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The Pseudomonas H6 lipopeptide is a surfactant which is able to eliminate various parasitic pathogens including the ciliate Ichthyophthirius multifiliis in vitro. This suggests an application for aquaculture purposes. However, further information on efficacy of the compound and possible immune modulation of surfactant exposed fish should be gathered before usage at farm level is considered. We performed an in vivo infection experiment using rainbow trout fry (mean weight 4.6 g, mean length 7.6 cm) as hosts and I. multifiliis theronts as the parasitic pathogen. We compared infection level, immune gene regulation and immune cell density in gills of 1) non-exposed control fish, 2) parasite exposed but untreated fish, 3) surfactant treated fish without parasite exposure, and 4) fish exposed both to parasites and surfactant. The surfactant concentration was 10 mg/L, the infection dosage 1000 theronts/fish and the exposure period 12 h. The parasite infection was recorded and samples were taken from rainbow trout gills at day 0 and 10 post-exposure. We performed an immunohistochemical investigation (detecting cells positive for MHC II, SAA, CD8, IgM, IgT and IgD) and measured the expression of genes encoding cathelidin-1, CD8, hepcidin, IFN γ, IgDs, IL-1β, IL-6 and SAA. Theront exposed fish (without surfactant treatment) became heavily infected whereas concomitant surfactant treatment (10 mg/l), along with parasite exposure, could prevent infection. A significant inflammation (upregulation of il-1β, il6, ifn γ, cathelicidin, hepcidin) was elicited in non-treated and parasite exposed fish but it was prevented by the surfactant treatment. When investigated 10 days after treatment no immune gene regulation was seen in fish exposed to surfactant only. The therapeutic effect may be due to a direct parasiticidal action of the surfactant, but it cannot be excluded that a modulation of the host immune reaction may influence the infection success.

Plastic pollution has become a major concern on a global scale. The plastic is broken down into minuscule particles, which have an impact on the biosystems, however long-term impacts through an entire generation is largely unknown. Here, we present the first whole generation study exposing fish to a 500 nm polystyrene plastic particle at environmentally relevant concentrations. Short- and long-term adverse effects were investigated in the zebrafish model organism using a holistic multi-omics approach. The particles accumulated in the yolk sac of young larvae and short-term biological impacts included immune-relevant gene regulation related to inflammation and tolerance as well as disruption of metabolic processes, such as the fatty acid and lipid pathways. The long-term effects comprised gene regulations pointing towards skin and/or gill inflammation, dysbiosis of the gut microbiota, a tendency towards decreased condition factor in adult males as well as a lowered reproductive capability. From this study, it can be concluded that exposures to plastic nanoparticles have an impact on population as well as ecosystem level in fish and likely also in other vertebrates.

Control of the skin parasitic ciliate Ichthyophthirius multifiliis is currently based on laborious chemical and mechanical approaches, thus advocating for alternative control measures. Here, we show that the early development of trophonts (feeding stage residing in the epidermis) in the Rainbow Trout Oncorhynchus mykiss epidermis was inhibited at 5 d postinfection after the administration of feed additives, including garlic Allium sativum, oregano Origanum onites, thyme Thymus vulgaris, coriander Coriandrum sativum, and astaxanthin (a carotenoid derived from the alga Haematococcus pluvialis). However, no inhibition was observed at 8 d postinfection. We investigated whether the early inhibition was caused by (1) the feed additives’ direct effect on the parasite and/or (2) stimulation of the Rainbow Trout immune responses. Garlic exhibited the highest in vitro killing capacity toward theronts (the infective stage of the parasite), followed by oregano, thyme, and astaxanthin, whereas coriander had no in vitro parasiticidal effect. Immune reactions were measured by plasma lysozyme activity in Rainbow Trout after feeding and by recording immune gene expression in trout leukocytes that were exposed to feed additives. Oregano-fed fish showed a significantly (P

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, has extensive negative effects on wild and farmed salmonids worldwide. Vaccination induces some protection under certain conditions but disease outbreaks occur even in vaccinated fish. Therefore, alternative disease control approaches are required to ensure the sustainable expansion of rainbow trout aquaculture. Selective breeding can be applied to enhance host resistance to pathogens. The present work used genome-wide association study (GWAS) to identify quantitative trait loci (QTL) associated with A. salmonicida resistance in rainbow trout. A total 798 rainbow trout exposed to A. salmonicida by bath challenge revealed 614 susceptible and 138 resistant fish. Genotyping was conducted using the 57 K single nucleotide polymorphism (SNP) array and the GWAS was performed for survival and time to death phenotypes. We identified a QTL on chromosome 16 and located positional candidate genes in the proximity of the most significant SNPs. In addition, samples from exposed fish were examined for expression of 24 immune-relevant genes indicating a systematic immune response to the infection. The present work demonstrated that resistance to A. salmonicida is moderately heritable with oligogenic architecture. These result will be useful for the future breeding programs for improving the natural resistance of rainbow trout against furunculosis.

Immersion vaccination of rainbow trout against Yersinia ruckeri infection is an established method to prevent enteric red mouth disease (ERM) but the effect is inferior to injection vaccination and the duration of protection is limited to less than six months. Adjuvants in vaccines may in general elevate the immune response and the present work elucidates how ERM immersion vaccination of trout in combination with exposure to soluble adjuvants, Montanide™ IMS 1312 VG PR and β-glucan, affects immune reactions. The former adjuvant, when used alone, induced a slightly increased protection (not statistically significant) whereas β-glucan did not increase protection. Adjuvant-treated and non-exposed groups showed elevated plasma lysozyme activity after challenge with Y. ruckeri. Specific antibody production was not positively affected by combining adjuvant and vaccine. Overall expression of immune genes tested was generally manifold higher in gills compared to skin. Only genes encoding SAA and IL-17C1 were expressed at a higher level in skin. Dynamic differences between the gill and skin compartments were also recorded for genes encoding cytokines (TNF-α, IL-1β, IL-6, IL-10, IL-12, IL-17A/F2, IL-17C1, IL-17C2, IL-22), immunoglobulins (IgM, IgD, IgT), cell markers (CD8α, TCR-β) and acute phase reactants (SAA, lysozyme). These genes were upregulated 24 h post-vaccination in fish gills exposed to both vaccine-adjuvant combinations when compared to fish exposed to vaccine alone. After a few weeks no vaccine induced reaction was seen and after challenge with bacteria mainly unvaccinated fish responded. Adjuvants used in combination with immersion vaccine clearly influences immune reactions and may improve duration and protection but further potency tests should be performed.

Selective breeding programmes involving marker assisted selection of innately pathogen resistant strains of rainbow trout rely on reliable controlled infection studies, extensive DNA typing of individual fish and recording of expression of relevant genes. We exposed juvenile rainbow trout (6 h bath to 2.6 × 105 CFU mL−1) to the fish pathogen Yersinia ruckeri serotype O1, biotype 2, eliciting Enteric Red Mouth Disease ERM, and followed the disease progression over 21 days. Cumulative mortality reached 42% at 12 days post challenge (dpc) after which no disease signs were recorded. All fish were sampled for DNA-typing (50 k SNP chip, Affymetrix®) throughout the course of infection when they showed clinical signs of disease (susceptible fish) or at day 21 when fish showed no clinical signs of disease (survivors - resistant fish). Genome-wide association analyses of 1027 trout applying single nucleotide polymorphisms (SNPs) as markers revealed an association between traits (susceptible/resistant) and certain regions of the trout genome. It was indicated that multiple genes are involved in rainbow trout resistance towards ERM whereby it is considered a polygenic trait. A corresponding trout group was kept as non-exposed controls and a comparative expression analysis of central innate and adaptive immune genes in gills, spleen and liver was performed for three fish groups: 1) moribund trout exhibiting clinical signs 7 dpc (CS), 2) exposed fish without clinical signs at the same sampling point (NCS) and 3) surviving fish at 21 dpc (survivors). Immune genes encoding inflammatory cytokines (IL-1β, IL-2A, IL-6A, IL-8, IL-10A, IL-12, IL-17A/F2A, IL-17C1, IL-17C2, IL-22, IFNγ, TNFα), acute phase reactants (SAA, C3, cathelicidins, lysozyme) were expressed differently in CS and NCS fish. Correlation (negative or positive) between expression of genes and bacterial load suggested involvement of immune genes in protection. Down-regulation of adaptive immune genes including IgDm, IgDs, IgT and TCR-β was seen primarily in CS and NCS fish whereas survivors showed up-regulation of effector molecule genes such as cathelicidins, complement and lysozyme suggesting their role in clearing the infection. In conclusion, SNP analyses indicated that ERM resistance in rainbow trout is a multi-locus trait. The gene expression in surviving fish suggested that several immune genes are associated with the trait conferring resistance.

The parasitic ciliate Ichthyophthirius multifiliis has a low host specificity eliciting white spot disease (WSD) in a wide range of freshwater fishes worldwide. The parasite multiplies rapidly whereby the infection may reach problematic levels in a host population within a few days. The parasite targets both wild and cultured fish but the huge economic impact of the protozoan is associated with mortality, morbidity and treatment in aquacultural enterprises. We have investigated the potential for genetic selection of WSD‐resistant strains of rainbow trout. Applying the DNA typing system Affymetrix® and characterizing the genome of the individual fish by use of 57,501 single nucleotide polymorphisms (SNP) and their location on the rainbow trout chromosomes, we have genetically characterized rainbow trout with different levels of natural resistance towards WSD. Quantitative trait loci (QTL) used for the selection of breeders with specific markers for resistance are reported. We found a significant association between resistance towards I. multifiliis infection and SNP markers located on the two specific rainbow trout chromosomes Omy 16 and Omy 17. Comparing the expression of immune‐related genes in fish—with and without clinical signs—we recorded no significant difference. However, trout surviving the infection showed high expression levels of genes encoding IgT, T‐cell receptor TCRβ, C3, cathelicidins 1 and 2 and SAA, suggesting these genes to be associated with protection.

Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of Vibrio anguillarum resistant rainbow trout (Oncorhynchus mykiss). Fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 × 107 cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish – including survivors – and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The possible genes associated with resistance are discussed.

Pseudomonas aeruginosa is one of the leading Gram-negative organisms associated with nosocomial infections. The increasing frequency of multidrug-resistant P. aeruginosa (MDRPA) strains affects the efficacious antimicrobial options which are severely limited. In this prospective study, forty two different types of samples collected from patients with multiple types of infections attainted to AL-Kadhyimia Teaching Hospital-Baghdad-Iraq, from February, 2012 to August, 2012. Morphological characteristics, biochemical testing by Vitek 2 Compact for Gram-negative Identification, card 2GN and amplification of species-specific 16SrDNA gene were used to identify P. aeruginosa. The antibiotic sensitivity profiles of the strains were determined against ten antibiotics belonging to different classes using the BioMerieux Vitek2 compact system AST card. In order to identify the genes implicated in antimicrobial resistance mechanisms, DNA was extracted from collected samples and resistance genes PstS, blaOXA-50, blaOXA-2 and bla IMP-13 were amplified using polymerase chain reaction (PCR). The results showed that there were difference in number of P. aeruginosa isolates that were identified using Vitek 2 Compact for Gram-negative Identification, card 2GN (25/42) and 16SrDNA gene(29/42), but it was not statistically significant (p value= 1.000). Also, it showed that the highest resistance percentages of strains to tested antibiotics was for Ceftriaxone (96.55%) and the lowest resistant percentages was (17.24%) for Meropenem. Seven strains out of twenty nine (24%) were resistant to all tested antibiotics and were identified as a multidrug resistance strain (MDR). The study detects the presence of PstS gene and blaOXA-50 in (65.5% and 93% of strains, respectively), but it did not identify the presence of blaOXA-2 or blaIMP 13 at any of the tested strains of P. aeruginosa. There was no significant relationship between the presence of blaOXA-50 and resistance to Meropenem, Imipenem, Ceftazidime and Cefepim (p value 1.000, 1.000, 0.474, and 0308, respectively). We concluded that Meropenem is the most effective antibiotic and can be considered as the drug of choice against P. aeruginosa. This study is the first report presence of PstS and blaOXA50 in P. aeruginosa in Iraq. The presence of blaOXA-50 is important in order to identify and track the spread of multidrug-resistant P. aeruginosa clones since blaOXA-50 may be potential clonality marker. Key words: Pseudomonas aeruginosa, molecular identification, antibiotic resistance genes.

Field experiment conducted during the winter agricultural season 2017-2018 at Al-Zafraniya Agricultural Research Station and Al-Tuwaitha Agricultural Research Station, according of Randomized Complete Block Design (RCBD) with three replications, to study maturity and productivity for accumulative temperature of rapeseed (Brassica napus L.). The experiment planting in Last week of September 2017 at Al-Zafraniya station and Last week of October 2017 at AL- Tuwaitha station. The results show increasing significantly in the yield (1110.11 kg. ha-1), percentage of oil (47.12 %), protein content in seeds (43.1%), carbohydrate (25.7%), nutrient content of seed (K, Ca) in Al-Zafraniya location; compared to Al-Tuwaitha location, yield (352.8 kg. ha-1), percentage of oil (41.1 %), protein content in seeds (41.2%), carbohydrate (26.2%), nutrient content of seed (Na, Ca, Mg, Fe) at fertilization, compared to no-fertilizer. It showed significant differences with nutrient content of soil (Na, Mg, Fe) for Al-Tuwaitha location and nutrient content of soil (K, Ca, Mg, Fe, S, Cu), pH, EC for Al-Zafraniya location at fertilization, compared to no-fertilizer. These results shows that the delay at time of cultivation leads to a decrease in seed yield, oil ratio, and percentage of protein in seeds, also high temperatures at seed filling stage leads decrease in seeds, oil and protein content.

Mariculture in Denmark is based on production of rainbow trout grown two years in fresh water followed by one growth season in sea cages. Although the majority of rainbow trout are vaccinated against the most serious bacterial pathogens - Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum and Yersinia ruckeri, by the use of commercially available vaccines, disease outbreaks requiring treatment with antibiotics still occur. The present study tested the potential of a new experimental multicomponent vaccine that is based on local bacterial strains, isolated from rainbow trout in Danish waters, and thus custom-designed for Danish rainbow trout mariculture. The vaccination with the multicomponent vaccine resulted in protection against three relevant bacterial diseases (yersiniosis, furunculosis, vibriosis) under experimental conditions. We showed that i.p. injection of the vaccine induced specific antibody responses in trout against the different bacterial antigens and regulated expression of genes encoding SAA, C3, IL-1β, IL-6, IL-8, IgD and MHCII.

A single immersion vaccination of rainbow trout fry using a Yersinia ruckeri bacterin confers immunity to reinfection but only for a shorter period. A longer protective period is needed in practical trout farming and we have shown that booster vaccination prolongs immunity. Due to economic considerations and management practices it is not possible to immersion vaccinate large trout (20–30 g) with the recommended high bacterin concentration. We here demonstrate that booster vaccination using dilutions of the bacterin (1:100, 1:1000 and 1:2000) with increased exposure time (1 h, 2 h) confers a higher and longer lasting immunity although a short term (30 s) booster in 1:10 bacterin proved superior. This has practical implications for fish farmers because this diluted booster vaccination over longer time can be applied when farmers handle and transport fish between ponds and farms. Therefore such a practice will not challenge farm management and economy. Increased antibody levels were recorded after challenge of vaccinated fish but not after immersion vaccination alone which suggests that immersion induces priming of memory cells.

Vaccination of rainbow trout against Enteric Redmouth Disease (ERM) caused by Yersinia ruckeri can be successfully performed by administering vaccine (a bacterin consisting of formalin killed bacteria) by immersion, bath or injection. Booster immunization is known to increase the protection of fish already primed by one of these vaccination methods. Oral vaccination of trout (administering vaccine in feed) is an even more convenient way of presenting antigen to the fish but the effect of an oral booster has not previously been described in detail. The present work describes to what extent protection may be enhanced by oral boostering following priming with different administration methods. The study confirms that vaccination by 30 s dip into a bacterin (diluted 1:10) may confer a significant protection compared to non-vaccinated fish. The immunity may be optimized by booster immunization either provided as dip (most effective), bath (less effective) or orally (least effective). Oral immunization may be used as booster after dip but applied as a single oral application it induced merely a slight and statistically non-significant response. It is noteworthy that primary oral immunization followed by an oral booster vaccination showed a trend for an even weaker response. It should be investigated if continued exposure to a low antigen concentration – as performed by two oral immunizations - may induce tolerance to the pathogen and thereby leave the fish more vulnerable.

Rainbow trout (Oncorhynchus mykiss) runts (n = 5) and harvest quality fish (n = 5) were sampled from each of all the net cage mariculture facilities in Denmark at the time of slaughter during autumns of 2012 and 2013. Thus, a total of 190 trout were obtained, represented by 95 runts and 95 fish of harvest quality. Trout were examined for macroscopic ectoparasites as well as helminths of the gastrointestinal tract and body cavity by careful visual inspection, and belly flap musculature by pepsin digestion. Stomach content analysis was performed in order to assess the risk of endoparasite transmission to the net cage cultured trout. Low numbers of salmon lice (Lepeophtheirus salmonis) (1–9 lice per fish) were found on 9 trout from western localities off the eastern coast of Jutland characterized by water salinity levels of 21–24‰, whereas no lice were detected on fish from areas of lower salinity. Body cavity and musculature of all trout were free from helminths, and the absence of medically important 3rd stage larvae of Anisakidae was thus confirmed. However, transmission of endoparasites was documented by the finding of the nematode Hysterothylacium aduncum in the intestines of 9.5% of the runts (mean intensity = 2.3) and 2.1% of the harvest quality trout (mean intensity = 2.0). A few cestodes (Eubothrium sp.) located in the pyloric caeca, and one acantocephalan (Neoechinorhynchus sp.) found in the intestine, were collected from four trout. The higher prevalence of H. aduncum among runts was associated with a markedly increased intake of parasite intermediate or paratenic hosts, i.e. small fish (mainly three-spined stickleback) and crustaceans (mainly amphipods), by these fish compared to those of harvest quality. Additionally, a higher number of runts than harvest quality fish had eaten biofouling organisms, e.g. mussels and algae, further indicating a difference in feeding behavior between the two quality classes of trout.

Background. Ichthyobodosis (“costiosis”) is induced by the flagellate Ichthyobodo necator in both wild and cultured host populations. Fish farmers for decades have been using formaldehyde (FA) for control but this compound is problematic due to its carcinogenic and allergenic nature. Alternative auxiliary substances for bath treatment are needed and therefore we decided to compare efficacies of sodium percarbonate (SPC), peracetic acid (PAA), and hydrogen peroxide (HP) for control of ichthyobodosis in rainbow trout, Oncorhynchus mykiss. Materials and methods. An experimental infection of Ichthyobodo necator on rainbow trout (2–3 g) was established (total parasite population up to 142 000 flagellates per fish) and duplicated controlled water bath treatments were conducted with specified water quality criteria. Four compounds: SPC (40, 80, and 120 mg · L–1), PAA (0.1, 0.2, and 0.3 mg · L–1), HP (15 and 30 mg · L–1), and FA (40 and 80 mg · L–1) were tested. The compounds were applied for 2 h (as static freshwater bath treatments at 13°C) in 20-L aerated tanks (containing 10 L of the treatment solution). Results. FA at a concentration of 80 mg · L–1 and PAA at 0.3 mg · L–1 showed almost complete elimination of parasites but it was found that also SPC at a concentration of the 120 mg · L– was highly effective in eliminating I. necator. In addition, SPC and PAA (80 mg · L–1 and 0.2 mg · L–1, respectively) also significantly reduced the parasite load compared to untreated control fish. HP significantly reduced parasite populations at a concentration of 30 mg · L–1, but the compound elicited adverse reactions in fish. Conclusion. It is suggested that SPC and PAA may serve as alternatives to formaldehyde for control of ichthyobodosis in rainbow trout farms.

Immune cells in rainbow trout Oncorhynchus mykiss comprise granulocytes (neutrophils, basophils and eosinophils), macrophages/monocytes and lymphocytes (B- and T-cells). These cellular elements occur early during the ontogenetic development of trout and allow both innate and adaptive responses towards an antigen to be initiated even in fry. The number of leukocytes inindividual fish at different developmental stages is likely to influence the capacity of the fish to respond simultaneously to several antigens (pathogens and vaccine components). This parameter may therefore be crucial for both wild and cultured fish and we show that the size of the leukocyte population increases exponentially with body size of rainbow trout. Four groups (5fish/group) of naive rainbow trout (Oncorhynchus mykiss) with a mean body weight of 2 - 4 g (group I), 4 - 6 g (group II), 25 - 30 g (group III), and 650 - 780 g (group IV) were investigated. The number of lymphocytes was generally higher in head kidney compared to blood and spleen but they dominated in all samples (blood, head kidney and spleen) and their numbers increasedexponentially with fish size. Percentages of lymphocytes in relation to neutrophils and macrophages were higher in spleen (98% - 99%) compared to blood and head kidney in all groups. Fish fry is therefore equipped to respond specifically against one or a few vaccine antigens, but the capacity to raise protective responses against a repertoire of pathogens may be limited untilthe larger fingerling stage has been reached. The implications for vaccination of early fry are discussed.

Various compounds have been applied for control of Ichthyophthirius multifiliis (commonly known as Ich) which is the parasitic pathogen, responsible for the white spot disease, in freshwater aquaculture worldwide. Available methods are based on disinfection of fish tank water, targeting infective free-swimming theronts and escaped tomonts. No legal drug is available and licensed for treatment or prevention of the disease. The presently reported study was performed to test the potential of toltrazuril (Baycox® vet.)—a drug licensed for treatment of coccidial infections in other animal species—to reduce Ich infections when administered orally. Commercial pelleted feed containing 5.0 or 2.5 mg toltrazuril per 1 g of feed was offered to rainbow trout, Oncorhynchus mykiss (Walbaum, 1792) (Actinopterygii: Salmoniformes: Salmonidae), during a three day feeding regime (1% of biomass offered per day). Two trials were performed: one with feeding before exposure to parasites and one where already infected fish were treated. Before the treatment it was tested if fish would eat feed containing different drug concentrations and it was found that feed with the high drug concentration was not eaten by the fish whereas all feed containing 2.5 mg per 1 g feed was eaten. Following the exposure to infective I. multifiliis theronts it was found that the fish treated with toltrazuril before challenge obtained a significantly lower parasite burden (number of trophonts in the skin) compared to untreated control. On the other hand, toltrazuril proved to be ineffective when administered to the fish which were already infected before the treatment.

Enteric redmouth disease (ERM) caused by the fish pathogen Yersinia ruckeri is a major threat to freshwater production of rainbow trout (Oncorhynchus mykiss) throughout all life stages. Injection vaccination of rainbow trout against Y. ruckeri infection has been shown to confer better protection compared to the traditionally applied immersion vaccination. It may be hypothesized, based on experience from other vaccines, that adjuvants may increase the protective level of ERM injection vaccines even more. Controlled comparative vaccination studies have been performed to investigate effects of the oil adjuvant Montanide™ ISA 763 A VG (Seppic) when added to an experimental Y. ruckeri bacterin (containing both biotype 1 and 2 of serotype O1). A total of 1000 fish with mean weight 19 g was divided into five different groups (in duplicated tanks 2 × 100 fish per group) 1) non-vaccinated control fish (NonVac), 2) fish injected with a commercial vaccine (AquaVac(®) Relera™) (ComVac), 3) fish injected with an experimental vaccine (ExpVac), 4) fish injected with an experimental vaccine + adjuvant (ExpVacAdj) and 5) fish injected with adjuvant alone (Adj). Injection of the experimental vaccine (both adjuvanted and non-adjuvanted) induced a significantly higher antibody (IgM) level, increased occurrence of IgM(+) cells in spleen tissue and significant up-regulation of several immune genes. Additional experiments using a higher challenge dosage suggested an immune enhancing effect of the adjuvant as the challenge produced 100% mortality in the NonVac group, 60% mortality in both of ComVac and Adj groups and only 13 and 2.5% mortalities in the ExpVac and the ExpVacAdj groups, respectively.

Enteric redmouth disease (ERM) caused by the fish pathogen Yersinia ruckeri is a major threat to freshwater production of rainbow trout throughout all life stages. Injection vaccination of rainbow trout against Y. ruckeri infection has been shown to confer better protection compared to the traditionally applied immersion vaccination. It may be hypothesized, based on experience from other vaccines, that adjuvants may increase the protective level of ERM injection vaccines even more. Controlled comparative vaccination studies conducted to investigate effects of the oil adjuvant MontanideTM ISA 763 A VG when added to an experimental Y. ruckeri bacterin (containing both biotype 1 and 2 of serotype O1). A total of 1000 fish with mean weight 19 g was divided into five different groups (in duplicated tanks 2 × 100 fish per group) 1) nonvaccinated control fish (NonVac), 2) fish injected with a commercial vaccine (AquaVac ReleraTM) (ComVac), 3) fish injected with an experimental vaccine (ExpVac), 4) fish injected with an experimental vaccine + adjuvant (ExpVacAdj) and 5) fish injected with adjuvant alone (Adj). Injection of the experimental vaccine (both adjuvanted and non-adjuvanted) induced a significantly higher antibody (IgM) level, increased occurrence of IgM cells in spleen tissue and significant up-regulation of several immune genes. Additional experiments using a higher challenge dosage suggested an immune enhancing effect of the adjuvant as the challenge produced 100% mortality in the NonVac group, 60% mortality in both of ComVac and Adj groups and only 13 and 2.5% mortalities in the ExpVac and the ExpVacAdj groups, respectively.

When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.

Gut microbiota (GM) composition of vertebrates are considered to influence health parameters including immunological parameters in the host but knowledge of these issues in fish are sparse. We have performed a 45 day experimental study elucidating effects of dietary supplementation of organic acids (Mera Cid) and immunostimulants on the GM composition of rainbow trout Oncorhynchus mykiss (Walbaum) and investigated its association with blood parameters and disease resistance (against enteric red mouth disease (ERM) caused the bacterium Yersinia ruckeri). A total of 640 fish received either 1) basic fish feed without organic acids or immunostimulants, 2) fish feed with immunostimulants (β6glucans, MOS, nucleotides, lactic acid bacteria, vitamin C and E) but no organic acids, 3) fish feed with immunostimulants and organic acids and 4) fish feed without immunostimulants but with organic acids. Ha lf of the fish (16 groups) were then Y. ruckeri challenged on day 21 and 28 of feeding. Haemoglobin, lysozyme and gut microbiota composition (16S rRNA gene based denaturing gradient gel electrophoresis DGGE) were determined at days 14, 30 and 45. Gut microbiota analyses showed that challenge of fish with Y. ruckeri and feeding with Mera Cid affected the intestinal content.

Infections with the parasitic flagellate Ichthyobodo necator (Henneguy, 1883) cause severe skin and gill disease in rainbow trout Oncorhynchus mykiss (Walbaum, 1792) juveniles. The epidermal disturbances including hyperplasia and mucous cell exhaustion caused by parasitization are known, but no details on specific cellular and humoral reactions have been presented. By applying gene expression methods and immunohistochemical techniques, further details of immune processes in the affected skin can be presented. A population of I. necator was established in the laboratory and used to induce an experimental infection of juvenile rainbow trout. The course of infection was followed by sampling for parasite enumeration, immunohistochemistry (IHC) and quantitative PCR (qPCR) on days 0, 5, 9 and 14 post-infection. IHC showed a significant increase in the occurrence of IgM-positive cells in the skin of the infected fish, whereas IgT-positive cells were eliminated and the number of CD8-positive cells declined. qPCR studies supported the IHC findings showing a significant increase in IgM and a decrease in the CD8 gene expression. In addition, genes encoding innate immune genes such as lysozyme, SAA and cathelicidin 2 were up-regulated. Expression of cytokines (IL-1β, IL-4/13A, IL-6, IL-8, IL-10), the cell marker CD4 and the transcription factor GATA3 showed a significant increase after infection. Cytokine profiling including up-regulation of IL-4/13A and IL-10 genes and transcription factor GATA3 connected to the proliferation of IgM producing lymphocytes suggests a partial shift towards a Th2 response associated with the I. necator infection.

Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.

s / Fish & Shellfish Immunology 34 (2013) 1635–1691 1641 predictor of vaccine success against yersiniosis.Wehave tested an effect of a novel inactivation method of Y. ruckeri on vaccine efficacy and showed that the transcriptional signaturewas a predictor of protection, thus confirming our previous results. * Corresponding author. E-mail address: B.Nowak@utas.edu.au (B.F. Nowak)