Effects of intrauterine infusion of Escherichia coli endotoxin in anestrous and steroid treated pony mares

This study was conducted to determine if Escherichia coli endotoxin was absorbed from the equine uterus and if exogenous progesterone and estrogen affected the absorption of intrauterine endotoxin. Six mature anestrous pony mares were used in three consecutive crossover experiments (Periods) with a 14 day recovery between each period. Mares were randomly assigned to one of two treatment groups (three mares per group) and received an intrauterine infusion of either saline or endotoxin. Treatment groups were reversed and readministered after 14 days completing a crossover design (Period 1). During Periods 2 and 3 the mares were treated with estradiol-17beta (4.0 mg/day) or progesterone (400 mg/day), respectively, for 5 days prior to receiving intrauterine infusions. On the day of intrauterine infusions rectal temperatures and jugular blood samples were obtained at 30 to 60 minute intervals. Blood samples were analyzed for total white blood cell counts and by Limulus Amebocyte Lysate Assays. There were no significant alterations in the observed parameters among the various treatment groups. These results indicated that intrauterine E. coli endotoxin was not absorbed from the uterus of anestrous pony mares in large enough amounts to produce a morbid condition and that prior exposure to exogenous progesterone or estradiol-17beta did not affect the absorption of endotoxin from the equine uterus.     

Changes in prostaglandin production and ovarian function in gilts during endometritis induced by Escherichia coli infection

The aim of this study was to determine the prostaglandins (PGs) production and ovarian function in gilts after intrauterine infusions of 10(6) and 10(9) colony-forming units (cfu)/ml of Escherichia coli (E. coli). In Experiments 1 and 2, 30 ml of saline or 30 ml of E. coli suspension containing 10(6) or 10(9)cfu/ml, were infused once into each uterine horn in three groups of gilts on day 3 of the estrous cycle, respectively. In Experiment 1, 17 days after treatment it was revealed that inoculation of E. coli 10(9)cfu/ml induced severe acute or subacute endometritis while 10(6)cfu of E. coli evoked moderate acute endometritis or resulted in no inflammatory changes. In the gilts receiving 10(9)cfu/ml of E. coli, the concentration of 13,14-dihydro-15-keto-PGF(2)alpha in blood from the jugular vein was elevated (P<0.05-0.001) compared to concentration in the gilts inoculated with 10(6)cfu on days 8-17 after treatment. Both the E. coli-treated groups had a lower (P<0.05, P<0.01) progesterone plasma level from days 10 to 14 after administration than the control group. On day 17 of the study, infusion of E. coli 10(9)cfu/ml, in comparison to 10(6)cfu, resulted in the greater (P<0.001) content of PGE(2) in the myometrium. The content of both PGs in the endometrium as well as PGF(2alpha) in the myometrium of gilts-treated with 10(9)cfu/ml of E. coli was lower (P<0.001) than in gilts-treated with 10(6)cfu of bacteria. Newly formed corpora lutea were found in the gilts infused with 10(6), but not those infused with 10(9)cfu/ml of E. coli on day 17 after infusion. On day 8 of the study (Experiment 2), the blood from utero-ovarian vein of the gilts-treated with 10(9)cfu/ml of bacteria had a higher (P<0.05) PGF(2alpha) level and lower (P<0.001) PGE(2) level than following infusion of E. coli 10(6)cfu/ml. Also on day 8 of the study, the content of PGE(2) in the endometrium, both the PGs in the myometrium as well as cyclooxygenase-2 in the endometrium and myometrium was greater (P<0.01, P<0.001) after applying 10(9)cfu/ml than 10(6)cfu/ml of E. coli. These results indicate that intrauterine infusions of 10(6) or 10(9)cfu/ml of E. coli lead to the development of inflammatory states of different intensities which is connected with different PGF(2alpha) and PGE(2) production and function of ovaries.     

The effect of the intramammary infusion of Escherichia coli endotoxin on ovulation in lactating dairy cows

The purpose of this experiment was to determine if intramammary inflammation during the periovulatory period affects the occurrence of ovulation in lactating dairy cows. Ten lactating, cyclic, Holstein dairy cows received 2 injections of prostaglandin F2alpha at eleven-day intervals, to synchronize luteolysis. The day of the second injection was designated as day 0. Ovulation was anticipated to occur 3-5 days later (on days 3-5). Beginning at the morning milking on day 1, cows received intramammary infusions of either Escherichia coli endotoxin (10 microg; n=5) or infusion vehicle (pyrogen free Hank's balanced salt solution; n=5) into 2 quarters immediately after milking. The same quarters were infused after each milking through day 4. Venous blood samples were collected daily from day -1 through 13 for determination of progesterone to monitor luteolysis and formation of a new corpus luteum. Blood samples were also collected at 4-h intervals (days -1 to 2), then at 2-h intervals (days 2 to 5) to measure concentrations of luteinizing hormone. Ovaries were examined ultrasonographically on days -1 through 5 and on day 12 to monitor follicular growth and formation of the corpus luteum. Collectively, these observations were used to determine if and when ovulation occurred. Intramammary infusion of E. coli endotoxin induced an immediate increase in the concentration of somatic cells in milk from treated quarters. However, this treatment had no effect on the occurrence or timing of ovulation. Based on ultrasonography and concentrations of progesterone, four of five cows in each treatment group appeared to have ovulated. Preovulatory surges of LH were detected within the intensive bleeding periods for three cows in each treatment group. The magnitude of the LH surge was reduced in cows receiving endotoxin.     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin.

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca2+ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl2 (0.1--2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.     

Lung vascular injury after Escherichia coli endotoxin and phorbol myristate acetate infusion in dogs

The effects of Escherichia coli endotoxin and phorbol myristate acetate (PMA), a potential stimulator of polymorphonuclear leukocyte (PMN), on circulating PMN counts, gas exchange, protein concentration of lavage fluid, pulmonary hemodynamics and pathology of the lung were studied in ten anesthetized dogs. Six dogs were infused with 1 microgram/kg endotoxin plus 10 micrograms/kg of PMA; four other dogs were infused with the same amount of endotoxin but 5 micrograms/kg of PMA. After administration of endotoxin plus 10 micrograms/kg PMA, the number of circulating PMN (per mm3) decreased dramatically from 4081 +/- 1041 to 303 +/- 119, arterial oxygen partial pressure (PaO2) dropped to 49.1 +/- 2.4 mmHg and the arterial alveolar oxygen partial pressure difference (A-a DO2) increased significantly above baseline. Lungs from this group appeared to be grossly damaged: edema with distinct petechial hemorrhage and areas of hemorrhagic consolidation; frothy edema fluid often emanated from the tracheas. The group infused with endotoxin plus 5 micrograms/kg PMA showed no significant decrease in the number of PMN; PaO2 and A-a DO2 maintained comparatively stable. Protein concentration of lavage fluid and lung wet/dry weight ratios in dogs of 10 micrograms/kg PMA group were significantly increased (P less than 0.05) as compared to those of 5 micrograms/kg PMA group. Our study showed that the magnitude of leukopenia after endotoxin and PMA was paralleled with the severity of lung vascular injury. These results support the potential role of PMN in the pathogenesis of acute edematous lung injury.     

Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (S. dysgalactiae) and five strains of Streptococcus agalactiae were used to identify lactoferrin-binding proteins (LBPs). LBPs from extracted surface proteins were detected by polyacrylamide gel electrophoresis and Western blotting. All strains of S. dysgalactiae evaluated had 52- and 74-kDa protein bands. All strains of S. agalactiae evaluated had 52-, 70- and 110-kDa protein bands. In addition, a 45-kDa band was detected in two of five S. agalactiae strains evaluated. This study demonstrated that S. dysgalactiae and S. agalactiae of bovine origin contain two and three major LBPs, respectively.     

Evaluation of the effectiveness of intrauterine treatment with formosulphathiazole of clinical endometritis in postpartum dairy cows

In cattle, elimination of bacterial contamination from the uterine lumen after parturition is often delayed or compromised, and pathogenic bacteria can persist, causing uterine disease and infertility. The aim of this study was to compare the clinical and bacteriologic recovery following a single intrauterine administration of formosulphatiazole, cephapirin or placebo in cows with clinical endometritis. Cows (n = 80), no less than 28 days postpartum, with clinical endometritis were enrolled in the study. Endometritis was diagnosed by a complete reproductive examination, including rectal palpation, ultrasonography, vaginoscopy and uterine swab. All cows were randomly assigned to receive one of three intrauterine treatments (T0): 2500 mg of formosulphatiazole (Group A); 500 mg of cephapirin (Group B); placebo (4250 mg of propylene glycol; Group C). Cows were examined at the first estrus after treatment or no more than 30 days after (T1). Bacteria isolated were E. coli, A. pyogenes, Pasteurella spp. and Streptococcus spp. After treatment, in Group A and B only 6/30 (20.0%) and 6/24 (25.0%) cows showed a positive bacteriologic culture (P > 0.05), while in Group C the number of positive animals was significantly higher (19/26; 73.1%; P < 0.05). At T0, total clinical scores were similar between the three groups (Group A: 5.84 ± 1.07; Group B: 5.91 ± 1.0; Group C: 5.62 ± 1.17; P > 0.05) and indicative of clinical endometritis. At T1, endometritis scores were significantly lower than those reported before uterine infusion (P < 0.05); however, Group A and B score, 0.4 ± 0.9 and 1.0 ± 2.1, respectively, correspond to no and slight endometritis, while animals in Group C reported a total endometritis score significantly higher (4.6 ± 3.5; P < 0.05) corresponding to endometritis. In the present study, a commercial formosulphatiazole preparation was as effective as cephapirin and more effective than placebo for the treatment of clinical endometritis.     

Alterations induced on cytoskeleton by Escherichia coli endotoxin in different types of rat liver cell cultures

Endotoxins (lipopolysaccharide, LPS) from Gram-negative bacteria are considered as the agents responsible for the induction of endotoxic shock, producing severe cellular metabolic dishomeostasis. Cytotoxic lesions, as well as functional and metabolic disturbances, occur mainly in the liver, which is one of the target organs and exerts an LPS clearance function. In an attempt to approach the molecular basis of endotoxic shock, and to propose an experimental model, we have focused this study on cytoskeleton (microtubules and microfilaments) alterations induced by different doses of endotoxin in different target liver cells. Microfilaments and microtubules were studied by immunofluorescence and different microscopy techniques (optic fluorescence microscopy and confocal laser scanning microscopy) in order to improve the cytoskeleton study resolution. Parenchymal and sinusoidal cell morphology, severely damaged by the LPS action, is related to a disturbance on the cytoskeletal organisation, even more evident in a particular proliferating rat liver cell culture. The most relevant changes are seen in the microtubule patterns in all liver cells tested, which could be related, depending on cell type, either to a direct LPS action or to [Ca+2]i dishomeostasis as well as free radical and cytokine (IL-1beta and TNF-alpha) production. Due to their features, proliferating rat liver cell cultures are used as a sensitive cell model to understand the effect of LPS on cytoskeleton organisation.     

Absorption of Escherichia coli endotoxin (lipopolysaccharide) from the uteri of postpartum dairy cows

An experiment was conducted to test the hypothesis that Escherichia coli (E. coli ) endotoxin is readily absorbed from uteri of early postpartum cows and that the absorbed endotoxin provokes systemic relcase of prostaglandins. Eleven postpartum Holstein dairy cows (aged 3 to 7 yr) with normal puerperium were selected and divided into a treatment group (n=7), which received intrauterine infusions of E. coli endotoxin, and a control group (n=4), which received intrauterine infusions of 10 ml of saline on Days 5 and 20 post partum. Blood samples were collected once every 30 min for 6 h starting from the time of infusion. Harvested sera samples were analyzed for concentrations of stable metabolites of prostacyclin (PCM), prostaglandin F(2alpha) (PGFM), and thromboxane A(2) (TXB(2)). Plasma samples were qualitatively tested for the presence of endotoxin. Endotoxin was detected in the plasma samples of cows that received endotoxin on Day 5 post partum 4 h after the infusion. Endotoxin was not detected in any of the samples from control cows on Days 5 and 20 post partum or from treatment group cows on Day 20 post partum. Cows treated on Day 5 post partum showed increases in serum PGFM concentrations from 710 +/-64pg/ml to peak concentrations of 1223 +/- 47 pg/ml within 2 h, followed by a decline to baseline concentrations within 4 h. The amount of PGFM released in treated cows on Day 5 post partum was higher (P < 0.05) than in control cows on Day 5 or in treated and control cows on Day 20 post partum. Serum PCM concentrations increased from 156+/-24 pg/ml to peak concentrations of 1348+/-127 pg/ml within 1 h. The amount of PCM released in treated cows on Day 5 postpartum was higher (P< 0.05) than in control cows on Day 5 or in treated and control cows on Day 20 post partum. The TXB(2) concentrations increased from 315+/-38 pg/ml to peak concentrations of 5043 +/- 242 pg/ml within 1 h and fell to baseline concentrations within 5 h. The amount of TXB(2) concentrations released in treated cows on Day 5 post partum was significant (P < 0.05) compared with those of cows in the other groups. The results support the hypothesis that uteri of early postpartum cows are capable of absorbing endotoxin, and the absorbed endotoxin provokes changes in the serum concentrations of prostanoids.     

Treatment of chronic endometritis in dairy cows with an intrauterine application of enzymes. A field trial

The use of proteolytic enzymes has been established in the non-antibiotic treatment of mastitis in dairy cattle. The objective of this study was to evaluate, if enzymes are efficacious in the treatment of chronic endometritis. In a controlled field trial, cows with vaginal discharge 21-27 days in milk (DIM) were randomly assigned to two treatment groups. Endometritis was classified into three categories, depending on the type of vaginal discharge: clear mucus with flakes of pus (E1), mucopurulent discharge or fluctuating contents in the uterus (E2), and purulent discharge (E3). In group ENZYMES (n=191), cows received an intrauterine treatment with a salve containing the enzymes trypsin (16 mg), chymotrypsin (16 mg), and papain (8 mg). Cows in group PGF (n=225) were treated with 0.5mg of cloprostenol. Cows that did not show any clinical signs of chronic endometritis were regarded as healthy control group (HC, n=699). In groups ENZYMES and PGF, all cows were re-examined 35-41 DIM. In group ENZYMES, cows were re-treated with enzymes if signs of endometritis were found, while in group PGF all cows received a second dose of cloprostenol, regardless of their clinical findings. Cure rate after the first treatment, defined as the absence of vaginal discharge at the re-examinations, was 59.7 and 68.0% in groups ENZYMES and PGF, respectively (P>0.05). Reproductive performance measures showed no significant differences between the two treatment groups. Service rate was significantly lower for ENZYMES and PGF, respectively, compared to HC. Conception rates to all services and percentages of cows pregnant by 250 DIM were significantly lower in group ENZYMES compared to HC, while no further differences were found between PGF and HC. In both treatment groups, cure rate and reproductive performance measures were better for cows categorized E1 or E2, than for cows categorized E3, respectively. Conception rate to all services for cows with endometritis category E1 was higher in group PGF than in group ENZYMES (P<0.05). The results of this field trial suggest that prostaglandin F(2alpha) is still the treatment of choice for chronic endometritis in dairy cattle.     

Humoral immune function and experimental Escherichia coli infection in splenectomized dogs

Intravenous administration of sheep red blood cells resulted in a deficient initial antibody response and provoked a much lower concentration of plaque forming cells in the blood of splenectomized dogs than in the control group. The in vitro serum opsonizing activity on Escherichia coli M-S-15 remained normal after splenectomy, and no difference was found between the test and the control group in the intravascular clearance rate of bacteria injected intravenously.     

Effect of intrauterine infusion of Escherichia coli on hormonal patterns in gilts during the oestrous cycle

The aim of this study was to determine the effect of intrauterine Escherichia coli infusion on the patterns of plasma LH, prolactin, progesterone, androstenedione, testosterone, oestrone, oestradiol-17beta, cortisol and 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM) in gilts during the oestrous cycle. On day 4 of the oestrous cycle (day 0), 25 mL of saline or 25 mL of Escherichia coli suspension, containing 10(7) colony forming units x mL(-1), was infused once into the each uterine horn in group I or II respectively. The control gilts developed a new oestrous cycle at the expected time but not bacteria-treated. Endometritis and vaginal discharge developed in all gilts after Escherichia coli infusion. The administration of Escherichia coli resulted in a reduction of plasma levels of LH, prolactin, oestrone and oestradiol-17beta (P < 0.05-0.001), mainly on days 15-18 after treatment (expected perioestrous period). During this time, the plasma androstenedione level was elevated (P < 0.05-0.001) after bacteria infusion. In the gilts receiving bacteria, progesterone concentration decreased from day 8 after treatment and was low until the end of the study (P < 0.05-0.001). On days 8-12 after bacteria administration, the level of PGFM was higher (P < 0.001) than that found in the control group. These results suggest that the developing inflammatory process of the endometrium in gilts following Escherichia coli infusion significantly affects the pituitary-ovarian axis function as well as prostaglandin production leading to anoestrus.     

PKC and RhoA signals cross-talk in Escherichia coli endotoxin induced alterations in brain endothelial permeability

Escherichia coli endotoxin LPS regulates blood-brain barrier permeability by disrupting the tight junction (TJ) complex between brain endothelial cells. This study used Bend.3 cells to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. The LPS-induced alterations in the brain endothelial barrier were associated with PKC (a, β, ζ) and RhoA, but were independent of PI3K and the tyrosine kinase pathway. Inhibition of PKC (a, β, ζ) and RhoA activity using shRNA and dominant negative mutants diminished the effects of LPS on the brain's endothelial TJs. The interactions between the PKC and Rho pathways were therefore examined. PKC-a and PKC-ζ, but not PKC-β interacted with RhoA in Bend.3 cells stimulated by LPS. PKC-a acted as the upstream molecule for Rho and PKC-ζ acted as the downstream target for Rho. Comparing the effect of double inhibition of "Rho and PKC" and single inhibition of "Rho" or "PKC" confirmed that this interaction is critical for LPS-induced brain endothelial cell hyperpermeability. Collectively these data are the first to suggest that LPS affects the brain's endothelial TJ barrier via PKC (a, β, ζ)- and RhoA, independent of the PI3K and tyrosine kinase pathways. In addition, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulator for RhoA in the process.     

Renal blood flow in normal dogs and in dogs with experimental liver cirrhosis following the acute continuous infusion of endotoxin

The purpose of this study was to determine if the renal circulation of normal and cirrhotic dogs behave similarly in response to an acute endotoxin infusion. Endotoxin was administered as a slow continuous infusion (13-26 micrograms/min) to a total of 20 normal dogs through the femoral vein, portal vein, or into the left renal artery. In each case, there was an initial increment in renal blood flow, of the order of 46%, while arterial blood pressure was actually declining. After 8-20 min, blood flow fell as perfusion pressure declined further. The initial increment in renal perfusion was not due to a hyperthermic response following the endotoxin. When similar doses were given to five dogs with chronic biliary cirrhosis and ascites, the biphasic response in renal perfusion was not observed, rather blood flow declined as perfusion pressure declined. When normal dogs were infused with bilirubin, bile salts, noradrenaline, and angiotensin in pressor doses, the subsequent infusion of endotoxin still produced the usual biphasic response in renal perfusion. Chronic elevation of portal pressure (but not acute elevation), volume contraction by diuresis or hemorrhage, and the infusion of bile intravenously, all abolished the biphasic response in renal perfusion and reproduced in normal dogs the response to endotoxin observed in cirrhotic dogs. Investigation of the factors causing the initial decrease in intrarenal vascular resistance in normal dogs following the endotoxin infusion implicated a role for histamine, kinins, and prostaglandins. We conclude there is a fundamental difference in the response of the renal circulation of normal and cirrhotic dogs to an endotoxin infusion, which may depend on failure of this latter group to release one or more humoral agents. This difference may be due to elevated portal pressure, a decreased effective arterial blood volume, or the products of bile having access to the circulation in cirrhotic dogs.