Septicemia Of Catfish Research Articles

The RNA chaperone Hfq has a multifaceted role in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qβ replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E.ictaluri by creating an hfq mutant (EiΔhfq) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella. The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, EiΔhfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, EiΔhfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. EiΔhfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the EiΔhfq vaccination completely protected catfish against subsequent EiWT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri, affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.

Chronic Exposure to Sublethal Concentrations of Aquatic Nitrite Change Nose and Gut Morphology and Associated Microbiomes in Channel Catfish

Nitrite is a common pollutant that can enter waterways via agricultural runoff or build up in aquaculture ponds when nitrogenous waste is converted to nitrite. Preliminary studies by the Huertas lab found that exposure to sublethal concentrations of aquatic nitrite alters the microbiome of goldfish and bioaccumulates in the olfactory epithelia, gills and brain. These studies also showed a negative impact on the olfactory tissue of exposed fish, which could lead to higher susceptibility to nasal pathogens. Our objective is to assess nitrite bioaccumulation in tissues and the alteration of the microbiome in the key aquaculture species channel catfish (Ictalurus punctatus). We hypothesize that the relative abundance and diversity of bacteria in the nose and gut microbiomes of channel catfish will be altered due to elevated nitrite exposure and that nitrite will bioaccumulate in tissues.Channel catfish were exposed to 0 mM (control), 0.2 mM, and 2 mM concentrations of nitrite. A total of six 500 liter tanks were used in a two‐phase experiment. For phase one, 16 fish per tank were placed into three 0 mM nitrite tanks and three 2 mM nitrite tanks. The 2 mM fish were kept for 12 hours before sampling. For phase two, 24 of the phase one control fish were placed into three 0.2 mM tanks and the remaining 24 control fish were left in the three control tanks for a total of 8 fish per tank. Phase two lasted 30 days with sub‐samplings at day five and 30. All fish were sampled for nose, gut, muscle, gill, brain, blood, liver, and kidney tissues for biochemical, microbial, or histological analysis. Upon dissection, fish from the 2 mM tanks exhibited marked methemoglobinemia due to nitrite‐induced oxidation of hemoglobin. The mortality rates were 0.04%, 0.08%, and 92% for the control, 0.2 mM, and 2 mM treatments respectively. The nose and gut microbiomes will be sequenced using whole genome shotgun sequencing to determine the microbial composition and measure changes in relative abundance. Histological analysis showed oxidative damage in all tissues from individuals exposed to 2 mM and partial damage in tissues of the 0.2 mM treatment.Because the microbiome is a powerful barrier against pathogens, disruption of the microbiome could lead to higher rates of disease infection and transmission in fish. Alteration of the nasal microbiome may increase mortality from the bacteria that cause septicemia in catfish, Edwardsiella ictaluri, whose common routes of infection are the nose or gut. Changes to the microbiome could be the missing link between nitrite and increased susceptibility to E. ictaluri infections in intensive farming. In the long term, this research aims to provide guidelines for water quality management in order to decrease the rate of bacterial infection and transmission in aquaculture systems.

Potentially new emerging diseases on two species of Indonesian freshwater aquaculture: giant gourami (Osphronemus gourami) and Asian catfish (Pangasius hypophthalmus)

Disease outbreaks and mortality in giant gourami and Asian catfish (pangasius) culture have been frequently reported during the last decade. Etiological agents of the disease have remained unknown, so it is needed to explore epidemiological studies to know the main causative agents. The study aimed to know the main pathogenic agents who were identified during disease outbreaks on giant gourami and pangasius. Data and information were collected through active and passive surveillance at the production centers of each species. Analysis was carried out according to standard fish disease diagnosis and eventually was focused on two suspected new emerging diseases (NED), namely Infectious Spleen and Kidney Necrosis Virus (ISKNV) belonging to Megalocytivirus on giant gourami and Enteric Septicemia of Catfish (ESC) caused by Edwardsiella ictaluri on pangasius. The results showed that ISKNV was detected on most of the diseased giant gourami samples and that E. ictaluri was identified on most diseased pangasius samples. This study concluded that both pathogenic agents, Megalocytivirus and E. ictaluri were suspected to be potentially NED on freshwater aquaculture, especially infecting giant gourami and pangasius respectively.

Synthesis of Pyranopyrans Related to Diplopyrone and Evaluation as Antibacterials and Herbicides.

Stereoselective syntheses of new pyranopyrans that are related to the natural product diplopyrone, which is a phytotoxin implicated in cork oak decline, have been achieved from carbohydrate starting materials in two approaches that are based on C-glycosides as key intermediates. A C-alkynyl glycoside prepared by Ferrier rearrangement was used as the precursor to a new pyranopyran alkyne that showed potent antibacterial activity against the common bacterial pathogen Edwardsiella ictaluri that causes enteric septicemia in catfish. The C-alkynyl glycoside also showed herbicidal activity. New bioassay data for the pyranopyran nitrile (4aR,6S,8aR)-6-cyano-6,8a-dihydropyrano-[3,2-b]pyran-2(4aH)-one, the most potent of the pyranopyrans synthesized to date, were obtained in greenhouse studies that revealed additional herbicidal activity. Other new analogues that were synthesized included desmethylpyranopyrans that were prepared by Isobe C-alkynylation-rearrangement/reduction and RCM-based pyranopyran construction. The antibiotic and phytotoxic activities of the new pyranopyrans synthesized in this study highlight the importance of substituents on the nonlactone ring and demonstrate the potential of such compounds as antibiotics and herbicides.

The Role of TonB Gene in Edwardsiella ictaluri Virulence.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen that causes enteric septicemia in catfish (ESC). Stress factors including poor water quality, poor diet, rough handling, overcrowding, and water temperature fluctuations increase fish susceptibility to ESC. The TonB energy transducing system (TonB-ExbB-ExbD) and TonB-dependent transporters of Gram-negative bacteria support active transport of scarce resources including iron, an essential micronutrient for bacterial virulence. Deletion of the tonB gene attenuates virulence in several pathogenic bacteria. In the current study, the role of TonB (NT01EI_RS07425) in iron acquisition and E. ictaluri virulence were investigated. To accomplish this, the E. ictaluri tonB gene was in-frame deleted. Growth kinetics, iron utilization, and virulence of the EiΔtonB mutant were determined. Loss of TonB caused a significant reduction in bacterial growth in iron-depleted medium (p > 0.05). The EiΔtonB mutant grew similarly to wild-type E. ictaluri when ferric iron was added to the iron-depleted medium. The EiΔtonB mutant was significantly attenuated in catfish compared with the parent strain (21.69 vs. 46.91% mortality). Catfish surviving infection with EiΔtonB had significant protection against ESC compared with naïve fish (100 vs. 40.47% survival). These findings indicate that TonB participates in pathogenesis of ESC and is an important E. ictaluri virulence factor.

Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System.

Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E.ictaluri, and in silico analysis of the E.ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E.ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E.ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000tons produced annually, and ESC is the leading cause of disease loss in the industry. We have demonstrated the survival and replication of E.ictaluri within channel catfish cells and identified a secretion system that is essential for E.ictaluri intracellular replication and virulence. We have also identified nine proteins encoded in the E.ictaluri genome that we believe are actively transferred from the bacterium to the cytoplasm of the host cell and act to manipulate host cell physiology to the advantage of the bacterium. The data presented here confirm that the proteins are actually transferred during an infection, which will lead to further work on approaches to preventing or controlling ESC.

Improved survival in channel catfish fed mannanoligosaccharides in an extruded diet

The high temperature and pressure achieved during cooking extrusion has been shown to affect nutrient availability. To determine the effects of extrusion temperature on the efficacy of mannanoligosaccharide (Bio-Mos?) in channel catfish, 4 experimental diets were fed for 9 wks and then challenged with Edwardsiella ictaluri bacterium. Catfish (9.9 ± 0.4 g) were randomly assigned to the following treatments: Low-None (low temperature process without additive); High-None (high temperature process without additive); Low-Bio (low temperature process with 4 g/kg diet Bio-Mos?); High-Bio (high temperature process with 4 g/kg diet Bio-Mos?). Although specific growth rate and food conversion ratio were similar among treatments (P > 0.10), survival after E. ictaluri challenge was highest (P ?-laden feed resulted in survival numbers similar to diets without Bio-Mos?. Extruding catfish diets supplemented with Bio-Mos? at lower temperatures may provide another strategy to control enteric septicemia of catfish.

Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3′,5′-monophosphate receptor protein (Crp) mutants in catfish host

Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3′,5′-adenosine monophosphate (cAMP) receptor protein ( crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δ crp mutants in a cAMP-dependent fashion. The E. ictaluri Δ crp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δ crp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish ( Danio rerio) and catfish ( Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δ crp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.

Expression of leptin-like peptide (LLP) mRNA in channel catfish (Ictalurus punctatus) is induced by exposure to Edwardsiella ictaluri but is independent of energy status

Leptin is a key pleiotropic cytokine involved in regulation of energy homeostasis and immunity in mammals. In channel catfish, the presence of a partial messenger RNA sequence that encodes a leptin-like peptide (LLP) has been identified and investigated. The objectives of the present studies were to clone and characterize full-length catfish LLP gene, examine tissue expression of LLP mRNA, and determine effects of prolonged fasting and exposure to Edwardsiella ictaluri (E. ictaluri), the bacteria that causes enteric septicemia in catfish, on LLP mRNA expression. Full-length catfish LLP gene was sequenced by genome walking and by 5′- and 3′-RACE. Catfish LLP gene contained three exons with the coding region located in exons 2 and 3. The amino acid sequence of the channel catfish LLP shared very low sequence similarities with leptin of other fish species or the mammalian leptin (24–49%). Using real-time polymerase chain reaction, LLP mRNA expression was detected in various tissues including brain, stomach, spleen, heart, liver, and trunk kidney and was especially high in the liver and trunk kidney. Expression of LLP mRNA in liver and brain was similar between fish that were fasted for 30days and those that received feed daily for 30days (P>0.10). Expression of LLP mRNA was increased in liver, spleen, and trunk kidney within 48h post-exposure to E. ictaluri compared to unexposed fish (P<0.05). Based on the results of the current studies, amino acid sequence of catfish LLP is highly dissimilar to mammalian and fish leptin. Unlike in most mammals, catfish LLP expression is independent of energy status. However, the expression of catfish LLP is increased after exposure to pathogenic bacteria, which is similar to mammals. Further investigations are required to clearly define the biological function and regulation of catfish LLP.

The Aspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and Its Use as Balanced-Lethal System in Fish Vaccinology

asdA mutants of Gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd+ plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd+ plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric Gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd+ expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd+ vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd+ plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.

Comparison of Growth, Body Composition, and Stress Responses of USDA103, USDA403, Industry, and Fast Growing Lines of Channel Catfish

Production traits such as growth, feed efficiency, survival, and fillet yield are economically important traits and an improvement in one or more of these traits will benefit the U.S. catfish farming industry. The U.S. Department of Agriculture (USDA) 103 line of channel catfish was developed jointly between the USDA Agricultural Research Service (ARS) Catfish Genetics Research Unit, Stoneville, Mississippi, and the Mississippi Agricultural and Forestry Experiment Station, Thad Cochran National Warmwater Aquaculture Center (NWAC), Stoneville. The USDA103 line of catfish (also referred to as the NWAC103 line) is being selected for increased growth, fillet yield, and resistance to enteric septicemia of catfish (ESC) and being compared with other genetic groups of catfish having potential commercial use. The USDA103 strain was developed from broodfish obtained in 1992 from the Uvalde National Fish Hatchery, Uvalde, Texas, USA. Many studies have compared growth rates of the USDA103 with other strains of channel catfish and have found that the USDA103 channel catfish exhibit superior growth characteristics (Silverstein et al. 1999; Wolters et al. 2000; Li et al. 2001; Jackson et al. 2003; Bosworth et al. 2004a; Peterson and Small 2006). The faster growth of the USDA103 strain of channel catfish is typically attributed to their ability to consume more feed (Li et al. 2001). The USDA103 strain has been selected for two generations for improved growth, fillet

The Edwardsiella ictaluri O polysaccharide biosynthesis gene cluster and the role of O polysaccharide in resistance to normal catfish serum and catfish neutrophils

Edwardsiella ictaluri, the causative agent of enteric septicaemia of catfish (ESC), expresses long O polysaccharide (OPS) chains on its surface. The authors previously reported the construction of an isogenic Ed. ictaluri OPS mutant strain and demonstrated that this strain is avirulent in channel catfish. This paper reports the cloning of the Ed. ictaluri OPS biosynthesis gene cluster and identification of the mutated gene in the OPS-negative strain. The sequenced region contains eight complete ORFs and one incomplete ORF encoding LPS biosynthesis enzymes. The mutated gene (designated wbiT) was similar to other bacterial galactose-4-epimerases. Glycosyl composition analysis indicated that wild-type Ed. ictaluri OPS contains higher amounts of galactose and N-acetylgalactosamine than the OPS mutant strain, which correlated well with predicted functions of the genes identified in the OPS biosynthesis cluster. The OPS mutant had a relatively small, but significant, decrease in its ability to survive in normal catfish serum compared to wild-type Ed. ictaluri, but it retained the ability to resist killing by catfish neutrophils.

Polymerase Chain Reaction Amplification of Genetic Loci from Diseased Channel Catfish Found Dead in Ponds

Abstract As part of a selective breeding program for farm-raised channel catfish Ictalurus punctatus, we screened diseased fish to identify genetic markers linked to disease resistance or susceptibility. Because many diseased fish in ponds are not detected until after death, we investigated the utility of DNA isolated from diseased channel catfish found dead in ponds. Channel catfish (4–25 g) diagnosed with enteric septicemia of catfish or saprolegniasis were sampled 24–48 h postmortem from infected ponds. Control fish were killed by anesthetic overdose and sampled immediately. Total DNA isolated from liver, muscle (with skin), and caudal fin was quantified and analyzed for degradation. Yield of purified DNA, measured as micrograms of DNA per milligram of tissue, was significantly (P < 0.05) lower in diseased fish than in controls. Two sets of DNA primers were used to amplify a portion of the channel catfish growth hormone gene and the mitochondrial D-loop region with the polymerase chain reaction. Degrad...

Specificity of Humoral Responses of Convalescent Channel Catfish toEdwardsiella ictaluri

Abstract Serum samples from 15 yearling channel catfish Ictalurus punctatus convalescing after experiencing enteric septicemia of catfish were distributed into three representative serum pools, each containing equal volumes of serum from five fish. Serologic recognition of each pooled serum sample against Edwardsiella ictaluri and Escherichia coli whole cells and against secretory antigen (exoantigen) derived from E. ictaluri was measured by enzyme-linked immunosorbent assays (ELISA). Serum samples were purified by affinity chromatography with a heterologous Ra,-mutant lipopolysaccharide that was derived from rough Salmonella typhimurium TV 119 and was covalently bound to an agarose matrix. Removal of antibodies recognizing the lipopolysaccharide by cross-reactive affinity purification caused significant decreases in serologic recognition of E. ictaluri (P < 0.10) and E. coli (P < 0.01) whole cells; however, serologic recognition of the E. ictaluri-specific exoantigen was not significantly decreased. Thes...

Electron Microscopic observations of channel catfish macrophages infected with Edwardsiella ictaluri

Edwardsiella ictaluri, a gram negative rod shaped enteric bacterium, is the etiological agent of Enteric Septicemia of Catfish (ESC). Little is known about the pathogenic mechanisms operative in ESC. It has been reported that infected macrophages are apparently unable to destroy the bacterium. The objective of this study was to detect differences in the way E. ictaluri virulent and avirulent strains associate with and are engulfed by channel catfish macrophages. This should help to characterize the possible role macrophages play in the disease process of ESC.The virulent bacterial strains used in this study were isolated from the kidneys of moribund channel catfish. Attenuated avirulent forms were derived from virulent strains by subsequent passages on Brain Heart Infusion Agar. The virulent and avirulent types were metabolically identical. Channel catfish, Ictalurus punctatus, were used as the susceptible host animals for experiments. For phagocytic studies, channel catfish macrophage suspensions were prepared as described by Trust et al.

Using Diagnostic Laboratory Records to Monitor Occurrence of Enteric Septicemia of Channel Catfish in Mississippi

Abstract Diagnostic laboratory information was used to monitor the temporal and spatial occurrence of enteric septicemia of catfish (ESC) in Mississippi. From 1980 through 1988, the Mississippi Cooperative Extension Service (MCES) entered information on 15,649 fish-kill investigations into a computerized data base. Analysis of the records revealed the following trends and patterns. From 1980 through 1985, there was an increase in laboratory submissions and in the ESC proportional morbidity rate (PMR; proportion of diagnosed epizootics that were specific for ESC. However, from 1986 to 1988, although the total yearly number of laboratory submissions had stabilized, there was a decrease in the PMR of ESC cases. During the 9-year interval, 427 producers from 56 counties had ESC diagnosed in their ponds. Twenty-eight producers from four counties were responsible for about 40% of all ESC cases.

Influence of Edwardsiella ictaluri septicemia on nitrite-induced methemoglobinemia in channel catfish (Ictalurus punctatus).

In a previous report, the authors showed that lack of acclimation to nitrite can result in abnormally high levels of methemoglobin in nitrite-exposed channel catfish. They also observed abnormal methemoglobin levels in fish when concurrent bacteremias are present. Enteric Septicemia of Catfish is an acute bacterial disease caused by Edwardsiella ictaluri. Nitrite-induced methemoglobinemia and Enteric Septicemia of Catfish are both economically important diseases of commercially cultured channel catfish. In the present study, the authors investigated the influence of acute infection with E. ictaluri on the level of methemoblobin in nitrite-exposed channel catfish fingerlings.