Feasibility of 16S rRNA gene qPCR for rapid detection of pathogenic bacteria in bovine cerebrospinal fluid

Bacterial meningoencephalitis (BME) in cattle is responsible for economic losses due to mortality, neurological impairment, and decreased productivity. The use of cerebrospinal fluid (CSF) bacterial culture-the gold standard for antemortem bacterial detection-is limited by its lengthy processing times and potential false negatives. With this study we wanted to determine whether 16S rRNA gene real-time quantitative PCR (16S rRNA qPCR) of DNA extracted from CSF could be used to detect BME in cattle. We applied a modified low-biomass microbial investigation protocol to extract CSF DNA from 15 healthy cattle and 14 cattle diagnosed with BME. Environmental, DNA extraction, and PCR controls were performed. Bacteria were detected by culture and/or direct smear microscopy in 4 of the 14 BME cases, in 3 of which 16S rRNA qPCR yielded positive results. Overall, there was no statistically significant difference between the bacterial load measured via 16S rRNA qPCR in the BME cases and that of the healthy controls and the DNA extraction controls. While 16S rRNA qPCR demonstrated its ability to detect bacterial DNA in the CSF samples with high microbial load, it was unable to reliably distinguish BME cases from healthy controls when bacterial load was low. The question remains whether this limitation is due to the absence of bacteria in the CSF-despite underlying infection-or whether a low bacterial load is masked by background contamination. More comprehensive sequencing approaches may provide an answer.