RESUMEN
Lactococcus garvieae is an important pathogen of fish, associated with high rates of mortality and infection recurrence in summer or stressful conditions. Chronic infection and disease recurrence have also been reported to be associated with biofilms. However, the impact of biofilm and planktonic bacterial infection on fish immune responses remains unclear. In this study, de novo sequencing was used to compare differences of the spleen transcriptome in planktonic- and biofilm-infected mullets. Among the 181,024 unigenes obtained, 3,392 unigenes were associated with immune response genes. Comparative analysis of the gene expression between infection with the L. garvieae planktonic type and biofilm type identified a total of 3,120 and 3,489 differentially expressed genes in response to planktonic and biofilm infection, respectively, of which 1,366 and 1,458 genes were upregulated, and 1,754 and 1,458 genes were downregulated, respectively. Gene ontology enrichment analysis of immune genes identified genes involved in the complement system, toll-like receptor signaling, and antigen processing, which were further verified by qPCR. Additionally, genes encoding TLR2, IL-1ß, TNF-α, C7, and MHC class II peptides were downregulated in response to biofilm infection. Importantly, the results show that biofilm infection induces a different immune pathway response compared with planktonic bacterial infection and, furthermore, illustrates that the prevention of biofilm formation may be a necessary and new strategy for controlling bacterial infection in aquaculture.
Asunto(s)
Infecciones Bacterianas , Enfermedades de los Peces , Smegmamorpha , Animales , Biopelículas , Enfermedades de los Peces/microbiología , Inmunidad Innata , Lactococcus , Plancton/genética , Smegmamorpha/genética , TranscriptomaRESUMEN
Photobacterium damselae subsp. damselae is a pathogen that mainly infects a variety of fish species. There are many antibiotic-resistant strains of Photobacterium damselae subsp. damselae. In a previously published article, we described the production method for a novel oral biofilm vaccine. In the study reported herein, we confirmed the protective effect of the oral biofilm vaccine against Photobacterium damselae subsp. damselae. Twenty-eight days after vaccination, phagocytosis increased by 256% relative to the control group. The mean albumin-globulin ratios of the vaccine groups were significantly lower than the mean albumin-globulin ratios of the control group. There were no significant intergroup differences in lysozyme activity. Mean IgM titers were significantly higher in the vaccine group than in the control group. There was a significant upregulation of the TLR 3, IL-1ß, and IL-8 genes in the spleen 28 days after vaccination. The cumulative mortality of the control fish was 84% after challenging fish with the Photobacterium damselae subsp. damselae, while the cumulative mortality of the oral biofilm vaccine (PBV) group was 32%, which was significantly higher than those of the whole-cell vaccine (PWV) and chitosan particle (CP) groups. There is minimal published research on the prevention and treatment of Photobacterium damselae subsp. damselae infection; therefore, this oral biofilm vaccine may represent a new method to fill this gap.
RESUMEN
Lactococcus garvieae (L. garvieae) is an important pathogen that causes enormous economic losses in both marine and freshwater aquaculture. At present, antibiotics are the only option for farmers to reduce the losses caused by L. garvieae. However, the usage of antibiotics leads to environmental pollution and the production of drug-resistant strains of bacteria. Therefore, vaccination is preferred as an alternative method to prevent infectious diseases. In this study, we describe an effective approach to the production of an oral biofilm vaccine, using bacteria grown on chitosan particles to form biofilms, and thus providing an inactive pathogen that enhances the immune response in fish. We observed the formation of a biofilm on chitosan particles and administered the novel oral biofilm vaccine to fish. We analyzed the immune responses, including antibody production, phagocytic ability, albumin/globulin ratio and immune-related genes, of vaccinated and control groups of black mullet. Our results show that the phagocytic ability of the biofilm vaccine group was 84%, which is significantly higher than that of the control group, and the antibody production in this group was significantly higher compared with the other group. The mRNA expression levels of immune-related genes (TLR2, IL-1ß, TNF-α) were significantly upregulated in the spleen after vaccination. In challenge experiments, the relative percent survival (RPS) was 77% in the biofilm vaccine group, 18% in the whole-cell vaccine group, and 0% in the chitosan particle group at 32 days post-vaccination. In addition, we also found that the relative percent survival (RPS) at 1 day post-vaccination was 74% in the biofilm vaccine group, 42% in the whole-cell vaccine group, and 26% in the chitosan particle group. In both long-term and short-term challenge experiments, the viability of the biofilm vaccine group was significantly higher than that of the whole-cell, chitosan particle and PBS groups. We conclude that based on its protective effect, the L. garvieae biofilm vaccine is better than the whole-cell vaccine when challenged several weeks after vaccination. In addition, the biofilm vaccine also has a greater protective effect than the whole-cell vaccine when challenged immediately after vaccination. Therefore, the biofilm vaccine might represent a novel method for the prevention and treatment of L. garvieae infection.
RESUMEN
Streptococcus agalactiae infects numerous fish species, causing considerable economic losses during fish cultivation. This study compared the phenotypic differences among S. agalactiae hemolytic variant isolates and investigated the genetic composition of their hemolysin genes. Hemolysin is encoded by the cyl operon and mainly regulated by covS/R, which also regulates encapsulation. In total, 45 S. agalactiae clinical isolates were collected from cultured fishes in Taiwan. Three different hemolytic phenotypes-α, ß, and γ-were identified. Of the 45 isolates, 39 were ß hemolytic, 3 were α hemolytic, and 3 were γ hemolytic. The γ-hemolytic isolates demonstrated significantly thicker encapsulation and slower growth rates than did the α- and ß-hemolytic isolates. However, no isolate had mutations in the regulatory gene covS/R. A 1252-bp insertion sequence (IS) in the cyl operon of α-hemolytic isolates, located at cylF region, was found. This IS interrupted cylF through insertion at 23â¯bp downstream of starting codon, causing incomplete mRNA transcription. The ß-hemolytic isolates showed no mutation in the cyl operon. By contrast, the γ-hemolytic isolates had lost the entire cyl operon; it had been replaced by a 14-kb genomic island containing genes for DNA recombinase and septum formation proteins. In summary, the differences in hemolysin genes between α- and ß-hemolytic isolates were due to the IS in the cylF region, whereas in the γ-hemolytic isolates, the entire cyl operon was deleted and replaced. These findings explain different hemolysin expressions of the clinical S. agalactiae isolates taken from fish ponds in Taiwan. IMPORTANCE: Streptococcus agalactiae infects both warm- and cold-blooded animals and causes major aquatic cultivation loss. Pathogenic isolates from the outbreak of fish ponds were examined their cyl operon gene. α-Hemolytic isolate with mutant cyl operon was observed for the first time in aquaculture animals and was compared to intact or entire cyl operon deletion of ß- and γ-hemolytic isolates. Hemolysis expression levels of Streptococcus agalactiae are explained.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mutación , Operón , Fenotipo , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/fisiología , Secuencia de Aminoácidos , Cápsulas Bacterianas , Secuencia de Bases , Genes Bacterianos , Genoma Bacteriano , Genómica/métodos , Hemólisis , Análisis de Secuencia de ADN , Streptococcus agalactiae/aislamiento & purificaciónRESUMEN
A monoclonal antibody (MAb) was generated against the capsid protein (ORF 72) of koi herpesvirus (KHV) isolated from diseased koi Cyprinus carpio in Taiwan. The clone of MAb-B2 was obtained by immunizing mice with whole virus particles and further identified using indirect enzyme-linked immunosorbent assay and Western blot assay. In addition, it detected KHV in KHV-infected cells but not in those of mock-infected cells as demonstrated by indirect immunofluorescence assay. The neutralization test showed that MAb-B2 neutralized KHV. Furthermore, we uncovered that MAb-B2 recognizes the ORF72 of KHV as revealed by liquid chromatography-tandem mass spectrometry and Western blot assays. Additionally, MAb-B2 has been used as a diagnostic tool for detection of KHV in clinical samples by immunohistochemistry. Collectively, our results indicated that MAb-B2 could be used in the development of a diagnostic kit for diagnosis of KHV infections and ORF72 protein of KHV might be a candidate for future vaccine development.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Carpas/virología , Herpesviridae/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Western Blotting , Cromatografía Liquida , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Herpesviridae/aislamiento & purificación , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Taiwán , Espectrometría de Masas en TándemRESUMEN
Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an emerging public health threat. The aims of this study were to identify nasal carriage of MRSA in finishing pigs from different geographical areas of Taiwan and to compare the genetic features of LA-MRSA with other countries. A total of 299 swine nasal samples were collected from 11 counties in western Taiwan with 220 identified MRSA isolates characterized by spa, SCCmec, MLST, pulsed-field gel electrophoresis (PFGE), antibiogram, and Panton-Valentine leukocidin (PVL) toxin genes. A widespread LA-MRSA clone, ST9-t899-PVL-negative, was detected as the predominant (91.6%) clone in Taiwan pigs, and a novel spa type t7616 was identified. Most isolates carried SCCmec type V but lacked ccrC gene (98.8%), a particular feature that was observed for the first time. The integration of minimum spanning trees (MST) based on a PFGE distance matrix and geographical information showed high genetic diversity among ST9-t899 isolates with three unique subpopulations identified in two counties that are nearby each other. Multidrug resistance (MDR) was prevalent in isolates from different counties with more than 80% resistance to erythromycin, ciprofloxacin, gentamicin, tetracycline, and clindamycin. In conclusion, this study reported the first identified LA-MRSA clone of MDR-ST9-t899-V lacking ccrC dominant in pig farms in western Taiwan that differed from ST398 in Europe and North America. Close monitoring of this MDR unique clone is warranted to survey its spread from livestock to humans.