ﺷﻨﺎﺳﺎﯾﯽ ﺗﯿﻠﺮﯾﺎ آﻧﻮﻻﺗﺎ از ﮔﺎوﻫﺎي ﻣﺸﮑﻮك ﺑﻪ ﺗﯿﻠﺮﯾﻮز در اﺳﺘﺎن ﻗﺰوﯾﻦ ﺑﺎ آزﻣﺎﯾﺶﻫﺎي ﻣﯿﮑﺮوﺳﮑﻮﭘﯽ و PCR

Diagnosis of theileria annulata from suspected bovine with theileriosis in Qazvin province using microscopic examination and PCR

ﺗﯿﻠﺮﯾﻮز ﮔﺎوي ﯾﮑﯽ از ﻣﻬﻢﺗﺮﯾﻦ ﺑﯿﻤﺎريﻫﺎي اﻧﮕﻠﯽ در اﯾﺮان اﺳﺖ ﮐﻪ ﺑﺎﻋﺚ ﺿﺮر و زﯾﺎن اﻗﺘﺼﺎدي ﺑﻪ ﺻﻨﻌﺖ داﻣﭙﺮوري ﺑﺎ ﮐﺎﻫﺶ وزن ﮐﺎﻫﺶ ﺷﯿﺮ و اﻓﺰاﯾﺶ ﻣﺮگ و ﻣﯿﺮ ﻣﯽ ﺷﻮد. ﻋﺎﻣﻞ ﺑﯿﻤﺎري ﺗﯿﻠﺮﯾﺎ آﻧﻮﻻﺗﺎ ﺑﻮده ﮐﻪ از ﻃﺮﯾﻖ ﮐﻨﻪ ﺟﻨﺲ ﻫﯿﺎﻟﻮﻣﺎ ﻣﻨﺘﻘﻞ ﻣﯽﮔﺮدد. ﺗﺸﺨﯿﺺ ﺗﯿﻠﺮﯾﻮز ﻋﻤﺪﺗﺎً ﺑﺮ اﺳﺎس ﻋﻼﯾﻢ ﺑﺎﻟﯿﻨﯽ و آزﻣﺎﯾﺶ ﻣﯿﮑﺮوﺳﮑﻮﭘﯽ اﺳﺖ. اﻣﺮوزه ﺑﺮاي ﺗﺸﺨﯿﺺ ﻗﻄﻌﯽ از آزﻣﻮن ﻣﻮﻟﮑﻮﻟﯽ PCRاﺳﺘﻔﺎده ﻣﯽﺷﻮد. ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻫﻤﯿﺖ ﺗﯿﻠﺮﯾﻮز ﮔﺎوي، ﻫﺪف از اﯾﻦ ﭘﮋوﻫﺶ ﺗﺸﺨﯿﺺ ﻣﻮارد ﺗﯿﻠﺮﯾﺎ آﻧﻮﻻﺗﺎ از ﮔﺎوﻫﺎي ﻣﺸﮑﻮك ﺑﻪ ﺗﯿﻠﺮﯾﻮز در اﺳﺘﺎن ﻗﺰوﯾﻦ ﺑﺎ روش ﻣﺸﺎﻫﺪه ﻣﯿﮑﺮوﺳﮑﻮ ﭘﯽ و PCRﺑﻮد. از ﻣﺠﻤﻮع 68 راس ﮔﺎو داراي ﻋﻼﯾﻢ ﺑﺎﻟﯿﻨﯽ ﺧﻮنﮔﯿﺮي ﺑﻪ ﻋﻤﻞ آﻣﺪ. از ﻧﻤﻮﻧﻪﻫﺎي ﺧﻮن اﺳﻤﯿﺮ ﺗﻬﯿﻪ و ﺑﻪ روش ﮔﯿﻤﺴﺎ رﻧﮓ آﻣﯿﺰي ﺷﺪ. ﻧﻤﻮﻧﻪﻫﺎي ﻣﺜﺒﺖ ﺑﺮاي ﺗﺸﺨﯿﺺ ﻣﻮﻟﮑﻮﻟﯽ ﺑﺮرﺳﯽ ﺷﺪﻧﺪ. از ﻧﻤﻮﻧﻪﻫﺎي ﺧﻮن ﺑﺎ روش ﻓﻨﻞ ﮐﻠﺮوﻓﺮم DNA اﺳﺘﺨﺮاج ﺷﺪ و آزﻣﻮن PCR ﺗﻮﺳﻂ ﭘﺮاﯾﻤﺮﻫﺎي Tams ،18s rRNA اﻧﺠﺎم ﺷﺪ، ﺳﭙﺲ ﻣﺤﺼﻮل PCR ﯾﮏ ﻧﻤﻮﻧﻪ ﺗﻌﯿﯿﻦ ﺗﻮاﻟﯽ ﺷﺪ. ﺑﺮ اﺳﺎس ﻧﺘﺎﯾﺞ ﺑﻪ دﺳﺖ آﻣﺪه ﺗﯿﻠﺮﯾﺎ آﻧﻮﻻﺗﺎ در ﮔﺴﺘﺮش ﺧﻮن 28 )41/18 درﺻﺪ( ﻧﻤﻮﻧﻪ از 68 ﻧﻤﻮﻧﻪ ﺧﻮن ﺑﺎ ﻣﯿﮑﺮوﺳﮑﻮپ ﻧﻮري ﻣﺸﺎﻫﺪه ﮔﺮدﯾﺪ، درﺣﺎﻟﯽ ﮐﻪ ﺗﻤﺎم ﻧﻤﻮﻧﻪﻫﺎ در آزﻣﻮن PCR ﻣﺜﺒﺖ ﺑﻮدﻧﺪ. آﻧﺎﻟﯿﺰ ﺑﻼﺳﺖ ﺑﺮ اﺳﺎس ﺗﻮاﻟﯽ ژن 18S rRNA در ﯾﮏ ﺟﺪاﯾﻪ اﯾﺮاﻧﯽ 99/1 درﺻﺪ ﺗﺸﺎﺑﻪ ﻧﺸﺎن داد. ﺑﺮ اﺳﺎس ﻧﺘﺎﯾﺞ ﺑﻪ دﺳﺖ آﻣﺪه، ﺗﯿﻠﺮﯾﺎ آﻧﻮﻻﺗﺎ ﺷﯿﻮع ﺑﺎﻻﯾﯽ در ﮔﺎوﻫﺎي اﺳﺘﺎن ﻗﺰوﯾﻦ دارد. ﭘﯿﺸﻨﻬﺎد ﻣﯽ ﺷﻮد ﻣﻄﺎﻟﻌﺎت ﺑﯿﺸﺘﺮ در اﯾﻦ زﻣﯿﻨﻪ ﺮاي ارزﯾﺎﺑﯽ ﺻﺤﯿﺢﺗﺮ ﺑﯿﻤﺎري در اﯾﺮان ﺻﻮرت ﮔﯿﺮد.

Bovine theileriosis is one of the most important parasitic diseases in Iran which causes severe economic losses through decreasing weight, milk production and increased mortality. The causative agent, Theileria annulata, is transferred by the ticks of genus Hyalomma. Diagnosis of theileriosis is based on clinical signs, microscopic examination. Nowadays, molecular biology methods like PCR are used for detection. Given the importance of bovine theileriosis, the aim of this study was diagnosis of T. annulata from suspected bovine with theileriosis in Qazvin province using microscopic examination and PCR. Out of 68 dairy cattle with clinical symptoms were taken from blood samples. Then the blood smear was prepared and stained with Giemsa dye. Positive samples were incorporated for molecular studies. DNA of blood was extracted using phenol/chloroform/isoamyl alcohol and performed PCR using specific primers of Tams1 and 18S rRNA genes. Then, the PCR product of one sample was sequenced. Based on the obtained results, out of 68 in 28 (41.18%) blood samples was observed Theileria annulata by light microscopy, whereas all of the samples were positive by PCR. BLAST analyses based on the 18S rRNA gene sequence of the Iranian isolate determined the 99.1%identity. Based on the obtained results, T. annulata had a high frequency in cattle of Qazvin province. It is suggested that further studies be conducted in this field in order to obtain more accurate estimates of disease in Iran.