The goal of this study was to characterize the Yersinia pestis KIM OmpX protein. Yersinia spp. provide a model for studying several virulence processes including attachment to, and internalization by, host cells. For Yersinia enterocolitica and Yersinia pseudotuberculosis, Ail, YadA and Inv, have been implicated in these processes. In Y. pestis, YadA and Inv are inactivated. Genomic analysis of two Y. pestis strains revealed four loci with sequence homology to Ail. One of these genes, designated y1324 in the Y. pestis KIM database, encodes a protein designated OmpX. The mature protein has a predicted molecular mass of 17.47 kDa, shares approximately 70 % sequence identity with Y. enterocolitica Ail, and has an identical homologue, designated Ail, in the Y. pestis CO92 database. The present study compared the Y. pestis KIM6 + parental strain with a mutant derivative having an engineered disruption of the OmpX structural gene. The parental strain (and a merodiploid control strain) expressed OmpX at 28 and 37 6C, and the protein was detectable throughout all phases of growth. OmpX was required for efficient adherence to, and internalization by, cultured HEp-2 cell monolayers and conferred resistance to the bactericidal effect of human serum. Deletion of ompX resulted in a significantly reduced autoaggregation phenotype and loss of pellicle formation in vitro. These results suggest that Y. pestis OmpX shares functional homology with Y. enterocolitica Ail in adherence, internalization into epithelial cells and serum resistance.

BackgroundThe diagnosis of congestive heart failure (CHF) in cats is challenging. Point‐of‐care (POC) thoracic ultrasound and NT‐proBNP testing are emerging tools that may aid in diagnosis.Hypothesis/ObjectivesTo assess the diagnostic accuracy of POC lung ultrasound (LUS), focused cardiac ultrasound (FCU), and NT‐proBNP in predicting a final diagnosis of CHF.AnimalsFifty‐one cats in respiratory distress.MethodsBlood NT‐proBNP, LUS, and FCU evaluating left atrial (LA) size and presence of pericardial effusion (PCEFF) were performed in all cats. Lung ultrasound findings including pleural effusion (PLEFF), number of B‐lines, and sub‐pleural abnormalities were noted. Medical records were evaluated for final diagnosis.ResultsThirty‐three of 51 (65%) cats were diagnosed with CHF. Lung ultrasound and blood NT‐proBNP were significant predictors of CHF in a multivariate model. The LUS criterion that maximized accuracy for CHF diagnosis was presence of >1 site strongly positive for B‐lines (>3 B‐lines per site), resulting in sensitivity of 78.8%, specificity of 83.3%, and area under the curve (AUC) of 0.833. Subjective LA enlargement was 97.0% sensitive and 100% specific for CHF (AUC 0.985). Presence of PCEFF also was 100% specific, but only 60.6% sensitive, for CHF (AUC 0.803). A positive blood NT‐proBNP test was 93.9% sensitive and 72.2% specific for the diagnosis of CHF (AUC 0.831).Conclusions and Clinical ImportancePoint‐of‐care diagnostic techniques of LUS, FCU, and NT‐proBNP are useful to diagnose CHF in cats with respiratory distress.

OBJECTIVE To characterize lung ultrasonography (LUS) findings in dogs with a primary clinical complaint of cough. ANIMALS 100 client-owned coughing dogs. PROCEDURES A standardized LUS examination was performed for all dogs to quantify the number of B lines and identify subpleural abnormalities at 4 sites on each hemithorax. The final clinical diagnosis (reference standard) was determined by medical record review, and sensitivity and specificity of LUS for the diagnosis of selected causes of cough was determined. RESULTS Common underlying causes of cough included dynamic airway collapse (n = 37), cardiogenic pulmonary edema (CPE; 12), and bronchitis (10). Compared with dogs with other causes of cough, dogs with bacterial pneumonia (n = 7) were more likely to have subpleural shred signs, whereas dogs with pulmonary neoplasia (4) were more likely to have subpleural nodule signs. Dogs with CPE had higher total B-line scores and higher numbers of LUS sites strongly positive for B lines (> 3 B lines/site) than other dogs. The LUS criteria of total B-line score ≥ 10 and presence of ≥ 2 sites strongly positive for B lines were each 92% sensitive and 94% specific for CPE diagnosis. Notably, 18% (16/88) of dogs with noncardiac causes of cough had been treated previously with diuretics because of prior CPE misdiagnosis. CONCLUSIONS AND CLINICAL RELEVANCE LUS profiles in dogs with cough differed by the underlying cause. In dogs with a clinical history of cough, this imaging modality could be diagnostically useful, particularly to help exclude the possibility of underlying CPE.

The Yersinia pestis PhoPQ gene regulatory system is induced during infection of the flea digestive tract and is required to produce adherent biofilm in the foregut, which greatly enhances bacterial transmission during a flea bite. To understand the in vivo context of PhoPQ induction and to determine PhoP-regulated targets in the flea, we undertook whole-genome comparative transcriptional profiling of Y. pestis WT and ΔphoP strains isolated from infected fleas and from temperature-matched in vitro planktonic and flow-cell biofilm cultures. In the absence of PhoP regulation, the gene expression program indicated that the bacteria experienced diverse physiological stresses and were in a metabolically less active state. Multiple stress response genes, including several toxin–antitoxin loci and YhcN family genes responsible for increased acid tolerance, were upregulated in the phoP mutant during flea infection. The data implied that PhoPQ was induced by low pH in the flea gut, and that PhoP modulated physiological adaptation to acid and other stresses encountered during infection of the flea. This adaptive response, together with PhoP-dependent modification of the bacterial outer surface that includes repression of pH 6 antigen fimbriae, supports stable biofilm development in the flea foregut.

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Background Point‐of‐care lung ultrasound (LUS) is an effective tool to diagnose left‐sided congestive heart failure (L‐CHF) in dogs via detection of ultrasound artifacts (B‐lines) caused by increased lung water. Hypothesis/Objectives To determine whether LUS can be used to monitor resolution of cardiogenic pulmonary edema in dogs, and to compare LUS to other indicators of L‐CHF control. Animals Twenty‐five client‐owned dogs hospitalized for treatment of first‐onset L‐CHF. Methods Protocolized LUS, thoracic radiographs (TXR), and plasma N‐terminal pro‐B‐type natriuretic peptide were performed at hospital admission, hospital discharge, and recheck examinations. Lung ultrasound findings were compared between timepoints and to other clinical measures of L‐CHF. Results From time of hospital admission to discharge (mean 19.6 hours), median number of LUS sites strongly positive for B‐lines (>3 B‐lines per site) decreased from 5 (range, 1‐8) to 1 (range, 0‐5; P < .001), and median total B‐line score decreased from 37 (range, 6‐74) to 5 (range, 0‐32; P = .002). Lung ultrasound indices remained improved at first recheck (P < .001). Number of strong positive sites correlated positively with respiratory rate (r = 0.52, P = .008) and TXR edema score (r = 0.51, P = .009) at hospital admission. Patterns of edema resolution differed between LUS and TXR, with cranial quadrants showing more significant reduction in B‐lines compared to TXR edema score (80% vs 29% reduction, respectively; P = .003). Conclusions and Clinical Importance Lung ultrasound could be a useful tool for monitoring resolution of pulmonary edema in dogs with L‐CHF.

A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1 ؉ ) Pgm ؊ (pigmentation negative)/, KIM6(pCD1 ؊ ) Pgm ؉ , and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 ⌬ilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). T he genus Yersinia is comprised of 12 species, three of which, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis, are pathogenic for humans and rodents (5). Y. pestis, the causative agent of plague, is typically transmitted subcutaneously to humans by the bite of an infected flea, causing either bubonic or septicemic plague, but can also be transmitted by aerosols, causing pneumonic plague (25). Pathogenesis of Y. pestis is dependent on the presence of three plasmids: pCD1 encoding a type III secretion system, pPCP1 encoding plasminogen activator (Pla), and pMT1 encoding the capsular antigen fraction 1 (Caf1) (25). Y. pestis has been considered an extracellular pathogen because it contains mutations in two major invasin and adhesin homologues (Inv and YadA) found in invasive Y. pseudotuberculosis (24, 29). However, recent studies demonstrate that Y. pestis is able to invade eukaryotic cells mediated in part by Pla, capsular F1 antigen, OmpX (Ail), and Psa fimbriae (3,18,20,21). None of these factors alone confers the full invasion phenotype.To identify additional Y. pestis surface proteins that may contribute to invasion and virulence, a comprehensive TnphoA insertion library of avirulent Y. pestis KIM6(pCD1 Ϫ ) Pgm ϩ (pigmentation positive) was generated. This avirulent strain was used because it lacks the well-characterized type III secretion system and effector Yop genes on pCD1, and therefore the phoA fusions would be biased toward uncharacterized chromosomal loci. The DNA sequence of transposon insertion junctions was determined, and insertionally inactivated gene DNA sequences were compared to bacterial DNA sequence databases. In this screen, DNA sequence analysis repeatedly identified insertions in a large gene of 9,042 bp (YPO3944) which we designated ilp (intimin/invasinlike protein), with predicted similarity to the class of Gram-negative bacterial intimin/invasin/autotransporter proteins. Because computer analysis of Ilp predicted a three-dimensional structure similar to the C-typ...