Investigating Bovine Blood Prokaryotic Microbial Populations Through 16S V4 Sequencing, qPCR, and dPCR, with a Specific Focus on Hemotrophic Mycoplasma wenyonii

Microbiomes have become an increasingly important field of study in the past decade, with data supporting microbial roles in disease control, metabolic efficiency, and more. Microbial DNA is detectable outside the digestive tract, including in blood. Bloodborne pathogens such as hemotrophic Mycoplasma are endemic in cattle. Hemoplasmas are associated with reduced male fertility and decreased milk production in dairy cattle, but their impact on beef cattle remains unclear. Strain variability, such as between the Massachusetts and INFAP01 strains of Mycoplasma wenyonii, may complicate detection. Coinfection with multiple species likely contributes to disease progression from latent to acute infection. To assess microbial DNA in blood and quantify erythrocytic M. wenyonii, blood was collected from 120 beef cattle in Erath County, Texas: 61 cows, 55 calves, and 4 bulls. DNA was extracted and used to prepare 16S rRNA V4 libraries and perform PCR. After rarefaction, ASVs were analyzed and separated into four groups: adult females (n = 61), adult males (n = 4), juvenile males (n = 27), and juvenile females (n = 28). Statistical analysis revealed differences in Actinobacteria by sex (p < 0.001) and higher Bartonella and Mycoplasma abundances in adults (p < 0.001). PCR revealed that M. wenyonii infection was more frequent in adult females (p = 0.006), suggesting age-related variation in infection.