Fabrication and Use of Paper-Based Tests for Diagnosis of Bacterial Pathogens of Veterinary Importance

Abstract

Bacterial pathogens cause variety of diseases in livestock and are becoming serious global health concern in veterinary science. Staphylococcus aureus and Escherichia coli are the most prevalent veterinary bacterial pathogens that are involved in different diseases in animals like mastitis, gasteroenteritis, skin infections etc. These diseases are usually treated with antibiotics, with β-lactams the most commonly used antibiotic in veterinary medicine. However, their frequent use in livestock lead to the emergence of β-lactam resistant bacteria, which cause difficulties for treatment of diseases in both animals and humans. Detection of pathogens as well as their antibiotic sensitivity is pre-requisite for successful treatment and this is generally achieved with laboratory-based techniques such as growth inhibition assays, enzyme-linked immunosorbent assays (ELISA) or polymerase chain reaction (PCR), which are unavailable in resource-limited areas. Microfluidics Paper-based analytical devices (μPADs) were investigated for the presumptive detection of S. aureus, E. coli and their antibiotic resistant bacterial strains in raw milk samples. The μPADs were fabricated on Whatman filter paper using wax printing. Initially efforts were made to develop paper-based immunoassays for detection of S. aureus using different strategies which proved to be unsuccessful in the present study. Then, μPADs impregnated with chromogenic substrates were used, which reacted with bacterial enzyme to produce coloured products. Initially, assay on μPAD was optimized using pure cultures and inoculated milk samples with S. aureus, E. coli and their antibiotic resistant strains. Limits of detection of S. aureus, E. coli and their antibiotic resistant strains in milk samples were found to be 106 cfu mL-1. Enrichment of the milk samples in selective medium for 12 h enabled detection to as low as 10 cfu mL-1. After successful optimization, paper-based devices were tested on a set of 1040 raw milk samples collected from dairy animals and milk vendors in Pakistan which demonstrated more than 90% sensitivity and 100% specificity compared to PCR; suggesting the promise to provide inexpensive and portable diagnostic solutions for pathogenic bacteria in resource-limited settings.