Mark Rishniw

Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum

Intestinal bacteria are implicated increasingly as a pivotal factor in the development of Crohns 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 Crohns 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 Crohns disease involving the ileum (13), Crohns 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 Crohns 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 Crohns disease involving the ileum.

Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs.

ABSTRACT 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 Crohns 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 Crohns 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.

Cardiac troponin I is elevated in dogs and cats with azotaemia renal failure and in dogs with non‐cardiac systemic disease

OBJECTIVE To determine if dogs and cats with renal failure, or other severe non-cardiac disease, and no antemortem evidence of cardiac disease on basic clinical evaluation, have elevated levels of cardiac troponin I (cTnI). DESIGN Cross-sectional study using 56 dogs and 14 cats with primary non-cardiac disease (39 dogs with azotaemic renal failure, 14 cats with azotaemic renal failure, 17 dogs with non-cardiac systemic disease); 7/25 dogs and 6/14 cats had murmurs detected on physical examination. Serum or heparinised plasma was collected and analysed for cTnI. RESULTS Cardiac troponin I concentrations were elevated above reference intervals in 70% of dogs and 70% of cats with azotaemic renal failure and in 70% of dogs with a variety of systemic non-cardiac diseases. Cardiac troponin I concentrations did not correlate with the degree of azotaemia, presence of murmurs, hypertension or type of non-cardiac illness. CONCLUSIONS Cardiac troponin I concentration is often elevated in dogs and cats with azotaemic renal failure and in dogs with other systemic non-cardiac illness, suggesting that these conditions often result in clinically inapparent myocardial injury or possibly altered elimination of cTnI.

Hypercoagulability in Cats with Cardiomyopathy

BACKGROUND Arterial thromboembolism (ATE) is a common complication of feline cardiomyopathy; however, the pathogenesis of ATE is unknown. HYPOTHESIS Systemic activation of the coagulation cascade (hypercoagulability) and endothelial injury promote ATE in cardiomyopathic cats. ANIMALS Healthy cats (n = 30) and 3 groups of cardiomyopathic cats: Group (1) left atrial enlargement only (LAE [n = 11]), ie, left atrial to aortic ratio >1.4; Group (2) LAE with spontaneous echocardiographic contrast, atrial thrombi or both (SEC-T [n = 16]); and Group (3) acute ATE with LAE (n = 16). METHODS Hypercoagulability was defined by 2 or more laboratory abnormalities reflecting coagulation factor excess (high fibrinogen concentration or Factor VIII coagulant activity), inhibitor deficiency (low antithrombin activity), or thrombin generation (high thrombin-antithrombin complex [TAT] and d-dimer concentrations). High von Willebrand factor antigen concentration (vWF : Ag) was considered a marker of endothelial injury. Data were analyzed using nonparametric statistics. RESULTS The 3 groups of cats with cardiac disease had higher median fibrinogen concentrations than did the healthy cats. Criteria of hypercoagulability were found exclusively in cats with SEC-T (50%) and ATE (56%). Hypercoagulability was not associated with left atrial size or congestive heart failure (CHF). ATE cats had significantly higher median vWF : Ag concentration than did the other groups. CONCLUSION AND CLINICAL IMPORTANCE Systemic hypercoagulability is evident in many cardiomyopathic cats, often without concurrent CHF or overt ATE. Hypercoagulabilty may represent a risk factor for ATE. High vWF : Ag in ATE cats was attributed to downstream endothelial injury from the occlusive thrombus.

A Multi-Institutional Study Evaluating the Diagnostic Utility of the Spec cPL™ and SNAP® cPL™ in Clinical Acute Pancreatitis in 84 Dogs

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 μg/L), 71.7-77.8% and 80.5-88.0% for SPEC (cutoff value of 400 μg/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.

Morphology of Ventricular Arrhythmias in the Boxer as Measured by 12-Lead Electrocardiography with Pace-Mapping Comparison

The QRS amplitude and polarity were determined in 12-lead electrocardiograms recorded from 22 Boxers with ventricular arrhythmias. Eighty-one percent (18/22) of dogs displayed a positive QRS morphology in the caudoventral leads (II, III, and aVF) and 77% (17/22) of dogs displayed a positive QRS morphology in the left precordial leads (V2-V6). In leads I and V1, the polarity of the QRS complex was variable (positive or negative). To determine if these morphologic features were suggestive of ventricular complexes arising from the right or left ventricle, a comparison was made to the QRS complexes in a pace-mapping study performed in 7 healthy mixed-breed dogs. A total of 3 right and 4 left ventricular sites were paced. None of the left ventricular paced sites resulted in a QRS morphology similar to the most common spontaneous ventricular arrhythmia in the Boxers. In contrast, QRS morphology in each of the 3 right ventricular sites was similar to that observed in the Boxers (P < .033). Each of these produced positive deflections in the caudoventral and left precordial leads, but both positive and negative QRS complexes were observed in leads I and V1 only when the right ventricular septum was paced. This finding suggested that the right ventricular septum might be a site of origin for the ventricular rhythm observed in the Boxers because in the Boxers the polarity of leads I and V1 also varied. Pacing the right ventricular outflow tract always resulted in a negative QRS complex in lead 1, whereas pacing the right ventricular apex always resulted in a positive QRS complex in lead I and a negative QRS complex in V1. However, these locations cannot be excluded as possible sites of origin for the spontaneous ventricular arrhythmias in the Boxers because the arrhythmias could be originating from both of these locations. The spontaneous ventricular arrhythmia of the Boxer is most similar to that of paced ventricular rhythms arising from the right ventricle. More precise localization to a region of the right ventricle such as outflow tract, septal, or apical could not be made.

Spatial Distribution of Helicobacter spp. in the Gastrointestinal Tract of Dogs

Background:  In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture‐independent methods.

Cloning and sequencing of equine cardiac troponin I and confirmation of its usefulness as a target analyte for commercial troponin I analyzers.

The analysis of cardiac troponin I (cTnI) in the diagnosis of myocardial injury in domestic animals is gaining popularity. In this study, equine cTnI was sequenced and compared with previously characterized cTnI from other species. A 6-amino-acid N-terminal deletion unique to the horse was identified. This deletion was outside the epitope region of cTnI recognized by most commercial immunoassays and did not affect the ability of a commercial analyzer system to detect recombinant equine cTnI. No function could be ascribed to the deleted portion. These data support the use of commercial analyzers in measuring equine cTnI.

The quality of veterinary in-clinic and reference laboratory biochemical testing

BACKGROUND Although evaluation of biochemical analytes in blood is common in veterinary practice, studies assessing the global quality of veterinary in-clinic and reference laboratory testing have not been reported. OBJECTIVE The aim of this study was to assess the quality of biochemical testing in veterinary laboratories using results obtained from analyses of 3 levels of assayed quality control materials over 5 days. METHODS Quality was assessed by comparison of calculated total error with quality requirements, determination of sigma metrics, use of a quality goal index to determine factors contributing to poor performance, and agreement between in-clinic and reference laboratory mean results. The suitability of in-clinic and reference laboratory instruments for statistical quality control was determined using adaptations from the computerized program, EZRules3. RESULTS Reference laboratories were able to achieve desirable quality requirements more frequently than in-clinic laboratories. Across all 3 materials, > 50% of in-clinic analyzers achieved a sigma metric ≥ 6.0 for measurement of 2 analytes, whereas > 50% of reference laboratory analyzers achieved a sigma metric ≥ 6.0 for measurement of 6 analytes. Expanded uncertainty of measurement and ± total allowable error resulted in the highest mean percentages of analytes demonstrating agreement between in-clinic and reference laboratories. Owing to marked variation in bias and coefficient of variation between analyzers of the same and different types, the percentages of analytes suitable for statistical quality control varied widely. CONCLUSION These findings reflect the current state-of-the-art with regard to in-clinic and reference laboratory analyzer performance and provide a baseline for future evaluations of the quality of veterinary laboratory testing.

Influence of Enterococcus faecium SF68 probiotic on giardiasis in dogs.

BACKGROUND Giardiasis is a common, potentially zoonotic disease, and dogs often harbor and shed cysts without showing clinical signs. Treatment with the probiotic Enterococcus faecium SF68 has been shown to stimulate mucosal and systemic immunity in a variety of animal models and in young dogs, and to reduce giardial cyst and antigen shedding in rodents. HYPOTHESIS Adult dogs with chronic naturally acquired giardiasis will have decreased giardial fecal cyst and antigen shedding and increased innate and adaptive immunity after 6 weeks probiotic treatment with E. faecium SF68. ANIMALS Twenty adult dogs. METHODS After a 6-week dietary equilibration period, dogs were randomized to receive E. faecium SF68 or placebo for 6 weeks, and then crossed over to the alternate treatment. We measured cyst shedding, fecal giardial antigen, fecal immunoglobulin A (IgA) concentration, and circulating leukocyte phagocytic activity at multiple timepoints to determine the effect of E. faecium SF68 on giardiasis and immune responses in these dogs. RESULTS No differences were observed between placebo or E. faecium SF68 treatment for giardial cyst shedding, fecal antigen shedding, fecal IgA concentration, or leukocyte phagocytic activity. CONCLUSIONS Short-term treatment with E. faecium SF68 of dogs with chronic naturally acquired subclinical giardiasis fails to affect giardial cyst shedding or antigen content and does not alter innate or adaptive immune responses.