Os principais marcadores sorológicos para o diagnóstico da toxoplasmose aguda ou recente são os anticorpos IgM específicos e anticorpos IgG de baixa avidez. Entretanto em alguns pacientes, anticorpos IgM e baixa avidez de anticorpos IgG podem persistir, ultrapassando o período da fase recente aguda contribuindo para erros de interpretação diagnóstica. No presente estudo, a eficiência diagnóstica do ensaio imunoenzimático foi avaliada, com o uso de frações antigênicas ou peptídeos sintéticos originados do antígeno ESA de T.gondii, denominados de SAG-1, GRA-1 e GRA-7. Foram estudadas frações isoladas e combinadas em múltiplos peptídeos antigênicos (MAP), visando estabelecer um perfil confiável para definição sorológica de toxoplasmose recente aguda em amostra única de soro. A melhor eficiência diagnóstica do ensaio foi encontrada com o uso da combinação de peptídeos SAG- 1,GRA-1 e GRA-7, denominada MAP1. A detecção de anticorpos IgG e IgM anti- MAP1 apresentou a melhor definição entre a fase recente aguda da fase recente não aguda na toxoplasmose. Nossos resultados mostraram que IgM anti-MAP1 poderá se constituir um marcador sorológico importante no aumento da eficiência diagnóstica da toxoplasmose recente aguda / The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis

A toxocaríase é considerada uma das cinco parasitoses negligenciadas pelo Centers for Disease Control and Prevention e recebe ainda pouca atenção. As metodologias diagnósticas conhecidas são bem estabelecidas, apresentando, porém, limitações caracterizadas, sobretudo, pela ocorrência de reações-cruzadas. A biologia molecular mostra grandes avanços para o diagnóstico eficaz de diversas parasitoses, mas ainda carece de estudos em amostras de fácil obtenção para o diagnóstico da toxocaríase. Para aprimorar o conhecimento sobre a importância da técnica da Reação em Cadeia da Polimerase Convencional (PCR) e sua relação com técnicas diagnósticas já conhecidas, foram utilizados 42 camundongos BALB/c, machos, entre 6 a 8 semanas de vida, divididos em três grupos, inoculados com 5, 50 ou 500 ovos larvados e sangrados pelo plexo orbital aos 15, 30, 60 e 90 dias pós infecção. Ainda, do total, 24 camundongos foram sangrados aos 120 dias pós infecção. Ao final do experimento, foi realizada a recuperação de larvas e a PCR de tecido hepático, cérebro e carcaça de camundongos dos grupos infectados. As amostras de soro foram processadas pelas técnicas de ELISA, Western-blotting e PCR. O ELISA e o Western-blotting mostraram resultados reagentes em todas as datas para a maioria dos inóculos de ovos, com relação diretamente proporcional entre a detecção de anticorpos e a carga parasitária. Durante o período da infecção, a detecção de IgG foi mais intensa próxima aos 60 dias pós-infecção para a maioria dos inóculos de ovos, por ambos os métodos imunológicos. Apesar de identificar DNA de larvas e vermes adultos, a PCR não foi capaz de detectar DNA do parasito em amostras de soro em todos os grupos e datas pós-infecção. Em contrapartida, foi detectado DNA do parasito em todos os órgãos com ao menos um dos primers utilizados. Foram recuperadas larvas na maioria dos órgãos com maior porcentagem de recuperação relatada nos animais inoculados com 50 ovos larvados. O diagnóstico molecular, utilizando sangue do paciente, ainda não pode ser considerado uma ferramenta para o diagnóstico dessa infecção / Toxocariasis is considered by the Centers for Disease Control and Prevention one of the five neglected diseases and still receives little attention. The diagnostic methods are well established, presenting, however, limitations characterized mainly by the occurrence of cross-reactions. Molecular biology shows great advance for the effective diagnosis of several parasitic infections, but still lacks studies using samples that are easily obtained for the diagnosis of toxocariasis. In order to refine the knowledge about the importance of Conventional Polymerase Chain Reaction (PCR) and its relation with known techniques, 42 BALB/c male mice, between 6-8 weeks of age were inoculated with 5, 50 and 500 embryonated eggs respectively and bled by the orbital plexus at 15, 30, 60 and 90 days post infection. Also, 24 of 42 animals were bled the same way at 120 days post-infection. At the end of the experiment, larval recovery and conventional PCR were performed in liver, brain and carcass of mice of the infected groups. Serum samples were processed by ELISA, Western-blotting and PCR. The ELISA and Western-blotting techniques showed positive results in all days post infection for most eggs inocula and showed a directly proportional dependence between the infective dose and the level of antibodies. During the course of the infection, IgG detection was most intense near 60 days post infection for most eggs inocula, for both diagnostic methods. Despite positive DNA identification in larvae and adult worms, PCR wasn\'t able to detect parasite DNA in serum samples in all infected groups and days post infection. In contrast, parasite DNA was detected in all organs with at least one of the primers. Larvae were recovered from most organs, and animals inoculated with 50 embryonated eggs showed the highest percentage of larval recovery. Molecular diagnosis using patient\'s blood is not the best tool for toxocariasis diagnosis so far

Philosophiae Doctor - PhD / Polyaniline nanocomposites doped with poly(vinylsulphonate) (PV-SO3) and nanostructured polystyrene (PSNP) latex beads functionalized with amine (PSNP-NH2) and sulphate ((PSNP-OSO3) were prepared and characterised for use as nitrite electro-catalytic chemosensors and ochratoxin A immunosensors. The resultant polyaniline electrocatalytic chemosensors (PANI, PANI|PSNP-NH2 or PANI|PSNP-OSO3 −) were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Brown-Anson analysis of the multi-scan rate CV responses of the various PANI films gave surface concentrations in the order of 10−8 mol/cm. UV-vis spectra of the PANI films dissolved in dimethyl sulphoxide showed typical strong absorbance maxima at 480 and 740 nm associated with benzenoid p-p* transition and quinoid excitons of polyaniline, respectively. The SEM images of the PANI nanocomposite films showed cauliflower-like structures that were <100 nm in diameter. When applied as electrochemical nitrite sensors, sensitivity values of 60, 40 and 30 μA/mM with corresponding limits of detection of 7.4, 9.2 and 38.2 μM NO2 −, were obtained for electrodes, PANI|PSNP-NH2, PANI and PANI|PSNP-SO3 −; respectively. Immobilisation of ochratoxin A antibody onto PANI|PSNP-NH2, PANI and PANI|PSNPSO3 - resulted in the fabrication of immunosensors. / South Africa

<p>Polyaniline nanocomposites doped with poly(vinylsulphonate) (PV-SO3 &minus / ) and nanostructured polystyrene (PSNP) latex beads functionalized with amine (PSNP-NH2) and sulphate (PSNP-OSO3 &minus / ) were prepared and characterised for use as nitrite electro-catalytic chemosensors and ochratoxin A immunosensors. The resultant polyaniline electrocatalytic chemosensors (PANI, PANI|PSNP-NH2 or PANI|PSNP-OSO3 &minus / ) were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Brown-Anson analysis of the multi-scan rate CV responses of the various PANI films gave surface concentrations in the order of 10&minus / 8 mol/cm. UV-vis spectra of the PANI films dissolved in dimethyl sulphoxide showed typical strong absorbance maxima at 480 and 740 nm associated with benzenoid p-p* transition and quinoid excitons of polyaniline, respectively. The SEM images of the PANI nanocomposite films showed cauliflower-like structures that were &lt / 100 nm in diameter. When applied as electrochemical nitrite sensors, sensitivity values of 60, 40 and 30 &mu / A/mM with corresponding limits of detection of 7.4, 9.2 and 38.2 &mu / M NO2 &minus / , were obtained for electrodes, PANI|PSNP-NH2, PANI and PANI|PSNP-SO3 &minus / , respectively. Immobilisation of ochratoxin A antibody onto PANI|PSNP-NH2, PANI and PANI|PSNPSO3 - resulted in the fabrication of immunosensors.</p>

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV)&nbsp / showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a&nbsp / group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in&nbsp / children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of&nbsp / HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified&nbsp / ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie&nbsp / Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the&nbsp / production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the&nbsp / Western Cape.</p>

Lectins are a class of proteins that bind to carbohydrates with a high degree of specificity. They are involved in various cellular processes such as, host - pathogen interactions, targeting of proteins within cells, cell - cell interaction, cellular segregation and development. They serve as important tools for probing the carbohydrate structures in biological systems such as cell membranes and also as model systems for elucidating protein - carbohydrate interactions. Lectins are distributed ubiquitously in nature ranging from microorganisms to the plants and animals. Plant lectins are a group of proteins that according to a recently updated definition comprise all plant proteins possessing at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharide. The majority of all currently known plant lectins may be classified into four major groups - (1) Legume lectins, (2) Chitin-binding lectins, (3) Type 2 Ribosome inactivating proteins and the (4) Monocot mannose binding lectins. The monocot mannose binding lectins are an extended superfamily of structurally and evolutionarily related proteins. Till now these proteins have been isolated from the following families, namely, Amaryllidaceae, Affiaceae, Araceae, Orchidaceae, Iridaceae and Li/iaceae. They exhibit marked sequence homology and a unique specificity for mannose. At present there is a wide interest in the monocot mannose-binding lectins because of: (1) their exclusive specificity towards mannose, (2) their anti - retroviral activity and (3) their potent entomotoxic properties. Of particular interest are lectins from the bulbs of garlic (Allium sativum) and ramson (A. ursinum), which contain more than one type of lectin. The first report of the presence of lectins in the bulbs of garlic {Allium sativum agglutinin, ASA) was made by Van Damme et al in 1991. Bulbs of garlic are known to accumulate two types of mannose binding lectins, the heterodimeric, ASAI and the hornodimeric, ASAII. Though these two lectins differ in the lengths of their polypeptide chains, they exhibit marked similarities with respect to their primary sequence, post translational modifications, serological properties, immunochemical attributes as well as carbohydrate binding properties. This thesis describes the successful cloning of the ASAI gene from the garlic genomic DNA and expression of the functional recombinant protein in insect cell lines. ASAI was subsequently characterized for its carbohydrate binding specificity by means of a sensitive enzyme based assay. Finer insights into this sugar binding topology of ASAI for its complementary ligands was obtained from the surface plasmon resonance studies. Lastly, the folding behaviour as well as an estimate of its conformational stability was investigated by differential scanning calorimetric and equilibrium solution denaturation studies. Chapter 1 provides a comprehensive review on lectins pertaining to their definition, historical background, occurrence in nature, three dimensional structure and architecture, modes of bonding, biological functions and implications as well as their applications in biomedical research. Chapter 2 describes the isolation and purification of the heterodimeric lectin, ASAI in two steps using affinity chromatography followed by gel filtration chromatography from the bulbs of garlic. The purified ASAI was then characterized for their serological, physico- and immuno-chemical properties by means of capillary electrophoresis, hemagglutination activity and generation of antisera against ASAI in rabbits. Chapter 3 revolves around the cloning of the gene encoding ASAI by PCR amplification from garlic genomic DNA. The authenticity of the ASA gene was established by means of gene sequencing, which in turn provided us with the primary sequence of this lectin. With the ASAI clone established innumerable attempts, as highlighted in the chapter, were made to express the functional protein in bacteria. All attempts yielded pure recombinant garlic lectin with no detectable activity. This prompted us to shift our efforts into expression of the recombinant protein in the baculovirus expression system using the Sf21 insect cell lines and the Autographa californica nuclear polyhedrosis virus (AcNPV). The choice of this system proved beneficial as we obtained functional recombinant garlic lectin with its hemagglutinating activity comparable to the native protein. Chapter 4 highlights the design of an elegant coupled enzyme-based colorimetric assay (Enzyme Linked Lectin Adsorbent Assay) for elucidation of the carbohydrate binding specificity of ASAI. This expansive and extensive study involved the assay of a wide range of mannooligosaccharides in order to gain an insight into the sugar binding details of ASAI. ASAI recognizes monosaccharides in the mannosyl configuration. The potencies of the ligands for ASAI is shown to increase in the following order: Mannobiose < Mannotriose Mannopentaose Man9 oligosaccharide. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied exhibited the greatest binding affinity suggesting ASAI to possess a preference for cluster of terminal αl-2-linked mannosyl residues at the non-reducing end. This kind of exquisite specificity is unique in the lectins described so far. Among the glycoproteins assayed, invertase, soyabean agglutinin and ovalbumin displayed high binding affinity. Chapter 5 unravels the fine specificity of the mannose containing carbohydrate moieties for binding to ASAI with emphasis on their kinetics of binding. This has been achieved by invoking the principle of surface plasmon resonance allowing measurement of bimolecular interactions in real time. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal a α1-2 linked mannose residues. Increase in binding propensity can be directly correlated to the addition of αl-2 linked mannose to the mannooligosaccharide at its non-reducing end. An analyses of these data reveals that the α1-2 linked terminal mannose on the α1-6 arm to be the critical determinant in the recognition of mannooligosaccharides by the lectin. While kI increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k-1 decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with ASAI with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition. Chapter 6 deals with the thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism which shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 2U). Moreover, its conformational stability has been determined as a function of temperature; GdnCl concentration and pH using a combination of thermal and isothermal GdnCl induced unfolding monitored by DSC, far-UV CD and fluorescence, respectively. Analysis of these data yielded the heat capacity change upon unfolding (∆CP) as also the temperature dependence of the thermodynamic parameters, namely, ∆G, ∆H, ∆S. The protein appears to attain a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates very well with its X-ray structure. The final chapter summarizes the findings reported in the thesis.

<p>Polyaniline nanocomposites doped with poly(vinylsulphonate) (PV-SO3 &minus / ) and nanostructured polystyrene (PSNP) latex beads functionalized with amine (PSNP-NH2) and sulphate (PSNP-OSO3 &minus / ) were prepared and characterised for use as nitrite electro-catalytic chemosensors and ochratoxin A immunosensors. The resultant polyaniline electrocatalytic chemosensors (PANI, PANI|PSNP-NH2 or PANI|PSNP-OSO3 &minus / ) were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Brown-Anson analysis of the multi-scan rate CV responses of the various PANI films gave surface concentrations in the order of 10&minus / 8 mol/cm. UV-vis spectra of the PANI films dissolved in dimethyl sulphoxide showed typical strong absorbance maxima at 480 and 740 nm associated with benzenoid p-p* transition and quinoid excitons of polyaniline, respectively. The SEM images of the PANI nanocomposite films showed cauliflower-like structures that were &lt / 100 nm in diameter. When applied as electrochemical nitrite sensors, sensitivity values of 60, 40 and 30 &mu / A/mM with corresponding limits of detection of 7.4, 9.2 and 38.2 &mu / M NO2 &minus / , were obtained for electrodes, PANI|PSNP-NH2, PANI and PANI|PSNP-SO3 &minus / , respectively. Immobilisation of ochratoxin A antibody onto PANI|PSNP-NH2, PANI and PANI|PSNPSO3 - resulted in the fabrication of immunosensors.</p>

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV)&nbsp / showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a&nbsp / group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in&nbsp / children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of&nbsp / HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified&nbsp / ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie&nbsp / Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the&nbsp / production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the&nbsp / Western Cape.</p>

Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns similar to those observed on western blotting, where anti-D. pinea, anti-F. circinatum and anti-F. oxysporum antibodies showed the same cross-reactivity relationships. Anti-F. circinatum and anti-F. oxysporumantibodies showed a significant difference when reacting with antigens isolated from other pathogens including D. pinea, F. circinatum, F. oxysporum, F. solani, F. graminearum and F. culmorum (P = 0.001). No significant difference was observed when the antigens were detected with anti-D. pinea antibodies. Reactivity of anti-F. circinatum-exo and anti-D. pinea34 kDa antibodies was mostly similar to that of non-immune antibodies and showed no significant difference between detection of different antigens. Pine seedlings were artificially infected with the three fungal pathogens using a spore concentration of 1 – 1 x 106conidiaml-1.Infection was monitored using scanning electron microscopy. Results showed increased levels of mycelium growth on the stem and roots of the F. circinatum and F. oxysporum infected seedlings and on the leaves and stem in the case of D. pinea infected seedlings. These plant parts were used in ELISA tests for the detection of antigens. Isolation of antigens from the plant materials involved crushing plant parts in buffer and centrifugation of the suspension. The supernatant obtained was directly used in the assay. ELISA tests prepared in this study were sensitive enough to detect infection caused by 1 conidium ml-1at two weeks post inoculation. A positive reaction for detection of F. circinatum and F. oxysporum was indicated by an ELISA reading above an optical density at 405 nm. The plant material used in ELISA tests were further analysed using PCR. Results indicated that there was no cross-infection between seedlings and served as a confirmation of the disease-causing pathogen. This indicated that cross-reactivity observed was due to other factors such as common epitopes on the major antigens. Use of an ELISA dip-stick or ELISA using these antibodies should provide an easy, fast field test to identify infections of pine, discriminating between F. circinatum, F. oxysporum and D. pinea. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and costimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.