Uterine Microbiota Progression from Calving until Establishment of Metritis in Dairy Cows

Abstract

The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n ‫؍‬ 72) collected at 0, 2, and 6 ؎ 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ؎ 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ؎ 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as "Candidatus Blochmannia," Escherichia, Sneathia, and Pedobacter. Metritis is a huge concern for the dairy industry worldwide because it is highly prevalent (25 to 40%) and negatively affects the productivity, survival, and welfare of dairy cows (1). Diverse bacteria, including anaerobes and facultative anaerobes, were observed in the uteri of dairy cows within the first 2 weeks postpartum, but they were naturally cleared out within 60 days postpartum (dpp) (1). Culture-based studies observed that Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, and Bacteroides spp. (e.g., Prevotella melaninogenica, formerly Bacteroides melaninogenicus) were commonly associated with endometritis or pyometra (1-3).Although culture-based studies have laid out the foundation of our understanding of the uterine microbiota, previous studies might have underestimated the microbial complexity of the intrauterine environment of cows postpartum, given that less than 1% of the microorganisms in many environments are readily cultured under standard laboratory conditions (4). In recent years, cultureindependent techniques such as clone library sequencing (5, 6) and pyrosequencing (7, 8) have been used to characterize the uterine microbiota of cows with metritis (5-7) and endometritis (7,8). Sequencing using the Illumina platform allows for deeper sequencing than has previously been feasible even with pyrosequencing (9). Indeed, evaluating the rarefaction curves from previous 16S rRNA sequencing studies ...

Uterine Microbiota and Immune Parameters Associated with Fever in Dairy Cows with Metritis

“…Metritis was diagnosed by examining the uterine discharge from 92 cows at 4, 6, and 8 days postpartum (6 ± 2 DPP) using a 5-point scale as previously described [15]: 1 = not fetid normal lochia, viscous, clear, red, or brown; 2 = cloudy mucoid discharge with flecks of pus; 3 = not fetid mucopurulent discharge with < 50% pus; 4 = not fetid mucopurulent discharge with ≥ 50% pus; 5 = fetid red-brownish, watery discharge. Cows with a discharge score ≤ 4 were classified as healthy (n = 40) and cows with a score of 5 were classified as metritic cows.…”

“…Uterine swab samples from 12 cows that developed metritis but did not have a fever at the time of metritis diagnosis (MNoFever) were randomly selected for metagenomic sequencing. The 16S data for metritic cows with a fever (MFever; n = 11) and healthy cows (Healthy; n = 11) were obtained from a previous study [15], and were used as reference groups for comparison with the MNoFever group. In the previous study, 12 samples from MFever and 12 from Healthy cows had been randomly selected; however, one sample from each group failed to amplify.…”

“…In cows with mastitis, infections with Gram-negative pathogens have also been shown to be more severe [14]. We recently observed that the uterine microbiota of metritic cows with a fever was dominated by Gram-negative bacteria such as Bacteroidetes and Fusobacteria [15]; therefore, the uterine microbiota could influence the development of fever.…”

“…Impairment of neutrophil function was observed in metritic cows starting 1 week before calving, and continuing until 3 weeks after calving [18]. Thus, the immunosuppression of pre- and post- partum dairy cows may be a determinant for the development of metritis given that all cows are naturally infected with bacteria during or shortly after parturition but not all cows develop disease [15]. Indeed, a previous study reported a decrease in the neutrophil function around the time of calving in cows that developed metritis [17–19].…”

Caviibacter abscessus gen. nov., sp. nov., a member of the family Leptotrichiaceae isolated from guinea pigs (Cavia porcellus)

“…More recently, it was shown that species of the genus Sneathia have the potential to cause infections outside the reproductive tract due to their involvement in a case of septic arthritis (Bachy et al, 2011). Sequences associated with the genus Sneathia have also occasionally been reported as genital microbiota in cows and macaques (Hu et al, 2015;Jeon et al, 2015;Machado et al, 2012;Spear et al, 2012). Different growth characteristics, as well as preliminary molecular data, cast doubt over whether respective micro-organisms from guinea pigs are indeed affiliated to the genera Streptobacillus or Sneathia.…”