[Troponins as biomarkers for myocardial injury and myocardial infarction]. / Troponine als Biomarker bei Myokardschaden und Myokardinfarkt.

Elevation of cardiac troponins above the 99th percentile of a healthy reference population is established as a marker for myocardial cell damage and is crucial for the diagnosis of myocardial infarction. In addition, corresponding clinical evidence of acute myocardial ischemia i.e. symptoms, changes in the electrocardiogram (ECG), wall motion abnormalities or suggestive angiographic findings are required for the diagnosis of myocardial infarction. Using modern highly sensitive assays myocardial infarction can be detected more frequently and earlier. On the other hand myocardial infarction can be ruled out with a higher diagnostic accuracy. Cardiac troponins are specific for myocardial cell damage but not for myocardial infarction and can be elevated in numerous other disease states. In these cases myocardial injury can be diagnosed independently of myocardial ischemia. Typical dynamics with rise and fall of troponin levels can distinguish acute myocardial injury (e. g. pericarditis/myocarditis and pulmonary embolism) from chronic myocardial injury (e. g. cardiomyopathy). Clinically, highly sensitive troponin assays are currently recommended in addition to the 0/3 h and 0/1 h algorithms for rapid inclusion or exclusion of myocardial infarction.

Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio).

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

Experimental infection by Yersinia ruckeri O1 biotype 2 induces brain lesions and neurological signs in rainbow trout (Oncorhynchus mykiss).

Pathological manifestations in rainbow trout (Oncorhynchus mykiss) following experimental waterborne infection with Yersinia ruckeri serotype O1 biotype 2 (strain 07111224) were investigated. Rainbow trout were exposed to 8 × 107  CFU/ml of Y. ruckeri by bath for 6 hr, and mortality was then monitored for 22 days post-infection (dpi). Organs were sampled at 3 dpi and also from moribund fish showing signs of severe systemic infection such as bleeding, exophthalmia or erratic swimming behaviour. Y. ruckeri was observed in the meninges and diencephalon of the brain, and lamina propria of olfactory organ at 3 dpi. At 12 dpi, Y. ruckeri had spread throughout the brain including cranial connective tissues and ventricles and the infection was associated with haemorrhages and an infiltration with leucocytes. Y. ruckeri infection and associated with leucocyte infiltration were observed at 13 dpi. In conclusion, Y. ruckeri strain 07111224 causes encephalitis in the acute phase of infection, which could explain why Y. ruckeri-affected fish show exophthalmia and erratic swimming known as signs of ERM.

Physical properties of hydrogel wound dressing and its use in low-level laser therapy (LLLT).

Hydrogel dressings are routinely used in the treatment of superficial skin wounds. Due to their excellent transparency, we decided to evaluate their usefulness in laser-based medical procedures. We focused on assessing selected physical properties of HydroAid hydrogel wound dressing, used for low-level laser therapy (LLLT) aka laser biostimulation procedures. For the two wavelengths of 660 and 808 nm used in the biostimulation laser POLARIS 2, a dressing transmittance of 92 and 98%, respectively, was determined. Using a FLIR i7 thermal imaging camera, the changes in temperature distribution across the surface of the dressing were assessed, during a 3-h period following its unpacking and placing on the skin of a patient or leaving it at the ambient temperature. The results of the thermal imaging, as well as temperature measurements using a digital thermometer, showed that the cooling properties of a hydrogel dressing were maintained throughout the entire experiment and that it was capable to keep the temperature at least 5° below the skin/ambient (room) temperature. During the 6-h observation using the holographic microscope, which provided indirect insight into the processes occurring within the hydrogel, only minimum topographical changes (observable at a micrometre scale) were recorded, although dressing thickness and its flexibility decreased significantly with time. Additionally, the possibility to regenerate the hydrogel dressing by treating it with distilled water or a physiological salt solution was tested.

Microencapsulation of a putative probiotic Enterobacter species, C6-6, to protect rainbow trout, Oncorhynchus mykiss (Walbaum), against bacterial coldwater disease.

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD), which has a major impact on salmonid aquaculture globally. An Enterobacter species, C6-6, isolated from the gut of rainbow trout, Oncorhynchus mykiss (Walbaum), has been identified as a potential probiotic species providing protection against BCWD. This study examined the effects of alginate microencapsulation on the protective efficacy of C6-6 against BCWD in vivo when administered to rainbow trout fry orally or by intraperitoneal (IP) injection. Viable C6-6 bacteria were microencapsulated successfully, and this process (microencapsulation) did not significantly deteriorate its protective properties as compared to the administration of non-microencapsulated C6-6 bacteria. Both oral and IP delivery of C6-6 achieved significantly better protection than control treatments that did not contain C6-6 bacteria. The highest relative percent survival (RPS) resulted from IP delivery (71.4%) and was significantly greater than the highest oral RPS (38.6%). Successful intestinal colonization was not critical to protective effects of C6-6. The study showed that C6-6 administration, with or without encapsulation, was a viable choice for protecting fry from BCWD especially when administered intraperitoneally.

Development and application of molecular methods (PCR) for detection of Tasmanian Atlantic salmon reovirus.

Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi-nested RT-PCR & RT-qPCR) and virus isolation in cell culture using finfish cell lines, CHSE-214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL-CSIRO and DPIPWE-Tasmania. A total of 144 fish from nine sites (12-33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south-east) during late spring to early summer of 2009, and the data were analysed using different statistical approaches. The prevalence of TSRV ranged from 6% to 22% in both regions. All the diagnostic methods (data from both laboratories) had high specificity, while the estimated sensitivity varied between tests with RT-qPCR being the most sensitive (95.2%) method followed by virus isolation and then conventional hemi-nested RT-PCR.

Changes in the interbranchial lymphoid tissue of Atlantic salmon (Salmo salar) affected by amoebic gill disease.

The interbranchial lymphoid tissue (ILT) was recently described in the gills of salmonids. This study examined changes in the ILT during a parasitic infection in marine environment, using amoebic gill disease (AGD) as a model. Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans were sampled at 0, 3, 7, 14 and 28 days post challenge. Transversal sections of three areas of the gills (dorsal, medial and ventral) were histologically assessed for morphological and cellular changes. AGD induced morphological changes and a cellular response in the ILT of affected fish. These changes included a significant increase in the ILT surface area in fish 28 days after AGD challenge, compared to control fish at the same time point. The length of the ILT increased significantly 28 days post exposure in the dorsal area of the gill arch in the fish affected by AGD. The lymphocyte density of the ILT increased after AGD challenge, peaking at 7 days post exposure; however, by 28 days post exposure, a reduction of lymphocyte density to values close to pre-infection levels was observed. PCNA immunostaining revealed that epithelial hyperplasia was the most likely factor contributing to the ILT enlargement in the affected fish.

Evidence of immune and inflammatory processes in the gills of AGD-affected Atlantic salmon, Salmo salar L.

Amoebic gill disease (AGD) is a disease caused by the ectoparasite Neoparamoeba perurans which affects several cultured marine fish worldwide. The characterisation of pro-inflammatory and immune related genes at the mRNA level in AGD-affected Atlantic salmon gills was performed at 10 days post-inoculation using 2D quantitative RT-PCR, a method of mapping transcriptional responses in tissues. The genes of interest were IL-1ß, TNF-α, TCR-α chain, CD8, CD4, MHC-IIα, MHC-I, IgM and IgT. A significant increase in expression of the mRNA of all the genes was observed in the gills of AGD-affected fish. Contrary to previous studies, our data suggest that the parasite, N. perurans, elicits a classical inflammatory response in the gills of AGD-affected fish and indicates that the mRNA expression of immune genes within gill lesions misrepresents the cellular immune response in the gills during AGD.

Enhanced accumulation of biologically active compounds in lichens with potential functional food applications.

Lichens have great potential as food, functional food additives or flavourings. The presence of specific substances with multiple biological activities is one of the characteristics of lichens. However, research on lichens as a food source or functional food additive is limited. The present study simulated, for the first time, the potential bioaccessibility of active compounds from 6 lichen species in simulated gastric and intestinal conditions. An in vitro digestion showed that the lichen substances had different bioaccessibility and stability during digestion. It was found that the application of some metabolic modulators significantly altered the accumulation of metabolites in most species. In addition, the study demonstrated the antimicrobial activity of the tested extracts as well as of 14 isolated lichen metabolites. These multi-directional studies demonstrate the potential of lichens in terms of their use as antimicrobial functional food additives.

Molecular characterization of "Candidatus Similichlamydia latridicola" gen. nov., sp. nov. (Chlamydiales: "Candidatus Parilichlamydiaceae"), a novel Chlamydia-like epitheliocystis agent in the striped trumpeter, Latris lineata (Forster).

Histological analysis of gill samples taken from individuals of Latris lineata reared in aquaculture in Tasmania, Australia, and those sampled from the wild revealed the presence of epitheliocystis-like basophilic inclusions. Subsequent morphological, in situ hybridization, and molecular analyses were performed to confirm the presence of this disease and discovered a Chlamydia-like organism associated with this condition, and the criteria set by Fredericks and Relman's postulates were used to establish disease causation. Three distinct 16S rRNA genotypes were sequenced from 16 fish, and phylogenetic analyses of the nearly full-length 16S rRNA sequences generated for this bacterial agent indicated that they were nearly identical novel members of the order Chlamydiales. This new taxon formed a well-supported clade with "Candidatus Parilichlamydia carangidicola" from the yellowtail kingfish (Seriola lalandi). On the basis of sequence divergence over the 16S rRNA region relative to all other members of the order Chlamydiales, a new genus and species are proposed here for the Chlamydia-like bacterium from L. lineata, i.e., "Candidatus Similichlamydia latridicola" gen. nov., sp. nov.

Molecular characterization of "Candidatus Parilichlamydia carangidicola," a novel Chlamydia-like epitheliocystis agent in yellowtail kingfish, Seriola lalandi (Valenciennes), and the proposal of a new family, "Candidatus Parilichlamydiaceae" fam. nov. (order Chlamydiales).

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported "Candidatus Piscichlamydia salmonis" (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as "Candidatus Parilichlamydia carangidicola" and that the new family be known as "Candidatus Parilichlamydiaceae."

Isolation of RAG-1 and IgM transcripts from the striped trumpeter (Latris lineata), and their expression as markers for development of the adaptive immune response.

A partial sequence of the recombination activating gene-1 (RAG-1) and several full length sequences of the immunoglobulin M (IgM) heavy chain mRNA were obtained from the striped trumpeter (Latris lineata). The RAG-1 fragment consisted of 205 aa and fell within the core region of the open reading frame. The complete IgM heavy chain sequences translated into peptides ranging between 581 and 591 aa. Both genes showed good homology to other vertebrate sequences. The expression of the two genes was assessed throughout the early developmental stages of striped trumpeter larvae (5-100 dph) and used as markers to follow the ontogeny of the adaptive immune response. Using RT-PCR, RAG-1 mRNA expression was detectable at 5 dph and remained so until 80 dph, before becoming undetectable at 100 dph. IgM expression was also detectable at 5 dph, and remained so throughout. These patterns of expression may suggest that the striped trumpeter possess mature B cells with surface IgM at 100 dph. However, complete immunological competence is likely not reached until some time later. The early detection of IgM mRNA at 5 dph led to the investigation of its presence in oocytes. Both RAG-1 and IgM mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. The biological significance of such a phenomenon remains to be investigated.

Culturable microbiota of ranched southern bluefin tuna (Thunnus maccoyii Castelnau).

AIMS: The Australian tuna industry is based on the ranching of wild southern bluefin tuna (SBT, Thunnus maccoyii). Within this industry, only opportunistic pathogens have been reported infecting external wounds of fish. This study aimed to identify different culturable bacteria present in three cohorts of SBT and to determine normal bacteria and potential pathogens in isolates from harvest fish and moribund/dead fish. Post-mortem changes in the microbiota were also studied. METHODS AND RESULTS: Moribund/dead showed a greater proportion of members from the family Vibrionaceae than harvested fish; the latter presented mainly non-Vibrio species. In harvested fish spleens, Vibrio splendidus I complex was the most commonly identified group among Vibrio isolates, while most groups from the family Vibrionaceae were isolated from gills. For moribund/dead, Vibrio chagasii and Photobacterium damselae subsp. damselae were common in gill, spleen and kidney samples. Non-Vibrio isolates from gills were characterized using 16S rRNA sequencing as Flavobacteriaceae and classes Gammaproteobacteria and Alphaproteobacteria, mainly from the genera Winogradskyella and Tenacibaculum. Post-mortem changes showed dynamic shifts in bacterial dominance in gills, with Vibrionaceae and non-Vibrio spp. found in similar proportions initially and types related to Pseudoalteromonas ruthenica prevailing after 27 h. Spleen samples showed little bacterial growth until 5 h post-mortem, while various Vibrio-associated species were isolated 27 h post-mortem. CONCLUSIONS: Bacterial isolates found include a range of potentially pathogenic bacteria that should be monitored though most of them have yet to be associated with disease in tuna. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms a foundation for future research into the bacterial population dynamics under different culture conditions of SBT. An understanding of the bacterial compositions in SBT is necessary to evaluate the effects of some bacterial species on their health.

Evaluation of fixation methods for demonstration of Neoparamoeba perurans infection in Atlantic salmon, Salmo salar L., gills.

Formaldehyde-based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross-linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistochemical stains widely used in disease diagnostics. Additionally, formaldehyde fixation greatly limits the ability to recover DNA and mRNA from fixed specimens to the detriment of further downstream molecular analyses. Amoebic gill disease (AGD) has been reliably diagnosed from histological examination of gills although complementary methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR) are required to confirm the presence of Neoparamoeba perurans, the causative agent of AGD. As molecular techniques are becoming more prevalent for pathogen identification, there is a need to adapt specimen collection and preservation so that both histology and molecular biology can be used to diagnose the same sample. This study used a general approach to evaluate five different fixatives for Atlantic salmon, Salmo salar L., gills. Neutral-buffered formalin and seawater Davidson's, formaldehyde-based fixatives commonly used in fish histopathology, were compared to formalin-free commercial fixatives PAXgene®, HistoChoice™MB* and RNAlater™. Each fixative was assessed by a suite of analyses used to demonstrate AGD including routine histochemical stains, immunohistochemical stains, ISH and DNA extraction followed by PCR. All five fixatives were suitable for histological examination of Atlantic salmon gills, with seawater Davidson's providing the best quality histopathology results. Of the fixatives evaluated seawater Davidson's and PAXgene® were shown to be the most compatible with molecular biology techniques. They both provided good DNA recovery, quantity and integrity, from fixed and embedded specimens. The capacity to preserve tissue and cellular morphology in addition to allowing molecular analyses of the same specimens makes seawater Davidson's and PAXgene® appear to be the best fixation methods for diagnosis and research on AGD in Atlantic salmon gills.

Laboratory evaluation of sample collection methods (organs vs swabs) for Tasmanian salmon reovirus detection in farmed Atlantic salmon, Salmo salar L.

The use of swabs relative to organs as a sample collection method for the detection of Tasmanian salmon reovirus (TSRV) in farmed Tasmanian Atlantic salmon, Salmo salar L., was evaluated by RT-qPCR. Evaluation of individual and pooled sample collection (organs vs swabs) was carried out to determine the sensitivity of the collection methods and the effect of pooling of samples for the detection of TSRV. Detection of TSRV in individual samples was as sensitive when organs were sampled compared to swabs, and in pooled samples, organs demonstrated a sensitivity of one 10-fold dilution higher than sampling of pooled swabs. Storage of swabs at 4 °C for t = 24 h demonstrated results similar to those at t = 0. Advantages of using swabs as a preferred sample collection method for the detection of TSRV compared to organ samples are evident from these experimental trials.

Laser-capture dissection and immunohistochemistry reveals chloride and mucous-cell specific gene expression in gills of seawater acclimated Atlantic salmon Salmo salar.

Laser-capture microdissection and immunohistochemistry were used to show that gene and protein expression varied in different cell types in the gills of Atlantic salmon Salmo salar, with chloride cells found to express high levels of sodium potassium ATPase and mucous cells expressing elevated levels of anterior gradient protein. It is therefore important that studies of gene expression in gill tissue take account of the proportion of the various cell types present.

Phagocytosis of latex beads by a human monocytic Mono Mac 6 cell line and effects of low-frequency electromagnetic field interaction.

Some studies have shown that electromagnetic fields (EMFs) may impact immune response cells and their functions. The first stage of the defense from pathogens is innate immunity encompassing phagocytosis and phagocytosis-related intracellular effects. Our work aimed to determine the influence of a low-frequency electromagnetic field (7 Hz, 30 mTrms) on the phagocytosis process of latex beads (LBs), the production of reactive oxygen species (ROS), and viability changes in a human monocytic Mono Mac 6 (MM6) cell line as an experimental model of the phagocytosing cells in in vitro cell culture conditions. For these purposes, cells were firstly activated with infectious agents such as lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB), or the proliferatory agent phytohaemagglutinin (PHA), and then a phagocytosis test was performed. Cell viability and range of phagocytosis of latex beads by MM6 cells were measured by flow cytometry, and the level of ROS was evaluated with the use of a cytochrome C reduction test. The obtained results revealed that applied EMF exposure mainly increased the necrosis parameter of cell death when they were pre-stimulated with SEB as an infectious factor and subsequently phagocytosed LBs (P=0.001). Prestimulation with other agents like LPS or PHA preceding phagocytosis resulted in no statistically significant changes in cell death parameters. The level of ROS depended on the used stimulatory agent, phagocytosis, and/or EMF exposure. The obtained effects for EMF exposure indicated only a slight decrease in the ROS level for cells phagocytosing latex beads and being treated with SEB or PHA, while the opposite effect was observed for LPS pre-stimulated cells (data not statistically significant). The results concerning the viability of phagocytosing cells, the effectiveness of the phagocytosis process, and the level of radical forms might result from applied EMF parameters like signal waveform, frequency, flux density, and especially single EMF exposure.

Description of an Atlantic salmon (Salmo salar L.) type II interleukin-1 receptor cDNA and analysis of interleukin-1 receptor expression in amoebic gill disease-affected fish.

Previously, we showed that IL-1ß transcription is induced in the gills of amoebic gill disease (AGD)-affected fish in an AGD lesion-restricted fashion. However, in this environment, there is very little evidence of inflammation on histopathological or transcriptional levels and we hypothesised that aberrant signalling may occur. As a first step in investigating this issue, we cloned and sequenced the Atlantic salmon IL-1 receptor type II (IL-1RII) mRNA, and then examined the expression of both the IL-1RI (IL-1 receptor-like protein) and II during Neoparamoeba perurans infection. In gill lesions from AGD-affected fish, a step-wise temporal increase in the relative expression of IL-1ß coincided with a significant reduction in IL-1RI, whereas the IL-1RII mRNA remained unchanged. Down-regulation of IL-1RI could explain the paucity of inflammation in affected tissue, although simultaneous up-regulation of IL-1ß-inducible transcripts indicated that this is not due to a complete blockage of the IL-1RI pathway. Rather, it appears that IL-1RI transcription is reduced and this rate limits the effects of chronic IL-1ß over-expression.

Preliminary success using hydrogen peroxide to treat Atlantic salmon, Salmo salar L., affected with experimentally induced amoebic gill disease (AGD).

Currently, the only effective and commercially used treatment for amoebic gill disease (AGD) in farmed Tasmanian Atlantic salmon is freshwater bathing. Hydrogen peroxide (H2O2), commonly used throughout the aquaculture industry for a range of topical skin and gill infections, was trialled in vitro and in vivo to ascertain its potential as an alternative treatment against AGD. Under in vitro conditions, trophozoites of Neoparamoeba perurans were exposed to three concentrations of H2O2 in sea water (500, 1000 and 1500 mg L⁻¹) over four durations (10, 20, 30 and 60 min) each at two temperatures (12 and 18 °C). Trophozoite viability was assessed immediately post-exposure and after 24 h. A concentration/duration combination of 1000 mg L⁻¹ for >10 min demonstrated potent amoebicidal activity. Subsequently, Atlantic salmon mildly affected with experimentally induced AGD were treated with H2O2 at 12 and 18 °C for 15 min at 1250 mg L⁻¹ and their re-infection rate was compared to freshwater-treated fish over 21 days. Significant differences in the percentage of filaments affected with hyperplastic lesions (in association with amoebae) and plasma osmolality were noted between treatment groups immediately post-bath. However, the results were largely equivocal in terms of disease resolution over a 3-week period following treatment. These data suggest that H2O2 treatment in sea water successfully ameliorated a clinically light case of AGD under laboratory conditions.