Prevalence,geographic distribution and phenotypic differences of Piscirickettsia salmonis EM‐90‐like isolates

Early reports accounted for two main genotypes of Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis, placing the single isolate EM‐90 apart from the prototypic LF‐89 and related isolates. In this study, we provide evidence that, contrary to what has been supposed, the EM‐90‐like isolates are highly prevalent and disseminated across Chilean marine farms. Molecular analysis of 507 P. salmonis field isolates derived from main rearing areas, diverse hosts and collected over 6 years, revealed that nearly 50% of the entire collection were indeed typed as EM‐90‐like. Interestingly, these isolates showed a marked host preference, being recovered exclusively from Atlantic salmon (Salmo salar) samples. Although both strains produce undistinguishable pathological outcomes, differences regarding growth kinetics and susceptibility to the antibiotics and bactericidal action of serum could be identified. In sum, our results allow to conclude that the EM‐90‐like isolates represent an epidemiologically relevant group in the current situation of piscirickettsiosis. Based on the consistency between genotype and phenotype exhibited by this strain, we point out the need for genotypic studies that may be as important for the Chilean salmon industry as the continuous surveillance of antimicrobial susceptibility patterns.     

Development of a Piscirickettsia salmonis immersion challenge model to investigate the comparative susceptibility of three salmon species

Piscirickettsia salmonis, the aetiological agent of salmonid rickettsial septicaemia (SRS), is a global pathogen of wild and cultured marine salmonids. Here, we describe the development and application of a reproducible, standardized immersion challenge model to induce clinical SRS in juvenile pink (Oncorhynchus gorbuscha), Atlantic (Salmo salar) and sockeye salmon (O. nerka). Following a 1‐hr immersion in 10⁵ colony‐forming units/ml, cumulative mortality in Atlantic salmon was 63.2% while mortality in sockeye salmon was 10%. Prevalence and levels of the bacterium in kidney prior to onset of mortality were lower in sockeye compared with Atlantic or pink salmon. The timing and magnitude of bacterial shedding were estimated from water samples collected during the exposure trials. Shedding was estimated to be 82‐fold higher in Atlantic salmon as compared to sockeye salmon and peaked in the Atlantic salmon trial at 36 d post‐immersion. These data suggest sockeye salmon are less susceptible to P. salmonis than Atlantic or pink salmon. Finally, skin lesions were observed on infected fish during all trials, often in the absence of detectable infection in kidney. As a result, we hypothesize that skin is the primary point of entry for P. salmonis during the immersion challenge.     

Development of piscirickettsiosis in Atlantic salmon (Salmo salar L.) smolts after intraperitoneal and cohabitant challenge using an EM90‐like isolate: A comparative study

Piscirickettsiosis, caused by the intracellular Gram‐negative bacteria Piscirickettsia salmonis, is at present the most devastating disease in the Chilean salmon industry. The aim of this study was to analyse disease development after challenge with a P. salmonis strain (EM90‐like) under a controlled environment by comparing intraperitoneal challenge with cohabitation challenge. The P. salmonis EM90‐like isolate was cultured in a liquid medium for the challenge of 400 Atlantic salmon (Salmo salar) smolts. Cumulative mortality was registered, necropsy was performed, and bacterial distribution in the tissues and histopathological changes were analysed. The results revealed a similar progression of the disease for the two different challenge models. Pathological and histopathological changes became more visible during the development of the clinical phase of the disease. Bacterial DNA was identified in all the analysed tissues indicating a systemic infection. Bacterial tropism to visceral organs was demonstrated by real‐time quantitative PCR and immunohistochemistry. Better knowledge of disease development during P. salmonis infection may contribute to further development of challenge models that mimic the field situation during piscirickettsiosis outbreaks. The models can be used to develop and test future preventive measures against the disease.     

Histology,immunocytochemistry and qRT‐PCR analysis of Atlantic salmon,Salmo salar L., post‐smolts following infection with infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post‐smolts. Post‐smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post‐infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fish’s metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up‐regulation of cytokine gene expression was found only in the IHC‐positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up‐regulated in liver and kidney, while only IFN and Mx were up‐regulated in gill. IL1β and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1β and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over‐produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.     

Tissue and organ distribution of 14C‐activity in dextrin‐adapted Atlantic salmon after oral administration of radiolabelled 14C1‐glucose

Accumulation of ¹⁴C in various tissues and organs was studied in three different groups of 0.8‐kg Atlantic salmon Salmo salar force‐fed with ¹⁴C₁‐glucose in order to evaluate if metabolism of glucose depended on adaptation to dietary carbohydrate level. The salmon had been fed diets supplemented with 0, 100 and 200 g maize dextrin kg^?1 for 10 months before the experiment. The fish were force‐fed 6.65 × 10⁴ Bq of ¹⁴C₁ glucose kg^?1 BW, in gelatin capsules. Fish for analysis were obtained 16 h later. ¹⁴C was measured in blood plasma, gill, kidney, liver and white muscle, and in lipid extract of liver. The liver contained most ¹⁴C, followed by heart, blood plasma, gill and liver lipid extract, while kidney and muscle contained the least ¹⁴C per gram or millilitre tissue. The muscle contained most radioactivity, on an estimated total tissue basis, followed by liver, blood plasma, gill, liver lipid extract, kidney and heart tissue. Thirty‐eight per cent of the orally administered ¹⁴C was recovered in the salmon adapted to the diet without dextrin after 16 h. This was significantly (P < 0.05) higher than the 30% and 32% recovered in the salmon adapted to diets with 10% and 20% dextrin. This effect on adaptation to dietary dextrin level in glucose uptake or metabolism was supported by a trend (P < 0.10) toward higher radioactivity per gram or millilitre of each individual tissue in the fish adapted to the diet without dextrin, when compared with the other two adaptation regimes.     

Cloning,characterization and transcription analysis of fatty acyl desaturase (Δ6Fad‐like) and elongase (Elovl4‐like,Elove5‐like) during embryos development of Strongylocentrotus intermedius

Strongylocentrotus intermedius has high nutritional value because it is rich in proteins, amino acids and long‐chain polyunsaturated fatty acids (LC‐PUFA). LC‐PUFA are essential nutrients that not only determine the nutritional value of sea urchins but also guarantee normal growth and reproduction performance. To better understand the molecular basis of LC‐PUFA biosynthesis in S. intermedius, the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were cloned and fatty acid compositions during the early developmental stages of sea urchins were detected. The full‐length of Δ6Fad‐like was 2,199 bp, with a putative open reading frame of 1,248 bp encoding a polypeptide of 415 amino acid (AA). The Elovl4‐like and Elovl5‐like genes encoded 310 and 234 AA, respectively, which exhibited all of the characteristics of the Elovl family, such as a histidine box motif and putative transmembrane‐spanning domains. Tissue distribution analysis revealed that Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were expressed at the highest levels in the gonads and intestine, and the expression levels gradually increased in embryos during development. These results can help to understand the role of the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes in the different developmental stages of the sea urchin and to clarify the biosynthetic pathways of LC‐PUFA during the development of the sea urchin and provide a theoretical basis for improving the quality and breeding of excellent traits in sea urchins.     

Occurrence of trypanosomiasis in net‐cage cultured groupers (Cromileptes altivelis and Epinephelus fuscoguttatus) in Nanshan port of Sanya,Hainan province,China

In the present study, reported was an unidentified trypanosome that caused massive mortalities of cultured humpback grouper, Cromileptes altivelis (Valenciennes, 1828) and brown‐marbled grouper, Epinephelus fuscoguttatus (Forsskål, 1775) in the net‐cages in Sanya, China, and some general pathological features of E. fuscoguttatus after experimental infection of the trypanosome. Before dying in the net cages of diseased fish, listlessness and anorexia were the observable symptoms. The blood of the diseased fish was light‐coloured in comparison with that of healthy one. E. fuscoguttatus experimentally infected with this trypanosome are with different degrees of engorgement in gill lamella, liver, spleen and kidney. Tissue lesion and necrosis were observed in liver and spleen of diseased fish.     

Aeromonas shuberti as a cause of multi‐organ necrosis in internal organs of Nile tilapia,Oreochromis niloticus

A disease with white spots in internal organs of Nile tilapia occurred in Zhanjiang, southern China. Multiple, white nodules, 0.8–2.2 mm in diameter, were scattered throughout the liver, spleen and kidney of diseased fish. Signs of nodules reproduced after artificial infection with the isolated strain. Isolated bacteria were Gram‐negative, facultative anaerobic, motile, short rod‐shaped, with a length of 1.2–2.2 μm. Morphological and biochemical tests, as well as phylogenetic analysis, all strongly indicated that the isolate from tilapia is identical to Aeromonas schubertii (A. schubertii) which temporary named LF1708 strain. Antibiotic sensitivity assays showed the LF1708 is sensitive to 24 of 27 tested antibiotics. Pathogenicity test revealed that the isolate at the dose of 3.75 × 10⁶ CFU/g killed 100% of experimental tilapia within 2 days and the dose of 1 × 10⁷ CFU/g killed 100% of experimental zebrafish within 1 day. Histopathology of diseased tilapia infected with A. schubertii showed numerous necrotic lesions widely distributed in spleen, liver and kidney, and infiltration with a large number of bacteria. To our knowledge, this was the first report that associated A. schubertii with mortality in tilapia.     

Susceptibility of freshwater rearing Asian seabass (Lates calcarifer) to pathogenic Streptococcus iniae

Asian seabass (Lates calcarifer) has been recognized as an economically important aquaculture species which can be adapted to and cultivated in wide range of salinities. The number of freshwater intensive seabass farms in Thailand is increasing annually. Here, we first describe the susceptibility of Asian seabass, which were cultured in freshwater, to Streptococcus inae (SI) and their pathological changes. Three isolates of putative SI were identified using a combination of standard biochemical assays and species‐specific PCR prior subjected to in vivo challenge. Accumulated mortalities of the fish which received 10⁷ CFU fish^?1 of either SI1J, SI SGSA or SI2J were 90%, 90% and 100% at 7 days‐post infection (dpi), respectively, and mortalities increased sharply between 3 and 5 dpi. Clinical signs such as erratic swimming and opaque eyes were identified from a few infected fish, while most died rapidly without any abnormal signs. Histopathological manifestations were observed in the multiple organs (kidney, liver and brain). Haemorrhage, hyperhemia, cellular degeneration and inflammatory cells infiltration were commonly found within the internal organs. Notably, the formation of numerous encyst‐like lesion aggregated by eosinophilic cells, resembling macrophages, were typically found in the brain of the infected fish. Summarily, this study first revealed that freshwater reared Asian seabass is highly susceptible to SI infection and haemorrhagic septicaemia was a major pathological change that could be found in the infected fish.     

New Zealand rickettsia‐like organism (NZ‐RLO) and Tenacibaculum maritimum: Distribution and phylogeny in farmed Chinook salmon (Oncorhynchus tshawytscha)

A total of 777 fish from three growing regions of New Zealand Chinook salmon farms comprising of five sites were tested. Quantitative PCR was used to determine the distribution of New Zealand rickettsia‐like organism and Tenacibaculum maritimum. Genetic information from these bacteria were then compared with strains reported worldwide. Using this information, suggested associations of pathogens with clinically affected fish were made. NZ‐RLO was detected in two of the three regions, and T. maritimum was detected in all regions. Three strains of NZ‐RLO were identified during this study. Based on analysis of the ITS rRNA gene, NZ‐RLO1 appears to be part of an Australasian grouping sharing high similarity with the Tasmanian RLO, NZ‐RLO2 was shown to be the same as an Irish strain, and NZ‐RLO3 was shown be closely related to two strains from Chile. Based on multi‐locus sequence typing, the New Zealand T. maritimum was the same as Australian strains. NZ‐RLOs were detected more frequently in fish with skin ulcers than fish without skin ulcers. While additional research is required to investigate the pathogenicity of these organisms, this is the first time that NZ‐RLOs have been associated with the development of clinical infections in farmed Chinook salmon.     

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non‐salmonid fish

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery‐propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment‐producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.     

In vivo growth and genomic characterization of rickettsia‐like organisms isolated from farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand

A rickettsia‐like organism, designated NZ‐RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ‐RLO2 was able to grow in CHSE‐214, EPC, BHK‐21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF‐89^T grew in all but BHK‐21 and Sf21. NZ‐RLO2 grew optimally in EPC at 15°C, CHSE‐214 and EPC at 18°C. The growth of LF‐89 ^T was optimal at 15°C, 18°C and 22°C in CHSE‐24, but appeared less efficient in EPC cells at all temperatures. Pan‐genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p‐value = 94%). NZ‐RLO2 was genetically different from previously described NZ‐RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p‐value = 88%), but were closely related to each other. TaqMan and Sybr Green real‐time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ‐RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes

Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A. salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A. salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A. salmonicida have an intraerythrocytic phase. Here, we evaluate whether A. salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A. salmonicida did not kill primary S. salar erythrocytes, even in the presence of high bacterial loads, but A. salmonicida invaded the S. salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A. salmonicida showed bacteraemia 5 days post‐infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.     

Synergistic osmoregulatory dysfunction during salmon lice (Lepeophtheirus salmonis) and infectious hematopoietic necrosis virus co‐infection in sockeye salmon (Oncorhynchus nerka) smolts

While co‐infections are common in both wild and cultured fish, knowledge of the interactive effects of multiple pathogens on host physiology, gene expression and immune response is limited. To evaluate the impact of co‐infection on host survival, physiology and gene expression, sockeye salmon Oncorhynchus nerka smolts were infected with the salmon louse Lepeophtheirus salmonis (V?/SL+), infectious hematopoietic necrosis virus (IHNV; V+/SL?), both (V+/SL+), or neither (V?/SL?). Survival in the V+/SL+ group was significantly lower than the V?/SL? and V?/SL+ groups (p = 0.024). Co‐infected salmon had elevated osmoregulatory indicators and lowered haematocrit values as compared to the uninfected control. Expression of 12 genes associated with the host immune response was analysed in anterior kidney and skin. The only evidence of L. salmonis‐induced modulation of the host antiviral response was down‐regulation of mhc I although the possibility of modulation cannot be ruled out for mx‐1 and rsad2. Co‐infection did not influence the expression of genes associated with the host response to L. salmonis. Therefore, we conclude that the reduced survival in co‐infected sockeye salmon resulted from the osmoregulatory consequences of the sea lice infections which were amplified due to infection with IHNV.     

Mycobacteria in aquarium fish: results of a 3‐year survey indicate caution required in handling pet‐shop fish

Fish are commonly infected with non‐tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real‐time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M. marinum, Mycobacterium fortuitum (M. fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well‐known human pathogen M. marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.