Dual transcriptome analysis reveals differential gene expression modulation influenced by Leishmania arginase and host genetic background.

The outcome of Leishmania infection is strongly influenced by the host's genetic background. BALB/c mice are susceptible to Leishmania infection, while C57BL/6 mice show discrete resistance. Central to the fate of the infection is the availability of l-arginine and the related metabolic processes in the host and parasite. Depending on l-arginine availability, nitric oxide synthase 2 (NOS2) of the host cell produces nitric oxide (NO) controlling the parasite growth. On the other hand, Leishmania can also use host l-arginine for the production of polyamines through its own arginase activity, thus favouring parasite replication. Considering RNA-seq data, we analysed the dual modulation of host and parasite gene expression of BALB/c or C57BL/6 mouse bone marrow-derived macrophages (BMDMs) after 4 h of infection with Leishmania amazonensis wild-type (La-WT) or L. amazonensis arginase knockout (La-arg-). We identified 12 641 host transcripts and 8282 parasite transcripts by alignment analysis with the respective Mus musculus and L. mexicana genomes. The comparison of BALB/c_La-arg-versus BALB/c_La-WT revealed 233 modulated transcripts, with most related to the immune response and some related to the amino acid transporters and l-arginine metabolism. In contrast, the comparison of C57BL/6_La-arg-vs. C57BL/6_La-WT revealed only 30 modulated transcripts, including some related to the immune response but none related to amino acid transport or l-arginine metabolism. The transcriptome profiles of the intracellular amastigote revealed 94 modulated transcripts in the comparison of La-arg-_BALB/c vs. La-WT_BALB/c and 45 modulated transcripts in the comparison of La-arg-_C57BL/6 vs. La-WT_C57BL/6. Taken together, our data present new insights into the impact of parasite arginase activity on the orchestration of the host gene expression modulation, including in the immune response and amino acid transport and metabolism, mainly in susceptible BALB/c-infected macrophages. Moreover, we show how parasite arginase activity affects parasite gene expression modulation, including amino acid uptake and amastin expression.

Author Correction: Differential immune response modulation in early Leishmania amazonensis infection of BALB/c and C57BL/6 macrophages based on transcriptome profiles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differential immune response modulation in early Leishmania amazonensis infection of BALB/c and C57BL/6 macrophages based on transcriptome profiles.

The fate of Leishmania infection can be strongly influenced by the host genetic background. In this work, we describe gene expression modulation of the immune system based on dual global transcriptome profiles of bone marrow-derived macrophages (BMDMs) from BALB/c and C57BL/6 mice infected with Leishmania amazonensis. A total of 12,641 host transcripts were identified according to the alignment to the Mus musculus genome. Differentially expressed genes (DEGs) profiling revealed a differential modulation of the basal genetic background between the two hosts independent of L. amazonensis infection. In addition, in response to early L. amazonensis infection, 10 genes were modulated in infected BALB/c vs. non-infected BALB/c macrophages; and 127 genes were modulated in infected C57BL/6 vs. non-infected C57BL/6 macrophages. These modulated genes appeared to be related to the main immune response processes, such as recognition, antigen presentation, costimulation and proliferation. The distinct gene expression was correlated with the susceptibility and resistance to infection of each host. Furthermore, upon comparing the DEGs in BMDMs vs. peritoneal macrophages, we observed no differences in the gene expression patterns of Jun, Fcgr1 and Il1b, suggesting a similar activation trends of transcription factor binding, recognition and phagocytosis, as well as the proinflammatory cytokine production in response to early L. amazonensis infection. Analysis of the DEG profile of the parasite revealed only one DEG among the 8,282 transcripts, indicating that parasite gene expression in early infection does not depend on the host genetic background.

Amino acid permease 3 (aap3) coding sequence as a target for Leishmania identification and diagnosis of leishmaniases using high resolution melting analysis.

BACKGROUND: The leishmaniases comprise a spectrum of clinical manifestations caused by different species of Leishmania. Identification of species is important for diagnosis, treatment and follow-up management. However, there is no gold standard for species identification. High resolution melting analysis (HRM) offers a possibility to differentiate Leishmania species without the need for processing of the PCR-product. The amino acid permease 3 (aap3) gene is an exclusive target for trypanosomatids and is conserved among Leishmania spp., thus it can be a valuable target for an HRM assay for diagnosis of the leishmaniases. RESULTS: The HRM dissociation profiles of three amplicons targeting the aap3-coding region allowed the discrimination of L. (Leishmania) donovani, L. (L.) infantum, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (L.) amazonensis, L. (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) naiffi and L. (V.) shawi using DNA from promastigote cultures. The protocol was validated with DNA samples from clinical infection in humans and a cat, naturally infected sand flies, and experimentally infected mice. CONCLUSIONS: HRM analysis using the aap3 coding sequence as target is a relatively cheap, fast and robust strategy to detect and discriminate Leishmania species from all the endemic regions worldwide. The target and method proved to be useful in clinical, field and experimental samples, thus it could be used as a tool in diagnosis as well as ecological and epidemiological studies.

RNA-seq transcriptional profiling of Leishmania amazonensis reveals an arginase-dependent gene expression regulation.

BACKGROUND: Leishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. METHODOLOGY/PRINCIPAL FINDINGS: A total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis. CONCLUSIONS/SIGNIFICANCE: All data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways.

Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

BACKGROUND: Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. CONCLUSIONS/SIGNIFICANCE: This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected by this protein in the parasite. Finally, these findings may be helpful for the development of alternative anti-leishmanial drugs that target purine pathway.

Detection of a broad range of Leishmania species and determination of parasite load of infected mouse by real-time PCR targeting the arginine permease gene AAP3.

Leishmaniasis is one of the world's most neglected infectious diseases, affecting around 12 million people and more than 350 million at risk of infection. The clinical picture varies from self-healing cutaneous lesions to severe visceral infections, but still no commercial vaccines for humans are available and the currently used drugs have unpleasant side effects. Here we report a real-time PCR assay targeting the arginine permease gene AAP3 that can be applied for all the nine different species of the Leishmania genus tested; 4 Old World species and 5 New World species, from both L. (Leishmania) and L. (Viannia) subgenera. No cross-reaction was seen with Trypanosoma cruzi, Trypanosoma brucei, human or mouse genomic DNA. The assay has a high sensitivity, with a limit of detection of 10fg DNA for L. (L.) major and L. (L.) donovani, and 100fg DNA for L. (V.) braziliensis, and can be used for both qualitative and quantitative purposes. This AAP3-Assay, run in duplex with a host specific gene-assay, was also successfully used for quantification of parasite load of footpads from L. (L.) major-infected mice. It can therefore be a valuable tool in applications like monitoring effects of drugs, the selection of vaccine candidates and in screening patients, including asymptomatic carriers.

Atlantic halibut (Hippoglossus hippoglossus L.) T-cell and cytokine response after vaccination and challenge with nodavirus.

Viral encephalopathy and retinopathy (VER), caused by nodavirus, is one of the major infectious diseases affecting the marine fish farming industry, yet no effective vaccine is available. In this study, we examined the halibut immune response following administration of an experimental vaccine comprising a recombinant nodavirus capsid protein in combination with an oil adjuvant (OA). Four groups of halibut were injected with either: PBS alone, PBS plus OA, 10µg recCP plus OA, or 50µg recCP plus OA. 15 weeks later, half the fish in each group were challenged with nodavirus and the immune response investigated by analysis of: serum levels of recCP-specific halibut immunoglobulins (Igs), and mRNA transcript levels of several T-cell markers (CD3ɛ, Lck, CD4, CD4-2, CD8α and CD8ß) and cytokines (IL-1ß, IL-6, IL-12ßc and IFNγ). Additionally, the presence of nodaviral RNA2 transcripts in the brains of infected halibut was analysed. After vaccination, the level of IL-6 was consistently elevated in the spleens of fish given injections containing the OA. The combination of recCP and OA increased the expression of IL-1ß and IFNγ, as well as the level of recCP-specific Igs in blood plasma. Following challenge with nodavirus, IL-1ß and IFNγ were elevated in halibut spleens after 24h in all groups that had received OA with or without recCP antigen. In brain, a general increase in the expression levels of all T-cell markers and IFNγ was observed following challenge with nodavirus. The viral load at 8 weeks post-challenge was lower in the fish that received 50µg recCP, with 5 out of 8 individuals being negative for nodavirus. Additionally, a better correlation between these markers (apart from the CD8 markers), and the viral RNA2 was also observed in this group, suggesting that the activation of CD4+T-cells might be important in reducing the viral load. In conclusion, this study identifies recCP as a promising candidate antigen for the future development of a vaccine against nodavirus.

Horizontal transmission of nervous necrosis virus between turbot Scophthalmus maximus and Atlantic cod Gadus morhua using cohabitation challenge.

Experimental horizontal transmission of nervous necrosis virus (NNV) originating from halibut Hippoglossus hippoglossus was studied through cohabitation of intraperitoneally (i.p.) injected fish with uninfected fish for 125 d. The experimental groups consisted of i.p. injected turbot Scophthalmus maximus or i.p. injected Atlantic salmon Salmo salar with turbot, salmon or Atlantic cod Gadus morhua cohabitants. The initial weights were cod 10 g, salmon 40 g and turbot 3 g. NNV was detected in brain, eye and spleen by real-time reverse transcriptase PCR (qRT-PCR) in cod cohabitated with i.p. injected turbot after 90 and 125 d, suggesting NNV infection was transmitted horizontally from the turbot to cod. NNV was not detected in salmon that were cohabitated with i.p. challenged turbot or salmon. This study shows that NNV strains belonging to the Barfin Flounder Nervous Necrosis Virus (BFNNV) clade may be transmitted from halibut to cod via water. Hence there is a potential risk of horizontal transmission of the virus from farmed halibut to farmed and wild cod. The lack of detection of NNV in cohabitant salmon suggests that this fish species is less susceptible than cod, or not susceptible, to horizontal NNV transmission. This result might be influenced by the size of salmon, viral load in i.p. injected cohabitants or insufficient duration of the experiment.

Characterisation and expression analysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1ß, IL-6, IL-11, IL-12ß and IFNγ.

Genes encoding the five Atlantic halibut (Hippoglossus hippoglossus L.) cytokines; interleukin (IL)-1ß, IL-6, IL-11b, IL-12ßc, and interferon (IFN) γ, were cloned and characterised at a molecular level. The genomic organisation of the halibut cytokine genes was similar to that seen in mammals and/or other fish species. Several mRNA instability motifs were found within the 3'-untranslated region (UTR) of all cytokine cDNA sequences. The putative cytokine protein sequences showed a low sequence identity with the corresponding homologues in mammals, avian and other fish species. Nevertheless, important structural features were presumably conserved such as the presence, or absence in the case of IL-1ß, of a signal peptide, secondary structure and family signature motifs. The relative expression pattern of the cytokine genes was analyzed in several halibut organs, revealing a constitutive expression in both lymphoid and non-lymphoid organs. Interestingly, the gills showed a relatively high expression of IL-1ß, IL-12ßc and IFNγ. The real time RT-PCR data also showed that the mRNA level of IL-1ß, IL-6, IL-12ßc and IFNγ was high in the thymus, while IL-11b was relatively highly expressed in the posterior kidney and posterior gut. Moreover, the halibut brain showed a relatively high level of IL-6 transcripts. Anterior kidney leucocytes in vitro stimulated with imiquimod showed a significant increase in mRNA level of the five halibut cytokine genes. The sequence and characterisation data presented here will be useful for further investigation of both innate and adaptive immune responses in halibut, and be helpful in the design of vaccines for the control of various infectious diseases.

Atlantic halibut experimentally infected with nodavirus shows increased levels of T-cell marker and IFNγ transcripts.

The transcript levels of viral RNAs, selected T-cell marker and cytokine genes, toll like receptor (TLR) 7, and two interferon stimulated genes (ISG) were analysed in sexually immature adult Atlantic halibut (Hippoglossus hippoglossus L.) experimentally infected with nodavirus. The expression of the T-cell markers, TLR7 and the cytokine genes was further explored in in vitro stimulated anterior kidney leucocytes (AK leucocytes) isolated from the experiment fish and from additional untreated non-injected fish. The levels of viral RNA1 and RNA2 were increasing in brain and eye at around 4 and 8weeks post injection (wpi), respectively, and still increasing at the end of the experiment, especially in eye. Immuno-positive cells and signs of vacuolisation in both brain and eye were seen at 14wpi. Increased transcript levels of TCRß, CD4-2, CD4, CD8α, and Lck in brain and eye of the experimentally infected halibut suggested an involvement of halibut T-cells in the immune response against nodavirus. Interestingly, a similar expression pattern of TCRß, CD4 and Lck was seen in both brain and eye. However, compared to brain that showed elevated transcript levels of TCRß, CD4 and Lck mainly at 10 and 14wpi, the increase appeared earlier between 3 and 4wpi in the eye. Yet, an increase in the transcript level of IFNγ was seen at 10 and 14wpi in both organs. Moreover, elevated levels of TLR7, IL-1ß, IL-6, ISG15 and Mx were detected in vivo. The in vitro experiments, stimulating AK leucocytes with ConA-PMA, imiquimod or nodavirus, further supported an involvement of IL-6 and IFNγ in the immune response against nodavirus and the involvement of CD8ß(+) cells. Results from the present study thus indicate an importance of T-cells, IFNγ and the analysed ISGs in the immune response against nodavirus in Atlantic halibut, and would be of great help in future vaccination trials giving the possibility to monitor the immune response rather than mortality during post-vaccination challenge experiments.

Expression of T-cell markers during Atlantic halibut (Hippoglossus hippoglossus L.) ontogenesis.

The immune system of Atlantic halibut is relatively undeveloped at the time of hatching, and thus larvae are vulnerable to bacterial and viral diseases that can result in high mortalities. To enable establishment of effective prophylactic measures, it is important to know when the adaptive immune system is developed. This depends on both B- and T-cell functions. In the present study the expression of RAG1, TCRα, TCRß, CD3γδ, CD3ɛ, CD3ζ, CD4, CD4-2, CD8α, CD8ß, Lck, and ZAP-70 was analyzed in larval and juvenile stages during halibut development. Using real time RT-PCR, low basal mRNA levels of all 12 genes could be detected at early stages. An increase in mRNA transcripts for the genes was seen at different time points, from 38 days post hatching (dph) about the time when the first anlage of thymus is found, and onwards. The transcription patterns of the 12 mRNAs were found to be similar throughout the developmental stages tested. In situ hybridization on larval cross-sections showed that RAG1 and Lck could be detected in lymphocyte like cells within the thymus at 42 dph. CD4 expression could not be detected within the thymus before 66 dph, however, positive cells were restricted to the cortical region. At 87 dph, the zonation of the thymus in a cortical, cortico-medullary, and a medullary region seemed to be more evident with CD8α expressing cells found in all regions, indicating the presence of mature T-cells. This correlates with previous results describing thymus development and the appearance of IgM(+) cells during halibut ontogenesis.

Cloning, characterization, and expression pattern of Atlantic halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70.

As known from mammalia, the co-receptors CD4 or CD8 associate with a lymphocyte cell-specific kinase (Lck) upon T-cell activation. Lck phosphorylates tyrosine residues within the CD3 chains, providing docking sites for a 70 kDa zeta-associated-protein (ZAP-70), a tyrosine protein kinase important for T-cell signaling. The sequences of a CD4-like gene (CD4-2), Lck, and ZAP-70 were cloned, characterized, and the relative expression pattern was explored in several organs of Atlantic halibut (Hippoglossus hippoglossus L.). Important structural features, as a signal peptide, two Ig-like domains followed by a connecting peptide, a transmembrane region, and a CxC motif within the cytoplasmic tail were conserved within the predicted halibut CD4-2 protein. The deduced halibut Lck protein sequence was found to be composed of a N-terminal Src homology (SH) 4 domain, required for membrane attachment and CD4/CD8 binding, SH3 and SH2 adapter domains, and a SH1 domain followed by a regulatory C-terminal tail (COOH-domain). Tyrosine residues important in Lck activation were conserved within the SH1 and COOH-domain. Structural features of ZAP-70 as tandem SH2 domains and a C-terminal SH1 domain were predicted within the halibut ZAP-70 sequence, having the highest level of conservation within these regions. Several important phosphorylation sites found to play a critical role in T-cell antigen receptor signaling in mammalian were conserved. The overall expression pattern of the three genes was highly similar, showing the highest mRNA level of all three genes in thymus. Some expression was seen in spleen, anterior and posterior kidney, gills, and fin, as seen for other halibut T-cell markers. This study will enable further experiments on halibut T-cell signaling and activation, and enhance understanding about the development of immunological memory T-cells of halibut.

Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes.

BACKGROUND: Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, beta-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes. RESULTS: The expression of all six genes was relatively stable from the unfertilized egg until 12 day degrees post fertilization (ddpf). However, none of the selected genes were found to be stably expressed throughout halibut development. The mRNA levels of the six genes increased from 18 ddpf, when zygotic transcription is likely to be activated, and stabilized at different time points. The Excel-based software programs BestKeeper, geNorm, and NormFinder ranked EF1A1 and UbcE as the best candidate reference genes before activation of zygotic transcription, and RPL7 and EF1A1 as the best candidates after hatching. EF1A1 and RPL7 were also listed as the best reference genes when exploring the expression levels of the six genes in various halibut organs, both in non-injected fish and in mock- and NNV-injected fish. None of the reference genes were found optimal for normalization of real time RT-PCR data from in vitro stimulated anterior kidney leucocytes. CONCLUSION: Generally, it was found that EF1A1 and RPL7 were the genes that showed least variation, with HPRT1 and UbcE as intermediate genes, and ACTB1 and Tubb2C as the least stable ones. None of the six reference genes can be recommended as reference gene candidates in ConA-PMA stimulated leucocytes. However, UbcE can be a good candidate in other experimental setups. This study emphasizes the need for reference gene evaluation, as universal reference genes have not been identified.

Cloning and expression analysis of Atlantic halibut (Hippoglossus hippoglossus) CD3 genes.

The CD3 complex is in higher vertebrates shown to be important for the activation of T-cells. The T-cell system in fish is believed to be similar to that in higher vertebrates, and the CD3 chains could therefore be an important marker for identification of T-cells in fish. Here, we report the cDNA and corresponding gene sequence of Atlantic halibut (Hippoglossus hippoglossus) CD3gammadelta, CD3varepsilon, and CD3zeta chains, and the tissue-specific expression pattern of CD3 and T- cell receptor (TCR) genes. Important structural characteristics defining the CD3 genes seemed to be conserved in the halibut CD3 chains, such as a signal peptide, an extracellular region, a transmembrane helix having a negatively charged residue, and an ITAM bearing cytoplasmic tail. The extracellular domain of halibut CD3gammadelta and CD3varepsilon included two cysteines presumably involved in Ig-fold stabilisation and the CxxCxE motif important for dimerization. A spliced variant of CD3varepsilon was identified, lacking the Ig-fold, but with the CxxCxE motif intact. The real time RT-PCR analysis revealed a highly similar expression pattern of the CD3 genes and the TCRalpha and TCRbeta genes, indicating that the functional relationship between the TCR and the CD3 genes are preserved in teleosts.

Identification of immune related genes in Atlantic halibut (Hippoglossus hippoglossus L.) following in vivo antigenic and in vitro mitogenic stimulation.

To identify and characterize genes and proteins of the Atlantic halibut (Hippoglossus hippoglossus) immune system, six cDNA libraries were constructed from liver, kidney, spleen, peripheral blood, and thymus. Halibut were injected with nodavirus, infectious pancreatic necrosis virus (IPNV), or vibriosis vaccine and tissue samples were collected at various time points. Leukocytes from peripheral blood and spleen from stimulated and mock-injected fish were isolated and further in vitro activated with the mitogens, concanavalin A (Con A) and phorbol myristate acetate (PMA) to facilitate activation and proliferation. A total of 5117 high quality expressed sequence tags (ESTs) were identified and assembled into 781 contigs and 2796 singletons. Amongst these ESTs, 147 different putative immune related genes were identified. Several genes involved in innate and adaptive immune responses such as complement proteins, immunoglobulins, cell surface receptors, and cytokines and chemokines were identified. Of the immune related genes identified in this study, 44% had no match against any of the publicly available sequence data for halibut and thus can be considered as novel identification in halibut species. The approach of combining in vivo antigenic with in vitro mitogen stimulation, in addition to preparation of cDNA libraries from thymus enabled identification of many of the interesting genes including those involved in T-cell receptor complex.

A CD4 homologue in Atlantic halibut (Hippoglossus hippoglossus): molecular cloning and characterisation.

CD4 is expressed mainly on the surface of T helper cells, where it functions as a co-receptor for the T-cell receptor (TCR) by binding to MHC class II proteins. In this study we describe the cloning and sequencing of a cDNA encoding a CD4 homologue from Atlantic halibut (Hippoglossus hippoglossus L.) and the subsequent characterisation of the CD4 genomic sequence. The predicted CD4 protein has four extracellular immunoglobulin-like (Ig-like) domains (D1-D4), and was structurally similar to other CD4 homologues previously described in fish and mammals. Real time RT-PCR analysis showed that the highest levels of CD4 mRNA were found in halibut thymus, while lower expression was also seen in the spleen, gills, anterior and posterior kidneys, pectoral fins, and skin. In situ hybridisation confirmed the relatively high expression of CD4 mRNA seen in thymus by real time RT-PCR analysis, and showed that CD4 expressing cells were localised mainly in the cortex region. Only moderate changes in the levels of CD4 mRNA expression were observed during the 48 h post-injection of either nodavirus or Vibrio anguillarum.

Ontogeny of lymphoid organs and development of IgM-bearing cells in Atlantic halibut (Hippoglossus hippoglossus L.).

In teleost fish, the head kidney, thymus, and spleen are generally regarded as important immune organs. In this study, the ontogeny of these organs was studied in Atlantic halibut (Hippoglossus hippoglossus), larvae at various stages of development. We observed that the kidney was present at hatching, the thymus at 33days post hatch (dph), while the spleen was the last organ to be detected at 49dph. All three lymphoid organs were morphologically well developed during late metamorphic stages. Real time RT-PCR analysis showed that IgM mRNA expression could be observed at 66dph and later, which correlates well with in situ hybridisation data showing that a few IgM positive cells could be detected in the anterior kidney and spleen from 66dph. Our data also showed that the highest levels of IgM mRNA could be detected in halibut spleen. Immunostaining using a monoclonal antibody against halibut IgM detected IgM positive cells at 94dph in both the head kidney and the spleen, which is much later than the IgM mRNA. Numerous cells expressing both IgM mRNA and protein could be detected in the spleen and anterior kidney and also to some extent in thymus specimens from adult halibut.

CD8alpha and CD8beta in Atlantic halibut, Hippoglossus hippoglossus: cloning, characterization and gene expression during viral and bacterial infection.

CD8 is expressed on cytotoxic T-cells where it functions as a co-receptor for the TCR by binding to MHC class I proteins that present peptides on the cell surface. In this study we describe the cloning and sequencing of full length cDNAs encoding CD8alpha and CD8beta from Atlantic halibut (Hippoglossus hippoglossus L.) and subsequent isolation and characterization of the CD8alpha and CD8beta genes. The predicted halibut CD8alpha and CD8beta proteins are similar to those of mammals and other fish. Real time RT-PCR revealed that the highest levels of CD8 mRNA were found in the thymus, while some expression was also seen in the spleen, the gills, and the anterior and posterior kidney. In situ hybridization confirmed that the halibut thymus contained numerous CD8alpha and CD8beta expressing cells, while the anterior kidney had no CD8alpha positive cells but only a few CD8beta expressing cells. Only moderate changes in CD8 mRNA expression in other organs during either nodavirus or Vibrio anguillarum infection were observed. Both CD8alpha and CD8beta were significantly (P<0.05) down-regulated in spleen at 48h compared to their levels at 12h post-infection with nodavirus and V. anguillarum.