Our study was focused on the effect of tick-borne encephalitis virus infection on global gene expression in two human neural cell lines (neuroblastoma and glioblastoma). Changes of gene expression were determined using microarray approach. We identified several genes with up-or down-regulated expression in neural cells following the infection. The changes in expression of some of them were similar in both cell lines,other exhibited different pattern.

The cattle tick, Rhipicephalus (Boophilus) microplus is a tick of veterinary and health importance globally, transmitting Babesia bovis and B. bigemina. Tick control is important and needed to prevent livestock diseases caused by tick-transmitted pathogens. Traditionally, tick control methods have resulted in development of acaricide-resistant ticks, environmental pollution and meat and milk contamination. Therefore, there is a need for alternative method and vaccines directed against tick feeding. The aim of this study was to identify proteins involved in tick feeding, tickhost-pathogen interactions and tick reproduction. Consequently, these will help in identification of antigens with the ultimate goal of developing anti-tick vaccines. R. (B.) microplus female ticks were collected at five different feeding stages. RNA was isolated from the salivary gland extracts (SGEs). The cDNA libraries were synthesized and sequenced with the Illumina MiSeq technology. Transcriptome data was analyzed with CLC Genomics Workbench, Trinity and Minia. The SGEs were also used to isolate the fractions: membrane, soluble and pellet protein for proteomic analysis. The proteomics data was analysed with Mascot, X!Tandem and Scaffold. Both the transcriptome and proteome analysis revealed the presence of major secretory protein families such as Kunitz, lipocalins, serpins, cement proteins and metalloproteases, while the majority of transcripts coded for housekeeping genes. / National Research Foundation (South Africa) / Life and Consumer Science / M. Sc. (Life Science)

Cattle ticks and tick-borne infections (TBIs) undermine cattle health and productivity in the whole of sub-Saharan Africa (SSA) including Nigeria. In this West African country, two thirds of the cattle population are reared in the central-northern regions, kept under the traditional pastoral husbandry of Fulani herders. Under the Fulanis’ management, cattle are grazed extensively, being exposed to infestation by several tick genera (i.e. Amblyomma, Hyalomma, and Rhipicephalus spp., sub-genus Boophilus spp. included), vectors of the causative agents of the most important bovine TBIs in West Africa: anaplasmosis, babesiosis and ehrlichiosis (cowdriosis). Nevertheless, the Fulani pastoralists do not usually employ chemicals to control ticks in their cattle, merely relying on traditional methods (i.e. manual removal of the most conspicuous specimens). This approach, however, does not prevent cattle from being re-infested, leaving the animals challenged by a broad variety of other tick species, most of which are vectors of economically relevant TBIs. Knowledge of tick and TBIs occurrence is an essential pre-requisite to assist field diagnosis and devising effective control strategies for a given area. Existing information on tick infestation of cattle in Nigeria is rather out-dated, mostly derived from studies carried out in the south of the country. Similarly, all studies published to date on cattle TBIs in the country do not include any molecular analysis, being based instead on cytological and/or serological diagnostics. Therefore, the aim of the present thesis was to assess the presence of cattle ticks and TBIs occurring in an area of Central Nigeria (i.e. Plateau State). This is a densely populated area with traditionally managed cattle, where no acarides have historically been employed on livestock. The work undertaken herein firstly reviews the information available to date on ticks and TBIs known to be endemic in Nigerian cattle, identifying gaps present in the existing knowledge, leading to the rationale of this study. An initial survey was conducted documenting the tick species infesting cattle in Central Nigeria, in order to assess the infestation rate of surveyed animals at the time of the year when the tick load on the host is known to be most abundant (i.e. the wet season). The survey provided novel information on tick populations in cattle in Nigeria disclosing the presence of a broad variety of species, most of which are vectors of hazardous TBIs. In order to conduct a molecular diagnosis of the TBIs within the study area, a novel methodology was developed (i.e. reverse line blotting, RLB). The application of this approach was based on a thorough review of its application to the diagnosis of TBIs worldwide as well as in SSA. The optimisation of the RLB at the University of Edinburgh to enable the detection of a broad-spectrum of TBIs in Nigeria, caused by an array of five genera of microorganisms (i.e. Ehrlichia and Anaplasma, Theileria and Babesia, Rickettsia spp.) is presented. The assessment of the analytical sensitivity of this technique for the detection of Anaplasma marginale, a highly endemic tick-borne pathogen in SSA, demonstrated a detection threshold of ≥ 7 infected cells (keeping the limit of a natural infection). The occurrence of TBIs in cattle in the study area was assessed during a large-scale epidemiological survey through the application of the validated RLB. This study disclosed the occurrence of a high prevalence of several bovine TBIs in Central Nigeria, some of which are of great veterinary and zoonotic concern. The RLB enabled the detection of carrier status as well as of numerous multiple infections (69.5%, 95% CI: 65.5–73.6%). Based on the findings presented, endemic stability for highly prevalent haemoparasites (i.e. Theileria mutans, Theileria velifera, Theileria taurotragi, Anaplasma marginale, Ehrlichia species Omatjenne) is postulated, whereas a more instable epidemiological scenario is hypothesized for other microorganisms (i.e. Anaplasma centrale and Babesia bovis), which might be connected with outbreaks of clinically apparent disease, sporadically seen in the study area. The effect of a monthly tsetse-borne trypanosomiasis-focused control programme (based on the application 0.005% deltamethrin spray formulation, applied only to the lower quarters of cattle) on the kinetics of bovine TBIs was assessed at the village level. Longitudinal monitoring of control and treated cattle was conducted over the period of eleven months. Results generated provide input to the improvement of future control strategies to be rolled out across SSA, aiming to achieve an integrated control of both trypanosomiasis and TBIs. The present thesis contributes to a better understanding of the epidemiology of bovine TBIs in Nigeria as well as in the rest of West Africa, using a highly sensitive tool of wide applicability. These findings will be shared with the local pastoralist communities to further promote effective yet sustainable, vector control, in tune with the traditional long-established practices.

This study is focused on changes in protein expression in a glioblastoma cell line during infection with tick-borne encephalitis virus. Newly synthesized proteins were distinguished from previously synthesized proteins using bioorthogonal chemistry (BONCAT method) to observe changes in protein synthesis. Labelled proteins were visualized using two-dimensional PAGE and western blotting followed by Click reaction on membrane. Differences in protein pattern between control and infected cells were observed.

This diploma thesis addresses the topic of informing people of tick-borne encephalitis and vaccination against this disease. Our diploma thesis has four objectives in total. The first objective was to ascertain how many of the respondents were vaccinated against tick-borne encephalitis. The second and the third objectives were to demonstrate the reasons of vaccinated and unvaccinated respondents. The fourth objective was to analyze the attitudes of the respondents to publicization of the tick-borne encephalitis issue. As an infection with the natural focus, the disease is caused by a tick-borne encephalitis virus belonging among flaviviruses. The carrier is a sheep tick (Ixodes ricinus). Ticks live as parasites on reservoir animals, such as small rodents, ungulates and domestic animals. The clinical picture of the disease has mostly two stages and the symptomatologic form affects in particular adults and seniors. South Bohemia is a significant focus of tick-borne encephalitis with the sickness rate considerably exceeding the average of the Czech Republic. The most efficient prevention of the disease is vaccination. Two vaccines have been registered in the Czech Republic at present, namely FSME-Immun of Baxter and Encepur of Novartis. Both the vaccines are intended for children from 1 year of age. The basic vaccination scheme consists of 3 doses and the vaccinated person needs to be revaccinated every 3-5 years. It follows from the questionnaire research that 49 % of the total number of 136 respondents are vaccinated. As expected, the most frequent reason for vaccination (65 %) is the fear of becoming infected with tick-borne encephalitis. Other reasons are the interventions of the surrounding persons persuading the person to become vaccinated (19 %) and the financial contribution of the health insurance company or the employer (16 %). 35 % of the unvaccinated respondents do not plan to become vaccinated in the future. Approximately 40 % of the unvaccinated respondents feel that they do not need the vaccination or do not trust vaccination as such. 42 % of the unvaccinated persons would agree to vaccination if they received a financial contribution of the health insurance company or the employer, but nothing at all would convince 35 % of the unvaccinated respondents. It follows from the research that most respondents are satisfied with the quantity and the content of the information about tick-borne encephalitis. They do not think that anything should be changed in the system of informing of the disease. This thesis contributes to monitoring the trends in thinking of the public, and will eventuate in the finding which way the education of people about tick-borne encephalitis should be directed.

Tick-borne encephalitis virus (strain Hypr) was serially subcultured in PS cells and tick cell line IRE/CTVM19, producing four different viral variants. Biological properties of these new variants were investigated in mouse model. Possible determinants of virulence were found by full-genome sequencing. The role of glycosylation for tick-borne encephalitis virus was evaluated.

This study attempted to characterise 5 field stocks of B. canis, collected from various geographic areas of southern Africa in terms of pathogenicity, immune response, cross resistance and antigenic composition. This was done by means of artificial infection of two to four year old Beagle dogs. Serological tests included indirect fluorescent antibody tests, polyacrylamide gel electrophoresis , Western blotting of B. canis antigen and immune serum transfers. The ZIM1, GHT1 , QTN1 and DBN1 stocks were found to be virulent and the JHB1 stock, which might be a cardiac form of babesiosis, was found to be highly virulent. Immune responses of dogs to primary challenges were poor. A second challenge or relapse was important in the development of a resistant immunity. Heterologous challenges showed that dogs which were immune to the ZIM1, QTN1 and DBN1 stocks were partially immune to the JHB1 stock. The dog that was immune to the GHT1 stock was the only one that was totally resistant to the JHB1 stock. Western blots revealed interstock antigenic variation between the JHB1 and the other four stocks. These differences were, however, not great enough to separate the stocks taxonomically.

The control of ticks, and to some extent tick-borne diseases, over much of South Africa is currently dependent on acaricides and will probably remain so for the foreseeable future. Resistance to these chemicals by ticks thus poses a major threat to the livestock industry especially as these chemicals constitute an ever-diminishing resource with fewer being discovered and the cost of their development becoming prohibitive. In order to determine the extent of acaricide resistance in the eastern region of the Eastern Cape Province one-, two- and three-host ticks were collected from cattle at 58 dip-tanks over a period of 2 years. The one-host tick selected was Rhipicephalus (Boophilus) microplus, the two-host tick Rhipicephalus evertsi evertsi and the three-host tick Rhipicephalus appendiculatus. The ticks were tested for resistance to three compounds, namely amitraz, cypermethrin and chlorfenvinphos. The Shaw Larval Immersion Test detected emerging resistance to amitraz in the one-host tick R. (Boophilus) microplus at two dip-tanks and resistance at a third. It also revealed resistance in this tick to cypermethrin at one dip-tank, and emerging resistance to chlorfenvinphos at eight dip-tanks and resistance at two. The two-host tick R. evertsi evertsi was susceptible to amitraz and cypermethrin at all dip-tanks, but showed emerging resistance to chlorfenvinphos at seven dip-tanks and resistance at four. The three-host tick R. appendiculatus was susceptible to amitraz and chlorfenvinphos at all dip-tanks and demonstrated emerging resistance to cypermethrin at one. With the exception of R. (Boophilus) microplus, in which emerging resistance to amitraz was detected at one dip-tank by the Reproductive Estimate Test, all three tick species at all dip-tanks at which sufficient numbers of ticks had been collected, were susceptible to the three acaracides in both the Egg Laying Test and the Reproductive Estimate Test. Thus despite its fairly long and widespread use in the eastern region of the Eastern Cape Province very little or no resistance to amitraz was detected in three tick species regularly encountered on cattle in this region, namely R. (Boophilus) microplus, R. evertsi evertsi and R. appendiculatus. On the other hand resistance to chlorfenvinphos was detected in both R. (Boophilus) microplus and R. evertsi evertsi at a number of dip-tanks even though it, or other organophosphorous-based compounds, had probably not been used for tick control in the region for a number of years. The localities at which ticks were collected had already been mapped and the localities at which acaricide resistant ticks were encountered were mapped during this study. The three tick species that were targeted for acaricide resistance testing were widespread throughout the study region, but no pattern of geographic distribution for the acaricide resistant strains of these species that were detected, emerged. The rapidity of selection for acaricide resistance appeared to be closely related to the life cycles of the three ticks and the number of days that they theoretically would spend annually on their preferred host animals. Thus a greater number of acaricide resistant strains were encountered amongst the one-host tickR. (Boophilus) microplus and the two-host tick R. evertsi evertsi than the three-host tick R. appendiculatus. The fist two ticks both complete more than one life cycle a year and hence spend a longer time on their cattle hosts than the three-host tick R. appendiculatus, which completes only one life cycle a year and in addition is a rapid feeder in all its stages of development. To counter selection for acaricide resistance it is proposed that regular testing for resistance should be carried out, and that as soon as emerging resistance is detected in ticks on cattle at a particular dip-tank, that the acaricide in use at that dip-tank should be changed to a compound belonging to a completely different group of chemicals. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

This observational study of 100 dogs naturally infected with Babesia rossi determined whether severity of parasitaemia was associated with outcome of infection and documented the relative distribution of parasitised red blood cells (pRBC) in capillary and venous circulation. The association between increased parasitaemias and outcome with a clinically compromised circulation was also investigated. Outcome was defined as either hospitalisation with death, or hospitalisation with eventual recovery or treatment as an outpatient. Dogs were enrolled if large babesias were found on stained thin capillary blood smears made from an ear prick. Thin venous smears were prepared from jugular or cephalic blood. Parasitaemias were manually counted and expressed as the percent pRBC. Ten dogs died, 50 recovered after hospitalisation and 40 were treated as outpatients. Venous sampling site did not affect venous parasitaemia (P = 0.6). Both capillary and venous parasitaemias of dogs that died were significantly higher than those of dogs that recovered after hospitalisation (P = 0.002) and dogs that were treated as outpatients (P < 0.0001). When assessing the whole group, capillary parasitaemia (median 0.61%, range <0.05-71.6%, interquartile range (IQR) 0.22-3.75%) was significantly higher than venous parasitaemia (median 0.14%, range 0-30.6%, IQR 0.046–0.52%) with P < 0.0001. The 21 dogs with a clinically compromised circulation were more likely to die (P <0.0001) and had significantly higher capillary (median 5.98%, range 0.09-71.6%, IQR 2.44-19.41%) and venous (median 2.81%, range <0.05-30.6%, IQR 0.17-9.03%) parasitaemias than the 79 dogs with a clinically normal circulation (capillary median parasitaemia 0.38%, range <0.05-12.87%, IQR 0.16-1.42%; venous median parasitaemia 0.096%, range 0-6.13%, IQR <0.05-0.33%; P < 0.0001). This study shows that high parasitaemia is significantly associated with death in B rossi infected dogs. Unfortunately, there was a wide overlap in the parasitaemias of the three outcome groups with the result that neither capillary nor venous parasitaemias appear prognostically useful. The previous clinical suspicion that capillary parasitaemias are usually higher than venous parasitaemias is confirmed. Thus capillary samples are the most appropriate diagnostic samples. Prior observations that a clinically compromised circulation is associated with death are confirmed. This association provides a rapid means of identifying patients in need of intensive monitoring and treatment. Despite the highly significant association between compromised circulation and higher parasitaemia, it is thought unlikely that parasite burden is the sole trigger for circulatory collapse. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Bohm, M 2006, The comparative assessment of capillary and venous Babesia rossi parasitaemias on thin blood smears and their association with disease manifestation, MMedVet dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-05042007-154527 / > / Dissertation (Master of Veterinary Medicine (Small Animal Medicine))--University of Pretoria, 2006. / Companion Animal Clinical Studies / unrestricted

The pathology of canine cerebral babesiosis was examined at the gross, histological and ultrastructural levels. Gross lesions could be categorised as either global or regional. Congestive brain swelling , diffuse cerebral congestion and diffuse cerebral pallor were classified as global lesions. Multifocal haemorrhage and malacia were classified as regional lesions. Oedema was inconsistently present and could be either focal or diffuse. The majority of histological changes were observed in both cerebral babesiosis and control cases. Regional lesions were unique to cerebral babesiosis and had specific histological features. Highly localised endothelial injury was the primary lesion. Early lesions were multifocal and strictly associated with the microvasculature. Intermediate lesions, with perivascular haemorrhage and neutrophil infiltration, were suggestive of reperfusion injury. Advanced lesions were locally extensive and similar in appearance to haemorrhagic infarction. It is likely that the pathogenesis of regional lesions is by a process of microvascular infarction, as venous thrombosis could not be demonstrated. Ultrastructural evidence for adherent contact between erythrocytes and capillary endothelium was demonstrated. Endothelial cell necrosis occurred early in the development of lesions, before neuronal and glial injury. It is postulated that endothelial injury is the primary event in the development of regional lesions and secondary lesions develop as a consequence of microvascular infarction. / Die patologie van die serebrale vorm van bosluiskoors in honde is ondersoek. Die letsels is makroskopies, histologies en elektronmikroskopies beskryf. Letsels kon makroskopies in twee groepe verdeel word: Globale letsels en gelokaliseerde letsels. Kongestiewe brein swelling, diffuse serebrale kongestie en serebrale anemie kom voor as globale letsels in serebrale babesiose. Multifokale bloeding en nekrose kom voor as gelokaliseerde letsels. Edeem was nie konsekwent teenwoordig nie, en was algemeen of verspreid. Die meeste algemene histologiese veranderinge was in beide serebrale en kontrole gevalle teenwoordig. Gelokaliseerde letsels waarin spesifieke hisotpatologiese veranderinge voorgekom het, was kenmerkend van serebrale babesiose. Die primere letsel is hoogs gelokaliseerde beskadiging van endoteelselle. Beskadiging van die kapillere bloedvate ontstaan vroeg in die ontwikkeling van letsels. Verdere ontwikkeling van die letsel word gekenmerk deur peri-vaskulere bloeding en neutrofiel infiltrasie wat aanduidend is van reperfusie beskadiging. Volontwikkelde letsels is plaaslik-ekstensief en het die voorkoms van hemoragiese infarkte Dit is waarskynlik dat mikrovaskulere infarksie 'n rol speel in die patogenese van die letsels, aangesien veneuse trombose nie ontstaan nie. Noue kontak tussen rooibloedselle en kapillere endoteel is elektronmikroskopies bevestig. Endoteelselnekrose ontstaan voordat tekens van beskadiging geidentifiseer kan word in neurone of gliaselle. Dit blyk dat kapillere endoteelselbeskadiging die primere letsel by die ontstaan van gelokaliseerde lese Is is, en dat sekondere lesels ontwikkel as gevolg van mikrovaskulere infarksie. / Dissertation (MSc)--University of Pretoria, 2000. / Paraclinical Sciences / Unrestricted