تشخيص و شناسايي گونه هاي كريپتوسپوريديوم با استفاده از روش هاي Nested PCR (Nested polymerase chain reaction) و (RFLP) Restriction fragment length polymorphism در گوساله هاي شهرستان شاهرود

The detection, and identification of Cryptosporidium species by nested polymerase chain reaction (nested-PCR) and restriction fragment length polymorphism (RFLP) of calves in Shahroud town

ﮔﻮﻧﻪﻫﺎي ﮐﺮﯾﭙﺘﻮﺳﭙﻮرﯾﺪﯾﻮم ﺑﻪ ﺷﺎﺧﻪ اﭘﯽ ﮐﻤﭙﻠﮑﺲﻫﺎ ﺗﻌﻠﻖ دارد و ﭘﺮوﺗﻮزواﻫﺎي ﻓﺮﺻﺖ ﻃﻠﺒﯽ ﻫﺴﺘﻨﺪ ﮐﻪ ﺳﯿﺴﺘﻢﻫﺎي ﺗﻨﻔﺴﯽ و ﮔﻮارﺷﯽ در اﻧﺴﺎن و ﺣﯿﻮانﻫﺎ را آﻟﻮده ﻣﯽﮐﻨﺪ. اﯾﻦ ﻣﻄﺎﻟﻌﻪ ﺑﻪ ﻣﻨﻈﻮر ﺑﺮرﺳﯽ ﺷﯿﻮع، ﺗﺸﺨﯿﺺ و ﺷﻨﺎﺳﺎﯾﯽ ﮔﻮﻧﻪﻫﺎي ﮐﺮﯾﭙﺘﻮﺳﭙﻮرﯾﺪﯾﻮم ﺑﺎ اﺳﺘﻔﺎده از روشﻫﺎي Nested PCR و )Restriction Fragment Length (RFLP Polymorphism در ﮔﻮﺳﺎﻟﻪﻫﺎي ﺷﻬﺮﺳﺘﺎن ﺷﺎﻫﺮود اﻧﺠﺎم ﺷﺪ. 256 ﻧﻤﻮﻧﻪي ﻣﺪﻓﻮع از ﮔﻮﺳﺎﻟﻪﻫﺎي زﯾﺮ 2 ﻣﺎه ﺟﻤﻊآوري ﺷﺪ و ﻧﻤﻮﻧﻪﻫﺎي ﻣﺜﺒﺖ ﺑﺎ اﺳﺘﻔﺎده از روش زﯾﻞ-ﻧﻠﺴﻮن رﻧﮓ آﻣﯿﺰي ﺷﺪﻧﺪ. ﭘﺮاﯾﻤﺮﻫﺎي اﺧﺘﺼﺎﺻﯽ ﺑﺮاي ﺑﺮرﺳﯽ Nested PCR اﺳﺘﻔﺎده ﺷﺪ و زﯾﺮﮔﻮﻧﻪﻫﺎ ﺗﻮﺳﻂ روش RFLP ﺑﺮرﺳﯽ ﺷﺪﻧﺪ. ﻧﺘﺎﯾﺞ ﻣﻄﺎﻟﻌﻪ ﻣﯿﮑﺮوﺳﮑﻮﭘﯽ ﻧﺸﺎن داد ﮐﻪ 27 ﻧﻤﻮﻧﻪ )10/54 %( از ﻧﻤﻮﻧﻪﻫﺎي ﻣﻮرد ﺑﺮرﺳﯽ ﻣﺜﺒﺖ ﺑﻮدﻧﺪ. ﻧﺘﺎﯾﺞ ﺑﺮاي روش Nested PCR ﻧﺸﺎن داد ﮐﻪ از ﻣﺠﻤﻮع ﻧﻤﻮﻧﻪﻫﺎي ﻣﻮرد ﺑﺮرﺳﯽ، ﺑﻪﺗﺮﺗﯿﺐ 92/59 % و 7/41 % ﺑﻪﺗﺮﺗﯿﺐ ﺑﺮاي ﮐﺮﯾﭙﺘﻮﺳﭙﻮرﯾﺪﯾﻮم ﭘﺎرووم و ﮐﺮﯾﭙﺘﻮﺳﭙﻮرﯾﺪﯾﻮم اﻧﺪرﺳﻮﻧﯽ ﻣﺜﺒﺖ ﺑﻮدﻧﺪ. ﻧﺘﺎﯾﺞ ﻧﺸﺎن داد ﮐﻪ 25 ﻧﻤﻮﻧﻪ ﺗﺤﺖ ﺗﺄﺛﯿﺮ آﻧﺰﯾﻢ VSP در ﻧﻮاﺣﯽ 104 و 628 ﺟﻔﺖ ﺑﺎز ﻗﺮار ﮔﺮﻓﺘﻨﺪ، ﮐﻪ ﻧﺸﺎن دﻫﻨﺪهي ﮐﻪ ﺗﺄﯾﯿﺪ ﮐﻨﻨﺪهي زﯾﺮﮔﻮﻧﻪﻫﺎي ﮐﺮﯾﭙﺘﻮﺳﭙﻮرﯾﺪﯾﻮم ﭘﺎرووم ﮔﺎوي و زﯾﺮﮔﻮﻧﻪي ژن A ﺑﻮدﻧﺪ. ﻧﻤﻮﻧﻪﻫﺎي ﺗﺤﺖ ﺗﺄﺛﯿﺮ آﻧﺰﯾﻢ Ssp I ﻗﺮار ﮔﺮﻓﺘﻨﺪ و ﻧﺘﺎﯾﺞ ﺑﺎﻧﺪﻫﺎﯾﯽ را در 385 و 448 ﺟﻔﺖ ﺑﺎز ﻧﺸﺎن داد ﮐﻪ ﻧﺸﺎن دﻫﻨﺪهي زﯾﺮ ﮔﻮﻧﻪﻫﺎي C. muris/C. andersoni ﺑﻮدﻧﺪ. ﻧﺘﺎﯾﺞ ﺗﻮﺳﻂ آﻧﺰﯾﻢ dde ﺑﺎﻧﺪﻫﺎﯾﯽ را در 186 ،156 و 224 ﺟﻔﺖ ﺑﺎز ﻧﺸﺎن داد ﮐﻪ ﻧﺸﺎن دﻫﻨﺪهي زﯾﺮ ﮔﻮﻧﻪي C. muris ﺑﻮد. ﮔﻮﻧﻪﻫﺎ و زﯾﺮﮔﻮﻧﻪﻫﺎي ﻣﺨﺘﻠﻒ ﺑﺎ اﺳﺘﻔﺎده از روشﻫﺎي ﻣﺨﺘﻠﻒ ﺷﻨﺎﺳﺎﯾﯽ ﺷﺪﻧﺪ ﮐﻪ ﻣﯽﺗﻮاﻧﺪ ﺑﻪ ﮐﻨﺘﺮل ﮐﺮﯾﭙﺘﻮﺳﭙﻮرﯾﺪﯾﻮز ﮐﻤﮏ كند

Cryptosporidium species belong to Apicomplexa phylum and are opportunistic protozoans that infect the gastrointestinal and respiratory systems of some animals and humans. This study was conducted to investigate the prevalence, detection, and identification of Cryptosporidium species by nested PCR and restriction fragment length polymorphism (RFLP) in calves in Shahroud town. A total number of 256 calves was collected from fecal samples of pre-weaned calves (≤2 months) in Shahroud town, and positive samples were stained by Ziehl-Neelsen (ZN) staining method. Specific primers were used to investigate the Nested-PCR and sub-species were investigated by RFLP method. The microscopic results showed that 27 samples (10.54 %) of the samples were positive. The results for nested-PCR showed that out of samples, 92.59 % and 7.41 % of samples were positive for C. parvum and C. Andersoi, respectively. The results showed that 25 samples were affected by VSP enzyme in regions of 104 and 628 bp that belong C. parvum bovine, and genotype A gene sub-species. The samples were affected by Ssp I enzyme and the results showed bands in regions of 385 and 448 bp that show C. muris/C. andersoni sub-species. The results for dde enzyme showed bands in regions of 156, 186, and 224 bp that confirm C. muris sub-specie. The different species and sub-species were identified by different methods and can help to control of the cryptosporidiosis.