RESUMO
An unbalanced composition of gut microbiota in fish is hypothesized to play a role in promoting bacterial infections, but the synergistic or antagonistic interactions between bacterial groups in relation to fish health are not well understood. We report that pathogenic species in the Piscirickettsia, Aeromonas, Renibacterium and Tenacibaculum genera were all detected in the digesta and gut mucosa of healthy Atlantic salmon without clinical signs of disease. Although Piscirickettsia salmonis (and other pathogens) occurred in greater frequencies of fish with clinical Salmonid Rickettsial Septicemia (SRS), the relative abundance was about the same as that observed in healthy fish. Remarkably, the SRS-positive fish presented with a generalized mid-gut dysbiosis and positive growth associations between Piscirickettsiaceae and members of other taxonomic families containing known pathogens. The reconstruction of metabolic phenotypes based on the bacterial networks detected in the gut and mucosa indicated the synthesis of Gram-negative virulence factors such as colanic acid and O-antigen were over-represented in SRS positive fish. This evidence indicates that cooperative interactions between organisms of different taxonomic families within localized bacterial networks might promote an opportunity for P. salmonis to cause clinical SRS in the farm environment.
Assuntos
Doenças dos Peixes , Infecções por Piscirickettsiaceae , Piscirickettsiaceae , Salmo salar , Humanos , Animais , Fatores de Virulência , Doenças dos Peixes/microbiologiaRESUMO
Caligidosis and Piscirickettsiosis are currently the most important sanitary challenges for the Chilean salmon industry. Caligidosis is caused by the ectoparasite, Caligus rogercresseyi and Piscirickettsiosis is caused by the intracellular bacterium, Piscirickettsia salmonis. Both diseases are highly prevalent and widely distributed in farming areas in Chile. The co-occurrence of the two diseases is frequently reported on salmon farms. However, there is little epidemiological evidence as to whether these two diseases are associated and generate interactive effects. This study was undertaken to evaluate the potential effects of C. rogercresseyi infestation on P. salmonis-attributed mortalities in farmed salmonids in Chile. Using a linear regression model, the potential association between the mean abundance of adult C. rogercresseyi in a period of 10 weeks and Piscirickettsiosis cumulative mortalities observed in the following 10 weeks was evaluated, while controlling for important confounders. These two 10-week windows were set around the time-point at which Piscirickettsiosis weekly mortality exceeded 0.1% for the first time in a production cycle. We found that the mean abundance of adult C. rogercresseyi was significantly associated with the Piscirickettsiosis cumulative mortality, suggesting the two diseases have a synergistic relationship. This relationship was of the same intensity in Atlantic salmon and rainbow trout. Our findings highlight the importance of taking effective control measures for C. rogercresseyi as a part of the strategies in place to reduce P. salmonis-attributed mortalities on salmon farms in Chile.
Assuntos
Doenças dos Peixes/microbiologia , Doenças dos Peixes/mortalidade , Infestações por Piolhos/veterinária , Infecções por Piscirickettsiaceae/veterinária , Salmonidae/microbiologia , Animais , Chile/epidemiologia , Doenças dos Peixes/parasitologia , Pesqueiros , Infestações por Piolhos/microbiologia , Infestações por Piolhos/mortalidade , Modelos Lineares , Ftirápteros , Piscirickettsiaceae/isolamento & purificação , Infecções por Piscirickettsiaceae/mortalidade , Infecções por Piscirickettsiaceae/parasitologiaRESUMO
More than 2,000 historic shipwrecks spanning 500 years of history, rest on the Gulf of Mexico seafloor. Shipwrecks serve as artificial reefs and hotspots of biodiversity by providing hard substrate, something rare in deep ocean regions. The Deepwater Horizon (DWH) spill discharged crude oil into the deep Gulf. Because of physical, biological, and chemical interactions, DWH oil was deposited on the seafloor, where historic shipwrecks are present. This study examined sediment microbiomes at seven historic shipwrecks. Steel-hulled, World War II-era shipwrecks and wooden-hulled, 19th century shipwrecks within and outside of the surface oiled area and subsurface plume were examined. Analysis of 16S rRNA sequence libraries, sediment radiocarbon age data, sedimentation rates, and hydrocarbons revealed that the German U-boat U-166 and the wooden-hulled sailing vessel known as the Mardi Gras Wreck, both in the Mississippi Canyon leasing area, were exposed to deposited oil during a rapid sedimentation event. Impacts to shipwreck microbiomes included a significant increase in Piscirickettsiaceae-related sequences in surface sediments, and reduced biodiversity relative to unimpacted sites. This study is the first to address the impact of the spill on shipwreck-associated microbiomes, and to explore how shipwrecks themselves influence microbiome diversity in the deep sea.
Assuntos
Monitoramento Ambiental , Sedimentos Geológicos/microbiologia , Microbiota/fisiologia , Água do Mar/microbiologia , Navios , Poluentes Químicos da Água/efeitos adversos , Archaea/genética , Sequência de Bases , Amplificação de Genes , Golfo do México , Hidrocarbonetos/análise , Petróleo/análise , Poluição por Petróleo/análise , Filogenia , Piscirickettsiaceae/genética , RNA Ribossômico 16S/genética , Datação Radiométrica , Poluentes Químicos da Água/análiseRESUMO
Piscirickettsia salmonis is a fish bacterium that causes the disease piscirickettsiosis in salmonids. This pathology is partially controlled by vaccines. The lack of knowledge has hindered its culture on laboratory and industrial scale. The study describes the metabolic phenotype of P. salmonis in culture. This study presents the first genome-scale model (iPF215) of the LF-89 strain of P. salmonis, describing the central metabolic pathway, biosynthesis and molecule degradation and transport mechanisms. The model was adjusted with experiment data, allowing the identification of the capacities that were not predicted by the automatic annotation of the genome sequences. The iPF215 model is comprised of 417 metabolites, 445 reactions and 215 genes, was used to reproduce the growth of P. salmonis (µmax 0.052±0.005h-1). The metabolic reconstruction of the P. salmonis LF-89 strain obtained in this research provides a baseline that describes the metabolic capacities of the bacterium and is the basis for developing improvements to its cultivation for vaccine formulation.
Assuntos
Aquicultura , Doenças dos Peixes/genética , Modelos Biológicos , Piscirickettsiaceae/genética , Salmonidae/microbiologia , Animais , Sequência de Bases , Infecções por PiscirickettsiaceaeRESUMO
We report here the protective effect against piscirickettsiosis elicited in fish by a mixture of recombinant proteins. A comparative genomics strategy was used on a genomic library of Piscirickettsia salmonis in order to select optimal candidates for a recombinant subunit vaccine to protect fish from rickettsial septicaemia (SRS). Based on this information, 15 P. salmonis ORFs encoding heat shock proteins, virulence factors, membrane bound and other surface exposed antigens, were isolated and expressed. Seven of the most promising antigens were formulated in three mixtures (V1-V3) containing two or three recombinant proteins each and injected into salmon to test their protective efficacy. Two of the three formulations (V1, V2) elicited a strong protective response in a challenge against the pathogen, which was coincident with the humoral response against the corresponding recombinant proteins present in each formulation. V1, formulated with recombinant chaperonines Hsp60, Hsp70 and flagellar protein FlgG of P. salmonis achieved the highest level of protection with a relative percent survival (RPS) of 95%.
Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Infecções por Piscirickettsiaceae/veterinária , Piscirickettsiaceae/imunologia , Animais , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/efeitos adversos , Feminino , Doenças dos Peixes/microbiologia , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Piscirickettsiaceae/prevenção & controle , Proteínas Recombinantes/imunologia , Salmo salarRESUMO
We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.
Assuntos
Chaperonina 60/biossíntese , DNA Bacteriano/genética , Proteínas de Choque Térmico HSP70/biossíntese , Piscirickettsiaceae/imunologia , Salmão/microbiologia , Animais , Anticorpos Antibacterianos/imunologia , Sequência de Bases , Chaperonina 60/genética , Chaperonina 60/imunologia , Biblioteca Genômica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Salmão/imunologiaRESUMO
We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80 percent homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.
Assuntos
Animais , /biossíntese , DNA Bacteriano/genética , /biossíntese , Piscirickettsiaceae/imunologia , Salmão/microbiologia , Anticorpos Antibacterianos/imunologia , Sequência de Bases , /genética , Biblioteca Genômica , /genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Salmão/imunologiaRESUMO
Piscirickettsiosis pathogenesis was examined using some tissues as entry portals of Piscirickettsia salmonis in coho salmon. Juvenile fish, weighing approximately 8.4 g, were used in this trial. Inocula were prepared using the strain SLGO-95 of P. salmonis. The micro-organism was cultured in the CHSE-214 cell line as described by Fryer et al. (1990) and doses containing 10(4.7) and 10(3.7) TCID50 were prepared. Each dose was used to infect the fish via skin, gills and intestine. Skin and gills were exposed by calibrated drops, and the intestine by an intubation through the anal opening. Some fish were injected intraperitoneally with the same P. salmonis doses, as positive virulence controls. Sham-inoculated fish for each of the tested routes were also included as negative controls. Piscirickettsiosis was experimentally reproduced with all the inoculation methods. Cumulative mortalities and survival analyses showed that the most effective entry portal was skin followed by intestinal intubation and finally by gill infection.
Assuntos
Doenças dos Peixes/microbiologia , Doenças dos Peixes/fisiopatologia , Infecções por Piscirickettsiaceae/veterinária , Piscirickettsiaceae , Animais , Linhagem Celular , Doenças dos Peixes/mortalidade , Oncorhynchus kisutch , Infecções por Piscirickettsiaceae/mortalidade , Infecções por Piscirickettsiaceae/fisiopatologia , Análise de Sobrevida , Fatores de TempoRESUMO
We have isolated and sequenced the genes encoding the membrane bound transglycosylase B (MltB) and the transferring binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis. The results of the sequence revealed two open reading frames that encode proteins with calculated molecular weights of 38,830 and 85,140. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from phylogenetically related microorganisms. Partial sequences coding the amino and carboxyl regions of MltB and a sequence of 761 base pairs encoding the amino region of TbpB have been expressed in E. coli. The strong humoral response elicited by these proteins in mouse confirmed the immunogenic properties of the recombinant proteins. A similar response was elicited by both proteins when injected intraperitoneally in Atlantic salmon. The present data indicates that these proteins are good candidates to be used in formulations to study the protective immunity of salmon to infection by P. salnonis.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Código Genético/genética , Glicosiltransferases/genética , Piscirickettsiaceae/enzimologia , Salmão/microbiologia , Proteína B de Ligação a Transferrina/genética , Animais , Sequência de Bases , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática , Imunidade Celular , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Piscirickettsiaceae/genética , Piscirickettsiaceae/imunologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes , Salmão/imunologiaAssuntos
Doenças dos Peixes/microbiologia , Técnicas de Sonda Molecular/veterinária , Oncorhynchus mykiss , Infecções por Piscirickettsiaceae/veterinária , Piscirickettsiaceae/genética , Animais , Aquicultura , Chile , Primers do DNA , Doenças dos Peixes/patologia , Hibridização In Situ , Rim/patologia , Fígado/patologia , Infecções por Piscirickettsiaceae/patologia , Baço/patologiaRESUMO
We have isolated and sequenced the genes encoding the membrane bound transglycosylase B (MltB) and the transferring binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis. The results of the sequence revealed two open reading frames that encode proteins with calculated molecular weights of 38,830 and 85,140. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from phylogenetically related microorganisms. Partial sequences coding the amino and carboxyl regions of MltB and a sequence of 761 base pairs encoding the amino region of TbpB have been expressed in E. coli. The strong humoral response elicited by these proteins in mouse confirmed the immunogenic properties of the recombinant proteins. A similar response was elicited by both proteins when injected intraperitoneally in Atlantic salmon. The present data indicates that these proteins are good candidates to be used in formulations to study the protective immunity of salmon to infection by P. salmonis.
Assuntos
Masculino , Animais , Camundongos , Código Genético/genética , Glicosiltransferases/genética , Piscirickettsiaceae/enzimologia , Salmão/microbiologia , Proteína B de Ligação a Transferrina , Sequência de Bases , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Piscirickettsiaceae/genética , Piscirickettsiaceae/imunologia , Proteínas de Membrana/genética , Salmão/imunologiaRESUMO
We have used the expression library immunization technology to study the protection of Coho salmon Oncorhynchus kisutch to the infection with Piscirickettsia salmonis. Purified DNA from this bacterium was sonicated and the fragments were cloned in the expression vector pCMV-Bios. Two libraries were obtained containing 22,000 and 28,000 colonies and corresponding to approximately 8 and 10 times the genome of the pathogen, respectively. On average, the size of the inserts ranged between 300 and 1,000 bp. The plasmid DNA isolated from one of these libraries was purified and 20 micrograms were injected intramuscularly into 60 fish followed by a second dose of 10 micrograms applied 40 days later. As control, fish were injected with the same amount of DNA of the vector pCMV-Bios without insert. The titer of IgM anti-P. salmonis of vaccinated fish, evaluated 60 days post-injection, was significantly higher than that of the control group injected with the vector alone. Moreover, this response was specific against P. salmonis antigens, since no cross reaction was detected with Renibacterium salmoninarum and Yersinia ruckeri. The vaccinated and control fish were challenged 60 days after the second dose of DNA with 2.5 x 10(7) P. salmonis corresponding to 7.5 times the LD50. At 30 days post-challenge, 100% mortality was obtained with the control fish while 20% of the vaccinated animals survived. All surviving fish exhibited a lower bacterial load in the kidney than control fish. The expression library was also tested in Balb/c mice and it was found that the humoral immune response was specific to P. salmonis and it was dependent on the amount of DNA injected.
Assuntos
Biblioteca Gênica , Imunização/veterinária , Oncorhynchus kisutch/imunologia , Piscirickettsiaceae/imunologia , Vacinas de DNA/imunologia , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , DNA Bacteriano/genética , DNA Bacteriano/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/prevenção & controle , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Oncorhynchus kisutch/microbiologia , Vacinas de DNA/genéticaRESUMO
The genes encoding the heat shock proteins HSP10 and HSP16 of the salmon pathogen Piscirickettsia salmonis have been isolated and sequenced. The HSP10 coding sequence is located in an open reading frame of 291 base pairs encoding 96 aminoacids. The HSP16 coding region was isolated as a 471 base pair fragment encoding a protein of 156 aminoacids. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from other prokaryotic organisms. Both proteins were expressed in E. coli as fusion proteins with thioredoxin and purified by chromatography on Nicolumn. A rabbit serum against P. salmonis total proteins reacts with the recombinant HSP10 and HSP16 proteins. Similar reactivity was determined by ELISA using serum from salmon infected with P. salmonis. The possibility of formulating a vaccine containing these two proteins is discussed.
Assuntos
Proteínas de Bactérias , Chaperonina 10/genética , Proteínas de Choque Térmico/genética , Piscirickettsiaceae/genética , Sequência de Aminoácidos , Animais , Chaperonina 10/metabolismo , Clonagem Molecular , Códon/genética , DNA Bacteriano/genética , Expressão Gênica/genética , Genes Bacterianos , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular , CoelhosRESUMO
Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.