Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (MA)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2015. / It is recognised that tooth loss as a consequence of oral diseases affects quality of life in humans. This has directed studies towards biological tooth replacement in vivo. In humans and other mammals, tooth replacement occurs only once (diphyodonty) as opposed to non-mammalian vertebrates where tooth replacement continues throughout life (polyphyodonty). Detailed knowledge of tooth initiation, development and morphology amongst vertebrates and especially amniotes, is necessary to understand the tooth replacement process. Crocodilians provide an interesting model for tooth replacement studies as they also exhibit thecodonty. Regulation of polyphyodonty has not been genetically defined, and it is uncertain whether the molecular mechanisms of continuous tooth replacement are similar to those involved in the primary dentition. The aim of this study was therefore to analyse crocodilian odontogenesis in detail, with the aid of light microscopy and CT scans, in order to provide a structural framework for molecular processes regulating polyphyodonty. Crocodile probes to bmp4 and pitx2 were designed, generated and labelled for use in in situ hybridisation. The expression patterns of pitx2 and bmp4 in embryos and hatchlings of the polyphyodont Nile crocodile (Crocodylus niloticus) were examined at different stages of tooth development. Histologically crocodilian tooth development appears similar to mammals. Interesting variations include the initiation of odontogenesis in the ectomesenchyme, the presence of dental placodes, the ‘null generation teeth’, the two different bell-stage tooth germs and the tooth-family organisation. A direct 1:1 relationship between the status of the erupted tooth and the developmental phase of the replacement tooth was not seen. However in more mature teeth, the replacement tooth germs were at a more advanced developmental stage than those associated with less mature teeth. Molecular data revealed that pitx2 was expressed in the oral epithelium and the dental placode. Bmp4 expression was not evident in the dental placode, but was localised in the odontoblasts of early bell stage tooth germs. Pitx2 and bmp4 were expressed in both the odontoblast and ameloblast layers in late bell stage tooth germs. Expression of pitx2 and bmp4 is conserved across vertebrates and pitx2 may play a role in initiation of primary and successional teeth.

Up until 1969 Nile Crocodiles (Crocodylus niloticus) were considered as vermin in South Africa and were actively persecuted throughout the country. In an effort to re-establish viable populations within protected areas in Zululand a restocking program was initiated in the late 1960‟s and early 1970‟s. Ndumo Game Reserve (NGR) in north eastern KwaZulu-Natal was one of the reserves involved in the restocking program and the Nile Crocodile population in the reserve increased from a minimum absolute abundance of 348 (SE ± 3.39; N = 3) in the early 1970‟s to a minimum absolute abundance of 992 (SE ± 58.70; N = 4) in the 1990‟s. However, in recent years there has been some concern that the NGR Nile Crocodile population may be on the decline, initiating the current investigation into the ecology and conservation of the NGR population. We examined changes in relative abundance using aerial survey data from 1971 – 2009. The precision and accuracy of population estimates was affected by water level, season, aircraft type and the use of different observers. A correction factor was applied to survey data and the current NGR Nile Crocodile population is estimated at an absolute abundance of 846 (± 263). Distribution data from the aerial surveys were also used to examine habitat use over the last 40 years and revealed that Nile Crocodiles were not evenly distributed in NGR and that crocodiles favoured the Phongola over the Usuthu floodplain systems. NGR is characterised by a floodplain mosaic landscape and crocodile distributions between the various habitat patches were influenced by landscape physiognomy and composition as well as connectivity and corridor quality. Anthropogenic disturbances influenced the functionality of the floodplain landscape negatively with impacts on habitat use and connectivity. To quantify the effects of environmental conditions on crocodile habitat use we conducted 40 diurnal counts at Lake Nyamithi between 2009 and 2012 and related changes in crocodile numbers here to temperature, rainfall and water level. Crocodile density in Lake Nyamithi was significantly and negatively related to average maximum ambient temperature and numbers increased in the lake over the cool, dry winter season. Water level and rainfall had strong but not significant (p >0.05) negative influence on crocodile density in Lake Nyamithi. Environmental variables influenced different size class of Nile Crocodiles differently and the density of crocodiles in the 1.5 – 2.5 m Total Length (TL) size class were significantly influenced by rainfall and average minimum monthly temperature. Movement patterns of 49 Nile Crocodiles between 202 – 472 cm total length (TL) were followed over 18 months using mark-resight (n = 36), radio (n = 10) and satellite (n = 3) telemetry. The duration of radio transmitter attachment (131 days, SE ± 11.35) was significantly related to TL and reproductive status. Satellite transmitters stopped functioning after 15 (SE ± 12.53) days and home range was calculated for 7 crocodiles ranging in size from 202 cm TL – 358 cm TL. Sub-adults (1.5 - 2.5 m TL) occupied smaller, more localized home ranges than adults (> 2.5 m TL). Home ranges overlapped extensively suggesting that territoriality, if present, did not cause Nile Crocodiles to maintain spatially discrete home ranges in NGR during the dry season. A single large scale migration event occurs every year between October and November whereby the majority of the NGR crocodile population leaves the reserve and enters the Rio Maputo floodplain in adjacent Mozambique and only return in April/May. Nesting effort (19 – 21 %) in NGR was comparable to other populations of Nile Crocodile in southern Africa. Nests are completely destroyed by floods once every 10 years and predation rates may range from 20 – 86 % per year. In addition to aerial surveys, nesting surveys and movement studies crocodiles (n = 103) were caught opportunistically to collect demographic data on population structure. The population structure of Nile Crocodiles in NGR is currently skewed towards sub-adults and adults suggesting an aging population that may decline naturally in the future. This could be due to low recruitment levels in NGR that are not able to sustain the artificially high population size created by the restocking program. Sex ratios were skewed towards females in the juvenile and sub-adult size classes and towards males in the adult size class while the overall sex ratio was even between males and females. It is predicted that the NGR Nile Crocodile population will decline in the future and that this decline should be considered as a natural process. However, the rate of decline will be accelerated at an unnatural speed and to an unnatural extent due to poaching, uncontrolled harvesting and destruction of nesting habitat within NGR. Based on the findings of the current study, management recommendations for the conservation of the combined NGR – Rio Maputo Nile Crocodile population were made. It is important that further research takes place in the Rio Maputo floodplain in Mozambique to better quantify the nesting ecology of the NGR Nile Crocodile population and to identify possible threats facing Nile Crocodiles in this region. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

The zoonotic potential of Trichinella zimbabwensis as supported by the clinical symptoms observed in experimentally infected, non-human primates (Mukaratirwa et al., 2001) necessitates research aimed at elucidating the distribution and epidemiology of this parasite. No controlled studies have been conducted to determine the predilection muscles of Trichinella zimbabwensis larvae in Nile crocodiles (Crocodylus niloticus) or the influence of infection intensity on the distribution of the larvae in crocodiles. Neither has the influence of Trichinella zimbabwensis on biochemical parameters in crocodiles been assessed previously. To determine the distribution patterns of Trichinella zimbabwensis larvae and predilection muscles and to assess the influence on selected biochemical parameters, fifteen crocodiles were randomly divided into three cohorts of five animals each to represent high infection (642 larvae/kg of body weight), medium infection (414 larvae/kg of bodyweight) and low infection (134 larvae/kg of bodyweight) cohorts. In the high infection cohort, high percentages of larvae were observed in the tricep muscles (26%) and hind limb muscles (13%). In the medium infection cohort, high percentages of larvae were found in the tricep muscles (50%), sternomastoid (18%) and hind limb muscles (13%). For the low infection cohort, larvae were mainly found in the intercostal muscles (36%), longissimus complex (27%), forelimb muscles (20%), and hind limb muscles (10%). Predilection muscles in the high and medium infection cohorts were similar to those reported in naturally infected crocodiles despite changes in infection intensity. The high infection cohort had significantly higher numbers of larvae in the intercostal, longissimus complex, external tibial flexor, longissimus caudalis and caudal femoral muscles (P < 0.05) compared to the medium infection cohort. In comparison to the low infection cohort, the high infection cohort harboured significantly higher numbers of larvae in all muscles (P < 0.05) except for the tongue and pterygoid. The high infection cohort harboured significantly higher numbers of larvae (P < 0.05) in the sternomastoid, tricep, intercostal, longissimus complex, external tibial flexor, longissimus caudalis and caudal femoral muscles compared to naturally infected crocodiles. The importance of host characteristics in determining predilection and the importance of leg musculature as a predilection site for Trichinella spp. in sylvatic carnivores were both confirmed in this study. Deviations from normal parameters of blood glucose, alanine transaminase (ALT), aspartate transaminase (AST), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) compared to observations in uninfected reptiles were observed. Hypoglycaemia was not observed in the infected groups in this study. The humoral immune response to Trichinella zimbabwensis infection was evaluated in all three groups by way of indirect ELISA. Peak values of blood glucose, LDH and AST were observed on day 56, 49 and 42 p.i. in the high, medium and low infection cohorts respectively. CPK values peaked on day 35 p.i. in all three cohorts. Peak ALT values were reached on day 56 in the high infection cohort and on day 28 p.i. in both the medium and low infection cohorts. No correlations between the biochemical parameters and infection intensity were observed. Peak antibody titres were reached on day 49 p.i. in the medium infection cohort and on day 42 p.i. in both the high and low infection cohorts. Infection intensity could not be correlated with the magnitude of the humoral immune response or time to seroconversion. The effect of infection intensity on time to seroconversion, magnitude and persistence of the humoral immune response was assessed. No significant differences in the titre levels between the three groups were observed. Infection intensity could not be correlated with the magnitude of the humoral response or time to seroconversion. Results of this study were in agreement with results reported in mammals (wild boars and horses) infected with other Trichinella species and showed that antibody titres could not be detected indefinitely. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

The objective of this study was to determine the dietary protein requirements of the Nile crocodile (Crocodylus niloticus) between the ages of 5 – 8 months, and to compare the results with documented protein requirements of the American alligator (Alligator mississippiensis). This was achieved by feeding the crocodiles 4 diets with varying amounts of crude protein (CP), including 62%, 56.6%, 51.6% and 46%. All four diets were iso-energetic with a metabolisable energy to protein ratio of 25.85KJ/g. The highest protein diet contained 44% raw minced chicken and 46.9% fish meal with minimal contribution (of 6.4%) made from vegetable protein sources (full fat soya). The lowest protein diet was made up of a majority of vegetable protein (30% soya bean oilcake, 9.8% full fat soya and 17.2% maize meal) and some contribution from protein of animal origin (4.3% carcass meal and 35% fish meal). This study was carried out for 12 weeks during the crocodiles first year of life. Body mass, total body length, head length and snout to vent length were measured five times at 3 weekly intervals. Twenty representative crocodiles in a pen of 200 were individually tagged for the duration of the trial. As there were 3 replicates for each of the 4 treatment diets, 240 crocodiles in 12 pens were tagged. During the early phase of the study, it was observed that crocodiles on the 46% CP treatment diet were not performing well, and that most of the crocodiles on this diet were losing mass. This treatment diet was discontinued at 9 weeks into the trial for both financial and ethical reasons. The remaining three diets were tested for the full 12 weeks. Chromium oxide was mixed into the diets at two time periods during the trial to determine the protein, energy and dry matter digestibility. It was determined that the 46%, the 51.6%, the 56.6% and the 62% CP diets had a digestible protein (DP) content of 246.44 g/kg, 294.80 g/kg, 381.32 g/kg and 468.65 g/kg and a digestibility coefficient of 53.50%, 57.00%, 69.15% and 75.65% respectively. Performance of crocodiles on the 46% CP treatment diet was found to be lower in all measurement categories than crocodiles on the three higher protein diets. However, crocodiles on the 62% CP treatment diet outperformed all the crocodiles (on all measurement criteria) on the lower protein diets. The poor performance of crocodiles on the lowest protein diet correlates with previous research indicating that crocodilians are unable to perform optimally when the majority of the diet’s protein is made up of vegetable protein sources. It was determined that juvenile American alligators would grow at optimal levels when the diet contained a DP content of 450 g/kg. As the Nile crocodiles in this study performed the best when the diet contained a DP value of 468.65 g/kg, it was concluded that juvenile Nile crocodiles have the same range of protein requirements as that determined for juvenile American alligators. / Dissertation (MSc(Agric))--University of Pretoria, 2011. / Animal and Wildlife Sciences / 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