Sensitive and rapid lateral-flow assay for early detection of subclinical mammary infection in dairy cows

Abstract

Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare. We have developed a semiquantitative lateral flow assay for the detection of SCM in dairy cows targeting myeloperoxidase (MPO) enzyme of milk neutrophils. A competitive immunoassay format was used, and colloidal gold nanoparticles (GNP) were prepared and used as a labelling agent. Monoclonal anti-MPO antibodies were used and assessed for its quality by enzymelinked immunosorbent assay and dot blot. Conjugation method for GNP and anti-MPO antibodies was standardised, and the conjugate was placed over the conjugate pad. MPO coupled with a carrier protein (OVA) and the species-specific secondary antibodies were placed on test and control lines, respectively. The developed assay was verified with 75 milk samples collected from healthy, SCM and clinical mastitis cows. It displayed a high sensitivity as it could detect MPO as low as 1.5 ng/ml, an accuracy greater than 97% and showed no crossreactivity when crosschecked with other milk proteins. The developed assay can be used as an alternative for SCM diagnostic tests where lab structure are available for obtaining the lysate of milk SCC.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows

“…However, the best strategy to combat mastitis is its prevention from occurrence. Following some proper management practices in dairy farms, diagnosing mastitis at the earliest and understanding the underlying mechanism of mammary immune response to invading pathogens are some key aspects for prevention ( 21 , 25 , 31 , 32 ). However, mastitis is usually caused by multiple infectious agents, and to fight against the infections, the mammary gland has a line of defense system called SCs, which comprise white blood cells and milk epithelial cells.…”

“…Several diagnostic tests such as CMT, milk SCC, and EC were performed to classify the experimental cows into various groups based on their udder health as described by Alhussien and Dang ( 25 ). For the CMT procedure, 2 mL of milk sample was loaded in the CMT paddle kit and mixed with 2 mL of CMT reagent (DeLaval Pvt.…”

Interaction between stress hormones and phagocytic cells and its effect on the health status of dairy cows: A review

“…Under normal physiological conditions, neutrophils circulate in the blood and survey for microbial-associated molecular patterns. Neutrophils are short-lived and are replaced every 24 h. During the periods of stress, phagocytes get activated and express their secretory and cell membrane activity [ 11 , 34 ]. Many receptors, adhesion molecules, and cytokines are involved in the activation, migration, and fighting ability of phagocytes in response to an inflammatory process ( Figure-1 ).…”

Inkjet Printing-Based Immobilization Method for a Single-Step and Homogeneous Competitive Immunoassay in Microchannel Arrays

“…However, these systems require complicated operational procedures such as the application of an electric field and step-by-step reactions. Although traditional lateral flow assays have undergone a number of improvements in recent years (Nishat et al, 2019 ; Zhan et al, 2019 ; Alhussien and Dang, 2020 ; Lee et al, 2020 ; Wu et al, 2020 ), they tend to limit multiplex detection. Lateral flow assays enabling multiplex detection was reported (Engels et al, 2018 ), however, this assay requires washing step.…”