Sequence analysis of TLR4 gene in river buffalo (Egyptian breed) and SNPs association with Mastitis

Abstract

Abstract Background: Mastitis is a major cause of economic loss for dairy farmers and industry. River buffalo play an economically significant role in Egypt. Buffaloes with mastitis have reduced milk yield and change in milk composition. Genetic variations in the TLR4 gene have been related to several diseases in farm animals and humans including mastitis. The present investigation aims to find the genotypic variations in the TLR4 gene and their relation to mastitis in the river buffalo, Egyptian breed. Results: Eighty-one buffaloes (Egyptian breed) were tested for mastitis using SCC. 45% of the investigated buffaloes had mastitis. DNA from 30 buffaloes' blood samples (15 healthy and 15 with mastitis) were extracted and the TLR4 gene was sequenced. Twenty-one SNPs were found from which four SNPs were associated with mastitis: one in 5'UTR (c.1-g27) and 3 SNPs in the coding region at c.87, c.575, and c.576. The nucleotide variations in SNPs c.1-g27(C>A) and c.87 (C>A) were only present in buffalo with mastitis, while buffaloes with genotype CC at both locations were healthy. The AA genotype at c.87 (P.29) results in a stop codon leading to an abnormally shortened protein. The nonsynonymous SNPs c.575 A>G, and c.576 T>G shared amino acid 192 resulting in three amino acids (His192Arg/Gln). The dominant genotypes AA at c.575 and TT at c.576 were associated with mastitis resistance (OR<1.00), while recessive genotype GG at c.575 was associated with mastitis susceptibility (OR> 1.00). These two SNPs may affect their role in ligand recognition since they were in the LRR4 domain (p.174-p.197) which is part of coreceptor binding region 1. Conclusion: The present study confirms the relation between TLR4 genotypes and mastitis resistance or mastitis susceptibility in river buffalo. The study suggested four SNPs c1-g27C>A, c.87 C>A, c.575A>G, c. 576T>G,) have the potential to be markers for assisted buffalo selection to improve milk production.