ABSTRACT
Bovine mastitis is primarily caused by a group of bacteria known as Staphylococcus and Streptococcus. However, additional types of bacteria, such as bovine non-aureus staphylococci and mammaliicocci (NASM) as well as lactic acid bacteria (LAB), are considered minor pathogens and have less impact on cows. Modulating bovine neutrophil activities and gene expressions in response to bacterial stimuli prompted the cells to execute effector functions to combat udder infections. Although neutrophils can manage major mastitis-causing bacteria, this strategy has not been tested against minor pathogens, i.e. NASM, Weissella spp. Our main objective was to investigate how neutrophils interacted with major and minor pathogens during in vitro bacterial stimulation. The results reveal that neutrophils performed offensive duties regardless of the type of bacteria encountered. Neutrophils generated high levels of reactive oxygen species, efficiently phagocytosed both types of bacteria, and facilitated extracellular killing by releasing NET structures against all bacteria. In addition, neutrophils migrated preferentially towards the majors rather than the minors, although myeloperoxidase (MPO) degranulation did not differ substantially across bacteria. Furthermore, the killing capacity of neutrophils was not dependent on any particular bacterium. The correlation of effector functions is intimately linked to the up-regulation of genes associated with the above functions, except for IL6, which was down-regulated. Furthermore, neutrophil apoptosis can be modulated by altering apoptosis-associated genes in response to harmful stimuli. These findings provide valuable information on how neutrophils react to major and minor mastitis-causing bacteria. However, future research should explore the interplay between minor pathogens and the host's responses.
ABSTRACT
Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.
