Thesis (Dr. P.H.)--University of Texas School of Public Health, 2008. Thesis (Dr. P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0973. Adviser: Kristy O. Murray. Includes bibliographical references.

Parmi les virus émergents transmis par des moustiques (arbovirus), le genre flavivirus est fortement représenté avec les virus Dengue, Zika, et le virus West Nile (WNV). Le WNV est responsable de nombreux cas de maladies neuroinvasives sévères, parfois mortelles, chez l'humain et les chevaux. Ce virus représente donc un problème de santé publique humaine et animale. Il n'existe pour le moment aucun vaccin humain ni aucun traitement spécifique anti-WNV.Parmi les déterminants viraux essentiels à l'infection par les flavivirus, la glycoprotéine non-structurale NS1 possède des propriétés multifonctionnelles. La forme sNS1, sécrétée dans le milieu extracellulaire, est fortement impliquée dans la dérégulation du système immunitaire de l'hôte. Ces mécanismes participent à l'évasion du virus à la réponse antivirale et, paradoxalement, à la pathogenèse observée dans les formes sévères de la maladie. L'essentiel de ces données concernant le virus de la Dengue, nous souhaitions étudier les propriétés fonctionnelles, in vitro, de la protéine sNS1WNV au cours de l'infection de cellules épithéliales, gliales et neuronales de mammifères. En effet, la structure des protéines sNS1 de flavivirus étant très similaire, notre hypothèse suppose un rôle de sNS1WNV dans les infections neuroinvasives.Si la protéine sNS1WNV ne semble pas moduler les étapes de l'infection virale, elle est cependant à l'origine d'un remodelage du cytosquelette d'actine dans les cellules épithéliales. Elle est aussi impliquée dans l'activation de voies antivirales chez les cellules neuronales non infectées. D'autre part, en ciblant sNS1 et la protéine d'enveloppe E du WNV, nous avons pu isoler, par criblage de molécules aRep (protéines artificielles à motifs répétés), des ligands de haute affinité pour ces déterminants viraux. Ces nouvelles molécules, capables de se lier spécifiquement aux protéines sNS1 et E, ont le potentiel pour servir de base au développement de nouveaux outils de diagnostics et d'agents thérapeutiques antiviraux / Among emerging mosquito-borne viruses (arboviruses), flaviviruses like Dengue, Zika and West Nile virus (WNV) are very often involved in outbreaks. WNV causes several neuroinvasive diseases, which can be lethal, in humans and horses each year. This virus is a threat for both, human and animal public health. Furthermore, there is no human vaccine currently or any specific antiviral treatments against WNV.Among viral factors which are essential for flavivirus infection, the nonstructural glycoprotein NS1 is a multifunctional protein. The secreted form sNS1, is released in the extracellular medium from infected cells and is strongly involved in immune system dysregulation. The functions of sNS1 play roles in immune escape and, paradoxically, in pathogenesis which is observed in severe forms of the disease. Because most of this data are about Dengue Virus, we would like to study, in vitro, functional properties of the sNS1WNV during infection of epithelial, glial and neuronal mammalian cells. Based on the high sNS1 protein structure similarities among flaviviruses, our hypothesis suggests a role of sNS1WNV in neuroinvasive infections.The sNS1WNV protein doesn’t seem to modulate viral infection steps. However, it is involved in actin cytoskeleton remodeling in epithelial cells. sNS1WNV is also involved in the activation of antiviral response pathways in non-infected neuronal cells. On the other hand, by targeting sNS1 and envelope protein E of WNV, we performed a screening of aRep molecules (artificial proteins with alphahelicoïdal repeats) and isolated ligands with high affinity for these viral factors. Because this new type of molecules is able to specifically bind to sNS1 and E, they have potential to be used for the development of new diagnostic tools and antiviral therapeutic agents

Le virus West Nile,WNV est responsable de milliers de cas de morbidité et de mortalité chez les oiseaux, les chevaux et l’homme. Le WNV se transmet par des moustiques du genre Culex. Les méthodes entomologiques ne permettent pas l’évaluation individuelle directe du contact hôte/vecteur. 5 protéines salivaires de Culex ont été sélectionnées, produites, et évaluées comme des candidats antigéniques de l'exposition aux piqûres de Culex. Des sérums humains du sud de France exposés à des densités de Culex distinctes et des sérums de chevaux exposés à l'infection par le WNV ont été testés. Une protéine 30kD est reconnue par les chevaux exposés à Culex. Cependant, pas de différence de réponse d’anticorps n’a été observée entre les animaux faiblement et fortement exposés. Concernant les processus physiopathologiques de la maladie causée par le WNV, la cinétique des profils d'expression de protéines de l’hôte dans le cerveau de souris infectées par le WNV, a été étudiée sur des échantillons prélevés avant et après l’apparition des signes cliniques, en utilisant 2D-DIGE et iTRAQ. 148 protéines différentiellement exprimées. Les voies de signalisation altérées au cours de l'infection précoce et tardive ont été identifiées. Les profils protéiques de LCR de patients atteints de WNND et des individus témoins ont été comparés, en utilisant l’approche iTRAQ. 47 protéines ont été trouvées différemment exprimées chez les patients WNND. Un candidat potentiel biomarqueur, la Defensine-alpha1, a été évalué par ELISA sur des échantillons humains de LCR/sérum. Les biomarqueurs putatifs identifiés dans cette étude peuvent être un outil précieux d’évaluation de la mesure de la gravité du WNV. / West Nile Virus,WNV is responsible for thousands of cases of morbidity and mortality in birds, horses and humans. WNV is transmitted mainly by mosquitoes by Culex species, to avian hosts. Entomological methods did not give direct individual evaluation of the host/vector contact. 5 salivary proteins from the Culex genus were selected for a production under recombinant forms for further evaluation as potential antigenic candidates of exposure to Culex bites. Sera from individuals living in south of France exposed to distinct Culex density and sera from horses exposed to WNV infection were tested. The recombinant protein30 kDa was recognized only by horses exposed to Culex. However, no difference of antibody response between low and high exposed to Culex. Concerning the pathophysiological processes of WNV disease, a kinetics host brain protein expression profiles of WNV-infected mice using samples collected prior and after clinical signs apparition was performed using proteomic approaches 2D-DIGE and iTRAQ. 148 distinct proteins was found altered following WNV infections. The functional signaling networks in samples collected during early and late infection have been identified. Un examination of CSF protein profiles between patients with neuroinvasive disease (WNND) and control individuals was performed using iTRAQ approach. 47 proteins were found differentially expressed in WNND patients compared to controls. A potential biomarker candidates, defensin-alpha1 was assessed by ELISA using other human paired CSF/serum samples. The putative biomarker identified in this study may potentially be a valuable tool in the assessment of the extent of WNV severity.

Background/Objectives: West Nile neuroinvasive disease (WNND) is a severe neurological illness that develops in approximately 1% of individuals infected with West Nile virus (WNV). Manifesting most frequently as encephalitis (WNE), meningitis (WNM), or acute flaccid paralysis (WNP), there is no cure for WNND beyond supportive care and rehabilitation, and death or permanent disability are common outcomes. As the virus arrived in North America less than 20 years ago, determinants of severe disease progression following infection are still being explored. This project is the first to examine comorbid conditions as risk factors of WNND in Ontario using a population-based study design. As prevention is the only avenue of defence against WNND, identifying comorbid risk factors of WNND would allow for public health prevention campaigns targeted to high-risk groups. The main objectives of this thesis were to explore whether pre-existing chronic diseases were associated with the development of WNND, or any of its three manifestations (i.e., encephalitis, meningitis, acute flaccid paralysis). Methods: This was a retrospective, population-based study including all Ontario residents with a confirmed diagnosis of WNV infection between January 1, 2002 and December 31, 2012. A cohort of individuals with WNV was identified from a provincial laboratory database and individually-linked to health administrative databases. In the WNV cohort, individuals with WNND and 13 comorbid conditions were identified using algorithms based on ICD-10-CA diagnostic codes. Incidence of WNND following WNV infection was then compared among individuals with and without comorbid conditions using relative risks estimated by log binomial regression. Additionally, risk ratios were calculated for associations between specific comorbid conditions and WNND neuroinvasive manifestation (i.e., encephalitis, meningitis, acute flaccid paralysis). Finally, associations between Charlson Comorbidity Index (CCI) scoring and development of WNND was examined through calculation of relative risk using log binomial regression. Results/Potential Impact: Risk factors for WNND included male sex (aRR: 1.21; 95% CI: 1.00-1.46) in addition to the combined effect of hypertension and increasing age (5-year intervals) (aRR: 1.16; 95% CI: 1.08-1.24); WNND was also associated with increasing CCI scores; individuals in low, medium, and high categories had increased risk compared to individuals with a score of zero, but the greatest risk was in the high CCI category (aRR: 3.45; 95% CI: 2.25-4.83) Male sex (aRR: 1.32; 95% CI: 1.00-1.76), increasing age (aRR: 1.02; 95% CI: 1.02-1.03), and being immunocompromised (aRR: 2.61; 95% CI: 1.23-4.53) were associated with development of WNE. No risk factors were identified for WNM and WNP. Identification of comorbid risk factors of WNND will allow public health officials to identify high-risk groups and to develop prevention strategies targeted for vulnerable individuals.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related Flaviviruses that are important arthropod-borne human pathogens. Both of these viruses can cause encephalitis with significant morbidity and mortality after infection. Flaviviruses co-circulate in many areas of the world, which raises the risk for sequential infection between heterologous viruses. Sequential infection between dengue virus serotypes can lead to cross-protection, but in some cases, it leads to a severe outcome, dengue hemorrhagic fever. Previous work in hamsters and non-human primates demonstrated that prior JEV immunity protects against a lethal WNV infection. However, the ability of prior WNV immunity to protect against a lethal JEV infection has been inconclusive. WNV-immune hamsters were fully protected from JEV viremia, but in non-human primates, prior WNV-immunity only reduced disease severity, with symptoms of encephalitis still observed. These differences in cross-protection led to further investigation on the directionality as well as the underlying mechanisms for this phenomenon. Previous work in our lab found that JEV-immune C57BL/6J (B6) mice were fully protected against a lethal WNV infection, and JEV-immune CD4+ and CD8+ T cells were required for this cross-protection. In other mouse models, memory cross-reactive CD4+ and CD8+ T cell responses may induce protection or immunopathology upon secondary heterologous viral challenge. We hypothesize that JEV/WNV cross-reactive CD4+and CD8+ T cells preferentially expand upon 2o infection and contribute to cross-protection. To elucidate the potential role of T cells in sequential flavivirus infection, we identified and characterized cross-reactive CD4+ and CD8+ T cell responses between JEV and WNV. A previously reported WNV NS4b CD8+ T cell epitope and its JEV variant elicited CD8+ T cell responses in both JEV- and WNV-infected mice. Despite similarities in viral burden for pathogenic JEV and WNV viruses, CD8+ T cells from pathogenic JEV-infected mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. We believe the differences in the CD8+ T cell responses during primary JEV and WNV infection are due at least in part to the low levels of peripheral replication seen in JEV-infected mice compared to WNV-infected mice. We also found that WNV-immune B6 mice were protected against a lethal JEV infection. Cross-reactive CD8+ T cells in JEV-immune mice rapidly expanded after WNV infection. Even though WNV-immune mice had higher frequencies of memory CD8+ T cells, cross-reactive CD8+ T cells did not expand after secondary JEV infection. Neutralizing antibodies to JEV were detected in WNV-immune mice; however, cross-reactive CD8+ T cells did not expand even in the absence of these cross-reactive neutralizing antibodies. We did not detect any differences in the CD8+ T cell repertoires between JEV- and WNV-infected mice nor were WNV-immune CD8+ T cells functionally exhausted. In fact, proliferation of memory CD8+ T cells did not correlate with the ability of WNV-immune CD8+ T cells to restrict recombinant vaccinia viruses expressing the cross-reactive epitope or lyse peptide-coated targets. These data suggest that the higher frequency of memory CD8+ T cells and cross-reactive antibodies in WNV-immune mice are better able to prevent neuroinvasion following 2o JEV infection.

Since 2008, large numbers of Nile crocodile (Crocodylus niloticus) carcasses were found in the Kruger National Park (KNP), South Africa. Most of the crocodile carcasses were found in the Olifants Gorge, which is situated below the Letaba and Olifants river confluence, before the Mozambique border and Massingir Dam. The Massingir Dam is an important resource and it plays a significant role in the welfare of the local Mozambican population. Autopsies performed on the crocodiles indicated that the adipose tissue colour changed from normal white to yellow and this is usually a sign of pansteatitis. Pansteatitis is caused by lipid peroxidation in an organism and it is characterised by the lack of vitamin E. This disease is recognisable by the hardening of the fatty tissue and yellow discolouration, and is mostly associated with aquatic organisms from polluted ecosystems. There are speculations that the crocodile fatalities may be associated with the Massingir Dam that backed up into the Olifants Gorge after flooding. After the dam was reconstructed, it flooded the Olifants Gorge, causing it to act like a localised sediment trap as the water flow slowed down and as a result, caused pollutants to build-up. Sediment samples were collected from selected rivers and ponds within the KNP. These samples were analysed for selected elements, persistent organic pollutants (POPs), and polycyclic aromatic hydrocarbons (PAHs). The sediment samples were analysed in Norway for POPs and PAHs with the use of a high-resolution gas chromatography/mass spectrometry (GC/MS) and the heavy metals were analysed in South Africa with the use of inductively-coupled plasma mass spectrometry (ICP/MS). In order to identify which elements may have affected the health of the crocodiles, a series of sediment quality indices were used. These indices made it possible to determine which elements may have been involved. The order of probability of heavy metals causing harm was Se>As>Ni>Cr>Cu>I>V>Mn>Co>Fe>Cd>Hg>Zn>Pb>Ba>U. The data was compared to selected international guidelines. All the information was used to determine which of the sampled sites had the highest contamination. The sites sampled with the highest concentrations were in the Crocodile, Nkomati, Olifants, and Letaba Rivers. Concentrations of the elements, POPs, and PAHs were also quantifiable in the Olifants Gorge. The following elements (Fe, Co, Cu, Cr, Pb, V, As, and Ni) were quantified at elevated levels and may therefore have caused negative effects on the crocodiles in the Olifants Gorge. These elevated concentrations, in combination with the dramatic change in the physical environment due to the dam, could have added additional stress that may have contributed to the observed crocodile mortalities in the Olifants Gorge. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Since 2008, large numbers of Nile crocodile (Crocodylus niloticus) carcasses were found in the Kruger National Park (KNP), South Africa. Most of the crocodile carcasses were found in the Olifants Gorge, which is situated below the Letaba and Olifants river confluence, before the Mozambique border and Massingir Dam. The Massingir Dam is an important resource and it plays a significant role in the welfare of the local Mozambican population. Autopsies performed on the crocodiles indicated that the adipose tissue colour changed from normal white to yellow and this is usually a sign of pansteatitis. Pansteatitis is caused by lipid peroxidation in an organism and it is characterised by the lack of vitamin E. This disease is recognisable by the hardening of the fatty tissue and yellow discolouration, and is mostly associated with aquatic organisms from polluted ecosystems. There are speculations that the crocodile fatalities may be associated with the Massingir Dam that backed up into the Olifants Gorge after flooding. After the dam was reconstructed, it flooded the Olifants Gorge, causing it to act like a localised sediment trap as the water flow slowed down and as a result, caused pollutants to build-up. Sediment samples were collected from selected rivers and ponds within the KNP. These samples were analysed for selected elements, persistent organic pollutants (POPs), and polycyclic aromatic hydrocarbons (PAHs). The sediment samples were analysed in Norway for POPs and PAHs with the use of a high-resolution gas chromatography/mass spectrometry (GC/MS) and the heavy metals were analysed in South Africa with the use of inductively-coupled plasma mass spectrometry (ICP/MS). In order to identify which elements may have affected the health of the crocodiles, a series of sediment quality indices were used. These indices made it possible to determine which elements may have been involved. The order of probability of heavy metals causing harm was Se>As>Ni>Cr>Cu>I>V>Mn>Co>Fe>Cd>Hg>Zn>Pb>Ba>U. The data was compared to selected international guidelines. All the information was used to determine which of the sampled sites had the highest contamination. The sites sampled with the highest concentrations were in the Crocodile, Nkomati, Olifants, and Letaba Rivers. Concentrations of the elements, POPs, and PAHs were also quantifiable in the Olifants Gorge. The following elements (Fe, Co, Cu, Cr, Pb, V, As, and Ni) were quantified at elevated levels and may therefore have caused negative effects on the crocodiles in the Olifants Gorge. These elevated concentrations, in combination with the dramatic change in the physical environment due to the dam, could have added additional stress that may have contributed to the observed crocodile mortalities in the Olifants Gorge. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Doctor of Philosophy / Department of Anatomy and Physiology / Elizabeth G. Davis / Activation of innate immunity through Toll-like receptor (TLR) signaling can also enhance antigen-specific adaptive immunity. TLR9 is an endosomal receptor for unmethylated bacterial and viral cytosine-phosphate-guanine DNA (CpG-DNA). West Nile virus (WNV) infection may result in meningitis and encephalitis in humans and horses, especially aged and immunocompromised individuals. Using flow cytometric analyses and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated equine cell-mediated immunity (CMI) to an inactivated West Nile virus vaccine in healthy yearling and adult horses. We also studied the potential of enhancing equine adaptive immunity to viruses and other pathogens by activation of innate immunity though TLR9 signaling pathway. We found vaccination with inactivated WNV vaccine induced strong WNV-specific T helper type 1 (Th1) and Th2 CMI with a Th1 bias, also effectively induced WNV-specific CTLs in yearling horses. In adult horses, the pre-existing Th1 CMI bias against WNV was enhanced following booster vaccination with inactivated WNV vaccine. Molecular characterization and flow cytometric analysis of TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in neutrophils (PMNs), CD4[superscript]+ and CD8[superscript]+ T cells and other leukocytes. Conservation of equine TLR9 and a high expression profile among leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity. Unmethylated CpG-DNA can significantly activate equine PMNs. It also induces expression of interferon (IFN)-[Alpha], IFN-[Beta], IFN-[Gamma], and interleukin (IL)-12p35 in PBMCs, as well as IFN-[alpha] and IFN-[gamma] in monocyte-derived DCs. Enhanced expression of IFNs in immune cells by CpG-DNA is not only crucial for host viral clearance, but also important in mediating host immune responses due to IFNs' anti-inflammatory effects. Compared to the relatively weaker activation of equine innate immunity by inactivated WNV, the tested CpG-DNA species showed potential as vaccine adjuvants for enhancement of CTLs and Th1 CMI against intracellular pathogens, characterized by significant induction of type I IFNs and Th1-specific cytokines such as IL-12p35 and IFN-γ. These data provide a basis for further investigation of these CpG-DNA species as potentially effective vaccine adjuvants in horses.

Aquaculture production is increasing annualy and wild fisheries for fishmeal production remain stagnant. As a consequence, extensive research has been deployed to reduce dietary fishmeal inclusion in feeds of farmed species. Usage of alternative protein sources derived from plants continues to increase with the most popular sources being oilseeds, legumes and cereal grains. The downside of these sources is that most of them could directly be used for human consumption arising legitimate criticism from voices referring to countries where protein shortages lead to malnutrition among the population. Jatropha curcas is a tropical oilseed with upcoming popularity for sustainable fuel sourcing. The plant is thought to thrive in semi-arid and arid areas, not just producing oil, but at the same time reclaiming previously eroded land for the local population. For these reasons, annual cultivation of Jatropha curcas is thought to rise over the next decades. After oil is extracted from the seed, the remaining press cake, is currently used as a fertilizer or energy source. This is mostly due to toxic phorbol esters that until recent- ly limited any nutritional applications. In 2011, a method to detoxify Jatropha press cake was developed and paved the way for nutritional research on the resulting detoxified Jatropha curcas kernel meal (JKM) to be launched. JKM offers very high protein content with a balanced amino acid composition suggesting opportunities for usage as a feedstuff in aquaculture diets. JKM further has higher mineral content than comparable oilseeds. However, potential anti-nutritional factors (ANFs) present in the meal could lead to impairment of nutrient availability or other adverse effects. Previous research has already started to evaluate JKM as a protein source for a variety of aquaculture species. This thesis attempts to further identify the potential of JKM as a protein source and assess the effects of JKM on the development of two model cultured teleost species, common carp (Cyprinus carpio) and Nile tilapia (Oreochromis niloticus), as they represent two of the most farmed species, particularly in the tropics. The work comprises growth trials on both species using fishmeal as a subject of replacement in solely fishmeal protein-based diets and on more practical fishmeal/plant protein-based diets. It engages with the effects of phytate, a prominent component of JKM with potential anti-nutritional attributes and commercially available phytase feed supplements to diminish these attributes, as well as oxalate, another plant-specific component with high concentrations in JKM and with limited attention in aquaculture nutrition research. Carp and tilapia showed varying results with different inclusion levels of JKM. For carp 50% fishmeal replacement was possible without losses in growth in diets where fishmeal was the only bulk protein source (Chapter 3.1), Tilapia showed slightly worse growth at a 30% replacement level (Chapter 3.2). A steep decline in growth could be observed when replacing 100% fishmeal with JKM in carp (Chapter 3.1), while tilapia showed no difference at that level compared to 30% replacement (Chapter 3.2). In practical diets, 100% of fishmeal could be replaced by JKM without any adverse effects on growth of carp (Chapter 3.3), while tilapia showed a slight, but significant linear negative correlation with higher inclusion levels of JKM (Chapter 3.4). Phytase addition in tilapia feeds was identified as having no obvious impact on growth in JKM based diets where enough available phosphorus was provided through mineral supplementation (Chapter 4.1). In JKM based diets where available phosphate was not added, phytase addition showed a tendency to increase growth and significantly increased mineral retention and decreased phosphorus effluent contamination (Chapter 4.2). Phytase application through pre-incubating JKM along with citric acid exerted a positive effect of growth on carp when fishmeal protein was replaced by 50% (Chapter 3.1). Phytase was further shown to completely hydrolyze phytate in vitro; however, incomplete hydrolysis was observed in vivo in tilapia (Chapter 4.3). Dietary soluble oxalate added to fishmeal based diets for carp showed better growth parameters, nutrient and mineral retention at inclusion levels 1.5% and higher (Chapter 5.1). For tilapia, a trial could demonstrate adverse effects of oxalate on potassium, calcium, manganese and zinc digestibilities, in this case without negative effects on growth (Chapter 5.2). For both, carp and tilapia, an impact of oxalate on lipid metabolism was evident, lowering body lipid content and blood cholesterol in inclusion levels from 1.5% or higher. JKM can become a valuable alternative to present dietary protein sources in aquaculture feeds. The nutritional attributes of JKM need further research, especially longer-term testing in a commercial scenario and application in commercially produced feeds. Results of this thesis pose a useful addition to previous research and can be referred to for realizing these next steps.

The bacterial pathogens Streptococcus agalactiae and S. iniae have the capacity to infect a wide range of fish species throughout the world, with Nile tilapia (Oreochromis niloticus) being particularly susceptible. Global tilapia aquaculture production was estimated to be 3.5 million tonnes in 2008, and has a significant contribution in the global farmed fish market. Due to their ability to adapt to a wide range of culture systems the commercialisation of tilapia production has occurred in more than 100 countries. However, countries such as China have suffered from severe and extensive outbreaks of streptococcosis in cultured tilapia continuously for many years. Such large-scale outbreaks in China have resulted in a loss of approximately US$0.4 billion in 2011. Fish are permanently exposed to a plethora of pathogens and natural disease outbreaks are complex host-pathogen interactions that seldom involve single pathogen infections. As a consequence, simultaneous infections, alternatively called concurrent or co-infections, are starting to receive interest from aquatic disease researchers. Streptococcus agalactiae and S. iniae infections can both occur in the same geographic area and both S. agalactiae and S. iniae have been found to be present on the same farm in a single disease outbreak. It has been found that a disease outbreak caused by one these pathogens can be followed by another outbreak from the other. These two pathogens have serious effects on the tilapia aquaculture industry yet there is no information regarding S. agalactiae and S. iniae co-infections. Such information would be valuable for understanding epidemiology and the development of improved treatment and control of aquatic streptococcosis infections. The overall aim of this study was to investigate the pathogenesis of S. agalactiae and S. iniae in Nile tilapia. One important aspect of investigating simultaneous infections was to examine if there was any competition or synergy between S. agalactiae and S. iniae in vitro or in vivo. It was found that competition between S. agalactiae and S. iniae in vitro was inconsistent between different experimental systems. Results indicated that there was either no interaction between bacterial species or they coexisted during in vitro competition assays. Whereas, an in vivo model utilising wax moth larvae (Galleria mellonella) suggested that during a simultaneous infection with S. agalactiae and S. iniae the total levels of larval mortality were lower than expected indicating that the pathogens may have interacted with one another in a competitive manner. Investigations were also conducted to identify the expression of virulence factors in vitro for S. agalactiae and S. iniae. Comparisons were then made to ascertain any inter- and intra-species variation. Results demonstrated that both S. agalactiae and S. iniae strains possessed a capsule but varied in their haemolytic activity, blood survival and resistance to complement-mediated killing. These variations suggested that the two bacterial species differed in their mechanisms of pathogenicity where aquatic S. agalactiae strains may initially have a more systemic spread of infection and aquatic S. iniae strains may utilise a more localised spread of infection within the host. This hypothesis was tested through the development of a robust and reliable challenge model for S. agalactiae and S. iniae in Nile tilapia. Through this work it was apparent that fish infected with S. iniae experienced an acute infection with morbidity/mortality occurring 1 – 3 days after exposure. Whereas, the S. agalactiae challenged fish showed a more chronic infection with morbidity/mortality occurring from 1 – 6 days after exposure. Findings clearly demonstrated a more systemic spread of infection during a S. agalactiae challenge with high bacterial loads in all the organs examined. Streptococcus iniae was observed in fewer organs of infected fish and bacterial numbers were substantially lower. Concurrent infections are complex in natural conditions and in experimental studies. As a result a substantial amount of research will be required to fully understand the nature of co-infection with these two streptococci. This study has provided a solid foundation upon which to base future work.