RESUMEN
A reverse vaccinology-based survey of potent antigens associated with fish nocardiosis was conducted using the largemouth bass, Micropterus salmoides, with an aim to develop subunit vaccines. The antigens selected from the virulent strain Nocardia seriolae 961113 include the gene products of NGL2579 (GAPDH), NGL5701 (MMP), NGL4377 (OCTase), NGL4486 (ABC transporter), NGL3372 (LLE), NGL3388 (GHf10), NGL6627 (Antigen-85), NGL6696 (Esterase), and NGL6936 (CBP). These antigens were heterologously expressed in E. coli BL21 (DE3) for recombinant protein production. Then fish were vaccinated was these antigens, boosted at 2 weeks, and challenged with N. seriolae at 6 weeks after vaccination. The relative protection survival assay revealed high and significant protection efficacies of 94.45, 50.00, and 44.45 in fish that received the NGL3388 (GHf10), NGL6936 (CBP), and NGL3372 (LLE) vaccines, respectively. There were no apparent relationships or differences in tissue lesions among the administered vaccines. The serum titers against the bacterial preparations were higher for all vaccinated groups than for the control group at 4 weeks after immunization. However, no significant difference in serum titer was found at 6 weeks after immunization. The results of this study demonstrate that subunit vaccines against fish nocardiosis have differential effects, but are highly promising for nocardial prophylaxis.
Asunto(s)
Vacunas Bacterianas/inmunología , Lubina/inmunología , Enfermedades de los Peces/prevención & control , Nocardiosis/veterinaria , Nocardia/inmunología , Animales , Escherichia coli/genética , Nocardiosis/prevención & control , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
In the present study, IL-1ß cDNA was identified and analyzed from largemouth bass (Micropterus salmoides). Full length IL-1ß mRNA was obtained using Rapid Amplification of cDNA Ends (RACE), which contains 78 bp 3'-UTR, a 455 bp 5'-UTR, and an open reading frame (ORF) of 702 bp coding for 233 amino acid residues. The molecular weight and theoretical isoelectric point of largemouth bass IL-1ß protein was predicted to be 26.7 kDa and 6.08 respectively. A largemouth bass IL-1ß phylogenetic analysis showed a close relation to the IL-1ßs of striped trumpeter (Latris lineata), Chinese perch (Siniperca chuatsi), and Japanese sea bass (Lateolabrax japonicus). Peptidoglycan upregulated IL-1ß in the spleen and head kidney, while lipopolysaccharide upregulated detectable levels of IL-1ß in the spleen only. Largemouth bass, challenged with Nocardia seriolae (1.0 × 106 cfu/mL), showed a significant increase in IL-1ß at 3 and 5 days post infection (dpi) in the spleen, while in the head kidney significant expression was found at 2 and 3 dpi, peaking at 3 dpi. Furthermore, tumor necrosis factor α (TNF-α) showed significantly higher expression in the spleen at 3 and 5 dpi, and in the head kidney at 1 and 3 dpi, with expression decreasing at 5 dpi in both tissues.
Asunto(s)
Lubina/genética , Lubina/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica , Interleucina-1beta/metabolismo , Nocardiosis/veterinaria , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Lubina/clasificación , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Enfermedades de los Peces/genética , Humanos , Interleucina-1beta/genética , Riñón/metabolismo , Ratones , Nocardia/inmunología , Nocardiosis/genética , Nocardiosis/inmunología , Filogenia , Estructura Secundaria de Proteína , Ratas , Alineación de Secuencia , Bazo/metabolismo , Transcriptoma , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Taura syndrome virus (TSV) has spread worldwide, causing significant economic losses since Taura syndrome was first described in Ecuador in 1992. To determine the prevalence and impact of TSV infection on the shrimp farming industry in Taiwan, Pacific white shrimp Litopenaeus vannamei B. were collected from 220 farms between 2004 and 2006 for viral detection by reverse transcription polymerase chain reaction. Data showed that the overall TSV prevalence rate was 20% (43/220 farms). Comparing shrimp growth stages, TSV prevalence rates were 4% for postlarvae, 24% for juveniles, 24% for subadults, 32% for adults, and 5% for brooders. Among TSV-positive farms, average infection incidence was 35% in postlarvae farms, 55% in juvenile farms, 39% in subadult farms, 31% in adult farms, and 20% in brooder farms. Notably, TSV was also detected in Exopalaemon orientis H. from 1 of 10 farms. Tail fans and appendages had red pigmentation, which is characteristic of TSV infection. Of shrimp with pathological lesions, 100% had lesions on tail fans, 88% on appendages, and 80% in gills. Sequence comparison using the TSV VP1 (structural protein) gene showed that 9 isolates from the farms had 92.3 to 99.5% nucleotide sequence identity with strains in the GenBank database from Taiwan (AF406789 and AY355310) and Venezuela (DQ212790). This is the first broad epidemiological study of TSV infection in L. vannamei in Taiwan.
Asunto(s)
Penaeidae , Virus ARN/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Filogenia , Virus ARN/aislamiento & purificación , TaiwánRESUMEN
The full-length complementary (c)DNA of the alpha2-macroglobulin (alpha2M) gene was cloned from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The 4875-bp cDNA contains an open reading frame (ORF) of 4419 bp, a 95-bp 5'-untranslated region (UTR), and a 361-bp 3' UTR containing the poly A tail. The ORF encodes a protein of 1472 amino acids (aa) with a 23-residue signal sequence. The molecular mass of the deduced amino acid sequence (1449 aa) was 163.29 kDa with an estimated pI of 4.88. The M. rosenbergii alpha2M sequence contains putative functional domains including a bait region and a GCGEQ internal thiol ester site, and a receptor-binding domain is present as in other aquatic arthropod alpha2Ms. Sequence comparison showed that alpha2M of this prawn had overall respective identities of 38.4%, 45.9%, 45.9%, and 46.0% to those of Scylla serrata, Litopenaeus vannamei, Penaeus monodon, and Marsupenaeus japonicus. A phylogenetic analysis revealed that M. rosenbergii alpha2M is the more-primitive genotype, and it showed significant differentiation from marine crustacean alpha2Ms. alpha2M was mainly expressed in haemocytes. The quantitative real-time RT-PCR analysis showed that alpha2M mRNA transcripts significantly increased in the A stage, achieved the highest level in the C stage, then declined in the D(0/1) stage, and reached the lowest level in the D(2/3) stage in haemocytes of prawn. alpha2M's expression in haemocytes of M. rosenbergii significantly increased at 24 h and 12 h after injection with heat-killed Lactococcus garvieae and Vibrio alginolyticus, respectively, which indicates that alpha2M is involved in the immune response of prawn.