Atlantic salmon (Salmo salar L.) serum vitellogenin neutralises infectivity of infectious pancreatic necrosis virus (IPNV).

Vitellogenin is a phosphoglycoprotein which represents the main precursor of the egg yolk in teleost fish. This reproductive protein was also demonstrated to play an important role in innate immunity by acting as a pattern recognition molecule capable of binding to bacteria, fungi and enhancing macrophage phagocytosis. The presented results demonstrate that, egg homogenate, ovarian fluid and serum of mature female Atlantic salmon have high neutralising ability for infectious pancreatic necrosis virus (IPNV). Vitellogenin from mature female Atlantic salmon serum, purified by immuno-affinity on a column matrix coated with monoclonal anti-Atlantic salmon vitellogenin antibody, was able to neutralise between 9.1 x 10(4) and 3.09 x 10(5) TCID(50) IPNV mg(-1) of protein. To the author's knowledge, this is the first time that the neutralising activity of vitellogenin on a teleost virus has been demonstrated. The results may explain why IPNV is difficult to detect by culture methods in ovarian fluid and egg homogenates from carrier mature females and suggest a possible means of vertical transmission via the egg.

Expression of interferon and interferon--induced genes in Atlantic salmon Salmo salar cell lines SHK-1 and TO following infection with Salmon AlphaVirus SAV.

Salmon AlphaVirus (SAV) is the aetiological agent of Salmon Pancreas Disease (SPD), a serious disease in farmed Atlantic salmon. Currently there is no available information on the ability of this virus to stimulate or suppress aspects of innate immunity in host cells. Two different Atlantic salmon cell lines (SHK-1 and TO), both derived from head kidney leucocytes, were infected with SAV and the kinetics and magnitude of gene expression were studied by real-time quantitative PCR. SAV nsP1 gene transcripts for strain P42P increased rapidly in TO cells with subsequent development of a cytopathic effect (CPE) while this virus strain hardly replicated at all SHK-1 cells causing no CPE. SAV P42P induced strong expression of type I IFN (IFN) and the antiviral IFN-induced gene MX transcripts in SHK-1 cells. Although the IFN response in infected TO cells was higher than in SHK-1 cells, the level of MX transcripts was lower. This may be because the virus was able to interfere with IFN-signaling and suppress MX transcription or that the TO cells are less able to transcribe the MX gene. Either way, it may account for why the SHK-1 cells suppress SAV replication while the TO cells are highly susceptible and succumb to the virus. The present results provide the first evidence for differential induction of expression of the interferon-induced antiviral gene, MX, correlating with resistant (SHK-1) and susceptible (TO) Atlantic salmon cell lines in response to infection by SAV.

Induction and persistence of Mx protein in tissues, blood and plasma of Atlantic salmon parr, Salmo salar, injected with poly I:C.

The expression of Mx transcripts and Mx protein was monitored at weekly intervals for 7 weeks, by qRT-PCR and immunohistochemistry, in the kidney, liver, gill and blood of Atlantic salmon parr following injection of poly I:C. Elevated levels of Mx transcripts compared to PBS injected control fish were found in the tissues at week 1. Background levels were then found up to week 7, with the exception of week 4 when high levels were again found in poly I:C injected fish as well as control fish. Immunostaining for Mx protein in the kidney, liver and gill of poly I:C injected fish was higher than in control fish from weeks 1-4, but little staining was found in the tissues of both poly I:C treated and control fish thereafter. Blood monocytes stained consistently in all fish, suggesting that this leucocyte type constitutively expressed Mx protein. From weeks 2-4, lymphocytes of both groups consistently stained for Mx protein but the consistency decreased at weeks 5-7. Staining of neutrophils was also inconsistent. Western blots of plasma showed an immunoreactive band of 76 kDa typical of salmon Mx protein. Semi-quantitative measurements of dot blots showed poly I:C injected fish to have higher levels of plasma Mx protein than controls on weeks 1-4 with very low levels on weeks 5-7. The results indicate that following induction of an interferon response with poly I:C, Atlantic salmon parr maintain elevated levels of Mx protein in tissues, leucocytes and blood plasma for about 4 weeks. Production of Mx protein by blood monocytes appears to be constitutive, though production by lymphocytes and neutrophils was less consistent.

Survival and replication of Piscirickettsia salmonis in rainbow trout head kidney macrophages.

Piscirickettsia salmonis is pathogenic for a variety of cultured marine fish species worldwide. The organism has been observed within host macrophages in natural disease outbreaks among coho salmon and European sea bass. In vitro studies, incorporating transmission electron microscopy (TEM) and ferritin loading of lysosomes, have confirmed that P. salmonis is capable of surviving and replicating in rainbow trout macrophages. Certain features of this intracellular survival underline its difference to other intracellular pathogens and suggest that a novel combination of defence mechanisms may be involved. Escape into the macrophage cytoplasm is not used as a means to avoid phago-lysosomal fusion and the organism remains at least partly enclosed within a vacuole membrane. While the piscirickettsial vacuole is often incomplete, survival and replication appear to require occupation of a complete, tightly-apposed, vacuolar membrane which does not fuse with lysosomes. Unlike some mammalian rickettsiae, actin-based motility (ABM) is not used as a means of intercellular spread. It is postulated that the presence of numerous small vesicles within vacuoles, and at gaps in the vacuolar membrane, may result from the blebbing of the piscirickettsial outer membrane seen early in the infection.

Infectious pancreatic necrosis virus suppresses type I interferon signalling in rainbow trout gonad cell line but not in Atlantic salmon macrophages.

RTG-P1 cells are a rainbow trout fibroblastic cell line permanently transfected with the luciferase gene under the control of the Mx promoter. On exposure to interferon (IFN) or IFN inducing agents, the cells produce luciferase. IPNV did not induce luciferase production up to 24h post-infection but did not suppress constitutive luciferase production. Furthermore, IPNV suppressed luciferase production induced by poly I:C. RT-PCR analysis of IPNV infected cells showed IFN gene transcription from 6h post-infection with increasing expression up to 24h. Housekeeping genes beta-actin and GAPDH were also expressed along with upregulation of IRF1 and slight upregulation of STAT1. When RTG-P1 cells were stimulated with IFN, Mx transcripts, measured by qRT-PCR, peaked at 3-6h and thereafter fell to low levels, but in the presence of IPNV, Mx transcription at this time was significantly suppressed but continued to rise gradually. Luciferase production was lower in infected cells at 12h post-infection but not significantly after 24h. These results indicate that, in non-stimulated RTG-P1 cells, while IPNV induces IFN transcription, activation of Mx expression is suppressed. Furthermore, when stimulated by IFN, the rate of Mx transcription is significantly suppressed by the virus. This would probably give time for the virus to replicate rapidly in the early phases of infection. Contrary to the fibroblastic cell line, IPNV stimulated IFN production by salmon macrophages in vitro at least as strongly as poly I:C, with no suppression of the IFN response to poly I:C, and the virus persisted for up to 9 days without causing CPE.

AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils.

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.

Characterisation of surface blebbing and membrane vesicles produced by Flavobacterium psychrophilum.

The surface of Flavobacterium psychrophilum was examined by electron microscopy to determine if previous findings of haemagglutination positive (HA+) and haemagglutination negative (HA-) abilities could be correlated with expression of pili or of a capsular layer. A thin capsular layer was observed in both HA+ and HA- strains but typical pili were absent. However, long, tubular blebs that released membrane vesicles (MVs) into the supernatant were observed on up to 94% of cells within 1 sample. The surface blebbing was increased for 1 strain following growth on media with restricted iron availability. The MVs had an intact membrane bilayer and were released from blebbing cells of both strains. The protein profiles of MVs, while containing some banding similarity with the profile of outer membrane preparations (OMPs) and of lysed whole cells (WCs), showed several bands that reacted strongly with rabbit anti-whole-cell antisera. Two distinct bands of approximately 62 and 58 kDA were highly expressed in the MVs and not seen in the OMP. MVs contained proteolytic activity towards gelatine but not towards casein and elastin, which were only degraded by live cells. Low molecular weight lipopolysaccharides (LPS) or lipooligosaccharides (LOS) were associated with the MVs. Only the MVs of the HA+ strain possessed haemagglutinin activity. These findings suggest that the F. psychrophilum may, through surface blebbing, release antigenic MVs that contain some proteolytic activity and may aid the bacterium in releasing nutrients from its surrounding environment as well as playing a role in impeding the immune response of its host.

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide.

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.

Role of capsule in serotypic differences and complement fixation by Lactococcus garvieae.

Seventeen geographically distinct isolates of Lactococcus garvieae, isolated from diseased fish, were compared serologically using antiserum raised against the various isolates in rainbow trout. Sera raised against a capsule deficient isolate did not agglutinate capsulated isolates, regardless of origin. In contrast, all antisera raised against capsulated isolates cross reacted strongly with non-capsulated isolates. Antisera raised against capsulated Japanese isolates cross reacted with other capsulated Japanese isolates including isolates from geographically distinct prefectures within Japan (Ehime and Oita). However, antisera against these virulent capsulated isolates did not cross react with European capsulated isolates. Antisera raised against European capsulated isolates cross reacted with other European isolates, regardless of origin within Europe (UK, Italy, Spain), but did not cross-react with Japanese capsulated isolates. Agglutination assays performed with a range of fifteen lectins revealed differences in surface carbohydrate structure: capsule deficient isolates agglutinated with concanavalin A, Ricinis communis agglutinin, Pisum sativum agglutinin, Lens culinaris agglutinin, wheat germ agglutinin and succinylated wheat germ agglutinin. European capsulated isolates agglutinated with concanavalin A only. The Japanese capsulated isolates were not agglutinated by any of the lectins used in this study. Representative isolates from each group (Japanese capsulated and non-capsulated, European capsulated and non-capsulated) were investigated for their ability to fix complement. Non-capsulated isolates fixed complement regardless of origin, and antibody did not markedly enhance complement fixation. In contrast, the capsulated isolates were less efficient at fixing complement, but complement fixation was markedly increased by homologous antibody.

Effect of intraperitoneally administered IL-1beta-derived peptides on resistance to viral haemorrhagic septicaemia in rainbow trout Oncorhynchus mykiss.

The present work provides the first information concerning the immunostimulatory activity of trout interleukin (IL)-1beta-derived peptides in vivo. Previous studies have demonstrated the ability of 2 such peptides, referred to as P1 and P3, to up-regulate a range of important immune parameters in vitro. P1 corresponds to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and is analogous to a biologically active mammalian IL-1beta-derived peptide, whilst P3 was synthesised to complex with the IL-1 receptor and corresponds to fragment 197-206 (YRRNTGVDIS) of the trout sequence. Optimal migration of peritoneal leucocytes, peptide induced phagocytosis and intracellular respiratory burst activity occurred following intraperitoneal injection of 3.0 micromol of P3. Furthermore, resistance to viral haemorrhagic septicaemia virus (VHSV) was soon augmented (2 d) post-injection of P3.

Streptococcus iniae expresses a cell surface non-immune trout immunoglobulin-binding factor when grown in normal trout serum.

Three capsulated isolates of S. iniae representing serotype I and II and being arginine dihydrolase positive, negative or variable (AD+ve, AD-ve, AD+-ve) were investigated for their ability to bind rainbow trout serum immunoglobulin by the Fc region. Using a coagglutination assay with bacteria grown in Todd-Hewitt broth (THB), no evidence of non-specific Fc-binding of trout immunoglobulin (Ig) was obtained. However, when grown in normal trout serum, all isolates produced similar protein patterns in SDS-PAGE, but they were markedly different from the patterns of the bacteria grown in THB. Some bands with MW 70 kDa and over 100 kDa were very intense in the profiles of the serum-grown isolates. In Western blots, these bands of all isolates were immunostained with the conjugated goat antiserum to trout Ig, after blocking with normal goat serum, demonstrating that the bacteria had bound the trout Ig during growth in the serum. When the isolates were grown overnight in trout antiserum against Lactococcus garvieae they coagglutinated with L. garvieae cells but S. iniae isolates grown in normal trout serum did not. These data indicate that S. iniae grown in serum express surface factors which can bind trout Ig by the Fc-region.

Streptococcus iniae: serological differences, presence of capsule and resistance to immune serum killing.

The biochemical profiles, presence of capsule, outer membrane protein profiles and serological interactions of isolates of Streptococcus iniae obtained from different geographical and fish host origins were examined. The isolates had very similar biochemical profiles using API 20 Strep but varied as to whether they were arginine dihydrolase-negative, -positive or -intermediate (AD-ve, AD+ve, AD+/-ve, respectively). Representatives of each AD type were compared in subsequent experiments. All types possessed a polysaccharide capsule. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of outer membrane proteins or whole cells revealed no difference in banding patterns between isolates. All isolates were resistant to trout normal and specific immune serum and grew well in the presence of added fresh normal serum. Serological analyses of the isolates revealed antigenic differences. Trout antiserum against the AD+ve isolate did not agglutinate the AD-ve or AD+/-ve isolates, while antisera against the latter 2 types showed low agglutinating activity with all 3 isolates. When whole live cells of AD-ve and AD+ve isolates were dot-blotted, antiserum to the AD+ve isolate did not stain the AD-ve isolate, but antiserum to the AD-ve isolate stained both AD types. However, if the cells were pre-treated with Proteinase K (to remove surface-exposed protein antigens), the AD+ve isolate was stained only by its homologous antiserum. These results suggest that while certain protein antigens of the different AD type strains are immunologically cross-reactive, the capsular antigens appear to be AD type-specific. Furthermore, the results suggest that the cross-reactive antigens on the AD-ve isolate are effectively hidden by the strain-specific capsule, while they are partially exposed on the AD+ve isolate.

Binding of haemin by the fish pathogen Photobacterium damselae subsp. piscicida.

Whole cells of virulent (DI 21 and B 51) and avirulent (ATCC 29690 and EPOY 8803-II) strains of Photobacterium damselae subsp. piscicida, grown under iron-supplemented or iron-restricted conditions, were able to bind haemin. Iron limitation resulted in an increased binding of haemin by DI 21, B 51 and ATCC 29690 cells but did not affect the haemin-binding ability of the EPOY 8803-II cells. Proteinase K treatment of whole cells markedly reduced the binding of haemin, indicating that protein receptors located at the cell surface are involved in the binding. This was confirmed by the observation that isolated total as well as outer membrane proteins from all the strains, regardless of the iron levels of the media, were able to bind haemin, with the outer membranes showing the strongest binding. Haemin binding by membrane protein extracts was not affected by heat treatment but was almost completely abolished by Proteinase K treatment, suggesting the presence of thermostable protein receptors for haemin. The capsular polysaccharide also appears to play a minor role in binding of haemin. It was concluded that constitutive as well as inducible mechanisms of haemin binding occur in P. damselae subsp. piscicida. These mechanisms would rely mainly upon the direct interaction between the haemin molecules and surface-exposed outer membrane protein receptors.

Resistance to serum killing may contribute to differences in the abilities of capsulate and non-capsulated isolates of lactococcus garvieae to cause disease in rainbow trout (Oncorhynchus mykiss L.).

Three capsulated and two non-capsulated isolates of Lactococcus garvieae were investigated in terms of their wall proteins, virulence and interactions with rainbow trout immunoglobulin (Ig). All isolates were similar in integral membrane protein profile, and all were able to bind non-immune rainbow trout Ig, although different proteins appeared to be involved in Ig binding. However, whilst capsulated isolates were highly virulent, non-capsulated isolates were avirulent. This appeared to correlate with susceptibility of the non-capsulated isolates to rainbow trout normal serum. In contrast, the capsulated isolates were resistant to both normal and immune serum killing. In spite of this, passive immunisation of rainbow trout with specific anti-serum to L. garvieae was able to protect against challenge by capsulated isolates of L. garvieae. This suggests the antibody may have some other role in protection against disease caused by this important Gram-positive bacterial fish pathogen.

Antibody increases phagocytosis and killing of Lactococcus garvieae by rainbow trout (Oncorhynchus mykiss, L.) macrophages.

The present study reports that specific antibody increased the bactericidal activity of rainbow trout head-kidney macrophages against virulent capsulated Lactococcus garvieae in the absence of complement. The observed increased bactericidal activity appeared to result from increased phagocytosis of capsulated L. garvieae in the presence of specific antibody and may in part explain the protective effect of antibody previously reported against this disease.

Invasion of fish epithelial cells by Photobacterium damselae subsp. piscicida: evidence for receptor specificity, and effect of capsule and serum.

Photobacterium damselae subsp. piscicida is a fish pathogen which causes serious disease in commercial warmwater fish species. Because information on the initial stages of the infection is scarce, an investigation of the invasion ability of this pathogen was undertaken utilizing a fish epithelial cell line (epithelioma papillosum carpio, EPC), a virulent capsulated strain of P. damselae (MT1415), an avirulent non-capsulated strain of P. damselae (EPOY-8803-ii) and Escherichia coli HB101 as a non-invasive control. P. damselae was found to be able to adhere to and invade fish epithelial cells and remain inside them for 6-9 h. There were no significant differences in invasiveness between the capsulated and non-capsulated strains. A kinetics study demonstrated that P. damselae invasiveness was more efficient at low m.o.i., reaching saturation at higher m.o.i., suggesting internalization may be receptor-mediated. Invasion efficiency (IE) was significantly higher than in the control E. coli HB101. Engulfment of bacteria was possibly by an endocytic process and was unaffected by killing the bacteria with UV light. However, heat-killed bacteria had significantly reduced invasion capability. Ultrastructural studies showed that inside the epithelial cells, the bacteria remained within large vacuoles for a few hours and no evidence of intracellular replication was found, by either fluorescence or electron microscopic studies. Normal sea bass serum slightly reduced the invasion capability of the MT1415 strain, but heat-inactivated normal serum had no effect. On the other hand, heat-inactivated fish antiserum raised against the same strain reduced the percentage of invaded epithelial cells by 50%. As for other pathogens, an intracellular phase of P. damselae may be a mechanism to delay or avoid phagocytosis and host immune responses, favouring the spread of infection.

Superoxide dismutase and catalase in Photobacterium damselae subsp. piscicida and their roles in resistance to reactive oxygen species.

Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) is the causative agent of pasteurellosis or pseudotuberculosis in warm water marine fish. Enzymes which neutralize reactive oxygen species, produced during aerobic metabolism or during respiratory burst in fish macrophages, are important virulence factors in many pathogens. This study characterizes a periplasmic superoxide dismutase (SOD) and a cytoplasmic catalase in P. damselae. Purification and partial amino-terminal sequencing confirmed the SOD to be iron-cofactored, with a high degree of homology to other bacterial FeSODs. The SOD was common to all strains analysed in terms of type, location and activity, whilst the catalase varied in activity between strains. The catalase was constitutively expressed, but the SOD appeared to be repressed under low oxygen conditions. In spite of the presence of a periplasmic SOD, P. damselae was susceptible to killing by exogenous superoxide anion generated in a cell-free system. Addition of exogenous SOD to this system did not abolish the bactericidal effect; however, addition of catalase was protective. These results suggest that lack of periplasmic catalase may be implicated in susceptiblity to killing by reactive oxygen species.

Utilization of transferrin and salmon serum as sources of iron by typical and atypical strains of Aeromonas salmonicida.

The ability of typical and atypical strains of Aeromonas salmonicida to utilize non-haem sources of protein-bound iron was evaluated. (i) In a plate bioassay, the suppression of growth imposed on typical and atypical A. salmonicida by addition of the high-affinity iron chelator ethylenediamine-di(o- hydroxyphenylacetic acid) (EDDA) to the growth medium was reversed by the addition of 30% or 90% iron-saturated bovine or human transferrin (Tf) or lactoferrin (Lf) to the growth medium. (ii) The mechanism of obtaining iron from Tf was investigated by the addition of bovine Tf contained within a dialysis bag. The reversal of iron-restricted growth suppression differed between the strains in that the atypical strains were unable to utilize Tf contained within a dialysis bag while the typical strains were able to do so. This suggested a siderophore-mediated uptake of iron from Tf by the typical strains, which are known to produce siderophores while atypical strains do not. (iii) A solid-phase binding assay using horseradish-peroxidase-conjugated or biotinylated Tf or Lf failed to detect Tf/Lf-binding activity using whole typical or atypical cells. (iv) When atypical extracellular products (ECP) plus bovine Tf or salmon serum were enclosed in a dialysis bag, diffusible products were released which could reverse the EDDA-imposed growth suppression of an atypical strain. This reversal was negated by inhibition of the ECP metalloprotease with EDTA. (v) Purified 70 kDa serine protease of a typical strain was able to digest bovine Tf to low molecular mass fragments as observed in SDS-PAGE. These results indicate that typical and atypical strains of A. salmonicida differ in their mechanism of utilization of non-haem protein- bound sources of iron. Typical strains utilize Tf via a siderophore-mediated mechanism and are also able to digest Tf with the extracellular serine protease. Atypical strains utilize Tf by a siderophore-independent mechanism probably involving the proteolytic degradation of Tf by the extracellular metalloprotease.