Single nucleotide polymorphism in TLR1 and TNFá genes and their association with susceptibility to bovine tuberculosis

Bovine tuberculosis (bTB) is a chronic debilitating infectious disease of cattle caused by Mycobacterium bovis (M. bovis) and characterized by granulomatous inflammation, necrosis, calcification and encapsulation of lung, intestines, lymph nodes and other tissues (Neill et al., 2001; Cousins, 2001). Although cattle are considered to be the true hosts of M. bovis, the disease has been reported in many other domesticated and non-domesticated animals (Corner, 2006; Hlokwe et al., 2014). Incidence and prevalence of bTB has been reported in bovine populations across the world but the infection is endemic in Africa and Indian sub-continent (Mukherjee, 2006; Perry et al., 2013; Prakash et al., 2015). An annual loss of $3 billion to the global livestock sector has been reported on account of bTB infection (Schiller et al., 2010). A typical attribute of M. bovis infection is that it has an insidious, chronic progression, which may take several weeks or months to become clinically potent (Neill et al., 2001) with a grave implication that the animals may become infectious long before they exhibit clinical signs or lesions. Therefore, the foundation of bTB eradication and control in cows lies in the early detection and removal of M. bovis-infected animals from the herd. Test and slaughter programmes have been used worldwide since several decades for the control of bTB using purified protein derivative (PPD) based tuberculin skin tests (Rua-Domenech et al., 2006). However, bTB remains intractable to eradication in many developed and some developing countries, (More et al., 2015). Genetic variation for susceptibility to bTB has been reported in cattle with heritability values ranging from 0.12 to 0.34 in different populations (Bermingham et al., 2009; Brotherstone et al., 2010; Richardson et al., 2014; Bermingham et al., 2014; Tsairidou et al., 2014; Raphaka et al., 2017). Higher resistance to bTB has been reported among Bos indicus than Bos taurus (Ameni et al., 2007). Candidate genes with specific roles in immune response to bTB can serve as a useful tool in identifying superior genotypes. A number of SNPs in genes encoding Pathogen Recognition Receptors (PRRs) viz. TLR1, TLR2, TLR4, TLR6, TLR9, CARD15, DC-SIGN, CD14 (Sun et al., 2012; Song et al., 2014; Bhaladhare et al., 2016; Zhao et al., 2017; Bhaladhare et al., 2018; Wang et al., 2015; Baqir et al., 2015; Xue et al., 2018), genes encoding cytokines and their receptors viz. TNFα, IL12RB1 and IFNGR1 (Cheng et al., 2016a; Baqir et al., 2014; Bhaladhare et al., 2019) and genes encoding other important immune response molecules viz. SLC11A1, NOS2 and SP110 (Baqir et al., 2016; Cheng et al., 2016b; Baqir et al., 2015) involved in recognition of components of M. bovis and subsequent activation of both innate and adaptive immune response have been investigated as potential strong candidates for genetic basis of resistance.
        
Toll Like Receptor 1 (TLR1) and Tumor Necrosis Factor- alpha (TNFα) mediated immune functions are essential for the clearance of invading pathogens and for the consequent initiation of the adaptive immune responses (Kawasaki et al., 2014a). TLRs are evolutionary conserved pattern-recognition receptors (PRRs) that recognize and interact with microbe-specific structural motifs known as pathogen-associated microbial patterns (PAMPs) leading to induction of immune response (Werling et al., 2003; Kawai et al., 2010). TNF-α serves important roles of augmenting phagocytic activity of neutrophils, stimulating reactive oxygen species apart from promoting other cytokines with an overall effect of increasing bactericidal and cytotoxic actions against invading pathogens (Roach et al., 2002; Bradley, 2008). In a Chinese Holstein population, G1596A polymorphism in the TLR1 gene was found to be associated with bTB infection (Sun et al., 2012). Cheng et al., (2006a) reported significant (p=0.02) association of g.27534932A >C polymorphism in exon 3 of TNF-α gene with bTB susceptibility in Holstein cattle. However, information on association of TLR1 and TNFα polymorphism with susceptibility or resistance to bTB in Bos indicus cattle/ crossbreds is unavailable. Therefore the objectives of this study were to genotype a case-control population and to investigate the potential association between bTB and SNPs in TLR1 and TNFα genes in cattle so as to find SNP that may act as a genetic marker.

Experimental cattle population
 
A total of 245 cattle including Indigenous (Koshi, Sahiwal, Gir)/Non-descript and crossbred from Shri Mataji Gaushala, Barsana comprised the resource population for the current investigation. All animals were maintained under similar feeding and managemental practices and had an equal opportunity of infection. Animals were screened for the presence of bTB by Single intradermal tuberculin test, wherein increase in thickness of skin after 72 h of intradermal injection of tuberculin antigen was noted to develop case (tuberculin positive) and Control (tuberculin negative) resource panel. An intradermal inoculation of 0.1 ml of tuberculin PPD antigen on neck region was carried out. The skin thickness was measured with vernier calipers before and 72 h after inoculation. Based on thickness, cattle were classified into three groups: Those showed marked swelling and skin thickness more than 4 mm (positive), skin thickness <4 mm and >2 mm (inconclusive), and no reaction >2 mm (negative). The inconclusive animals were not included in the present investigation. A case and control resource panel of 35 positive and 45 negative animals was developed. All the procedures performed in the study involving animals were in accordance with the ethical standards of the Institutional Animal Ethics Committee (IAEC) of ICAR-Indian Veterinary Research Institute (IVRI), Bareilly, India (196/GO/RE/SL/2000/CPCSEA).
 
Sample collection and isolation of Genomic DNA
 
From each of case and control animals, 5 ml of blood was collected from a jugular vein in tubes containing 2.7% EDTA and stored at -20°C. Genomic DNA was isolated from whole blood using Promega Wizard® Genomic DNA Purification Kit as per recommended protocols. The DNA concentration was determined using Qubit Fluorometer. Those DNA samples having a minimum concentration of 50 µg/ml were used for further study. DNA quality was also assessed by 1% agarose gel electrophoresis. 1 µl of genomic DNA was resolved on 1% agarose gel stained with ethidium bromide or SYBR® Safe DNA gel stain and quantification was made by comparing the intensity of the band with the intensity of a known quantity of lambda DNA. Only thick DNA band and without smearing were chosen for further processing.
 
SNP Genotyping
 
Primers for the 3 SNPs in TLR1 gene (rs43702940, rs68343175 and rs55617317) from Sun et al., (2012) and 1 SNP in TNFα (rs109967811) gene designed using OligoAnalyzer (Integrated DNA Technology software) software were used for amplification of the loci. The detail of primers and restriction enzymes are being presented in Table 1. Concerned amplicons were amplified under the optimized PCR condition. The PCR reaction was carried out in 25 µl volume which included 1 µL of each primers (forward and reverse), 1.5 µL MgCl₂, 5 µl buffer, 0.2 µL dNTPs, 0.125 µl to 0.25 µl Taq polymerase, 1 µl genomic DNA and Nuclease free water 15.05 to 15.175 µl. The cycling program used for amplification having following steps; initial denaturation (94°C for 4 min), followed by 35 cycles of 30 s at 94°C, 30 s at annealing temperature (Table 1), 30 s at 72°C and final extension of 5 min at 72°C. The PCR products were resolved in 2.4% agarose gel and visualized under UV light after staining with ethidium bromide. Restriction digestion was carried out in 25 µL reaction volume which included 20 µL of PCR product, 1.5 U of restriction enzyme, 2.5 µL of 10x buffer and Nuclease free water to make volume up to 25 µL and incubated at recommended temperature as proscribed by manufacturer for 16 hours. The restriction enzyme digestion was made at the optimized conditions and the restriction digested products were resolved in 3.5% agarose gel and visualized under UV light after staining with ethidium bromide. Mass genotyping of all case-control resource population for all four SNPs was done by using PCR- Restriction Fragment Length Polymorphism (PCR-RFLP).
 

 
Statistical analysis
 
Initially in univariate logistic regression analysis, the non-genetic factors like age (two levels), sex (two levels) and breed (two levels) were fitted and found that none of these effects were significantly affecting the Single intradermal tuberculin test result. The genotypes were determined by reading the restriction fragment patterns of each digested sample in the gels. The gene and genotype frequencies were estimated by the standard procedure given by Falconer and Mackay (1996). Genotype of every animal was recorded manually from the autoradiograph. Genotyping involved the recording of the homozygous or heterozygous state of the animal, as well as the size of the respective alleles in base pairs.  On population basis, the number of alleles, their size and frequencies for different markers was recorded. The association between various allelic variants with bTB tolerance/susceptibility was worked out by suitable statistical techniques using different procedures of SAS 9.3. The PROC LOGISTIC procedure of SAS 9.3 was used to find association of allelic and genotypic frequencies with bTB. The Odds Ratio (OR) of genotypes was calculated in affected population versus their contemporary genotypes. The PROC ALLELE procedure of the SAS 9.3 used for the estimation of polymorphism information content (PIC), Hardy Weinberg Equilibrium (HWE) and heterozygosity.

In the cattle resource population, all non-genetic factors (breed, age and sex) had non-significant (p < .05) effect on the tuberculin test. The case-control population was genotyped by using PCR-RFLP for the three SNPs (rs43702940, rs68343175 and rs55617317) in TLR1 gene and one SNP (rs109967811) in TNFα gene. One SNP viz. rs43702940 was not found to be present in the study population i.e. it revealed monomorphism, while the other three SNPs under investigation displayed polymorphism. The chi square test revealed that the population was not in HWE for all three SNP loci investigated. All three loci revealed moderate estimates of PIC and allelic diversity while medium (rs109967811) to high (rs68343175, rs55617317 and rs55617193) estimates were found for heterozygosity. PIC, Heterozygosity, Allelic diversity and probabilities of the population being in HWE for the three SNPs is presented in Table 2. The allelic frequencies and the genotypic frequencies in Case and Control populations at three SNP loci and their effect on susceptibility to infection along with ODDs Ratio (OR) have been shown in Table 3 and 4 respectively.
 

 

 

 
Polymorphism and genetic association analysis at the rs68343175 locus in TLR1 gene
 
At SNP locus rs68343175, two alleles i.e. A and G and three genotypes were identified i.e. AA (179 bp), AG (179 bp, 86 bp and 93 bp) and GG (86 bp, 93 bp) (Fig 1). The frequency of A allele was 0.486 in case and 0.50 in control whereas G allele had frequency of 0.514 and 0.50 in case and control population respectively. Similarly the frequency of genotype AA, AG and GG were 0.057, 0.857 and 0.086 in case and 0.082, 0.837 and 0.082 respectively in control. The probability values showed that the genotype (P =0.91) as well as allele (P = 0.86) had non-significant effect on occurrence of bTB. The OR of A verses G was 0.94(0.51-1.74; 95% CI), where as OR of AA verses GG and AG verses GG were 0.67 (0.07 - 6.41; 95% CI) and 0.98 (0.2 - 4.69; 95% CI) respectively (Table 3 and 4).
 

        
Polymorphism and genetic association analysis at the rs55617317 locus in TLR1 gene
 
At SNP locus rs55617317, the mass PCR-RFLP revealed polymorphism within as well as between case and control population. At this SNP site two alleles i.e. A and G and three genotypes i.e. AA (334 bp, 20 bp), AG (334 bp, 73 bp, 261 bp, 20 bp) and GG (73 bp, 261 bp, 20 bp) were observed (Fig 2). The frequency of A allele was 0.529 in case and 0.612 in control where as C allele had frequency of 0.471 and 0.388 in case and control population respectively. Similarly the frequency of genotypes AA, AG, and GG were 0.114, 0.829, and 0.057 in case and 0.245, 0.735 and 0.020 respectively in control. The probability values showed that the genotype (P =0.23) as well as allele (P= 0.27) had non-significant effect on occurrence of bTB. The OR of A verse G was 0.71(0.38-1.32; 95% CI) whereas OR of AA verses GG and AG verse GG were 0.17 (0.01 – 2.37: 95% CI) and 0.4 (0.03 – 4.67; 95% CI) respectively (Table 3 and 4).
 

 
Polymorphism and genetic association analysis at SNP locus in TNFα gene
 
At rs109967811 locus in TNFα gene, the mass PCR-RFLP revealed polymorphism within as well as between case and control population. At this SNP site two alleles i.e. A and G and three genotypes i.e. AA, AG and GG were observed (Fig 3). The AA genotype showed the restriction fragments of 348 bp; AG genotype showed the restriction fragments of 348 bp, 224 bp and 124 bp and GG genotype showed the restriction fragments of 224 bp and 124 bp. While the frequency of A allele was 0.514 in case and 0.204 in control, the G allele had frequency of 0.486 and 0.796 in case and control population respectively. Similarly the frequency of genotypes AA and AG were 0.029, and 0.971 in case and the frequency of genotype AA, AG, and GG were 0.020, 0.367 and 0.612 respectively in control. The probability values showed that the genotype (P <0.01) as well as allele (P <0.01) had non-significant effect on occurrence of bTB.  The OR of A verses G was 4.13(2.09-8.14; 95% CI), whereas OR of AA verses GG and AG verses GG were >999.99 (<0.01 - >999.99; 95% CI) and >999.99 (<0.01 - >999.99; 95% CI) respectively (Table 3 and 4).
 

        
In the present investigation, an attempt was made at genotyping SNPs in TLR1 and TNFα genes and to study their association with susceptibility to bovine tuberculosis in a case-control population of cows established using single intradermal tuberculin test. All the four SNP loci investigated under the present investigation were in exonic region of TLR1/ TNFα genes thus directly governing the encoded proteins with important roles in host immune response to bTB infection. The SNP loci rs68343175 and rs55617317 in TLR1 gene showed variability in the Case: Control population, while one SNP i.e. rs43702940 revealed monomorphic PCR-RFLP pattern suggesting the absence of variability for this locus in the population. However the polymorphism observed at the three SNPs in TLR1 gene was not significantly association with susceptibility to bovine tuberculosis. SNPs of TLR1 were earlier studied by Sun et al., (2012) for their association with bTB in Chinese Holstein cattle and genetic variability was reported at all the SNPs, however only one SNP i.e. A1569C was significantly associated with bTB susceptibility. Sun et al., 2012 observed that GH or HH genotypes at A1569C locus had a larger relative risk of having bTB incidence [Odds Ratio (OR) = 2.431, 95% Confidence Interval (CI) (1.465–4.034); OR = 1.490, 95% CI (0.848–2.618) respectively] than the GG genotype. This study indicated that the GG genotype might be protective against bTB infection. Absence of rs43702940 in our population as compared to that of Sun et al., (2012) might be due to the different genetic composition and herd history. Further, an SNP at TLR1 (+1380 G/A) was significantly (P <0.05) associated with bovine brucellosis in a case control population for bovine brucellosis (Prakash et al., 2014) established from same resource population as our study. At TLR1 (+1380 G/A) locus ‘A’ allele was significantly (P = 0.01) lower than ‘G’ allele in brucellosis positive animals with its odds ratio of 0.43 (0.22-0.83; 95 % CI). Although SNPs in TLR2, TLR4, TLR9 and other PRRs have been investigated in Indian cattle breeds and crossbreds for association with bTB, PTB and mastitis (Wakchaure et al., 2012; Bhaladhare et al., 2016; Bhaladhare et al., 2018; Mishra et al., 2017; Yadav et al., 2014; Kumar et al., 2017; Kumar et al., 2018a; Kumar et al., 2018b; Kumar et al., 2019a; Kumar et al., 2019b) but no previous report exists on association study of SNPs in TLR1 gene with bTB in Indian cattle breeds and crossbreds. These results also show presence of high genetic diversity in the TLR1 gene among different kinds of cattle. Although none of the studied SNPs in the TLR1 gene were found to be associated with susceptibility to bTB in our population, other SNPs in TLR1/ signalling pathway genes might influence BTB susceptibility and needs investigation.
        
Under the present investigation, one SNP from TNFα gene i.e. rs109967811 was also evaluated for any possible association with susceptibility to bTB. This SNP existed in our resource population and revealed significant effect on susceptibility to bTB in cattle (P<0.01). The ODDs of AA and AG genotype vs GG genotype were close to infinity revealing that while the AG genotype was more related with susceptibility to bTB, the GG genotype was specific to resistance to bTB. Odd of A vs G allele was 4.13 (2.09 – 8.14; 95% CI), showing that A allele was more associated with susceptibility to bTB in comparison to G allele. The ODDs of AA and AG genotype vs GG genotype were close to infinity revealing that AA and AG genotype was more related with susceptibility to bTB. Odd of A vs G allele was 4.13 (2.09 – 8.14; 95% CI), showing that A allele was more associated with susceptibility to bTB in comparison to G allele. SNP locus rs109967811 in TNFα gene showed significant association with the susceptibility to bovine tuberculosis in cows. Cheng et al., (2016a) reported significant (p=0.02) association of g.27534932A>C polymorphism in exon 3 of TNF-α gene with bTB susceptibility in Holstein cattle. CA genotype cattle had 4.11-fold (95% CI, 1.27–13.36) higher susceptibility for bTB compared with the CC genotype.  Additionally, cows with A allele had 3.84-fold higher risk of bTB than those having C allele (95% CI, 1.21–12.17). Polymorphisms in coding regions of the TNF-α gene have been reported to affect the TNF-α mRNA expression and immune function in dairy cows, and allele A was seen to be favourable and related to stronger immunity against infection than other allele in this locus (Kawasaki et al., 2014b; LeRoex et al., 2013; Wojdak-Maksymiec et al., 2013). Alteration of transcriptional regulation caused by polymorphism in TNF-α gene is responsible for the association with susceptibility/resistance to bTB (Qidwai et al., 2011). Based on transcriptional profiling of host macrophage mRNA repertoire upon in-vitro M. bovis infection, TNF-α has been found as among the putative biomarkers for M. bovis infection in cattle (Magee et al., 2012; Lin et al., 2015; Shukla et al., 2017). This is first report of association of SNP in TNFα gene with the susceptibility to bTB in Indian cattle breeds. This SNP might play a significant role in the bTB risk in cattle warranting validations in a larger population along with other important SNPs in immune response genes to reveal biomarkers for susceptibility to bTB. Present investigation revealed that SNP locus rs109967811 in exonic portion of TNFα gene was found to be significantly associated with the susceptibility to bTB. Further, this study demonstrated the presence of a significant genetic variation in the population for the TLR1 and TNFα genes, which are critical for mounting an effective immune response against invading M. bovis. These findings emphasize upon the scope for genetic improvement of cattle for bTB susceptibility trait and worthy of validations and further investigation in immune response genes to reveal the mechanisms of disease resistance in cattle.

The authors sincerely acknowledge the help and support in the form of funds and important facilities provided by the Director and Joint Director (Research), ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India to perform this research.