Characterisation of extracellular products produced by Mycobacterium spp. and their effects on the fish immune system

Mycobacterium spp. isolated from food and ornamental fish in Thailand (strains TB 1, TB40, TB267, TB268), and the type strains Mycobacterium marinum (NCIMB 1298), Mycobacterium fortuitum (NCIMB 1294), and Mycobacterium chelonae (NClMB 1474) were cultured in Long's medium, Eagle's minimum essential medium, Sauton's medium and modified Sauton's medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB40, TB267, TB268 and M marinum in particular, whereas growth and production of ECP for all strains was limited in Long's medium. SDS-PAGE protein profiles of ECPs from 14 day culture supernatants showed major bands at 65, and <14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M marinum averaged approximately 4 to 10 fold higher than from strains cultured for 14 days at 28°C. The major fibronectin binding proteins from ECP of Mycobacterium spp. isolated from infected fish were identified at 21-25 kDa. Cross reactivity was detected between ECP from Mycobacterium spp. and MAb anti-heat shock protein (60 kDa) and MAb anti-M Tuberculosis. The 65 kDa antigen of TB267 is a strongly immunogenic protein eliciting antibodies in fish, rabbits and mice. Cross-reactivity was found between rabbit anti-65 kDa antibody and sonicated proteins from many other bacterial species. Therefore, the 65 kDa protein from Mycobacterium sp. isolated from snakehead fish may be a common protein in fish bacterial pathogens. Eighteen MAbs to TB 267 and M chelonae were produced. The epitopes to which the MAbs are against located on molecules susceptible to protease treatment. All MAbs recognized the 65 kDa protein. It is one of major proteins in the ECP, whole cell sonicates and lysates from Jv1ycobacterium spp. and is located in the peri plasmic space or cell wall, and is secreted in the medium during culture. A pnmary intraperitoneal (IP) immunisation of extracellular products (ECP) from Jv1ycobacterium spp., (strains TB40, TB267 or M marinum) mixed with Freund's incomplete adjuvant (FIA), followed by a secondary IP injection at 8 wks, resulted in the elevation of both the non-specific immune response (by measuring nitroblue tetrazolium, lysozyme and phagocytosis activity) and the specific immune responses of rainbow trout, Oncorhynchus mykiss (by measuring specific antibody levels). Nile tilapia were immunised by injecting extracellular products (ECP) of Jv1ycobacterium spp. (strain TB40, TB267 or the type strain M marinum) into their swimbladders and this resulted in the elevation of the non specific immune response. The cytological response of rainbow trout head kidney macrophages to ingested Mycobacterium spp was examined in vitro. The bacteria had previously been opsonised with either fresh rainbow trout serum (FS), or serum which had been heat-inactivated (IDS), or rainbow trout antiserum against the extracellular products (ECP) of Mycobacterium strains TB267 or M marinum. MAbs against the ECP were also used as opsonins. Opsonisation of the mycobacteria was found to greatly enhance the phagocytic and killing activity of the rainbow trout macrophage

Comparative Study of Immune Reaction Against Bacterial Infection From Transcriptome Analysis

Transcriptome analysis is a powerful tool that enables a deep understanding of complicated physiological pathways, including immune responses. RNA sequencing (RNA-Seq)-based transcriptome analysis and various bioinformatics tools have also been used to study non-model animals, including aquaculture species for which reference genomes are not available. Rapid developments in these techniques have not only accelerated investigations into the process of pathogenic infection and defense strategies in fish, but also used to identify immunity-related genes in fish. These findings will contribute to fish immunotherapy for the prevention and treatment of bacterial infections through the design of more specific and effective immune stimulants, adjuvants, and vaccines. Until now, there has been little information regarding the universality and diversity of immune reactions against pathogenic infection in fish. Therefore, one of the aims of this paper is to introduce the RNA-Seq technique for examination of immune responses in pathogen-infected fish. This review also aims to highlight comparative studies of immune responses against bacteria, based on our previous findings in largemouth bass (Micropterus salmoides) against Nocardia seriolae, gray mullet (Mugil cephalus) against Lactococcus garvieae, orange-spotted grouper (Epinephelus coioides) against Vibrio harveyi, and koi carp (Cyprinus carpio) against Aeromonas sobria, using RNA-seq techniques. We demonstrated that only 39 differentially expressed genes (DEGs) were present in all species. However, the number of specific DEGs in each species was relatively higher than that of common DEGs; 493 DEGs in largemouth bass against N. seriolae, 819 DEGs in mullets against L. garvieae, 909 in groupers against V. harveyi, and 1471 in carps against A. sobria. The DEGs in different fish species were also representative of specific immune-related pathways. The results of this study will enhance our understanding of the immune responses of fish, and will aid in the development of effective vaccines, therapies, and disease-resistant strains

Exploration of the memory effect on the photon-assisted tunneling via a single quantum dot: A generalized Floquet theoretical approach

The generalized Floquet approach is developed to study memory effect on electron transport phenomena through a periodically driven single quantum dot in an electrode-multi-level dot-electrode nanoscale quantum device. The memory effect is treated using a multi-function Lorentzian spectral density (LSD) model that mimics the spectral density of each electrode in terms of multiple Lorentzian functions. For the symmetric single-function LSD model involving a single-level dot, the underlying single-particle propagator is shown to be related to a 2 x 2 effective time-dependent Hamiltonian that includes both the periodic external field and the electrode memory effect. By invoking the generalized Van Vleck (GVV) nearly degenerate perturbation theory, an analytical Tien-Gordon-like expression is derived for arbitrary order multi- photon resonance d.c. tunneling current. Numerically converged simulations and the GVV analytical results are in good agreement, revealing the origin of multi- photon coherent destruction of tunneling and accounting for the suppression of the staircase jumps of d.c. current due to the memory effect. Specially, a novel blockade phenomenon is observed, showing distinctive oscillations in the field-induced current in the large bias voltage limit

A Critical Examination of Hypernova Remnant Candidates in M101. II. NGC 5471B

NGC 5471B has been suggested to contain a hypernova remnant because of its extraordinarily bright X-ray emission. To assess its true nature, we have obtained high-resolution images in continuum bands and nebular lines with the Hubble Space Telescope, and high-dispersion long-slit spectra with the Kitt Peak National Observatory 4-m echelle spectrograph. The images reveal three supernova remnant (SNR) candidates in the giant HII region NGC 5471, with the brightest one being the 77x60 pc shell in NGC 5471B. The Ha velocity profile of NGC 5471B can be decomposed into a narrow component (FWHM = 41 km/s) from the background HII region and a broad component (FWHM = 148 km/s) from the SNR shell. Using the brightness ratio of the broad to narrow components and the Ha flux measured from the WFPC2 Ha image, we derive an Ha luminosity of (1.4+-0.1)x10^39 ergs/s for the SNR shell. The [SII]6716,6731 doublet ratio of the broad velocity component is used to derive an electron density of ~700 cm^-3 in the SNR shell. The mass of the SNR shell is thus 4600+-500 Mo. With a \~330 km/s expansion velocity implied by the extreme velocity extent of the broad component, the kinetic energy of the SNR shell is determined to be 5x10^51 ergs. This requires an explosion energy greater than 10^52 ergs, which can be provided by one hypernova or multiple supernovae. Comparing to SNRs in nearby active star formation regions, the SNR shell in NGC 5471B appears truly unique and energetic. We conclude that the optical observations support the existence of a hypernova remnant in NGC 5471B.Comment: 27 pages, 9 figures, to appear in May 2002 issue of The Astronomical Journa

Memory effect on the multiphoton coherent destruction of tunneling in the electron transport of nanoscale systems driven by a periodic field: A generalized Floquet approach

This is the published version, also available here: http://dx.doi.org/10.1103/PhysRevB.79.235323.Time-dependent electron-transport processes are often studied in the wide-band limit. In this paper, a generalized Floquet approach beyond the wide-band limit is developed for the general treatment of memory effect on the virtually unexplored multiphoton (MP) coherent destruction of tunneling (CDT) phenomenon of periodically driven electrode-wire-electrode nanoscale systems. As a case study, we apply the approach for a detailed analysis of the electron-transport dc current in the electrode-quantum double dot-electrode system, showing the significance of memory effect as well as illustrating the origin of the MP-CDT phenomenon