Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Animal Science
Non Technical Summary
As long as we have known about the existence of bacteria, we believed that certain environments need to be bacteria-free and pristine for them to be functional. The womb of a mammal - where a new life forms - is one such environment. Work done by us and others have shown that the mammalian uterus harbors a rich and permanent collection of bacteria and that they play an important role in mammalian reproduction.In horses, infertility is a significant problem compounded by the fact that horses have a very short "open period" where they need to conceive so they can give birth to a foal every year. It is estimated that 40-60% of mares exhibit some form of inflammation-associated infertility. The traditional practice of veterinary medicine is to treat those animals that show signs of uterine inflammation with broad-spectrum antibiotics.Our lab, in previous studies identified over sixty previously unreported bacteria from uteri of healthy mares. Surprisingly some of those bacteria belonged to families that contained well-known uterine pathogens like Pseudomonas. We propose to individually isolate, culture, and characterize some of the major species of bacteria that inhabit the uterus as a first step towards using these organisms for therapeutic purposes in infertile mares.Up to now, no one has described a similar commensal viral population in the uteri of mammals. We have some indirect evidence that the equine uteri also harbors a native virome. We propose to identify the core virome of the equine uterus.Lastly, we propose to identify the differences in microbiomes between diseased and healthy equine uteri to facilitate more educated therapy for equine endometritis.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The long-term goal of our research is to better understand the role microorganisms play in health and dysbiosis of the equine uterus. The overall objective of this proposal is to characterize the dominant microorganisms that inhabit the uteri of healthy mares and to establish that a resident virome exists in the mammalian uterus. The central hypothesis of the research is that the equine uterus is the home to a thriving community of previously undescribed bacteria and viruses that play a crucial role in the maintenance of the reproductive health of a mare. This hypothesis rests on our preliminary data on microbiome profiles generated from horses across the world and from a few metagenomes generated from the same animals. The rationale that underlies the proposed research is that a comprehensive understanding of the microbial composition of a healthy uterus would help in understanding perturbations associated with dysbiosis and give us tools to mitigate the perturbations.The specific aims of the proposal are;Isolate, culture, and characterize the major resident species of bacteria inhabiting the equine uterus. The working hypothesis for this objective is that the core microbiome of a healthy mare consists of previously uncharacterized organisms belonging to known genera that also consist of established pathogens. Recent advances in culturomic techniques should enable us to cultivate and individually characterize the most predominant bacteria in the uterus.Establish the presence of a native virome in the equine uterus. The working hypothesis is that the equine uterus, like most other environments with a resident microbiome, has its own virome consisting of bacteriophages, etc. A limited metagenomic sequencing effort supports this hypothesis. We plan to methodically analyze the environment for the presence of viruses and partially characterize them.Determine the microbial dysbiosis of the endometriotic equine uterus, and the response of the microbiome to antibiotic therapy. The working hypothesis is that the normal microbiome of the uterus is significantly altered in endometritis and comparison of the diseased to normal microbiomes would facilitate the management and treatment of chronic endometritis.
Project Methods
Specific Aim 1Selection of Animals. Since one of our major interests is in Pseudomonas spp. We would collect small volume lavage samples from mares that were shown to harbor the uncharacterized Pseudomonas species.Bacterial Culture. Lavage samples would be immediately plated on Pseudomonas isolation plates (Fisher Scientific), or Pseudomonas isolation plates +CFC medium. Both sets of plates would be split for incubation under aerobic and anaerobic conditions. Plates would be incubated for 7 days while observing them every day for growth. Any observed cultures would be colony-picked and grown in liquid media.Sequence Analysis DNA would be extracted from the liquid culture of a single colony and the full-length 16S rRNA gene would be PCR amplified and sequenced to verify the identity of the organism."Culturomics"-based isolation. We would prepare 18-different culture media developed for a wide array of culture conditions including anaerobic, fastidious, and slow-growing bacteria and several alcohol conditions whose purpose is to selectively isolate spore-forming bacteria (Lagier et al. 2016; Diakite et al. 2019). Bacterial cultures would be grown both aerobically and anaerobically for 30 days and observed every 3 days. Plates would be observed under a dissecting microscope for micro-colonies known to exist in environment samples (Diakite et al. 2019). Once growth is observed, they would be colony isolated onto the same media and identified using 16s rDNA sequencing.A subset of isolated species would be grown in large-volume liquid culture, DNA extracted, and fully-sequenced (Novogene Inc.) for further characterization.Specific Aim 2Selection of Animals. Small-volume lavage samples would be collected from the same animals used in specific aim 1 for comparison purposes.Removal of eukaryotic and bacterial DNA. Lavage samples would be iteratively filtered through filter paper, 0.45 uM filter, and 0.22 uM filter. Free nucleic acids would be removed by DNAse and RNAse treatment.Viral DNA and RNA isolation viral DNA and RNA would be purified using the Qiagen QIAmp Ultrasense Virus kit and manufacturer's protocol.Viral RNA would be reverse-transcribed to make cDNAWe expect the amount of viral nucleic acids to be extremely small. To obtain sufficient nucleic acids, whole genome amplification would be performed using Syngis True Prime WGA kit (Lucigen).Metagenomic library preparation would be accomplished using NEBNext Ultra II Library preparation kit (#E7645L) and Index primers (#E7500S and #E7335L) (New England Biolabs).Samples would then be diluted to equivalent molarities, pooled and outsourced to be sequenced on an Illumina MiSeq® (Novogene Inc.)Specific Aim 3Small volume lavage samples would be obtained from 30 mares presented to the theriogenology services at the College of Veterinary Medicine 1. At the time of presentation, 2. After the standard antibiotic treatment regime, and 3. After parturition, if they become pregnant or at the next visit to the unit if they were chronically infected.DNA would be extracted, V4 region of the 16S rDNA fragment amplified and all samples would be sequence analyzed to a depth of ~50,000 reads per sample at Novogene Inc. as previously described (Holyoak et al. 2018).Endometrial/antibiotic-treated/chronic/healthy microbiomes would be compared as previously described (Lyman et al. 2019).