Molecular characterization, expression analysis and function identification of Pf_IL-12p35, Pf_IL-23p19 and Pf_IL-12p40 genes in yellow catfish (Pelteobagrus fulvidraco).

The interleukin-12 (IL-12) family is a class of heterodimeric cytokines that play crucial roles in pro-inflammatory and pro-stimulatory responses. Although some IL-12 and IL-23 paralogues have been found in fish, their functional activity in fish remains poorly understood. In this study, Pf_IL-12p35a/b, Pf_IL-23p19 and Pf_IL-12p40a/b/c genes were cloned from yellow catfish (Pelteobagrus fulvidraco), four α-helices were found in Pf_IL-12p35a/b and Pf_IL-23p19. The transcripts of these six genes were relatively high in mucus and immune tissues of healthy individuals, and in gill leukocytes. Following Edwardsiella ictaluri infection, Pf_IL-12p35a/b and Pf_IL-23p19 mRNAs were induced in brain and kidney (or head kidney), Pf_IL-12p40a mRNA was induced in gill, and Pf_IL-12p40b/c mRNAs were induced in brain and liver (or skin). The mRNA expression of these genes in PBLs was induced by phytohaemagglutinin (PHA) and polyinosinic-polycytidylic acid (poly I:C), while lipopolysaccharides (LPS) induced the mRNA expression of Pf_IL-12p35a and Pf_IL-12p40b/c in PBLs. After stimulation with recombinant (r) Pf_IL-12 and rPf_IL-23 subunit proteins, either alone or in combination, mRNA expression patterns of genes related to T helper cell development exhibited distinct differences. The results suggest that Pf_IL-12 and Pf_IL-23 subunits may play important roles in regulating immune responses to pathogens and T helper cell development.

Iron supplementation in the diets of hybrid catfish (Ictalurus punctatus × I. furcatus) juveniles affected haematocrit levels and potentially decreased disease resistance to Edwardsiella ictaluri.

To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.

Influence of probiotic and prebiotic supplementation on intestinal microbiota and resistance to Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus) following florfenicol administration.

Enteric septicemia of catfish (ESC), caused by the gram-negative enteric bacteria Edwardsiella ictaluri, is a significant threat to catfish aquaculture in the southeastern United States. Antibiotic intervention can reduce mortality; however, antibiotic use results in an imbalance, or dysbiosis, of the gut microbiota, which may increase susceptibility of otherwise healthy fish to enteric infections. Herein, recovery of the intestinal microbiota and survivability of channel catfish in response to ESC challenge was evaluated following a 10-day course of florfenicol and subsequent probiotic or prebiotic supplementation. Following completion of florfenicol therapy, fish were transitioned to a basal diet or diets supplemented with a probiotic or prebiotic for the remainder of the study. Digesta was collected on Days 0, 4, 8 and 12, beginning on the first day after cessation of antibiotic treatment, and gut microbiota was characterized by Illumina sequencing of the 16S rRNA gene (V4 region). Remaining fish were challenged with E. ictaluri and monitored for 32 days post-challenge. Florfenicol administration resulted in dysbiosis characterized by inflated microbial diversity, which began to recover in terms of diversity and composition 4 days after cessation of florfenicol administration. Fish fed the probiotic diet had higher survival in response to ESC challenge than the prebiotic (p = .019) and negative control (p = .029) groups.

Synergistic infection of Edwardsiella ictaluri and Flavobacterium oreochromis in cage cultured tilapia (Oreochromis sp.).

Widespread distribution of a highly pathogenic Edwardsiella ictaluri strain in farmed tilapia in northern Vietnam has recently been reported. The subsequent investigation noticed a disease outbreak occurred at five nearby tilapia farms with floating cages, in which the clinical signs of both edwardsiellosis and columnaris diseases were observed on the same infected fish and caused 65% to 85% fish mortality. Naturally diseased fish (n = 109) were sampled from the five infected farms for bacterial identification and conducting challenge tests. The two bacteria Edwardsiella ictaluri and Flavobacterium oreochromis were identified by a combination of biochemical tests, PCR and 16SrRNA sequencing methods. Experimental challenge tests on Nile tilapia resulted in the median lethal dose (LD50 ) of E. ictaluri and F. oreochromis at 70 CFU/fish by intraperitoneal (i.p.) injection and 3.6 × 106 CFU/mL by immersion, respectively. The experimentally co-infected challenged fish exposed to LD50 doses resulted in 83% ± 6% mortality, with the infected fish exhibiting clinical signs of both edwardsiellosis and columnaris diseases, mimicking the naturally diseased fish. This finding suggests that the co-infection of E. ictaluri and F. oreochromis may interact in a synergistic manner, to enhance the overall severity of the infection and elevates the need for efficient methods to control both pathogens.

Molecular Characterization of Nine TRAF Genes in Yellow Catfish (Pelteobagrus fulvidraco) and Their Expression Profiling in Response to Edwardsiella ictaluri Infection.

The yellow catfish (Pelteobagrus fulvidraco) is an economic fish with a large breeding scale, and diseases have led to huge economic losses. Tumor necrosis factor receptor-associated factors (TRAFs) are a class of intracellular signal transduction proteins that play an important role in innate and adaptive immune responses by mediating NF-κB, JNK and MAPK signaling pathways. However, there are few studies on the TRAF gene family in yellow catfish. In this study, the open reading frame (ORF) sequences of TRAF1, TRAF2a, TRAF2b, TRAF3, TRAF4a, TRAF4b, TRAF5, TRAF6 and TRAF7 genes were cloned and identified in yellow catfish. The ORF sequences of the nine TRAF genes of yellow catfish (Pf_TRAF1-7) were 1413-2025 bp in length and encoded 470-674 amino acids. The predicted protein structures of Pf_TRAFs have typically conserved domains compared to mammals. The phylogenetic relationships showed that TRAF genes are conserved during evolution. Gene structure, motifs and syntenic analyses of TRAF genes showed that the exon-intron structure and conserved motifs of TRAF genes are diverse among seven vertebrate species, and the TRAF gene family is relatively conserved evolutionarily. Among them, TRAF1 is more closely related to TRAF2a and TRAF2b, and they may have evolved from a common ancestor. TRAF7 is quite different and distantly related to other TRAFs. Real-time quantitative PCR (qRT-PCR) results showed that all nine Pf_TRAF genes were constitutively expressed in 12 tissues of healthy yellow catfish, with higher mRNA expression levels in the gonad, spleen, brain and gill. After infection with Edwardsiella ictaluri, the expression levels of nine Pf_TRAF mRNAs were significantly changed in the head kidney, spleen, gill and brain tissues of yellow catfish, of which four genes were down-regulated and one gene was up-regulated in the head kidney; four genes were up-regulated and four genes were down-regulated in the spleen; two genes were down-regulated, one gene was up-regulated, and one gene was up-regulated and then down-regulated in the gill; one gene was up-regulated, one gene was down-regulated, and four genes were down-regulated and then up-regulated in the brain. These results indicate that Pf_TRAF genes might be involved in the immune response against bacterial infection. Subcellular localization results showed that all nine Pf_TRAFs were found localized in the cytoplasm, and Pf_TRAF2a, Pf_TRAF3 and Pf_TRAF4a could also be localized in the nucleus, uncovering that the subcellular localization of TRAF protein may be closely related to its structure and function in cellular mechanism. The results of this study suggest that the Pf_TRAF gene family plays important roles in the immune response against pathogen invasion and will provide basic information to further understand the roles of TRAF gene against bacterial infection in yellow catfish.

Development of a hyper-adhesive and attenuated Edwardsiella ictaluri strain as a novel immersion vaccine candidate in yellow catfish (Pelteobagrus fulvidraco).

Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJei, and the ΔeseJei strain fails to replicate in the epithelioma papillosum of carp cells. The type 1 fimbria plays a pivotal role in the adhesion of E. ictaluri, and it was found in this study that deletion of -245 to -50 nt upstream of fimA increases its adhesion to around five times that of the WT strain. A hyper-adhesive and highly attenuated double mutant (ΔeseJeiΔfimA-245--50 strain) was constructed, and it was used as a vaccine candidate in yellow catfish via bath immersion at a dosage of 1 × 105 CFU/mL. It was found that this vaccine candidate can stimulate protection when challenged with E. ictaluri HSN-1 at 5 × 107 CFU/mL (∼20 × LD50). The survival rate was 83.61% for the vaccinated group and 33.33% for the sham-vaccinated group. The RPS (relative percent of survival) of the vaccination trial reached 75.41%. In conclusion, the ΔeseJeiΔfimA-245--50 strain developed in this study can be used as a vaccine candidate. It excels in terms of ease of delivery (via bath immersion) and is highly efficient in stimulating protection against E. ictaluri infection.

Response of cecropin transgenesis to challenge with Edwardsiella ictaluri in channel catfish Ictalurus punctatus.

Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (ß-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F3 channel catfish family transgenic for cecropin transgene driven by the CMV promoter, and one F1 channel catfish family transgenic for cecropin transgene driven by the common carp ß-actin promoter were produced. F3 and F1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F1 and F3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection.

Pathology and virulence of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella anguillarum in channel (Ictalurus punctatus), blue (Ictalurus furcatus), and channel × blue hybrid catfish.

In the mid-2010s, Edwardsiella tarda was reaffiliated into three discrete taxa (E. anguillarum, E. piscicida, and E. tarda), obscuring previous descriptions of E. tarda-induced pathology in fish. To clarify ambiguity regarding the pathology of E. tarda, E. piscicida, and E. anguillarum infections in US farm-raised catfish, channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and channel × blue catfish hybrids were challenged with comparable doses of each bacterium. The most severe pathology and mortality occurred in fish challenged with E. piscicida, supporting previous reports of increased pathogenicity in commercially important ictalurids, while E. anguillarum and E. tarda warrant only minimal concern. Acute pathologic lesions among bacterial species were predominantly necrotizing and characteristic of gram-negative sepsis but became progressively granulomatous over time. After 100 days, survivors were exposed to the approximate median lethal doses of E. piscicida and E. ictaluri, revealing some cross-protective effects among E. piscicida, E. anguillarum, and E. ictaluri. In contrast, no fish that survived E. tarda challenge demonstrated any protection against E. piscicida or E. ictaluri. This work supports reports of increased susceptibility of channel, blue, and hybrid catfish to E. piscicida, while highlighting potential cross-protective affects among fish associated Edwardsiella spp.

A multi-epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia.

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E. ictaluri immunogenic proteins previously reported. The 49.32 kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E. ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E. ictaluri infections.

Cross-protective efficacy of a live-attenuated Edwardsiella ictaluri vaccine against heterologous Edwardsiella piscicida isolates in channel and channel × blue catfish hybrids.

Edwardsiella piscicida is a growing problem for catfish aquaculture in the southeastern United States, particularly in channel (Ictalurus punctatus) x blue (I. furcatus) catfish hybrids. Research has shown E. piscicida isolates recovered from farmed catfish in Mississippi form at least five discrete phyletic groups, with no apparent differences in virulence in channel and hybrid catfish. Laboratory trials have shown a live-attenuated E. ictaluri vaccine (340X2) cross-protects against at least one E. piscicida isolate (S11-285) in channel and hybrid catfish, although it is unknown if this protection exists for other E. piscicida variants. To this end, channel and hybrid catfish were immunized by immersion with E. ictaluri 340X2. Thirty days later, fish were challenged by intracoelomic injection with representative E. piscicida variants from each phyletic group. Relative percent survival (RPS) for hybrids ranged from 54.7% to 77.8%, while RPS in channels ranged from 80.5% to 100%. A second study investigated whether channel and hybrid catfish exposed to heterologous E. piscicida isolates were similarly protected against wild-type E. ictaluri. Fish were exposed by bath immersion to representative E. piscicida isolates from each phyletic group. Thirty days post-immunization, fish were challenged by immersion with wild-type E. ictaluri isolate S97-773. Regardless of variant, previous exposure to heterologous E. piscicida isolates significantly improved survival following E. ictaluri challenge. These findings suggest the presence of shared and conserved antigens among E. piscicida and E. ictaluri that could be exploited by application of polyvalent or cross-protective vaccines.

Evaluation of Edwardsiella piscicida basS and basR mutants as vaccine candidates in catfish against edwardsiellosis.

Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E. piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E. piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E. piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish ♀ × blue catfish ♂) challenges demonstrated long-term protection against subsequent challenges with E. piscicida and E. ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E. piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E. piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.

Pathological and Ultrastructural Characterization of an Edwardsiella ictaluri Triple hemR Mutant.

Enteric septicemia of catfish, which is caused by Edwardsiella ictaluri, is detrimental to farmed Channel Catfish Ictalurus punctatus. The hemin receptor HemR is involved in binding and uptake of heme into bacteria. Here, we explored pathological and ultrastructural changes in catfish fry that were immunized with a triple hemR mutant of E. ictaluri and challenged with wild-type E. ictaluri (EiWT) 28 d after immunization. Following immunization, pathological changes in the triple hemR-immunized fry were less severe compared to the EiWT-exposed control fry. Widely disseminated bacteria and severe necrosis in most organs, especially the kidney and spleen, were detected in both groups at days 4, 5, and 6. Multifocal granulomatous encephalitis with bacteria was seen in hemR-immunized fry at days 21 and 28 and in EiWT-exposed control fry at day 14. Phagocytic cells in the kidney and spleen of EiWT-exposed control fry contained more replicating bacteria compared to hemR-immunized fry. During the EiWT challenge of immunized fry, a robust immune response was observed in the triple hemR-immunized fry compared to the sham-vaccinated group. Many activated phagocytic cells were detected in the kidney and spleen with fragmented or no bacteria in the triple hemR-immunized fry. Our data suggested that virulence of triple hemR was lower and the onset of the lesions was delayed compared to EiWT. Additionally, triple hemR-immunized fry could mount an immune response and had milder lesions compared to the sham control after EiWT exposure.

Edwardsiella ictaluri evpP is required for colonisation of channel catfish ovary cells and necrosis in anterior kidney macrophages.

Edwardsiella ictaluri is a Gram-negative facultative anaerobe that can survive inside channel catfish phagocytes. E. ictaluri can orchestrate Type VI Secretion System (T6SS) for survival in catfish macrophages. evpP encodes one of the T6SS translocated effector proteins. However, the role of evpP in E. ictaluri is still unexplored. In this work, we constructed an E. ictaluri evpP mutant (EiΔevpP) and assessed its survival under complement and oxidative stress. Persistence of EiΔevpP in catfish as well as attachment and invasion in catfish macrophage and ovary cells were determined. Further, virulence of EiΔevpP in catfish and apoptosis it caused in macrophages were explored. EiΔevpP behaved same as wild type (EiWT) under complement and oxidative stress in complex media, whereas oxidative stress affected mutant's survival significantly in minimal media (p < .05). Persistence of EiΔevpP in live catfish and uptake and survival inside peritoneal macrophages were similar. The attachment and invasion capabilities of EiΔevpP in catfish ovary cells were significantly less than that of EiWT (p < .05). Although EiΔevpP showed reduced attenuation in catfish, causing decreased catfish mortality compared with EiWT (44.73% vs. 67.53%), this difference was not significant. The apoptosis assay using anterior kidney macrophages indicated that the number of live macrophages exposed to EiΔevpP was significantly higher compared with EiWT exposed macrophages at 24-hr post-treatment (p < .05). However, there were no significant differences in the early and late apoptosis. Remarkably, necrosis in EiΔevpP exposed macrophages was significantly less than that of EiWT exposed macrophages at 24 hr (p < .05). Our results demonstrated that evpP is required for colonisation of catfish ovary cells and increased apoptosis and necrosis in anterior kidney macrophages.

Interspecies transmission of Edwardsiella ictaluri in Brazilian catfish (Pseudoplatystoma corruscans) from exotic invasive fish species.

Infections caused by Edwardsiella ictaluri are one of the biggest problems in the catfish industry in North America and have been reported in fishes around the world. E. ictaluri was detected in juvenile pintado Pseudoplatystoma corruscans-a Brazilian catfish-in a farm in Paraná State, Brazil; diseased animals showed ascites and neurological signs of infection, with more than 50% mortality. Exotic invasive species susceptible to this bacterium have been reported in this area. We assessed the susceptibility of pintado to E. ictaluri with experimental infection via intraperitoneal and immersion methods as well as a cohabitation experiment with Nile tilapia Oreochromis niloticus and African walking catfish Clarias gariepinus, 2 exotic invasive species. All pintados challenged by intraperitoneal and immersion routes and those cohabiting with infected C. gariepinus died within 17 d of the challenge. Mortality of Nile tilapia reached 71.42% after the intraperitoneal and 35.71% in the immersion challenges within 28 d, whereas African walking catfish showed zero mortality. Observed clinical signs were comparable to those in the farm and those described in the literature as enteric septicemia of catfish. With this study, we demonstrated the susceptibility of P. corruscans to E. ictaluri, as well as interspecies transmission of this bacterium.

Genetic correlations between challenge tested susceptibility to bacillary necrosis, caused by Edwardsiella ictaluri, and growth performance tested survival and harvest body weight in Mekong striped catfish (Pangasianodon hypophthalmus).

The aim was to carry out a joint genetic analysis of survival and harvest body weight, recorded in a growth performance test in Mekong striped catfish (Pangasianodon hypophthalmus), and susceptibility to bacillary necrosis (caused by Edwardsiella ictaluri), recorded in challenge tests. Data were from two challenge tested year-classes (~6,000 fish in both) that both had growth test data available for survival and body weight (~13,000 fish each year). Data were analysed with a linear tri-variate sire-dam model without the common environmental effect because otherwise genetic correlations were estimated with large standard errors. Susceptibility to bacillary necrosis was found weakly genetically correlated to both growth and survival in the growth test, while growth was found with moderate favourable genetic correlation to growth survival. To defend continued challenge testing of striped catfish in Vietnam, a strong genetic relationship needs to be established between bacillary necrosis and survival under a natural disease outbreak. This requires another field test (in addition to the growth test) with siblings, without antibiotic treatment and the cause of death continuously monitored. Meanwhile, the routine challenge testing with the aim to indirectly improve field survival through selection should continue.

Environmental factors affecting Edwardsiella ictaluri-induced mortality of riverine ayu, Plecoglossus altivelis (Temminck & Schlegel).

We analysed the predisposing factors for Edwardsiella ictaluri infection in the riverine ayu Plecoglossus altivelis on the basis of environmental and epidemiological data obtained in a tributary to and the lower reaches of the Tama River, Japan, in July and August 2011-2015. Mortality of ayu due to E. ictaluri infection was observed only in the tributary in August 2012 and 2013; both periods were unusually hot. During these mortality events, daily average water temperatures rose approximately 3-4°C over 4-8 days, reaching the optimum temperature for E. ictaluri infection (>25°C) and approaching the upper tolerable limit for ayu (30°C). Diurnal water temperature ranges (DWTRs) in the tributary during the mortality events exceeded 6°C, which was 1-2°C greater than in the lower reaches. Experimental infection of ayu with E. ictaluri resulted in higher mortality when exposed to 6°C DWTR than to 4°C DWTR. Furthermore, water levels in the tributary were generally low in August 2012 and 2013 because of low rainfall. From these results, we conclude that unusually high-water temperatures combined with high DWTRs and low water levels drove riverine ayu mortality from E. ictaluri infection.

Genetic analysis of resistance in Mekong striped catfish (Pangasianodon hypophthalmus) to bacillary necrosis caused by Edwardsiella ictaluri.

The aim of this study was to analyse four cohabitation challenge-test experiments with Mekong striped catfish (Pangasianodon hypophthalmus) against the bacterium Edwardsiella ictaluri. The data were genetically analysed per experiment by three models: 1) a cross-sectional linear model; 2) a cross-sectional threshold model; and 3) a linear survival model, at both 50% mortality (for models 1 and 2) and at the end of the test (for all three models). In two of the experiments (3 and 4) that were carried out in two replicated tanks, the predicted family effects (sum of sire, dam and common environmental effects) in each tank were correlated with the family survival in the other replicated tank (cross-validation). The heritability estimates of resistance to E. ictaluri infection were ≤ 0.012 with the survival model, and up to 0.135 - 0.220 (50% survival) and 0.085 and 0.174 (endpoint survival) for the cross-sectional linear and threshold models, respectively. The challenge test should aim for an endpoint survival that ceases naturally at 50%. Then, genetic analysis should be carried out for survival at the endpoint (reflecting susceptibility) with a simple cross-sectional linear model.

Case study development of a challenge test against Edwardsiella ictaluri in Mekong striped catfish (Pangasianodon hypophthalmus), for use in breeding: Estimates of the genetic correlation between susceptibilities in replicated tanks.

Bacillary necrosis is a problematic disease in farming of Mekong striped catfish (Pangasianodon hypophthalmus). The pathogenic bacterium is Edwardsiella ictaluri, causing numerous white spots in swelled liver, kidney and spleen. An alternative to antibiotic treatment and vaccine is to select for improved genetic resistance to the disease that requires to establish a proper challenge test. Here, four challenge tests of Mekong striped catfish against E. ictaluri are reported proposing 3 days of acclimatization of test fish prior to the challenge, with restricted water level in the test, keeping a temperature of 26°C. In the challenge, cohabitant shedders should be released directly into the test tank and make up around ⅓ of the fish, and bacteria should be added directly to water. The last two experiments, with the highest mortality, suggest that any factor involving the dead cohabitants should be removed and that additional experimentation should focus on bacteria (density) and timing for addition of bacteria to water. Genetic analyses revealed that resistance to bacillary necrosis tested in replicated tanks in the same experiment can be considered the same genetic trait.

Proteomic profiling of yellow catfish (Pelteobagrus fulvidraco) skin mucus identifies differentially-expressed proteins in response to Edwardsiella ictaluri infection.

Fish mucus acts as a physiological and immunological barrier for maintaining normal fish physiology and conferring defense against pathogens infection. Here we report proteomic profiling of skin mucus of yellow catfish before and after E. ictaluri infection by Label-free LC-MS/MS approach. A total of 918 non-redundant proteins were identified from 54443 spectra referring to yellow catfish genome database. Further annotation via GO and KEGG database revealed complex protein composition of yellow catfish mucus. Besides structural proteins in mucus, a lot of immune-related proteins were retrieved, such as lectins, complement components, antibacterial peptides and immunoglobins. 133 differentially-expressed proteins (DEPs), including 76 up-regulated and 57 down-regulated proteins, were identified, most of which were enriched into 17 pathways centering on "immune system" category with 33 proteins involved. Consistently, significant proliferation of mucus-secreting goblet cells and CYPA-expressing cells were observed along outside of yellow catfish skin after E. ictaluri infection, indicating an enhanced immune response to E. ictaluri infection in yellow catfish skin mucus. The proteomic data provide systematic protein information to comprehensively understand the biological function of yellow catfish skin mucus in response to bacterial infection.

Hematological and immune genes responses in yellow catfish (Pelteobagrus fulvidraco) with septicemia induced by Edwardsiella ictaluri.

Yellow catfish (Pelteobagrus fulvidraco) has been an economically important freshwater species in China because of its good meat quality. In present, the high-density breeding industry has suffered great damage from bacterial infections, in especial, the rapid illness and death of fish caused by bacterial septicemia leads to huge economic losses. Therefore, it is urgent and important to identify pathogenic bacteria and study its pathogenicity. In this study, we isolated a bacterial strain from the yellow catfish with typical septicemia and named it E. 719, then, by morphological observations, regression infection, biochemical identification, 16S rDNA sequence analysis and triple PCR identification, E. 719 was determined to be Edwardsiella ictaluri. Further, we infected yellow catfish with E. ictaluri to study its effects on mortality rate, hematological, histopathological disturbances and expression of immune genes. The mortality results showed that E. ictaluri was highly pathogenic, all infected fish died after 14 days post injection, and the distribution of bacteria in body kidney, spleen, liver, head kidney and brain of fish was continuously detected by measuring the amount of bacteria in the tissues. In addition, the number of red blood cells decreased significantly with the time of infection, while the number of white blood cells and thrombocytes increased. In particular, the number of monocytes and neutrophils increased significantly in the differential leucocyte count (DLC). Histopathologic changes observed by HE staining showed similar results, gill, intestine, spleen and head kidney showed obvious inflammation, bleeding and necrosis. Besides, checking by real time quantitative RT-PCR assays, in both spleen and head kidney tissues which were the major immune organs, mRNA expressions of immune gene IL-1ß, TNF-α, and MR significantly increased in the early and middle stages of infection, which suggested that the infection of E. ictaluri caused a strong immune response in yellow catfish. This study provides a preliminary basis for the diagnosis and treatment of pathophysiology septicemia in yellow catfish induced by E. ictaluri.