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.

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.

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.

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.

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.