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Current breastfeeding guidelines promote initiating breastfeeding ≤1 h after birth to establish long-term breastfeeding. Previous studies dichotomized initiation to ≤1 h versus subsequent hours combined. There are limited data evaluating the effect of initiation in each subsequent hour on breastfeeding duration. Our objective was to evaluate the association between breastfeeding initiated at ≤1 h versus the subsequent 23 hours after birth and outpatient breastfeeding duration.In this retrospective cohort study, we analyzed real-time, discretely documented electronic health record (EHR) breastfeeding data for 3315 infants born at a university center and followed to age ≥12 mo at 27 university primary care clinics. The primary outcome was breastfeeding duration. The exposure variable was hour of breastfeeding initiation within 24 h postnatally. Data were analyzed by univariable and multivariable linear regression separately for infants born by vaginal versus cesarean delivery.In adjusted models, initiating breastfeeding during each hour from age1 to ≤6 h and during ages6 to ≤24 h was not associated with decreased breastfeeding duration versus initiating breastfeeding at ≤1 h after birth for infants born via vaginal or cesarean delivery.Delaying breastfeeding initiation to1 to ≤24 h after birth is not associated with decreased breastfeeding duration compared with initiating breastfeeding at ≤1 h after birth. Integration of breastfeeding measures into inpatient and outpatient EHR discrete data fields may clarify best practices that support long-term breastfeeding as a public health imperative.

Despite known health benefits of breastfeeding, the Navajo have low reported frequency of breastfeeding initiation and support. We evaluated breastfeeding frequencies and practices in the predominately Navajo community of rural San Juan County, Utah, to identify factors that affect breastfeeding decisions and duration. We performed retrospective chart review for 135 infants aged 0 to 12 months, and surveys of 85 mothers of infants aged 0 to 2 years, and eight primary care providers. We characterized demographic factors using counts/percentages and medians/inter-quartile ranges, and compared mothers who breastfed for 6 months or less versus greater than 6 months. In 96 infants with complete feeding documentation, 86 infants (90%) received some breast milk and 36 infants (38%) were exclusively breastfed at age 2 months. In 67 infants with complete feeding documentation at ≥ 6 months, 22 infants (33%) were exclusively breastfed 6 months. Most mothers knew about breastfeeding benefits. In 56 mothers whose infants were aged ≥ 6 months at the time of the survey, breastfeeding for more than 6 months had been planned by 44 mothers (79%) but performed by only 29 mothers (52%). Mothers who breastfed for > 6 months were more likely to have been influenced by WIC and less likely to have introduced formula at an early age. Barriers to breastfeeding included maternal pain, latch difficulties, and concerns about inadequate milk supply. Primary care providers reported limited confidence in providing breastfeeding support but would support telehealth-driven interventions. Practical, culturally sensitive interventions, including telehealth and improved provider education, may improve breastfeeding outcomes and community health in this underserved population.

Despite known health benefits of breastfeeding, the Navajo have low reported frequency of breastfeeding initiation and support. We evaluated breastfeeding frequencies and practices in the predominately Navajo community of rural San Juan County, Utah, to identify factors that affect breastfeeding decisions and duration.We performed retrospective chart review for 135 infants aged 0 to 12 months, and surveys of 85 mothers of infants aged 0 to 2 years, and eight primary care providers. We characterized demographic factors using counts/percentages and medians/inter-quartile ranges, and compared mothers who breastfed for 6 months or less versus greater than 6 months.In 96 infants with complete feeding documentation, 86 infants (90%) received some breast milk and 36 infants (38%) were exclusively breastfed at age 2 months. In 67 infants with complete feeding documentation at ≥ 6 months, 22 infants (33%) were exclusively breastfed 6 months. Most mothers knew about breastfeeding benefits. In 56 mothers whose infants were aged ≥ 6 months at the time of the survey, breastfeeding for more than 6 months had been planned by 44 mothers (79%) but performed by only 29 mothers (52%). Mothers who breastfed for 6 months were more likely to have been influenced by WIC and less likely to have introduced formula at an early age. Barriers to breastfeeding included maternal pain, latch difficulties, and concerns about inadequate milk supply. Primary care providers reported limited confidence in providing breastfeeding support but would support telehealth-driven interventions.Practical, culturally sensitive interventions, including telehealth and improved provider education, may improve breastfeeding outcomes and community health in this underserved population.

Objective To determine whether the 2011 guidelines for universal routine screening for dyslipidemia in children aged 9 to 11 years, published by the Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents and National Heart, Lung, and Blood Institute, are being followed by pediatric primary-care providers. Methods Retrospective data were obtained for 63,951 well-child visits (WCV) in children aged 9 to 11 years from 2 health care systems and 1 insurance program from 2009 to 2015. The proportion of WCV that had a lipid panel or total cholesterol test performed within 1 year of the visit was compared for 2009–2011 versus 2013–2015. Associations between demographic variables and lipid screening were evaluated with logistic regression. The frequency of tested children who had abnormal lipid results was evaluated. Results Only 3.5% of 9- to 11-year WCV had lipid tests performed in association with the visit before and after the guidelines. Of those tested, 43% had an abnormal lipid result. Conclusions Utah clinicians rarely follow guidelines for universal lipid screening of children aged 9 to 11. This represents a missed opportunity to identify children at risk for early-onset cardiovascular disease.

Qualitative studies have suggested that food insecurity adversely affects infant feeding practices. We aimed to determine how household food insecurity relates to breastfeeding initiation, duration of exclusive breastfeeding and vitamin D supplementation of breastfed infants in Canada.We studied 10 450 women who had completed the Maternal Experiences - Breastfeeding Module and the Household Food Security Survey Module of the Canadian Community Health Survey (2005-2014) and who had given birth in the year of or year before their interview. We used multivariable Cox proportional hazards models and logistic regression to examine the relation between food insecurity and infant feeding practices, adjusting for sociodemographic characteristics, maternal mood disorders and diabetes mellitus.Overall, 17% of the women reported household food insecurity, of whom 8.6% had moderate food insecurity and 2.9% had severe food insecurity (weighted percentages). After adjustment for sociodemographic factors, women with food insecurity were no less likely than others to initiate breastfeeding or provide vitamin D supplementation to their infants. Half of the women with food insecurity ceased exclusive breastfeeding by 2 months, whereas most of those with food security persisted with breastfeeding for 4 months or more. Relative to women with food security, those with marginal, moderate and severe food insecurity had significantly lower odds of exclusive breastfeeding to 4 months, but only women with moderate food insecurity had lower odds of exclusive breastfeeding to 6 months, independent of sociodemographic characteristics (odds ratio 0.60, 95% confidence interval 0.39-0.92). Adjustment for maternal mood disorder or diabetes slightly attenuated these relationships.Mothers caring for infants in food-insecure households attempted to follow infant feeding recommendations, but were less able than women with food security to sustain exclusive breastfeeding. Our findings highlight the need for more effective interventions to support food-insecure families with newborns.

In 2011, an expert National Institutes of Health panel published the “Integrated Guidelines for CV Health and Risk Reduction in Children and Adolescents,” which recommended screening all children aged 9 to 11 years for dyslipidemia. It is unknown if this guideline is being followed. We surveyed members of the Utah chapter of the American Academy of Pediatrics to determine whether they performed universal lipid screening at well-child visits (WCV) on their patients at 9,10, or 11 years and how comfortable they were with evaluating and/or managing children with dyslipidemia. Of the 118 respondents who practiced primary care, only 18 (15%) screened all children at WCV; 86 (73%) tested “some,” most commonly children who were obese or had a positive family history. 18% were unfamiliar with the guidelines; 28% were familiar with the guidelines but felt they were “inappropriate;” 98 (84%) of the respondents said they were “very or somewhat comfortable” evaluating children with dyslipidemia.

18 páginas, 3 tablas, To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirusinfected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection, This work was supported by the NRC-CSIC Canadian-Spanish Cooperation Program. A. Montes gratefully acknowledges the Ministerio de Educación y Ciencia, the CSIC (I3P program) for a research fellowship.

Atypical furunculosis caused by atypical Aeromonas salmonicida, is an emerging problem in farming of Atlantic cod (Gadus morhua) in Norway, and vaccines are needed. Atypical A. salmonicida comprises a heterogeneous group of bacteria differing in surface antigens such as the A-layer protein and lipopolysaccharides (LPS). Except for one of the experimental oil-adjuvanted whole cell vaccines based on various isolates they all resulted in moderate protection. No clear correlation between vaccine efficacies and the A-protein group or LPS type of the vaccine isolates was revealed, while a correlation between efficacy and the presence of cross-reacting LPS-specific antibodies is indicated.

We report genetic differences for resistance to the pathogen Listonella anguillarum within and among one cultured and two wild Canadian populations of Atlantic salmon, Salmo salar, using a common-garden experimental protocol. Following exposure to the causative agent for vibriosis, parr originating from the endangered Stewiacke River population experienced significantly higher mortality than cultured parr, four generations removed from the Saint John River population, and wild parr from Tusket River. Pathogen resistance differed between sexes; males consistently experienced higher survival than females. There was no evidence that maturity influenced pathogen resistance in male parr. The population and sex differences in pathogen resistance documented here have implications for risk assessments of the demographic consequences of interbreeding between wild and farmed Atlantic salmon.

O-acetylation is one of the major modifications of sialic acids that significantly alters biological properties of the parent molecule. These O-acetylated forms are components of the cellular membrane and can affect physiological and pathological responses. Understanding the role of N-glycans in physiology is of increasing relevance to cellular biologists in various disciplines who study glycoproteomics yet lack information regarding the function of the attached glycans. It is well known that stress may decrease immune function in fish; however, there are only few suitable biomarkers available to monitor the physiological responses under the stress conditions. This study is the first report on the effect of stress on the profile of O-acetylation of sialic acids in fish serum. In order to preserve the relevant structural characteristics as much as possible, native N-glycans were directly analyzed using CE-MS. We have characterized the N-glycans in serum of salmon (Salmo salar) exposed to long-term handling stress (15 s out of the water, daily for 4 wk) and compared with the results obtained from sera of control fish. The results indicated that major N-glycans in salmon serum contained mono-acetylated sialic acids (83%), and that the O-acetylation pattern of sialic acids could be altered by long-term stress.

The cell envelope of Aeromonas salmonicida contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of bacterial cell membrane. Using a recently developed in-source fragmentation technique, we screened 39 typical and atypical isolates of A. salmonicida and established their O-chain polysaccharide structure by capillary electrophoresis–mass spectrometry (CE–MS), compositional and linkage analyses and comparison to the previously determined O-chain polysaccharide structure of A. salmonicida strain A449. These studies have demonstrated that A. salmonicida isolates fall into three distinct structural types, types A–C, based on chemical structures of their respective O-chain polysaccharide components. Subsequent immunoblotting and serological studies with salmon polyclonal antisera produced to formalin-fixed cells of A. salmonicida strains A449, N4705 and 33659 representing three structural types A–C revealed that variations in the O-chain polysaccharide structure have led to significant serological differences between strains belonging to type A and non-type A, where non-type A species include chemically separated structural types B and C. Due to the presence of common antigenic determinants shared by their respective O-chain polysaccharide components, serological cross-reactions were observed between A. salmonicida strains belonging to structural types B and C. These findings suggest the possibility of developing LPS-based classification system of A. salmonicida sub-species consisting of two serologically distinct types, type A and non-type A.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The ΔascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or ΔascC. The experimental infection by immersion with ΔaopO was indistinguishable from that of the parental strain, that of ΔaexT was delayed, whilst the virulence of ΔaopH was reduced significantly but not abolished. By i.p. injection, ΔaexT, ΔaopH and ΔaopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The ΔascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.

Toll-like receptors (TLRs) are involved in the innate immune response against microbial pathogens in vertebrates and insects. The extracellular region of a TLR recognizes pathogen-associated molecules, while the intracellular region initiates the signaling pathway leading to immune response. Membrane-bound TLRs have been found in most vertebrates, but few soluble forms have been reported. A novel transcript corresponding to a portion of a soluble TLR was identified in liver of infected Atlantic salmon. The complete coding sequence of this TLR was obtained and BLASTN analysis showed the highest sequence identity to a recently described full-length cDNA sequence of a soluble TLR5 from rainbow trout (GenBank Accession No.: AB062504). The deduced protein is 40% identical to the mammalian counterpart of the leucine-rich repeat (LRR)/LRR-like motifs of TLR5. Based on the structure of human TLRs, it contains 21 LRRs with conserved LxxLxLxxNx*xx*xxxxFxxL pattern. Since TLR5 is essential for the recognition of bacterial flagellins, we hypothesize that flagellin and perhaps some other pathogen-derived factors from Aeromonas salmonicida bind to this soluble TLR through an unknown binding domain within the LRR.

Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3-deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated.

Infectious salmon anemia virus (ISAV) is a very important fish virus in the Northern hemisphere and there is continued interest in understanding the mechanisms of its pathogenesis and persistence in fish. In this study, the permissive fish cell lines SHK-1, CHSE-214 and TO were used to determine whether ISAV-induced cytopathic effect (CPE) is due to apoptosis or necrosis. Characteristic apoptotic DNA fragmentation was observed only in ISAV-infected SHK-1 and CHSE-214 cells. Apoptosis in ISAV-infected SHK-1 cells was confirmed by fragment end-labelling assay, suggesting that CPE in these cells is associated with apoptosis. ISAV-infected TO cells did not undergo apoptosis, but showed leakage of high-mobility group 1 (HMGB1) protein from the nucleus, which is characteristic of cells undergoing necrosis; this suggests that CPE in these cells is associated with necrosis. ISAV-infected SHK-1 cells did not show leakage of HMGB1 protein. Infection with two different strains of ISAV showed that induction of apoptosis was correlated with the appearance of CPE in SHK-1 cells. ISAV-induced apoptosis was inhibited by a pan-caspase inhibitor, Z-VAD-fmk, indicating a caspase-activation pathway. The ISAV putative PB2 protein and proteins encoded by RNA segment 7 bound caspase-8 specifically in vitro, suggesting that these viral proteins may have a role in ISAV-induced apoptosis. These findings demonstrate for the first time that the mechanism of cell death during ISAV infection is dependent on the cell type, which may have implications for ISAV pathogenesis and persistence.

Commercially available human complementary DNA microarrays were used to compare differential expression in the livers of Atlantic salmon ( Salmo salar) infected with Aeromonas salmonicida and of healthy fish. Complementary DNA probes were prepared from total RNA isolated from livers of control salmon and infected salmon by reverse transcription in the presence of (33)P-dCTP and independently hybridized to human GENE-FILTERS GF211 microarrays. Of the 4131 known genes on the microarray, 241 spots gave clearly detectable signals using labeled RNA from the control salmon liver. Of these, 4 spots were consistently found to have a greater than 2-fold increase in infected salmon compared with controls when using the same pair of filters to generate hybridization data from triplicates. These up-regulated genes were ADP/ATP translocase (AAT2), Na(+)/K(+) ATPase, acyloxyacyl hydrolase (AOAH), and platelet-derived growth factor (PDFG-A). A BlastN search revealed an AAT2 homolog from Atlantic salmon, and a reverse transcriptase polymerase chain reaction assay using primers based on this sequence confirmed its up-regulation (approx. 1.8-fold) during early infection. This work demonstrates the feasibility of using human microarrays to facilitate the discovery of differentially expressed genes in Atlantic salmon, for which no homologous microarrays are available.

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A . salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB . The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O 2 − -catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A . salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.

Bacterial kidney disease (BKD) in salmonids is caused by Renibacterium salmoninarum (Rs) which is transmitted vertically through salmonid eggs. This study investigated the effect of vertical transmission of one protein of Rs, p57, on the development of the immune system of coho salmon ( Oncorhynchus kisutch ) and on the susceptibility of the fish to subsequent challenge with Rs. It was shown that exposure of coho salmon at the egg stage to the p57 protein of Rs induces at least partial immunosuppression. Coho salmon eggs were injected with varying amounts of p57. The eggs were fertilized, the progeny were vaccinated against Rs, and then challenged with live, virulent Rs. Fish that had been injected as eggs with 100 ng of p57 demonstrated a significantly higher cumulative percent mortality (50 %) than those from the saline-injected eggs (controls) (14 %). The same fish also produced significantly lower levels of antibodies against p57, although not against whole Rs cells, than those derived from saline-injected eggs. It appeared, therefore, that there was some specificity to the immune suppression. A decreased proportion of phagocytic cells isolated from fish exposed as eggs to 100 ng p57, showed respiratory burst activity (0 ·4 %) compared with the proportion of cells isolated from the saline-injected (control) group (16 ·5 %). These data suggest that early (egg stage) exposure of coho salmon to p57 results in long-term immunosuppression and a decreased ability in the animal to resist subsequent challenges with Rs.

The sustained systems performance (SSP) test has been implemented on certain High Performance Computing Modernization Program (HPCMP) HPC systems in order to quantitatively evaluate updates to system software, hardware repairs, job queuing policy modifications, and revisions to the job scheduler as necessary. The test employs codes used in the system acquisition cycle with proven migration capability to HPCMP HPC systems and non-empirical tests for numerical accuracy. Metrics such as compilation time, queue wait time, benchmark execution time, and total test throughput time are gathered and compared against metric data from previous tests to monitor the systems under test while minimizing impact to the users. Jobs failing to execute properly or in anomalously short or long times are investigated, and the results are reported to system administrators and center directors at each center for appropriate actions. During the past year, the SSP test has been instrumental in surfacing configuration issues with the PBS scheduler and performance issues on several HPC systems. Additionally, the frequency of the SSP test on systems procured in Technology Insertion 2009 (TI-09) and thereafter has increased, with attendant changes in the test cases comprising the test. The SSP test continues to play an important role in monitoring the quality of service delivering HPC to HPCMP users at the system, DoD Supercomputing Resource Center, and vendor levels.

Introduction. Aquaculture‐Relevant Microarray Research on Salmonids. Microarray Research on Catfish. Aquaculture‐Relevant Microarray Research on Flatfish. Aquaculture‐Relevant Microarray Research on Zebrafish. The Use of DNA Microarrays to Investigate Aquatic Animal Pathogens. Examples of Novel Microarray Platforms for Aquaculture‐Related Research. Our First Glimpses of the Frontier. A Case Study—Application of Microarray Technology to the Squid–Vibrio Symbiosis. Future Directions. References

Time-to-solution and scalability of an application can vary greatly from one computer architecture to another. It is important then to consider the appropriateness of architectures with respect to applications to make the most efficient use of available resources. However, the number of applications currently used on systems within the High Performance Computing Modernization Program (HPCMP) is very large. This paper, therefore, focuses solely on codes within the HPCMP Technology Insertion 2009 (TI-09) Applications Benchmarking Suite. These codes include Adaptive Mesh Refinement (AMR), Air Vehicles Unstructured Solver (AVUS), CTH, General Atomic and Molecular Electronic Structure System (GAMESS), HYbrid Coordinate Ocean Model (HYCOM), Improved Concurrent Electromagnetic Particle In Cell (ICEPIC), and Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS). This paper evaluates the parallel performance-per-processor of each of these seven codes on major systems within the Program. This, in turn, can guide users and developers in selecting appropriate architectures for each code.

Moritella viscosa is the causative agent of winter ulcer disease in salmonids reared in North-Atlantic countries. In this study the effects of selected M. viscosa antigens on cytotoxicity and pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) macrophage-like cell line (SHK-1) were examined. SHK-1 cells were stimulated with live and heat-killed bacterial cells, extracellular products (ECP) and an extracellular vibriolysin, termed MvP1. Following incubation, cytotoxicity and expression levels of interleukin-1 beta (IL-1 beta) and interleukin-8 (IL-8) were examined at different time points. Both live M. viscosa cells and ECP were cytotoxic, but neither heat-killed cells, nor the MvP1 peptidase caused cell death. Expression levels of both IL-1 beta and IL-8 increased significantly after stimulation with live cells, but heat-killed cells only caused increased IL-8 expression. ECP did not affect IL-1 beta expression, but did stimulate IL-8 expression. The isolated MvP1 peptidase stimulated both IL-1 beta and IL-8 expression at the highest concentration tested. This study reveals a difference in the induction of pro-inflammatory gene expression in salmon SHK-1 cells between live and heat-killed M. viscosa cells, and also that an unknown secreted factor is the main stimulant of IL-beta and IL-8 expression.

Aeromonas salmonicida subsp. salmonicida , a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae . The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.

Background Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations. Results Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested. Conclusion We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research.

Tetrameric immunoglobulin (lg) in most teleosts is a fully disulphide cross-linked molecule. The multimeric lg from four species from the order Gadiformes; haddock (Melanogrammus aeglefinus L.), Atlantic cod (Gadus morhua L), pollock (Pollachius pollachius L.) and tusk (Brosme bromse A.) was examined by denaturing, non-reducing gel electrophoresis and gel filtration chromatography. Despite having a native molecular mass equivalent to that of other tetrameric teleost lg, gadid lg was never observed to exist as a fully disulphide cross-linked molecule; existing instead as a covalently cross-linked trimer with a non-covalently associated monomer. It is unknown whether this unique cross-linking is associated with the limited or absent specific humoral responses reported for members of this order.

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.

In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314-BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription-polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.

Chiari I malformation is characterized by downward displacement of cerebellar tonsils through the foramen magnum.1-3 In the era of magnetic resonance imaging (MRI), specific radiological criteria for the diagnosis of Chiari I malformation has emerged.1,2 In both children and adults, diagnosis is established by displacement of one or both tonsils 5 mm below the plane of the foramen magnum.4 However, Mikulis et al examined a group of 221 patients ranging from 5 months to 89 years, and found that tonsils tend to ascend with age.5 They recommend that criteria be modified for age, such that younger patients are permitted a higher degree of ectopia. The most commonly reported symptom in both adults and children is headache, occurring in more than half of patients and is often precipitated by Valsalva maneuvers.1,2 Younger children tend to present more commonly with dysphagia and respiratory difficulties.6,7 Before children can communicate verbally, symptoms like headache may present with crying or irritability.2,8 Neurological signs of Chiari I malformation can be codified into 3 categories: a brainstem syndrome (eg, cranial nerve dysfunction, respiratory impairment), a spinal cord syndrome (eg, motor and sensory loss, hyperreflexia or hyporeflexia), and a cerebellar syndrome (eg, ataxia).3 Syringomyelia is associated with Chiari I malformation and deserves special mention as it often warrants surgical intervention.9 We report an unusual presentation of Chiari I in an infant, associated with developmental regression, which resolved on surgical treatment.

Background Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish. Results The nucleotide sequences of the A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids are characterized. The chromosome is 4,702,402 bp and encodes 4388 genes, while the two large plasmids are 166,749 and 155,098 bp with 178 and 164 genes, respectively. Notable features are a large inversion in the chromosome and, in one of the large plasmids, the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integron encoding genes for resistance to streptomycin/spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. A large number of genes encoding potential virulence factors were identified; however, many appear to be pseudogenes since they contain insertion sequences, frameshifts or in-frame stop codons. A total of 170 pseudogenes and 88 insertion sequences (of ten different types) are found in the A. salmonicida genome. Comparison with the A. hydrophila ATCC 7966T genome reveals multiple large inversions in the chromosome as well as an approximately 9% difference in gene content indicating instances of single gene or operon loss or gain. A limited number of the pseudogenes found in A. salmonicida A449 were investigated in other Aeromonas strains and species. While nearly all the pseudogenes tested are present in A. salmonicida subsp. salmonicida strains, only about 25% were found in other A. salmonicida subspecies and none were detected in other Aeromonas species. Conclusion Relative to the A. hydrophila ATCC 7966T genome, the A. salmonicida subsp. salmonicida genome has acquired multiple mobile genetic elements, undergone substantial rearrangement and developed a significant number of pseudogenes. These changes appear to be a consequence of adaptation to a specific host, salmonid fish, and provide insights into the mechanisms used by the bacterium for infection and avoidance of host defence systems.

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.