مطالعه فیلوژنی جدایه های Theileria annulata از استان البرز با استفاده از توالی ژن های 18S rRNA و ITS1-2

نوع مقاله: مقاله کامل

نویسندگان

1 کارشناس ارشد موسسه تحقیقات واکسن وسرم سازی رازی

2 استادیار موسسه تحقیقات واکسن وسرم سازی رازی

3 استاد دانشکده دامپزشکی دانشگاه تهران

چکیده

تیلریوز گرمسیری بیماری انگلی ناشی از تک یاخته T.annulata بوده و بوسیله کنه های جنس هیالوما منتقل می شود. تشخیص بیماری معمولاً با مشاهده انگل در گسترش های خون محیطی، طحال و غدد  لنفاوی صورت می گیرد.  امروزه روش  های ملکولی با حساسیت و ویژگی بالائی به منظور تشخیص و شناسائی عامل بیماری در دام های مبتلا و ناقل، مطالعات همه گیری شناسی  و بررسی های فیلوژنی مورد استفاده قرار می گیرد.  با توجه به حضور تیلریوز گاوی در کشور، مطالعه فیلوژنی T.annulata با استفاده از بررسی توالی ژن 18S rRNA و نواحی ITS شامل (5.8S rRNA , ITS2, ITS1) مربوط به دو جدایه استان البرز به همراه رده سلولی سویه واکسن تیلریوز گاوی مورد توجه قرار گرفت.  توالی های مورد نظر ابتدا با PCR تکثیر شده و تعیین توالی گردید. درخت فیلوژنی بر اساس توالی های ناحیه ITS و ژن 18S rRNA ترسیم شد.  نتایج این مطالعه نشان داد که ژن 18S rRNA دارای ارزش شناسائی در حد جنس و توالی ITS در حد گونه می باشد.  یافته های حاصل از بررسی درخت فیلوژنی  توالی کامل ژن 18S rRNA نشان داد گونه های بیماری زای T.lestoquardi، T.annulata و T.parva در یک خوشه و مستقل از سایر گونه های تیلریا قرار می گیرند.

عنوان مقاله [English]

Phylogenetic study of Theileria annulata isolates from Alborz province by using 18S rRNA gene and ITS regions sequences

نویسندگان [English]

  • A. Afshari 1
  • Gh.R. Habibi 2
  • K. Esmaeilnia 2
  • P. Shayan 3
1 Msc Razi Vaccine and Serum Research
2 Assistant Professor Razi Vaccine and Serum Research
3 Professor College of Veterinary Tehran University
چکیده [English]

Tropical theileriosis is a tick-transmitted disease which caused by obligative intracellular protozoan Theileria annulata. Diagnosis is usually based on identification of parasite in Giemsa stained smears of peripheral blood, spleen or lymph nodes. Today, the molecular methods with high sensitivity and specificity are used for diagnosis of infected and carrier animals, epidemiological and phylogenetic studies. Due to the presence of bovine theileriosis in Iran, T. annulata phylogenetic analysis has been focused using 18S rRNA gene and ITS region sequences (ITS1, 5.8S rRNA, ITS2) for two Alborz Theileria annulata isolates and bovine theileriosis vaccine strain S15 cell line. The target sequences were amplified by PCR and further PCR product sequencing. The phylogenetic tree were constructed based on 18S rRNA complete gene sequences and ITS region, then the sequences of Iranian isolates and registered Theileria sequences from other countries registered in GenBank were compared. The obtained results showed that the 18S rRNA gene and ITS sequence are valuable for detection and classification at the species and subspecies levels, respectively. Other important finding obtained from the present study was the result from phylogenetic tree of complete 18S rRNA gene sequences that revealed the highly pathogenic Theileria spp. (lymphoproliferative Theileria) including T. annulata, T. lestoquardi and T. parva were placed in a separate cluster away from the other Theileria spp.

1- Ahmed, J.S., Luo, J., Schnittger, L., Seitzer, U., Jongejan, F. and Yin, H. (2006) Phylogenetic position of small ruminant infecting piroplasms. Annals of the NewYork Academy of Sciences, 1081: 498–504.
2- Aktas, M., Bendele, K.G., Altay, K., Dumanli, N., Tsuji, M. and Holman, P.J. (2007) Sequence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species, Veterinary Parasitology, 147(3-4): 221–230.
3-Allsopp, M.T.E.P., Cavalier-Smith, T., De Waal, D.T. and Allsopp, B.A. (1994) Phylogeny and evolution of the piroplasms. Parasitology, (108): 147-153.
4-Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.D. and Struhl, K. (1987) Current Protocols in Molecular Biology. (John Wiley and Sons, New York).
5-Chansiri, K., Kawazu, S., Kamio, T., Terada, Y., Fujisaki, K., Philippe, H. et al. (1999) Molecular phylogenetic studies on Theileria parasites based on small subunit ribosomal RNA gene sequences. Veterinary Parasitology, 83: 99-105.
6-Ellis, J., Hefford, C., Baverstock, P.R., Dalrymple, B.P. and Johnson, A.M. (1992) Ribosomal DNA sequence comparison of Babesia and Theileria. Molecular and Biochemical Parasitology, 54:87–95.
7- Gardner, M.J., Bishop, R., Shah, T., de Villiers, E.P., Carlton, J.M., Hall, N., et al. (2005) Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes. Science, 309(5731):134-7.
8-Habibi, G.R. (2012) Phylogenetic Analysis of Theileria annulata Infected Cell Line S15 Iran Vaccine Strain. Iranian Journal of Parasitology, 7(2): 73-81.
9-Habibi, G.R., Esmaeil-Nia, K., Bozorgi. S., Najjar, E., Hashemi-Fesharki, R., and Bordbar, N. (2007) PCR-based detection of Theileria infection and molecular characterization of Tams1T. annulata vaccine strain. Archives of Razi Institute, 62(2):83-89.
10-Hashemi-Fesharki, R., Habibi, G.R. and Ahouri, P. (1998) Delayed type hypersensitivity theilerin test in cattle vaccinated against Theileria annulata infection. Veterinary Parasitology, 75: 261-263.
11-Herrmann, B.G. and Frischauf, A.M. (1987) Isolation of genomic DNA. Methods in Enzymology, 152: 180–3.
12-Hillis, D.M. and Dixon, M.T. (1991) Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology, 66(4): 411-53.
13- Jalali, S.M., Khaki, Z., Kazemi, B., Rahbari, S., Shayan, P., Bandehpour, M., Yasini, S.P. (2014) Molecular Detection and Identification of Theileria Species by PCR-RFLP Method in Sheep from Ahvaz, Southern Iran. Iranian Journal of Parasitology, 9(1): 99-106
14-Kakuda, T., Shiki, M., Kubota, S., Sugimoto, C., Brown, W.C., Kosum, C., et al. ( 1998) Phylogeny of benign Theileria species from cattle in Thailand, China and the USA based on the major piroplasm surface protein and small ribosomal RNA genes. International Journal for Parasitology, 28: 1261-7.
15-Katzer, F., McKellar, S., Kirvar, E. and Shiels, B. (1998) Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests. Molecular and Biochemical Parasitology, 95: 33–44.
16-Levine, N.D., (1985) Veterinary Protozoology. Iowa State University Press, Ames. P: 414.
17-Marquardt W.C., Demaree R.S. and Grieve R.B. (2000), Parasitology Vector Biology, 2nd edition. P: 221.
18-Moretti, A., Mangili, V., Salvatori, R., Maresca, C., Scoccia, E., Torina A. et al. (2010) Prevalence and diagnosis of Babesia and Theileria infections in horses in Italy: A preliminary study. Veterinary Journal, 184(3): 346-350.
19-Namavari, M., Ezhdehakosh-pour, S., Habibi, G.R., Rahimian, A. and Namazi, F. (2013) First isolation and establishment of in vitro culture of Theileria lestoquardi from a naturally infected cow. Journal of Parasitic Diseases, DOI: 10.1007/s12639-013-0309-2
20-Nijhof, A.M., Pillay, V., Steyl, J., Prozesky, L., Stoltsz, W.H., Lawrence, J.A. et al. ( 2005) Molecular characterization of Theileria species associated with mortality in four species of African antelopes. Clinical Microbiology, 43(12): 5907-5911.
21-Radostits, O.M., Gay, C.C., Hinchcliff, K.W. and Constable, P.D. (2007) Veterinary Medicine. 10th Ed. Saunders, Philadelphia, pp: 1484-97.
22-Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning : A laboratory Manual , Volume 3 , Cold Spring Harbor Laboratory ; NY,B.16
23-Schnittger, L., Yin, N., Jianxun, L., Luding, W., Shayan, P., Rahbari, S. et al. (2000) Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and Theileria species highly pathogenic for small ruminants in China. Parasitology Research, 86: 352-358.
24-Taha, K.M., Salih, D.A., Ali, A.M., Omer, R.A. and El Hussein, A.M. (2013) Naturally occurring infections of cattle with Theileria lestoquardi and sheep with Theileria annulata in the Sudan. Veterinary Parasitology, 191(1-2): 143-5.