Development of Loop-Mediated Isothermal Amplification for the Detection of Prototheca bovis Directly from Milk Samples of Dairy Cattle.

Abstract

Prototheca bovis is an algal emerging pathogen in dairy farms causing refractory protothecal mastitis with increasing incidence worldwide and significant economic impact. P. bovis infects cows throughout the lactation cycle, including dry periods, and can persist in the udder and the environment for a long time. Since P. bovis does not respond to treatments with antibiotics, the suggested sanitary measure to restrict the spread is culling infected animals. A point-of-care test for early detection of the causative agent is critically needed to guide farm management and the appropriate treatment of mastitis. Loop-mediated isothermal amplification (LAMP) is a highly specific molecular method, time-saving, cost-effective and easy to perform in limited settings. This study aimed to develop a LAMP assay for P. bovis detection directly from milk samples; it was employed in conjunction with a commercial DNA extraction kit which was previously used to extract DNA from milk specimens containing microbes. The LAMP assay detected P. bovis DNA within 1h in milk samples spiked with P. bovis at a concentration of 50 cells/μL, enabling on-farm disease monitoring and decision-making based on a reliable diagnosis. The LAMP method will contribute to the accurate and rapid identification of P. bovis in asymptomatic or recurrent mastitis cases and consequently aid the implementation of targeted control measures and the reduction of losses in milk production.