Acute phase proteins: A seasonal perspective on mastitis in Sahiwal cows

Introduction

Bovine mastitis is a chronic inflammatory condition of the parenchyma of the mammary gland that is typically accompanied by pathological alterations in the glandular tissue and modifications to the physicochemical characteristics of milk (Ali et al., 2022) [1] . Intramammary infections can also cause changes in the milk, such as an increase in somatic cell counts and types (Wever and Emanuelson 1989) [24] , inflammatory proteins like leukotrienes (Rose and others 1989) [19] , or interleukins (Shuster and Kehrli 1993) [21] . These changes can happen either before or after the onset of clinical symptoms. Therefore, it is crucial to find specific mastitis biomarkers for early identification in order to provide therapeutic intervention at an earlier stage and lessen the severity of the disease as well as significant financial losses to the dairy industry. A number of systemic events occur after tissue damage, most notably the acute phase response (APR), which is triggered by macrophages or blood monocytes of the injured tissue. These cells secrete a variety of mediators, mainly cytokines and other pro-inflammatory molecules like interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-) (Gruys et al., 2005) [9] . The most notable changes linked to APR are metabolic alterations that boost the production of a class of plasma proteins called acute phase proteins (APPs), which are mostly produced by the liver's hepatocytes (Ceciliani et al., 2012) [3] . Any type of illness, swelling or inflammation, and any related trauma or stress can alter APP concentration (Murata et al., 2004) [16] . The blood-milk barrier gets altered during mastitis as a result of increased permeability, allowing plasma proteins, immune cells, and inflammatory mediators to leak into the mammary gland and milk. The presence of APPs in milk suggests that SCC and APP have a self-regulating connection (Wellnitz et al., 2012) [23] . The most sensitive APPs in cattle are amyloid A and haptoglobin (Hp), whose blood concentrations can rise by more than 100 times, especially in reaction to acute inflammation (Eckersall et al., 1999) [4] . Because Hp is produced inside the mammary gland, measuring it in milk could be a useful technique for examining inflammatory activities within the gland as well as an early indicator of mastitis (Hiss et al, 2004) [12] . The objectives of the current study was to evaluate APPs in the milk and serum of Sahiwal cows with SCM and CM, as well as to investigate the seasonal impact, which may serve as useful indicators for the early detection of SCM.

Materials and Methods

At the Livestock Research Centre (LRC) of NDRI in Karnal, Haryana, 73 Sahiwal cows were initially screened for clinical or subclinical mastitis. The modified California Mastitis Test (mCMT) was used to detect subclinical mastitis in the milk of these cows. Clinical symptoms were used to diagnose cows with clinical mastitis. Out of 73 animals, 24 Sahiwal cows were selected, which included 12 clinical and 12 subclinical cases. Blood samples (~10ml) were drawn in sterile heparinised vacutainer from each cow by jugular vein puncture. The plasma was separated by centrifugation at 3000 rpm for 15 minutes. The samples of composite milk (representing all four quarters) were collected from SCM and CM-affected cows. A part of whole milk and skimmed milk (obtained after centrifugation at 3000 rpm for 20 minutes at 4°C) was stored at -20°C for analysis of acute phase proteins. Bacteriological analyses were performed as per the standard procedures. The "Bovine SAA ELISA kit" (catalogue No. E0023Bo) from Bioassay Technology Laboratory was used to measure the amount of SAA in milk and plasma. The "Bovine Hp ELISA kit" (catalogue No. E0022Bo) from Bioassay Technology Laboratory was used to measure haptoglobin in milk and plasma. EMB agar was used to isolate E. coli for microbiological evaluations, and MSA (Mannitol salt agar) was used to isolate Staphylococcus aureus. These products were purchased from Hi-Media and TM Media Company, respectively. The GraphPad Prism software program was used to analyze the data using an unpaired t-test.

Result and Discussion

The findings of microbiological analysis indicated that Staphylococcus aureus is predominantly identified bacteria in the milk samples (Surya et al., 2021) [22] . thermoneutral). The seasonal variations were found to be significant P(<0.05) for plasma SAA but not for plasma Hpt. SAA levels were lowest in thermoneutral conditions (21.60 ± 3.01 µg/ml), slightly higher in winter (26.11 ± 2.47 µg/ml), and much higher in summer (32.14 ± 1.76 µg/ml). Summer values were statistically different from those in thermoneutral and winter groups. Haptoglobin concentrations showed a similar increasing trend, with values of 0.80 ± 0.16 mg/ml under thermoneutral conditions, 0.99 ± 0.23 mg/ml during winter, and the highest concentration (1.14 ± 0.23 mg/ml) during summer. The results unequivocally indicate that acute phase protein levels are influenced by seasonal stress. A higher inflammatory or stress response under heat-stress circumstances is indicated by the notable summertime rise of SAA. The considerably increased SAA levels seen during this season may be explained by the fact that heat stress is known to impair immunological function, increase oxidative stress, and predispose animals to subclinical infections. Lamp et al., 2015 [15] found that increased protein catabolism and changes in food utilization are two metabolic adaptations to heat stress in dairy cows that might cause acute phase reactions and inflammatory pathways. Heat-stressed transition cows, for instance, have elevated plasma indicators of tissue protein breakdown and an adrenergic response that prioritizes the oxidation of carbohydrates over the catabolism of fats, which may lead to systemic inflammation and elevated APP levels. [7] , who discovered mean peak levels of haptoglobin and SAA in healthy calves over the first six weeks in a novel environment. They observed that SAA reacts fast to stimuli and is extremely sensitive. Furthermore, SAA might rise in reaction to nonpathological conditions like stress in addition to illness. (Horadagoda et al., (1999) [13] , Alsemgeest et al., (1995) [2] . Serum Amyloid A (SAA) and haptoglobin (Hpt) levels in plasma showed clear and significant variations across seasons and mastitis status in Sahiwal cows. During the thermoneutral season, SAA concentrations increased from 20.27 ± 6.06 µg/ml in subclinical mastitis (SCM) cows to a significantly higher 22.93 ± 2.09 µg/ml in clinical mastitis (CM) cows, while Hpt values rose significantly from 0.51 ± 0.11 mg/ml (SCM) to 1.09 ± 0.22 mg/ml (CM). In winter, SAA increased from 21.38 ± 3.54 µg/ml in SCM to a markedly higher 30.84 ± 1.08 µg/ml in CM, accompanied by a significant rise in Hpt from 0.49 ± 0.08 mg/ml (SCM) to 1.49 ± 0.28 mg/ml (CM). The summer season showed the highest acute phase protein response, with SAA increasing to 31.31 ± 2.90 µg/ml in SCM and further to a significantly elevated 32.97 ± 2.37 µg/ml in CM cows. Similarly, Hpt levels peaked at 0.64 ± 0.07 mg/ml in SCM and reached a significantly higher 1.64 ± 0.26 mg/ml in CM cows. In the thermoneutral season, SAA levels increased from 0.40 ± 0.02 µg/ml in subclinical mastitis (SCM) cows to a significantly higher 0.53 ± 0.02 µg/ml in clinical mastitis (CM) cows, while Hpt rise sharply from 2.37 ± 0.54 mg/ml (SCM) to 7.26 ± 0.96 mg/ml (CM), as indicated by different superscripts. During winter, SAA increased slightly from 0.43 ± 0.04 µg/ml in SCM to 0.53 ± 0.02 µg/ml in CM cows, reflecting a significant difference, and Hpt showed a similar pattern, rising significantly from 1.83 ± 0.17 mg/ml (SCM) to 8.75 ± 1.48 mg/ml (CM). Summer values showed the highest acute phase response, with SAA increasing from 0.47 ± 0.03 µg/ml in SCM to a significantly elevated 0.59 ± 0.01 µg/ml in CM cows, while Hpt increased from 3.19 ± 0.53 mg/ml (SCM) to a markedly higher 12.22 ± 1.36 mg/ml (CM). SAA is a helpful diagnostic tool for mastitis because it has been demonstrated to rise much earlier in milk than in plasma (Petersen et al., 2004) [17] . In this investigation, we discovered that clinical mastitis cases in Sahiwal cows had much higher plasma and milk Hpt and SAA values than subclinical cases. These findings are consistent with a previous study by Pyorala et al. (2011) [18] , which linked the quantities of Hp and SAA in milk to isolated pathogens in addition to finding differences between and subclinical cases. Eckersall et (2001) [6] showed a strong correlation between the concentrations of haptoglobin in milk and serum, but not between the concentrations of SAA in milk and serum. Overall, SAA and Hp concentrations are higher in clinical mastitis compared to subclinical mastitis. These results align with a prior investigation by Eckersall, P.D. & Young, F.J. (2006) [5] , Hirvonen, J. & Pyörälä, S. (1998) [11] , Safayi et al., (2015) [20] , Grönlund et al., (2003) [8] , Kumar et al., (2011) [14] .

Conclusion

The current study demonstrates that seasonal fluctuations in Sahiwal cows have a significant impact on acute phase proteins, especially Serum Amyloid A (SAA) and Haptoglobin (Hp), which are reliable markers of mastitis severity. SAA and Hp concentrations were consistently much greater in clinical mastitis than in subclinical instances, demonstrating their diagnostic utility in determining the severity of the disease. Additionally, both biomarkers were most elevated during the summer, underscoring the compounding effect of heat stress on the inflammatory response. These results highlight the need to include APP testing in routine mastitis monitoring and imply that better environmental control, particularly during summer months, may help to reduce inflammation and improve udder health.

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