2019 Reproductive microbiome alterations: canine pyometra   Reproductive microbiome alterations: canine pyometra Michelle Kutzler Oregon State University, Corvallis, OR Abstract Pyometra is a life-threatening uterine infection that affects 20 - 25% of reproductively intact bitches. Canine pyometra is characterized by accumulation of leukocytes and exudate within the uterine lumen. Although exact temporal and progressive mechanisms underlying its pathogenesis are not fully understood, a combination of endocrine, structural, inflammatory and bacterial factors are likely involved in most cases. Pyometra develops during luteal phase; progesterone has a key role in establishment of infection with opportunistic bacteria. Cystic endometrial hyperplasia results from proliferation of endometrial glands and is a predisposing factor for development of pyometra. Endometrial proliferation factors exacerbate uterine innate immune responses. Whereas Escherichia coli is the most common organism isolated in up to 90% of cases with a positive culture, genes encoding for adhesins, toxins, and other factors increase virulence of certain strains. A presumptive diagnosis of pyometra is based on clinical signs and laboratory tests, with a confirmatory diagnosis based on B-mode ultrasonography, with or without Doppler ultrasonography, detecting an enlarged, fluid-filled uterus with increased blood flow. Broad-spectrum antibiotics with minimal nephrotoxicity are needed to prevent septicemia, but antibiotics alone will not resolve pyometra. Safest and most effective treatment is surgical removal of infected uterus and ovaries. Medical management of pyometra may be indicated to improve general status of the bitch prior to surgery or treatment in valuable breeding bitches. Protocols using prostaglandin F2α or aglepristone are discussed in detail. Keywords: Aglepristone, cystic endometrial hyperplasia, Escherichia coli, prostaglandin F2α, virulence factors Introduction Pyometra is a life-threatening bacterial infection of the uterus that affects 20 - 25% of reproductively intact bitches.1-5 However, incidence of pyometra may be > 50% in certain high-risk breeds, indicating that there may be genetic factors predisposing to development of pyometra.2 Mean age for pyometra diagnosis in bitches is 7 years (range: 4 months to 18 years).2 Etiopathogenesis Canine pyometra is characterized by an accumulation of leukocytes and exudate within uterine lumen. Although exact temporal and progressive mechanisms underlying pathogenesis are not fully understood, a combination of endocrine, structural, inflammatory and bacterial factors are likely involved in most cases. Endocrine factors Pyometra develops during luteal phase; progesterone has a key role for establishment of infection with opportunistic bacteria. During diestrus, progesterone induces endometrial proliferation, endometrial gland secretion, myometrial quiescence and cervical closure.6-7 Progesterone not only inhibits endometrial bactericidal activity, it also specifically increase the affinity of endometrium to Escherichia coli (E. coli).8 In addition, repeated exposure to endogenous and/or exogenous progestogens has an important role in pathogenesis of cystic endometrial hyperplasia, which also has a key role in development of pyometra.9-12 Structural factors Cystic endometrial hyperplasia (CEH) results from proliferation of endometrial glands and subsequent endometrial cyst formation. CEH is accompanied by severe changes in endometrial cell proliferation and apoptosis, coinciding with dysregulation in expression of apoptosis regulatory proteins (e.g. Bcl2, Bax).13 These alterations cause excessive proliferation, combined with deregulation of 639 Clinical Theriogenology • Volume 11, Number 4 • December 2019   apoptosis, which then causes insufficient endometrial regeneration.13 Role of progestogens in pathogenesis of CEH is exacerbated by estradiol 17β.6-7 CEH is the most common uterine disease in dogs.14 Incidence of CEH increases with age from < 4% in bitches under 3 years of age to > 50% in bitches by 7 years.15 It is important to note that not all cases of pyometra involve CEH. Normal uterine defence mechanisms against pyometra include: (1) a potent local immune response with leukocytes and antibodies, (2) well-regulated endometrial secretions and (3) myometrial contractions.7 However, functional patency of the cervix also has an important structural role. With a functionally patent cervix (open pyometra), mucopurulent to hemorrhagic exudate can exit the uterus. When cervix is not patent (closed pyometra), bacterial laden exudate accumulates within uterine lumen, resulting in endometrial atrophy.16 Closed pyometras are associated with more severe illness compared to open pyometras, but not poorer outcomes as measured by postoperative hospitalization.3 With respect to incidence, open pyometra occurs more commonly (65 - 75%) than closed pyometra (25 - 35%).3,5 Inflammatory factors Lipopolysaccharide (LPS) from bacterial cell walls elicits a strong inflammatory response. Uterine inflammation in response to LPS is associated with upregulation of endometrial genes related to the innate immune response. In response to this inflammation, endometrial proliferation factors exacerbate the inflammatory reaction.17 These factors are: (1) insulin-like growth factor (IGF1), (2) endometrial remodelling (via matrix metalloproteinases) and (3) inflammatory response genes (e.g. chemokine ligand 2 (CXCL2), secretory leukocyte peptidase inhibitor (SPLI)). Based on global endometrial transcriptomic profiling by microarray, many genes are upregulated ~ 10 - 77 times in uteruses of bitches with pyometra.18 Pyometra can be histologically classified as either hyperplastic or atrophic.19 Endometrial thickness and ratio of endometrial to myometrial thickness are increased 3-fold in hyperplastic pyometra compared to a normal uterus.19 Histologically, a uterus associated with atrophic pyometra has severe thinning of endometrial mucosa (with thin cuboidal epithelial cells) with concurrent hypertrophy of myometrium.19 These 2 forms have differential regulation of inflammatory cytokines and enzymes in the prostaglandin synthetic pathway in the endometrium.19 For example, a relative fold increase in interleukin 8 (IL8) of 1.82 times occurs in atrophic pyometra compared to hyperplastic pyometra.19 In addition, expression of cyclooxygenase type-2 (COX2) and prostaglandin F synthase (PGFS) are significantly upregulated (3.75 and 3.15 times in atrophic pyometra compared to hyperplastic pyometra, respectively).19 This results in greater expression of proliferative Ki-67 marker in endometrial stromal cells in response to the bacterial agent.13 During inflammation, COX2 is an important source of prostaglandins and thromboxane A2. COX-2 interacts directly with chemokines that are overexpressed in pyometra (e.g. CXCL8/IL8, CXCL14).17 Overexpression of CXCL14 contributes to inflammatory cell infiltration into uterine lumen, due to its chemoattractant action in monocytes and natural killer cells.20 However, CXCL10 has antimicrobial activity against E. coli.21 In stromal cells, CXCL10 may be involved in recruitment and potentiation of T helper 1 response.22 Because IL8 and CXCL14 are induced by COX2, selective COX2 inhibitors may mitigate inflammatory response during pyometra.17 COX2 is also downstream of toll-like receptor (TLR) signalling after activation by endogenous S100 proteins and other stimuli (e.g. LPS). Conserved pathogen-associated molecular patterns synthesized by microorganisms (LPS) are recognized by TLR to initiate an innate immune response. Normal canine endometrial epithelial and stromal cells express TLR4, but both TLR2 and TLR4 are upregulated in pyometra. TLR mediated immune surveillance is an important component in uterine defence mechanisms.23 Continuous activation of TLR by both endogenous and exogenous stimulus can lead to an exacerbated inflammatory response in pyometra. LPS interacts with lactoferrin (LTF) on the surface of bacteria to activate TLR4 on surfaces of phagocytes and epithelial cells.17 Both LTF gene and protein are overexpressed in pyometra.24 Calcium-binding proteins of the S100 family (e.g. S100A8, S100A9, S100A12) have been identified as endogenous danger-associated molecular patterns (DAMPs).17 DAMPs are intracellular 640Clinical Theriogenology • Volume 11, Number 4 • December 2019   molecules released following cell death and are recognized by the innate immune system. S100A8 and S100A9 stimulate production of several pro-inflammatory cytokines (e.g. tumor necrosis factor (TNF), IL6, IL1B, IL8).17 Another op upregulated gene in pyometra is secretory leukocyte peptidase inhibitor (SLPI) gene.17 SLPI encodes for an antimicrobial peptide secreted by epithelial tissues. SPLI modulates infection and inflammation by neutralizing LPS and reducing activation of TLRs.17 Bacteria Positive uterine cultures have been obtained during various stages of the reproductive cycle in bitches without reproductive problems.25 Common bacteria in uteruses of healthy bitches reflect bacterial flora of vagina and cervix.25 Presence of mixed bacteria in uterus during proestrus and estrus is not uncommon in dogs, presumably due to cervical dilation.25 Presence of intrauterine bacteria cultured from uterine lumen following elective ovariohysterectomies during diestrus is counterintuitive. In 1 study, > 10% of “normal” diestrous uteruses yielded positive bacterial cultures.25 Organisms isolated included Enterococcus sp., Bacillus sp., and undefined gram positive species.25 Furthermore, ~ 16% of bitches believed to have pyometra have negative bacteriologic findings following ovariohysterectomy.25 However, there were no attempts to detect anaerobic organisms or isolate other bacteria such as Mycoplasma sp., Leptospira sp., or Chlamydia sp.25 With respect to pyometra, E. coli is the most common organism isolated in 90% of cases with positive culture.26-27 However, other bacteria are also cultured (Table 1), with Enterococcus avium isolated from the uterus of a dog with an emphysematous pyometra. In > 90% of cases of canine pyometra, only a single bacterial species was cultured.25 It is important to mention that when > 1 bacterial species is isolated from canine pyometra, E. coli is always isolated.25 Escherichia coli Strains of these that are isolated from pyometra are those that normally inhabit canine intestine and those present in urinary tract infections.28 Escherichia coli isolated from canine pyometra is mainly assigned to phylogenetic group B2 and characterized by a high number of Table 1. Bacteria other than Escherichia coli cultured from cases of canine pyometra.25,72 Bacteria Frequency (%) Β-hemolytic Streptococcus 15 Citrobacter spp. 10 Unidentified Enterobacteriaceae 5.9 - 33.3 Streptococcus spp. 5.1 Klebsiella spp. 4.2 Enterococcus spp. 3.4 Actinomycetaceae spp. 2.5 Staphylococcus spp. 2.5 - 16.7 Pseudomonas spp. 1.7 - 2.0 Undefined Gram negatives 0.8 Undefined Gram positives 0.8 Proteus 0.8 - 16.7 uropathogenic E. coli (UPEC) virulence factor (VF) genes and pathogenicity-associated island markers.28 Escherichia coli have VF genes encoding for adhesins (including fimH, papC and papGIII), toxins (including hlyA/F, usp and astA), and other factors (e.g. those involved in escape from host defences and iron uptake).28 Frequency of VF genes in E. coli isolates from canine pyometra are summarized in Table 2. Escherichia coli isolates with > 2 adhesin genes imply that a combination of genes confers an 641 Clinical Theriogenology • Volume 11, Number 4 • December 2019   advantage.28 Strongly virulent strains of E. coli (e.g. nau-b) contain 7 virulence genes. When strongly virulent strains of E. coli are inoculated into female dogs, they produce symptoms of pyometra with an earlier onset and greater severity than weakly virulent strains of E. coli (e.g. nau-i) with fewer virulence genes.28 Alpha-hemolysin (hlyA) VF gene was detected in 35 - 52% of E. coli pyometra cases.27,29-30 HlyA is an RTX pore-forming exotoxin that contributes to virulence of E. coli by inducing tissue damage and a compromised early uterine immune response.27 Table 2. Virulence genes frequency in Escherichia coli isolates from canine pyometra.28,73 Gene Encodes for Frequency expressed (%) fimH adhesin 91.3 irp-2 yersiniabactin 91.3 fim Type 1 fimbriae 90.9 fyuA yersiniabactin 82.6 hlyA hemolysin 30.4 - 63.6 sfa S fimbriae 63.6 pap P fimbriae 63.6 papC P fimbriae 30.4 - 63.6 papGIII adhesin 26.1 - 63.6 iroN salmochelin 56.5 usp Uropathgenic-specific protein 39.1 - 54.5 traT Escape from host defenses 47.8 sfaD/E adhesin 34.8 tsh adhesin 34.8 cnf-1 toxin 26.1 papE/F adhesin 21.7 iss Escape from host defenses 21.7 iutA aerobactin system 17.4 ompT Escape from host defenses 17.4 cvaC Escape from host defense 17.4 hlyF toxin 17.4 iucD aerobactin system 17.4 iucC aerobactin system 13.0 astA toxin 13.0 papGII adhesin 4.3 At high concentrations, HlyA is able to lyse erythrocytes and nucleated host cells. At low (sublytic) concentrations, HlyA can disrupt the immune signalling and cytoskeletal components.31 Endometrial epithelial cells had high sensitivity to cytotoxic effect of HlyA, suggesting that β-hemolytic E. coli induces earlier damage to the epithelial glandular cells than nonhemolytic E. coli strains.32 Diagnosis A presumptive diagnosis of pyometra is based on clinical signs and laboratory test results, with a confirmatory diagnosis made with B-mode ultrasonography, with or without Doppler ultrasonography, detecting an enlarged, fluid-filled uterus with increased blood flow. Clinical signs and laboratory test results Clinical signs common in pyometra include depression, anorexia, polydipsia/polyuria, vomiting and vaginal discharge.5,33-34 Anorexia, polydipsia/polyuria, vomiting, and moderate to severe dehydration did not differ significantly between cases of open and closed pyometra.3 Leukocytosis, with neutrophilia and left shift, lymphopenia and monocytosis are characteristic findings in pyometra, accompanied by normocytic, normochromic regenerative anemia.5 If present, leukopenia was the most important predictive factor for prolonged postoperative hospitalization, as it was associated with an 18-fold increased risk of peritonitis.5 642Clinical Theriogenology • Volume 11, Number 4 • December 2019   Concomitant cystitis, proteinuria, and hypoalbuminemia usually resolve after treatment of the pyometra, but severe proteinuria that remains may predispose to renal failure.35 Renal dysfunction is common. Contributing factors to renal dysfunction are: endotoxemia, glomerular dysfunction, renal tubular damage and decreased response to antidiuretic hormone.35-36 Blood urea nitrogen > 30 mg/dl and creatinine concentrations > 1.5 mg/dl have been associated with death.33 Hypercholesterolemia, increased serum alkaline phosphatase (ALP) and coagulation impairment have also been reported.36-38 Systemic inflammatory response syndrome In ~ 50% of cases, pyometra can progress into systemic inflammatory response syndrome (SIRS), a form of sepsis and endotoxemia that causes multi-organ dysfunctions.4,39-41 SIRS is more common in dogs with closed pyometra than an open pyometra.3 Clinical diagnostic criteria for SIRS are nonspecific but consist of tachycardia and tachypnea, with a similar hemogram as previously described for pyometra. In pyometra cases diagnosed with SIRS, there are increases in blood concentrations of: C-reactive protein (CRP), prostaglandin 15-ketodihydro-PGF2α metabolite, serum amyloid A and haptoglobin. In addition, there were high serum IL8 concentrations in dogs SIRS, suggesting IL8 may contribute to development of systemic disease in dogs with pyometra. Despite being potentially life-threatening, mortality due to pyometra is relatively low (3 - 10%).5,42 Ultrasonography In cases of hyperplastic pyometra, proliferative response can be detected with B-mode ultrasonography as a thickened uterine wall.15 In addition, increased expression of VEGF-A and its receptors during pyometra cause increased blood flow (both peak systolic flow and end diastolic flow) from reduced vascular resistance.15 Females diagnosed with CEH and mucometra should be monitored by Doppler ultrasound during diestrus for early pyometra identification, to prevent development of systemic disease.15 Treatment Pyometra is an emergency condition that requires rapid medical and/or surgical intervention to minimize long-term health complications and prevent death. Early intervention increases chances of survival.43 Surgical management Safest and most effective treatment is surgical removal of infected uterus and ovaries, (ovariohysterectomy). Anesthetic risks associated with this surgery are heightened due to a compromised status. Uterus may be large, friable, and prone to rupture, so it is important to handle it carefully. Abdominal cavity should be protected from accidental leakage of purulent material from uterine laceration or uterine tubes/ovarian bursa opening. This is accomplished by packing off uterus with moistened laparotomy pads.43 Prior to surgery, patient is stabilized with adequate intravenous fluid therapy to correct hypotension (60 ml/kg + % dehydration x body weight/100), hypoperfusion, shock, dehydration, acid-base balance and electrolyte abnormalities, coagulation disturbances, and organ dysfunctions.44 Intensive postoperative monitoring is essential and in uncomplicated cases, 1 to 2 days of postoperative hospitalization is usually adequate.43 Broad-spectrum antibiotics with minimal nephrotoxicity are needed to prevent septicemia (e.g. amoxicillin-clavulanic acid 12.5 mg/kg twice a day). Antibiotics alone for treatment of pyometra will improve general status and may prevent progression but will not resolve the condition. Antibiotics should be continued for > 2 weeks.45 Antibiotic drug selection and route of administration should be based on bacterial culture, sensitivity tests and pharmacokinetics for achieving optimal effect.43 The initial choice of antibiotics should be effective against the most likely pathogen (E. coli) and adjusted after culture and sensitivity results to a narrow-spectrum alternative.43,46 In 1 study, 90% of E. coli pyometra isolates were sensitive to ampicillin.47 Intravenous administration may be superior to prevent systemic effects of sepsis.46 643 Clinical Theriogenology • Volume 11, Number 4 • December 2019   Medical management Medical management of pyometra may be indicated to improve the general status of the bitch prior to surgery or in the bitch that cannot undergo general anesthesia for other reasons. In addition, medical management may be indicated for treatment of pyometra in valuable breeding bitches. However, the efficacy of medical management depends on clinical presentation (e.g. stability of patient, patency of cervix) and presence of underlying problems (e.g. cystic endometrial hyperplasia, ovarian cyst or tumor).48 Goal of medical management is to evacuate uterine contents by dilating cervix and stimulating myometrial contractility. Protocols using prostaglandin F2α (PGF2α) and progesterone antagonists (aglepristone) are described in Table 3. Aglepristone can be used to successfully treat either open or closed pyometras, with cervical opening occurring within 25.8 ± 12.3 hours after initial treatment.16,48-50 However, PGF2α treatment is contraindicated in closed pyometra due to its potential for forcing purulent material retrograding to uterine tubes through ovarian bursae and into peritoneal cavity or through rupture of uterine wall.51 Administration of prostaglandin E2 (either intravaginally or orally) may result in cervical relaxation sufficient to allow PGF2α but this remains to be studied.45,52 Intrauterine drainage and lavage via transcervical catheterization followed by instillation of intrauterine antibiotics may facilitate recovery in refractory cases.53-56 With respect to complications of medical management, ~ 20% of pyometra patients experienced complications, most commonly peritonitis (10 - 12% of cases) followed by chronic pyelonephritis, urinary tract infection, myocarditis, and disseminated intravascular coagulation.3,5,40,57-59 Persistent proteinuria and urinary protein-creatinine indicate renal disease that requires special attention.35 Other reported complications include uveitis, intracranial thromboemboli, bacterial osteomyelitis, pericarditis, septic arthritis, incisional swelling, dehiscence, urethral trauma, recurrent estrus, uterine stump pyometra, fistulous tracts, urinary incontinence, septic shock and death.35,58-59 Severe complications associated with pyometra include sepsis, septic shock, peritonitis, disseminated bacterial infection organ dysfunctions and death.58,60-61 Proportion of dogs with complications did not differ significantly between open and closed pyometras.3 Despite this, it is prudent to obtain signed owner consent prior to treatment, regarding potential risks necessary to obtain prior to extra label drug usage.3 In addition, aglepristone is not currently approved for use in North America. Prognosis Following medical management of pyometra with PGF2α or aglepristone, prognosis for survival and fertility is considered guarded to good. Average fertility rate following medical management is 70% (range, 14 - 100%).43,51,62-64 Fertility rates are even higher in younger (< 5 years) bitches and those that have no other uterine or ovarian pathology.50 Average reported long-term success (resolution of clinical illness) following medical management of canine pyometra with PGF2α is 86% (range, 46 - 100%).48- 49,54,62-70 Combining PGF2α with cabergoline (a dopamine agonist) results in resolution rates from 83 - 90.5%.66-67 Resolution rate with aglepristone alone is lower (60%) than with a combination of aglepristone and cloprostenol (84%).16 Breeding on subsequent estrus is frequently recommended after medical management of pyometra in attempt to avoid recurrence.43 Mean recurrence rate following medical management with either treatment is ~ 20% (range, 0 - 85%).43,49-50,63,71 It is important to mention that medical management of bitches with recurrence has been successful. At least in some breeds, pregnancy is considered to slightly reduce the risk for pyometra recurrence, which points to genetic differences in protective factors.10,39 However, pregnancy is not completely protective, and developing pyometra during pregnancy has been reported in several cases.15 644Clinical Theriogenology • Volume 11, Number 4 • December 2019   Table 3. Medical management of canine pyometra using prostaglandin F2α (PGF2α) or aglepristone. Mechanism of action Protocol Efficacy Adverse effects PGF2α PGF2α is both luteolytic and uterotonic. Efficacy of treatment is correlated to repeated administration rather than overall total dose administered. Comparison of uterotonic effect in diestrus bitches of PGF2α at a dosage of 250 µg/kg compared to 50 µg/kg resulted in uterine contractility for 32 and 23 minutes, respectively.51,75 PGF2α is most effective when administered in repeated low doses for 8 - 10 days. Administer natural PGF2α at a dosage of 10 - 50 µg/kg SC every 4 - 6 hours.7,45,68-70 Administer cloprostenol at a dosage of 1 - 3 µg/kg SC every 12 - 24 hours.62,74 Following first treatment, copious discharge will be observed coming from vulva for first 48 hours and then will start to decrease. Hemorrhagic discharge may be observed 4 - 5 days into treatment. Evacuation of uterine contents greatly improves bitch’s physical condition. Treatment efficacy is confirmed by ultrasonographic clearance of uterine contents 10 - 14 days after first treatment. Adverse effects are dose-dependent and develop within minutes and last for ~ 1 hour.62 Adverse effects include hypothermia and shivering, increased frequency of defecation (± diarrhea), hypersalivation and vomiting. Administer atropine (25 µg/kg SC) 15 minutes before PGF2α and walk bitch for 15 - 20 minutes after administration to lessen symptoms. Brachycephalic breeds may also be predisposed to bronchospasm.45,52 Dosage calculations must be done correctly to prevent serious adverse effects, including hypovolemic shock, ventricular tachycardia and death.43 Aglepristone Aglepristone is a progesterone receptor antagonist that competitively binds progesterone receptors and decreases intrauterine progesterone concentration. Aglepristone does not have direct uterotonic activity. Administer aglepristone at a dosage of 10 mg/kg SC on days 1, 3, 6 and 9 of treatment.63 If uterine contents are still visible on day 15, administer another injection on day 15 and on day 30. Results can be improved by giving cloprostenol (1 µg/kg SC) from days 3 - 7.16 When a combination of aglepristone and cloprostenol is administered, there was no significant difference in success rates between bitches with open versus closed pyometra.16 Adverse effects have not been reported when using aglepristone alone. Vomiting has been reported when used in combination with cloprostenol.16 645 Clinical Theriogenology • Volume 11, Number 4 • December 2019   References 1. Wallace GB, Casal ML: A review of pyometra in small animal medicine: incidence, pathophysiology, clinical diagnosis, and medical management. Clinical Theriogenology 2018;10:435-452 2. Jitpean S, Hagman R, Strom Holst B, et al: Breed variations in the incidence of pyometra and mammary tumours in Swedish dogs. Reprod Domestic Anim 2012;47 Suppl 6:347-350. 3. Jitpean S, Ambrosen A, Emanuelson U, et al: Closed cervix is associated with more severe illness in dogs with pyometra. BMC Vet Res 2017;13:11. doi: 10.1186/s12917-016-0924-0. 4. Jitpean S, Pettersson A, Höglund OV, et al: Increased concentrations of serum amyloid A in dogs with sepsis caused by pyometra. BMC Vet Res 2014;10:273. 5. 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Cole AM, Ganz T, Liese AM, et al: Cutting edge: IFN-inducible ELR-CXC chemokines display defensin-like antimicrobial activity. J Immunol 2001;167:623-627. 22. Dufour JH, Michelle D, Liu MT, et al: IFN-gamma inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol 2002;168:3195-3204. 23. Silva E, Leitão S, Henriques S, et al: Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia-pyometra complex. J Reprod Immunol 2010;84:66-74. 24. Kida K, Baba E, Torii R, et al: Lactoferrin expression in the canine uterus during the estrous cycle and with pyometra. Theriogenology 2006;66:1325-1333. 25. Rubio A, Boyen F, Tas O, et al: Bacterial colonization of the ovarian bursa in dogs with clinically suspected pyometra and in controls. Theriogenology 2014;82:966-971. 26. Dhaliwal GK, Wray C, Noakes DE: Uterine bacterial flora and uterine lesions in bitches with cystic endometrial hyperplasia (pyometra). Vet Rec 1998;143:659-661. 27. Mateus L, Henrique S, Merino C, et al: Virulence genotypes of Escherichia coli canine isolates from pyometra, cystitis and fecal origin. Vet Microbiol 2013:166:590-594. 28. Maluta RP, Borges CA, Berldo LG, et al: Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra. Vet J 2014;202:393-395. 29. Chen YMM, Wright PJ, Lee C-S, et al: Uropathogenic virulence factors in isolates of Escherichia coli from clinical cases of canine pyometra and feces of healthy bitches. Vet Microbiol 2003;94:57-69. 30. Siqueira A, Ribeiro M, Leite D, et al: Virulence factors in Escherichia coli strains isolated from urinary tract infection and pyometra cases and from feces of healthy dogs. Res Vet Sci 2009;86:206-210. 646Clinical Theriogenology • Volume 11, Number 4 • December 2019   31. Wiles TJ, Mulvey MA: The RTX pore-forming toxin alpha-hemolysin of uropathogenic Escherichia coli: progress and perspectives. Future Microbiol 2013;8:73-84. 32. Henriques S, Silva E, Silva MF, et al: Immunomodulation in the canine endometrium by uteropathogenic Escherichia coli. Vet Res 2016;47:114. doi 10.1186/s13567-016-0396-z. 33. Kuplulu S, Vural MR, Demirel A, et al: The comparative evaluation of serum biochemical, haematological, bacteriological and clinical findings of dead and recovered bitches with pyometra in the postoperative process. Acta Vet-Beograd 2009;59:193-204. 34. Borresen B: Pyometra in the dog- a pathophysiological investigation. II. Anamnestic, clinical and reproductive aspects. Nord Vet Med 1979;31:251-257. 35. Maddens B, Heiene R, Smets P, et al: Evaluation of kidney injury in dogs with pyometra based on proteinuria, renal histomorphology, and urinary biomarkers. 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