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.     

Associations between intrauterine bacterial infection,reproductive tract inflammation,and reproductive performance in pasture-based dairy cows

Reproductive tract bacterial infections, particularly those caused by Escherichia coli and Trueperella pyogenes, can have a negative impact on reproductive performance. It has been hypothesized that the presence of E coli early postpartum may increase the risk of isolation of T pyogenes later postpartum. The objective of the present study was to examine associations between intrauterine bacterial infections with E coli and T pyogenes and any bacterial growth (irrespective of bacterial species), purulent vaginal discharge (PVD), cytologic evidence of endometritis (an increased proportion of polymorphonuclear cells [PMNs]), and reproductive performance. Dairy cows (n = 272) from six herds were examined at Days 0 (median, 2 days in milk), 21 and 42 postpartum. From each cow two intrauterine samples were collected via triple-guarded cytobrush at Days 0 and 21. The first cytobrush was used for bacteriologic culture. Escherichia coli and T pyogenes were isolated by culture, and E coli isolates were assigned to one of four phylogenetic groups using a two-step triplex polymerase chain reaction. In addition, T pyogenes was confirmed by polymerase chain reaction. The second cytobrush was used to prepare a cytology slide. Nucleated cells (n = 200) were categorized as epithelial cells, PMNs, or macrophages. Cows were also assessed for body condition score, PVD score, the presence of a CL, and pregnancy. Statistical analysis was performed using multivariable models. There was no association between the presence of E coli at Day 0 and probability of isolation of T pyogenes 3 weeks later; however, E coli positive cows at Day 0 were more likely to be diagnosed with E coli at Day 21 (relative risk [RR] = 2.0, P < 0.01). Escherichia coli at Day 0 or T pyogenes at Day 21 increased the risk of PVD diagnosis 3 weeks later (RR = 1.9; P = 0.04 and RR = 3.0; P = 0.05, respectively). Cows with any bacterial growth at Day 21, irrespective of species, were less likely to conceive within 3 weeks after the start of the seasonal breeding program (RR = 0.8; P = 0.05). Interestingly, cows with 25% PMNs or greater at Day 0 had shorter time to pregnancy (hazard ratio = 1.32; P = 0.05). Intrauterine bacterial infection may impair reproductive performance but the presence of E coli was not associated with isolation of T pyogenes 3 weeks later. Increased endometrial flux of PMNs in cows early postpartum may be a physiological process and improve reproductive performance.     

Risk factors for uterine diseases on small- and medium-sized dairy farms determined by clinical,bacteriological, and cytological examinations

The involution process of the postpartum bovine uterus is usually accompanied by invasion of various bacteria. The objectives of this study were to identify the relationship between the postpartum findings as risk factors for clinical endometritis (CE) and subclinical endometritis (SE). Furthermore, the effects of CE or SE on reproductive performance in small- and medium-sized dairy herds were investigated. A total of 400 cows were examined by vaginoscopy for CE at 20 to 30 days postpartum, and samples were collected for cytological examinations for SE and for bacteriology by cytobrush technique. The vaginoscopic and cytological examinations showed that 27.3% and 21.0% of the cows were found with CE and SE, respectively. The bacterial community analyses revealed a large variety of bacteria. Overall, bacteria from the order Actinomycetales, Lactobacillales, Bacillales, Burkholderiales, Caulobacteriales Enterobacteriales, Pasteurellales, and Pseudomonadales were detected, whereas in 39.5% of the samples no bacterial growth was detectable. The uterine pathogens Escherichia coli and Trueperella pyogenes were found in 16.8% and 13.0% of the samples cultivated under aerobic conditions. Other frequently isolated bacteria were Streptococcus spp. (31.3%), Staphylococcus spp. (20.0%), Corynebacterium spp. (16.5%), and Bacillus spp. (10.5%). The infection with T. pyogenes was the most important bacteriological risk factor for the occurrence of CE (odds ratio (OR) = 5.72; 95% CI = 3.07–10.83) and had a detrimental effect on the hazard of nonpregnancy by 200 days postpartum (hazard ratio = 1.66; 95% CI = 1.12–2.46). Calving assistance (OR = 1.79; 95% CI = 1.16–2.98) and farm (OR = 1.11; 95% CI = 1.02–1.20) were indicated as further risk factors for CE and SE. Effects of CE and SE on reproductive performance parameters could not be demonstrated.     

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.     

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.     

Evaluation of the effectiveness of intrauterine treatment with formosulphathiazole of clinical endometritis in postpartum dairy cows

In cattle, elimination of bacterial contamination from the uterine lumen after parturition is often delayed or compromised, and pathogenic bacteria can persist, causing uterine disease and infertility. The aim of this study was to compare the clinical and bacteriologic recovery following a single intrauterine administration of formosulphatiazole, cephapirin or placebo in cows with clinical endometritis. Cows (n = 80), no less than 28 days postpartum, with clinical endometritis were enrolled in the study. Endometritis was diagnosed by a complete reproductive examination, including rectal palpation, ultrasonography, vaginoscopy and uterine swab. All cows were randomly assigned to receive one of three intrauterine treatments (T0): 2500 mg of formosulphatiazole (Group A); 500 mg of cephapirin (Group B); placebo (4250 mg of propylene glycol; Group C). Cows were examined at the first estrus after treatment or no more than 30 days after (T1). Bacteria isolated were E. coli, A. pyogenes, Pasteurella spp. and Streptococcus spp. After treatment, in Group A and B only 6/30 (20.0%) and 6/24 (25.0%) cows showed a positive bacteriologic culture (P > 0.05), while in Group C the number of positive animals was significantly higher (19/26; 73.1%; P < 0.05). At T0, total clinical scores were similar between the three groups (Group A: 5.84 ± 1.07; Group B: 5.91 ± 1.0; Group C: 5.62 ± 1.17; P > 0.05) and indicative of clinical endometritis. At T1, endometritis scores were significantly lower than those reported before uterine infusion (P < 0.05); however, Group A and B score, 0.4 ± 0.9 and 1.0 ± 2.1, respectively, correspond to no and slight endometritis, while animals in Group C reported a total endometritis score significantly higher (4.6 ± 3.5; P < 0.05) corresponding to endometritis. In the present study, a commercial formosulphatiazole preparation was as effective as cephapirin and more effective than placebo for the treatment of clinical endometritis.     

Diagnosis of acute puerperal metritis by electronic nose device analysis of vaginal discharge in dairy cows

The objective of this study was to estimate the diagnostic accuracy of an electronic nose device using vaginal discharge samples to diagnose acute puerperal metritis (APM) in dairy cows. Uterine fluid was sampled manually with a gloved hand and under sterile conditions for electronic nose device analysis (day in milk (DIM) 2, 5, and 10) and bacteriologic examination (DIM 5), respectively, and on additional days, if APM was diagnosed during the daily clinical examinations. A dataset containing samples from 70 cows was used to create a model and to validate the APM status predicted by this model, respectively. Half of the dataset (n = 35; 14 healthy and 21 metritic cows) was provided with information regarding the APM diagnosis and contained all three measurements (DIM 2, 5, and 10) for each cow and was used as a training set whereas the second half was blinded (n = 35; 14 healthy and 21 metritic cows) and contained only the samples collected on DIM 5 of each cow and was used to validate the created prediction model. A receiver operating characteristic curve was calculated using the prediction results of the validation test. The best observed sensitivity was 100% with specificity of 91.6% when using a threshold value of 0.3. The calculated P-value for the receiver operating characteristic curve was less than 0.01. Overall, Escherichia coli was isolated in eight of 28 (28.6%) and 22 of 42 (52.4%) samples collected from healthy and metritic cows, respectively. Trueperella pyogenes and Fusobacterium necrophorum were isolated in 14 and six of 28 (50.0% and 21.4%) and 17 and 16 of 42 (40.5% and 38.1%) samples collected from healthy and metritic cows, respectively. The prevalence of Escherichia coli and Trueperella pyogenes was similar in the samples obtained from metritic cows used for the training set and the validation test. The results are promising especially because of the objective nature of the measurements obtained by the electronic nose device.     

Reproductive tract defense and disease in postpartum dairy cows

This paper briefly reviews recent data and concepts on the development and mitigation of infection and inflammation in the reproductive tract of dairy cows during the first 2 mo after calving. The incidence of metritis is typically between 10 and 20%, of clinical endometritis or purulent vaginal discharge (PVD) approximately 15%, and of subclinical or cytological endometritis a further 15%. Worse postpartum negative energy balance is associated with more severe or prolonged uterine inflammation. Changes in feed intake, expression of genes for pro-inflammatory cytokines, notably interleukin (IL) 1, IL6 and IL8, circulating concentrations of beta-hydroxybutyrate (BHBA) or nonesterified fatty acids (NEFA), and innate immune function precede both metritis and endometritis by several weeks. Infections with Escherichia coli and Arcanobacterium pyogenes are associated with both metritis and PVD. There are new data to suggest that specific virulence factors in E. coli associated with adherence may be important in metritis and PVD. Cytological endometritis and PVD are overlapping but largely distinct conditions, and there are emerging data that cervicitis exists both concurrent with and separate from endometritis. Much remains to be learned about what initiates and sustains harmful inflammation of the reproductive tract. Such information is necessary to develop effective treatments for the various forms of disease and, more importantly, to develop means to prevent endometritis and cervicitis. In particular, vaccination against specific uterine pathogens and interventions to modulate innate immune response appear to be important avenues for investigation. Presently, commonly recommended best management practices for cows in the transition period are likely to be helpful to mitigate the risk of reproductive disease.     

Reproductive tract defense and disease in postpartum dairy cows

This paper briefly reviews recent data and concepts on the development and mitigation of infection and inflammation in the reproductive tract of dairy cows during the first 2 mo after calving. The incidence of metritis is typically between 10 and 20%, of clinical endometritis or purulent vaginal discharge (PVD) approximately 15%, and of subclinical or cytological endometritis a further 15%. Worse postpartum negative energy balance is associated with more severe or prolonged uterine inflammation. Changes in feed intake, expression of genes for pro-inflammatory cytokines, notably interleukin (IL) 1, IL6 and IL8, circulating concentrations of beta-hydroxybutyrate (BHBA) or nonesterified fatty acids (NEFA), and innate immune function precede both metritis and endometritis by several weeks. Infections with Escherichia coli and Arcanobacterium pyogenes are associated with both metritis and PVD. There are new data to suggest that specific virulence factors in E. coli associated with adherence may be important in metritis and PVD. Cytological endometritis and PVD are overlapping but largely distinct conditions, and there are emerging data that cervicitis exists both concurrent with and separate from endometritis. Much remains to be learned about what initiates and sustains harmful inflammation of the reproductive tract. Such information is necessary to develop effective treatments for the various forms of disease and, more importantly, to develop means to prevent endometritis and cervicitis. In particular, vaccination against specific uterine pathogens and interventions to modulate innate immune response appear to be important avenues for investigation. Presently, commonly recommended best management practices for cows in the transition period are likely to be helpful to mitigate the risk of reproductive disease.     

Using vaginal discharge score (VDS) grading system to evaluate the effect of clinical endometritis on reproductive performance of dairy cows in China

Clinical endometritis (CE) is a major cause in affecting the reproductive performance of dairy cows. The objectives of this study were to ascertain the prevalence of CE and to evaluate the effect of CE on reproductive performance in dairy cows using vaginal discharge score (VDS) grading system. 803 dairy cows were examined by vaginoscope with 4-point VDS at 26 ± 3 days in milk (DIM) and classified into six groups: non-endometritis with VDS 0 (control; CON), endometritis with VDS 1 (MEM), non-treated endometritis with VDS 2 (NTME), treated endometritis with VDS 2 (TME), non-treated endometritis with VDS 3 (NTPE), and treated endometritis with VDS 3 (TPE). Cows in TME and TPE groups were treated with 200 mL of 50% dextrose solution by intrauterine infusion. The prevalence of CE was 33% at 26 ± 3 DIM. Binary logistic regression analysis revealed cows in MEM, NTME and NTPE groups had a less likelihood of first artificial insemination (AI) pregnancy than those in CON group (P < 0.05). Kaplan-Meier survival curves for days open were statistically different (P = 0.004). In Cox regression model, cows in NTME and NTPE groups had a reduced pregnancy rate than those in CON group (P < 0.05). The hazard of pregnancy in NTME group was lower than that in TME group (P = 0.044). Similarly, it was lower for the hazard of pregnancy in NTPE group than in TPE group (P = 0.048). Cows in MEM, NTME, and NTPE groups required more services per pregnancy than those in CON group (P < 0.05). In conclusion, CE examined by the VDS grading system impaired reproductive performance, and mild endometritis with VDS 1 should be treated in the early postpartum period to ameliorate fertility in dairy herds.     

Non-specific uterine infection and bovine fertility. I. Infection patterns and endometritis during the first seven weeks post-partum

From a total of 93 dairy cows which were sampled repetitively throughout the non-gravid period, 14 cows developed persistent purulent endometritis, failed to complete uterine involution and were chronically infertile. The remaining 79 cows had completed uterine involution prior to 50 days post-partum. Forty-seven of these cows subsequently proved to be fertile and 32 cows were infertile. Fertility was not influenced by the variable uterine flora or by the endometritis which was found in all the cows within the first two weeks post-partum. Cows which had uterine infection with $\text{Corynebacterium pyogenes}$ subsequent to Day 21 developed severe endometritis and were almost invariably infertile to the first service. Fertility to subsequent services was not necessarily impaired in those cows which eliminated $\text{C. pyogenes}$ sufficiently early to allow complete uterine involution prior to Day 50. Cows with abnormal oestrous cycles were not particularly susceptible to uterine infection.In all the cows the composition of the uterine flora fluctuated constantly throughout the first 7 weeks pp. as a result of spontaneous contamination, clearance and recontamination. Contamination of the uterus by other non-specific bacteria during the 3 to 4 week period which is required for resolution of severe endometritis induced by $\text{C. pyogenes}$ could lead to a false correlation between the endometritis and the bacterial flora as determined by a single sample. It is concluded that the information which is derived from $\text{in vivo}$ sampling of the uterus in clinically normal cows during the first 7 weeks pp. is of little value in predicting subsequent fertility.     

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.     

Baogong decoction treats endometritis in mice by regulating uterine microbiota structure and metabolites

Endometritis is persistent inflammation caused by bacteria, which can lead to infertility. Although traditional Chinese medicine (TCM) has been used to treat endometritis, the underlying mechanism is still unclear. Here, Baogong Decoction (BGD), a TCM compound, was used to treat mouse endometritis induced by Escherichia coli (E. coli), and then 16S rRNA sequencing and non-targeted metabolomics were used to investigate the change of uterine microbiota and metabolomes in serum and uterine after BGD treatment. Finally, the therapeutic effect of potential metabolites for treating mouse endometritis screened by combined omics analyses was verified using pathological model. The results showed that BGD treatment could effectively treat endometritis associated with the increasing relative abundance of Firmicutes, Bacteroides, Lactobacillus and Lactococcus, and the decreasing relative abundance of Cupriavidus and Proteobacteria. 133 and 130 metabolites were found to be potential biomarkers in serum and uterine tissue respectively. In serum and tissues, dehydroepiandrosterone (DHEA) and catechol were significantly increased in the BGD treatment versus the inflammation group. Results of combined omics analyses demonstrated that DHEA was positively correlated with changes in microbiota. Results of pathological model demonstrated that DHEA could cure endometritis effectively associated with the decreasing infiltration of inflammatory cells and expression of inflammatory factors in the uterus. In summary, our results demonstrated that BGD could cure endometritis in mice by modulating the structure of the uterine microbiota and its metabolites, in which DHEA may be one of the main components of the therapeutic effect of BGD.     

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.     

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.     

Spontaneous recovery or persistence of postpartum endometritis and risk factors for its persistence in Holstein cows

It has been stated that postpartum endometritis in dairy cows has a tendency to cure without intervention. The objectives of this field study, therefore, were to determine the proportions of cows with spontaneous clinical recovery or persistence of postpartum endometritis and to determine some risk factors for its persistency in dairy cows (Bos taurus). Holstein-Friesian cows (n = 441 lactations) from seven dairy herds were examined monthly by vaginoscopy and transrectal palpation. A cow was considered to have “postpartum endometritis” if it had pus in the cervico-vaginal discharge at the first postpartum examination during Days 15 to 60 (Day 0 = day of calving); this was classified as mild, mucopurulent, or purulent endometritis, or endometritis with fluid in uterus. Furthermore, a cow with evidence of endometritis at least once during Days 61 to 150 was considered to have “persistence (or recurrence) of endometritis.” A total of 104 (23.6%) lactations had postpartum endometritis, of which 25.3% had persistence or recurrence of clinical endometritis. Cows with persistence or recurrence of endometritis became pregnant at a slower rate (hazard ratio [HR] = 0.28; P < 0.001) than those with no endometritis until Day 150. Calving in summer (odds ratio [OR] = 7.00; P = 0.04), early postpartum complications (OR = 6.58; P = 0.05), moderate (OR = 4.03; P = 0.08) and severe (OR = 30.99; P = 004) degrees of urovagina, and mucopurulent (OR = 9.54; P = 0.02) and purulent (OR = 5.70; P = 0.04) endometritis were risk factors for the persistence or recurrence of endometritis. Furthermore, 10.6% of cows that had not shown signs of postpartum endometritis had a new diagnosis of endometritis during Days 61 to 150. Some risk factors for the new diagnosis of endometritis beyond Day 60 were early postpartum complications (OR = 2.82; P = 0.03) and moderate (OR = 5.00; P = 0.001) or severe (OR = 12.63; P < 0.001) degrees of urovagina. In conclusion, approximately one quarter of cows with postpartum endometritis had persistence of endometritis until or beyond the breeding period. Risk factors for the persistence of clinical endometritis were summer calving, early postpartum complications, clinically relevant urovagina, and clinically relevant endometritis within 2 mo postpartum.     

Some observations on Corynebacterium pyogenes infection of the bovine uterus

Throughout the non-gravid period, bacteriological samples and endometrial biopsy specimens were taken repetitively from the uteri of 93 cows in 9 dairy herds. The genital organs of 7 of the 14 cows which developed chronic purulent endometritis or pyometra were examined at slaughter 8 to 9 months after parturition. $\text{C.}$$\text{pyogenes}$ was recovered at least once from 61 (65.6 $\text{per}$$\text{cent.}$) of the cows. The highest rate of infection was found during the second week post-partum. Intrauterine infection with $\text{C.}$$\text{pyogenes}$ invariably induced endometritis. The severity of the endometrial reaction was determined by the duration of the infection but the lesions never progressed to the “gland site mass” lesions and extensive stromal sclerosis which have been described as the “significant lesions” in endometritis.The duration of the infection also determined the effect of $\text{C.}$$\text{pyogenes}$ on fertility. Transient infection during the puerperium did not affect fertility; transient infection at a later period reduced fertility to one service, rarely to a second or third service; persistent infection induced chronic purulent endometritis or pyometra accompanied initially by functional anoestrum and, after some months, by organic anoestrum. The factors which determined the duration of the intrauterine infection with $\text{C.}$$\text{pyogenes}$ were not identified.     

Indicators of immunosuppression peripartum in dual purpose cows in the tropics affected health,productive and reproductive parameters

The objective of the study was to identify immunosuppression peripartum indicators in dual purpose cows in the tropics and determine their effects on productive and reproductive parameters. The indicators used were: changes in leukocyte and neutrophils population, concentrations of energy metabolites (β-hydroxybutyrate and glucose) and body condition scores (BCS). Blood sampling and BCS (scale 1 – 5) were taken weekly during the peripartum. Uterine health was assessed (3 weeks postpartum) by ultrasonography and using a vaginal score (0-3) described by Sheldon et al. (2006). Cows (n=30) were classified as healthy or clinical endometritis (CE). CE prevalence was as high as 29.6%. Leukocyte and neutrophils populations diminished while in the peripartum and were lower (P<0.05) in cows suffering CE. Healthy cows had higher (P<0.05) daily milk production than those with CE (18.84±0.63 vs 14.76±0.84 kg). CE cows had lower (P<0.05) reproductive performance compared with healthy cows (open days: 244.40 ± 35.00 vs 178.00 ± 23.33 and services by conception 3.33 ± 0.51 vs 1.83 ± 0.34). BCS similarly (P>0.05) decreased following parturition in both groups. Concentrations of energy metabolites during peripartum fluctuated in a similar (P>0.05) manner in healthy and CE cows. In summary, dual purpose cows in tropical conditions, presented peripartum immunosuppression indicators, characterized by a decline in the leukocyte population, mainly neutrophils, as well as decreased glucose concentrations and BCS postpartum. In addition to it, there was a rise in the β-hydroxybutyrate concentrations and cows presenting CE had a negative effect in the productive and reproductive parameters.     

Degree of variation and reproducibility of different methods for the diagnosis of subclinical endometritis

Endometrial cytology as a reliable diagnostic technique has been established for the diagnosis of subclinical endometritis (SE) in cows. Several counting techniques have been used to determine polymorphonuclear neutrophils (PMN) in endometrial samples. Information on the agreement between different techniques, however, is limited. Therefore, the objective of this study was to analyze the degree of variation in the percentage of endometrial cells and PMN determined by six different counting techniques. A second objective was to evaluate the interobserver reproducibility of the cell counting by two different examiners. One hundred samples were examined by the different counting techniques. The applied methods counted a total of 100, 300, or 500 cells (C100, C300, C500), respectively. In addition, method HPF100 and HPF300 counted 100 and 300 cells in 10 high-power fields per slide. Finally, one method estimated (EST) the percentage of PMN by screening the slide under the microscope. The interobserver reproducibility between two examiners was analyzed for method C300. The comparison between the six different methods showed a strong compliance (r = 0.77–0.90) with greatest correlation coefficient between C100 and C300. The results of Kappa statistics revealed agreement between methods varying from ? = 0.30–0.85, with the greatest agreement between HPF300 and EST. Furthermore, the impact of the different methods on the resulting prevalence of SE was calculated, with the greatest prevalence determined by C100 (33.0%) and the least by HPF300 (10.0%). The results of the interobserver reproducibility showed good correlation and agreement (r = 0.86, ? = 0.79). In conclusion, all examined methods were suitable for the cytological evaluation of PMN, with method C100 showing lowest agreement with the other methods. This confirms the hypothesis that a suitable threshold for PMN is not only influenced by, for example, time of sampling postpartum, but also by the diagnostic method. A threshold of 5% PMN seems to be useful when C300 and HPF100 are used, whereas counting 100 cells or estimating the percentage of PMN seems to overestimate or underestimate the prevalence of SE, respectively. In conclusion, method C300 and HPF100 can be recommended as methods of choice for evaluating the percentage of PMN in endometrial samples to diagnose SE.     

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.