RESUMEN
The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.
Asunto(s)
Infecciones por Actinomycetales/veterinaria , Enfermedades de los Peces/prevención & control , Proteínas de Peces/genética , Infecciones por Bacterias Gramnegativas/veterinaria , Ictaluridae/genética , Ictaluridae/inmunología , Muramidasa/genética , Infecciones por Actinomycetales/genética , Infecciones por Actinomycetales/prevención & control , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Aeromonas hydrophila/inmunología , Aeromonas hydrophila/patogenicidad , Aeromonas hydrophila/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Escherichia coli/genética , Enfermedades de los Peces/genética , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/prevención & control , Ictaluridae/metabolismo , Micrococcus/inmunología , Micrococcus/fisiología , Datos de Secuencia Molecular , Muramidasa/química , Muramidasa/metabolismo , Filogenia , Plásmidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia/veterinariaRESUMEN
To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with A. hydrophila by bath immersion. Quantitative PCR revealed that the transcription levels of Lys-c in infected catfish were significantly (P < 0.05) induced in all five tissues tested as well as in blood cells. Recombinant CC-Lys-c produced in Escherichia coli expression system (R-CC-Lys-c) exhibited significant (P < 0.05) lytic activity to Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-c (pcDNA-Lys-c) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-c offered significant (P < 0.05) protection to G1B against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-Lys-c and challenged with a highly virulent A. hydrophila strain AL-09-71 at 1-, 2-, 14-, and 28-days post treatment, pcDNA-Lys-c offered 75%, 100%, 60%, and 77% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-Lys-c produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. Taken together, our results suggest that pcDNA-Lys-c could be used as a novel immunostimulant to protect channel catfish against A. hydrophila infection.
Asunto(s)
Aeromonas hydrophila , Enfermedades de los Peces/microbiología , Regulación de la Expresión Génica/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Ictaluridae , Muramidasa/metabolismo , Animales , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/microbiología , Óxido Nítrico , Filogenia , Especies Reactivas de OxígenoRESUMEN
The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P<0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P<0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity.