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.     

Differential resistance to edwardsiellosis in rohu (<Emphasis Type="Italic">Labeo rohita</Emphasis>) 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.     

Spleen size predicts resistance of rainbow trout to Flavobacterium psychrophilum challenge

Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.     

Salmonella carrier state in chicken: comparison of expression of immune response genes between susceptible and resistant animals

Asymptomatic Salmonella enterica serovar Enteritidis carrier state in poultry has serious consequences on food safety and public health due to the risks of food poisoning following consumption of contaminated products. An understanding the mechanisms of persistence of Salmonella in the digestive tract of chicken can be achieved by a better knowledge of the defects in the control of infection in susceptible versus resistant animals. The gene expression of innate immune response factors including anti-microbial molecules, inflammatory and anti-infectious cytokines was studied in the caecal lymphoid tissue associated with the carrier state. Expression levels of these genes were assessed by real-time PCR and were compared in two inbred lines of chickens differing in resistance to the carrier state following oral inoculation of S. enterica serovar Enteritidis at 1 week of age. No correlation was observed between resistance/susceptibility to caecal carrier state and level of interleukin (IL)-1beta, IL-8, IL-18, inducible NO synthase (iNOS) and natural resistance associated macrophage protein 1 (NRAMP1). A high baseline level of defensin gene expression was recorded in young animals from the susceptible line. In contrast, a significantly low expression of interferon-gamma (IFN-gamma) gene was observed in these susceptible infected animals in comparison to resistant ones and healthy counterparts. IFN-gamma expression level represents a valuable indication of immunodeficiency associated with persistence of Salmonella in the chicken digestive tract, and IFN-gamma thus represents a factor to consider in the development of prophylactic measures for the reduction of Salmonella carrier state.     

Effects of polyherbal formulation 'ImmuPlus' on immunity and disease resistance of Indian major carp, Labeo rohita at different stages of growth

A series of experiments were performed to determine the impact of polyherbal immunomodulatory formulation 'ImmuPlus' (AquaImmu) on growth, immunity and disease resistance of rohu (Labeo rohita), one of the Indian major carp at different stages of growth. Rohu larvae were fed on plankton, ImmuPlus-mixed compound feed, and plankton plus ImmuPlus-mixed compound feed (ImmuPlus added at three dose levels of 0.25, 0.50, and 0.75 g/kg feed) from 4th day of hatching to 14th day. ImmuPlus-mixed diets enhanced growth of larvae, survival and disease resistance against Aeromonas hydrophila challenge, compared to only plankton-fed group. In two other experiments, advanced rohu larvae and fingerlings were fed with ImmuPlus-mixed compound feed (at 0, 0.5, 1.0 and 2.0 g/kg) for 15, 30 and 45 days. At the end of 45 days for advanced larvae and 30 days for fingerlings, the fish fed with ImmuPlus at 1.0 g/kg level showed significantly higher growth and disease resistance against A. hydrophila challenge. In a separate experiment, juveniles of rohu were fed with 1 g/kg of ImmuPlus incorporated feed for 15 and 30 days. At the end of the trial, the ImmuPlus fed fish showed enhanced non-specific immunity (as measured through nitroblue tetrazolium reduction assay, serum lysozyme activity, serum haemolysin titre and resistance against A. hydrophila challenge in non-vaccinated fish as well as specific immunity levels (as measured through bacterial agglutination titre against A. hydrophila in vaccinated fish). Incorporation of ImmuPlus at 1 g/kg level in the diet of rohu may be beneficial for enhancing disease resistance.     

Genetic resistance to rhabdovirus infection in teleost fish is paralleled to the derived cell resistance status

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction--that was not observed in the susceptible cells--and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses.     

In vivo and in vitro studies of genetic resistance to systemic salmonellosis in the chicken encoded by the SAL1 locus

A number of inbred lines of chickens have been shown to be resistant or susceptible to systemic salmonellosis caused by Salmonella enterica serovar Gallinarum in adult birds, or by S. enterica serovar Enteritidis and S. enterica serovar Typhimurium in young chicks. Resistant lines show only moderate pathology and low mortality rates, whereas susceptible lines display extensive pathological changes and higher levels of mortality following Salmonella infection. Genetic resistance to salmonellosis is dominant and not linked to sex, MHC or Slc11a1 (formerly known as Nramp1), which leads to resistance in mice and other species. A novel locus encoding resistance to salmonellosis has been identified on chicken chromosome 5, and designated SAL1. The nature of the differences in pathology found between resistant and susceptible chicken lines in vivo indicates that resistance is expressed at the level of the mononuclear phagocyte system. Macrophages from adult resistant line birds cleared Salmonella serovar Gallinarum from infected macrophages within 24 h, whereas Salmonella bacteria persisted within macrophages from susceptible line birds for at least 48 h. Clearance of Salmonella by macrophages was accompanied by a strong and reproducible respiratory burst response in resistant lines, but little or no response in susceptible lines. Macrophages from an outbred chicken line showed variable responses. No differences were seen in macrophage nitric oxide production in cells from resistant or susceptible lines. These differences suggest that increased macrophage antimicrobial activity correlates with resistance and that macrophage activity plays an important role in genetic resistance to systemic salmonellosis in the chicken.     

Wide range of susceptibility to rhabdoviruses in homozygous clones of rainbow trout

Inbred lines differentially susceptible to diseases are a powerful tool to get insights into the mechanisms of genetic resistance to pathogens. In fish, chromosome manipulation techniques allow a quick production of such homozygous lines. Using gynogenesis, we produced nine homozygous clones of rainbow trout from a domestic population (INRA Sy strain). We examined the variability between clones for resistance to two rhabdoviruses, the viral haemorrhagic septicaemia virus (VHSV) and the infectious haematopoietic necrosis virus (IHNV). Intraperitoneal injections and waterborne infections were performed in parallel for both viruses. No survival was recorded after intraperitoneal injection of VHSV or IHNV, indicating that fish from all clones were fully susceptible to both viruses by this route of infection. In contrast, the different clones showed a wide range of survival frequency after waterborne infection. The resistance levels to VHSV ranged from 0 to 99% and resistance was not abrogated when resistant and sensitive animals were mixed and subjected to waterborne infection. VHSV was recovered from 10% of resistant fish after waterborne infection, confirming that virus replication was possible in this context but effective only in a low proportion of the population. The different clones also exhibited a wide range of survival (0-68%) after a waterborne infection with IHNV. Although VHSV-resistant clones were not fully resistant to IHNV, the susceptibility to IHNV and VHSV tended to be correlated, suggesting that non-specific mechanisms common to both viruses were involved.     

Host plant resistance of different chickpea genotypes against Ascochyta rabiei (Pass.) Lab. under tunnel conditions

Host plant resistance is the most efficient and easy way to manage chickpea blight caused by Ascochyta rabiei (Pass.) Lab. For this purpose, 374 chickpea lines/varieties from various research organisations were evaluated in plastic tunnels. None of the line showed immune response against the blight; however, one line (K-01005) was found to be highly resistant. Moreover, 15 entries were resistant, 136 exhibited moderate resistant reaction, 150 were susceptible and 72 showed highly susceptible response. The genotypes found that resistance against blight can serve as a source of resistance for breeding programmes, and they could be released for commercial production directly.     

Cryptobiosis and its control in North American fishes

Cryptobiosis is caused by the haemoflagellates Cryptobia bullocki and Cryptobia salmositica. These parasites infect food fishes (e.g. flounders, salmon) on both the Atlantic and Pacific coasts of North America and clinical signs of the disease include anaemia, and abdominal distention with ascites. The virulent factor in salmonid cryptobiosis, caused by C. salmositica, is a secretory metalloprotease (200 kDa). Fish mortality may be up to 100% in the absence of treatment, consequently strategies have been developed to protect them from disease/mortality. A single dose of a live vaccine protects fish for at least 2 years, and it is via the production of complement-fixing antibodies, enhanced phagocytosis and cell-mediated cytotoxicity. Inhibition of the parasite's cysteine protease by a monoclonal antibody reduces multiplication, infectivity and survival of the parasite. Consequently, the recombinant cysteine protease (49 kDa) of the parasite will be tested as a potential vaccine. The trypanocidal drug, isometamidium chloride (1.0 mg/kg), is effective (therapeutic and prophylactic) against C. salmositica in chinook salmon. Its efficacy is significantly enhanced if it is conjugated either to a monoclonal antibody or to polyclonal antibodies from immune fish. Selective breeding of Cryptobia-resistant brook charr (innate resistance to infection) is possible, and the resistant factor(s) is controlled by a dominant Mendelian locus. In these resistant charr the parasite is lysed via the alternate pathway of complement activation (innate immunity to infection). There are also Cryptobia-tolerant charr, fish that are susceptible to infection but have no clinical disease (innate resistance to disease). In these fish, one of the natural anti-proteases, alpha2-macroglobulin, neutralises the metalloprotease secreted by C. salmositica. Production of transgenic Cryptobia-tolerant salmon is an option to vaccination and or chemotherapy. Also, transgenic pathogen-tolerant animals may be an alternate strategy against other pathogens where the disease mechanism is similar to cryptobiosis.     

Response to vaccination of Atlantic cod (Gadus morhua L.) progenies from families with different estimated family breeding values for vibriosis resistance

The purpose of the study was to elucidate whether responses to vibriosis vaccination and gene expressions in parts of the innate immune system were different in families of Atlantic cod (Gadus morhua). The fish were progenies of families with differences in estimated breeding values (EBV) for vibriosis resistance. Families of coastal cod (CC) and northeast Arctic cod (AC) responded well to vaccination with a relative percent survival of 72–95. No correlation between response to vaccination and vibriosis resistance were found (p = 0.146). The AC family with medium low (M) resistance had significant (p ≤ 0.019) lowest mortality among all the unvaccinated fish but the CC-M family. Further, when comparing the vaccinated fish the AC family with very high (VH) resistance had significant (p ≤ 0.004) higher mortality than all except the CC-VL and CC-H families.Parts of the innate immune response were studied by measuring the gene expression of innate immune genes 2 and 4 days post dip vaccination. Vaccinated fish from two families had a weak but significant higher innate immune response compared to control fish of the same family. In vaccinated fish, the gene expression of interleukin (IL) 1b, IL-10, IL-12p40 and hepcidin were significant up-regulated. While, no measureable activations of interferon gamma (IFNγ), IL-8, cathelicidin, LBP/BPI and G-type lysozyme were found.     

Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu (Labeo rohita)

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various TLR types, TLR2 is involved in recognizing specific microbial structures such as peptidoglycan (PGN), lipoteichoic acid (LTA), zymosan etc., and after binding them it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce various cytokines. In this report, TLR2 gene was cloned and characterized in rohu (Labeo rohita), which is highly commercially important fish species in the farming-industry of Indian subcontinent. Full-length rohu TLR2 (rTLR2) cDNA comprised of 2691 bp with a single open reading frame (ORF) of 2379 bp encoding a polypeptide of 792 amino acids (aa) with an estimated molecular mass of 90.74 kDa. Structurally, it comprised of one leucine-rich repeat region (LRR) each at N-terminal (LRR-NT; 44-55 aa) and C-terminal (LRR-CT; 574-590 aa), 21 LRRs in between C and N-terminal, one trans-membrane (TM) domain (595-612 aa), and one TIR domain (645-790 aa). Phylogenetically, rohu TLR2 was closely related to common carp and exhibited significant similarity (93.1%) and identity (88.1%) in their amino acids. During embryogenesis, rTLR2 expression was detected as early as ∼7 h post fertilization indicating its importance in embryonic innate immune defense system in fish. Basal expression analysis of rTLR2 showed its constitutive expression in all the tissues examined, highest was in the spleen and the lowest was in the eye. Inductive expression of TLR2 was observed following zymosan, PGN and LTA exposure and Streptococcus uberis and Edwardsiella tarda infections. Expression of immunoregulatory cytokine interleukin (IL)-8, in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with inductive expression of TLR2. In vitro studies showed that PGN treatment induced TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) expression, NF-κB (nuclear factor kappa B) activation and IL-8 expression. Blocking NF-κB resulted in down-regulation of PGN mediated IL-8 expression indicating the involvement of NF-κB in IL-8 induction. Together, these findings highlighted the important role of TLR2 in immune surveillance of various organs, and in augmenting innate immunity in fish in response to pathogenic invasion. This study will be helpful in developing preventive measures against infectious diseases in fish.     

Some immune parameters in carp cyprinus Carpio susceptible and resistant to the haemoflagellate Trypanoplasma borreli

The present study addresses aspects of the (specific) immune response of carp to the haemoflagellate Trypanoplasma borreli. Sera of resistant carp contained antibodies, which agglutinated the flagellates in vitro. When flagellates were incubated in fish sera from resistant carp, binding of antibodies to flagellates could be demonstrated by flow cytometry, and T. borreli were effectively killed. Heat-treatment of the sera prevented killing, indicating that complement activation is important for the control of a T. borreli infection. Sera of carp that were highly susceptible to infection with T. borreli contained no antibodies capable of binding to or killing the parasite. Furthermore, specific antibodies were not generated after experimental infection. This lack of antibody production in susceptible carp is not due to a general unresponsiveness of lymphoid cells, since peripheral blood leukocytes (PBL) from susceptible and resistant carp responded to mitogenic stimuli in vitro with lymphocyte proliferation in a similar manner. However, viable flagellates were significantly less able to stimulate proliferation of PBL from susceptible carp. In vitro-produced culture supernatants of freshly isolated PBL from both carp lines (but not those of head kidney cells) differentially modulated the mitogen-induced proliferation of PBL from susceptible and resistant carp. The supernatants enhanced the proliferation of leukocytes obtained from individuals from the same carp line, but suppressed the mitogen-induced proliferation of PBL from the other line tested. This indicates that lymphoid cells from susceptible and resistant carp differ in their spectrum of spontaneously produced immunomodulatory mediators. Whether this is decisive for a T. borreli-specific and successful immune response is discussed.