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.     

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.     

Effects of stocking density and feeding duration in cage‐cum‐pond‐integrated system on growth performance,water quality and economic benefits of Labeo victorianus (Boulenger 1901) culture

We evaluated the effect of varying cage stocking density (60, 90 and 120 fish m^?3) and feeding duration (10, 30 and 60 min) in a cage‐cum‐pond‐integrated system on growth performance, water quality and economic benefits in Labeo victorianus culture. Interactions between stocking density and feeding duration significantly (P < 0.05) affected the fish growth performance and yields in the cages‐cum‐pond system. Stocking density of 60 fish m^?3 resulted in the highest growth in cages and in ponds regardless of the feeding duration, but produced lower yields than at stocking density 90 fish m^?3_. The lowest Apparent Food Conversion Ratio (AFCR) in cages occurred at stocking density of 60 fish m^?3 and feeding duration of 30 min. Growth performance in the open ponds declined with increased feeding duration of the caged fish. Survival in cages and in the open ponds decreased with increased cage density, but was not affected by feeding duration. Low dissolved oxygen were recorded, at stocking density of 120 fish m^?3, the lowest DO occurred when feeding of caged fish lasted 60 min. Growth performance, water quality and economic benefits in Labeo victorianus culture positively respond to interaction between stocking density and feeding durations.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real‐time bioluminescence imaging

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual‐labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild‐type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24‐h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.     

Aeromonas salmonicida infectivity studies in goldsinny wrasse, Ctenolabrus rupestris (L.)

Goldsinny wrasse, Ctenolabrus rupestris (L.). were experimentally and naturally infected with Aeromonas salmonicida. Goldsinny wrasse were found to be susceptible to infection with A. salmonicida but significantly more resistant to infection than Atlantic salmon, Salmo salar L. The pathology of the acute infection is described. Following infection significantly higher levels of agglutinating antibody were detected in the sera of recovered wrasse than those observed in the Atlantic salmon controls. However, a large proportion of the recovered wrasse were culture positive for A. salmonicida and appeared to have entered a chronic stage of infection quite distinct from the carrier status that can develop in Atlantic salmon. This study indicates that, although goldsinny wrasse are susceptible to A. salmonicida, these fish are unlikely to become primarily infected, but contract furunculosis from infected Atlantic salmon. However, goldsinny wrasse may act as a reservoir for subsequent infections of cultivated Atlantic salmon because of the development of a chronic form of the disease. The potential for the vaccination of goldsinny wrasse against furunculosis is discussed.     

Detection and quantification of Aeromonas salmonicida in fish tissue by real‐time PCR

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg^?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida.     

Systemic infection in European perch with thermoadapted virulent Aeromonas salmonicida (Perca fluviatilis)

In non‐salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold‐water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.     

Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida

A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10⁴ ± 1 × 10⁴ CFU g^?1 (without enrichment) and 40 ± 10 CFU g^?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.     

Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real‐time quantitative PCR

Aeromonas schubertii is a major epidemiological agent that threatens cultured snakeheads (Channidae) and has caused great economic losses in fish‐farming industries in China in recent years. In present study, a specific TaqMan minor groove binder (MGB) probe fluorescence real‐time quantitative PCR (qPCR) assay was developed to rapidly detect and quantify A. schubertii. A pair of qPCR primers and a TaqMan MGB probe were selected from the rpoD gene, which were shown to be specific for A. schubertii. A high correlation coefficient (R² = 0.9998) in a standard curve with a 103% efficiency was obtained. Moreover, the qPCR method's detection limit was as low as 18 copies/μl, which was 100 times more sensitive than that of conventional PCR. The detection results for the A. schubertii in pond water and fish tissue were consistent with those of the viable counts. Bacterial load changes detected by qPCR in different tissues of snakeheads infected with A. schubertii showed that the gills and intestines may be the entry for A. schubertii, and the spleen and kidney are major sites for A. schubertii replication. The established method in present study should be a useful tool for the early surveillance and quantitation of A. schubertii.     

Colonization of Aeromonas salmonicida subsp. masoucida strains in Atlantic salmon (Salmo salar L.) during infection

Aeromonas salmonicida subsp. masoucida (ASM) is classified as atypical A. salmonicida and brought huge economic damages to the local salmonid aquaculture in China. An ASM strain named AS‐C4 was used to investigate the colonization of ASM in Atlantic salmon (Salmo salar L.) by an immersion challenge with the control group (T0, no AS‐C4), group T1 (2.67 × 10⁴ CFU/ml AS‐C4) and group T2 (2.67 × 10⁷ CFU/ml AS‐C4). The numbers of AS‐C4 copies in different fish tissues (gill, intestine, skin, blood, muscle, spleen, liver and kidney) were determined at different time points post challenge using the quantitative real‐time PCR (qRT‐PCR). AS‐C4 were detected in the gill and intestine as early as 0 hr after the challenge both in T1 and T2 groups, suggesting that the gill and intestine were probably the portals of entry of AS‐C4 into salmon. Although AS‐C4 could not be detected in the skin until 24 hr after the challenge in T1 group, it could be detected in the skin as early as 0 hr after the challenge in T2 group, indicating that the skin may also be a portal of entry of AS‐C4 into salmon. AS‐C4 was immediately detected in the blood within 3 hr after it entered the host, suggesting that AS‐C4 successfully invaded the bloodstream of fish. After AS‐C4 colonized the host, it colonized the internal tissues, such as the spleen, liver, kidney and muscle. The results of this study will contribute to the understanding of the pathogenesis of the ASM strains and give a broader understanding of the infection route of ASM in it's host, providing more information for the development of new therapeutic strategies to protect against this pathogen in aquaculture.     

Cold‐water vibriosis. The current status of knowledge

The current review for the first time summarizes the findings of the 30 years of research on cold‐water vibriosis (CWV). The diseased caused by Aliivibrio salmonicida (earlier known as Vibrio salmonicida) was for the first time described in 1986 and became one of the most important bacterial diseases in salmon aquaculture. The lack of appropriate vaccine hampered development of Atlantic salmon aquaculture until the late 1980s when a novel vaccine allowed dramatic increase in the Atlantic salmon farming. In December 2007, the genus Vibrio was split into two genera and several bacterial species including V. salmonicida were transferred to genus Aliivibrio. The change of the names create significant difficulties with the designation of the CWV disease agent since its abbreviation A. salmonicida became similar to another well‐known salmon pathogen Aeromonas salmonicida (A. salmonicida). The disease was considered as controlled by vaccination, but reappeared at Atlantic salmon farms in 2011, this time affecting vaccinated Atlantic salmon. The current review summarizes the knowledge on pathogenesis, vaccination and treatment of CWV and proposes further directions for studying the disease.     

vapA (A‐layer) typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes

Sequence variation in a region of the virulence array protein gene (vapA; A‐layer) was assessed in 333 (‘typical’ and ‘atypical’) isolates of the fish pathogenic bacterium Aeromonas salmonicida. Resulting similarity dendrograms revealed extensive heterogeneity, with nearly all isolates belonging to either of 14 distinct clusters or A‐layer types. All acknowledged A. salmonicida subspecies (except ssp. pectinolytica, from which no vapA sequence could be obtained) were clearly separated, and notably, all isolates phenotypically identified as ssp. salmonicida formed a distinct and exclusive A‐layer type. Additionally, an array of un‐subspeciated atypical strains formed several equally prominent clusters, demonstrating that the concept of typical/atypical A. salmonicida is inappropriate for describing the high degree of diversity evidently occurring outside ssp. salmonicida. Most representatives assessed in this study were clinical isolates of spatiotemporally diverse origins, and were derived from a variety of hosts. We observed that from several fish species or families, isolates predominantly belonged to certain A‐layer types, possibly indicating a need for host‐/A‐layer type‐specific A. salmonicida vaccines. All in all, A‐layer typing shows promise as an inexpensive and rapid means of unambiguously distinguishing clinically relevant A. salmonicida subspecies, as well as presently un‐subspeciated atypical strains.     

Ca. Branchiomonas cysticola,Ca. Piscichlamydia salmonis and Salmon Gill Pox Virus transmit horizontally in Atlantic salmon held in fresh water

Elucidation of the role of infectious agents putatively involved in gill disease is commonly hampered by the lack of culture systems for these organisms. In this study, a farmed population of Atlantic salmon pre‐smolts, displaying proliferative gill disease with associated Candidatus Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Atlantic salmon gill pox virus (SGPV) infections, was identified. A subpopulation of the diseased fish was used as a source of waterborne infection towards a population of naïve Atlantic salmon pre‐smolts. Ca. B. cysticola infection became established in exposed naïve fish at high prevalence within the first month of exposure and the bacterial load increased over the study period. Ca. P. salmonis and SGPV infections were identified only at low prevalence in exposed fish during the trial. Although clinically healthy, at termination of the trial the exposed, naïve fish displayed histologically visible pathological changes typified by epithelial hyperplasia and subepithelial inflammation with associated bacterial inclusions, confirmed by fluorescent in situ hybridization to contain Ca. B. cysticola. The results strongly suggest that Ca. B. cysticola infections transmit directly from fish to fish and that the bacterium is directly associated with the pathological changes observed in the exposed, previously naïve fish.     

Evaluation of sodium percarbonate as a bath treatment for amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a major health challenge for Atlantic salmon aquaculture globally. While freshwater bathing for 2 hr is effective in reducing infection severity, there is need for more rapid and lower cost alternatives. To this end, a combination of sodium percarbonate (SPC) in freshwater was examined for its treatment efficacy. Initial in vitro studies showed a reduction in amoeba viability when exposed for 30 min to freshwater containing >500 mg/L SPC. Subsequently, AGD‐affected salmon were bathed for 30 min in 16°C freshwater containing 100, 500 or 1,000 mg/L SPC, or for 2 hr in 16°C freshwater to mimic industry practice. Treatment at the highest SPC concentration caused extensive gill damage and substantial mortality. Neither occurred to a significant extent at lower SPC concentrations. Gill pathology of surviving fish 10 days post‐treatment (dpt) was comparable to or more severe than pre‐treatment, and significantly (p < .001) more severe than in 2 hr freshwater bathed fish. N. perurans DNA was confirmed by qPCR in all treatment groups at 10 dpt. The data indicate that a 30‐min exposure to SPC in freshwater is not a suitable alternative to existing freshwater treatment of AGD.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.