Osteoid osteomas of the hip: a well-recognized entity with a proclivity for misdiagnosis.

OBJECTIVES: The diagnosis of osteoid osteomas (OO) about the hip can be challenging as presenting symptoms can mimic other, more common, periarticular pathologies. Our aims were to identify the most common misdiagnoses and treatments, mean delay in diagnosis, characteristic imaging features and provide tips for avoiding diagnostic imaging pitfalls for patients with OO of the hip. METHODS: We identified 33 patients (34 tumors) with OO about the hip who were referred for radiofrequency ablation between 1998 and 2020. Imaging studies reviewed included radiographs (n = 29), CT (n = 34), and MRI (n = 26). RESULTS: The most common initial diagnoses were femoral neck stress fracture (n = 8), femoroacetabular impingement (FAI) (n = 7), and malignant tumor or infection (n = 4). The mean time from symptom onset to diagnosis of OO was 15 months (range, 0.4-84). The mean time from initial incorrect diagnosis to OO diagnosis was 9 months (range, 0-46). CONCLUSIONS: The diagnosis of OO of the hip is challenging, with up to 70% of cases initially misdiagnosed as a femoral neck stress fracture, FAI, bone tumor, or other joint pathology in our series. Consideration of OO in the differential diagnosis of hip pain in adolescent patients and awareness of the characteristic imaging findings are critical for making an accurate diagnosis. KEY POINTS: • The diagnosis of osteoid osteoma of the hip can be challenging, as demonstrated by long delays in time to initial diagnosis and high rates of misdiagnoses which can lead to inappropriate interventions. • Familiarity with the spectrum of imaging features of OO, especially on MRI, is imperative given the increase in the utilization of this modality for the evaluation of young patients with hip pain and FAI. • Consideration of OO in the differential diagnosis of hip pain in adolescent patients and awareness of the characteristic imaging findings, including bone marrow edema and the utility of CT, are critical for making a timely and accurate diagnosis.

Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Detecting Flavobacterial Fish Pathogens in the Environment via High-Throughput Community Analysis.

Diseases caused by the fish pathogens Flavobacterium columnare and Flavobacterium psychrophilum are major contributors of preventable losses in the aquaculture industry. The persistent and difficult-to-control infections caused by these bacteria make timely intervention and prophylactic elimination of pathogen reservoirs important measures to combat these disease-causing agents. In this study, we present two independent assays for detecting these pathogens in a range of environmental samples. Natural water samples were inoculated with F. columnare and F. psychrophilum over 5 orders of magnitude, and pathogen levels were detected using Illumina MiSeq sequencing and droplet digital PCR. Both detection methods accurately identified pathogen-positive samples and showed good agreement in quantifying each pathogen. Additionally, the real-world application of these approaches was demonstrated using environmental samples collected at a rainbow trout (Oncorhynchus mykiss) aquaculture facility. These results show that both methods can serve as useful tools for surveillance efforts in aquaculture facilities, where the early detection of these flavobacterial pathogens may direct preventative measures to reduce disease occurrence. IMPORTANCE Early detection of a deadly disease outbreak in a population can be the difference between mass mortality or mitigated effects. In the present study, we evaluated and compared two molecular techniques for detecting economically impactful aquaculture pathogens. We demonstrate that one of these techniques, 16S rRNA gene sequencing using Illumina MiSeq technology, provides the ability to accurately detect two freshwater fish pathogens, F. columnare and F. psychrophilum, while simultaneously profiling the native microbial community. The second technique, droplet digital PCR, is commonly used for pathogen detection, and the results obtained using the assays we designed with this method served to validate those obtained using the MiSeq method. These two methods offer distinct advantages. The MiSeq method pairs pathogen detection and microbial community profiling to answer immediate and long-term fish health concerns, while the droplet digital PCR method provides fast and highly sensitive detection that is useful for surveillance and rapid clinical responses.

Type IX Secretion System Effectors and Virulence of the Model Flavobacterium columnare Strain MS-FC-4.

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here, we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted 10 genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding 10 secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture.

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.

Disruption of the Francisella noatunensis subsp. orientalis pdpA Gene Results in Virulence Attenuation and Protection in Zebrafish.

Several Francisella spp., including Francisella noatunensis, are regarded as important emerging pathogens of wild and farmed fish. However, very few studies have investigated the virulence factors that allow these bacterial species to be pathogenic in fish. The Francisella pathogenicity island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus Francisella. pdpA is a member of the pathogenicity-determining protein genes carried by the FPI that are implicated in the ability of the mammalian pathogen Francisella tularensis to escape and replicate in infected host cells. Using a sacB suicide approach, we generated pdpA knockouts to address the role of PdpA as a virulence factor for F. noatunensis. Because polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity (the F. noatunensis subsp. orientalis ΔpdpA1 and ΔpdpA2 strains). Both mutants were attenuated (P < 0.0001) in zebrafish challenges and displayed impaired intracellular replication (P < 0.05) and cytotoxicity (P < 0.05), all of which could be restored to wild-type (WT) levels by complementation for the ΔpdpA1 mutant. Importantly, differences were found for bacterial burden and induction of acute-phase and proinflammatory genes for the F. noatunensis subsp. orientalis ΔpdpA1 and ΔpdpA2 mutants compared to the WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with the F. noatunensis subsp. orientalis ΔpdpA1 mutant led to protection (P < 0.012) against an acute 40% lethal dose (LD40) challenge with WT F. noatunensis in the zebrafish model of infection. Taken together, the results from this study further demonstrate physiological similarities within the genus Francisella relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.

Genetic characterization of heterologous Edwardsiella piscicida isolates from diverse fish hosts and virulence assessment in a Chinook salmon Oncorhynchus tshawytscha model.

Edwardsiella piscicida is an emergent global fish pathogen with a wide host range, although host associations driving genetic diversity remain unclear. This study investigated the genetic and virulence diversity of 37 E. piscicida isolates recovered from 10 fish species in North America. Multilocus sequence analysis (MLSA) was conducted using concatenated alignments of the gyrB, pgi and phoU sequences. MLSA clustered the tested isolates into six discrete clades. In light of recent disease outbreaks in cultured salmonids, the virulence of each clade was evaluated in Chinook salmon Oncorhynchus tshawytscha fingerlings following intracoelomic challenge of ~106  CFU/fish. Challenged and control fish were monitored for 21d, and microbiological and histological examination was performed on dead and surviving fish. Peak mortality occurred 3-5 days post-challenge (dpc) regardless of isolate or genetic group. Edwardsiella piscicida was recovered from all moribund and dead animals. At 21 dpc, fish challenged with isolates from clades II, III and IV presented cumulative mortality ≥83.3%, whereas isolates from clade I, V and VI resulted in cumulative mortality ≤71.4%. This study suggests an underlying genetic basis for strain virulence and potential host associations. Further investigations using other fish models and variable challenge conditions are warranted.

Awareness of implicit bias mitigates discrimination in radiology resident selection.

OBJECTIVES: Implicit bias is common and is thought to drive discriminatory behaviour. Having previously demonstrated discrimination against specific applicant demographics by academic radiology departments in a simulated resident selection process, the authors sought to better understand the relationship between implicit bias and discrimination, as well as the potential and mechanisms for their mitigation. METHODS: A total of 51 faculty reviewers at three academic radiology departments, who had participated in a 2017 audit study in which they were shown to treat applicants differently based on race or ethnicity and physical appearance, were invited to complete testing for implicit racial and weight bias using the Implicit Association Test in 2019. Respondents were also surveyed regarding awareness of their own personal racial and weight biases, as well as any prior participation in formal diversity training. Comparisons were made between implicit bias scores and applicant ratings, as well as between diversity training and self-awareness of bias. RESULTS: A total of 31 out of 51 faculty reviewers (61%) completed and submitted results of race and weight Implicit Association Tests. A total of 74% (23/31) reported implicit anti-obese bias, concordant with discrimination demonstrated in the resident selection simulation, in which obese applicants were rated 0.40 standard deviations (SDs) lower than non-obese applicants (P < .001). A total of 71% (22/31) reported implicit anti-Black bias, discordant with application ratings, which were 0.47 SDs higher for Black than for White applicants (P < .001). A total of 84% (26/31) of participants reported feeling self-aware of potential racial bias at the time of application review, significantly higher than the 23% (7/31) reporting self-awareness of potential anti-obese bias (P < .001). Participation in formal diversity training was not associated with implicit anti-Black or anti-fat bias, nor with self-reported awareness of potential racial or weight-based bias (all P > .2). CONCLUSIONS: These findings suggest that implicit bias, as measured by the Implicit Association Test, does not inevitably lead to discrimination, and that personal awareness of implicit biases may allow their mitigation.

Characterization of a novel Yersinia ruckeri serotype O1-specific bacteriophage with virulence-neutralizing activity.

A lytic bacteriophage (φNC10) specific to serotype O1 Yersinia ruckeri has been identified and evaluated as a model to assess the potential use of bacteriophages and their products for disease control in aquaculture. Electron microscopy of purified φNC10 revealed a virion particle with a small (70 nm) polyhedral head and short tail. φNC10 infected only serotype O1 strains of Y. ruckeri and failed to bind a defined Y. ruckeri mutant strain lacking O1 lipopolysaccharides (O1-LPS), suggesting that φNC10 uses O1-LPS as its receptor. In addition, spontaneous φNC10-resistant mutants of Y. ruckeri exhibited defects in O1-LPS production and were sensitive to rainbow trout serum. Purified φNC10 displayed a polysaccharide depolymerase activity capable of degrading Y. ruckeri O1-LPS and thereby sensitizing Y. ruckeri to the bactericidal effects of rainbow trout serum. The φNC10-associated polysaccharide depolymerase activity also reduced the ability of Y. ruckeri cells to cause mortality following intraperitoneal injection into rainbow trout. These data demonstrate a potential utility of φNC10 and its associated polysaccharide depolymerase activity for Y. ruckeri disease prevention.

Flagellar regulation mediated by the Rcs pathway is required for virulence in the fish pathogen Yersinia ruckeri.

The flagellum is a complex surface structure necessary for a number of activities including motility, chemotaxis, biofilm formation and host attachment. Flagellin, the primary structural protein making up the flagellum, is an abundant and potent activator of innate and adaptive immunity and therefore expression of flagellin during infection could be deleterious to the infection process due to flagellin-mediated host recognition. Here, we use quantitative RT-PCR to demonstrate that expression of the flagellin locus fliC is repressed during the course of infection and subsequently up-regulated upon host mortality in a motile strain of Yersinia ruckeri. The kinetics of fliC repression during the infection process is relatively slow as full repression occurs 7-days after the initiation of infection and after approximately 3-logs of bacterial growth in vivo. These results suggests that Y. ruckeri possesses a regulatory system capable of sensing host and modulating the expression of motility in response. Examination of the master flagellar operon (flhDC) promoter region for evidence of transcriptional regulation and regulatory binding sites revealed potential interaction with the Rcs pathway through an Rcs(A)B Box. Deletion of rcsB (ΔrcsB) by marker-exchange mutagenesis resulted in overproduction of flagellin and unregulated motility, showing that the Rcs pathway negatively regulates biosynthesis of the flagellar apparatus. Experimental challenge with ΔrcsB and ΔrcsBΔfliC1ΔfliC2 mutants revealed that mutation of the Rcs pathway results in virulence attenuation which is dependent on presence of the flagellin gene. These results suggest that the inappropriate expression of flagellin during infection triggers host recognition and thus immune stimulation resulting in attenuation of virulence. In addition, RNAseq analyses of the ΔrcsB mutant strain verified the role of this gene as a negative regulator of the flagellar motility system and identified several additional genes regulated by the Rcs pathway.

Multilocus Variable-Number Tandem-Repeat Analysis of Yersinia ruckeri Confirms the Existence of Host Specificity, Geographic Endemism, and Anthropogenic Dissemination of Virulent Clones.

A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen Yersinia ruckeri, which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484 Y. ruckeri isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, Y. ruckeri strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse Y. ruckeri isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains.IMPORTANCE This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context.

Comparative Phenotypic and Genotypic Analysis of Edwardsiella Isolates from Different Hosts and Geographic Origins, with Emphasis on Isolates Formerly Classified as E. tarda, and Evaluation of Diagnostic Methods.

Edwardsiella spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as Edwardsiella tarda actually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each Edwardsiella species using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the Edwardsiella spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of Edwardsiella spp. and advantages of employing alternative single-copy genes gyrB and sodB for molecular identification and classification of Edwardsiella were demonstrated. Last, sodB sequencing confirmed that isolates previously defined as typical motile fish-pathogenic E. tarda are synonymous with Edwardsiella piscicida, while atypical nonmotile fish-pathogenic E. tarda isolates are equivalent to Edwardsiella anguillarum Fish-nonpathogenic E. tarda isolates are consistent with E. tarda as it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of Edwardsiella isolates in clinical and research settings.

Global proteomic profiling of Yersinia ruckeri strains.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) of salmonids. There is little information regarding the proteomics of Y. ruckeri. Herein, we perform whole protein identification and quantification of biotype 1 and biotype 2 strains of Y. ruckeri grown under standard culture conditions using a shotgun proteomic approach. Proteins were extracted, digested and peptides were separated by a nano liquid chromatography system and analyzed with a high-resolution hybrid triple quadrupole time of flight mass spectrometer coupled via a nano ESI interface. SWATH-MS technology and sophisticated statistical analyses were used to identify proteome differences among virulent and avirulent strains. GO annotation, subcellular localization, virulence proteins and antibiotic resistance ontology were predicted using bioinformatic tools. A total of 1395 proteins were identified in the whole cell of Y. ruckeri. These included proteases, chaperones, cell division proteins, outer membrane proteins, lipoproteins, receptors, ion binding proteins, transporters and catalytic proteins. In virulent strains, a total of 16 proteins were upregulated including anti-sigma regulatory factor, arginine deiminase, phosphate-binding protein PstS and superoxide dismutase Cu-Zu. Additionally, several virulence proteins were predicted such as Clp and Lon pro-teases, TolB, PPIases, PstS, PhoP and LuxR family transcriptional regulators. These putative virulence proteins might be used for development of novel targets for treatment of ERM in fish. Our study represents one of the first global proteomic reference profiles of Y. ruckeri and this data can be accessed via ProteomeXchange with identifier PXD005439. These proteomic profiles elucidate proteomic mechanisms, pathogenicity, host-interactions, antibiotic resistance ontology and localization of Y. ruckeri proteins.

Shotgun proteomic analysis of Yersinia ruckeri strains under normal and iron-limited conditions.

Yersinia ruckeri is the causative agent of enteric redmouth disease of fish that causes significant economic losses, particularly in salmonids. Bacterial pathogens differentially express proteins in the host during the infection process, and under certain environmental conditions. Iron is an essential nutrient for many cellular processes and is involved in host sensing and virulence regulation in many bacteria. Little is known about proteomics expression of Y. ruckeri in response to iron-limited conditions. Here, we present whole cell protein identification and quantification for two motile and two non-motile strains of Y. ruckeri cultured in vitro under iron-sufficient and iron-limited conditions, using a shotgun proteomic approach. Label-free, gel-free quantification was performed using a nanoLC-ESI and high resolution mass spectrometry. SWATH technology was used to distinguish between different strains and their responses to iron limitation. Sixty-one differentially expressed proteins were identified in four Y. ruckeri strains. These proteins were involved in processes including iron ion capture and transport, and enzymatic metabolism. The proteins were confirmed to be differentially expressed at the transcriptional level using quantitative real time PCR. Our study provides the first detailed proteome analysis of Y. ruckeri strains, which contributes to our understanding of virulence mechanisms of Y. ruckeri, and informs development of novel control methods for enteric redmouth disease.

Yersinia ruckeri lipopolysaccharide is necessary and sufficient for eliciting a protective immune response in rainbow trout (Oncorhynchus mykiss, Walbaum).

Enteric redmouth disease (ERM), caused by Yersinia ruckeri, has been controlled successfully using immersion-applied bacterin vaccines for several decades. While the host response to vaccination and the mechanism of protection of this vaccine have been elucidated, the bacterial components eliciting protection have remained unclear. Here we show that highly purified serotype O1 Y. ruckeri lipopolysaccharide (LPS) is sufficient to induce a protective response to experimental challenge in rainbow trout (Oncorhynchus mykiss). Dose response experiments demonstrated that Y. ruckeri LPS at doses of 1 ng/fish and above resulted in essentially complete protection and doses as low as 0.01 ng/fish (1.38 ng/kg) resulted in significant protection, thus demonstrating the extremely high potency of this immunogen. Analysis of the Y. ruckeri genome identified a cluster of putative O-antigen biosynthetic genes specific to serotype O1 strains. This cluster primarily consisted of genes encoding proteins predicted to function in the biosynthesis of legionamic acid, a nonulosonic acid known to be part of the O-polysaccharide repeat of O1 Y. ruckeri. Mutation of the nab2 gene, a nonulosonic acid biosynthesis gene (nab gene), resulted in production of severely truncated forms of LPS. Vaccination with bacterin vaccines derived from the nab2 mutant and its wild type parent strain demonstrated that LPS is a required component of the whole-cell bacterin vaccine and suggests that LPS is the only cellular component contributing to the protective response elicited by this vaccine. We speculate that the exceptionally high potency of Y. ruckeri LPS accounts for the unusual success of this vaccine when delivered by immersion.

The Timing and Presentation of Major Hemorrhage After 18,947 Image-Guided Percutaneous Biopsies.

OBJECTIVE: The objective of our study was to characterize the temporal and clinical manifestation of major bleeding events after biopsy to guide clinicians in the institution of appropriate surveillance. MATERIALS AND METHODS: We performed a retrospective review of percutaneous image-guided biopsies performed between September 1, 2005, and May 31, 2012, including 18,947 biopsy events. According to routine protocol, follow-up telephone calls were made to patients 24 hours after biopsy, and chart review was performed 3 months after biopsy. Bleeding complications were defined using the Common Terminology Criteria for Adverse Events (CTCAE, version 4.0) established by the National Cancer Institute. In patients with a grade 3 or greater bleeding complication, a retrospective chart review was performed to characterize the details of the complication including the timing of the complication and the primary clinical presentation of the event. RESULTS: Grade 3 hemorrhage was associated with 64 of 18,947 (0.3%) procedures, and there were three deaths associated with the biopsy event (0.02% or ≈ 2/10,000). Hemorrhage was most commonly associated with biopsy of a native kidney (17/1407, 1.2%). Twenty patients (31%) presented with a bleeding complication within 1 hour of biopsy. Twenty-seven patients (42%) presented within 2 hours of biopsy. Fifty-two patients (81%) presented within 24 hours, and the remaining 12 patients (19%) presented more than 24 hours after biopsy. Pain was the most common presentation of patients with bleeding complications, occurring in 39 (61%) patients. CONCLUSION: The incidence of major bleeding after percutaneous biopsies is very low, but delayed complications occur more frequently than anticipated. Pain is the most common clinical presentation of major bleeding complications.

Comparative Evaluation of Booster Vaccine Efficacy by Intracoelomic Injection and Immersion with a Whole-Cell Killed Vaccine against Lactococcus petauri Infection in Rainbow Trout (Oncorhynchus mykiss).

Lactococcus petauri is an important emergent bacterial pathogen of salmonids in the USA. The purpose of this study was to evaluate the protection conferred to rainbow trout (Oncorhynchus mykiss) against L. petauri by formalin-killed vaccines in immersion and injectable forms, as well as the enhanced protection afforded by booster vaccination. In the first challenge, fish were immunized via intracoelomic injection (IC) or immersion (Imm) routes alone. Approximately 418 degree days (Temperature in degree Celsius × days post-immunization) (dd) Imm, or 622 dd IC post-vaccination, fish were challenged via IC with wild-type L. petauri. In the second experiment, initial Imm vaccination was followed by booster vaccination via Imm or IC routes 273 dd post-immunization along with appropriate PBS controls. The various vaccination protocol efficacies were evaluated by challenging fish with L. petauri by cohabitation with diseased fish 399 dd post-booster administration. A relative percent survival (RPS) of 89.5% and 28% was recorded in the IC and Imm single immunization treatments, respectively. In the second study, an RPS of 97.5%, 10.2%, 2.6% and -10.1% plus approximately 0%, 50%, 20%, and 30% bacterial persistence was recorded in the Imm immunized + IC boosted, Imm immunized + mock IC boosted, Imm immunized + Imm boosted, and Imm immunized + mock Imm boosted treatments, respectively. Only the Imm immunized + IC injection boosted treatments provided significant protection when compared to unvaccinated and challenged treatments (p < 0.05). In conclusion, although both Imm and IC vaccines appear safe for trout, the inactivated Imm vaccines seem to provide only mild and temporary protection against lactococcosis; whereas IC immunized trout develop a significantly stronger protective response in both challenges.

Pan-genome survey of the fish pathogen Yersinia ruckeri links accessory- and amplified genes to virulence.

While both virulent and putatively avirulent Yersinia ruckeri strains exist in aquaculture environments, the relationship between the distribution of virulence-associated factors and de facto pathogenicity in fish remains poorly understood. Pan-genome analysis of 18 complete genomes, representing established virulent and putatively avirulent lineages of Y. ruckeri, revealed the presence of a number of accessory genetic determinants. Further investigation of 68 draft genome assemblies revealed that the distribution of certain putative virulence factors correlated well with virulence and host-specificity. The inverse-autotransporter invasin locus yrIlm was, however, the only gene present in all virulent strains, while absent in lineages regarded as avirulent. Strains known to be associated with significant mortalities in salmonid aquaculture display a combination of serotype O1-LPS and yrIlm, with the well-documented highly virulent lineages, represented by MLVA clonal complexes 1 and 2, displaying duplication of the yrIlm locus. Duplication of the yrIlm locus was further found to have evolved over time in clonal complex 1, where some modern, highly virulent isolates display up to three copies.

Cryoablation of recurrent sacrococcygeal tumors.

PURPOSE: To review the safety and efficacy of cryoablation of recurrent sacrococcygeal tumors. MATERIALS AND METHODS: The radiology departmental ablation database was retrospectively searched for cases of cryoablation performed to treat recurrences of sacrococcygeal tumors between January 1, 2010, and August 1, 2011. Patient demographics, procedure technical parameters, and patient outcomes were reviewed. RESULTS: Five cases of recurrent chordoma and one recurrent myxopapillary ependymoma were treated with cryoablation in six patients whose ages ranged from 31 to 80 years. The tumors measured 14-39 mm in maximal dimension. Cryoablation was performed with the use of computed tomography guidance (n = 5) or a combination of ultrasound and magnetic resonance imaging guidance (n = 1). Sterile fluid was instilled to displace adjacent bowel and/or vagina in four cases, and electromyography monitoring was performed in two cases with adjacent nerve roots. Two patients with recurrent chordoma were treated for palliation of pain, with complete pain relief in one patient (pain recurred after 6 wk) and immediate reduction in pain from a score of six to a score of two on a 10-point scale in the other (pain recurred after 7 mo). Four tumors were treated for local control, with no evidence of recurrence on follow-up imaging at 3, 6, 12, and 15 months. No serious complication occurred. CONCLUSIONS: Limited results suggest cryoablation to be a safe and relatively effective means of treating recurrent sacrococcygeal neoplasms for local control or palliation of pain in this small series with short-term follow-up.

Can the use of deception be justified in medical education research? A point/counterpoint and case study.

Deception is a common feature of behavioral research design, although not commonly employed in the medical literature. It can promote scientific validity but is ethically controversial because it compromises subject autonomy and incurs additional costs.  In this Point/Counterpoint monograph, we review the nature of deception in research and present arguments for and against its ethical use as a research methodology in behavioral studies.  We describe the necessary guidelines, safeguards, and oversight, when deceptive methodology is considered, and report our experiences and lessons learned from conducting a multi-institutional audit study that relied upon deception of academic radiology faculty.