The objectives of this study were to determine the effect of bacterial culture conditions on adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 in vivo to pig enterocytes and to compare the results with adherence in vitro to cultured HEp-2 and IPEC-J2 cells. Growth of O157:H7 in MacConkey broth (MB) resulted in almost no adherence to both HEp-2 and IPEC-J2 cells; prior exposure of the bacteria to pH 2.5 reduced adherence. There was greater adherence by bacteria from static cultures than by those from shaken cultures and by bacteria cultured in brain-heart infusion (BHI) plus NaHCO3 (BHIN) than by bacteria cultured in BHI. In contrast, in pig ileal loops, bacteria cultured in MB adhered well to enterocytes, and prior exposure to pH 2.5 had no effect on adherence. Among several media tested for their effect on bacterial adherence in the pig intestine, MB and BHIN proved to be the best. Bacterial adherence was dose-dependent and was more extensive in the ileum than in the colon. This study demonstrated that there are remarkable differences between culture conditions that promote adherence of an EHEC O157:H7 strain in vitro and in vivo, that culture conditions profoundly affect adherence to epithelial cells in vitro and in vivo, and that pig ileal loops are better suited to adherence studies than are colon loops.

Objective—To identify a causative mutation for dilated cardiomyopathy (DCM) in Doberman Pinschers by sequencing the coding regions of 10 cardiac genes known to be associated with familial DCM in humans. Animals—5 Doberman Pinschers with DCM and congestive heart failure and 5 control mixed-breed dogs that were euthanized or died. Procedures—RNA was extracted from frozen ventricular myocardial samples from each dog, and first-strand cDNA was synthesized via reverse transcription, followed by PCR amplification with gene-specific primers. Ten cardiac genes were analyzed: cardiac actin, α-actinin, α-tropomyosin, β-myosin heavy chain, metavinculin, muscle LIM protein, myosinbinding protein C, tafazzin, titin-cap (telethonin), and troponin T. Sequences for DCM-affected and control dogs and the published canine genome were compared. Results—None of the coding sequences yielded a common causative mutation among all Doberman Pinscher samples. However, 3 variants were identified in the α-actinin gene in the DCM-affected Doberman Pinschers. One of these variants, identified in 2 of the 5 Doberman Pinschers, resulted in an amino acid change in the rod-forming triple coiled-coil domain. Conclusions and Clinical Relevance—Mutations in the coding regions of several genes associated with DCM in humans did not appear to consistently account for DCM in Doberman Pinschers. However, an α-actinin variant was detected in some Doberman Pinschers that may contribute to the development of DCM given its potential effect on the structure of this protein. Investigation of additional candidate gene coding and noncoding regions and further evaluation of the role of α-actinin in development of DCM in Doberman Pinschers are warranted.

Objective—To determine effects of a continuous rate infusion of lidocaine on the minimum alveolar concentration (MAC) of sevoflurane in horses. Animals—8 healthy adult horses. Procedures—Horses were anesthetized via IV administration of xylazine, ketamine, and diazepam; anesthesia was maintained with sevoflurane in oxygen. Approximately 1 hour after induction, sevoflurane MAC determination was initiated via standard techniques. Following sevoflurane MAC determination, lidocaine was administered as a bolus (1.3 mg/kg, IV, over 15 minutes), followed by constant rate infusion at 50 μg/kg/min. Determination of MAC for the lidocaine-sevoflurane combination was started 30 minutes after lidocaine infusion was initiated. Arterial blood samples were collected after the lidocaine bolus, at 30-minute intervals, and at the end of the infusion for measurement of plasma lidocaine concentrations. Results—IV administration of lidocaine decreased mean ± SD sevoflurane MAC from 2.42 ± 0.24% to 1.78 ± 0.38% (mean MAC reduction, 26.7 ± 12%). Plasma lidocaine concentrations were 2,589 ± 811 ng/mL at the end of the bolus; 2,065 ± 441 ng/mL, 2,243 ± 699 ng/mL, 2,168 ± 339 ng/mL, and 2,254 ± 215 ng/mL at 30, 60, 90, and 120 minutes of infusion, respectively; and 2,206 ± 329 ng/mL at the end of the infusion. Plasma concentrations did not differ significantly among time points. Conclusions and Clinical Relevance—Lidocaine could be useful for providing a more balanced anesthetic technique in horses. A detailed cardiovascular study on the effects of IV infusion of lidocaine during anesthesia with sevoflurane is required before this combination can be recommended.

Objective—To investigate the effect of opsonization of Rhodococcus equi with R equi-specific antibodies in plasma on bacterial viability and phagocyte activation in a cell culture model of infection. Sample—Neutrophils and monocyte-derived macrophages from 6 healthy 1-week-old foals and 1 adult horse. Procedures—Foal and adult horse phagocytes were incubated with either opsonized or nonopsonized bacteria. Opsonization was achieved by use of plasma containing high or low concentrations of R equi-specific antibodies. Phagocyte oxidative burst activity was measured by use of flow cytometry, and macrophage tumor necrosis factor (TNF)-α production was measured via an ELISA. Extracellular and intracellular bacterial viability was measured with a novel R equi-luciferase construct that used a luminometer. Results—Opsonized bacteria increased oxidative burst activity in adult horse phagocytes, and neutrophil activity was dependent on the concentration of specific antibody. Secretion of TNF-α was higher in macrophages infected with opsonized bacteria. Opsonization had no significant effect on bacterial viability in macrophages; however, extracellular bacterial viability was decreased in broth containing plasma with R equi-specific antibodies, compared with viability in broth alone. Conclusions and Clinical Relevance—The use of plasma enriched with specific antibodies for the opsonization of R equi increased the activation of phagocytes and decreased bacterial viability in the extracellular space. Although opsonized R equi increased TNF-α secretion and oxidative burst in macrophages, additional factors may be necessary for effective intracellular bacterial killing. These data have suggested a possible role of plasma antibody in protection of foals from R equi pneumonia.

Objective—To investigate the in vitro effect of the combination of lignan enterolactone (ENL) or lignan enterodiol (END) with melatonin on steroid hormone secretion and cellular aromatase content in human adrenal carcinoma cells. Sample—Human adrenocortical carcinoma cells. Procedures—Melatonin plus ENL or END was added to cell culture medium along with cAMP (100μM); control cells received cAMP alone. Medium and cell lysates were collected after 24 and 48 hours of cultivation. Samples of medium were analyzed for progesterone, 17-hydroxyprogesterone, androstenedione, aldosterone, estradiol, and cortisol concentration by use of radioimmunoassays. Cell lysates were used for western blot analysis of aromatase content. Results—The addition of ENL or END with melatonin to cAMP-stimulated cells (treated cells) resulted in significant decreases in estradiol, androstenedione, and cortisol concentrations at 24 and 48 hours, compared with concentrations in cells stimulated with cAMP alone (cAMP control cells). The addition of these compounds to cAMP-stimulated cells also resulted in higher progesterone and 17-hydroxyprogesterone concentrations than in cAMP control cells; aldosterone concentration was not affected by treatments. Compared with the content in cAMP control cells, aromatase content in treated cells was significantly lower. Conclusions and Clinical Relevance—The combination of lignan and melatonin affected steroid hormone secretion by acting directly on adrenal tumor cells. Results supported the concept that this combination may yield similar effects on steroid hormone secretion by the adrenal glands in dogs with typical and atypical hyperadrenocorticism.

Objective—To examine the expression and distribution of tight junction (TJ) and adherens junction (AJ) proteins in canine duodenal and colonic mucosa. Sample—Mucosa obtained from 4 healthy Beagles. Procedures—Biopsy specimens of the duodenum and colon were obtained via endoscopy from 4 healthy dogs. The expression patterns and subcelluar localization of claudin-1, -2, -3, -4, -5, -7, and -8; E-cadherin; and β-catenin in the duodenum and colon were analyzed by use of immunoblotting and immunofluorescence microscopy. Results—In the duodenum, there was clear expression of claudin-3 and -5, E-cadherin, and β-catenin proteins and weak expression of claudin-7 protein. In contrast, there was clear expression of claudin-2 and -3, E-cadherin, and β-catenin proteins and weak expression of claudin-5 and -7 proteins in the colon, as determined by use of immunoblotting. As determined by the use of immunofluorescence microscopy, the duodenum and colon had staining for claudin-3 and -5, E-cadherin, and β-catenin in the most apical region and staining for claudin-7 in the basolateral region. Staining for claudin-2 was also observed in the colon. Conclusions and Clinical Relevance—Information was provided about the expression patterns of TJ and AJ proteins in the duodenum and colon of clinically normal dogs. These results may provide valuable information for use in evaluating the importance of these TJ and AJ proteins in the pathogenesis of inflammatory bowel disease in dogs.

Objective—To characterize and compare the urine protein content in cats without urinary tract disease and cats with idiopathic cystitis (IdC), bacterial urinary tract infection (UTI), or urolithiasis. Animals—Control cats (n = 18) and cats with IdC (18), UTI (12), and urolithiasis (12) from which urine samples were obtained and 2 cats with obstructive IdC and 4 additional control cats from which postmortem urinary bladder biopsy specimens were obtained. Procedures—Protein contents in urine samples obtained via cystocentesis or catheterization were measured via the Bradford method. Urine proteins were separated by means of 1-dimensional gel electrophoresis. Evaluation of fibronectin content was performed via western blotting and immunohistochemical analysis. Urinary bladder biopsy specimens were examined histologically and analyzed immunohistochemically for fibronectin. Results—Urine fibronectin content was significantly greater in cats with IdC, compared with control cat findings. Urine fibronectin contents did not differ significantly among controls and cats with UTI or urolithiasis. Histologic examination of bladder biopsy specimens obtained from 2 cats with obstructive IdC revealed destruction of the urothelial lining of the urinary bladder and severe fibrosis; immunohistochemical analysis revealed few fluorescence signals for fibronectin, unlike findings in control bladder biopsy specimens. Conclusions and Clinical Relevance—Results indicated that urine fibronectin content in cats with IdC was greater than that in controls, cats with UTI, or cats with urolithiasis. In cats with IdC, increased permeability of damaged urothelium may result in detachment and leakage of fibronectin into urine. Urine fibronectin might serve as a biomarker for diagnosis of IdC in cats.

Objective: To compare echocardiographic variables of dogs with postmortem anatomic measurements and histologic characteristics of the mitral valve (MV). Animals: 21 cardiologically normal dogs. Procedures: The MV was measured echocardiographically by use of the right parasternal 5-chamber long-axis view. Dogs were euthanized, and anatomic measurements of the MV annulus (MVa) were performed at the level of the left circumflex coronary artery. Mitral valve leaflets (MVLs) and chordae tendineae were measured. Structure of the MVLs was histologically evaluated in 3 segments (proximal, middle, and distal). Results: Echocardiographic measurements of MVL length did not differ significantly from anatomic measurements. A positive correlation was detected between body weight and MVa area. There was a negative correlation between MVa area and the percentage by which the MVL area exceeded the MVa area. Anterior MVLs had a significantly higher number of chordae tendineae than did posterior MVLs. Histologically, layering of MVLs was less preserved in the distal segment, whereas the muscular component and adipose tissue were significantly more diffuse in the proximal and middle segments. Conclusions and Clinical Relevance: The MV in cardiologically normal dogs had wide anatomic variability. Anatomic measurements of MVL length were correlated with echocardiographic measurements.

Objective—To evaluate effects of imaging plane, flexion and extension, patient weight, and observer on computed tomographic (CT) image measurements of the area of the lumbosacral (L7-S1) intervertebral foramen (LSIF) in dogs. Sample—12 dog cadavers (2 were excluded because of foraminal stenosis). Procedures—In each cadaver, sagittal, sagittal oblique, transverse oblique, and double oblique CT images were obtained at 3 zones (entrance, middle, and exit zones) of the region of the lateral lumbar spinal canal that comprises the LSIF while the lumbosacral junction (LSJ) was positioned in flexion or extension. Barium-impregnated polymethylmethacrylate was used to fill the intervertebral foramina to aid boundary detection. Measurements of interest were obtained. Results—Among the dog cadavers, there was large variability in LSIF cross-sectional areas (range, 0.12 to 0.44 cm2; SD, 0.1 cm2) and in foraminal angles required to obtain a double oblique plane in LSJ extension (SD, 8° to 9°). For LSIF area measurements in standard sagittal CT images, interobserver variability was 23% to 44% and intraobserver variability was 4% to 5%. Sagittal oblique images obtained during LSJ extension yielded smaller mean LSIF areas (0.30 cm2), compared with findings in sagittal images (0.37 to 0.52 cm2). The exit and middle zone areas were smaller than the entrance zone area in sagittal images obtained during LSJ extension. Conclusions and Clinical Relevance—Repeated measurements of the LSIF area in images obtained during LSJ extension may be unreliable as a result of interobserver variability and the effects of dog positioning and CT slice orientation.

Objective—To evaluate the ability of 2-D time-of-flight (ToF) magnetic resonance angiography (MRA) to depict intracranial vasculature and compare results obtained with 3.0- and 7.0-T scanners in dogs. Animals—5 healthy Beagles. Procedures—2-D ToF-MRA of the intracranial vasculature was obtained for each dog by use of a 3.0-T and a 7.0-T scanner. Quantitative assessment of the images was obtained by documentation of the visibility of major arteries comprising the cerebral arterial circle and their branches and recording the number of vessels visualized in the dorsal third of the brain. Qualitative assessment was established by evaluation of overall image quality and image artifacts. Results—Use of 3.0- and 7.0-T scanners allowed visualization of the larger vessels of the cerebral arterial circle. Use of a 7.0-T scanner was superior to use of a 3.0-T scanner in depiction of the first- and second-order arterial branches. Maximum-intensity projection images had a larger number of vessels when obtained by use of a 7.0-T scanner than with a 3.0-T scanner. Overall, image quality and artifacts were similar with both scanners. Conclusions and Clinical Relevance—Visualization of the major intracranial arteries was comparable with 3.0- and 7.0-T scanners; the 7.0-T scanner was superior for visualizing smaller vessels. Results indicated that ToF-MRA is an easily performed imaging technique that can be included as part of a standard magnetic resonance imaging examination and should be included in the imaging protocol of dogs suspected of having cerebrovascular disease.