Temperature-dependent expression of immune-relevant genes in rainbow trout following Yersinia ruckeri vaccination

The gene expression of immune-relevant genes in rainbow trout Oncorhynchus mykiss following vaccination with a bacterin of Yersinia ruckeri, a bacterial pathogen causing enteric red mouth disease (ERM), was investigated at 5, 15, and 25 degrees C. Rainbow trout were immunized by i.p. injection of a water-based Y. ruckeri (serotype O1) bacterin, and gene expression profiles were compared to control groups injected with phosphate buffered saline (PBS). Blood and tissue samples (spleen and head kidney) were taken for subsequent analysis using solid phase enzyme-linked immunosorbent assay (ELISA) and real-time PCR, respectively. The up-regulation of cytokine genes was generally faster and higher at high water temperature, with major expression at 25 degrees C. The proinflammatory cytokine interleukin (IL)-1beta and interferon (IFN)-gamma were significantly up-regulated in all immunized groups, whereas the cytokine IL-10 was only up-regulated in fish kept at 15 and 25 degrees C. The gene encoding the C5a (anaphylatoxin) receptor was expressed at a significantly increased level in both head kidney and spleen of immunized fish. The secreted immunoglobulin M (IgM)-encoding gene was significantly up-regulated in the head kidney of immunized trout reared at 25 degrees C, and a positive correlation (r = 0.663) was found between gene expression of secreted IgM in the head kidney and Y. ruckeri-specific antibodies in plasma measured by ELISA. However, no regulation of the teleost specific immunoglobulin T (IgT), which was generally expressed at a much lower level than IgM, could be detected. The study indicated that expression of both innate and specific adaptive immune-response genes are highly temperature-dependent in rainbow trout.     

Rhabdovirus infection induces public and private T cell responses in teleost fish.

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian alphabeta TCRs have been recently characterized in rainbow trout and in several teleost species, but the alphabeta T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRbeta chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VbetaJbeta combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vbeta4Jbeta1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.     

Inflammatory response of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) larvae against Ichthyophthirius multifiliis

At hatching, the immune system of the rainbow trout larva is not fully developed. The larva emerges from the egg and is exposed to the aquatic freshwater environment containing pathogenic organisms. At this early stage, protection from disease causing organisms is thought to depend on innate immune mechanisms. Here, we studied the ability of young post-hatch rainbow trout larvae to respond immunologically to an infection with Ichthyophthirius multifiliis and also report on the localization of 5 different immune relevant molecules in the tissue of infected and uninfected larvae. Quantitative RT-PCR (qPCR) was used to analyze the genetic regulation of IL-1β, IL-8, IL-6, TNF-α, iNOS, SAA, cathelicidin-2, hepcidin, IL-10, IL-22, IgM and IgT. Also, a panel of 5 monoclonal antibodies was used to investigate the presence and localization of the proteins CD8, SAA, MHCII, IgM and IgT. At 10 days (84 degree days) post-hatching, larvae were infected with I. multifiliis and sampled for qPCR at 3, 6, 12, 24, 48 and 72 h post-infection (p.i.). At 72 h p.i. samples were taken for antibody staining. The first of the examined genes to be up-regulated was IL-1β. Subsequently, IL-8 and cathelicidin-2 were up-regulated and later TNF-α, hepcidin, IL-6, iNOS and SAA. Immunohistochemical staining showed presence of CD8 and MHCII in the thymus of both infected and non-infected larvae. Staining of MHCII and SAA was seen at sites of parasite localization and weak staining of SAA was seen in the liver of infected larvae. Staining of IgT was seen at site of infection in the gills which may be one of the earliest adaptive factors seen. No positive staining was seen for IgM. The study illustrates that rainbow trout larvae as young as 10 days (84 degree days) post-hatch are able to regulate important immune relevant cytokines, chemokines and acute phase proteins in response to infection with a skin parasitizing protozoan parasite.     

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.     

Cloning, expression analysis and bioactivity studies of rainbow trout (Oncorhynchus mykiss) interleukin-22

This report describes the cloning and characterisation of rainbow trout (Oncorhynchus mykiss) interleukin (IL)-22, and presents studies of the functional activity of its recombinant protein for the first time in a non-mammalian species. The predicted IL-22 coding region consists of 522 nucleotides which translates into a 173 amino acid protein, that contains an IL-10 family signature which is reasonably well conserved with other vertebrate IL-22 molecules. Expression analysis in tissues from healthy fish revealed a higher constitutive expression of IL-22 in mucosal tissues, suggesting a potentially important role in mucosal immunity. In vitro studies demonstrated that IL-22 expression was induced significantly by PHA and PMA in splenocyte primary cultures 4h post-stimulation. Expression was also induced in the spleen upon infection of fish with the Gram-negative bacterium Yersinia ruckeri, suggesting a potential role of IL-22 in vivo in defence against bacterial diseases. The Escherichia coli produced recombinant IL-22 enhanced the expression of a number of antimicrobial peptides, promoting host innate immunity against microbes and revealing a biological similarity with its mammalian counterpart.     

IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence

IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1β, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1β and IL-8 did not rise in the spleen although an increase of IL-1β, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1β and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1β and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.     

The expression analysis of inflammatory and antimicrobial genes in the goldfish (Carassius auratus L.) infected with Trypanosoma carassii

We report results of a comprehensive analysis of inflammatory gene expression during the course of infection of Trypanosoma carassii in the goldfish. We observed significant increases in mRNA levels of genes encoding pro-inflammatory cytokines IFN-γ, TNFα1 and TNFα2; IL-1β-1 and IL-1β-2; IL-12-p35 and IL-12-p40; CCL1; CXCL8, anti-inflammatory cytokines IL-10 and TGFβ and iNOS A and iNOS B, using quantitative PCR. Expression levels and profiles of these cytokines and iNOS isoforms varied in the different tissues (kidney, spleen, liver) of goldfish during the course of T.?carassii infection. The expression of majority of genes that encode pro- and anti-inflammatory cytokines were up-regulated during the acute phase of infection (days 7-21 post-infection). The mRNA levels of these cytokines returned to normal levels or were down-regulated during the elimination phase of infection (days 28-56), with exception of IL-10 in the spleen and liver of infected fish. A parallel up-regulation of IFN-γ and IL-10 mRNA levels were observed in all tissues of infected fish during the acute phase of the infection. The expression of iNOS genes (iNOS A and B) was significantly delayed (day 14?pi) in the kidney, liver and spleen of infected fish. These results provide insights into the interaction between T.?carassii and goldfish, and suggest that Th1/Th2-like responses may be important for controlling T.?carassii infection in the goldfish.     

The expression of immune-regulatory genes in rainbow trout, Oncorhynchus mykiss, during amoebic gill disease (AGD)

Amoebic gill disease (AGD) is an ectoparasitic disease caused by infection with the protozoan Neoparamoeba sp. and is characterised by epithelial hyperplasia that manifests as gill lesions. In order to examine the nature of the immune response to AGD, the expression of a range of immune-regulatory genes was examined in naïve uninfected rainbow trout, Oncorhynchus mykiss, and naïve rainbow trout subjected to a laboratory-induced AGD infection. The immune-regulatory genes examined were interleukin-1 beta isoform 1 (IL-1β1), tumour necrosis factor alpha isoforms 1 and 2 (TNF-α1, TNF-α2), interleukin-8 (IL-8), transforming growth factor beta isoform 1 (TGF-β1), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), major histocompatibility complex beta chain (MHC-II β-chain) and T-cell receptor beta chain (TCR β-chain). Immune-regulatory genes that were up/down-regulated in AGD-infected trout compared to uninfected controls at 0, 7, and 14 days post-inoculation (p.i.) in gill, liver and anterior kidney tissue were initially identified by means of semi-quantitative RT–PCR. Up/down-regulated immune-regulatory genes were subsequently quantitated and validated by real-time RT–PCR (qRT–PCR). The extent of AGD-associated pathology was consistent amongst all AGD-infected trout at 7 days p.i. and increased considerably by 14 days p.i. At both 7 and 14 days p.i. IL-1β1 and iNOS gene expression was significantly up-regulated in the gills, and IL-8 was significantly up-regulated in the liver of AGD-infected trout at 7 days p.i. These data demonstrate the involvement of the immune response to AGD at the molecular level, and indicate the importance of this response at the site of infection and the possible involvement of a systemic immune response.     

Interleukin-4 causes delayed virus clearance in influenza virus-infected mice.

Two different subsets of T cells, Th1 and Th2 cells, have been demonstrated to secrete different profiles of cytokines and to influence various infections in different ways. Whereas cytokines secreted by Th1 cells, particularly gamma interferon, promote the generation of cell-mediated immunity, Th2 cells and their cytokines (interleukin-4 [IL-4], IL-5, IL-10, and IL-13) have been shown to function in recovery from parasitic infections and in antibody responses. In this study, we analyzed the effects of the dominant Th2 cytokine, IL-4, on immunity to virus infection. We assessed the effects of IL-4 on both secondary immune responses by an adoptive transfer assay and primary immune responses by in vivo treatment of influenza virus-infected mice with IL-4. The results demonstrated that IL-4 can function to inhibit antiviral immunity at both stages. We found that IL-4 treatment of sensitized cells during secondary stimulation in vitro had little effect on their ability to lyse virus-infected target cells in a 51Cr release assay. Nevertheless, the clearance of influenza A/PR/8/34 (H1N1) virus from the lungs of infected BALB/c mice was significantly delayed after the transfer of virus-specific T cells secondarily stimulated in the presence of IL-4 in comparison to virus clearance in recipients of cells stimulated in the absence of IL-4. In contrast to the adoptive transfer results, the treatment of PR8 virus-infected mice with IL-4 during primary infection greatly suppressed the generation of cytotoxic T-cell precursors, as assessed by secondary stimulation in vitro. In addition, culture supernatants of secondarily stimulated spleen cells from IL-4-treated mice contained significantly less gamma interferon and more IL-4 than did spleen cells from controls. More importantly, the treatment of mice with IL-4 resulted in an extremely significant delay in virus clearance. Thus, IL-4 can inhibit both primary and secondary antiviral immune responses.     

Cloning of two rainbow trout nucleotide-binding oligomerization domain containing 2 (NOD2) splice variants and functional characterization of the NOD2 effector domains

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic pattern recognition receptor (PRR), which is involved in innate antibacterial and antiviral responses. Here, two NOD2 splice variants, trNOD2a and trNOD2b, are reported in rainbow trout Oncorhynchus mykiss, that share 63% and 61% similarity with human NOD2, respectively. These two trout NOD2 splice variants were shown to be constitutively expressed in thymus, gills, skin, muscle, liver, spleen, head kidney, intestine, heart, and brain, with the expression of trout NOD2 (trNOD2) mainly contributed by trNOD2a in all the examined tissues. PolyI:C transfection up-regulated the expression of trNOD2a and trNOD2b in RTG-2 cells. The expression of trNOD2a/b was modulated by the inflammatory stimulant interferon-γ (IFN-γ) or interleukin-1β (IL-1β). Overexpression of trout NOD2 effector domains resulted in induced expression of proinflammatory cytokines including IL-1β, tumor necrosis factor-α (TNF-α), IL-6 and IL-8, the antibacterial peptide cathelicidin-2, a variety of caspases including caspase-2, -6, -7, -8, -9, and type I and type II IFN. These results suggest that fish NOD2 functions in inflammatory events, possibly via NF-κB activation, regulation of apoptosis, and triggering of antibacterial and antiviral defences.