General and comparative aspects of endometritis in domestic species: A review.

Endometritis is a leading cause of sub- and infertility in domestic animal species. The healthy uterus is colonized by commensal bacteria, viruses and yeast/fungi that represent the nonpathogenic microbiota. A shift in the number or type of organisms accompanied by immune dysfunction, however, may trigger uterine infection and inflammation. Metritis is associated with inflammation of all uterine layers (endometrium, myometrium and perimetrium), whereas endometritis is a more superficial inflammation involving solely the endometrium. Endometritis generally occurs at two time points in domestic animal species, postpartum and postmating. Postpartum endometritis may chronically persist, either as a low-grade disease that often manifests as a vaginal discharge but not a systemic illness (in some species termed clinical endometritis) or sometimes subclinical where features are only detected by endometrial sampling. Contamination of the uterus at the time of mating occurs by direct deposition of semen (ejaculated or artificially inseminated) into the uterus. Improper drainage of the ejaculatory fluid or an inadequate immune response may result in persistent mating-induced endometritis. Both postpartum and postmating endometritis interferes with fertility by creating a suboptimal environment for embryo development and placentation, and chronic endometritis may have an impact on sperm survival and fertilization ability. In the postpartum animal, there may also be changes in milk production and maternal behaviour, which can affect offspring health and survival. Preventive strategies for endometritis largely depend on monitoring their known risk factors, which are sometimes specific with regard to the species. Effective, nonantibiotic therapy for endometritis is not available to date. Overall, extensive research has been performed in cattle and horses to unravel key aspects of endometritis, but in sows and bitches, the available literature is scant. Thus, the need and opportunity to investigate the condition vary considerably among domestic species and necessitate their comparative assessment. This article reviews general and comparative aspects of the diagnosis and classification, pathogenesis, preventive strategies and therapeutics of endometritis in domestic species with a specific focus on cows, mares, sows and bitches.

Effect of overconditioning on the hepatic global gene expression pattern of dairy cows at the end of pregnancy.

Overconditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that overconditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of normal- versus overconditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were killed 2 wk before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before cows were killed. After cows were killed, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (median BCS = 3, range 2.75-3.5) and 5 as overconditioned (median BCS = 4, range 4-5). Regression models confirmed that normal-conditioned cows had lower BFT (1.29 ± 0.29 cm; least squares means ± standard error) and serum NEFA (0.16 ± 0.04 mmol/L) in comparison to overconditioned cows (3.14 ± 0.43 cm and 0.38 ± 0.07 mmol/L for BFT and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- versus overconditioned cows (4.63 ± 0.40% and 6.06 ± 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal- and overconditioned cows, 1 overconditioned cow had a relatively low (5.21%) and 1 normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes analysis (edgeR quasi-likelihood method) showed that normal-conditioned cows presented 11 upregulated and 12 downregulated genes in comparison to overconditioned cows. Linear discriminant analysis effects size revealed 133 differentially expressed genes between normal- versus overconditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (ALB, SELENOP, IGF1, and IGF2). On the other hand, overconditioned cows had upregulated genes associated with the acute-phase response (C3, HPX, and, LBP). High basal lipolysis in overconditioned cows at the end of pregnancy increased liver lipid content, and this may alter the hepatic gene expression pattern to a pro-inflammatory state.

Retrospective study of factors associated with bovine infectious abortion and perinatal mortality.

Abortion and perinatal mortality, leading causes of economic loss in cattle industry, are the consequence of both non-infectious and a wide range of infectious causes. However, the relative contribution of pathogens to bovine abortion and perinatal mortality is poorly documented, since available studies involved only a limited number of pathogens. Therefore, the objectives of the present monitoring study were to determine the prevalence of infectious agents associated with bovine abortion and perinatal mortality, and to identify differences in production type, gestation length, parity and seasonality by using mixed effect models (logistic regression). A pre-established sampling protocol based on the collection of the aborted fetus/calf and a corresponding maternal blood sample, involving diagnostic testing for 10 pathogens, was performed. At least one potential causal agent of the abortion or perinatal mortality was detected in 39 % of cases. In these diagnosed cases, Neospora caninum was the most detected pathogen, followed by Trueperella pyogenes, BVDv, Escherichia coli, and Aspergillus fumigatus. Neospora caninum [odds ratio (OR): 0.4; 95 % confidence interval (CI): 0.3-0.7] and Aspergillus fumigatus (OR: 0.1; 95 % CI: 0.1-0.3) were detected less in late versus early gestation. Aspergillus fumigatus was less common in dairy in comparison to beef abortion cases (OR: 0.2; 95 % CI: 0.1-0.6). Winter was associated with a lower positivity for Neospora caninum and BVDv in comparison to warmer seasons. Despite extensive diagnostic testing, an etiological diagnosis was not reached in 61 % of cases, highlighting the need for even more extensive (non-)infectious disease testing or more accurate tests.

Dynamics of uterine microbiota in postpartum dairy cows with clinical or subclinical endometritis.

Our objectives were to describe and compare the uterine bacterial composition of postpartum Holstein cows diagnosed as healthy (n = 8), subclinical endometritis (SCE; n = 8), or clinical endometritis (CE; n = 5) in the fifth week postpartum. We did metagenomic analyses of 16S rRNA gene sequences from endometrial cytobrush samples at 10, 21, and 35 days in milk (DIM), and endometrial bacterial culture at 35 DIM. Uterine bacterial composition in healthy, SCE, and CE was stable at 10, 21, and 35 DIM. Alpha and beta diversities showed a different uterine microbiome from CE compared to healthy or SCE, but no differences were found between healthy and SCE cows. At the phylum level, the relative abundance of Bacteroidetes and Fusobacteria, and at genera level, of Trueperella was greater in CE than healthy or SCE cows. Trueperella pyogenes was the predominant bacteria cultured in cows with CE, and a wide variety of bacterial growth was found in healthy and SCE cows. Bacteria that grew in culture were represented within the most abundant bacterial genera based on metagenomic sequencing. The uterine microbiota was similar between SCE and healthy, but the microbiome in cows with CE had a loss of bacterial diversity.

Effect of anti-inflammatory treatment on systemic inflammation, immune function, and endometrial health in postpartum dairy cows.

Systemic inflammation (SI) is increasingly studied in several species because it may be central in many metabolic disturbances and be a risk factor for clinical disease. This proof-of-concept study evaluated the effects of the anti-inflammatory drug meloxicam on markers of SI and energy metabolism, polymorphonuclear neutrophil (PMN) function, and endometritis in clinically healthy postpartum dairy cows. Cows received meloxicam (0.5 mg/kg of body weight; n = 20) once daily for 4 days (10-13 days postpartum) or were untreated (n = 22). Blood samples were collected -7, 1, 3, 5, 7, 10, 11, 12, 13, 14, 18, 21, 28, and 35 days relative to calving to measure serum concentrations of metabolic and inflammatory markers. Function of peripheral blood PMN were evaluated at 5, 10, 14, and 21, and proportion of PMN in endometrial cytology were performed at 5, 10, 14, 21, 28 and 35 days postpartum. Meloxicam decreased serum haptoglobin from the second until the last day of treatment, and improved indicators of energy metabolism (lesser ß-hydroxybutyrate and greater insulin-like growth factor-1 during treatment, and greater glucose at the end of treatment than control cows). This improved PMN function at 14 days postpartum, but the endometrial inflammatory status was not affected.

Feed restriction to induce and meloxicam to mitigate potential systemic inflammation in dairy cows before calving.

Most dairy cows experience a transient decrease in feed intake in the 1 to 2 wk before calving, which has been associated with systemic inflammation (SI), indicated by increased blood haptoglobin (Hp) concentration. We aimed to characterize the association between prepartum decrease in feed intake and the onset of SI and, if present, the ability of meloxicam (MEL), a non-steroidal anti-inflammatory drug, to mitigate SI. Holstein cows (n = 45) were assigned to control (n = 13), feed restriction (FR) untreated (FR-U; n = 15), and FR treated with MEL (FR-T; n = 17) groups. Daily feed intake was measured from -22 d from expected parturition until 35 d postpartum. Control cows were fed ad libitum, whereas FR-U and FR-T cows were reduced to 60% of their average intake for 4 consecutive days (-15 to -12 d from expected calving). The FR-T cows received MEL (0.5 mg/kg of body weight) once daily for 4 consecutive days (-13 to -10 d from expected calving). Blood samples were collected -22, -15, -14, -13, -12, -10, -7, -5, -3, 0, 1, 3, 5, 7, 15, 22, and 35 d relative to calving to measure serum concentrations of total calcium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, ß-hydroxybutyrate, nonesterified fatty acids, Hp, and insulin-like growth factor-1. Serum concentrations of lipopolysaccharide-binding protein were measured -22, -15, -14, -13, -12, and -10 d from expected calving. Simplified glucose tolerance tests were performed on -15, -12, -5, 1, and 5 d relative to calving. Mixed linear regression models were used to assess the effects of FR and MEL on each metabolite. The interaction between treatment group and blood sampling day was forced into each model. All models accounted for body condition score, parity, and the cow as a random effect. Nonesterified fatty acids concentrations in both the FR-U and FR-T groups significantly increased from the second until the last day of FR. Feed restriction increased urea concentrations compared with the control group on -14 d but decreased urea concentrations on -10 d from expected calving. Control cows had greater ß-hydroxybutyrate concentrations compared with FR cows on 15, 21, and 35 d postpartum. For all other metabolites, no differences were found. This model of FR produced substantial fat mobilization but based on serum Hp and lipopolysaccharide-binding protein concentrations did not generate measurable SI; therefore, we were unable to evaluate the ability of MEL to mitigate SI.

Technical note: Assessment of neutrophil endocytosis and proteolytic degradation and its relationship with phagocytosis and oxidative burst in dairy cows.

Conventional assays of polymorphonuclear cell (PMN, neutrophil) function such as oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. Oxidative burst is commonly evaluated by measuring PMN median fluorescence intensity (MFI) involving the release of reactive oxygen species after in vitro stimulation. Phagocytosis can be measured by engulfment of fluorescent beads by PMN. DQ-ovalbumin (DQ-OVA) is a molecule suitable for the assessment of intracellular proteolytic degradation, so it might be informative about an additional pathway of pathogen handling by PMN. In this study, we evaluated the use of the DQ-OVA assay for the assessment of PMN function and the relationships among OB, PC, and DQ-OVA results in PMN isolated from blood of dairy cows between 5 and 21 d post partum. Results of the DQ-OVA validation assay were assessed with mixed linear regression models. Pearson correlation tests and kappa values for agreement were used to associate the MFI between each PMN function assay (OB, PC, and DQ-OVA). For the validation assay (9 cows in 3 replicates), PMN incubated with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D, and fluorescence was compared with untreated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units) and lesser (593 ± 55 units) MFI relative to untreated PMN (791 ± 154 units), respectively, indicating that DQ-OVA fluorescence reflected enhanced or reduced endocytic and proteolytic function. To associate the MFI outcomes among OB, PC, and DQ-OVA, 153 samples from 40 cows were analyzed. Results showed significant, although weak association between DQ-OVA and PC MFI (Pearson r = 0.16). When values of MFI were categorized according to the first ("high" PMN functionality), second and third ("moderate" PMN functionality), or fourth ("low" PMN functionality) quartiles, agreement beyond chance (κ) was moderate: κ = 0.38 for DQ-OVA and OB, κ = 0.43 for DQ-OVA and PC, and κ = 0.43 for OB and PC. The DQ-OVA assay may complement traditional PMN functional assays because it provides additional information regarding the combination of endocytosis and proteolytic degradation, but it is not a substitute for assessment of OB or PC.

Association between metabolic diseases and the culling risk of high-yielding dairy cows in a transition management facility using survival and decision tree analysis.

The objective of this study was to assess the association between individual metabolic diseases (MD) and multiple MD (MD+) in the transition period (±3 wk relative to calving) and the culling risk in the first 120 d in milk (DIM) in Holstein-Friesian dairy cows. Health records from a transition management facility in Germany with 1,946 calvings were analyzed in a 1-yr cohort via survival analysis and a decision tree model. The recorded MD were milk fever (MF), retained placenta (RP), metritis (METR), ketosis (KET), displaced abomasum (DA), twinning (TWIN), and clinical mastitis (MAST). The overall culling within 120 DIM was 18%. The 120 DIM culling risk for healthy cows (64.8% of the total) was 13%, whereas it was 25% for MD (24.5%) and 33% for MD+ (10.7%) cows. The 120 DIM culling risk (%) for each MD and MD+, respectively, was 34.6 and 48 for MF and MF+, 15 and 31.5 for RP and RP+, 9.4 and 22.2 for METR and METR+, 30.7 and 37.3 for KET and KET+, 56.1 and 46.8 for DA and DA+, 30.3 and 34 for TWIN and TWIN+, and 36.6 and 27.8 for MAST and MAST+. Moreover, the incidence risk (%) for each MD and MD+, respectively, was 4 and 2.6 for MF and MF+, 1 and 2.8 for RP and RP+, 8.7 and 6 for METR and METR+, 4.5 and 6.1 for KET and KET+, 0.8 and 2.4 for DA and DA+, 1.7 and 2.7 for TWIN and TWIN+, and 3.6 and 1.8 for MAST and MAST+. Setting the healthy cows as the referent, the 120 DIM hazard ratios (HR) for culling were MD 2.1, MD+ 2.9, MF 3.3, MF+ 4.6, RP+ 2.7, METR+ 1.8, KET 2.6, KET+ 3.3, DA 5.5, DA+ 4.5, TWIN 2.8, TWIN+ 3.0, MAST 3.1, and MAST+ 2.3. According to both decision tree and random forest analyses, MF was the most significant disease influencing survival, followed by DA, MAST, METR, and TWIN. In conclusion, the presence of MD or MD+ during the transition period was associated with increased culling risk in the first 120 DIM. The culling hazard was greater when an MD was complicated with another MD. In this study performed in a well-managed large farm, uncomplicated cases of RP (HR = 1.2) and METR (HR = 0.7) did not have an influence on the 120 DIM culling risk. Interestingly, both decision tree and random forest analyses pointed to MF and DA as main culling reasons in the first 120 DIM in the present dairy herd.

Risk factors associated with cytological endometritis diagnosed at artificial insemination in dairy cows.

In this study, we aimed to determine risk factors associated with cytological endometritis (CYTO) diagnosed at artificial insemination (AI) in dairy cows. The CYTO risk factors were evaluated based on 1.625 AI-CYTO samples obtained from 873 Holstein-Friesian cows from in total 18 dairy herds in Flanders (Belgium). The endometrial cytology samples were obtained using the cytotape technique, which consisted of adapting a 1.5 cm piece of paper tape to a standardly loaded AI catheter, covered with a double guard sheet. The polymorphonuclear cells' (PMNs) cut-off point for CYTO at AI was set at ≥ 1%. We constructed multilevel generalized mixed effect models in order to identify the risk factors associated with the presence of CYTO at AI. The CYTO prevalence at AI was 27.8% at the animal level, while the within-herd level prevalence ranged from 10.7 to 39.7%, with an average of 28.1%. Risk factors associated with the occurrence of CYTO were parity ≥2 [odds ratio (OR) = 1.8], days in milk (DIM) at AI ≥ 124 (OR = 0.4), and warm months of the year [July (OR = 2.9), August (OR = 2.3), and September (OR = 1.4)]. In conclusion, the present study supports that multiparous cows and cows that are inseminated in the summer months have a higher risk to suffer from CYTO at insemination, while the risk for CYTO is lower when the insemination is taking place at ≥ 124 DIM.

Prevalence of cytological endometritis and effect on pregnancy outcomes at the time of insemination in nulliparous dairy heifers.

The objectives of the present study were to assess the prevalence of cytological endometritis (CYTO) at the time of artificial insemination (AI) and its effect on pregnancy outcomes in nulliparous dairy heifers. In total, 512 endometrial cytology samples were taken during AI from 351 nulliparous Holstein-Friesian heifers using cytotape (a 1.5-cm piece of paper tape rolled on the top of an AI catheter covered with a double guard sheet). After sampling, the top of the AI catheter was gently rolled onto a glass slide, air-dried, and stained using Diff-Quick (Fisher Diagnostics, Newark, DE). For each slide, 300 nucleated cells were counted, and the polymorphonuclear cell ratio (% PMN) was assessed at 400× magnification. We constructed a receiver operating characteristic curve to find the cutoff point at which sensitivity and specificity (% PMN) affected pregnancy outcomes. The receiver operating characteristic curve revealed that the threshold level for diagnosing CYTO in nulliparous dairy heifers was 1% PMN. An insemination was considered successful when pregnancy was confirmed by rectal palpation at least 45d post-AI. Heifers were considered not pregnant when they received a subsequent insemination or were diagnosed empty by rectal palpation. We built multilevel generalized mixed-effect models to test factors affecting pregnancy outcomes and the occurrence of CYTO at AI. We excluded 16 samples harvested from 12 heifers due to poor sample quality or unavailability of reproductive data. Of the 496 AI samples, the prevalence of CYTO at AI was 7.86% (n=39). The conception rate was 62.8% (n=287) in CYTO-negative samples (n=457) and 38.46% (n=15) in CYTO-positive samples. Risk factors for non-pregnancy were a previous AI (odds ratio 2.96; 95% confidence interval: 1.21-7.26) and the interaction between CYTO and previous AI. The only risk factor identified as being associated with the occurrence of CYTO was a previous AI (odds ratio 4.7; 95% confidence interval: 2.15-10.34). The performance of unsuccessful inseminations significantly affects reproductive outcomes in subsequent AI and may lead to CYTO in nulliparous dairy heifers.

Effect of uterine lavage on neutrophil counts in postpartum dairy cows.

Subclinical endometritis affects approximately 30% of lactating dairy cows, causing significant economic losses to the dairy industry. Yet, there is no efficient treatment available for this condition. The present study examines the effect of uterine lavage in clinically normal cows with sterile saline solution at 30 days in milk (DIM) on the percentage of polymorphonuclear cells (PMNs) detected with endometrial cytology as an indicator of subclinical endometritis. It was hypothesized uterine lavage would be a technique to reduce the number of PMNs in the uterus, and hence be beneficial for cows affected by subclinical endometritis. Cytology samples were taken by low-volume flushing from 50 Holstein Friesian cows on 30 and 40 DIM. On Day 30, cows were clinically examined and randomly assigned into a treatment and control group. In the treatment group, the cytology sampling on Day 30 was immediately followed by uterine lavage with 500-600 mL of sterile physiological saline (35-40°C). Cytology sampling was repeated in all cows at 40 DIM. Lactation numbers >2, peripheral progesterone concentrations >1 ng/mL and uterine lavage at 30 DIM all were significantly associated with lesser PMN percentages at 40 DIM (P=0.0041; 0.0187 and 0.0043, respectively). Uterine lavage might, therefore, be a useful and practical method to decrease the number of PMNs in the uterus of cattle. Results from the current study can be used as preliminary data for designing in depth therapeutic protocols for treatment of subclinical endometritis in cattle.