Comparison of Tissue Digestion Techniques for Detecting Toxoplasma gondii in Cockles (Anadara granosa) Along the Coast of Thailand
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Toxoplasmosis, a parasitic infection due to the protozoan Toxoplasma gondii, is considered one of the most common diseases around the world. It is believed that humans are infected through intermediate hosts, often livestock and marine animals. Due to a decreased number of sea otters (Enhdra lutris neris) in California as a result of this parasitic infection, it is assumed that cockles (Anadara granosa), which otters prey upon, may act as vehicles carrying the parasite. They may also have an ability to infect humans directly if humans ingest raw cockles. In this study, we performed a comparison between two methods in order to optimize the analysis of T. gondii contamination in cockles along the coast of Thailand. We analyzed tissues from the gills and digestive tract, the locations where the parasites are most likely to be found. By using B1 and SAG1 specific primers, the detection sensitivities toward the parasite for both methods were obtained through real time polymerase chain reaction (qPCR) and compared by the one-tailed t-test. Matrix spikes of the known amount in an ascending order of T. gondii DNA into cockle gills and digestive tracts were applied to both methods. The pepsin digestion method was found to be the better option as it could detect as few as two T. gondii parasites, while the fat extraction by ethyl acetate could only detect the lowest of 80 parasites. Moreover, the gills appeared to be a better location for finding this parasite than the digestive tract. With this knowledge, the optimized method can be used to analyze cockles along the coast without the matrix spikes. The results obtained will further confirm us whether cockles can be possible vehicles for the parasite to infect intermediate hosts and humans.