Theileria parva is a tick-borne intracellular protozoan parasite which infects cattle and African buffalo in Eastern and Southern Africa. Cattle may be immunised against T. parva by the infection and treatment method (ITM), which involves inoculation with live sporozoites and simultaneous treatment with oxytetracycline. One such ITM vaccine is the Muguga Cocktail, which is composed of a mixture of three parasite stocks: Muguga, Serengeti-transformed and Kiambu 5. Although the vaccine has been used with success in the field in several areas in Eastern Africa, there is evidence that vaccination using cattle-derived parasites does not always provide adequate protection against buffalo-derived T. parva. A number of T. parva antigens recognised by CD8+ T cells from cattle immunised by ITM have been identified in previous studies. A proportion of these antigens show a high degree of sequence polymorphism and allelic diversity is believed to be much greater in buffalo-derived T. parva than in cattle-derived parasites. The present study focussed on the development and application of a deep sequencing technique for characterising genotypically heterogeneous T. parva DNA samples. A panel of genes encoding CD8+ T cell antigens was used as the basis of a multi-locus sequence typing system (MLST) built upon Roche 454 amplicon sequencing technology. This system was validated using parasite stocks of known composition and then utilised to investigate genetic and antigenic diversity in vaccine stabilates and samples derived from African buffalo. The MLST profile obtained for the Muguga Cocktail stocks was compared to those of African buffalo in two geographically separated sites and was also compared with micro/mini-satellite DNA profiles of Muguga Cocktail stocks. The three components of the T. parva Muguga Cocktail vaccine were found to have limited genotypic and antigenic diversity using both methods. The composition of vaccine batches produced in a single production run (ILRI0801-ILRI0804) was shown to be relatively consistent. In contrast, the composition of the component stocks was shown to alter following passage through cattle and ticks. The deep multi-locus sequence profile and satellite DNA profile established in this study may be used as a reference for comparison with future vaccine batches. It is suggested that formulation of a new cocktail vaccine containing three parasite clones selected on the basis of genotypic and antigenic divergence may well provide protection comparable to that obtained with the Muguga Cocktail. The components of such a vaccine could readily be distinguished and the composition of vaccine batches monitored, thus allowing improved quality control and greater consistency of the vaccine. Genetic and antigenic diversity was found to be very high in parasite populations from African buffalo from the Kruger National Park, South Africa and the Ol Pejeta conservancy, Kenya. The estimated average genetic ‘distance’ between any two alleles in the Kruger National Park and within the Ol Pejeta conservancy was very similar for all six genes investigated. Many of the identified alleles were ‘private’ to either the buffalo from Ol Pejeta or the Kruger National Park and many of these alleles were present in several individuals in one location. Principal co-ordinate analysis and phylogenetic investigation of several antigen-encoding loci indicated that extant buffalo parasite populations are geographically sub-structured although some of the underlying diversity may reflect ‘ancient’ polymorphism in an ancestral population. A subset of the CD8+ T cell antigens examined exhibited extensive antigenic polymorphism while others were highly conserved at the amino acid level. These conserved genes may represent good candidates for the development of next generation vaccines, as strain specificity may be overcome if protective CD8+ T cell responses could be generated against these conserved antigens. This would enable the use of sub-unit vaccines in areas where cattle co-graze with buffalo. Theileria sp (buffalo) was identified in cell lines isolated from cattle, indicating that this parasite can transform bovine lymphocytes and may therefore be implicated in pathology in cattle. Phylogenetic analysis of T. parva and T. sp (buffalo) clones using the 5S subunit ribosomal RNA gene, Tp6, Tp7 and Tp8 showed a clear distinction between the two parasite species. These genes could thus be considered as candidates for an improved diagnostic test for T. parva in South Africa.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy. / The aim of this thesis was to develop DNA probes to distinguish between the protozoan parasites Theileria parva parva and T. p. lawrencei which cause East Coast fever (ECF) and Corridor disease respectively. ECF was eradicated from South Arrlca in 1954, and today Corridor disease has become the most important form of theileriosis. Although ECF has been eradicated, the vector ticks are still prevalent in South Africa and the cattle population would be highly susceptible to a recurrence of the disease, At present there is no reliable means of distinguishing between T.p. parva and T. p. lawrencei. Sequence differences between T. parva and other Theileria species have previously been found in the small subunit ribosomal RNA (rRNA) gene; probes designed to detect these sequence differences Can be used to distinguish between Theileria species. We therefore decided to search for differences in the rRNA genes of T. p. parva and T.p. lawrencei. To this end, the entire "RNA transcription unit was amplified from a cloned T. p, lawrence; parasite; the unit comprises the small subunit rRNA (SSUrRNA) gene, the internal transcribed spacer (ITS) and the large subunit rRNA (LSUrRNA) gene. The amplification products were cloned and sequenced, and the T.p, lawrencei rRNA sequence was compared to that of T. p, parva, While there was little variation in their SSUrRNA and LSUrRNA gene sequences, there was major sequence variation in the ITS The ITSs from twelve T. parva isolates were amplified, cloned and sequenced, and eleven characterisation oligonucleotide probes were identified. The T. p, parva isolates screened in this study hybridised with a limited subset of the probes, While the T. p. lawrencei isolates, hybridised with many more of the probes, indicating that the T. parva population in cattle is more homogenous than that in buffalo. There thus appears to have been a selection in cattle of a relatively homogenous subpopuiation of T. parva from a much larger, more diverse gene pool in buffalo. Although most T.p. parva isolates (93.5%) were detected by probe TPPI, and most T.p, lawrencei isolates (81.8%) were detected by / AC2017

Non-native species invasions threaten the structure, function and biodiversity of ecosystems worldwide, and those of non-native fishes pose amongst the greatest threats to inland waters of the U.K. This PhD investigated the establishment, dispersal and ecological implications of introducing the two non-native fish species, sunbleak Leucaspius delineatus (Heckel, 1843) and topmouth gudgeon Pseudorasbora parva (Temminck and Schlegel, 1842) to inland waters of England. The introduction and initial dispersal of both species can be attributed to the commercial fish trade. Species-specific variability of life history, growth and morphological traits was examined in sunbleak (12 sites) and topmouth gudgeon (3 sites) to assess their role in establishment success. The drift dynamics, i.e. timing and intensity (propagule pressure), of sunbleak and topmouth gudgeon were assessed for source populations to determine dispersal potential. Potential risks for native species posed by these two alien cyprinids were assessed with respect to the parasite fauna and overlaps in resource use. For sunbleak, these were also examined in terms of social integration of this species into a native fish assemblage. Biological resistance to topmouth gudgeon invasion was evaluated by stomach flushing and gut content analysis of native piscivorous fishes. Inter-population variability in life histories and morphological characters were observed in both sunbleak and topmouth gudgeon. Populations of both species matured at small body sizes and between the ages 1 and 2. The fish were of good body condition and exhibited high reproductive investment. In both species, dispersal from source waters followed a diel pattern, with higher rates at night than during the day (e.g. maximum drift densities during May of 2004 and 2005: 9-10 sunbleak per 1000 m -3 at about 23:00 hrs; 40-52 topmouth gudgeon per 1000 m -3 at about 05:00 hrs). Downstream of one source population, microhabitat use of topmouth gudgeon was found to overlap with native species (brown trout Salmo trutta L., European chub Leuciscus cephalus (L.), bullhead Cottus gobio L., stoneloach Barbatula barbatula (L.); both brown trout and chub were observed to prey on topmouth gudgeon. However, predation intensity may be density-dependent and of insufficient level to impede topmouth gudgeon establishment, which was facilitated in the receiving stream by the consistent propagule pressure from on-line source populations. Sunbleak diet and microhabitat use also overlapped with native species (roach Rutilus rutilus (L.) and common bream Abramis brama (L.)) as young larvae, but this decreased with age. Social network analysis of sunbleak-native species interactions revealed that sunbleak creates significantly stronger social bonds with the native species than do natives amongst themselves. No macro-parasites were found in topmouth gudgeon, but two ‘Category II’ non-native parasites Neoergasilus japonicus (Harada, 1930) and Ergasilus briani (Markewitsch, 1932) were found in some populations of sunbleak. The potential for sunbleak to spread beyond their current distribution in England and the species’ social integration behaviour may facilitate the dispersal of these parasites, which may spread faster among communities invaded by sunbleak than in those where this non-native species is absent. The results of this PhD study are discussed within their wider context and their relevance to non-native species risk analysis and management.

M.Sc. / Refer to full text to view abstract

In South Africa, the diagnosis of Theileria parva in cattle and buffalo has been complicated by the presence of mildly pathogenic and non-pathogenic Theileria spp. This can lead to inaccurate diagnostic results and confuse the epidemiology of theileriosis. The aims of this study were to identify and characterize the 18S rRNA genes of novel Theileria spp. of the African buffalo, as well as to test new gene targets that will allow for the development of more accurate diagnostic tests for the identification of T. parvainfections in cattle and buffalo. Buffalo blood samples originating from different geographical regions in South Africa and from Mozambique were screened for the presence of Theileria spp. by the reverse line blot (RLB) hybridization assay. A total of six Theileria spp., namely T. parva, Theileria sp. (buffalo), Theileria mutans, Theileria velifera and Theileria buffeli, were identified from the buffalo samples. These occurred mainly as mixed infections. Some of the samples hybridized only with the Theileria/Babesia genus specific probe that is used in the RLB assay, and not with any of the species-specific probes used, suggesting the presence of novel genotypes or species. The full-length 18S rRNA genes of parasites from selected samples were characterized by cloning and sequencing. In addition to the identification of 18S rRNA gene sequences that were similar to published Theileria spp. of cattle and buffalo, we identified Theileria sp. (bougasvlei), and novel 18S rRNA gene variants of T. mutans, T. velifera, T. bufJeli. This variation explained why the RLB hybridization assay failed to detect these species in some of the analysed samples. As extensive variation was observed within the T. mutan group, specific RLB oligonucleotide probes were designed from the V 4 hypervariable region of the T. mutans-like 1 and 2/3 18S rRNA gene sequences. Unfortunately these cross-hybridized with T. mutans target DNA and could not be used to screen buffalo samples to determine the occurrence of these genotypes in buffalo in South Africa. This problem could be solved by designing probes from a more variable area of the 18S rRNA gene of the T. mutans groups. Alternatively, a quantitative real-time PCR (qPCR) assay could be used for differentiation of these genotypes as it is more sensitive than the RLB assay. Despite the variation observed in the full-length T parva 18S rRNA gene sequences, the area in the V 4 hypervariable region where the T parva RLB and real-time PCR hybridization probes were developed was relatively conserved between sequences obtained in this study. The existing T parva-specific qPCR assay was able to successfully detect all T parva variants identified in this study and, although amplicons were obtained from Theileria sp. (buffalo) and Theileria sp. (bougasvlei) DNA, these species were not detected by the T parva-specific hybridization probes. The sequences of the other Theileria spp. and the novel genotypes identified in this study under the probes were also different from that of T parva and therefore these species do not compromise the specificity of the T parva 18S qPCR assay. In order to determine the sequence variation and phylogenetic positions of T buffeli spp. of the African buffalo, we cloned and sequenced their 18S rRNA gene and complete internal transcribed spacer (ITS). We identified novel T buffeli-like and T sinensis-like 18S rRNA and ITS genotypes from buffalo originating from two different geographical regions in South Africa. There was extensive sequence variation between these novel South African genotypes and known T buffeli-like and T sinensis-like genotypes. The presence of organisms with T buffeli-like and T. sinensis-like genotypes in the African buffalo is of significant importance, particularly to the cattle industry in South Africa as these animals might act as sources of infections to naIve cattle. Recently, a qPCR assay based on the cox III gene was developed for the diagnosis of Theileria spp. in cattle. This test detects and differentiates six Theileria spp. in cattle. We evaluated the use of this assay for the detection of Theileria spp. in buffalo. The results of the cox III qPCR were compared to those of the RLB and 18S qPCR for the simultaneous detection and differentiation of Theileria spp. of the African buffalo, and for the specific detection of T parva, respectively. The cox III genes from selected samples with non-specific melting peaks were characterized by cloning and sequencing. Extensive sequence variation in the cox III gene was observed between and within species. The T mutans group was the most variable. The qPCR assay could be further improved by designing new primers and probes using all known cox III gene sequences of Theileria spp. Of buffalo and cattle. This study highlights the complexity of the diagnosis of T parva in cattle and buffalo in South Africa. It provides invaluable information towards the development of an improved molecular diagnostic assay for T parva and co-infecting species in cattle and buffalo in South Africa which will assist the veterinary regulatory authorities in the control of Corridor disease in South Africa. / Thesis (PhD)--University of Pretoria, 2011. / Veterinary Tropical Diseases / Unrestricted

The aim of this study was to improve the official diagnostic test package in South Africa for detection of Theileria parva infections in cattle and Cape buffalo (Syncerus caffer) and to investigate the presence of cattle-type T. parva parasites in buffalo and cattle in South Africa. To improve diagnosis of T. parva infections, a T. parva-specific real-time polymerase chain reaction (PCR) assay based on hybridization probe technology was developed. Oligonucleotide primers and hybridization probes used in the assay were designed based on the 18S ribosomal RNA (rRNA) gene. The primers amplify T. parva and Theileria sp. (buffalo) DNA but the hybridization probes specifically detect T. parva amplicons. Because of the high sequence similarity between the T. parva and Theileria sp. (buffalo) 18S rRNA genes, amplification of Theileria sp. (buffalo) DNA could not be avoided; no other bovine blood pathogens tested were amplified by these primers. The real-time PCR assay demonstrated superior sensitivity compared to other molecular tests used in detection of T. parva infections, reliably detecting the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79x10-4% with minute template DNA input. The assay requires less time to perform with a low risk of contamination because of the closed-tube system that does not require handling of amplicons for post-PCR analysis. The presence of cattle-typeT. parva parasites in buffalo and cattle was investigated using restriction fragment length polymorphism (RFLP) profiles of PCR products and sequences of the parasite genes which code for the antigenic proteins p67, p104, and the polymorphic immunodominant molecule (PIM). Cattle-type p67, p104 and PIM alleles were identified from three T. parva samples obtained from cattle from a farm near Ladysmith in the KwaZulu-Natal Province. These cattle-type alleles were identical to those previously identified from a cattle-derived T. parva stock, T. parva Muguga, a parasite stock that causes East Coast fever (ECF) in Kenya; however, ECF was not diagnosed in animals in this farm. Cattle-type alleles identical to those previously reported were not identified from T. parva buffalo samples, but variants of p67 allele 1 as well as p104 allele 1, both previously obtained from T. parva Muguga, were identified. It is not known if parasites that possess these variants can cause disease, and the risk of their adapting to cattle as in the case of ECF and January disease needs to be evaluated. Furthermore, these findings suggest that cattle-like alleles may not be exclusively associated with cattle-derived T. parva parasites. Most of the p67, p104 and PIM gene sequences obtained in this study were not identical to known sequences; furthermore, novel alleles were identified, demonstrating extensive genetic diversity in the South African T. parva parasite population in buffalo. The significance of the parasites that possess ‘novel’ alleles in the epidemiology of theileriosis in South Africa still needs to be determined. The identification of variants and novel alleles reveals that p67, p104 and PIM gene PCR-RFLP profiles are more complex than previously thought and the classification of buffalo- and cattle-derived T. parva parasites in South Africa based on p67, p104 and PIM gene profiles would not be possible. Identification of more reliable markers that can be directly associated with the theilerial disease syndromes remains a challenge. / Thesis (PhD)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

The work presented in this thesis is focused upon the community of endemic pathogens of African cattle in Sub-Saharan Africa, which has long constrained livestock production in these areas. The first aim of this work is to investigate whether the pathogen community as a whole shapes the ensuant epidemiology and morbidity which are currently attributed to any of its individual pathogens. The second aim is to determine if a greater understanding of the interactions present amongst genetically distinct parasites of the same species can be used to better explain epidemiological features that are at present poorly understood. Emphasis is placed on examining spatial variation in the epidemiology of Theileria parva, a tick-transmitted protozoan that causes East Coast Fever. To achieve these aims, this work examines field data collected from a large and comprehensive study conducted in south east Uganda. Through application of apposite statistical techniques and mathematical modelling, aspects of the complex relations amongst the pathogen community and their environment are explored. Evidence is presented that demonstrates the paramount role of the pathogen community as a whole in shaping the infection dynamics and pathogenicity of any of its individual components. By focusing on a single member of this pathogen community (Theileria parva), some of the influences of host, vector, geographical location, temporal dynamics and intra-species pathogen interactions are elucidated. Application of a polymorphic molecular marker to Theileria parva infected blood samples and the use of Cox proportional hazard analysis, show variability in the survival of infections in cattle in high and low tick challenge areas. Moreover infection survival, which plays a pivotal role in parasite transmission, is shown to be a function of the interactions established amongst genetically distinct co-infective parasites. In consequence, vector intensity alone is insufficient to develop reliable transmission models which can accurately predict the epidemiology of the parasite inside and outside enzootic belts. Finally, a theoretical model is developed which, based upon the field evidence obtained throughout this work, provides a possible explanation for the mechanics of T. parva survival in cattle. In summary, this thesis makes a case that consideration of both inter- and intra-species pathogen interactions, can greatly augment understanding of the epidemiology of these pathogen communities. An integrated approach to pathogen dynamics can better equip an integrated approach to control of important diseases of African cattle.

Previous research has provided evidence that CD8 T cells mediate immunity against infection with Theileria parva. However, the immunity induced by one parasite strain doesn‟t give complete protection against other strains and this is associated with parasite strain specificity of the CD8 T cell responses. There is evidence that such strain specificity is a consequence of the CD8 T cell responses of individual animals being focused on a limited number of immunodominant polymorphic peptide-MHC determinants. Dominant responses to the Tp2 antigen have been demonstrated in animals homozygous for the A10 MHC haplotype. Three Tp2 epitopes recognised by A10+ animals (Tp249-59, Tp250-59 and Tp298-106) have been defined. This project set out to investigate the dominance of these epitopes and to examine the T cell receptor (TCR) repertoires of the responding T cells. The specific objectives were to: (i) Determine the dominance hierarchies of the three defined Tp2 epitopes in both A10-homozygous and -heterozygous cattle. (ii) Examine the clonal repertoires of epitope-specific responses by analysis of TCR gene expression. (iii) Isolate full-length cDNAs encoding TCR α and β chain pairs from T cell clones of defined epitope specificity and use them to generate cells expressing the functional TCRs. Using MHC class I tetramers the relative dominance of CD8 T cell responses were found to differ between A10-homozygous and heterozygous cattle. All A10-homozygous cattle examined had detectable responses to all 3 Tp2 epitopes, the Tp249-59 epitope consistently being the most dominant. By contrast, only some A10-heterozygous cattle had detectable responses to Tp2 and when present the response was specific only for the Tp298-106 epitope. Analyses of the sequences of expressed TCR β chains showed that the responses in individual animals were clonotypically diverse, but often contained a few large expanded clonotypes. The TCRs of Tp298-106–specific T cells showed preferential usage of the Vβ13.5 gene and the frequent presence of a “LGG” motif within the CDR3 of the B chain. A conserved (public) TCRβ clonotype shared by the Tp250-59-specific CD8 T cells from all A10-homozygous cattle was identified. The TCRα chains co-expressed with this public TCRβ clonotype were identified for a number of T cell clones. Lentivirus transduction of Jurkat cells with three full-length TCR α and β chain pairs resulted in successful expression of one of the α/β chain pairs as a functional TCR, thus providing the basis for future work to generate bovine T cells expressing defined TCRs in vitro.

Small cyprinid fish, the topmouth gudgeon (Pseudorasbora parva Schlegel, 1842) is considered as an undesirable fish species because it represents an important food competitor for commercial non-predatory fishes. This study was performed under pond conditions (four ponds in South Moravia and two ponds in South Bohemia) during the growing seasons in 2006 {--} 2007. The purpose of my work was to determinate the level of food competition between topmouth gudgeon and common carp (Cyprinus carpio) as a dominating pond fish species. Food selectivity was evaluated using Ivlev´s electivity index (Jacobs, 1974). The level of food competition between common carp and topmouth gudgeon was used to evaluate the index of food similarity according to Shorygin (1952). The diet of topmouth gudgeon consisted mainly of chironomid larvae and zooplankton, mostly cladocerans (Daphnia, Bosmina), detritus and periphyton (Oscillatoria, Scenedesmus, Sphaerotilus). Macrophytes, copepods and Brachionus were also ingested by P.parva but in comparatively low proportions. In contrast, carp diet consisted mainly of bottom items including chironomid larvae, macrophytes and organic debris, mainly detritus and periphyton. The food items of lesser importance were dragon fly (Anisoptera) larvae, cladocerans (Bosmina, Daphnia) and ostracods. Topmouth gudgeon competed with common carp for chironomid larvae, periphyton and detritus, for zooplankton (mainly cladocerans Daphnia, Bosmina). The highest valuation the food competition was registered in fish from the Vracovický pond (South Moravia) and the Podsedek pond (South Bohemia), when it amounted from 24.02 to 34.78 % food similarity.

Thesis (MSc (Veterinary Science)--University of Pretoria, 2007. / Includes bibliographical references.