Understanding acute phase proteins: Key players in E. coli and S. aureus mastitis in Sahiwal and Karan fries cows

Introduction

A group of proteins known as "acute phase proteins" show changes in their blood plasma levels during inflammatory reactions; some of these proteins increase (referred to as positive acute phase proteins) while others decrease (referred to as negative acute phase proteins). The term "acute phase response" describes a collection of nonspecific host reactions to different stimuli that are typified by variations in the concentrations of certain acute phase proteins, which are plasma proteins produced by the liver (Baumann and Gauldie, 1994) [2] . The degree of tissue damage or infection is closely correlated with the amount of APP in the blood (Murata et al., 2004) [11] . It is crucial for preserving or regaining homeostasis and preventing microbial growth in animals prior to the host producing antibodies following trauma, inflammation, or infection (Murata et al., 2004) [11] . The importance of haptoglobin and serum amyloid A in the diagnosis and prognosis of mastitis, endometritis, enteritis, peritonitis, pneumonia, and endocarditis was emphasized by Eckersall and Bell (2010). Additionally, the infected mammary gland secretes SAA and Hp (Eckersall et al., 2006) [5] . Eckersall and Bell (2006) [5] demonstrated and cited APP in serum and milk samples as potential markers of mastitis under both natural and experimental circumstances. Their low concentration in healthy animals, their quick rise during the acute phase of inflammation, and their quick fall as the illness resolves make them potentially helpful as disease markers (Gruys et al., 1994) [10] . The two most significant acute phase proteins in cattle are serum amyloid A and haptoglobin, which are either absent or produced in relatively small amounts in healthy cows but in large quantities in bacterial or viral diseases (Murata et al., 2004) [11] . Although these biomarkers lack specificity, they are very sensitive indicators of inflammation. [12] and Gronlund et al. (2005) [8] investigated APP concentrations in milk with various infections. The quantities of Hp were highest in mastitis produced by A. pyogenes, and SAA was highest in mastitis induced by E. coli. The concentrations of APPs were low in cases of CNS-induced mastitis. In general, the streptococcal groupings had higher APP concentrations than the S. aureus group.

Materials and Methods

Initial screening of infected cows for clinical or subclinical mastitis was carried out on 92 KF and 73 Sahiwal cows at the Livestock Research Centre (LRC) of NDRI in Karnal, Haryana. The milk of these cows was tested by modified California Mastitis Test (mCMT) for identification of subclinical mastitis. Cows suffering from clinical mastitis were diagnosed on the basis of clinical symptoms. Out of 165 animals, we randomly selected 24 Sahiwal and 24 KF 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) were stored at -20 °C for analysis of acute phase proteins. Bacteriological analyses were performed as per the standard procedures. Plasma and milk SAA was determined by "Bovine SAA ELISA kit" (catalogue No: E0023Bo) obtained from Bioassay Technology Laboratory. Plasma and milk Haptoglobin was determined by "Bovine Hp ELISA kit" (Catalogue No E0022Bo) obtained from Bioassay Technology Laboratory. For microbiological assessments, EMB agar was utilized for the isolation of E. coli, while MSA (Mannitol salt agar) was used for isolating Staphylococcus aureus, with both products obtained from Hi-Media and TM Media Company, respectively. Data were analysed by unpaired t-test utilizing the GraphPad Prism software package.

Results and Discussion

The findings of microbiological analysis indicated that two types of bacteria were predominantly identified in the milk samples of both breeds: Staphylococcus aureus and Escherichia coli (Surya et al., 2021) [19] . A prevalent type of bovine mastitis in the world is staphylococcal mastitis, which is typically brought on by Staphylococcus aureus. Staphylococcus aureus was found to be responsible for around 90% of new udder infections, including both clinical and subclinical mastitis.

Distribution of mastitogens in NDRI farm

Staphylococcus aureus was identified by Fox and Gay (1993) [6] as a primary cause of infectious cow mastitis. Sentitula et al. (2012) [16] discovered that 43.3% of mastitic milk samples from the NDRI herd tested positive for Staphylococcus utilizing biochemical and PCR-based techniques. The aforementioned table clearly shows that cows infected with E. coli had numerically larger levels of acute phase proteins in both milk and plasma. Coliform mastitis is the most dangerous type of mastitis, according to the levels. One of the main causes of acute mastitis in dairy cows is E. coli (Suriyasathaporn et al., 2000) [18] . The findings of Suriyasathaporn et al. (2000) [18] and Pyorala and Taponen (2009) [13] are consistent with our data. Hpt and SAA levels differed according to the type of pathogen in the current study. [20] . The mean peak concentrations of Hp and SAA in the milk increased in a S. aureus subclinical mastitis, peaking with a mean of 115 mg/l and 540 mg/l, respectively (Eckersall et al., 2006) [5] . In experimental mastitis caused by E. coli, mean peak concentrations of Hpt and SAA in the milk were as high as 600 mg/l and 1315 mg/l, respectively (Suojala et al., 2008) [17] .

Variations in plasma and

Conclusion

The current study shows that in both Sahiwal and Karan Fries cows, acute phase proteins specifically, Serum Amyloid A (SAA) and Haptoglobin (HP) serve as extremely sensitive markers of mastitis severity and pathogen type. Compared to Staphylococcus aureus, Escherichia coli infections produced noticeably larger amounts of these biomarkers in milk and plasma, supporting the more severe inflammatory response linked to coliform mastitis. The results are consistent with previous studies that demonstrate Gram-negative pathogens elicit a more potent acute-phase response than Gram-positive organisms. The distinct differences in APP levels among pathogens demonstrate their usefulness in identifying clinical severity, facilitating early diagnosis, tracking the course of disease, and directing more focused therapeutic measures. Therefore, by enabling prompt and pathogen-specific responses to mastitis outbreaks, integrating APP profiling into routine mastitis surveillance may improve herd health management.

Conflict of Interest: Not available

Financial Support: Not available

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