Genetic Association of Bovine TNF-α Gene Polymorphism with Clinical and Sub-clinical Mastitis in Sahiwal Cows

For the identification of polymorphism within TNF- α gene, a total of 150 Sahiwal cows (clinical mastitis n= 50; subclinical mastitis n=50; non-mastitis n=50) were selected. For subclinical mastitis detection, Surf Field mastitis test (Muhammad et al., 1995) was performed at the animal site. For this purpose, 3% solution of household detergent (Lever Brothers, Surf Excel, and Arial) was mixed with quarter foremilk sample in equal volume and was swirled for 15-20 seconds and looked for thickening of the mixture (i.e. gel formation). About 5-10 mL blood sample was collected from jugular vein aseptically in blood vacutainer tubes. Organic DNA extraction method was used for DNA extraction from blood samples (Sambrook et al., 2001).

For amplification of TNF- α gene, six sets of primer were designed using already reported sequence of B. taurus from NCBI (NCBI GenBank; Accession no. AC_ 000180.1) (Table I). Optimization of primers was carried out with following temperature profile; initial denaturation-95°C for 5 min, denaturation-94°C for 30 s, annealing of TNF1 at 56ºC, TNF2 at 60ºC, TNF3 at 55ºC, TNF4 at 52ºC, TNF5 at 56ºC and TNF6 at 58ºC for 30 s, extension-72ºC for 60 s, final extension-72ºC for 10 min, repeat step 1-3 for 30 cycle.

Purified amplicon was subjected for commercial sequencing. The sequencing result was analyzed manually using BioEdit software followed by sequence alignment through NCBI BLAST. Protein analysis was done using ExPasy translate tool along with the development of 3D structure using PAYMOl software.

In this present study, the TNF- α gene of Pakistani Sahiwal cow was sequenced for identification of polymorphism. Comparative sequence analysis with reference sequence revealed SNPs at different position of the gene. In TNF- α gene sequence of clinical and subclinical mastitic cows, six transition SNPs were identified at location 268 (A>G), 383 (G>A), 750 (C>T), 2075 (A>G), 2444 (T>C), 2511(C>T). Three changes were identified at position 538 (G>C), 1139 (T>G) and 2512 (T>G) that show transversion polymorphism (Table II). The protein analysis shows missense mutation in amino acid sequence i.e., Leucine is changed with Tryptophan at 215 (Fig.1).

SNPs were identified in TNF- α gene in both clinical and subclinical cases but not in non-mastitic samples so, this candidate gene are associated with cow immune response against invading microorganism. A total of nine changes including transition (n=6) and transversion (n=3) were found at different positions (exon and intron) of this gene. The polymorphism at position 111 (C/T), 209 (C/T) and 308 (A/G) in exon 4 of TNF-α were reported by Shirasuna (2011) and Maksymiec (2013) in Holstein-Friesian cows and Firyal (2018) in Sahiwal cows (Shirasuna et al., 2010; Wojdak-Maksymiec et al., 2013; Firyal et al., 2018) but the other SNPs are not reported.

The protein analysis performed indicates that there is a change in amino acid from Leucine to tryptophan at amino acid position 215. These mutational changes in polar groups produce a change in the overall 3D protein structure of TNF- α gene (Fig. 2).

All these changes were found in both clinical and subclinical mastitis samples of Sahiwal cows at different intronic and exonic region. A comparison of the TNF-α gene in various type of mastitis in the present study indicates variations within the TNF-α gene that is responsible for mastitis. So, TNF-α is a potential candidate gene for the screening of the mastitis susceptible and resistant dairy cows.

The present investigation demonstrates the presence of nucleotide changes at various positions in bovine TNF- α gene that leads to change in amino acid sequence. Our result suggests that these SNPs may be used as a potential genetic marker for screening of mastitis resistant and susceptible Sahiwal cows. This can be useful in the selective breeding of cattle for an enhanced immune response as a tool to improve inherent animal health.

The authors declare no conflict of interest.