Reference values for gut microbiota parameters in rhesus macaques established by real-time polymerase chain reaction

Introduction. Reference values for the gut microbiota are essential for a valid assessment of the health status of laboratory primates and for monitoring their keeping conditions. The obtained ranges of gut microbiota parameters are species-specific for rhesus macaques and cannot be directly extrapolated to humans. The results highlight the need to use age-specific norms for key taxa when interpreting the state of the microbiota in biomedical research. Aim. To establish age-specific reference ranges for key taxa of the gut microbiota in rhesus macaques, detected by real-time PCR with the Kolonoflor-16 kit. Materials and methods. The study utilized 120 fecal samples from clinically healthy rhesus macaques (Macaca mulatta) kept at the Kurchatov Complex of Medical Primatology. DNA extraction from fecal samples was performed using the Express-DNA-Bio kit (Alkor Bio) following a thermal lysis protocol. Quantitative analysis of 24 microbiological markers was conducted by real-time PCR using the Kolonoflor-16 kit (AlfaLab) on a CFX-96 thermocycler (Bio-Rad). Results. Comprehensive age-specific reference ranges for the gut microbiota of rhesus macaques were established. Statistically significant ontogenetic trends reflecting consistent processes of microbial-host coadaptation were identified: a progressive decrease in the abundance of Lactobacillus spp. and Escherichia coli against an increase in Bacteroides spp. The stability of the total bacterial mass and the level of Faecalibacterium prausnitzii as a key butyrate producer throughout life was demonstrated. A pronounced age-related dynamics was observed for taxa that are commensals of the rhesus macaques gut. The detection rate of Akkermansia muciniphila increased from complete absence in infants to stabilization within species-typical limits in adults and elderly individuals, reflecting the maturation of the immune system and mucosal barrier. Enterococcus spp. was detected in all age groups and demonstrates variability in its quantitative values. The presence of Fusobacterium nucleatum in the studied sample is not associated with pathological conditions and is a species norm, not a marker of dysbiosis. Conclusion. The data obtained using the real-time PCR method (KOLONOFLOR-16 kit) prove the inapplicability of human reference values for assessing the microbiota of monkeys and emphasize the need to use species- and age-specific criteria in scientific research.