Ram Savan

Genomics of fish cytokines

Cytokines are important regulators of immune system and finding these genes in fish have potential applications for the development of vaccines and or immunostimulants for aquaculture. Major hurdle in discovering cytokines from fish is the low identities of these molecules when compared to its mammalian counterparts. In fish, prior to pufferfish (Fugu rubripes and Tetraodon nigroviridis) and zebrafish (Danio rerio) genome sequencing, only a handful of cytokines like IL-1beta, TNF-alpha, TGFbeta, some CXC (including IL-8) and CC chemokine genes were identified. Post genome release of pufferfish and zebrafish, the rate of discovery of fish cytokines has accelerated tremendously. Fish and human share a certain degree of synteny in genomes. Exploiting this characteristic, cytokines have been searched for in the genome databases of pufferfish (http://fugu.hgmp.mrc.ac.uk) and zebrafish. Pro-inflammatory cytokines like TNFs, IL-6 and IL-17 family have been cloned. A new TNF gene has been found adjacent to TNFalpha gene from fugu and zebrafish. Among the T(H)1 type interleukins, IL-2, IL-15, IL-12alpha, IL-12beta, IL-18 have been cloned. Among IL-10 and its family members, IL-10, IL-19/20, IL-22 and IL-26 have been discovered. Interferon genes have also been identified by from fish. However, T(H)2 cytokines (IL-4, IL-5 and IL-13), IL-3, IL-7 and IL-9 are yet to be discovered from fish. This study shows that the type and diversification of fish cytokines are very similar to that found in mammals.

Discovery of a new class of immunoglobulin heavy chain from fugu

In teleosts, the genomic organization of the immunoglobulin (Ig) heavy (H)‐chain locus was thought to follow a typical translocon‐type multigene structure; however, recent studies have indicated a variation in the structure and this might be teleost specific. Isotypes of the Ig H‐chain, namely IgM, IgD, IgZ and IgT, have been identified. In this study, we report the discovery of a new class of IgH from fugu. This isotype was first identified from the genomic sequence of the fugu IgH locus. This novel IgH gene is composed of two constant (C) domains, a hinge region, and two exons encoding membrane regions. Surprisingly, the new IgH gene is present between the variable (V)H and Cµ regions of the locus. The C domains of the new isotype do not show any significant similarity to mammalian or fish IgH genes. The cloned cDNA from the new isotype has typical Ig H‐chain characteristics and is expressed as both secretory and membrane form. Transcript analyses suggest that the new IgH from fugu might only use the joining (J)H segments present in front of the new CH domains and that the usage of DH and JH segments is specific to the isotype expressed. The expression pattern of the gene has been confirmed by in situ hybridization and PCR studies.

Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.

Abstract Fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early diagnosis plays a vital role in management of fish and shellfish diseases. Traditionally, various biochemical and serological tests have been used for fish disease diagnosis. However, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. Polymerase chain reaction and probe‐based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. Recently, a novel technique called loop‐mediated isothermal amplification (LAMP) has been developed, which is highly sensitive and rapid. LAMP has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. In aquaculture, LAMP‐based detection of pathogens like Edwardsiella tarda, E. ictaluri, Nocardia seriolae, Tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. In this review, the application of LAMP for the detection of aquaculture‐associated pathogens is discussed.

Structural conservation of interferon gamma among vertebrates

Interferon gamma (IFN-gamma), being the hallmark of the T-cell T(H)1 response, has been extensively studied with respect to its expression and regulation of immune function. This gene has been extensively characterized in many mammalian species, making it one of the most widely cloned immunoregulatory genes. Recently, the gene has been identified in avian and piscine species and we have identified the gene in the frog genome. Based on these identified DNA sequences, we have constructed an evolutionary history of IFN-gamma that shows this molecule can be traced back more than 450 million years ago. Our analysis shows that type II interferon (IFN-gamma) function evolved before the tetrapod-fish split, a finding that contrasts earlier studies showing its origins in tetrapods. The IFN-gamma gene has undergone a further duplication event in teleosts after the tetrapod-fish split suggesting a specific-evolutionary adaptation in fish. The analyses of IFN-gamma, IL-22 and IL-26 genomic region in mammals, chicken, frog and fish reveal an evolutionary conservation of the loci and several regulatory elements controlling IFN-gamma gene transcription. Furthermore, across the vertebrata, the first intron of IFN-gamma gene contains a polymorphic microsatellite that has been closely correlated with disease susceptibility. Comparative-modeling of IFN-gamma structure revealed differences among the representative species but with an overall conservation of the fold, dimer interface and some interactions with the receptor. The structural and functional conservation of IFN-gamma suggests the presence of an innate, natural killer (NK) like response or even an adaptive T(H)1 immune response in lower vertebrates.

Discovery of a novel immunoglobulin heavy chain gene chimera from common carp (Cyprinus carpio L.)

In fish, two types of immunoglobulin heavy chain (IGH) genes, namely, IgM and IgD, have been cloned and characterized. Recently, a new IGH isotype specific to teleosts had been identified from zebra fish, rainbow trout, and fugu. In zebra fish, the domains of this new gene are present upstream of the μ region along the IGH locus. During this study, a novel IGH chimera (IgM-IgZ) has been discovered from common carp. The cloned cDNA encodes a typical leader peptide, a variable region, two constant regions, and a secretory tail. The first constant region is made up of the CH1 domain of carp IgM, while the second constant region shares a high similarity to the CH4 domain of the IgZ from zebrafish. Southern hybridization studies of the μ and ζ domains, conducted separately, revealed the presence of at least three copies of the respective genes, and μ and ζ domains might be present on the same loci, although far apart. Expression studies of the IGH genes suggest that there is an increase in chimeric immunoglobulin gene transcription when stimulated with lipopolysaccharide.

Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

ABSTRACT Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.

A novel role for IL-22R1 as a driver of inflammation

The interleukin (IL)-22R1 chain of the heterodimeric IL-22 receptor is not expressed on normal leukocytes, but this receptor is expressed on T cells from anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large cell lymphoma (ALCL) patients. To investigate the consequences of aberrant expression of this receptor on lymphocytes, we generated transgenic mice that express IL-22R1 on lymphocytes. The health of these animals progressively deteriorated at 8 to 12 weeks of age, as they displayed respiratory distress, rough coat and sluggish movement, and subsequent lethality due to multiorgan inflammation. The IL-22R1 transgenic animals developed neutrophilia that correlated with increased levels of circulating IL-17 and granulocyte colony-stimulating factor. In addition, these mice had increased serum IL-22 levels, suggesting that T cells expressing IL-22R1 generate IL-22 in a positive autoregulatory loop. As a result of the mouse model findings, we analyzed circulating cytokine levels in ALK(+)ALCL patients and detected elevated levels of IL-22, IL-17, and IL-8 in untreated patient samples. Importantly, IL-22 and IL-17 were undetectable in all patients who were in complete remission after chemotherapy. This study documents a previously unknown role of IL-22R1 in inflammation and identifies the involvement of IL-22R1/IL-22 in ALK(+)ALCL.

Analysis of expressed sequence tags (EST) obtained from common carp, Cyprinus carpio L., head kidney cells after stimulation by two mitogens, lipopolysaccharide and concanavalin-A☆

A representative cDNA library from mRNA obtained from lipopolysaccharide and concanavalin-A-induced head kidney cells of carp, Cyprinus carpio, was constructed. Two hundred single pass and partially sequenced clones (AU183343 to AU183542) were generated from expressed sequence tags (ESTs) and these were searched for homology in the DDBJ/GENBANK with blastN and blastX programs. Clones matching known genes were classified according to their function and distribution. One hundred and twenty-nine genes showed homology with known genes in databases, whereas 71 (35.5%) clones did not show any significant homology to sequences in the public database. Known genes also showed homology to fish genes deposited in the database. Twenty-two clones (11%), encoding 16 different sequences, were identified as putative biodefense and oncogenes, associated with an immune response. High expression of lysozyme (3%) was detected. Putatively identified biodefense-related sequences such as Lectin type 2, MHC class II invariant chain, mcl-1a and lysozyme were aligned with known homologues from the database and the percentage identity determined. A time course evaluation of gene expression due to mitogen stimulation by RT-PCR revealed the above mentioned gene homologues were switched on early during the cell proliferation.

Common carp have two subclasses of bonyfish specific antibody IgZ showing differential expression in response to infection

Immunoglobulin heavy chains identified in bony fish are broadly classified into three classes namely IgM, IgD and IgZ. The most recently described isotype is IgZ, a teleosts-fish specific isotype that shows variations in gene structure across teleosts. In this study we have identified two IgZ subclasses in common carp. IgZ1 is a four constant heavy chain domains containing antibody isolated across teleosts and IgZ2 is a two constant domains containing heavy chain chimera with a μ1 and ζ4 domain. Sequence analyses suggest that these subtypes are expressed from two separate genomic loci. Expression analyses show that IgZ1 is more abundant in systemic organs and IgZ2 chimera is preferentially expressed at mucosal sites. The basal expression level of IgM in fish is much higher than of the other isotypes. We show that IgZ1 expression in systemic and mucosal organs is responsive to blood parasites, while mucosal parasite infection induces IgM and IgZ2 gene expression. This report is the first to show differential expression of the IgZ variants in response to pathogens and suggests that the IgZ subtypes in carps may have mutually exclusive humoral functions.

Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP)

Abstract Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the structural glycoprotein gene of yellow head virus (YHV). The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. The whole procedure is very simple and rapid, and reaction time and temperatures were optimized for 60min at 65°C, respectively. Detection of gene amplification could be accomplished by agarose gel electrophoresis. The standardized RT-LAMP procedure was used to detect YHV in the heart and gill from infected shrimp. Thus, the RT-LAMP assay is extremely rapid, cost-effective, sensitive and specific and has potential usefulness for rapid diagnosis for YHV detection in shrimp.