David T. Gauthier

Mycobacteriosis in fishes: A review

Mycobacterium species have long been recognised as a significant source of morbidity and mortality in finfish aquaculture, as well as in wild finfishes. Mycobacteria infecting fishes also include zoonotic pathogens that can cause protracted illness, especially in immunocompromised individuals. Several basic aspects of mycobacterial pathobiology in aquatic animals remain poorly understood, although a number of important recent developments have been made, especially with respect to identification of novel Mycobacterium spp. infecting fishes and a new group of mycobacteria closely related to the human pathogen Mycobacterium ulcerans. This review will encompass important aspects of mycobacterial disease in fishes, discuss recent research including studies of mycobacteriosis in striped bass (Morone saxatilis) of Chesapeake Bay, USA, and suggest directions for future work.

dc electrokinetic transport of cylindrical cells in straight microchannels

Electrokinetic transport of cylindrical cells under dc electric fields in a straight microfluidic channel is experimentally and numerically investigated with emphasis on the dielectrophoretic (DEP) effect on their orientation variations. A two-dimensional multiphysics model, composed of the Navier-Stokes equations for the fluid flow and the Laplace equation for the electric potential defined in an arbitrary Lagrangian-Eulerian framework, is employed to capture the transient electrokinetic motion of cylindrical cells. The numerical predictions of the particle transport are in quantitative agreement with the obtained experimental results, suggesting that the DEP effect should be taken into account to study the electrokinetic transport of cylindrical particles even in a straight microchannel with uniform cross-sectional area. A comprehensive parametric study indicates that cylindrical particles would experience an oscillatory motion under low electric fields. However, they are aligned with their longest axis parallel to the imposed electric field under high electric fields due to the induced DEP effect.

MYCOBACTERIOSIS‐ASSOCIATED MORTALITY IN WILD STRIPED BASS (MORONE SAXATILIS) FROM CHESAPEAKE BAY, USA

The striped bass (Morone saxatilis) is an economically and ecologically important finfish species along the Atlantic seaboard of the United States. Recent stock assessments in Chesapeake Bay (U.S.A.) indicate that non-fishing mortality in striped bass has increased since 1999, concomitant with very high (>50%) prevalence of visceral and dermal disease caused by Mycobacterium spp. Current fishery assessment models do not differentiate between disease and other components of non-fishing mortality (e.g., senescence, predation); therefore, disease impact on the striped bass population has not been established. Specific measurement of mortality associated with mycobacteriosis in wild striped bass is complicated because the disease is chronic and mortality is cryptic. Epidemiological models have been developed to estimate disease-associated mortality from cross-sectional prevalence data and have recently been generalized to represent disease processes more realistically. Here, we used this generalized approach to demonstrate disease-associated mortality in striped bass from Chesapeake Bay. To our knowledge this is the first demonstration of cryptic mortality associated with a chronic infectious disease in a wild finfish. This finding has direct implications for management and stock assessment of striped bass, as it demonstrates population-level negative impacts of a chronic disease. Additionally, this research provides a framework by which disease-associated mortality may be specifically addressed within fisheries models for resource management.

Apolipoprotein A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro.

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.

Bacterial zoonoses of fishes: A review and appraisal of evidence for linkages between fish and human infections

Human contact with and consumption of fishes presents hazards from a range of bacterial zoonotic infections. Whereas many bacterial pathogens have been presented as fish-borne zoonoses on the basis of epidemiological and phenotypic evidence, genetic identity between fish and human isolates is not frequently examined or does not provide support for transmission between these hosts. In order to accurately assess the zoonotic risk from exposure to fishes in the context of aquaculture, wild fisheries and ornamental aquaria, it is important to critically examine evidence of linkages between bacteria infecting fishes and humans. This article reviews bacteria typically presented as fish-borne zoonoses, and examines the current strength of evidence for this classification. Of bacteria generally described as fish-borne zoonoses, only Mycobacterium spp., Streptococcus iniae, Clostridium botulinum, and Vibrio vulnificus appear to be well-supported as zoonoses in the strict sense. Erysipelothrix rhusiopathiae, while transmissible from fishes to humans, does not cause disease in fishes and is therefore excluded from the list. Some epidemiological and/or molecular linkages have been made between other bacteria infecting both fishes and humans, but more work is needed to elucidate routes of transmission and the identity of these pathogens in their respective hosts at the genomic level.

Mycobacterium-Inducible Nramp in Striped Bass (Morone saxatilis)

ABSTRACT In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized an Nramp gene in this species and obtained evidence that there is induction following Mycobacterium exposure. The striped bass Nramp gene (MsNramp) and a 554-amino-acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely grouped MsNramp with other teleost Nramp genes and revealed high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection of Mycobacterium marinum, MsNramp expression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels of MsNramp were three- and sixfold higher on days 3 and 15, respectively. Injection of Mycobacterium shottsii resulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of an Nramp gene by mycobacteria in a poikilothermic vertebrate.

The isoprenoid perillyl alcohol inhibits telomerase activity in prostate cancer cells

Isoprenoids are recognized for their ability to suppress carcinogenic processes in vivo and in vitro. We previously established that the isoprenoid, perillyl alcohol, acted mechanistically on translation of specific proteins through modulation of mechanistic target of rapamycin (mTOR) signaling. Telomerase-the enzyme responsible for immortalizing cells through the addition of telomeric repeats-is de-repressed early in an aspiring cancer cell. Here the effects of biologically-relevant concentrations and short incubations (1-16 h) of perillyl alcohol or the mTOR inhibitor, rapamycin, on telomerase activity were examined in prostate cancer cell lines. A rapid suppression of telomerase activity was observed (from ∼65% to >95%) determined by real-time quantitative telomerase repeat amplification protocol and confirmed by polyacrylamide gel-analysis. Using real-time reverse transcriptase-PCR, we demonstrated that human telomerase reverse transcriptase (hTERT) mRNA levels were unaltered. Western blot analysis revealed that hTERT protein levels decreased in response to perillyl alcohol or rapamycin. This decrease was partially blocked by pretreatment with a proteasome inhibitor MG-132, indicating that proteasomal degradation contributed to the loss of hTERT protein. No change in hTERT phosphorylation at Ser824 was observed, indicating the absence of cellular hTERT protein redistribution. These findings provide evidence for a unique link between nutrient- and macrolide-mediated regulation of mTOR and hTERT, a key enzyme that regulates DNA structure and stability.

A Novel Method of Microsatellite Genotyping-By-Sequencing Using Individual Combinatorial Barcoding

This study examines the potential of next-generation sequencing based ‘genotyping-by-sequencing’ (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data.

Quantitative PCR Assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and Application to Environmental Samples and Fishes from the Chesapeake Bay

ABSTRACT Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.

An eDNA assay for Irish Petromyzon marinus and Salmo trutta and field validation in running water.

This pilot study presents an environmental DNA (eDNA) assay for sea lamprey Petromyzon marinus and brown trout Salmo trutta, two species of economic and conservation importance in the Republic of Ireland. The results demonstrate the effectiveness of eDNA for assessing presence of low-abundance taxa (here, P. marinus) for environmental managers, and they highlight the potential for assessing relative abundance of rare or invasive freshwater species.