RESUMEN

Tilapia is one of the most important farmed fish in the world and the most cultivated in Brazil. The increase of this farming favors the appearance of diseases, including bacterial diseases. Therefore, the aim of this study was to evaluate the bactericidal activity of copaiba oil, Copaifera duckei, against Streptococcus agalactiae and Flavobacterium columnare and the dietary effect of copaiba oil on zootechnical performance, hematological, biochemical, immunological, and histological analysis before and after an intraperitoneal infection (body cavity) with S. agalactiae in Nile tilapia. For this, fish were randomly distributed into 15 fiber tanks in five treatments (0, 0.25, 0.50, 0.75, and 1.0%) and fed with a commercial diet supplemented with copaiba oil for 30 days. After this period, the fish were randomly redistributed for the experimental challenge with S. agalactiae into six treatments (T0, T1, T2, T3, T4, and T5), the fish were anesthetized, and blood samples were collected to assess hematological, biochemical, immunological, and histological parameters. Copaiba oil showed bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro. In addition, concentrations of 0.75 and 1.0% of copaiba oil have an anti-inflammatory effect and improve hematological and immunological parameters, increasing leukocyte numbers, albumin, and serum lytic activity. Furthermore, there is an increase in the intestinal villus length and tissue damage in groups at concentrations of 0.75 and 1.0% of copaiba oil. In conclusion, copaiba oil presented bactericidal activity against Streptococcus spp. and Flavobacterium spp. in vitro, and oral supplementation at concentrations of 0.75 and 1.0% compared to the control group enhanced non-specific immune parameters and digestibility in Nile Tilapia.

Asunto(s)

RESUMEN

TonB-dependent siderophore receptors play a critical transport role for Flavobacterium columnare virulence formation and growth, and have become valuable targets for the development of novel antimicrobial agents. Traditional Chinese medicine has demonstrated notable efficacy in the treatment of fish diseases and includes potential antibacterial agents. Herein, we performed molecular docking-based virtual screening to discover novel TonB-dependent siderophore receptor inhibitors from traditional Chinese medicine and provide information for developing novel antibacterial agents. Firstly, we efficiently obtained 11 potential inhibitors with desirable drug-like characteristics from thousands of compounds in the TCM library based on virtual screening and property prediction. The antibacterial activity of Enoxolone, along with its interaction characteristics, were determined via an MIC assay and molecular dynamic simulation. Transcriptional profiling, along with validation experiments, subsequently revealed that an insufficient uptake of iron ions by bacteria upon binding to the TonB-dependent siderophore receptors is the antibacterial mechanism of Enoxolone. Finally, Enoxolone's acceptable toxicity was illustrated through immersion experiments. In summary, we have used virtual screening techniques for the first time in the development of antimicrobial agents in aquaculture. Through this process, we have identified Enoxolone as a promising compound targeting the TonB-dependent siderophore receptor of F. columnare. In addition, our findings will provide new ideas for the advancement of innovative antimicrobial medications in aquaculture.

RESUMEN

Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB, sprF, and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ563, which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCEFlavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies.

Asunto(s)

RESUMEN

Eutrophication of aquatic ecosystems is associated with an increased risk of pathogen infection via increased pathogen growth and host exposure via increased pathogen doses. Here, we studied the effect of nutrients on the virulence of an opportunistic bacterial pathogen of fish, Flavobacterium columnare, in challenge experiments with rainbow trout fingerlings. We hypothesized that removing all nutrients by washing the bacteria would reduce virulence as compared to unwashed bacteria, but adding nutrients to the tank water would increase the virulence of the bacterium. Nutrient addition and increase in bacterial dose increased virulence for both unwashed and washed bacteria. For unwashed bacteria, the addition of nutrients reduced the survival probability of fish challenged with low bacterial doses more than for fish challenged with higher bacterial doses, suggesting activation of bacterial virulence factors. Washing and centrifugation reduced viable bacterial counts, and the addition of washed bacteria alone did not lead to fish mortality. However, a small addition of nutrient medium, 0.05% of the total water volume, added separately to the fish container, restored the virulence of the washed bacteria. Our results show that human-induced eutrophication could trigger epidemics of aquatic pathogens at the limits of their survival and affect their ecology and evolution by altering the dynamics between strains that differ in their growth characteristics.

Asunto(s)

RESUMEN

BACKGROUND: The bacterial pathogen, Flavobacterium columnare causes columnaris disease in Labeo rohita globally. Major effects of this bacterial infection include skin rashes and gill necrosis. Nimbolide, the key ingredient of the leaf extract of Azadirachta indica possesses anti-bacterial properties effective against many microorganisms. Nano-informatics plays a promising role in drug development and its delivery against infections caused by multi-drug-resistant bacteria. Currently, studies in the disciplines of dentistry, food safety, bacteriology, mycology, virology, and parasitology are being conducted to learn more about the wide anti-virulence activity of nimbolide. METHODS: The toxicity of nimbolide was predicted to determine its dosage for treating bacterial infection in Labeo rohita. Further, comparative 3-D structure prediction and docking studies are done for nimbolide conjugated nanoparticles with several key target receptors to determine better natural ligands against columnaris disease. The nanoparticle conjugates are being designed using in-silico approaches to study molecular docking interactions with the target receptor. RESULTS: Bromine conjugated nimbolide shows the best molecular interaction with the target receptors of selected species ie L rohita. Nimbolide comes under the class III level of toxic compound so, attempts are made to reduce the dosage of the compound without compromising its efficiency. Further, bromine is also used as a common surfactant and can eliminate heavy metals from wastewater. CONCLUSION: The dosage of bromine-conjugated nimbolide can be reduced to a non-toxic level and thus the efficiency of the Nimbolide can be increased. Moreover, it can be used to synthesize nanoparticle composites which have potent antibacterial activity towards both gram-positive and gram-negative bacteria. This material also forms a good coating on the surface and kills both airborne and waterborne bacteria.

Asunto(s)

RESUMEN

OBJECTIVE: Columnaris disease is a leading cause of disease-related losses in the catfish industry of the southeastern United States. The term "columnaris-causing bacteria" (CCB) has been coined in reference to the four described species that cause columnaris disease: Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry; however, there is a lack of knowledge of which CCB species are most prevalent in farm-raised catfish. The current research objectives were to (1) sample columnaris disease cases from the U.S. catfish industry and identify the species of CCB involved and (2) determine the virulence of the four CCB species in Channel Catfish Ictalurus punctatus in controlled laboratory challenges. METHODS: Bacterial isolates or swabs of external lesions from catfish were collected from 259 columnaris disease cases in Mississippi and Alabama during 2015-2019. The DNA extracted from the samples was analyzed using a CCB-specific multiplex polymerase chain reaction to identify the CCB present in each diagnostic case. Channel Catfish were challenged by immersion with isolates belonging to each CCB species to determine virulence at ~28°C and 20°C. RESULT: Flavobacterium covae was identified as the predominant CCB species impacting the U.S. catfish industry, as it was present in 94.2% (n = 244) of diagnostic case submissions. Challenge experiments demonstrated that F. covae and F. oreochromis were highly virulent to Channel Catfish, with most isolates resulting in near 100% mortality. In contrast, F. columnare and F. davisii were less virulent, with most isolates resulting in less than 40% mortality. CONCLUSION: Collectively, these results demonstrate that F. covae is the predominant CCB in the U.S. catfish industry, and research aimed at developing new control and prevention strategies should target this bacterial species. The methods described herein can be used to continue monitoring the prevalence of CCB in the catfish industry and can be easily applied to other industries to identify which Flavobacterium species have the greatest impact.

Asunto(s)

RESUMEN

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish. Columnaris disease can cause heavy economic losses in aquaculture. In this study, whole-genome sequencing was used to characterize this pathogen. F. columnare isolate AH-01 had a circular chromosome and plasmid that encoded a total of 3,022 genes. Isolate GX-01 only had a circular chromosome and encoded 2,965 genes. Genomic islands, prophage regions, and CRISPR/Cas systems were identified in both genomes. Both genomes presented evidence of gene variation and horizontal transfer, both of which are the essential components of genetic diversity, genome plasticity, and functional evolution. Single-gene phylogeny and comparative genome analyses were performed to investigate the variation and evolution of this pathogen. Genetic analysis of 16S rRNA and housekeeping gene sequences significantly clustered 55 F. columnare isolates into four clades. The intragroup identity of the 16S rRNA gene exceeded 99%, while the intergroup identity was below the species delineation threshold. We discovered significant translocation, inversion, and rearrangement events that influenced local synteny within each group. Notably, the observed alignments varied considerably among all the studied groups. The core genomes of all strains with available sequences comprised 747 genes, corresponding to approximately 25% of the genome. Core genome multilocus sequence typing, genome-wide orthology and phylogenetic analyses, and average nucleotide identity suggested that the currently existing F. columnare was an assemblage of several distinct species, with levels of divergence at least equivalent to those between recognized bacterial species. The present investigation provided genomic evidence of gene variation and horizontal transfer, which were the basis of genetic diversity, genome plasticity, and functional evolution. The findings supported a proposed new taxonomic perspective on F. columnare.

RESUMEN

Recent work has identified pituitary adenylate cyclase activating polypeptide (PACAP) as a potential antimicrobial and immune stimulating agent which may be suitable for use in aquaculture. However, its effects on teleost immunity are not well studied and may be significantly different than what has been observed in mammals. In this study we examined the effects of PACAP on the Atlantic salmon macrophage cell line SHK-1. PACAP was able to increase the expression of LPS-induced il-1ß in at concentrations of 1 uM when administered 24h prior to LPS stimulation. Furthermore, concentrations as low as 40nM had an effect when administered both 24h prior and in tandem with LPS. PACAP was also capable of increasing the expression of il-1ß and tnf-α in SHK-1 cells challenged with a low dose of heat-killed Flavobacterium columnare. We attempted to get a better understanding of the mechanism underlying this enhancement of il-1ß expression by manipulating downstream signaling of PACAP with inhibitors of phosphodiesterase and phospholipase C activity. We found that inducing cAMP accumulation with phosphodiesterase inhibitors failed to recapitulate the effect of PACAP administration on LPS-mediated il-1ß expression by PACAP, while use of a phospholipase C inhibitor caused a PACAP-like enhancement in LPS-mediated il-1ß expression. Interestingly, the VPAC1 receptor inhibitor PG97-269, but not the PAC1 inhibitor max.d.4, also was capable of causing a PACAP-like enhancement in LPS-mediated il-1ß expression. This suggests that fish do not utilize the PACAP receptors in the same manner as mammals, but that it still exerts an immunostimulatory effect that make it a good immunostimulant for use in aquaculture.

RESUMEN

Flavobacterium columnare, which causes columnaris disease, is responsible for significant mortality in grass carp. Vaccination is a safe and effective measure to combat this disease, and this study aimed to investigate the immune protective effects of different treatments using an inactivated F. columnare vaccine. The vaccine was prepared by inactivating the bacteria with 0.05% formaldehyde at 4°C for 24 hours. The experiments involving grass carp were divided into two parts. In Experiment 1, the immune effects of two isolates, JX-01 (genomovar I) and MU-04 (genomovar II), were compared, along with the impact of white oil adjuvant and the number of immunizations. The results showed that when the white oil adjuvant was used as a booster, the relative percent survival (RPS) of the JW2 group and MW2 group after 8 weeks of the first immunization was 34% and 61%, respectively. In Experiment 2, only the MU-04 (genomovar II) isolate was used as an antigen, with the white oil adjuvant as a booster. The effects of different doses (CFU=108, 107, and 106 bacteria/mL) on immune responses were compared, and the RPS values in the MW6, MW7, and MW8 groups after 4 weeks of the first immunization were found to be 38%, 57%, and 71%, respectively. Furthermore, in the cross-antigen protection experiment, the MW2 group exhibited an RPS of 55% against the JX-01 isolate, which was significantly higher than the control group (33%). These findings suggest that an inactivated vaccine comprising an appropriate antigen isolate when administered with a white oil adjuvant as a booster, can provide effective protection in grass carp.

RESUMEN

TLR3 and TRIF (adaptor protein for TLR3) are vital to the MyD88-independent pathway mediated by Toll-like receptors (TLRs). In order to identify the role of TLR3 and TRIF in Micropterus salmoides, the Ms_TLR3 and Ms_TRIF (Ms: abbreviation for M. salmoides) were cloned and characterized in this study. The open reading frames (ORFs) of Ms_TLR3 and Ms_TRIF genes were 2736 bp and 1791 bp in length, encoding 911 and 596 amino acids, respectively. The protein structure of Ms_TLR3 includes a signal peptide, 18 LRR-related domains, a low complexity region, a transmembrane region, and a TIR domain. However, only a TIR domain and a coiled coil domain were found in Ms_TRIF. Both Ms_TLR3 and Ms_TRIF showed the highest homology to that of M. dolomieu. Ms_TLR3 and Ms_TRIF showed similar expression patterns in various tissues, with the highest expression level in the head kidney. After stimulation of Flavobacterium columnare, the mRNA expressions of Ms_TLR3 and Ms_TRIF were significantly up-regulated at 1 dpi in the gill, spleen and head kidney, and at 6 hpi in the trunk kidney. Furthermore, morphological changes in the gills of largemouth bass challenged with F. columnare suggested that F. columnare infection can destroy the gill filament. Taken together, Ms_TLR3 and Ms_TRIF are indeed involved in F. columnare infection and the subsequent immune response in largemouth bass. Moreover, Ms_TLR3 and Ms_TRIF might respectively play their potential roles in mucosal (mainly in the gill) and systemic (mainly in the head kidney) immune response to bacterial infection.

Asunto(s)

RESUMEN

Interleukin (IL) 4 and 13 are signature cytokines orchestrating Th2 immune response. Teleost fish have two homologs, termed IL-4/13A and IL-4/13B, and have been functionally characterized. However, what cells express IL-4/13A and IL-4/13B has not been investigated in fish. In this work, the recombinant IL-4/13A and IL-4/13B proteins of grass carp (Ctenopharyngodon idella) were produced in the Escherichia coli (E. coli) cells and purified. Monoclonal antibodies (mAbs) against the recombinant CiIL-4/13A and CiIL-4/13B proteins were prepared and characterized. Western blotting analysis showed that the CiIL-4/13A and CiIL-4/13B mAbs could specifically recognize the recombinant proteins expressed in the E. coli cells and HEK293T cells and did not cross-react with each other. Confocal microscopy revealed that the CiIL-4/13A+ and CiIL-4/13B+ cells were present in the gills, intestine and spleen and could be upregulated in fish infected with Flavobacterium columnare (F. columnare). Interestingly, the cells expressing CiIL-4/13A and CiIL-4/13B were mostly CD3γ/δ+ cells. The CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells were significantly upregulated in the gill filaments and the intestinal mucosa after F. columnare infection. Our results imply that the CD3γ/δ+/IL-4/13A+ and CD3γ/δ+/IL-4/13B+ cells are important for homeostasis and the regulation of mucosal immunity.

Asunto(s)

RESUMEN

Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted heme-binding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare.

Asunto(s)

RESUMEN

Flavobacterium columnare represent one of the most important bacterial pathogens of cultured sturgeon. However, at present there are no commercially available vaccines to prevent infection and treatment options are limited. ß-glucans have been shown to be potent immunostimulants that can provide fish protection against infectious disease. In this study, the effects of dietary ß-glucan supplementation on disease susceptibility were examined by exposing 0.3% ß-glucan-fed white sturgeon (Acipenser transmontanus) to Flavobacterium columnare in laboratory-controlled challenges. Morbidity and mortality were monitored for 15 days post-challenge (dpc). Additionally, transcript levels for pro-inflammatory cytokines, regulatory cytokines and acute phase proteins (APP) were investigated in the spleen and gills at different time points post-challenge. No evidence of protection was observed in ß-glucan-fed fish challenged with the bacteria. Moreover, significantly greater mortalities were observed in ß-glucan-fed fish challenged with F. columnare (p<0.05), likely associated with acute inflammatory response as haptoglobin and serotransferrin transcripts in the gills were significantly higher in fish within this group at 1 dpc. Transcript levels for all tested cytokines and APP in the spleen were similar amongst treatment groups. The results from this study suggest that ß-glucan supplementation at the concentration and rate investigated provides no-benefit to white sturgeon against F. columnare.

RESUMEN

Flavobacterium columnare, the causative agent of columnaris disease in a large variety of freshwater fish, is a major problem in commercial aquaculture. A limited number of antimicrobial therapies are available to control this disease; therefore, these agents must be used judiciously. To facilitate effective monitoring for changes in susceptibility, the Clinical Laboratory Standards Institute (CLSI) has a standard broth microdilution test method specific for F. columnare. However, there are no CLSI-approved criteria (termed epidemiological cutoff values [ECVs]) to interpret results. Nevertheless, researchers have developed provisional ECVs based on testing by one laboratory. To satisfy CLSI data requirements, three laboratories used the standard method to generate additional antimicrobial susceptibility data against ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, oxolinic acid, oxytetracycline, sulfadimethoxine/ormetoprim, and sulfamethoxazole-trimethoprim using 109 F. columnare isolates. The new data combined with previously published data from 120 F. columnare isolates were analyzed and ECVs proposed to CLSI. Of the 10 antimicrobials, ECVs were approved for ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid, and oxytetracycline, which were published in the 2020 edition of the CLSI document VET04 performance standards. These ECVs will help microbiologists categorize decreased antimicrobial susceptibility among F. columnare and will help in surveillance efforts to ensure judicious antimicrobial use.

Asunto(s)

RESUMEN

A disease outbreak occurred in Murray cod Maccullochella peelii peelii in a recirculating aquaculture farm in Tianjin city, China, in 2019. Strain MRX-2019 was isolated and considered to be the etiological pathogen; it was identified as Flavobacterium columnare based on a 16S rDNA gene sequence analysis and physiological and biochemical tests. The effect of salinity on the growth of MRX-2019 was investigated in vitro. Salinity >4‰ (i.e. 6‰) inhibited MRX-2019 growth, whereas 8 and 10‰ salinity killed it. The effect of 4‰ salinity on F. columnare was not significant (p > 0.05). When MRX-2019-infected Murray cod were treated with 4, 6, or 8‰ salinity, the mortality rate was reduced by 8.9, 67.76, or 75.56%, respectively, compared with that of the control. However, the mortality rate increased by 7.77% at 10‰ salinity. In this study, we found that maintaining the fish in freshwater with 6-8‰ salinity effectively reduced the mortality of these fish when infected with F. columnare. The findings provide an environmentally friendly control strategy for columnaris disease in Murray cod.

Asunto(s)

RESUMEN

In teleost fish, the nucleotide polymorphisms of histone H2A significantly affect the resistance or susceptibility of zebrafish to Edwardsiella piscicida infection. Whether histone H2A variants can enhance the resistance of grass carp to Flavobacterium columnare infection remains unclear. Here, the effects of 7 previously obtained variants (gcH2A-1~gcH2A-7) and 5 novel histone H2A variants (gcH2A-11, gcH2A-13~gcH2A-16) in response to F. columnare infection were investigated. It was found that these histone H2A variants could be divided into type I and II. Among them, 5 histone H2A variants had no any effects on the F. columnare infection, however 7 histone H2A variants had antibacterial activity against F. columnare infection. The gcH2A-4 and gcH2A-11, whose antibacterial activity was the strongest in type I and II histone H2A variants respectively, were picked out for yeast expression. Transcriptome data for the samples from the intestines of grass carp immunized with the engineered Saccharomyces cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 revealed that 5 and 12 immune-related signaling pathways were significantly enriched by gcH2A-4 or gcH2A-11, respectively. For the engineered S. cerevisiae expressing gcH2A-4, NOD-like receptor and Toll-like receptor signaling pathways were enriched for up-regulated DEGs. Besides NOD-like receptor and Toll-like receptor signaling pathways, the engineered S. cerevisiae expressing gcH2A-11 also activated Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway and C-type lectin receptor signaling pathway. Furthermore, grass carp were immunized with the engineered S. cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 for 1 month and challenged with F. columnare. These grass carp immunized with gcH2A-4 or gcH2A-11 showed lower mortality and fewer numbers of F. columnare than did the control group. All these results suggest that gcH2A-4 and gcH2A-11 play important roles in evoking the innate immune responses and enhancing disease resistance of grass carp against F. columnare infection.

Asunto(s)

RESUMEN

The major histocompatibility complex (MHC) is an important component of the immune system of vertebrates, which plays a vital role in presenting extrinsic antigens. In this study, we cloned and characterized the mhc ⅡA and mhc ⅡB genes of yellow catfish Pelteobagrus fulvidraco. The open reading frames (ORFs) of mhc ⅡA and mhc ⅡB genes were 708 bp and 747bp in length, encoding 235 and 248 amino acids, respectively. The structure of mhc ⅡA and mhc ⅡB includes a signal peptide, an α1/ß1 domain, an α2/ß2 domain, a transmembrane region and a cytoplasmic region. Homologous identity analysis revealed that both mhc ⅡA and mhc ⅡB shared high protein sequence similarity with that of Chinese longsnout catfish Leiocassis longirostris. mhc ⅡA and mhc ⅡB showed similar expression patterns in different tissues, with the higher expression level in spleen, head kidney and gill and lower expression in liver, stomach, gall bladder and heart. The mRNA expression level of mhc ⅡA and mhc ⅡB in different embryonic development stages also showed the similar trends. The higher expression was detected from fertilized egg to 32 cell stage, low expression from multicellular period to 3 days post hatching (dph), and then the expression increased to a higher level from 4 dph to 14 dph. The mRNA expression levels of mhc ⅡA and mhc ⅡB were significantly up-regulated not only in the body kidney and spleen, but also in the midgut, hindgut, liver and gill after challenge of Flavobacterium columnare. The results suggest that Mhc Ⅱ plays an important role in the anti-infection process of yellow catfish P. fulvidraco.

Asunto(s)

RESUMEN

Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.

RESUMEN

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

Asunto(s)

RESUMEN

Yellow River carp (Cyprinus carpio) is an economically-important freshwater fish. It is the common host of the epizootic bacterium Flavobacterium columnare, a common fish pathogen that causes columnaris disease resulting in aquacultural losses. However, information on the functions and mechanisms of the immune system of Yellow River carp infected with F. columnare is limited. Therefore, the aim of this study is to evaluate the genetic and histopathological effects of an experimentally-induced F. columnare infection in Yellow River carp. Sixty fish were divided into control (CT group) and challenged groups. The gills were collected for histological and transcriptome analysis to understand the host immune response after challenge with F. columnare. The infected fish of the IF group presented typical columnaris disease symptoms and higher mortality, as well as histological changes. However, some challenged fish showed asymptomatic infection (IC group). Additionally, there were 1776 significant differentially-expressed genes (DEGs) between the IC and CT groups, 1853 DEGs between the IF and CT groups, and 1836 DEGs between the IF and IC groups, All the DEGs were classified into three gene ontology categories, which were allocated to 158 KEGG pathways. Moreover, immune-related genes were confirmed by qRT-PCR. we quantified the level of IL-1, IL-6, TNF-α and IL-8 by ELISA. The results showed the highest expression levels of inflammatory cytokines as well as stress proteins and the adhesion molecules in the lF group, which may contribute to severe infection, and a higher case fatality rate, while the high expression of chemokines, costimulatory molecules and the up regulation of antigen presentation function could help the carp resist F. columnare infection.

Asunto(s)