Characterizing the prevalence of <i>Fusobacterium necrophorum</i> subsp. <i>necrophorum, Fusobacterium necrophorum</i> subsp. <i>funduliforme,</i> and <i>Fusobacterium varium</i> in bovine and ovine semen, bovine gut, and vagino-uterine and fetal mic

<i>Fusobacterium necrophorum</i> is an important pathogen associated with several infectious diseases in cattle. However, recent sequencing-based studies reported that <i>F. necrophorum</i> may be positively associated with pregnancy in beef cows and that <i>Fusobacterium</i> is highly abundant in bull seminal microbiota with potential involvement in reproductive health and fertility. Here, we performed a comprehensive screening to (i) determine the prevalence of <i>Fusobacterium necrophorum</i> (subspecies <i>necrophorum</i> [FNN] and <i>funduliforme</i> [FNF]) and <i>Fusobacterium varium</i> (FV) in the reproductive microbiota of cattle and sheep as well as bovine digestive tract ecosystems, and (ii) explore whether these <i>Fusobacterium</i> spp. colonize calf prenatally. For this, we screened 11 different sample types including bovine and ram semen, bovine vaginal and uterine swabs, and bull fecal samples, as well as samples from 180- and 260-day-old calf fetuses and their respective dams using both quantitative PCR (qPCR; 514 samples) and targeted culturing (499 samples). By qPCR, all the targeted <i>Fusobacterium</i> spp. were detected across all sample types with varying prevalence rates and viability. FNF was highly prevalent in the bull semen (66.7%) and maternal ruminal fluids (87.1%), and its viability was confirmed through culturing. All the targeted <i>Fusobacterium</i> spp. were identified in vaginal and uterine swab samples (3.1%-9.4%), caruncles, fetal fluids, rumen, and meconium samples (2.7%-26.3%) by qPCR but were not isolated by culture method. Overall, our results, for the first time, suggest that <i>F. necrophorum</i> is a commensal member of healthy male reproductive microbiota, and that FNF, FNN, and FV are present in bovine vagino-uterine microbiota and calf intestine prenatally.IMPORTANCERecent sequencing-based studies suggest that <i>Fusobacterium,</i> including <i>F. necrophorum</i>, a known primary etiological agent for several important infectious diseases in cattle, may be non-pathogenic members of the reproductive microbiota with pro-fertility effects. However, further information regarding the absolute abundance, viability, and higher taxonomic resolution of the <i>Fusobacterium</i> species and subspecies which cannot be achievable by the amplicon sequencing approach is needed to confirm the commensal and non-pathogenic status of the <i>Fusobacterium</i> spp. in cattle. Here, we performed a comprehensive screening of <i>F. necrophorum</i> subspecies <i>necrophorum</i>, <i>F. necrophorum</i> subspecies <i>funduliforme</i>, and <i>Fusobacterium varium</i> from over 500 samples from 11 different sample types using targeted culturing and qPCR. Overall, our results provide novel insights into the prevalence and viability of <i>Fusobacterium</i> spp. in bovine male and female reproductive tracts and their presence in calf fetuses, which will serve as the basis for further research into understanding the role of <i>Fusobacterium</i> in cattle fertility.