Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. / Author: Mirinda van Kleef neé Rossouw

The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibody-reactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics.

The main objective of this study was to use the Attraction-aggregation-attachment¬pheromone/carbon dioxide (AAAP/C02) trap on a sustainable basis at six different field sites in South Africa. This trap was developed in Zimbabwe, but had not been used successfully in the field for the collection of free-living adult and nymphal A. hebraeum. A two-year collection survey was carried out at one of the sites, the Rietgat communal grazing area (CGA) where 1 196 adult and 292 nymphal A. hebraeum were trapped with the AAAP/CO2. Only free-living, unfed adult and nymphal A. hebraeum were collected, as these ticks were considered to be epidemiologically more credible than ticks collected off hosts. A distinct seasonal appearance of adult ticks was noted in both 1996 and 1997, and this could explain the difficulty experienced in collecting these ticks in the field in the past. Peak numbers of adult ticks were collected from late spring (September/October) to midsummer (November - January). This was followed by a sharp decline to very low counts for the remainder of the year (February - August). Field work was also conducted at five other sites in South Africa. At three of these sites, the AAAP/CO2 trap was used successfully, these included a farm near East London (n = 187 adults, 17 nymphs) Kruger National Park (KNP) (n = 447 adults) and the Songimvelo Game Reserve (SGR) (n = 48 adults). At the two other sites, namely the MEDUNSA campus (n = 31 adults) and at a farm near Warmbaths (n = 25 adults), the AAAP/CO2 trap was not really successful. A total of 1 934 adult and 309 nymphal A. hebraeum were collected with the AAAP/CO2 trap. A sample (n = 570) of the adult ticks collected from the Rietgat CGA (n = 434), the KNP (n = 88) and the SGR (n = 48) was tested for C. ruminantium with a specific PCR assay developed at the UFIUS AID/SADC Healtwater Research Project in Harare, Zimbabwe. Nearly nine per cent (8.9%) of the ticks from the Rietgat CGA, 5.7% from the KNP and 25% from the SGR were positive for C. ruminantium. The overall infection rate of 9.8% for the total sample (n = 570) is similar to others recorded in southern Africa. This was the first time that a large, statistically-relevant sample of free-living, unfed adult A. hebraeum collected with a AAAP/C02 trap, from a variety of different ecological areas has been processed with a C. ruminantium-specific PCR. The epidemiological data from this project should be more credible than those from many of the previous surveys, where feeding ticks were collected off hosts, and indirect methods used to determine C. ruminantium prevalence. / Dissertation (MMedVet)--University of Pretoria, 2000. / Veterinary Tropical Diseases / unrestricted

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Heartwater is a major threat to livestock in Africa due to its high mortality rate. The intracellular nature of the causative organism, Cowdria ruminantium, makes it difficult to study, hence an effective and user-friendly vaccine has been extremely difficult to obtain. Two C. ruminantium DNA libraries have recently been constructed, the lambda GEM11 bacteriophage DNA library and the lambda ZAPII bacteriophage DNA library, and this has lead to a renewed search for protective genes that could be used as a vaccine against heartwater. In this study, several molecular techniques including PCR, cloning and sequencing were used to identify genes in the lambda GEM11 bacteriophage DNA library that code for proteins, which could be used as vaccines to protect susceptible animals against heartwater. The lambda GEM11 library was screened with a rickettsial secretory protein gene sequence, known as seeD. One positive colony was selected from which the bacteriophage DNA was isolated. The C. ruminantium DNA was amplified from the bacteriophage DNA by using PCR and C. ruminantium-specific primers. The C. ruminantium DNA was screened with Mycoplasma, bovine and Cowdria DNA probes. The amplified DNA was subeloned into two vectors and the clones were screened by restriction analysis to identify clones containing inserts. The appropriate clones were sequenced and overlapping sequences matched, ordered and aligned. Two sequences were continuous with a short sequence of unidentified bases in between. Oligonucleotide primers were designed to amplify the DNA sequence between the two contiguous sequences. This led to the identification of the entire sequence of the C. ruminantium genome contained within the bacteriophage plaque. The single contiguous sequence was analysed and the putative protein-coding sequences were obtained and compared to DNA sequences of known organisms using the BLAST program. Five open reading frames were identified with homology to genes encoding specific proteins in bacteria. Two open reading frames showed homology to the genes encoding the transporter proteins, FtsY and the ABC transporter, and three open reading frames were found to be homologous to genes encoding the essential enzymes dethiobiotin synthetase, pro lipoprotein diacylglycerol transferase and the putative NADH-ubiquinone oxidoreductase subunit. The five open reading frames encode for genes, which are essential for the normal functioning of the C. ruminantium organism. However, these open reading frames might not be effective for use in a DNA vaccine since none of the open reading frames showed homology to obvious genes that could play a role in immunity and therefore confer protection. The open reading frames can be used in mutagenesis studies to produce attenuated strains of the organism that possess mutated versions of these proteins. These attentuated strains could be used for the vaccination of cattle, and thereby confer protection against viable pathogenic C. ruminantium isolates. / AFRIKAANSE OPSOMMING: Hartwater is 'n bedreiging vir vee in Afrika weens die hoë mortaliteitssyfer verbonde aan die siekte. Die intrasellulêre aard van die organisme wat hartwater veroorsaak, Cowdria ruminantium, bemoeilik navorsing aangaande die organisme. Dit het tot gevolg dat 'n effektiewe en gebruikersvriendelike entstof moeilik bekombaar is. Daar is onlangs sukses behaal met die konstruksie van twee C. ruminantium DNA genoteke, die lambda GEM11 bakteriofaag genoteek en die lambda ZAPII bakteriofaag genoteek. Dit het gelei tot 'n herlewing in die soektog na beskermende gene, wat in 'n entstof teen hartwater gebruik kan word. In hierdie studie is verskeie molekulêre tegnieke insluitende PKR, klonering en geenopeenvolging bepaling, gebruik om gene te identifiseer in die lambda GEM11 bakteriofaag genoteek wat kodeer vir proteïene wat in entstowwe gebruik kan word as beskerming teen hartwater. Die secD geen is gebruik om die lambda GEM11 bakteriofaag genoteek te sif. Een positiewe plaak is gevind waarna die DNA uit die bakteriofaag plaak geïsoleer en die C. ruminantium DNA vanuit die bakteriofaag plaak geamplifiseer is deur gebruik te maak van PKR en spesifieke C. ruminantium inleiers. Die C. ruminantium DNA is gesif met Mycoplasma, bees en Cowdria radioaktief gemerkte DNA peilers. Die C. ruminantium DNA is vervolgens in twee vektore gekloneer. Die klone is gesif deur middel van restriksie analise. Die DNA volgorde van die klone is bepaal en twee ononderbroke sekwense is geïdentifiseer met 'n gaping in die middel tussen die twee sekwense. Oligonukleotied inleiers is daarna ontwerp om die geenopeenvolging van die gaping tussen die twee sekwense te vul. Hierdeur kon die volledige geenopeenvolging van die genoom van C. ruminantium wat in die lambda GEM 11 bakteriofaag plaak voorkom, bepaal word. Hierdie volledige geenopeenvolging is vervolgens geanaliseer en die oop leesrame wat daarin voorkom geïdentifiseer. Vyf leesrame is gevind om homologie met gene wat kodeer vir proteïene wat in bakterieë voorkom, te toon. Twee leesrame het homologie met die gene wat kodeer vir transport proteïene, FtsYen die ABC transporter getoon, en drie leesrame het homologie met gene wat kodeer vir die essensiële ensieme detiobiotin sintetase, prolipoproteïen diasielgliserol transferase en die NADHubikinoon oksidoreduktase subeenheid getoon. Dié vyf leesrame het die potensiaal om as entstowwe gebruik te word aangesien al vyf leesrame kodeer vir gene wat 'n belangrike rol speel in die oorlewing van die C. ruminantium organisme. Alhoewel die leesrame moontlik nie so effektief sal wees in 'n DNA entstof nie, toon dit potensiaal om in mutasieeksperimente gebruik te word. Organismes wat die gemuteerde weergawe van die geen besit sal nie-funksionele proteïene produseer, wat 'n invloed kan hê op die normale fisiologiese funksies van die organisme en dus sal lei tot 'n minder virulente organisme. Die geattenueerde organisme kan moontlik gebruik word om diere te immuniseer en daardeur immuniteit aan diere lewer wat beskerming sal bied teen patogeniese C. ruminantium isolate.

The rickettsial pathogen Ehrlichia ruminantium, transmitted by ticks of the genus Amblyomma, causes heartwater, an economically important, often fatal disease of domestic and wild ruminants in sub-Saharan Africa and in the Caribbean. The studies described in this thesis have contributed to understanding several aspects of heartwater. First, a real-time PCR method was developed in order to study the kinetics of infection with E. ruminantium in the mammalian host. The assay was validated for specificity and sensitivity and was used to estimate numbers of the organisms in the blood of infected sheep. However, organisms were only detected during the clinical phase of infection, indicating that the way in which it was applied did not provide sufficient sensitivity to follow the early stages of infection. This PCR assay was then used, together with transcription and proteomic analyses, to investigate differential gene expression of E. ruminantium in the arthropod and mammalian hosts, in order to identify genes that may allow the organisms to successfully adapt to different environments. These studies used in vitro tick and mammalian cell culture systems, as well as tissues from infected A. variegatum ticks, and initially focused on the map1 multigene family. Although transcripts for most of the map1 paralogs were detected in organisms grown in vitro, in both mammalian and tick cells, only transcripts from map1 and map1-1 were detected in infected ticks. Moreover, map1-1 transcripts were more abundant in midguts than in salivary glands whereas map1 transcripts were most abundant in salivary glands and were expressed at higher levels following several days of tick feeding on a mammalian host. Because of the quantities of material required, proteomic analysis was only possible using in vitro-cultured organisms. Comparison of proteins encoded by the map1 cluster in E. ruminantium grown in tick or bovine endothelial cell cultures, using 2D gels and MALDI-TOF analysis, revealed that different proteins predominated in the corresponding spots in 2D gels from the different cultures; products of the map1-1 gene were abundant in tick cells, while products of map1 were abundant in endothelial cells. The detection of higher levels of map1 transcripts in salivary glands than in midguts of infected ticks, together with the presence of abundant MAP1 protein in organisms grown in mammalian but not in tick cell lines, suggest that expression of this protein may be associated with infectivity for mammalian cells. In contrast, map1-1 transcripts were abundant both in midguts of infected ticks and in tick cell lines, and the protein was expressed at high levels in infected tick cell cultures. Since both of these stages have low infectivity for sheep, these results suggest that the MAP1-1 protein may play an important role within the vector, possibly associated with colonisation and replication of E. ruminantium in the tick midgut. Collectively these findings suggest that this multigene family is involved in functions of biological relevance in different stages of the life cycle of E. ruminantium. Lastly the suppression subtractive hybridisation (SSH) technique was applied to RNA extracted from E. ruminantium-infected endothelial and tick cell cultures in an attempt to sample a large portion of the E. ruminantium genome for differentially expressed genes; although not resulting in identification of any differentially transcribed genes in the present study, this method was shown to work in principle.

This degree has been obtained at the Faculty of Veterinary Science, Medical University of South Africa, now part of the new Faculty of Veterinary Medicine of the University of Pretoria Saanen and South African Indigenous goats were bred to kid at twelve months and annually thereafter. Milk production was recorded. Conception rates were generally more than 90 %, except for Indigenous goats in their first year. Few Indigenous goats (12 %) had twins at the first parturition, whereas 45% of Saanens had twins at 12 months of age. Twinning increased with age, and Saanen and Indigenous goats had kidding rates of 182% and 174% respectively in their third year, with Saanens later exceeding 200%. Triplets were infrequent, except in mature Saanens (9% of parturitions), and in Crossbreds (16%). Mean lactation yields were 579, 838, and 758kg for Saanens in first, second and third lactations, respectively. Lactation lengths were 283, 293 and 290 days respectively (excluding milk production beyond 300 days). Mean lactation yields for Crossbreds were 317, 446 and 438kg for first, second and third lactations. Lactation lengths were slightly shorter for Crossbreds than for the Saanens at 236, 248 and 257 days respectively. Indigenous goats were recorded at a mean milk yield of 23kg per lactation, and a mean lactation length of 94 days. Milk composition analyses for Saanens averaged 3.43, 2.88, and 4.49% for milk fat, protein and lactose, respectively. The analyses for Crossbred goats were 5.47, 3.88 and 4.81%, and for Indigenous goats were 9.33, 5.04 and 5.12%, respectively. These results showed that Crossbred goats gave less milk than Saanens, but significantly more than Indigenous goats. Milk production of Crossbred goats was found to be adequate for household requirements (subsistence purposes). In this way, the Crossbred goats were shown to be able to fulfil one of the objectives of the crossbreeding programme. The main disease identified was coccidiosis, acccompanied by pneumonia, which caused unacceptably high mortality among goat kids: 31% of Saanen, 24% of Crossbred, 38% of Three-quarter Saanen and 28% of Indigenous female kids. It is believed that this problem is largely management related, and worsened by overcrowding and the consequent poor hygiene; but the presence of rotavirus might also be significant. These aspects warrant further investigation. The main disease problem identified in mature goats was mastitis, which caused deaths of goats from peracute cases. Another important problem which became apparent after four years of age, was the incidence of squamous cell carcinoma on the udders of Saanens. Reduced exposure to the sun, by the provision of adequate shade should alleviate this problem; but the crossbreeding programme was seen to be of benefit, since no cases occurred in Crossbred goats. The experiment on heartwater aimed to assess resistance to this disease. Saanen, Indigenous and Crossbred goats were reared in a tick-free environment. In Year 1, eight goats of each type at eight months of age were given 5ml virulent heartwater blood of the Ball 3 stock. Temperatures and clinical sign were monitored. All eight Saanens were overcome by the disease, but only one Indigenous goat and two Crossbreds. In Year 2, Phase 1 of the experiment included six males and six females each of Indigenous and Crossbred goats at 11 months of age. Seven Crossbreds, but no Indigenous goats died. In Phase 2, nine Saanens were treated with tetracycline and compared to two untreated Saanens and nine untreated Three-quarter Saanen goats at 12 months of age. Both of the untreated and one of the treated Saanens died, and seven of the Three-quarter Saanens died. There were only small differences in temperature reactions; but Indigenous goats showed less clinical signs than other breeds. No differences of gender or year were apparent. These experiments indicated that Saanen goats show no genetic resistance, but that South African Indigenous goats appear to be genetically resistant to heartwater, and can transmit this resistance to a good proportion of Crossbred progeny. It has been shown therefore that it is feasible to develop a dairy goat resistant to heartwater, which could contribute significantly to the reduction of human malnutrition in rural and peri-urban communities in Southern Africa. / Dissertation (PhD)--University of Pretoria, 1997. / Production Animal Studies / 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

The general antibacterial activity of Drimia delagoansis, Petalidium oblongifolium and Ipomoea adenioides was determined using selected Gram-positive and Gram-negative bacteria. Only extracts or compounds with high antibacterial activity were then tested against the causative agent of heartwater, Ehlrichia ruminantium, since the latter requires specialised culturing conditions. The crude aqueous extract of D. delagoansis had low antibacterial activity with its highest MIC against Gram-negative bacteria being 20.0 mg ml-1 while the crude methanolic extracts of P. oblongifolium and I. adenioides had their highest antibacterial activity against Gram-negative bacteria at MIC's of 5.0 and 10.0 mg ml-1 respectively. Two compounds were isolated and identified from I. adenioides and an unidentified one was isolated from P. oblongifolium. The two compounds from I. adenioides proved to be caffeic acid with MIC's of 0.8 and 1.0 mg ml-1 against Gram-positive and Gram-negative bacteria respectively; and ethyl caffeate with MIC's of 0.4 and 1.0 mg ml-1 against Gram-positive and Gram-negative bacteria respectively. Synergism between the two compounds increased the respective MIC's to 0.4 and 0.2 µg ml-1 against Gram-positive and Gram-negative bacteria. The unidentified compound isolated from P. oblongifolium had a very low MIC of 2.5 µg ml-1 against E. ruminantium. / Thesis (PhD (Botany))--University of Pretoria, 2007. / Plant Science / 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