Identification of single nucleotide polymorphisms in Toll-like receptor candidate genes associated with tuberculosis infection in water buffalo (Bubalus bubalis)

Abstract

BackgroundToll-like receptors play a key role in innate immunity by recognizing pathogens and activating appropriate responses. Pathogens express several signal molecules (pathogen-associated molecular patterns, PAMPs) essential for survival and pathogenicity. Recognition of PAMPs triggers an array of anti-microbial immune responses through the induction of various inflammatory cytokines. The objective of this work was to perform a case-control study to characterize the distribution of polymorphisms in three candidate genes (toll-like receptor 2, toll-like receptor 4, toll-like receptor 9) and to test their role as potential risk factors for tuberculosis infection in water buffalo (Bubalus bubalis).ResultsThe case-control study included 184 subjects, 59 of which resulted positive to both intradermal TB test and Mycobacterium bovis isolation (cases) and 125 resulted negative to at least three consecutive intradermal TB tests. The statistical analysis indicated that two polymorphisms exhibited significant differences in allelic frequencies between cases and controls. Indeed, the TT genotype at TLR9 2340 C > T locus resulted significantly associated with susceptibility to bovine tuberculosis (P = 0.030, OR = 3.31, 95% CI = 1.05-10.40). One polymorphism resulted significantly associated with resistance to the disease, and included the CC genotype, at the TLR4 672 A > C locus (P = 0.01, OR = 0.26, 95% CI = 0.08-0.80). Haplotype reconstruction of the TLR2 gene revealed one haplotype (CTTACCAGCGGCCAGTCCC) associated with disease resistance (P = 0.04, OR = 0.51, 95% CI = 0.27–0.96), including the allelic variant associated with disease resistance.ConclusionsThe work describes novel mutations in bubaline TLR2, TLR4 and TLR9 genes and presents their association with M. bovis infection. These results will enhance our ability to determine the risk of developing the disease by improving the knowledge of the immune mechanisms involved in host response to mycobacterial infection, and will allow the creation of multiple layers of disease resistance in herds by selective breeding.

Investigation into the genetic diversity in toll-like receptors 2 and 4 in the European badger Meles meles

“…However, population level data on the variation in either TLR2 or TLR4 genes in wildlife species is scarce. In comparison to other studies conducted, haplotype diversity in the badger was lower than that in other species, for example, TLR 2 from bank voles (Tschirren et al, 2013), TLR4 from grey partridges (Vinkler et al, 2015), pigs (Palermo et al, 2009) and both TLRs from water buffalo (Alfano et al, 2014), humans (Mukherjee et al, 2014). Furthermore, studies on other TLRs have also shown greater diversity in other species than in the badger genes reported here (e.g.…”

“…Single nucleotide polymorphisms (SNP) in TLR genes have been linked to pathogen susceptibility in some host species. For example, there is a relationship between TLR variation and Borrelia afzelii susceptibility (Tschirren et al, (2013)) and TLR polymorphism and susceptibility to bovine tuberculosis (bTB) in both Chinese Holstein cattle (Sun et al 2012) and water buffalo (Alfano et al, 2014).…”

The SNP g.4667G>A at 3′‐UTR of IFNG gene is associated with susceptibility to bovine tuberculosis in Mediterranean water buffalo (Bubalus bubalis)

“…Our findings confirm the existence of a high level of trans-species polymorphisms at MHC Class II loci, probably due to ongoing balancing selection, leading to conservation and sharing of favourable, functionally important alleles between Bovidae species. 4,5…”

“…To test our hypothesis, we collected peripheral blood from 183 water buffalo reared in Campania region (south Italy) and grouped into cases (n = 59 bTB-positive animals) and controls (n = 124 bTB-negative animals), according to previous work. 4 Then, we amplified the four exons and the flanking regions of the IFNG gene (Gene-Bank NW_005783995.1 complement 106206-110946; Table S1) in 10 each, randomly chosen, cases and controls. Alignment analysis showed a G>A point mutation in position 4467 in the 3 0 -untranslated region (UTR).…”

The tumor necrosis factor g1022G>A polymorphism is associated with resistance to tuberculosis in water buffalo (Bubalus bubalis)

“…Selective breeding for disease-resistant genotypes therefore represents an approach supporting disease control, 4,5 and different studies have identified several genes important for controlling the disease. [6][7][8] The role of tumor necrosis factor (TNF) in TB has been confirmed by the unrestrained growth of mycobacteria in TNF knockout mice caused by unpaired T-cell recruitment and unstructured granuloma development. 9 Thus, using samples (59 cases and 125 controls) recruited by Alfano and colleagues, 6 we amplified and sequenced in 20 subjects (10 cases and 10 controls) a DNA fragment of 554 bp (forward primer: 5 0 -AACACAC CCCTGAGAGACAG-3 0 ; reverse primer: 5 0 -AGCAGAGATA CAGACATAAAGA-3 0 ) that spans exon 1 and part of intron 1 of water buffalo TNF (GenBank accession no.…”