Molecular characterisation of the uterine microbiome of dairy cows suffering from endometritis, metritis, and pyometra

Postpartum uterine disease is a problem in dairy herds. Approximately 90% of dairy cows experience postpartum bacterial contamination of the uterus. Most of the cows are able to clear the infection within 8 weeks in the process of involution, but up to 20% of the cows develop metritis, which is infection throughout the uterine wall; and in some herds, 30-50% of cows develop endometritis, which is infection in the inner lining of the uterus. Pyometra is a related postpartum uterine disease, which is thought to occur when a cow with endometritis ovulates, and the cervix closes. The diseases are negatively correlated to reproductive performance, and in combination with the high incidence rate, they are costly for the farmers. Traditional culturebased studies are biased towards bacteria that thrive in a laboratory environment. In this project the bacterial flora were investigated by molecular microbiology methods, primarily 16S rRNA PCR and next generation sequencing.
The study included uterine flush samples from the lumen as well as endometrial samples, to evaluate the correlation between the uterine flush samples, which are commonly used sample type in the area, and the bacteria found adhering to the mucosal layer of the uterus, the endometrium. It was hypothesised that pathogenic bacteria in the uterus initially adhere to the endometrium to cause disease, and that the chance of identifying pathogens is higher in examinations of endometrial biopsies than in uterine flush samples. In order to investigate the expression patterns of the bacteria in the endometritic uterus, a metatransgenomic study was performed. This method is based on mRNA sequencing, and provides a snapshot of the expression profile of the bacteria at the time of sampling. Previous studies of virulence factors have been performed with quantitative PCR, which requires prior knowledge of gene sequence.
It was found that there was an association between the Fusobacteriaceae and Porphyromonadaceae families and metritis in week 1 postpartum. For endometritis in weeks 4 and 7, there was not a bacterial family consistently associated with the disease across time points and sample types. There were large differences between the uterine flush samples and the endometrial biopsies, and although the sample types were correlated, the diversity of the microbiota in the biopsy samples was higher than the diversity of the microbiota of the uterine flush samples. Furthermore, the bacterial families that made out the majority of the population were the same over time.
The most abundant family observed in cows with pyometra was the Fusobacteriaceae family, which contain F. necrophorum, a pathogen previously known to be associated with pyometra, whereas evidence of the association of T. pyogenes with pyometra was less convincing. The previously unidentified Gram-negative bacteria observed in other studies of pyometra are likely to belong to the Porphyromonadaceae, Pasteurellaceae, and Mycoplasmataceae families identified.
It was found that the 50 most up-regulated transcripts of the microbiota from the uterus of cows with metritis and endometritis were primarily involved in DNA replication, transcription, translation, and metabolic processes. This indicates an active multiplication phase in the infection, and an adaption to the host environment. Furthermore, an up-regulation was observed of genes potentially involved in the synthesis of LPS, lipid A, haemagglutinin, and several genes that code for proteases. These genes are putative virulence genes. The majority of the most differentially expressed transcripts mapped most closely to proteins from the F. necrophorum and P. levii species. This indicates that these species were the most metabolically active in the uterus of the cows with uterine disease, and that these may be the primary pathogens of uterine disease. Transcripts from other species were also observed to be highly expressed in the uterus of cows with uterine disease, among others from M. bovigenitalium.
The results in this thesis underline the high number of bacteria found in the bovine postpartum uterus. F. necrophorum and P. levii, were observed to be the most important pathogens in uterine disease, but the association of other bacterial species, perhaps contained within the Mycoplasmataceae, Pasteurellaceae, Ruminociccaceae, Bacteroidaceae, Leptotrichiaceae families, and an uncharacterised family that belong to class Bacteroidia seems likely. The data presented in this thesis does not support a role of the E. coli and T. pyogenes species, that have been identified as possible pathogens with traditional culture methods.