Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Large Animal Clinical Sciences
Non Technical Summary
Metritis is an acute inflammatory disease of multiple layers of the uterine lining with systemic implications that affects ~ 20% of postpartum dairy cows and has marked welfare, health, production, reproduction, and economic consequences to the individual animal and the herd. The dairy cow is unique in the sense that virtually all cows are colonized by bacteria in the first days after calving, and failure to eliminate pathogenic bacteria leads to the establishment of disease. Our previous work showed that the microbiota is identical between cows that develop metritis and healthy cows up until 2 days postpartum, after which the bacterial number and structure deviate in favor of greater abundance of pathogenic gram-negative anaerobic bacteria in metritic cows, particularly Fusobacterium, Bacteroides and Porphyromonas. Interestingly, we saw that a sub-community of uterine pathogens that included Fusobacterium, Bacteroides and Porphyromonas was present in blood shortly after calving, which indicate that a hematogenous route of uterine infection was possible in cows. Therefore, uterine pathogens could be transferred to the uterus shortly after calving or even before calving. Indeed, a recent study has identified a uterine microbiome in the placenta and amniotic fluid of pregnant cows, and another group of researcher were able to visualize important bacteria such as Fusobacterium necrophorum and Porphyromonas levii within the endometrium of pregnant cows using fluorescence in situ hybridization (FISH). Meanwhile, our group has shown that thethe first stool of calves had a microbiome, and that the abundance of Bacteroidaceae and Prevotellaceae, two known families of uterine pathogens, in calves' blood was positively correlated with abundance of these bacteria in cows' blood. These findings beg the question: If bacteria can colonize the amniotic fluid of the dam and the gastrointestinal (GI) tract of the fetus, and can be found in the fetal circulation, could it also colonize the uterus of the fetus? If the answer is yes, we would like to study possible routes of bacterial translocation from the dam to the fetal uterus. If the answer is no, then we would like to know when in the life of a calf/heifer does the uterus become colonized with bacteria. We would also like to determine the progression of the uterine microbiome from fetal life to the pregnant state. This knowledge would give us a better understanding of the uterine microbiota and uterine disease etiology and could completely change the way we approach uterine infection in cows, from prevention of uterine infection to prevention of uterine microbiome dysbiosis. Our long-term goal is to develop solutions for improving animal health and welfare. Herein, we will investigate the uterine microbiome from fetal life until pregnancy establishment with the overall goal of determining when and how a uterine microbiome is established and determining the progression of the uterine microbiome until the first pregnancy. Additionally, we will investigate the establishment and progression of a microbiome in other major organs such as the GI tract, lungs and the mammary gland. Our central hypothesis is that bacteria from the dam colonize the GI tract of the fetus and then translocate to the uterus of the fetus or that bacteria from the dam translocate to the blood circulation of the calf and then colonize the uterus of the fetus. Alternatively, a calf would be born without a uterine microbiome but would acquire a microbiome before the establishment of its first pregnancy. Our rationale for this proposal is that developing an understanding of the origin and progression of the uterine microbiome will provide insight on uterine disease etiology and allow for the development uterine microbiome modulation strategies. Although our project is focused on the uterine microbiome, we will also develop an understanding of the microbiome of major organs such as the GI tract, lungs and the mammary gland from fetal life until adulthood, which will provide insight into the etiology of GI disease, pneumonia and mastitis, the major diseases of dairy calves and cows. This is expected to improve animal health, welfare, and fertility which is expected to enhance farm profitability and sustainability. Furthermore, reducing disease burden will decrease antibiotic use, which is expected to delay the development of bacterial resistance to antibiotics used in human medicine, which will improve public sentiment towards animal agriculture which will ultimately improve the sustainability of the dairy industry.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Our long-term goal is to develop solutions for improving animal health and welfare. Herein, we will investigate the uterine microbiome from fetal life until pregnancy establishment with the overall goal of determining when and how a uterine microbiome is established and determining the progression of the uterine microbiome until the first pregnancy.We propose the following specific aims:Aim 1) To determine if the uterine microbiome is established during fetal life, and to determine the progression of the uterine microbiome from fetal life to the pregnant state. To determine if the fetus has an established uterine microbiome, we will collect the fetuses from primigravid Holstein heifers slaughtered at 90 (n=6) and 260 d of gestation (n=6) and multigravid cows at 260 d of gestation (n=6). We will collect the fetal uterus to characterize the uterine microbiota using 16S rRNA gene sequencing, quantitative real-time PCR (qPCR), bacterial culture, and bacteria visualization using FISH. Samples of meconium, small and large intestines will be collected and expected to have an established microbiome. Lungs are also expected to have an established microbiome because of fetal breathing of amniotic fluid. Blood, liver, heart, and the mammary gland of the fetus will also be sampled and evaluated for the presence of a resident microbiota. To determine the progression of the uterine microbiome, we will collect the uteri from heifers slaughtered at 6 months of age (prepubertal; n=6), virgin heifers at 12 months of age (pubertal; n=6), and bred but open heifers at 16-17 months of age (n=6). The uteri collected from pregnant heifers (90 and 260 d pregnant) and cows (260 d pregnant) will also contribute to our understanding of the progression of the uterine microbiome during pregnancy. The uterine microbiota will be characterized using 16S rRNA gene sequencing, qPCR, bacterial culture, and FISH. Blood, fecal, and vaginal samples from heifers and cows will also be collected for comparison.Aim 2) To investigate possible routes of bacterial translocation from the dam to the fetal uterus. Bacteria from the dam could colonize the fetal GI tract then translocate to the fetal uterus or bacteria from the dam could translocate to the blood circulation of the fetus then colonize the fetal uterus. To investigate these routes, we will collect samples from several tissues including blood from heifers/cows and fetuses, placentomes, intercaruncular endometrium, intercotyledonary plancenta, umbilical cord, and allantoic and amniotic fluid. The fornix, cervix and feces of heifers/cows will also be sampled for comparison. If the uterine microbiome is established after birth, then a hematogenous or ascending uterine colonization are possible. Blood mononuclear (PBMC) and polymorphonuclear cells (PMN) and plasma will be separated and investigated as possible routes of transmission of bacteria from the heifers/cows to the fetuses. All tissues will be used for microbiota characterization using 16S rRNA gene sequencing, qPCR, bacterial culture, and FISH. To establish the likely source of the uterine microbiome we will use the SourceTracker software in QIIME2 and Network analysis. We will also use 16S rRNA gene sequencing data to predict possible genes involved in bacterial translocation to the uterus such as adhesion and invasion of cells, and immune cells evasion and hijacking mechanisms using PICRUSt2. Moreover, tissues showing evidence of bacterial translocation will be used for duo in situ hybridization and immunofluorescence (ISH-IF) using RNAscopeTM to visualize bacteria within monocytes/macrophages to test whether bacteria translocate using the Trojan horse mechanism.
Project Methods
Animals:Cows and heifers pregnant with a female calf will be used in this project.Blood Sample Collection and Processing: A blood sample will be collected before slaughtering. Blood will be collected from the jugular vein following surgical preparation of the collection site, and will be used for separation of PBMC, PMN and plasma . PBMC and PMN will be used for molecular microbiology and bacterial culture.Slaughtering: Cows and heifers will be transportedthe USDA certified slaughterhouse in the department of Animal Sciences at the University of Florida for slaughtering. Slaughter will be performed using a non-penetrative stunner to achieve immediate insensibility, followed by exsanguination.Heifer and Cow Tissue Sampling: After slaughter, the reproductive tract of pregnant cows and heifers will be tied at the mid-portion of the vagina, severed, and transported to a sterile laminar flow hood. Then, the uterine surface will be wiped with 70% ethanol and flame sterilized. At this point, study personnel will wear surgical gloves. For pregnant cows and heifers, the uterus will be opened using sterile surgical instruments with care not to puncture the chorioallantoic membrane. A sample of the allantoic fluid (~ 5 mL) will be collected using a sterile syringe and needle and transferred to a sterile cryogenic vial. Then the chorioallantoic membrane will be opened with care not to puncture the amniotic membrane. A sample of the amniotic fluid (~ 5 mL) will be collected using a sterile syringe and needle and transferred to a sterile cryogenic vial. The allantoic and amniotic fluids will be swabbed using ESwabsfor bacterial culture, then the fluids will be divided in four 1 mL aliquots and stored at -80 oC for molecular microbiology. Full thickness uterine specimens will be taken from the placentomes, intercotyledonary plancenta, and intercaruncular uterine wall at the major curvature of the pregnant horn. Full thickness uterine specimens will be taken from the major curvature of both uterine horns in non-pregnant animals. Separate specimens will be placed in 1.5 mL sterile tubes and stored at -80 oC for molecular microbiology, in anaerobic transport medium for bacterial culture, and in 10% neutral buffered formalin for microscopy. Swab samples from the fornix and cervix will then be collected for molecular microbiology using a Dacron swaband for bacterial culture using an ESwabs swab from all animals. Tissue or swab samples from the mesenteric lymph nodes, lungs, liver, mammary gland, and the GI tract will be collected for molecular microbiology and bacterial culture from all animals.Fetal Tissue Sampling: Samples from the fetuses will be collected before sampling of the vagina and cervix. Fetuses will be opened by a midline incision and full thickness uterine specimens will be collected for molecular microbiology and bacterial culture as previously described. Samples of blood, umbilical cord, lungs, liver, heart, the mammary gland, GI tract, and meconium (at 260 days of gestation only) will be collected for molecular microbiology, histology and bacterial culture as described before.Bacterial culture: ESwabs and tissues collected for bacterial culture will be plated on blood and chocolate agar and incubated at 37°C under oxic, hypoxic (5% CO2, 5% O2), and anoxic (5% CO2, 10% H, 85% N) atmospheres. Tissue samples will be homogenized using a sterilized Wheaton glass tissue grinder. All samples will be additionally plated on MacConkey agar, and also added to SP4 broth with urea and SP4 broth with arginine and incubated at 37°C under an oxic atmosphere. All samples will be cultured in duplicate under all growth conditions (medium type × atmosphere) and incubated for 7 days. The PBS stock used for tissue homogenization will be used as negative controls. E. coli, F. necrophorum, P. levii and B. pyogenes from our laboratorywill be used as positive controls.Histopathology: Samples of uterine and placental specimens will be trimmed, fixed in 4% paraformaldehyde buffered solution for 24 h, dehydrated and embedded in paraffin wax. Sections of 4 μm, mounted on SuperFrost Plus slides (Menzel-Gläser, Braunschweig, Germany) and stained with hematoxylin and eosin (H&E). Specimens will be blindly evaluated by a single pathologist and graded for presence of inflammation. Degree of inflammation will be correlated with bacterial abundance observed in tissue Gram-stained slides.Fluorescence in situ hybridization: FISH will be performed in all specimens using a bacterial probe targeting an overall bacterial domain (EUB338) and species-specific probes targeting F. necrophorum, P. levii and B. pyogenes. Briefly, tissue sections will be deparaffinized and rehydrated for 2 × 3 min in xylene, 2 × 3 min in 99% ethanol and then hybridized with a mixture of 99 μL in situ hybridization buffer (1 M Tris (pH 7.2), 5 M NaCl, 10% SDS, H2O) and 1 μL probe. The slides will be incubated at 46 °C for 16-24 h. After hybridization, the sections will be washed three times in prewarmed (45 °C) hybridization buffer for 9 min and subsequently washed three times in prewarmed (45 °C) washing solution (10 μL of 1 M Tris [pH 7.2], 18 μL of 5 M NaCl, 72 μLl of H2O). The sections will be rinsed in water, air dried, and mounted in Vectashield for epifluorescence microscopy. The probes will be 5′labeled with either FITC or Cy3 fluorophores.Microbiome characterization: DNA will be extracted from swabs, tissues, and controls (i.e., sterile Dacron swabs and blank DNA extraction kits) using the DNeasy PowerLyzer PowerSoil kitfollowing tissue homogenization using a bead beater. Then, the V4 region of the 16S rRNA gene will be amplified by PCR with dual-index primers and the amplicons will be sequenced using the Illumina platform (MiSeq v2, 2 × 250 cycle cartridge). Metagenomic DNA will also be used to quantify total bacterial load, F. necrophorum, P. levii, and B. pyogenes using qPCR.Blood Sample Processing for ISH-IF: The PBMC and PMN cell pellets will be fixed in 4% paraformaldehyde buffered solution for 24h, snap-frozen in Tissue-Tek OCT embedding compound and sectioned for FISH-IF protocol to investigate the Trojan horse mechanism.Samples from Heifers and Cows for ISH-IF: Samples collected fromcows and heifers showing evidence of bacterial colonization in Aim1, will be used to track the source of bacterial translocation using ISH-IF. Tissue samples or cell pellets will be fixed in 4% paraformaldehyde buffered solution for 24 h, snap-frozen in Tissue-Tek OCT embedding compound and stored at -20°C until use.Fetal Samples for ISH-IF: Samples showing evidence of bacterial colonization using molecular microbiology techniques will be used for investigation of translocation to the uterus using ISH-IF. Tissue and cell pellet samples will be fixed in 4% paraformaldehyde buffered solution for 24 h, snap-frozen in Tissue-Tek OCT embedding compound and stored at -20°C until use.Duo ISH-IF for bacterial DNA (ISH) and CD14 and CD68 (IF): The duo ISH-IF will be performed in all specimens in which the presence of bacteria was identified by either qPCR or microbiome analysis. The whole section will be analyzed to identify colocalization of bacteria and monocytes/macrophages by ISH-IF to test whether the Trojan horse mechanism is used for bacterial translocation from the mother to the fetus. The Dual RNAscopeTM ISH-IF will be performed using the RNAscope Multiplex Fluorescent V2 assay (Advanced Cell Diagnostics).Data Management, Bioinformatics, and Biostatistics: We will use PCoA, discriminant analysis, network analysis, pathway analysis, and source tracking analysis to: 1 - Determine if the fetus has an established microbiome in the uterus and other major organs; 2 - Determine the progression of the microbiome in the uterus and other major organs from fetal life to the pregnant state.