Indian Journal of Animal Research 780 RESEARCH ARTICLE Indian Journal of Animal Research, Volume 58 Issue 5: 780-784 (May 2024) Relative Expression of Toll-like Receptors, Cytokines and Acute Phase Protein by Real-Time PCR in Milk Somatic Cells of Subclinical Mastitis Affected Cattle Rajesh Singathia 1 , Deepak Kumar Sharma 1 , Abhishek Gaurav 2 , Arpita Sain 1 , Karishma Rathore 1 10.18805/IJAR.B-5124 ABSTRACT Background: Subclinical mastitis is one of the most important diseases of economic importance affecting dairy animals worldwide. The present study was planned to evaluate the level of expression of toll-like receptors, cytokines and acute phase protein in milk somatic cells during subclinical mastitis of cattle. Methods: The milk samples of subclinical mastitis-positive and healthy cattle were collected. After that, extraction of total RNA was done from milk somatic cells followed by cDNA synthesis by the process of reverse transcription. Following that qPCR was carried out and relative transcript levels were determined. Result: In the present study, a relative up-regulated expression was found in TLR-2, IL-1β, IL-10 and Hp and down-regulation was found in TLR-4, TNF-α, IFN-γ and IL-6 in the milk of cattle with subclinical mastitis as compared to healthy ones. Monitoring of cytokines entangled in the modulation of immune responses during the infection is useful in deciding cytokine markers that could be employed as a forecasting tool in the early diagnosis of subclinical mastitis. Key words: Acute phase protein, Cytokine, Subclinical mastitis, Toll-like receptors. 1 Department of Veterinary Microbiology, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences, Navania, Udaipur-313 601, Rajasthan, India. 2 Department of Veterinary Public Health and Epidemiology, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences, Navania, Udaipur-313 601, Rajasthan, India. Corresponding Author: Rajesh Singathia, Department of Veterinary Microbiology, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences, Navania, Udaipur-313 601, Rajasthan, India. Email: drrajeshsingathia@gmail.com How to cite this article: Singathia, R., Sharma, D.K., Gaurav, A., Sain, A. and Rathore, K. (2024). Relative Expression of Toll-like Receptors, Cytokines and Acute Phase Protein by Real- Time PCR in Milk Somatic Cells of Subclinical Mastitis Affected Cattle. Indian Journal of Animal Research. 58(5): 780-784. doi: 10.18805/IJAR.B-5124. Submitted: 05-04-2023 Accepted: 05-10-2023 Online: 10-10-2023 INTRODUCTION 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. MATERIALS AND METHODS 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