Relative Expression of Toll-like Receptors, Cytokines and Acute Phase Protein by Real-Time PCR in Milk Somatic Cells of Subclinical Mastitis Affected Cattle

Mastitis is one of the most important diseases of economic importance affecting dairy animals worldwide (Gulbe et al., 2020). It is an inflammatory response in the mammary gland, which is predominantly a result of the infectious challenge and is the most frequent disease of dairy animals (Fonseca et al., 2015). Subclinical mastitis (SCM) is subtle and more difficult to detect than clinical mastitis. SCM usually remains unnoticed because the milk and udder appear normal (Tanamati et al., 2019). In addition, SCM constitutes a reservoir of microorganisms that spreads the infection of other animals within a herd (Bhatt et al., 2012). So, the subclinical form of the disease is important because it is 15 to 40 times more prevalent than its clinical form (Singh et al., 2015a) and therefore usually persists longer in the herd, causing production losses (Kumar et al., 2014).
       
Early diagnosis of SCM is extremely important to check its development in clinical cases. Changes in the expression patterns of toll-like receptors, cytokines and acute phase proteins of the mammary glands in healthy and diseased animals can help in detecting early infection. Studies indicated variations in toll-like receptors, cytokines and acute phase proteins expression in mastitis cases were associated with disease activity (Bhatt et al., 2012). But, little information is available on toll-like receptors (TLR-2 and TLR-4) and IL-10 expression in natural SCM in cattle. Therefore, the present study was undertaken for the quantification of relative transcription levels of toll-like receptors, cytokines and acute phase protein in milk somatic cells of cattle suffering from subclinical mastitis.

Place of study
 
The study was conducted in the Department of Veterinary Microbiology, College of Veterinary and Animal Science, Navania, Udaipur from 2019-2021.
 
Source of milk samples
 
The milk samples were collected aseptically from the lactating cattle in and around the Udaipur district of Rajasthan. Screening of the SCM was conducted by the California mastitis test (CMT) and somatic cell count (SCC).
 
Bacteriological examination
 
The milk samples found positive (based on CMT and SCC) were subjected to bacteriological examination as per standard procedures by Markey et al., (2013).
 
Extraction of total RNA from milk and cDNA synthesis
 
Total RNA was extracted from the milk sample (5×10⁶ somatic cells) using the Trizol reagent (Genetix Biotech Asia Pvt. Ltd., New Delhi, India) as per the manufacturer’s protocol. Total RNA was quantified by biospectrometer (Eppendorf, Hamburg, Germany) and A260/A280 was measured for RNA quality. RNA of different milk samples was used as a template for the synthesis of first-strand cDNA using reverse transcriptase as per the standard procedures by Singh et al., (2016). Synthesized cDNA was stored at -80°C until further use.
 
Real-time PCR assay
 
First of all the optimization of Real-Time PCR was done as per the standard procedures by Singh et al., (2016). The real-time PCR assay was carried out using a real-time PCR machine (Biorad Pvt. Ltd., California, U.S.A.) in triplicate for each sample. In the present study, toll-like receptors (TLR-2, TLR-4), cytokines (TNF-α, IL-1β, IFN-γ, IL-6 and IL-10) and acute phase protein i.e. Haptoglobin (Hp) were chosen as the target and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acted as endogenous control. The specific oligonucleotide sequence of these genes has been presented in Table 1. Reactions were performed in a 20 μl volume reaction mixture comprised of various components (Table 2). Non-template control (NTC) comprises all other components except the template cDNA.
 

 

       
The thermal cycling conditions were performed as an initial step of denaturation at 95°C for 5 min, followed by 35 cycles at 95°C for 30 sec (cyclic denaturation), annealing (annealing temperature as described in Table 1 for each gene) for 30 sec. and extension at 72°C for 30 sec. For each sample, a dissociation curve was generated after the completion of amplification and analyzed to determine the specificity of the qPCR assay. Quantitative RT-PCR data were analyzed with the comparative Cq method (^ΔΔCq) (Livak and Schmittgen, 2001).

As per International Dairy Federation criteria, quarters having SCC of more than 5 lakhs and bacteriologically positive, were identified as subclinically infected. Milk samples of 4 healthy and 4 SCM cattle were considered for this study. Staphylococcus was the microbe found associated with SCM in all these samples. From infected and healthy milk somatic cells, total RNA extracted was in the range of 300-400 ng/µl and A260/280 ratio of >1.75-1.8 indicated that the purity of RNA was good. The amplification plots and dissociation curves for the genes of interest showed a single peak indicative of primers specificity. Relative transcript levels of these genes in SCM are presented (Table 3).
 

 
TLR-2
 
In the present study, TLR-2 gene expression level was found to be upregulated (p<0.01) in diseased quarters compared to healthy quarters. These observations were in corroboration with the earlier report of a significant up regulation in the expression of the TLR-2 in SCM caused by S. aureus. The presence of S. aureus organism would have triggered the TLR-2 expression in milk somatic cells of SCM-affected cows (Karthikeyan et al., 2016).
 
TLR-4
 
In the current study, TLR-4 gene expression level was found to be down regulated (p>0.05) in diseased quarters. In our study, there was down-regulation of TLR-4 which was the prime TLR reported to be activated by gram-negative bacteria (Kayagaki et al., 2013). However, some studies reported an increased mammary abundance of TLR-4 in experimental induced mastitis with gram-positive bacteria (Beecher et al., 2012; Fonseca et al., 2015), natural infection with gram-positive bacteria (Tanamati et al., 2019), induced infection with both gram-positive and gram-negative bacteria (Lee et al., 2006). The up regulation and down regulation of genes are under the control of various regulatory mechanisms in the host immune response. Any type of negative feedback will bring down the TLR expression and positive feedback will up-regulate the TLR level (Mitra et al., 2014).
 
TNF-α
 
In the present study, TNF-α gene expression level was found to be down regulated (p>0.05) in diseased quarters. Similar to the present study observations, Alluwaimi et al., (2003) observed down regulation of TNF-α at 32 h post-infection of S. aureus in the bovine mammary gland. The present study results are also in conformation with the finding of Bruno et al., (2010) who found a low levels of TNF-α associated with subclinical streptococcal-infected bovine mammary glands at dry-off. Low conc. of TNF-α mRNA transcript in diseased quarters could be due to timings as this TNF-α cytokine can be released in a pulse-like mode during inflammatory reactions to pathogens (Sordillo and Peel, 1992). Alluwaimi et al., (2003) were also of the opinion that differences in expression might be due to cyclic rise and decline in the number of viable pathogens. In the present study, there was a high level of IL-10 was found and it was able to inhibit the production of TNF-α by macrophages (Fiorentino et al., 1991). In contrast, multiple studies (Beecher et al., 2012; Fonseca et al., 2015; Singh et al., 2016) reported higher mRNA expression of TNF-α in animals with mastitis. 
 
IL-1β
 
IL-1 has been designated as an important mediator of neutrophil appointment at the place of inflammation and regulating the functions of infiltrating neutrophils, monocytes and the cytokines released by these cells (Fonseca et al., 2009). In our study, up regulation of IL-1β was recorded. Our result is consistent with the finding of Beecher et al., (2012) who also reported higher mRNA expression of IL-1β in cattle with induced mastitis.
 
IFN-γ
 
In the present study, the IFN-γ gene expression level was found to be down regulated (p>0.05) in diseased quarters. This down regulation might be due to the Staphylococcus spp. infection. These results were in accordance with previous studies (Alluwaimi et al., 2003; Lee et al., 2006) which also reported down regulation of mRNA of IFN-γ after staphylococcal mastitis. S. aureus could deploy mechanisms to suppress IFN-γ expression in somatic cells to survive intracellularly (Lee et al., 2006). In the present study, there was an increase in the level of IL-10 found and IL-10 was able to suppress the secretion of IFN-γ by Th1 lymphocytes (Mosmann and Moore, 1991). In contrast, results of up regulation were observed by different researchers (Bhupal, 2007; Fonseca et al., 2009; Bhatt et al., 2012) in experimentally induced mastitis, natural mastitis and SCM.
 
IL-6
 
In the present study, the relative level of the IL-6 gene was down regulated (p>0.05). The findings in our study are in accordance with the previous study (Bruno et al., 2010). In the current study, Staphylococcus spp. infections were predominant and Staphylococcus caused a chronic type of mastitis. Perhaps this explains why expression of IL-6 was downregulated in our milk samples of SCM. Although IL-6 mRNA transcription has been detected in chronic S. aureus infected quarters, its transcriptional activity changed only slightly (Alluwaimi et al., 2003). The present study results contrast with the findings of many studies (Bhatt et al., 2012; Bochniarz et al., 2017) that reported IL-6 concentration to be significantly higher in subclinical mastitic milk.
 
IL-10
 
In the present study, the IL-10 gene expression level was found to be up regulated (p>0.05). The present study results are consistent with the finding of other studies (Fonseca et al., 2009; Bochniarz et al., 2017) reported higher expression of the IL-10 gene in animals with mastitis. In contrast, a study (Beecher et al., 2012) observed no change in the transcript abundance of IL-10.
 
Hp
 
In the present study, the Hp gene expression level was upregulated. It is consistent with the previous studies (Kumar et al., 2014; Singh et al., 2015b) which also showed increased concentrations of Hp in the milk of animals suffering from mastitis and SCM.
       
Mastitis is a multi-etiological and multifactorial disease that is influenced by many genes. Divergent results were observed in the fold of expression level of target genes which can be explained by the fact that immune response differed according to the stage of SCM, bacterial strain and host with the observation of wide individual variation. Further studies including a larger number of genes and animals in different steps of infection are necessary to better understand the immune response mechanism and to develop a more systematic scheme for the control and eradication of SCM.

In the present study, a relative upregulated expression was found in TLR-2, IL-1β, IL-10, Hp and downregulation was found in TLR-4, TNF-α, IFN-γ and IL-6 in the milk somatic cells of subclinical mastitis affected cattle.

The authors thank the Dean, CVAS, Navania, Udaipur for providing the necessary facilities during the study.