Corridor disease (Theileria parva infection in cattle associated with carrier buffaloes) was not reported to cause serious outbreaks prior to 1994. From 2002-2004, outbreaks in cattle have increased in the areas where the disease is endemic in buffalo populations. In this study, the occurrence of Corridor disease outbreaks in the Zululand district municipality was closely monitored from 2004-2009. The observations included the number of cattle involved in the outbreaks, clinical signs, parasitological and post-mortem examinations while blood for serum and in EDTA were collected for serological (IFA test) and molecular (real-time PCR) tests specific for T. parva. Samples were collected from cattle involved in the outbreak, the sick and presumed recovered cattle. Recovered cattle from the farms were brought to the laboratory at the Onderstepoort Veterinary Institute for further investigations. This included tick pick-up and transmission attempts to demonstrate their carrier status as well as assessing their immunity to further experimental challenge using virulent T. parva stabilate. Results were obtained on Corridor disease outbreaks in the study area and ad hoc locations comprising a total of 15 commercial farms and community diptanks in the district from 2004 to 2009. A total of 31 outbreaks were recorded during the study period. The number of outbreaks per year was stable, being 3 or 4 from 2004 to 2007. A 100 percent increase was recorded in the subsequent years, 2008-2009. In one location, Morgenzon farm comprising a commercial and community farmers, had experienced regular outbreaks from 2004-2009. It is also noted that some farms experienced outbreaks for three consecutive years. Three other farms had experienced outbreaks for the first time in either 2008 or 2009. The most severe outbreak occurred in Nyalisa in 2009 where the disease was experienced for the first time in one herd in which 202 cattle were involved and 57 died within 30-40 days after the onset of the disease. Using all the tools mentioned above, the cause of death was confirmed to be due to T. parva infection. The Corridor disease outbreaks that were investigated, have mostly been reported during the months from March-May (88 %) but some (8 %) were encountered during the winter months (June-August). The distribution of outbreaks mainly coincided with the activity period of adult R. appendiculatus. During the investigation period, a total of 846 cattle were tested for Corridor disease and the prevalence was found to be 27 %. The percentage of cattle which were found positive by PCR was 16.5. Seven percent were found positive on both PCR and IFA tests, an indication of the development of a carrier state. However, 10 % of the cattle remained sero-positive with no indication of being parasite-carriers (real-time PCR negative). Five cattle which recovered from an apparent severe T. parva infection in the field and confirmed to be positive by PCR, all became negative before they were used in the transmission experiments. Ticks derived from these cattle were used to infect susceptible bovines but only T. taurotragi was transmitted. The xeno-diagnosis failed to demonstrate the carrier state in these field cattle. The five Corridor disease recovered cattle obtained from different study locations mentioned above, received lethal challenge using T. parva buffalo-derived stabilate. All challenged animals, including the susceptible control, showed schizont parasitosis as detected by the T. parva</i. real-time PCR test starting day 11 to 23. All animals also developed significant antibody titer to T. parva by day 28. Of the field cattle, only one bovine which showed mild reactions manifested by high temperature on day 11 for two consecutive days and schizonts parasitosis in lymph nodes on day 15 for only two days and recovered. The rest of the field cattle did not show any clinical or parasitological reactions during the observation period (103 days). The control bovine had high fever and showed schizonts parasitosis by day 11 for seven consecutive days. The reaction was classified as severe and had to be treated. Unfed R. appendiculatus collected off grass from one of the study sites were applied to feed on a susceptible bovine and only T. taurotragi was transmitted. There were no apparent clinical signs and the animal behavior kept normal during the observation period (60 days). This study suggests that Corridor disease should be considered as an “emerging disease” and more stringent control methods should be implemented. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Between closely related species there are genomic regions with a higher level of differentiation compared to the rest of the genome. For a time it was believed that these regions harbored loci important for speciation but it has now been shown that these patterns can arise from other mechanisms, like recombination. The aim of this project was to estimate the recombination landscape for red-breasted flycatcher (Ficedula parva) and taiga flycatcher (F. albicilla) using patterns of linkage disequilibrium. For the analysis, 15 red-breasted and 65 taiga individuals were used. Scaffolds on autosomes were phased using fastPHASE and the population recombination rate was estimated using LDhelmet. To investigate the accuracy of the phasing, two re-phasings were done for one scaffold. The correlation between the rephases were weak on the fine-scale, and strong between means in 200 kb windows. 2,176 recombination hotspots were detected in red-breasted flycatcher and 2,187 in taiga flycatcher. Of those 175 hotspots were shared, more than what was expected by chance if the species were completely independent (31 hotspots). Both species showed a small increase in the rate at hotspots unique to the other species. The low number of shared hotspots might indicate that the recombination landscape is less conserved between red-breasted and taiga flycatchers than found between collared and pied flycatcher. However, the investigation of the phasing step indicate that the fine-scale estimation, on which hotspots are found, might not be reliable. For future analysis, it is important to use high-quality data and carefully chose methods.

The aim of this study was to investigate three key epidemiological aspects of Theileria parva infections in cattle and African buffalo (Syncerus caffer) in South Africa. The first of these was the possible behavioural change (i.e. transformation) of buffalo-derived T. parva (causing classical Corridor disease in cattle) to what might be considered cattle-derived T. parva (causing classical East Coast fever in cattle) after repeated tick-passage in cattle. For the first time a South African isolate of buffalo-derived T. parva was successfully transmitted using Rhipicephalus zambeziensis for eight passages in non-splenectomised cattle. This was achieved despite most animals developing fatal infections with extremely low piroplasm parasitaemias, and without chemotherapeutic intervention. This finding indicates that, contrary to earlier belief, Corridor disease is not a self-limiting disease in cattle, and given the opportunity, could well become established in a cattle population in the absence of buffalo. Despite repeated tick transmission in cattle of the South African buffalo isolate of T. parva used in this study, it did not exhibit the behavioural changes associated with “transformation” to typical cattle-derived T. parva. Secondly, the potential role of the common waterbuck (Kobus ellipsiprymnus) in the selection of cattle-adapted subpopulations of parasites from buffalo-derived T. parva was investigated. Waterbuck captured in Kruger National Park (KNP) were screened by conventional and molecular diagnostic techniques for Theileria spp. infections. Laboratory-reared R. zambeziensis were fed on captive buffalo confirmed to be naturally infected with T. parva. The ensuing adult ticks were fed on captive waterbuck and cattle. All the waterbuck were found to carry microscopically detectable Theileria sp. piroplasm infections, found by polymerase chain reaction (PCR) diagnosis to belong to a hitherto uncharacterised Theileria species. R. zambeziensis adults which fed as nymphs on the buffalo transmitted fatal T. parva infections to cattle. However, no transmission of T. parva to the waterbuck could be demonstrated clinically or by PCR diagnosis. Also, R. zambeziensis nymphs that were subsequently fed on the waterbuck failed to transmit T. parva to cattle in the ensuing adult stage, confirming the absence of T. parva-group infections in the waterbuck. The results suggest that buffalo in KNP probably do not carry T. parva-group parasites which are readily transmissible to common waterbuck and waterbuck are therefore unlikely to play an important role in the epidemiology of T. parva-group infections in cattle in South Africa. Thirdly, to investigate the carrier state of buffalo-derived T. parva infections in cattle, blood from infected non-splenectomised and splenectomised carrier cattle was subinoculated to splenectomised cattle. T. parva infections were successfully transmitted by subinoculation of 1000 ml of blood at various intervals after infection to splenectomised recipient cattle. Donor animals comprised of recovered intact cattle, reacting intact cattle or splenectomised recovered cattle. Microscopically detectable piroplasm parasitaemias were detected in all recipients after inoculation. One splenectomised recipient developed a moderate clinical reaction, accompanied by a moderate schizont parasitosis, but recovered spontaneously, confirming persistence of schizonts in some T. parva carrier animals. By contrast, a T. parva piroplasm infection, persisting in a treated recovered splenectomised bovine, in the apparent absence of circulating schizonts, was serially (consecutively) passaged in splenectomised cattle. Seroconversion occurred in all recipient cattle. With the exception of the recipient which developed a clinical reaction and circulating schizonts, none of the recipients showed any clinical signs of T. parva infection. Upon homologous sporozoite challenge with T. parva, two out of three recipient animals with only microscopically detectable piroplasm parasitaemias developed fatal T. parva infections and one recovered after exhibiting severe clinical signs. These findings confirm the stage-specific immunity in T. parva and, contrary to popular belief, the possibility of long-term maintenance of piroplasm parasitaemias in the absence of schizonts in carrier cattle. The technique of subinoculating and establishing virulent T. parva carrier infections in splenectomised cattle also provides a method whereby buffalo-derived parasite stocks may be isolated and maintained for characterisation and the preparation of sporozoite stabilates for inclusion in T. parva vaccines. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Veterinary Tropical Diseases / unrestricted

Theileria parva is an intracellular protozoan parasite and the causative agent of the lethal livestock disease East Coast fever (ECF). Research has shown that a protective cell-mediated immune response against parasite-infected lymphocytes is capable of clearing the host of T. parva (Pearson et al. 1979), leaving the host solidly immune to reinfection. The work presented in this thesis describes my attempts to develop a method for identification of major histocompatibility complex class I-associated T. parva peptides involved in eliciting this protective cell-mediated immune response. The soluble chaperone gp96 interacts with peptides destined for association with major histocompatibily complex class I molecules and is therefore a source of T. parva peptides that interact with extracellular immune effectors. Using sensitive mass spectrometry methods the gp96-chaperoned peptide proteome from model parasite infected T lymphocytes was compared to an uninfected T cell line. With our findings we have demonstrated proof of concept for a highly sensitive method for the elucidation of potentially immunogenic peptides capable of initiating a protective immune response against the intracellular parasite T parva.

Modeling is a powerful tool that can be used to identify important relationships between organisms and their habitat (Guisan & Zimmermann, 2000). Understanding the dynamics of how the two relate to one another is important for conserving and managing ecosystems, but the extreme complexity of those ecosystems makes it very difficult to fully diagram. Unlike many other modeling techniques, Multivariate Regression Trees (MRTs) are not limited by a prior assumptions, pre-determined relationships, transformations, or correlations. MRTs have the power to provide both explanation and prediction of ecological data by producing simple models that are easy to interpret. This study proposed to use MRTs to evaluate and model relationships between Lucania parva and the environment and habitat of Florida Bay. Counts were transformed to presence-absence and abundance groupings. Models were first run using a variety of combination of response variables and all explanatory variables. Results of these models were used to select the best combination of response and explanatory variables in an effort to create a best fit model. Models indicated that Lucania parva populations are found in the dense (cover ≥50%), shallow water (<1.8 m) grass beds that occur in the western portion of Florida Bay. A best fit model was able to explain 63.7% of the variance with predictive error of 0.43.

Attività antropiche e pressioni di mercato stanno rapidamente riducendo la biodiversità. Per questa ragione, conservare il patrimonio ecosistemico, tassonomico e genetico risulta fondamentale al fine di garantire potenziale adattativo alle specie, e, in ultima analisi, un futuro sostenibile per il pianeta. Al fine di minimizzare la perdita di biodiversità, numerosi metodi sono stati proposti per priorizzare ecosistemi, specie e popolazioni. Il presente lavoro di tesi fornisce in primo luogo una revisione di tali approcci, proponendo un albero decisionale volto a favorirne un corretto utilizzo. Secondariamente, la variabilità genomica neutrale del bufalo d’acqua (Bubalus bubalis L.) è investigata per mezzo di un pannello di marcatori SNP a media densità, rivelando due centri di domesticazione (India Nord-occidentale, Cina-Indocina) e possibili rotte di migrazione per gli ecotipi ‘river’ e ‘swamp’. L’adattamento locale ad East Coast Fever, patologia endemica delle popolazioni bovine in Africa Sub-sahariana, è stato inoltre studiato in bovini autoctoni Ugandesi (Bos taurus L.) combinando tecniche di modellizzazione delle nicchie ecologiche e di genomica del paesaggio. L’approccio ha portato ad indentificare PRKG1 e SLA2 come possibili geni di adattamento. I risultati sono discussi alla luce delle possibili implicazioni nella conservazione del bufalo e nella gestione delle risorse genetiche animali Ugandesi. / Biodiversity is quickly disappearing due to human impact on the biosphere, and to market pressure. Consequently, the protection of both wild and domestic species needs to become a priority in order to preserve their evolutionary potential and, ultimately, guarantee a sustainable future for coming human generations. To date, tens of methods have been proposed to prioritize biodiversity for conservation purposes. Here, an ontology for priority setting in conservation biology is provided with the aim of supporting the selection of the most opportune methodologies given specific conservation goals. Further, two case studies are presented characterizing neutral and adaptive genomic diversity in water buffalo (Bubalus bubalis L.) and indigenous Ugandan cattle (Bos taurus L.), respectively. In particular, two independent domestication centres (North-western India and Indochina) and separate migration routes are suggested for the ‘river’ and ‘swamp’ water buffalo types. In the case of indigenous Ugandan cattle, the integration of species distribution modelling and landscape genomics techniques allowed the identification of PRKG1 and SLA2 as candidate genes for local adaptation to East Coast Fever, a vector-borne disease affecting bovine populations of Sub-Saharan Africa. Results are discussed for their implications in water buffalo conservation and Ugandan cattle adaptive management.

Text in English / Corridor disease is an economically important tick-borne disease of cattle in southern Africa. The disease is caused by Theileria parva and transmitted by the vectors, Rhipicephalus appendiculatus and R. zambeziensis. There is currently no vaccine to protect cattle against T. parva that is permitted in South Africa. To develop recombinant anti-tick vaccines against Corridor disease, comprehensive databases of genes expressed in the tick’s salivary glands are required. Therefore, in Chapters 2 and 3, mRNA from the salivary glands of R. appendiculatus and R. zambeziensis was sequenced and assembled using next generation sequencing technologies. Respectively, 12 761 and 13 584 non-redundant protein sequences were predicted from the sialotranscriptomes of R. appendiculatus and R. zambeziensis and uploaded to public sequence domains. This greatly expanded the number of sequences available for the two vectors, which will be invaluable resources for the selection of vaccine candidates in future. Further, in Chapter 3, differential gene expression analysis in R. zambeziensis revealed dynamic expression of secretory protein transcripts during feeding, suggestive of stringent transcriptional regulation of these proteins. Knowledge of these intricate expression profiles will further assist vaccine development in future. In Chapter 4, comparative sialotranscriptomic analyses were performed between R. appendiculatus and R. zambeziensis. The ticks have previously shown varying vector competence for T. parva and this chapter presents the search for correlates of this variance. Phylogenetic analyses were performed using these and other publically available tick transcriptomes, which indicated that R. appendiculatus and R. zambeziensis are closely related but distinct species. However, significant expression differences were observed between the two ticks, specifically of genes involved in tick immunity or pathogen transmission, signifying potential bioinformatic signatures of vector competence. Furthermore, nearly four thousand putative long non-coding RNAs (lncRNAs) were predicted in each of the two ticks. A large number of these showed differential expression and suggested a potential transcriptional regulatory function of lncRNA in tick blood feeding. LncRNAs are completely unexplored in ticks. Finally, in Chapter 5, concluding remarks are given on the potential impact the R. appendiculatus and R. zambeziensis sialotranscriptomes may have on future vaccine developments and some future research endeavours are discussed. / Life and Consumer Sciences / Ph. D. (Life Sciences)

The mango (Mangifera indica L.) is an appealing subtropical fresh fruit with a pleasant flavor and taste, high nutritional value, beneficial medicinal properties and various processing options. However, as is the case with most subtropical fruit, it is a sensitive commodity, prone to losses postharvestly. The South African mango industry is highly dependent on a hot water and cold prochloraz dip treatment, to control postharvest anthracnose and soft brown rot on fruit destined for export. However, negatve public perceptions of synthetic fungicides and its use on fresh produce for disease control has been increasing in major export markets such as the European Union. This growing concern from a public point of view is forcing industry to consider more environmentally acceptable methods to maintain quality of mangoes during extended export periods. ‘Tommy Atkins’ and ‘Kent’ mangoes either uninoculated or artificially inoculated with Colletotrichum gloesporioiedes, Botryosphaeria parva or sterile agar, were used to evaluate softer, greener alternatives, in this study. Fruit were subjected to either a hot, Bacillus amyloliquefaciens (PPCB004) containing dip treatment for two minutes or a 24 hour 1-methylcyclopropene (1-MCP) gas treatment at 16 ºC or no treatment. Fruit were then stored at 10 ºC under either 5% O2 and 5% CO2 (CA-1) or 3% O2 and 8% CO2 (CA-2) controlled atmospheres (CA) for 18 days and allowed to ripen for five days at 25 ºC. Similarly, uninoculated or artificially inoculated fruit subjected to B. amyloliquefaciens, 1-MCP or a combination of the two treatments was stored at 10 ºC for 18 days under conventional storage. ‘Tommy Atkins’ fruit were packed into bags made from four different film types, untreated or after being subjected to a cold B. amyloliquefaciens dip treatment and stored for 23 days at 10 ºC. Overall, ‘Kent’ fruit were more susceptible to anthracnose and SBR after artificial inoculation. In vivo inoculated ‘Tommy Atkins’ fruit, storage under CA-1 gave the best control of soft brown rot whilst CA-2 storage gave the best control of anthracnose. For quality retention no definite conclusion could be made for both cultivars after CA storage or the combination of 1-MCP pre-treatment and CA storage. The combination of 1-MCP pre-treatment and B. amyloliquefaciens maintained the quality of ‘Kent’ mangoes under conventional storage the best. Anthracnose severity on both cultivars was reduced with 1-MCP treated fruit combined with the biocontrol pre-treatment. Modified atmosphere packaging in this study was found to be ineffective in maintaining quality of mangoes. Copyright / Dissertation (MSc(Agric))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted