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The European brown shrimpCrangon crangonis an abundant and commercially important species in the North Sea. Currently, there is interest in landing live shrimp to provide fresh animals for a growing market in live brown shrimp. During 4 survival studies between 2014 and 2016, shrimp were collected from commercial trawlers and maintained alive in off-shore facilities. From Day 1 onwards, a minority of shrimp (~2.24%) developed a white discoloration of the abdominal muscles and a depigmentation of the distal part of the abdomen, along with paralysis of the affected tissues and appendages. As the symptoms progressed, a circumferential blackish delineation appeared, creating a distinct boundary between healthy and necrotic tissue. Affected shrimp survived up to 3 wk, although in several animals the distal part of the tail was completely lost. Histological and electron microscopical examination confirmed the myonecrosis. A secondary bacterial invasion of the necrotic muscle was observed in some animals. RT-PCR for infectious myonecrosis virus was negative. The condition appears not to be contagious, based on the feeding of healthy shrimp with necrotic tissue of affected shrimp. Based on these observations, a mechanical cause inflicted during the catching process is proposed.

This study reports the comparative analyses of four Flavobacterium columnare isolates that have different virulence and antimicrobial resistance patterns. The main research goal was to reveal new insights into possible virulence genes by comparing the genomes of bacterial isolates that could induce tissue damage and mortality versus the genome of a non-virulent isolate. The results indicated that only the genomes of the virulent isolates possessed unique genes encoding amongst others a methyl-accepting chemotaxis protein possibly involved in the initial colonization of tissue, and several VgrG proteins engaged in interbacterial competition. Furthermore, comparisons of genes unique for the genomes of the highly virulent (HV) carp and trout isolates versus the, respectively, low and non-virulent carp and trout isolates were performed. An important part of the identified unique virulence genes of the HV-trout isolate was located in one particular gene region identified as a genomic island. This region contained araC and nodT genes, both linked to pathogenic and multidrug-resistance, and a luxR-gene, functional in bacterial cell-to-cell communication. Furthermore, the genome of the HV-trout isolate possessed unique sugar-transferases possibly important in bacterial adhesion. The second research goal was to obtain insights into the genetic basis of acquired antimicrobial resistance. Several point-mutations were discovered in gyrase-genes of an isolate showing phenotypic resistance towards first and second-generation quinolones, which were absent in isolates susceptible to quinolones. Tetracycline-resistance gene tetA was found in an isolate displaying acquired phenotypic resistance towards oxytetracycline. Although not localized on a prophage, several flanking genes were indicative of the gene’s mobile character.

Environmental changes or stressors can result in the development of diseases. Through regular fish disease surveys in the Belgian part of the North Sea, attention was drawn to a sudden increase of skin ulceration prevalence between 2011 and 2014 in common dab (Limanda limanda). Information on prevalence, ulceration, bacteriology, fish-related (e.g., length, age, and sex) and (spatial and temporal) environmental factors, and fishing intensity were gathered. This detailed investigation was framed within a long-term monitoring program, executed every spring-autumn from 2000 to present. Ulcerations were observed in 1.3% of fish (n=3,999). Spatial and temporal differences were evident, and highest prevalence was found in summer. Vibrio was the dominant cultivated bacterial genus present in the lesions. Skin ulcerations appeared to be correlated with length and body condition of the fish, as well as with temperature and pH of the seawater and fishing vessel density. Our research suggested the involvement of multiple factors in the development of skin ulcerations in common dab and endorsed the effects of changing environment and human influence on the marine ecosystem through activities such as fishing.

European brown shrimp (Crangon crangon) have great commercial value. Recently, an increasing demand for fresh shrimp has led fishermen to land part of their catch alive. In the present study, the effects of various parameters on the post-catch survival of brown shrimp were investigated by housing them in natural seawater and monitoring survival for two weeks. In a first experiment shrimp survival was tested under high population densities at different temperature settings ranging between 8 degrees C and 21 degrees C. A second experiment investigated the effect of a sand layer in the holding tanks when shrimp were housed at low population densities. Finally, the impact of onboard sorting with a rotary sorting riddle on subsequent survival was assessed. A higher survival rate was found at lower water temperatures. The use of a sand substrate did not significantly improve survival but did result in a more even distribution of shrimp across the bottom of the holding tanks. Onboard sorting with a mechanical rotary sorting riddle did not significantly affect the survival of brown shrimp during the first four days of holding although injuries sustained during onboard handling resulted in high mortality at the time of landing.

Plasma cortisol is the classical parameter to assess acute stress in vertebrates, while in fish larvae whole body cortisol is used. Immuno assays are commonly adopted for cortisol determination in a pool of fish larvae. At present no method exists for quantification of a full glucocorticoid profile of a single fish larva providing a detailed snap-shot of hypothalamic-pituitary-interrenal (HPI) axis activity. To date, a substantial amount of studies has confirmed the potential of probiotics to positively impact fish welfare by alleviating the general stress response. However, no study investigated how probiotics impact the glucocorticoid profile in an individual fish larva. In this respect, this study assessed the effect of the administration of the probiotic candidate Vibrio lentus, previously shown to protect European sea bass (Dicentrarchus labrax) larvae, on the HPI axis activity. Therefore, V. lentus was administered to germ-free European sea bass larvae and a ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for quantifying cortisol, its precursors (17α-hydroxyprogesterone and 11-deoxycortisol) and phase I metabolites (cortisone, 20β-dihydrocortisone, tetrahydrocortisol and tetrahydrocortisone) in the whole body of a single fish larva was developed in an EN ISO/IEC 17025 regulated environment and validated according the requirements of the Commission Decision No. 2002/657/EC and applied, making this the first study in its kind. In the supplemented larvae, the administration of the probiotic candidate V. lentus significantly decreased glucocorticoid baseline levels. This study provides a better insight into the effect of the probiotic candidate V. lentus on the HPI axis activity in European sea bass larvae.

Despite electric pulse fishing being regarded as a promising environmentally friendly fishing method, very little is known about possible negative effects on early life stages of bottom-dwelling species such as Sole Solea solea. Electrotrawling for brown shrimp Crangon crangon is increasingly used in shallow coastal zones and estuaries of the southern North Sea. As these fishing grounds are often important nurseries and spawning areas for various marine species, electrotrawling could therefore harm inhabiting fishes during their early life stages. Hence, this research aims to investigate the effect of electric pulses used to catch brown shrimp on the survival and development of Sole embryos and larvae. Exposure of Sole embryos at 2d postfertilization and larvae at 11d posthatching to pulsed DC used to catch brown shrimp did not result in a lower survival 8d after exposure. Additionally, no differences in yolk sac resorption and morphometric length measurements of the notochord, muscle, eye, and head were observed in the developing larvae.

Electrotrawling using electric pulse stimulation is a promising alternative to beam trawling in the brown shrimp Crangon crangon and Dover Sole Solea solea (also known as Solea vulgaris) fisheries of the North Sea. In the sole fishery, a 40-80-Hz pulse stimulation induces tetany in the muscles, which may result in injuries. Whereas no injuries have been reported in flatfish or selachian sharks and rays, electrically induced spinal injuries have been observed in gadoids such as Atlantic Cod Gadus morhua and Whiting (also known as European Whiting) Merlangius merlangus. This may indicate that fish species with a fusiform shape are more susceptible to electric pulses. Similar variation among species in electrically induced spinal injuries has been observed in freshwater electrofishing, although large variability in vulnerability has been reported among different freshwater fusiform species. Therefore, we aimed to assess the vulnerability of another, nongadoid, fusiform osteichthyan: Sea Bass Dicentrarchus labrax (also known as European Bass Morone labrax). Two length groups of Sea Bass (31.3 +/- 2.2 and 42.1 +/- 2.5cm) were exposed to electric pulses as used in commercial electrotrawls targeting Sole (80 bipolar pulses per second, 2% duty cycle). Thereafter, the fish were monitored daily and then euthanized 14d after exposure for gross, radiographic, and histologic examination. No injuries were found in fish exposed to the electrical pulses. Differences in vertebral morphology among fusiform species may result in varying vulnerabilities to electrically induced spinal injuries. As a result, electrically induced spinal injuries and/or their variability in both marine and freshwater species may be determined by similar morphological parameters.

Skin ulcerations rank amongst the most prevalent lesions affecting wild common dab (Limanda limanda) with an increase in prevalence of up to 3.5% in the Belgian part of the North Sea. A complex aetiology of these ulcerations is suspected, and many questions remain on the exact factors contributing to these lesions. To construct the aetiological spectrum of skin ulcerations in flatfish, a one-day monitoring campaign was undertaken in the North Sea. Fifteen fish presented with one or more ulcerations on the pigmented and/or non-pigmented side. Pathological features revealed various stages of ulcerations with loss of epidermal and dermal tissue, inflammatory infiltrates and degeneration of the myofibers bordering the ulceration, albeit in varying degrees. Upon bacteriological examination, pure cultures of Vibrio tapetis were retrieved in high numbers from five fish and of Aeromonas salmonicida in one fish. The V. tapetis isolates showed cross-reactivity with the sera against the representative strain of serotype O2 originating form a carpet-shell clam (Ruditapes descussatus). Moreover, the A. salmonicida isolates displayed a previously undescribed vapA gene sequence (A-layer type) with possible specificity towards common dab. Further research is necessary to pinpoint the exact role of these agents in the development of skin ulcerations in common dab.

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L-rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L-rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L-rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L-rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.

Aeromonas salmonicida was isolated from ulcerations in common dab (Limanda limanda). An experiment was performed to pinpoint its role in ulceration development, considering the importance of the skin barrier and the pigmented and non-pigmented sides. The skin of dab was treated in three zones, one where scales and epidermis were removed, one where mucus was discarded and one non-treated zone. Fish were tagged to allow individual identification and challenged with A. salmonicida. Mortality and severity of the developing lesions were recorded for 21 days post-inoculation. Starting 12 days post-inoculation, mortality occurred gradually in challenged fish; however, no direct cause could be established. Both control fish and challenged fish developed ulcerations containing A. salmonicida. Sequencing of vapA gene revealed that isolates retrieved from both groups were distinct, suggesting the presence of A. salmonicida prior to the trial. Most ulcerations developed in zones where skin was removed, suggesting that abrasion might be a predisposing factor in ulceration development. Ulcerations were also observed at the insertion site of the tag, where exposed muscle tissue might have favoured the development of ulcerations. In conclusion, A. salmonicida seems to be involved in the development of skin ulcerations in dab, although the exact pathogenesis needs to be elucidated.

Recently, Vibrio tapetis was isolated for the first time from skin ulcerations in wild-caught common dab (Limanda limanda). To further examine its role in the development of these skin lesions, an in vivo experiment was performed. The significance of the skin barrier and in addition the difference between pigmented and non-pigmented side were investigated. Hence, the skin of common dab was treated in three different ways on both the pigmented and non-pigmented side. On a first “treatment zone”, the scales and overlying epidermal tissue were removed whereas in a second zone only the mucus was discarded. The third zone served as a non-treated zone. Thereafter, fish were challenged with V. tapetis. The control group was sham treated. Mortality, clinical signs, severity and size of the developing lesions were recorded. All animals were sacrificed and sampled 21 days post-inoculation. Significantly more fish of the group challenged with V. tapetis died compared to the control group with the highest incidence occurring 4 days post-inoculation. Fish challenged with V. tapetis developed more severe skin ulcerations. In zones where scales and epidermal tissue were removed, the ulcerations were more severe compared to zones where only mucus was eliminated. Ulcerations occurred more frequently, were more severe and larger on the pigmented side. Our data represents prove of V. tapetis as causative agent of ulcerative skin lesions although prior damage of the skin seems to be a major contributing factor. Furthermore, the pigmented side seemed predisposed to the development of skin ulcerations. The research was funded by the European Fisheries Fund (EVF-project VIS/15/A03/DIV), the Flemish Government and the Research Foundation—Flanders (FWO). This work makes use of resources, facilities and/or services provided by UGent and Flanders Marine Institute as part of the Belgian contribution to EMBRC-ERIC SI

Vibrio tapetis, the etiological agent of Brown Ring Disease, mainly affects the Manila clam Ruditapes philippinarum. Although this bacterium is mainly known as a clam pathogen, it has been isolated from several fish species. The main aim of the present study was to further explore the variability of 27 V. tapetis isolates from bivalves and fish, considering three different aspects; in vitro virulence based on the loss of clam hemocyte adhesion properties, detection of the gene virB4 encoding for an essential component of the Type IV Secretion System, and MALDI-TOF MS characterization based on whole cell extracts. Finally, these approaches were compared and evaluated for their ability to differentiate the potential pathogenicity of the 27 isolates against the Manila clams. Among the 11 V. tapetis isolates from the common dab isolated in 2018 in Belgium, only one (2BB) showed intermediate in vitro virulence against the Manila clam, and seven carried the virB4 gene while none of the V. tapetis previously isolated from fish in 2003 showed the presence of this particular gene. Finally, the peak protein profiles generated with MALDI-TOF MS analysis from all 27 V. tapetis strains showed a clear clustering of clam pathogenic and nonpathogenic isolates suggesting that a new isolate of V. tapetis that would cluster within the clam pathogenic isolates could be potentially pathogenic to the Manila clam. Thereupon, MALDI-TOF MS typing allows rapid and cost-efficient identification of V. tapetis isolates and can be defined as a complementary method of the traditional qPCR that opens new perspectives to study the virulence of V. tapetis isolates but also to perform environmental monitoring in order to prevent outbreaks.

BACKGROUND In this study the growth kinetics of Chlorella and Scenedesmus, isolated from a Waste Stabilization Pond were investigated under different conditions of light intensity and temperature. Experimental data were collected by means of a combined respirometric and titrimetric set-up and used to extend a mathematical model. RESULTS The experimental results illustrated the interdependent relationship of light intensity and temperature, which had a significant influence on the microalgal growth. Consequently, a previously developed model was extended with a mathematical function that describes this relationship. The maximum specific growth rate and oxygen mass transfer coefficient were considered for model calibration. The optimized parameter values for Chlorella were μmax = 0.56 ± 0.0008 d−1 and KL a= 10.02 ± 0.02 d−1. For Scenedesmus, the optimized parameter values were μmax = 0.19 ± 0.0004and KL a = 7.71 ± 0.004 d−1. The model with optimized parameter settings described the dissolved oxygen production (derived from the respirometric data) and the related proton consumption (derived from the titrimetric data) reasonably well for both microalgal species. The threshold value for Theil's Inequality Coefficient of 0.3 was never exceeded. In addition, model validation for both species was performed indicating good correspondence between model prediction and experimental values. CONCLUSIONS Based on the experimental observations, a previously developed mathematical model was extended with a function that describes the interaction between light intensity and temperature. After model calibration a difference in maximum specific growth rate between the two microalgal species was observed. This might be explained by differences in cell metabolism.

Electric pulses in fishing gear are increasingly used in the North Sea and are considered a promising alternative to ameliorate the sustainability of demersal trawl fisheries. The electrotrawl for brown shrimp employing low frequency pulsed direct current (PDC) selectively induces a startle response in shrimp engendering decreased environmental impact and reduced by-catch. Prior to commercially introducing this fishing technique, data on its impact on marine organisms are crucial. The aim of this study was to evaluate the short-term effects of this pulse used for electrotrawling for brown shrimp on five marine fish species inhabiting shrimp fishery areas. For this purpose, 25 European plaice (Pleuronectes platessa), 30 Dover sole (Solea solea), 20 Atlantic cod (Gadus morhua), 19 bull-rout (Myoxocephalus scorpius) and 20 armed bullhead (Agonus cataphractus) were exposed to the shrimp pulse for 5 s. Before, during and till 20 min following exposure, the behaviour of the fish was monitored. Twenty-four hours post-exposure, all fish were sacrificed, inspected and samples for histological analysis were taken from the gills, dorsal muscle and internal organs. To investigate possible spinal injuries radiographs were taken. Behavioural responses were variable and species dependant. Roundfish species, cod in particular, were displaying more active and agitated fast swimming activity during exposure. The majority of flatfish showed only minor reactions and remained close to the bottom throughout the observation period. However, 15% of the exposed sole actively swam upwards during exposure. Mild multifocal petechial haemorrhages and suffusion, encountered mainly in plaice and sole, were not significantly different between exposed and control groups. Upon histological examination, in two exposed plaice, a focal small haemorrhage between muscle fibres was found, which was not encountered in control animals. In addition, the number of melanomacrophage centres in the spleen of exposed cod was significantly higher than in the non-exposed animals. In conclusion, under the circumstances as adopted in this study, the electrical field seemed to have only limited immediate impact on the exposed animals.

Electrotrawling is currently the most promising alternative for conventional beam trawls targeting sole and shrimp, meeting both the fisherman’s aspirations and the need for more environmentally friendly fishing techniques. Before electrotrawling can be further developed and implemented on a wider scale, more information is needed about the effects of electrical pulses on marine organisms. Adult Dover sole (Solea solea L.) and Atlantic cod (Gadus morhua L.) were used in the present study as model species for flatfish and roundfish, respectively. These animals were exposed to homogeneously distributed electrical fields with varying values of the following parameters: frequency (5–200 Hz), electrical field strength (100–200 V/m), pulse polarity, pulse shape, pulse duration (0.25–1 ms) and exposure time (1–5 s). The goal was to determine the range of pulse parameters which can be regarded as safe and thereby also to evaluate the effect of the pulses already being used in commercial electrotrawls. Fish behaviour during and shortly after exposure, 14-days post exposure mortality rates, as well as gross and histological examination were used to evaluate possible effects. During exposure, both species showed an escape response below a frequency of 20 Hz and a cramp reaction above 40 Hz. These reactions were immediately followed by post-exposure escape behaviour and at high electrical loads cod showed tonic-clonic epileptiform seizures. No mortality was observed and histological examination did not reveal any abnormalities, except for one cod showing a spinal injury. These data reveal the absence of irreversible lesions in sole as a direct consequence of exposure to electric pulses administered in the laboratory, while in cod, more research is needed to assess cod’s vulnerability for spinal injuries when exposed to the cramp pulses.

Pulse trawling is currently the best available alternative to beam trawling in the brown shrimp Crangon crangon and Sole Solea solea (also known as Solea vulgaris) fisheries. To evaluate the effect of repetitive exposure to electrical fields, brown shrimp were exposed to the commercial electrodes and pulse settings used to catch brown shrimp (shrimp startle pulse) or Sole (Sole cramp pulse) 20 times in 4 d and monitored for up to 14 d after the first exposure. Survival, egg loss, molting, and the degree of intranuclear bacilliform virus (IBV) infection were evaluated and compared with those in stressed but not electrically exposed (procedural control) and nonstressed, nonexposed (control) brown shrimp as well as brown shrimp exposed to mechanical stimuli. The lowest survival at 14 d (57.3%) occurred in the Sole cramp pulse treatment, and this was significantly lower than in the group with the highest survival, the procedural control (70.3%). No effect of electrical stimulation on the severity of IBV infection was found. The lowest percentage of molts occurred in the repetitive mechanical stimulation treatment (14.0%), and this was significantly lower than in the group with the highest percentage of molts, the procedural control (21.7%). Additionally, the mechanically stimulated brown shrimp that died during the experiment had a significantly larger size than the surviving individuals. Finally, no effect of the shrimp startle pulse was found. Therefore, it can be concluded that repetitive exposure to a cramp stimulus and mechanical stimulation may have negative effects on the growth and/or survival of brown shrimp. However, there is no evidence that electrical stimulation during electrotrawls would have a larger negative impact on brown shrimp stocks than mechanical stimulation during conventional beam trawling.

International audience; AbstractSevere economic losses due to diseases in marine larviculture may be linked to vibriosis. To better understand the pathogenesis of vibriosis and evaluate new ways to prevent and combat this important disease, there is a great need for reliable and reproducible experimental infection models. The present study aimed at developing a challenge model for vibriosis in Dover sole larvae and testing its applicability to study the effect of the probiotic treatment. For that purpose, larvae were challenged at 10 days post hatching with Vibrio anguillarum WT, V. anguillarum HI610 or V. harveyi WT. Following administration of V. anguillarum WT via immersion at 1 × 107 colony forming units/mL, a larval mortality of 50% was observed at 17 days post-inoculation. In a next step, the probiotic potential of 371 isolates retrieved from Dover sole was assessed by screening for their inhibitory effects against Vibrio spp. and absence of haemolytic activity. One remaining isolate (V. proteolyticus) and V. lentus, known for its protective characteristics in seabass larvae, were further tested in vivo by means of the pinpointed experimental infection model. Neither isolate provided via the water or feed proved to be protective for the Dover sole larvae against challenge with V. anguillarum WT. This developed challenge model constitutes a firm basis to expedite basic and applied research regarding the pathogenesis and treatment of vibriosis as well as for studying the impact of (a)biotic components on larval health.

In aquaculture hatcheries, egg disinfection is commonly employed as a mortality mitigation and disease management tool. In addition, disinfection protocols are utilized in research facilities, as a means to create axenic and gnotobiotic larval models. For the latter, a complete sterilization of the eggs is warranted. Multiple research groups have tested various disinfectants in fish eggs adopting different protocols and a range of parameters to evaluate their efficacy and safety. However, there is a clear lack of a critical review listing these different studies and stressing the advantages and shortcomings of the protocols and adopted disinfectants. This review provides a commented survey of the findings/experiments on the most frequently used and upcoming disinfecting products, the protocols in which they are included and their purposes with emphasis on decreasing bacterial and/or fungal load, underscoring the benefits and the limitations of each product. Supplemented with a critical note, this review assists both aquaculturists and researchers in making an informed choice. Moreover, it will help stakeholders to identify the gaps in knowledge where future research on egg disinfection is needed.

This study was executed to investigate the effect of dietary β-alanine (BA) on amino acid (AA) metabolism and voluntary feed intake in carp (Cyprinus carpio) at mildly elevated temperature to exert AA catabolism. Twenty-four fish in 12 aquaria were randomly assigned to either a control diet or the same diet with 500 mg BA/kg. A 14-day period at an ideal temperature (23 °C) was followed by 15 days at chronic mildly elevated temperature (27 °C). After the 15 days, all fish were euthanised for muscle analysis on histidine-containing dipeptides (HCD), whole blood on free AA and carnitine esters. The carnosine and anserine analysis indicated that all analyses were below the detection limit of 5 µmol/L, confirming that carp belongs to a species that does not store HCD. The increases in free AA concentrations due to BA supplementation failed to reach the level of significance. The effects of dietary BA on selected whole blood carnitine esters and their ratios were also not significant. The supplementation of BA tended to increase body weight gain (P = 0.081) and feed intake (P = 0.092). The lack of differences in the selected nutrient metabolites in combination with tendencies of improved growth performance warrants further investigation to unravel the mechanism of BA affecting feed intake. This first trial on the effect of BA supplementation on AA catabolism showed that its metabolic effect in carp at chronic mildly elevated temperature was very limited. Further studies need to evaluate which conditions are able to exert an effect of BA on AA metabolism.

This study is the first to describe the ultrastructural morphology of the envelope of Solea solea eggs from fertilisation until hatching. Defining the ultrastructural morphology of fish eggs is important for species identification and may assist in predicting the effect of external influences on these early life stages. In first instance, various fixation and embedding protocols were assessed to explore the morphology of the egg envelope, whereby the encountered difficulties were highlighted. The successful protocol for SEM proved to be combined fixation with 4% glutaraldehyde in 0.1M cacodylate buffer for minimum 4h with post-fixation of 2h with 1% OsO4 in 0.1M cacodylate buffer. For TEM, puncturing the egg envelope during the first steps of the fixation protocol was necessary to allow the embedding medium to penetrate through the egg envelope. Based on both scanning and transmission electron microscopical examination, three distinct layers were discerned in the egg envelope. During the development of the fish embryo, a change in the outer structure of the egg was observed. Scanning electron microscopical examination of one day post-fertilisation eggs (DPF) revealed a homogeneous outer layer, displaying a large number of pores uniformly distributed on the surface of the egg envelope. Starting from 2 DPF parts of the outermost layer or two outer layers peeled off. The second deeper layer showed larger pores, with less defined edges. In the third innermost layer irregular indentations were noted. On transmission electron microscopy the first outermost layer of 1 DPF eggs clearly folded into the pores. The second layer was more electron dense, had a uniform appearance and did not cover the surface of the pores. The third innermost layer was much thicker and possessed indentations. A total number of 12 undulating zones were discriminated based on different degrees of electron density. Prior to hatching, the compact structure of the innermost layer was distorted by dispersed holes and tears.

Due to the increasing importance of the aquaculture sector, diversification in the number of cultured species imposes itself. Dover sole Solea solea L. is put forward as an important new aquaculture candidate due to its high market value and high flesh quality. However, as for many other fish species, sole production is hampered by amongst others high susceptibility to diseases and larval mortality, rendering the need for more research in this area. In this respect, in first instance, a housing system for Dover sole larvae was pinpointed by keeping the animals individually in 24-well plates for 26 days with good survival rates and initiating metamorphosis. This ensures a standardised and reliable experimental set-up in which the possible death of one larva has no effect on the other larvae, rendering experiments adopting such a system more reproducible. In addition to proving valuable in many other applications, this multi well system constitutes a firm basis to enable the gnotobiotic rearing of larvae, which hitherto is non-existing for Dover sole. In this respect, secondly, a large number of disinfection protocols were tested, making use of widely employed disinfectants as hydrogen peroxide, glutaraldehyde and/or ozone whether or not combined with a mixture of antimicrobial agents for 24 h. Although none of the tested protocols was sufficient to reproducibly generate a gnotobiotic model, the combination of glutaraldehyde and hydrogen peroxide resulted in hatchable, bacteria-free eggs in some cases.

The application of electrical pulses in fishing gear is considered a promising option to increase the sustainability of demersal trawl fisheries. In the electrotrawl fishery for brown shrimp Crangon crangon, an electrical field selectively induces a startle response in the shrimp. Other benthic organisms remain mainly on the seafloor and escape underneath a hovering trawl. Previous experiments have indicated that this pulse has no short-term major harmful effects on adult fish and invertebrates. However, the impact on young marine life stages is still unknown. Because brown shrimp are caught in shallow coastal zones and estuaries, which serve as important nurseries or spawning areas for a wide range of marine species, electrotrawling on these grounds could harm embryos, larvae, and juveniles. We carried out experiments with different developmental stages of Atlantic Cod Gadus morhua, which are considered vulnerable to electrical pulses. Three embryonic stages, four larval stages, and one juvenile stage of Atlantic Cod were exposed to a homogeneous electrical field of 150 Vpeak/m for 5 s, mimicking a worst-case scenario. We detected no significant differences in embryo mortality rate between control and exposed groups. However, for the embryonic stage exposed at 18 d postfertilization, the initial hatching rate was lower. Larvae that were exposed at 2 and 26 d posthatch exhibited higher mortality rates than the corresponding nonexposed control groups. In the other larval and juvenile stages, no short-term impact of exposure on survival was observed. Morphometric analysis of larvae and juveniles revealed no differences in measurements or deformations of the yolk, notochord, eye, or head. Although exposure to a worst-case electrical field did not impact survival or development for six of the eight young life stages of Atlantic Cod, the observed delayed hatching rate and decreased survival for larvae might indicate an impact of electric pulses and warrant further research. Received July 20, 2016; accepted April 14, 2017

Pulse fishing may pose a promising alternative for diminishing the ecosystem effects of beam trawling. However, concerns about the impact on both target and non-target species still remain, amongst others the possible damage to the electro-receptor organs, the Ampullae of Lorenzini, of elasmobranchs. The current study aimed to examine the role of pulsed direct current (PDC) used in pulse trawls on the electro-detection ability of the small-spotted catshark, Scyliorhinus canicula. The electroresponse of the sharks to an artificially created prey-simulating electrical field was tested before and after exposure to the pulsed electrical field used to catch flatfish and shrimp. No statistically significant differences were noted between control and exposed animals, both in terms of the number of sharks exhibiting an electroresponse prior to and following exposure as well as regarding the timing between onset of searching behaviour and biting at the prey simulating dipole. These results indicate that, under the laboratory circumstances as adopted in this study, the small-spotted catshark are still able to detect the bioelectrical field of a prey following exposure to PDC used in pulse trawls. However, to fully grasp the impact of PDC on elasmobranchs, further studies are imperative, including examining the effect on reproduction and young life stages, the longer-term and indirect influences and experiments under field conditions.

The present study aimed at evaluating the cellular and transcriptomic responses induced by the probiotic candidate Vibrio lentus with gnotobiotic European sea bass (Dicentrarchus labrax, Linnaeus 1785) larvae. For this, a histomorphological analysis was performed using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and the anti-proliferating cell nuclear antigen (PCNA) assay. In addition, a global transcriptomic approach was adopted to study the whole body mRNA changes upon administration of V. lentus by microarrays with the custom Agilent sea bass oligonucleotide-microarray v2.0 (4 × 44 K). Following V. lentus administration, the apoptotic and cell proliferative indexes did not show significant differences between treatments for hindgut nor for midgut. However, V. lentus treatment did significantly modify the gene expression related not only to cell proliferation and cell death, but also to cell adhesion, reactive oxygen species metabolism, iron transport, and immune response. Our data represent the first global analysis of the effects of the probiotic candidate V. lentus on the gene expression profile in gnotobiotic European sea bass, and as such, provides a first delineation of the mechanisms by which this agent interacts with its host and exerts its beneficial effects.

Teleost fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal axis to release glucocorticoids, in particular cortisol, into the blood. For the majority of bacterial fish pathogens, stress is considered a key factor in disease outbreaks. Based upon studies in mammals, there is considerable evidence to suggest that, besides impairing the immune system, cortisol can have a direct effect on bacterial cells. Hitherto, this intriguing field of microbial endocrinology has remained largely unexplored in aquatic diseases. The present study investigated in vitro the impact of cortisol on phenotypic traits of the fresh water fish pathogen Flavobacterium columnare. Colonies obtained from the highly virulent (HV) isolates resulted in significantly larger and more spreading colonies compared to those from the low virulent (LV) isolates. High cortisol doses added displayed a direct effect on the bacterial cells and induced a significant decrease in colony size. An additional intriguing finding was the inverse relationship between cortisol concentrations added to the broth and the spreading character of colonies retrieved, with higher cortisol doses resulting in less rhizoid to rough and even smooth colony formation (the latter only in the LV trout isolate), suggesting a dose–response effect. The loss of the rhizoid appearance of the F. columnare colonies upon administration of cortisol, and hence the loss of motility, might indicate a phenotypic change to the biofilm state. These findings form the basis for further research on the impact of glucocorticoids on other virulence factors and biofilm formation of F. columnare. Electronic supplementary material The online version of this article (doi:10.1186/s13567-016-0370-9) contains supplementary material, which is available to authorized users.

Pulse trawling is the most promising alternative to conventional beam trawls targeting Sole Solea solea (also known as Solea vulgaris), but due to the electric fields created by electrotrawls spinal injuries are reported in gadoid round fishes such as Atlantic Cod Gadus morhua. This study aimed to investigate the variability in the occurrence of electric-induced spinal injuries in cod. Four groups of cod, each originating from a different wild or farmed stock, were exposed to the pulses used by electrotrawls targeting Sole. Effects were analyzed based on behavior, mortality, and lesions up to 14 d after exposure, and morphological characteristics such as size, somatic weight, muscularity, the number of vertebral bodies, and the vertebral mineral contents of animals were compared among different cohorts. Second, the influence of factors such as water temperature, electrode diameter, and pulse type and amplitude were tested. Electrode diameter and pulse amplitude showed a positive correlation with the intensity of the fish’s reaction. However, the present experiments confirmed that cod also show variable vulnerability, with injury rates ranging from 0% to 70% after (almost) identical exposures near the electrode. This indicates that these injuries are not only determined by the pulse parameter settings but also by subtle, fish-specific factors. Although the absence of a sensitive group of cod did not enable the elucidation of the conclusive factor, the effect of physiological and morphological factors such as intervertebral ligaments and rearing conditions during early life merit further attention in future research.

A thorough understanding of host-microbe interactions is crucial for more efficient disease management in the marine larviculture industry. As demonstrated in terrestrial animal research, gnotobiotic systems (involving animals cultured in germ-free conditions or inoculated with known microorganisms) are excellent tools to extend our understanding of the mechanisms involved in host-microbe interactions and allow the evaluation of new treatments for diseases. In this study, we introduce a germ-free European sea bass Dicentrarchus labrax larval model, independent of the continuous addition of antimicrobial agents. This model has an experimental set-up that allows addition of live feed to the larvae without compromising the germ-free status. This model will facilitate and render aquaculture research more effective in terms of mitigation fish larval diseases.

A study was conducted to evaluate whether dietary mannanoligosaccharides (MOS) modulate the effect of mildly elevated water temperature on intestinal morphology and some selected whole blood amino acids and acylcarnitines in common carp ( Cyprinus carpio carpio ). Twenty fish in 10 aquaria were randomly assigned to either a control diet or the same diet with 4 g/kg MOS. A 35-day period at ideal temperature (23.2 °C) was followed by 14 days at elevated temperature (26.1 °C). After both temperature periods, one fish per aquarium was euthanized for the intestinal morphology and whole blood analysis on free amino acids and carnitine esters. The elevated temperature period decreased the number of neutral (P = 0.031) and acid (P = 0.004) mucin-secreting goblet cells and tended to reduce (P = 0.056) the fold height in the midgut, irrespective of MOS supplementation. In both periods, supplementation of MOS increased (P = 0.035) the number of goblet cells in the hindgut, but other histomorphometries were unaffected. Free concentrations of whole blood amino acids were increased after the mildly elevated temperature period: valine (P = 0.002), leucine (P = 0.001), methionine (P = 0.001) and tyrosine (P = 0.001), but not affected by MOS supplementation. None of the carnitine esters were altered by the elevated temperature except propionyl carnitine that was higher (P = 0.003) after the mildly elevated temperature of 14 days. Supplementation of MOS only tended to reduce the tiglyl carnitine (P = 0.069) and methylmalonyl carnitine (P = 0.078) concentrations. The analysis of free amino acids and carnitine esters could not support the hypothesis that MOS counteracts depressing effects of elevated temperature on amino acid catabolism. In conclusion, moderate elevation of water temperature could lead to considerable changes in gut histology and metabolism.

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards - amongst others - ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.

Luminescent vibrios are amongst the most important pathogens in aquaculture, affecting almost all types of cultured organisms. Vibrio campbellii is one of these most important pathogens. In this study, the effects of feeding mnn9 yeast cell wall mutant and wild type yeast strain were investigated in the digestive tract of brine shrimp nauplii, Artemia franciscana, after experimental infection with V. campbellii (LMG 21363). Gnotobiotic A. franciscana nauplii were fed daily with dead Aeromonas hydrophila LVS3, and with either wild type strain of baker's yeast, Saccharomyces cerevisiae, or mutant strain mnn9, of which the cell wall contains elevated chitin and glucan and lower mannose levels. After three days of feeding, some nauplii were challenged with V. campbellii. Mean survival (%), individual length (mm) and total length (mm) at one day and two days after challenge were significantly higher in the group fed mnn9 than in the group fed wild type yeast (81 ± 1.50 and 63 ± 0.49, 1.56 ± 0.07 and 1.13 ± 0.02, 38.21 ± 3.11 and 21.26 ± 0.81 respectively for one day and 50 ± 2.37 and 20 ± 1.41, 2.33 ± 0.01 and 1.24 ± 0.04, 34.97 ± 5.56 and 7.45 ± 1.63 for two days after challenge). Histological examination revealed that the luminal diameter and enterocyte height of both mid- and hindgut were larger in the mnn9-fed group. Colonization of the gut lumen by V. campbellii could be observed by transmission electron microscopy for the group of nauplii fed with wild type yeast. Furthermore, it was observed that V. campbellii caused damage to the gut epithelium including shortening and disappearance of the microvilli, destruction of the apical cell membrane and cell lysis in the nauplii fed wild type yeast. The gut epithelium remained intact in challenged nauplii fed mnn9 yeast. The morphological findings of the present study further substantiate previous studies reporting a protective effect of this yeast cell wall mutant.

Aquacultuur is een zeer snel groeiende sector, maar er zijn nog talrijke knelpunten bij het opkweken van larven (larvicultuur) en van adulte vissen. Veel van deze knelpunten zijn van nutritionele aard. Het voedingsregime is nog vaak onvoldoende afgesteld op de behoeften van specifieke vissoorten waardoor uithongering een grote invloed blijft hebben. Deze problemen kunnen opgelost worden door de ontwikkeling van het gastro-intestinale stelsel per vissoort in kaart te brengen. Parameters, zoals de enterocytenhoogte, de aanwezigheid van supranucleaire vacuoles in de enterocyten en ‘the point of no return’ kunnen als maatstaf gebruikt worden om uithongering te monitoren en te voorkomen. Aan de hand van deze gegevens kan een optimaal voedingsregime met rotiferen (radardiertjes), Artemia (pekelkreeftjes) en Copepoda (eenoogkreeftjes) uitgewerkt worden.

Commercial Pangasianodon catfish production is heavily impacted by Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri. This study aimed to investigate the early bacterium-host interactions following immersion challenge and to compare the retrieved data with the invasion ability of the used isolates in fish cell lines. Firstly, Pangasianodon hypophthalmus fingerlings were challenged via immersion using E. ictaluri isolate HO2 or 223. At different times post inoculation, fish were sacrificed and gill and internal organ samples were taken for bacteriological, histological and immunohistochemical evaluation. The bacterial load was higher for fish inoculated with isolate HO2 compared with 223. Histological and immunohistochemical analysis revealed multifocal necrotic areas in kidney, spleen and liver of HO2 inoculated fish at 72 h post inoculation with short rod-shaped immunoperoxidase positive bacteria clustered inside cells respectively. Bacteria especially were present in the gills and intestinal tract of HO2 inoculated fish, suggesting the gastrointestinal tract and gills act as portals of entry. Following, the ability of HO2, 223 and four additional isolates to invade a Chinook salmon embryo cell line, a fat head minnow cell line and a rainbow trout liver cell line was tested. All E. ictaluri isolates were invasive in all cell lines albeit at different degrees. Isolate HO2 was highly invasive in all cell lines with a significantly higher invasion capacity than isolate 223 in the Chinook salmon embryo cell line. A correlation between in vivo virulence and in vitro invasiveness hence is suggested although further studies are needed to confirm this hypothesis.

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin–F. columnare interactions warranting further research. Electronic supplementary material The online version of this article (doi:10.1186/s13567-015-0164-5) contains supplementary material, which is available to authorized users.

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non-virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE-214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non-virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin-D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non-virulent strains were generally serum sensitive.

Helicobacter pullorum infections have been associated with several enterohepatic diseases, but the mechanism of action is currently undefined. The present study was therefore set up to investigate possible cytotoxic effects of this pathogen on liver cells. A mouse hepatic cell line was exposed to H. pullorum sonicate and cytotoxicity was observed for all isolates after incubation for 72 h. Features characteristic for mitotic catastrophe characterized by chromatin condensation, formation of multinuclear distended cells and micronucleation, were recorded. In addition, intranuclear pseudoinclusions were seen in sonicate-treated cells. Finally, cells exposed to sonicate eventually underwent cell death with the morphological features of necrosis, which occurred without activation of caspase-3. The toxic factor involved in the cytotoxic activity proved to be soluble, trypsin–sensitive and stable at 56°C and at −70°C with a molecular weight to be over 50 kDa. The current study shows for the first time that H. pullorum causes mitotic catastrophe resulting in primary necrosis in mouse hepatocytes. J. Morphol., 2009. © 2009 Wiley-Liss, Inc.

The purpose of this study was to assess the in vitro susceptibility of 64 Vietnamese isolates of Edwardsiella ictaluri, the causal agent of the infectious disease Bacillus Necrosis Pangasius in Pangasianodon hypophthalmus, using the agar dilution technique. All isolates originated from different farms and were collected between 2002 and 2005. None of the isolates displayed acquired resistance to amoxicillin, amoxicillin-clavulanic acid, chloramphenicol, florfenicol, gentamicin, kanamycin, neomycin, and nitrofurantoin. Acquired resistance to streptomycin was detected in 83%, to oxytetracycline in 81%, and to trimethoprim in 71%, of the isolates, as indicated by a bimodal distribution of the minimal inhibitory concentrations (MICs) of these antimicrobials. The MICs of enrofloxacin displayed a monomodal distribution with tailing toward the higher MIC values, possibly indicating reduced susceptibility of a minority of isolates (3 Out of the 64). For the quinolone antimicrobial agents flumequin and oxolinic acid, acquired resistance was encountered in 8% and 6% of the strains, respectively. All strains were intrinsically resistant to the polypeptide antimicrobial agent colistin. Seventy-three percent of the isolates were shown to have acquired resistance to at least three antimicrobial agents. The results of this study emphasize the strict need to control both the prophylactic and curative use of antimicrobial agents in Vietnamese aquaculture.

In this study, the virulence heterogeneity of Listeria monocytogenes serotype 4b strains of different origins was analysed on different levels. On one hand, the survival of L. monocytogenes strains in synthetic gastric fluid was studied. On the other hand, the pathogenic potential of strains with different inlB expression levels was analysed in an A/J mouse model for gastrointestinal listeriosis. Differences in survival capacity in gastric fluid and in in vivo virulence potential were observed between the tested strains. No clear correlation between the origin and the obtained data could be made. However, these results confirm the existence of heterogeneity in virulence potential of L. monocytogenes serotype 4b strains.

A new cultivation method was successfully applied for the in vitro isolation of a hitherto uncultured spiral Helicobacter species associated with ulceration of the non-glandular stomach and gastritis in pigs and formerly described as 'Candidatus Helicobacter suis'. Three isolates, HS1(T), HS2 and HS3, were subcultured from the stomach mucosa of three pigs after slaughter and were analysed using a polyphasic taxonomic approach. The novel isolates grew on biphasic culture plates or very moist agar bases in microaerobic conditions and exhibited urease, oxidase and catalase activities. Sequencing of the 16S rRNA gene, the 23S rRNA gene, the partial hsp60 gene and partial ureAB genes confirmed that the strains present in the gastric mucosa of pigs constituted a separate taxon, corresponding to 'Helicobacter heilmannii' type 1 strains as detected in the gastric mucosa of humans and other primates. For all genes sequenced, the highest sequence similarities were obtained with Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis, Helicobacter species isolated from the gastric mucosa of dogs and cats, which have also been detected in the human gastric mucosa and which are commonly referred to as 'Helicobacter heilmannii' type 2. SDS-PAGE of whole-cell proteins of strains HS1(T), HS2 and HS3 differentiated them from other Helicobacter species of gastric origin. The results of the polyphasic taxonomic analysis confirmed that the novel isolates constitute a novel taxon corresponding to 'Helicobacter heilmannii' type 1 strains from humans and to 'Candidatus H. suis' from pigs. The name Helicobacter suis sp. nov. is proposed for the novel isolates with the type strain HS1(T) (=LMG 23995(T) =DSM 19735(T)).

A Gram-negative, microaerophilic slender rod, measuring approximately 10 mum long and approximately 1 microm wide, isolated from the gastric mucosa of a cat and designated strain M50(T), was subjected to a polyphasic taxonomic study. Despite its apparent lack of helical coils, the organism showed a corkscrew-like motion by means of multiple sheathed flagella located at both ends of the cell and by a periplasmic fibril coiled around the body. Strain M50(T) grew preferably on biphasic culture plates or on very moist agar. Coccoid forms predominated in cultures older than 4 days as well as in growth obtained on dry agar plates. The strain grew at 37 degrees C, but not at 25 or 42 degrees C and exhibited urease, oxidase and catalase activities. On the basis of 16S rRNA gene sequence analysis, the novel isolate was identified as a member of the genus Helicobacter and showed about 98 to 99 % sequence similarity to Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis, Helicobacter cynogastricus and 'Candidatus Helicobacter heilmannii', five highly related species previously detected in the feline or canine gastric mucosa. Protein profiling of strain M50(T) using SDS-PAGE revealed a pattern different from those of other Helicobacter species of mammalian gastric origin. Additionally, the urease and HSP60 gene sequences of strain M50(T) were different from those of H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus and 'Ca. H. heilmannii'. It is thus proposed that strain M50(T) (=LMG 23839(T)=CCUG 53816(T)) represents a novel species within this genus, for which the name Helicobacter baculiformis sp. nov. is proposed.

The quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE of ten Mycoplasma hyopneumoniae field isolates that were either sensitive (5) or resistant (5) to the fluoroquinolones flumequine and enrofloxacin were characterized. In all five resistant isolates, one point mutation (C A) in parC was found, resulting in an amino acid change from serine to tyrosine at position 80 (Escherichia coli numbering). For four of these isolates, this was the only mutation found. These isolates had a minimum inhibitory concentration (MIC) of enrofloxacin of 0.5 g/ml, whereas for sensitive isolates the MIC of enrofloxacin was 0.06 g/ml. One resistant isolate (Mh 20) had an extra mutation (C T) in gyrA resulting in an amino acid change from alanine to valine at position 83 (E. coli numbering), leading to a further increase in the MIC of enrofloxacin (1 g/ml). No mutations resulting in an amino acid change were detected in the QRDR of the gyrB and parE genes of the selected isolates. This is the first description of the mechanism of stepwise resistance against fluoroquinolones in M. hyopneumoniae. ispartof: Microbial Drug Resistance vol:13 issue:3 pages:166-170 ispartof: location:United States status: published

The accuracy of antimicrobial susceptibility determinations relies on the bacterial species identification and the test methodology that is being used. In the present study, both aspects were investigated for 141 Pasteurella multocida and 34 Mannheimia haemolytica sensu lato isolates recovered from the upper respiratory tract of healthy calves. This was performed on the one hand by comparing of classical phenotyping with tDNA-PCR genotyping and on the other hand by pairwise comparison of disk diffusion with agar dilution results for seven antimicrobial compounds (ampicillin, ceftiofur, oxytetracycline, gentamicin, florfenicol, enrofloxacin, and the combination trimethoprim-sulfonamides). Phenotyping and genotyping correlated well (90%). The pairwise comparisons of the susceptibility methods were investigated traditionally by means of error binding rates and sensitivity and specificity test characteristics. Obtained sensitivities (indication for absence of false susceptible results) were often lower than 85%, especially for older antimicrobial agents (oxytetracycline, gentamicin, trimethoprim-sulfonamides) and when M. haemolytica sensu lato was considered. Specificities (indication for absence of false-resistant results) exceeded 90% for almost all antimicrobial-bacterial combinations. The calculated test characteristics (sensitivities and specificities) were subsequently used in a second dataset of Pasteurellaceae from intensively (n = 99) and extensively housed calves (n = 196), to modify the apparent prevalence of antimicrobial resistance based upon disk diffusion results into an estimated true prevalence. It was concluded that the disk diffusion method is reliable in epidemiological studies like surveillance programs if resistance is sparse, whereas it needs to be interpreted with caution in situations where resistance is abundantly present.

Samples from the antrum and fundus of the stomachs of 457 pigs from 22 different herds were screened for the presence of 'Candidatus Helicobacter suis' by pcr, and samples from the antrum and/or fundus of 222 of the stomachs were tested for urease activity. The prevalence of the infection was very low before weaning, increased rapidly after weaning and reached 90 per cent in the adult boars and sows. The agreement between the results obtained with the pcr test and the urease test was very good for some age groups and sampling sites, but poor for other age groups and sampling sites.

The aim of the present study was to evaluate differences in resistance patterns of Escherichia coli in different parts of the digestive tract of veal calves. Therefore, after slaughter, the lower intestinal tract of 19 calves was sampled at five locations (duodenum, jejunum, cecum, colon, and rectum), and up to three E. coli isolates per sample underwent susceptibility testing for seven antimicrobial agents (gentamicin, amoxycillin + clavulanic acid, tetracycline, trimethoprim + sulfamethoxazole, ampicillin, nalidixic acid, and enrofloxacin), using the Kirby-Bauer disk diffusion method. Multiresistance (resistance to more than two compounds) was present in 93.5% of all isolates (n = 179). For gentamicin, nalidixic acid, and enrofloxacin, the percentage of resistant E. coli isolates was significantly lower in the duodenum and jejunum than in the cecum, colon, and rectum. For ampicillin, the percentage of resistance was significantly lower in the jejunum, compared to the other segments of the intestinal tra...

Recently, a new enterohepatic Helicobacter species, H. equorum, was isolated from faecal samples of two clinically healthy horses. At the onset of this study, nothing was known about the prevalence of this organism in horses, nor was there any information available on the possible zoonotic character of this agent. This study aimed to determine the prevalence of H. equorum in faecal samples from equine and human origin. Therefore, faecal samples of 120 healthy privately owned horses, 227 healthy riding-school horses and 239 hospitalised horses were screened for H. equorum-DNA by means of a PCR amplifying a 1074-bp fragment of the 23S rRNA gene with primers specific for H. equorum. The vast majority of the hospitalised horses were under treatment with an antimicrobial agent at the moment of sampling, while the other horses had not been treated with an antimicrobial agent in the 14 days preceding the sampling. Stool samples of 531 humans suffering from gastro-intestinal disease and 100 clinically healthy humans were likewise examined. H. equorum-DNA was demonstrated in faeces from 0.8% of the privately owned horses, 3.1% of the riding-school horses and 7.9% of the hospitalised horses. The prevalence of H. equorum was significantly higher in hospitalised than in healthy, privately owned horses (P = 0.02). H. equorum-DNA was not detected in human samples. These results indicate that the prevalence of H. equorum in horses may be influenced by the health status of the investigated horse population and/or by antimicrobial treatment. We may additionally assume that this micro-organism does not commonly infect humans.

The purpose of the present study was to identify Moraxella ( M. )—like organisms recovered from calves suffering from respiratory disease down to species level by means of tDNA-intergenic spacer length polymorphism analysis (tDNA-PCR), and to perform antimicrobial susceptibility testing of these isolates using an agar dilution technique. A total of 16 isolates originating from 12 unrelated occasions were identified as Moraxella ovis , and tDNA fingerprinting showed clear delineation from other Moraxella species. The minimal inhibitory concentrations (in μg/mL) for 90% of the investigated isolates were ≤0.03 for ampicillin; 0.25 for ceftiofur; 0.5 for oxytetracycline; 8 for gentamicin; 64 for spectinomycin; 0.5/9.5 for the combination trimethoprim-sulfonamides; 4 for erythromycin; 8 for tilmicosin; 1 for florfenicol and 0.125 for enrofloxacin.

Recently, strains of Streptococcus bovis were reclassified as Streptococcus gallolyticus . In the present study we describe for the first time an outbreak of S. gallolyticus in a broiler flock. Mortality during the first week was normal (S. bovis - S. equinus complex, the 16S rRNA-derived phylogeny of the strains investigated in this study demonstrated a paraphyletic group ( S. gallolyticus ) well separated from two monophyletic groups: (i) S. equinus - S. bovis plus S. infantarius and (ii) S. alactolyticus plus S. intestinalis . According to information in GenBank, none of the strains included from the two monophyletic groups have been isolated from birds. Further biochemical analyses, including tannase activity, identified for the first time avian isolates belonging to S. gallolyticus subsp. gallolyticus . However, these investigations also demonstrated a clear heterogeneity with pigeon isolates.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. To obtain better insight in the mechanisms responsible for differences in virulence between highly and low virulent M. hyopneumoniae isolates, 23 caesarean-derived, colostrum-deprived piglets were randomly assigned to three groups. Groups 1 and 2 consisted of nine animals each, which were intratracheally inoculated at 1 week of age with a highly or a low virulent isolate of M. hyopneumoniae, respectively. The remaining five animals were inoculated with sterile culture medium. Animals were euthanized at 5, 10, 15 and 28 days post-inoculation (DPI). Animals inoculated with the highly virulent isolate had more neutrophils in BAL fluid at 10, 15 and 28DPI compared to the other groups. At 10 and 15DPI, animals in the highly virulent group had significantly higher concentrations of TNF-alpha in BAL fluid. IL-1beta concentration in this group was higher at 5 and 28DPI compared to the other groups. From 10DPI onwards, significantly higher titres of M. hyopneumoniae were detected in the BAL fluid of animals inoculated with the highly virulent isolate compared to animals inoculated with the low virulent isolate. Additionally, the in vitro generation time of the highly virulent M. hyopneumoniae isolate was significantly shorter than that of the low virulent isolate. The present study indicates that the difference in pathogenicity between the highly and low virulent isolates is associated with a faster in vitro growth, a higher capacity to multiply in the lungs and the induction of a more severe inflammation process by the highly virulent isolate.