The Corinnidae is one of 76 families of spiders (Arachnida: Araneae) presently recognised in the Afrotropical Region. By the end of the last century their taxonomy and systematics had been very poorly studied and no modern revisions existed on the group. At that time, 110 species in 22 genera were known from the region, making it a family with moderate species richness. The description of the new genus Hortipes Bosselaers & Ledoux, 1998 in the family Liocranidae signalled the start of modern systematics studies in that family, and following the transfer of Hortipes to the Corinnidae, of that family by default too. Since that time, 20 taxonomic papers have been published on the Afrotropical Corinnidae and 10 new genera (all endemic to the region) and 164 new species have been described, of which three species form part of the current study (Chapters 7 and 9). Several genera have also been transferred to or from the Corinnidae in those papers. Presently there are 35 genera and more than 270 species known from the region, with the Corinnidae now ranking eighth in species richness in the region. Most of the revisionary work so far has focused on the subfamilies Trachelinae and Phrurolithinae, while the Corinninae sensu lato and Castianeirinae have largely remained neglected. The broad aim of the current study was to focus on the systematics of the latter group, treat the taxonomy of each of the currently known genera, at least in part, and provide a basis for future work on the subfamily. As such, many of the smaller genera in the subfamily were revised in the Afrotropical Region and two new genera were described. The genus Apochinomma Pavesi, 1881, the only described genus of accurate antmimicking castianeirines from the region, is revised and separated into two species groups based on genitalic and abdominal morphology. The type species, A. formicaeforme Pavesi, 1881, is redescribed and three new species are described in the A. formicaeforme species group: A. malkini sp. nov., A. parva sp. nov. and A. tuberculata sp. nov.. Two new species, A. decepta sp. nov. and A. elongata sp. nov., are described in the A. decepta species group, although an additional species only known from juveniles can also be placed in the latter group. Members of the A. formicaeforme species mimic Polyrhachis ants and are mainly arboreal, while members of A. decepta species group are ground- or grass-dwelling and probably mimic ponerine ants. The genus Cambalida Simon, 1909 is revised and three species are transferred from Castianeira Keyserling, 1879 to Cambalida: C. deminuta (Simon, 1909) comb. nov., C. fulvipes (Simon, 1896) comb. nov. and C. loricifera (Simon, 1885) comb. nov.. An additional species is transferred from Brachyphaea Simon, 1895 to Cambalida: C. fagei (Caporiacco, 1939) comb. nov.. All of these species are redescribed, as is Cambalida coriacea Simon, 1909. Two species, Castianeira depygata Strand, 1916 syn. nov. and C. mestrali Lessert, 1921 syn. nov., are considered junior synonyms of C. fulvipes. The type material of the type species of the genus, C. insulana Simon, 1909 from Annobon Island, is lost, and only immature specimens have been subsequently collected from a nearby island. The species is regarded as a nomen dubium until fresh adult material can be collected. A replacement name, Cambalida simoni nom. nov., is proposed for Cambalida fulvipes Simon, 1909, the latter being a secondary junior homonym of Cambalida fulvipes (Simon, 1896) comb. nov.. The type material of C. simoni is also lost and it too is considered a nomen dubium. Five new species are described: C. compressa sp. nov., C. dippenaarae sp. nov., C. griswoldi sp. nov., C. lineata sp. nov. and C. unica sp. nov.. Castianeira Keyserling, 1879 is the largest genus in the Corinnidae with 131 described species, of which 22 are presently known from the Afrotropical Region. There is a very rich undescribed fauna known from the region, and the variable morphology of its component species would suggest it is polyphyletic and should be divided into several genera. For example, six species are misplaced and have been transferred to or synonymised with species in Cambalida or the new genus Copuetta gen. nov.. In the present study, five species are redescribed and illustrated for the first time based on the type material: C. delicatula Simon, 1909, C. formosula Simon, 1909, C. majungae Simon, 1896, C. phaeochroa Simon, 1909 and C. thomensis Simon, 1909. The female holotype of C. bicolor (Simon, 1890) lacks an abdomen and the species is considered a nomen dubium. The types of several Afrotropical species could not be traced as yet and the species should be redescribed, if possible, based on recently collected material from near their type localities. The ground-dwelling genus Copa Simon, 1885 is one of four genera in the Afrotropical Region that have cryptic colouration that bears a resemblance to that of wolf spiders (Lycosidae), hereafter referred to as cryptic lycosiform colouration. The type species of the genus, C. flavoplumosa Simon, 1885, is redescribed and proposed as a senior synonym of C. benina Strand, 1916 syn. nov. and C. benina nigra Lessert, 1933 syn. nov.. This is possibly the most widespread corinnid in the Afrotropical Region albeit that is has not yet been recorded from any of the islands. A new species, C. kei sp. nov., is described from South Africa. Copa agelenina Simon, 1910, originally described from a subadult female from southern Botswana, is considered a nomen dubium. Although the Madagascan fauna was not included in this revision, nearly 30 new species have been distinguished from museum collections, and once that fauna is revised it will provide an exceptional example of island radiation. In a revision of the Afrotropical species of the ant-mimicking genus Corinnomma Karsch, 1880, Apochinomma semiglabrum Simon, 1896 is redescribed from both sexes, and based on these descriptions it is transferred to Corinnomma as C. semiglabrum (Simon, 1896) comb. nov.. A new species, C. lawrencei sp. nov., is described from Mozambique, Tanzania and South Africa. The taxonomic status of C. olivaceum Simon, 1896 is discussed and the first illustrations of the female genitalic structures are presented. Since no fresh material of this species is available and the female holotype is badly faded, it is not thoroughly redescribed. An English translation of Simonâs (1896) Latin description of C. olivaceum is provided with the intention of more accurately describing the colouration of this species. The arboreal cryptic lycosiform castianeirine genus Echinax Deeleman-Reinhold, 2001, previously known only from South-East Asia, is recorded from the Afrotropical Region for the first time. Copa longespina Simon, 1909 is redescribed and the species is transferred to Echinax as E. longespina (Simon, 1909) comb. nov.. Six new species are described from both sexes: E. clara sp. nov., E. hesperis sp. nov., E. natalensis sp. nov., E. scharffi sp. nov., E. similis sp. nov. and E. spatulata sp. nov.. The genus Graptartia Simon, 1896, presently known only from Africa, is revised. The type species, G. granulosa Simon, 1896, is redescribed and the first genitalic sketches of the species are provided. Two new species, G. mutillica sp. nov. and G. tropicalis sp. nov., are described. Unique amongst African castianeirines, all species of Graptartia are mimics of wingless female velvet ants (Mutillidae). Although the genus Merenius Simon, 1909 is not revised, a single common species, Merenius alberti Lessert, 1923, is redescribed. The species was previously known only from South Africa, and is recorded for the first time from Mozambique, Swaziland and Zimbabwe. While most populations of M. alberti comprise the typical black morph of the species, a red morph is described for the first time here. As part of a field study to identify the potential models of the two colour morphs of M. alberti, spiders were collected by hand and ants by pitfall trapping in the Ndumo Game Reserve in northern KwaZulu-Natal, South Africa. The ants assemblages sampled at 20 sites in the reserve seem to indicate that the black morph is a generalised mimic of black ground-dwelling ants, most likely Camponotus cinctellus (Gerstäcker, 1859), Streblognathus peetersi Robertson, 2002 and Polyrhachis gagates F. Smith, 1858, while the red morph is a mimic of Anoplolepis custodiens (F. Smith, 1858) ants. are inadequate to support any systematic changes in the Corinnidae, but future analyses need to include a more diverse range of castianeirine genera from outside the Afrotropical Region to better understand the relationships of the Afrotropical fauna. In the final chapter, the role of Castianeirinae as components of arthropod mimicry complexes is described for three species of ants, Anoplolepis custodiens (F. Smith, 1858), Polyrhachis gagates F. Smith, 1858 and Camponotus fulvopilosus (De Geer, 1778). There are respectively two out of 10, four out of six, and zero out of five species of Castianeirinae forming part of the arthropod complexes associated with these ants. All of these castianeirines are inaccurate (weak/ generalised) mimics of their models except for Apochinomma formicaeforme, which is an accurate (good/specialised) mimic of P. gagates. Colour polymorphism is also described for the first time in four species of Afrotropical Castianeirinae, i.e. Corinnomma semiglabrum, Merenius alberti, Castianeira cf. venustula (Pavesi, 1895) and Copa flavoplumosa. Three of these species are inaccurate mimics of ants, while C. flavoplumosa is a species with a widespread variant with cryptic lycosiform colouration and a nigrito form restricted mainly to tropical forests. High Castianeirinae biodiversity and endemism corresponds to most of the main Biodiversity Hotspots and Centres of Endemism (CE) in the Afrotropical Region: Maputaland-Pondoland-Albany CE (five endemics), Madagascar and Indian Ocean Islands CE (>30 endemics), East African Afromontane Forests CE (four endemics), East African Coastal Forests CE (five endemics), Guinean Forests of West Africa CE (seven endemics) and the Horn of Africa CE (one endemic). No endemic castianeirines have been recorded in the Succulent Karoo and Cape Floristic Region CEâs in southern Africa, although this corinnid fauna of these two CEâs is largely dominated by Trachelinae, most of which are endemics.

The study of blood parasites of reptiles is a relatively new and unexplored field in South Africa. Therefore, the general aims of this research were to explore the haemoparasite fauna of cordylid lizards, Pseudocordylus melanotus, Pseudocordylus subviridis and Pseudocordylus langi, and especially to search for the definitive hosts and likely vectors of their haemogregarines. Surveys of lizard blood were conducted at various altitudinal gradients on the Sentinel Trail in the escarpment area of the Drakensberg, and at the top of Platberg, near Harrismith, both in the Free State. Five species of haemogregarines were identified, all suspected to belong to the genus Hepatozoon, and none was known from previously published records. These occurred in the blood of P. melanotus, P. langi and P. subviridis from the two disjunct study sites, and mostly were accompanied by other haemoparasites including a saurian malaria, so-called Sauroplasma, and filarial nematodes. Developmental stages of two of the Hepatozoon spp. were documented in the internal organs of P. melanotus and P. subviridis by means of light and confocal microscopy, histology, and transmission electron microscopy. Life stages were also observed in ectoparasitic lizard mites, by means of stained histological sections, and in stained squashes of mites and mosquitoes. Three species of experimentally reared mosquitoes were found to act as likely definitive hosts for Hepatozoon spp. of P. melanotus and P. subviridis, while wild caught Culex (Afroculex) lineata appeared to serve as a definitive host, and therefore possible vector for an Hepatozoon species of P. melanotus at the top of Platberg. A saurian malaria that appeared to have features of two previously described species was recorded in P. melanotus and P. subviridis. New locality records for so-called Sauroplasma and filarial nematodes were also documented for the three species of crag lizards. Some aspects of the fine structure of two haemogregarines, the Plasmodium sp. and so-called Sauroplasma infections were recorded for the first time in the erythrocytes of the Pseudocordylus spp. Differential leucocyte and thrombocyte counts were performed on the three crag lizard species and, with erythrocyte characteristics, compared with those of cordylid lizards in previous studies. Several types of leucocytes were characterized ultrastructurally, as well as by light microscopy, and attempts were made to correlate statistically leucocyte counts, and host and environmental data, with parasite loads. Finally, mites and mosquitoes associated with crag lizards were explored further as possible vectors of lizard haemoparasites, and studied using light and scanning electron microscopy.

Culicoides midges are involved in the transmission of a variety of pathogens, the most economically important of these are the orbiviruses that cause African horse sickness (AHSV) and bluetongue (BTV). The identification of vectors of these viruses and monitoring of their occurrence and activity plays an important role in the control measures and disease risk analysis. The primary tool used for monitoring these midges through collection is various models of light traps. In order to standardise collection data to be comparable between laboratories, a variety of factors that affect the light trap collections were assessed. Comparisons of different light traps (Onderstepoort trap and the Free State trap), the influence of light colour, trapping height and the distance a trap is operated from the host animals were assessed. Comparisons were done using either three replicates of a 4 x 4 or two replicates of a 6 x 6 randomised Latin square design. The most significant variables were the trap type, with the Onderstepoort trap collecting significantly more Culicoides than the Free State trap, and the sampling distance from the host animals. The proportion of C. imicola (the most frequent species collected) was the highest when collected right next to host animals and decreased rapidly as collections moved further from host animals. Trap height also proved to be an important variable, although no significant differences were observed when collecting midges at two metres to three metres above the ground. The occurrence, abundance and seasonality of the midges frequenting livestock in the central Free State were also assessed by collecting midges using light traps over a four-year period from April 2007 up to May 2010. Twenty Culicoides species were collected, the most abundant and important species was C. imicola, a confirmed vector of both the AHSV and BTV. The midges showed a distinct seasonal pattern, but were also collected year round, identifying periods of high risk, as well as a year-round risk of disease transmission. The midge populations almost disappeared when temperatures dropped during the winter months, the build-up and abundance during favourable conditions, however, indicated a high risk for disease transmission. Culicoides imicola also showed a considerable preference for livestock animals when assessing collections made near horses sheep and cattle as opposed to collections made in the absence of livestock host animals. An effort was made to identify possible breeding sites of Culicoides species. No midges were, however, collected in the tent type traps or the dung and soil samples collected and placed in emergence boxes. This again emphasised the diversity of the midgesâ breeding habitats and the enormous task still ahead to identify these sites to aid in possible reduction of midge numbers.

Equine piroplasmosis is a protozoan disease of horses caused by two parasites, Babesia caballi and Theileria equi. Both parasites are transmitted by ixodid ticks belonging to the genera Boophilus, Hyalomma, Dermacentor and Rhipicephalus. Equine piroplasmosis has a worldwide distribution and is endemic in tropical and sub-tropical regions, including Central and South America, Africa, Asia and Southern Europe. The economic impact of equine piroplasmosis in South Africa is assumed to be millions of Rands due to a combination of direct losses, convalescence period and incidental costs such as vaccinations, treatment and veterinary fees. There is little information on parasite strains in South Africa. The objectives of this study were to determine (i) the prevalence of equine piroplasmosis in Free State (FS) and KwaZulu Natal (KZN) of South Africa, using molecular and serological techniques, and (ii) strain variation of equine piroplasmosis parasites, namely, B. caballi and T. equi, using 18S rRNA DNA sequences analysis. Diagnostic methods used in this study include microscopy (thin blood smears), Polymerase Chain Reaction (PCR), Indirect Fluorescent Antibody Test (IFAT) and Enzyme-Linked Immunosorbent Assay (ELISA). Blood samples were collected from a total of 534 horses in the Free State and KwaZulu-Natal (444 were collected from FS and 90 from KZN). No B. caballi was detected from all samples collected from both provinces (FS and KZN) by microscopy and PCR. Of 507 serum samples tested for B. caballi by IFAT, a seroprevalence of 61% was detected. A mean value of 34% of samples was positive for T. equi using a PCR test and sero-prevalence of 94% was detected by IFAT. Fifty two percent of the 250 tested sera samples were positive for T. equi by ELISA. Together these results suggest high levels of exposure to parasites, high levels of current infections and uncertainty in current serological tests for these parasites. Sequencing and phylogenetic analysis of the 18S rRNA gene showed considerable diversity of T. equi strains in South Africa. T. equi is highly prevalent in South Africa with the parasite appearing to be more prevalent in KZN than FS. This study also confirmed the distribution of this disease as described in previous studies, and the disease was found also in the area which previously was declared disease-free. There is considerable variation in T. equi genotypes in the country and no clear phylogeographic structuring of these genotypes. These results may indicate that there is much movement of infected carrier horses within the country and even to and from other countries. B. caballi prevalence is still not clear as only IFAT seems to detect antibodies to infection by this parasite. There is a need for the development of highly sensitive assays for the detection of B. caballi, thereby enabling determination of prevalence and strain diversity studies of this parasite.

Anaplasma marginale is a virulent intra-erythrocytic pathogen that causes bovine anaplasmosis, its closely related species, Anaplasma centrale causes mild sickness. The pathogen is transmitted biologically by tick vectors and mechanically through blood contaminated fomites. It has a worldwide distribution extending from tropical to subtropical regions in correlation with the vector distribution. Bovine anaplasmosis is often characterised by progressive anaemia, jaundice, decreased milk production, abortion and a sudden death. The commonly used method for the diagnosis of A. marginale of infected cattle in South Africa is microscopic examination of Giemsa stained blood smears and detection of antibodies from serum using cELISA. However the diagnostic methods have limitations in cases of low parasitemia and in carrier cattle (microscopy) and they fail to differentiate closely related Anaplasma spp due to antigenic similarity (serology), the detection limitations of the diagnostic methods influenced the aim of this study which is to develop molecular species -specific assays for the detection of A. marginale strains in South Africa, specifically Including conventional polymerase chains reaction, real-time polymerase chain reaction and loop-mediated isothermal amplification assay. Chapter one of this study discusses bovine anaplasmosis and its causative agent A. marginale, the diversity of the strains transmission, distribution, clinical signs, treatment and economic importance of the disease. The first objective of this study was to develop a species specific conventional PCR for detection of A. marginale in cattle in South African regions based on msp1b gene. The conventional PCR primers were designed through visual inspection and were named F3 and B3 primers. In the specificity test, the primers were specific whereby the amplified only A. marginale DNA and did not amplify control DNAâs: A. centrale, Babesia bovis, B. bigemina and Ehrlihia rumunantium. The sensitivity of the conventional PCR primers was examined using a 10 ng/ul DNA and the detection limit of the assay was 0.01 ng/ul, The assay was validated on field samples to confirm the infection of the cattle with A. marginale, out of 144 samples, (60%) infection rate was obtained with the newly developed conventional PCR, the homogeneity of the sequences were confirmed with the GenBank, the maximum similarity varied from 94 - 100%. The second objective of this study was to develop a species-specific real-time PCR for detection of A. marginale in cattle in South African regions based on msp1b gene. The real-time PCR primers and probes were designed using Genescript program, one set of primer (Prf 2, PrR2, and PrB2) was chosen to carry out the study as it showed high sensitivity with the detection limit of 0.001 ng/ul .The specific and sensitive TaqMan based real-time PCR was successfully developed for the of A. marginale infections in South Africa. Validation of the assay on field samples showed that the rate of infection was 74% in different sampled provinces of South Africa. The third objective of this study was to develop loop-mediated isothermal amplification for the detection of A. marginale in South African regions based on msp1b gene. The LAMP primers were designed using primer Explorer version 4, the LAMP primers were named LAF3, LA-B3, LA-FIP, LA-BIP,LA-LF and LA-LB. The LAMP assay showed positive results with specific amplification, but as far as the validation of the assay false positive results were obtained, troubleshooting involved the addition of additives, changing of primer purification and manufacturers, however the results were not consistent, false positive results were obtained, speculations were that it could be possible contamination of the laboratory resulting in the amplification of control DNA and distilled water. The first three objectives of this study were achieved. The newly developed assays were further compared for specificity, sensitivity and detection performance on field derived samples. The developed assays are specific and sensitive; they form a good tool of diagnosis of bovine anaplasmosis, with each assay having its own unique characteristic over the other, they are sensitive giving a correct determination of the infection status, aiding in compiling of epidemiological information. These assays will aid in understanding the major constraint to develop control measures due to the genetic diversity of A. marginale, and will also help in constructing of phylogenetic tree between strains from South Africa and other countries.

Ticks and tick-borne diseases are a burden in the livestock industry, decreasing productivity and compromising food security, leading to high socioeconomic impacts on agro-exporting nations. Apart from being agricultural pests they can transmit pathogens of zoonotic significance. The aim of the study was to therefore detect and determine with PCR the prevalence of tick-borne zoonotic pathogens i.e. Coxiella burnetii, Ehrlichia spp., Rickettsia spp., Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato from ticks collected from livestock. The sampling areas included both commercial and communal farms as well as domestic animals from KwaZulu-Natal, Free State, Eastern Cape, North West and Mpumalanga Provinces. As a result a total of 1947 tick samples were collected which were then identified and further processed for PCR amplification. Tick species collected included Rhipicephalus spp. (n = 570), R. sanguineus (n = 275), R. evertsi evertsi (n = 650), R. decoloratus (n = 228), R. appendiculatus (n = 10), Amblyomma hebraeum (n = 171), Hyalomma marginatum rufipes (n = 4), and Haemaphysalis elliptica (n = 38). The overall prevalence of infection with B. burgdorferi and A. phagocytophilum was 8±1.4% and 9±1.2% respectively, this was an unexpected finding since only one positive PCR confirmation of A. phagocytophilum has been reported in the country, since then no other studies have been successful in detecting this pathogen. There have been anecdotal cases of B. burgdorferi but the pathogen has, to the best my knowledge, not been detected and characterized by molecular methods. Both pathogens have not been isolated from ticks in South Africa previously. The tick vectors for these pathogens are not known to occur in the country, however this study managed to isolate A. phagocytophilum from R. sanguineus, R. appendiculatus, R. evertsi evertsi and H. elliptica and isolated B. burgdorferi from Rhipicephalus spp., R. evertsi evertsi, R. decoloratus and A. hebraeum which may act as main vectors and reservoir for livestock infections. I am however uncertain about their transmissibility neither to human hosts nor of their vectorial capacity, nevertheless tick species of Amblyomma readily bite humans and may be able to transmit both pathogens and could therefore pose a serious threat to the public. C. burnetii incidence was 16±1.6% amongst the ticks, this was also a first detection from ticks in the country but the findings seem to be consistent with previous serological studies, also all the tick species in the collection were found harboring the pathogen. The prevalence of E. ruminantium, E. canis and Ehrlichia/Anaplasma was determined to be 29±2.2%, 20±3.6% and 18±3.8% respectively. No significant Ehrlichia/Anaplasma species were characterized except for A. phagocytophilum as reported above. Rickettsia species that were isolated and characterized in the current study were R. africae and R. conorii as expected and the prevalence was 26±1.7%. All in all the target pathogens were successfully isolated, characterized and validated through sequencing reactions, however there still remains a task of determining the vectorial capacity of the ticks and evaluation of factors that could lead to their transmission to the public. In conclusion, these pathogens should be considered as part of routine screening in patients presenting with fevers of unknown origin especially amongst tourists where pathogens like Rickettsia seem to have become problematic in South Africa.

Helminths belonging to the genera Haemonchus and Trichostrongylus as well as those of the genus Fasciola are the causative agents of various helminthiases in animals and sometimes in humans. In both host types infections are often acquired through the ingestion of infected food as well as drinking contaminated water. In animals they disrupt the efficient conversion of food material and absorption of nutrients resulting in weakness and death. Infected humans often suffer from intestinal obstruction, insomnia, vomiting, weakness and stomach pains and sometimes temporary asthma. Infections are associated with huge economic losses globally. Diagnosed is achieved through the observation of clinical manifestations. In low infections alternative diagnosis is based on the recovery and identification of faecal eggs and cultivation of L3 by microscopy. More accurate diagnosis has recently been achieved through the use of molecular diagnostic tools such as PCR and LAMP. The aim of this study was to develop rapid, sensitive, specific and accurate molecular diagnostic assays. In particular the study focused on LAMP and PCR for the detection of H. contortus, Fasciola and Trichostrongylus spp. infections in four provinces of South Africa. The first study emphasized on the development of LAMP and PCR assays for detection of H. contortus infections in livestock. LAMP primers that specifically amplify the ITS2 gene of H. contortus were designed from this target gene. This set of primers was used to develop a PCR assay for species-specific gene amplification. Both assays were tested at various reaction conditions to optimize for primer annealing temperature. Sensitivity reactions were conducted using 10 fold serial dilutions of target DNA while primer specificity was determined using DNA extracted from closely related species. For the LAMP assay, the optimum annealing temperature was found to be 60°C and 55°C for the PCR assay. When tested for specificity both assays only amplified target DNA thereby proving to be specific. The sensitivity reactions for both the LAMP and PCR assay yielded a detection limit of 0.42 ng and 10-3 ng respectively as the lowest amount of the target DNA that can be detected by the assays. Screening of field samples by PCR yielded negative results on several occasions while the positive control amplified the target gene as expected. Failure to validate the assay using field samples was attributed to poor quality or lack of DNA. Validation of these assays is central to determining their efficacy and potential importance in diagnosing natural infections with high sensitivity and rapidity. Therefore we recommend close evaluation of DNA extraction from faecal material as well as ways of reducing or eliminating PCR inhibitors. The second study was aimed at developing a LAMP and PCR assay for the detection of Trichostrongylus spp. infections in livestock. In this study, the target gene used for LAMP primer design for genus-specific amplification was the ITS2 gene. Two primer sets were designed and from these primers, two PCR assays were developed. All these assays were subjected to various LAMP and PCR conditions respectively in order to determine suitable annealing temperatures for each assay. Various methods of DNA extraction were evaluated for troubleshooting together with the use of already published PCR primers. Both LAMP and PCR assays did not amplify the target gene at different ranges of annealing temperatures tested. Furthermore, no amplification was achieved from control DNA samples extracted using different methods. Negative results were obtained when the PCR was troubleshot using already published primers. The results achieved with the designed assays may be a direct consequence of improperly designed primers; however, the inability of all primer sets to function for LAMP and PCR as well as for already published primers suggests problems with the extraction of DNA. This can be attributed to ineffective disruption of worms or the presence of DNases that may degrade DNA and inhibit its amplification. The results of this study suggest the need to evaluate pre-treatment of worms prior to DNA extraction. Evaluation of the methods used to reduce the effects of PCR inhibitors during extraction and amplification of DNA may also be considered. The third study was aimed at developing a LAMP and PCR assay for detection of Fasciola spp. infections in livestock. Two sets of primers for species-specific amplification by LAMP were designed by targeting the ITS2 gene of both F. hepatica and F. gigantica. Species-specific PCR assays were developed from the latter primers and gene of the parasites and the assays were then tested at various reaction conditions. A genus-specific PCR assay was subsequently developed and tested. No amplification of DNA was observed with the F.hepatica-specific LAMP assay whereas the assay developed for specific detection of F. gigantica produced false positive results. All PCR assays yielded negative results following many attempts to optimize for primer annealing temperature. Reagent contamination was eliminated as the source of non-specific amplification with LAMP suggesting that improper primer design was a possible cause of this problem. On the other hand failed attempts to optimize all the other assays suggest that there is need to evaluate pre-treatment of worms prior to DNA extraction as well as the methods reducing the effects of PCR inhibitors during amplification. Overall, this study successfully achieved the development and optimization of a LAMP and PCR assay for detection of H. contortus DNA. However, subsequent validation of the assays using field derived samples was not possible. The overall results of this study mostly point to faulty primer design and the presence of PCR inhibitors in extracted DNA samples, extracted from either tissue or faecal samples. According to previous studies the presence of inhibitory substances may interfere with the lysis step, inactivate the thermostable DNA polymerase and even interfere with nucleic acids. Therefore evaluation of more accurate methods of mechanical disruption of the worms, DNA extraction, primer design and the use of amplification facilitators may yield desired results. Therefore, these factors should be taken into account for successful development and validation of the molecular diagnostic tool for detection of helminth infections.

Forensic entomology is the study of arthropods associated with bodies. Arthropod successional studies have been successfully used to estimate a postmortem interval. This research was to determine the influence of a) seasons, b) clothing, c) wrapping and d) knife wounds on carcass decomposition and arthropod succession. The experimental site consisted of a 26 hectares grass field interspersed with trees. For the wrapped trials, six pig (Sus scrofa) carcasses were divided into three sample groups, each with a clothed carcass and an unclothed carcass wrapped in sheeting. Arthropod sampling was done (i) daily, (ii) five day intervals and (iii) ten day intervals. Two additional unwrapped carcasses, one with clothes and one without, were sampled daily as controls. For the wounds trials six carcasses were divided into three groups. Each group consisted of a carcass with clothes and one without clothes. The wounds consisted of (i) a knife wound to the throat, (ii) three deep knife wounds, in the back, in the front thoracic and in the front abdominal region. The controls, were without any wounds. Oviposition occurred simultaneously and was not delayed or hastened by the presence of wrapping, clothing or wounds. However, during the winter wrapped trials there was a delay of four days. In winter, the carcasses remained acceptable to Diptera for oviposition over an extended period. Oviposition continued up to two months after placement, whilst in the warmer seasons oviposition occurred within the first few days. The Diptera did not select the wounds as oviposition sites. Calliphoridae and Sacrophagidae were the dominant Diptera recorded during all the trials. In the autumn and summer seasons Chrysomya marginalis and Chrysomya albiceps were the dominant species. In the spring seasons, the dominant species were Chrysomya chloropyga and C. albiceps. In the winter seasons, Sarcophaga cruentata , C. chloropyga, Calliphora vicina, and Lucilia spp. were the species breeding on the carcasses. Muscidae adults were present during all the seasons, but no maggots of this family were recorded. Due to the short oviposition time during warmer seasons, the maggots were of a similar age at any time. Due to the extended oviposition that occurred during winter, different instar groups, often the same species, were present at any time. In all seasons the Coleoptera community present on the carcasses were dominated by Dermestes maculatus (adults and larvae) and Necrobia rufipes. In the summer Thanatophilus micans (adults and larvae) and Histeridae spp. were also recorded on the carcasses. There was no overall difference in arthropod succession between any of the carcasses. During the autumn seasons, noticeable predation by C. albiceps maggots on C. marginalis maggots was observed. There was limited maggot predation during the spring trials and some predation observed during the summer trials. Presence of clothing, wrapping and wounds had no influence the Coleoptera community. In the winter seasons, D. maculatus larvae were found while the maggots were still present on the carcasses. In summer seasons, they were only present after maggot migration. Significant maggot mortality was associated with the wrapped carcasses during the warmer seasons. The presence of the sheets or clothing did allow the maggots to move more freely on the surface of the carcasses, especially in the summer. Less skin remained on the wrapped or clothed carcasses after the maggots migrated to pupate.

A study was conducted on the feeding behaviour of giraffe within the Bloemfontein vicinity, central Free State. Full-day field surveys were done on a monthly basis over a period of 12 months (March 2003 â February 2004) on four different study areas. Night observations were only conducted on a seasonal basis during full moon periods, to make use of maximum vision. Utilisation frequencies and âdurations for each plant species, as utilised by the giraffe, were carefully documented. The daily activity patterns of giraffe were determined by means of the momentary-scanning method. Plant surveys were conducted through circle, quadrants distributed in transects over each of the four study areas to determine the woody composition for each area. Approximately 100 g of leaf material of the most important food plants of giraffe were gathered on a monthly basis for subsequent chemical analyses (calcium and cru-protein). Prehistoric signs of giraffe by means of fossils and rock art have been noted by several writers since the nineteen thirties and forties. The first signs of the presence of giraffe in South Africa by whites were detected in rock art. The Limpopo Province lies in the savannah biome and therefore contains the best habitat for giraffe. It is thus self-evident that most conservation areas with giraffe can be found in the Limpopo Province. There is no earlier physical evidence of giraffe in the Free State. This province is also not part of the current range of giraffe. The biggest concentration of established giraffe presently occurs in the Boshof-district in the western Free State. This can be attributed to the vegetation which correlates with the savannah biome, namely the occurrence of Acacia trees. The extent of the adaptation of giraffe in the central Free State is mainly unknown. As a whole the active browsing of giraffe in this study was responsible for more than half (53 %) of the daily activities. Giraffe only spend 4 % of the total daily time in the lying position, usually when environmental temperatures are high. In contrast with the day-activities, browsing was responsible for less than a third (31 %) of their activity during night time. Peaks in the lying position where all individuals were involved, occurred long before and shortly after midnight alternating with browsing. During this study period giraffe in the central Free State browsed a total of 28 plant species. According to the utilization frequency and -time, the sweet-thorn, asparagus and buffalo-thorn are the most important components in the diet of giraffe. Collectively these three plant species constitute approximately 74 % of all observations and were consumed through out the year. Leaves of deciduous trees and shrubs are the staple food during the wet season but, as the plants shed their leaves and the food stock decrease, there is a change in the food preference. In this connection an increase in the utilisation of evergreen trees, woody- and offshoot fodder during the dry season, is significant. Unidentified grasses constitute nearly 2 % of the total diet of giraffe. Grasses were utilised mainly in August and September, the most critical time of the year which can possibly suggest an imbalance in the diet, which relates to calcium deficiencies. Osteophagia was detected in especially cows and younger individuals during the dry season, probably due to the fact that either cows in calf, nursing cows or growing individuals are more susceptible to mineral deficiencies in their diet. Although the availability of leaves in the dry season is limited, the asparagus leaves are available for utilization until late in this season. Sweet-thorn legumes and buffalo-thorn fruit are also available during the dry season and are utilised together with the sprigs. An increase occurred in the cru-protein contents for some of the leaves, including the sweet-thorn, during the dry season. There is a noticeable increase in the calcium content of the three dominant preferable plant species during the dry season. It seems as if the chemical composition and availability of the three dominant plant species per se play a less important role in the giraffeâs diet compared to the preference these animals show towards certain plant species. Giraffe are mainly browsers of thorny, deciduous trees, especially the Acacia species. Exotic plant species such as eucalyptus-, pine-, poplar- and willow trees as well as conyza weeds, cotoneaster and prickly pear are seasonally utilised in the central Free State. This phenomenon is more significant during the dry season when leaf materials of deciduous plants of the preference plants are absent and thus show a shortage. Critical periods for giraffe feeding in the central Free State are during the late dry- and early wet season (August â October), and not the dry season as a whole. The topography and composition of vegetation in the central Free State have to allow for seasonal habitat selection. Thus there is a correlation between the occurrence of the giraffe in the Free State and the occurrence of their preference food, namely Acacia-tree species.

The mango seed weevil (MSW), Sternochetus mangiferae (Fabricius) (Coleoptera: Curculionidae), generally causes few problems on early-season cultivars, since the fruit are marketed and consumed before adult emergence from the fruit. Adult emergence from late-hanging cultivars, however, results in unattractive lesions that influence the marketability of the fruit. There is little evidence that MSW influences yield, although some authors argue that MSW development in the seed may lead to premature fruit drop. The economic impact of the MSW is primarily based on the fact that it is a major phytosanitary pest, restricting access to new foreign markets and contributing to substantial rejections of fruit destined for existing export countries. The MSW has no natural enemies, is monophagous on mango and completes its entire life cycle within the mango seed. The impact of this pest can, therefore, be greatly reduced by orchard sanitation. Sanitation practices, however, are labour intensive, necessitating producers to rely on alternative or additive control measures. Several semi-penetrant and contact pesticides are registered for MSW control. However, with trans-laminar products it is imperative that treatments coincide with, or are applied just after, the onset of weevil oviposition. This requires intensive and accurate scouting programmes, with an in-depth knowledge regarding the duration of oviposition necessary to ensure seasonal control. When using contact insecticides, applications should coincide with seasonal and daily activity peaks to ensure direct contact. Since adult weevils are extremely inactive, this necessitates an in-depth knowledge of MSW activity patterns. It is also imperative to understand the development cycle and life strategies of the insect in order to know at which time intervention would prove to be the most effective. The product most generally used for MSW control in the Hoedspruit magisterial district of the Limpopo Province is fenthion (Lebaycid® EC 500g/â a.i.). This product is very effective, but does not provide 100% control and can lead to secondary infestations of mango scale, Aulacaspis tubercularis (Newstead) (Hemiptera: Diaspididae), and mealybug (various species). The use of organophosphates on fruit destined for certain overseas markets is also under investigation by the EU. It is for this reason that Westfalia Technological Services, over the past four years, investigated various aspects of MSW general biology, reproduction and control. The investigation into the activity patterns of adult weevils indicated that MSW were crepuscular â nocturnal insects. For this reason, applications with contact insecticides aimed at controlling the adult weevil would be expected to be more efficacious when applied at dusk. During the study investigating MSW development, it was found that the majority of MSW eggs hatched between 7 and 14 days, with some of the first instar larvae already having penetrated into the seeds between 7 and 14 days after oviposition, depending on whether the eggs were laid early or late in the season. This implies that chemical control with contact and semi-penetrant chemicals, aimed at controlling the MSW larvae, should preferably not commence later than 7 days after observing the first eggs in the orchards. However, it was found during the course of this study that MSW oviposition commenced during the latter part of September and continued up to the latter part of January, a period considerably longer than previously stated in the literature. For this reason, more than one chemical application would be warranted. While investigating alternative chemical control measures, it was found that a single application with the systemic insecticide, thiamethoxam (Actara⢠SC 240g/â a.i.), applied during flowering in the root zone, rendered seasonal MSW control. The use of this product, therefore, negates the necessity of tedious fruit inspections and an in-depth understanding of the pest in order to determine the most appropriate time for chemical intervention.