Metrisor: A novel diagnostic method for metritis detection in cattle based on machine learning and sensors.

The Metrisor device has been developed using gas sensors for rapid, highly accurate and effective diagnosis of metritis. 513 cattle uteri were collected from abattoirs and swabs were taken for microbiological testing. The Metrisor device was used to measure intrauterine gases. The results showed a bacterial growth rate of 75.75 % in uteri with clinical metritis. In uteri positive for clinical metritis, the most commonly isolated and identified bacteria were Trueperella pyogenes, Fusobacterium necrophorum and Escherichia coli. Measurements taken with Metrisor to determine the presence of metritis in the uterus yielded the most successful results in evaluations of relevant machine learning algorithms. The ICO (Iterative Classifier Optimizer) algorithm achieved 71.22 % accuracy, 64.40 % precision and 71.20 % recall. Experiments were conducted to examine bacterial growth in the uterus and the random forest algorithm produced the most successful results with accuracy, precision and recall values of 78.16 %, 75.30 % and 78.20 % respectively. ICO also showed high performance in experiments to determine bacterial growth in metritis-positive uteri, with accuracy, precision and recall values of 78.97 %, 77.20 % and 79.00 %, respectively. In conclusion, the Metrisor device demonstrated high accuracy in detecting metritis and bacterial growth in uteri and could identify bacteria such as E. coli, S. aureus, coagulase-negative staphylococci, T. pyogenes, Bacillus spp., Clostridium spp. and F. necrophorum with rates up to 80 %. It provides a reliable, rapid and effective means of detecting metritis in animals in the field without the need for laboratory facilities.

[Factors influencing inflammatory diseases in the bitch and the survival of her offsprings]. / Einflussfaktoren auf entzündliche Erkrankungen der Mutterhündin und das Überleben ihrer Nachkommen.

INTRODUCTION: Feeding raw meat is becoming increasingly common among dog owners. This feeding practice can pose a hygienic risk and can lead to health risks for dogs and their owners. Hygienically sound food rations, that are balanced in terms of all nutrients and energy, must be feed to breeding dogs. The aim of this study was to record the influence of raw feeding on the occurrence of mastitis, metritis and the survival of puppies. An online questionnaire was sent to kennel club registered dog breeders in Germany and Switzerland. A total of 531 litters were recorded and evaluated. Mastitis and/or metritis were present in 9,2 % and 2,8 % (n = 49 and n = 15, respectively) of the breeding bitches. 29 % (n = 154) of the breeders reported loss of puppies in the litter. The most common cause were stillbirths (n = 105 litters with 187 stillborn puppies), and/or early mortality (n = 50 litters with 73 puppies that died in the first 48 hours). The occurrence of puppy losses in the litter and/or a higher proportion of puppy losses in the litter was favored by increased body weight of the bitch in larger dog breeds, existing previous illnesses, previously reported gestation or postpartum problems, increasing length of the parturition phase and/or a cesarean section. A higher total number of surviving puppies was associated with increased weight and anamnestic reported health of the bitch and the use of milk powder instead of other milk substitutes. A relationship between raw feeding and the occurrence of mastitis, metritis, the total number of surviving puppies and/or the occurrence or proportion of pup losses in the litter and was not found in this study. However very few breeders in this cohort fed their bitches raw, which in turn was due to the random selection of breeders. Many years of breeding experience and the increasing parity of the bitch had a clearly positive influence on the health of the bitch.

INTRODUCTION: L'alimentation à base de viande crue est de plus en plus répandue parmi les propriétaires de chiens. Cette pratique alimentaire peut présenter un risque hygiénique et entraîner des dangers pour la santé des chiens et de leurs propriétaires. Des rations alimentaires hygiéniques et équilibrées en termes de nutriments et d'énergie doivent en particulier être données aux chiens reproducteurs. L'objectif de cette étude était d'enregistrer l'influence de l'alimentation crue sur l'apparition de mammites, de métrites et sur la survie des chiots. Un questionnaire en ligne a été envoyé aux éleveurs de chiens enregistrés auprès d'un club d'élevage en Allemagne et en Suisse. Au total, 531 portées ont été enregistrées et évaluées. Une mammite et/ou une métrite étaient présentes chez 9,2 % et 2,8 % (n = 49 et n = 15, respectivement) des chiennes reproductrices. 29 % (n = 154) des éleveurs ont signalé la perte de chiots dans la portée. La cause la plus fréquente était la mortinatalité (n = 105 portées avec 187 chiots mort-nés) et/ou la mortalité précoce (n = 50 portées avec 73 chiots morts dans les 48 premières heures). Les pertes de chiots dans la portée et/ou une proportion plus élevée de pertes de chiots dans la portée était favorisée par un poids corporel plus élevé de la chienne dans les grandes races de chiens, des maladies antérieures existantes, des problèmes de gestation ou de post-partum déjà signalés, une durée plus longue de la phase de parturition et/ou une césarienne. Un nombre total plus élevé de chiots survivants a été associé à un poids plus élevé et à un état de santé anamnestique de la chienne, ainsi qu'à l'utilisation de lait en poudre plutôt que d'autres substituts du lait. Cette étude n'a pas mis en évidence de lien entre l'alimentation crue et l'apparition de mammites, de métrites, le nombre total de chiots survivants et/ou l'apparition ou la proportion de pertes de chiots dans la portée. Cependant, très peu d'éleveurs de cette cohorte ont nourri leurs chiennes avec des aliments crus, ce qui est dû à la sélection aléatoire des éleveurs. Une longue expérience de l'élevage et l'augmentation de la parité de la chienne ont eu une influence clairement positive sur la santé de la chienne.

Shifts in uterine microbiome associated with pregnancy outcomes at first insemination and clinical cure in dairy cows with metritis.

Objectives were to assess differences in uterine microbiome associated with clinical cure and pregnancy outcomes in dairy cows treated for metritis. Cows with metritis (reddish-brownish, watery, and fetid vaginal discharge) were paired with cows without metritis based on parity and days postpartum. Uterine contents were collected through transcervical lavage at diagnosis, five days later following antimicrobial therapy (day 5), and at 40 days postpartum. Uterine microbiome was assessed by sequencing the V4 hypervariable region of the 16S rRNA gene. Although alpha-diversity based on Chao1, Shannon, and inverse Simpson indexes at diagnosis did not differ between cows with and without metritis, disease was associated with differences in beta-diversity. Prevalence of Porphyromonas, Bacteroides, and Veillonella was greater in cows with metritis. Streptococcus, Sphingomonas, and Ureaplasma were more prevalent in cows without metritis. Differences in beta-diversity between cows with and without metritis persisted on day 5. Uterine microbiome was not associated with clinical cure. Richness and alpha-diversity, but not beta-diversity, of uterine microbiome 40 days postpartum were associated with metritis and pregnancy. No relationship between uterine microbiome and pregnancy outcomes was observed. Results indicate that factors other than changes in intrauterine bacterial community underlie fertility loss and clinical cure in cows with metritis.

Supplementation of rumen-protected lysine during the close-up period improves vaginal discharge clearance in Holstein dairy cows.

We aimed to evaluate the effects of rumen-protected lysine (RPL) supplementation during the close-up period on uterine involution and the resumption of ovarian function in dairy cows. Fifty-two multiparous Holstein cows were categorized based on parity and expected calving date and randomly assigned to the RPL or control (CON) groups. The RPL group received 80 g of RPL daily from day 21 before the expected calving date until parturition. Blood samples were obtained twice weekly from pre-supplementation to 6 weeks postpartum. The onset of luteal activity postpartum was determined via ultrasonography twice weekly for up to 6 weeks postpartum. Uterine involution was tracked at 3 and 5 weeks postpartum through the vaginal discharge score, percentage of polymorphonuclear cells (PMN) in endometrial cytology samples, presence of intrauterine fluid, and gravid horn diameter via ultrasonography. Before supplementation, the RPL group showed amino acid imbalance, which was improved by RPL supplementation. There were no significant differences in the onset of luteal activity, percentage of PMN, intrauterine fluid, or the diameter of the uterine horn between the two groups. The vaginal discharge score in the RPL group decreased from 3 to 5 weeks postpartum, whereas that in the CON groups did not decrease. The number of cows with clinical endometritis was lower in the RPL group. Overall, RPL supplementation during the close-up period enhanced vaginal discharge clearance, potentially averting clinical endometritis, but did not affect the first ovulation in dairy cows.

Changes in bacterial community composition in the uterus of Holstein cow with endometritis before and after treatment with oxytetracycline.

It is important to study the bacteria that cause endometritis to identify effective therapeutic drugs for dairy cows. In this study, 20% oxytetracycline was used to treat Holstein cows (n = 6) with severe endometritis. Additional 10 Holstein cows (5 for healthy cows, 5 for cows with mild endometritis) were also selected. At the same time, changes in bacterial communities were monitored by high-throughput sequencing. The results show that Escherichia coli, Staphylococcus aureus and other common pathogenic bacteria could be detected by traditional methods in cows both with and without endometritis. However, 16S sequencing results show that changes in the abundance of these bacteria were not significant. Endometritis is often caused by mixed infections in the uterus. Oxytetracycline did not completely remove existing bacteria. However, oxytetracycline could effectively inhibit endometritis and had a significant inhibitory effect on the genera Bacteroides, Trueperella, Peptoniphilus, Parvimonas, Porphyromonas, and Fusobacterium but had no significant inhibitory effect on the bacterial genera Marinospirillum, Erysipelothrix, and Enteractinococcus. During oxytetracycline treatment, the cell motility, endocrine system, exogenous system, glycan biosynthesis and metabolism, lipid metabolism, metabolism of terpenoids, polyketides, cofactors and vitamins, signal transduction, and transport and catabolism pathways were affected.

Up-regulation of inflammatory, oxidative stress, and apoptotic mediators via inflammatory, oxidative stress, and apoptosis-associated pathways in bovine endometritis.

Endometritis is the inflammation of the endothelial lining of the uterine lumen and is multifactorial in etiology. Escherichia (E.) coli is a Gram-negative bacteria, generally considered as a primary causative agent for bovine endometritis. Bovine endometritis is characterized by the activation of Toll-like receptors (TLRs) by E. coli, which in turn triggers inflammation, oxidative stress, and apoptosis. The objective of this study was to investigate the gene expression of inflammatory, oxidative stress, and apoptotic markers related to endometritis in the uteri of cows. Twenty uterine tissues were collected from the abattoir. Histologically, congestion, edema, hyperemia, and hemorrhagic lesions with massive infiltration of neutrophil and cell necrosis were detected markedly (P < 0.05) in infected uterine samples. Additionally, we identify E. coli using the ybbW gene (177 base pairs; E. coli-specific gene) from infected uterine samples. Moreover, qPCR and western blot results indicated that TLR2, TLR4, proinflammatory mediators, and apoptosis-mediated genes upregulated except Bcl-2, which is antiapoptotic, and there were downregulations of oxidative stress-related genes in the infected uterine tissue. The results of our study suggested that different gene expression regimes related to the immune system reflex were activated in infected uteri. This research gives a novel understanding of active immunological response in bovine endometritis.

Streptococcus equi subsp. zooepidemicus: High molecular diversity of Argentinian strains isolated from mares with endometritis.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a mucosal commensal of the lower genital tract in horses and is the most isolated bacterium causing endometritis in mares. The aim of this study was to determine the molecular diversity of S. zooepidemicus obtained from endometritis in mares in Buenos Aires province, Argentina. Thirty isolates obtained from the uterus of mares in 2005 and 2017 were studied. The MLST scheme was applied to identify the Argentinian genotypes and the clonal relationships and patterns of evolutionary descent were identified using the eBURST algorithm - goeBURST. Twenty six different Sequence types (STs) were identified, being only 11 of them previously reported in horses and also, from several host species and tissues. The other 15 STs were reported in Argentinian reproductive strains of mares in our study for the first time. The genotypes obtained from uterus in Argentina were not evenly distributed when all the published S. zooepidemicus STs were analysed, thus, it was not possible to establish that the same lineage circulates in our equine population. The fact that the identified genotypes were also reported in other countries, diverse samples and host species suggest that there is not a host, and an anatomical niche adaptation. Finally, the isolation of the same genotype in the vagina/clitoris and the uterus of the same mare highlights the versatility of S. zooepidemicus and its role as an opportunistic pathogen.

Analysis of blood biochemistry and non-targeted metabolomics of endometritis in dairy cows.

The incidence of bovine endometritis, which has a negative impact on the reproduction of dairy cows, has been recently increasing. In this study, the differential markers and metabolites of healthy cows and cows with endometritis were analyzed by measuring blood biochemical indicators and immune factors using biochemical and enzyme-linked immunosorbent assay kits combined with nontargeted metabolomics. The LC-QTOF platform was used to evaluate the serum metabolomics of healthy cows and cows with endometritis after 21-27 days of calving. The results showed that glucose, free fatty acid, calcium, sodium, albumin, and alanine aminotransferase levels were significantly lower in the serum of cows with endometritis than in healthy cows (P < 0.05). However, the serum potassium, interleukin-1, interleukin-6, and tumor necrosis factor levels were significantly higher in cows with endometritis (P < 0.05). In addition, the serum metabolome data analysis of the two groups showed that the expression of 468 metabolites was significantly different (P < 0.05), of which 291 were upregulated and 177 were downregulated. These metabolites were involved in 78 metabolic pathways, including amino acid, nucleotide, carbohydrate, lipid, and vitamin metabolism pathways; signal transduction pathways, and other biological pathways. Taken together, negative energy balance and immune activation, which are related to local abnormalities in amino acid, lipid, and carbohydrate metabolism, were the important causes of endometritis in dairy cows. Metabolites such as glucose, carnosine, dehydroascorbic acid, L-malic acid, tetrahydrofolic acid, and UDP-glucose may be used as key indicators in the hematological diagnosis and treatment of endometritis in dairy cows.

Selecting potential biomarkers of plasma proteins in mares with endometritis.

BACKGROUND: Endometritis is a common condition in mares that causes significant economic loss. Lacking obvious clinical signs, the clinical diagnosis of endometritis in mares relies on case-by-case clinical examinations, which can be particularly inefficient in large-scale farms. Therefore, the identification of potential biomarkers can serve as a non-invasive and efficient screening technique for endometritis in mares. OBJECTIVES: To compare the blood proteome between fertile mares and mares with endometritis to identify biomarkers potentially associated with the development of endometritis and validate their predictive potential. STUDY DESIGN: Observational and experimental study. METHODS: Differentially expressed proteins were identified via Data Independent Acquisition (DIA) proteomic profiling in a screening cohort composed of eight healthy mares and eight mares with endometritis. Subsequently, enzyme-linked immunosorbent assay was employed that included a validation cohort of 40 healthy mares and 40 mares with endometritis to verify the accuracy and sensitivity of the identified proteins, thereby establishing a diagnostic threshold. RESULTS: In the screening cohort, 12 proteins were significantly differentially expressed between endometritis mares and healthy controls (p < 0.05, outside the 1/1.2 to 1.2-fold). In the validation experiment, all six screened proteins were assessed with area under the curve (AUC) >0.8. MAIN LIMITATIONS: The samples displayed certain levels of individual heterogeneity, and the number of samples analysed was limited. Additionally, the identified biomarkers were primarily associated with generalised inflammation, which potentially limited their specificity for endometritis. CONCLUSION: Levels of plasma proteins are sensitive indicators of equine endometritis and potential tools for endometritis screening. In plasma, fetuin B, von Willebrand factor, vitamin K-dependent protein C, insulin-like growth factor binding protein 3, interleukin 1 receptor accessory protein, and type II cell cytoskeleton showed great predictive ability, with fetuin B being the best predictor (AUC = 0.93, 95% CI: 0.89-0.98), which performs better when combined with all six detected proteins (AUC = 1, 95% CI: 0.99-1.00).

Comparison between intrauterine application of an antibiotic and an herbal product to treat clinical endometritis in dairy cattle - A randomized multicentre field study.

Uterine diseases are main indications for antibiotic use in dairy cows. To test a non-antibiotic treatment option, we compared the effect of an intrauterine cephapirin (Metricure®; cefapirin benzathin 500 mg per dosis; CEPH) with an intrauterine applied herbal product (25 ml of EucaComp® PlantaVet containing alcoholic extracts of Calendula officinalis L., Mellissa officinalis L., Origanum majorana L. and Eucalyptus globulus Labill. (EUC)) on the clinical cure of endometritis. Examinations of 816 cows between 21 and 35 days after calving were performed and cases of clinical endometritis (n = 169) were included. Diagnosis based on a scoring system for vaginal discharge. Study animals were randomly assigned to one of two treatment groups and treated immediately. After excluding animals with incomplete datasets, 136 cows (EUC: n = 61; CEPH: n = 75) remained for the final analysis. In total, 64% (EUC: 61%, CEPH: 67%) of analysed endometritis cases were considered as clinically cured 14 ± 2 days after the first treatment, 15% stayed uncured after the application of a maximum of two consecutive treatments, leading to an overall clinical cure rate of 85% (EUC: 82%, CEPH: 88%). No statistically difference in clinical cure rates could be observed between both treatment groups nor 14 ± 2 days after the first treatment (p = 0.956) neither regarding the overall cure rate (p = 0.923). In conclusion, the clinical cure of dairy cows' endometritis after the intrauterine application of the herbal product was non-inferior to the intrauterine application of the antibiotic cephapirin. These results could contribute to reduce the antimicrobial use in the daily veterinary routine treatment of endometritis.

Molecular Mechanisms Associated with the Development of the Metritis Complex in Dairy Cattle.

The metritis complex (MC), a group of post-partum uterine diseases, is associated with increased treatment costs and reduced milk yield and fertility. The goal of this study was to identify genetic variants, genes, or genomic regions that modulate MC disease. A genome-wide association study was performed using a single-locus mixed linear model of 1967 genotypes (624,460 SNPs) and metritis complex records. Then, in-silico functional analyses were performed to detect biological mechanisms and pathways associated with the development of MC. The ATP8A2, COX16, AMN, and TRAF3 genes, located on chromosomes 12, 10, and 21, were associated with MC at p ≤ 0.0001. These genes are involved in the regulation of cholesterol metabolism in the stromal tissue of the uterus, which can be directly associated with the mode of transmission for pathogens causing the metritis complex. The modulation of cholesterol abundance alters the efficiency of virulence factors and may affect the susceptibility of the host to infection. The SIPA1L1, DEPDC5, and RNF122 genes were also significantly associated with MC at p ≤ 0.0001 and are involved in the PI3k-Akt pathway, responsible for activating the autophagic processes. Thus, the dysregulation of these genes allows for unhindered bacterial invasion, replication, and survival within the endometrium.

Antimicrobial efficiency of bromhexine hydrochloride against endometritis-causing Escherichia coli and Trueperella pyogenes in bovines.

In this study, the main agents associated with endometritis in cows in the state of Santa Catarina, Brazil, were identified and the resistance profile and virulence mechanisms of the bacterial isolates were evaluated. Isolates of Escherichia coli and Trueperella pyogenes were tested for their biofilm forming ability and the antimicrobial action of bromhexine hydrochloride in combination with other antimicrobials. A total of 37 uterine lavage samples were collected from cows with endometritis. Of the 55 bacteria isolated, 25.4% were identified as T. pyogenes and 16.3% as E. coli. The bacterial isolates showed greater resistance to sulfamethoxazole + trimethoprim (58.2%) and tetracycline (56.3%). Among the species, E. coli showed the highest resistance rates, with 100% of isolates showing resistance to amoxicillin, streptomycin, and gentamicin. The results of the minimum inhibitory concentration for the T. pyogenes isolates showed that 91.6% of the isolates were resistant to enrofloxacin and tetracycline, and 75% were resistant to ceftiofur and sulfamethoxazole + trimethoprim. All E. coli and T. pyogenes isolates showed biofilm forming ability. The plo, fimA, and nanH genes were identified in 100% of T. pyogenes isolates. In parallel, 100% of E. coli isolates had the fimH gene, and 11.1% had the csgD gene. Bromhexine hydrochloride showed antimicrobial activity against 100% of E. coli isolates and 66.6% of T. pyogenes isolates. Furthermore, when associated with antimicrobials, bromhexine hydrochloride has a synergistic and additive effect, proving to be an option in the treatment of endometritis in cows and an alternative for reducing the use of antimicrobials.

Phenotypic Characteristics, Antimicrobial Susceptibility and Virulence Genotype Features of Trueperella pyogenes Associated with Endometritis of Dairy Cows.

Trueperella pyogenes can cause various infections in the organs and tissues of different livestock (including pigs, cows, goats, and sheep), including mastitis, endometritis, pneumonia, or abscesses. Moreover, diseases induced by T. pyogenes cause significant economic losses in animal husbandry. In recent large-scale investigations, T. pyogenes has been identified as one of the main pathogens causing endometritis in lactating cows. However, the main treatment for the above-mentioned diseases is still currently antibiotic therapy. Understanding the impact of endometritis associated with T. pyogenes on the fertility of cows can help optimize antibiotic treatment for uterine diseases, thereby strategically concentrating the use of antimicrobials on the most severe cases. Therefore, it is particularly important to continuously monitor the prevalence of T. pyogenes and test its drug resistance. This study compared the uterine microbiota of healthy cows and endometritis cows in different cattle farms, investigated the prevalence of T. pyogenes, evaluated the genetic characteristics and population structure of isolated strains, and determined the virulence genes and drug resistance characteristics of T. pyogenes. An amount of 186 dairy cows were involved in this study and 23 T. pyogenes strains were isolated and identified from the uterine lavage fluid of dairy cows with or without endometritis.

Doppler ultrasonographic scan, gene expression and serum profile of immune, APPs and antioxidant markers in Egyptian buffalo-cows with clinical endometritis.

The objective of this study was to elaborate Doppler ultrasonographic scan, genetic resistance and serum profile of markers associated with endometritis susceptibility in Egyptian buffalo-cows. The enrolled animals were designed as; twenty five apparently healthy buffalo-cows considered as a control group and twenty five infected buffalo with endometritis. There were significant (p < 0.05) increased of cervical diameter, endometrium thickness, uterine horn diameter, TAMEAN, TAMAX and blood flow through middle uterine artery with significant decrease of PI and RI values in endometritis buffalo-cows. Gene expression levels were considerably higher in endometritis-affected buffaloes than in resistant ones for the genes A2M, ADAMTS20, KCNT2, MAP3K4, MAPK14, FKBP5, FCAMR, TLR2, IRAK3, CCl2, EPHA4, and iNOS. The RXFP1, NDUFS5, TGF-ß, SOD3, CAT, and GPX genes were expressed at substantially lower levels in endometritis-affected buffaloes. The PCR-DNA sequence verdicts of healthy and affected buffaloes revealed differences in the SNPs in the amplified DNA bases related to endometritis for the investigated genes. However, MAP3K4 elicited a monomorphic pattern. There was a significant decrease of red blood cells (RBCs) count, Hb and packed cell volume (PCV) with neutrophilia, lymphocytosis and monocytosis in endometritis group compared with healthy ones. The serum levels of Hp, SAA, Cp, IL-6, IL-10, TNF-α, NO and MDA were significantly (P˂0.05) increased, along with reduction of CAT, GPx, SOD and TAC in buffalo-cows with endometritis compared to healthy ones. The variability of Doppler ultrasonographic scan and studied genes alongside alterations in the serum profile of investigated markers could be a reference guide for limiting buffalo endometritis through selective breeding of natural resistant animals.

In vitro effects of different ozone preparations on microorganisms responsible for endometritis in the mare.

Infectious endometritis is considered one of the major causes of infertility and it can affect up to 60% of barren mares. It is characterized by the presence of one or more microorganisms in the reproductive tract and it is treated with the administration of antibiotics, ecbolic agents and uterine lavages. Ozone, thanks to its antimicrobial properties that are based on its high oxidative potential, could represent an effective alternative treatment for endometritis. The aim of this study was to test in vitro the bactericidal and fungicidal properties of different ozone formulations, either as gas (experiment 1) or dissolved in two liquid matrices (experiment 2), specifically distilled water or oil (Neozone 4000, Cosmoproject, Parma, Italy), onto 6 different species of microorganisms isolated from mares with clinical endometritis, namely Escherichia coli, Staphylococcus aureus, Streptococcus equi subsp. Zooepidemicus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans. In the first experiment, 3 clinical antibiotic-resistant strains per each species were exposed to different conditions: to O2O3 gas mixtures (15 and 40 µg/ml for 1, 3 and 5 min), to 100 % O2 or left untreated. The results showed a reduction of the microbial count of over 99,9% for every pathogen, time and concentration of O2O3 gas mixtures tested. Furthermore, gaseous ozone showed both a time-dependant effect (5 vs 3 vs 1 min of exposure) and a concentration-dependant effect (40 vs 15 µg/ml) at 1 and 3 min, while after 5 min no differences were observed. In the second experiment, minimum inhibitory concentration (MIC), and minimum bactericidal/fungicidal concentration (MBC, MFC) of ozonated distilled water and ozonated oil were evaluated. Ozonated oil showed a bactericidal/fungicidal activity against all the strains tested (MIC range 12.5-25 % v/v, MBC/MFC range 12.5-50 % v/v) while ozonated distilled water didn't show an observable antimicrobial effect, discouraging its use as an antimicrobial agent for the treatment of endometritis. The results of this in vitro study indicate that both gaseous ozone and ozonated oil exerted remarkable antimicrobial activities and are promising alternative treatments for infectious endometritis, even when caused by antibiotic-resistant bacteria, and encourage further experiments in an effort to scale down or even prevent the use of antibiotics in equine reproduction.

Exploring vaginal discharge scoring to assess clinical metritis severity: Comparison between intrauterine dextrose and systemic antibiotics treatments.

The objectives of this study were to assess: 1) differences in the metabolic status, systemic inflammation, daily milk yield, and daily rumination time between Holstein dairy cows with different vaginal discharge scores (VDS) in the first 7±3 DIM, and 2) effects of intrauterine dextrose infusion on metabolic status, systemic inflammation, daily milk yield and daily rumination time in dairy cows with VDS4 and VDS5. Cows (n=641) from a farm located in central Pennsylvania were screened at 7±3 DIM (study d 0) to assess vaginal discharge scores. Vaginal discharge was scored using a five-point scale (i.e., 1- clear fluid, 2- <50% white purulent fluid, 3- >50% white purulent fluid, 4- red-brownish fluid without fetid smell, and 5- fetid red-brownish watery fluid). Cows with VDS4 and VDS5 were blocked by parity and randomly assigned to one of two treatment groups: 1) CONV (VDS4 n=15; VDS5 n= 23): two injections of ceftiofur (per label; 6.6 mg/Kg) 72 h apart; and 2) DEX (VDS4 n=15; VDS5 n=22): three intrauterine infusions of a 50% dextrose solution (1 L/cow) every 24 h. Cows that presented a VDS 1, 2, and 3 were categorized as normal vaginal discharge animals (NOMVDS; n=35) and were randomly selected and matched by parity to CONV and DEX cows. Daily milk yield and rumination time for the first 150 DIM were collected from on-farm computer records. Blood samples were collected to assess haptoglobin (HP) and ß-hydroxybutyrate (BHB) concentrations at study d 0, d 7, and d 14 relative to enrollment. Subclinical ketosis was defined as having a BHB concentration >1.2 mmol/dL at any of the sampling points. The data were analyzed using the MIXED and GLIMMIX procedures of SAS as a randomized complete block design. When comparing cows with different VDS (i.e., NOMVDS, VDS4, VDS5) separately, cows with VDS5 had the highest concentration of HP at enrollment compared to cows with VDS4 and NOMVDS; however, cows with VDS4 had higher concentrations of HP compared to cows with NOMVDS. Cows with VDS4 or VDS5 had a higher incidence of subclinical ketosis compared to cows with NOMVDS (p=0.005; VDS4= 62.08±9.16%; VDS5=74.44±6.74%; NOMVDS=34.36±8.53%). Similarly, daily milk yield (p<.0001; VDS4=30.17±1.32 kg/d; VDS5=27.40±1.27 kg/d; NOMVDS=35.14±1.35 kg/d) and daily rumination time (p=0.001; VDS4=490.77±19.44 min; VDS5=465±16.67 min; NOMVDS=558.29±18.80 min) was lower for cows with VDS4 and VDS5 compared to cows with NOMVDS at 7±3 days in milk. When analyzing HP concentration between treatment groups in cows with VDS4 (p=0.70), VDS5 (p=0.25), or VDS4 and VDS5 combined (p=0.31), there was no difference in HP concentration by study d 14 between treatment groups. Interestingly, when only cows with VDS4 were considered for treatment, both treatments, DEX and CONV, increased the daily milk yield to the levels of NOMVDS cows by 14 days in milk. On the other hand, when only cows with VDS5 were considered for treatment, cows treated with DEX produced, on average, 4.48 kg/d less milk in the first 150 days in milk compared to cows treated with CONV or cows that had NOMVDS. Similarly, when cows with either VDS4 or VDS5 were considered for treatment, DEX treatment also impaired milk yield. These results suggest that cows with either VDS 4 or 5 have an altered inflammatory status, and decreased milk yield and rumination compared to cows with NOMVDS. Furthermore, DEX treatment may have similar effects on daily milk yield and metabolic status compared to CONV in cows with VDS4, while DEX is not recommended for cows with VDS5.

Profile of uterine flush lipid mediators in cows with subclinical endometritis: pilot study.

Subclinical endometritis affects reproductive outcomes and causes economic losses in dairy cows, thus, it is important to understand disease progression mechanisms and develop diagnostic procedures for better disease management. We measured the levels of 146 lipid mediators in uterine flush samples using lipid chromatography-mass spectrometry. We detected 25 lipid mediators in the uterine flush of both the control and subclinical endometritis cows; 15 of the 25 lipid mediators were AA-derived metabolites. Among the AA-derived metabolites, cyclooxygenase (COX)-generated mediators were the most abundant. Specifically, levels of 11ß-13,14-dihydro-15-keto prostaglandin (PG) F2α, PGE2, PGA2, 13-hydroxyoctadecadienoic acid, and PGD1 were elevated in all the cows with subclinical endometritis. This study may provide new insights for the management of subclinical bovine endometritis.

Endometrial expression of antimicrobial peptides as markers of subclinical endometritis in mares.

BACKGROUND: Endometritis is a major cause of subfertility in mares. Multiparous old mares are more susceptible to developing endometritis given that ageing is associated with an altered immune response and with inadequate physiological uterine clearance after breeding, which can lead to degenerative changes in the endometrium. Molecules such as antimicrobial peptides (AMPs) have been proposed as endometritis markers in the equine species. STUDY DESIGN: Cross-sectional. OBJECTIVES: To investigate the endometrial expression of defensin-beta 4B (DEFB4B), lysozyme (LYZ) and secretory leukocyte peptidase inhibitor (SLPI) genes in mares either affected or not by subclinical endometritis, due to the role of these AMPs in the immune response to bacteria and inflammatory reactions. METHODS: Endometrial biopsy for histopathological and gene expression examinations was performed on 26 mares. The inclusion criteria for the normal mare group (NM, N = 7) were 2-4 years of age, maiden status, no clinical signs of endometritis and a uterine biopsy score of I, while for mares affected by subclinical endometritis (EM, N = 19) the inclusion criteria were 10-22 years of age, barren status for 1-3 years, no clinical signs of endometritis and a uterine biopsy score between IIA and III. RESULTS: A significantly higher expression of LYZ (NM: 0.76 [1.84-0.37] vs. EM: 2.78 [5.53-1.44], p = 0.0255) and DEFB4B (NM: 0.06 [0.11-0.01] vs. EM: 0.15 [0.99-0.08], p = 0.0457) genes was found in endometritis mares versus normal mares. Statistically significant moderate positive correlations were found between the level of expression of LYZ gene and both the age (r = 0.4071, p = 0.039) and the biopsy grade (r = 0.4831, p = 0.0124) of the mares. MAIN LIMITATIONS: The study investigated a limited number of genes and mares, and the presence/location of the proteins coded by these genes was not confirmed within the endometrium by IHC. CONCLUSIONS: If the results of this study are confirmed, LYZ and DEFB4B genes can be used as markers to identify mares that are affected by subclinical endometritis.

Effect of estradiol after bacterial infection on the Wnt/ß-catenin pathway in bovine endometrium epithelial cells and organoids.

Endometritis is a disease caused by a postpartum bacterial infection with a poor prognosis that primarily affects dairy cows. Three-dimensional organoids have been used as a model for endometritis, because they exhibit a structure comparable to that of the endometrium, demonstrating both expansibility and hormone responsiveness. These characteristics render them an ideal platform for in vitro investigations of endometrial diseases. Estradiol (E2) is an endogenous steroid hormone with demonstrated anti-inflammatory properties, and the objective of this study was to determine the mechanism by which E2 modulates the inflammatory response and the Wnt signal transduction pathway in bovine endometrial epithelial cells and organoids following E. coli infection. We present the techniques for isolating and culturing primary bovine endometrial epithelial cells (BEECs), and producing endometrial organoids. For the experiments, the endometrial epithelial cells and organoids were infected with E. coli for 1 h, followed by incubation with E2 for 12 h. The mRNA and protein expressions of the inflammation-related genes, IL-1ß, IL-6, TLR4, and NF-κB, as well as the Wnt pathway-related genes, Wnt4, ß-catenin, c-Myc, and CyclinD1, were assessed using real-time quantitative-PCR and western blotting, respectively. The CCK8 viable cell counting assay was utilized to determine the optimal concentration of the Wnt inhibitor, IWR-1. The mRNA and protein expression of Wnt pathway-related genes was assessed following IWR-1 treatment, while the expression levels of proliferation-associated genes (Ki67, PCNA) and barrier repair genes (occludin, claudin, and Zo-1) in BEECs and organoids were evaluated after E2 treatment. The results of this study show that mRNA expression of the inflammatory genes, IL-1ß, TLR4, and NF-κB (P < 0.05) decreased in BEECs following E2 treatment compared to the E. coli group. The protein expression of the IL-1ß, IL-6, TLR4 and NF-κB genes was also inhibited (P < 0.05). Similar results were observed in tests on the organoids. Our findings demonstrate that E2 significantly upregulates the expression of Wnt-related genes, including ß-catenin and c-Myc, while concurrently downregulating the expression of GSK3ß (P < 0.05). Next, we treated E. coli-infected BEECs and organoids with the Wnt inhibitor, IWR-1. Compared with E. coli and E. coli + E2, the expression of mRNA and protein from Wnt 4, ß-catenin, and CyclinD1 in E. coli + E2 and E. coli + IWR-1 was down-regulated (P < 0.05). The expression of the proliferation genes, Ki67, PCNA, and the tight junction genes, occludin, claudin1, and Zo-1, in organoids was significantly higher than that in BEECs (P < 0.05). In summary, we found strong potential for E2 mitigation of the E. coli-induced inflammatory response in BEECs and organoids, through activation of the Wnt pathway. In addition, the proliferation and repair capacity of organoids was much higher than that of BEECs.

Metagenomic analysis unravels novel taxonomic differences in the uterine microbiome between healthy mares and mares with endometritis.

BACKGROUND: The application of high throughput technologies has enabled unravelling of unique differences between healthy mares and mares with endometritis at transcriptomic and proteomic levels. However, differences in the uterine microbiome are yet to be investigated. OBJECTIVES: The present study was aimed at evaluating the differences in uterine microbiome between healthy mares and mares with endometritis. METHODS: Low-volume lavage (LVL) samples were collected from the uterus of 30 mares classified into healthy (n = 15) and endometritis (n = 15) based on their reproductive history, intrauterine fluid accumulation, gross appearance of LVL samples, endometrial cytology and bacterial culture. The samples were subjected to 16S rRNA sequencing. RESULTS: Notable differences in the uterine microbiome were observed between healthy mares and mares with endometritis at various taxonomic levels. In healthy mares, the most abundant phylum, class, order and family were Firmicutes, Bacilli, Bacillales and Paenibacillaceae, respectively. In contrast, the most abundant corresponding taxonomic levels in mares with endometritis were Proteobacteria, Gammaproteobacteria, Enterobacterales and Enterobacteriaceae, respectively. At the genus level, Brevibacillus and Paenibacillus were more abundant in healthy mares, whereas Escherichia, Salmonella and Klebsiella were more abundant in mares with endometritis. In healthy mares, Brevibacillus brevis was the most abundant species, followed by Brevibacillus choshinensis and Paenibacillus sp JDR-2. However, in mares with endometritis, Escherichia coli was the most abundant species, followed by Salmonella enterica and Klebsiella pneumoniae. CONCLUSIONS: These results confirmed the previously reported presence of a uterine microbiome in healthy mares and helped unravel some alterations that occur in mares with endometritis. The findings can potentially help formulate new approaches to prevent or treat equine endometritis.