New insights into subclinical mastitis in buffaloes: B-mode and Doppler ultrasonography, molecular expression and clinicopathological correlates.

BACKGROUND

Subclinical mastitis in buffaloes is a major hidden constraint to milk quality and herd productivity, as it develops without obvious clinical signs and frequently escapes early diagnosis. The vascular and molecular alterations accompanying early disease remain poorly characterized.

OBJECTIVES

The objective of this study was to provide a comprehensive assessment of subclinical mastitis in buffaloes by integrating ultrasonographic imaging (B-mode and Doppler), inflammatory gene expression (TLR4, IL-6, and SAA), and clinicopathological parameters, aiming to enhance early diagnosis, clarify disease pathogenesis, and support effective mastitis control strategies in buffalo dairy systems.

METHODS

Buffaloes with subclinical mastitis were investigated alongside healthy and clinically mastitic animals. Leukogram profiles, serum biochemical indicators of hepatic and renal function, and mineral-electrolyte levels (Ca, P, Na, and K) were evaluated. Mammary gland structure and blood flow were examined using B-mode ultrasonography and pulsed-wave Doppler imaging. Relative gene expression of Toll-like receptor 4 (TL4), interleukin-6 (IL-6), and serum amyloid A (SAA) was analyzed to assess innate immune activation.

RESULTS

Subclinically mastitic buffaloes showed significantly increased somatic cell counts without obvious inflammatory signs. Ultrasonography revealed increased parenchymal echogenicity, mild ductal enlargement, and early loss of tissue homogeneity. Doppler analysis demonstrated altered mammary blood flow, characterized by increased resistive and pulsatility indices and reduced mean velocity. Leukogram, biochemical, and electrolyte analyses indicated mild systemic disturbances, particularly hypocalcemia and sodium-potassium imbalance. Marked upregulation of innate immune and acute-phase genes confirmed early inflammatory activation.

CONCLUSION

Integrating ultrasonography with clinicopathological and molecular biomarkers provided a sensitive strategy for early identification of subclinical mastitis in buffaloes, enabled timely intervention, improved udder health, and enhanced productivity.