Knudsen, Lif Rødtness Vesterby

Postpartum uterine sygdomme er et problem i malkekvægsbesætninger. Omtrent 90% af alle malkekøer bliver kontaminerede af bakterier i uterus efter kælvning. De fleste køer kommer selv af med infektionen indenfor otte uger i involutionsprocessen, men op mod 20% of alle køer udvikler metritis, hvor hele uterusvæggen er inficeret; og i nogle besætninger udvikler 30%-50% af køerne endometritis, som er infektion i det inderste lag af uterus. Pyometra er en relateret postpartum uterin sygdom, som forekommer når en ko med endometritis får ægløsning, og cervix lukkes om infektionen. Sygdommene er negativt korrelerede med reproduktions ydelsen, og dette i tillæg til den høje prævalens gør sygdommene dyre for landmændene. Traditionelle dyrkningsmetoder til identifikation af bakterier er tilbøjelige til at indføre bias mod bakterier, der trives i et laboratoriemiljø. I dette projekt blev den bakterielle flora undersøgt ved molekylære mikrobiologiske metoder, primært 16S rRNA PCR og næste generations sekventering. Studiet inkluderer skylleprøver fra uterus lumen, såvel som endometrielle biopsier, for at evaluere korrelationen mellem de bakterier der observeres i de uterine skylleprøver, som hyppigst anvendes i feltet, og de bakterier som adhærer til det mucosale lag i uterus, endometriet. For at undersøge ekspressionsmønstret af bakterier tilstede i en uterus med endometritis blev et metatranskriptom studie udført. Denne metode er baseret på mRNA sekventering, og giver et øjebliksbillede af ekspressionsprofilen fra bakterier på prøve udtagnings tidspunktet. Tidligere studier af virulensfaktorer i postpartum uterine sygdomme har været baseret på kvantitativ PCR, hvilket kræver forhåndsviden om gensekvens.Der blev fundet en signifikant association mellem Fusobacteriaceae og Porphyromonadaceae familierne og metritis i uge 1 postpartum. For endometritis i uge 4 og 7 var der ingen bakterielle familier der var signifikant associeret med sygdommen konsistent over prøvetidspunkter og prøvetyper. Der var store forskelle mellem de uterine skylleprøver og de endometrielle biopsier, og selvom prøvetyperne var korrelerede, så var diversiteten af mikrobiotaen i uterus højere i biopsierne end i skylleprøverne.Den mest observerede bakterielle familie i uterus fra køer med pyometra var Fusobacteriaceae familien, som indeholder Fusobacterium necrophorum; en kendt patogen, der allerede er associeret med pyometra. Derimod støtter vores data ikke sammenhængen mellem pyometra og Trueperella pyogenes, det andet tidligere identificerede patogen. De uidentificerede Gram-negative bakterier som tidligere studier har observeret i pyometra, tilhører formentlig de Gram-negative familier Porphyromonadaceae, Pasteurellaceae og Mycoplasmataceae, som er identificeret i dette studie.Det blev observeret at de 50 mest opregulerede transkripter i uterus mikrobiotaen fra køer med metritis of endometritis primært var involveret i DNA replikation, transkription, translation og metaboliske processer. Dette indikerer en aktiv multiplikationsfase i infektionen, og en tilpasning til værtens miljø. Derudover blev en opregulering observeret i gener, der potentielt er involverede i syntese af LPS, lipid A, hæmagglutinin samt flere gener, der koder for proteaser. Disse gener er mulige virulens gener. Størstedelen af de mest differentielt udtrykte transkripter havde størst similaritet med proteiner fra F. necrophorum og P. levii arterne. Dette indikerer at disse arter er de mest metabolisk aktive i uteri fra køerne med metritis og endometritis, og at de måske er de primære patogener i uterine sygdomme. Transkripter fra andre arter blev også obseveret, blandt andet fra Mycoplasma bovigenitalium.Resultaterne i denne afhandling understreger diversiteten i den bovine mikrobiota i postpartum uterus. F. necrophorum og P. levii blev identificeret som de mest vigitge patogener i uterine sygdomme, men en association med andre bakterie arter synes sandsynligt, muligvis indenfor familierne Mycoplasmataceae, Pasteurellaceae, Ruminococcaceae, Bacteroidaceae og Leptotrichiaceae. Den rolle i uterine sygdomme, som ved tidligere studier, der benyttede traditionelle dyrkningsmetoder, er blevet tilskrevet Escherichia coli og T. pyogenes kunne ikke understøttes af data i denne afhandling.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

Online Research Database In Technology

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

http://orbit.dtu.dk/ws/files/119955402/PhD_Thesis_Lif_Knudsen.pdf

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

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