Clinical vs. Subclinical mastitis in Sahiwal Cows: The role of season and pathogens in metabolic disruption

The present study investigated the metabolic alterations associated with intramammary infections (IMI) caused by Staphylococcus aureus and Escherichia coli in early-lactating Sahiwal cows, with emphasis on β-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA) in plasma and milk. A total of 24 mastitis-affected cows (12 clinical, 12 subclinical) were selected after screening 73 animals using mCMT and clinical examination. Results showed that cows infected with E. coli exhibited significantly higher (P

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R Q -hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA) in plasma and milk. A total of 24 mastitis-affected cows (12 clinical, 12 subclinical) were selected after screening 73 animals using mCM T and clinical examination. Results showed that cows infected with E. coli exhibited significantly higher (P<0.05) plasma and milk BHBA and NEFA concentrations compared to S. aureus. Seasonal variations were also significant (P<0.05), with both plasma and milk BHBA and NEFA reaching their highest levels during summer, indicating pronounced metabolic stress under heat load. Clinical mastitis cows demonstrated markedly elevated (P<0.05) BHBA and NEFA values in both plasma and milk across all seasons compared to subclinical cases, revealing greater lipid mobilization and impaired energy balance in severe infections. The study overall underscores the joint impact of mastitogen type, the severity of the disease, and seasonal heat stress on metabolic dysregulation in Sahiwal cows. It highlights the importance of BHBA and NEFA as potential biomarkers for evaluating the severity of mastitis and metabolic stress.

K eywor ds: Sahiwal, M astitis, Season, M etabolic parameters-NEFA , BHBA

I ntr oduction

During the initial phase of lactation, dairy cows experience a negative energy balance to satisfy the substantial demands for milk production, resulting in the rapid mobilization of fat and protein (M acrae et al., 2019) [17] . During this period, the cow transitions from a nonlactating state to the beginning of abundant colostrum and milk synthesis, resulting in major nutritional, physiological, metabolic, and immunological changes (Sundrum, 2015) [27] . Cows must adapt metabolically to the significant increase in nutritional needs for high milk production and the subsequent lactation's return of reproductive function (Bisinotto et al., 2012) [1] . Inadequate intake of dry matter, at the beginning of lactation, results in insufficient adaptation and metabolism regulation (Fiore et al., 2018) [3] . High-yielding dairy cows face a significant metabolic challenge that causes drastic changes in their metabolic profiles. These changes can be used to assess the health of cows (M acrae et al., 2006) [18] and identify risk factors for metabolic and periparturient clinical diseases (Hammon et al., 2006; M cA rt et al., 2013) [7,19] , out of which mastitis is the costliest disease in the dairy industry. Certain periparturient illnesses disrupt metabolic processes, leading to changes that may be interpreted as metabolic markers. The concentration of these metabolites changes in a diseased state, revealing details about the pathophysiology of the illness (Shin et al., 2015) [26] . The majority of research has concentrated on the impact of metabolic status on the immune response in dairy cows (M oyes et al., 2009; Zarrin et al., 2014) [21,34] . During mastitis, the immunometabolic responses mainly concentrate on the transcription-level reactions in both liver and mammary tissues (Loor et [16,21,31] . Prior research suggests that the liver's capacity to metabolize fatty acids diminishes, and essential genes linked to metabolic functions are down-regulated following an intramammary E. coli challenge (Jorgensen et al., 2012) [11] , as well as after an intramammary endotoxin challenge (Jiang et al., 2008) [9] .

A dditionally, alterations in circulating non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA ), and glucose have been observed before the reductions in feed intake and milk production during an intramammary infection (IM I) in dairy cows (Graugnard et al., 2013; Waldron et al., 2006; Waldron et al., 2003) [4,32] The objective of this study was to characterize the changes in various metabolic parameters (NEFA and BHBA)in blood and milk during IM I with E. coli and S. aureus in Sahiwal cows during early lactation.

M ater ials and methods

A t the Livestock Research Centre (LRC) of NDRI located in K arnal, Haryana, a total of 73 Sahiwal cows were initially assessed for either clinical or subclinical mastitis. The modified California M astitis Test (mCM T) was employed to identify subclinical mastitis in the milk of these cows. Clinical symptoms were utilized to diagnose cows suffering from clinical mastitis. From the 73 animals, 24 Sahiwal were chosen, comprising 12 clinical and 12 subclinical cases. Blood samples (approximately 10ml) were collected in sterile heparinised vacutainers from each cow via jugular vein puncture. The plasma was isolated through centrifugation at 3000 rpm for 15 minutes. Composite milk samples (representing all four quarters) were gathered from cows affected by SCM and CM . A portion of whole milk and skimmed milk (obtained after centrifugation at 3000 rpm for 20 minutes at 4°C) was preserved at -20°C for the analysis of metabolic profiles. Bacteriological examinations were conducted following standard procedures. EM B agar was utilized to isolate E. coli for microbiological assessments, while M SA (M annitol salt agar) was employed to isolate Staphylococcus aureus. These products were procured from Hi-M edia and TM M edia Company, respectively. The copper soap solvent extraction method modified by Shipe et al., (1980) was adopted for the

-HBA in samples was estimated by ‡ -Hydroxy butyrate (K etone Body) A ssay K it -• (catalogue No:201-04-0182) obtained from Sun Red Laboratory. The GraphPad Prism software program was used to analyze the data using an unpaired t-test.

Result and Discussion

M icrobiological analysis indicated that Staphylococcus aureus was the bacterium most commonly isolated from the milk samples that were examined (Surya et al., 2021) [30] . In Sahiwal cows, both Staphylococcus aureus and Escherichia coli mastitogens had an impact on circulating

BHBA ) and non-esterified fatty acids (NEFA ). Cows that were infected with E. coli exhibited significantly higher plasma BHBA levels (0.62 ± 0.08 mM /L) and plasma NEFA levels (478.59 ± 102.31 µM /L) compared to those infected with S. aureus, which showed levels of 0.51 ± 0.05 mM /L and 413.32 ± 25.16 µM /L, respectively. A similar pattern was noted in milk, where E. coli mastitis led to increased BHBA (0.59 ± 0.03 mM /L) and NEFA (277.25 ± 39.33 µM /L) levels in comparison to S. aureus infections, which recorded 0.45 ± 0.02 mM /L and 223.60 ± 13.31 µM /L, respectively. Elevated BHBA levels in milk and serum increase the risk of mastitis in dairy cows (Suriyasathaporn et al., 2000) [28] . Grinberg et al., (2008) [5] reported that BHBA reduces neutrophil phagocytosis and NET formation, weakening bactericidal activity against E. coli. These negative effects on immune function help explain the higher susceptibility of ketotic cows to mastitis. K ornalijnslijper et al., (2003) [13] further noted that severe E. coli mastitis was associated with -OH-butyrate levels above 1.4 mmol/L. Cows with E. coli mastitis show significantly higher serum NEFA levels (Jirillo et al., 2002; Katoh, 2002) [10,12] , and Hagiwara et al., (2014) found that non-survivors of acute E. coli mastitis had the highest NEFA concentrations. This indicates that NEFA levels rise with infection severity. Increased NEFA and BHBA, along with reduced glucose, characterize severe negative energy balance. Such metabolic stress makes cows more susceptible to diseases like mastitis (M oyes et al., 2009; Perkins et al., 2002; Suriyasathaporn et al., 1999) [21,25,29] . V alues with different superscripts for different seasons in a row differ at 5% level. Significant seasonal variations were observed in plasma metabolic parameters of mastitisinfected Sahiwal cows. Plasma BHBA levels exhibited a progressive increase from the thermoneutral season (0.45 ± 0.06 mM /L) to winter (0.53 ± 0.07 mM /L), with the peak value observed in summer (0.60 ± 0.08 mM /L), indicating a notable rise during periods of heat stress. Similarly, plasma NEFA concentrations reached their highest levels in summer (523.47 ± 48.77 µM /L), significantly surpassing those recorded during the thermoneutral (384.56 ± 29.80 µM /L) and winter seasons (364.56 ± 37.21 µM /L). The metabolic indicators of milk exhibited a similar pattern. The concentration of BHBA in milk experienced a slight seasonal rise, increasing from 0.42 ± 0.03 mM /L during the thermoneutral season to 0.49 ± 0.04 mM /L in winter and reaching 0.50 ± 0.03 mM /L in summer. A dditionally, the levels of NEFA in milk peaked in summer at 277.89 ± 27.41 µM /L, which was significantly greater than the values recorded during the thermoneutral period (200.94 ± 15.39 µM /L) and winter (218.80 ± 15.92 µM /L). Demonstrated that the levels of BHBA and NEFA fluctuated based on breed, season, and lactation stage, with elevated values observed in winter. Conversely, K umar et al., (2018) and Gulab Chandra et al., (2012) [14,2] reported that NEFA and BHBA levels increased during the summer months in both indigenous and K F cows. These findings indicate that metabolic responses vary among different breeds and in response to environmental stressors. Our results are consistent with observations in indigenous cattle, revealing significantly higher levels of NEFA and BHBA during the summer in mastitis-affected Sahiwal cows. [20] reported significantly elevated (P

-HBA in cows with mastitis compared to healthy cows during the early postpartum period, which supports our findings. The variations in BHBA levels were also observed in the milk of infected cows. Clinical cases of mastitis exhibited significantly higher (P<0.01) BHBA levels when compared to subclinical mastitis cases in both Sahiwal cows. Oltenacu and Ekesbo (1994) [24] indicated that there was no variation in milk BHBA at the onset of mastitis; however, subsequent research revealed a more significant correlation. M oyes et al., (2009) [21] discovered that increased BHBA levels during the transition phase heightened the likelihood of mastitis occurring in early lactation. Comprehensive data indicated

greater probability of developing mastitis within a fourweek period. A dditionally, M oyes et al., (2009) [21] affirmed that elevated BHBA levels make cows more susceptible to clinical mastitis during calving and early lactation.

Conclusion

This study shows that NEFA and BHBA are useful metabolic indicators for evaluating the severity of mastitis, the impact of pathogens, and seasonal stress in Sahiwal cows. M etabolite levels were considerably higher in E. coli infections than in S. aureus infections, suggesting more metabolic disturbance. NEFA and BHBA concentrations in milk and plasma were significantly higher (P<0.05) in clinical mastitis than in subclinical instances. The effects of heat stress and negative energy balance were highlighted by these metabolic changes, which were most noticeable in the summer. In general, monitoring these metabolites can help detect mastitis in dairy herds earlier and manage it better.

Refer ences

Refer ences

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Season	Thermo neutral	Winter	Summer
BHBA (mM/L)	0.45A ±0.06	0.53A±0.07	0.60B ±0.08
NEFA (µM/L)	384.56X ±29.80	364.56X ±37.21	523.47Y ±48.77

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Season	Thermo neutral	Winter	Summer
BHBA (mM/L)	0.42 ±0.03	0.49 ±0.04	0.50 ±0.03
NEFA (µM/L)	200.94 X ±15.39	218.80 X ±15.92	277.89 Y ±27.41

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Breed	Mastitogen	n	Plasma	—	Milk	—
			BHBA (mM/L)	NEFA (µM/L)	BHBA (mM/L)	NEFA (µM/L)
Sahiwal	S. aureus	20	0.51 ±0.05	413.32 ±25.16	0.45 ±0.02	223.60 ±13.31
	E.coli	4	0.62 ±0.08	478.59 ±102.31	0.59 ±0.03	277.25 ±39.33

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Variations in plasma and milk Acute Phase Proteins of

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Season	Treatment	BHBA (mM/L)	NEFA(µM/L)
Thermoneutral	SCM	0.32 a ±0.02	378.65±48.20
	CM	0.57bA ±0.08	390.48±42.40
Winter	SCM	0.36 c ±0.05	324.28±39.35
	CM	0.70 d ±0.06	404.83±61.89
Summer	SCM	0.39 e ±0.03	458.87±41.83
	CM	0.81fB ±0.05	588.07±81.04

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Plasma metabolic parameters in Sahiwal cows suffering

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Season	Treatment	BHBA (mM/L)	NEFA(µM/L)
Thermoneutral	SCM	0.32 a±0.06	165.17 r±5.99
	CM	0.48 b±0.04	236.71sA±14.71
Winter	SCM	0.40 c±0.03	179.38 u±4.25
	CM	0.58 d±0.03	258.21 v±11.35
Summer	SCM	0.39 e±0.03	208.12 x±8.85
	CM	0.47 f±0.03	347.67yB±13.45

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Variations in milk metabolic parameters in Sahiwal cows

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