Cloning, characterization and expression of cDNA containing major histocompatibility complex class I, IIα and IIβ genes of Japanese flounder Paralichthys olivaceus

ABSTRACT:   The nucleotide sequences of Japanese flounder Paralichthys olivaceus , major histocompatibility complex (MHC) cDNA, classical MHC class Iα, non-classical MHC class Iβ, MHC class IIα and IIβ, were determined. The domain structures and antigen binding motifs of vertebrate MHC are conserved in the Japanese flounder MHC. A phylogenetic analysis supports the classification of these genes into class I and class II MHC. Classical MHC class Iα was ubiquitously expressed, whereas the non-classical MHC class Iβ was expressed mainly in lymphoid organs, gills, intestine and stomach. The MHC classes IIα and IIβ were also ubiquitously expressed.     

A multi-epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E. ictaluri immunogenic proteins previously reported. The 49.32 kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E. ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E. ictaluri infections.     

Genetic parameters for resistance against Flavobacterium columnare in Nile tilapia Oreochromis niloticus (Linnaeus, 1758)

Columnaris disease is a major cause of mortality in tilapia hatcheries and commonly occurs during the summer season in Thailand. One way of reducing the problem is by selective breeding for increased disease resistance. The objective of this study was to estimate quantitative genetic parameters for resistance against columnaris in the Chitralada 4 strain of Nile tilapia. Data from 43 full‐sib families (2,580 records) of fry (age = 32 ± 4 days post‐hatch) were used in the analyses. Initially, fry were subjected to bath challenge with Flavobacterium columnare (LD₅₀ concentration = 1.2 × 10⁶ CFU/ml) for 14 days. Disease resistance was defined as the number of days from challenge until death (DD) or as a binary trait (dead/alive) on day 14. Linear animal and sire‐dam models were used for DD, while threshold animal, threshold sire‐dam, binary linear animal and binary linear sire‐dam models were used for binary outcomes. Covariate effect of age, fixed effect of challenge day and random effects of the individual animals or sires and dams were included in the models. Mean survival was 32.4 ± 11.6%, and survival rates of the best and poorest families were 70% and 8%, respectively. The highest estimate of heritability (0.30 ± 0.025) was obtained under the threshold sire‐dam model. Heritability estimates for DD (0.16 ± 0.034 and 0.17 ± 0.046) were comparable to those obtained from the threshold animal (0.15 ± 0.031) and the binary linear (0.14 ± 0.045 and 0.15 ± 0.044) models. The linear animal and sire‐dam models for DD and the threshold sire‐dam models performed equally with similar values of r_EBV (0.629, 0.628 and 0.627) and accuracy of selection (0.793, 0.793 and 0.791). This study reveals the potential of selective breeding to increase disease resistance to F. columnare in the studied population of Nile tilapia.     

Molecular characterization and increased expression of the Nile tilapia, Oreochromis niloticus (L.), T-cell receptor beta chain in response to Streptococcus agalactiae infection

The complete cDNA sequence of the Nile tilapia T-cell receptor (TCR) β chain was cloned using 5' RACE. The full-length, 1263-bp cDNA contained a 942-bp open reading frame (ORF) encoding a 314-amino-acid protein. Sequence analyses revealed that the Nile tilapia TCR β chain contains four conserved cysteine residues involved in the formation of disulphide bridges and a conserved amino acid motif believed to be important for assembly and signalling of the TCR αβ/CD3 complex, both of which are normally found in the TCR β chain of other vertebrates. As detected using semi-quantitative and quantitative RT-PCR, the highest expression level of TCR β was detected in the thymus. Interestingly, Streptococcus agalactiae significantly induced the up-regulation of the TCR β chain, and the strongest up-regulation was detected in the brain and peripheral blood leucocytes (PBLs). In in vitro experiments, concanavalin A and Aeromonas hydrophila were found to significantly increase the expression of the TCR β chain in PBLs after 48 h (P < 0.01) and 72 h (P < 0.05), respectively. Furthermore, real-time PCR analysis showed that intraperitoneal injection (IP) of 10(7) cfu mL(-1) of S. agalactiae could induce TCR β expression that was greater than the expression observed following administration of 10(9) cfu mL(-1). The presence of the TCR β chain in fish detected in this study suggests the presence of T-cell populations that have been found in higher vertebrates, which may play a crucial functional role in the response to fish pathogens.     

Growth of giant tiger prawn, <Emphasis Type="Italic">Penaeus monodon</Emphasis> Fabricius,under co-culture with a discarded filamentous seaweed, <Emphasis Type="Italic">Chaetomorpha ligustica</Emphasis> (Kützing) Kützing,at an aquarium-scale

Growth of juvenile giant tiger prawn, Penaeus monodon Fabricius, was evaluated at an aquarium-scale in co-culture with a discarded filamentous seaweed, Chaetomorpha ligustica (Kützing) Kützing. Juveniles at different ages in days were examined, designated as J 16, J 44, J 58, J 93 and J 128, where a 1-day-old juvenile (J 1) is equivalent to a 20-day-old post-larva (PL 20)). Juveniles at every age group grazed directly on live C. ligustica, even those fed an artificial shrimp diet to satiation. Mean specific growth rate (SGR: % day⁻¹) was higher in early age juveniles. Compared to mono-culture, significant differences in growth were observed at J 16 (4.44% day⁻¹) and J 44 (1.60% day⁻¹); however, no significant differences were recorded at J 58 (1.16% day⁻¹), J 93 (0.75% day⁻¹) or J 128 (0.45% day⁻¹). It was concluded that co-culture of giant tiger prawn with C. ligustica has a dietary advantage, especially in early age juveniles.     

Characterization and expression analysis of the transferrin gene in Nile tilapia (Oreochromis niloticus) and its upregulation in response to Streptococcus agalactiae infection

In this study, full-length tilapia transferrin (OnTF) isolated from liver cDNA of Nile tilapia (Oreochromis niloticus) was found to have an open reading frame of 2,091-bp encoding 696 amino acid residues. Two additional amino acids: Gly³⁶⁹ and Gly³⁷⁰ were observed compared with the reported Nile tilapia transferrin protein sequence. Pre-mature protein has a predicted molecular weight of 78.2 kDa, while mature protein is 73.28 kDa in size. Comparative sequence analysis with transferrin from other species revealed two major putative iron-binding domains designated as the N-lobe and the C-lobe in accordance with the transferrin protein characteristics. The predicted tertiary structure of tilapia transferrin confirmed the presence of iron and anion-binding sites on both lobes that are conserved among transferrins from other species. Quantitative real-time PCR analysis showed significantly higher expression of tilapia transferrin gene in liver than in other tissues (p < 0.05). Transferrin expression in tilapia experimentally infected with 10⁶ and 10⁸ colony-forming units mL^?1 of Streptococcus agalactiae was significantly upregulated at 24 and 12 h post-infection (hpi), respectively, and decreased afterward. Iron-deficiency in serum of bacterially infected fish was detected at 48 and 24 hpi, respectively. The expression pattern of the transferrin gene and the iron levels of infected tilapia in this study were consistent with the function of transferrin in innate immunity.     

Pharmacokinetics,optimal dosages and withdrawal time of florfenicol in Asian seabass (Lates calcarifer) after oral administration via medicated feed

Asian seabass (Lates calcarifer) is an economically important fish in Asian and Australian markets, but few pharmacokinetic (PK) data of antimicrobial drugs in this species is available. The present study investigated the PK behaviour of florfenicol (FF) through medicated feed in Asian seabass cultured at 25°C. The serum and muscle/skin concentrations of FF and its metabolite florfenicol amine (FFA) were determined by the HPLC-FLD method and analysed by one-compartmental model. The optimal dosages were determined by pharmacokinetic-pharmacodynamic (PK-PD) approach and the linear regression analysis was used to determine the withdrawal time (WDT). The PK study following a single oral administration of 15 mg/kg FF via medicated feed revealed that the absorption half-life (t_1/2Ka), elimination half-life (t_1/2K), peak concentration (C_max), area under the concentration-time curve (AUC), volume of distribution (Vd/F) and clearance (CL/F) were 1.47 h, 8.07 h, 8.61 μg/ml, 146.41 h·μg/ml, 1.19 L/kg and 0.102 L/kg/h, respectively. The muscle/skin concentration-time profile was similar to that of the serum, suggesting well distribution but only a small fraction of FF was metabolized to FFA. The optimal dosage for a minimum inhibitory concentration of 2 μg/ml was calculated as 13.38 mg/kg/day. The appropriate WDT after multiple oral medications with 15 mg/kg FF once daily for 7 days was determined as 8 days. Information obtained from the current study can potentially be applied for the treatment of bacterial diseases in farming Asian seabass.     

Selection response for Streptococcus agalactiae resistance in Nile tilapia Oreochromis niloticus

The potential of selection to improve resistance to streptococcosis was evaluated in a commercial population of Nile tilapia in Thailand. The base generation (G0) consisted of offspring from 98 sires and 149 dams using a partly nested design. At 60 days post‐hatch, 30 fish from each family were injected intraperitoneally with a Streptococcosis agalactiae solution (1 × 10⁹ CFU/ml) and evaluated for 14 days. Disease resistance was recorded as the number of days from challenge until death (DD) and as a binary (BIN) trait (dead/alive) on day 14. Three models were used for genetic analyses: Cox frailty model for DD; animal model for DD; and animal model for BIN. Age at challenge was fitted as a covariate and contemporary group as fixed or random effect, depending on the model. Fish from the 18 most resistant families were selected to produce the first generation (G1). Heritability estimates for G0 were 0.22, 0.14 ± 0.02 and 0.11 ± 0.02 for the Cox, linear DD and linear BIN models, respectively. Selection response indicated that the risk of death decreased to 54%, survival time increased to 3.4 days and survival rate increased to 21%. These results suggest that genetic improvement is possible for this population.