A study on the correlation between intrauterine microbiota and uterine pyogenesis in dogs.

Pyometra is a common and high-incidence reproductive system disease in female dogs, and its development involves both hormonal and bacterial factors. Characterization of the endometrial microbiome in healthy dogs and diseased dogs with pyometra remains unclear at present, however. In this study, dogs with pyometra were identified based on the clinical examinations, hematology examinations, vaginal smears and uterine histopathology. The endometrial samples of healthy dogs (n = 30) and diseased dogs (n = 41) were then collected and sequenced by 16S rRNA high-throughput sequencing technology. Dogs with pyometra suffered from inflammation, and their endometrial microbial diversity (ACE and Chao 1 indices) was significantly lower than that of healthy dogs (P < 0.05). The endometrial samples of both groups were enriched in four phyla (Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria), with a greater abundance of Firmicutes in diseased dogs (P < 0.05). At the genus level, the most prevalent microbes in diseased dogs belonged to Pseudomonas, Escherichia-Shigella, Mycoplasma, Enterococcus, Haemophilus, Vibrio and Ralstonia, with lower levels of Mycoplasma, Enterococcus and Haemophilus in the healthy control. Principal co-ordinates analysis and non-metric multi-dimensional scaling showed that the endometrial microbiome of diseased dogs clustered separately from that of the healthy controls (P < 0.05). In the LDA effect size analysis, 18 members of the endometrial microbiome were screened. Of these, the bacterial species Pseudomonas_aeruginosa and microbes within the genera Mycoplasma, Enterococcus and Haemophilus were found to be enriched in the uteruses of diseased dogs. Furthermore, the Random Forests model further confirmed that Mycoplasma and Haemophilus could be considered as biomarkers of diseased endometrium. In conclusion, this study provided a theoretical basis for the development of probiotic preparation in the future.

Identification of Canine Pyometra-Associated Metabolites Using Untargeted Metabolomics.

Canine pyometra frequently occurs in middle-aged to older intact bitches, which seriously affects the life of dogs and brings an economic loss to their owners. Hence, finding a key metabolite is very important for the diagnosis and development of a new safe and effective therapy for the disease. In this study, dogs with pyometra were identified by blood examinations, laboratory analyses and diagnostic imaging, and fifteen endometrium tissues of sick dogs with pyometra and fifteen controls were collected and their metabolites were identified utilizing a UHPLC-qTOF-MS-based untargeted metabolomics approach. The results indicated that the elevated inflammatory cells were observed in dogs with pyometra, suggesting that sick dogs suffered systemic inflammation. In the untargeted metabolic profile, 705 ion features in the positive polarity mode and 414 ion features in the negative polarity mode were obtained in endometrium tissues of sick dogs with pyometra, with a total of 275 differential metabolites (173 in positive and 102 in negative polarity modes). Moreover, the multivariate statistical analyses such as PCA and PLS-DA also showed that the metabolites were significantly different between the two groups. Then, these differential metabolites were subjected to pathway analysis using Metaboanalyst 4.0, and Galactose metabolism, cAMP signaling pathway and Glycerophospholipid metabolism were enriched, proving some insights into the metabolic changes during pyometra. Moreover, the receiver operating characteristic curves further confirmed kynurenic acid was expected to be a candidate biomarker of canine pyometra. In conclusion, this study provided a new idea for exploring early diagnosis methods and a safe and effective therapy for canine pyometra.

Phylogeny and Virulence Factors of Escherichia coli Isolated from Dogs with Pyometra.

We aimed to investigate the occurrence, phylogeny, and virulence of E. coli in the uterine contents and urine of female dogs with pyometra, through the presence of virulence genes and their genetic similarity. Uterine secretions and urine samples from 52 female dogs with pyometra were collected and cultured. Strains identified as E. coli from 25 uterine and 7 urine samples were tested for virulence genes by PCR. Genetic similarity between the isolates was studied using RAPD-PCR. E. coli was observed in 48.07% uterine samples with pyometra and 20.0% urine samples. The strains showed high percentages for the presence of virulence genes: 96.9% had the gene sfa, 59.4% afa, 46.9% pap, 53.1% hly, and 68.75% cnf. Even with the high prevalence of virulence genes, the samples were not submitted to DNA sequencing to confirm the results. Analysis showed high genetic diversity in E. coli, however, strains isolated from the same animal indicate that cystitis and pyometra could be related. Our study indicated the association between E. coli in dogs with pyometra and cases of urinary tract infection and the pathogenic potential of strains increasing with animal age.

The endometrium histopathology and cell ultrastructure in bitches with pyometra induced using progesterone and Escherichia coli.

BACKGROUND: We investigated uterine histopathological and ultrastructural changes in female dogs with pyometra induced by Escherichia coli (E. coli) inoculation using progesterone and/or estradiol. METHODS: Dogs were ovariectomized and classified into six groups: Groups 1-6 corresponding to estradiol treatment followed by progesterone supplementation, progesterone supplementation only, estradiol supplementation only, simultaneous treatment using estradiol and progesterone, similar to Group 1 but with a double dose, and control group, respectively. RESULTS: Pyometra was successfully induced in Groups 1, 2, 4, and 5, but not in Group 3. An uneven endometrial surface was observed, along with a purulent discharge, bleeding, inflammatory lesions, cystic endometrial hyperplasia (CEH) or cystic endometrial atrophy. Endometrial thickness percentage, uterine wall thickness, and the percentage of endometrial cyst area increased. Endometrial epithelial mushroom-like hyperplasia and the honeycomb-like structure exposed under the epithelium after flaky exfoliation were found, and the glandular epithelial villi became longer or shorter. Mitochondria expansion and increased lysosome were observed. Endoplasmic reticulum dilation and swelling and many inflammatory cells, especially plasma cell infiltration in the stroma, were found. CONCLUSIONS: Endometrial histopathology and ultrastructural changes in affected dogs were accompanied by induction of pyometra, and they were affected by different hormonal patterns and E. coli.

The plasma proteome and the acute phase protein response in canine pyometra.

Canine pyometra is a common inflammatory disease of uterus in sexually mature bitches caused by secondary bacterial infection, leading to change in plasma proteins associated with the innate immune system. Proteomic investigation is increasingly being applied to canine diseases in order to identify and quantify significant changes in the plasma proteome. The aim of the study was to assess and quantify changes in plasma proteome profiles of healthy dogs and pyometra affected bitches using a TMT-based high-resolution quantitative proteomic approach. As a result, 22 proteins were significantly down-regulated including transthyretin, antithrombin, retinol-binding protein, vitamin D binding protein, paraoxonase 1, and kallikrein, while 16 were significantly up-regulated including haptoglobin light chain, alpha-1-acid glycoprotein, C-reactive protein precursor, and lipopolysaccharide-binding protein in dogs with pyometra. Pathway analysis indicated that acute inflammatory response, regulation of body fluid levels, protein activation cascade, the humoral immune response, and phagocytosis were affected in pyometra. Validation of biological relevance of the proteomic study was evident with significant increases in the concentrations of haptoglobin, C-reactive protein, alpha-1-acid glycoprotein, and ceruloplasmin by immunoassay. Pyometra in bitches was shown to stimulate an increase in host defence system proteins in response to inflammatory disease including the acute phase proteins. SIGNIFICANCE: The label-based high-resolution quantitative proteomics analysis and bioinformatic approach used in this study provide insight into the complex pathophysiology of inflammation associated with pyometra revealing proteins with biomarker potential. Early diagnosis and therapeutic intervention may prevent severe complications associated with advancing sepsis in dogs with pyometra. Therefore the identification of diagnostic biomarkers that, after clinical validation may be used in veterinary practice and protein relevant to pathways responding to disease are important findings of the study. Data are available via ProteomeXchange with identifier PXD015951.

The development of a silicone vaginal ring with a prostaglandin analogue for potential use in the treatment of canine reproductive disorders.

In veterinary medicine, vaginal rings (VRs) are rarely used. However, there are diseases of female dogs' reproductive system which represent a suitable possibility for their usage. An example of such a disease is canine pyometra which can be treated by lipophilic prostaglandin drugs, unfortunately with harmful side effects after systemic administration. The aim of the study was to prove that the matrix VR based on silicone and channel-forming substance can be successfully used as a carrier for a three-day delivery of prostaglandin E2 (PGE2). Based on an in-vitro release study, an optimum channel-forming substance and its concentration were selected. The results were implemented during the construction of VR from the medical grade silicone DDU-4840 with PGE2 (5 mg). Glucose anhydrous in the 30% concentration was chosen as the most functional channel-forming substance due to synergism of osmotic activity and solubility. The DDU-VR containing PGE2 and 30% of glucose anhydrous exhibited excellent mechanical characteristics and ensured 29% drug release through water-filled channels in first-order kinetic manner. This is eight times higher than a sample without glucose where molecular diffusion through the silicone matrix was dominating the release mechanism. Moreover, drug-free VRs were tested for mechanical resistance and the design of removal thread.

Protein-to-creatinine urinary in the early diagnosis of renal injury in canine pyometra / Relação proteína-creatinina-urinária no diagnóstico precoce de lesão renal em cadelas com piometra

Kidney disease that affects bitches with pyometra may lead patients to develop chronic renal failure even after pyometra treatment. Therefore, several studies have sought to clarify the gaps in the understanding of the pathogenesis of renal injury in pyometra. Identification of early detection markers for renal damage, which can predict and identify the prognosis of the disease, is very important. Proteinuria analysis can diagnose kidney damage, since proteins such as albumin are not filtered through the glomerulus and those that undergo glomerular filtration are almost completely reabsorbed by tubular cells. The objective of this study was to evaluate whether the urinary protein-to-creatinine ratio (UPC) can detect renal injury in bitches with pyometra before development of azotemia. For this, 44 bitches with pyometra were divided into two groups: bitches with azotemic piometra (A, n=15, creatinine >1.7) and bitches with non-azotemic pyometra (NA, n=29). The two groups were compared to the control group (CG, n=12), which had no signs of systemic disease. All animals underwent blood and urine tests. Leukocytosis was more evident in bitches in the A group than in the other groups. This shows that the inflammatory response may be associated with the pathogenesis of renal injury. The median UPC in bitches with pyometra was significantly higher than in the CG, with a median above the reference values. In conclusion, the UPC can be used in bitches with pyometra to detect renal damage before the development of azotemia. It has been suggested that the UPC of bitches with pyometra should be followed through during the postoperative period so that permanent renal lesions secondary to pyometra can be diagnosed and treated properly before the development of azotemia.(AU)

A doença renal que afeta cadelas com piometra pode levar a insuficiência renal crônica mesmo após o tratamento. Portanto, vários estudos procuraram esclarecer as lacunas na compreensão da patogênese da lesão renal na piometra. A identificação de marcadores de lesão renal precoce, que podem prever e identificar o prognóstico da doença é muito importante. A análise da proteinúria pode diagnosticar lesão renal, uma vez que proteínas como a albumina não são filtradas através do glomérulo e aquelas que sofrem filtração glomerular são quase completamente reabsorvidas pelas células tubulares. O objetivo deste estudo foi avaliar se a relação proteína-creatinina urinária (UPC) pode detectar lesão renal em cadelas com piometra antes do desenvolvimento de azotemia. Para isso, 44 cadelas com piometra foram divididas em dois grupos: cadelas com piometra azotêmica (A, n=15, creatinina >1,7) e cadelas com piometra não azotêmica (NA, n=29). Os dois grupos foram comparados ao grupo controle (CG, n=12), que não apresentaram sinais de doença sistêmica. Todos os animais foram submetidos a exames de sangue e urina. A leucocitose foi mais evidente nas cadelas do grupo A do que nos outros grupos. Isso mostra que a resposta inflamatória pode estar associada à patogênese da lesão renal. A mediana da UPC em cadelas com piometra foi significativamente maior que no CG, com uma mediana acima dos valores de referência. Em conclusão, a UPC pode ser usada em cadelas com piometra para detectar lesões renais antes do desenvolvimento de azotemia. Sugeriu-se que a UPC de cadelas com piometra deve ser acompanhada durante o pós-operatório, de modo que as lesões renais permanentes secundárias à piometra possam ser diagnosticadas e tratadas adequadamente antes do desenvolvimento de azotemia.(AU)

Escherichia coli virulence influences the roles of sex hormone receptors in female dogs with simulated pyometra.

The present study aimed to investigate whether Escherichia coli virulence affects the roles of sex hormone receptors in female dogs with simulated pyometra. A total of 33 healthy, nulliparous, crossbred female dogs were divided into four groups, with 10 dogs in each of the three experimental groups and 3 dogs in the control group. Estradiol was administrated to female dogs in group 1 continuously at 0.6-4.8 mg/kg twice daily for 12 days (the dose doubled every three days), followed by intramuscular injection of 0.2-1.8 mg/kg progesterone. The progesterone was administrated with an initial dose of 0.2 µg/kg and increased 0.2 mg/kg every three days, twice daily until the maximum of 1.8 mg/kg for 24 days and maintained at 1.8 mg/kg for 19 days. Progesterone only was administrated at 1.8 mg/kg in group 2 (twice daily) for 55 continuous days and only estradiol was administered with an initial dose of 0.6 µg/kg (dose doubled every 3 days for 12 days) in group 3 twice daily and maintained at 4.8 mg/kg for the following 43 days. A strongly virulent E. coli strain, nau-b, and a weakly virulent strain, nau-i, were screened. On the 12th day of diestrus, 5 female dogs in each of the experimental groups were inoculated with E. coli nau-i strain, while the other five in each group were inoculated with nau-b strain. Histopathological changes of uterine tissues were microscopically observed 50 days after E. coli inoculation and hormone receptor expression levels were detected by quantitative polymerase chain reaction. Simulated pyometra was observed in dogs administrated with progesterone alone or progesterone combined with estradiol. The clinical symptoms and histopathological observation demonstrated that inoculation with strongly virulent E. coli strain, nau-b, caused earlier onset of pyometra symptoms and more severe pyometra symptoms compared with the weakly virulent E. coli strain, nau-i. Furthermore, estrogen and progesterone receptor levels in dogs with pyometra inoculated with E. coli strain nau-i was higher than those in dogs with pyometra inoculated with E. coli strain nau-b. These results indicated that E. coli affects the roles of sex hormone receptors in female dogs with simulated pyometra.

Phylo-typing of clinical Escherichia coli isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR.

BACKGROUND: Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs. OBJECTIVES: In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method. RESULTS: Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates. CONCLUSIONS: The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.

Genotypic and phenotypic comparison of Escherichia coli from uterine infections with different outcomes: clinical metritis in the cow and pyometra in the bitch.

Escherichia coli uterine infection originates different clinical outcomes in the canine and bovine species. Here, E. coli strains isolated from bovine clinical metritis and canine pyometra cases were analyzed by PFGE, screened for 33 virulence factor (VF) genes and for phylogenetic grouping. Bovine and canine E. coli isolates presented a low degree of genetic similarity. Canine E. coli strains belonged to phylogenetic group B2 and presented a high number of VF genes, whereas bovine E. coli strains belonged to phylogenetic groups B1 and A and had a low number of VF genes. In conclusion, E. coli strains isolated from cow clinical metritis had a low potential of virulence. In contrast, bitch pyometra E. coli isolates had a high virulence potential, which might be relevant in the pathogenesis of pyometra. These differences between canine and bovine E. coli isolates may partially explain the different outcomes of the uterine infection in the two species.

Virulence genotypes of Escherichia coli canine isolates from pyometra, cystitis and fecal origin.

Pyometra is the most common diestrual uterine disease of bitches. Escherichia coli is the most frequent bacterium isolated from the uterine content of pyometra uteri and it is associated with the most severe clinical signs, leading to endotoxemia and sepsis. In this study, canine E. coli isolates from pyometra (n=31), cystitis (n=23) and fecal (n=26) origin were compared regarding the prevalence of 23 potential virulence traits (15 virulence factor (VF) genes and 8 pathogenicity associated islands-PAIs), detected by PCR assays. Overall, there was a considerable overlap between pyometra, cystitis and fecal isolates regarding the phylogenetic grouping and virulence traits. Virulence traits more prevalent in pyometra than in cystitis and fecal isolates included two PAIs (PAI IV536 and PAI ICFT073) and three VF genes (sfa/focDE, fyuA and chuA). Regardless the isolates' origin, the average number of virulence traits per strain was higher in B2 than in the other phylogenetic groups (A, B1 and D). The prevalence of phylogenetic group B2 was significantly higher in pyometra (94%) than in cystitis (48%) and fecal (39%) isolates. In conclusion, pyometra isolates have a high potential of virulence and a broad virulence genotype, although being similar to a subset of cystitis and fecal isolates. This leads to the suggestion that cystitis and fecal isolates may be able to induce pyometra in receptive hosts.