# Food and Drinking Water as Sources of Pathogenic Protozoans: An Update
## 17 Methods for the Detection/Identification of Protozons in Food and Drinking Water
axytenensis_ DNA in surface water samples [237]. Methods were made available to detect pathogenic protozoans from challenging food matrices, such as meat, milk, seafood, and cheese, and are available for validation. Molecular detection in meat was carried out for _T. gondii_ by homogenizing tissue samples and extracting DNA from 25 mg of homogenate using commercial kits [168; 175; 176; 177; 295]. Detection of _T. gondii_ from milk was accomplished by centrifugation, separation of fat, washing, and resuspension of the concentrated pellet before DNA extraction. The detection in cheese was carried out by homogenization in stomacher, washing the pellet from 1 mL of the homogenate, and using a commercial kit for DNA extraction from 200 uL of pellet suspension [177]. A method used for serological analysis in meats of _T. gondii_, applied to wild boar, is based on freezing at \(-20\)\({}^{\circ}\)C in a plastic bag and meat juice collection after thawing for subsequent analysis [175]. A method of detection from oysters was described for _Cryptosporidium_ spp. and comprises the homogenization of gills and gastrointestinal tract, lipid extraction with diethyl ether concentration by centrifugation, and detection with fluorescent anti-_C. parvum_ monoclonal antibodies [137]. _Cryptosporidium_ spp. in fish was detected in gastrointestinal tissue scrapings mixed with intestinal contents used for direct DNA extraction with a commercial kit [136]. Detection from mussels was carried out for _Cryptosporidium_ and _Giardia_ on the entire contents of the shells including the intravascular liquid. Alternatively, water and coarse-sieving, diethyl ether lipid removal, and digestion with pepsin, followed by IFA or IMS+IFA, were carried out. The digestion method was the most effective protocol to detect both parasites [296]. Detection from blue crabs was carried out for _C. axytenensis_ and _T. gondii_ on the homogenate obtained from gills, hepatopancreas, stomach, and gonads sieved through a double layer of gauze, pelleted via centrifugation, washed with an appropriate buffer, and lysed through freeze-thawing cycles before DNA extraction [176]. Reported methods for the detection of _Sarccystis_ spp. are the most varied and include the isolation of individual sarcocysts from the heart and diaphragm with tweezers and needles for microscope examination [173; 266], homogenization of tissues, and extraction/decantation methods with or without pepsin/HCl digestion, microscope observation with or without staining with different dyes on sediments or directly on tissues not stained, stained, paraffin-embedded, or not [258; 266; 267; 297]. DNA extraction for molecular identification was carried out from individual sarcocysts or infected tissues by using commercial DNA extraction kits [173; 265; 297]. PCR methods of detection were targeted on the SSU rDNA for _Cryptosporidium_[4; 122; 123; 132], _C. cayetanensis_[237], and _Sarcocystis_ species [258; 260; 263; 297; 173]. Specific genetic targets were the internal transcribed spacer 2 (ITS 2), the _lsp70_ gene and the ITS1, the mitochondrial cytochrome oxidase gene for _C. cayetanensis_[37; 176; 248; 291] and a specific real-time PCR detection method using mitochondrial primers (Mit1C qPCR) [292], the ITS1188-5.8S rRNA, ROP5 and the 529 bp repetitive element for _T. gondii_, [37; 173; 176; 295], the ITS1 region, and the mtDNA _cox1_ gene for _Sarcocystis_ species [258; 260; 263; 264; 265; 266; 297]. The sensitivity of detection was found to be affected by the target gene chosen for PCR. As an example, the LODs of _T. gondii_ in lettuce were 10 and 100 ocysts per uL, with PCR targeted on the B1 gene and the 529 bp fragment, respectively, since the B1 gene is present in 35 copies while the 529 bp fragment is repeated 200-300 times in the genome [37]. Limits of detection (LODs) of 3 oocytes/g of _G. duodenalis_ and _C. parvum_ and less than 1 oocyst/g for _T. gondii_, were obtained in the analysis of basil using quantitative PCR (qPCR). _T.