Detection of Toxoplasma Gondii in Fresh New Zealand Farmed Meat by the Polymerase Chain Reaction
Anderson, M. A. (2015). Detection of Toxoplasma Gondii in Fresh New Zealand Farmed Meat by the Polymerase Chain Reaction (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/10118
Permanent Research Commons link: https://hdl.handle.net/10289/10118
Toxoplasma gondii is an obligate intracellular parasite that is estimated to infect one third of the world’s human population. Upon infection this parasite causes the disease toxoplasmosis which in most healthy humans is chronic and often asymptomatic. If infection occurs in a human host without a competent immune system such as an unborn baby or infant or someone suffering from AIDS, toxoplasmosis can lead to permanent disability or death. A growing amount of evidence indicates a statistical relationship between chronic infection in healthy adults and neurological disorders leading to behavioural and psychological problems. Toxoplasma gondii can be spread through contact with cat faeces. This was thought to be the main mode of human infection. Subsequent testing with murine and Felidae models has shown that the bradyzoite of Toxoplasma gondii within intracellular cysts in muscle and neural tissue are far more infectious than the oocyst morphology shed in cat faeces. This indicates that consumption of infected meat products may also play a major contributing role in the world’s high rate of toxoplasmosis infection. The chief aim of this study was to develop a sensitive PCR assay to detect the presence of Toxoplasma gondii in fresh raw meat products. Beef, chicken, lamb and pork mince were chosen as convenient forms of fresh raw meat as each pack available for purchase contains the meat from several animal’s tissues (increasing the likelihood of contamination) and it has been partially homogenised allowing for easier sampling and DNA extraction. Secondary aims of this study were to evaluate PCR primer sets targeting polymorphic regions to gain some understanding of what strains of Toxoplasma gondii are present in our meat and the prevalence in the local cat populations. To accomplish these aims positive and negative PCR controls were also developed using a live toxoplasmosis vaccine and DNA extracted from Zebrafish. DNA extraction techniques were developed to purify DNA from both muscle tissue and feline faecal samples and primer sets were selected from published literature as well as designed using NCBI primer BLAST. Toxoplasma gondii specific DNA was detected in only one of the 25 meat samples purchased. This was achieved using a primer set targeting the P30 region of the SAG1 gene and confirmed by DNA sequencing. Using primer sets targeting polymorphic sites within the Toxoplasma gondii genome, sequencing and restriction enzyme digests it was shown that Toxoplasma gondii present in the feral Felis catus population in the raglan area is type II at the GRA6 and SAG3 loci. Amplification of polymorphic PCR targets from DNA extracted from meat samples could not be achieved. Non-specific amplification occurred with a majority of primer sets trialed with meat DNA extracts indicating improvements are needed in primer design for the purpose of PCR detection of Toxoplasma gondii in meat. Nested PCR increased specificity however resulted in contamination in several instances.
University of Waikato
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