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Molecular Characterization of Cryptosporidium spp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

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ABSTRACT

Background. Rodents could act as reservoir for Cryptosporidium spp. specially C. parvum, a zoonotic agent responsible for human infections. Since there is no information about Cryptosporidium infection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization of Cryptosporidium spp. in this region. Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method using SspI and VspI restriction enzymes was carried out to genotype the species and then obtained results were sequenced. Results. Three out of 100 samples were diagnosed as positive and overall prevalence of Cryptosporidium spp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples and C. parvum pattern was identified. Finally PCR-RFLP findings were sequenced and presence of C. parvum was confirmed again. Conclusions. Our study showed rodents could be potential reservoir for C. parvum. So an integrated program for control and combat with them should be adopted and continued.

No MeSH data available.


Secondary stage of nested-PCR findings on agarose gel. Lane M, DNA size marker. Lane 1, positive control for Cryptosporidium. Lanes 2–4, positive Cryptosporidium samples (830 bp).
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fig1: Secondary stage of nested-PCR findings on agarose gel. Lane M, DNA size marker. Lane 1, positive control for Cryptosporidium. Lanes 2–4, positive Cryptosporidium samples (830 bp).

Mentions: Three out of 100 samples were detected as positive for Cryptosporidium spp. using modified Ziehl-Neelsen staining under light microscope. In addition, nested-PCR was done and showed 830 bp band which confirmed only 3 samples as Cryptosporidium spp. (Figure 1). So, overall prevalence of Cryptosporidium infection in rodents of Ahvaz city was calculated at 3% using both methods. All positive samples belonged to R. norvegicus (3/73) and from M. musculus (0/6) and R. rattus (0/21) no positive cases were observed. In order to determine the genotype, PCR-RFLP technique using SspI and VspI restriction enzymes was utilized. With SspI restriction enzyme, three cuttings were seen in locations of 108, 258, and 421 bp visible on agarose gel after electrophoresis. Also after using from VspI enzyme, three cuttings happened in locations 104, 106, and 600 bp (Figure 2) and indicate C. parvum pattern. The amplified 18s rRNA genes from PCR-RFLP products of three C. parvum were sequenced. After submitting the results to the DDBJ/GenBank at accession numbers AB986579, AB986580, and AB986581, the nucleotide sequences were aligned with nucleotide sequences of C. parvum certified in GenBank with accession number AB986578 (Figure 3). Based on findings, C. parvum was recognized as infectious agent of Ahvaz rodents.


Molecular Characterization of Cryptosporidium spp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques
Secondary stage of nested-PCR findings on agarose gel. Lane M, DNA size marker. Lane 1, positive control for Cryptosporidium. Lanes 2–4, positive Cryptosporidium samples (830 bp).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5121462&req=5

fig1: Secondary stage of nested-PCR findings on agarose gel. Lane M, DNA size marker. Lane 1, positive control for Cryptosporidium. Lanes 2–4, positive Cryptosporidium samples (830 bp).
Mentions: Three out of 100 samples were detected as positive for Cryptosporidium spp. using modified Ziehl-Neelsen staining under light microscope. In addition, nested-PCR was done and showed 830 bp band which confirmed only 3 samples as Cryptosporidium spp. (Figure 1). So, overall prevalence of Cryptosporidium infection in rodents of Ahvaz city was calculated at 3% using both methods. All positive samples belonged to R. norvegicus (3/73) and from M. musculus (0/6) and R. rattus (0/21) no positive cases were observed. In order to determine the genotype, PCR-RFLP technique using SspI and VspI restriction enzymes was utilized. With SspI restriction enzyme, three cuttings were seen in locations of 108, 258, and 421 bp visible on agarose gel after electrophoresis. Also after using from VspI enzyme, three cuttings happened in locations 104, 106, and 600 bp (Figure 2) and indicate C. parvum pattern. The amplified 18s rRNA genes from PCR-RFLP products of three C. parvum were sequenced. After submitting the results to the DDBJ/GenBank at accession numbers AB986579, AB986580, and AB986581, the nucleotide sequences were aligned with nucleotide sequences of C. parvum certified in GenBank with accession number AB986578 (Figure 3). Based on findings, C. parvum was recognized as infectious agent of Ahvaz rodents.

View Article: PubMed Central - PubMed

ABSTRACT

Background. Rodents could act as reservoir for Cryptosporidium spp. specially C. parvum, a zoonotic agent responsible for human infections. Since there is no information about Cryptosporidium infection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization of Cryptosporidium spp. in this region. Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method using SspI and VspI restriction enzymes was carried out to genotype the species and then obtained results were sequenced. Results. Three out of 100 samples were diagnosed as positive and overall prevalence of Cryptosporidium spp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples and C. parvum pattern was identified. Finally PCR-RFLP findings were sequenced and presence of C. parvum was confirmed again. Conclusions. Our study showed rodents could be potential reservoir for C. parvum. So an integrated program for control and combat with them should be adopted and continued.

No MeSH data available.