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.     

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.     

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.     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

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.     

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.     

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin‐killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin‐killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710‐1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60‐s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.     

Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3′,5′‐monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss)

Precise deletion of genes related to virulence can be used as a strategy to produce attenuated bacterial vaccines. Here, we study the deletion of the cyclic‐3′,5′‐adenosine monophosphate (cAMP) receptor protein (Crp) in Aeromonas salmonicida, the aetiologic agent of furunculosis in marine and freshwater fish. The Crp protein is a conserved global regulator, controlling physiology processes, like sugar utilization. Deletion of the crp gene has been utilized in live attenuated vaccines for mammals, birds and warm water fish. Here, we characterized the crp gene and reported the effect of a crp deletion in A. salmonicida virulent and non‐virulent isolates. We found that A. salmonicida Δcrp was not able to utilize maltose and other sugars, and its generation time was similar to the wild type. A. salmonicida ?crp showed a higher ability of cell invasion compared to the wild type. Fish challenges showed that A. salmonicida ?crp is ~6 times attenuated in Oncorhynchus mykiss and conferred protective immunity against the intraperitoneal challenge with A. salmonicida wild type. We concluded that deletion of A. salmonicida crp influences sugar utilization, cell invasion and virulence. Deletion of crp in A. salmonicida could be considered as part of an effective strategy to develop immersion live attenuated vaccines against furunculosis.     

The protective effect of humic‐rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.)

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic‐rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic‐rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite‐derived humic‐rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic‐rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic‐rich water from the recirculating system, the synthetic humic acid or the Leonardite humic‐rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic‐rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.     

Dietary β‐glucan modulate haematological parameters,cytokines and gene expression in TLR and ERK pathways of rainbow trout (Oncorhynchus mykiss) during infection by Aeromonas salmonicida

To explore the role of β‐glucan (0, 0.05%, 0.1% and 0.2%) in resisting bacteria, rainbow trout (Oncorhynchus mykiss) was challenged with Aeromonas salmonicida after 42‐day β‐glucan administration, and then sampled at 0, 2, 4 and 6 days post infection (dpi). The data showed that 0.2% β‐glucan reduced the accumulated mortality rates compared with the ICG (infected control group) (p < .05). The white blood cells, red blood cells and haemoglobin were higher in the 0.2% β‐glucan group (BG) than the ICG (p < .05). 0.1% and 0.2% β‐glucan elevated serum total antioxidative capability and glutathione activity but alleviated the increase of serum alkaline phosphatase activity and glucose concentration (p < .05) during infection. Serum TNF‐α, IL‐1β, IL‐8 and IgM in three BGs elevated remarkably on 6 dpi compared with the ICG (p < .05). Expression of tnf, il1b and cxcl8 of the head kidney in the 0.1% and 0.2% BGs was higher than the ICG on 4 dpi while ighm expression in the 0.2% BG was higher than in the ICG on 2 and 6 dpi (p < .05). 0.1% and 0.2% β‐glucan increased the expression of tlr5m, tlr5s, tmek and myd88 in the spleen after infection (p < .05). Similarly, 0.2% β‐glucan up‐regulated the expression of tmek, myd88, oncmyk‐dab and c3 in head kidney (p < .05). Overall, 0.2% dietary β‐glucan effectively decreased accumulated mortality rate by modulating the biochemistry process, cytokines, and activating TLR and ERK signalling pathways during A. salmonicida infection.     

Development and validation of a real‐time PCR assay for the detection of anguillid herpesvirus 1

Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody‐based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real‐time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real‐time PCR is faster, less labour‐intensive and has a reduced risk of cross‐contamination. The real‐time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r²‐value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real‐time PCR. The developed real‐time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.     

Development of real‐time and quantitative QCM immunosensor for the rapid diagnosis of Aeromonas hydrophila infection

Since bacterial infection cause a significant economic loss in fish farms, it is necessary to develop rapid diagnostic tools. Interests on label‐free biosensors have been raised for the rapid detection of aquatic pathogenic bacteria but have not been extensively studied yet. Here we report a quartz crystal microbalance (QCM) immunosensor system for the rapid and simple detection of Aeromonas hydrophila, a pathogen for fish and human, in comparison with a conventional indirect ELISA method. In QCM immunosensor system, an antibody against A. hydrophila was covalently cross‐linked to the gold surface of sensor chip and bacterial attachment was monitored as real‐time frequency shifts within 5 min. The frequency shifts were very positively related to the amounts of bacterial cells between 6.25 and 100 μg corresponding to 6 × 10⁶ to 10⁸ CFU with a high specificity. The QCM immunosensor was also able to detect bacterial cells in fish tissue extract in a dose‐dependent manner. Indirect ELISA also showed the dose‐dependent reaction and the amplified signal may allow a lower detection limit. However, QCM immunosensor system showed a more linear and reliable standard curve with R² value of almost 1 (0.9999). Moreover, detection of the bacteria was much quicker and simpler.     

Development of a real‐time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 10¹ amplicon copies per assay (equivalent to 2 × 10^–9 ng/µl) using bacterial DNA and of 1.44 × 10¹ gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.     

Investigations on the role of the salmon louse,Lepeophtheirus salmonis (Caligidae), as a vector in the transmission of Aeromonas salmonicida subsp. salmonicida

A bacteria–parasite challenge model was used to study the role of sea lice, Lepeophtheirus salmonis (Copepoda), as a vector of Aeromonas salmonicida subsp. salmonicida. Three hypotheses were tested: (i) L. salmonis can acquire A. salmonicida subsp. salmonicida via water bath exposure; (ii) L. salmonis can acquire the bacteria via parasitizing infected Atlantic salmon, Salmo salar; and (iii) L. salmonis can transmit the bacteria to naïve Atlantic salmon via parasitism. Adult L. salmonis exposed to varying A. salmonicida subsp. salmonicida suspensions (10¹–10⁷ cells mL^?1) for 1.0, 3.0 or 6.0 h acquired the bacteria externally (12.5–100%) and internally (10.0–100%), with higher prevalences associated with the highest concentrations and exposures. After exposure to 10⁷ cells mL^?1, viable A. salmonicida subsp. salmonicida could be isolated from the external carapace of L. salmonis for 120 h. Lepeophtheirus salmonis also acquired the bacteria externally and internally from parasitizing infected fish. Bacterial transmission was observed only when L. salmonis had acquired the pathogen internally via feeding on ‘donor fish’ and then by parasitizing smaller (<50 g) ‘naive’ fish. Under specific experimental conditions, L. salmonis can transfer A. salmonicida subsp. salmonicida via parasitism; however, its role as a mechanical or biological vector was not defined.     

Antibiotic resistance and repetitive‐element PCR fingerprinting in Aeromonas veronii isolates

This study evaluated antibiotic resistance and the related genes in total 47 Aeromonas veronii isolates from pet fish, eel (Anguilla japonica) and koi (Cyprinus carpio) in Korea. In comparison with the antibiotic susceptibilities of isolates from eel and koi, those of pet fish were more resistant to ceftiofur, aminoglycosides, tetracycline and nitrofurantoin. And isolates from pet fish showed high prevalences of class 1 integron, quinolones and tetracycline resistance determinants than those from eel and koi. Repetitive‐element palindromic PCR (rep‐PCR) showed larger diversities among A. veronii isolates. Collectively, pet fish may be a reservoir for multiple clones of A. veronii involved in antibiotic resistance. In this aspect, imported fish in the aquaculture trade should be steadily and continually screened for bacterial antibiotic resistance and related genes.