We examined the effect of infusion of lipopolysaccharide (LPS) on serum tumor necrosis factor alpha (TNF alpha) concentration and clinical attitude in 2- to 3-day-old colostrum-fed (CF) and colostrum-deprived (CD) foals. Eleven CF and 8 CD neonatal foals were given a bolus IV infusion of Escherichia coli 055:B5 lipopolysaccharide (0.5 microgram/kg of body weight) in sterile saline (0.9% NaCl) solution. Four CF and 2 CD foals were given saline solution alone. Serum IgG concentration and serum anti-LPS IGG(T) antibody titer were determined for each foal prior to infusion. A depression index was used to score clinical abnormalities. Serum TNF alpha concentration was estimated by use of an in vitro cytotoxicity bioassay that used WEHI 164 clone 13 cells as targets. The cytotoxic serum factor was identified as TNF alpha by immunoprecipitation with caprine antisera raised against the 15 NH2-terminal amino acids of human TNF alpha. Tumor necrosis factor alpha was not detected in any preinfusion serum samples nor in any samples from foals given saline solution alone. Serum TNF alpha concentration increased in all LPS-infused foals and peaked between 60 and 90 minutes after infusion. Serum TNF alpha concentrations, expressed as mean percentage of peak serum TNF alpha concentration, persisted longer in CD foals given LPS than in CF foals given LPS. All LPS-infused foals displayed clinical signs of endotoxemia, but mean depression index scores of the CF and CD foals given LPS were not significantly different at any time. Serum TNF alpha concentrations were correlated with depression index scores in both LPS-infused groups. Mean rectal temperature increased by 1 hour and remained high for 4 hours after infusion in CF foals given LPS. Mean rectal temperature in CD foals given LPS was significantly less than that for CF foals given LPS 1 and 2 hours after infusion and was higher than mean rectal temperature prior to infusion 3 and 4 hours after.

Fifteen isolates of Escherichia coli obtained from the blood and tissues of septic foals had plasmid DNA of size ranging from 2.5 to 93 megadaltons. These isolates grew in normal equine serum (serum resistant), a trait previously documented to be expressed by isolates obtained from blood and tissues of septic foals, but not by isolates obtained from the feces of clinically normal horses. Of these isolates, 3 contained conjugal plasmids that encoded resistance to multiple antimicrobial agents linked to serum resistance and, in 1 isolate, to production of aerobactin as well. Serum resistance and production of aerobactin are related to virulence of septicemic E coli from non-equine sources.

In each of 4 horses, sterile synovitis was induced by intra-articular injection of 3 micrograms of Escherichia coli endotoxin (lipopolysaccharide, LPS) into one antebrachiocarpal joint; an equal volume (2 ml) of phosphate-buffered saline solution (PBSS) was injected into the opposite, control carpus. Blood and 1.5 ml of synovial fluid were obtained at postinjection hours (PIH) 0, 2, 4, 8, 12, 18, 42, 66, and 144. Synovial fluid sample collection was accomplished by use of an indwelling, intra-articular catheter through PIH 12, and by arthrocentesis subsequently. Joint fluid samples were analyzed for cell counts, protein concentration, cytologic variables, and tumor necrosis factor (TNF), interleukin 6 (IL-6), and prostaglandin E2 (PGE2) values. Tumor necrosis factor and IL-6 activities and WBC count were also measured in blood. To monitor local inflammation, skin temperature of each carpus was imaged, using a thermographic scanner prior to each sample collection time. Horses had minimal systemic effects. Mean (SEM) rectal temperature increased significantly to 39.02 +/- 0.15 C only at PIH 18 after intra-articular injection of LPS. One horse had signs of mild depression from PIH 7 to 18, but its vital signs did not change appreciably. Each horse had mild signs of discomfort in the LPS-injected limb from PIH 1 to 3 until PIH 8 to 10. Mean peak surface temperature of the LPS-injected carpi was significantly higher than that of control carpi from PIH 8 to 144 (P < 0.05). Mean synovial fluid WBC count in the LPS-injected and control carpi increased after injection and peaked by PIH 8 (193,125 +/- 8,528 cells/microliter, LPS-treated; 16,425 +/- 8,336 cells/microliter, controls). Mean values for LPS-treated (principal) joints were significantly greater than values for control joints from PIH 2 until after PIH 66 (P < 0.05). Mean synovial fluid protein concentration increased in the principal and control joints, with values for the principal joints remaining significantly greater than values for control joints from PIH to 144 (P < 0.05). Mean TNF activity in synovial fluid was maximal at PIH 2 (10,573 +/- 5,924 U/ml). Interleukin-6 activity increased more gradually and peaked at PIH 8 (1.78 +/- 0.71 X 10(6) U/ml). Tumor necrosis factor activity did not increase above the minimal detectable value of 6 U/ml in the control joints. Mean PGE2 concentration in the principal joints peaked by PIH 2 (3.6 +/- 0.37 ng/ml) and remained significantly (P < 0.05) greater than the value for control joints from PIH 2 through 66. These results indicate that a humane model of synovitis was created by intra-articular injection of LPS and can be used to establish the basic responses of synovial TNF, IL-6, and PGE, in horses with early inflammatory joint disease.

Twenty newborn Holstein calves were allotted at random to 4 groups: group A received 0.9% sterile saline solution; group B received phenylbutazone (5 mg/kg of body weight, IV) and 0.9% sterile saline solution; group C received progressively increasing doses of endotoxin (0.1 to 15 micrograms/kg); and group D received phenylbutazone and endotoxin similarly as did calves of groups B and C, respectively. Phenylbutazone was given once daily and saline solution or endotoxin were given every 8 hours for 5 days. Clinical variables-PCV, plasma total protein and fibrinogen concentrations, platelet count, prothrombin time, activated partial thromboplastin time, and fibrin degradation products concentration were measured at 24-hour intervals. Necropsy was performed on each calf. Phenylbutazone suppressed the clinical response to endotoxin challenge until large doses (7.5 to 15 micrograms/kg) were administered. Calves of groups C and D remained stable until they abruptly developed severe dyspnea necessitating euthanasia. Thrombocytopenia and leukopenia developed after the initial endotoxin dose. Prothrombin time was prolonged and PCV suddenly decreased at 96 hours. Necropsy revealed consistent lesions in the vascular endothelium and lungs. Phenylbutazone administration did not enhance or ameliorate endotoxin-induced hemostatic alterations or pathologic lesions.

Saline (0.9% NaCl) solution or 1 of 3 nonsteroidal anti-inflammatory drugs (NSAID) was administered IV to 5 neonatal calves 15 minutes after the start of a 3-hour IV infusion of Escherichia coli lipopolysaccharide (LPS; 2 micrograms/kg/h). Four additional calves were given a 3-hour IV infusion of saline solution alone. Clinical attitude, mean arterial blood pressure, PCV, WBC, and plasma lactate, glucose, and eicosanoid concentrations (thromboxane B2, 6-keto-PGF(1 alpha)) were monitored for 12 hours. Flunixin meglumine (1.1 mg/kg of body weight, IV), ketoprofen (2.2 mg/kg, IV), and ketorolac tromethamine (1.1 mg/kg, IV) each ameliorated the clinical signs of endotoxemia and LPS-induced lacticemia, but failed to significantly alter the degree of leukopenia or hypoglycemia associated with infusion of LPS. Although the 3 NSAID prevented eicosanoid production, they provided only partial protection against LPS-induced hypotension. Each NSAID modified the response to LPS, but none was clearly superior to the others in modulating the clinical signs or physiologic alterations induced by infusion of LPS in neonatal calves.

We evaluated the biochemical and hemodynamic response to hypertonic saline solution plus dextran in isoflurane-anesthetized pigs infused IV with Escherichia coli endotoxin (5 micrograms/kg of body weight for 0 to 1 hour + 2 micrograms/kg for 1 to 4 hours). After 120 minutes of endotoxemia, pigs were treated with a bolus (4 ml/kg over 3 minutes) of either normal saline solution (NSS; 0.9% NaCl), or hypertonic saline solution plus dextran (HSSD; 7.5% NaCl + 6% dextran-70). Administration of HSSD significantly (P < 0.05) increased serum osmolality and concentrations of sodium and chloride for approximately 2 hours during endotoxemia. Plasma total protein concentration decreased significantly (P < 0.05) for 2 hours after treatment with HSSD, indicating hemodilution and increased plasma volume. Although HSSD transiently increased cardiac index (CI) for approximately 15 minutes, this effect was not sustained; however, the endotoxin-induced decrease in CI was ameliorated from 120 to 180 minutes. In pigs of the endotoxin + NSS group from 180 to 240 minutes, CI decreased significantly (P < 0.05), compared with baseline and control values. The endotoxin-induced increases in mean pulmonary arterial pressure and pulmonary vascular resistance were not attenuated by HSSD. At 135 minutes, total peripheral vascular resistance was transiently lower (for approx 15 minutes) in pigs treated with HSSD, compared with control pigs. The endotoxin-induced increase in plasma lactate concentration was not attenuated by HSSD, indicating continued peripheral O2 debt. We conclude that, despite sustained increases in serum osmolality and concentrations of sodium and chloride, HSSD has only transiently beneficial cardiopulmonary effects during endotoxemia in pigs.

The role of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor a during endotoxin-induced mastitis in cows was characterized. Six cows had 10 microgram of Escherichia coli lipopolysaccharide infused into 1 mammary gland. Three other cows served as nontreated controls. Within 1.5 to 2.5 hours after infusion, endotoxin caused obvious edema of the mammary gland and increased serum albumin concentration in milk of infused glands 6 times. Milk somatic cell count began to increase 3 to 5 hours after infusion in all treated glands. At 7 hours after infusion, somatic cell counts were increased > 10 times, compared with counts in milk from control cows. Pyrexia of > 1 C developed in only 1 cow, but all treated cows had serum cortisol concentrations > 50 ng/ml in response to endotoxin treatment. High concentrations of IL-1 (10 to 600 U/ml) and IL-6 (2 to 22 U/ml) were detected in milk of infused glands beginning 2.5 to 4 hours after infusion. Endotoxin did not induce detectable amounts of tumor necrosis factor activity in milk or serum. Swelling and mammary gland permeability changes preceded any detectable increase in IL-1 and IL-6 activity, indicating that these clinical signs of inflammation were not mediated by these cytokines. Systemic responses and the leukocytic influx into endotoxin-infused glands developed after or concurrently with initial increases in IL-1 and IL-6 activities in milk. These results suggested that IL-1 and IL-6 may have a role in mammary gland defenses and in the pathophysiologic changes during endotoxin-induced mastitis.

To assess the role of enterotoxigenic (ETEC), verotoxigenic (VTEC), and necrotoxigenic (NTEC) Escherichia coli in cattle with diarrhea, 1,524 colonies of E coli isolated from 197 calves with diarrhea and from 112 healthy controls were investigated for production of heat-labile and heat-stable enterotoxins, verotoxins, and cytotoxic necrotizing factors (CNF1 and CNF2). The ETEC were isolated from only 2 (1%) calves with diarrhea and from 5 (4%) healthy controls. In contrast, VTEC and NTEC that produced CNF2 were frequently identified. The VTEC were isolated from 18 (9%) calves with diarrhea and from 21 (19%) healthy cattle (P < 0.05), whereas NTEC that produced CNF2 were detected in 39 (20%) ill calves and in 38 (34%) controls (P < 0.01). Therefore, VTEC and NTEC that produced CNF2 were isolated significantly more frequently from healthy than diseased calves. Serogroups to which VTEC belonged differed considerably from the O groups involved with NTEC. Although, VTEC belonged to 18 serogroups, only 4 (O26, O103, O113, and O157) accounted for 56% (25 of 45) of verotoxigenic strains. The NTEC that produced CNF2 belonged to 26 serogroups; however, 64% (69 of 108) were from 6 serogroups (O1, O3, O15, O55, O88, and O123). Our results are compatible with cattle being a reservoir of VTEC that are pathogenic for human beings and with ETEC being an unusual cause of bovine colibacillosis in Galicia (northwestern Spain). Furthermore, results of this study indicate that VTEC and NTEC that produced CNF2 may be part of the normal intestinal flora of cattle.

We examined the effect of infusion of lipopolysaccharide (LPS) on serum tumor necrosis factor alpha (TNF alpha) concentration and clinical attitude in 2- to 3-day-old colostrum-fed (CF) and colostrum-deprived (CD) foals. Eleven CF and 8 CD neonatal foals were given a bolus IV infusion of Escherichia coli 055:B5 lipopolysaccharide (0.5 microgram/kg of body weight) in sterile saline (0.9% NaCl) solution. Four CF and 2 CD foals were given saline solution alone. Serum IgG concentration and serum anti-LPS IGG(T) antibody titer were determined for each foal prior to infusion. A depression index was used to score clinical abnormalities. Serum TNF alpha concentration was estimated by use of an in vitro cytotoxicity bioassay that used WEHI 164 clone 13 cells as targets. The cytotoxic serum factor was identified as TNF alpha by immunoprecipitation with caprine antisera raised against the 15 NH2-terminal amino acids of human TNF alpha. Tumor necrosis factor alpha was not detected in any preinfusion serum samples nor in any samples from foals given saline solution alone. Serum TNF alpha concentration increased in all LPS-infused foals and peaked between 60 and 90 minutes after infusion. Serum TNF alpha concentrations, expressed as mean percentage of peak serum TNF alpha concentration, persisted longer in CD foals given LPS than in CF foals given LPS. All LPS-infused foals displayed clinical signs of endotoxemia, but mean depression index scores of the CF and CD foals given LPS were not significantly different at any time. Serum TNF alpha concentrations were correlated with depression index scores in both LPS-infused groups. Mean rectal temperature increased by 1 hour and remained high for 4 hours after infusion in CF foals given LPS. Mean rectal temperature in CD foals given LPS was significantly less than that for CF foals given LPS 1 and 2 hours after infusion and was higher than mean rectal temperature prior to infusion 3 and 4 hours after infusion. Neither preinfusion total serum IgG concentration nor serum anti-LPS IgG(T) antibody titer correlated with peak serum TNF alpha concentration in the 19 LPS-infused foals.

The cellular response induced in the host animal by endotoxin contributes greatly to the morbidity and mortality of gram-negative infections in bovine neonates. We characterized the temporal sequence, magnitude, and duration of mediator release during endotoxemia and evaluated the effect of endotoxin dose and method of administration. Thromboxane B2 (TxB2), and 6-keto prostaglandin F(1 alpha) (PGF 1 alpha) concentrations and tumor necrosis factor (TNF), and interleukin-1 beta (IL-1 beta) activities were measured in 34 newborn calves given Escherichia coli endotoxin at dosage of 0 (saline solution), 0.2, 2.0, or 20 micrograms/kg of body weight, either by IV administered bolus or infusion over 50 minutes. In all groups and at each lipopolysaccharide dosage, mediators peaked in this sequence; TxB2 and TNF, followed by PGF 1 alpha, then IL-1 beta. Neither dose nor method of administration affected the sequence of mediator release. The magnitude of eicosanoid response to endotoxin was dose-dependent. During induced endotoxemia, duration and/or magnitude of mediator response reflected the dose of endotoxin administered, indicating that the outcome of endotoxemia, in neonatal calves, may be related to the amount of circulating endotoxin.