Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.)

The esrB gene of Edwardsiella tarda, which encodes a regulator protein of the type III secretion system, was mutated by the unmarked deletion method and reintroduced by allelic exchange into the chromosome of E. tarda LSE40 by means of the suicide vector pRE 112. The LSE40 esrB mutant was highly attenuated when inoculated intraperitoneally into turbot Scophthamus maximus L., showing a 50% lethal dose of 10(8.1) cfu/fish. The esrB mutants were not recoverable from the internal organs at 14 days post-inoculation. Vaccination with a single dose of 10(5)-10(7) cfu/fish of the esrB mutant elicited significant protection against the wildtype strain of E. tarda LSE40 (relative percentage survival > 50%). The protection correlated well with the antibody titres in the serum of vaccinated fish. (c) 2006 Elsevier Ltd. All rights reserved.

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

A Trabecular Bone Explant Model of Osteocyte–Osteoblast Co-Culture for Bone Mechanobiology

The osteocyte network is recognized as the major mechanical sensor in the bone remodeling process, and osteocyte-osteoblast communication acts as an important mediator in the coordination of bone formation and turnover. In this study, we developed a novel 3D trabecular bone explant co-culture model that allows live osteocytes situated in their native extracellular matrix environment to be interconnected with seeded osteoblasts on the bone surface. Using a low-level medium perfusion system, the viability of in situ osteocytes in bone explants was maintained for up to 4 weeks, and functional gap junction intercellular communication (GJIC) was successfully established between osteocytes and seeded primary osteoblasts. Using this novel co-culture model, the effects of dynamic deformational loading, GJIC, and prostaglandin E-2 (PGE(2)) release on functional bone adaptation were further investigated. The results showed that dynamical deformational loading can significantly increase the PGE(2) release by bone cells, bone formation, and the apparent elastic modulus of bone explants. However, the inhibition of gap junctions or the PGE(2) pathway dramatically attenuated the effects of mechanical loading. This 3D trabecular bone explant co-culture model has great potential to fill in the critical gap in knowledge regarding the role of osteocytes as a mechano-sensor and how osteocytes transmit signals to regulate osteoblasts function and skeletal integrity as reflected in its mechanical properties.

Effects of live food and formulated diets on survival, growth and protein content of first-feeding larvae of Plelteobagrus fulvidraco

In recent years, much progress has been made in the rearing of fish larvae fed only artificial diets. A preliminary study was made in an attempt to evaluate the effects of live food and formulated diets on survival, growth and body protein content of first-feeding larvae of Plelteobagrus fulvidraco. Three test diets varying in protein level were formulated: Feed 1 containing 45% protein, Feed 2 with 50% protein and Feed 3 with 55% protein. Larvae fed live food (newly hatched Artemia, unenriched) were the control. The experiment started 3 days post-hatch and lasted for 23 days. At the end of the 23-day trial, survival was best in the control group (65.6%) whereby the final body weight and specific growth rate (SGR) were significantly lower than those in the test feed groups. At the same time, coefficients of variation for SGR and final body weight in the test groups were significantly higher than those in the control. Whole body protein content in all treatments showed a similar tendency during development: significantly higher 3 days post-hatch, then decreasing significantly, and then increasing unstatistically 10 days post-hatch. All results suggest that live food is still better for first-feeding larvae of P. fulvidraco, since live food leads to healthier larvae growth.

Growth of juvenile Chinese sturgeon Acipenser sinensis fed live and formulated diets

Triplicate groups of 3.8-g juvenile Chinese sturgeon Acipenser sinensis were reared for 8 weeks in indoor flow-through systems on one of four diets: a natural diet consisting solely of live tubificid worms, a semimoist practical diet, a dry practical diet, and a purified diet. The formulated diets were prepared in the laboratory and had protein contents of 47-50%. Except for the group fed the purified diet, fish showed high survival (94-96%) and growth (final weight, 41-45 g). Survival and specific growth rate did not differ significantly between groups fed the natural, semimoist, and dry practical diets, but were significantly (P < 0.05) lower in fish fed the purified diet. Proximate analysis showed that fish fed purified diet had lower protein and lipid levels but a higher moisture content than fish fed other diets. Our results demonstrated that growth and survival of cultured juvenile Chinese sturgeon fed practical diets were comparable with those fed live tubificid worms. However, Chinese sturgeon fed a purified diet showed inferior growth and survival.

Live-weight gain, apparent digestibility, and economic benefits of yaks fed different diets during winter on the Tibetan plateau

Our goal was to determine the effect of diets with different crude protein (CP) contents and metabolizable energy (W) levels on daily live-weight gain, apparent digestibility, and economic benefit of feedlot yaks on the Tibetan plateau during winter. Yaks were either 2- or 3-years old and randomly selected from the same herd. The 3-year-olds were placed into one of two experimental groups (A and B) and a control (CK1), and the two-year-olds were placed into one of three experimental groups (C, D and E) and a control (CK2) (N per group = 5). Yak in the control groups were allow graze freely, while those in the experimental groups yaks were fed diets higher in contains crude protein and metabolizable energy through a winter period inside a warming shed. Results indicated that live-weight gain of treatment groups was higher than their respective controls during experiment, and that daily live-weight gain of every 10 days among different treatments was significant difference (P < 0.05). In addition, apparent digestibility of different diets was linearly and positively related to feedlotting time, and feed conversion efficiency for A, C, D and E groups was quadratically related to feedlotting time (P < 0.01), however, feed conversion efficiency for B group was linearly and positively related to feedlotting time (P < 0.05). The economic benefit was 1.15 for A, 1.89 for B, 1.16 for C, 1.54 for D, and 4,52 for E. (c) 2005 Elsevier B.V. All rights reserved.