Comparative assessment of Vibrio virulence in marine fish larvae.

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single-egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty-nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high-virulent strains had acquired virulence genes from other pathogenic Vibrio.

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes--a novel early defence strategy in larval fish.

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

An aerolysin-like enterotoxin from Vibrio splendidus may be involved in intestinal tract damage and mortalities in turbot, Scophthalmus maximus (L.), and cod, Gadus morhua L., larvae.

Vibrio splendidus is a pathogen that can cause major losses during the early stages of larval turbot rearing when live feed (rotifers or Artemia) is used. As haemolytic bacteria have often been associated with larval rearing losses, we studied the role of the V. splendidus haemolysin in infection of larvae. From a bank of over 10,000 transposon mutants of V. splendidus, two different types of haemolysin-negative mutants were obtained. Both had lost virulence for larval fish, and immunohistochemistry showed that the transposon mutant studied colonized the turbot larval intestinal tract at a similar level to the wild-type organism but did not cause damage or signs of enteritis found with the wild-type organism. One transposon insertion site was located within a gene with high homology to aerolysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we have termed vibrioaerolysin, had properties similar to aerolysin and osmotic protection studies showed that it formed pores in the membranes of erythrocytes of similar diameter to those of aerolysin. The Tn10 insertion site of the second transposon mutant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaerolysin. The gastroenteritis caused by Aeromonas spp. in humans is considered to be due to production of aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine fish larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial flora of the larval fish gut can secrete aerolysin-like toxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on blood agar plates of live feed is recommended with measures to reduce the concentrations of haemolytic bacteria in rearing systems.

Novel application of nitrifying bacterial consortia to ease ammonia toxicity in ornamental fish transport units: trials with zebrafish.

AIMS: To evaluate whether two commercial nitrifying bacterial consortia can function as biocontrol agents in ornamental fish transporting systems. METHODS AND RESULTS: The consortia were applied in a simulated set-up using zebrafish as the model organism in three trials. The efficacy of the bacterial consortia in controlling the ammonia level was validated by measuring water quality parameters such as total ammonia, nitrate and pH of the transport water. The bacterial community structure in the transport unit was studied using denaturing gradient gel electrophoresis. The consortia tested improved the nitrifying activity that in turn facilitated the reduction of ammonia that had accumulated during the transport. Bacterial profiles revealed the presence of both ammonia-oxidizing and nitrite-oxidizing bacteria in the transport bags. CONCLUSIONS: The application of the consortia during the transportation of zebrafish could profoundly improve the water quality by curbing ammonia accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of applying nitrifying bacteria as a bioremediation practice during the transport of ornamental fish has been demonstrated and this innovative approach contributes to the amelioration of current fish welfare in ornamental fish trade.

Treating primary insomnia: clinical effectiveness and predictors of outcomes on sleep, daytime function and health-related quality of life.

Primary insomnia, as defined by DSM-IV-TR, refers to a persistent sleep disturbance which is not connected to a current psychiatric or physical condition, but significantly impairs social and occupational functioning. This study explored the impact of Cognitive Behavioral Therapy for Insomnia (CBT-i) on sleep, daytime functioning and health-related quality of life (HRQoL). Next, we investigated which factors predicted positive treatment outcome by examining demographics, insomnia characteristics, baseline levels of daytime function, HRQoL, sleep-disruptive beliefs and psychological health on post-treatment sleep quality, daytime function and HRQoL. 138 consecutive primary insomnia patients completed questionnaires pre- and post-treatment and at 6 months follow-up. After CBT-i, robust clinical improvements were observed in sleep, daytime function and HRQoL, regardless of age, gender, type or duration of the complaint. Patients with pre-treatment severe insomnia, pronounced daytime impairment and low psychological well-being benefited most.

Antigen-presenting properties of human vascular endothelial cells.

Human endothelial cells were obtained by collagenase digestion of the umbilical vein wall, explanted into tissue culture, and grown as monolayers of cells. Endothelial cells extracted from these monolayers were specifically lysed by anti-HLA-DR alloantisera. Moreover, the stimulating capacity of these endothelial cells toward allogeneic peripheral blood mononuclear lymphocytes was specifically and significantly inhibited by the presence of relevant anti-HLA-DR antisera. Endothelial cells that expressed HLA-DR determinants were also capable of substituting for macrophages in the lymphoproliferative response of purified T cells to soluble protein antigens. Moreover, in concordance with the results previously reported in which macrophages were employed, the endothelial cell donor should share HLA-DR determinants with the T cell donor for an optimal response to occur.

Clonal distribution of HLA-restricted antigen-reactive T cells in man.

The [3H]thymidine suicide technique was used to test the hypothesis that the response of immune human T cells from HLA-D/DR heterozygous donors to the soluble protein antigen purified protein derivative (PPD) is clonally expressed and consists of the concurrent proliferation of at least two separable subpopulations of lymphocytes. The results showed that each of the two subpopulations react to one or the other of the HLA-D/DR antigens presented together with PPD by allogeneic monocytes. In addition, using in vitro priming techniques of in vivo sensitized lymphocytes from heterozygous donors, it was possible to generate specific memory cells capable of recognizing the priming soluble protein antigen together with the HLA-D/DR determinant present in the initial sensitizing culture.

[Cognitive-behavioural therapy for primary insomnia: effectiveness in a clinical setting]. / Cognitieve gedragstherapie voor primaire insomnia: effectiviteit in een klinische setting.

BACKGROUND: The effectiveness of cognitive behavioural therapy for insomnia (cbt-i) has been demonstrated in randomised controlled trials (rct's) with primary insomnia patients and, more recently, with comorbid insomnia patients. The clinical impact of the treatment is mainly on sleep quality and the use of medication and to a lesser extent on daytime functioning. So far there have been very few studies of the effectiveness of cbt-i in clinical settings. AIM: To examine the effectiveness of cbt-i for primary insomnia via an uncontrolled prospective study in a clinical tertiary care setting. METHOD: We included 76 patients with primary insomnia, most of whom having been referred by their gp or medical specialist to the Leuven University Centre of Sleep, who followed a 6-week course of cbt-i as a group. Effects of cbt-i on primary and secondary outcome measures were studied. results cbt-i resulted in a significant improvement in all primary sleep variables such as sleep onset, sleep efficiency and sleep quality. The increase in total sleep time was less substantial, probably as a result of the specific sleep restriction guidelines. There was also a significant improvement on several secondary parameters, in particular dysfunctional cognitions, affective state, general health and use of medication. CONCLUSIONS: cbt-i has a significant impact on sleep quality, medication use and daytime functioning in primary insomniacs in a clinical tertiary care setting.

Occurrence of Francisella piscicida in farmed and wild Atlantic cod, Gadus morhua L., in Norway.

Francisellosis, caused by the bacterium Francisella piscicida, has become one of the most serious diseases in Atlantic cod production in Norway. The major aim of this study was to determine the distribution of F. piscicida in farmed and wild fish in areas with cod farming along the Norwegian coast, and its occurrence in cod from areas without cod farming. Two real-time PCR assays, targeting the 16S rRNA gene and the FopA gene of F. piscicida, were developed since sensitive and specific diagnostic tools are required for detecting asymptomatic carriers of the bacterium. A total of 422 wild cod from 13 sampling areas and 955 farmed cod from 10 areas along the coast of Norway were examined. Using the real-time polymerase chain reaction (PCR) assays, F. piscicida was detected in wild populations of cod from all counties examined south of Sogn og Fjordane in southern Norway (overall prevalence 13%, n = 221). Wild cod north of Sogn og Fjordane were negative for the bacterium (n = 201). Farmed cod from most parts of Norway were F. piscicida positive. The apparent absence of the bacterium in wild populations of cod in the northern parts of Norway and its widespread occurrence in wild cod from southern parts of Norway is believed to relate to differences in seawater temperatures.

The role of physical workload and pain related fear in the development of low back pain in young workers: evidence from the BelCoBack Study; results after one year of follow up.

AIMS: To study the influence of work related physical and psychosocial factors and individual characteristics on the occurrence of low back pain among young and pain free workers. METHODS: The Belgian Cohort Back Study was designed as a prospective cohort study. The study population of this paper consisted of 716 young healthcare or distribution workers without low back pain lasting seven or more consecutive days during the year before inclusion. The median age was 26 years with an interquartile range between 24 and 29 years. At baseline, these workers filled in a questionnaire with physical exposures, work related psychosocial factors and individual characteristics. One year later, the occurrence of low back pain lasting seven or more consecutive days and some of its characteristics were registered by means of a questionnaire. To assess the respective role of predictors at baseline on the occurrence of low back pain in the following year, Cox regression with a constant risk period for all subjects was applied. RESULTS: After one year of follow up, 12.6% (95% CI 10.1 to 15.0) of the 716 workers had developed low back pain lasting seven or more consecutive days. An increased risk was observed for working with the trunk in a bent and twisted position for more than two hours a day (RR 2.2, 95% CI 1.2 to 4.1), inability to change posture regularly (RR 2.1, 95% CI 1.3 to 3.5), back complaints in the year before inclusion (RR 1.7, 95% CI 1.1 to 2.8), and high scores of pain related fear (RR 1.8, 95% CI 1.0 to 3.1). Work related psychosocial factors and physical factors during leisure time were not predictive. CONCLUSION: This study highlighted the importance of physical work factors and revealed the importance of high scores of pain related fear in the development of low back pain among young workers.

The latent structure of the functional dyspepsia symptom complex: a taxometric analysis.

OBJECTIVES: Rome III introduced a subdivision of functional dyspepsia (FD) into postprandial distress syndrome and epigastric pain syndrome, characterized by early satiation/postprandial fullness, and epigastric pain/burning, respectively. However, evidence on their degree of overlap is mixed. We aimed to investigate the latent structure of FD to test whether distinguishable symptom-based subgroups exist. METHODS: Consecutive tertiary care Rome II FD patients completed the dyspepsia symptom severity scale. Confirmatory factor analysis (CFA) was used to compare the fit of a single factor model, a correlated three-factor model based on Rome III subgroups and a bifactor model consisting of a general FD factor and orthogonal subgroup factors. Taxometric analyses were subsequently used to investigate the latent structure of FD. KEY RESULTS: Nine hundred and fifty-seven FD patients (71.1% women, age 41 ± 14.8) participated. In CFA, the bifactor model yielded a significantly better fit than the two other models (χ² difference tests both p < 0.001). All symptoms had significant loadings on both the general and the subgroup-specific factors (all p < 0.05). Somatization was associated with the general (r = 0.72, p < 0.01), but not the subgroup-specific factors (all r < 0.13, p > 0.05). Taxometric analyses supported a dimensional structure of FD (all CCFI<0.38). CONCLUSIONS AND INFERENCES: We found a dimensional rather than categorical latent structure of the FD symptom complex in tertiary care. A combination of a general dyspepsia symptom reporting factor, which was associated with somatization, and symptom-specific factors reflecting the Rome III subdivision fitted the data best. This has implications for classification, pathophysiology, and treatment of FD.

[Clinical application of portable spirometry in asthma].

OBJECTIVE: To investigate the clinical applications of portable spirometry in asthma. METHODS: Twenty patients with asthma were recruited from Peking Union Medical College Hospital. Flow-volume loop, simultaneous asthma symptoms, and mood were monitored three times a day for consecutive 14 days. RESULTS: In patients with a normal daytime spirometry, marked decline of forced expiratory volume in one second (FEV1) and peak expiratory flow (PEF) were observed at night and/or in the early morning. A within subject correlation analysis between FEV1, PEF, and asthma symptoms showed that the correlation between symptoms and airway obstruction was found only in seven out of twenty patients (35%). Four patients (20%) reported many symptoms with nearly normal portable spirometry. Accordingly, their symptoms were not correlated with FEV1 and PEF. This group of patients was defined as over-perceivers. On the contrary, another two patients (10%) did not report any symptoms while obvious airways obstruction was recorded by a portable spirometry. These patients were defined as under-perceivers. CONCLUSIONS: Dynamic monitoring of flow-volume loop with a portable spirometry is more accurate than routine lung function test in assessment of asthma severity. In addition, combined with simultaneous monitoring of symptoms, it would be of particularly helpful in identifying two specific types of asthma patients, e.g. over-perceivers and under-perceivers.

Acquiring symptoms in response to odors: a learning perspective on multiple chemical sensitivity.

In this chapter, a learning account is discussed as a potential explanation for the symptoms in multiple chemical sensitivity. Clinical evidence is scarce and anecdotal. A laboratory model provides more convincing results. After a few breathing trials containing CO2-enriched air as an unconditioned stimulus in a compound with harmless odor substances as conditioned stimuli, subjective symptoms are elicited and respiratory behavior is altered by the odors only. Also, mental images can become conditioned stimuli to trigger subjective symptoms. The learning effects cannot be explained by a response bias or by conditioned arousal, and they appear to involve basic associative processes that do not overlap with aware cognition of the relationship between the odors and the CO2 inhalation. Learned symptoms generalize to new odors and they can be eliminated in a Pavlovian extinction procedure. In accordance with clinical findings, neurotic subjects and psychiatric cases are more vulnerable to learning subjective symptoms in response to odors. Consistent with a learning account, cognitive-behavioral treatment techniques appear to produce beneficial results in clinical cases. Several criticisms and unresolved questions regarding the potential role of learning mechanisms are discussed.

Memory effects on symptom reporting in a respiratory learning paradigm.

With odors as conditioned stimuli (CSs) and CO2-enriched air as the unconditioned stimulus, participants learned to exhibit respiratory responses and somatic complaints on presentation of only the odor CS+. Studied was whether complaints during CS+-only trials were inferred from the conditioned somatic responses or were based on activated memory of the complaints during acquisition. Participants (N = 56) were either attentionally directed away or not from the complaints during acquisition, and the effects on somatic complaints during test were studied. Respiratory responses, heart rate, and somatic complaints were measured. No physiological conditioning effects were found. However, more complaints were reported to the CS+ than to the CS- odor, but only when the CS+ was foul smelling. This effect was modulated by the attention manipulation, showing that the learned complaints during the test phase were based on memory of the acquisition complaints and not on physiological responses during the test.

Fear-relevant images as conditioned stimuli for somatic complaints, respiratory behavior, and reduced end-tidal pCO2.

Two fear-relevant imagery scripts were used as conditioned stimuli (CSs) in a differential learning paradigm with 5.5% CO2-enriched air as unconditioned stimulus (US). In another condition, 2 neutral scripts were used as CSs (N = 56). Within each condition, one script was imagined while breathing the CO2-enriched air (CS+/US trial), the other while breathing room air (CS- trial). Three CS+ and 3 CS- trials were run in an acquisition phase, followed by 2 CS+ and 2 CS- test trials (imagining the scripts while breathing air). Respiratory behavior, end-tidal CO2, and heart rate were measured throughout the experiment; subjective symptoms were measured after each trial. The type of imagery had strong effects on symptoms and physiological responses. A selective conditioning effect was also observed: CS+ imagery produced more symptoms and altered respiratory behavior compared with CS- imagery, but only in the fear-relevant script condition. The findings are discussed as to their relevance for panic and agoraphobic anxiety.

Hyperventilation and attention: effects of hypocapnia on performance in a stroop task.

This study aimed to investigate the effect of hypocapnia on attentional performance. Hyperventilation, producing hypocapnia, is associated with physiological changes in the brain and with subjective symptoms of dizziness, concentration problems and derealization. In this study (N=42), we examined cognitive performance on a Stroop-like task, following either 3 min of hypocapnic or normocapnic overbreathing. Both overbreathing trials were run on separate days, each preceded by a baseline trial with the same task during normal breathing. More and other symptoms were reported after hypocapnia compared to normocapnia. Also, more errors were made and progressively slower reaction times (RT's) were observed during recovery from hypocapnia. These performance deficits were only found in participants characterized by apneas. The number of symptoms did not correlate with RT's or errors. The pattern of data suggested that hypoxia, as a result of apneas during recovery from hypocapnia, caused the cognitive performance deficit.

Negative affect, respiratory reactivity, and somatic complaints in a CO2 enriched air inhalation paradigm.

Subjects scoring high on negative affectivity (NA) are known to report more psychosomatic complaints than subjects scoring low. According to the symptom perception hypothesis, high NA subjects attend more to somatic sensations and interpret these as more threatening. We investigated the relationship between NA and psychosomatic complaints in a group of high and low NA subjects (N = 72) in, (a) a questionnaire study, and (b) in a laboratory setting. The latter involved the inhalation of three different gas mixtures (room air, 5.5% and 7.5% CO2-enriched air) while respiratory responses were registered. Subjective complaints were measured after each trial. High NA subjects reported more complaints than low NA subjects in the questionnaire study. However, NA had no main effects on complaints in the laboratory study and did not interact with the effects of gas mixture on complaints. During room air trials, NA correlated only with general arousal complaints when a strong respiratory challenge had not been given before. The pattern of results suggests that experimental inductions of complaints may largely wipe out NA-related differences in attentional/interpretative processes that may mediate the NA complaints link.

Psychosomatic symptoms and breathing pattern.

OBJECTIVE: Study of the links between breathing pattern, negative affectivity, and psychosomatic complaints at rest and following hyperventilation. METHODS: In 819 patients with anxiety and somatoform disorders and 159 healthy subjects, self-reported symptoms, breathing pattern, and end-tidal CO(2) concentration (FetCO(2)) were recorded during rest and following a hyperventilation provocation test (HVPT). The relationship between disorder category, symptoms, age, and score of STAI-trait (as a measure of negative affectivity) on the one hand, and breathing pattern on the other was investigated, separately in men and women. RESULTS: Anxiety disorders, and to a lesser extent, somatoform disorders, were characterized by breathing instability (progressive decrease of FetCO(2) at rest during mouthpiece breathing, delayed recovery of FetCO(2) following HVPT), the mean values of respiratory frequency, and FetCO(2) being modulated by STAI-trait. After grouping the symptoms into independent factors, links were observed between symptoms and breathing pattern, independently from the presence of an anxiety or somatoform disorder. CONCLUSION: Some symptom factors appeared to be related to a lower FetCO(2) during hyperventilation, others likely directly influenced the breathing pattern. Among those, mainly respiratory symptoms were accompanied by a reduction of FetCO(2) at rest, with slower recovery of FetCO(2) following HVPT. The latter was observed also in the presence of marked anxiety. In contrast, subjects complaining of dizziness, fainting, and paresthesias in daily life presented higher values of FetCO(2) following HVPT, probably due to a voluntary braking of ventilation during HVPT.