Quantitation of TLR-2 mRNA Expression in Bovine Mastitis Caused by E. coli

Aims: This study aim was to assay the expression of an important TLR2 gene in naturally infected cows with sub-clinical and clinical mastitis caused by E. coli . Methodology: cDNA from E. coli infected samples were obtained from hind quarters of three animals with sub-clinical mastitis, three animals with clinical mastitis along with a healthy animal, were used for TLR2 expression analysis using commercial kit and oligo (dT) primers. Quantitative reverse transcription PCR (RT-qPCR) were standardized for the TLR2 and β -actin gene, genes expression level was analyzed by Illumina Eco® Q- RT PCR system. Results: The mRNA expression of TLR2 gene in animals sub-clinical mastitis was significant (P ≤ 0.01) higher (3.63 fold) than clinical case (1.89 fold) when compared to healthy bovine. Conclusion: The expression of TLR2 gene during early stage of infection was at high level, therefore most of the sub-clinical mastitis subsided by itself without precipitating into clinical mastitis. It is suggested that TLR2 may consider as candidate gene for mastitis screening in cattle population.


INTRODUCTION
In dairy industry, mastitis is one of the most important disease conditions and it is caused various groups of bacteria, among which a gram negative bacteria E. coli plays a considerable role in bovine mastitis [1].These infection lead to severe milk loss and are a major cost to dairy farmers. For this reason, there is a need to understand the pathogenesis of mastitis. E. coli has the ability to grow in mammary secretions and to liberate lipopolysaccharide (LPS) is crucial in the pathogenesis of mastitis [2].The innate immune response to pathogen in the mammary gland is crucial for effective clearance of the pathogen [3]. It is the predominant defense strategy during the early stages of an infection [4]. There is difference in host response towards experimentally induced infection of gram positive and gram negative bacteria [5]. Toll-Like Receptors are vital to immune function through the sensing of pathogenic agents through pattern recognition receptors [6]. In mammals, 13 TLRs are reported that recognize some specific ligands from pathogens to trigger immunological responses [7]. Of these, TLR2 is the main pathogen recognition receptors (PRR), recognizes lipoteichoic acid (LTA) from Gramnegative bacteria [8]. The activation of TLRs may commonly lead to early activation of the transcription factors and the release of several proinflammatory mediators [9,10]. Assessing the expression of an important TLR2 will be vital for improving our understanding the pathogenesis of mastitis caused by E. coli. Hence, the objective of this study was to investigate the mRNA expression of TLR2 gene in naturally infected E. coli cases of sub-clinical and clinical bovine mastitis.

Milk Sample Collection and Identification of Bacteria
Milk samples were collected from hind quarters of eighty animals at University farm and Veterinary Dispensaries, Thrissur, Kerala. The milk samples were categorized into sub-clinical, clinical and healthy based physical examination, California mastitis test (CMT) and somatic cell count (SCC). During sampling, the samples were collected aseptically and put into sterile centrifuge tube and kept in an ice box containing ice packs and taken immediately to laboratory for bacteriological analysis [11]. The milk samples were streaked on Mcconkey's agar plates for identification of coliforms. The positive cultures were further subjected to biochemical tests for identification of E. coli [12].

RNA Isolation and cDNA Synthesis
Total RNA was obtained from milk somatic cells using TRIzol reagent of SIGMA (as per the manufacturer's protocol). The quality of extracted RNA was determined by agarose gel electrophoresis (1.5%) and ethidium bromide staining. Residual DNA was removed from extracted total RNA by treatment with DNase1. The concentration of RNA was assessed by using nanodrop spectrophotometer. Total RNA was subjected to cDNA synthesis using oligo dT primers (Thermo Scientific, K1622).

Primers Design and Synthesis
Primers for RT-qPCR of TLR2 and β-actin were designed from published bovine mRNA sequences available from GenBank. Designing and checking of primers were done with Primer3 software (Table 1).

RT-qPCR
Before real-time quantification, the PCR was optimized for TLR2 and β-actin genes. Expression level of mRNA of TLR2 was analysed by Illumina Eco® RT-qPCR system. The β-actin was selected as housekeeping gene because it showed a stable expression from all milk samples. Real time PCR was carried out in triplicate for each sample in a total volume of 20 microlitre, 10 microlitre of 2X SYBR Green PCR mastermix, 10 pmole (1microlitr) of each genespecific primers, 2 microlitre of cDNA template and 7 microlitre of nuclease free water. Real time PCR cyclic conditions were 95°C for 10 min then 40 cycles of 95 for 30 sec, 58 for 30 sec and 72 for 1 min with fluorescence recording at the end of each cycle. The final step was to obtain a dissociation curve analysis PCR products to determine the specificity of the amplicons. The protocol for melt curve analysis was 95°C for 15 sec, 55°C for 15 sec followed by 95°C for 15 sec. Data acquisition was performed during the final denaturation step. The result was expressed at threshold cycle values (Ct). The relative expression of each sample was calculated using the 2 -∆∆Ct method [13].

RESULTS AND DISCUSSION
Lactating crossbred cows were physical examination for clinical mastitis and also screened for sub-clinical mastitis by using SCC and CMT [14]. Based on biochemical test, 8 (eight) revealed the presence of E. coli in subclinical case of mastitis and 5 (five) samples showed the presence of E. coli in clinical mastitis. For RT-qPCR assay, milk from three animals from each group of sub-clinical and clinical mastitis caused by E. coli was selected (randomly) for expression study. Milk samples from three apparently healthy crossbred cows were also selected as control for expression studies. Analysis of variance for TLR2 gene revealed significant difference (P ≤ 0.01) for expression level between the groups ( Table 2).
The mean values of C q , ∆C q, ∆∆Cq along with standard error and relative quantification of TLR2 expression in E. coli caused mastitis are given in Table 3.
The relative expression of TLR2 gene was ranged between 3.02 and 4.49 fold for subclinical mastitis and it was ranged from 1.52 to 2.15 fold, when compared with healthy crossbred cows (Fig. 1). E. coli infected mammary gland shows mRNA levels of TLR2 was higher in subclinical mastitis (3.63 fold) followed by clinical mastitis (1.89 fold) when compared to normal animal. Relative expression of TLR2 gene was significantly (P ≤ 0.01) higher in the sub-clinical mastitis, and also exhibits significant difference (P ≤ 0.01) between sub-clinical and clinical mastitis (Fig. 1). In general, TLR2 is involved in the recognition of PGN of Gram-positive bacteria, although many authors also reported the expression of TLR2 in response to E. coli infection [8,15,16]   Toll-like receptors are key component of innate immune system that recognize a variety of pathogen through pattern recognition receptor [17]. TLR2 is principal receptor for peptidoglycan and lipotechoic acid from gram positive gram negative bacteria. In our study TLR2 was expressed with 3.63 fold upregulation in subclinical case of mastitis caused by E. coli infection. TLR2 mRNA expression were increased by mastitis infected by E. coli [18]. Similarly Ibeagha-Awemu et al. [19] also reported increased mRNA expression of TLR2 in mammary glands infected with LPS. Similarly, TLR2 and TLR4 mRNA expression increased in infected mammary glands of cows with mastitis caused by either S. aureus or E. coli [16]. Stimulation of TLR2 consequently induces a series of signalling cascades that ultimately result in the activation of NF-kB. The ability of the TLR2-induced NF-κB pathway to cross-talk with other signaling molecules is a key element in shaping the overall pattern of host responses, such as production of cytokines and chemokines, antimicrobial killing mechanisms, maturation of antigen presenting cells, and recruitment of the adaptive immune response [20,21,22].

CONCLUSION
In our study the expression of TLR2 gene was significantly higher in sub-clinical mastitis compared to clinical case of mastitis infected by E. coli. Sub-clinical mastitis is an early stage of infection, and hence the expression of TLR2 is relatively higher at this stage compared to clinical stage. The result suggested that mastitis increases the expression of TLR2 contribute to innate immune system in the cow udder Therefore, the sub-clinical stage of mastitis may be subsided by itself without precipitating into clinical mastitis. It is suggested that TLR2 might be considered as a candidate gene for screening mastitis resistance in cattle population.