| Transmissible Spongiform Encephalopathies (TSEs) are fatal group of diseases which are neurodegenerative and it is also included chronic wasting disease in deer and elk, Creutzfeldt-Jakob disease (CJD) in humans, transmissible mink encephalopathy (TME) in mink, mad cow disease in cattle and Scrapie in sheep and goats. The accumulation of abnormal form of the cellular prion protein (PrPC) is common in all diseases related to TSE. This abnormal form of PrP called PrPSc which is not only partly resistant to proteolysis but also found infectious in nature. In this study, blood samples were taken from 129 selected sheep of 12 breeds from all provinces of Pakistan. Sequence analysis of prion gene were carried out and compared with other mammalian species. Firstly,genomic DNA was extracted from blood samples using standard protocol which involved RBCs lysis, protein digestion and precipitation. The PrP gene was amplified using specific primers. Both sense and antisense strands of the amplified DNA was sequenced in an automated DNA sequencer (ABI Prism 3130xL Genetic Analyzer, Applied Biosystems, USA) according to the instruction of manufacturer. Sequences were analyzed through Codon Code aligner manually followed by the identification of novel SNPs detection. Codon Code aligner software was used to edit and align the PrP gene sequences using MEGA v.6 software in order to construct the phylogenetic trees. "R" statistical package was used to draw the multidimensional scaling plot, genetic distance and principle component analysis. Single nucleotide polymorphism was observed at different positions including 379,380,404,407,414,428,437,455,461, 511,512,513,566,572,691,711 and 718. These mutations were either transitional or transversional in nature. The sequence analysis results showed that an aggregate of 7 amino acid polymorphism were recognized in the PrP gene of sheep. The amino acid polymorphism was joined in 13 alleles and 15 genotypes in sheep. The general recurrence of the most sheep scrapie resistance polymorphism (Q171R) was ascertained to be 0.107. The most scrapie-susceptible polymorphism (A 136V) was not identified in any of the prion gene in selected sheep.Subsequent studies were conducted to explore PrP gene in 94 selected goats belonging to 11 Pakistani goat breeds from all provinces of Pakistan. Here PCR amplification of 771 bp coding region of PrP gene was carried out revealing six polymorphic sites (one novel) with amino acid substitutions were observed which includes 126 (A-> G),304 (G-> T),379 (A-> G),414 (C-> T),428 (A-> G) and 718 (C-> T). The locus C.428 was found highly mutant in all breeds as compare to other 10 breeds. One of the breed, namely Beetal was observed with carrying more variable on this locus as compared to other breeds. On the basis of these PrP variants NJ phylogenetic tree was constructed which showed that all goat breeds along with domestic sheep and Mouflon sheep appeared as in one clade and sharing its most recent common ancestors (MRCA) with deer species. Protein analysis has shown that these polymorphisms may lead to varied primary, secondary and tertiary structure of protein. Multidimensional scaling plot represents the distances graphically which explains that all goat breeds are explicitly separated into different group as compare to other mammals.Principle component analysis findings on prion protein gene in sheep and goat may help to provide assistance for further studies on pathogenesis, cross species transmission, development of effective breeding programs and mechanism studies on resistance and susceptibility to Scrapie. In conclusion, all these results on polymorphism of prion protein gene of Pakistani sheep and goat breeds will provide basic data for the surveillance of scrapie disease in Pakistan and will be beneficial for the Pakistani economy. |
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