Reclassification of Francisella noatunensis subsp. orientalis Ottem et al. 2009 as Francisella orientalis sp. nov., Francisella noatunensis subsp. chilensis subsp. nov. and emended description of Francisella noatunensis.

Francisella noatunensis is a fastidious facultative intracellular bacterial pathogen that causes 'piscine francisellosis', a serious disease affecting both marine and fresh water farmed and wild fish worldwide. Currently two F. noatunensis subspecies are recognized, i.e. F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis. In the present study, the taxonomy of F. noatunensis was revisited using a polyphasic approach, including whole genome derived parameters such as digital DNA-DNA hybridization, whole genome average nucleotide identity (wg-ANIm), whole genome phylogenetic analysis, whole genome G+C content, metabolic fingerprinting and chemotaxonomic analyses. The results indicated that isolates belonging to F. noatunensis subsp. orientalis represent a phenotypically and genetically homogenous taxon, clearly distinguishable from F. noatunensis subsp. noatunensis that fulfils requirements for separate species status. We propose, therefore, elevation of F. noatunensis subsp. orientalis to the species rank as Francisella orientalis sp. nov. with the type strain remaining as Ehime-1T (DSM 21254T=LMG 24544T). Furthermore, we identified sufficient phenotypic and genetic differences between F. noatunensis subsp. noatunensis recovered from diseased farmed Atlantic salmon in Chile and those isolated from wild and farmed Atlantic cod in Northern Europe to warrant proposal of the Chilean as a novel F. noatunensis subspecies, i.e. Francisella noatunensis subsp. chilensis subsp. nov. with strain PQ1106T (CECT 9798T=NCTC14375T) as the type strain. Finally, we emend the description of F. noatunensis by including further metabolic information and the description of atypical strains.

Establishing bacterial infectivity models in striped Catfish Pangasianodon hypophthalmus (Sauvage) with Edwardsiella ictaluri.

A bacterial infectivity challenge model of Edwardsiella ictaluri in striped catfish was developed. All experiments were conducted using a bacterial isolate of E. ictaluri that had been recovered during a natural outbreak of bacillary necrosis of Pangasianodon (BNP) in farmed striped catfish Pangasianodon hypophthalmus in Vietnam. Time of immersion in 107  CFU.ml-1 had a significant effect on mortality. The immersion bacterial dose of 107  CFU/ml for 30 s resulted in a cumulative percentage mortality of 63%. Three to four days post-bacterial challenge, fish showed gross clinical signs of natural BNP and E. ictaluri was recovered and identified from these fish. Moreover, a cohabitation challenge was evaluated as an alternative challenge method, although the mortalities among the infected fish were lower at around 15%-40%. This study confirmed the horizontal transmission of E. ictaluri in striped catfish and elucidated that cohabitation challenge could be used in reproducing the disease under controlled conditions.

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection.

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm-water fish for which no vaccines are commercially available. In this study, a whole cell formalin-inactivated vaccine was developed for the first time using the highly virulent isolate STIR-GUS-F2f7 and the oil-based adjuvant Montanide™ ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post-vaccination, all fish were i.p. injected with 4.0 × 103 CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia.

Characterisation of proteins in excretory/secretory products collected from salmon lice, Lepeophtheirus salmonis.

BACKGROUND: The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic copepod which feeds on the mucus, skin and blood of salmonid fish species. The parasite can persist on the surface of the fish without any effective control being exerted by the host immune system. Other ectoparasitic invertebrates produce compounds in their saliva, excretions and/or secretions which modulate the host immune responses allowing them to remain on or in the host during development. Similarly, compounds are produced in secretions of L. salmonis which are thought to be responsible for immunomodulation of the host responses as well as other aspects of crucial host-parasite interactions. METHODS: In this study we have identified and characterised the proteins in the excretory/secretory (E/S) products of L. salmonis using LC-ESI-MS/MS. RESULTS: In total 187 individual proteins were identified in the E/S collected from adult lice and pre-adult sea lice. Fifty-three proteins, including 13 serine-type endopeptidases, 1 peroxidase and 5 vitellogenin-like proteins were common to both adult and pre-adult E/S products. One hundred and seven proteins were identified in the adult E/S but not in the pre-adult E/S and these included serine and cysteine-type endopeptidases, vitellogenins, sphingomyelinase and calreticulin. A total of 27 proteins were identified in pre-adult E/S products but not in adult E/S. CONCLUSIONS: The assigned functions of these E/S products and the potential roles they play in host-parasite interaction is discussed.

Impact of Salmonid alphavirus infection in diploid and triploid Atlantic salmon (Salmo salar L.) fry.

With increasing interest in the use of triploid salmon in commercial aquaculture, gaining an understanding of how economically important pathogens affect triploid stocks is important. To compare the susceptibility of diploid and triploid Atlantic salmon (Salmo salar L.) to viral pathogens, fry were experimentally infected with Salmonid alphavirus sub-type 1 (SAV1), the aetiological agent of pancreas disease (PD) affecting Atlantic salmon aquaculture in Europe. Three groups of fry were exposed to the virus via different routes of infection: intraperitoneal injection (IP), bath immersion, or cohabitation (co-hab) and untreated fry were used as a control group. Mortalities commenced in the co-hab challenged diploid and triploid fish from 11 days post infection (dpi), and the experiment was terminated at 17 dpi. Both diploid and triploid IP challenged groups had similar levels of cumulative mortality at the end of the experimental period (41.1% and 38.9% respectively), and these were significantly higher (p < 0.01) than for the other challenge routes. A TaqMan-based quantitative PCR was used to assess SAV load in the heart, a main target organ of the virus, and also liver, which does not normally display any pathological changes during clinical infections, but exhibited severe degenerative lesions in the present study. The median viral RNA copy number was higher in diploid fish compared to triploid fish in both the heart and the liver of all three challenged groups. However, a significant statistical difference (p < 0.05) was only apparent in the liver of the co-hab groups. Diploid fry also displayed significantly higher levels of pancreatic and myocardial degeneration than triploids. This study showed that both diploid and triploid fry are susceptible to experimental SAV1 infection. The lower virus load seen in the triploids compared to the diploids may possibly be related to differences in cell metabolism between the two groups, however, further investigation is necessary to confirm this and also to assess the outcome of PD outbreaks in other developmental stages of the fish when maintained in commercial production systems.

Draft Genome Sequence of Francisella noatunensis subsp. orientalis STIR-GUS-F2f7, a Highly Virulent Strain Recovered from Diseased Red Nile Tilapia Farmed in Europe.

A highly virulent strain of Francisella noatunensis subsp. orientalis, STIR-GUS-F2f7, was isolated from moribund red Nile tilapia (Oreochromis niloticus) farmed in Europe. In this communication, the complete genome sequencing of this bacterium is reported.

A Polyphasic Approach for Phenotypic and Genetic Characterization of the Fastidious Aquatic Pathogen Francisella noatunensis subsp. orientalis.

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis, an emerging infectious disease in Asia and Latin America. In this study two outbreaks of francisellosis were diagnosed in the UK on the basis of histopathology, electron microscopy, PCR, bacterial isolation and fulfillment of Koch's postulates. Furthermore, a phenotypic fingerprint based on biochemical analyses, metabolic activity, chemotaxonomic composition, and antimicrobial assays was generated for the novel isolates, the Fno type strain Ehime-1 from Asia and other Fno from Latin America. The genetic relatedness between the novel Fno and other Francisellaceae species was investigated by sequencing and comparing the 16SrRNA gene, 8 housekeeping genes (individually and concatenated) and the 16SrRNA-ITS-23SrRNA sequence. The phenotypic profiling indicated a high degree of similarity among the Fno strains as all were able to metabolize dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate, and glucose-6-phosphate. The chemotaxonomic analyses indicated that 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%), and 18:0 (5.5%) were the predominant structural fatty acids in Fno. The antimicrobial assays showed little variation between the isolates and high susceptibility to enrofloxacin, gentamicin, neomycin, streptomycin, amikacin, ciprofloxacin, gatifloxacin, nitrofurantoin, tobramycin, kanamycin, tetracycline, oxytetracycline, florfenicol, oxolinic acid, and streptomycin in all the Fno analyzed. In all the phylogenetic trees the Fno strains clustered together in independent branches confirming a high degree of homogeneity. Interestingly in five of the 11 trees i.e., mutS, putA, rpoB, 16SrRNA-ITS-23SrRNA, and concatenated sequence the two Francisella noatunensis ssp. diverged more from each other than from the closely related Francisella philomiragia (Fp). The phenotypic and genetic characterization confirmed the Fno isolates represent a solid phylo-phenetic taxon that in the current context of the genus seems to be misplaced within the species Fn. We propose the use of the present polyphasic approach in future studies to characterize strains of Fnn and Fp and verify their current taxonomic rank of Fno and other aquatic Francisella spp.

Pathogenesis of experimental salmonid alphavirus infection in vivo: an ultrastructural insight.

Salmonid alphavirus (SAV) is an enveloped, single-stranded, positive sense RNA virus belonging to the family Togaviridae. It causes economically devastating disease in cultured salmonids. The characteristic features of SAV infection include severe histopathological changes in the heart, pancreas and skeletal muscles of diseased fish. Although the presence of virus has been reported in a wider range of tissues, the mechanisms responsible for viral tissue tropism and for lesion development during the disease are not clearly described or understood. Previously, we have described membrane-dependent morphogenesis of SAV and associated apoptosis-mediated cell death in vitro. The aims of the present study were to explore ultrastructural changes associated with SAV infection in vivo. Cytolytic changes were observed in heart, but not in gill and head-kidney of virus-infected fish, although they still exhibited signs of SAV morphogenesis. Ultrastructural changes associated with virus replication were also noted in leukocytes in the head kidney of virus-infected fish. These results further describe the presence of degenerative lesions in the heart as expected, but not in the gills and in the kidney.

The effect of dietary n-3/n-6 polyunsaturated fatty acid ratio on salmonid alphavirus subtype 1 (SAV-1) replication in tissues of experimentally infected rainbow trout (Oncorhynchus mykiss).

Salmon pancreas disease (SPD) is one of the most commercially significant viral diseases of farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Europe. In this study, the potential for dietary mitigation of the disease using different polyunsaturated fatty acid (PUFA) profiles was assessed in rainbow trout. We experimentally infected fish with salmonid alphavirus subtype 1 (SAV-1), the causative agent of SPD. These fish were fed two diets with different n-3/n-6 PUFA ratio (high omega 3, 3.08, and high omega 6, 0.87). We assessed the influence of the diets on the fatty acid composition of the heart at 0 days post infection (d.p.i.) (after 4 weeks of feeding the experimental diets prior to SAV-1 infection), and sampled infected and control fish at 5, 15 and 30d.p.i. Viral E1 and E2 glycoprotein genes were quantified by two absolute real-time PCRs in all the organs sampled, and significantly lower levels of the virus were evident in the organs of fish fed with high omega 6. Characteristic pathological lesions were identified in infected fish as early as 5d.p.i., with no significant differences in the pathology lesion scores between the two dietary regimes. This study shows that decreasing the n-3/n-6 PUFA ratio in experimental diets of rainbow trout changes the fatty acid content of the fish, and is associated with reduced SAV-1 replication in rainbow trout.

Triploid and diploid Atlantic salmon show similar susceptibility to infection with salmon lice Lepeophtheirus salmonis.

BACKGROUND: Sea lice infection is the most expensive disease factor for Atlantic salmon sea-cage farming. For triploid salmon to be accepted as a commercial possibility, investigation of susceptibility of triploid salmon to sea lice infection is a fundamental milestone. The susceptibility of diploid and triploid salmon to infection with Lepeophtheirus salmonis was examined in a tank trial in Scotland, a tank trial in Norway and a cage trial in Scotland. RESULTS: Following a single infection challenge, results indicated a significant correlation between fish size and the number of attached sea lice. Triploid fish were larger than diploids at the smolt stage. In the tank trials, no difference was found between infection levels on diploids and triploids after a single infection challenge. The tank trial in Scotland continued with a second infection challenge of the same fish, which also showed no infection differences between ploidies. A borderline correlation between first infection and re-infection intensity was found for PIT-tagged diploid salmon examined after each challenge. No significant difference in louse infection between diploid and triploid salmon (∼2 kg) was found in the cage trial undertaken under commercial conditions. CONCLUSION: This study concludes that triploid Atlantic salmon are not more susceptible to sea louse infection than diploid fish.

Interferon-mediated host response in experimentally induced salmonid alphavirus 1 infection in Atlantic salmon (Salmo salar L.).

Salmonid alphavirus (SAV) infection in cultured salmonids causes severe economic losses across Europe. Immune protection and antiviral mechanisms of the host against SAV are poorly characterised in vivo. Analysis of immune gene expression in head kidney of Atlantic salmon (Salmo salar L.) experimentally infected with SAV 1, using a quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealed rapid induction of interferon I (INF-I), interferon II (INF-II) and INF-I associated Mx genes in SAV 1 infected fish compared to control fish injected with tissue culture supernatant. Mx protein was found to be highly expressed in the heart and mucosal membranes of infected fish by immunohistochemistry (IHC). Interestingly, the pathological changes that were observed in the target tissues of the virus became visible some time after peak expression of genes associated with the INF-I-pathway in head kidney tissue. These findings suggest that a non-specific antiviral immune response is rapidly induced during the early stages of SAV infection in salmon.

Transcriptomic analysis of the host response to early stage salmonid alphavirus (SAV-1) infection in Atlantic salmon Salmo salar L.

Salmon pancreas disease, caused by salmonid alphavirus (SAV) of the family Togaviridae, is an economically important disease affecting farmed Atlantic salmon (Salmo salar L.) in Scotland, Norway, and Ireland. The virus causes characteristic lesions in the pancreas, heart, kidney and skeletal muscle of infected fish. The mechanisms responsible for the pathology and the immune responses elicited in infected Atlantic salmon are not fully understood. A microarray-based study was therefore performed to evaluate the host transcriptomic response during the early stages of an experimentally-induced SAV-1 infection. Atlantic salmon parr were injected intra-peritoneally with viral cell culture supernatant or cell culture supernatant without virus. RNA, extracted from head kidney sampled from infected and control fish at 1, 3 and 5 days post-injection (d.p.i.), was interrogated with the 17 k TRAITS/SGP cDNA microarray. The greatest number of significantly differentially expressed genes was recorded at 3 d.p.i., mainly associated with immune and defence mechanisms, including genes involved in interferon I pathways and Major Histocompatibility Complex Class I and II responses. Genes associated with apoptosis and cellular stress were also found to be differentially expressed between infected and uninfected individuals, as were genes involved in inhibiting viral attachment and replication. The microarray results were validated by follow-on analysis of eight genes by real-time PCR. The findings of the study reflect mechanisms used by the host to protect itself during the early stages of SAV-1 infection. In particular, there was evidence of rapid induction of interferon-mediated responses similar to those seen during mammalian alphavirus infections, and also early involvement of an adaptive immune response. This study provides essential knowledge to assist in the development of effective control and management strategies for SAV-1 infection.

Aquaculture: global status and trends.

Aquaculture contributed 43 per cent of aquatic animal food for human consumption in 2007 (e.g. fish, crustaceans and molluscs, but excluding mammals, reptiles and aquatic plants) and is expected to grow further to meet the future demand. It is very diverse and, contrary to many perceptions, dominated by shellfish and herbivorous and omnivorous pond fish either entirely or partly utilizing natural productivity. The rapid growth in the production of carnivorous species such as salmon, shrimp and catfish has been driven by globalizing trade and favourable economics of larger scale intensive farming. Most aquaculture systems rely on low/uncosted environmental goods and services, so a critical issue for the future is whether these are brought into company accounts and the consequent effects this would have on production economics. Failing that, increased competition for natural resources will force governments to allocate strategically or leave the market to determine their use depending on activities that can extract the highest value. Further uncertainties include the impact of climate change, future fisheries supplies (for competition and feed supply), practical limits in terms of scale and in the economics of integration and the development and acceptability of new bio-engineering technologies. In the medium term, increased output is likely to require expansion in new environments, further intensification and efficiency gains for more sustainable and cost-effective production. The trend towards enhanced intensive systems with key monocultures remains strong and, at least for the foreseeable future, will be a significant contributor to future supplies. Dependence on external feeds (including fish), water and energy are key issues. Some new species will enter production and policies that support the reduction of resource footprints and improve integration could lead to new developments as well as reversing decline in some more traditional systems.

Mycobacterium stomatepiae sp. nov., a slowly growing, non-chromogenic species isolated from fish.

Slowly growing, non-chromogenic mycobacteria were isolated from striped barombi mbo cichlids (Stomatepia mariae) maintained at the London Zoo Aquarium, UK. The isolates could be differentiated from other slowly growing, non-pigmented mycobacteria by a combination of phenotypic features including their inability to grow at 37 degrees C, positive tests for heat-stable catalase, tellurite reduction and arylsulfatase activity, and the absence of urease activity, Tween 80 hydrolysis, nitrate reductase, iron uptake and semiquantitative catalase. The almost full-length 16S rRNA gene sequence, together with partial sequences from the 65 kDa heat-shock protein (hsp65) and the beta-subunit of the bacterial RNA polymerase (rpoB) genes and the 16S-23S internal transcribed spacer 1 (ITS 1) region were identical for all three novel strains, but distinct from those of all known mycobacterial species. Phylogenetic analysis based on 16S rRNA gene sequences placed the novel isolates within the slowly growing mycobacteria group in close proximity to Mycobacterium florentinum. Based on genotypic and phenotypic findings, it is proposed that these isolates represent a novel species of the genus Mycobacterium, for which the name Mycobacterium stomatepiae sp. nov. is proposed with strain T11(T) (=DSM 45059(T)=CIP 109275(T)=NCIMB 14252(T)) as the type strain.

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification.

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA).

Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.