Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan.
AbstractBackground: The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp.) in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan. Methods: Reverse line blot (RLB) assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products. Results: Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa. Conclusion: Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), ani-mals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites.
Zulfiqar S, Shahnawaz S, Ali M, Bhutta AM, Iqbal S, Hayat S, Iqbal F. Detection of Babesia bovis in blood samples and its effect on the hematological serum biochemical profile in large ruminants from Southern Punjab (Paki-stan). Asi Pac J Trop Biomed. 2012; 2(2): 104.
Livestock census. Data on Punjab livestock. Punjab, Pakistan: Livestock Census Organiza-tion, Government of the Punjab. 1996.
Shahnawaz S, Ali M, Aslam MA, Fatima R, Chaudhry ZI, Hassan MU, Iqbal F. A study on the prevalence of a tick transmitted patho-gen, Theileria annulata, and hematological pro-file in cattle from Southern Punjab (Pakistan). Parasitol Res. 2011; 109: 1155-1160.
Khattak RM, Rabib M, Khan Z, Ishaq M, Hameed H, Taqddus A, Faryal M, Durrani S, Gillani QUA, Allahyar R, Shaikh RS, Khan MA, Ali M. Iqbal F. A comparison of two different techniques for the detection of blood parasite, Theileria annulata, in cattle from two districts in Khyber Pukhtoon Khwa Province (Pakistan). Parasit. 2012; 19(1): 91-95.
Li Y, Guan G, Liu A, Peng Y, Luo J, Hong Yin. Experimental transmission of Theileria ovis by Hyalomma anatolicum anatolicum. Parasitol Res. 2010; 106: 991–994
Ahmed J, H Yin, M Bakheit, Z Liu, H Mehlhorn and U Seitzer. Small ruminant theileriosis. Parasitol Res. 2011; 2: 135-153.
Durrani AZ, Younus M, Kamal N, Mehmood N, Shakoori AR. Prevalence of ovine Theileria species in district Lahore, Pakistan. Pak J Zool. 2011; 43(1): 57-60.
Fornelio CA, Martinez-Marcos A, Bulinga-Sarana A, Barba-Carretero JC. Molecular studies on Babesia, Theileria and Hepatozoon in Southern Europe Part 1. Epizootological aspects. Vet Parasitol. 2003; 113: 189.
Iqbal F, Fatima M, Shahnawaz S, Naeem M, Shaikh RS, Shaikh AS. A study on the deter-mination of risk factors associated with babesiosis and prevalence of Babesia sp., by PCR amplification, in small ruminants from Southern Punjab (Pakistan). Parasit. 2011; 18: 229.
Dumanli N, Altay K, Holman PJ, Aktas M. Detection of Theileria ovis in naturally in-fected sheep by nested PCR. Vet Parasitol. 2005; 127: 99–104.
Qayyum M, Farooq U, Samad HA, Chauhdry HR. Prevalence, clinicotherapeutic and prophylactic studies on theileriosis in district Sahiwal (Pakistan). J Anim Plant Sci. 2010; 20: 266.
Aktas M, Dumanli NC, Etinkaya B, Cakmak A. Field evaluation of PCR in detecting Theileria annulata infection in cattle in the east of Turkey. Vet Rec. 2002; 150: 548–549.
Aktas M, Altay K, Dumanlı N. Development of a polymerase chain reaction method for di-agnosis of Babesia ovis infection in sheep and goats. Vet Parasitol. 2005; 133: 277-281.
Altay K, Dumanli N, Holman PJ, Aktas M. Molecular identification, genetic diversity and distribution of Theileria and Babesia spp. infect-ing small ruminants. Vet Parasitol. 2007; 147: 121-127 .
Altay K, Aktas M, Dumanli N, Aydin MF. Evaluation of a PCR and comparison with RLB for detection and differentiation of Theileria sp. MK and other Theileria and Babesia species of small ruminants. Parasitol Res. 2008; 103(2): 319-323.
Heidarpour BM, Khazraiinia P, Haddadzadeh HR, Kazemi B. Identification of Theileria spe-cies in sheep in the eastern half of Iran using nested PCR-RFLP and microscopic tech-niques. Iran J Vet Res. 2010; 11(32): 262-265.
Georges K, Loria GR, Riili S, Greco A, Caracappa S, Jongejan F, Sparagano O. Detec-tion of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribu-tion of ticks in Sicily. Vet Parasitol. 2001; 99: 273–286.
Nagore D, García-Sanmartín J, García-Pérez AL, Juste RA, Hurtado A. Identification, ge-netic diversity and prevalence of Theileria and Babesia spp. in sheep population from Nortern Spain. Int J Parasitol. 2004; 34: 1059-1067.
Gubbels JM, de Vos AP, van der Weide M, Viseras J, Schouls LM, de Vries and E, Jongejan F. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J Clin Microbiol. 1999; 37: 1782–1789.
Durrani S, Khan Z, Khattak RM, Ali M, Hameed H, Taqddas A, Faryal M, Kiran S, Riaz M, Sajid M, Sheikh RS, Ali M, Iqbal F. A comparison of the presence of Theileria ovis by PCR amplification of their ssu rRNA gene in small ruminants from two provinces of Paki-stan. Asi Pac J Trop Dis. 2012;. 2(1): 43-47.