Genotyping of Cryptosporidium spp. from a Sewage-Contaminated River in Guilan, Iran

The role of water in Cryptosporidium transmission is now well recognized. In the present study, 19 water samples were collected from the river contaminated with wastewater in Guilan, Iran. This province lies along the Caspian Sea and has a Mediterranean-like climate. The species of Cryptosporidium were identified by polymerase chain reaction (PCR) assay using two set primers: (CPB-DIAGF/R and N-DIAGF2/R2) and (Xiao F1/R1 and F2/R2). A total of 10/19 water samples were positive for Cryptosporidium species (C. parvum, C. muris and C. hominis). The use of sewage-contaminated river water for the irrigation of agriculture farms is a threat of infection to the local population.


Introduction
Recent molecular studies of cryptosporidiosis in animals and the environment helped researchers to better understand the transmission of cryptosporidiosis in humans and the public health significance of Cryptosporidium spp. [1]. Waterborne cryptosporidiosis has been reported worldwide, and the ingestion of oocyst-contaminated drinking water has led to a large number of outbreaks, mainly reported from North America, the United Kingdom, Japan, and Australia [2][3][4]. The use of molecular techniques in epidemiological studies has shown that five Cryptosporidium species (C. hominis, C. parvum, C. meleagridis, C. felis, and C. canis) are responsible for most human infections and two species, C. parvum and C. hominis, are the most common [5]. The distribution of Cryptosporidium species in environmental water and wastewater in Iran is unclear. The objective of this study was to identify the genotype of Cryptosporidium spp. in two important rivers in Iran.

Geography and Sample Collection
Guilan Province (37.2809° N, 49.5924° E) lies along the Caspian Sea and has a moderate, mild, and Mediterranean-like climate. Goharrood river is a branch of the Sefidrood River that crosses Rasht City and finally meets the Bandar-e Anzali Lagoon. In the present study, 19 water samples were collected from the Goharrood River. Many pollutants, such as rural, urban, and agricultural runoff and animals' raw sewage threaten water supplies in the cities of Guilan [6]. About 5 L of each sample was filtered through a cellulose nitrate filter of pore size 1.2 μm using a vacuum pump. The sediment pellet was subjected to sucrose flotation [7], then the supernatant was subject to DNA extraction and Polymerase Chain Reaction (PCR).

DNA Extraction and PCR
DNA was extracted using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions with some modifications. Briefly, the suspension, including amoeba cysts or trophozoites, was frozen and thawed at −196 °C for 5 min ten times, followed by 5 min in boil water [8]. Two primer sets were used in this study. The first was a genus-specific primer targeting the 18 rRNA gene-specifically for Cryptosporidium spp.-used for the amplification of the approximately 435 bp fragment using CPB-DIAGF/R and N-DIAGF2/R2 primers [9,10].
The second set, XIAOF1/R1 and XIAO F2/R2, were used to amplify the approximately 850 bp fragment of the gene encoding the 18SSU rRNA of the genus Cryptosporidium as described previously [11,12]. Each reaction was performed in 50 μL and both PCR reactions were performed in PCR tubes according to a previous study [9][10][11][12]. The PCR products electrophoresis was performed on 1.5% gel agarose stained with ethidium bromide solution and visualized under UV light. The PCR products were then sequenced and all sequences data were manually edited and analyzed with reference sequences using the Chromas software program. Genotype identification was performed by the available Cryptosporidium DNA sequences in GenBank.

Results
In the present study, 10/19 of the samples were positive for Cryptosporidium spp. two nested PCR primers. DNA sequencing of five PCR products revealed the presence of three species of Cryptosporidium, C. parvum (3), C. muris (1) and C. hominis (1), respectively. C. parvum was the most common species.

Discussion
The detection of pathogens and the safety of drinking water are topics of great concern to health authorities. Accurate identification of Cryptosporidium species requires the use of genotyping tools. Currently, most Cryptosporidium genotyping tools use PCR targeting the small subunit (SSU) rRNA gene [13]. Molecular studies have shown that five Cryptosporidium species (C. hominis, C. parvum, C. meleagridis, C. felis, and C. canis) are responsible for most human infections, but two species-C. hominis and C. parvum-are the most common, and the role of water in disease transmission is now well recognized [5]. Numerous outbreaks of human cryptosporidiosis all over the world have been reported, mainly from North America, the United Kingdom, Japan, and Australia [2][3][4]14]. In the present study, C. parvum, C. muris and C. hominis were detected. The occurrence of species other than C. parvum in the environment is well recognized. Xiao et al. [15,16] reported the presence of Cryptosporidium spp. in 27 of 29 storm water samples in the United States, mainly wildlife Cryptosporidium genotypes. In contrast, the most common species found in surface waters were C. parvum, C. hominis, and C. andersoni, with the last reported to be the most commonly found in wastewater (eight samples). Mahmoudi et al. [11,12] analyzed surface and river water samples in Iran by different methods, and showed that C. parvum was the most prevalent species, followed by C. hominis and C. canis, respectively [11,12]. Another study [17] reported this parasite from untreated water samples in Iran. In a comprehensive study by Keshavarz et al. (2009), Iranian cattle were mainly found to be infected with C. parvum (72.6%), C. andersoni (17.7%), and C. bovis (7.8%) [18]. C. parvum was the predominant species in children with diarrhea and AIDS patients in other studies in Iran [19,20]. Other studies in Iran have shown a frequent occurrence of C. hominis in children or AIDS patients [21,22]. C. muris was detected in the present study, which is a well-known rodent parasite [23]; the results of the present study indicate that these animals are also a source of Cryptosporidium oocysts in river water in Iran. The differences in the distribution of Cryptosporidium species in humans are considered an indication of differences in infection sources [1]. C. hominis is responsible for far more infections than C. parvum in humans in developing countries, where genotyping studies have been conducted [24][25][26][27][28][29]. In the United Kingdom, Europe, and New Zealand, C. parvum was as common as C. hominis in humans [30][31][32][33][34]. The results of the present study indicate that the investigated river water supplies were contaminated by pathogenic species of Cryptosporidium from humans and animals.

Conclusions
The use of long-term sewage-contaminated river water for the irrigation of crops and agriculture farms and their subsequent transfer to the food chain is a threat of infection to the population. More future studies with a systematic sampling of water in the study area would provide a better picture of the extent of water contamination with Cryptosporidium species. Periodic determination of Cryptosporidium spp. in river water can help to develop strategies for the protection of human health.

Conflicts of Interest:
The authors declare no conflict of interest.