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Background Flatfish live in a diverse marine ecosystem that is changing due to natural variations and anthropogenic influences. These changes can evoke a stress response mainly resulting in production of the glucocorticoid cortisol, which mediates effects on various levels of biological organization. The finding that cortisol accumulates in fish scales, offering a retrospective view on cortisol production, provides opportunities to use this matrix for chronic stress assessment. The present study is the first to gather information on scale cortisol concentration in wild-caught common dab (Limanda limanda), based on a two-pronged approach using (1) field measurements and (2) a laboratory in vivo-study where wild-caught dab were fed by cortisol-spiked feed during 30 or 90 days to demonstrate the possible accumulation of cortisol in the scales and to evaluate its impact on fish health. Results Based on the field measurements, the average scale cortisol concentration in wild-caught fish was 0.0034 ± 0.0046 µg kg−1 scale (n = 67). This indicates that wild common dab is indeed able to incorporate cortisol in the scales. Based on the experimental data, the cortisol-fed fish showed an increased plasma cortisol concentration (80.16 ± 82.58 µg L−1) compared to the control group (4.54 ± 9.57 µg L−1) after 30 days of cortisol feeding. The increase in plasma cortisol concentration was positively correlated with an increased cortisol concentration in the scale after 30 days of cortisol-spiked feeding. This correlation was, however, no longer observed after 90 days of cortisol-spiked feeding. Interestingly, cortisol concentration of the scales on the pigmented side was significantly higher compared to the non-pigmented side. Some health parameters such as epidermal thickness, body condition and Ichthyobodo sp. parasitic infection showed a correlation with scale cortisol concentration after 30 days. Conclusions We have demonstrated that common dab is able to accumulate cortisol in its scales. This seems to occur proportionally to circulating concentrations of plasma cortisol in fish fed with cortisol supplemented feed after 30 days.

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.

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

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.

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.

Sinds het begin van de massaproductie van plastics vormt de contaminatie van het mariene milieu met deze persisterende synthetische stoffen een alsmaar groter probleem. De laatste decennia wordt aangenomen dat microplastics hierbij een grote rol spelen. Deze kleine plastic partikels (< 5mm) komen voor in het mariene milieu en kennen diverse bronnen, waaronder fragmentatie van macroplastics en de afvoer van primaire microplasticscrubs via afvalwater. Recent onderzoek heeft uitgewezen dat microplastics door talrijke mariene organismen opgenomen worden. Dit zou kunnen leiden tot gezondheidsproblemen, onder meer door mechanische schade en door cellulaire toxiciteit. Hiernaast wordt de microplasticproblematiek gecompliceerd door de contaminatie van deze partikels met toxische chemicaliën. Bovendien zouden microplastics en microplastic-gebonden contaminanten kunnen accumuleren in de voedselketen en uiteindelijk bij de mens terechtkomen. Ondanks het toenemend aantal publicaties omtrent microplastics zijn er nog veel onbeantwoorde vragen. In dit artikel wordt de actuele kennis besproken aangaande microplastics in het mariene milieu.

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.

Het belang van zoönosen neemt de laatste jaren toe, ondermeer ten gevolge van de internationalisering van handel. Zoönosen kunnen via opname van voedsel (voedselzoönose) of via huidcontact (contactzoönose) optreden. Specifiek voor de toename van viszoönosen is de uitbreiding van de aquacultuur. Voornamelijk vissers en vishandelaars maar ook vishobbyisten lopen het grootste risico om aan vis, als mogelijke zoönotische infectiebron, te worden blootgesteld. Vaak zijn zij zich daar onvoldoende van bewust. Voorzorgsmaatregelen tegen zoönotische infecties zijn dan ook cruciaal. De belangrijkste bacteriële contactzoönosen van vis worden veroorzaakt door Mycobacterium marinum, Vibrio vulnificus, Edwardsiella tarda en Streptococcus iniae. Minder bedreigend zijn onder andere Aeromonas hydrophila en Erysipelothrix rhusiopathiae. Infectie van de mens gebeurt via contact van huidwonden met de bacterie en voor sommige van de genoemde bacteriën ook via orale weg. De diagnose van deze infecties kan meestal worden vermoed op basis van de anamnese waaruit contact met vissen blijkt. De diagnose van de specifieke kiem kan worden bevestigd met behulp van bacterie-isolatie en –identificatie, bijvoorbeeld via “polymerase chain reaction”. De behandeling van de infectie geschiedt meestal met antibiotica. Soms is tevens een chirurgische behandeling vereist.

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, 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.

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.

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.

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.

Due to the mounting awareness of the risks associated with the use of antibiotics in aquaculture, treatment with probiotics has recently emerged as the preferred environmental-friendly prophylactic approach in marine larviculture. However, the presence of unknown and variable microbiota in fish larvae makes it impossible to disentangle the efficacy of treatment with probiotics. In this respect, the recent development of a germ-free culture model for European sea bass (Dicentrarchus labrax L.) larvae opened the door for more controlled studies on the use of probiotics. In the present study, 206 bacterial isolates, retrieved from sea bass larvae and adults, were screened in vitro for haemolytic activity, bile tolerance and antagonistic activity against six sea bass pathogens. Subsequently, the harmlessness and the protective effect of the putative probiotic candidates against the sea bass pathogen Vibrio harveyi were evaluated in vivo adopting the previously developed germ-free sea bass larval model. An equivalence trial clearly showed that no harmful effect on larval survival was elicited by all three selected probiotic candidates: Bacillus sp. LT3, Vibrio lentus and Vibrio proteolyticus. Survival of Vibrio harveyi challenged larvae treated with V. lentus was superior in comparison with the untreated challenged group, whereas this was not the case for the larvae supplemented with Bacillus sp. LT3 and V. proteolyticus. In this respect, our results unmistakably revealed the protective effect of V. lentus against vibriosis caused by V. harveyi in gnotobiotic sea bass larvae, rendering this study the first in its kind.

Primaten worden vaak gebruikt als diermodel in het biomedisch onderzoek wegens hun sterke overeenkomsten met de mens. Voornamelijk penseelapen (Callithrix jacchus) en resusapen (Macaca mulatta) zijn frequent gebruikte apensoorten in het wetenschappelijk onderzoek. In dit artikel wordt een systematisch overzicht gegeven van de belangrijkste botaandoeningen bij primaten. Botaandoeningen kunnen spontaan voorkomen maar ook experimenteel worden geïnduceerd in het kader van onderzoek naar humane ziekten. Een gedegen kennis van deze pathologieën is niet enkel belangrijk om preventieve of therapeutische interventies te onderbouwen bij zowel de aap als de mens, maar draagt ook bij tot een optimale keuze van het diermodel. De, voornamelijk historisch gezien, belangrijkste groep van aandoeningen die hier worden besproken zijn de metabole aandoeningen, waarbij de nadruk ligt op rachitis/osteomalacie en osteoporose. Congenitale aandoeningen, ontwikkelingsstoornissen, intoxicaties en deficiënties met effect op het skelet worden ook besproken. Tenslotte zijn er nog de bottumoren, die weliswaar minder frequent gerapporteerd worden, maar die toch een niet te verwaarlozen groep vormen.

'Candidatus Helicobacter suis' is a spiral-shaped bacterium that colonizes the stomach of more than 60% of slaughter pigs. The role of 'Candidatus Helicobacter suis' in gastric disease of pigs is still unclear. Experimental studies in pigs are lacking because this bacterium is unculturable until now. An inoculation protocol using 'Candidatus Helicobacter suis' infected mouse stomach homogenate was used to reproduce the infection in pigs. Control animals were inoculated using negative mouse stomach homogenate. Pigs were inoculated three times with one-week intervals and euthanized 6 weeks post inoculation. Tissue samples were taken from different mucosal stomach regions to detect 'Candidatus Helicobacter suis' by PCR and urease test. Mucosal inflammation was evaluated on formalin-fixed tissue samples. Lesions in the pars oesophagea were scored macroscopically. Infection was successful in all challenged animals, with the antrum and the fundus being predominantly positive. Infection was associated with infiltration of lymphocytes and plasma cells in the antral mucosa, evolving to follicular gastritis. No apparent inflammation of the fundic stomach region was detected in the infected animals. A clear link between 'Candidatus Helicobacter suis' and pars oesophageal lesions could not be found.

The aim of the present study was to determine which Pasteurella and Mannheimia species are present in the upper respiratory tract of healthy calves with no history of antimicrobial treatment prior to sampling. The presence of subpopulations of tetracycline-resistant Pasteurellaceae was also investigated. Nasal swabs from 61 loose group-housed, clinically healthy calves, 1 to 4 months old, from 16 dairy herds were inoculated aerobically on a selective medium (Columbia agar with 5% ovine blood and 16 mg/L bacitracin) with or without 4 mg/L oxytetracycline (OTC). A total of 43 strains belonging to the family Pasteurellaceae were isolated from 38 calves (62.3%) out of 13 herds (81.3%). The predominant organisms were Pasteurella multocida subsp. multocida (57.4%), Mannheimia varigena (4.9%) and M. haemolytica (3.2%). Growth of Pasteurellaceae on the OTC-containing medium was seen only with samples from two herds (6 animals; 9.8%), and on only one farm this proved to be an OTC-resistant subpopulation. Minimum inhibitory concentration (MIC) determinations by means of agar dilution confirmed a low prevalence of OTC-resistant Pasteurellaceae, with overall MIC(50) and MIC(90) values of 0.25 and 32 mg/L, respectively. These data do not support the hypothesis that the relative high frequency of tetracycline-resistant P. multocida isolates from fatal cases of bovine respiratory disease is related to the presence of minor tetracycline-resistance subpopulations within this species.

The aim of this study was to investigate the prevalence of acquired antimicrobial resistance in the resident intestinal microbiota of cats and to identify significant differences between various cat populations. Escherichia coli, Enterococcus faecalis, E. faecium and Streptococcus canis were isolated as faecal indicator bacteria from rectal swabs of 47 individually owned cats, 47 cattery cats and 18 hospitalised cats, and submitted through antimicrobial sensitivity tests. The results revealed that bacteria isolated from hospitalised and/or cattery cats were more frequently resistant than those from individually owned cats. E. coli isolates from hospitalised cats were particularly resistant to ampicillin, tetracycline and sulfonamide. Both enterococci and streptococci showed high resistance to tetracycline and in somewhat lesser extent to erythromycin and tylosin. Most E. faecium isolates were resistant to lincomycin and penicillin. One E. faecalis as well as one E. faecium isolate from hospitalised cats showed ‘high-level resistance’ (MIC > 500 μg/ml) against gentamicin, a commonly used antimicrobial agent in case of human enterococcal infections. The results of this research demonstrate that the extent of acquired antimicrobial resistance in the intestinal microbiota of cats depends on the social environment of the investigated population. It is obvious that the flora of healthy cats may act as a reservoir of resistance genes.

"Helicobacter (H.) heilmannii" type 1 colonizes the human stomach. It has been shown to be identical to "Candidatus H. suis", a Helicobacter species colonizing the stomach of more than 60% of slaughter pigs. This bacterium is, until now, not isolated in vitro. The effect of vaccination on "Candidatus H. suis" infection was studied in a mouse model. Mice were vaccinated intranasally or subcutaneously with whole bacterial cell lysate of Helicobacter pylori or Helicobacter felis and subsequently challenge infected with "Candidatus H. suis". Intranasal and subcutaneous immunisation caused a decrease in faecal excretion of "Candidatus H. suis" DNA. Urease tests on stomach tissue samples at 16 weeks after challenge infection were negative in all H. felis intranasally immunized animals and in the majority of the animals of the other immunisation groups. Since PCR on stomach tissue samples at 16 weeks after challenge infection could still detect "Candidatus H. suis DNA" in all immunisation-challenge groups, complete clearance of challenge bacteria was not achieved.

This paper discusses what can be expected with regard to efficacy of antibacterial vaccines used in swine, based on the present knowledge of pathogen-host interactions. First, vaccination against bacteria that mainly cause disease by production of exotoxins is considered. Vaccines containing the inactivated toxin or a non-toxic but antigenic recombinant protein derived from the exotoxin can be expected to provide protection against disease. The degree of protection induced by such vaccines varies, however, depending amongst other things on the pathogenesis of the disease. Vaccination against clostridial infections, Actinobacillus pleuropneumoniae infections, progressive atrophic rhinitis and enterotoxigenic Escherichia coli, is considered. The second part of the article deals with vaccination against extracellular bacteria. Protection against these bacteria is generally mediated by antibodies against their surface antigens and certain secreted antigens, but cellular immunity may also play a role. Efficacy of vaccines against swine erysipelas, Streptococcus suis infections, Mycoplasma hyopneumoniae infections and swine dysentery is discussed. Finally, vaccination against facultatively intracellular bacteria is considered. For protection against these bacteria cell-mediated immunity plays an important role, but antibodies may also be involved. It is generally accepted that live-attenuated vaccines are more suitable for induction of cell-mediated immunity than inactivated vaccines, although this also depends on the adjuvant used in the vaccine. As an example, vaccination against Salmonella enterica serotype Typhimurium is discussed.

Mycobacterium marinum, M. fortuitum and M. chelonae are the etiological agents of fish mycobacteriosis. Fish mycobacteriosis is a disseminated infection reported in more than 150 fish species and is usually accompanied by emaciation and death over a period of months to years. Granulomas are formed both externally and scattered throughout the internal organs. Treatment is in most cases unsatisfactory and the overall recommendation is to destroy the diseased stock, particularly since these pathogens are capable of affecting man as well as fish. Especially fish handlers and aquarium hobbyists are infected and the disease is mostly confined to the superficial, cooler body tissues, most often the extremities. Dissemination is apparently rare but has been reported.

tRNA-intergenic spacer PCR (tDNA-PCR) was evaluated for its effectiveness in differentiating Pasteurella and Mannheimia (sub)species predominantly of ruminant origin. For this purpose, 38 reference strains and 13 field isolates belonging to both genera were investigated. tDNA-PCR enabled discrimination of all Pasteurella species tested ( Pasteurella ( P. ) aerogenes , P. avium , P. canis , P. lymphangitidis , P. multocida , P. trehalosi ). For the differentiation of the subspecies of P. multocida , an additional dulcitol reaction was required. Two of the five so far-defined Mannheimia species, M. granulomatis and M. varigena , had a distinct fingerprinting profile. The remaining three phylogenetically highly related species ( M. haemolytica , M. glucosida , and M. ruminalis ) clustered together. Nevertheless, M. ruminalis is non-haemolytic, and M. haemolytica and M. glucosida can be differentiated on the basis of two additional phenotypic characteristics (β-glucosidase and aesculin hydrolysis). In conclusion, tDNA-PCR is a useful tool in differentiating organisms belonging to the genera Pasteurella and Mannheimia .

This study was carried out in order to try to establish an efficacious and reliable experimental infection model for Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome, using contact, oral and anal challenges. Ten F psychrophilum strains of different origin were included. The influence of water temperature, scarification, water quality, stress and growth conditions of the pathogen on the experimental infection was assessed. For each challenge protocol, all strains failed to reproduce disease signs or mortality in rainbow trout (Oncorhynchus mykiss L.) fry. Histological and bacteriological examination of the skin, gills and internal organs of the fish 3 weeks following inoculation were found to be negative. Different hypotheses to explain the inability of the challenge models to reproduce the disease experimentally are discussed.

Callithrix jacchus (common marmoset) is regularly used in biomedical research, including for studies involving the skeleton. To support these studies, skeletons of healthy animals that had been euthanized for reasons not interfering with skeletal anatomy were prepared. The marmoset dental formula 2I-1C-3P-2M of each oral quadrant is atypical for New World monkeys which commonly possess a third molar. Seven cervical, 12–13 thoracic, 7–6 lumbar, 2–3 sacral and 26–29 caudal vertebrae are present, the thoracolumbar region always comprising 19 vertebrae. A sigmoid clavicle connects the scapula with the manubrium of the sternum. Depending on the number of thoracic vertebrae, 4–5 sternebrae are located between the manubrium and xiphoid process. Wide interosseous spaces separate the radius from the ulna, and the tibia from the fibula. A small sesamoid bone is inserted in the m. abductor digiti primi longus at the medial border of the carpus, a pair of ovoid sesamoid bones is located at the palmar/plantar sides of the trochleae of each metapodial bone, and round fabellae articulate with the proximal surfaces of the femoral condyles. Male marmosets possess a small penile bone. Both the front and hind feet have five digits. The hallux possesses a flat nail, whereas all other digits present curved claws. Interestingly, a central bone is present in both the carpus and tarsus. This study provides a description and detailed illustrations of the skeleton of the common marmoset as an anatomical guide for further biomedical research.

IntroductionTo reduce the unwanted by-catch in shrimp trawling, alternative stimulation techniques such as electricity, which selectively invokes a reaction in shrimp, might be used. The purpose of this study was to evaluate the effects of such electric pulses in sea fish.Materials and MethodsPlaice (n = 21), sole (n = 22), dragonet (n = 21), pogge (n = 21), codfish (n = 20), armed bullhead (n = 14) and fivebeard rockling (n = 8) were exposed to electric pulses (60 V, 0.5 milliseconds, 5 Hz) for 10 sec. Behaviour and mortality were recorded. All fish were killed and subjected to necropsy examination 24 h after exposure. Control animals consisted of equal numbers of the same species not exposed to electric pulses.ResultsAll fish survived the experiment. In almost all exposed fish, minor and brief fright reactions were observed. In general, gross and microscopical abnormalities were rarely present in exposed and control fish. In one control and two electrical-exposed plaice, small multifocal cutaneous haemorrhages were observed. In one electrical-exposed sole, a small focal interstitial haemorrhage in the muscle was present.ConclusionAlternative techniques such as electric pulses for catching shrimp could be promising, since this appears to have low impact on fish, at least under experimental conditions.

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.

The natural occurrence of established Helicobacter species was investigated in the stomachs of 65 laboratory rabbits, by means of polymerase chain reaction (PCR) (65/65) and histological analysis (51/65). The degree of inflammation in the different regions of the rabbits' stomach was evaluated on haematoxylin and eosin (HE)-stained histological slides. Four rabbits were found positive for Helicobacter species by PCR. Based on 16S ribosomal ribonucleic acid (rRNA) sequences, H. canadensis/H. pullorum organisms were identified in three animals. Bacteria were seen on merely one histological slide from one of these animals. H. felis was identified in one rabbit. Histological examination revealed no inflammation in the stomachs of 40 rabbits, while moderate gastric inflammation was seen in 11 animals, mainly in the antrum. In conclusion, the stomach of the laboratory rabbits included in the study was occasionally found positive for Helicobacter species, which were mostly identified as enterohepatic helicobacters, probably reflecting a mere passage of these bacteria through the stomach.

Experimental groups of 15 susceptible 3-week-old turkeys were inoculated oculonasally with avian metapneumovirus (APV) subtype A and susceptible Escherichia coli O2:K1 and Ornithobacterium rhinotracheale (ORT) bacteria, with a 3 days interval between viral and bacterial inoculation and approximately 8h between the two bacterial inoculations. The aims of the present study were to assess the efficacy of drinking-water administration of enrofloxacin for 3 and 5 days, amoxicillin for 5 days and florfenicol for 5 days for the treatment of the resulting respiratory disease, based on clinical and bacteriological examinations. Antimicrobial treatment started 1 day after dual bacterial inoculation. After infection, the birds were examined and scored for clinical signs daily, weighed at different times, and their tracheae swabbed daily. Five birds were euthanised and examined for macroscopic lesions at necropsy at 5 days post-bacterial inoculation (dpbi) and the remainder at 15dpbi. Samples of the turbinates, trachea, lungs, sinuses, air sacs, heart, pericardium and liver were collected for bacteriological examination. Recovery from respiratory disease caused by an APV/E. coli/ORT triple infection in 3-week-old turkey poults was overall most successful after enrofloxacin treatment, irrespective of treatment duration, followed by florfenicol treatment. Compared with the untreated group, clinical signs as well as ORT and E. coli multiplication in the respiratory tract were significantly reduced by both enrofloxacin treatments and the florfenicol treatment, with the enrofloxacin treatments showing significantly better reductions than the florfenicol treatment. Five-day treatment with amoxicillin, compared with the untreated group, did not cause a significant reduction in any of the aforementioned parameters.

In contrast to Helicobacter(H.) pylori, little is known about the pathogenic mechanisms of gastric non-H. pylori Helicobacter species. Mongolian gerbils were inoculated intragastrically with H. felis or H. bizzozeronii and killed at different timepoints post-inoculation (p.i.), stomach tissue being taken for light and transmission electron microscopy (TEM) and polymerase chain reaction (PCR) analysis. Parietal cells (PCs), apoptosis, cell proliferation and nuclear factor-kappaB (NF-kappaB) activation were "visualized" immunohistochemically. Inflammation consisted of neutrophilic granulocytes, mainly in the antrum, and lymphocytic infiltrates around the limiting ridge and throughout the stomach mucosa and submucosa. From day 11 p.i. onwards, H. felis-inoculated animals showed moderate to severe loss of PCs extending from the limiting ridge into the fundus. Apoptotic cells, spiral bacteria, cell proliferation, and NF-kappaB activation were detected at the transition zone between affected and normal PCs. TEM revealed interaction of H. felis flagella with PCs and chief cells. Moreover, H. felis was seen in proximity to, and inside, necrotic cells. At 10 weeks p.i., some H. felis-infected gerbils showed complete loss of fundic glands, and mucous metaplasia of the epithelium. H. bizzozeronii, which made no flagellar contact with epithelial cells, was associated with only mild PC loss. The mechanism by which H. felis induces PC necrosis and apoptosis remains unclear. The observed flagellar contact and NF-kappaB activation may play an important role in H. felis-associated inflammation.

Helicobacter pullorum has been associated with diarrhoea, gastroenteritis and liver disease in humans and with hepatitis and enteritis in poultry. The purpose of the present study was to examine whether cytolethal distending toxin was present among 10 poultry and three human H. pullorum isolates and whether a different level of cytolethal distending toxin activity was noted. A PCR assay was performed to detect the cdtB gene. In addition, epithelial Hep-2 cells inoculated with sonicate from all strains were observed microscopically and DNA analysis of these cells was done by flow cytometry. All H. pullorum isolates harboured the cdtB gene, but functional cytolethal distending toxin activity was only demonstrated in the human H. pullorum strain CCUG 33839. A significant number of cells treated with sonicate from this strain were enlarged. The nuclei were distended proportionally. Giant cells and multinucleated cells were observed as well. In addition, stress fibers accumulated. DNA analysis by flow cytometry revealed 31.0% of these cells at the S/G2 stage of the cell cycle. The tested poultry and human H. pullorum isolates all possess the cdtB gene, but under the circumstances adopted in this study only the human strain CCUG 33839 seems to show biological activity typically for CDT in vitro.

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The objectives of the present study were to evaluate the accuracy of broad range 16S rRNA gene PCR compared to bacterial culture for the detection of synovial infection in horses. The study included 57 synovial fluid samples from horses with presumed synovial infection and a control group consisting of 31 synovial fluid samples originating from clinically normal horses and horses with aseptic synovial inflammation. All samples were analysed by 16S PCR with reverse line blot (RLB) hybridisation. Synovial fluid samples were cultured using conventional agar plate methods (APM) and/or blood culture medium (BCM). The results of the study showed a superior detection rate (89.5%) for 16S PCR with RLB. Bacterial culture had lower sensitivity, but highly acceptable detection rates (77.6%) were observed using BCM. APM had very low sensitivity (37.8%) and infection was never detected by plate isolation without positive incubation in BCM. The highest sensitivity (91.8%) for the detection of synovial infection was achieved when the results of incubation in BCM and 16S PCR were combined. For all the tests, the specificity was higher than 90%.

An alternative fish model with the principal aim of studying the interaction between fish pathogens and the intestinal tissue was developed. The preparation consisted of an excised gut tractus from rainbow trout ( Oncorhynchus mykiss), perfused through cannulation of the aorta intestinalis ventralis with filtered and heparinized Cortland + dextran 1% as the perfusion fluid. This perfusion fluid was delivered by means of a drip. The perfused gut tractus was suspended in a circular bath filled with Ringer solution, which was aerated and kept at a constant temperature of 12°C. Unperfused gut placed in Ringer solution at the same temperature served as the negative control. Perfusion was effective in maintaining the gut in a healthy condition for at least 60 min with only slight oedema and sloughing of the epithelium. Conversely, the unperfused gut revealed excessive tissue degeneration and severe necrosis.

Gastric Helicobacter spp. have been described in a wide range of animal species, including dogs, cats, primates, swine, cattle and rodents. However, in lagomorphs--more specifically rabbits--gastric Helicobacter infections have never been reported. Biopsy specimens were collected from different stomach regions of 23 rabbits, including 10 pet rabbits, 10 industrial animals and 3 research animals. These were subjected to a PCR assay for the detection of Helicobacter DNA. Identification up to the species level was based on 16S rRNA sequence analysis and a recently developed multiplex PCR. Seven rabbits (four pet, one research animal and two industrial animals) tested positive in the Helicobacter genus-specific PCR in the stomach, with the corpus being predominantly positive. H. felis and H. salomonis, hitherto presumed to be naturally hosted by cats and dogs, were detected in three animals and one animal, respectively. One of these animals had been completely devoid of any form of contact with cats or dogs. A H. pullorum/H. rappini-like organism (96% 16S rDNA sequence similarity) was found in an industrially held rabbit. The helicobacters of the two remaining rabbits could not be identified up to the species level. To conclude, this is the first report on the occurrence of Helicobacter spp. in the stomach of rabbits. In view of the fact that H. felis and H. salomonis are put forward as having zoonotic potential, further research is necessary to investigate the implications of these findings not only for the rabbit but also for human health.

The inflammatory response in the mouse stomach was evaluated as a means of distinguishing different non-pylori Helicobacter (H.) strains in terms of virulence. Mice of four strains (BALB/c, SJL, C57BL/6 and CFW) were infected intragastrically with four bacterial strains (H. felis ATCC 49179 and CCUG 37471, H. bizzozeronii and H. salomonis). The animals were killed for gastric examination at 3, 9 or 16 weeks post-inoculation. H. salomonis could not be detected by the polymerase chain reaction, but the other three organisms were detected in all stomach samples at all timepoints. SJL mice consistently showed particularly severe gastric inflammation regardless of bacterial strain. Lymphocytes and occasionally neutrophils were seen in submucosa and lamina propria mucosae. BALB/c mice showed the least severe inflammatory changes. H. bizzozeronii differed from the two H. felis strains in producing less striking pathological changes in mice. Of the two H. felis strains, ATCC 49179 produced the more severe inflammatory changes in SJL mice.

This study describes the development of a trout gill perfusion model, consisting of an excised branchial arch from rainbow trout ( Oncorhynchus mykiss L.), perfused via the afferent branchial artery and suspended in a circular organ chamber filled with Ringer solution. Different perfusion fluids were tested: Ringer, Cortland, Ringer + procaine, Ringer + adrenalin, Cortland + procaine, Cortland + adrenalin and Cortland + dextran 1% . The latter perfusion fluid proved to be satisfactory, maintaining the gill tissue in a healthy condition outside the body of the fish for at least 180 min. Using this model, the interaction of damaging agents with the trout gill tissue may be studied under carefully controlled conditions. The trout gill perfusion model leads effectively to a reduction in the number of experimental animals to be used and also involves an elimination of pain and/or suffering, which is as good as complete.

Eighty-seven Streptococcus suis isolates recovered in 1999-2000 from diseased pigs, all from different farms, were screened for resistance against macrolide and lincosamide antibiotics by the disk diffusion and agar dilution test and a PCR assay, amplifying the ermB gene and the mefA/E gene. Seventy-one percent of the isolates showed constitutive resistance to macrolide and lincosamide antibiotics (MLS(B)-phenotype). All these isolates were positive for the ermB gene in the PCR, but negative for the mefA/E gene. For all strains minimum inhibitory concentrations (MIC) against five other antimicrobial agents were determined. All strains were susceptible to penicillin. Ninety-nine percent of the isolates were susceptible to enrofloxacin and tiamulin. Eighty-five percent of the strains were resistant to doxycycline. A 540bp fragment of the ermB genes of eight S. suis strains was sequenced and compared with ermB genes of five S. pneumoniae and five S. pyogenes strains of human origin. A 100% homology was found between these fragments in seven S. suis, one S. pneumoniae and three of the S. pyogenes isolates. This study demonstrates that resistance against macrolides, lincosamides and streptogramin B is widespread in S. suis and mediated by ribosome methylation, encoded by the ermB gene.

The ability of a high virulence strain (AJS 1) and a low virulence strain (AJS 4) of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of black mollies (Poecilia sphenops) was investigated. For that purpose, two groups of 25 black mollies each were immersed in a bath containing 10(6) CFU/ml of F. columnare AJS 1 or AJS 4. At regular intervals from 1 to 12 h after the contact infection, fish were sacrificed and gills, skin, spleen and heart were sampled for bacteriology. Samples of the gills were taken for immunohistochemical and electron microscopic examination. Bacteriological examination proved that the number of gill-associated F. columnare was higher for AJS 1 than for AJS 4. Strain AJS 1 was isolated from the heart and spleen of 6 and 1 of the 16 examined animals, respectively. Strain AJS 4 was not isolated from the internal organs of any fish. When examined immunohistochemically, strain AJS 1 was found closely associated with gill epithelium whereas this was not the case for strain AJS 4. The adherence of bacteria to the gill tissue challenged with the virulent strain AJS 1 was also clearly demonstrated using scanning and transmission electron microscopy. These results indicate that adhesion of F. columnare to the gill tissue constitutes an important step in pathogenesis.