10/25/2012 1 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 Metritis Abnormally enlarged uterus and a fetid watery red-brown uterine discharge, associated with signs of systemic illness
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trace minerals and the intrauterine microbiology 2012.pdf · Endometritis • Inflammation of the uterus without systemic illness (Sheldon et al., 2006) Only the endometrium is affected
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10/25/2012
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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.
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
• 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.