West Nile virus (WNV) belongs to the Flaviviridae family, a virus family of which many members are known as human pathogens. WNV has a worldwide distribution and strains that cluster in lineage II is endemic to sub-Saharan Africa. The complete nucleotide sequence of four lineage II West Nile virus strains, isolated in South Africa from patients with mild or severe WNV infections, were determined. Using a murine model, these strains had been shown to produce either highly or less neuroinvasive infections and induced similar genes to corresponding highly or less neuroinvasive lineage I strains. Nucleotide and amino acid sequence comparison between highly and less pathogenic lineage II strains demonstrated that the non-structural genes and in particular the gene coding for the NS5 proteins were the most variable. All the lineage II strains sequenced in this study were found to possess the E-protein glycosylation site previously postulated to be associated with virulence. Comparison of the signalase cleavage sites suggested that lineage II strains may be cleaved slightly more efficiently than lineage I strains in the C-prM junction, but less efficiently between prM and E genes. Relative to the highly neuroinvasive strains sequenced in this study major deletions were found in the 3’ noncoding region of 2 lineage II strains shown in previous studies to be either less- or not at all neuroinvasive. This is the first report of full genome sequences of highly neuroinvasive lineage II WNV strains. Currently available commercial WNV ELISA kits were developed with lineage I WNV strains and are expensive to use. For these reasons the development of a potential ELISA diagnostic assay based on the South African lineage II strain, H442, was envisaged. Such assay, if reliable and efficacious would be a useful tool towards WNV surveillance. The prM and E genes were selected to be expressed as recombinant antigens because of their co-expression nature and because the envelope protein is the principal target for neutralization. After cloning of the respective genes and verification of integrity, a mammalian expression system was utilized. Different mammalian cells and transfection media were tested and BHK 21 cells with SuperFect transfection medium were found to be best. Attempted expression of proteins was tested with immunofluorescent antibody testing as well as SDS-PAGE and Western blot analysis. Expression of recombinant WNV antigens were also tested in indirect and sandwich ELISA’s systems. It was however not possible to perform these two ELISA systems at a satisfactory level or clearly indicated if expression of proteins was successful. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Urine samples are routinely used in human and animal patients to diagnose health problems; often to investigate or monitor specific health-related problems that essentially may remain silent for extended periods. However, not much work has been performed on crocodilian urine for diagnostics. In general, crocodilian species lack a bladder as a separate storage organ (as found in mammals), possess metanephric kidneys (unable to concentrate urine) and have functional salt excreting glands. Collection of urine from the Nile crocodile (Crocodylus niloticus) is a simple and atraumatic procedure where a dog urinary catheter is used to collect relatively clean urine from the urinary chamber in the crocodile’s cloaca. Unfortunately, in-depth investigations of urine variables, and establishing baseline concentrations, have not been performed on Nile crocodile urine samples before. The specific focus areas of this research project were: (1) determination of urine and plasma biochemical concentrations by means of a standard veterinary clinical pathology profile and the establishment of the ratio between urine and blood biochemical parameters; (2) the validation of a gas chromatographic–mass spectrometric (GC/MS) method for the determination of steroid metabolite concentrations in urine; and (3) using this established analytical method to determine the presence (identify) and concentrations of steroid metabolites in the urine of individual crocodilians. / Urine and plasma samples collected at Izintaba Crocodile Farm during the period November 2005 to July 2006, from captive bred, healthy young Nile crocodiles, were analysed for standard biochemistry variables. The urine samples (n = 101) were analysed for sodium, potassium, chloride, urea, creatinine, calcium, magnesium, phosphate, uric acid, osmolality, and ammonium ion, while the plasma samples (n = 101) were screened for total protein, glucose, sodium, potassium, chloride, urea, creatinine, total calcium, ionised calcium, magnesium, phosphate, uric acid and osmolality. Means, medians and standard deviations were statistically determined, as well as urine to plasma (U/P) ratios for corresponding variables. The value of this project is the establishment of reference concentrations for Nile crocodile urine samples that may become useful for interpretation of laboratory results, in future. / The clinical validation of a GC/MS method for the analysis of urinary steroids in the Nile crocodile was achieved using urine samples from two-year-old Nile crocodiles. The main objective of this investigation was to develop, optimize and validate the laboratory analysis of urinary steroid metabolites. Steroid profiling was performed on individual and pooled Nile crocodile urine samples. Ascending concentrations of representative steroid standards: androsterone, etiocholanolone, dehydroepiandrosterone, 11-OH androsterone, pregnanediol, pregnanetriol, 11-deoxytetrahydrocortisol, tetrahydrocortisone, tetrahydrocortisol and tetra-hydrocorticosterone, were spiked into aliquots of the pooled urine samples, to obtain calibration samples ranging from 0.2 to 20 μg. Sample preparation and analysis methodology were based on a well-established, validated GC/MS method for determination of human urinary steroid metabolites. The validation of the GC/MS method for Nile crocodile urine was successfully completed, by determining lower limits of quantitation and limits of detection for each analyte, obtaining linearity up to the highest calibration level, correlations exceeding 0.90, and recoveries of 82% and more. / Steroid profiling was performed on urine samples collected from a number of mature crocodilian species, namely Nile crocodile, American alligator (Alligator mississippiensis), spectacled caiman (Caiman crocodilus) and dwarf crocodile (Osteolaemus tetraspis). Steroid metabolites were identified and were quantitated and reported per urinary creatinine. Qualitative reporting was conducted in cases where creatinine concentrations were not available. Results included identification and quantitation of the steroid metabolites: androsterone, etiocholanolone, 11-hydroxy androsterone, pregnanediol, pregnanetriol, and the tetrahydro- metabolites of cortisone (THE), cortisol (THF), and corticosterone (THB). In some urinary steroid profiles, several prominent peaks were observed which could not be identified. The study findings confirmed that crocodile urine could successfully be used, as it is commonly used in humans, to determine steroid metabolite profiles. A follow-up study to identify the unknown peaks by structure elucidation with more sophisticated equipment is recommended - this could lead to valuable information about liver metabolism of steroids in crocodilians. / An adrenocorticotrophic hormone (ACTH) stimulation test was conducted on 18 captive Nile crocodiles. The experimental animals were temporarily housed in separate enclosures at Le Croc Crocodile Farm for four weeks, to ensure controlled conditions and easy and frequent access to the animals. Twenty-seven urine samples were collected both pre- and post-ACTH or saline injections. Steroid profiling was performed on 24 of the 27 urine samples to assess the corticosterone and tetrahydrocorticosterone concentrations following the ACTH treatment. Quantitation relative to urine creatinine levels was recorded following analyses with a standardised liquid chromatography/mass spectrometry (LC/MS) method, reporting the concentrations in nmol steroid/μmol creatinine. Unfortunately, a significant increase in urinary corticosterone concentrations 6 h after the injection of Synacthen® (5 μg/kg) was not observed. A possible explanation for this could be that the 6 h period was too short for a significant increase in urinary glucocorticoid metabolite excretion in the Nile crocodile. / In conclusion, this is the first in-depth study that focused, specifically, on Nile crocodile urine for analyses as diagnostic tools and for indices of health. The screening of the urine samples, collected from healthy Nile crocodiles, for a large array of biochemical variables contributed significantly to the database of “normal” concentrations. The establishment of a validated urinary steroid profiling method may significantly contribute to future validation and implementation of innovative diagnostic methods to monitor the health status and endocrine systems of wild Nile crocodiles in Africa. / Thesis (PhD)--University of Pretoria, 2017. / The Norwegian Council for Higher Education’s Programme for Development, Research and Education (NUFU) / Royal Netherlands Embassy in South Africa / Crocodile Specialist Group / SAVF / Paraclinical Sciences / PhD / Unrestricted

The Nile crocodile (Crocodylus niloticus) is one of 23 extant crocodilian species, and has been farmed in southern Africa since the 1960s. For the crocodile industry, chronic stress and its often negative consequences are a concern, since stressors can negatively affect animal production as well as the health of the crocodiles. When confronted with a stressor, an individual displays a stress response consisting of a suite of physiological and behavioral alterations to cope with the challenge. So far, however, no method for determining stress-related responses in Nile crocodiles has been established. In other crocodilians, the assessment of physiological responses to stress, like the related alterations in glucocorticoid concentrations, has already been done, but only by using an invasive approach, with the disadvantage of a possible handling-induced stress response. By establishing a non-invasive technique to monitor glucocorticoid levels in captive Nile crocodiles based on faecal hormone analysis, this study not only made an important contribution to a better understanding of stress and related hormonal changes in Nile crocodiles, but also provided a solid basis for developing similar non-invasive tools to collect information on the level of stress experienced by other crocodilians. Specifically the study aimed 1) to assess adrenocortical activity in Nile crocodiles by measuring faecal glucocorticoid metabolite (FGM) concentrations, and 2) to characterise changes in FGM levels in captive Nile crocodiles in relation to different housing conditions. An adrenocorticotropic hormone (ACTH) challenge was performed on 10 sub-adult crocodiles at Le Croc crocodile farm, South Africa, resulting in serum corticosterone levels of up to ~1200 %, 1 - 5 hours post-injection, above the pre-injection levels. An additional 8 individuals were exposed to electric immobilisation and handling only (control group), which resulted in a 20 – 2700 % elevation in serum corticosterone concentrations, indicating that handling was already a sufficient stressor. FGM levels in 3 singly housed animals (2 ACTH challenge; 1 handling only) reached peaks of 136 – 380 % above pre-injection levels at about 7 to 15 days following treatment, demonstrating that non-invasive hormone monitoring can be used for assessing adrenocortical function in captive Nile crocodiles based on FGM analysis. By assessing the impact of group size (n = 1, 2, or 4 individuals) on FGM levels, highest mean hormone values were found in the paired animals. A possible explanation for this finding could be that the necessary re-grouping for the study resulted in an unstable group composition, especially for the paired animals of similar size, which is reflected in comparable higher FGM concentrations. However, future research would be necessary to investigate this potential relationship in more detail. My study created opportunities to improve the management and welfare of farmed crocodiles in terms of more appropriate housing conditions and husbandry for these animals. Finally, the now established non-invasive method for monitoring adrenocortical function in Nile crocodiles provides a solid basis for further studies focusing on monitoring factors influencing adrenocortical function in populations of Nile crocodiles in the wild. / Dissertation (MSc)--University of Pretoria, 2012. / Paraclinical Sciences / unrestricted

Since the first report of Mycoplasma-associated polyarthritis in farmed Nile crocodiles in 1995, the disease has spread across Zimbabwe and South Africa and has resulted in significant economic losses on infected farms. Due to poor response to antimicrobial treatment and frequent relapses, the use of an autogenous vaccine to manage disease outbreaks was evaluated. Two previous trials had been performed with a similar vaccine and the results suggested that the vaccine could be effective in alleviating disease, although the numbers of animals were limited in both. This trial aimed to evaluate an inactivated, alum-adjuvanted M. crocodyli whole-cell vaccine in a large group of yearling crocodiles under field conditions on a farm in Zimbabwe where repeated M. crocodyli outbreaks have been reported. The safety of the vaccine was assessed by administrating the vaccine intraperitoneally to a subset of crocodiles. No adverse clinical reactions were observed in any of these crocodiles. A group of two thousand two hundred crocodiles received two intramuscular vaccinations four weeks apart in the autumn of 2011, while another group of two thousand two hundred crocodiles served as unvaccinated controls. Serum was collected from a subset of the vaccinated and unvaccinated crocodiles at different time-points before and after vaccination to evaluate the humoral response to vaccination. Latex slide agglutination tests (LAT) were performed on all samples and positive samples were titrated with the latex slide agglutination test and metabolism inhibition assay. A low percentage of sera were positive with serological tests done prior to vaccination, suggesting either circulating Mycoplasma or maternal immunity. Statistically significant increase in sero-positivity was detected with LAT four weeks after primary vaccination, although the titre remained low. Six weeks after the booster vaccination the percentage seropositive vaccinated crocodiles had decreased and there were no statistically significant difference between the percentage seropositive vaccinated and unvaccinated crocodiles. A significant outbreak of Mycoplasma-like polyarthritis was encountered 6 months after vaccination, in October 2011. Both vaccinated and unvaccinated crocodiles were affected. Serum samples from different subsets of crocodiles were collected and evaluated similar to the vaccine trial. The results indicated that a similar rate of sero-positivity was present in all crocodiles, irrespective of vaccination- or disease status Sera collected during this trial was used to evaluate the performance of the latex slide agglutination assay compared to the metabolism inhibition assay (“Gold standard” assay), as the performance of the LAT had not been evaluated previously. The calculated diagnostic sensitivity was 72%, diagnostic specificity was 32%, the predictive value of the positive test was 36% while the predictive value of the negative test was 69%. This trial indicated that the autogenous, inactivated, alum-adjuvanted, whole-cell vaccine against M. crocodyli was not able to protect farmed Nile crocodiles on an infected farm against clinical Mycoplasma-associated polyarthritis. It was also found that the latex slide agglutination assay could be useful as a robust, pen-side assay to evaluate exposure to M. crocodyli, although other assays, such as PCR, bacterial culture or growth inhibition assays, has to be performed to confirm the presence of disease. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Objectives. Acute flaccid paralysis (AFP) has recently emerged as a major central nervous system complication associated with West Nile virus (WNV) infection. The spectrum of clinical presentations of AFP in WNV infection and its sequelae have not been well-studied. Methods. We describe three patients with AFP due to WNV infection and review the clinical presentations of 56 patients with this complication derived from published studies. Results. Patients with AFP and WNV presented with a spectrum of illness ranging from single extremity paralysis to quadriparalysis with cranial nerve involvement. Patients commonly developed respiratory failure (54%) and bladder dysfunction (22%). While fever was nearly universal (92%), signs of meningismus were less common (17%). Cerebrospinal fluid (CSF) analysis generally revealed a modest pleocytosis, and imaging studies were not diagnositic. Persistent neurologic impairment occurred in all survivors; overall mortality rate was high (22%) and was associated with both the extent of paralysis and advanced age. Conclusion. AFP in the setting of WNV is associated with significant mortality and long-term morbidity.

In view of the paucity of detailed information in the literature relevant to the upper digestive tract of the Nile crocodile, this study describes the morphological and histological features of the oral cavity (gingivae, palate and tongue), pharyngeal cavity and oesophagus of the Nile crocodile, Crocodylus niloticus (Laurenti, 1768) using light microscopy. The findings, which were supplemented by scanning electron microscopy, were compared with published information. The ciliated component of the oesophagus was also examined using transmission electron microscopy. The oral cavity had the form of a triangle and was dorso-ventrally flattened. The dorsal limit was formed by the palate and the ventral limit by the broad-based tongue. The close proximity of the tongue and palate severely limited the space within the cavity. The caudal border of the cavity was formed by the dorsal and ventral components of the gular valve. The epithelium of the palate, gingivae and tongue was stratified squamous in nature and appeared lightly keratinised. Specialised epithelial structures in the palate, gingivae and tongue, revealed by both light microscopy (LM) and scanning electron microscopy (SEM), bore characteristics resembling structures responsible for pressure and taste reception. Glandular tissue in the tongue was arranged in a triangular formation in the posterior region and displayed morphological features ascribed to salt secreting glands described in other Crocodilia. There were no palatine glands in the oral region of the palate, except that the oral surface of the dorsal gular fold contained branched tubular mucus secreting glands. The pharyngeal cavity was also dorso-ventrally flattened and was bordered rostrally by the flaccid dorsal gular fold, which displayed a median apical notch, and the ventral gular fold, which was supported internally by the broad rostral tip of the basihyal plate (hyaline cartilage). In the occluded mouth, the dorsal gular fold and the more rostrally positioned ventral component of the gular valve isolated the pharyngeal cavity. This arrangement is essential in preventing the crocodile from drowning (flooding of the pharyngeal cavity) while capturing prey. The roof of the pharyngeal cavity was characterised by the opening to the internal nares (an extension of the nasal passage from the external nares), the fibrous Eustachian plug sealing the common opening to the paired Eustachian ducts and a nodular tonsillar region, which was situated caudo-laterally to the Eustachian plug. Throughout this region, the epithelium was typically ciliated with goblet cells. However, the tonsillar nodules displayed regions of partial or no ciliation on their surface. SEM and stereomicroscopic observations showed fine longitudinal mucosal folding throughout the pharynx the distension of which, together with the large capacity for mucus production (produced by intraepithelial glands and mucus secreting glands), would facilitate the swallowing of large chunks of food in the living state. The ventrally situated laryngeal mound containing the slit-like glottis also displayed longitudinal folds and a ciliated epithelium. Anatomically, the oesophagus could be divided into two clear regions. The cranial, approximate two-thirds appeared broad and flabby. At the tracheal bifurcation, the oesophagus narrowed significantly and indicated a greater muscular content, confirmed by light microscopy. LM and SEM examination of the oesophagus, however, revealed three regional components, viz., the cranial, mid- and caudal regions. In the cranial region, the epithelium was densely ciliated with intervening goblet cells being present. In the mid-region the ciliated component decreased with a concomitant increase in the goblet cell component. In the caudal region there was a further decrease in the number of ciliated cells and a higher concentration of goblet cells. Transmission electron microscopy (TEM) of the ciliated component of the oesophagus showed typical ultrastructural features of both the ciliated and goblet cells. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2002. / Anatomy and Physiology / unrestricted

This investigation illustrates the topography, gross anatomy, histology and ultrastructure of the liver and gallbladder of the Nile crocodile in order to fill the gap that exists in the literature regarding this important crocodilian. For the topographical and macroscopical descriptions the livers and gallbladders were obtained from the carcasses of slaughtered juvenile Nile crocodiles. Perfusion and immersion fixation of tissues for histology and transmission electron microscopy were performed on juvenile Nile crocodiles donated to the university. Published descriptions of other vertebrates were inevitably relied upon for comparison due to the lack of information on these two organs of the Nile crocodile. The liver was located in its own coelomic cavity with the post-pulmonary and the post-hepatic membranes intimately associated with the cranial and caudal surfaces of the bi-lobed liver respectively. The right lobe was larger than the left lobe and they were located at the level of the third to seventh intercostal spaces with their extremities extending to the ninth intercostal space. The triangular shaped liver lobes were joined dorso-medially by a narrow isthmus consisting of liver tissue. The liver was covered by Glisson’s capsule. Central veins, sinusoids and portal tracts were distributed haphazardly with no visible lobulation. The parenchymal component occupied the largest part of the liver and was formed by anastomosing and branching cell cords consisting of two-cell-thick plates in the longitudinal sectional plane and at least five hepatocytes in the cross-sectional plane. Central bile canaliculi contained microvilli originating from apical hepatocyte surfaces and were sealed off by junctional complexes. Hemosiderin granules, bile pigments, melanin pigments, lipid droplets, cholesterol ester slits and glycogen granules were observed in addition to the normal hepatic cytoplasmic organelles. Non-parenchymal cells consisted of endothelial cells, Kupffer cells, stellate cells and pit cells localized in and around the angular sinusoids. The space of Disse existed between endothelial cells and the base of the hepatocytes which was lined by microvilli. Endothelial cells were flat cells with long fenestrated cytoplasmic extensions that lined the sinusoidal wall and contained numerous endocytotic vesicles and many lysosomes. Pleomorphic Kupffer cells were located in the sinusoidal lumen, in the space of Disse and within groups of hepatocytes. They were often situated between groups of hepatocytes, connecting two adjacent sinusoids. Large phagosomes were present in the Kupffer cells and contained a combination of melanin and hemosiderin granules as well as ceroid. Phagocytosis of apoptotic and dying cells was evident. Conspicuous groups of membrane-bound tubular organelles with a filamentous or crystalline interior were present in the Kupffer cells. Stellate cells occupied a subendothelial position in the space of Disse and contained prominent lipid droplets that indented the nuclei. A solitary cilium was infrequently found projecting into the space of Disse. Myofibroblastic cells were found in the same region as stellate cells. Pit cells with indented eccentric nuclei were found in the sinusoidal lumen and in close contact with endothelial and Kupffer cells. Numerous small electron-dense membrane-bound cytoplasmic granules were present. Occasional intercalated cells resembling lymphocytes were seen in the space of Disse and forming part of the groups of hepatocytes. Glisson’s capsule extended collagenous trabeculae into the parenchymal interior and variably sized trabeculae randomly traversed the liver tissue. Portal tracts were enmeshed by a collagenous network that contained fibroblasts, lymphocytes, plasma cells and phagocytes. Portal triads consisted of branches of the portal vein, hepatic artery and bile duct with lymphatic vessels sometimes in accompaniment. Reticular fibres were positioned around hepatocyte tubules and a basal lamina supported the hepatocytes adjacent to Glisson’s capsule. Occasional unmyelinated nerve axons were present. The isthmus contained liver tissue with similar parenchymal and a non-parenchymal components. Three anatomical zones were identified in the pouch-like gallbladder that was attached caudally to the right liver lobe in the dorso-medial region. The gallbladder wall consisted of pseudostratified columnar epithelium, a lamina propria, a muscularis externa and a serosal layer. The accumulation of apical secretory granules, apical bulging, exocytosis of mucous granules and the desquamation of the apical portions of the epithelial cells into the lumen indicated different stages of the mucus secretory cycle. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Anatomy and Physiology / Unrestricted

No description available.

Because little is known about juvenile American White Pelicans (Pelecanus erythrorhynchos) this study was conducted to gather more information on disease, general ecology and growth of American White Pelicans from hatching to fledging. In July 2011, American White Pelican regurgitate samples from North and South Dakota sub-colonies were collected/analyzed in preparation for a captive trial. Nutrient content compared between the colonies was found to be significantly different. Concentrations of Immunoglobulin Y and A in regurgitate samples were significantly different between colonies. A captive trial began 29 May 2012 and ended 30 July 2012, in which 16 American White Pelicans were hand raised from hatching to fledging. During the captive trial, various growth parameters, intake and fecal output were examined to determine the effect of the parasite Bolbophorus damnificus in 8 infected and 8 non-infected (parasite free) pelicans. Growth data collected on B. damnificus infected (n = 8) American White Pelicans was compared to previously mentioned parasiteree pelicans (n = 8) to determine effects of the parasite. There were no differences between groups for culmen length (P= 0.214), tarsal length (P = 0.306), body weight (P = 0.884) or intake (P = 0.963). There was also no effect of the parasite on body temperature. Towards the end of the captive trial, several pelicans both on (n = 16) and off (n = 11) trial became naturally infected with West Nile Virus. Clinical symptoms ranged from lethargy and/or wing droop to total paralysis. Progression of disease is detailed in two well-defined case studies with additional information included on clinical signs, physiological parameters, and a review of the pathology of disease for other infected birds.

Analysis of seaweed as a vector for microplastics is an integral part of understanding the formation and deposition of micro-sized plastic waste in seawater. The project itself originated due to the influx of seaweed (and mismanaged plastic waste) residing on the shores of St. Kitts and Nevis and the constant deposition of plastic pollution intertwined within the seaweed. The natural occurrence of the two together lead to the consideration of fragmented plastics remaining on the seaweed. The objective of this research is to stain, identify, and quantify the concentration of microplastics sourced from both store-bought and environmental seaweed samples. A Nile red solution dissolved in either acetone or methanol was used to stain the microplastics, as per a proven method. The fluorescence of the stained microplastics was measured (excitation: 523-543 nm and emission: 580-640 nm) to identify potential dissolution. The seaweed was washed of microplastics and the solid particles collected were evaluated using infrared (IR) spectroscopy. The fluorescence and infrared spectrum results were compared to spectra within the Spectral data base system (SDBS) for the most common plastics: polyethylene (PE), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polypropylene (PP), polycarbonate (PC). The use of a fluorescence microscope allowed for direct quantification of microplastics over a specific area of the sample and therefore allowed for further identification of microplastic presence.