The diagnosis and prevalence of subclinical endometritis in cows evaluated by different cytologic thresholds

The aims of our study were to determine (1) how the prevalence of cytologically determined subclinical endometritis varies when using three different cytological threshold ratios to categorize cows as either with or without endometritis, (2) how the number of animals categorized as having endometritis changes from the fourth to the sixth wk postpartum when using each threshold, (3) how subclinical endometritis influences the number of days open, and (4) how the results of cytological and bacterial examinations correlate. To answer these questions, 222 clinically healthy cows in two herds were examined in the fourth (Exam 1) and the sixth wk (Exam 2) postpartum, when endometrial surface scrapings for bacteriologic and cytologic examination were collected by cytobrush from their uterine horns. After each examination, all cows were categorized using three different thresholds: (1) > 18% polymorphonuclear leucocytes in Exam 1 and > 10% in Exam 2, (2) > 8% in both exams, and (3) > 5% in both exams. It was found that: (1) The number of cows categorized as having endometritis increased as the threshold was lowered, and ranged from 18.9% to 75.4% according to herd, time of examination, and the threshold used; (2) with all three thresholds and in both herds, the number of cows categorized as having endometritis in Exam 1 was approximately double that in Exam 2; whereas depending on the herd and the threshold used, 6.1% to 17.0% of the cows that were negative in the first exam were positive in the second, and 7.4% to 33.3% were positive in both exams; (3) cows were open for a significantly greater number of days if categorized as having endometritis with the first threshold in Exam 1 (mean ± SEM 151.5 ± 9.5 vs. 115.9 ± 7.8; P < 0.01), or with either the first or the second threshold in Exam 2 (mean ± SEM 155.0 ± 15.0 vs. 125.1 ± 6.6; P < 0.05); and (4) the most common bacteria were Streptococcus acidominimus and Escherichia coli, and the correlation between cytologic and bacteriologic findings was low (Φ = 0.08 to 0.17 for different tested thresholds). Subclinical endometritis seems to be associated more with the postpartum recovery of the endometrium than with bacterial infection.     

Correlations between periparturient serum concentrations of non-esterified fatty acids,beta-hydroxybutyric acid,bilirubin, and urea and the occurrence of clinical and subclinical postpartum bovine endometritis

Background  

Postpartum endometritis in cattle is a multifactorial disease with high economic impact. Both, clinical endometritis (CE) and subclinical endometritis (SCE) result in decreased reproductive performance. Results from in vitro studies led to the implication that non-esterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), bilirubin, and urea could be used as predictors for endometritis in veterinary practice. In this field study, we set out to establish optimal predictor cut points of these metabolic parameters for the detection of CE and SCE. Serum samples were collected one week prior to parturition (wk -1), in the first week postpartum (wk +1) and between 28 and 35 days postpartum (wk +5) from 209 Holstein-Friesian cows. At wk +5, all cows were examined for signs of CE and SCE.     

Secretion of prostaglandins and leukotrienes by endometrial cells in cows with subclinical and clinical endometritis

The aims of this study were (1) to measure the secretion of prostaglandin F_2α (PGF_2α), prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄), and leukotriene C₄ (LTC₄) by endometrial cells collected by a cytobrush from healthy cows and cows with subclinical and clinical endometritis in the fourth week postpartum, and (2) to evaluate the relationship between the mediators' levels of secretion and the number of polymorphonuclear neutrophils (PMNs) in the uterine smears of cows with subclinical endometritis. The study included cows without any signs of clinical endometritis (n = 63) and cows with clinical endometritis as a positive control (ENDOM, n = 12). Two different threshold ratios (>5% and >18% of PMNs) were used to categorize the cows without clinical signs as with or without cytologic endometritis (CE). Considering the first or second threshold, the animals with CE were included in group CE POS I or CE POS II, whereas the healthy cows were assigned to group CE NEG I or CE NEG II, respectively.     

Postpartum endometrial cytology in beef cows

The objectives were to characterize postpartum endometrial cytology and to determine the prevalence of subclinical endometrial inflammation and its impact on reproduction in beef cows. Samples for endometrial cytology (low-volume uterine lavage) were obtained from 135 of 137 Angus cows (2-87 d postpartum) in northern Minnesota, 26 d before breeding started. Agreement between examiners for subjective inflammation scores was very high (kappa = 0.971); the correlation between these scores and PMN counts was high (r = 0.83; P < 0.001), validating subjective categorization. The proportion of PMN and large mononuclear cells (principally macrophages) declined with postpartum interval (P < 0.001), whereas small mononuclear cells were consistently present (and not significantly affected by postpartum interval). Pregnancy rate to fixed-time AI was 29% and overall pregnancy rate was 89%. There was no association between cell type and ultimate pregnancy status or day of conception (P > 0.10). Although inflammation later in the postpartum period apparently impaired subsequent reproduction in dairy cows, in cows >50 d postpartum at sample collection in the present study, no cytological parameter significantly predicted final pregnancy status or day of conception. Previous twinning increased the risk of subclinical endometritis (P = 0.02), but not the probability of becoming pregnant (P = 0.14). In conclusion, we inferred that beef cows had the ability to clear uterine inflammation after resumption of ovarian cyclicity.     

Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate

IntroductionDNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results.MethodFive major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR).ResultsDisease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.ConclusionsOur results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0748-5) contains supplementary material, which is available to authorized users.     

A clinical approach to determine false positive findings of clinical endometritis by vaginoscopy by the use of uterine bacteriology and cytology in dairy cows

Clinical endometritis in dairy cows is defined as mucopurulent or purulent vulvar discharge 21 days or more after parturition. The diagnosis of clinical endometritis is commonly based on vaginal examination. Techniques to reduce the proportions of false negative findings have been described. This paper discusses a clinical approach to determine the proportion of false positive findings that might occur by vaginal inspection. The consequences of false positive findings in dairy practice are unnecessary or inadequate treatments. In research, incorrect diagnoses have an impact on the interpretation of studies on the diagnosis and treatment of clinical endometritis. The objective of the present study was to compare intrauterine bacteriology and endometrial cytology in cows diagnosed with clinical endometritis with findings obtained by vaginoscopy. Clinical endometritis was defined as mucopurulent or purulent vulvar discharge. On two commercial dairy farms, cows were examined 21 to 28 d postpartum. Uterine samples (n = 230) were collected from cows with clinical endometritis with the cytobrush technique to determine the proportion of polymorphonuclear neutrophils (PMN) and to culture smears for aerobic bacteria. Two threshold values for the proportion of PMN (5 and 18%) were chosen as possible indicators for an inflamed endometrium. Common uterine pathogens A. pyogenes and E. coli were found in 33.5 and 10.4% of the samples, respectively. With increasing vaginal discharge score, proportion of samples positive for A. pyogenes increased significantly. The proportion of cows exceeding the thresholds for PMN increased with vaginal discharge score and the presence of A. pyogenes.Considering only the presence of aerobic uterine pathogens and a proportion of PMN above the threshold values of 5 and 18% as indicative for endometritis, a proportion of 17.3 and 28.5%, respectively, of diagnoses by vaginoscopy were false positive.     

Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.     

Phenotyping of leukocytes and granulocyte and monocyte phagocytic activity in the peripheral blood and uterus of cows with endometritis

This study was a comparative evaluation of selected immunological parameters in peripheral blood and uterine wash samples from cows with a normal postpartum period compared with cows with endometritis. We aimed to determine the usefulness of these parameters in monitoring the puerperium. In total, 40 cows were included in the study: 20 had endometritis (experimental group), and 20 did not have uterine inflammation (control group). Animals were chosen on the basis of cytological and bacteriological test results. The tests were conducted 5, 22, and 40 days postpartum. In both groups, flow cytometric analysis of the surface molecules CD4, CD8, CD21, CD25, and CD14 in the peripheral blood and uterine washings was performed. Granulocyte and monocyte phagocytic activity was determined using a commercial Phagotest kit that was adapted for flow cytometry. The percentage of phagocytic granulocytes and monocytes in both the peripheral blood and the uterine washings was significantly lower for cows in the experimental group compared with the control group (P < 0.01). A significant decrease (P < 0.01) in the percentage of CD4+, CD25+, CD14+, and CD4 + CD25^high leukocyte subpopulations was also observed in the peripheral blood of cows with endometritis. A significant decrease (P < 0.01) in CD21+ lymphocytes and an increase in CD8+ lymphocytes was detected in uterine washings. The results of this work indicate that cell immunity dysfunction may be the main factor causing advanced inflammation of the uterus in endometritis. Knowledge of the immunological mechanisms observed in cows with endometritis might aid in choosing the correct immunomodulating agent–based adjuvant therapy.     

Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis

The objective of this study was to compare the concentrations of inflammatory cytokines in uterine flush and serum from healthy postpartum dairy cows and cows with clinical or subclinical endometritis. Clinical endometritis was diagnosed by observation of vaginal discharges (>50% pus) and subclinical endometritis was diagnosed by evaluation of uterine cytology (neutrophils >18%) at 4 weeks postpartum. Uterine flush was obtained from 48 cows at 4, 6, and 8 weeks postpartum for evaluation of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10 concentrations. Serum samples were obtained from 34 cows just after calving and at 1, 2, 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β, and IL-6 concentrations. Concentrations of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than in cows with subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows with clinical and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-10 concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum, concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant effects of group, sampling time, or interaction on serum cytokine concentrations. In conclusion, cows with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows, but no differences were observed in serum.     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

Phenotypic and Functional Heterogeneity of Bovine Blood Monocytes

Murine and human peripheral blood monocytes are heterogeneous in size, granularity, nuclear morphology, phenotype and function. Whether and how bovine blood monocytes follow this pattern was analyzed in this study. Flow cytometrically, classical monocytes (cM) CD14⁺ CD16⁻, intermediate monocytes (intM) CD14⁺ CD16⁺ and nonclassical monocytes (ncM) CD14⁺ CD16⁺ were identified, with cM being the predominant subset (89%). cM showed a significant lower expression of CD172a, intM expressed the highest level of MHC class II molecules, and ncM were low positive for CD163. Compared to cM and intM, ncM showed a significantly reduced phagocytosis capacity, a significantly reduced generation of reactive oxygen species, and reduced mRNA expression of CXCL8, CXCL1 and IL-1β after LPS stimulation. Based on IL-1β secretion after LPS/ATP stimulation, the inflammasome could be activated in cM and intM, but not in ncM. IFNγ increased the expression of CD16 selectively on cM and induced a shift from cM into intM in vitro. In summary, bovine CD172a-positive mononuclear cells define three monocyte subsets with distinct phenotypic and functional differences. Bovine cM and intM share homologies with their human counterparts, whereas bovine ncM are not inflammatory monocytes.     

Evaluation of cytokine expression by blood monocytes of lactating Holstein cows with or without postpartum uterine disease

Whereas neutrophils are the main phagocytic leukocytes, monocytes and macrophages are actively involved in immunomodulation after infection. Recent studies have demonstrated that neutrophil function is impaired by the state of negative energy balance around parturition, and that cows that develop uterine disease have a greater degree of negative energy balance than healthy cows. The objectives of this study were to compare monocyte gene expression and protein secretion of selected cytokines from calving to 42 d after calving in Holstein cows that did or did not develop uterine disease. Real time quantitative RT-PCR (Tumor necrosis factor-α (TNFα), Interleukin (IL)-1β, IL-6, IL-8 and IL-10) and ELISA (TNFα, IL-1β and IL-8) were used to evaluate cytokine response following in vitro stimulation of blood-derived monocytes with irradiated E. coli. Relative to unstimulated cells, E. coli-stimulated monocytes from cows with metritis had lower gene expression of key pro-inflammatory cytokines than healthy cows from calving to 14 d after calving (TNFα at 0, 7, and 14 d after calving, IL-1β and IL-6 at 7 and 14 d after calving; P < 0.05). There were no significant differences between groups for expression of IL-8 or the anti-inflammatory cytokine IL-10. This was due, in part, to higher gene expression in unstimulated monocytes (TNFα, IL-1β, IL-6 and IL-10) in early lactation from cows with metritis. Expression of mRNA in stimulated cells (relative to housekeeping genes) was lower for TNFα (7 and 14 d postpartum) and for IL-10 (7 and 14 d postpartum) in cows with metritis. Concentration of TNFα was lower in the culture medium of E. coli-stimulated monocytes from cows with metritis than healthy cows at calving and 7 and 21 d after calving (P < 0.05). Circulating cytokine concentrations were not different between groups for IL-8 and were below the limits of detection for TNFα and IL-1β. Cytokine gene expression and production were similar between healthy cows and cows that developed endometritis, diagnosed cytologically at 42 d after calving. We concluded that altered levels of expression and production of pro-inflammatory cytokines postpartum could contribute to impaired inflammatory response and predispose cows to development of metritis.     

Endometrial expression of selected transcripts involved in prostaglandin synthesis in cows with endometritis

Several cytokines and prostaglandins play an important role in preparing the endometrium for implantation and mediating pro-inflammatory events. The aim of the present study was to examine mRNA expression of interleukin 1α (IL-1α), interleukin receptor antagonist (IL-1-RN), cytosolic prostaglandin E synthase (cPGES), microsomal PGES (mPGES-1 and mPGES-2) and lipocalin-type PGDS (L-PGDS) in the bovine endometrium. Endometrial epithelium samples were collected ex vivo from cows with different status of health at day 21-27 postpartum on a dairy farm. Three groups (n = 9 animals each) were defined: (1) healthy cows with no signs of endometritis (control group), (2) cows with subclinical endometritis, and (3) cows with signs of clinical endometritis. Oestrous cycle-dependent mRNA expression pattern was investigated using bovine endometrial epithelial cells from healthy uteri collected at the abattoir. These uteri were classified into post-ovulatory, early-to-mid luteal, late luteal or pre-ovulatory phase (n = 8 animals for each cycle phase). After collecting endometrial epithelium using the cytobrush-method, mRNA analysis was performed by real-time RT-PCR. L-PGDS, IL-1α and IL-1-RN mRNA were expressed significantly higher (P < 0.05) in the endometrium of cows with subclinical or clinical endometritis compared with healthy cows. A twofold lower cPGES mRNA expression (P < 0.05) was detected in cows with subclinical endometritis compared to healthy cows. L-PGDS and IL-1-RN mRNA expression was increased (P < 0.05) after ovulation compared with the pre-ovulatory or luteal phase, respectively. These results support the hypothesis that a dys-regulated cytokine and/or prostaglandin profile in the uterus could be induced by subclinical endometritis or clinical endometritis.     

Lipopolysaccharide (LPS) suppresses follicle development marker expression and enhances cytokine expressions,which results in fail to granulosa cell proliferation in developing follicle in cows

Postpartum endometritis is known to be associated with ovarian dysfunction in cows. Lipopolysaccharide (LPS) generated by Gram-negative bacteria is recognized by toll-like receptor 4 (TLR4), which leads to an inflammatory response by the generation of cytokines such as tumor necrosis factor-α (TNF-α) and interleukins. In this study, we investigated the effect of endometrial LPS on granulosa cell functions during early follicular development in cows. Uteri and follicles were obtained from a slaughterhouse and classified into either clinical endometritis (CE) or normal groups by vaginal mucus test. TLR4 mRNA and protein in normal cows were expressed in granulosa cells collected from follicles measuring 1–3 and 4–7 mm in a diameter, respectively. LPS content in endometrium and follicular fluid of CE cows was significantly higher than that in normal cows. Compared to normal cows, CE cows showed lower expression of follicular development markers (FSHR, CYP19A1, CCND2, and LHCGR) in granulosa cells, lower estradiol-17β concentrations in follicular fluid, and lower granulosa cell proliferation. CE contraction significantly increased cytokine expressions (TNF, IL-1A, and IL-1B) in granulosa cells and suppressed apoptosis of granulosa cells compared to normal cows. LPS significantly suppressed the expression of follicular development markers and the production of estradiol-17β in granulosa cells and reduced granulosa cells proliferation compared to cells cultured without LPS. LPS significantly increased cytokine expressions and suppressed granulosa cell apoptosis. Thus, the present results suggest that the existence of LPS in developing follicles is one of the causes of ovarian quiescence in cows.     

Prevalence of endometritis and its effects on reproductive performance of dairy cows

Hostein cows (n=141) in five commercial dairy herds in central New York were examined for endometritis by examination of endometrial aspirates for presence of inflammatory cells, principally neutrophils, by endometrial cytology at 40-60 days postpartum. The prevalence of cytologically-diagnosed endometritis was 53%; within herds the prevalence varied from 37 to 74% (P=0.02). There was excellent agreement between two examiners (Kappa=0.864; P<0.0001). Parity did not influence prevalence of endometritis (P=0.53). Cytologically diagnosed endometritis was associated with profoundly impaired reproductive performance; Kaplan-Meier survival analysis revealed lower overall pregnancy rate (P<0.0001). Median days open was 206 for cows with endometritis and 118 for cows free of the condition. Overall, 76% of cows in this study became pregnant by 300 days postpartum; 63% of cows with endometritis and 89% of cows without endometritis were confirmed pregnant by 300 days postpartum (P<0.003). (For these two groups, 69, and 90% respectively, became pregnant during the duration of the study). Pregnancy to first service percentage was lower (11 versus 36%; P=0.001) for cows with than without endometritis, and these cows required more services before 50% became pregnant (3 versus 2; P=0.006). In a second study using 22 cows in a university-owned herd, the prevalence of cytological evidence of inflammation was 100% at 2 weeks postpartum, and dropped to 89, 58, and 41% at 4, 6, and 8 weeks, respectively. Endometritis diagnosed by endometrial cytology late in the voluntary waiting period was highly prevalent and exerted a profoundly detrimental effect on subsequent reproductive performance, making this condition potentially extremely costly to the North American dairy industry.     

Some aspects of immunology of the bovine uterus related to treatments for endometritis

Endometritis in breeding cattle occurs during the postpartum period, and is associated primarily with contamination of the reproductive tract involving Arcanobacter pyogenes (formerly Actinomyces pyogenes) together with Gram-negative anaerobes. Polymorphonuclear inflammatory cells (PMNs) contribute partly to the defense mechanisms against micro-organisms contaminating the vagina and uterine lumen, whose phagocytic activity depends on bacterial opsonisation by humoral antibodies; significant numbers of lymphocytes are also present. Whilst leukocyte numbers in the uterine lumen are relatively high during metoestrus and dioestrus compared to other phases of the oestrous cycle, their functional activity is unaffected. Humoral antibody concentrations in the reproductive tract are stimulated following exposure to local antigen, and the response is site dependent; of the several different classes of immunoglobulins, IgG predominates in the uterus and IgA the vagina. Only a portion of the total IgG1 found on the uterine lumen is synthesised locally in the endometrium, the remainder and all of the IgG2 is derived from the local uterine blood supply. Generally, concentrations of immunosuppressant proteins present in the uterine lumen increase under progesterone dominance, and these inhibit lymphocyte proliferation, making the uterus more susceptible to infection. The relationship between uterine susceptibility to micro-organism contamination and the luteal phase of the oestrous cycle is still unclear. Intrauterine infusion of immunomodulators such as E. coli lipopolysaccharides (LPS) or oyster glycogen, in healthy cows and those with endometritis, stimulates leukocytes to migrate into the uterine lumen. At a dosage rate of 100 microg, lipopolysaccharides are not absorbed by the healthy endometrium and do not alter the oestrous cycle length. It is unknown, whether a similar dose can be absorbed through an inflamed endometrium in naturally occurring cases of endometritis to cause systemic illness. Currently, prostaglandin F2alpha is recommended for treating endometritis in both cycling and non-cycling cows, but its mode of action in non-cycling cows is not fully understood. The efficacy of endometritis treatment using an intrauterine infusion of an immunomodulator in cases occurring naturally has not been determined on a large scale.     

Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows

The objectives of the study were to validate the use of endometrial cytology (EC) and ultrasonography (US) to diagnose subclinical endometritis in clinically normal postpartum dairy cows, and to measure the impact of subclinical endometritis on reproductive performance. Holstein cows from two dairy farms were examined at Visit 1 (V1) at 20-33 days in milk (DIM), and clinically normal cows (n = 228), based on the absence of abnormal discharge on external inspection and vaginoscopy, were selected. The reproductive tract of selected cows was evaluated by transrectal palpation, US and EC. All cows in the study were re-examined at Visit 2 (V2) at 34-47 DIM (2 weeks after V1) and were subsequently followed for a minimum of 8 months (until pregnant or culled). Survival analysis was used to derive a case definition of subclinical endometritis, based on factors associated with decreased relative pregnancy rate. Positive EC at V1 (>18% polymorphonuclear leukocytes; PMN) or fluid in uterus at V1 (FIU1) were associated with a significant reduction in the relative pregnancy rate and identified cows with subclinical endometritis. Similarly, a positive EC (>10% PMN) at V2 or fluid in the uterus at V2 (FIU2), identified cows with subclinical endometritis. Cows with subclinical endometritis at V1 and at V2 had a relative pregnancy rate of 41 and 51% (hazard ratio for pregnancy of 0.59 and 0.49), respectively, compared to cows without subclinical endometritis. Given EC or US findings, no diagnostic criteria based on transrectal palpation of the uterus had predictive value for risk of pregnancy. In conclusion, subclinical endometritis, diagnosed by EC or US, was associated with reduced relative pregnancy rate.     

Prevalence of bovine subclinical endometritis 4h after insemination and its effects on first service conception rate

The objective of this study was to investigate the prevalence of subclinical endometritis 4 h after AI and its effect on first service conception rate (FSCR) in dairy cows.A total of 201 Holstein-Friesian cows with no signs of clinical endometritis were examined 4 h after first AI for signs of subclinical endometritis. Endometrial samples were collected from the uterus using the cytobrush technique. The proportion of polymorphonuclear cells (PMN) in the cytological sample was used to characterize an inflammation of the endometrium. Cows were categorized into three groups according to the proportion of PMN in the sample. Cows with 0% PMN (n = 115) were assigned to group Zero, cows with >0-15% PMN (n = 59) to group Medium, and cows with >15% PMN (n = 27) to group High. Pregnancy diagnosis was performed between days 38-44 after AI by palpation of the uterus and its contents per rectum.The FSCR was significantly higher in group Medium than in groups Zero and High (57.6% vs. 39.1% and 29.6%). Statistical analysis revealed an interaction between parity and PMN group. Primiparous cows were at higher risk of being classified into group Medium than multiparous cows (OR = 2.27, P = 0.01). Primiparous cows in group Zero had lower odds of pregnancy after first AI than primiparous cows in group Medium (OR = 0.3, P = 0.02). A comparison with cows that were not examined for subclinical endometritis showed that the collection of endometrial samples itself had no effect on FSCR.     

A Rapid Crosstalk of Human γδ T Cells and Monocytes Drives the Acute Inflammation in Bacterial Infections

Vγ9/Vδ2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vγ9/Vδ2 T cells is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vγ9/Vδ2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vγ9/Vδ2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL)-6, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and oncostatin M (OSM); the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL). Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs) with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan) induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4⁺ effector αβ T cells expressing IFN-γ and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD) patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vγ9/Vδ2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe-responsive γδ T cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity.