title: Detection of Toxoplasma Gondii in Fresh New Zealand Farmed Meat by the Polymerase Chain Reaction

authors: Anderson, Michael Andrew

subjects: Toxoplasma

abstract: 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.

text: Detection of Toxoplasma Gondii in Fresh New Zealand Farmed Meat by the Polymerase Chain Reaction 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.

year: 2015

title: The Control Points of Lactose Synthesis in the Lactating Ruminant

authors: Eichler, Stephen J.

abstract: It has been established that lactose is the major osmole in the milk of many mammalian species, and hence that the amount of water secreted is approximately proportional to the amount of lactose secreted. It is envisaged that reduction of the amount of lactose synthesised would allow a reduction of the amount of water in milk. 
This thesis studied the regulation of lactose synthesis in the mammary glands of goats by altering the flux of glucose and other intermediates of lactose synthesis. The consequent effects were determined by measuring the levels of various metabolites in milk. 
The mechanism of the reduction of milk yield brought about by twenty-four hour fasting in goats was explored by looking for perturbations in some of these parameters measured. Also, known differences between certain bovine genetic groups in water secretion were investigated by measurements in milk of trace metabolites involved in lactose synthesis. 
It was intended to further substantiate the paradigm that availability of glucose within mammary secretory cells of ruminants normally regulates lactose synthesis during midlactation. This widely held belief has not been supported by this research.

text: The Control Points of Lactose Synthesis in the Lactating Ruminant It has been established that lactose is the major osmole in the milk of many mammalian species, and hence that the amount of water secreted is approximately proportional to the amount of lactose secreted. It is envisaged that reduction of the amount of lactose synthesised would allow a reduction of the amount of water in milk. 
This thesis studied the regulation of lactose synthesis in the mammary glands of goats by altering the flux of glucose and other intermediates of lactose synthesis. The consequent effects were determined by measuring the levels of various metabolites in milk. 
The mechanism of the reduction of milk yield brought about by twenty-four hour fasting in goats was explored by looking for perturbations in some of these parameters measured. Also, known differences between certain bovine genetic groups in water secretion were investigated by measurements in milk of trace metabolites involved in lactose synthesis. 
It was intended to further substantiate the paradigm that availability of glucose within mammary secretory cells of ruminants normally regulates lactose synthesis during midlactation. This widely held belief has not been supported by this research.

year: 1993

title: Military Settlement in the Middle Waikato Basin

authors: Allen, Peter D.H.

abstract: Military settlement formed a brief but distinctive phase in the European occupation of the Middle Waikato Basin. Prior to the 1863 - 1864 Waikato War, few Europeans, other than a small number of missionaries and traders, were settled in the region. The setting of the Middle Waikato Basin was largely unmodified by Man, except along the major rivers where a dense Maori population was settled. 
During the 1850's, the encroachment of European settlers upon Maori tribal lands in many North Island regions, including the Waikato, led to an increase in tension between the two populations and open conflict. This conflict slowed down the progress of colonization in the North Island. To enable colonization to continue in those regions disturbed by Maori unrest, a scheme of military settlement was devised. The aim of this scheme was the formation of compact, self-sufficient defensive settlements to act as a deterrent to Maori unrest. These settlements would also provide an assurance to settlers of security for their life and property.
 Military settlement in the Middle Waikato Basin consisted of two phases. In the first phase, the nodal points of the military settlements, the townships, were established and occupied by the military settlers. In the second phase, farm districts were surveyed around the nodal points and the military settlers moved out from the townships to occupy and develop the land they were allocated. A number of factors, relating to deficiencies in the scheme of military settlement and to the particular problems of settlement in the region, contributed to the failure of the military settlers to successfully establish farms on the land they were allocated. 
The scheme of military settlement largely failed as a method of colonization in the Middle Waikato Basin. However, the pattern of human occupation, established in the initial phase of military settlement, remained when the need for defensive settlements had gone, and forms the basis of the present pattern of settlement.

text: Military Settlement in the Middle Waikato Basin Military settlement formed a brief but distinctive phase in the European occupation of the Middle Waikato Basin. Prior to the 1863 - 1864 Waikato War, few Europeans, other than a small number of missionaries and traders, were settled in the region. The setting of the Middle Waikato Basin was largely unmodified by Man, except along the major rivers where a dense Maori population was settled. 
During the 1850's, the encroachment of European settlers upon Maori tribal lands in many North Island regions, including the Waikato, led to an increase in tension between the two populations and open conflict. This conflict slowed down the progress of colonization in the North Island. To enable colonization to continue in those regions disturbed by Maori unrest, a scheme of military settlement was devised. The aim of this scheme was the formation of compact, self-sufficient defensive settlements to act as a deterrent to Maori unrest. These settlements would also provide an assurance to settlers of security for their life and property.
 Military settlement in the Middle Waikato Basin consisted of two phases. In the first phase, the nodal points of the military settlements, the townships, were established and occupied by the military settlers. In the second phase, farm districts were surveyed around the nodal points and the military settlers moved out from the townships to occupy and develop the land they were allocated. A number of factors, relating to deficiencies in the scheme of military settlement and to the particular problems of settlement in the region, contributed to the failure of the military settlers to successfully establish farms on the land they were allocated. 
The scheme of military settlement largely failed as a method of colonization in the Middle Waikato Basin. However, the pattern of human occupation, established in the initial phase of military settlement, remained when the need for defensive settlements had gone, and forms the basis of the present pattern of settlement.

year: 1969