Intestinal bacteria are implicated increasingly as a pivotal factor in the development of Crohn's disease, but the specific components of the complex polymicrobial enteric environment driving the inflammatory response are unresolved. This study addresses the role of the ileal mucosa-associated microflora in Crohn's disease. A combination of culture-independent analysis of bacterial diversity (16S rDNA library analysis, quantitative PCR and fluorescence in situ hybridization) and molecular characterization of cultured bacteria was used to examine the ileal mucosa-associated flora of patients with Crohn's disease involving the ileum (13), Crohn's disease restricted to the colon (CCD) (8) and healthy individuals (7). Analysis of 16S rDNA libraries constructed from ileal mucosa yielded nine clades that segregated according to their origin (P<0.0001). 16S rDNA libraries of ileitis mucosa were enriched in sequences for Escherichia coli (P<0.001), but relatively depleted in a subset of Clostridiales (P<0.05). PCR of mucosal DNA was negative for Mycobacterium avium subspecies paratuberculosis, Shigella and Listeria. The number of E. coli in situ correlated with the severity of ileal disease (ρ 0.621, P<0.001) and invasive E. coli was restricted to inflamed mucosa. E. coli strains isolated from the ileum were predominantly novel in phylogeny, displayed pathogen-like behavior in vitro and harbored chromosomal and episomal elements similar to those described in extraintestinal pathogenic E. coli and pathogenic Enterobacteriaceae. These data establish that dysbiosis of the ileal mucosa-associated flora correlates with an ileal Crohn's disease (ICD) phenotype, and raise the possibility that a selective increase in a novel group of invasive E. coli is involved in the etiopathogenesis to Crohn's disease involving the ileum.

The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n ‫؍‬ 13) and controls (n ‫؍‬ 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a "splash"-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohn's disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohn's disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile.

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Background: Pancreas-specific lipase is reported to aid in diagnosing acute pancreatitis (AP) in dogs but has not been rigorously evaluated clinically.Hypothesis/Objectives: To describe variability of disease in dogs with suspected clinical AP, and to evaluate accuracy of 2 pancreatic-specific lipase immunoassays, Spec cPL (SPEC) and SNAP cPL (SNAP), in diagnosing clinical AP. We hypothesized that SPEC and SNAP provide better diagnostic accuracy than serum amylase or total lipase.Animals: A total of 84 dogs; 27 without AP and 57 with clinical signs associated with AP. Methods: Multicenter study. Dogs were prospectively enrolled based upon initial history and physical examination, then retrospectively classified into groups according to the likelihood of having clinical AP by a consensus of experts blinded to SPEC and SNAP results. Bayesian latent class analyses were used to estimate the diagnostic accuracy of SPEC and SNAP.Results: The estimates for test sensitivities and specificities, respectively, ranged between 91.5-94.1% and 71.1-77.5% for SNAP, 86.5-93.6% and 66.3-77.0% for SPEC (cutoff value of 200 lg/L), 71.7-77.8% and 80.5-88.0% for SPEC (cutoff value of 400 lg/L), and were 52.4-56. 0% and 76.7-80.6% for amylase, and 43.4-53.6% and 89.3-92.5% for lipase.Conclusions and Clinical Importance: SNAP and SPEC have higher sensitivity for diagnosing clinical AP than does measurement of serum amylase or lipase activity. A positive SPEC or SNAP has a good positive predictive value (PPV) in populations likely to have AP and a good negative predictive value (NPV) when there is low prevalence of disease.

Two-dimensional (2D) echocardiography is the cornerstone of noninvasive evaluation of the cardiac patient, and often involves estimating left atrial (LA) size. However, 2D echocardiographic methods of estimating LA size have been inadequately described, and most reference intervals are based on M-mode echocardiographic measurements. We determined reference intervals for 4 different 2D echocardiographic methods of estimating LA size in adult (> or =9-month-old) dogs without cardiovascular disease. Thirty-six dogs, placed in right lateral recumbency, were examined by 2D echocardiography. The left atrium was measured at specific time points in the cardiac cycle. Measurement methods were LA diameter in short axis, LA diameter in long axis, LA circumference in short axis, and LA cross-sectional area in short axis. Comparisons of these LA dimensions to appropriate aortic dimensions provided body weight-independent estimates of LA size. We found strong associations of LA dimensions with body weight (r2 = .76-.88). Comparable body weight-independent 2D echocardiographic estimates of LA size in short axis exceeded historical M-mode reference intervals. These data provide echocardiographers with reference intervals for 2D echocardiographic estimates of LA size in adult dogs.

No abstract

We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.

Two-dimensional (2D) echocardiography is the cornerstone of noninvasive evaluation of the cardiac patient, and often involves estimating left atrial (LA) size. However, 2D echocardiographic methods of estimating LA size have been inadequately described, and most reference intervals are based on M-mode echocardiographic measurements. We determined reference intervals for 4 different 2D echocardiographic methods of estimating LA size in adult (Ն9-month-old) dogs without cardiovascular disease. Thirtysix dogs, placed in right lateral recumbency, were examined by 2D echocardiography. The left atrium was measured at specific time points in the cardiac cycle. Measurement methods were LA diameter in short axis, LA diameter in long axis, LA circumference in short axis, and LA cross-sectional area in short axis. Comparisons of these LA dimensions to appropriate aortic dimensions provided body weight-independent estimates of LA size. We found strong associations of LA dimensions with body weight (r 2 ϭ .76-.88). Comparable body weight-independent 2D echocardiographic estimates of LA size in short axis exceeded historical Mmode reference intervals. These data provide echocardiographers with reference intervals for 2D echocardiographic estimates of LA size in adult dogs.Key words: Canine; Cardiac; Echocardiography; Heart; Left atrium; Normal.E chocardiography is the standard method for noninvasive assessment of cardiac function, anatomy, and pathology in domestic animals and humans. Standard imaging planes have been described for 2-dimensional (2D) echocardiography in dogs. 1 Similarly, cardiologists have developed guidelines for assessing function using M-mode echocardiography.Evaluation of left-heart disease usually includes assessment of the size of the left atrium. 2 This evaluation allows the investigator to gauge the severity of the disease and the risk of developing left-sided congestive heart failure. In dogs, the risk of developing congestive heart failure increases with increasing left atrial (LA) size, because LA hypertrophy and stretch reflect increased LA pressure. 3 Several investigators have examined an M-mode method of estimating LA size in small animals based on a method used in human medicine. [4][5][6] Those investigators correlated LA short-axis diameter to body weight or body surface area, and also derived a body weight-independent measure of LA size (left atrium : aorta; [LA : Ao]). 6 A body weightindependent measure of LA size (such as LA : Ao) does not require a body weight measurement and, more importantly, provides a more consistent measure of LA size for any individual, because the aortic diameter in an adult dog would be expected to change less over time than body weight.Unfortunately, the current M-mode method has inherent limitations. These include difficulty in reliably imaging the maximum diameter of the aorta and potentially transecting the left auricle (rather than the LA body) with the M-mode cursor. Consequently, many cardiologists and echocardi- ographers have resorted to measuring LA pa...