Detection and Enumeration of Cryptosporidium sp. Oocysts in Sewage Sludge Samples from the City of Florianópolis (Brazil) by Using Immunomagnetic Separation Combined with Indirect Immunofluorescence Assay

Abstract

In the last few decades, agricultural reclamation of sewage sludge has increased in many countries. However, this practice must be adopted under recommended conditions to limit the risks associated with the presence of potential pathogens, including protozoan parasites such as Cryptosporidium. In the present work, we evaluated immunomagnetic separation (IMS) combined with an indirect immunofluorescence assay (IFA) in order to detect Cryptosporidium oocysts in sewage sludge samples obtained from the Municipal Wastewater Treatment Plant (MWTP) operating in the city of Florianópolis, State of Santa Catarina, Brazil. Integrated application of spontaneous sludge sedimentation and centrifugation of the supernatant, purification and concentration of oocysts through immunomagnetic separation as well as detection and quantification by means of indirect immunofluorescence using highly specific monoclonal antibodies, proved to be an effective multiple-analysis tool for monitoring the presence of Cryptosporidium oocysts in urban sewage sludge samples. Among 27 samples collected from MWTP, 20 (74.07%) were positive for Cryptosporidium sp. The efficiency of recovery by using the internal control ColorSeed® (Biotechnology Frontiers Inc., Sydney, NSW, Australia) was medially 39.96 ± 11.20%. Actually, oocysts were recovered from 100% of the positive samples tested and the number of Cryptosporidium oocysts found in the samples ranged from one to four. These results demonstrated a high recovery rate of Cryptosporidium oocysts in sewage sludge samples by using IMS-IFA analysis. In conclusion, such an approach can be applied to monitor the presence of the parasite and to determine the potential contamination of sludge destined for soil application.