Karen Wagener

Dynamics of uterine infections with Escherichia coli, Streptococcus uberis and Trueperella pyogenes in post-partum dairy cows and their association with clinical endometritis

The diversity and dynamics of the uterine microbiota of dairy cows are poorly understood although it is becoming increasingly evident that they play a crucial role in the development of metritis and endometritis. Fourier-transform infrared (FTIR) spectroscopy was used to monitor the bovine microbiota of 40 cows on the day of calving and days 3, 9, 15, and 21 after parturition, and to investigate the associations of selected species with clinical endometritis (CE). Trueperella pyogenes (43.5%), Escherichia coli (21.5%), Bacillus spp. (21.0%) and Streptococcus uberis (18.5%) were the most frequently isolated microbes. Analyses of different sampling time points revealed that the presence of S. uberis on day 3 increased the risk of subsequent T. pyogenes infection on day 9 (odds ratio [OR] = 5.1, 95% confidence interval [CI] = 1.2-22.6). T. pyogenes infection (OR = 36.0, 95% CI = 3.8-343.2) and retained fetal membranes (RFM) (OR = 12.4, 95%CI = 1.4-112.7) were significant risk factors for CE. Cows with S. uberis on day 3 tended to have greater odds of CE than S. uberis-negative cows (OR = 7.1, 95% CI = 0.9-55.6). Chemometric analysis revealed significant differences in the metabolic profile of S. uberis strains isolated from cows with different vaginal discharge scores. This is the first study showing the association of specific S. uberis subtypes with the uterine health status of post-partum dairy cows. The study demonstrates that uterine clearance is a highly dynamic process, during which time bacteria show distinct patterns of progression, and provides information about interactions between bacterial species involved in the occurrence of CE.

Diversity and health status specific fluctuations of intrauterine microbial communities in postpartum dairy cows.

For the interpretation of clinical findings of endometritis and the development of disease prevention and intervention strategies a better understanding of the dynamics and interactions within intrauterine bacterial communities in healthy and diseased cows is required. To gain deeper insights into fluctuations within the uterine microbiota, intrauterine samples were collected from 122 cows at the day of calving, days 3, 9, 15, 21 and 28 postpartum. A total of 2052 bacterial isolates were identified by Fourier-transform-infrared spectroscopy. This culturomics-based approach showed that the aerobic uterine microflora comprised a huge diversity of bacteria belonging to 202 different species, representing 76 genera, with members of the genus Staphylococcus (24.2%) being predominant. On species level the uterine microflora was dominated by Trueperella pyogenes (13.2%), Escherichia coli (11.2%), Staphylococcus xylosus (5.4%), Bacillus pumilus (5.2%) and Streptococcus uberis (4.9%). Comparative analysis of uterine bacteria from cows with different vaginal discharge scores (VDS) revealed health status specific temporal microbial diversifications. Although the intrauterine flora of all VDS groups was dominated by T. pyogenes, E. coli and Staphylococcus spp., the relative number of bacteria differed between VDS groups. The presence of T. pyogenes on days 15 and 21 significantly increased the risk of VDS 2 and 3 at day 21, whereas Staphylococci at day 9 reduced the likelihood of VDS 3 (P<0.05). This study demonstrates that intrauterine bacterial infections are highly dynamic processes and that bacterial species follow specific patterns of progression, which require further research to decipher their potential role in uterine disease development.

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.

Dynamics of bacteriologic and cytologic changes in the uterus of postpartum dairy cows

The objectives of this study were to characterize clinical, intrauterine, bacteriologic and cytologic changes during the first month after parturition in healthy dairy cows and in cows with subclinical endometritis (SE) or clinical endometritis (CE). Furthermore, risk factors related to clinical bacteriologic and cytologic findings were determined. A total of 170 calvings were enrolled, and intrauterine samples were collected on Days 0, 3, 9, 15, 21, and 28 postpartum using the cytobrush technique. The presence of Escherichia coli and Trueperella pyogenes was determined by Fourier transform infrared spectroscopy. The cows were categorized according to their uterine health status (UHS) on Day 21 as healthy (clear or absent vaginal discharge and <5% polymorphonuclear cells [PMN] in the cytologic sample), SE (clear or absent vaginal discharge and ≥5% PMN), or CE (vaginal mucus containing any signs of pus). The prevalence of SE and CE on Day 21 was 27.9% and 58.4%, respectively. Generally, samples from cows with SE and CE showed a greater bacterial growth density (BGD) than those from healthy cows. The BGD tended to be affected by the interaction of time by UHS (P = 0.057). Differences between healthy, SE, and CE cows were found from Day 3 to the last sampling day. Furthermore, the percentage of PMN differed between healthy, SE, and CE cows and was affected by time in a cubic way (decrease/increase/decrease). Overall, E coli was found in 25.4% of the samples, and T pyogenes was identified in 30.2% of the samples. The risk for CE was increased by BGD and the presence of T pyogenes. Conversely, the presence of E coli had no effect on the risk of CE or the risk of SE. The risk for an infection with T pyogenes was greater in the first-parity cows and in cows with assisted calving. In conclusion, changes in BGD and proportion of PMN varied with the UHS (healthy, SE, and CE), which was affected by the presence of T pyogenes but not E coli.

A review of the ongoing discussion about definition, diagnosis and pathomechanism of subclinical endometritis in dairy cows

In the last decade, several new aspects of the inflammation of the bovine endometrium have been investigated and described, including a new definition of subclinical endometritis. This review summarizes the recent discussion about the definition, diagnosis and pathomechanism of subclinical endometritis. Subclinical endometritis also referred to as cytological endometritis is defined by findings of endometrial cytology, which is usually performed with the cytobrush-technique or by low-volume flushing of the uterus. The sampling procedure is minimally invasive and has no negative impact on subsequent conception rate. The suggested threshold value for polymorphonuclear cells (PMN) as diagnostic for subclinical endometritis depends on the time postpartum and varies from 5 to 18%. It has also been shown that a general threshold of 5% PMN is eligible for all cows between 21 and 62 days postpartum. Accuracy and repeatability of counting PMN under the microscope have been evaluated and can be regarded as reliable. The impact of subclinical endometritis on reproductive performance is characterized by decreased conception rates, and prolonged days to first service and days open. In addition, it has been demonstrated that subclinical endometritis has an impact on survival and quality of the embryo. Some studies, however, did not confirm this negative effect of subclinical endometritis on fertility. More detailed analyses of the cytobrush samples revealed higher mRNA expression of several cytokines in cows with subclinical endometritis compared with healthy cows, and contributed to the understanding of detrimental effects of subclinical endometritis on fertility. In contrast to clinical endometritis, there are no predominant bacteria related to subclinical endometritis, but associations between the presence of α-hemolytic streptococci and Trueperella pyogenes and subclinical endometritis have been found. For the treatment of subclinical endometritis, intrauterine infusions with cephapirin as well as the administration of PGF2α have been recommended. Other studies, however, did not confirm the efficiency of these treatments.

The prevalence of subclinical endometritis and intrauterine infections in repeat breeder cows

The objectives of this study were to assess the prevalence of subclinical endometritis and the presence of common uterine pathogens in repeat breeder cows. A total of 121 cows with three or more consecutive artificial inseminations without conception and no clinical signs of disease were defined as repeat breeder cows and were enrolled in this trial. Intrauterine samples were collected with the cytobrush technique to determine the prevalence of subclinical endometritis and bacteriologic infections. Blood samples were analyzed for concentrations of progesterone and estradiol in plasma to assess ovarian activity. Furthermore, breed, parity, history of calving and postpartum uterine infection, clinical findings of transrectal palpation, and backfat thickness were analyzed as potential factors for the prevalence of subclinical endometritis in repeat breeder cows. The prevalence of subclinical endometritis in repeat breeder cows was 12.7%; but common uterine pathogens, Escherichia coli and Trueperella pyogenes, were found in only one and three cows, respectively. Ovarian activity was determined in 95.0% of all cows. Recorded variables had no effect on the prevalence of subclinical endometritis in repeat breeder cows. In conclusion, subclinical endometritis and uterine infections linked to common pathogens were playing a minor role as a cause for repeat breeder cows in this study. Alternative reasons for failure to conceive in these cows are discussed.

Bovine Endometrial Epithelial Cells Scale Their Pro-inflammatory Response In vitro to Pathogenic Trueperella pyogenes Isolated from the Bovine Uterus in a Strain-Specific Manner

Among different bacteria colonizing the bovine uterus, Trueperella pyogenes is found to be associated with clinical endometritis (CE). The ability of cows to defend against T. pyogenes infections depends on the virulence of invading bacteria and on the hosts innate immunity. Therefore, to gain insights into bacterial factors contributing to the interplay of this host pathogen, two strains of T. pyogenes were included in this study: one strain (TP2) was isolated from the uterus of a postpartum dairy cow developing CE and a second strain (TP5) was isolated from a uterus of a healthy cow. The two strains were compared in terms of their metabolic fingerprints, growth rate, virulence gene transcription, and effect on bovine endometrial epithelial cells in vitro. In addition, the effect of the presence of peripheral blood mononuclear cells (PBMCs) on the response of endometrial epithelial cells was evaluated. TP2, the strain isolated from the diseased cow, showed a higher growth rate, expressed more virulence factors (cbpA, nanH, fimE, and fimG), and elicited a higher mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, and IL8) in bovine endometrial epithelial cells compared with TP5, the strain isolated from the healthy cow. The presence of PBMCs amplified the mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, IL1A, IL6, and IL8) in bovine endometrial epithelial cells co-cultured with live TP2 compared with untreated cells, especially as early as after 4 h. In conclusion, particular strain characteristics of T. pyogenes were found to be important for the development of CE. Furthermore, immune cells attracted to the site of infection might also play an important role in up-regulation of the pro-inflammatory response in the bovine uterus and thus significantly contribute to the host-pathogen interaction.

A robust high-throughput fungal biosensor assay for the detection of estrogen activity

&NA; Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which &bgr;‐galactosidase gene expression is controlled by an estrogen‐responsive‐element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre‐treatment by chemical extraction was needed. For 17&bgr;‐estradiol, the biosensor showed a limit of detection of 1 ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high‐throughput formats without the necessity for organic solvents. Graphical abstract Figure. No caption available. HighlightsAdaptation and validation of a fungal biosensor for detection of estrogen activity.Development of a highly sensitive and economic high‐throughput method.With green chemistry assay no chemical extraction and clean‐up procedure.Detection of estrogen activity in feces for pregnancy diagnosis in cows and mares.