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Molecular Characterization of Cryptosporidium Species in Children with Diarrhea in North West of Iran.

Cryptosporidium is one of the most common causes of childhood diarrhea in developing countries. The aim of this randomized pilot study was to detect and characterize infective species and determine the genotypes of Cryptosporidium parasites in pediatric patients suffering from diarrhea in North West of Iran. A total of 113 fecal samples were collected from diarrheic children hospitalized in Tabriz Pediatric Hospital. The amplification of small subunit ribosomal RNA gene was performed using a nested polymerase chain reaction protocol and its products were digested using two restriction enzymes for Cryptosporidium species and genotype differentiation. Cryptosporidium oocysts were found in 2 (1.76%) children with diarrhea and restriction pattern revealed the presence of C.parvum bovine genotype in both positive fecal samples. The findings indicate that Cryptosporidium parvum is responsible for cryptosporidiosis in children in the study region and probably zoonotic transmission is the predominant route of parasite transmission.

hominis (previously known as the C. parvum human genotype), C. parvum (bovine genotype), C. meleagridis, C. canis and C. felis have been found to be responsible for most human infections. C. hominis is an anthroponotic genotype exclusively found in humans, while C. parvum (zoonotic genotype) is found in humans and wide range of domestic and wild animals (4).
Cryptosporidiosis occurs by consumption of ubiquitous oocysts which are transmitted indirectly via contaminated drinking and recreational water or directly via contact with infected humans or animals (1). The oocysts of parasites are environmentally highly resistant to common disinfectants and can survive in water for weeks (5). They are excreted in large numbers by infected humans and animals, and by means of discharging of waste, can reach surface water and consequently contaminate drinking water.
The oocyst transmission via contaminated drinking water, recreational water and municipal water is well -proven, but probably drinking water has the most important role in the transmission of the infection, and proper treatment of drinking water leads to significant reduction in cases of disease (1,6).

Samples collection
Fecal samples were collected from 113 children (age range: 3 months-12 years) with acute gastroenteritis hospitalized in Tabriz Pediatric Hospital, a referral center in the North West of Iran.
The average age of children was 3 years old. Each sample was divided into two parts. One part of the specimen was concentrated by formalin-ether concentration method and the other part stored in 2.5% potassium dichromate solution and kept at 4 °C for further molecular analysis. The concentrated specimens were stained with acid-fast staining method and examined microscopically (7)(8).

DNA extraction
Positive fecal samples and forty negative fecal samples which were randomly selected, were subjected to DNA extraction. About 200 mg of fecal materials that were stored in 2.5% potassium dichromate were rinsed five times with a solution of phosphate buffered saline (PBS, pH = 7.2) and centrifuged at 14000 g for 10 min at 4 °C to remove potassium dichromate completely. The pellets were subjected to ten freeze -thaw cycles (three min freeze in liquid nitrogen followed by three min at 65 °C). DNA was extracted by using the modified proteinase K (Fermentas, Lithuania), sodium dodecyl sulfate (SDS) (Merck, Germany) and cetyltrimethylammonium bromide (CTAB) (Merck, Germany) method (9). The extracted DNA pellets were dissolved in 20 µl of Tris-EDTA (TE) buffer and stored at -20 °C before application in PCR (2).

PCR amplification and RFLP
The amplification of small ribosomal subunit Characterizations of the species and genotypes were performed according to the restriction patterns described previously (6).

Results
The Cryptosporidium oocysts were found in 2 in gel electrophoresis (6), (Fig.1a).To distinguish human and bovine genotypes of C. parvum, the products of second PCR step were digested with VspI. C. parvum bovine genotype showed two visible bands of 628 and 104 bp (6) (Fig.1b).

Discussion
The present pilot study attempted to detect The disease in humans is mainly caused by two species of C.parvum and C.hominis (15) (18)(19).