تعيين ميزان آلودگي به تيلريا آنولاتا در كنه‌هاي ايكسوديده در منطقه اروميه به روش واكنش زنجيره‌اي پليمراز (PCR)

Determination of infestation rate of Ixodidae ticks to Theileria annulata in Urmia region by polymerase chain reaction (PCR)

ﺗﯿﻠﺮﯾﻮز ﯾﮑﯽ از ﺑﯿﻤﺎريﻫﺎي ﻣﻬﻢ اﻧﮕﻠﯽ ﺗﮏﯾﺎﺧﺘﻪاي در اﯾﺮان ﻣﯽﺑﺎﺷﺪ ﮐﻪ ﺑﺎﻋﺚ ﺑﺮوز ﺿﺮرﻫﺎي اﻗﺘﺼﺎدي و ﻣﺮگوﻣﯿﺮ در دامﻫﺎ ﻣﯽﺷـﻮد. اﯾﻦ ﺑﯿﻤﺎري ﺗﻮﺳﻂ ﮐﻨﻪﻫﺎي ﺧﺎﻧﻮاده اﯾﮑﺴﻮدﯾﺪه ﻣﻨﺘﻘﻞ ﻣﯽﺷﻮد و ﺑﺎ ﺗﺐ، ﮐﻢﺧﻮﻧﯽ، آﻧﻮﮐﺴﯽ و ﻧﻬﺎﯾﺘﺎً ﻣﺮگ دام ﻫﻤـﺮاه اﺳـﺖ. ﺗﺸـﺨﯿﺺ ﺑﯿﻤﺎري ﺳﺎﺑﻘﺎً ﺑﺮ اﺳﺎس رﻧﮓآﻣﯿﺰي ﻧﻤﻮﻧﻪﻫﺎي ﺧﻮﻧﯽ و ﻏﺪد ﻟﻨﻔﺎوي ﺑﺎ روش ﮔﯿﻤﺴﺎ اﻧﺠﺎم ﻣﯽﭘﺬﯾﺮﻓﺖ. اﺳﺘﻔﺎده از روشﻫﺎي ﻣﻮﻟﮑﻮﻟﯽ ﺑـﻪ دﻟﯿﻞ ﺑﺎﻻ ﺑﻮدن ﺣﺴﺎﺳﯿﺖ و وﯾﮋﮔﯽ ﻧﺴﺒﺖ ﺑﻪ روشﻫﺎي ﻗﺒﻠﯽ ارﺟﺤﯿﺖ دارد. ﺑﻪدﻟﯿـﻞ اﯾـﻦﮐـﻪ در داﻣـﺪاريﻫﺎي ﺳـﻨﺘﯽ اﯾـﺮان ﮐﻨـﻪﻫﺎي اﯾﮑﺴﻮدﯾﺪه ﮐﻪ ﻣﯽﺗﻮاﻧﻨﺪ ﻧﺎﻗﻞ ﺑﯿﻤﺎري ﺑﺎﺷﻨﺪ ﺑﻪﻓﺮاواﻧﯽ ﯾﺎﻓﺖ ﻣﯽﺷﻮﻧﺪ، ﺷﻨﺎﺳﺎﯾﯽ اﯾﻦ ﮐﻨﻪﻫﺎ اﻫﻤﯿﺖ ﻓﺮاوان دارد. اﯾـﻦ ﻣﻄﺎﻟﻌـﻪ ﺑـﺎ ﻫـﺪف ﺷﻨﺎﺳﺎﯾﯽ ﻣﻮﻟﮑﻮﻟﯽ آﻟﻮدﮔﯽ ﺑﻪ ﺗﯿﻠﺮﯾﺎ در ﮐﻨﻪﻫﺎي ﺟﺪاﺷﺪه از ﮔﺎوﻫﺎي ﻣﻨﻄﻘﻪ اروﻣﯿﻪ اﻧﺠﺎم ﭘﺬﯾﺮﻓﺖ. ﺗﻌﺪاد 100 ﮐﻨﻪ از ﮔﻠﻪﻫﺎي ﺑﺎ ﺗﺎرﯾﺨﭽﻪ آﻟﻮدﮔﯽ ﺑﻪ ﺗﯿﻠﺮﯾﺎ در ﻣﻨﻄﻘﻪ ﺟﻤﻊآوري و ﺑﺎ اﺳـﺘﻔﺎده از ﮐﻠﯿـﺪﻫﺎي ﺗﺸـﺨﯿﺺ ﺷﻨﺎﺳـﺎﯾﯽ ﺷـﺪﻧﺪ. از ﻣﺠﻤـﻮع 100 ﮐﻨـﻪ ﻣـﻮرد آزﻣـﺎﯾﺶ، ﺗﮏﯾﺎﺧﺘﻪﻫﺎي اﻧﮕﻠﯽ ﻫﯿﺎﻟﻮﻣﺎ آﻧﺎﺗﻮﻟﯿﮑﻮم آﻧﺎﺗﻮﻟﯿﮑﻮم )33 درﺻﺪ (، ﻫﯿﺎﻟﻮﻣﺎ دﯾﺘﺮﯾﺘﻮم )16 درﺻـﺪ(، ﺑﻮوﻓﯿﻠـﻮس آﻧﻮﻻﺗـﻮس )16 درﺻـﺪ(، درﻣﺎﺳﻨﺘﻮر ﻣﺎرژﯾﻨﺎﺗﻮم )14 درﺻﺪ(، رﯾﭙﯿﺴﻔﺎﻟﻮس ﺑﻮرﺳﺎ )9 درﺻﺪ(، ﻫﯿﺎﻟﻮﻣﺎ آﻧﺎﺗﻮﻟﯿﮑﻮم اﮐﺴﮑﺎواﺗﻮم )7 درﺻﺪ(، درﻣﺎﺳـﻨﺘﻮر ﻧﯿـﻮوس )4 درﺻﺪ( و ﻫﻤﺎﻓﯿﺰاﻟﯿﺲ ﭘﻮﻧﮑﺘﺎﺗﺎ )1 درﺻﺪ( ﺷﻨﺎﺳﺎﯾﯽ ﺷﺪﻧﺪ. ﮐﻨﻪ ﻏﺎﻟﺐ ﻫﯿﺎﻟﻮﻣﺎ آﻧﺎﺗﻮﻟﯿﮑﻮم آﻧﺎﺗﻮﻟﯿﮑﻮم ﺗﺸﺨﯿﺺ داده ﺷﺪ. ﻏﺪد ﺑﺰاﻗﯽ ﮐﻨﻪﻫﺎ ﭘﺲ از ﺟﺪاﺳﺎزي ﺑﺎ اﺳﺘﻔﺎده از ﺟﻔﺖ ﭘﺮاﯾﻤﺮ اﺧﺘﺼﺎﺻﯽ ﻣﺮﺑﻮط ﺑﻪ ﮔﻮﻧﻪ ﺗﯿﻠﺮﯾﺎ آﻧﻮﻻﺗﺎ )N516 وN517( ﻣﻮرد آزﻣﺎﯾﺶ PCR ﻗﺮار ﮔﺮﻓﺘﻨﺪ. آزﻣﺎﯾﺶ PCR وﺟﻮد ﺑﺎﻧﺪ اﺧﺘﺼﺎﺻﯽ ﺑﺎ اﻧﺪازه bp 721ﻧﺎﺷﯽ از وﺟﻮد ﺗﯿﻠﺮﯾﺎ آﻧﻮﻻﺗﺎ را روي ژل آﮔﺎرز در 62 درﺻﺪ ﮐﻨﻪ ﻫـﺎ ﻣﺸـﺨﺺ ﺳﺎﺧﺖ. ﻣﯿﺰان ﺑﺎﻻي آﻟﻮدﮔﯽ در ﮐﻨﻪﻫﺎي ﻧﺎﻗﻞ ﻣﻮﺟﻮد در ﻣﻨﻄﻘﻪ ﭘﺘﺎﻧﺴﯿﻞ ﺑﺎﻻي آنﻫﺎ را در راه آﻟﻮدﮔﯽ ﮔﺎوﻫﺎ ﺑﻪ ﻋﺎﻣﻞ ﺗﯿﻠﺮﯾـﺎ آﻧﻮﻻﺗـﺎ را ﻣﯽرﺳﺎﻧﺪ.

Theileriosis is one of the most important protozoan parasitic diseases in Iran that causes economic losses and mortality in livestock. The disease is transmitted by Ixodid ticks and is characterized by fever, anemia, anoxia and eventually death. Diagnosis of the disease was previously based on staining of blood and lymph node samples by Giemsa method. The use of molecular methods is preferred over previous methods due to their high sensitivity and specificity. Due to the fact that Ixodid ticks that can carry the disease are found in many traditional Iranian farms, it is very important to identify and introduce these ticks. The aim of this study was molecular identification of Theileria annulata infection in ticks isolated from cows in Urmia region. A total of 100 ticks from herds with a history of Theileria annulata infection in the area were collected and transferred to the laboratory. Ticks were identified using diagnostic keys. Salivary glands of ticks were separated and analyzed by PCR after isolation using Theileria annulata specific primer (N516, N517). Out of 100 ticks, ten genera and species including Hyalomma anatolicum anatolicum (33%), Hy. detritum (16%), Boophylus anulatus (16%), Dermacentor marginatum (14%), Rhipicephalus bursa (9%), Hy. anantolicum excavatum (7%), D. niveus (4%) and Haemaphysalis punctate (1%) were identified. The predominant tick was identified as Hyalomma anatolicum anatolicum. PCR test showed the presence of 721 bp specific band on agarose gel in 62% of tick samples. The high rate of infection in vector mites in the area indicates their high potential for infection of cows with the agent of Theileria annulata.