Detection and subtype identification of Blastocystis isolates from wastewater samples in the Philippines Jan Ervin G. Banaticla and Windell L. Rivera ABSTRACT To provide further evidence of waterborne transmission of Blastocystis, a total of 31 wastewater treatment plants from geographically distinct locations across the Philippines were sampled for influent and effluent sewage samples. In vitro cultivation was the method of choice to increase sensitivity of detection. Blastocystis cysts were detected in 15% (9/62) of the samples using in vitro culture. Moreover, influent and effluent samples were 23% (7/31) and 7% (2/31) positive for the parasite, respectively. The presence of viable cysts in treated samples may be an indication of the inefficiency of the treatment process in preventing Blastocystis from entering the environment. Polymerase chain reaction and sequencing of the full-length small subunit ribosomal RNA (SSU rRNA) genes of the nine wastewater isolates were performed. The SSU rRNA gene sequences of the isolates showed very high similarity (98 to 99%) to homologous sequences of Blastocystis described previously. The phylogenetic tree constructed showed that the wastewater isolates clustered with each other with good bootstrap support and belonged to two subtypes (ST) – ST1 and ST2. This is the first report of subtyping Blastocystis isolates from wastewater samples and gives further emphasis to the remarkable genetic diversity of the parasite. Key words 9 Blastocystis, Philippines, small subunit ribosomal RNA (SSU rRNA) gene, subtype, wastewater, waterborne transmission INTRODUCTION Blastocystis sp. is one of the most frequently reported proto- zoan parasites in faecal samples. The parasite thrives in the human gastrointestinal tract and is strictly anaerobic in axenic culture (Tan 2008; Tan et al. 2010). Recent data suggest that it has a worldwide distribution with prevalence ranging from 30 to 50% in developing countries, while a lower rate exists in developed countries (Yoshikawa et al. 2004a; O ¨ zyurt et al. 2008; Tan 2008; Wong et al. 2008; Souppart et al. 2010; Tan et al. 2010). Owing to its highly zoonotic potential, Blastocystis sp. has been isolated in a wide array of hosts including, but not limited to, the following: monkeys, pigs, cattle, birds, amphibians, rodents, reptiles and insects (Noe¨l et al. 2005; Rivera 2008; Yoshikawa et al. 2009). More recently, three new subtypes (ST) of Blastocystis have been reported: ST11, ST12 and ST13 from elephants, giraffes and quokkas, respectively (Parkar et al. 2010). Extensive polymorphism is exhibited by Blastocystis, with four major forms observed in culture studies: vacuolar, gran- ular, amoeboid and cyst. The cyst form, which has a small size (2–5 mm) and is mostly ovoid and spherical in shape, was discovered more than a decade ago and is easily confused with faecal debris (Moe et al. 1996, 1999). It has been demonstrated that the cyst is the transmissible form of the parasite and that as few as 10 to 100 cysts are sufficient to establish an infection (Yoshikawa et al. 2004b; Tanizaki et al. 2005). Apparently, there is a paucity of studies reported in the literature regarding the analysis of Blastocystis isolates from sewage samples. Suresh et al.(2005) reported that viable cysts Jan Ervin G. Banaticla Windell L. Rivera (corresponding author) Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101, Philippines and Molecular Protozoology Laboratory, Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines Fax: þ 63 2 920 5471 E-mail: wlrivera@science.upd.edu.ph doi: 10.2166/wh.2010.127 128 & IWA Publishing 2011 Journal of Water and Health 9 09.1 9 2011 Downloaded from http://iwaponline.com/jwh/article-pdf/9/1/128/397575/128.pdf by guest on 22 February 2023