Genetic variation is crucial for the long-term survival of the species as it provides the potential for adaptive responses to environmental changes such as emerging diseases. The Major Histocompatibility Complex (MHC) is a gene family that plays a central role in the vertebrate’s immune system by triggering the adaptive immune response after exposure to pathogens. MHC genes have become highly suitable molecular markers of adaptive significance. They synthesize two primary cell surface molecules namely MHC class I and class II that recognize short fragments of proteins derived respectively from intracellular (e.g. viruses) and extracellular (e.g. bacteria, protozoa, arthropods) origins and present them to immune cells. High levels of MHC polymorphism frequently observed in natural populations are interpreted as an adaptation to detect and present a wide array of rapidly evolving pathogens. This variation appears to be largely maintained by positive selection driven mainly by pathogenic selective pressures. For my doctoral research I focused on MHC I and II variation in free-ranging cheetahs (Acinonyx jubatus) and leopards (Panthera pardus) on Namibian farmlands. Both felid species are sympatric thus subject to similar pathogenic pressures but differ in their evolutionary and demographic histories. The main aims were to investigate 1) the extent and patterns of MHC variation at the population level in both felids, 2) the association between levels of MHC variation and disease resistance in free-ranging cheetahs, and 3) the role of selection at different time scales in shaping MHC variation in both felids. Cheetahs and leopards represent the largest free-ranging carnivores in Namibia. They concentrate in unprotected areas on privately owned farmlands where domestic and other wild animals also occur and the risk of pathogen transmission is increased. Thus, knowledge on adaptive genetic variation involved in disease resistance may be pertinent to both felid species’ conservation. The cheetah has been used as a classic example in conservation genetics textbooks due to overall low levels of genetic variation. Reduced variation at MHC genes has been associated with high susceptibility to infectious diseases in cheetahs. However, increased disease susceptibility has only been observed in captive cheetahs whereas recent studies in free-ranging Namibian cheetahs revealed a good health status. This raised the question whether the diversity at MHC I and II genes in free-ranging cheetahs is higher than previously reported. In this study, a total of 10 MHC I alleles and four MHC II alleles were observed in 149 individuals throughout Namibia. All alleles but one likely belong to functional MHC genes as their expression was confirmed. The observed alleles belong to four MHC I and three MHC II genes in the species as revealed by phylogenetic analyses. Signatures of historical positive selection acting on specific sites that interact directly with pathogen-derived proteins were detected in both MHC classes. Furthermore, a high genetic differentiation at MHC I was observed between Namibian cheetahs from east-central and north-central regions known to differ substantially in exposure to feline-specific viral pathogens. This suggests that the patterns of MHC I variation in the current population mirrors different pathogenic selective pressure imposed by viruses. Cheetahs showed low levels of MHC diversity compared with other mammalian species including felids, but this does not seem to influence the current immunocompetence of free-ranging cheetahs in Namibia and contradicts the previous conclusion that the cheetah is a paradigm species of disease susceptibility. However, it cannot be ruled out that the low MHC variation might limit a prosperous immunocompetence in the case of an emerging disease scenario because none of the remaining alleles might be able to recognize a novel pathogen. In contrast to cheetahs, leopards occur in most parts of Africa being perhaps the most abundant big cat in the continent. Leopards seem to have escaped from large-scale declines due to epizootics in the past in contrast to some free-ranging large carnivore populations in Africa that have been afflicted by epizootics. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, I characterized genetic variation at MHC I and MHC II genes in free-ranging leopards from Namibia. A total of six MHC I and six MHC II sequences were detected in 25 individuals from the east-central region. The maximum number of sequences observed per individual suggests that they likely correspond to at least three MHC I and three MHC II genes. Hallmarks of MHC evolution were confirmed such as historical positive selection, recombination and trans-species polymorphism. The low MHC variation detected in Namibian leopards is not conclusive and further research is required to assess the extent of MHC variation in different areas of its geographic range. Results from this thesis will contribute to better understanding the evolutionary significance of MHC and conservation implications in free-ranging felids. Translocation of wildlife is an increasingly used management tool for conservation purposes that should be conducted carefully as it may affect the ability of the translocated animals to cope with different pathogenic selective pressures. / Genetische Variabilität ist entscheidend für das langfristige Überleben von Arten, denn es ermöglicht dem Organismus sich Umweltveränderungen, wie z.B. neu aufkommende Krankheiten, schneller anzupassen. Der Haupthistocompatibilitätskomplex (MHC) ist eine Familie von Genen, der eine zentrale Rolle im Immunsystem von Wirbeltieren zukommt, da sie nach Pathogenkontakt das adaptive Immunsystem aktivieren. Zudem sind MHC Gene geeignete molekulare Marker um Anpassungsfähigkeiten aufzuzeigen. MHC Gene kodieren primär für Zelloberflächenmoleküle, die kurze Peptidfragmente erkennen und den Immunzellen präsentieren, die im Falle der Klasse I Gene intrazellulären (z.B. von Viren) oder im Falle der Klasse II Gene extrazellulären (z.B. von Bakterien, Protozoen, Arthropoden) Ursprungs sein können. In der Regel wird in natürlich vorkommenden Populationen ein hoher Grad an Polymorphismus im MHC beobachtet, was als Anpassung an das Erkennen und Präsentieren einer großen Anzahl sich schnell entwickelnder Pathogene interpretiert wird. Das Bestehen vieler MHC Varianten über große Zeiträume hinweg wird hauptsächlich durch positive Selektion bewirkt, der ein pathogengetriebener Selektionsdruck zugrunde liegt. In meiner Doktorarbeit habe ich mich mit der Variation von MHC I and MHC II in freilebenden Geparden (Acinonyx jubatus) und Leoparden (Panthera pardus) in Farmgebieten innerhalb Namibias beschäftigt. Beide Felidenarten leben sympatrisch und sind so demselben Pathogendruck ausgesetzt, sie unterscheiden sich allerdings in ihrem evolutionären und demographischen Hintergrund. Mein Hauptziel war es 1) das Ausmaß und Muster der MHC Variation auf Populationsebene beider Feliden zu untersuchen; 2) einen möglichen Zusammenhang zwischen dem Grad der MHC Variation und der Krankheitsresistenz in frei lebenden Geparden aufzudecken und 3) zu untersuchen, welche Rolle der Selektion auf die MHC Variabilität beider Arten in der Vergangenheit wie auch gegenwärtig zukommt. Geparden und Leoparden repräsentieren die größten frei lebenden Carnivoren Namibias. Beide Arten kommen hauptsächlich in Farmgebieten vor, die sich in Privatbesitz befinden, und können dort mit anderen Wild- aber auch Haustieren zusammentreffen und potentiell Krankheitserreger austauschen. Die Kenntnis über die adaptive genetische Variation, die für Krankheitsresistenzen mitverantwortlich ist, kann für den Schutz beider Felidenarten von Bedeutung sein. Geparden werden häufig in Lehrbüchern als klassische Beispiele für eine Tierart mit einer generell geringen genetischen Diversität verwendet. Neben neutralen Markern ist bei Geparden auch eine geringe Variabilität der MHC Gene beschrieben worden, die als Ursache einer hohen Anfälligkeit für infektiöse Krankheiten gesehen wird. Bisher wurde allerdings eine erhöhte Krankheitsanfälligkeit nur bei Geparden aus Gefangenschaft beschrieben, wohingegen neuste Studien an frei lebenden Geparden diesen einen guten Gesundheitsstatus attestierten. Dadurch stellt sich die Frage, ob die MHC I und II Diversität in frei lebenden Geparden nicht höher sein könnte als bisher angenommen. In dieser Arbeit konnten insgesamt 10 MHC I und vier MHC II Allele in 149 frei lebenden Geparden aus ganz Namibia nachgewiesen werden. Die Zugehörigkeit zu funktionellen MHC Genen wurde durch Expressionsanalysen bei allen Allelen, außer einem, bestätigt. Durch phylogenetische Analysen konnten die Allele vier MHC I und drei MHC II Genen zu geordnet werden. Das Wirken von positiver Selektion in der Vergangenheit konnte an spezifischen Aminosäuren des Proteins, die in direktem Kontakt zu den pathogenen Antigenen stehen, festgestellt werden. Dies traf für beide MHC Klassen zu. Des Weiteren konnte eine starke genetische Differenzierung des MHC I zwischen Geparden aus einer nord-zentralen und einer ost-zentralen Region festgestellt werden, von denen auch bekannt ist, dass sie unterschiedlichen, felidenspezifischen, viralen Pathogenen ausgesetzt sind. Das lässt vermuten, dass die unterschiedlichen Muster der MHC I Variation in der gegenwärtigen Population den unterschiedlichen pathogengetriebenen Selektionsdruck durch Viren in den beiden Regionen widerspiegelt. Verglichen mit anderen Säugetierarten, insbesondere andere Feliden, zeigen Geparden einen geringen Grad an MHC Diversität, doch das scheint die derzeitige Immunkompetenz frei lebender Geparden in Namibia nicht einzuschränken und widerspricht der bisherigen Meinung dass Geparden ein typisches Beispiel für eine krankheitsanfällige Tierart sind. Es kann allerdings nicht ausgeschlossen werden, dass bei neu auftauchenden Krankheiten die geringe MHC Variation eine erfolgreiche Immunkompetenz verhindert, da möglicherweise keines der gegenwärtigen Allele die Fähigkeit besitzt neue Pathogene zu erkennen. Im Gegensatz zu Geparden kommen Leoparden in allen Teilen Afrikas vor und sind wahrscheinlich die am weitverbreiteste Großkatze des afrikanischen Kontinents. Es scheint, dass Leoparden, im Gegensatz zu anderen afrikanischen Großkatzen, einer ausgedehnten Dezimierung durch Tierseuchen in der Vergangenheit, der einige Populationen afrikanischer Großkatzen ausgesetzt waren, entkommen sind. Bisher fehlten Information über die MHC Variabilität in afrikanischen Leoparden. In dieser Studie konnte ich die genetische Variation der MHC I und MHC II Gene frei lebender namibischer Leoparden charakterisieren. In 25 Tieren aus einer Population der ost-zentralen Region konnten sechs MHC I sowie sechs MHC II Sequenzen nachgewiesen werden. Aus der maximalen Anzahl Allele pro Tier kann auf drei MHC I und auf drei MHC II Gene geschlossen werden. Außerdem konnten die typischen Kennzeichen einer variationserhaltenden MHC Evolution betätigt werden, wie positive Selektion in der Vergangenheit, Rekombination und über Artgrenzen hinaus bestehender Polymorphismus. Der geringe Grad an MHC Variation in namibischen Leoparden ist jedoch noch nicht endgültig und weitere Untersuchungen in unterschiedlichen Regionen aus der gesamten geographischen Verbreitung des Leoparden sind notwendig um die MHC Variation der Leoparden in Gänze einschätzen zu können. Die Ergebnisse dieser Arbeit werden zu einem besseren Verständnis des evolutionären Stellenwerts des MHC und in Folge zu einem besseren Schutz von frei lebenden Feliden beitragen. Die Umsiedelung von Wildtieren ist ein zunehmend angewendetes Hilfsmittel im Natur- und Artenschutz, welches jedoch mit Sorgfalt eingesetzt werden sollte, da die umgesiedelten Tiere möglicherweise einem anderen pathogenen Selektionsdruck ausgesetzt sind, dem sie nichts entgegenzusetzen haben.

Cheetah are difficult to census and as a result there is only a limited amount of information regarding the status of cheetah populations across South Africa making it difficult to identify and address potential threats and to plan effective conservation strategies. The aim of this study was to determine if scatology could be used as a non-invasive method for genetic analysis of cheetah in South Africa. Corresponding blood and faecal samples were collected from cheetah at the De Wildt Cheetah Centre. The faecal samples were aged and then collected and stored in ethanol after four days and after two weeks. Faecal samples of suspected wild cheetah were also collected in the Thabazimbi area in the Limpopo Province. DNA was than isolated from all the blood and faecal samples. A species-specific primer set was developed in the mitochondrial control region to reliably identify cheetah faecal samples. The PCR amplification success of the species-specific marker was high, all fresh faecal samples had a positive result (100%), four-day-old samples were 83% positive and two-week-old samples were 94% positive. The species-specific primer was found to be effective in accurate cheetah identification and had greater amplification success on fresh samples. Twelve microsatellite markers were used to profile the blood and faecal samples. Multiple PCRs were performed per faecal sample per marker to ensure that the genotype obtained was correct. Amplification success of the 12 microsatellites was 59% for fresh samples, 20% for four-day-old samples and 2.4% for two-week-old samples. Difficulties associated with the technique included poor DNA quality and quantity and genotyping errors, resulting in allelic dropout and false alleles. Genotyping errors were determined by comparing the genotype of the faeces with that of the matching blood sample. Overall, allelic dropout experienced was 16% and 7% for false alleles. These values correspond to the results of similar studies. Samples of suspected wild cheetah were collected in the Thabazimbi area and were analysed. The alleles obtained were similar to those from the pilot study. Data of 45 De Wildt cheetah samples obtained from the NZG were analysed and compared with the results of the pilot study, as expected, all alleles in the De Wildt population occurred in the NZG samples. The alleles found in this study and in the NZG samples can be used as the standard when using molecular scatology techniques. The results of this study provide valuable guidelines for future studies of cheetah using the non-invasive technique of scatology. The genotypic information from the 12 microsatellites can now be used as a standard when analyzing samples of cheetah origin. Using this information, additional markers can now be tested for improved amplification success and decreased rates of allelic dropout and occurrences of false alleles. The results prove the feasibility of using cheetah faeces as a potential source of DNA for population studies of cheetah in South Africa in addition to the methods currently employed. / Dissertation (MSc)--University of Pretoria, 2010. / Animal and Wildlife Sciences / unrestricted

The objectives of this study were(1) to analyse a data-set on cheetah home range size, habitat utilisation, prey selection and hunting behaviour in the Kruger National Park (KNP), and (2) conduct a comparative study on the above aspects of cheetah ecology across a variety of African savanna ecosystems. Cheetah home range sizes in the KNP for territorial male cheetahs were 173 km2, 438 km2 for a nomadic male cheetah coalition, and 193 km2 and 179 km2 for two female cheetahs. Cheetahs preferred open savanna habitat, although females used areas with thicker bush such as drainage lines) more than males, probably because this is the preferred habitat of their main prey. Analysis showed that while females were closely associated with drainage lines, males were closely associated with roads. A male cheetah coalition preferred scent marking along roads rather than in the bush. Impala occurred most frequently in the cheetahs’ diet, although males took larger prey than females. Cheetahs hunted and killed more frequently in open savanna, even though impala densities were higher in adjacent habitats. Mean chase distance for successful hunts was 18l9 m and unsuccessful hunts was 96 m. Cheetah hunting success was 20.7%, kleptoparasitism was 11.8%, mean kill retention time was 165 min, kill rated averaged 1 kill per 4.61 days, and consumption rate of a three-male cheetah coalition was 1,4 kg meat/cheetah/day. Across African savanna ecosystems, female cheetah home range size was determined by the dispersion patterns and biomass of medium-sized prey. Female home range size was larger in areas with migratory (833 km2) than sedentary prey (105 km2) and increase as prey biomass decreased in areas with sedentary prey only. Conversely, male cheetah territory size was smaller in areas with migratory (37 km2) than sedentary prey (108 km2). Although it was predicted that male cheetah territory size would be determined by female cheetah density, no relationship was found here. Across a range of African savannas, cheetahs showed distinct preferences for open habitats that provided some woody cover. Adults (40%) and juveniles (20%) of medium-sized prey made up the largest proportion in the cheetahs’ diet. There were, however, significant variations in the size and age groups of prey taken by cheetahs in different areas. Areas with the least amount of cover appeared to have the longest mean chase distance, and the greatest percentage hunting success and incidents of kleptoparasitism (%). A population viability analysis, using VORTEX, found that cheetah population viability was greater in a woodland savanna than a grassland savanna, particularly at small population sizes. While the grassland savanna population was most affected by changes in juvenile mortality the woodland savanna population with exceptionally high cub mortality may be a sink for cheetahs while the woodland population with lower cub mortality and predicted high dispersal rates may be a source. Maximum annual litter size and female mortality rates had large impacts on population persistence. / Dissertation (MSc (Zoology))--University of Pretoria, 2006. / Zoology and Entomology / unrestricted

Please read the abstract (Uittreksel) in the section, 00front of this document. See also 10summary / Dissertation (MSc (Zoology))--University of Pretoria, 2007. / Zoology and Entomology / MSc / unrestricted

The last 50 years was characterized by a dramatic decrease in free-roaming cheetah populations and consequently the cheetah now appears on the IUCN Red List for Threatened Species. In order to save cheetahs from extinction, a number of projects were launched to breed cheetahs in captivity. Captive cheetahs, however, receive fundamentally different diets than their free-roaming counterparts, which necessitates feed supplementation to fulfill their unique dietary needs. The Hoedspruit Endangered Species Centre (HESC) is one such project aiming to breed captive cheetahs. At the HESC, a number of juvenile cubs were diagnosed with a form of relaxed carpal joints, namely metacarpal deformity of the front legs. Literature suggests that the condition is due to a magnesium deficiency, which is a consequence of an unbalanced diet. Supplementing magnesium to the diet of cheetahs can, however, affect the urinary system negatively: such as the formation of urolithiasis. V The aim of this study was to determine whether dietary magnesium supplementation in the diets of captive cheetahs will remedy metacarpal deformity and also to investigate the influence of magnesium supplementation on the formation of urolithiasis. The study was divided into two phases. Phase 1 was conducted to determine the influence of dietary magnesium supplementation on metacarpal deformity, identified in juvenile cheetahs at the HESC. To determine the degree of deformity, a leg deformity scoring system was developed. On a scale from 1-3, the cheetahs were scored twice to determine the Flexed Deformity Score (FDS) and Rotational Deformity Score (RDS) values before and after dietary magnesium supplementation. Phase 2 was conducted to determine the influence of magnesium supplementation on different physiological parameters that have an influence on the formation of urolithiasis. Phase 2 was divided into three periods. During each period, the cheetahs received a different diet. During period C, the experimental period, the cheetahs were divided into two groups. One group received a meat-only diet, whereas the other group received a meat-Mg diet. At the end of each of the three periods, blood- and urine samples were collected and analyzed to determine the concentration of minerals in the cheetah’s blood plasma and urine. Based on the FDS and RDS scores, a 25.5% response rate to dietary magnesium supplementation on rotational deformities was found, whereas a 60.8% response rate on flexural deformation was found. It is thus concluded that dietary supplementation of magnesium in juvenile cheetahs that experience metacarpal deformities, will remedy the deformity. By analyzing the changes of different blood- and urine parameters in the cheetahs it was observed that dietary magnesium supplementation do influence the formation of urolithiasis. The physiological state of the cheetahs can influence these parameters. The results obtained from the study can be utilized by nutritionists, veterinarians and institutions to enhance the health of captive cheetahs. Copyright / Dissertation (MSc(Agric))--University of Pretoria, 2012. / Animal and Wildlife Sciences / unrestricted

The conservation benefits of animals in captivity are limited to education and genetic preservation. However, where species or sub-species are critically endangered, the release of captive bred animals into the wild can be used as a strategy to supplement existing populations or to form new founder populations. Cheetahs Acinonyx jubatus born in captivity have no prior experience of survival in wild circumstances. Captive bred cheetahs are currently the greatest source of individuals. This project worked in conjunction with IUCN reintroduction guidelines and pre-existing recommendations in an attempt to develop an ideal methodology of reintroducing captive bred cheetah into the wild. Three captive bred cheetahs, one female and two males, were reintroduced onto Makulu Makete Wildlife Reserve, a predator proofed reserve in the Limpopo province of South Africa, as part of an experimental rehabilitation project. The cheetahs had to lose their homing instinct, be habituated to researchers, become accustomed to eating venison, chewing skin, infrequent large meals and strength development within the three months spent in the enclosures. The cheetahs were habituated to the field researchers on foot and to research vehicles for ease of monitoring. A soft release methodology was used to ease the cheetahs into the environment. Upon release they were tracked twice daily to observe behavior and interaction with their environment. The duration of the project was 20 months which includes enclosure time. The establishment of a home range indicated that the resource need of the cheetahs was satisfied. Eight habitat types were identified in the study area and all cheetahs encompassed part of each in their home range. Though the hunting instinct was present in all the cheetahs, the appropriate hunting techniques and prey selection had to be learned overtime. There was no difference between what was stalked and what was caught. From the observed kills, prey weight ranged from small <40 kg to medium <160 kg. This weight range is comparable to wild cheetahs that select prey within the same range. Each cheetah hunted every four to five days and consumed three to five kilograms of meat per day. Nine prey species were identified, two of which were not detected during direct observations. In the collection of scat, the trained dog found approximately a scat per hour while each direct observation by the researchers took approximately 10 hours. These captive bred cheetahs had to learn how to hunt and how to select appropriate prey thus addressing learned behavior versus instinct. Given the frequency and intensity of injuries sustained and the extensive human influence in the form of veterinary treatment, the reintroduction was not a success. However the techniques developed and modified are relevant for future endeavors‟. / Dissertation (MSc)--University of Pretoria, 2011. / Animal and Wildlife Sciences / unrestricted

Objectives: The aims of this study was to investigate the RC microbiota in CCF canine teeth in the domestic dogs (Canis familiaris) and cheetahs (Acinonyx jubatus), identify the possible factors related to the presence of aerobic or anaerobic bacteria and evaluate and evaluate antibiotic susceptibility of bacteria isolated. Animals: Thirty nine animals suffering from CCF of their canine teeth were included in this study, of which 20 were dogs and 19 were cheetahs. Procedures: Evaluation of the oral cavity of animals while under general anaesthesia was performed and those without necrotic pulps or those that had received antibiotic therapy in the previous two weeks were excluded. Microbial samples were taken from 63 RC of which 27 were from dogs and 36 were from cheetahs. Strict anaerobic and aerobic techniques were used in parallel for plating, incubation and identification of the bacteria isolated in this manner. In an attempt to evaluate the sensitivity of the culture media and anaerobic technique used, additional samples were collected after the samples for bacterial isolation had been taken from the last eight pulps. These comprised those from six cheetahs and two dogs and were analysed using culture techniques and an initial screening with the 16S rRNA-specific PCR. Results: • Dogs: A total of 49 cultivable isolates were recovered belonging to 19 different bacterial species and 13 different genera. Individual RC yielded a maximum of four bacterial species. Of the bacterial isolates, 4.08 % were strict anaerobes, being represented by Clostridium acetobulitycum (2.04 %) and Prevotella melalinogenica (2.04 % ). The incidence of aerobic bacteria and facultative anaerobic bacteria in this study were 18.36 % and 77.56 %respectively of all the bacterial isolates. Of these Pasteurella multocida ( 10.20 % ), Corynebacterium spp. (10.20 %), Moraxella spp. (8.17 %), Bacillus spp. (6.12 %), Aeromonas salmonicida (6.12 %), Escherichia coli (6.12 %) and Pseudomonas aeruginosa (6.12 %) were the bacteria most frequently isolated. In summary, the RC microflora was found to be predominantly Gram negative facultative anaerobic microorganisms. The antibiotic agents that showed the highest efficacy in vitro against the different bacteria isolates were Enrofloxacin (85.21 % ), Gentamicin (92.39 %), Chloramphenicol (89.13 %). • Cheetahs: A total of 59 cultivable isolates, belonging to 19 different microbial species and 13 different genera were recovered from 36 RC sampled. Thirty-two (54.49 %) of the cultivable isolates were Gram positive while 27 (45.71 %) were Gram negative. Individual root canals each yielded a maximum of six species. Four RC had no cultivable bacteria. The bacterial micro flora recovered from the RC of the animals showed a higher number of facultative anaerobes (62.72 % of all the bacterial isolates). Aerobic isolates were 28.81 %, and strict anaerobes 8.47 % of all the isolates. The latter species comprised Clostridium sordelli (5.08 % ), and Clostridium septicum (3.38 % ). The species with the highest isolation frequency were Bacillus spp. (22.13 %), Pasteurella multocida (10.16 %), Corynebacterium spp. (8.47 %), Enterococcus spp. (8.47 %), Moraxella spp. (8.47 %) and Pseudomonas aeruginosa (5.25 %). In summary, the bacteria isolated from the RC were Gram positive facultative anaerobic bacteria. The antibiotics, which showed the highest efficacy in vitro against the different bacteria isolates, were Enrofloxacin (91.96 %), Gentamicin (86.37 %) and Orbifloxacin (86.28 %). • Nucleic Acid-Base detection: In dogs, Gram negative and Gram positive bacterial species were equally represented. Anaerobic bacterial species predominated at 83.3 % (5/6) of the species detected. On the other hand, in cheetahs, the bacterial species isolated by the PCR method showed a prevalence of anaerobic bacteria (60.8 %, 14/23), while facultative anaerobes were isolated in 30.2 % (7 /23) of cases and aerobic bacteria in 8.6 % (2/23). Conclusions and Clinical Relevance: This study has indicated that the microbial flora in any single infected RC is much more diverse than it has been shown using cultural techniques alone and can contain potentially uncultivable bacterial species. Some of these species may represent potentially new phylotypes, which may be involved in endodontic infections and ultimatelyin periradicular periodontitis, and should therefore be considered in any future studies involved in defining endodontic pathogens. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Companion Animal Clinical Studies / restricted