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Oestrogens are pivotal in ovarian follicular growth, development and function, with fundamental roles in steroidogenesis, nurturing the oocyte and ovulation. Infections with bacteria such as Escherichia coli cause infertility in mammals at least in part by perturbing ovarian follicle function, characterised by suppression of oestradiol production. Ovarian follicle granulosa cells produce oestradiol by aromatisation of androstenedione from the theca cells, under the regulation of gonadotrophins such as FSH. Many of the effects of E. coli are mediated by its surface molecule lipopolysaccharide (LPS) binding to the Toll-like receptor-4 (TLR4), CD14, MD-2 receptor complex on immune cells, but immune cells are not present inside ovarian follicles. The present study tested the hypothesis that granulosa cells express the TLR4 complex and LPS directly perturbs their secretion of oestradiol. Granulosa cells from recruited or dominant follicles are exposed to LPS in vivo and when they were cultured in the absence of immune cell contamination in vitro they produced less oestradiol when challenged with LPS, although theca cell androstenedione production was unchanged. The suppression of oestradiol production by LPS was associated with down-regulation of transcripts for aromatase in granulosa cells, and did not affect cell survival. Furthermore, these cells expressed TLR4, CD14 and MD-2 transcripts throughout the key stages of follicle growth and development. It appears that granulosa cells have an immune capability to detect bacterial infection, which perturbs follicle steroidogenesis, and this is a likely mechanism by which ovarian follicle growth and function is perturbed during bacterial infection.

Background Escherichia coli are widespread in the environment and pathogenic strains cause diseases of mucosal surfaces including the female genital tract. Pelvic inflammatory disease (PID; metritis) or endometritis affects ∼40% of cattle after parturition. We tested the expectation that multiple genetically diverse E. coli from the environment opportunistically contaminate the uterine lumen after parturition to establish PID.Methodology/Principal FindingsDistinct clonal groups of E. coli were identified by Random Amplification of Polymorphic DNA (RAPD) and Multilocus sequence typing (MLST) from animals with uterine disease and these differed from known diarrhoeic or extra-intestinal pathogenic E. coli. The endometrial pathogenic E. coli (EnPEC) were more adherent and invasive for endometrial epithelial and stromal cells, compared with E. coli isolated from the uterus of clinically unaffected animals. The endometrial epithelial and stromal cells produced more prostaglandin E2 and interleukin-8 in response to lipopolysaccharide (LPS) purified from EnPEC compared with non-pathogenic E. coli. The EnPEC or their LPS also caused PID when infused into the uterus of mice with accumulation of neutrophils and macrophages in the endometrium. Infusion of EnPEC was only associated with bacterial invasion of the endometrium and myometrium. Despite their ability to invade cultured cells, elicit host cell responses and establish PID, EnPEC lacked sixteen genes commonly associated with adhesion and invasion by enteric or extraintestinal pathogenic E. coli, though the ferric yersiniabactin uptake gene (fyuA) was present in PID-associated EnPEC. Endometrial epithelial or stromal cells from wild type but not Toll-like receptor 4 (TLR4) null mice secreted prostaglandin E2 and chemokine (C-X-C motif) ligand 1 (CXCL1) in response to LPS from EnPEC, highlighting the key role of LPS in PID.Conclusions/SignificanceThe implication arising from the discovery of EnPEC is that development of treatments or vaccines for PID should focus specifically on EnPEC and not other strains of E. coli.

Two point mutations were identified within the gene encoding bovine CD18 in a Holstein calf afflicted with leukocyte adhesion deficiency (LAD). One mutation causes an aspartic acid to glycine substitution at amino acid 128 (D128G) in the highly conserved extracellular region of this adhesion glycoprotein, a region where several mutations have been found to cause human LAD. The other mutation is silent. Twenty calves with clinical symptoms of LAD were tested, and all were homozygous for the D128G allele. In addition, two calves homozygous for the D128G allele were identified during widespread DNA testing, and both were subsequently found to exhibit symptoms of LAD. The carrier frequency for the D128G allele among Holstein cattle in the United States is approximately 15% among bulls and 6% among cows. This mutation is also prevalent among Holstein cattle throughout the world, placing this disorder among the most common genetic diseases known in animal agriculture. All cattle with the mutant allele are related to one bull, who through the use of artificial insemination sired many calves in the 1950s and 1960s. The organization of the dairy industry and the diagnostic test described herein will enable nearly complete eradication of bovine LAD within 1 year. These results also demonstrate that bovine LAD is genetically homologous and phenotypically similar to human LAD, thus providing a useful animal model for studies of LAD and P2 integrin function.Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease characterized by greatly reduced expression of the heterodimeric 32 integrin adhesion molecules on leukocytes, resulting in multiple defects in leukocyte function (1-3). Neutrophil extravasation requires 132 integrin interaction with endothelial intercellular adhesion molecules (2, 4, 5). Without (2 integrins, neutrophils are unable to enter the tissues to destroy invading pathogens. Consequently, LAD patients suffer frequent and recurrent bacterial infections (1-3). The P32 integrins include LFA-1, Mac-i, and p150,95, which consist of unique a subunits, CD11a, CD11b, and CD11c, respectively, and a common 13 subunit, CD18. Because P2 integrin expression requires intracellular association of the CD11 and CD18 subunits, defects in CD18 prevent expression of all (32 integrins (2,4,6). All human cases of LAD have been traced to defects in the gene encoding CD18 (2, 4, 7-11).A granulocytopathy syndrome had been described in Holstein cattle (12-14), and Kehrli et al. (15) showed that this disease results from a lack of leukocyte (32 integrin expression, indicating that this disease is equivalent to LAD. The etiologic mutation for bovine LAD had not been determined, and efforts to study and control this disease were seriously limited by the lack of a diagnostic test to identify carner cattle. We now show that LAD in Holstein cattle is caused by a point mutation in the gene encoding CD18 ¶ and that this mutation is prevalent among Holstein cattle throughout the world. MATERIALS AND METHODSCattle. Two ...

Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3␣, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1␤ release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.

The objective of this study was to evaluate the association between uterine disease and indicators of neutrophil (PMN) and systemic energy status in dairy cows. Peripheral blood (120 mL) was collected weekly from 84 Holstein cows for PMN isolation and plasma collection from calving until 42 d in milk (DIM). The final analysis included 80 cows. Of those, 20 cows were classified as having metritis (fetid uterine discharge and fever), 15 as having subclinical endometritis (SCE; >or=10% PMN on uterine cytology), and 45 as healthy controls. Plasma haptoglobin concentration was increased only in cows that developed metritis. Neutrophil glycogen content was reduced in cows developing metritis compared with healthy cows on the day of calving and at 7 and 42 DIM. Cows with SCE had lower PMN glycogen content than healthy cows at 7, 28, and 42 DIM. Blood glucose was affected by disease status within parity. Primiparous metritis cows had greater blood glucose concentrations than healthy primiparous cows. Multiparous metritis cows tended to have lower blood glucose concentration than multiparous SCE cows. Cows that developed metritis and SCE had or tended to have greater NEFA and BHBA than healthy cows, mainly around calving. At calving, cows that developed metritis had higher plasma estradiol concentration than healthy cows and greater plasma cortisol than cows that had SCE. Plasma insulin was not affected. Plasma glucagon was increased for SCE cows. Cows that developed uterine disease experienced a greater degree of negative energy balance and had decreased lower intracellular PMN glycogen levels, which could be a major predisposing factor for disease because of decreased availability of oxidative fuels.

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