In vivo innate immune responses of groper (Polyprion oxygeneios) against Miamiensis avidus infection and lack of protection following dietary vitamin C administration.

Scuticociliates are extracellular histophagous parasites that affect farmed fish worldwide. One of the most common pathogenic species is Miamiensis avidus, a pathogen of New Zealand groper (Polyprion oxygeneios). The aim of this study was to characterise both the host (groper)-parasite (M. avidus) immune interactions and the possible protective role of dietary sodium ascorbate. Head-kidney leucocytes (HKLs) from naturally infected adult groper showed decreased respiratory burst response and peroxidase (Px) levels than healthy individuals. Infected groper also had significantly higher serum Px levels compared to controls. Myeloperoxidase (MPO) was inhibited in the head-kidney (HK) whereas MPO(+) cells were observed in the skin and muscle lesions. The inhibition of the innate immune responses was further studied in experimental infections with M. avidus, which confirmed depletion of Px inside leucocytes and marked increases in serum Px in infected individuals. Groper juveniles were fed a diet supplemented with sodium ascorbate (Vitamin C) (2g Kg(-1)) for 21 days and then challenged by subcutaneous injection or immersion exposure with live M. avidus cells. No protection was observed in the sodium ascorbate fed groper compared to the control diet following challenge by either injection or immersion. In vitro assays showed that sodium ascorbate itself results in the inhibition of Px and respiratory burst of groper HKLs, supporting the results obtained in vivo. Our results show that histophagous protozoa such as M. avidus hamper innate immune defences of fish hosts and that dietary sodium ascorbate does not protect groper against experimental infection with this parasite.

Polyketide assembly lines of uncultivated sponge symbionts from structure-based gene targeting.

There is increasing evidence that uncultivated bacterial symbionts are the true producers of numerous bioactive compounds isolated from marine sponges. The localization and heterologous expression of biosynthetic genes could clarify this issue and provide sustainable supplies for a wide range of pharmaceuticals. However, identification of genes in the usually highly complex symbiont communities remains a challenging task. For polyketides, one of the most important groups of sponge-derived drug candidates, we have developed a general strategy that allows one to rapidly access biosynthetic gene clusters based on chemical moieties. Using this method, we targeted polyketide synthase genes from two different sponge metagenomes. We have obtained from a sponge-bacterial association a complete pathway for the rare and potent antitumor agent psymberin from Psammocinia aff. bulbosa. The data support the symbiont hypothesis and provide insights into natural product evolution in previously inaccessible bacteria.

Variation in bacterial DGGE patterns from human saliva: over time, between individuals and in corresponding dental plaque microcosms.

OBJECTIVE: Eubacterial 16S rDNA fingerprints of human saliva and dental plaque microcosm biofilms grown in the multi-plaque artificial mouth (MAM) were characterised using denaturing gradient gel electrophoresis (DGGE). DESIGN: The stability of the bacterial community in the saliva of one individual collected over 7 years was assessed and compared with bacterial patterns in the saliva of 10 different individuals. DGGE was also used to assess changes in bacterial composition between saliva and mature plaque microcosms developed in the MAM from these 10 individual saliva samples. RESULTS: A relatively stable bacterial community (>87% concordance) was maintained within the individual oral environment of the standard donor over 7 years of monitoring. By comparison, DGGE fingerprint patterns of saliva from 10 different donors displayed greater variability (66% concordance). Variability between individual DGGE profiles increased further in mature plaque microcosms grown from the saliva of the 10 donors (52% concordance) with an increase in detected species diversity and evidence for conserved similarity and hence the maintenance of organisation during community development. CONCLUSIONS: These results suggest that stable ecological conditions were maintained long-term within the oral environment of the individual saliva donor but that transient fluctuations also occurred. The ecology and predominating microbiota in different individuals was host-specific and these differences were maintained to a degree during development into mature plaque microcosms. These findings also demonstrate the potential usefulness of applying DGGE to monitor temporal and developmental changes and possibly pathogenic patterns in oral bacterial communities from saliva and plaque.

Spatial and temporal variability of the bacterial community in different chemotypes of the New Zealand marine sponge Mycale hentscheli.

Molecular fingerprinting of 16S rRNA genes using terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) was used to characterize the temporal and spatial variability among sponge-associated bacteria from Mycale hentscheli having distinct bioactive chemotypes. Cluster analysis of T-RFLP and DGGE profiles from M. hentscheli chemotypes largely grouped sponge microbial diversity to their distinct chemotype pattern. Repeat sampling of individual M. hentscheli at one location over a 21-month period showed that the T-RFLP profiles from individual sponges had similarity indices ranging from 60% to 82% and calculated DGGE similarities between 23% and 95%. However, a portion (>35% from DGGE and >19% from T-RFLP) of the microbial community from M. hentscheli appeared to be spatially conserved through all M. hentscheli populations. Sequence analysis of DGGE band fragments showed a similarity among the bands originating from different individuals, different times, and different locations. The sponge-associated relationship of these bands was confirmed, with sequences having similarity to sponge-associated bacteria reported from global locations. This study highlights the spatial and temporal complexity in the distribution of bacterial communities associated with different chemotypes of the marine sponge M. hentscheli.

In vitro culture and cryopreservation of Uronema marinum isolated from farmed New Zealand groper (Polyprion oxygeneios).

An in vitro culture method was developed for the ciliated protozoa Uronema marinum isolated from New Zealand aquacultured groper (Polyprion oxygeneios). Both formulated media and sterile seawater supplemented with homogenised fish tissue as a food source supported growth of U. marinum achieving cell densities of up to 1 x 10(5)cells/mL in culture. A cryopreservation method based on a cryomix formula of 20% glycerol, 10% fetal bovine serum and 70% cultured U. marinum, incorporating a slow freeze method to -80 degrees C, then liquid nitrogen storage, allowed cryogenic storage of cells and successful re-culture up to 12 months in storage.

Application of carbon source utilization patterns to measure the metabolic similarity of complex dental plaque biofilm microcosms.

Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H2-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (Streptococcus mutans plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.