Canine babesiosis is a tick-borne disease caused by Babesia spp., which infects and destroys healthy erythrocytes, leading to mortality and morbidity in dogs. The diagnosis of babesiosis is tedious and time-consuming, especially in latent and chronic infections. Here, a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay was developed for rapid and accurate detection of Babesia spp. in canine blood specimens based on the 18S rRNA region. The RPA-LFD assay using rpaBab264 gave specificity to Babesia spp. in dogs (B. vogeli and B. gibsoni) without cross-amplification to other parasites (apicomplexans and non-apicomplexans), with detection limit of at least 22.5 copies/μl (0.1 fg/µl) at 40 °C for at least 10 min. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. To determine the performance of the RPA-LFD assay, a total of 30 clinical samples was examined and compared with conventional PCR (cPCR) and multiplex HRM (mHRM). Eight dogs (26.67%) were detected as positive by RPA-LFD, while seven and six were found positive by cPCR and mHRM, respectively. RPA-LFD and cPCR showed high agreement with Babesia spp. detection with kappa > 0.9. We confirmed that the dogs were infected by B. vogeli from sequences of positive PCR results. Our findings suggested that RPA-LFD using the rpaBab264 assay offered a rapid, accurate, cost-effective and simple method for Babesia spp. detection that is feasibly applicable to be rapid kit at a pet hospital or point-of-care testing.

Microbial production of hydrogen (future ideal fuel and important gas for industries) under anoxic conditions has limited ATP availability and low efficiency. We engineered E. coli K12 to acquire a flavin-based electron bifurcation (FBEB) system, a bioenergetic route typically found in strict anaerobes, which uses NADH to generate low potential reduced ferredoxin and high potential butyryl-CoA. The oxygen-tolerant FBEB-E. coli showed higher H2 and succinate production (2-4 folds), lower cellular reduction potentials, greater accumulation of cellular reductants and various metabolites, including ATP (up to a 7-fold increase). It could better tolerate prolonged and recycled usage of the engineered cell for H2 and succinate production than the native strain. FBEB-E. coli could also use various substrates such as formate, D-glucose and food waste for H2 and succinate production. This is a promising pathway to sustainable H2 and succinate production. This work also demonstrates that E. coli with an extra electron bifurcation system is a robust synthetic biology host.