Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Veterinary Pathobiology
Non Technical Summary
In the field of equine dentistry, dental extractions are becoming more and more common as equine dental abnormalities and disease are recognized more frequently. As veterinary medicine progresses and horses are used more for pleasure and less for work, they are living longer and needing more care as they age into a geriatric population. Remarkably, significant dental disease is present in up to 75% of horses and the prevalence of dental disease increases with increasing age of the horse. Horses needing dental extraction are being identified by veterinarians commonly and there is little known about the immediate post-operative consequences of tooth removal to the horse's systemic health. In humans and other veterinary species (dogs and cats), dental disease is intimately linked to systemic health. The process of extracting a tooth(exodontia) causes incursion of bacteria into the circulation and may lead to inflammation and infection at remote locations in the body, affecting systemic health. Until recently, demonstrating the presence of bacteria in blood relied on bacteriological culturing, a method that is very limited by poor sensitivity, thus vastly underestimating the true extent of bacterial bacteremic showering. More recently, molecular techniques (PCR-based Next Generation Sequencing) have allowed identification of even uncultivable bacteria in biological media such as blood. Using this approach, we will identify specific bacterial 16S rRNA signatures, a much more highly sensitive technique than conventional bacterial culturing.For the proposed project, horses in need of exodontia will be identified and a venous blood sample will be collected just prior to the start of the procedure and one hour after the termination of the procedure. The gums/gingiva surrounding the afflicted tooth/teeth will also be swabbed just prior to beginning the surgery. Blood and oral swab samples will be analyzed to identify specific (and unique) bacterial 16s rRNA signatures present in the mouth and to characterize the bacterial signature in the blood just prior to and one hour after the completion of exodontia. The ultimate goal of this project is to demonstrate that there is translocation of oral bacteria into the horse's systemic circulation up to one hour post-operatively. Identification and characterization of these bacteria will help to inform antimicrobial drug choices for the perioperative period and increase awareness of the risks and benefits of perioperative antimicrobial usage in equine dentistry.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Long-term goals - Our major long-term goal is to document that bacteremic showering occurs during dental extraction in horses affected with diseased teeth. The secondary goal is to document the extent and the nature of bacterial communities that enter the circulation during routine dental surgical procedures.Specific objectives -Objective 1: Quantify and characterize the bacterial 16S rRNA signature on and in proximity to the affected tooth, in horses affected with dental disease and for which dental extraction will be undertaken.Objective 2: Quantify and characterize the bacterial 16S rRNA signature in blood obtained via aseptic jugular venipuncture immediately prior to and one hour following conclusion of dental extraction.
Project Methods
Study population - The study population will consist of 26 horses presenting to the MU-VHC with dental disease necessitating exodontia; signalment data and details about the outcome of dental examination will be recorded; the reason for exodontia will be summarized; the nature of the exodontia process for each horse will be recorded(commonly encountered justifications for exodontia in horses include: dental fracture, peripheral caries, periapical abscess, equine odontoclastic tooth resorption and hypercementosis syndrome). All procedures will be performed by the same veterinarian (Dr. Alison LaCarrubba, equine dental specialist, co-investigator). All samples will be collected with the owner's permission.Sampling - Following aseptic preparation of the left jugular vein (LJV), 20 mL of blood will be collected into an EDTA tube and stored frozen. A temporary IV catheter will then be placed in the LJV for standing sedation for the dental procedure and all horses will be sedated using a combination of detomidine hydrochloride (Dormosedan® [10 mg/mL]) and butorphanol tartrate (Torbugesic® [10 mg/mL])). Following sedation, an intra-oral speculum will be placed to facilitate access to the oral cavity and the gingiva in proximity to the affected tooth will be swabbed with a sterile cotton tip applicator (Thermo Scientific™ Remel BactiSwab™ Liquid Stuart, Plastic Shaft) and placed in a culture medium. At conclusion of exodontia and after the last tooth is delivered (if >1), a timer will be started for one hour. One hour following surgical conclusion, 20 mL of blood will be aseptically collected into EDTA tubes from the IV catheter. All samples will be placed in the freezer until analysis as a batch.Sample processing (DNA purification) - (swabs, blood) - DNA purification will be undertaken using methodological approaches that have been established and validated in our laboratory (MU Metagenomics Center). Briefly, samples that had been stored frozen at -10°C will be thawed to room temperature (15-25°C) and DNA will be extracted using the QIAmp® PowerFecal® DNA Kit, according to the manufacturer's instructions: 750 μL of blood or the tip of the culture swab will be added to a Dry Bead Tube. 750 μL of PowerBead Solution will be added to the Dry Bead Tube. 60 μL of Solution C1 will be added to each tube and the tubes will each be vortexed briefly. The tubes will then be heated at 65°C for 10 minutes. The tubes will then be secured horizontally using a Vortex Adapter tube holder (cat. No. 13000-V1-24) and vortexed at maximum speed for 10 minutes. Each tube will then beremoved from the Vortex Adapter and centrifuged at 13,000 x g for 1 minute. The supernatant from each tube will be transferred to a clean 2 mL collection tube. 250 μL of Solution C2 will be added to teach tube and the tubes will each be vortexed briefly and then incubated at 2-8°C for 5 minutes. The tubes will then be centrifuged at 13,000 x g for 1 minute. In each tube, the pellet will be avoided and up to 600 μL of supernatant will be transferred to a clean 2 mL collection tube. 200 μL of Solution C3 will be added to each tube and the tubes will be vortexed briefly and incubated at 2-8°C for 5 minutes. The tubes will then be centrifuged at 13,000 x g for 1 minute. From each tube, not more than 750 μL of supernatant will be transferred to a clean 2 mL collection tube. 1,200 μL of Solution C4 will be transferred into each tube with the supernatant and vortexed for 5 seconds. 650 μL of supernatant will be transferred into a MB Spin Column and the tubes will be centrifuged at 13,000 x g for 1 minute. The flow-through from each tube will be discarded and this step will be repeated until all the supernatant has been processed (each sample processed requires a total of three loads). 500 μL of Solution C5 will be added to each tube and the tubes will be centrifuged at 13,000 x g for 1 minute. The flow-through from each tube will be discarded and the tubes will be centrifuged again at 13,000 x g for 1 minute. The MB Spin Column will then be placed carefully into a clean 2 mLollection tube. 100 μL of Solution C6 will be added to the center of the white filter membrane of each spin column. The tubes will be centrifuged at 13,000 x g for 1 minute and the spin filter basket will be discarded. The DNA in each tube will then be ready for downstream processing. Each sample will then be individually mixed with Qubit® dsDNA High Sensitivity Assay Kit solutions and a Qubit® Fluorometer will be used to measure µL/mL of dsDNA in each sample.DNA analysis - 16S rRNA library preparation, sequencing and informatics - Library construction and sequencing will be performed at the University of Missouri DNA Core facility as previously described [22, 23]. Assembly, filtering, binning, and annotation of DNA sequences will be performed at the MU Informatics Research Core Facility as previously reported [23].Statistical analysis - Statistical analysis will be performed using Sigma Plot 12.3 (Systat Software Inc., Carlsbad, CA). Differences between sample collection sites and sample times regarding DNA yield, coverage, richness and relative abundance will be determined using repeated measures ANOVA on ranks with post hoc comparisons performed via Tukey's test. Testing via PERMANOVA, using Past 3.13, will be performed to identify differences in β-diversity between samples sites and sample times. Results will be considered significant for p values <0.05.