The flagellar master operon flhDC is a pleiotropic regulator involved in motility and virulence of the fish pathogen Yersinia ruckeri.

AIMS: To investigate the function of the master flagellar operon flhDC in the fish pathogen Yersinia ruckeri and compare the effect of a constructed flhD mutation to a naturally occurring fliR mutation causing loss-of-motility in emergent biotype 2 (BT2) strains. METHODS AND RESULTS: Yersinia ruckeri flhD and fliR mutants were constructed in a motile strain. Both mutations caused loss-of-motility, ablation of flagellin synthesis and phospholipase secretion, similar to naturally occurring BT2 strains. Transcriptome analysis confirmed flhDC regulation of flagellar, chemotaxis and phospholipase loci as well as other genes of diverse function. The flhD mutation confers a competitive advantage within the fish host when compared with its parent strain, while this advantage was not seen with the naturally occurring fliR mutation. CONCLUSIONS: An intact flhD is necessary for expression of the flagellar secretion system as well as other diverse loci, consistent with a role for flhD as a pleiotropic regulator. The maintenance of the flhD locus in Y. ruckeri strains suggests its importance for aspects of Y. ruckeri biology other than virulence, since the flhD mutation conferred a competitive advantage during experimental challenge of rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Yersinia ruckeri is the causative agent of enteric red mouth disease, an invasive septicaemia that affects farmed salmonid fish species. Disease outbreaks can cause severe economic losses in aquaculture. BT2 variants, which have independently emerged worldwide, are an increasing threat to farmed fish production. Knowledge of mechanisms involved in virulence, conserved functions and gene regulation among strains may be exploited for the development of novel disease control strategies to prevent pathogen growth or virulence phenotypes within aquaculture.

Diagnostic tools for rapid detection and quantification of Weissella ceti NC36 infections in rainbow trout.

Weissellosis of rainbow trout is caused by the Gram-positive bacteria Weissella ceti and has been reported in China, Brazil and the United States. This disease can result in high mortality in market-sized fish and thus can cause significant economic loss. Thus far, phenotypic characterization and 16S rRNA sequencing have been used to confirm a Weissellosis diagnosis. Here, we present the development of PCR-based diagnostic tools for the rapid identification and quantification of W. ceti within bacteriological culture and infected tissues. A duplex PCR, which amplifies both genus- and strain-specific targets, positively identifies isolates as W. ceti NC36. A qPCR assay was also developed to quantify pathogen load from infected tissues, using a W. ceti NC36 unique locus. A proof of concept study was performed to demonstrate that quantification using traditional plate count methods and qPCR were significantly correlated when assessed from infected brain and spleen tissue. These tools were also used to confirm diagnosis of Weissellosis in a commercial rainbow trout farm during an outbreak investigation. These are the first diagnostic tools developed for identification and quantification of W. ceti infection within rainbow trout, contributing to rapid Weissellosis diagnosis, enhanced pathogen surveillance and epidemiological studies.

Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

Rare case of occupational pulmonary hemorrhage in a firefighter.

Alveolar hemorrhage associated with physical exertion, known as exercise-induced pulmonary hemorrhage (EIPH), is a rare condition linked to strenuous exertion. This can be an unusual form of respiratory and occupational illness. We present the case of a healthy firefighter who developed fatal pulmonary hemorrhage after participating in a strenuous physical training exercise regimen. This case represents a severe presentation of EIPH, which results from the disruption of the pulmonary blood-gas barrier as a result of strenuous exertion. Clinicians caring for those in vocations and recreation involving extremely vigorous exercise should be aware that such activities can cause EIPH.

Rapid genotyping assays for the identification and differentiation of Yersinia ruckeri biotype 2 strains.

AIMS: To establish PCR-based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype-causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States. METHODS AND RESULTS: Novel assays were developed relying on detection of mutant allele-specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing. CONCLUSIONS: The described methods were specific, rapid and simple to perform and interpret. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.

Myoglobin production in emperor penguins.

Increased oxygen storage is essential to the diving capacities of marine mammals and seabirds. However, the molecular mechanisms underlying this adaptation are unknown. Myoglobin (Mb) and Mb mRNA concentrations were analyzed in emperor penguin (Aptenodytes forsteri) adults and chicks with spectrophotometric and RNase protection assays to evaluate production of their large Mb-bound O(2) stores. Mean pectoral Mb concentration and Mb mRNA content increased throughout the pre-fledging period and were 15-fold and 3-fold greater, respectively, in adults than in 3.5 month old chicks. Mean Mb concentration in 5.9 month old juveniles was 2.7+/-0.4 g 100 g(-1) muscle (44% that of wild adults), and in adults that had been captive all their lives it was 3.7+/-0.1 g 100 g(-1) muscle. The Mb and Mb mRNA data are consistent with regulation of Mb production at the level of transcription as in other animals. Significant Mb and Mb mRNA production occurred in chicks and young juveniles even without any diving activity. The further increase in adult Mb concentrations appears to require the exercise/hypoxia of diving because Mb concentration in captive, non-diving adults only reached 60% of that of wild adults. The much greater relative increase in Mb concentration than in Mb mRNA content between young chicks and adults suggests that there is not a simple 1:1 relationship between Mb mRNA content and Mb concentration. Nutritional limitation in young chicks and post-transcriptional regulation of Mb concentration may also be involved.

Cloning, sequencing and overexpression of the gene encoding malate dehydrogenase from the deep-sea bacterium Photobacterium species strain SS9.

The gene encoding malate dehydrogenase (mdhA) was obtained from the psychrophilic, barophilic, deep-sea isolate Photobacterium species strain SS9. The SS9 mdhA gene directed high levels of malate dehydrogenase (MDH) production in Escherichia coli. A comparison of SS9 MDH to three mesophile MDHs, a MDH sequence obtained from another deep-sea bacterium, and to other psychrophile proteins is presented.

Imaging-guided biopsy.

In the past 10 years, imaging-guided biopsy has become an accepted technique of tissue retrieval for pathologic analysis. Fluoroscopy, ultrasonography, computed tomography, and magnetic resonance imaging are the current methods of guiding biopsy procedures. Each modality has advantages and disadvantages for various organ systems. High accuracy rates and low complication rates can be expected. Cost savings and rapid reporting of results add to the value of these procedures.

Current applications of duplex and color Doppler ultrasound imaging: carotid and peripheral vascular system.

Recent advances in ultrasound technology have made possible the development of diagnostic instruments that combine cross-sectional imaging and Doppler analysis. These instruments have expanded the role of diagnostic ultrasonography to the assessment of carotid and peripheral vascular disease. The current applications of duplex Doppler and color Doppler imaging in evaluating the extracranial carotid arteries, vertebral artery, peripheral venous system, and peripheral arterial system are reviewed. The indications for and limitations of these examinations, as well as the potential future uses, are discussed.

Percutaneous placement of the Greenfield vena caval filter.

During the period from August 1986 to August 1987, 50 patients underwent percutaneous placement of a Greenfield vena caval filter from the right femoral vein, left femoral vein, or right internal jugular vein at our institution. All 50 patients had a contraindication to anticoagulation therapy or had complications of anticoagulation for deep venous thrombosis or pulmonary emboli. The percutaneous placement was accomplished in the angiographic suite with use of local anesthesia and was well tolerated by all patients. Only three complications related to the percutaneous approach occurred during the short-term follow-up (3 months to 1 year). These complications were deep venous thrombosis of the leg in two patients and misplacement of the filter in one patient. The three patients tolerated these complications well. We conclude that placement of Greenfield vena caval filters can be readily accomplished by means of percutaneous entry. Our experience demonstrated minimal associated morbidity and no mortality.

Directed intravascular precipitation of bisantrene for pelvic malignant lesions: preclinical studies.

Bisantrene, a clinically active anticancer drug with limited solubility at physiologic pH, was delivered by selective injection into the internal iliac artery of male calves. The percutaneous transfemoral angiographic techniques used in the calves were identical to those used in adult human patients. Directed intravascular precipitation of bisantrene at the maximal tolerable clinical dose for intravenous administration (260 mg/m2) caused severe tissue damage in 5 of 10 animals that received these intra-arterial injections. (One calf in this study group died of unknown causes 10 days after the drug infusion). A reduced intra-arterial dose (50 mg/m2) was used in seven calves, and no local tissue damage was evident on gross or microscopic examination. Nevertheless, resultant concentrations of bisantrene deposited in the ipsilateral bladder wall were 10- to 100-fold those concentrations found after intravenous administration of a dose 5 times higher. These animal toxicology and pharmacology data support initiation of a phase I clinical trial of directed intravascular precipitation of bisantrene in humans. This clinical trial will be developed for patients with advanced refractory cancers of the anatomic true pelvis, such as those originating in the urinary bladder, prostate, rectum, and uterine cervix.

Renal cell carcinoma metastatic to the pancreas: clinical and radiological features.

OBJECTIVE: To review the clinical features, computed tomographic (CT) appearance, and treatment outcomes in a case series of patients with renal cell carcinoma (RCC) metastatic to the pancreas. PATIENTS AND METHODS: We retrospectively reviewed the records of 23 patients (15 men and 8 women) with RCC metastatic to the pancreas, detected by CT examination between 1986 and 1996. All patients had undergone a previous nephrectomy for RCC. RESULTS: Isolated mild elevation in liver function test results (in 5 patients) or in serum amylase level (in 8 patients) was observed. New-onset diabetes was detected in 3 patients. The CT characteristics of the pancreatic metastases generally resembled those of primary RCC with well-defined margins and greater enhancement than normal pancreas with a central area of low attenuation. The mean interval between resection of the primary RCC and detection of the pancreatic metastases was 116 months (range, 1-295 months). In 18 patients (78%), the pancreatic metastases were diagnosed more than 5 years after nephrectomy. The pancreas was the initial metastatic site in 12 patients (52%). Survival was shortened with higher tumor grade (mean survival time of 41 months and 10 months in patients with grade 2 and 3, respectively). Surgical resection was carried out in 11 patients (7 distal and 3 total pancreatectomies and 1 distal pancreatectomy followed 4 years later by total pancreatectomy), with 8 patients alive at a mean follow-up of 4 years, 6 of whom remained free of recurrence. Overall, 12 patients (52%) were alive at a mean of 42 months after diagnosis of metastatic disease. CONCLUSIONS: The appearance of metastatic RCC lesions in the pancreas closely resembles the appearance of primary RCC on CT images. Pancreatic metastases from RCC are frequently detected many years after nephrectomy. Patient survival correlates with tumor grade. Histologic analysis of pancreatic masses in patients with a history of resected primary RCC is important since the prognosis for RCC metastatic to the pancreas is much better than that for primary pancreatic adenocarcinoma.

Renal artery angioplasty: technical results and clinical outcome in 320 patients.

OBJECTIVE: To review the results of percutaneous transluminal renal artery angioplasty (PTRA), including technical success and clinical outcome, at Mayo Clinic Rochester. DESIGN: We retrospectively reviewed our experience with 320 patients who underwent PTRA for stenosis of 396 arteries during a 14-year period. MATERIAL AND METHODS: We reviewed medical records and angiograms of patients who underwent PTRA at Mayo Clinic Rochester between January 1980 and December 1993. The patients were divided into four groups, based on clinical history and angiographic appearance of the stenosing lesion: renal artery atherosclerosis (ASO group), fibromuscular dysplasia (FMD group), previous renal artery bypass or endarterectomy, and renal artery stenosis in a solitary kidney. Technical results of the PTRA were determined by evaluation of angiograms obtained before and after the procedure. Data on patient demographics, blood pressure, antihypertensive medications, and serum creatinine were recorded for the period preceding PTRA, after the procedure, and at last follow-up. RESULTS: All groups had statistically significant reductions in mean arterial pressure and antihypertensive medications after PTRA. The percentage of patients who benefited after renal artery angioplasty was 70% for patients with ASO (8.4% cured), 63% for patients with FMD (22% cured), 53.8% for patients with prior surgical revascularization (23.1% cured), and 91.7% for patients with a solitary kidney (0% cured). No significant overall change in serum creatinine level was noted after the procedure in any group. Complications were comparable to those reported in other studies. The 30-day all-cause mortality rate was 2.2% for the current study, all deaths occurring in the ASO group. CONCLUSION: PTRA rarely leads to a "cure" of renovascular hypertension but provides effective control of blood pressure and decreases the medication requirements in selected patients.

Pituitary desensitization to gonadotropin-releasing hormone increases abdominal adiposity in hyperandrogenic anovulatory women.

OBJECTIVE: To determine whether hyperandrogenism in anovulatory women affects body fat distribution. DESIGN: Prospective nonrandomized study. SETTING: An academic research environment. PATIENT(S): Ten hyperandrogenic anovulatory patients and 10 healthy women matched by body mass index. INTERVENTION(S): Regional body fat analysis was performed before and after 3 months of GnRH analogue (GnRH-a) therapy. MAIN OUTCOME MEASURE(S): Body fat distribution was measured by waist-to-hip circumference ratio, single-slice computed tomography imaging (L2-3 interspace), and total body dual-energy x-ray absorptiometry. RESULT(S): Weight, body mass index, waist-to-hip circumference ratio, total body and leg fat mass, and subcutaneous adipose area were unaffected by the presence of hyperandrogenism or the use of GnRH-a therapy. Basal abdominal fat mass, abdomen-to-leg fat mass ratio, visceral adipose area, and total visceral adipose volume were comparable in both study groups. The abdominal fat mass increased in both groups during GnRH-a therapy, whereas the abdomen-to-leg fat mass ratio rose significantly only in the hyperandrogenic patients. During GnRH-a therapy, the hyperandrogenic patients demonstrated a significant increase in visceral adipose area compared with the healthy women so that total visceral adipose volume increased significantly in the former but not the latter. CONCLUSION(S): Three months of GnRH-a administration preferentially increased abdominal fat, as measured by single-slice computed tomography imaging and total body dual-energy x-ray absorptiometry, in hyperandrogenic anovulatory women.

Hemorrhage in major pelvic fractures.

Significant hemorrhage following major pelvic fractures should always be expected. Early recognition of such fractures during the resuscitation of any multiply injured patient is essential before instituting measures that might combat blood loss. In the majority of patients, simple resuscitative measures, including employment of the pneumatic antishock garment, will suffice. With certain types of fracture geography, the early application of external fixation devices may also play an important role. Increasingly popular has been the technique of diagnostic angiography and therapeutic embolization, applicable to approximately 3 per cent of all pelvic fracture patients. With exsanguinating hemorrhage, even the best equipped and most sophisticated major trauma centers can be taxed. The decision whether a patient should be taken directly to the operating room or to the angiography suite remains one of the most difficult for even the most highly skilled trauma surgeon. Patients with rapidly expanding or free rupture of pelvic hematomas noted at the time of celiotomy, or those with large open wounds, usually leave no recourse but to attempt direct operative control, to include even the most morbid option of a life-saving hemipelvectomy or corpectomy. More often, however, once other sources of surgically correctable hemorrhage are controlled or ruled out, diagnostic angiography followed by therapeutic embolization is a mainstay in the modern-day management of pelvic fracture hemorrhage.

Fast CT for pulmonary embolus.

Recent technical advances in CT have led to the ability to scan a volume in a shorter period with optimal contrast opacification of blood vessels, including the pulmonary arteries. Initially, there were isolated instances of an accidental detection of a filling defect because of pulmonary embolus in the pulmonary arteries. Gradually, directed examinations were introduced to diagnose or exclude pulmonary embolus. These examinations were successful. Radiologists and clinicians now are turning to fast CT for this purpose for a variety of reasons, including the proven accuracy and reliability of the method and the relative deficiencies of traditional noninvasive methods. Accurate interpretation of CT scans for possible pulmonary embolus depends on the ability to conduct a high-quality examination and to recognize the spectrum of findings for both acute and chronic emboli. This article reviews the history of fast CT pulmonary angiography, and it presents both technical and diagnostic information germaine to the performance of this technique.

Evidence of major genes affecting resistance to bacterial cold water disease in rainbow trout using Bayesian methods of segregation analysis.

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. We previously detected genetic variation for BCWD resistance in our rainbow trout population, and a family-based selection program to improve resistance was initiated at the National Center for Cool and Cold Water Aquaculture (NCCCWA). This study investigated evidence of major trait loci affecting BCWD resistance using only phenotypic data (without using genetic markers) and Bayesian methods of segregation analysis (BMSA). A total of 10,603 juvenile fish from 101 full-sib families corresponding to 3 generations (2005, 2007, and 2009 hatch years) of the NCCCWA population were challenged by intraperitoneal injection with Flavobacterium psychrophilum, the bacterium that causes BCWD. The results from single- and multiple-QTL models of BMSA suggest that 6 to 10 QTL explaining 83 to 89% of phenotypic variance with either codominant or dominant disease-resistant alleles plus polygenic effects may underlie the genetic architecture of BCWD resistance. This study also highlights the importance of polygenic background effects in the genetic variation of BCWD resistance. The polygenic heritability on the observed scale of survival status is slightly larger than that previously reported for rainbow trout BCWD resistance. These findings provide the basis for designing informative crosses for QTL mapping and carrying out genome scans for QTL affecting BCWD resistance in rainbow trout.