Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

AIMS: Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterization, including antimicrobial resistance and virulence profiles, of endometrial pathogenic (EnPEC) and uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. METHODS AND RESULTS: Sixteen E. coli strains, from eight different animals, were analyzed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. CONCLUSIONS: Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

KC-like chemokine as a biomarker of sepsis in dogs with pyometra.

BACKGROUND: Sepsis, defined as a dysregulated inflammatory response to infection inducing organ dysfunction, is a common cause of mortality in both humans and animals. Early detection and treatment is essential for survival, but accurate diagnosis is challenging due to the lack of specific biomarkers for sepsis. This study explored the potential of the keratinocyte-derived chemokine (KC)-like protein in dogs as a biomarker of sepsis in dogs with bacterial uterine infection (pyometra). The aim was to compare KC-like concentrations in dogs with pyometra with or without sepsis and to assess associations between KC-like and clinical variables, including days of hospitalization as an outcome. RESULTS: A mouse KC ELISA was validated and used to determine the concentrations of KC-like in serum from 34 dogs with pyometra and 18 healthy controls. Dogs with pyometra were classified as having sepsis based on two different criteria for systemic inflammatory response syndrome (SIRS), resulting in 74% and 30% sepsis-positive, respectively. The concentration of KC-like protein was higher in pyometra dogs with sepsis than in pyometra dogs without sepsis (p < 0.05) and in healthy controls (p < 0.0001) when using either of the two SIRS criteria. Moreover, KC-like was slightly increased in dogs with pyometra without sepsis compared with healthy controls when using the more stringent SIRS criteria (p < 0.05). Analyses of all dogs showed that KC-like concentrations correlated positively with hospitalization days, C-reactive protein (CRP) concentrations, white blood cells, and percentage of band neutrophils; however, KC-like correlated negatively with hemoglobin and did not correlate with circulating creatinine. CONCLUSIONS: Our results suggest that circulating KC-like protein increases in dogs with sepsis in pyometra and that KC-like is associated with more severe clinical illness. These findings support a potential role of KC-like as a biomarker of sepsis; however, the true identity of KC-like in dogs has yet to be uncovered.

First report of a blaNDM-5-carrying Escherichia coli sequence type 12 isolated from a dog with pyometra in Japan.

Carbapenemase-producing Enterobacterales (CPE) are a serious concern in human clinical settings. Companion animal-origin CPE have been only rarely identified in several countries, but they have not yet been identified in Japan. In this study, we present the first case of a canine infected with CPE in Japan. The patient was hospitalized due to pyometra. The pus discharged from the patient's uterus was subjected to bacteriological analysis. As a result, E. coli was identified in the pus and exhibited resistance to piperacillin, amoxicillin-clavulanic acid, cefazolin, ceftazidime, cefepime, meropenem, amikacin, and sulfamethoxazole-trimethoprim and susceptibility to aztreonam, minocycline, and levofloxacin. Results of the sodium mercaptoacetic acid double-disk synergy test showed that the E. coli isolate was positive for metallo-ß-lactamases. Next-generation sequencing identified the blaNDM-5 gene, which was located in the IncFII-type plasmid together with blaTEM-1b, rmtB, aadA2, bleMBL, sul1, qacE, and dfrA12. The case was treated successfully with doxycycline and orbifloxacin. Our finding emphasizes that close attention should be paid to the significance of CPE harboring multidrug-resistance plasmid in companion animals, based on the perspective of One Health approach in Japan as well as in other countries.

Renal biomarkers, clinical parameters, and renal Doppler velocimetry in bitches with cystic endometrial hyperplasia-pyometra complex.

Cystic endometrial hyperplasia (CEH)-pyometra complex is the most common uterine infection in adult and elderly bitches and can cause renal dysfunction. The aim of this study was to measure and compare urinary creatinine, urea, symmetric dimethylarginine (SDMA), urinary protein-creatinine ratio (UPC), measurement of systolic blood pressure (SBP), and Doppler velocimetry of renal arteries in patients with CEH-pyometra complex before and after an average of 6 months of treatment, evaluating the possibility of the changes persisting. The evaluation was conducted at two moments: M1 (at the diagnosis of CEH-pyometra, n = 36) and M2 (after an average of six months of treatment, n = 16). For the control group, eight bitches with no changes in blood tests or history of conditions underwent Doppler ultrasound evaluation of the renal arteries. At both M1 and M2, we measured creatinine, urea, and serum SDMA, UPC, SBP, and Doppler ultrasound of the renal arteries. Patients were evaluated according to the following groups: azotemic (AZO) and non-azotemic (NAZO), and open and closed cervix pyometra. The parameters were compared between animals present in both moments presented as M1R (bitches that were in M1 and M2) and M2. Statistical significance was considered when p < .05. The medians found for creatinine in M1 were as follows: 1.15 mg/dL, being 1.8 mg/dL for AZO (12/36) and 0.95 mg/dL for NAZO (24/36); and in M2: 0.85 mg/dL (16/16), being 1.15 mg/dL for AZO (4/16) and 0.8 mg/dL for NAZO (12/36). For urea, in M1 it was 36 mg/dL (32/36), with AZO being 103 mg/dL (11/32) and 33 mg/dL in NAZO (21/32); and in M2 32 mg/dL (16/ 16), being 29 mg/dL for AZO (4/36), and 31 mg/dL for NAZO (3/15). The median SDMA measured in M1R was 17 µg/dL (15/16), with AZO being 31 µg/dL (3/15), and NAZO being 16.5 µg/dL (12/15); and in M2, SDMA was 12 µg/dL (16/16), with AZO being 12.5 µg/dL (4/16), and NAZO being 12 µg/dL (12/16). The median UPC measured in M1 was 1.15 (10/36), with AZO being 0.25 (1/10), and NAZO being 1.38 (9/10); and in M2, it was 0.2 (13/16), being 0.1 in AZO (4/13), and 0.2 (9/16) in NAZO. For SBP, in M1, it was 118 mmHg (30/36), with AZO being 102 mmHg (10/30) and 133 mmHg in NAZO (20/30); and in M2 142.5 mmHg (12/16), being 155 mmHg for AZO (4/12), and 140 mg/dL for NAZO (8/12). When comparing animals with open and closed cervixes, a difference was found between SDMA measurements (p = .001). There was a distinction between PI and RI of the left and right kidneys consecutively (p = .007; p = .033; p = .019; p = .041). Correlations found in M1: SDMA × PI RIM DIR (r = 0.873; p = .002), SDMA × PSV RIM ESQ (r = 0.840; p = .004), SDMA × EDV RIM ESQ (r = 0.675; p = .046). With this study, we conclude a return to normality of renal biomarkers and clinical parameters after six months. Yet, there is a persistence of Doppler velocimetric measurements between the two moments. Thus, this parameter is not suitable for identifying and classifying chronic kidney injury in bitches with pyometra.

Exploring oxidative stress, immunological and metabolic biomarkers in dairy cows with postpartum pyometra.

Pyometra is a prevalent and severe infectious disease that affects the reproductive systems of cattle worldwide. This study's main goal was to investigate the biomarkers for oxidative stress (OS), adiponectin, leptin and neopterin (NPT) in cows suffering from postpartum pyometra. The study also aimed to determine which bacteria were most commonly implicated in the development of the disease. A total of 74 cows with pyometra were examined and compared to a control group of healthy cows (n = 20). In comparison to the healthy control and post-treatment groups, the pyometra group showed higher mean values of leptin, adiponectin and malondialdehyde (MDA). In contrast, the glutathione (GSH) and superoxide dismutase (SOD) mean values were lower in the pyometra group as compared to the post-treatment and control groups. NPT levels in the post-treatment groups were lower than those in cows with pyometra but comparable to the healthy control group (p > .05). When compared to the other biomarkers, NPT, leptin and adiponectin showed higher sensitivity and specificity in identifying pyometra cases (AUC ≥0.99). The predominant bacterial isolates from the ptomtra-affected cows consisted of Escherichia coli (N = 29; 39.2%), Arcanobacterium pyogenes (N = 27; 36.5%) and Fusobacterium necrophorum (N = 13; 17.6%). Mixed infection was determined in nine samples (12.2%). Conclusively, OS, adiponectin, leptin and NPT play crucial roles in comprehending the development of postpartum pyometra in cows and have the potential to serve as biomarkers for the disease.

The presence of virulence factor genes downregulates uterine AQP3 and alters glutathione peroxidase activity and uterine histopathology in canine pyometra.

Present study was designed to evaluate the role of virulence factor genes (papG, cnf1 and hylA) in the pathogenesis of canine pyometra. Antimicrobial susceptibility test and detection of virulence genes were performed Escherichia coli (E. coli) detected in uterine swab samples. Animals were divided into two groups based on the presence (VF+, n:14) or absence (VF-, n:7) of the virulence factor genes papG, cnf1 and hylA. Blood and tissue glutathione peroxidase activity, uterine histopathologic analysis and AQP3, ESR1, PGR, OXTR gene expressions were determined in both groups. Statistical analyses were performed using Stata version 15.1. All E. coli isolates were susceptible to amikacin, whereas resistant to ampicillin, amoxicillin/clavulanic acid and lincomycin. None of the isolates were susceptible to cefotaxime. E. coli isolates had at least one virulence gene. The most prevalent gene was fimH (100%), followed by fyuA (95.8%), usp (83.3%), sfa (75%), cnf1 and hlyA (70.8%) genes. Blood GPx activity was greater in VF+ animals. On the other hand, uterine tissue GPx activity was lower in VF+ group compared to the control group. Expression levels of AQP3 were upregulated more than fivefold in VF-dogs compared to the control group. In addition, AQP3 expression levels were found approximately threefold higher in VF (-) than VF (+) group (p < .05). Varying degree of inflammation noted for all animals with pyometra, but the presence of bacteria noted only in VF+ animals. In conclusion, the presence of virulence factor genes does not play a role in the histopathological degree of inflammation, the presence of bacteria was found to vary. Serum GPx activity increased in VF+ animals. While the hormone receptor expressions were similar, AQP expression was upregulated in the absence of virulence factor genes.

Nonfunctional Incomplete Infected Ureteral Duplication in a French Bulldog.

A 7 yr old female French bulldog exhibited recurrent purulent vulvar discharge following an episode of pyometra treated by ovariohysterectomy. The diagnosis of ureteral duplication was established through a combination of ultrasonography, computed tomography scanning, and cystoscopy/vaginoscopy. Despite initial medical intervention, the dog's clinical condition did not improve. Consequently, surgical treatment was pursued to remove the duplicated ureter while preserving the integrity of the urinary tract. Resection of the duplicated ureter was performed from the kidney to the bladder, and the blind ends left in place in the kidney and in the bladder wall were omentalized. Histopathological analysis confirmed the presence of the duplicated ureter. Postoperatively, the dog made a full recovery with no complications or urinary tract dysfunction. Long-term follow-up (11 mo) revealed complete resolution of all clinical signs. Ureteral duplication should be included in the differential diagnosis of vulvar purulent discharge in dogs. Removal of the abnormal ureter resulted in complete resolution of the clinical signs. This case underscores the importance of individualized treatment plans for ureteral duplications in dogs and the potential for successful surgical treatment in selected cases.

Emphysematous pyometra and emphysematous hepatitis caused by Klebsiella pneumoniae infection in a diabetic dog.

A 14-year-old intact female diabetic dog presented with seizures and hyperglycemic hyperosmolar syndrome. Radiographs revealed gas-filled tubular structures in the right and left caudal abdomen, raising concerns of emphysematous pyometra or small intestinal ileus. Ultrasonography played a pivotal role in confirming emphysematous pyometra, a technique previously documented only once in veterinary practice. This report also presents the first documented case of emphysematous pyometra in a diabetic dog attributed to Klebsiella pneumoniae and complicated by emphysematous hepatitis.

Relationships between haptoglobin and inter-alpha trypsin inhibitor heavy chain 4 levels in local and peripheral blood and systemic inflammatory response syndrome in bitches with pyometra.

We aimed to assess the usefulness of monitoring inter-alpha trypsin inhibitor heavy chain 4 (ITIH4) and haptoglobin (Hp) in peripheral and local blood in canine pyometra, and evaluation the relationships among acute phase proteins (APPs), systemic inflammatory response syndrome (SIRS) and the presence of bacteria. The material was collected from bitches with pyometra and from healthy ones. Blood was taken from the cephalic and uterine veins. APPs levels were quantified by ELISA. In the peripheral circulation, the Hp was higher in animals with open-cervix pyometra (OCP) than in the closed-cervix pyometra (CCP) and the control group. The Hp concentration was not correlated with age, with the presence of SIRS or with the type of bacteria (Gram-negative, Gram-positive or mixed flora). The ITIH4 concentrations in the peripheral blood did not differ significantly in the cases of pyometra. The Hp concentration in the local circulation increased in the OCP but not in the CCP groups, although the histopathological changes in the endometrium were similar. Peripheral Hp concentrations may be a useful tool in differentiating between the types of pyometra.

Divergences between serum C-reactive protein and ferritin concentrations in canine pyometra.

The main aim of this report was to investigate and compare the response of serum C-reactive protein (CRP) and ferritin, two positive acute phase proteins (APPs) which usually show an increase in inflammatory processes, in dogs with pyometra. For this purpose, two different studies were made. In the first one , both proteins were measured together in an APPs profile in 25 dogs with pyometra, 25 dogs with pancreatitis (as an example of a positive inflammatory control group), and in 25 healthy dogs. In the second study, to advance the knowledge of the changes and evolution of serum ferritin and CRP in dogs with pyometra after treatment, the concentrations of both APPs were analyzed in 30 dogs with pyometra at diagnosis and after ovariohysterectomy and in 10 clinically healthy female dogs before and after elective spaying. In both studies, bitches with pyometra showed significant increases in serum CRP, indicating an inflammatory condition, but not in serum ferritin despite being a moderate positive APP. This divergence between the dynamics of these APPs could be a useful tool for the suspicion of cases of canine pyometra.

Abdominal perfusion in canine patients with pyometra and sepsis evaluated by Doppler and contrast-enhanced ultrasound.

BACKGROUND: Sepsis is a condition characterized by organic dysfunction, leading to hemodynamic instability and high morbidity and mortality rates in humans and animals. Early identification of perfusion changes and appropriate management of sepsis are crucial for improving patient prognosis. Currently, the Systemic Inflammatory Response Syndrome (SIRS) and Sequential Organ Failure Assessment (SOFA) scores are widely studied for sepsis identification and evaluation of organ dysfunction. However, these scores do not assess gastrointestinal involvement, which is common in this condition. Contrast-enhanced ultrasound (CEUS) and Doppler have been considered promising diagnostic techniques for detecting changes in vascularization and microcirculation in a non-invasive and safe manner, particularly in the gastrointestinal system. This study aimed to evaluate duodenal perfusion using CEUS, as well as abdominal aortic and cranial mesenteric artery blood flow using Doppler ultrasound, and systolic arterial pressure (SAP) in 17 bitches with pyometra and in 10 healthy animals. RESULTS: The variables were compared between the pyometra and control groups, as well between patients with and without sepsis determined by the SOFA or SIRS scores. Pyometra was found to cause a reduction in abdominal aortic blood flow volume, aortic peak systolic velocity, and resistivity index as evaluated by Doppler ultrasound. Patients with sepsis according to the SOFA criteria only presented lower SAP. In contrast, sepsis animals identified by the SIRS score exhibited lower SAP, aortic peak systolic velocity, aortic blood flow volume, and aortic resistivity index and additionally, higher peak intensity of contrast in the duodenal wall. CONCLUSIONS: Pyometra causes a reduction in abdominal aortic blood flow, which is more pronounced in animals with sepsis identified by the SIRS criteria. These animals also exhibited a decrease in systolic blood pressure and an increase in duodenal perfusion, as evident by CEUS. However, these changes were not observed in patients with sepsis identified by the SOFA criteria. The alterations in intestinal perfusion observed in animals with sepsis indicate the presence of inflammation or dysfunction. In this regard, CEUS proves to be a valuable technique for detecting subtle changes in tissue hemodynamics that may not be apparent in conventional exams.

A double-blinded randomized placebo-controlled non-inferiority trial protocol for postoperative infections associated with canine pyometra.

BACKGROUND: Pyometra is a common infectious condition, especially in elderly bitches. In addition to an infected uterus, dogs may have concurrent urinary tract infection (UTI). The preferred treatment is surgical removal of the ovaries and uterus, whereupon the general prognosis is excellent. In addition, antimicrobial therapy is frequently prescribed for postoperative treatment. However, no research exists on the benefit of postoperative antimicrobial treatment in uncomplicated canine pyometra. Antimicrobial resistance has become a major challenge in treatment of bacterial infections. Diminishing overuse of antimicrobial agents is essential for controlling the development of antimicrobial resistance in both animals and humans. METHODS: This double-blinded, randomized, placebo-controlled two-arm clinical trial is designed to compare the incidence of postoperative infections associated with surgical treatment of uncomplicated pyometra followed by two different treatment protocols. For the study, 150 dogs presenting with an uncomplicated pyometra and that are to undergo surgical treatment will be recruited. Dogs with body weight < 3 or > 93 kg, complicated pyometra, primary disease increasing the risk of infection, or immunosuppressive medication will be excluded. All dogs will receive one dose of sulfadoxine-trimethoprim intravenously as an antimicrobial prophylaxis. Postoperatively, dogs will be randomized to receive either a five-day course of placebo or an active drug, sulfadiazine-trimethoprim orally. During the surgery microbiological samples will be taken from urine and uterine content. The follow-up includes a control visit in 12 days and an interview of the owner 30 days after surgery. If bacteriuria is detected at the time of surgery, a urinary sample will be cultured for bacterial growth at the control visit. The primary outcome is the incidence of a postoperative surgical site infection (SSI), and the secondary outcome is the occurrence of clinical UTI with bacteriuria. Intention-to-treat and per-protocol analyses will be performed to compare outcome incidences between the treatment groups. DISCUSSION: Research-based evidence is necessary to create treatment guidelines for judicious use of antimicrobials. The goals of this study are to provide evidence for reducing the use of antimicrobials and targeting the treatment to patients proven to benefit from it. Publishing the trial protocol will increase transparency and promote open science practices.

Secretory leucocyte protease inhibitor (SLPI) as an adjunct prognostic biomarker for canine pyometra.

Pyometra is a potentially life-threatening condition that affects intact female dogs in their middle to advance age. Timely diagnosis and appropriate treatment are critical for the survival of patients, especially when pyometra advances to sepsis. This study aimed to investigate the prognostic potential of certain haematology, serum biochemical and inflammatory biomarker, secretory leucocyte protease inhibitor (SLPI) for pyometra in bitches (n = 41). Blood samples were collected after clinical diagnosis of pyometra for haematology and serum biochemistry. Based on the prognosis following medical/surgical treatment, animals were retrospectively categorized into survivor (n = 29) and dead (n = 12). Endometrial tissue sections were obtained from the bitches undergoing ovariohysterectomy (n = 21). Serum concentration of SLPI was quantified using sandwich ELISA and its expression in the endometrium was investigated using RT-qPCR. A marked increase in the total leucocyte count (TLC), neutrophils, blood urea nitrogen (BUN) and serum creatinine was observed in the female dogs that did not survive. Significant elevation in the serum SLPI concentration (3.49 ± 0.44 vs. 2.38 ± 0.13 ng/mL) was observed in the bitches those died after the treatment, in comparison to those survived (p < .01). Additionally, there was a notable upregulation of SLPI in the endometrium in the bitches those died due to pyometra. Based on the ROC analysis results, it was observed that a cut-off concentration of 2.93 ng/mL for SLPI, 27.77 mg/dL for BUN and 16.3 × 103 /µL for TLC could effectively distinguish the prognosis of pyometra-affected dogs. From this study, it can be concluded that upregulation of SLPI in the endometrium and its elevated concentration in peripheral circulation along with TLC and BUN concentration could serve as valuable indicators for predicting the prognosis of pyometra in bitches.

Serum concentrations of secretory leukocyte protease inhibitor and IL6 can predict the onset of sepsis in pyometra bitches.

As onset of sepsis adversely affects the prognosis of canine pyometra, finding biomarkers that would distinguish sepsis status would be useful in the clinical management. Accordingly, we hypothesized that differential expression of endometrial transcripts and circulating concentration of certain inflammatory mediators would discriminate pyometra-led sepsis (P-sepsis+) from those of pyometra without sepsis (P-sepsis-). Bitches with pyometra (n = 52) were classified into P-sepsis+ (n = 28) and P-sepsis- (n = 24) based on vital clinical score and total leukocyte count. A group of non-pyometra bitches (n = 12) served as control. The relative fold changes in the transcripts of IL6, IL8, TNFα, IL10, PTGS2, mPGES1 and PGFS, SLPI, S100A8, S100A12 and eNOS were determined by quantitative polymerase chain reaction. Furthermore, the serum concentrations of IL6, IL8, IL10, SLPI and prostaglandin F2α metabolite (PGFM) were assayed by ELISA. The relative fold changes in S100A12 and SLPI and mean concentrations of IL6 and SLPI were significantly (p < .05) higher in P-sepsis+ than that of P-sepsis- group. Receiver operating characteristic analysis revealed that serum IL6 had a diagnostic sensitivity of 78.6% and a positive likelihood ratio (LR+) of 2.09, at a cut-off value of 15.7 pg/mL to diagnose P-sepsis+ cases. Similarly, serum SLPI had a sensitivity of 84.6% and an LR+ of 2.23, at a cut-off value of 2.0 pg/mL. It was concluded that SLPI and IL6 would serve as putative biomarkers for pyometra-led sepsis in bitches. Monitoring SLPI and IL6 would be a useful adjunct to the established haemato-biochemical parameters in customizing the treatment strategies and arriving at the decision for management of pyometra bitches with critical illness.

Sonographic features of the uncomplicated postoperative abdomen in dogs treated for pyometra by ovariohysterectomy.

Pyometra is a prevalent disease in intact bitches and the standard treatment is ovariohysterectomy (OHE). Published descriptions of normal sonographic findings after OHE are currently lacking. The aims of this prospective observational study were to describe and compare postoperative abdominal sonographic features for three timepoints following OHE in a group of dogs with pyometra and an uneventful recovery. A total of 22 dogs had sequential focused abdominal ultrasound examinations on days 1, 4-6, and 10-15 postsurgery. Recorded sonographic features for each examination time point and characteristics of the cervical stump and the mesovarium, size, and echogenicity of medial iliac lymph nodes (MILNs), presence of free peritoneal fluid, and pneumoperitoneum. The cervical stump appeared as a heterogenous area with a hypoechoic center surrounded by hyperechogenic tissue in all dogs. The cervical stump transverse-sectional area was larger on day 4-6 compared with day 1 and day 10-15 (P = .0009). Mesovarium ligature reactions were identified as heterogeneous and hyperechoic areas with central and/or edge shadowing in all dogs. The size and echogenicity of MILNs and the mesovarium reactions did not significantly differ among time points. Free peritoneal fluid was detected in 45%, 41%, and 9% and pneumoperitoneum in 95%, 82%, and 14% of dogs at sequential time points. Findings from this sample of dogs with an uneventful recovery following OHE due to pyometra can be used to assist veterinarians in interpreting postoperative abdominal ultrasonographic characteristics in future dogs treated surgically for pyometra.

Lipid droplet dynamics in healthy and pyometra-affected canine endometrium.

BACKGROUND: Accumulation of lipid droplets (LDs) was recently observed in pyometra-affected uteri. As data about their nature and function are missing we intended to compare the localization, quality and quantity of LDs in canine healthy and pyometra-affected tissues and in an in vitro model. METHODS AND RESULTS: We characterized LDs in healthy and pyometra uterine tissue samples as well as in canine endometrial epithelial cells (CEECs) in vitro by means of histochemistry, immunohistochemistry, transmission electron microscopy, western blot, and RT-qPCR. Oil Red O (ORO) staining and quantification as well as p-phenylenediamine staining showed a higher number of LDs in epithelial cells of pyometra samples. Immunohistochemistry revealed that the amount of LDs coated by perilipin2 (PLIN2) protein was also higher in pyometra samples. Transmission electron microscopy showed an increase of LD size in surface and glandular epithelial cells of pyometra samples. In cell culture experiments with CEECs, supplementation with oleic acid alone or in combination with cholesterol lead to an increased LD accumulation. The expression of PLIN2 at protein and mRNA level was also higher upon oleic acid supplementation. Most LDs were double positive for ORO and PLIN2. However, ORO positive LDs lacking PLIN2 coating or LDs positive for PLIN2 but containing a lipid class not detectable by ORO staining were identified. CONCLUSIONS: We found differences in the healthy and pyometra-affected endometrium with respect to LDs size. Moreover, several kinds of LDs seem to be present in the canine endometrium. In vitro studies with CEECs could show their responsiveness to external lipids. Since epithelial cells reacted only to oleic acid stimulation, we assume that the cyclic lipid accumulation in the canine endometrium is based mainly on triglycerides and might serve as energy provision for the developing early embryo. Further studies are necessary to verify the complex role of lipids in the healthy and pyometra-affected canine endometrium.

Endometrial hyperplasia and pyometra in captive lions (Panthera leo) and tigers (Panthera tigris).

Endometrial hyperplasia (EH) is a pathologic condition of the uterus with increased endometrial gland to stroma ratio compared to normal cyclic uterine proliferation. In domestic animals, EH often involves cystic distension of proliferating endometrial glands and may be concurrent with pyometra. In large captive nondomestic felids, an association between EH and pyometra is common; however, detailed species differences between the histological uterine findings in lions (Panthera leo) and tigers (Panthera tigris) and clinical manifestations have yet to be described. Uterine sections from 14 lions and 24 tigers with EH and/or pyometra were scored for several histological parameters and clinical histories were recorded. The percentage of endometrium affected by hyperplasia, endometrial gland to stroma ratio, and adenomyosis were significantly (P = .0385, P = .0008, and P = .0463, respectively) more severe in lions compared to tigers as univariate analytes. Although tubular complexity was not statistically significant (P = .3254), when combined as a proposed EH grading scheme, these 4 features confirmed lions had significantly (P = .0068) more severe EH compared to tigers. Endometrial hyperplasia severity significantly correlated with inflammation/pyometra severity when controlling for species (P = .0203). A significant correlation exists between pyometra-associated clinical sign severity and the presence of pyometra in tigers, (P = .0026) but not in lions (P = .1144). There was no statistical difference in the severity of clinical signs associated with pyometra between these species (P = .1986). This proposed grading scheme may have clinical utility in providing a more consistent and objective evaluation of EH in large captive felids.

Analysis of the concentrations of some haematological parameters, C-reactive protein and anti-Müllerian hormone in bitches affected by pyometra.

The work was aimed to research into the vital signs, haematological and biochemical parameters, acute-phase protein concentrations (CRP, albumin), progesterone (PG) and anti-Müllerian hormone (AMH) concentrations in bitches with pyometra and their comparison with those in healthy bitches. In addition, the goal of this work was to assess if some of the tested parameters may be used as a biomarker in the diagnostics of pyometra and the monitoring of the postoperative recovery. Forty 3-6-year-old bitches of various breeds were included in the research and allotted to two groups: the control (C, n = 20) and the diseased - bitches diagnosed with pyometra (P, n = 20). Blood samples for the analyses were taken immediately before surgery (0 h), and 12 h, 24 h, 48 h and 72 h after, excepting for AMH and PG concentrations, which were determined only at 0 h. Clinical examination detected significantly higher respiration rates in bitches with pyometra in comparison to healthy ones at all time points, higher heart rates before and 12 h after surgery, and higher body temperature before and 12 h, 48 h and 72 h post operation (p < .01). Significantly higher concentrations of CRP (p < .01) and lower concentrations of albumin were detected in bitches with pyometra in comparison to their healthy counterparts at all time points. Progesterone and AMH concentration analysis immediately before surgery detected significantly higher levels of the hormone in bitches with pyometra than in the control (p < .01). The inflammatory reaction of the endometrium resulted in a significant AMH concentration increase, whilst increased CRP concentrations and lower albumin concentrations throughout the research may be used as biomarkers of proinflammatory activities, that is of the acute-phase response.

Antimicrobial susceptibility and production of virulence factors by bacteria recovered from bitches with pyometra.

Pyometra is one of the most common diseases in adult female dogs, characterized by a suppurative bacterial infection of the uterus with accumulation of inflammatory exudate and a variety of local and systemic clinical manifestations. This study aimed to identify the bacteria within the uterine content and vaginal canal of bitches with pyometra and evaluate their antimicrobial susceptibility and production of virulence factors. Uterine and vaginal content were collected with sterile swabs from 30 bitches diagnosed with pyometra. Bacteria were identified and assessed for their antimicrobial susceptibility and production of virulence factors, including biofilms, siderophores, proteases and hemolysins, both in planktonic and biofilm forms. A total of 82 bacterial isolates (35 uterus, 47 vagina), belonging to 21 species, were identified, with Escherichia coli as the most prevalent species (32/82, 39%). As for susceptibility, 39/79 (49.4%) isolates were resistant to one or more drugs, with resistance proportion among Gram-positive bacteria (87.5%) higher (p < .05) than that observed for Gram-negative bacteria (32.7%). Four coagulase-negative Staphylococcus species were resistant to methicillin. Regarding virulence, the isolates had low production of biofilms, siderophores, proteases and hemolysins, suggesting that the occurrence of pyometra might be more associated with host-related factors than bacterial virulence.

Peroxisome proliferator-activated receptor expression in the canine endometrium with cystic endometrial hyperplasia-pyometra complex.

The most common uterine diseases in bitches occurring during diestrus are cystic endometrial hyperplasia (CEH) and pyometra. These diseases can coexist as CEH-pyometra complex (CEH-P). Their pathogenesis has not been fully explained. Peroxisome proliferator-activated receptors (PPARs) are important factors regulating mammalian reproductive function and inflammatory processes. Although there is a lack of data concerning the expression of PPARs in the canine endometrium during CEH and CEH-P, we hypothesized that they might be involved in the development of pathological disorders of the canine endometrium. Therefore, the current study was conducted to evaluate and compare PPARα, PPARδ and PPARγ mRNA expression using quantitative real-time PCR (qPCR) and their immunolocalization using immunohistochemistry (IHC) staining in the endometrium of clinically healthy bitches (control group; n = 8) and those with CEH (n = 8) or CEH-P (n = 8). For quantification, the arithmetic means of all intensities of immunostaining from the cells were measured with the optical density. PPARα, PPARδ and PPARγ were detected in the luminal epithelium, glandular epithelium and stromal cells. The mRNA transcription of PPARα was higher in the CEH group than in the control group (p < .05). Additionally, the mRNA expression and immunostaining intensities of PPARδ and PPARγ in the endometrium in the CEH-P group were downregulated relative to those in the control group (p < .05). Moreover, the serum progesterone concentration measured by direct radioimmunoassay was decreased in the CEH-P group compared to the control group (p < .001) and CEH group (p < .05). The obtained results indicate that PPARs are present in the canine endometrium and that their mRNA profile and intensity levels change under pathological conditions such as CEH and CEH-P. This finding may suggest a correlation between changes in the PPAR expression profile and hormonal disturbances, as well as the potential involvement of PPARs in signal transduction during inflammatory processes occurring in the endometrium during CEH-P. These results pave the way to further research into the role of PPARs in the pathogenesis of CEH and CEH-P in female dogs.