Characterization of Bacillus pumilus Strains Isolated from Bovine Uteri.

Uterine infections are a major source of economic losses to dairy farmers. The uterine microbiota as well as opportunistic uterine contaminants can contribute to the development of endometritis in dairy cows during the postpartum period. Therefore, it is important to characterize potential pathogens and to further elucidate their role in the disease. In this study, we aimed to characterize Bacillus pumilus field isolates to obtain more details regarding their effect on uterine cells by using an in vitro endometrial epithelial primary cells model. We found that B. pumilus isolates possessed the keratinase genes ker1 and ker2 and therefore may produce keratinases. When primary endometrial epithelial cells were infected with 4 different B. pumilus strains, an effect on cellular viability was observed over the course of 72 h. The effect was dose-dependent and time-dependent. Nevertheless, significant differences between the strains were not observed. All tested strains reduced the viability of the primary cells after 72 h of incubation, indicating that B. pumilus potentially has a pathogenic effect on endometrial epithelial cells.

Dynamics and Diversity of Intrauterine Anaerobic Microbiota in Dairy Cows with Clinical and Subclinical Endometritis.

The aim of the study was to characterize the dynamics of anaerobic cultivable postpartum microbiota in the uterus of dairy cows. In total, 122 dairy cows were enrolled and sampled on day 0 (day of calving) and on days 3, 9, 15, 21, and 28 postpartum (pp). Samples were cultivated anaerobically and analyzed by MALDI-TOF MS. In total, 1858 isolates were recovered. The most prevalent facultative anaerobic genera were Trueperella (27.8%), Streptococcus (25.4%), and Escherichia (13.1%). The most prevalent obligate anaerobes were Peptoniphilus (9.3%), Bacteroides (3.3%), and Clostridium (2.4%). The microbial communities were highly dynamic and diverse. On the animal level, Trueperella pyogenes on day 21 and 28 pp was associated with clinical endometritis, and E. coli on day 21 pp was associated with subclinical endometritis. The occurrence of Streptococcus pluranimalium on day 28 was related to uterine health. The presence of T. pyogenes, Streptococcus, and Peptoniphilus was significantly associated with an increased risk for purulent vaginal discharge. Primiparous cows showed a higher prevalence of T. pyogenes, Fusobacterium necrophorum, Porphyromonas levii, and Peptoniphilus spp. than multiparous cows but were not more susceptible to uterine diseases. This study might provide a suitable basis for future co-cultivation studies to elucidate potential synergistic interactions between microbiota.

Identification of genes associated with susceptibility to persistent breeding-induced endometritis by RNA-sequencing of uterine cytobrush samples.

This study aimed to investigate the susceptibility to persistent breeding-induced endometritis (PBIE). Cytobrush samples were collected from 81 broodmares 1-3 days before artificial insemination (AI). Susceptibility to PBIE was evaluated by the presence of ≥ 2 cm of intrauterine fluid 24 h after AI, besides the fertility was determined by a sonographic pregnancy diagnosis 2 weeks after ovulation. RNA expressions were compared between susceptible non-pregnant (SNP) mares (n=9) and resistant pregnant (RP) mares (n=9) as well as between susceptible pregnant (SP) mares (n=9) and susceptible non-pregnant (SNP) mares. 66 differentially expressed genes (DEGs) were identified between SNP and RP mares and 60 DEGs between SP and SNP mares. In SNP compared to RP mares, transcript levels of genes regulating steroid hormone metabolism and neutrophil chemotaxis were lower, while higher for genes participating in uterine inflammation.Transcripts of genes related to extracellular matrix degradation, tissue adhesions, and fibrosis were lower in SP mares than in SNP mares, while higher for genes related to uterine cell proliferation, differentiation, and angiogenesis in SP mares than SNP mares. In conclusion, increased transcript levels of apolipoprotein E (APOE) and roundabout 2 (ROBO2), cluster domain 44 (CD44), integrin beta 3 (ITGB3), and epidermal growth factor (EGF) are possible biomarkers for susceptibility to PBIE. While higher expression of fibroblast growth factor 9 (FGF9), kinase domain receptor (KDR), and C-X-C motif chemokine ligand (CXCL) 16, collagen type V alpha 2 (COL5A2) and fibronectin (FN1) are suggested indicators of fertility in susceptible mares if they receive proper breeding management.

Endometrial Inflammation at the Time of Insemination and Its Effect on Subsequent Fertility of Dairy Cows.

Our objective was to investigate the level of endometrial immune response at artificial insemination (AI) and to relate it to subsequent fertility. From 71 healthy cows, endometrial cytobrush samples were taken at the first AI for cytological and mRNA analyses. Total RNA isolated from the cytobrushes was used for reverse transcription qPCR for selected transcripts. Animals were grouped into pregnant (PREG; n = 32) and non-pregnant (non-PREG; n = 39) cows following their first AI. The mRNA abundance of the neutrophil-related factor CEACAM1 and the chemokine CXCL5 was 1.2- (p = 0.03) and 2.0-fold (p = 0.04) greater in PREG than in non-PREG cows, respectively. Animals were further subdivided according to the number of inseminations until pregnancy (PREG1, n = 32; PREG2-3, n = 19) and in repeat breeder cows (RBC, n = 13). CEACAM1 and CXCL8 mRNA expression was 1.7- (p = 0.01) and 2.3-fold (p = 0.03) greater in PREG1 than in RBC, respectively. Cox regression showed that cows with PMN ≥ 1% had a 1.8-fold increased chance of pregnancy within 150 days postpartum compared with cows with fewer PMNs. We conclude that a certain level of inflammation before the stimulus of AI might be beneficial for subsequent fertility.

A comparative analysis of the intrauterine transcriptome in fertile and subfertile mares using cytobrush sampling.

BACKGROUND: Subfertility is a major problem in modern horse breeding. Especially, mares without clinical signs of reproductive diseases, without known uterine pathogens and no evidence of inflammation but not becoming pregnant after several breeding attempts are challenging for veterinarians. To obtain new insights into the cause of these fertility problems and aiming at improving diagnosis of subfertile mares, a comparative analysis of the intrauterine transcriptome in subfertile and fertile mares was performed. Uterine cytobrush samples were collected during estrus from 57 mares without clinical signs of uterine diseases. RNA was extracted from the cytobrush samples and samples from 11 selected subfertile and 11 fertile mares were used for Illumina RNA-sequencing. RESULTS: The cytobrush sampling was a suitable technique to isolate enough RNA of high quality for transcriptome analysis. Comparing subfertile and fertile mares, 114 differentially expressed genes (FDR = 10%) were identified. Metascape enrichment analysis revealed that genes with lower mRNA levels in subfertile mares were related to 'extracellular matrix (ECM)', 'ECM-receptor interaction', 'focal adhesion', 'immune response' and 'cytosolic calcium ion concentration', while DEGs with higher levels in subfertile mares were enriched for 'monocarboxyl acid transmembrane transport activity' and 'protein targeting'. CONCLUSION: Our study revealed significant differences in the uterine transcriptome between fertile and subfertile mares and provides leads for potential uterine molecular biomarkers of subfertility in the mare.

Characterization of intrauterine cultivable aerobic microbiota at the time of insemination in dairy cows with and without mild endometritis.

It is generally accepted that postpartum uterine infections decrease conception rates in dairy cows. Whereas clinical endometritis (CE) has been studied intensively, only little information about CE at the time of artificial insemination (AI) is available. The aim of this study was to characterize the intrauterine cultivable aerobic microbiota in healthy cows and in cows with mild CE at AI and to investigate its effect on the subsequent insemination success. The vaginal discharge score (VDS) of 120 Holstein Frisian dairy cows was assessed shortly after AI using the Metricheck device and pregnancy diagnosis was performed 39 days after AI. On average, cows received their second insemination (2.0 ± 1.2 standard deviation (SD)) and were 120.5 ± 40.2 days in milk. Intrauterine cytobrush samples were taken from cows with clear mucus (VDS 0, n = 58) and from cows with flecks of pus (VDS 1, n = 62), which was regarded as mild CE. Bacteria collected with the cytobrush were cultivated aerobically and identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 358 bacterial isolates were recovered, comprising 49 bacterial genera and 116 species. Bacillus, Staphylococcus, Corynebacterium and Streptococcus were the most prevalent genera with relative abundances of 19.6%, 14.2%, 10.1% and 8.1%, whereas common uterine pathogens, such as Trueperella pyogenes and Escherichia coli, were rarely detected. Bacillus spp. were more frequently detected in group VDS 0 (51.7%) than in VDS 1 (22.6%; P = 0.001) and there was a higher prevalence of Corynebacterium spp. in VDS 0 than in VDS 1 (37.9% vs 19.3%; P = 0.03). Cows with VDS 1 at the time of AI harbored neither Bacillus licheniformis nor Bacillus subtilis, while these species were detected in 27.6% and 8.6% of VDS 0 (P < 0.001 and P = 0.024 respectively). The insemination success in VDS 0 and 1 was 43.1 and 25.8% (P = 0.046). The diagnosis of mild CE at AI and the total bacterial load decreased the chance of pregnancy (P = 0.03) but no significant correlation between a distinctive bacterial species and insemination success was observed. In summary, cultivable uterine bacteria at AI presumably represent a part of the physiological microbiota, which is not directly linked to impaired fertility. These findings question intrauterine antimicrobial treatment shortly after AI and could represent the basis for the development of future treatment strategies.

[Bovine uterine diseases: Aspects of microbiology, molecular biology, and immunology]. / Gebärmuttererkrankungen des Rindes: mikrobiologische, molekularbiologische und immunologische Aspekte.

Postpartum uterine diseases, such as puerperal metritis and clinical endometritis may affect over 40 % of cows in dairy farms. Regardless of their severity, these diseases are one of the main reasons for impaired fertility, causing declines in dairy cow productivity and hence, resulting in economic losses. Although uterine diseases have been the topic of scientific discussion for many years, until now it was not possible to agree on uniform definitions for the different kinds of manifestation. By including technical innovations and testing procedures, enormous scientific progress and a deeper knowledge of the physiology as well as the pathologic mechanisms have been achieved. Bovine metritis and endometritis may be regarded as multifactorial diseases caused by a combination of microbial infection, the dysregulation of the immune system, and additional risk factors. These interactions have been analyzed on microbial and molecular biological levels as well as by the use of bioinformatics and molecular genetics. As a result, new species of bacteria and inflammatory mediators possibly contributing to the development of uterine diseases have recently been described. Additionally, metabolic and genetic risk factors and their roles in leading to fertility impairment have been evaluated. In conclusion, it was possible to identify new approaches for possible therapeutic and preventive methods, a subset of which may already be implemented into daily practical routine. This article provides an overview of recent scientific results concerning bovine metritis and endometritis with a focus on microbial, microbiological and immunological studies.

Field trial on the post-insemination intrauterine treatment of dairy cows with mild endometritis with cephapirin.

Cows in estrus but with signs of clinical endometritis (CE) are often not inseminated or undergo an intrauterine treatment after artificial insemination (AI). Decades ago, the so-called Aström method was described as intrauterine infusion of iodine-potassium solution 2-4 days after AI. Nowadays, it is common to use antibiotics instead of iodine solution and the treatment is performed only a few hours after AI. Although widespread in practice, there is only little information about the efficacy of this treatment. Thus, this study evaluated the effect of a post-breeding intrauterine treatment with cephapirin on insemination success in cows with signs of mild CE. In total, 281 cows subjected to an Ovsynch program with fixed-time AI and examined for vaginal discharge straight after AI by use of the Metricheck device were included. Cows with cloudy discharge or flecks of pus in the mucus were assigned to a treatment or a control group. The treatment group (MET; n = 87) received 6 ± 1 h after AI an intrauterine treatment with 500 mg of cephapirin (Metricure, Intervet Deutschland GmbH). Control cows (CON; n = 91) remained untreated. Animals with clear discharge were assigned to a healthy comparison group (HE; n = 103). Pregnancy diagnosis was performed 39 days after AI. The proportion of pregnant cows after the AI directly preceding the enrollment did not differ significantly (P > 0.05) between HE (35.0%), CON (27.5%) and MET (32.2%). Cephapirin treatment had also no positive effect on other reproductive performance measures, i.e, the percentage of pregnant cows 200 days after enrollment (HE: 64.1%, CON: 73.6%, and MET: 73.6%) or the mean interval from enrollment to conception (HE: 25.4 days, CON: 30.0 days, and MET: 29.7 days). The binary logistic regression showed that the only risk factors with a detrimental effect on fertility were a history of CE 28-34 days postpartum and season. Although cows in MET and HE were 1.74 and 1.37 times more likely to conceive after AI than CON, this effect was not significant. Uterine sampling of a subset of cows with CE (n = 50) revealed 127 bacterial isolates. The most frequently found genera were Staphylococcus (19.7%), Bacillus (12.6%), Streptococcus (10.2%), Corynebacterium (8.7%), and Lysinibacillus (7.9%). The finding that common uterine pathogenic bacteria were rarely detected additionally questions an intrauterine antibiotic treatment of cows with mild CE at AI.

Streptococcus uberis strains originating from bovine uteri provoke upregulation of pro-inflammatory factors mRNA expression of endometrial epithelial cells in vitro.

Streptococcus uberis is an opportunistic pathogen involved in various infections of cattle. It is a well-known etiological agent of bovine mastitis and has recently also been linked to postpartum endometritis in dairy cows. S. uberis is frequently isolated from the uterus of postpartum cows but its actual contribution to host pathophysiology is unknown and information on S. uberis virulence factors potentially involved in the disease is lacking. To gain first insights into the role of S. uberis in the pathology of bovine endometritis, a cell-culture-based infection model was employed to study inflammatory host responses and investigate cytotoxic effects. A comprehensive strain panel, comprising 53 strains previously isolated from bovine uteri, was compiled and screened for known virulence factor genes. Isolates showing distinct virulence gene patterns were used to study their impact on cellular viability and influence on mRNA expression of pro-inflammatory factors in endometrial epithelial cells. Our study revealed that S. uberis negatively impacts the viability of endometrial epithelial cells and provokes an upregulation of specific pro-inflammatory factors, although with certain strains having a greater effect than others. Especially, mRNA expression of IL1A and CXCL8 as well as CXCL1/2 and PTGS2 was found to be stimulated by S. uberis. These results suggest that S. uberis might indeed contribute to the establishment of bovine endometritis.

Corynebacterium urogenitale sp. nov. isolated from the genital tract of a cow.

A Gram-stain-positive bacterial isolate, designated LMM-1652T, was isolated from an intrauterine cytobrush sample originating from a postpartum Holstein Friesian dairy cow. The strain had a rod to coccoid-shape, was catalase-positive and oxidase-negative. 16S rRNA gene sequence similarity analyses revealed that its closest relatives were Corynebacterium falsenii (97.05 % similarity), Corynebacterium jeikeium (96.83 %) and Corynebacterium urealyticum (96.82 %). Subsequent whole genome analysis showed that the genome-to-genome distance of strain LMM-1652T to its closest relatives was in the range of 23.2-24.8 %, while the average nucleotide identity values ranged from 73.7 to 74.3%, thus confirming that this isolate represents a novel species. Strain LMM-1652T was characterized by a quinone system mainly consisting of MK-9(H2) and MK-10(H2). The polar lipids profile of the strain consisted mainly of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol-mannoside, as well as one unidentified lipid lacking any functional group. Smaller amounts of four unidentified phospholipids, four unidentified glycolipids, ß-gentiobiosyl diacylglycerol and four unidentified lipids lacking a functional group were also found. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan. The fatty acid profile was mainly composed of C18 : 1 ω9c, C18 : 0 and C16 : 0. We propose a novel species of the genus Corynebacterium with the name Corynebacterium urogenitale LMM-1652T (=LMG 31163T=DSM 108747T).

Corynebacterium endometrii sp. nov., isolated from the uterus of a cow with endometritis.

A Gram-stain-positive bacterial strain, designated LMM-1653T, was isolated from a uterus swab from a Holstein Frisian dairy cow in the frame of a clinical sampling trial. The isolated strain, which showed a rod to coccoid shape, was catalase-positive and oxidase-negative. Based on 16S rRNA gene sequence similarity, its closest relatives were Corynebacterium flavescens and Corynebacterium argatoratense (96.50 % similarity each), suggesting that this isolate represents a novel species. Strain LMM-1653T had a quinone system consisting mainly of menaquinones MK-8(H2) and MK-9(H2). The polar lipid profile showed presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol-mannoside as well as one unidentified glycolipid and one unidentified aminoglycolipid. Moderate to minor amounts of three unidentified glycolipids, ß-gentiobiosyl diacylglycerol, one unidentified aminoglycolipid and three unidentified lipids without a functional group were also found. The cell wall contained meso-diaminopimelic acid and the strain also contained corynemycolic acids. The fatty acid profile was predominantly composed of straight-chain, saturated and mono-unsaturated fatty acids, dominated by C18 : 1ω9c and C16 : 0. Since this isolate differs from the nearest related established Corynebacterium species in its genetic and phenotypic traits, a novel species named Corynebacterium endometrii LMM-1653T (=LMG-31164T=CCM 8952T) of the genus Corynebacterium is proposed.

Supplementing rumen-protected methionine to lactating multiparous dairy cows did not improve reproductive performance.

There is evidence that supplementing methionine has positive effects on uterine environment, oocyte quality and embryo development in cattle. Thus, the objective of this study was to evaluate reproductive traits of cows supplemented with rumen-protected methionine (RPM) during early to mid-lactation in comparison with an untreated control group (CON). An additional focus was on the effect of puerperal diseases on reproductive performance parameters in RPM-supplemented group MET and in CON. A total of 1,709 multiparous Holstein-Friesian cows were enrolled in this field trial conducted on a commercial dairy farm in Slovakia. Cows were allocated at approximately 12 days post-partum (dpp) to either CON or MET, the latter supplemented with 25.0 g-27.2 g RPM per cow per day incorporated into the total mixed ration (TMR) until leaving the study pen at approximately 140 dpp. The amount of RPM was calculated based on individual feed ingredients analysis and adjusted during the study period when TMR changed. Cows were monitored during the post-partum period by vaginal examination (day 5 pp), measuring of beta-hydroxybutyrate in blood (3, 5, and 8 dpp) and by vaginal examination, uterine cytology and measuring of back fat thickness by ultrasound (all at 31 ± 3 dpp). Compared with CON, cows supplemented with RPM did not show better reproduction performance parameters (first service submission rate, days to first service, conception risk, days open 140). Results from binary logistic regression model for the risk of conception showed that metritis had a significant effect, but the supplementation of methionine had not. Results of Cox regression analysis for the odds of conception within 140 dpp revealed only metritis and clinical endometritis as significant factors. In conclusion, supplementation of RPM had no beneficial effect on reproductive performance in this study farm compared with an untreated control group.

Pathogenesis of uterine diseases in dairy cattle and implications for fertility.

Uterine diseases in cattle occur at all stages of the reproduction cycle but the majority of cases is found in the postpartum period. The inflammation of the uterus is generally defined as metritis or endometritis, with several graduations, e.g. puerperal metritis, clinical metritis, clinical or subclinical endometritis. Whether uterine diseases have a negligible, moderate or detrimental effect on fertility is still under discussion and depends on definitions and classification. In the past, it was assumed that the pregnant uterus is free of pathogens, but recent studies found several species including pathogens in the uterus and endometrium of pregnant cows. After parturition, a broad diversity of bacteria with >200 different species has been found in the early postpartum period. Not all of these bacteria, however, are considered as pathogens. Furthermore, bacteriological findings provide only evidence for infection but not for inflammation. For some bacteria, particularly Escherichia coli and Trueperella pyogenes pathogenic mechanism resulting in metritis and endometritis have been elucidated in detail. The role of bacteria that can be regarded as opportunistic or potential pathogens, e.g. Bacillus pumilus, is still under investigation. The understanding of the uterine microbiota and its interactions is increasing with the use of modern high-resolution techniques such as Fourier- transform infrared spectroscopy. Endometrial cytology provides additional information about alterations in the endometrium. Knowledge of innate uterine defense mechanism in cattle has increased a lot in the recent past. It can be speculated that improving or modulating uterine defense mechanism will be part of future prevention and treatment approaches beyond the use of antimicrobials. In this context, cellular and molecular defense mechanisms have been in the focus of interest, e.g. the role of interleukins or mucins. This review gives a short overview on some aspects of recent research on uterine diseases in cattle.

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

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 ß-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 17ß-estradiol, the biosensor showed a limit of detection of 1ng/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.

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 host's 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 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.

Identification of Escherichia coli and Trueperella pyogenes isolated from the uterus of dairy cows using routine bacteriological testing and Fourier transform infrared spectroscopy.

BACKGROUND: Uterine disorders are common postpartum diseases in dairy cows. In practice, uterine treatment is often based on systemic or locally applied antimicrobials with no previous identification of pathogens. Accurate on-farm diagnostics are not available, and routine testing is time-consuming and cost intensive. An accurate method that could simplify the identification of uterine pathogenic bacteria and improve pathogen-specific treatments could be an important advance to practitioners. The objective of the present study was to evaluate whether a database built with uterine bacteria from European dairy cows could be used to identify bacteria from Argentinean cows by Fourier transformed infrared (FTIR) spectroscopy. Uterine samples from 64 multiparous dairy cows with different types of vaginal discharge (VD) were collected between 5 and 60 days postpartum, analyzed by routine bacteriological testing methods and then re-evaluated by FTIR spectroscopy (n = 27). RESULTS: FTIR spectroscopy identified Escherichia coli in 12 out of 14 samples and Trueperella pyogenes in 8 out of 10 samples. The agreement between the two methods was good with a Kappa coefficient of 0.73. In addition, the likelihood for bacterial growth of common uterine pathogens such as E. coli and T. pyogenes tended to increase with VD score. The odds for a positive result to E. coli or T. pyogenes was 1.88 times higher in cows with fetid VD than in herdmates with clear normal VD. CONCLUSIONS: We conclude that the presence of E. coli and T. pyogenes in uterine samples from Argentinean dairy cows can be detected with FTIR with the use of a database built with uterine bacteria from European dairy cows. Future studies are needed to determine if FTIR can be used as an alternative to routine bacteriological testing methods.

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.

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.