RESUMO
Lactic acid bacteria are a powerful vehicle for releasing of cytokines and immunostimulant peptides at the gastrointestinal level after oral administration. However, its therapeutic application against pathogens that affect rainbow trout and Atlantic salmon has been little explored. Type II interferon in Atlantic salmon activates the antiviral response, protecting against viral infection, but its role against bacterial infection has not been tested in vivo. In this work, through the design of a recombinant lactic acid bacterium capable of producing Interferon gamma from Atlantic salmon, we explore its role against bacterial infection and the ability to stimulate systemic immune response after oral administration of the recombinant probiotic. Recombinant interferon was active in vitro, mainly stimulating IL-6 expression in SHK-1 cells. In vivo, oral administration of the recombinant probiotic produced an increase in IL-6, IFNγ and IL-12 in the spleen and kidney, in addition to stimulating the activity of lysozyme in serum. The challenge trials indicated that the administration of the IFNγ-producing probiotic doubled the survival in fish infected with F. psychrophilum. In conclusion, our results showed that the oral administration of lactic acid bacteria producing IFNγ managed to stimulate the immune response at a systemic level, conferring protection against pathogens, showing a biotechnological potential for its application in aquaculture.
Assuntos
Proteínas de Peixes/metabolismo , Infecções por Flavobacteriaceae/prevenção & controle , Flavobacterium/patogenicidade , Interferon gama/metabolismo , Lactococcus lactis/metabolismo , Oncorhynchus mykiss/microbiologia , Probióticos/administração & dosagem , Administração Oral , Animais , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/metabolismo , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/imunologia , Interações Hospedeiro-Patógeno , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/metabolismo , FilogeniaRESUMO
Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.
Assuntos
Bacteriófagos/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/virologia , Salmonidae , Animais , Bacteriófagos/classificação , Doenças dos Peixes/mortalidade , Doenças dos Peixes/virologia , Infecções por Flavobacteriaceae/mortalidade , Infecções por Flavobacteriaceae/prevenção & controle , Flavobacterium/patogenicidade , Variação Genética , Genoma Bacteriano , FilogeniaRESUMO
Eighty-four marine gliding bacteria were isolated from specimens collected in the Gulf of Thailand and the Andaman Sea. All exhibited gliding motility and swarm colonies on cultivation plates and they were purified by subculturing and micromanipulator techniques. Their 16S rRNA genes were amplified by the polymerase chain reaction (PCR) technique. The phylogenetic analysis indicated that the represented isolates can be separated into six different clads (gr 1 - gr 6) within the Cytophaga-Flavobacterium-Bacteriodes (CFB) group. Group 1 formed a remote linear, with only 90 percent sequence similarity, from Flavobacteriaceae bacterium which indicated a potentially novel taxonomic group. Groups 2 and 3 were identified as the recently proposed Tenacibaculum mesophilum and Fulvivirga kasyanovii respectively. Groups 4, 5 and 6, consisting of the largest number of the members, were identified as Rapidithrix thailandica, Aureispira marina and Aureispira maritima respectively. The isolates were cultivated in four different cultivation media (Vy/2, RL 1, CY and SK) and the crude extracts were submitted to screen cytotoxicity using a sulphorodamine B (SRB) assay. The results from cytotoxic screening showed that groups 2, 4 and 6 were capable of producing the cytotoxic metabolites against selected human cell lines (breast adenocarcinoma (MCF-7), colon cancer (HT-29), cervical cancer (HeLa) and oral cancer (KB)). However, groups 1, 3 and 5 did not produce metabolites with cytotoxicity when cultivated in the same cultivation media as the previous groups. CY medium was the only cultivation medium which could yield the cytotoxic metabolites against MCF-7.
Assuntos
Bactérias/citologia , Bactérias/patogenicidade , Citotoxinas/biossíntese , Citotoxinas , Cytophaga/citologia , Cytophaga/patogenicidade , Flavobacterium/citologia , Flavobacterium/patogenicidade , Citotoxinas/análise , Reação em Cadeia da Polimerase , TailândiaRESUMO
Characterization of 20 Flavobacterium psychrophilum strains isolated from farmed Atlantic salmon and rainbow trout in Chile was done using phenotypic, antigenic and genetic techniques. Experimental infections were also performed to assess the virulence of two representative isolates and of the type strain. Biochemical and physiological analyses showed that Chilean F. psychrophilum strains, regardless of the host species, constitute a phenotypically very homogeneous group matching with previous descriptions of this pathogen. However, serological assays indicated the existence of antigenic heterogeneity with four patterns of serological reactions. The first group contained most (14 of 20) of the F. psychrophilum isolates showing cross-reaction with the antisera obtained against Atlantic salmon and rainbow trout isolates. Group 2 corresponded to four other rainbow trout isolates (1658, 1731, 1762 and 29009) that did not agglutinate with anti-1150 serum. Two minor serological groups were identified for the remaining isolates (Groups 3 and 4). Marked homogeneity was also revealed by genetic studies including 16S rRNA alleles, random amplified polymorphic DNA and REP-PCR showing that a major genetic group of F. psychrophilum may be dominant in disease outbreaks in farms. Restriction fragment length polymorphism of PCR analysis showed that gyrase genotypes B-S or B-R were found in Chilean isolates from rainbow trout and Atlantic salmon, whereas genotype A was not found. Virulence assays using Atlantic salmon indicated no relationship between the degree of pathogenicity and the host origin of the F. psychrophilum strains.