Rahim Gul

Isolation Of Local Strain Of Toxoplasma Gondii Through In-Vivo Cultivation In Mice - 2013

Toxoplasma gondii is an obligate apicomplexan, intracellular, parasite that infects all warm-blooded vertebrates, including mammals and birds. Human beings can be infected by ingestion of oocysts from cat faeces or through the consumption of meat containing Toxoplasma gondii cysts. Thus, food animals can be the source of transmission of Toxoplasmosis in human population especially among people who consume undercooked meat in the forms of barbecues, beef steaks, kebabs, burgers and shawarmas. Oocysts of T. gondii from cat faeces were identified by using direct microscopy and flotation technique. The positive oocysts were confirmed by micrometry having diameter of 9-13 ìm. The oocysts were then sporulated in aerated condition. After sporulation oocyst were inoculated in Swiss albino mice for in-vivo culturing. After 56-70 days brain tissue was collected from infected mice and subjected to DNA extraction and PCR amplification. Similarly DNA was also extracted from sporulated oocyst for copro-PCR. Out of 200 faecal samples only three were found positive for Toxoplasma gondii through direct microscopic examination and flotation technique. From positive faecal sample and brain tissue DNA was extracted by QIAGEN mini stool kit and QIAGEN DNA mini kit. After DNA extraction the samples were examined through PCR by using specific Toxoplasma gondii B1 gene primer having 529 bp size. Two hundred faecal samples were examined for T. gondii using direct microscopy, flotation technique, bioassay and polymerase chain reaction. Out of 200 samples 3 (1.5%) were found infected through direct microscopy and flotation technique. Toxoplasmosis was more prevalent in adult cats (1.65%) as compared to young ones. Prevalence was also found high in females (2.08%) as compared to males. Similarly healthy cats have higher prevalence rate (1.30%) as compared to diseased ones. A further confirmation was done through polymerase chain reaction and brain tissue cyst Bioassay give 1 positive amplification while Copro-PCR gives 2 positive amplifications. Therefore it can be concluded that the copro-PCR is can be used for the confirmation of Toxoplasma oocysts from cat faeces and tissue cysts from bioassay in mice. Therefore, we propose that the copro-PCR can be used as the new gold standard for determining potential cat infectivity and tissue cysts from bioassayed mice or contaminated meat samples of livestock.

Department of Parasitology


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