Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita).

Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open reading frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4-91 aa and 111-200 aa), one NACHT domain (at 271-441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenetically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distribution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-γ were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preventive measures against infectious diseases in fish.

Selection for improved resistance to Aeromonas hydrophila in Indian major carp Labeo rohita: survival and innate immune responses in first generation of resistant and susceptible lines.

Selection for disease resistance in fish may be performed directly on basis of survival data obtained in controlled challenge trials, or indirectly using information from immunological or molecular markers linked to differential survival. In the present study, several key innate immune parameters were measured in aeromoniasis resistant and susceptible lines of rohu Labeo rohita to assess their suitability as immune markers for use in indirect selection for increased resistance. Experimental infection with Aeromonas hydrophila (9.55 × 10(6) cfu g(-1) fish) through the intraperitoneal route produced higher survival in the resistant line (73.33%) as compared to the susceptible line (16.67%). Blood and liver tissue samples from both lines were collected to study some of the innate immune parameters and immune-related gene expression. The respiratory burst activity of blood phagocytes, serum myeloperoxidase activity and ceruloplasmin level were significantly (p < 0.05) higher in the resistant line compared to the susceptible line. Lower level of blood glucose and serum natural haemolysin titre were marked in the resistant line as compared to the susceptible line. No significant difference was measured in total serum protein concentration, antiprotease activity and bacterial agglutinin level between two lines, while the expression of transferrin, complement factor C3 and TLR 22-like transcripts were significantly (P < 0.05) higher in liver samples of the susceptible line. However, no such difference was found in ß(2)-microglobulin and lysozyme gene expression between lines. The study demonstrated the possibility of using some of the investigated innate immune parameters as indirect marker traits for selection for improved resistance to aeromoniasis in rohu.

Ontogeny and tissue-specific expression of innate immune related genes in rohu, Labeo rohita (Hamilton).

The innate immune response in fish represents an early and rapid defense against pathogens. The present study aims at looking into ontogeny of innate immune system in the teleost, Labeo rohita using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs, and hatchlings (hatched at 28 ± 2 °C) at 0, 1, 3, 6, 12, 24 h, and 3, 7, 16, 21, 31 days post-fertilization were subjected to RT-PCR using self-designed or earlier published primers to amplify some innate immune relevant genes (lysozyme C, lysozyme G, beta-2 microglobulin, toll-like receptor 22-like and transferrin). The constitutive expression of ß-actin was detected in unfertilized eggs and further developmental stages. Transferrin and TLR22-like mRNA transcripts were detected by RT-PCR from 6 h post-fertilization to 31 day post-fertilization, whereas ß-2 microglobulin transcripts were detected only from 7 day post-fertilization onwards. Lysozyme C mRNA transcripts were detected from 24 h post-fertilization to 31 day post-fertilization. Lysozyme G mRNA transcripts were detected early from unfertilized egg stage onwards. Similarly, tissues viz. intestine, heart, ovary, gill, spleen, muscle, liver, brain, skin, anterior kidney, posterior kidney, and blood collected from juveniles of rohu were subjected to detection of all above mentioned gene transcripts by RT-PCR. ß2-microglobulin mRNA transcript was expressed in all tissues. Lysozyme C mRNA expression is confined to blood and posterior kidney only whereas lysozyme G mRNA is expressed in all tissues. TLR22-like mRNA is expressed in all tissues except ovary and liver whereas transferrin mRNA transcript is detected only in liver. Finally, all these information taken are likely to shed light on the ontogeny of innate immunity in L. rohita, which offers new insights to developmental biology when compared to higher vertebrates and also helpful in the development of preventive measures against problems concerning infectious diseases.

Comparative susceptibility of carp fingerlings to Lernaea cyprinacea infection.

Study was conducted to find out the comparative susceptibility of fingerlings of seven species of carps (Labeo fimbriatus, L. rohita, L. calbasu, Catla catla, Ctenopharyngodon idella, Cyprinus carpio and Hypophthamichthys molitrix) grown under both mono and polyculture to Lernaea cyprinacea infection. Under monoculture, C. carpio, L. rohita and L. calbasu, did not acquire Lernaea infection and were thus considered resistant, whereas C. idella, H. molitrix, C. catla and L. fimbriatus were susceptible. Even challenge with higher infective doses of copepodids under monoculture did not result in infection in the resistant fish species. The resistance of L. rohita and C. carpio to Lernaea infection under monoculture was not sustained when these two fish species were maintained in polyculture along with susceptible fish species. Labeo calbasu, even under polyculture, however, did not acquire Lernaea infection indicating that this fish species is the most resistant and least preferred host for this parasite. Similarly, C. carpio, L. rohita and L. calbasu when grown together in polyculture and exposed to a higher infective dose (120 copepodids/fish) also did not develop the infection. The possible reasons for differences in susceptibility shown by these carp species in monoculture and the loss of resistance by rohu and common carp while in polyculture with susceptible species are discussed. The ability of resistant fish species to prevent establishment of anchor worms on them under monoculture can be utilized to control this parasitic infection commonly encountered in culture ponds.

Toxicity of Crude Extracellular Products of Aeromonas hydrophila on Rohu, Labeo rohita (Ham.).

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD(50) 1.7 × 10(4) cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.

Derivation and characterization of embryonic stem-like cells of Indian major carp Catla catla.

Embryonic stem (ES)-like cells were derived from mid-blastula stage embryos of a freshwater fish, catla Catla catla, under feeder-free condition and designated as CCES cells. The conditioned media was optimized with 10% foetal bovine serum (FBS), fish embryo extract (FEE) having 100 µg ml(-1) protein concentration, 15 ng ml(-1) basic fibroblast growth factor (bFGF) and basic media containing Leibovitz-15, DMEM with 4·5 g l(-1) glucose and Ham's F12 (LDF) in 2:1:1 ratio using a primary culture of CCES cells. Cells attached to gelatin-coated plates after 24 h of seeding and ES-like colonies were obtained at day 5 onwards. A stable cell culture was obtained after passage 10 and further maintained up to passage 44. These cells were characterized by their typical morphology, high alkaline phosphatase activity, positive expression of cell-surface antigen SSEA-1, transcription factor Oct4, germ cell marker vasa and consistent karyotype up to extended periods. The undifferentiated state was confirmed by their ability to form embryoid bodies and their differentiation potential.