Dietary Glutamine Inclusion Regulates Immune and Antioxidant System, as well as Programmed Cell Death in Fish to Protect against Flavobacterium columnare Infection

The susceptibility of animals to pathogenic infection is significantly affected by nutritional status. The present study took yellow catfish (Pelteobagrus fulvidraco) as a model to test the hypothesis that the protective roles of glutamine during bacterial infection are largely related to its regulation on the immune and antioxidant system, apoptosis and autophagy. Dietary glutamine supplementation significantly improved fish growth performance and feed utilization. After a challenge with Flavobacterium columnare, glutamine supplementation promoted il-8 and il-1β expression via NF-κB signaling in the head kidney and spleen, but inhibited the over-inflammation in the gut and gills. Additionally, dietary glutamine inclusion also enhanced the systematic antioxidant capacity. Histological analysis showed the protective role of glutamine in gill structures. Further study indicated that glutamine alleviated apoptosis during bacterial infection, along with the reduced protein levels of caspase-3 and the reduced expression of apoptosis-related genes. Moreover, glutamine also showed an inhibitory role in autophagy which was due to the increased activation of the mTOR signaling pathway. Thus, our study for the first time illustrated the regulatory roles of glutamine in the fish immune and antioxidant system, and reported its inhibitory effects on fish apoptosis and autophagy during bacterial infection.

Does the freshwater mussel Anodonta anatina remove the fish pathogen Flavobacterium columnare from water?

Global decline of freshwater mussels (Unionoida) is threatening biodiversity and the essential ecosystem services that mussels provide. As filter-feeding organisms, freshwater mussels remove phytoplankton and suspended particles from the water. By filtering bacteria, freshwater mussels also decrease pathogen loads in the water. The objective of this study was to evaluate whether the common freshwater bivalve Anodonta anatina (duck mussel) could remove the bacterial fish pathogen Flavobacterium columnare from the water. Mussels reduced bacteria in both of the two experiments performed, so that the bacterial concentration at the end of the 96-h monitoring in mussel treatments was only 0.3–0.5 times that of the controls. Surprisingly, mussels did not reduce algal cell concentration statistically significantly. Mussel behavior (shell openness, foot position, and movement) was not affected by the presence of bacteria or algae, except for biodeposition formation, which was greatest in algal-fed treatments, followed by bacterial-fed treatments and controls, respectively. The intestines of bacteria-incubated A. anatina harbored F. columnare, suggesting that mussels ingested the bacteria. Present results suggest that freshwater mussels may also have a potential to mitigate aquaculture pathogen problems, as well as play a role in water quality management.

Type IX secretion system effectors and virulence of the model Flavobacterium columnare strain MS-FC-4

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and for gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and to other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted ten genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE: Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding ten secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

Deciphering the Molecular Basis for Attenuation of Flavobacterium columnare Strain Fc1723 Used as Modified Live Vaccine against Columnaris Disease

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.

A Review of Bacterial Co-Infections in Farmed Catfish: Components, Diagnostics, and Treatment Directions

Catfish production is a major aquaculture industry in the United States and is the largest sector of food fish production. As producers aim to optimize production yields, diseases caused by bacterial pathogens are responsible for high pond mortality rates and economic losses. The major bacterial pathogens responsible are Edwardsiella ictaluri, Aeromonas spp., and Flavobacterium columnare. Given the outdoor pond culture environments and ubiquitous nature of these aquatic pathogens, there have been many reports of co-infective bacterial infections within this aquaculture sector. Co-infections may be responsible for altering disease infection mechanics, increasing mortality rates, and creating difficulties for disease management plans. Furthermore, proper diagnoses of primary and secondary pathogens are essential in ensuring the correct treatment approaches for antimicrobials and chemical applications. A thorough understanding of the interactions and infectivity dynamics for these warm water bacterial pathogens will allow for the adoption of new prevention and control methods, particularly in vaccine development. This review aims to provide an overview of co-infective pathogens in catfish culture and present diagnostic case data from Mississippi and Alabama to define prevalence for these multiple-species infections better.

Immersion Vaccination by a Biomimetic-Mucoadhesive Nanovaccine Induces Humoral Immune Response of Red Tilapia (Oreochromis sp.) against Flavobacterium columnare Challenge

Immersion vaccination with a biomimetic mucoadhesive nanovaccine has been shown to induce a strong mucosal immune response against columnaris disease, a serious bacterial disease in farmed red tilapia caused by Flavobacterium columnare. However, the induction of a systemic immune response by the vaccine is yet to be investigated. Here, we examine if a specific humoral immune response is stimulated in tilapia by a biomimetic-mucoadhesive nanovaccine against Flavobacterium columnare using an indirect-enzyme-linked immunosorbent assay (ELISA), serum bactericidal activity (SBA) and the expression of immune-related genes within the head-kidney and spleen, together with assessing the relative percent survival of vaccinated fish after experimentally infecting them with F. columnare. The anti-IgM antibody titer of fish at 14 and 21 days post-vaccination was significantly higher in chitosan complex nanoemulsion (CS-NE) vaccinated fish compared to fish vaccinated with the formalin-killed vaccine or control fish, supporting the serum bactericidal activity results at these time points. The cumulative mortality of the unvaccinated control fish was 87% after challenging fish with the pathogen, while the cumulative mortality of the CS-NE vaccinated group was 24%, which was significantly lower than the formalin-killed vaccinated and control fish. There was a significant upregulation of IgM, IgT, TNF α, and IL1-β genes in the spleen and kidney of vaccinated fish. Significant upregulation of IgM and IgT genes was observed in the spleen of CS-NE vaccinated fish. The study confirmed the charged-chitosan-based mucoadhesive nanovaccine to be an effective platform for immersion vaccination of tilapia, with fish generating a humoral systemic immune response against columnaris disease in vaccinated fish.

Molecular Characterization and Defense Functions of the Nile Tilapia (Oreochromis niloticus) DnaJ B9b and DnaJ C3a Genes in Response to Pathogenic Bacteria under High-Temperature Stress Conditions

DnaJ proteins or heat shock protein 40s (HSP40s) form one of the largest heat shock protein families. In this study, 2 cDNAs encoding Nile tilapia (Oreochromis niloticus) DnaJ proteins (On-DnaJ B9b and On-DnaJ C3a) were successfully cloned and characterized. The structures and organizations of these two genes are first reported in the present study. On-DnaJ B9b is approximately 2.1 kb long and contains 2 exons and 1 intron, while On-DnaJ C3a is approximately 12 kb long and contains 12 exons and 11 introns. Under normal conditions, On-DnaJ B9b mRNA is highly expressed in gonad and trunk kidney tissues, while On-DnaJ C3a transcripts are abundantly expressed in gills, intestine, liver, and trunk kidney tissues. Following pathogenic infections, the expression of both genes is induced in the liver, spleen and head kidney tissues of Nile tilapia that were infected with two virulent pathogenic bacteria, Streptococcus agalactiae and Flavobacterium columnare. Silencing of these two genes was first carried out, and the results clearly indicated their crucial roles under both heat and bacterial stress conditions. The fundamental knowledge obtained from this study indicates the characteristic basic biofunctions of heat shock proteins in the regulation of intracellular proteins during infection, which involve preventing protein aggregation, promoting protein refolding, and activating unfolded protein degradation.