Effects of white mustard (Sinapis alba) oil on growth performance, immune response, blood parameters, digestive and antioxidant enzyme activities in rainbow trout (Oncorhynchusmykiss).

A study was conducted to evaluate the effects of dietary supplementation of white mustard (Sinapis alba) oil (WMO) on growth performance, immune responses, digestive and antioxidant enzyme activities in juvenile rainbow trout (Oncorhynchus mykiss). For this purpose, fish (initial weight: 25.77 ± 0.13 g) were divided into four experimental groups in triplicate and fed ad libitum twice a day with diets containing WMO at 0 (control), 0.5, 1, and 1.5% of diet for 9 weeks. Three fish from each tank (n:9 per treatment) were sampled on 21st, 42nd, and 63rd days for further analyses. At the end of the feeding period, fish were challenged with Aeromonas hydrophila and Yersinia ruckeri in two separate experimental setups. Results showed that final weight, weight gain, and specific growth rate were significantly increased in all experimental groups compared to the control. Feed conversion ratio was similar among treatments. Respiratory burst and potential killing activity decreased in all experimental groups compared to the control (P < 0.05). Lysozyme and myeloperoxidase activities were elevated in all experimental groups at the end of the experiment compared to the control (P < 0.05). Cytokine gene expressions in the head kidney and intestine were elevated in all experimental groups compared to that of the control in general (P < 0.05). Hematological responses of the experimental fish groups were similar to that of the control (P > 0.05). Pepsin and trypsin levels decreased in all experimental groups (P < 0.05). In terms of antioxidant enzyme activities, significant improvement in liver superoxide dismutase, catalase, and glutathione s-transferase activities in all treatment groups were determined (P < 0.05). In addition, a significant decline in liver lipid peroxidation levels was recorded in all treated groups at all sampling times compared to the control (P < 0.05). At the end of this feeding trial, no significant differences (P > 0.05) were observed in survival against A. hydrophila among experimental groups compared to the control (P > 0.05). However, increased survival against Y. ruckeri was determined in experimental fish groups (P < 0.05). This study suggests that white mustard oil had a favorable effect on the overall health and growth of rainbow trout.

Long-term efficacy of nasal vaccination against enteric red mouth (ERM) disease and infectious hematopoietic necrosis (IHN) in juvenile rainbow trout (Oncorhynchus mykiss).

Previous research demonstrated that bacterial and viral vaccines delivered via the nasal route in rainbow trout (Oncorhynchus mykiss) at 7 and 28 days post-vaccination are highly protective (>95% protection). Long-term protection following nasal vaccination in teleosts has not been evaluated. The goal of this study was to assess efficacy and immune responses at 6 months (mo) post-vaccination (mpv), and long-lasting immune responses at 12 mpv of two different vaccines: an inactivated enteric red mouth disease (ERM) Yersinia ruckeri bacterin and a live attenuated infectious hematopoietic necrosis virus (IHNV) vaccine. Juvenile rainbow trout were vaccinated for Y. ruckeri via intraperitoneal (I.P.) and intranasal (I.N.) routes, and for IHNV by intramuscular (I.M.) and I.N. routes, then challenged at 6 mpv. Immune responses were determined at 6 and 12 mpv. ERM vaccine I.P. delivery elicited significantly higher serum IgM-specific titers that remained elevated compared to mock-vaccinated fish at 6 mpv. By 12 mpv, antibody titers to Y. ruckeri were not significantly different across all treatments. Following Y. ruckeri challenge at 6 mpv, a significant difference in cumulative percent mortality (CPM) was found for I.P.-vaccinated fish but not I.N.-vaccinated fish. I.M. and I.N. vaccination with live attenuated IHNV did not result in significant specific serum IgM titers at 6 or 12 mpv. Yet, I.N.-vaccinated fish showed the lowest CPM 6 mpv indicating long-term protection that does not correlate with systemic IgM responses. Repertoire analyses confirmed unique expansions of VH-JH rearrangements in the spleen of rainbow trout 12 mpv that varied with the type of vaccine and route of vaccination. Combined, these data demonstrate that I.N. vaccination with a live attenuated viral vaccine confers long lasting protection, but I.N. ERM vaccination does not and booster before 6 mpv is recommended.

Supplementation of zebrafish (Danio rerio) diet using a short antimicrobial peptide: Evaluation of growth performance, immunomodulatory function, antioxidant activity, and disease resistance.

Short-chain bioactive peptides are new and promising antimicrobial, immune moderating, and antioxidant agents. Therefore, the present study was conducted to evaluate in vitro antibacterial activity of CM11, a short antimicrobial peptide (AMP), against Streptococcus iniae and Yersinia ruckeri as fish pathogenic bacteria using standard disk diffusion and microdilution assays. In addition, in vivo effects of CM11 on fish growth, immunity, antioxidant activity, and disease resistance were evaluated using zebrafish (Danio rerio) as an animal model. For in vivo study, based on in vitro susceptibility results, four diets were designed to include zero (as control), 10, 20, and 50 µg of CM11 per g diet referred to as control, P1, P2, and P3 treatments, respectively. After eight weeks of dietary trial, fish were challenged with Streptococcus iniae, and the survival rate was calculated for a period of two weeks. Results showed that CM11 effectively inhibited the growth of S. iniae and Y. ruckeri on agar plates at a concentration of eight µg/ml. Minimum inhibitory and minimum bactericidal concentrations of CM11 were measured at 8 and 32 µg/ml for S. iniae and 16 and 64 µg/ml Y. ruckeri, respectively. In vivo results showed no noticeable effects on fish growth parameters, however, feed conversion ratio (FCR) was found lower in P3 and P2 compared to control (P < 0.05). Immunological and antioxidant responses were found strongly affected by CM11 in all treatment groups in which the highest values were found in the P3 treated group. Key immune and antioxidant genes were up-regulated particularly in fish receiving the highest level of CM11 (P3). Fish receiving the CM11 peptide showed better survival when challenged with S. iniae. These findings suggest the potential of CM11 for use in aquaculture as an antibacterial and immunostimulant agent.

Sepsis due to Yersinia enterocolitica in an aborted equine fetus: case report / Sepse por Yersinia enterocolitica em um feto equino abortado: relato de caso

Yersinia enterocolitica is a bacterium with zoonotic potential and there are no previous records of this bacteria being isolated from aborted foals. This report aims to describe a case of sepsis due to Y. enterocolitica in a seven month old aborted equine. The fequinoetus was submitted to necropsy and samples of all the organs were collected for the histological exam. Samples of liver, lung, placenta, and stomach contents were collected for bacterial culture. Macroscopically, the liver was enlarged with yellowish heterogeneous color, heart with pale myocardial areas; lungs not collapsed, heavy and shiny, thickened umbilical cord covered with fibrin and pus. Histopathologically, there was moderate multifocal necrosuppurative myocarditis and thrombosis, moderate diffuse suppurative bronchopneumonia, mild multifocal fibrinonecrotic hepatitis, and moderate diffuse necrosuppurative omphalitis with intralesional bacterial myriads and thrombosis. Mild multifocal suppurative placentitis, nephritis, myositis, cystitis, and dermatitis were also observed, in addition to mild diffuse lymphoid rarefaction. The microbiological evaluation identified Y. enterocolitica in the liver, lung, and stomach fluid. This is the first report of sepsis due to Y. enterocolitica causing an abortion in a horse. This bacterium has zoonotic importance; therefore, it should be investigated in abortion in this species, serving as a differential diagnosis in reproductive disorders.(AU)

Yersinia enterocolitica é uma bactéria com potencial zoonótico, e não há informações desse agente como causa de abortamento em equinos. O objetivo deste relato é descrever um caso de sepse por Y. enterocolitica em um feto equino abortado aos sete meses. O feto foi submetido à necropsia, e amostras de todos os órgãos foram processadas para histopatologia. Para microbiologia, foram coletadas amostras de fígado, pulmão, placenta e conteúdo estomacal. Macroscopicamente, observou-se fígado aumentado com coloração amarelada heterogênea; coração com áreas pálidas no miocárdio; pulmões não colabados, pesados e brilhantes; e cordão umbilical espessado e recoberto por fibrina e pus. Na análise histopatológica, havia miocardite necrossupurativa multifocal moderada e trombose, broncopneumonia supurativa difusa moderada, hepatite fibrinonecrótica multifocal discreta e onfalite necrossupurativa difusa moderada com miríades bacterianas intralesionais e trombose. Observou-se também placentite, nefrite, miosite, cistite e dermatite supurativa multifocal discreta, além de rarefação linfoide difusa discreta. A avaliação microbiológica identificou Y. enterocolitica no fígado, no pulmão e no líquido estomacal. Este é o primeiro relato de sepse por Y. enterocolitica causando abortamento na espécie equina. Essa bactéria tem importância zoonótica, portanto deve ser investigada em casos de abortamento nessa espécie, servindo como diagnóstico diferencial em tal distúrbio reprodutivo.(AU)

Toll/IL-1 receptor-containing proteins STIR-1, STIR-2 and STIR-3 synergistically assist Yersinia ruckeri SC09 immune escape.

Immune escape is a common feature of bacteria, viruses, parasites and even cancer cells. Our earlier work on an integrative and conjugative element (ICEr2) of Yersinia ruckeri SC09 demonstrated contributory roles of stir-1, stir-2 and stir-3 in bacterial toxicity and ability to code for immune evasion. Here, we further examined the ability of stir-4 in ICE (r2) and its encoded STIR-4 protein to mediate immune evasion using comparative genomic analysis. Additionally, the mechanisms underlying the synergistic activities of STIR-1, STIR-2, STIR-3 and STIR-4 in immune evasion were examined. Our results showed that STIR-4 did not contribute to bacterial toxicity, either in vivo nor in vitro, or show the ability to assist in bacterial immune escape. STIR-1, STIR-2, and STIR-3 formed heterotrimers in bacteria while facilitating immune evasion, which we speculate may be essential to maintain their stability. This discovery also partially explains the previous finding that a single gene can mediate immune evasion. Our data provide further knowledge on the distribution of ICE (r2)-like elements in bacteria, validating the prevalence of large-scale gene transfer in pathogens and its potential for enhancing virulence levels. Further studies are necessary to establish the biological significance of the ICE (r2) component.

Growth performance, haematological changes, immune response, antioxidant activity and disease resistance in rainbow trout (Oncorhynchus mykiss) fed diet supplemented with ellagic acid.

The present study was performed to investigate the effects of various levels of dietary ellagic acid (EA) on growth performance, haematological values, immune response, protection against Yersinia ruckeri infection, and oxidant/antioxidant status in rainbow trout, Oncorhynchus mykiss. Fish were fed with the control diet and three different experimental diets containing three graded levels of EA (50, 100 and 200 mg kg-1 diet) for 8 weeks. At the end of the experiment, the growth performance [weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR)], haematological values [the red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (Ht) level and erythrocyte indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)], immune response [white blood cell (WBC) count, oxidative radical production (nitroblue tetrazolium (NBT) assay), phagocytic activity (PA) and phagocytic index (PI), total protein (TP) and immunoglobulin M (IgM) levels, serum bactericidal activity (BA), lysozyme (LYZ) and myeloperoxidase (MPO) activities] and oxidant/antioxidant status [tissue malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities] were analysed. In addition, fish were challenged by Y. ruckeri and survival rate was recorded for 14 days. In the groups fed with EA the growth parameters such as WG, SGR, and FCR did not vary significantly. The RBC count, Hb concentration, and Ht level increased in the groups fed with EA when compared with the control group. However, there were no significant differences in the MCV, MCH and MCHC values among the groups. The results demonstrated enhancement in all the immunological parameters in the groups fed with EA compared to the control group. The results obtained from challenge with Y. ruckeri revealed reduction in the mortalities in the groups fed with EA. The dietary EA stimulated the SOD, CAT and GSH-Px activities in liver, head kidney and spleen as compared to the control group; however, a reverse trend was observed in the MDA levels of tissues. The present study suggest that EA can effectively enhance the haematological values, immune response, antioxidant capacity, and disease resistance in rainbow trout.

Modulation of posterior intestinal mucosal proteome in rainbow trout (Oncorhynchus mykiss) after Yersinia ruckeri infection.

Yersinia ruckeri is the causative agent of enteric redmouth disease in salmonids. In fish, the intestine represents an important site of nutrient uptake, host-pathogen interactions, and defense. The posterior intestine can be inflamed, reddened, and filled with an opaque, yellowish fluid during Y. ruckeri infection. Herein, we report an investigation on the proteome alteration in the posterior intestinal mucosa of rainbow trout (Oncorhynchus mykiss) after exposure to Y. ruckeri. The intestinal mucosal proteins were identified and quantified by a shotgun proteomic approach by applying data-independent quantification with sequential windowed acquisition of all theoretical mass spectra (SWATH). A total of 437 proteins were found to be differentially up- or downregulated in the posterior intestine. Gene ontology of upregulated proteins pointed to their involvement into exopeptidase, endopeptidase, and hydrolase activities, while the downregulated proteins were involved in lipid metabolism, actin binding, and translation processes. Additionally, upregulated proteins were predicted to be involved in lysosome, oxidative phosphorylation, and metabolic pathways, while downregulated proteins were implicated in focal adhesion, regulation of actin cytoskeleton, protein digestion and absorption pathways. This study showed that Y. ruckeri infection can alter protein abundance involved in serine-type carboxypeptidase, cysteine and aspartic-type endopeptidases, metallopeptidases, antioxidant defense, calcium ion binding, glycolytic and carbohydrate metabolic processes in the proteome of the intestinal mucosa of rainbow trout.

Possible effect of hala extract (Pandanus tectorius) on immune status, anti-tumour and resistance to Yersinia ruckeri infection in rainbow trout (Oncorhynchus mykiss).

The possible effect of dietary administration of hala extract (Pandanus tectorius) on rainbow trout (Oncorhynchus mykiss) immune status as well as its effect as an anti-tumour agent was studied. Fish were divided into 4 groups before feeding with commercial diet (0%, control; 0.5%, 1% and 2% of hala extract) for 2 weeks. The effect of diet on the humoral immune parameters, ie total protein, myeloperoxidase content, antiproteases, lysozyme and bactericidal activities were studied. Also, the effect of the diets on the expression of some immune-related genes in rainbow trout head-kidney (TNF, LYZ2, IL-8 and CD-4) as well as tumour suppressor gene (WT-1a) was investigated. At the end of the feeding trial fish groups were challenged with Yersinia ruckeri. The results demonstrated enhancement in all the immune parameters in fish fed hala extract diets compared to control fish especially with the highest dose (2%) which recorded the highest significant increase (p < 0.05) in some parameters (total protein, myeloperoxidase content, antiproteases, and bactericidal activities) compared to the control. The results obtained from challenge with Y. ruckeri revealed reduction in the mortalities in fish groups fed with 1% and 2% doses of hala extract. Feeding with hala extract provoked upregulation in all immune- related genes. Again, the highest dose of hala extract showed a significant upregulation in WT1a expression (p < 0.05). The current study suggest that the hala extract, especially the highest dose, could be considered a good food additive to improve the immune status, resist tumour formation and to resist or control infectious diseases of rainbow trout.

Characterisation of arginase paralogues in salmonids and their modulation by immune stimulation/ infection.

In this study we show that four arginase isoforms (arg1a, arg1b, arg2a, arg2b) exist in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). We have characterised these molecules in terms of a) sequence analysis, b) constitutive expression in different tissues, and modulated expression following c) stimulation of head kidney macrophages in vitro, or d) vaccination/infection with Yersinia ruckeri and e) parasite infection (AGD caused by Paramoeba perurans and PKD caused by Tetracapsuloides bryosalmonae). Synteny analysis suggested that these arginase genes are paralogues likely from the Ss4R duplication event, and amino acid identity/similarity analyses showed that the proteins are relatively well conserved across species. In rainbow trout constitutive expression of one or both paralogues was seen in most tissues but different constitutive expression patterns were observed for the different isoforms. Stimulation of rainbow trout head kidney macrophages with PAMPs and cytokines also revealed isoform specific responses and kinetics, with arg1a being particularly highly modulated by the PAMPs and pro-inflammatory cytokines. In contrast the type II arginase paralogues were induced by rIl-4/13, albeit to a lesser degree. Vaccination and infection with Y. ruckeri also revealed isoform specific responses, with variation in tissue expression level and kinetics. Lastly, the impact of parasite infection was studied, where down regulation of arg1a and arg1b was seen in two different models (AGD in salmon and PKD in trout) and of arg2a in AGD. The differential responses seen are discussed in the context of markers of type II responses in fish and paralogue subfunctionalization.

Shotgun proteomic analysis of Yersinia ruckeri strains under normal and iron-limited conditions.

Yersinia ruckeri is the causative agent of enteric redmouth disease of fish that causes significant economic losses, particularly in salmonids. Bacterial pathogens differentially express proteins in the host during the infection process, and under certain environmental conditions. Iron is an essential nutrient for many cellular processes and is involved in host sensing and virulence regulation in many bacteria. Little is known about proteomics expression of Y. ruckeri in response to iron-limited conditions. Here, we present whole cell protein identification and quantification for two motile and two non-motile strains of Y. ruckeri cultured in vitro under iron-sufficient and iron-limited conditions, using a shotgun proteomic approach. Label-free, gel-free quantification was performed using a nanoLC-ESI and high resolution mass spectrometry. SWATH technology was used to distinguish between different strains and their responses to iron limitation. Sixty-one differentially expressed proteins were identified in four Y. ruckeri strains. These proteins were involved in processes including iron ion capture and transport, and enzymatic metabolism. The proteins were confirmed to be differentially expressed at the transcriptional level using quantitative real time PCR. Our study provides the first detailed proteome analysis of Y. ruckeri strains, which contributes to our understanding of virulence mechanisms of Y. ruckeri, and informs development of novel control methods for enteric redmouth disease.

Genomic organization, expression and antimicrobial activity of a hepcidin from taimen (Hucho taimen, Pallas).

Hepcidin, an antimicrobial peptide, plays a crucial role in innate immune system of teleost fish. As a cysteine-rich peptide, hepcidin possesses a dual function including iron regulation and innate immunity. In the present study, a full-length hepcidin cDNA (HtHep) was cloned and characterized by RT-PCR and RACE techniques from taimen (Hucho taimen, Pallas), which is a type of rare, precious and cold-water fish species in China. The cDNA contains an open reading frame (ORF) of 267 bp encoding 88 amino acid (aa), with 170 bp located in the 5(') untranslated region (UTR) and 151 bp in the 3' UTR. The genomic sequences analysis showed that the HtHep gene consisted of three exons and two introns (with the length 94 and 251 bp, respectively). With a predicted molecular mass of 2881.4 Da and a theoretical pI of 8.53, the deduced amino acid encodes a signal peptide of 24 aa, prodomain of 39 aa and mature peptide of 25 aa. The signal peptidase (SA-VP) and the motif RX (K/R)R of propeptide convertase suggested the cleavage site of signal and mature peptide. Eight conserved cysteine residues were also identified and formed four disulfide bonds. Pair-wise alignments showed that HtHep clustered together with two fish species of Salmonidae family (Salmo salar and Oncorhynchus mykiss) in HAMP1 branch. Quantitative RT-PCR analysis indicated that the mRNA levels of HtHep were detected in a wide range of tissues and the highest level was detected in the liver. Its expression was also detected early during embryonic stage and could be up-regulated in the liver when challenged with pathogenic bacteria (Yersinia ruckeri). The recombinant HtHep (rHtHep) had antimicrobial activity against both gram-positive (Micrococcus lysodeikticus and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). Our results suggested that HtHep might be involved in the innate immune defense against bacterial pathogens in taimen.

Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens.

Identification and expression analysis of two interleukin-23α (p19) isoforms, in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar.

Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 ß-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule. In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8-29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial (Yersinia ruckeri) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1ß in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish.

Evaluation of dual nasal delivery of infectious hematopoietic necrosis virus and enteric red mouth vaccines in rainbow trout (Oncorhynchus mykiss).

Farmed fish are susceptible to different infectious disease agents including viruses and bacteria. Thus, multivalent vaccines or vaccination programs against two or more pathogens are valuable tools in aquaculture. Recently, nasal vaccines have been shown to be very effective in rainbow trout. The current study investigates, for the first time, the use of the nasal route in dual vaccination trials against two important aquatic diseases, infectious hematopoietic necrosis virus (IHN) and enteric red mouth (ERM) disease. Rainbow trout received live attenuated IHN virus (IHNV) vaccine and the ERM bacterin using four different vaccine delivery methods and were challenged with virulent IHNV or Yersinia ruckeri 7 (100 deg day) and 28 (400 deg day) days post-vaccination. The highest survival rates against IHNV at day 7 were obtained by nasal vaccination either when IHNV and ERM were delivered separately into each nare or when they were premixed and delivered to both nasal rosettes (group D). Protection at 28 days against IHNV was similar in all four vaccinated groups. Early protection against ERM was highest in fish that received each vaccine in separate nares (group B), whereas protection at 28 days was highest in the i.p. vaccinated group (group E), followed by the nasally vaccinated group (group B). Survival results were supported by histological observations of the left and right olfactory organ which showed strong immune responses one day (14 deg days) after vaccination in group B vaccinated fish. These data indicate that dual vaccination against two different pathogens via the nasal route is a very effective vaccination strategy for use in aquaculture, particularly when each nare is used separately during delivery. Further long-term studies should evaluate the contribution of adaptive immunity to the protection levels observed.

Influence of adult Anguillicoloides crassus load in European eels swimbladder on macrophage response.

Anguillicoloides crassus has become one of the most important threats to the European eel (Anguilla anguilla). Adult parasites colonize the swimbladder leading to an impaired functioning of this organ. The infection is also responsible for an increased in the stress level of infected eels, that could produce an altered immune response as well. Differences in parasite loads and effects in the European and Japanese eel (Anguilla japonica) have been described. We have studied the influence of the number of adult parasites present in the swimbladder of wild eels on the macrophage response (phagocytosis and respiratory burst) as part of the first immune response to pathogens. Our results show an increased phagocytozed bacterial survival 24 h post-infection in macrophages of eels infected with more than ten adult parasites compared to macrophages from eels infected with less than those ten adult parasites. Respiratory burst results also showed a less efficient response in macrophages from eels infected with more than ten adult parasites, although in this case results were not found to be significant.

Unique virulence properties of Yersinia enterocolitica O:3--an emerging zoonotic pathogen using pigs as preferred reservoir host.

Enteropathogenic Yersinia enterocolitica bioserotype 4/O:3 are the most frequent cause of human yersiniosis worldwide with symptoms ranging from mild diarrhea to severe complications of mesenteric lymphadenitis, liver abscesses and postinfectious extraintestinal sequelae. The main reservoir host of 4/O:3 strains are pigs, which represent a substantial disease-causing potential for humans, as they are usually asymptomatic carriers. Y. enterocolitica O:3 initiates infections by tight attachment to the intestinal mucosa. Colonization of the digestive tract is frequently followed by invasion of the intestinal layer primarily at the follicle-associated epithelium, allowing the bacteria to propagate in the lamina propria and disseminate into deeper tissues. Molecular characterization of Y. enterocolitica O:3 isolates led to the identification of (i) alternative virulence and fitness factors and (ii) small genetic variations which cause profound changes in their virulence gene expression pattern (e.g. constitutive expression of the primary invasion factor InvA). These changes provoke a major difference in the virulence properties, i.e. reduced colonization of intestinal tissues in mice, but improved long-term colonization in the pig intestine. Y. enterocolitica O:3 strains cause also a considerably lower level of proinflammatory cytokine IL-8 and higher levels of the anti-inflammatory cytokine IL-10 in porcine primary macrophages, as compared to murine macrophages, which could contribute to limiting inflammation, immunopathology and severity of the infection in pigs.

In vitro cytotoxicity and multiplex PCR detection of virulence factors of Yersinia ruckeri isolated from rainbow trout in North West Germany.

The aim of this study was to investigate differences in presence and expression of virulence factors between biotype 1 and 2 strains of 82 Yersinia (Y.) ruckeri isolates, collected from North West Germany during the period of 2004-2012, and to analyze the cytotoxicity of these strains to different fish cell lines. The common virulence factor genes, such as yhlA and yhlB encoding for hemolysin YhlA, rucC and rupG encoding for ruckerbactin, yrp1 and yrpDEF for ABC exporter protein system, and two flagellar genes, including flgA for flagellar secretion chaperones and flhA for flagellar secretion apparatus, were found present in both biotype 1 and 2 isolates of Y. ruckeri collected from North West Germany using multiplex PCR. mRNA expression of these genes was compared between the two biotypes of Y. ruckeri. There was no significant diversity (p > 0.05) in the expression of these genes between biotype 1 and 2 strains. 27 Y. ruckeri isolates from different typing groups were analysed in cytotoxicity tests to common carp brain (CCB), epithelioma papulosum cyprini (EPC), fathead minnow epithelial (FHM) and rainbow trout gonad-2 (RTG-2) cells, respectively. In vitro cytotoxicity of the isolates to CCB, EPC and FHM was higher than that to RTG-2 (p < 0.05). At 15 degrees C the maximum cytotoxicity to FHM and EPC was higher in non-motile strains than in motile stains after an incubation of 24 h (p < 0.05), however, after 48 h, there was no significant difference (p > 0.05) of cytotoxicity between those two biotypes. Our results suggest that biotype 2 strains from North West Germany are homogenous with biotype 1 strains on the basis of genetic virulence factor genes. At lower temperature non-motile Y. ruckeri isolates were found more active than motile strains, which could explain why in winter non-motile strains were found more often responsible for ERM outbreaks than motile strains.

Detection, enumeration and characterization of Yersinia enterocolitica 4/O:3 in pig tonsils at slaughter in Northern Italy.

Tonsils from 150 pigs slaughtered at 270 days or older were tested for Yersinia enterocolitica with different cultural methods. Samples were collected in three different abattoirs of Northern Italy between April and November 2012 and were analysed by direct plating on cefsulodin-irgasan-novobiocin (CIN) agar and by enrichment procedures following the ISO 10273:2003 reference method. Twenty-three (15.3%) samples were positive: 22 tonsils (14.7%) were positive for human pathogenic Y. enterocolitica bio-serotype 4/O:3 and one tonsil (0.7%) for Y. enterocolitica bio-serotype 1A/7,8-8,8,19. Seventeen samples out of 23 (73.9%) were positive by direct plating method. Among the enrichment procedures, the best recovery rate (8 positives out of 23; 34.8%) was obtained by the two-day enrichment in peptone-sorbitol-bile (PSB) broth followed by plating on CIN agar plates. The two-day enrichment in PSB followed by potassium hydroxide (KOH) treatment before plating onto CIN agar gave 7 positives out of 23 (30.4%), decreasing to 3 positives (13.0%) without KOH treatment. The worst results were obtained by prolonged (five days) enrichment in PSB, with or without KOH treatment, followed by plating on CIN agar: 4.3% (1 out of 23) and 0.0% recovery rates, respectively. The mean concentration was 1.9 × 10(4)CFU/g, with a minimum of 1.0 × 10(2)CFU/g and a maximum of 5.8 × 10(4)CFU/g, thus demonstrating that tonsils may play an important role in contamination of pluck sets, carcasses, and slaughterhouse environment. Prevalence of virulence genes among the Y. enterocolitica 4/O:3 isolates was as follows: 12/22 (54.5%) for yadA, 21/22 (95.5%) for ail, 21/22 (95.5%) for inv and 22/22 (100%) for ystA. All Y. enterocolitica 4/O:3 isolates were sensitive to amoxicillin/clavulanic acid, ciprofloxacin and ceftazidime and resistant to ampicillin and cephalotin. High proportions of 4/O:3 isolates (95%) were sensitive to cefotaxime, gentamicin, kanamicin and nalidixic acid. High levels of resistance were observed to sulphonamide compounds (91%), streptomycin (64%) and chloramphenicol (55%). Multi-resistant isolates were very common; resistance to three or more antimicrobials was observed in 91% (20/22) of 4/O:3 isolates. High level of resistance to chloramphenicol was possibly due to coresistance to tiamphenicol, which was detected in 100% of the isolates. XbaI-PFGE detected four clusters among the 22 Y. enterocolitica 4/O:3 isolates. The most represented accounted for 77% (17/22) of the isolates, the second most common was found in 14% (3/22) of the isolates and the two other profiles were observed in single isolates. The comparison with a selection of human isolates supported the role of the pig as reservoir of 4/O:3 Y. enterocolitica.

Within-batch prevalence and quantification of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis in tonsils of pigs at slaughter.

Yersiniosis is a common bacterial zoonosis in Europe and healthy pigs are known to be the primary reservoir of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis. However, little information is available about the prevalence of these pathogens within pig batches at time of slaughter. The tonsils of 7047 fattening pigs, belonging to 100 farms, were aseptically collected immediately after evisceration in two Belgian slaughterhouses. The batch size varied between 70 and 930 pigs. On average, 70 pigs were sampled per batch. The tonsils were examined by direct plating on cefsulodin-irgasan-novobiocin (CIN) agar plates and the number of suspect Yersinia colonies was counted. Pathogenic Y. enterocolitica serotype O:3 were found in tonsils of 2009 pigs (28.5%), originating from 85 farms. The within-batch prevalence in positive farms ranged from 5.1 to 64.4%. The number of Y. enterocolitica in positive pigs varied between 2.01 and 5.98 log10 CFU g(-1) tonsil, with an average of 4.00 log10 CFU g(-1) tonsil. Y. pseudotuberculosis was found in seven farms, for which the within-batch prevalence varied from 2 to 10%. In five of these farms, both Y. enterocolitica and Y. pseudotuberculosis were simultaneously present. Human pathogenic Yersinia spp. are widespread in slaughter pig batches in Belgium as 87% of the tested batches were infected with these pathogens at the time of slaughter. The large variation of the prevalence between batches may lead to different levels of contamination of carcasses and risks for public health.

Prevalence of pathogenic Yersinia enterocolitica in slaughter-aged pigs during a one-year survey, 2010-2011, France.

The prevalence of pathogenic Yersinia enterocolitica in French slaughter-aged pigs was estimated by sampling 3120 pigs from 96 batches in 16 slaughterhouses from January 2010 to February 2011. Respectively, 36 batches (20 pigs/batch) and 60 batches (40 pigs/batch) were considered during the cold period and the warm period. Tonsils were swabbed before the chilling step. Pathogenic Y. enterocolitica was detected after enrichment in ITC and streaking on CIN and YeCM media. Typical isolates were confirmed as Y. enterocolitica and biotyped by biochemical tests as described in the ISO 10273:2003 method. Of the tested pigs, 13.7% (CI95% [10.1-17.3]) were found positive for pathogenic Y. enterocolitica and 74.3% (CI95% [64.8-83.8]) of the pig batches contained at least one positive pig. The percentage of positive pigs per batch was generally low; 60.3% of positive batches contained fewer than 5 positive pigs. The prevalence of the pathogen at the batch level remained unchanged throughout this one-year study, but the prevalence in pigs was significantly higher during the warm period than during the cold period. Biotype 4 was the most prevalent biotype among the 827 isolated strains (91.9% of the isolates), followed by biotype 3 (7.25% of the isolates). Six isolates were of biotype 5 and one of biotype 2. Biotype 4 was found in all the 16 participating slaughterhouses, biotype 3 in ten slaughterhouses and biotype 5 in four. This study provides valuable recent figures for the prevalence of pathogenic Y. enterocolitica in French pigs. It also highlights the seasonal aspect of the carriage of this pathogen by pigs, a pattern which differs from those in other countries.