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Research Detail

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Md. Abdul Khaleque
Department of Parasitology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Farjana Boby
Department of Parasitology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Shahiduzzaman*
Department of Parasitology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

The aim of this study was molecular detection and characterization of Cryptosporidium isolated from animal and human stool sample. A polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique based on the small-subunit rRNA gene was used for the analysis of 10 positive samples (9 samples from human and 1 sample from animal). Nine human samples were collected from Shurjo Kanti (S.K.) Hospital, Mymensingh that has a diarrheal disease section and 1 animal sample was collected from Bangladesh Agricultural University campus. The samples were employed for DNA extraction using Purelink™ Genomic DNA Mini Kit (Invitrogen, USA) and then PCR was performed. Five PCR positive Cryptosporidium DNA were genotyped by using VspI (AseI) restriction enzyme (Lithuania, EU). Cryptosporidium parvum, Bovine A gene was detected in 2 samples, Cryptosporidium parvum, Bovine B gene was detected in 1 sample and Cryptosporidium hominis was detected in 2 samples. The present study confirms the Cryptosporidium genotype (Cryptosporidium parvum, Bovine A gene, Cryptosporidium parvum, Bovine B gene and Cryptosporidium hominis) in the patient admitted to Shurjo Kanti (S.K.) Hospital, Mymensingh, indicating the existence of Cryptosporidium genotyes in the study areas. However, further studies are needed to better understanding the transmission dynamics of these genotypes and thereafter taking necessary measure to control and/or prevent the disease.

  Cryptosporidium, Human child, Animal, Bangladesh
  Laboratory at the Department of Parasitology, Bangladesh Agricultural University, Mymensingh
  
  
  Pest Management
  Diseases

This study assists to confirm the types (genotypes) of Cryptosporidium affected both animal and human in the study area and thereafter taking necessary measure to control and prevent the disease in Bangladesh.

Samples: Stool samples were collected from Shurjo Kanti (S.K.) Hospital, Mymensingh district of Bangladesh and fecal sample from cattle of Bangladesh Agricultural University, Mymensingh. The stool and fecal samples were initially processed at the collection sites and then brought to the Laboratory at the Department of Parasitology, Bangladesh Agricultural University, Mymensingh. Part of the work was done at the Laboratory of Department of Microbiology & Hygiene, and Department of Pathology, Bangladesh Agricultural University Mymensingh. The study was conducted during the period of January 2016 to June 2016. The samples were initially screened by by Ziehl-Neelsen technique and positive samples (9 samples from human and 1 sample from animal) were subjected to concentration of oocysts and DNA extraction.

DNA extraction Purelink™ Genomic DNA Mini Kit (Invitrogen, USA) was used to extract the DNA following manufacturer instructions.

Polymerase chain reaction (PCR) A primary PCR was performed by targeting the Small Sub Unit (SSU) rRNA gene of Cryptosporidium to confirm the detection of Cryptosporidium from stool samples according to the protocol described by Xiao et al., (1999) with some modifications. A PCR product of 1,325 bp from target gene was amplified by forward primer (5'-TTCTAGAGCTAATACATGCG-3) and reverse primer (5'-CCCATTTCCTTCG AAACAGGA -3'), (IDT, USA). PCR reaction was performed in a total 50 µl reaction volume containing PCR Master mix (Promega, USA) 45 µl, forward primer 1 µl, reverse primer 1 µl, deionized water 1 µl, and DNA template 2 µl. A total of 35 cycles were carried out, each consisting of 94°C for 3 min in case of initial denaturation, 94°C for 45 sec in case of denaturation, 54°C for 45s in case of annealing, 72°C for 1 min in case of extension and 72°C for 7 min in case of final extension, one kb DNA ladder (Promega, USA) was used to compare the band size of target DNA.

Agarose gel electrophoresis PCR products were analyzed in 1.5% agarose gel, stained with ethidium bromide and examined against UV light using an image documentation system. Genotyping For genotyping PCR products of different DNA samples were subjected to digestion by restriction enzyme. VspI (AseI) restriction enzyme (Lithuania, EU). For restriction fragment analysis, 10 µl of the PCR product was digested in a total of 25µl of reaction mixture, consisting of 1µl of VspI (AseI) restriction enzyme (Lithuania, EU), 5µl of respective restriction buffer and 9µl of distilled water at 37° C for 1 h, according to manufacturer’s instructions. The digested products were fractionated on 2.0% agarose gel and visualized by ethidium bromide staining.

 

  2016 © International Journal of Applied Research 2(3) 122-126
  
Funding Source:
1.   Budget:  
  

The present study detected the genotypes of Cryptosporidium in human and cattle in the study area by PCR-RFLP analysis. The methodologies adopted for isolation, identification of Cryptosporidium can be used for further investigation of the stool and faecal samples for better understanding the molecular characterization of Cryptosporidium in Bangladesh and thereafter increase understanding the transmission dynamics of Cryptosporidium in order to take necessary measures for prevention and control of this disease in human and animal in Bangladesh.

  Journal
  


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