BACKGROUND
Bovine mastitis is a major inflammatory disease of dairy cattle with important economic and one-health implications. This study focused on whether serum immunologic and oxidative biomarkers could help identify clinical and subclinical mastitis earlier, while also characterizing the bacterial causes and antimicrobial resistance patterns. The investigators note that clinical mastitis shows obvious udder and milk abnormalities, whereas subclinical mastitis can be difficult to detect without laboratory testing. They specifically evaluated acute-phase proteins (APPs), cytokines, and oxidative stress markers in relation to bacteriologically confirmed disease.
METHODS
The study was conducted in January 2021 and included 100 Holstein Friesian dairy cows from two farms in Gamasa city, Dakahlia Governorate, Egypt. Cows were divided into healthy controls (n = 20), clinical mastitis (n = 30), and subclinical mastitis (n = 50). Ages ranged from 4 to 7 years. Cows had not received medication in the week before sampling. Clinical mastitis was diagnosed by physical signs such as udder inflammation, systemic illness, or abnormal milk. Subclinical mastitis was diagnosed using the California mastitis test and somatic cell count, with cows considered affected if they had positive CMT or SCC > 200,000 cells/mL without clinical signs.
Milk and blood samples were obtained from all 100 cows. Milk underwent bacteriologic culture, and samples containing more than two bacterial species were considered contaminated and discarded. Suspected Staphylococcus aureus isolates were confirmed by PCR amplification of the nuc gene, and suspected Escherichia coli isolates were confirmed by PCR targeting the 16S-rRNA gene. Antimicrobial susceptibility testing used disc diffusion according to CLSI. The investigators tested 21 E. coli isolates against 12 antimicrobial compounds and 38 S. aureus isolates against 10 antimicrobial agents. Multidrug resistance (MDR) was defined as resistance to more than two antibiotics from different antimicrobial classes, and the paper also describes this as resistant to ≥3 antimicrobial class. Hematologic, biochemical, APP, cytokine, and oxidative stress markers were measured in serum or plasma using established assays. Statistical analysis used SPSS Version 21, with normally distributed data reported as means and standard mean of error, and significance set at p < 0.05.
KEY RESULTS
Among mastitic milk samples, E. coli was identified in 70% (21/30) of clinical mastitis samples, while S. aureus was identified in 76% (38/50) of subclinical mastitis samples. Across all mastitic samples, the bacterial agent was detected in 73.75% (59/80). All 20 healthy milk samples were culture negative.
Antimicrobial resistance was striking. E. coli isolates were resistant to penicillin, cefuroxime, cefoperazone, azithromycin, nalidixic acid, trimethoprim-sulfamethoxazole, rifamycin, and gentamycin. They showed intermediate resistance to neomycin (61.9%), amikacin (52.4%), and chloramphenicol (47.6%), and were more sensitive to streptomycin (38.1%). MDR was detected in 100% of tested E. coli isolates. For S. aureus, resistance was highest to ampicillin, oxacillin, and gentamycin at 76.3%, 76.3%, and 73.7%, respectively. Intermediate resistance was reported for ceftazidime (52.6%), kanamycin (47.4%), and streptomycin (50%). S. aureus was more sensitive to norfloxacin (34.2%), ciprofloxacin (36.8%), chloramphenicol (26.3%), and tetracycline (23.7%). MDR was detected in 94.74% of S. aureus isolates, and only two strains were sensitive to all antimicrobials tested.
Hematologic changes were directionally consistent with systemic inflammation and illness. Compared with controls and subclinical mastitis cows, clinical mastitis cows had significantly lower RBCs count (p < 0.01), Hb (p < 0.001), and PCV (p < 0.01). WBCs (p < 0.01) and neutrophil count (p < 0.001) were significantly diminished in clinical and subclinical mastitic cows compared to controls. Exact mean values were not clearly reported in the provided text.
Biochemically, both mastitic and subclinical mastitic cows had significantly higher AST (p < 0.001, p < 0.01), LDH (p < 0.001, p < 0.01), total protein (p < 0.001, p < 0.05), and globulin (p < 0.001, p < 0.05) than controls. Albumin declined only in mastitic cows (p < 0.05).
APPs were elevated in affected cows. Haptoglobin (p < 0.001), fibrinogen (p < 0.01), and amyloid A (p < 0.001, p < 0.01) were significantly increased in both mastitic and subclinical mastitic cows versus controls. Ceruloplasmin increased in mastitic cows (p < 0.05), while ferritin showed no significant differences.
Inflammatory cytokines also shifted toward a pro-inflammatory pattern. TNF-α (p < 0.001), IL-1β (p < 0.001, p < 0.01), and IL-6 (p < 0.001, p < 0.01) were increased in mastitic and subclinical mastitic cows relative to healthy controls. IL-10 decreased only in mastitic cows (p < 0.01).
Oxidative stress findings supported increased reactive oxygen species activity in disease. MDA was higher in mastitic cases (p < 0.001), while TAC (p < 0.05, p < 0.001) and catalase (p < 0.01, p < 0.001) were reduced in mastitic and subclinical mastitic cows compared with controls. SOD decreased only in mastitic cows (p < 0.05).
CLINICAL IMPLICATIONS
This study suggests that bovine mastitis, including subclinical disease, is associated with coordinated changes in APPs, cytokines, and oxidative stress markers that may complement traditional tests such as CMT, SCC, and culture. The most clinically relevant candidate biomarkers from the study are haptoglobin, fibrinogen, amyloid A, TNF-α, IL-1β, IL-6, MDA, TAC, and catalase, because these changed significantly in both mastitic and subclinical mastitic cows. The findings also underscore a major antimicrobial stewardship issue, since MDR occurred in 100% of E. coli and 94.74% of S. aureus isolates. Although this was an observational single-region study and exact biomarker means were not clearly reported in the provided text, the results support using immunologic and oxidative biomarkers as early adjunctive indicators of mastitis and highlight the need for surveillance of resistant pathogens in dairy herds.