Uterine infection alters the transcriptome of the bovine reproductive tract three months later

Infection of the postpartum uterus with pathogenic bacteria is associated with infertility months later in dairy cattle. However, it is unclear whether these bacterial infections lead to long-term changes in the reproductive tract that might help explain this infertility. Here we tested the hypothesis that infusion of pathogenic bacteria into the uterus leads to changes in the transcriptome of the reproductive tract 3 months later. We used virgin Holstein heifers to avoid potential confounding effects of periparturient problems, lactation, and negative energy balance. Animals were infused intrauterine with endometrial pathogenic bacteria Escherichia coli and Trueperella pyogenes (n = 4) and compared with control animals (n = 6). Three months after infusion, caruncular and intercaruncular endometrium, isthmus and ampulla of the oviduct, and granulosa cells from ovarian follicles >8 mm diameter were profiled by RNA sequencing. Bacterial infusion altered the transcriptome of all the tissues when compared with control. Most differentially expressed genes were tissue specific, with 109 differentially expressed genes unique to caruncular endometrium, 57 in intercaruncular endometrium, 65 in isthmus, 298 in ampulla, and 83 in granulosa cells. Surprisingly, despite infusing bacteria into the uterus, granulosa cells had more predicted upstream regulators of differentially expressed genes than all the other tissues combined. In conclusion, there were changes in the transcriptome of the endometrium, oviduct and even granulosa cells, 3 months after intrauterine infusion of pathogenic bacteria. These findings imply that long-term changes throughout the reproductive tract could contribute to infertility after bacterial infections of the uterus.

Experimentally Induced Endometritis Impairs the Developmental Capacity of Bovine Oocytes†

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Delayed elevation of ED1-cellular fibronectin in plasma following postsurgical bacteremia

Fibronectin (Fn) exists in both a soluble form in plasma and lymph as well as an insoluble form in the extracellular matrix. Matrix-localized cellular fibronectin (cFn) contains extra domains (ED1 and/or ED2) not found in plasma Fn (pFn). Very little (< 1-2%) ED1-containing cFn exists in normal blood, and its rapid release into plasma and/or lymph is believed to reflect acute vascular injury. We used a polyclonal antibody to sheep pFn and a monoclonal antibody to ED1 domain of cFn to measure both pFn and ED1-cFn in relationship to lung lymph flow (QL), lung lymph-to-plasma (L/P) total protein concentration ratio, and lung protein clearance (LPC). Unanesthetized sheep (n = 7) were injected intravenously with Pseudomonas aeruginosa (5 x 10(8)) at both 2 and 7 days following surgical preparation of a lung lymph fistula. After both bacterial challenges, we observed an early increase in QL and a small decline in the L/P ratio (0-2 h), reflecting increased fluid filtration in the presence of an intact vascular barrier. This was followed by a further increase (P < 0.05) in QL; an elevation in the L/P ratio; and a marked (P < 0.05) increase in LPC over 3-6 h, indicative of an increase in lung endothelial protein permeability. Before the first bacterial infusion, ED1-cFn in plasma was 9.97 micrograms/ml or approximately 2% of the total Fn antigen in plasma; whereas ED1-cFn in lung lymph was 6-8% of total lymph Fn.(ABSTRACT TRUNCATED AT 250 WORDS)

ED1-containing cellular fibronectin release into lung lymph during lung vascular injury with postoperative bacteremia

Fibronectin (Fn) exists in both a soluble and insoluble form. Soluble Fn in plasma and lymph is an opsonic molecule that enhances phagocytic host defense. Insoluble Fn in the subendothelial and extracellular matrix is an adhesive molecule that mediates cell adhesion to substratum. The extracellular matrix of tissues such as the lung contains a mixture of both plasma-derived fibronectin (pFn) as well as locally synthesized cellular fibronectin (cFn). cFn is antigenically related to pFn, but cFn has extra domains (ED1 and ED2) that do not exist in liver synthesized pFn. The purpose of this study was to determine whether ED1-Fn was released into lung lymph before an increase in lung vascular permeability following postoperative bacteremia. Male sheep (n = 8) with surgically prepared lung lymph fistulae were infused intravenously with a sublethal dose (5 x 10(8)) of Pseudomonas aeruginosa 2 days following surgery. Lymph flow (QL), lymph-to-plasma (L/P) total protein ratio, lung protein clearance (QL x L/P), and hemodynamics were measured over 48 h following bacterial challenge. The lymph and plasma ED1-Fn concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using a murine monoclonal antibody specific to the ED1 region of human cFn. There was a rapid rise of ED1-Fn flux in lung lymph which was evident 60 min after the start of bacterial infusion, resulting in a maximum three- to fourfold increase (P < 0.05) in this parameter. In contrast, the ED1-Fn concentration in plasma before bacterial infusion was less than lung lymph and it did not increase over the initial 6 h following bacterial infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary hemodynamics and tissue damage after one lung infusion of Pseudomonas aeruginosa in sheep

The relative roles of hematogenous mediators and direct bacterial toxicity due to phagocytosis by pulmonary intravascular macrophages were determined by selective bacterial infusion into the left pulmonary artery and comparison of right and left lungs at 24 h. Chronically instrumented sheep received 15-min pulmonary arterial infusions of live Pseudomonas aeruginosa (0.35–2.9 x 10(9), n = 6) or saline (n = 5). The saline group demonstrated stable cardiopulmonary function over time. Left lung blood flow, measured by Doppler flow probe, decreased 15 min into the bacterial infusion, with a concomitant sevenfold increase in left lung pulmonary vascular resistance index. The right lung pulmonary vascular resistance index doubled at 1 h, in association with increased plasma thromboxane B2 levels. An increase in cardiac index and decrease in systemic vascular resistance occurred at 12 h. The wet-to-dry weight ratio of the Pseudomonas-infused left lung was increased compared with that of the sham-infused lung. The tissue count of neutrophils in the lungs was doubled in both sides, but neutrophils on the left were more degranulated. The left lung tissue damage was caused by direct bacterial toxicity, including activation of phagocytic cells. Hematogenous mediators induced pulmonary and systemic hemodynamic changes and right lung neutrophil sequestration, but they did not damage the noninfused lung.

Sepsis in sheep reduces pulmonary microvascular sieving capacity

The changes in pulmonary microvascular permeability in sheep, after infusion of live Escherichia coli, were studied using estimations of the osmotic reflection coefficients (sigma) for total protein, albumin, immunoglobins (Ig) G and M and based on these estimations equivalent pore dimensions were calculated. A chronic lung lymph fistula was prepared in seven sheep. After a base-line period, left atrial pressure (Pla) was increased. E. coli (10(9) X kg body wt) were given after attaining filtration independent L/P values. The sigma's for the normal lung were calculated to 0.73 for total protein and to 0.65, 0.76, and 0.91 for albumin, IgG, and IgM, respectively. The equivalent pore radii were determined to 50 and 175 A with 35% of the filtration accounted for by the large pores. After bacterial infusion, the sigma's for total protein, albumin, IgG, and IgM decreased significantly from preseptic values to 0.58, 0.50, 0.64, and 0.83, respectively. After sepsis the small pores were 50 A and the large pores 200 A with 49% of total volume flow at maximum lymph flows occurring through the large pores. Assuming a constant small-pore population the large-pore number increased 32% after bacterial infusion. These results indicate that pulmonary microvascular permeability may have increased due to the sepsis.

Hyperdynamic severe intravascular sepsis depends on fluid administration in cynomolgus monkey

A new model of high cardiac output septic shock in primates is presented that includes hemodynamic and metabolic effects of separate and combined infusions of normal saline and live Escherichia coli. In monkeys receiving ketamine anesthesia, cardiac output (QT) increased with saline loading but not significantly with bacterial infusion, and bacterial infusion without saline never significantly increased QT. Depressed oxygen consumption (VO2) was reversed with saline loading. In spite of continuing E. coli infusion, radiolabeled microspheres showed no increase in systemic anatomic shunt when QT was elevated. It is perceived that the tissue dysfunction associated with sepsis, which is called septic shock, and elevation of cardiac output are related only indirectly. High cardiac output only reflects an adequate volume status in a stressed individual; some of the afferent stress signals can be related to the septic state, but no specific or direct relationship is involved. In general, shock as indicated by a depression of VO2 does not appear to occur when QT is increased.