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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Effects of immunostimulants on ranched southern bluefin tuna Thunnus maccoyii: immune response, health and performance.

Ranched southern bluefin tuna Thunnus maccoyii were fed baitfishes supplemented with vitamins (predominantly E and C) or vitamins and immunostimulants, nucleotides and ß-glucans, over 12 weeks after transfer and monitored for enhancement in immune response, health and performance through their 19 week grow-out period. Fish from two different tows were sampled separately at three different sampling points: at transfer to grow-out pontoons, at 8 weeks post-transfer and at harvest, 19 weeks post-transfer. Lysozyme activity was enhanced during vitamin supplementation compared to control fish. Performance (i.e. survival, condition index and crude fat), health (i.e. blood plasma variables including pH, osmolality, cortisol, lactate and glucose) and alternative complement activity were not commonly improved through diet supplementation. There were some tow-specific improvements in performance through vitamin supplementation including survival, selected parasite prevalence and intensity, and alternative complement activity. Immunostimulant supplementation also showed a tow-specific improvement in plasma cortisol level. Tow-specific responses may suggest that life history, previous health condition and husbandry can affect the success of vitamin and immunostimulant enhancement of immune response, health and performance of ranched T. maccoyii.

Multilocus phylogenetic analyses reveal that habitat selection drives the speciation of Didymozoidae (Digenea) parasitizing Pacific and Atlantic bluefin tunas.

Parasite communities of wild and reared bluefin tuna display remarkable diversity. Among these, the most prevalent and abundant are the Didymozoidae (Monticelli, 1888) (Trematoda, Digenea), considered one of the most taxonomically complex digenean families. The aim of this study was to evaluate phylogenetic structure of Didymozoidae occurring in Pacific (Thunnus orientalis) and Atlantic bluefin tuna (T. thynnus) in order to increase our knowledge of didymozoid zoogeography and identify species that could successfully be employed as biological tags for stock assessment studies. For the present analyses we used 2 nuclear ribosomal DNA loci, part of the 28S gene and the second internal transcribed spacer (ITS-2) as well as a portion of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). In most parasitic groups, morphology is the primary factor in the structuring of phylogenetic relationships. In rare examples, however, habitat has been suggested as a primary factor affecting parasite evolution. During their evolution, didymozoids have spread and inhabited a remarkable number of different sites in their hosts, colonizing exterior as well as strictly interior niches. Our data suggest that habitat selection has been the leading force in shaping didymozoid phylogenetic relationships. For 2 didymozoid species (D. wedli and D. palati), cox1 sequences indicate intraspecific differences between Mexican and Adriatic populations.

Cloning and expression analysis of three striped trumpeter (Latris lineata) pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-8, in response to infection by the ectoparasitic, Chondracanthus goldsmidi.

This study reports the cloning and sequencing of three striped trumpeter (Latris lineata Forster) pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-8, as well as their differential expression in response to an infection by the ectoparasite Chondracanthus goldsmidi. The striped trumpeter TNF-alpha transcript consisted of 1093 bp, including a 759 bp ORF which translated into a 253 aa transmembrane peptide. The sequence contained a TACE cut site, that would produce a 167 aa soluble peptide containing the TNF ligand family signature. The IL-1beta sequence consisted of 963 bp, including a 774 bp ORF which translated into a 258 aa protein. The protein lacked both a signal peptide and an ICE cleavage site, but did contain the IL-1 family signature. The sequence for the chemokine IL-8 contained 906 bp, with an ORF of 297 bp, which translated into a 99 aa protein. The protein lacked an ELR motif as is common with many teleost IL-8 sequences. The differential expression of the three cytokine genes in parasitized fish was investigated via quantitative real-time PCR. A significant up-regulation of all three pro-inflammatory cytokines was found in the gills, which were the site of parasite attachment. Examination of head kidney cells revealed a significant up-regulation of TNF-alpha, but not IL-1beta or IL-8. Conversely, the spleen cells showed significant up-regulation of both IL-1beta and IL-8, but not TNF-alpha. These findings allow for more detailed investigations of the striped trumpeter immune response.

Seasonal epizootics of sea lice, Caligus spp., on southern bluefin tuna, Thunnus maccoyii (Castelnau), in a long-term farming trial.

Within the typical 2-8 month (January to August inclusive) farming cycle for southern bluefin tuna, Thunnus maccoyii (Castelnau), in Spencer Gulf, South Australia, counts of a sea louse, Caligus chiastos Lin et Ho, 2003, were strongly statistically associated with both fish condition and severity of eye damage. During a trial examining the feasibility of maintaining T. maccoyii in farms for more than 1 year, including over the summer season when temperatures may exceed 24 degrees C, we collected additional epidemiological data on burdens of sea lice over a 17-month period (April 2005 to August 2006 inclusive), on a total of 200 T. maccoyii and 40 'control'T. maccoyii farmed and harvested within 2006. In the first farming season, an epizootic of C. chiastos was characterized by a significant increase in prevalence from 0% to 55% in the first 6 weeks after transfer to farms from the wild, which was followed by a significant decline to zero over the next 12 weeks. A single specimen of a second species of Caligus was also detected within this 4.5-month period. In the second farming season, we recorded a third species of sea louse, C. amblygenitalis Tripathi, 1961. In March 2006, a second epizootic peak occurred, this time with mixed infections of C. chiastos and C. amblygenitalis, with a combined prevalence of 100%. The prevalence of both sea lice species then declined significantly over the second winter period (June to August inclusive). On all but one date that sea lice were detected, sea lice counts were significantly associated with the severity of gross eye damage. Because both peaks in infection occurred in summer months (December to February inclusive), we conclude that infections of sea lice pose a risk to the farming of T. maccoyii under certain summer conditions within Spencer Gulf.

Coordinated down-regulation of the antigen processing machinery in the gills of amoebic gill disease-affected Atlantic salmon (Salmo salar L.).

Several important cultured marine fish are highly susceptible to an ectoparasitic condition known as amoebic gill disease (AGD). In AGD-affected fish, modulation of IL-1beta, p53 and p53-regulated transcripts is restricted to the (multi)focal AGD-associated gill lesions. To determine whether this lesion-restricted modulation of transcripts occurs on a transcriptome-wide scale and to identify mechanisms that underpin the susceptibility of fish to AGD, we compared the transcriptome of AGD lesions with "normal" tissue from AGD-affected and healthy individuals. Global gene expression profiling using a 16K salmonid microarray, revealed a total of 176 significantly regulated annotated features and of those, the modulation of 99 (56%) was lesion-restricted. Annotated transcripts were classified according to functional gene ontology. Within the immune response category, transcripts were almost universally down-regulated. In AGD-affected tissue, significant, coordinated down-regulation of the major histocompatibility complex class I (MHC I) pathway-related genes occurred during the later stages of infection and appeared to be mediated by down-regulation of interferon-regulatory factor (IRF)-1, independent of interferon-alpha, interferon-gamma and IRF-2 expression. Within this micro-environment, suppression of the MHC I and possibly the MHC II pathways may inhibit the development of acquired immunity and could explain the unusually high susceptibility of Atlantic salmon to AGD.

Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.

MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease.

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II beta chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II beta chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II+ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II+ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II+ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II+ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.