trace minerals and the intrauterine microbiology 2012.pdf · endometritis • inflammation of the...
TRANSCRIPT
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New insights into the bacterial pathogenesis of Uterine Infections
Rodrigo Bicalho DVM, PhD
Assistant Professor of Dairy Production Medicine
Diseases of the post-partum uterus
• Metritis
Perimetritis
• Clinical endometritis
• Subclinical endometritis
MetritisAbnormally enlarged uterus and a fetid watery red-brown uterine discharge, associated with signs of systemic illness
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Endometritis
• Inflammation of the uterus without systemic illness (Sheldon et al., 2006)
Only the endometrium is affected
• Characterized by the presence of purulent uterine discharge detectable in the vagina 21 days or more after parturition, or mucopurulentdischarge detectable in the vagina after 26 days post partum
Endometritis
Subclinical endometritis
• Subclinical inflammation of the endometrium
• Diagnosis is made by cytological examination of uterine lavage
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Subclinical endometritis
Disease triangle
The host; dairy cow
10-20 kg colostrum, then 30-40 kg/d
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The environment
(Hammon et al., 2006)
Killing ability is associated with EB and cows that develop
Uterine Dz have greater NEB___ Healthy
----- Metritis
….. SCE
Immunosuppression in cows that develop uterine Dz
Loss of neutrophil killing ability :
(a): Myeloperoxidase activity (b): Cytochrome c reduction
(Hammon et al., 2006)
___ Healthy
----- Metritis
….. SCE
Week Around Calving
-6 -4 -2 0 2 4 6
Imm
nu
ne F
un
ctio
n, %
Co
ntr
ols
20
40
60
80
100
120
140
160
PMN functionSMN function
(Kehrli et al., 1989)
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Links with Immunology; Retained placenta
Retention of placenta is a major risk factor for metritis and is caused by a decrease in chemotaxis of neutrophils to the site of placental attachment (cotyledons).
Kimura et al., 2002
• Common bacterial isolated from clinical cases:
E. coli
A. pyogenes
Fusobacterium necrophorum
Prevotela melaninogenica
Bacteroids
The pathogens
Escherichia coli and bovine uterine infection
• The role of E. coli in the pathogenesis of metritis and endometritis is poorly understood (Silva et al., 2009)
• Recently three different research group have partially characterized different collections of E. coli:
1. Genomic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of puerperal dairy cows. J. Dairy Sci. 92:6000-6010
2. Specific strains of Escherichia coli are pathogenic for the endometrium of cattle and cause pelvic inflammatory disease in cattle and mice. PLoS One. 5:e9192.
3. Molecular and epidemiological characterization of bovine intrauterine Escherichia coli. J. Dairy Sci. 93: 5818-5830
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• Characterization of 35 isolates from 12 healthy cows and 37 isolates from 18 metritic cows.
• 15 VFs were screened for and only 4 were detected (hlyE, hlyA, iuc, and eaeA)
• None of the evaluated E.coli characteristics were significantly related to the establishment of the uterine infection “This corroborates the putative role of the
bacterium in the pathogenesis of the puerperal uterine infection of the cow”
• Evaluated the effect of 17 VF genes and none were found to be associated with uterine disease
• Clonal groups of E. coli associated with metritis were most adherent to endometrial cells
• Clonal groups of E. coli associated with metritis were most invasive for endometrial cells
• Concluded that endometrial pathogenic E. coliwere expressing the fimH gene because Mannose treatment of E. coli isolates decreased their ability to adhere to endometrial cells
• Uterine swabs were performed by the research team (5 veterinarians) a total of 374 cows (200 cows in farm A, 70 in farm B, 63 in farm C, and 41 in farm D).
• DIM at sampling ranged from 2 to 7.
• Uterine swabs were collected as follows: cows were restrained and the perineum area was cleansed and disinfected with a 70% ethyl alcohol solution.
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Molecular and epidemiological characterization of bovine intrauterine
E.coli
Multiplex PCRs
• cdt PCR using 4 primer pairs was performed to detect E. coli isolates having sequences common to cdtB genes, as described by (Toth et al., 2003).
• 16-plex PCR genes related to diarrheagenic E. coli; eaeA, escV, ent, bfpB, EHEC-hly, stx1, stx2, ipaH, invE, astA, aggR, pic, elt, estIa, estIb, and uidA as described (Antikainen et al., 2009).
• 15-plex PCR VF associated with UPEC; papAH, PapG (allele I), PapG (allele II), PapG (allele III), fimH, afa/draBC, sfa/focDE, hlyA, cnf1, iutA, fyuA, kpsMII, traT, ibeA, malX, PAI] as described (Moreno et al., 2005)
DNA Gyrase amplification, sequencing, and phylogenetic analysis
• For this molecular analysis, only genetically distinct bacteria (based on RAPD-PCR gels) presenting at least one VF gene were used.
• gyrB gene was amplified by PCR as described previously (Fukushima et al., 2002)
• PCR products were sequenced using DNA lluminaPaired-End sequencing at the Cornell University Life Sciences Core Laboratory Center
• Sequences were align, trimmed, and phylogenetic analyzes performed using Geneious4. 8. 4.
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Results
• A total of 374 cows were enrolled in the study, of which 33.4% were E. coli positive
• Five E. coli were isolated from each of the 125 positive cows, totaling an initial pool of isolates of 625
• 14 isolates were negative for rDNA amplification and were excluded
• RAPD-PCR revealed that, on average, 2.8 genetically distinct E. coli isolates were found per contaminated cow
Results
Risk factor n E. coli %Adjusted
O.R.P-value
Twin 18 60 (35 - 80) 4.4
< 0.01Stillborn 18 55 (32 - 77) 3.7
Male alive 138 35 (27 - 32) 1.6
Female alive 200 25 (19 - 31) Ref.
BCS < 3 142 36 (26 - 47) 2.3
< 0.01BCS = 3 98 42 (32 - 52) 2.8
BCS > 3 134 20 (13 - 29) Ref.
Retained placenta
35 65 (44 – 81) 4.7
< 0.01Non- retained
placenta339 29 (24 – 34) Ref
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VF n Metritis % O. R. P - Value
Culture negative 249 11 (8 – 15) Ref.
fimH absent 16 22 (18 – 27) 2.6<0.001
fimH present 109 39 (30 – 49) 4.6
astA absent 102 28 (24 – 38) 3.7< 0.001
astA present 23 63 (46 – 74) 10.2
ibeA absent 101 29 (24 – 36) 4.1< 0.001
ibeA present 24 57 (40 – 72) 8.4
cdt absent 93 28 (22 – 35) 4.0< 0.001
cdt present 32 46 (39 – 67) 6.7
hlyA absent 109 31 (25 – 39) 4.7< 0.001
hlyA present 16 63 (47 – 76) 9.7
kpsMII absent 109 31 (24 – 38) 4.7<0.001
kpsMII present 16 61 (45 – 74) 9.4
Synergetic relationship was observed between fimH and astA, cdt, kpsMII, ibeA, or hlyA.
The incidence of metritis was highest (>52%) when fimHoccurredconcurrently with 1 of those 5 VF
FimH
• In the present study, fimH was highly prevalent in E. coli-infected cows and was the most important predictor of metritis and endometritis
• FimH was present in 87% of the E. coli positive cows
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UTI and uterine infection
• UTI - Hours after contamination the bladder environment will quickly select for bacteria expressing type1 fimbriae, and tissue colonization and damage follows (Kaper et al., 2004)
FimH- mutant is incapable of binding to bladder mucosa (Langermann, S. 1997)
IbeA
• neonatal meningitis E. coli (NMEC),
• ibeA (invasion of brain endothelium) plays an important role in neonatal gram-negative meningitis in humans, which is mainly caused by vertical transmission of E. coli (Huang et al., 2001).
• ibeA contributes to the invasiveness of E. coli into brain microvascular endothelial cells (BMECs) via a ligand–receptor interaction (Huang et al., 2001)
• ibeA is also important in other ExPEC infections
IbeA
• APEC = aerosacculitis, polyserositis, septicemia and other mainly extraintestinal diseases in chickens, turkeys and other avian species Germon et al. demonstrated that the virulence of
an ibeA-free mutant APEC was reduced compared to APEC expressing ibeA.
• Prevalence of ibeA was 19.2%
• Cows infected with FimH and IbeA positive E. coli were 4.6 times more likely to have metritis
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kpsMII
• kpsMII encodes the capsule proteins K1 or K5 and has been associated with cellulitis in chickens (de
Brito et al., 2003), and UTI in women (Moreno et al., 2005; Moreno et al., 2009).
• kpsMII was found in 12.8% of the E. coli-positive cows, which is comparable to the 16% prevalence found in chickens with cellulitis (de Brito et al., 2003) and the 21% found in UTI in humans (Johnson et al., 2002).
hlyA
• Another significant VF gene encountered encodes hly, which is a heat-labile extracellular protein synthesized by a large proportion of ExPEC isolates (Smith et al., 2008).
• The hly toxin is responsible for poring the membrane and lysing a number of different mammalian cells (Lally et al., 1999).
• A total of 12.8% of the E. coli-positive cows in this study were carriers of bacteria with the hlyAgene.
Other VFs
• astA - important characteristic of enteroaggregative E. coli (EAEC) encodes a 38-amino-acid protein named
enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1)
• cdt (cytolethal distending toxin) unique family of toxins which causes characteristic
enlargement of specific mammalian cells
cdt was initially identified in an EPEC it is now known that cdt is widely present in other E. colipathotypes, mainly in ExPECs
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ExPECS
• The group of ExPECs is diverse and includes several pathotypes, including urinary pathogenic E. coli (UPEC), neonatal meningitis E. coli(NMEC), avian pathogenic E. coli (APEC), necrotoxigenic E. coli (NTEC), and the newly described IUEC.
• All ExPECs will encounter similar challenges during the process of establishing extraintestinalinfections, and as a consequence they are likely to share similar VF genes (Johnson et al., 2008)
Interplay between innate host defenses and UPEC within the bladder.
Mulvey M A et al. PNAS 2000;97:8829-8835
©2000 by The National Academy of Sciences
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???
• 12% of E. coli culture negative cows developed metritis
• E. coli alone does not explain the whole story!
• Compare the uterine bacterial composition in healthy and metritic postpartum Holstein dairy cows.
• Construction and Sequencing of 16S rRNA Clone Library
• Group-specific 16S rDNA PCR-DGGE
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Metagenomic DNA extraction
OTUsLibrary Totalb Specificc % Coveraged Chao1e H’f
Healthy 1 2 2 100 NDg 0.65Healthy 2 8 8 97.1 8.5 1.90Metritic 1 11 7 94.5 15 1.55Metritic 2 21 17 91.3 51.25 2.12
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F. Necrophorum and the uterus:a hypothesis
Association between virulence factors of Escherichia coli, Fusobacterium necrophorum, and Arcanobacterium
pyogenes and uterine diseases of dairy cows.
• 115 Holstein cows were sampled at 4 ± 2, 12 ± 2, and 35 ± DIM
• Uterine swab was used for the first two samples and uterine lavage was performed for the 35 dim sample
• Isolation of total DNA was performed from 400 μL of the suspension by using a QIAmp DNA minikit ( Quiagen ,Santa Clara, CA) according to manufacturer’s instructions for DNA purification from blood and body fluids
Prevalence of VFs by stage of lactation
0
10
20
30
40
50
60
70
80
90
100
4 DIM 12 DIM 35 DIM
Fre
qu
ency
dis
trib
uti
on %
Days in milk (DIM) at sample collection
Leuk
fimH
fimA
plo
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Multivariable logistic regressionmetritis
Virulence factors Metritis % Odds ratio P-value
FimH 4 DIM
positive 76.2 4.70.008
negative 32.2 Reff.
Leukotoxin 12 DIM
positive 54.1 2.60.031
negative 24.0 Reff.
Multivariable logistic regressionclinical endometritis
Virulence factors Metritis % Odds ratio P-value
FimH 4 DIM
positive 38.1 5.40.010
negative 15.6 Reff.
FimA 12 DIM
positive 33.3 5.60.005
negative 12.5 Reff.
FimA 35 DIM
positive 61.5 8.80.001
negative 14.3 Reff.
Effect of Fimh at 4 dim on reproductive performance
Solid black lines represent FimHnegative cows and the interrupted line the FimHpositive cows.
FimHnegative cows were at a 2.1 times increased hazard of pregnancy
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Injectable trace mineral and the health of post-partum dairy cows
• The study was conducted in 4 large commercial dairy farms in upstate NY.
• A total of over 1,500 Holstein cows were randomly allocated into 2 treatment groups The treatment group received 3 injections of
Multimin 300 mg of Zinc 50 mg of Manganese 25 mg of Selenium 75 mg of Copper
Negative controls did not receive treatment
Randomized clinical trial
• Treatment group were injected at dry-off, 15 pre-partum, and 35 days post-partum
• Uterine secretion samples were collected at 35 DIM from all research cows and clinical endometritis was diagnosed
• All farms were on DHI program and milk production, components and somatic cell were monitored
NRC 2011 recommendations
Farm 1 Farm 2 Farm 3
Pre-fresh Lactation Pre-fresh Lactation Pre-fresh Lactation Pre-fresh Lactation
Calcium % 0.45 0.67 1.37 0.83 1.35 0.80 1.52 0.88
Phosphorus% 0.23 0.36 0.30 0.36 0.31 0.42 0.34 0.38
Magnesium% 0.12 0.2 0.42 0.32 0.37 0.35 0.39 0.33
Potassium% 0.52 1.06 1.11 1.24 1.07 1.28 1.55 1.54
Sodium% 0.10 0.22 0.12 0.46 0.11 0.47 0.14 0.53
Chloride% 0.15 0.28 0.52 0.50 0.36 0.50 0.45 0.59
Sulfur% 0.20 0.20 0.41 0.25 0.36 0.24 0.45 0.25
Cobalt ppm 0.11 0.11 0.96 1.49 0.87 1.13 0.75 1.90
Copper ppm 13.0 11.0 19.25 20.02 16.29 21.61 16.47 25.67
Iodine ppm 0.40 0.44 0.96 0.99 0.18 0.69 0.92 1.08
Iron ppm 13.0 17.0 225.45 205.4 219.68 189.78 316.06 212.85
Manganese ppm 18.0 13.0 108.59 72.85 80.99 81.49 95.83 90.19
Selenium ppm 0.30 0.30 0.38 0.47 0.36 0.53 0.55 0.57
Zinc ppm 22.0 52.0 63.77 80.81 53.73 84.45 70.90 91.76
Vitamin A kIU/d 82.6 75.0 178 170 138 172 178 187
Vitamin D kIU/d 21.5 21.0 35 39 27 43 34 42
Vitamin E IU/d 1202 545 1772 726 1560 611 1760 874
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ControlsTrace mineral supplemented
Average age (days) at enrollment (± SE) 1335 (±420) 1320 (±411)
Average body condition score at enrollment (± SE) 3.64 (±0.56) 3.65 (±0.54)
Average days carried calf at enrollment (± SE) 228.2 (±19.2) 230.2 (±17.5)
Enrolled animals on farm 1 (%) 198 (56) 153 (44)
Enrolled animals on farm 2 (%) 140 (48) 151 (52)
Enrolled animals on farm 3 (%) 350 (48) 371 (52)
Total enrolled animals (%) 710 (50.1) 706 (49.9)
Effect of trace mineral supplementation on the stillbirth
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
parity 1 parity > 1
Multimin
Control
diseases
Control TMS1Adjusted odds ratio
95% C. I.P-value
Stillbirth 6.1 % 4.3 % 1.69 (1.03 – 2.80) 0.039
Endometritis 34.2 % 28.6% 1.30 (1.03 – 1.64) 0.028
Metritis 11.5 % 11.8 % 1.04 (0.74 – 1.46) 0.827
Retained Placenta 6.7 % 6.8 % 1.00 (0.65 – 1.53) 0.999
Displaced abomasum 2.6 % 1.3 % 1.73 (0.76 – 3.93) 0.194
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Effect of trace mineral supplementation calf survival
Reproduction
Survival
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Effect of trace mineral supplementation on clinical mastitis
0
2
4
6
8
10
12
14
16
18
parity 1 parity 2 parity > 2
Multimin
Control
Incidence ofClinical mastitis
AdjustedOdds ratio
P-value
Treatment
Control 22.4 % 1.25
0.14
TMS1 18.7 % Reff.
Parity
Primiparous 22.6 % 1.82
< 0.01
Multiparous 13.8% Reff.
Treatment*parity2
Control and primiparous
11.8 % 0.72
0.33
TMS and primiparous 15.6 % Reff.
Control and multiparous
25.4 % 1.39
0.03
TMS and multiparous 19.7 % Reff.
Effect of trace mineral supplementation on clinical endometritis
0
5
10
15
20
25
30
parity 1 parity > 2
Multimin
Control
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LSM of linear
scores (95% C.I.)2P-value
TreatmentControls 2.3 (2.2 - 2.4)
0.021TMS3 2.1 (2.0 – 2.2)
Parity
1 2.0 (1.9 – 2.2)
< 0.0012 1.9 (1.8 – 2.0)
>2 2.6 (2.5 – 2.7)
Month of lactation
1 2.5 (2.4 – 2.6)
< 0.001
2 1.9 (1.8 – 2.0)
3 2.0 (1.8 – 2.1)
4 2.2 (2.1 – 2.3)
5 2.4 (2.2 – 2.5)
Treatment*parity*month1
Refer to figures 1, 2, and 3 < 0.001
LS for primiparous
1.5
1.7
1.9
2.1
2.3
2.5
2.7
2.9
1 2 3 4 5
LS lactation=2
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
1 2 3 4 5
control parity 2
treatment parity 2
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LS lactation > 2
1
1.5
2
2.5
3
3.5
1 2 3 4 5
control parity >2
treatment parity > 2
What about SCC??
Incidence ofsubclinical mastitis
AdjustedOdds ratio
P-value
TreatmentControl 10.4 1.31
0.005TMS1 8.0 Reff.
Parity
>2 14.2 2.6
< 0.0012 7.4 1.2
1 6.0 Reff.
Month of lactation
5 10.3 1.3
< 0.001
4 11.2 1.4
3 9.6 1.2
2 7.0 0.8
1 8.2 Reff.
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Milk and components
Treatment P-value
TMS1 Control
Milk (Kg/d) 40.3 (39.6 – 41.0) 40.5 (39.7 – 41.3) 0.66
Fat corrected milk(Kg/d) 43.0 (42.4 – 43.5) 43.0 (42.4 – 43.6) 0.95
Fat (%) 3.7 (3.6 – 3.7) 3.7 (3.6 – 3.7) 0.75
Protein (%) 3.1 (3.07 – 3.11) 3.1 (3.07 – 3.11) 0.84
Economic analysis
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Second lactating dairy cows
• A total of 251 cows were enrolled in the study.
• A total randomized field trial study design was used; cows were randomly allocated into one of two treatments: trace mineral supplemented (TMS) or control.
• All dry cows (Lactation>1) that were available during the enrollment period were included in the study.
Peripheral blood neutrophil function and serum SOD activity
• Serum SOD activity was assessed using Superoxide Dismutase Assay Kit (Cayman Chemical Company, Ann Arbor, MI) following the manufacturer’s instructions. Serum SOD activity was measured at at 230 ± 3 days of gestation, 10 ±1 DIM, 60 ±3 DIM and 100 ± 3 DIM.
BHB concentration
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 1
β‐BHB concentrations mmol/L
Treatment groups
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Superoxide dismutase and mastitis
8
9
10
11
12
13
14
15
16
17
-60 10 60 100
mastitis
no mastitis
Multimin supplementation and superoxide dismutase
10
12
14
16
18
20
22
-60 10 60 100
control
TMS
Pyrosequencing
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Pyrosequencing
Pyrosequencing results
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.900
1.000
H1 H2 H3 ME1 ME2 ME3
Fusobacteria
Bacteroidetes
Firmicutes
Proteobacteria
Tenericutes
Actinobacteria
Synergistetes
Spirochaetes
SR1
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Percentage of phylum
Phylo Mean
Firmicutes 48
Bacteroides 14
Proteobacteria 17
Tenericutes 10.00
Fusobacteria 3
Actinabacteria 5
Spirochaetes 0.1
Others 0.2
Percentage of phylum
0
5
10
15
20
25
30
35
40
45
50
%
%
Proportion of phylum by multimintreatment
Phylo Treatment % Std Error Lower 95% Upper 95% P‐value
Actinobacteria No 6.00 1.04 3.95 8.06
0.40Yes 4.74 1.07 2.62 6.86
Bacteroidetes No 16.18 1.93 12.35 20.01
0.07Yes 11.09 2.01 7.10 15.08
Firmicutes No 50.80 3.31 44.24 57.37
0.25Yes 45.28 3.41 38.51 52.06
Fusobacteria No 3.99 1.50 1.02 6.96
0.73Yes 3.26 1.54 0.20 6.33
others No 0.14 0.06 0.02 0.27
<0.03Yes 0.35 0.06 0.22 0.48
Proteobacteria No 13.94 2.96 8.07 19.80
<0.02Yes 24.29 3.05 18.24 30.35
Spirochaetes No 0.20 0.04 0.11 0.28
0.14Yes 0.10 0.04 0.02 0.19
Tenericutes No 8.75 3.27 2.26 15.24
0.61Yes 11.12 3.37 4.42 17.81
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Proportion of phylum by multimintreatment
Proportion of phylum by multimintreatment
0.00
5.00
10.00
15.00
20.00
25.00
30.00
others Proteobacteria
No
Yes
Proportion of phylum by mucus scorePhylos Mucus Score Mean Std Error Lower 95% Upper 95% P‐value
Firmicutes0 52.29 2.38 47.56 57.02
<0.00011 35.81 6.15 23.60 48.012 11.08 9.52 ‐7.83 29.99
Bacteroidetes 0 12.25 1.40 9.48 15.03
<.00011 12.40 3.58 5.28 19.522 40.44 5.55 29.41 51.46
Proteobacteria0 20.13 2.37 15.42 24.84
0.1371 18.85 6.12 6.70 31.002 0.42 9.48 ‐18.41 19.25
Tenericutes 0 7.00 2.50 2.04 11.97
0.0251 23.18 6.46 10.35 36.002 24.34 10.01 4.47 44.20
Fusobacteria 0 2.00 1.07 ‐0.13 4.13
<.00011 7.02 2.77 1.52 12.522 21.74 4.29 13.22 30.26
Actinobacteria 0 6.03 0.81 4.42 7.65
0.1681 2.50 2.09 ‐1.66 6.662 1.98 3.24 ‐4.46 8.43
Spirochaetes 0 0.17 0.03 0.10 0.23
0.4661 0.12 0.09 ‐0.06 0.292 0.00 0.14 ‐0.27 0.27
others 0 0.28 0.05 0.18 0.38
0.2641 0.13 0.13 ‐0.13 0.382 0.00 0.20 ‐0.40 0.40
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Proportion of phylum by mucus scoreProportion of Phylos by mucus score
0
0.2
0.4
0.6
0.8
1
0 1 2
mucus score
Legend
Actinobacteria
Bacteroidetes
Firmicutes
Fusobacteria
others
Proteobacteria
Spirochaetes
Tenericutes
Proportion of phylum by mucus score
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Firmicutes Bacteroidetes Tenericutes Fusobacteria
0
1
2
Proportion of phylum by endometritisPhylos Endometritis Mean Std Error Lower 95% Upper 95% P‐value
FirmicutesNo 50.14 2.28 45.61 54.67
<0.001Yes 11.08 9.78 ‐8.34 30.50
Bacteroidetes No 12.27 1.29 9.70 14.84
<0.001Yes 40.44 5.52 29.48 51.40
Proteobacteria No 19.96 2.20 15.60 24.33
0.046Yes 0.42 9.43 ‐18.31 19.15
Tenericutes No 9.11 2.39 4.37 13.85
0.15Yes 24.34 10.24 4.01 44.66
Fusobacteria No 2.65 1.01 0.65 4.66
<0.0001Yes 21.74 4.33 13.13 30.35
ActinobacteriaNo 5.57 0.76 4.06 7.09
0.288Yes 1.98 3.27 ‐4.51 8.47
Spirochaetes No 0.16 0.03 0.10 0.22
0.259Yes 0.00 0.14 ‐0.27 0.27
others No 0.26 0.05 0.16 0.35
0.217Yes 0.00 0.20 ‐0.40 0.40
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Proportion of phylum by endometritisProportion of Phylos by Endometritis
0
0.2
0.4
0.6
0.8
1
0 1
Endometritis
Legend
Actinobacteria
Bacteroidetes
Firmicutes
Fusobacteria
others
Proteobacteria
Spirochaetes
Tenericutes
Proportion of phylum by endometritis
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Firmicutes Bacteroidetes Proteobacteria Fusobacteria
No
Yes
Proportion of phylum by metritisPhylos Metritis Mean Std Error Lower 95% Upper 95% P‐value
FirmicutesNo 51.70 2.80 46.13 57.26
0.0087Yes 36.82 4.78 27.32 46.32
BacteroidetesNo 12.67 1.71 9.26 16.08
0.0669Yes 18.93 2.90 13.15 24.70
ProteobacteriaNo 20.33 2.51 15.33 25.32
0.0514Yes 10.50 4.29 1.98 19.03
TenericutesNo 5.75 2.74 0.30 11.20
>0.001Yes 24.43 4.68 15.12 33.73
FusobacteriaNo 3.45 1.34 0.79 6.11
0.534Yes 5.10 2.28 0.57 9.63
ActinobacteriaNo 5.94 0.92 4.10 7.77
0.2623Yes 3.88 1.57 0.75 7.01
SpirochaetesNo 0.12 0.04 0.04 0.19
0.0636Yes 0.25 0.06 0.13 0.38
othersNo 0.24 0.04 0.16 0.32
0.0601Yes 0.09 0.07 ‐0.05 0.23
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Proportion of phylum by metritis
Proportion of phylum by metritis
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Firmicutes Tenericutes
No
Yes
Proportion of phylum by RPPhylos RP Mean Std Error Lower 95% Upper 95% P‐value
Firmicutesno 48.09 2.57 42.99 53.20
0.7156yes 43.64 11.91 19.96 67.32
Bacteroidetesno 14.43 1.54 11.37 17.49
0.6633yes 11.26 7.09 ‐2.84 25.35
Proteobacteriano 18.47 2.24 14.01 22.92
0.1724yes 3.82 10.40 ‐16.85 24.50
Tenericutesno 9.25 2.50 4.29 14.21
0.0174yes 37.94 11.58 14.94 60.94
Fusobacteriano 4.00 1.18 1.65 6.35
0.6091yes 1.13 5.47 ‐9.75 12.01
Actinobacteriano 5.57 0.82 3.95 7.19
0.3562yes 1.98 3.78 ‐5.54 9.50
Spirochaetesno 0.15 0.03 0.08 0.21
0.6457yes 0.22 0.15 ‐0.08 0.52
othersno 0.21 0.04 0.14 0.28
0.2367yes 0.01 0.17 ‐0.32 0.34
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Proportion of phylum by RP
Proportion of phylum by RP
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Tenericutes
no
yes
Proportion of phylum by Reproduction
Phylos Pregnant Mean Std Error Lower 95% Upper 95% P‐value
Firmicutesno 43.97 4.56 34.90 53.04
0.3065yes 49.58 2.99 43.64 55.51
Bacteroidetesno 15.63 2.78 10.10 21.15
0.5678yes 13.73 1.79 10.18 17.28
Proteobacteriano 11.21 3.96 3.34 19.08
0.0493yes 20.65 2.59 15.49 25.80
Tenericutesno 18.44 4.49 9.52 27.36
0.0379yes 7.13 2.94 1.29 12.97
Fusobacteriano 6.43 2.09 2.28 10.57
0.1466yes 2.78 1.37 0.06 5.49
Actinobacteriano 4.79 1.46 1.89 7.69
0.6122yes 5.68 0.96 3.78 7.58
Spirochaetesno 0.07 0.06 ‐0.05 0.18
0.0881yes 0.19 0.04 0.11 0.26
othersno 0.05 0.06 ‐0.08 0.17
0.0028yes 0.27 0.04 0.19 0.35
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34
Proportion of phylum by Reproduction
Proportion of phylum by Reproduction
-5.00
0.00
5.00
10.00
15.00
20.00
25.00
Proteobacteria Tenericutes others
no
yes
Questions?