Environmental DNA from multiple pathogens is elevated near active Atlantic salmon farms.

The spread of infection from reservoir host populations is a key mechanism for disease emergence and extinction risk and is a management concern for salmon aquaculture and fisheries. Using a quantitative environmental DNA methodology, we assessed pathogen environmental DNA in relation to salmon farms in coastal British Columbia, Canada, by testing for 39 species of salmon pathogens (viral, bacterial, and eukaryotic) in 134 marine environmental samples at 58 salmon farm sites (both active and inactive) over 3 years. Environmental DNA from 22 pathogen species was detected 496 times and species varied in their occurrence among years and sites, likely reflecting variation in environmental factors, other native host species, and strength of association with domesticated Atlantic salmon. Overall, we found that the probability of detecting pathogen environmental DNA (eDNA) was 2.72 (95% CI: 1.48, 5.02) times higher at active versus inactive salmon farm sites and 1.76 (95% CI: 1.28, 2.42) times higher per standard deviation increase in domesticated Atlantic salmon eDNA concentration at a site. If the distribution of pathogen eDNA accurately reflects the distribution of viable pathogens, our findings suggest that salmon farms serve as a potential reservoir for a number of infectious agents; thereby elevating the risk of exposure for wild salmon and other fish species that share the marine environment.

High-volume culture and quantitative real-time PCR for the detection of Aspergillus in sputum.

OBJECTIVES: Sputum culture is an insensitive method for the diagnosis of pulmonary aspergillosis. Growth of the organism allows identification of the causative species and susceptibility testing, both of which can inform treatment choices. The current practice is to culture an aliquot of diluted sputum. We assessed the value of culturing large volumes of unprocessed sputum, a method that we have termed high-volume culture (HVC). METHODS: Specimens were processed by conventional culture (using an aliquot of homogenized, diluted sputum on Sabouraud agar at 37°C and 45°C for up to 5 days) and HVC (using undiluted sputum on Sabouraud agar at 30°C for up to 14 days). A separate specimen was tested by quantitative real-time PCR. Antifungal susceptibility testing was performed by the EUCAST standard. RESULTS: We obtained sputum specimens from 229 individuals with the following conditions: chronic pulmonary aspergillosis (66.8%, 153/229), allergic bronchopulmonary aspergillosis (25.3%, 58/229) and Aspergillus bronchitis (7.9%, 18/229). Individuals with invasive pulmonary aspergillosis were not included. The positivity rate of conventional culture was 15.7% (36/229, 95% CI 11.6%-21.0%) and that of HVC was 54.2% (124/229, 95% CI 47.7%-60.5%) (p < 0.001). The higher positivity rate of HVC was demonstrated regardless of administration of antifungal treatment. Quantitive real-time PCR had an overall positivity rate of 49.2% (65/132, 95% CI 40.9%-57.7%), comparable to that of HVC. CONCLUSION: Detection of Aspergillus spp. in sputum is greatly enhanced by HVC. HVC allows for detection of azole-resistant isolates that would have been missed by conventional culture. This method can be performed in any microbiology laboratory without the need for additional equipment.

Experimental Hendra virus infection of dogs: virus replication, shedding and potential for transmission.

OBJECTIVE: Characterisation of experimental Hendra virus (HeV) infection in dogs and assessment of associated transmission risk. METHODS: Beagle dogs were exposed oronasally to Hendra virus/Australia/Horse/2008/Redlands or to blood collected from HeV-infected ferrets. Ferrets were exposed to oral fluids collected from dogs after canine exposure to HeV. Observations made and samples tested post-exposure were used to assess the clinical course and replication sites of HeV in dogs, the infectivity for ferrets of canine oral fluids and features of HeV infection in dogs following contact with infective blood. RESULTS: Dogs were reliably infected with HeV and were generally asymptomatic. HeV was re-isolated from the oral cavity and virus clearance was associated with development of virus neutralising antibody. Major sites of HeV replication in dogs were the tonsils, lower respiratory tract and associated lymph nodes. Virus replication was documented in canine kidney and spleen, confirming a viraemic phase for canine HeV infection and suggesting that urine may be a source of infectious virus. Infection was transmitted to ferrets via canine oral secretions, with copy numbers for the HeV N gene in canine oral swabs comparable to those reported for nasal swabs of experimentally infected horses. CONCLUSION: HeV is not highly pathogenic for dogs, but their oral secretions pose a potential transmission risk to people. The time-window for transmission risk is circumscribed and corresponds to the period of acute infection before establishment of an adaptive immune response. The likelihood of central nervous system involvement in canine HeV infection is unclear, as is any long-term consequence.

The implementation of rare events logistic regression to predict the distribution of mesophotic hard corals across the main Hawaiian Islands.

Predictive habitat suitability models are powerful tools for cost-effective, statistically robust assessment of the environmental drivers of species distributions. The aim of this study was to develop predictive habitat suitability models for two genera of scleractinian corals (Leptoserisand Montipora) found within the mesophotic zone across the main Hawaiian Islands. The mesophotic zone (30-180 m) is challenging to reach, and therefore historically understudied, because it falls between the maximum limit of SCUBA divers and the minimum typical working depth of submersible vehicles. Here, we implement a logistic regression with rare events corrections to account for the scarcity of presence observations within the dataset. These corrections reduced the coefficient error and improved overall prediction success (73.6% and 74.3%) for both original regression models. The final models included depth, rugosity, slope, mean current velocity, and wave height as the best environmental covariates for predicting the occurrence of the two genera in the mesophotic zone. Using an objectively selected theta ("presence") threshold, the predicted presence probability values (average of 0.051 for Leptoseris and 0.040 for Montipora) were translated to spatially-explicit habitat suitability maps of the main Hawaiian Islands at 25 m grid cell resolution. Our maps are the first of their kind to use extant presence and absence data to examine the habitat preferences of these two dominant mesophotic coral genera across Hawai'i.

Lessons from sea louse and salmon epidemiology.

Effective disease management can benefit from mathematical models that identify drivers of epidemiological change and guide decision-making. This is well illustrated in the host-parasite system of sea lice and salmon, which has been modelled extensively due to the economic costs associated with sea louse infections on salmon farms and the conservation concerns associated with sea louse infections on wild salmon. Consequently, a rich modelling literature devoted to sea louse and salmon epidemiology has been developed. We provide a synthesis of the mathematical and statistical models that have been used to study the epidemiology of sea lice and salmon. These studies span both conceptual and tactical models to quantify the effects of infections on host populations and communities, describe and predict patterns of transmission and dispersal, and guide evidence-based management of wild and farmed salmon. As aquaculture production continues to increase, advances made in modelling sea louse and salmon epidemiology should inform the sustainable management of marine resources.

DL-2-hydroxyisocaproic acid attenuates inflammatory responses in a murine Candida albicans biofilm model.

Chronic biofilm infections are often accompanied by a chronic inflammatory response, leading to impaired healing and increased, irreversible damage to host tissues. Biofilm formation is a major virulence factor for Candida albicans and a challenge for treatment. Most current antifungals have proved ineffective in eradicating infections attributed to biofilms. The biofilm structure protects Candida species against antifungals and provides a way for them to evade host immune systems. This leads to a very distinct inflammatory response compared to that seen in planktonic infections. Previously, we showed the superior efficacy of dl-2-hydroxyisocaproic acid (HICA) against various bacteria and fungi. However, the immunomodulatory properties of HICA have not been studied. Our aim was to investigate the potential anti-inflammatory response to HICA in vivo. We hypothesized that HICA reduces the levels of immune mediators and attenuates the inflammatory response. In a murine model, a robust biofilm was formed for 5 days in a diffusion chamber implanted underneath mouse skin. The biofilm was treated for 12 h with HICA, while caspofungin and phosphate-buffered saline (PBS) were used as controls. The pathophysiology and immunoexpression in the tissues surrounding the chamber were determined by immunohistochemistry. Histopathological examination showed an attenuated inflammatory response together with reduced expression of matrix metalloproteinase 9 (MMP-9) and myeloperoxidase (MPO) compared to those of chambers containing caspofungin and PBS. Interestingly, the expression of developmental endothelial locus 1 (Del-1), an antagonist of neutrophil extravasation, increased after treatment with HICA. Considering its anti-inflammatory and antimicrobial activity, HICA may have enormous therapeutic potential in the treatment of chronic biofilm infections and inflammation, such as those seen with chronic wounds.

2-hydroxyisocaproic acid is fungicidal for Candida and Aspergillus species.

The amino acid derivative 2-hydroxyisocaproic acid (HICA) is a nutritional additive used to increase muscle mass. Low levels can be detected in human plasma as a result of leucine metabolism. It has broad antibacterial activity but its efficacy against pathogenic fungi is not known. The aim was to test the efficacy of HICA against Candida and Aspergillus species. Efficacy of HICA against 19 clinical and reference isolates representing five Candida and three Aspergillus species with variable azole antifungal sensitivity profiles was tested using a microdilution method. The concentrations were 18, 36 and 72 mg ml(-1) . Growth was determined spectrophotometrically for Candida isolates and by visual inspection for Aspergillus isolates, viability was tested by culture and impact on morphology by microscopy. HICA of 72 mg ml(-1) was fungicidal against all Candida and Aspergillus fumigatus and Aspergillus terreus isolates. Lower concentrations were fungistatic. Aspergillus flavus was not inhibited by HICA. HICA inhibited hyphal formation in susceptible Candida albicans and A. fumigatus isolates and affected cell wall integrity. In conclusion, HICA has broad antifungal activity against Candida and Aspergillus at concentrations relevant for topical therapy. As a fungicidal agent with broad-spectrum bactericidal activity, it may be useful in the topical treatment of multispecies superficial infections.

Allee effect from parasite spill-back.

The exchange of native pathogens between wild and domesticated animals can lead to novel disease threats to wildlife. However, the dynamics of wild host-parasite systems exposed to a reservoir of domesticated hosts are not well understood. A simple mathematical model reveals that the spill-back of native parasites from domestic to wild hosts may cause a demographic Allee effect in the wild host population. A second model is tailored to the particulars of pink salmon (Oncorhynchus gorbuscha) and salmon lice (Lepeophtheirus salmonis), for which parasite spill-back is a conservation and fishery concern. In both models, parasite spill-back weakens the coupling of parasite and wild host abundance-particularly at low host abundance-causing parasites per host to increase as a wild host population declines. These findings show that parasites shared across host populations have effects analogous to those of generalist predators and can similarly cause an unstable equilibrium in a focal host population that separates persistence and extirpation. Allee effects in wildlife arising from parasite spill-back are likely to be most pronounced in systems where the magnitude of transmission from domestic to wild host populations is high because of high parasite abundance in domestic hosts, prolonged sympatry of domestic and wild hosts, a high transmission coefficient for parasites, long-lived parasite larvae, and proximity of domesticated populations to wildlife migration corridors.

Production of carcinogenic acetaldehyde by Candida albicans from patients with potentially malignant oral mucosal disorders.

OBJECTIVES: Production of carcinogenic acetaldehyde by Candida has been suggested to contribute to epithelial dysplasia and oral carcinogenesis. Oral lichen planus (OLP), oral lichenoid lesion (OLL) and oral leukoplakia (OL) are potentially carcinogenic oral diseases where colonisation by Candida is common, but acetaldehyde production by Candida has not been studied. STUDY DESIGN: Acetaldehyde production in ethanol (11 mM), glucose (100 mM), ethanol-glucose (11 mM and 100 mM) or red wine (1200 mM ethanol) incubation by Candida albicans from patients with OLL (n = 6), OLP (n = 16), OL (n = 6) and controls (n = 6) was measured by gas chromatography. Participants completed a questionnaire regarding their smoking habits and alcohol consumption. RESULTS: All Candida albicans isolates produced potentially carcinogenic levels of acetaldehyde (>100 µM) in all incubations containing ethanol. The control group isolates produced the highest acetaldehyde levels. Isolates from smokers produced more acetaldehyde in all incubations than those from non-smokers. The difference was significant in ethanol-glucose incubation. Isolates from patients who were both smokers and drinkers produced the highest amounts when incubated in ethanol, ethanol-glucose and wine. CONCLUSIONS: Candida albicans isolated from potentially carcinogenic oral diseases can produce mutagenic amounts of acetaldehyde. Cigarette smoking and alcohol consumption may favour adaptational changes resulting in the upregulation of candidal acetaldehyde metabolism.

Critical thresholds in sea lice epidemics: evidence, sensitivity and subcritical estimation.

Host density thresholds are a fundamental component of the population dynamics of pathogens, but empirical evidence and estimates are lacking. We studied host density thresholds in the dynamics of ectoparasitic sea lice (Lepeophtheirus salmonis) on salmon farms. Empirical examples include a 1994 epidemic in Atlantic Canada and a 2001 epidemic in Pacific Canada. A mathematical model suggests dynamics of lice are governed by a stable endemic equilibrium until the critical host density threshold drops owing to environmental change, or is exceeded by stocking, causing epidemics that require rapid harvest or treatment. Sensitivity analysis of the critical threshold suggests variation in dependence on biotic parameters and high sensitivity to temperature and salinity. We provide a method for estimating the critical threshold from parasite abundances at subcritical host densities and estimate the critical threshold and transmission coefficient for the two epidemics. Host density thresholds may be a fundamental component of disease dynamics in coastal seas where salmon farming occurs.

Tissue-specific expression of FLOWERING LOCUS T in Arabidopsis is maintained independently of polycomb group protein repression.

The Polycomb Group (PcG) pathway represses transcription through a mechanism conserved among plants and animals. PcG-mediated repression can determine spatial territories of gene expression, but it remains unclear whether PcG-mediated repression is a regulatory requirement for all targets. Here, we show the role of PcG proteins in the spatial regulation of FLOWERING LOCUS T (FT), a main activator of flowering in Arabidopsis thaliana exclusively expressed in the vasculature. Strikingly, the loss of PcG repression causes down-regulation of FT. In addition, our results show how the effect of PcG-mediated regulation differs for target genes and that, for FT expression, it relies primarily on tissue differentiation.

Dynamic regulation of H3K27 trimethylation during Arabidopsis differentiation.

During growth of multicellular organisms, identities of stem cells and differentiated cells need to be maintained. Cell fate is epigenetically controlled by the conserved Polycomb-group (Pc-G) proteins that repress their target genes by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although H3K27me3 is associated with mitotically stable gene repression, a large fraction of H3K27me3 target genes are tissue-specifically activated during differentiation processes. However, in plants it is currently unclear whether H3K27me3 is already present in undifferentiated cells and dynamically regulated to permit tissue-specific gene repression or activation. We used whole-genome tiling arrays to identify the H3K27me3 target genes in undifferentiated cells of the shoot apical meristem and in differentiated leaf cells. Hundreds of genes gain or lose H3K27me3 upon differentiation, demonstrating dynamic regulation of an epigenetic modification in plants. H3K27me3 is correlated with gene repression, and its release preferentially results in tissue-specific gene activation, both during differentiation and in Pc-G mutants. We further reveal meristem- and leaf-specific targeting of individual gene families including known but also likely novel regulators of differentiation and stem cell regulation. Interestingly, H3K27me3 directly represses only specific transcription factor families, but indirectly activates others through H3K27me3-mediated silencing of microRNA genes. Furthermore, H3K27me3 targeting of genes involved in biosynthesis, transport, perception, and signal transduction of the phytohormone auxin demonstrates control of an entire signaling pathway. Based on these and previous analyses, we propose that H3K27me3 is one of the major determinants of tissue-specific expression patterns in plants, which restricts expression of its direct targets and promotes gene expression indirectly by repressing miRNA genes.

Sea-cage aquaculture, sea lice, and declines of wild fish.

A sea cage, sometimes referred to as a net pen, is an enclosure designed to prevent farm fish from escaping and to protect them from large predators, while allowing a free flow of water through the cage to carry away waste. Farm fish thus share water with wild fish, which enables transmission of parasites, such as sea lice, from wild to farm and farm to wild fishes. Sea lice epidemics, together with recently documented population-level declines of wild salmon in areas of sea-cage farming, are a reminder that sea-cage aquaculture is fundamentally different from terrestrial animal culture. The difference is that sea cages protect farm fish from the usual pathogen-control mechanisms of nature, such as predators, but not from the pathogens themselves. A sea cage thus becomes an unintended pathogen factory. Basic physical theory explains why sea-cage aquaculture causes sea lice on sympatric wild fish to increase and why increased lice burdens cause wild fish to decline, with extirpation as a real possibility. Theory is important to this issue because slow declines of wild fish can be difficult to detect amid large fluctuations from other causes. The important theoretical concepts are equilibrium, host-density effect, reservoir-host effect, and critical stocking level of farmed fish (stocking level at which lice proliferate on farm fish even if wild fish are not present to infect them). I explored these concepts and their implications without mathematics through examples from salmon farming. I also considered whether the lice-control techniques used by sea-cage farmers (medication and shortened grow-out times) are capable of protecting wild fish. Elementary probability showed that W ≈ W* - εF (where W is the abundance of wild fish, W* is the prefarm abundance, F is the abundance of farm fish, and ε is the ratio of lice per farm fish to lice per wild fish). Declines of wild fish can be reduced by short growing cycles for farm fish, medicating farm fish, and keeping farm stocking levels low. Declines can be avoided only by ensuring that wild fish do not share water with farmed fish, either by locating sea cages very far from wild fish or through the use of closed-containment aquaculture systems. These principles are likely to govern any aquaculture system where cage-protected farm hosts and sympatric wild hosts have a common parasite with a direct life cycle.

Sea-lice infection models for fishes.

As free-living sea-lice larvae are difficult to sample directly, lice abundances on fish have recently been used to study larvae in the water. In the KLV problem, juvenile wild salmon migrate past a salmon farm, and the change of infection with distance along the migration route is used to estimate larvae production from the farm. In the farm problem, time-varying infection of sea-cage fish is used to estimate the time-variation of free-living larvae in waters near the farm. Both inverse problems require good forward models for infection. In the farm problem, hosts are relatively large and lice pathogenesis is seldom mortal, whereas in the KLV problem hosts are small and lice-induced host mortality can affect lice abundance; thus, infection models for the farm problem are special cases of models for the KLV problem. Here I give an infection model for the KLV problem that explicitly includes lice clumping and host mortality, showing that Krkosek et al. (Proc R Soc B 272:689-696, 2005) (KLV) probably underestimated larvae production by the salmon farm, and further, that if lice development rates were known from laboratory data, lice abundance field data could be directly inverted for lice-induced host mortality during migration. If lice-induced host mortality is negligible, or if lice are Poisson distributed, infection models of arbitrary complexity reduce to Erlang models. I give two useful Erlang models with their solutions for non-zero initial conditions.

Sperm whale three-dimensional track, swim orientation, beam pattern, and click levels observed on bottom-mounted hydrophones.

In an earlier paper [Nosal and Frazer Appl. Acoust. 61, 1187-1201 (2006)], a sperm whale was tracked in three-dimensions using direct and surface-reflected time differences (DRTD) of clicks recorded on five bottom-mounted hydrophones, a passive method that is robust to timing errors between hydrophones. This paper refines the DRTD method and combines it with a time of (direct) arrival method to improve the accuracy of the track. The position and origin time of each click having been estimated, pitch and yaw are then obtained by assuming the main axis of the whale is tangent to the track. Roll is then found by applying the bent horn model of sperm whale phonation, in which each click is composed of two pulses, p0 and p1, that exit the whale at different points. With instantaneous pitch, roll, and yaw estimated from time differences, amplitudes are then used to estimate the beam patterns of the p0 and p1 pulses. The resulting beam patterns independently confirm those obtained by Zimmer et al. [J. Acoust. Soc. Am. 117, 1473-1485 (2005); 118, 3337-3345 (2005)] with a very different experimental setup. A method for estimating relative click levels is presented and used to find that click levels decrease toward the end of a click series, prior to the "creak" associated with prey capture.

Epizootics of wild fish induced by farm fish.

The continuing decline of ocean fisheries and rise of global fish consumption has driven aquaculture growth by 10% annually over the last decade. The association of fish farms with disease emergence in sympatric wild fish stocks remains one of the most controversial and unresolved threats aquaculture poses to coastal ecosystems and fisheries. We report a comprehensive analysis of the spread and impact of farm-origin parasites on the survival of wild fish populations. We mathematically coupled extensive data sets of native parasitic sea lice (Lepeophtheirus salmonis) transmission and pathogenicity on migratory wild juvenile pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon. Farm-origin lice induced 9-95% mortality in several sympatric wild juvenile pink and chum salmon populations. The epizootics arise through a mechanism that is new to our understanding of emerging infectious diseases: fish farms undermine a functional role of host migration in protecting juvenile hosts from parasites associated with adult hosts. Although the migratory life cycles of Pacific salmon naturally separate adults from juveniles, fish farms provide L. salmonis novel access to juvenile hosts, in this case raising infection rates for at least the first approximately 2.5 months of the salmon's marine life (approximately 80 km of the migration route). Spatial segregation between juveniles and adults is common among temperate marine fishes, and as aquaculture continues its rapid growth, this disease mechanism may challenge the sustainability of coastal ecosystems and economies.

Localization of marine mammals near Hawaii using an acoustic propagation model.

Humpback whale songs were recorded on six widely spaced receivers of the Pacific Missile Range Facility (PMRF) hydrophone network near Hawaii during March of 2001. These recordings were used to test a new approach to localizing the whales that exploits the time-difference of arrival (time lag) of their calls as measured between receiver pairs in the PMRF network. The usual technique for estimating source position uses the intersection of hyperbolic curves of constant time lag, but a drawback of this approach is its assumption of a constant wave speed and straight-line propagation to associate acoustic travel time with range. In contrast to hyperbolic fixing, the algorithm described here uses an acoustic propagation model to account for waveguide and multipath effects when estimating travel time from hypothesized source positions. A comparison between predicted and measured time lags forms an ambiguity surface, or visual representation of the most probable whale position in a horizontal plane around the array. This is an important benefit because it allows for automated peak extraction to provide a location estimate. Examples of whale localizations using real and simulated data in algorithms of increasing complexity are provided.