3 of in vitro incubation. These microparticles did not release any protein in week 2 (day 7-14). Microparticles with 0.5% cetyltrimethylammonium bromide (CTAB) on their surfaces adsorbed DNA and released more than 40% of DNA on day 1 with 100% release by day 14. RG502H microparticles formed with PVA as the internal phase viscosity enhancer released intact DNA only from day 12 to day 21. A cocktail of these microparticles could therefore be used as an autobooster vaccine thus reducing the need for repeated immunizations needed to obtain protective immunity. Potential scientific publication Tshikhudo, N.P., Pretorius, A., Putterill, J., and van Kleef, M. 2009, “Biodegradable microparticles as a single dose delivery system for Ehrlichia ruminantium vaccines”, Journal of Controlled Release, (draft manuscript). Publication of results in conference proceedings / abstracts NanoAfrica 2009: Biodegradable microspheres as a single dose delivery system for Ehrlichia ruminantium vaccines: N. Tshikhudo, A. Pretorius, J. Putterill and M. van Kleef. / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Ehrlichia ruminantium is an obligate intracellular pathogen and, as such, cell mediated immunity plays a key role in the control of bacterial replication and subsequent protection against heartwater in ruminants. IFN-γ has been shown to be a potent inhibitor of E. ruminantium growth in endothelial cells in vitro. Thus, identification of antigens that preferentially activate T cells to proliferate and secrete IFN-γ needs to be done so that they can be evaluated as vaccine candidates. Previously, a cocktail of four open reading frames (ORFs) that induce 100% protection after needle challenge have been identified. However, only 20% protection was obtained after tick challenge in the field, when administered as a DNA vaccine in sheep. Because only limited protection was obtained during a field vaccine trial, our research focused on the identification of additional ORFs as a mean to improve the efficacy of this vaccine. Because secreted proteins are reported to be major targets in a specific immune response we hypothesize that they may be potential heartwater vaccine candidates. Five ORFs (Erum5000, Erum5010, Erum7760, Erum8060 and Erum8610) encoding secreted E. ruminantium proteins were selected from the Welgevonden stock genome sequence using bioinformatics tools. The ORFs were cloned into a pET 102/D-TOPO® vector. The corresponding recombinant (r) proteins were expressed in a bacterial expression system and the expression was confirmed by immunoblots using anti-His antibodies and sheep sera. Proteins were purified by immobilized metal ion affinity chromatography and resulted in a yield of 200 μg-2000 μg per 100 ml and 1L cultures respectively. Four of the five recombinant proteins (rErum5000, rErum7760, rErum8060 and rErum8610) could be expressed. Recombinant proteins were assayed to determine whether they induce recall cellular immune responses in vitro. The peripheral blood mononuclear cells used in the assays were obtained from a naïve and four heartwater immune sheep. Significant proliferative responses (p ≤ 0.01) were evident for 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610). IFN-γ production was determined using an ELISPOT assay and 3/4 recombinant proteins (rErum5000, rErum8060 and rErum8610) induced IFN-γ production. Each recombinant protein had its own optimum concentration for inducing immune responses and the responses differed between animals. In addition, real-time PCR was used to measure IFN-γ and IL-4 gene expression by antigen stimulated immune PBMC. The real-time PCR results correlated with the ELISPOT assay results. Based on the results obtained, it can be concluded that a Th1 type immune response was elicited. Thus these proteins that induced proliferation and IFN-γ production may be important in protection against heartwater and will be tested in future vaccine studies. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / Unrestricted

A shotgun genome sequencing project was undertaken in the expectation that access to the entire protein coding potential of E. Ruminantium (Welgevonden) will facilitate the identification of vaccine candidate genes against heartwater. The 1,516,355 bp sequence is predicted to encode 888 proteins and 41 stable RNA species. The most prominent feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number. These repeats have mediated numerous translocation and inversion events and seem to be responsible for the generation of both new full and partial protein coding sequences. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Of the 13 members of the order Rickettsiales compared in this study, E. Ruminantium has the lowest coding capacity (62%), lowest GC content (27.5%), but the highest proportion of repetitive sequences, which comprise 8.5% of the genome. Metabolic reconstruction of E. Ruminantium revealed the metabolic and biosynthetic capabilities typical of an obligate intracellular organism. We identified a number of genes unique to E. Ruminantium, most of which are not functionally characterised in any organism, and those shared with 12 other members of the Rickettsiales. Bioinformatic tools were used to identify possible vaccine candidates from the annotated genome sequence. The protective properties of seven open reading frames (ORFs), which induced cellular immune responses in vitro, were tested in vivo Only 20% survival was obtained in sheep immunised with a DNA formulation consisting of three ORFs. We found that the levels of peripheral blood mononuclear cell proliferation and interferon-gamma (IFN-γ) production did not correlate with each other, nor with the levels of protection, suggesting that the current assays are just not reliable and that IFN-γ expression alone is not an indicator of protection. Therefore more cytokines and different assays will have to be investigated to define in detail what constitutes a protective immune response against E. Ruminantium infection. However, the data generated from the genome sequence will continue to facilitate novel approaches to study the organism and to develop an efficacious vaccine against heartwater. / Thesis (PhD)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

AU COURS DE LA THESE, L'ETUDE DU TRANSCRIPTOME DE SOUCHES GARDEL ET SENEGAL VIRULENTES ET ATTENUEES D'E. RUMINANTIUMA ETE REALISEE. UNE ANALYSE DU TRANSCRIPTOME A DIFFERENTS STADES DE DEVELOPPEMENT, A D'ABORD ETE EFFECTUEE POUR LA SOUCHE GARDEL VIRULENTE. AU STADE CORPS RETICULE (FORME INTRACELLULAIRE NON INFECTIEUSE), UNE SUREXPRESSION DES GENES CODANT POUR DES PROTEINES IMPLIQUEES DANS LE METABOLISME, LE TRANSPORT ET L'ECHANGE DE NUTRIMENTS ET DANS LA RESISTANCE AU STRESS OXYDATIF ETAIT OBSERVEE. IL SEMBLERAIT QUEE. RUMINANTIUMMETTE EN PLACE UN PANEL DE MECANISMES POUR SA SURVIE ET SON DEVELOPPEMENT A L'INTERIEUR DE LA CELLULE HOTE. AU STADE CORPS ELEMENTAIRE (FORME EXTRACELLULAlRE INFECTIEUSE), LE GENE DKSA CODANT POUR UN FACTEUR DE TRANSCRIPTION ETAIT SUREXPRIME. CE GENE A ETE MONTRE COMME ETANT IMPLIQUE DANS LA REGULATION DE FACTEURS DE VIRULENCE. IL SEMBLERAIT . DONC, QU'AU STADE CORPS ELEMENTAIRE, IL Y AIT UNE INDUCTION DE MECANISMES DE VIRULENCE. LA COMPARAISON DE L'EXPRESSION DES GENES AU STADE CORPS ELEMENTAIRE ENTRE SOUCHES VIRULENTES ET ATTENUEES A AUSSI ETE EFFECTUEE. NOS RESULTATS ONT MONTRE UNE MODIFICATION IMPORTANTE DE LA MEMBRANE POUR LES SOUCHES VIRULENTES ET ATTENUEES. POUR LES SOUCHES ATTENUEES, IL A ETE MONTRE UNE SUREXPRESSION DES GENES IMPLIQUES DANS LA BIOGENESE MEMBRANAlRE ET UNE SOUS-EXPRESSION·DES PROTEINES DE LA FAMILLE MULTIGENIQUE MAP. CES RESULTATS SUGGERENT QUE LES PROTEINES MAP JOUENT UN ROLE DE LEURRE VIS-A-VIS DE LA REPONSE IMMUNITAIRE PROTECTRICE. DES PROTEINES MEMBRANAlRES HYPOTHETIQUES SONT SUREXPRIMEES A LA FOIS CHEZ LES SOUCHES VIRULENTES ET ATTENUEES. CERTAINES D'ENTRE ELLES SUREXPRIMEES CHEZ LES SOUCHES ATTENUEES SEMBLENT ETRE DE BONS CANDIDATS VACCINAUX ET DEVRAIENT ETRE ETUDIEES / Transcriptomic study of gardel and senegal both virulent and attenuated e. ruminantium strains was conducted during my phd. an analysis of transcriptome at different stages of development has been first conducted for virulent gardel strain. at reticulate body stage (intracellular form non-infectious), over-expression of genes coding for proteins involved in metabolism, transport and exchange of nutrients and resistance to oxidative stress was observed. at this stage of development, e. ruminantium seems to activate mechanisms for its survival and development within the host cell. at elementary body stage, dksa the gene encoding for a transcription factor was over-expressed. this gene has been shown to be involved in the regulation of virulence factors. it seems, therefore, at the elementary body stage, e. ruminantium induces its virulence factors. secondly, we compare the transcriptome of elementary body between virulent and attenuated strains. our results showed an important membrane modification of attenuated and virulent strains. for attenuated strains, we observed an over-expression of genes involved in membrane biogenesis and a diminution of expression of map multigenic family. it seems that map proteins subvert the protective immune response. hypothetical membrane proteins are over-expressed in both virulent and attenuated strains. some over-expressed proteins in attenuated strains seem to be good vaccine candidates and willstudied.

Ehrlichia ruminantium, the causative organism of heartwater infections, places severe economic constraint on the livestock industry wherever Amblyomma tick vectors are present. Angora goats are particularly susceptible to this disease and the current live blood vaccine cannot safely be used to protect these animals. An attenuated E. ruminantium (Welgevonden) experimental vaccine has previously shown promising results in Merino sheep and Boer goats. The vaccine was administered by intravenous route (i/v). The general objective of this study was to test the efficacy and safety of the attenuated heartwater vaccine E. ruminantium (Welgevonden) in Angora goats. The specific objectives were, firstly to assess the intra-muscular route of administration of the attenuated vaccine as compared to the standard i/v route and, secondly, to study the haematological changes in Angora goats before, during and after vaccination under controlled conditions at the Onderstepoort Veterinary Institute tick-free stables. A total of 55 Angora goats were used in this trial. They were purchased from an area in South Africa which is known to be Amblyomma-free and heartwater-free. Furthermore, on arrival, the goats were screened for E. ruminantium infection by the immunofluorescent antibody (IFA) test to confirm their disease-free status. The Angora goats were divided into 3 groups: In Group 1, ten were vaccinated by the standard i/v route, in Group 2, 31 received the vaccine by i/m route and 10 served as untreated controls for Group 3. Five of the 10 i/v vaccinated group, 20/31 of the i/m vaccinated and 5 controls were challenged by feeding of known infected adult A hebreaum. The other remaining animals within the three groups were challenged using a known infected blood stabilate administered by the standard i/v route (dose 5xLD50). All animals were challenged 42 days after vaccination. The vaccine did not produce any inflammatory reactions at the site of injection. However, 3/31 (9.7%) of i/m and 7/10 (70%) of i/v vaccinated goats developed febrile reactions starting on Day 11 post-immunisation and were treated. All vaccinated goats were fully protected against either needle i/v or tick challenge, while the control non-vaccinated goats reacted severely to the challenge materials and required oxytetracycline treatment. Despite treatment, two of the unvaccinated goats died from the challenge material. 9 Haematological values (packed cell volume, differential blood cells count) were obtained on blood samples taken from the treatment and control groups at different times during the course of the trial. Wide within group variations as shown by the high standard deviation values were found. As no significant changes were found between vaccinated and control animals, it is likely that the attenuated vaccine does not cause significant clinical haematological changes. This study has demonstrated that the attenuated E. ruminantium (Welgevonden) vaccine is safe in 90.3% and efficacious (100% efficacy) for intramuscular administration in Angora goats. However, further laboratory and on-farms studies are needed in order to establish the lowest effective and safety dose, duration of immunity, and the vaccine’s safety in young and pregnant animals. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

Ehrlichia ruminantium est l'agent causal de la cowdriose, une maladie tropicale mortelle des ruminantstransmis par les tiques Amblyomma. Jusqu'à présent, il n'existe pas de vaccin efficace dû à la faible protection croisée des souches vaccinales vis-à-vis des isolats de terrain. Ceci est principalement lié àdiversité génétique d'E. ruminantium au sein les zones géographiques. Par conséquent, la caractérisation de lastructure génétique de la population d'E. ruminantium à l'échelle mondiale et régionale est importante pour définir les meilleures stratégies de contrôle et améliorer les stratégies de surveillance de la cowdriose. / Ehrlichia ruminantium is the causal agent of heartwater, a ruminant tropical fatal diseasetransmitted by Amblyomma ticks. Up to now, no effective vaccine is available due to a limitedcross protection of vaccinal strains on field isolates mainly associated to a high geneticdiversity of E. ruminantium within geographical locations. Thus, both characterization of E.ruminantium genetic population structure at worldwide and regional scale and estimation of E.ruminantium tick prevalence are important to delimitate better control strategies and improveheartwater monitoring strategies

Little is understood about off-host behavior and ecology of immature Amblyomma maculatum Koch (Gulf Coast tick). A more complete understanding of this tick is essential to protect human and animal health. My research focused on seasonality and distribution of immatures in Mississippi, potential suitability of some insect and human hosts to larvae, and aspects of nymphal questing behavior. A single larva was collected (third off-host collection reported) when sampling A. maculatum habitat using a novel device. Collection of this larva in November expands the stage’s known seasonality and confirmed a prediction concerning seasonality of larval A. maculatum. Low frequency of immatures (8.3%) confirmed that they’re incredibly difficult to collect off-host. Nymphal collections peaked in March, and known seasonality was extended for both nymphs and adults. I examined known records, elucidating seasonality and distribution of A. maculatum in Mississippi. Either multiple generations per year or diapause are responsible for observed bi-modal distribution of immature collections. Additionally, I compiled the most extensive host record of immature A. maculatum in Mississippi and investigated seasonality patterns using USDA plant hardiness zones. I compiled the most complete record of ticks found on arthropods. Amblyomma americanum and A. maculatum were both confirmed to crawl onto arthropods, giving support to occasional, unintentional dispersal by phoresy. There was no conclusive evidence that larval A. maculatum feed on arthropods, however data supported feeding by larval A. americanum. These results have interesting implications regarding evolution of pathogens/endosymbionts. I provided the first evidence that larval A. maculatum can attach to humans. Rickettsia parkeri, a human pathogen transmitted by this species has recently been shown to be capable of transovarial transmission. Therefore, larval A. maculatum may provide another avenue of transmission. I have demonstrated that A. maculatum are difficult to collect off-host in part because they prefer to quest low to the ground. In choice studies, 5-cm-tall stems were most likely to be occupied by nymphs released into an array of stems. Low vapor pressure deficit encouraged questing, while higher VPD and warmer temperature increased questing height. These results may have implications in understanding host-seeking behavior in other tick species as well.