PSLBI-12 Screening and isolation of <i>Fusobacterium necrophorum</i> and <i>Fusobacterium varium</i> from seminal, and vagino-uterine microbiota of healthy cattle and sheep

Abstract Fusobacteria necrophorum is an important opportunistic bacterial pathogen associated with liver abscesses, foot rot, necrotic laryngitis and endometritis in both dairy and beef cattle. Recently, we identified F. necrophorum as one of the taxa in the uterine microbiota that are positively associated with pregnancy outcome in beef cows, and that Fusobacterium was identified as the most dominant genus in the bovine seminal microbiota. These findings indicate that Fusobacterium spp. may have a role in reproductive health and cattle fertility. Our objectives were to detect and isolate the F. necrophorum subsp necrophorum (FNN), F. necrophorum subsp. funduliforme (FNF) and F. varium (FV) in seminal, vaginal and uterine microbial ecosystems as well as fetal samples by culture and real-time PCR (qPCR) methods. The qPCR was performed on the DNA (n = 215) extracted from bull semen (n = 37), ram semen (n = 100), and vaginal (n = 25) and uterine swabs (n = 32), caruncles (n = 9), allantoic fluid (n = 6), and amniotic fluid (n = 6) from pregnant beef cows. The qPCR targeted the hgdA gene, and leukotoxin promoter regions (lktA-n and lktA-f) to detect and quantify FV, FNN and FNF, respectively. Cryopreserved samples (n = 174; 57 bull semen, 10 ram semen, 38 vaginal, and 47 uterine swabs) were cultured to detect and isolate FNN, FBF and FV by direct plating and after culturing in anaerobic peptone-yeast extract broth containing sodium lactate broth with josamycin, vancomycin and norfloxacin for selective enrichment of FN and FV. Also, 28 samples of 6-mo-old bovine fetuses caruncles, allantoic and amniotic fluids, and intestinal tissue) were screened for Fusobacterium species. By qPCR, FNF was detected in 75.7% of bull semen, and 8.0% of vaginal swabs as well as 33.3% of both allantoic fluid and caruncles, but was undetected in ram semen, uterine swabs, and amniotic fluids. Subspecies necrophorum was identified in 37.8% of bull semen, 3% of ram semen, 8.0% of vaginal and 15.6% of uterine 55.6 % of caruncles, 50% of allantoic fluid samples, but was not detected in amniotic fluid samples. Meanwhile FV was only detected in vaginal, uterine swabs and caruncles at 4%, 6.3%, and 11.1%, respectively. The culture method detected FNF in 63.2% of bull semen and 5.3% of vaginal samples, while FNN was identified in 1.8% of bull semen and 2.1% of uterine samples. F. varium was not isolated from any of the samples. Overall, FNF was the most prevalent subspecies present in the seminal microbiome of bulls. Identification of FNN subspecies and FV in seminal, vaginal, and uterine microbiota of healthy cattle, as well as in healthy calf fetus-associated samples suggests potential transfer from bulls to cows and then on to offspring, and potential involvement of the Fusobacterium spp. in cattle fertility.