Differential resistance to edwardsiellosis in rohu (Labeo rohita) families selected previously for higher growth and/or aeromoniasis-resistance.

Selection in fish for disease resistance is one of the most useful approaches to solve disease problems. Genetic variation in resistance to edwardsiellosis in fullsib families of rohu, Labeo rohita was investigated in the present study. A large variation in the susceptibility pattern (0 to 94.74 percent survival) against Edwardsiella tarda challenge was observed among 57 families. Additive genetic variation showed a heritability of 0.38 ± 0.08 across the year-class survival. The apparent resistant families showed more delayed mortality than the apparent susceptible ones. The cross-protection provided by aeromoniasis-resistant lines of rohu to edwardsiellosis was also studied to evaluate the possibility of selection for both diseases simultaneously. Challenge of F1-generation aeromoniasis-resistant and -susceptible lines with E. tarda showed significant difference in survival between the lines with higher percent survival in resistant line. This study suggests that direct selection method may be used reliably in selection programs and selection for multiple diseases simultaneously can be considered for rohu.

Pathophysiology of experimental Aeromonas hydrophila infection in Puntius sarana: early changes in blood and aspects of the innate immune-related gene expression in survivors.

A decline in production of Puntius sarana worldwide and subsequent intensification of its culture as a measure for conservation and increase food fish production have led to emergence of diseases. Investigations on innate immunological changes in fish that survives an acute bacterial infection are a few or lacking. An attempt was made to study the detailed changes in innate immune responses in P. sarana following an intraperitoneal challenge with Aeromonas hydrophila at 2.24 × 10(7)colony-forming unit (CFU)/fish. Sequential changes in transcription of immune related genes in liver and kidney, innate immune status, haematological profile and histopathology of major organs were studied after different time intervals post-challenge in the survivors. A significant increase in serum myeloperoxidase, ceruloplasmin activities and total leucocyte count, and decline in alternative complement activity were observed at early infection periods. A significant decline in plasma glucose level, total erythrocyte count, haemoglobin content and packed cell volume in blood was noticed after challenge. Superoxide radical production and serum antiprotease activity increased significantly at later period of challenge. In addition, few immune related genes pertinent to acute phase reactants and inflammatory response viz., complement component 3 (C3), lysozyme G, lysozyme C, ß2-microglobulin (B2M), transferrin, interleukin 1ß, interleukin 8, manganese superoxide dismutase (MnSOD), natural killer cell enhancing factor-B and chemokine CXCa were modulated after bacterial challenge. In liver, a significant up-regulation of C3 and transferrin, and down-regulation of lysozyme G, interleukin 8, MnSOD and B2M were noticed in survivors. On the contrary, lysozyme G, lysozyme C, interleukin 1ß, interleukin 8, CXCa, and MnSOD were found to be significantly up-regulated and C3 was down-regulated in the kidney. All these data suggest that the innate immune factors were highly modulated during infection process to render protection of P. sarana to this bacterium. This study also generated the primary information on many innate immune molecules/genes for this important medium carp species for the first time.

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.

Expression of immune-related genes in rohu Labeo rohita (Hamilton) by experimental freshwater lice Argulus siamensis (Wilson) infection.

The crustacean ectoparasite, Argulus poses one of the major threats to carp culture due to absence of any suitable control measure. The study was undertaken to determine the expression of immune-related genes in three major immunocompetent organs viz., kidney, skin and liver of rohu (Labeo rohita) during experimental freshwater lice Argulus siamensis infection. Results showed that the expression of TLR 22-like, lysozyme G and ß2-microglobulin genes in kidney was significantly (P ≤ 0.05) down-regulated in lice-infected fish. On the other hand, no significant difference (P>0.05) in CXCa, lysozyme C, TNFα and complement component 3 (C3) expression was found between uninfected control and different degrees of lice infected fish. In the skin, the expression of TLR 22-like and TNFα genes were significantly up-regulated whereas that of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish with respect to control fish. The expression of CXCa, lysozyme C and transferrin was not detected in the skin samples of fish. In the liver, the expression of CXCa, lysozyme G, ß2-microglobulin and transferrin was significantly (P≤0.05) up-regulated in lice-infected fish with respect to control fish whereas expression of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish. The expression of TLR 22-like, lysozyme C, TNFα genes was not detected in the liver samples of fish. This study indicates that majority of the genes showed down-regulation in kidney tissue whereas up-regulation in liver and skin tissues except C3 in Argulus-infected fish. We show that infection with this parasite irrespective of intensity can also result in immune gene expression changes in tissues situated away from the site of parasite attachment and feeding. The information obtained here could be useful towards understanding the susceptibility of rohu to argulosis and mechanisms involved in protection of rohu to ectoparasitic infections, which is causing immense economic losses to freshwater aquaculture sector.

Immune responses and expression profiles of some immune-related genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection.

Edwardsiella tarda is an important Gram-negative bacterium that causes systemic infections in a wide range of hosts including fish. The pathogenic mechanisms in this disease are still poorly understood in fish. Indian major carp, Labeo rohita were intraperitoneally challenged with a pathogenic isolate of E. tarda to measure sequential changes in immunity level. A significant decrease in the superoxide production, myeloperoxidase, alternative complement activity, total protein levels and antiprotease activity of serum was marked in the infected fish. However, the serum lysozyme activity and haemagglutination titre were raised in the infected fish. Similarly, a significant rise in specific antibody titre was noticed on and after 10 days post-challenge. This study also elucidates the changes in the relative expression of some immune-related genes viz., interleukin 1-beta (IL-1beta), inducible nitric oxide synthase (iNOS), complement component C3, beta(2)-microglobulin, CXCa, tumor necrosis factor-alpha (TNFalpha), and C-type and G-type lysozymes during the infection. Significant up-regulation of IL-1beta, iNOS, C3, CXCa and expression of both types of lysozyme genes was noticed at 6-12 h post-challenge (h.p.c.) whereas down-regulation of beta(2)-microglobulin and TNFalpha genes was observed after 48 h p.c. The results obtained here strengthen the understanding on molecular pathogenesis of edwardsiellosis in L. rohita.

Sequence information, ontogeny and tissue-specific expression of complement component C3 in Indian major carp, Labeo rohita (Hamilton).

The complement system is one of the first line of immune defence mechanisms as well as a modifier of acquired immunity. C3 is the central complement component primarily synthesized in liver. The local synthesis of C3 in tissues other than liver may play an important role in local inflammatory processes. The present study aims at looking into ontogeny of C3 in Labeo rohita and its tissue-specific expression that is yet to be explored for Indian carps. Unfertilised eggs, and eggs after 0, 1, 3, 6, 12 h post-fertilization and hatchlings at 24 h, and 3 and 7 days post-fertilization were collected from three brood fish of L. rohita (rohu). Total RNA was extracted from approximately 50 mg of tissue and subjected to RT-PCR using heterologous carp primers to amplify C3 fragment. A product of 155 bp size of rohu C3 was amplified, the deduced amino acid sequence of which had 91.1% similarity to that of Cyprinus carpio C3. C3 mRNA was not detected in unfertilized and 6 h post-fertilised eggs. C3 transcripts were detected 12 h post-fertilisation. Similarly, tissues from liver, spleen, kidney, muscle, brain, gonads, intestine, blood, heart and gills collected from juveniles of rohu were subjected to detection of C3 transcripts by RT-PCR and C3 mRNA was detected in all the tissues. Thus, it is concluded that there is extra-hepatic synthesis of complement (C3) in L. rohita and the synthesis of this component occurs only 6 h post-fertilisation.

Cloning, nucleotide sequence and phylogenetic analyses, and tissue-specific expression of the transferrin gene in Cirrhinus mrigala infected with Aeromonas hydrophila.

Transferrin partial complementary DNAs were cloned from the livers of five species in four genera of Indian carps (Indian major carp species: Labeo rohita, Catla catla and Cirrhinus mrigala; medium carp: Puntius sarana; minor carp: Labeo bata) subsequent to polymerase chain reaction amplification with published heterologous primers or self-designed primers derived from conserved regions of transferrin cDNA sequences. The partial transferrin cDNAs of the five species of carps had sizes from 624 to 633bp (487bp for L. rohita) and encoded an open reading frame consisting of 206-211 (162 for L. rohita) amino acids. The alignments of carp cDNA sequences showed 85-97% homology and 71-93% homology in deduced amino acid sequences. A phylogenetic tree of amino acid sequences of transferrin cDNAs from carps showed that the relationship among the four genera of Indian carps is well correlated with that derived from classic morphologic analyses. The hypothesized cleavage site and interdomain bridge of transferrin molecule were predicted for the above carp species and interestingly the cleavage site amino acid sequence was found to be conserved among all the carps. To study the tissue-specific expression of the transferrin gene, various tissues (liver, kidney, spleen, brain, muscle, testis, heart, intestine, gill and fin) from apparently healthy (control), moribund and survived C. mrigala experimentally infected with Aeromonas hydrophila infection were analyzed. Transferrin mRNA was detected only in liver RNA and to lesser extent in brain tissue out of the 10 tissues analyzed irrespective of bacterial infection.

Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita.

Seven innate immune parameters were investigated in 64 full-sib families (the offspring of 64 sires and 45 dams) from two year-classes of farmed rohu carp (Labeo rohita). Survival rates were also available from Aeromonas hydrophila infection (aeromoniasis) recorded in controlled challenge tests on a different sample of individuals from the same families. Due to strong confounding between the animal additive genetic effect and the family effects (common environmental+non-additive genetic), reliable additive (co)variance components and hence heritabilities and genetic correlations could not be obtained for the investigated parameters. Therefore, estimates of the association of challenge test survival with the studied immune parameters were obtained as product moment correlations between family least square means. These correlations revealed statistically significant (p<0.05) negative correlations of survival with bacterial agglutination titre (-0.48), serum haemolysin titre (-0.29) and haemagglutination titre (-0.34); and significant positive correlation with ceruloplasmin level (0.51). The correlations of survival to aeromoniasis with myeloperoxidase activity, superoxide production and lysozyme activity were found to be not significantly different from zero (p>0.05). Assuming that the negatively correlated candidate traits are not favourable as indirect selection criteria, the results suggest that ceruloplasmin level could potentially be a marker for resistance to aeromoniasis in rohu. The use of this immune parameter as an indirect selection criterion for increased resistance to aeromoniasis in rohu will, however, require that the parameter shows significant additive genetic variation and a significant genetic correlation with survival. Further studies are therefore needed to obtain a reliable heritability estimate for ceruloplasmin and its genetic correlation with survival from aeromoniasis.

Edwardsiellosis in fish: a brief review.

Edwardsiellosis is one of the most important bacterial diseases in fish. Scientific work on this disease started more than forty years ago and numerous workers around the world are continually adding to the knowledge of the disease. In spite of this, not a single article that reviews the enormous scientific data thus generated is available in the English language. This article briefly discusses some of the recent research on edwardsiellosis, describing the pathogen's interaction with the host and environment, its pathogenesis and pathology as well as diagnostic, preventive and control measures.