Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8

CD14, the leukocyte co-receptor for lipopolysaccharide (LPS), is important in the response of bovine polymorphonuclear neutrophil leukocytes (PMN) to Gram-negative bacteria. In other species, the expression of CD14 on the surface of PMN was shown to increase after exposure to inflammatory stimuli. These newly expressed molecules may originate from either an intracellular pool or through new gene expression. We sought to characterize bovine PMN cell surface expression and shedding of CD14 molecules, and CD14's effect on secretion of the chemoattractants IL-8 and IL-1beta by PMN. Bovine PMN were incubated in RPMI for 20 h at 37 degrees C with LPS (1, 10, 100 microg/mL). IL-8 release increased with treatment of 1 microg/mL LPS, but decreased 41.5 and 95% at the 10 and 100 microg/mL concentrations of LPS, respectively. In contrast, shedding of CD14 from the surface of PMN only increased at the highest concentration of LPS (100 microg/mL). Secretion of IL-1beta was similar regardless of the LPS concentration used to stimulate PMN. The effect of PMN concentration (1 x 10(7), 2.5 x 10(7), 5 x 10(7), and 10 x 10(7)/mL) on CD14 cell surface expression and shedding of IL-8 and IL-1beta were also determined. Shedding of CD14 by PMN increased with increasing concentration of PMN after exposure to 0.1 and 10 microg/mL of LPS, while secretion of IL-8 decreased. IL-1beta increased at the highest concentration of PMN. The use of real time polymerase chain reaction showed that CD14 mRNA expression was not different between control and LPS-stimulated cells, indicating that the sCD14 came from either membrane bound CD14 or a preformed pool. Our results demonstrate that release of CD14 from PMN suppresses secretion of IL-8, and may be an important regulatory mechanism for controlling excessive migration of PMN into the bovine mammary gland.     

Measurement of phagocytosis of 32P-labeled Staphylococcus aureus by bovine leukocytes: lysostaphin digestion and inhibitory effect of cream.

A procedure to measure phagocytosis by blood and milk neutrophils was developed. One milliliter of heat-killed 32P-labeled Staphylococcus aureus ([32P]SA) (180-200 X 10(6) CFU), 1 ml of phosphate-buffered saline solution (PBSS), and 2 ml of serum, whole milk, skimmed milk, whey, or PBSS were incubated in duplicate for 60 minutes at 37 C. Isolated blood or milk nuetrophils (polymorphonuclear leukocytes (PMN), 25 X 10(6) cells/ml; 1 ml) were added and incubated at 37 C for 30 minutes. Unphagocytosed [32P]SA organisms were lysed by incubation with 5 ml of lysostaphin (10 U) at 37 C for 30 minutes, and the PMN and phagocytosed T2P]SA were removed by centrifugation. Radioactivity of the supernatant was determined in a scintillation spectrometer and was used in estimate the percentage of [32P]SA phogocytosed. With this procedure, 25 assays in duplicate could be conducted each day with an expected coefficient of variation between duplicates of 5.6%. Blood PMN phagocytosed 80, 44, 74, 72, and 11% of the [32P]SA when incubated in serum, whole milk, skimmed milk, whey, and PBSS, respectively. Mik PMN phagocytosed 78, 44, 72, 74, and 22%, respectively. The addition of cream to either skimmed milk or serum reduced phagocytosis of [32P]SA by both blood and milk PMN. The inhibitory effect of cream was verified by the microscopic observation that PMN containing large quantities of ingested fat contained fewer S aureus. Seemingly, PMN upon entering the alveoli of the mammary gland become less efficiently phagocytic for bacteria, because of the presence of milk fat globules. This phef intramammary infection by invading mastitic pathogens.     

In vitro study of polymorphonuclear leukocyte damage to mammary tissues of lactating cows

Effects of polymorphonuclear neutrophil leukocytes (PMN) on mammary tissue of lactating cows were studied in vitro. The PMN were isolated from mammary glands of nulliparous heifers given an injection of 5 micrograms of Escherichia coli endotoxin. Mammary tissue was obtained from noninfected quarters of 5 lactating Holstein cows and was cultured in supplemented medium 199. Mammary explants were treated by addition of intact or lysed PMN (10(5), 10(6), 10(7)/ml) or PMN (10(5), 10(7)/ml) which were allowed to phagocytose opsonized zymosan. Controls included cultures of mammary tissue alone, PMN alone, and mammary tissue plus zymosan. Cultures were incubated at 37 C for 3, 8, or 24 hours. Tissue from 1 randomly selected culture/treatment was weighed and processed for microscopy. Tissue from remaining cultures was incubated with [3H]amino acids or [14C]acetate to determine rates of protein and fatty acid synthesis. Media from all cultures were assayed for activity of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. An increase (P less than 0.02) in the activity of this enzyme was detected in the medium of explant cultures treated with 10(7) phagocytosing PMN/ml at 3 and 8 hours and with 10(7) intact or lysed PMN/ml at 8 hours. Treatment did not inhibit (P greater than 0.05) rates of protein or fatty acid synthesis. Microscopic examination indicated that epithelial cell damage resulted from treatment with 10(6) and 10(7) intact, lysed, or phagocytosing PMN/ml. Greatest morphologic damage resulted from treatment with phagocytosing PMN.     

Duck lymphocytes--III. Transformation responses to some common mitogens

The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6 degrees C, 8 x 10(5) cells in 200 microliters medium (= 4 x 10(6) cells/ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1-100 micrograms/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of 1 microCi [3H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40-100 micrograms/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3-5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2-10 micrograms/ml in cultures harvested after 4-7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.     

Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils

Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli. During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function. In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma. Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha. Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E. coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN.Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C. The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies. When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity. This is the first study reporting the effects of apoptosis on bovine PMN function. These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection. Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland.     

Pharmacokinetics of azithromycin in foals after i.v. and oral dose and disposition into phagocytes

The properties of azithromycin suggest that it may be an alternative to erythromycin for treatment of Rhodococcus equi pneumonia in foals. To investigate this possibility, the disposition of azithromycin in plasma, polymorphonuclear leukocytes (PMN), and alveolar cells was examined after a single administration in foals. Azithromycin suspension was administered orally (p.o.) at a dose of 10 mg/kg to five healthy 2-3-month-old foals. Two weeks later, azithromycin for injection was administered by intravenous (i.v.) infusion at a dose of 5 mg/kg to the same foals. Plasma samples were collected after p.o. and i.v. administration. Peripheral blood PMN and bronchoalveolar lavage fluid and alveolar cells were collected after p.o. administration. Azithromycin concentrations were determined by reverse-phase high-performance liquid chromatography (HPLC) with coulometric electrochemical detection. Azithromycin p.o. absorption was variable with a mean systemic availability of 39% (+/-20%). The plasma half-life was 16 and 18.3 h after i.v. and p.o. administration, respectively. Azithromycin had a very large volume of distribution (V(d)) of 11.6 L/kg [V(d(ss))] and 12.4 L/kg [V(d(area))]. The large V(d) can be attributed to high tissue and intracellular concentrations, exhibited by the high concentration of azithromycin in PMN and alveolar cells. The PMN half-life was 49.2 h. Dosage of 10 mg/kg of azithromycin p.o. once daily for foals with R. equi pneumonia is recommended for further study.     

Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide.

Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.     

Failure of Lipopolysaccharides to Directly Trigger the Chemiluminescence Response of Isolated Equine Polymorphonuclear Leukocytes

Divergent results have been reported on the effects of lipopolysaccharides (LPS) on the activation of equine polymorphonuclear leukocytes (PMN). We therefore attempted to determine whether LPS alone can stimulate equine PMN or whether plasma factors are necessary. PMN were isolated from citrated blood on a discontinuous density gradient of Percoll. The luminol (10-3 mol/L)-enhanced chemiluminescence (CL) of 1.25 x 106 cells was measured after addition of Escherichia coli LPS (0.001-10 µg/ml) alone or after incubation in autologous plasma (1 h, 37°C). After direct stimulation with LPS, there were random variations of CL in 16 horses that were not reproducible from one sample to the next for the same horse. LPS which had been incubated in plasma gave a dose-dependent stimulation of the CL of the PMN, which did not occur if the plasma had been heat inactivated (1 h, 56°C). These results indicated a role for plasma factors, which were unlikely to be cytokines, as there were no monocytes or lymphocytes in the plasma incubated with LPS, but might have been complement fragments or LPS ligands, such as LPS binding protein. Studies using specific antibodies against these factors are needed to clarify this question.     

Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-alpha, IL-1beta, IL-12 and IFN-gamma

After intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-12, and interferon (IFN)-gamma by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-alpha or IL-1beta. Bovine PMN were shown to secrete TNF-alpha , IL-1beta, IL-12, IL-8 and IFN-gamma in response to LPS. Co-incubation of PMN with LPS and TNF-alpha increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-alpha had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines.     

Pharmacokinetics, bioavailability, and in vitro antibacterial activity of rifampin in the horse

The pharmacokinetics and bioavailability of rifampin were determined after IV (10 mg/kg of body weight) and intragastric (20 mg/kg of body weight) administration to 6 healthy, adult horses. After IV administration, the disposition kinetics of rifampin were best described by a 2-compartment open model. A rapid distribution phase was followed by a slower elimination phase, with a half-life (t1/2[beta]) of 7.27 +/- 1.11 hours. The mean body clearance was 1.49 +/- 0.41 ml/min.kg, and the mean volume of distribution was 932 +/- 292 ml/kg, indicating that rifampin was widely distributed in the body. After intragastric administration of rifampin in aqueous suspension, a brief lag period (0.31 +/- 0.09 hour) was followed by rapid, but incomplete, absorption (t1/2[a] = 0.51 +/- 0.32 hour) and slow elimination (t1/2[d] = 11.50 +/- 1.55 hours). The mean bioavailability (fractional absorption) of the administered dose during the first 24 hours was 53.94 +/- 18.90%, and we estimated that 70.0 +/- 23.6% of the drug would eventually be absorbed. The mean peak plasma rifampin concentration was 13.25 +/- 2.70 micrograms/ml at 2.5 +/- 1.6 hours after dosing. All 6 horses had plasma rifampin concentrations greater than 2 micrograms/ml by 45 minutes after dosing; concentrations greater than 3 micrograms/ml persisted for at least 24 hours. Mean plasma rifampin concentrations at 12 and 24 hours after dosing were 6.86 +/- 1.69 micrograms/ml and 3.83 +/- 0.87 micrograms/ml, respectively. We tested 162 isolates of 16 bacterial species cultured from clinically ill horses for susceptibility to rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phagocytosis and intracellular killing of Staphylococcus aureus by bovine blood polymorphonuclear leukocytes after migration in vitro

Phagocytic activity and intracellular killing of opsonized staphylococci (Smith strain) by bovine blood polymorphonuclear leukocytes (PMNL) was studied after their migration in vitro in blind well chambers. The results indicate that migration of PMNL to RPMI-1640 medium without chemotactic factor significantly (P less than 0.01) increased the percentage of rosette-forming PMNL (from 11 +/- 2 to 49 +/- 4%), as well as phagocytic activity mediated through Fc receptors (FcRs) (from 25 +/- 3 to 81 +/- 4% phagocytizing PMNL and from 151 +/- 22 to 942 +/- 54 number of phagocytized staphylococci/100 PMNL), and intracellular killing of bacteria (from 13 +/- 2 to 59 +/- 7%). On the other hand, PMNL migration to RPMI-1640 medium with the chemotactic factor (serum activated with zymosan [AS] or lipopolysaccharide [LPS] or formyl-L-methionyl-L-leucyl-L-phenylalanine [fMLP]) did not significantly (p greater than 0.05) increase either the FcRs number on the PMNL surface or the phagocytic and bactericidal activity connected with these receptors. The possible mechanisms are discussed.     

ZD制剂对RAW264.7细胞分泌TNF-α水平及TNF-α诱导L929细胞死亡的影响

[目的]研究ZD制剂对炎症模型细胞中TNF-α含量及对TNF-α诱导小鼠成纤维细胞L929死亡的影响。[方法]通过脂多糖（LPS）刺激小鼠腹腔巨噬细胞RAW264.7细胞系来构建炎症细胞模型,使用5种不同浓度（100、10、1、0.1、0.01μg/m L）的ZD制剂进行试验,并用ELISA法检测ZD制剂干预前后细胞上清液中TNF-α浓度。通过TNF-α刺激小鼠成纤维细胞L929细胞系来构建细胞毒性模型,采用MTT法检测ZD制剂干预前后L929细胞活力。[结果]ZD制剂浓度为100、10、1、0.1μg/m L时,TNF-α浓度与LPS模型组相比,差异极显著（P〈0.01）;ZD制剂浓度为0.01μg/m L时,TNF-α浓度与LPS模型组相比,差异显著（P〈0.05）。经100、10μg/m L ZD制剂干预后的小鼠成纤维细胞L929细胞活力与TNF-α组相比显著提高。[结论]ZD制剂可以通过降低TNF-α的分泌量和降低TNF-α的生物学活性来达到抗炎的目的。     

Substance P: a neurogenic mediator of acute cellular inflammation in the dog?

Substance P (SP) is a neuropeptide that has recently been implicated in the pathogenesis of neurogenic inflammation. SP has been shown to activate polymorphonuclear leukocytes (PMN) as well as other inflammatory cells. The present study investigated the direct stimulatory and priming effects of SP on canine PMN aggregation and migration. Direct stimulation of cell migration by SP was present at an unphysiologically high concentration of the mediator. However, when micromolar concentrations of SP were added to PMN prior to stimulation with sub-optimal concentrations of leukotriene B4 (LTB4), the cells exhibited enhanced aggregation and migration, i.e. priming, when stimulated with the latter. Since SP has been reported to act via the formyl-Met-Leu-Phe (fMLP) chemotaxin receptor, this mediator was also studied and found not to possess any effects similar to SP. Thus, the results indicate that SP acts as a primer of canine PMN functions in vitro via a receptor different from that for fMLP. Before ascribing SP a mediator role in canine neurogenic inflammation, in vivo studies determining the concentrations of, and responses to SP in inflamed tissue should be performed.     

Goat cathelicidin‐2 is secreted by blood leukocytes regardless of lipopolysaccharide stimulation

It has been reported that goat cathelicidin‐2, an antimicrobial peptide, localizes in leukocytes and is present in milk. Here, we examined whether cathelicidin‐2 is secreted by leukocytes. Different concentrations (10⁵–10⁸ cells/mL) of blood leukocytes were cultured for 0–48 h with or without lipopolysaccharide (LPS). After culture, the concentrations of cathelicidin‐2 in the conditioned media were measured. Blood was collected from male goats 0–24 h after the intravenous injection of Escherichia coli O111:B4 LPS. The plasma cathelicidin‐2 concentrations were determined and the blood leukocytes immunostained with anti‐cathelicidin‐2 antibody to calculate the proportion of cathelicidin‐2‐positive cells in the total leukocytes. When higher concentrations of leukocytes were cultured, the cathelicidin‐2 concentrations in the media increased significantly, whereas the addition of LPS to the media caused no further increase. The plasma cathelicidin‐2 concentrations did not increase with time after LPS infusion. The proportion of cathelicidin‐2‐positive cells in the total leukocytes was significantly reduced 1 h after LPS injection compared with that at 0 h, but increased again at 6 h and thereafter. These results suggest that cathlicidin‐2 is secreted by leukocytes even without LPS stimulation, whereas LPS may be required for cathelicidin‐2‐containing leukocytes to be recruited from the blood to tissues showing inflammation.     

Analysis of synovial fluid from clinically normal alpacas and llamas

OBJECTIVE: To establish reference range values for synovial fluid from clinically normal New World camelids. ANIMALS: 15 llamas and 15 alpacas. PROCEDURE: Llamas and alpacas were anesthetized with an IM injection of a xylazine hydrochloride, butorphanol tartrate, and ketamine hydrochloride combination. Synovial fluid (1 to 2 ml) was obtained by aseptic arthrocentesis from the radiocarpal and tarsocrural joints. Synovial fluid evaluation included determination of total nucleated cell count (NCC), absolute number and percentage of polymorphonuclear (PMN) and mononuclear leukocytes, total protein, and specific gravity. RESULTS: Synovial fluid evaluation revealed a total NCC of 100 to 1,400 cells/microl (mean +/- SD, 394.8+/-356.2 cells/microl; 95% confidence interval [CI], 295.2 to 494.6 cells/microl). Mononuclear leukocytes were the predominant cell type with lymphocytes, composing 50 to 90% (mean, 75.6+/-172%; 95% CI, 70.8 to 80.4%) of the mononuclear leukocytes. Approximately 0 to 12% (mean, 1.3+/-2.9%; 95% CI, 0.49 to 2.11%) of the cells were PMN leukocytes. Total protein concentrations ranged from 2.0 to 3.8 g/dl (mean, 2.54+/-0.29 g/dl; 95% CI, 2.46 to 2.62 g/dl); the specific gravity ranged between 1.010 and 1.026 (mean, 1.017+/-0.003; 95% CI, 1.016 to 1.018). CONCLUSION AND CLINICAL RELEVANCE: In llamas and alpacas, significant differences do not exist between species or between limbs (left vs right) or joints (radiocarpal vs tarsocrural) for synovial fluid values. Total NCC and absolute number and percentage of PMN and mononuclear leukocyte are similar to those of other ruminants and horses. However, synovial fluid total protein concentrations in New World camelids are high, compared with other domestic species.     

Development of a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, using an antigen of Brucella abortus

A study was conducted to develop a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, with an antigen of Brucella abortus. Different concentrations of mononuclear leukocytes were prepared by the Ficoll-Hypaque technique from the blood of nonvaccinated calves and from calves previously vaccinated with strain 19. Concentrations of 0.5, 1, 2, 3, 4, and 5 x 10(6) leukocytes were suspended in RPMI-1640 medium and various dilutions (20, 10, 1, and 0.1 microgram) of B abortus-soluble antigen, dispensed in triplicate wells cut in 1% agarose containing minimal essential medium and 10% bovine fetal serum. These agarose plates were incubated for 4-, 8-, 12-, 16-, 20-, and 24-hour periods and then were fixed; leukocytes were stained with Wright's stain. Migration distances were measured, and statistical analyses of the data revealed a concentration of 2 x 10(6) cells/well and an antigen concentration of 10 microgram/well. An incubation period of 20 hours was optimal for the assay.     

Effect of probenecid administration on cephapirin pharmacokinetics and concentrations in mares

Cephapirin (20 mg/kg of body weight, IV) was administered before and after 3 doses of probenecid (25, 50, or 75 mg/kg, intragastrically, at 12-hour intervals) to 2 mares. Clearance and apparent volume of distribution, based on area under the curve, were negatively correlated with probenecid dose. Clearance of cephapirin was decreased by approximately 50% by administration of 50 mg of probenecid/kg. Serum, synovial fluid, peritoneal fluid, CSF, urinary, and endometrial concentrations of cephapirin were determined after 5 doses of cephapirin (20 mg/kg, IM, at 12-hour intervals) without and with concurrently administered probenecid (50 mg/kg, intragastrically) to 6 mares, including the 2 mares given cephapirin, IV. Highest mean serum cephapirin concentrations were 16.1 +/- 2.16 micrograms/ml at 0.5 hour after the 5th cephapirin dose [postinjection (initial) hour (PIH) 48.5] in mares not given probenecid and 23.7 +/- 1.30 micrograms/ml at 1.5 hours after the 5th cephapirin dose (PIH 49.5) in mares given probenecid. Mean peak peritoneal fluid and synovial fluid cephapirin concentrations were 6.2 +/- 0.57 micrograms/ml and 6.6 +/- 0.58 micrograms/ml, respectively, without probenecid administration and 12.3 +/- 0.46 micrograms/ml and 10 +/- 0.78 micrograms/ml, respectively, with concurrent probenecid administration. Mean trough cephapirin concentrations for peritoneal and synovial fluids in mares given probenecid were 2 to 3 times higher than trough concentrations in mares not given probenecid. Overall mean cephapirin concentrations were significantly higher for serum, peritoneal fluid, synovial fluid, and endometrium when probenecid was administered concurrently with cephapirin (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume-controlled bronchopulmonary lavage of normal and pneumonic calves

Saline bronchopulmonary lavage of the right lung of 16 anesthetized calves was performed using a single-lumen cuffed endotracheal tube. The initial volume of saline introduced was based on the functional residual capacity (FRC) of the right lung lobes as determined from the proportional weights of the right (58% of total FRC) and left (42% of total FRC) lung lobes. Calves were divided into "pneumonic" and "normal" groups based on clinical signs. Five sequential washes were done on each calf. There was no difference in the percentage of total lavage fluid volume recoverable between normal (83.8 +/- 4.2%) and pneumonic (81.1 +/- 8.2%) calves. Cell yield in the initial wash was consistently greater than in subsequent washes for both normal (12.7 +/- 6.6 X 10(6) cells/kg body weight) and pneumonic (58.1 +/- 37.6 X 10(6) cells/kg body weight) calves, and constituted 62.0% (normal) and 75.4% (pneumonic) of the total recoverable cell yield. Total cell yields were higher (P less than 0.05) in pneumonic calves, primarily due to neutrophil leukocytes (PMN). Neutrophils constituted 53.7 +/- 25% of the total cell yield in the pneumonic calves, but only 12.3 +/- 9.5% in the normal calves. The pulmonary alveolar macrophage (PAM) was the major recoverable cell in normal calves (85.7 +/- 8.7% of total lavage cells). Macrophages constituted a smaller (42.9 +/- 23.5) percentage of the total lavage cells in the pneumonic group due to increased PMN numbers. Viability of recovered cells from the pneumonic calves (91.5 +/- 4.8%) was lower than for the normal calves (94.1 +/- 2.5%), but the difference was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)