Stewart C. Johnson

The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen

BackgroundAeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish.ResultsThe nucleotide sequences of the A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids are characterized. The chromosome is 4,702,402 bp and encodes 4388 genes, while the two large plasmids are 166,749 and 155,098 bp with 178 and 164 genes, respectively. Notable features are a large inversion in the chromosome and, in one of the large plasmids, the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integron encoding genes for resistance to streptomycin/spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. A large number of genes encoding potential virulence factors were identified; however, many appear to be pseudogenes since they contain insertion sequences, frameshifts or in-frame stop codons. A total of 170 pseudogenes and 88 insertion sequences (of ten different types) are found in the A. salmonicida genome. Comparison with the A. hydrophila ATCC 7966T genome reveals multiple large inversions in the chromosome as well as an approximately 9% difference in gene content indicating instances of single gene or operon loss or gain.A limited number of the pseudogenes found in A. salmonicida A449 were investigated in other Aeromonas strains and species. While nearly all the pseudogenes tested are present in A. salmonicida subsp. salmonicida strains, only about 25% were found in other A. salmonicida subspecies and none were detected in other Aeromonas species.ConclusionRelative to the A. hydrophila ATCC 7966T genome, the A. salmonicida subsp. salmonicida genome has acquired multiple mobile genetic elements, undergone substantial rearrangement and developed a significant number of pseudogenes. These changes appear to be a consequence of adaptation to a specific host, salmonid fish, and provide insights into the mechanisms used by the bacterium for infection and avoidance of host defence systems.

The immune and stress responses of Atlantic cod to long-term increases in water temperature.

Sea-caged cod are limited in their movements in the water column, and thus can be exposed to large seasonal ( approximately 0-20 degrees C) temperature fluctuations. To investigate the physiological response of Atlantic cod to summer-like increases in temperature, we exposed 10 degrees C acclimated juvenile cod to a graded thermal challenge (1 degrees C increase every 5 days) and measured: (1) plasma cortisol and glucose levels; (2) the respiratory burst activity of blood leukocytes; and (3) the expression of specific immune-related genes [MHC Class I, Interleukin-1beta (IL-1beta), beta2-microglobulin (beta2-M), Immunoglobulin M (IgM)-light (L) and -heavy (H) chains] in the blood using quantitative reverse transcription-polymerase chain reaction (QRT-PCR). The experiment was stopped at 19.1 degrees C, with 26.7% of the fish surviving to this point. Plasma glucose levels increased slightly at 16 and 18 degrees C (by 1.39- and 1.74-fold, respectively), in contrast, cortisol levels were elevated significantly (by 2.9-fold) at 16 degrees C but returned to control levels thereafter. The effect of increasing temperature on the expression of immune related genes in blood cells (leukocytes) was variable and depended on the gene of interest. The expression of IgM-H remained stable for the duration of the experiment. In contrast, IL-1beta expression was increased significantly (by approximately 25-fold) at 19 degrees C as compared to time-matched control fish, and changes in the expression of beta2-M, MHC Class I and IgM-L followed a pattern similar to that seen for cortisol: increasing at 16 degrees C (by 4.2-, 5.3- and 17-fold, respectively), but returning to pre-stress levels by 19 degrees C. Interestingly, increasing temperatures had no effect on respiratory burst activity. This study is the first to examine the effects of a chronic regimen of increasing temperature on the stress physiology and immunology of a marine teleost, and suggests that immune function is influenced by complex interactions between thermal effects and temperature-induced stress (elevated circulating cortisol levels).

Functional genomic analysis of the response of Atlantic cod (Gadus morhua) spleen to the viral mimic polyriboinosinic polyribocytidylic acid (pIC).

In order to improve our understanding of how Atlantic cod (Gadus morhua) respond to viruses, we characterized immune-related gene expression in spleen tissues following stimulation with a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid (pIC). We used reciprocal suppression subtractive hybridization (SSH) cDNA libraries and quantitative RTPCR (QPCR) to identify and quantify pIC-responsive transcripts. A total of 3874 expressed sequence tags (ESTs) were generated from SSH libraries enriched for genes responsive to pIC. Thirteen immune-relevant genes from the libraries were subjected to QPCR. Genes confirmed as up-regulated by pIC included interferon stimulated gene 15, a small inducible cytokine, interferon regulatory factors (1, 7, and 10), MHC class I, viperin, and ATP-dependent helicase LGP2. Alpha-1-microglobulin (bikunin) was down-regulated, suggesting that pIC may suppress the acute phase response. Since the SSH libraries built for this study identified genes involved in the antiviral response, they are important resources for studying the responses of Atlantic cod to viruses. Evidence is provided for the existence of a RIG-I-like RNA helicase viral recognition pathway in Atlantic cod. Taken together, our data show that Atlantic cod can recognize double-stranded RNA and mount a rapid and potent interferon pathway response that is similar to that observed in other fish species and higher vertebrates.

Characterization of proteases in the skin mucus of Atlantic salmon (Salmo salar) infected with the salmon louse (Lepeophtheirus salmonis) and in whole-body louse homogenate.

As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17–22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.

Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida

Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod (Gadus morhua) challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Of 4,154 ESTs from four cDNA libraries, 10 genes with immune-relevant functional annotations were selected for QPCR studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as upregulated by A. salmonicida included interleukin-1 beta, interleukin-8, a small inducible cytokine, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin. This study is the first large-scale discovery of bacteria-responsive genes in cod and the first to demonstrate upregulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that bacterial antigen stimulation caused changes in many biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. Moreover, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.

Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin

We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml(-1) to virulent A. salmonicida failed to significantly reduce (P> 0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P < or = 0.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (P < or = 0.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0-20.0 ng ml(-1) but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml(-1)), a significant decrease (P < or = 0.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida.

Contribution of Type IV Pili to the Virulence of Aeromonas salmonicida subsp. salmonicida in Atlantic Salmon (Salmo salar L.)

ABSTRACT Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.

ENZYMES RELEASED FROM LEPEOPHTHEIRUS SALMONIS IN RESPONSE TO MUCUS FROM DIFFERENT SALMONIDS

Adult and mobile preadult sea lice Lepeophtheirus salmonis were incubated with mucus samples from rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), Atlantic salmon (Salmo salar), and winter flounder (Pseudopleuronectes americanus) to determine the response of L. salmonis to fish skin mucus as assessed by the release of proteases and alkaline phosphatase. There was variation in the release of respective enzymes by sea lice in response to different fish. As well, sea lice collected from British Columbia responded differently than New Brunswick sea lice to coho salmon mucus. Fish mucus and seawater samples were also analyzed using protease gel zymography to observe changes in the presence of low molecular weight (LMW) proteases after L. salmonis incubation. Significantly higher proportions of sea lice secreted multiple bands of L. salmonis–derived LMW proteases after incubation with rainbow trout or Atlantic salmon mucus in comparison with seawater, coho salmon, or winter flounder mucus. Susceptibility to L. salmonis infections may be related to the stimulation of LMW proteases from L. salmonis by fish mucus. The resistance of coho salmon to L. salmonis infection may be due to agents in their mucus that block the secretion of these LMW proteases or factors may exist in the mucus of susceptible species that stimulate their release.

Identification and Characterization of a Piscine Neuropathy and Nodavirus from Juvenile Atlantic Cod from the Atlantic Coast of North America

Abstract In 1999, disease outbreaks in juvenile Atlantic cod Gadus morhua that showed the classic signs of viral encephalopathy and retinopathy (VER) were reported in Nova Scotia. Brain and retinal tissues from moribund cod showed diffuse degenerative vacuolative encephalopathy and degenerative histiocytic retinitis. The affected brain and retinal tissues were observed to be positive for nodaviral antigens by means of immunohistochemical techniques. We partially characterized a nodavirus-like agent from brain and eye tissues and cell culture using reverse-transcriptase polymerase chain reaction and primer sets originally designed for amplification of white trevally Caranx dentex (also known as striped jack Pseudocaranx dentex) and Atlantic halibut Hippoglossus hippoglossus nervous necrosis virus coat (capsid) proteins. Sequencing of the T2 region of the coat protein revealed high similarities (>85% nucleotide identity) to the coat protein genes of other fish nodavirus strains, especially those of Atlantic...

Lepeophtheirus salmonis secretory/excretory products and their effects on Atlantic salmon immune gene regulation

We have previously shown that Lepeophtheirus salmonis produces trypsin and prostaglandin E2 (PGE2) that are most likely responsible for the limited inflammatory response of Atlantic salmon to infection. After removal of the dopamine and PGE2, the immunomodulatory activity of unfractionated and pools of the fractionated secretions was determined by examining the effects of the secretions on Atlantic salmon immune gene expression. Incubation of macrophage‐enriched isolates of Atlantic salmon head kidney cells with the unfractionated secretion + PGE2 revealed a significant inhibition of interleukin‐1β (IL‐1β) and major histocompatibility class I gene expression. Inhibition of lipopolysaccharide‐induced IL‐1β expression in the Atlantic salmon head kidney cell line (SHK‐1) was observed when three pools of the secretory/excretory products were tested. Further purification of products within these pools revealed that fraction 1‐2 could account fully for the inhibition of IL‐1β expression in SHK‐1 cells observed in pooled fraction 1. This study demonstrates that there are other immunomodulatory compounds produced by L. salmonis, in addition to PGE2 and trypsin, that can inhibit the expression of Atlantic salmon immune‐related genes in vitro.