Insights into Migration and Development of Coral Black Band Disease Based on Fine Structure Analysis

In many diverse ecosystems, ranging from natural surfaces in aquatic ecosystems to the mammalian gut and medical implants, bacterial populations and communities exist as biofilms. While the process of biofilm development has been well-studied for those produced by unicellular bacteria such Pseudomonas aeruginosa, little is known about biofilm development associated with filamentous microorganisms. Black band disease (BBD) of corals is characterized as a polymicrobial biofilm (mat) community, visually-dominated by filamentous cyanobacteria. The mat migrates across a living coral host, completely lysing coral tissue and leaving behind exposed coral skeleton. It is the only known cyanobacterial biofilm that migrates across a substratum, thus eliciting questions about the mechanisms and unique characteristics of this system. Fragments of the coral Montastraea annularis, five artificially infected with BBD and two collected from a naturally BBD-infected colony, were used to address these questions by detailed examination using scanning and transmission electron microscopy (SEM and TEM). In areas close to the interface of coral tissue and the mature disease band two types of clusters of cyanobacteria were observed, one with random orientation and one with parallel orientation of filaments. The latter exhibited active secretion of extracellular polysaccharide (EPS) while the randomly oriented clusters did not. Within the well developed band cyanobacterial filaments were observed to be embedded in EPS and were present as layers of filaments in parallel orientation. These observations suggest that BBD cyanobacteria orient themselves and produce EPS in a sequential process during migration to form the complex BBD matrix

Pseudomonas aeruginosa Secretion of Exopolymeric Substances (EPS) and Acanthamoeba sps. Adherence to Contact Lenses

Microbial attachment to contact lenses is utilized in this study as a model to examine microbial/substrate interactions. These interactions commonly occur on a biofilm, containing microbes and exopolymeric secretions (EPS). EPS commonly contains sugars for attachment. Silica hydrogel contact lenses were studied with relation to microbial attachment. Scanning and transmission electron microscopy (SEM, TEM) and Fourier Transform Infared Spectrometry (FTIR) were utilized to examine this mechanism. A novel method was developed to prepare lenses and attached microbial communities for SEM imaging. A common marine fixative was utilized with extended fixation times which resulted in fixation osmolarities that preserved the delicate lens polymers as well as the attached microbial communities. In addition, a 24 hemisphere shaped well assaylike tray was developed that aided in lens shape retention during dehydration. SEM analysis of P. aeruginosa EPS yielded information on its 3-dimentional morphology on the attached lens. TEM analysis using gold nanoparticles confirmed that active sites on EPS secreted for attachment were comprised mainly of highly supported structures on the periphery of the EPS, with strut like supports crossing the surface. FTIR analysis of the EPS revealed chemical signatures related to the nature of its bonds as spectral peaks. When heated to 34 °C and then cooled to 20 °C the EPS did not return to its original chemistry. Compared to some polymers, which retain “memory” and return to original chemistry and shape, the bacteria EPS appeared to denature to the point that major components of its molecular structure went into solution. These results have possible implication to the in vivo behavior of bacteria. Assessment of several Acanthamoeba species attachment success found that cysts were in generally more successful in attachment compared to trophozoite stages. Results indicate that in all species and experimental conditions, cyst forms of Acanthamoeba sps. were more successful at attachment then the trophozoite forms. Furthermore, there was a suggested differential pattern in attachment if pathogen and non-pathogen species are compared. The results suggest that in pathogen forms (between 1/2 and 12 hours after inoculation) attachment was greatest at 8 hours compared with non-pathogen species, which had lower success attachment at 8 hr, and higher relative success attachment at ½ and 12 hours. It was also found that one species in which pathogeniety is not known (FL 32) was intermediate in attachment success. These findings warrant further study of pathogen Acanthamoeba sps. in terms of attachment success

Cyanobacterial Associated Colored Band Diseases of the Atlantic/Caribbean

Coral disease is quickly becoming a crisis to the health and management of the world’s coral reefs. There is a great interest from many in preserving coral reefs. Unfortunately, the field of epizootiology is disorganized and lacks a standard vocabulary, methods, and diagnostic techniques, and tropical marine scientists are poorly trained in wildlife pathology, veterinary medicine, and epidemiology. Diseases of Coral will help to rectify this situation.https://nsuworks.nova.edu/occ_facbooks/1061/thumbnail.jp

Fine-Structural Analysis of Black Band Disease-Infected Coral Reveals Boring Cyanobacteria and Novel Bacteria

Examination of coral fragments infected with black band disease (BBD) at the fine- and ultrastructural levels using scanning (SEM) and transmission electron microscopy (TEM) revealed novel features of the disease. SEM images of the skeleton from the host coral investigated (Montastraea annularis species complex) revealed extensive boring underneath the BBD mat, with cyanobacterial filaments present within some of the bore holes. Cyanobacteria were observed to penetrate into the overlying coral tissue from within the skeleton and were present throughout the mesoglea between tissue layers (coral epidermis and gastrodermis). A population of novel, as yet unidentified, small filamentous bacteria was found at the leading edge of the migrating band. This population increased in number within the band and was present within degrading coral epithelium, suggesting a role in disease etiology. In coral tissue in front of the leading edge of the band, cyanobacterial filaments were observed to be emerging from bundles of sloughed-off epidermal tissue. Degraded gastrodermis that contained actively dividing zooxanthellae was observed using both TEM and SEM. The BBD mat contained cyanobacterial filaments that were twisted, characteristic of negative-tactic responses. Some evidence of boring was found in apparently healthy control coral fragments; however, unlike in BBD-infected fragments, there were no associated cyanobacteria. These results suggest the coral skeleton as a possible source of pathogenic BBD cyanobacteria. Additionally, SEM revealed the presence of a potentially important group of small, filamentous BBD-associated bacteria yet to be identified

Microscopy and Microanalysis in Marine Invertebrate Biology

The study of marine organisms can provide insight of exceptional clarity to past, current and future climatic, geological, and biological conditions. The use of transmission electron microscopy (TEM), scanning electron microscopy (SEM), and light microscopy are essential in advancing our ability to detect and assess the effects of regional and global environmental change on marine invertebrates. Coccolithophorids are small (~5 μm) unicellular mainly marine algae, covered with calcified plates of diverse and intricate morphologies known as coccoliths. They can be an important component of the phytoplankton, and are a major producer of calcium carbonate in the world ocean. TEM studies have shown that coccoliths are produced intracellularly within vesicles, are extruded, and adhere to the cell surface (Fig. 1). Variability in Ca, Mg and Sr in growth media of cultured representatives have effects on cell ultrastructure and coccolith mineralogy, facilitating interpretation of the effects of variability in ocean ionic composition over geologic time [1]. Ostracods are small laterally compressed crustaceans, usually \u3c 1 mm in length, protected by a bivalve-like, calcareous carapace. Marine ostracods are typically benthic organisms, are commonly found as microfossils, and have a long, well-documented fossil record particularly valuable in paleoreconstruction studies. SEM and TEM studies of ostracods include the morphology of sieve pores and carapace nodes demonstrating that ostracods may be sensitive salinity indicators (Fig. 2). This data coupled with community analyses may be used to reconstruct natural rainfall patterns and anthropogenic effects such as water management practices and documented anthropogenic usage [2]. Coral reefs are extensive biomineralized structures and are dominant features of tropical coastlines worldwide, covering ~15% of seabed \u3c 30 m deep. They have a significant oceanic geochemical impact, and the specific ratios of their skeletal chemistry provide excellent chronological records of environmental conditions. Histologic techniques are used to assess overall tissue and cellular condition, and extending histological observations to the ultrastructural level enables detection of initial changes and variability in coral tissue, skeleton and symbiotic dinoflagellate cellular structure and morphology resulting from anthropogenic environmental factors and disease (Fig. 3) [3]. Microscopic study and analysis of marine invertebrates can provide valuable data on organismal response to changes in environmental parameters such as seawater composition, temperature, CO2 partial pressure and nutrification. In turn, this data provides valuable information useful for establishment of “proxies” in prediction and management of the ever-changing marine environment

Platelet Satellitism and Dual Surface Immunoglobulin Light-Chain Expression in Circulating Splenic Marginal Zone Lymphoma Cells

Platelet satellitism is believed to be an in vitro phenomenon induced at room temperature in ethylenediamine tetraacetic acid–anticoagulated blood. Most reports involve neutrophils; involvement with circulating lymphoma cells are exceedingly rare. Normally, mature B cells exhibit allelic exclusion in which a single class of surface immunoglobulin light chains (either κ or λ ) is expressed. The simultaneous expression of both κ and λ immunoglobulin light chains is rare. Herein, we report the unusual case of a patient with splenic marginal zone lymphoma in which circulating lymphoma cells express dual surface immunoglobulin light chains and exhibit platelet satellitism. In addition to clinical findings, a comprehensive analysis of the peripheral blood including correlated light and electron microscopy as well as flow cytometry are described

Peptide-Conjugated PAMAM Dendrimer as a Universal DNA Vaccine Platform to Target Antigen-Presenting Cells

DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics. This platform is based on conjugation of fifth generation polyamidoamine (G5-PAMAM) dendrimers, a DNA-loading surface, with MHC class II–targeting peptides that can selectively deliver these dendrimers to APCs under conditions that enhance their immune stimulatory potency. DNA conjugated with this platform efficiently transfected murine and human APCs in vitro. Subcutaneous administration of DNA-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (DC) in the draining lymph nodes, promoted generation of high affinity T cells, and elicited rejection of established tumors. Taken together, our findings show how PAMAM dendrimer complexes can be used for high transfection efficiency and effective targeting of APCs in vivo, conferring properties essential to generate effective DNA vaccines