Comparison of Chemical and Biological Methods of Filtering Cryptosporidia from Water

Despite the fact that Cryptosporidium spp. is a parasite which commonly causes diarrhea, it still receives little attention. In our experiment, we focused on comparing the biological (N. davidi shrimp) and physical (zeolite with different thicknesses) possibility of filtering cryptosporidia from a small volume of water, which could contribute to increasing the catchability of this parasite. We monitored the ability to capture oocysts of the parasite Cryptosporidium parvum, genotype IIaA11G2R1, found in water samples. We infected drinking water with feces with a known number of cryptosporidial oocysts. One gram of sample contained ±28 oocysts. We filtered eight water samples with different concentrations of oocysts (0.1-2 g of infected stool per 15 L of water) using zeolite with a particle thickness of 0.2-0.6 mm and 0-0.3 mm. This was followed by purification, centrifugation and isolation utilizing the isolation kit AmpliSens^® DNA-sorb-B, which is intended for stool. In total, 120 shrimp were divided into four aquariums (A, B, C, n = 30) including the control (K), while drinking water with the same parameters was infected with different concentrations of oocysts (A: 2.5 g, B: 2 g, C: 1 g of infected stool per 15 L of water). We took 10 individual shrimp and processed them in three time intervals (6 h, 12 h and 24 h). We processed them whole, and we isolated the DNA utilizing the isolation kit AmpliSens^® DNA-sorb-AM, which is intended for tissues. Detection was carried out by molecular methods, namely the Nested PCR targeting of the region of the GP60 gene (60 kD glycoprotein). Gel electrophoresis showed the presence of C. parvum in seven zeolite-filtered water samples, and the parasite was not found in the water sample with the lowest number of oocysts filtered through the smaller-particle zeolite. There were 67 C. parvum-positive shrimp. Whereas the most positive shrimp were identified at 12 h of sampling, the least were identified at the 24 h mark. No shrimp positive for C. parvum was found in the control group. By sequencing, we confirmed the presence of C. parvum, genotype IIaA11G2R1, in all positive samples. We thus proved that the filtration capabilities of zeolite and N. davidi can be used for the rapid diagnosis of the presence of protozoa in a small amount of studied water.