Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals, This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 X 10(6) viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 degrees C and 5% CO2. Histologic and histochemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22. Glycosaminoglycan (GAG) assay by dimethylmethylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P < 0.0001) increase in total GAG content was observed by day 3 (0.329 micrograms/mg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 +/- 0.062 micrograms/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations. The CS content significantly (P = 0.0002) increased for the first 14 days of incubation, then a plateau was observed for the remainder of the study. Peak CS content was 0.354 +/- 0.037 micrograms/mg. Concentration of KS reached a plateau earlier than did CS content, with peak amount of 0.193 +/- 0.027 micrograms/mg on day 10. Fluctuations in KS content were not significant until an increase on day 22. Chondrocytes actively populated the collagen implants, increasing in number and synthesizing matrix GAG epitopes over the 22 days of incubation. These results indicate that chondrocyte-laden porous collagen matrices may be suitable cartilage analogue materials and the optimal metabolic time for transfer to cartilage defects is 10 to 14 days.

Effects of intraluminal distention (25 cm of H2O, 120 minutes) and subsequent decompression (60 minutes) on intramural vascular patterns of the small intestine was evaluated in 7 anesthetized horses. Intraluminal distention (25 cm of H2O, 120 minutes) was created in 2 jejunal segments in each horse. Experimental and control segments were removed either immediately after the experimental period or after 60 minutes of decompression. The vascular system of experimental and control jejunal segments was lavaged with NaCl, then was injected with a blue-colored radiopaque medium for microangiography or with a diluted methyl methacrylate for scanning electron microscopy of microcorrosion vascular casts. After angiographic evaluation, tissue sections were prepared for light microscopic evaluation to assess vascular filling and tissue morphology. The distended segments had short villi, which were separated by expanded crypts, and had mesothelial cell loss, neutrophil infiltration, and edema in the seromuscular layer. The number of perfused vessels was significantly (P < 0.05) decreased in the seromuscular layer and, to a lesser extent, in the mucosal layer of the distended segments, compared with controls. After decompression, the morphologic lesions progressed in mucosal and serosal layers and the number of observed vessels increased in all intramural layers; however, vascular density did not return to the predistention state. These results identify altered intramural vascular patterns in the equine jejunum during luminal distention and subsequent decompression.

Bordetella avium is an important respiratory tract pathogen of turkeys. In common with other pathogenic bordetellae, B avium manifests a tissue tropism for cilia of the respiratory tract epithelium. To determine the molecular characteristics of the host cell receptors for B avium, we used hemagglutination and in vivo adherence assays. Carbohydrates, mucus, sialic acid-specific lectin, and other glycoconjugates were evaluated for their ability to competitively inhibit binding of B avium to host cells. The gangliosides, GD(1a) and GT(1b), completely inhibited hemagglutination, whereas N-acetylneuraminic acid (sialic acid) partially inhibited hemagglutination. Adherence to turkey tracheal mucosa in vivo was significantly (P < 0.01) inhibited by GD(1a) and GT(1b) gangliosides, N-acetylneuraminic acid, bovine sub-maxillary mucin, and horseshoe crab (Limulus polyphemus) lectin. Treatment of the tracheal mucosa with neuraminidase also inhibited adherence of B avium. We conclude that N-acetylneuraminic acid and the gangliosides, GD(1a) and GT(1b) may be important components of the tracheal mucosa receptor for B avium in turkeys.

Procedures were developed to count neutrophils in milk using a flow cytometer. Milk samples from 2 experiments were counted: 1 with 4 noninfected cows and a second with 5 noninfected cows that were injected with endotoxin in 2 mammary quarters. Thus, the procedures were evaluated on normal milk and on that with high somatic cell count. Flow cytometric procedures involved fluorescence detection (from the dye carboxydimethylfluorescein diacetate) to distinguish intact and viable from fragmented cells, forward light scatter to detect cell size differences, and right-angle side scatter to detect cellular granularity. High fluorescence, large size, and high degree of granularity identified viable neutrophils. For all samples, neutrophils were also counted manually, using the cytologic centrifugation approach to create the slides; manual counts were used as the standard for comparison. In experiment 1 (normal milk), mean values for percentage of viable neutrophils estimated by manual and flow cytometry procedures agreed closely (26% vs 25.8% for foremilk and 28.8% vs 26.6% for bucket milk). Sources of variation in manual and flow cytometric estimates of percentage of neutrophils were examined. Cow variation was significant (P < 0.01) for manual and flow cytometric counts, but was larger for flow cytometric counts. Day-to-day variation in counts on milk from the same cow was negligible for manual counts, but was significant (P < 0.01) for flow cytometric counts. Coefficients of variation were considerably Larger for manual counts than for flow cytometry. In experiment 2 (milk with high cell count, foremilk), agreement between mean values obtained by flow cytometry and by manual counting was somewhat less. However, predicting manual percentage of neutrophils, using the flow cytometric estimate, had R2 of 0.77. Regression of the manual percentage of neutrophils value on the flow cytometric percentage of neutrophil value was close to 1.0, with only a small negative intercept. Some additional refinement of flow cytometric procedures may be required before flow cytometric estimates of percentage of neutrophils can be accepted without simultaneous validation by use of manual counting. In particular, causes of day-to-day variation in flow cytometric results should be identified and reduced.

One indication for referral of horses to veterinary hospitals is for diagnosis of the microbiologic cause of pneumonia, particularly when the initial treatment fails. Although endoscopic methods have long been available for microbiologic sample collection, accuracy of these methods under these conditions have not been studied in detail. We compared the bacteria isolated from samples obtained by bronchoalveolar lavage (BAL) with those obtained by protected catheter brush (PCB) from foals with unilateral pneumonia induced by inoculation with Klebsiella pneumoniae. As part of previously described clinical trials, foals were administered antimicrobial therapy IM (n = 15) or vehicle IM (n = 7), and collection of distal airway secretion samples was conducted during the treatment period. Sensitivity and specificity of the sample collection methods were assessed by comparison of the isolates from BAL or PCB samples with isolates from tissue of the inoculated lung lobe, which was the most severely affected lung region. Sensitivity and specificity of BAL for recovery of K pneumoniae (challenge strain) and Streptococcus zooepidemicus (common secondary pathogen) was 90 and 69%, respectively, compared with 76 and 85%, respectively, for the PCB method. Sensitivity was significantly (P = 0.03) higher for BAL (100%) than for PCB (69%) for recovery of K pneumoniae (P = 0.03) from lungs. However, difference in the sensitivity of these methods for recovery of S zooepidemicus was not significant. In conclusion, BAL was a more reliable method for recovery of bacteria from the lungs in chronically infected foals that received antimicrobial treatment.

The antibiotic polymyxin B sulfate is a cationic polypeptide with a unique cyclical configuration and distinct cationic characteristics. In this investigation, polymyxin B was evaluated to determine its ability to prevent synthesis of lactic acid and tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide-stimulated strain RAW 2647 macrophage-like cell populations. In this context, gradient concentrations of polymyxin B were formulated in the presence of fixed concentrations of lipopolysaccharide fractions from Escherichia coli (B4:0111), E. coli (J5), Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and S. typhimurium (Re). Quantitation of TNF-alpha was established by the application of a tissue culture-based biological assay system, using the WEHI 164 clone 13 indicator cell line. Investigations also included evaluation of the ability of gradient concentrations of lipopolysaccharide fractions from E. coli (B4:0111), E. coli (J5), K. pneumoniae, P. aeruginosa, S. minnesota, and S. typhimurium (Re) to form a complex with polymyxin B. This was established through application of high-performance thin-layer chromatography techniques. On the basis of the known molecular characteristics of lipopolysaccharide, its lipid A-core subfractions, and polymyxin B, these results imply that cytoprotective properties of polymyxin B are attributable to direct interaction and subsequent complex formation. More specifically, the mechanism by which polymyxin B exerts affinity for lipopolysaccharide fractions is proposed to occur through attractive ionic interactions established between the cationic diaminobutyric acid residues of polymyxin B and the mono- or diphosphate group(s) of the lipid A-core moiety. It is highly probable that this molecular phenomenon is accompanied by hydrophobic interactions established between the terminal methyloctanoyl or methylheptanoyl groups of polymyxin B and the saturated carbon chains of the lipid A-core subfraction of lipopolysaccharide fractions.

Accumulation of D-xylose by jejunal mucosa from healthy horses and rabbits was studied in Vitro. When tissue sheets were incubated with 1 mM D-xylose for 60 minutes, mucosa from horses and rabbits accumulated D-xylose against a concentration gradient. There was no accumulation when equine specimens were incubated with 5 mM D-xylose. By comparison, equine jejunum accumulated D-glucose against a concentration gradient when incubated in 5 mM D-xylose glucose. In equine and rabbit jejunum, 13.3 +/- 7.0% and 36 +/- 11.0%, respectively, of accumulated D-xylose was phosphorylated when sheets were incubated in 1 mM D-xylose. Short-circuit current and potential difference were lower in equine jejunum than in rabbit jejunum, possibly because of differences in tissue thickness. None of the transmucosal electrical measurements increased after addition of D-xylose (1 mM and 5 mM) or D-glucose (5 mM). The active transport system for D-xylose has a low affinity for this sugar and becomes saturated at low intraluminal concentrations. Therefore, abnormal D-xylose absorption test results in horses are more likely caused by abnormalities in mucosal surface area and mucosal permeability than by abnormalities of nutrient carbohydrate absorption.

Cardiovascular and at accompany markedly long periods (12 hours) of halothane anesthesia were characterized. Eight spontaneously breathing horses were studied while they were positioned in left lateral recumbency and anesthetized only with halothane in oxygen maintained at a constant end-tidal concentration of 1.06% (equivalent to 1.2 times the minimal alveolar concentration for horses). Results of circulatory and respiratory measurements during the first 5 hours of constant conditions were similar to those previously reported from this laboratory (ie, a time-related significant increase in systemic arterial blood pressure, cardiac output, stroke volume, left ventricular work, PCV, plasma total solids concentration, and little change in respiratory system function). Beyond 5 hours of anesthesia, arterial blood pressure did not further increase, but remained above baseline. Cardiac output continued to increase, because heart rate significantly (P < 0.05) increased. Peak inspiratory gas flow increased significantly (P < 0.05) in later stages of anesthesia. There was a significant decrease in inspiratory time beginning at 4 hours. Although PaO2, and PaCO2, did not significantly change during the 12 hours of study, PVO2 increased significantly P < 0.05) and progressively with time, beginning 6 hours after the beginning of constant conditions. Metabolic acidosis increased with time significantly [P < 0.05] starting at 9 hours), despite supplemental IV administered NaHCO3. Plasma concentrations of eicosanoids: 6-ketoprostaglandin F1 alpha (PGF1 alpha, a stable metabolite of PGI2), PGF2 alpha, PGE, and thromboxane (TxB2, a stable metabolite of TxA2) were measured in 5 of the 8 horses before and during anesthesia. Significant changes from preanesthetic values were not Significant changes from preanesthetic values were not detected. Dynamic thoracic wall and lung compliances decreased with time.

Critical tests were conducted in horses (n = 11) with naturally acquired infections of benzimidazole (BZ)-resistant population-B small strongyles in 1989 and 1990. Anthelmintics administered were thiabendazole (44 mg/kg of body weight, n = 4), oxibendazole (10 mg/kg, n = 3), and oxfendazole (OFZ, 10 mg/kg; n = 4). All compounds were paste formulations administered orally except for 1 of the OFZ treatments, which was a suspension formulation given by stomach tube. Aggregate mean efficacy was calculated for all species of small strongyles, drug-resistant and nonresistant. The highest efficacy was for oxibendazole (98%) and OFZ 94%); efficacy for thiabendazole was 63%. Five genera and 16 species of small strongyles were recovered from the 11 horses, ranging from 7 to 13 species (mean, 11). Of these, 7 species were found to have resistance in variable degrees to most of the anthelmintics. These strongyles were Cyathostomum catinatum, Cyathostomum coronatum, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus goldi, Cylicostephanus longibursatus, and Cylicostephanus minutus. The large strongyle, Strongylus vulgaris, was present in afl 11 test horses, and efficacy was 100% for all drugs. Seven of the BZ-treated foals (at least 1 horse from each BZ-treatment group), were infected with S edentatus; removal was 100%.

The effect of furosemide-induced weight loss on the energetic responses of horses to running was examined in a 3-way crossover study. Eight 2- to 3-year-old Standardbred mares received, in random order, 10 ml of saline solution 4 hours before running on a treadmill (control trial, C); or, during 2 trials, 1 mg of furosemide/kg of body weight, IV, 4 hours before running. During one of the trials when the horses received furosemide, they carried weight equal to that lost over the 3.75 hours after furosemide administration while running (furosemide-loaded, FL), and during the other trial they did not carry weight equal to that lost after furosemide administration (furosemide-unloaded, FU). Horses performed an incremental exercise test on a treadmill during which rates of oxygen consumption (V(O2)) and carbon dioxide production (V(CO2)) were measured, respiratory exchange ratio was calculated, and blood samples were collected for determination of mixed venous plasma lactate concentration and arterial and mixed venous oxygen saturation. Furosemide treatment caused significantly (P < .001) greater weight loss than did saline administration; mean +/- SEM weight loss (exclusive of fecal loss) was 1.6, 8.8, and 10.2 kg (SEM = 2.0) for C, FL, and FU trials, respectively. The speed at which peak V(O2) was achieved was 9.31, 9.56, and 9.50 (SEM = 0.16) m/s, respectively, time to fatigue was 547, 544, and 553 (SEM = 26) seconds, respectively, and the highest speed attained was 10.3, 10.2, and 10.2 (SEM = 0.2) m/s, respectively. Mean peak rate of oxygen consumption was 130.7, 129.6, and 129.6 (SEM = 1.9) ml/min/kg, respectively. There was a significant (P = 0.070) group X speed interaction for V(CO2); during trial FU, horses had significantly (P < 0.05) lower rate of CO2 production at speed of 9 m/s and at the speed that caused peak V(O2), than during trial C. The respiratory exchange ratio during the FU trial was significantly (P < 0.05) less than that during the C trial at the speed that caused peak V(O2). Plasma lactate concentration at speed of 9 m/s for C, FL, and FU trials was 15.4, 16.5, and 13.3 (SEM = 0.8) mmol/L, respectively; values for the FL and C trials were not significantly different, whereas the mean value for the FU trial was significantly (P < 0.05) less than that for the C trial. Thus, administration of furosemide to horses altered the energetic response to exertion. Replacement of the furosemide-induced weight loss resulted in V(CO2), plasma lactate, and respiratory exchange values indistinguishable from those during the control trial.