Abstract:
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.
