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

Two species of Haemaphysalis occurring in the western United States were studied with respect to geographical distribution, seasonal occurrence, host-parasite association and external anatomy and morphology. H. leporispalustris is widely distributed in the west. Abundant records for some states are lacking and should be provided to extend the knowledge of this tick's distribution.

Invertebrates do not possess an adaptive immune system, but rely on several mechanisms similar to the innate immune system of mammals. The synthesis and release of a host of potent antimicrobial proteins is an important component of this immune response. The antibacterial activity in the hemolymph of Ornithodoros savignyi is specific for Gram-positive bacteria, and the synthesis and release of the antibacterial factors need to be induced by challenging the ticks with heat-killed Gram-negative bacterial suspensions. The induction of the factors is very rapid, leading to a maximal response within one hour following bacterial challenge. The factors are stable at high temperatures, and were found to be protein in nature. By using reverse phase high performance liquid chromatography, four fractions exhibiting antibacterial activity were identified in the hemolymph of immune challenged ticks. Four antibacterial peptides were isolated from these fractions, and the mass analyses of the peptides indicate that there are at least two different antibacterial peptides present in the hemolymph. The N-terminal amino acid sequence of one of the peptides was determined, and the analysis showed that the peptide has high homology with defensin peptides isolated from other tick species. This led to the putative classification of the peptides as part of the invertebrate defensin family. The presence of lysozyme in O. savignyi was studied using molecular biological methods. Vertebrate and invertebrate lysozyme sequences were used to design a lysozyme-specific primer, which was used to amplify specific DNA products from whole tick cDNA using the polymerase chain reaction (PCR). The conditions for the amplification reaction were optimized, the products of the optimized reaction were cloned into a cloning vector and the nucleotide sequences of the products were determined. The nucleotide sequences were used for similarity searches of sequence databases to determine homology with sequences of known proteins. It is deduced the degenerate primer was not specific for lysozyme and did not playa significant role in the amplification of the PCR products. This method is thus not feasible for the investigation of the lysozyme of O. savignyi. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

The saliva of hematophagous ectoparasites contains a cocktail of vasodilators, anticoagulants and immunosuppressors that maintain blood in a liquid state at the site of the lesion and evade the host's defense mechanisms in suppressing the immune response. Since ticks have evolved to utilize mammals as a source of food, our understanding of the tick material, especially the salivary glands will enhance the control of tick infestation and allow the exploitation of the tick's natural resources. SGE protease activity was determined by measuring the degradation of azocasein. Proteolytic activity was found in the pH range of 3 to 11 with the highest activity at pH 9 followed by pH 7. At pH 3-5 the activity was mainly due to aspartic proteases, whereas at pH 7-9 the activity was due to the action of metallo- and serine proteases. At pH 11, the activity was mainly ascribed to metallo- and aspartic proteinase activity The fibrinogenolytic activity was determined by incubating human fibrinogen in the presence of SGE and monitoring the fibrinogen degradation by SDS-PAGE. SGE degraded the Au-chain of fibrinogen within 2 hours of incubation and even after 24 hours incubation there was no hydrolysis of the Bβ and γ-chains of fibrinogen. Characterization of the fibrinogenolytic activity revealed that metalloprotease activity was present over pH range of 3-9 and at pH 3-5, the cysteine proteases were active. No serine protease activity was found under similar experimental conditions. CE-HPLC separation of the SGE revealed three regions of proteolytic activity. Further characterization of the activity containing fractions using protease inhibitors at various pH values showed that the activity associated with region A is mainly due to the presence of aspartic and cysteine proteases in the lower pH range (< 5). Region B was mainly due to the activity of the metallo- and serine proteases, while the activity in region C was mainly due to the metalloproteinases which were more active in the higher pH range (> 9). CE-HPLC separation of SGE resulted in three regions exhibiting fibrinogenolytic activity at pH 7-9. In region A all four enzyme classes were found while in regions B and C, serine, cysteine and metalloproteinases were found to be responsible for the activity. Region A was further purified on the HIC-column and activity eluted in several peaks which after individual application on SE-HPLC column had similar retention times. The pooled samples were analyzed for purity using C5 RP-HPLC and reducing tricine SDS-PAGE and three bands of relative molecular masses 15, 22 and 12 kDa, respectively were found. In an attempt to purify the proteins in region C, four individual CE-HPLC runs were combined and applied to a fibrinogen affinity column. Reducing SDS-PAGE analysis of bound material showed two bands of relative molecular masses of 31 and 39 kDa, respectively. CE-HPLC region C as well as the SGE control was found to disaggregate platelets aggregated by ADP, epinephrine, collagen as well as TRAP. No disaggregation was observed for the saline negative control. The disaggregation is most probably due to the hydrolysis of the fibrinogen cross-linking platelets by the metalloproteinase activity in region C. Understanding of the proteolytic activities present in the salivary gland and therefore identifying molecules crucial for tick feeding could aid in the development of experimental vaccines. Even though the fibrinogenolytic activity was not purified to homogeneity, this study has laid the groundwork for further experiments in this field. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

Thesis (M.Sc. (Agric.)) --University of Limpopo, 2010 / The objectives of the study were to estimate genetic parameters for tick resistance and to evaluate the effect of the level of tick infestation on the estimates of genetic parameters in South African Bonsmara cattle. Field data of repeated tick count records (n = 11 280) on 1 176 animals were collected between 1993 and 2005 by ten breeders participating in the National Beef Recording and Improvement Scheme. The distribution of tick count records were normalized using a Box-Cox transformation. Data were divided into 7 sub-data sets based on the mean tick count per contemporary group, to facilitate the investigation of the effect of level of tick infestation on the derived genetic parameters. A repeatability animal model including the fixed effects of contemporary group and age of animal at tick counting and random effects of the direct additive genetic, permanent environmental and residual effects was used to estimate genetic parameters using REML procedures. The additive genetic variances for tick count ranged from 0.01 to 0.08. Variances for the permanent environment ranged from 0.00 to 0.03. Phenotypic variance decreased with increasing mean tick count level while additive genetic variance increased with increasing mean tick count level. The heritability also increased with mean tick count level until a mean tick count level of ≥30. The highest heritability estimate obtained in the current study was 0.17 for data with mean tick count level ≥25. These results suggest that sufficient genetic variation for tick count exists in the Bonsmara cattle. Therefore genetic selection for tick resistance is feasible even though genetic progress may be slow. / the Limpopo Department of Agriculture (LDA) and the Department of Science and Technology (DST)

The main objective oft his dissertation was to identify the causes of the rise of the tick population in KwaZulu-Natal in 1999. After 100 years of intensive chemical tick control, tick numbers remain high and the stock losses caused by tick borne diseases are still significant. In South Africa legislation was introduced to support intensive chemical tick control. Ticks have consistently shown themselves to possess a genetic pool containing the potential to resist a wide range of chemical poisons. The introduction of new chemicals followed by widespread use, has often resulted in the appearance of a tick population resistant to those chemicals. The problem is compounded by the fact that some farmers are also found to be helping ticks to multiply by not following instructions given by the chemical industry on how to use dips. Chemicals which are used to control ticks are also beyond the financial means of many cattle owners especially in resource- poor communities. Apart from the high cost of intensive tick control, the chemicals that are used to destroy ticks are very poisonous, not only to ticks but to the birds which are natural predators of ticks. The negative effects of these chemicals on the environment combined with the high cost of tick control has forced a revision of intensive chemical tick control strategy. There is now a shift to use methods of tick control which are friendly to the environment and affordable to the resource-poor communities. This dissertation provides a historical overview of the problem in KwaZulu-Natal and recommendations on how to deal with the problem in future. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

Arthropod-borne viruses, arboviruses, have the ability to replicate in both vertebrates and invertebrates and are transmitted to susceptible vertebrate hosts by vectors such as mosquitoes and ticks. Ticks are important vectors of many highly pathogenic arboviruses, including the flavivirus tick-borne encephalitis virus (TBEV) and the nairovirus Crimean-Congo haemorrhagic fever virus. In contrast, alphaviruses are principally mosquito-borne and have been isolated only rarely from ticks; ticks have not been implicated as their vectors. Nevertheless, the alphavirus Semliki Forest virus (SFV) replicates in cell lines derived from many different tick species, including those of the genus Ixodes, which includes vectors of TBEV and its lesspathogenic relative Langat virus (LGTV). In vertebrate cells, arboviruses generally cause cytopathic effects; however, arbovirus infection of arthropod cells usually results in a persistent low-level infection without cell death. While little is known about antiviral immunity in tick cells, the immune system of other arbovirus vectors such as mosquitoes has been studied extensively over the last decade. In insects, pathways such as RNA interference (RNAi), JAK/STAT, Toll, Imd and melanisation have been implicated in controlling arbovirus infection, with RNAi being considered the most important antiviral mechanism. In tick cells, RNAi has been shown to have an antiviral effect, but current knowledge of other immunity pathways is limited and none have been implicated in the antiviral response. In the present study, SFV and LGTV replication in selected Ixodes spp. tick cell lines was characterised and the Ixodes scapularis-derived cell line IDE8 was identified as a suitable cell line for this project. Potential antiviral innate immunity pathways were investigated; putative components of the tick JAK/STAT, Toll and Imd pathways were identified by BLAST search using available sequences from well-studied arthropods including the fruit fly Drosophila melanogaster. Using gene silencing, an attempt was made to determine whether these pathways play a role in controlling SFV and LGTV infection in tick cell lines. Selected genes were silenced in IDE8 cells using long target-specific dsRNA and cells were subsequently infected with either SFV or LGTV. Effects of gene silencing on virus replication were assessed by quantitative real time PCR (qPCR) or luciferase reporter assay. Effects on infectious virus production were measured by plaque assay. Replication of the orbivirus St Croix River virus (SCRV), which chronically infects IDE8 cells, was also quantified by qPCR after silencing of selected genes. Interestingly, SFV or LGTV infection of IDE8 cells resulted in a significant increase in SCRV replication, possibly as a result of interference with antiviral pathways by SFV and LGTV or possibly due to diversion of cellular responses from sole control of SCRV. No evidence for an antiviral role for the JAK/STAT or Toll pathways was found in IDE8 cells. However, an antiviral effect was observed for protein orthologues putatively involved in the RNAi response. Argonaute proteins play an important role in translation inhibition and target degradation mediated by RNAi, and silencing of selected Argonaute proteins resulted in a significant increase in SFV and SCRV replication. The carboxypeptidase CG4572 is essential for an efficient antiviral response in D. melanogaster, and supposedly involved in the systemic RNAi response. A putative tick orthologue of CG4572 was identified and this appeared to be involved in the antiviral response in IDE8 tick cells. When expression of CG4572 was silenced and cells subsequently infected with SFV or LGTV, replication of both viruses was significantly increased. In addition, it was shown that three mosquito orthologues of CG4572 also had an antiviral role against SFV in Aedes mosquito cells. In conclusion, of the tick cell lines investigated, IDE8 provided a suitable model system for investigating tick cell responses against arboviruses and new insight into the nature of the tick cell antiviral response was gained.

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