Aquatic polymers can drive pathogen transmission in coastal ecosystems

Gelatinous polymers including extracellular polymeric substances (EPSs) are fundamental to biophysical processes in aquatic habitats, including mediating aggregation processes and functioning as the matrix of biofilms. Yet insight into the impact of these sticky molecules on the environmental transmission of pathogens in the ocean is limited. We used the zoonotic parasite Toxoplasma gondii as a model to evaluate polymer-mediated mechanisms that promote transmission of terrestrially derived pathogens to marine fauna and humans. We show that transparent exopolymer particles, a particulate form of EPS, enhance T. gondii association with marine aggregates, material consumed by organisms otherwise unable to access micrometre-sized particles. Adhesion to EPS biofilms on macroalgae also captures T. gondii from the water, enabling uptake of pathogens by invertebrates that feed on kelp surfaces. We demonstrate the acquisition, concentration and retention of T. gondii by kelp-grazing snails, which can transmit T. gondii to threatened California sea otters. Results highlight novel mechanisms whereby aquatic polymers facilitate incorporation of pathogens into food webs via association with particle aggregates and biofilms. Identifying the critical role of invisible polymers in transmission of pathogens in the ocean represents a fundamental advance in understanding and mitigating the health impacts of coastal habitat pollution with contaminated runoff.     

Effect of Estuarine Wetland Degradation on Transport of Toxoplasma gondii Surrogates from Land to Sea

The flux of terrestrially derived pathogens to coastal waters presents a significant health risk to marine wildlife, as well as to humans who utilize the nearshore for recreation and seafood harvest. Anthropogenic changes in natural habitats may result in increased transmission of zoonotic pathogens to coastal waters. The objective of our work was to evaluate how human-caused alterations of coastal landscapes in California affect the transport of Toxoplasma gondii to estuarine waters. Toxoplasma gondii is a protozoan parasite that is excreted in the feces of infected felids and is thought to reach coastal waters in contaminated runoff. This zoonotic pathogen causes waterborne toxoplasmosis in humans and is a significant cause of death in threatened California sea otters. Surrogate particles that mimic the behavior of T. gondii oocysts in water were released in transport studies to evaluate if the loss of estuarine wetlands is contributing to an increased flux of oocysts into coastal waters. Compared to vegetated sites, more surrogates were recovered from unvegetated mudflat habitats, which represent degraded wetlands. Specifically, in Elkhorn Slough, where a large proportion of otters are infected with T. gondii, erosion of 36% of vegetated wetlands to mudflats may increase the flux of oocysts by more than 2 orders of magnitude. Total degradation of wetlands may result in increased Toxoplasma transport of 6 orders of magnitude or more. Destruction of wetland habitats along central coastal California may thus facilitate pathogen pollution in coastal waters with detrimental health impacts to wildlife and humans.Estuaries are recognized as being critically endangered worldwide. Pollution of estuarine waters is a significant threat to the health of aquatic life, as well as to humans who depend on coastal habitats (23). Contamination of nearshore waters with terrestrially derived, zoonotic pathogens has received little attention in the field of marine water pollution, which has primarily focused on chemical and nutrient pollutants (22, 42, 46, 55). Yet, studies have documented the presence of fecal pathogens from terrestrial animals in coastal waters and filter-feeding shellfish (7, 37, 48), as well as infections and deaths in aquatic wildlife and humans who become exposed through recreation activities or seafood (4, 18, 39). The zoonotic parasite Toxoplasma gondii is emerging as an important waterborne pathogen in both human and marine wildlife populations (2, 3, 6, 11, 15, 38). Consumption of raw oysters, clams, or mussels has recently been determined to be a risk factor for human exposure to T. gondii (24). Moreover, this parasite is an important cause of death in threatened Southern sea otters (Enhydra lutris nereis) (10, 29). Sea otter infection appears most likely to result from ingestion of environmentally resistant T. gondii oocysts that reach coastal waters in contaminated freshwater runoff (35, 36). These oocysts are shed in the feces of infected wild and domestic felids, with an individual cat capable of shedding up to 1 billion oocysts over several days postinfection (12).Elkhorn Slough, within Monterey Bay in California, is one of the high-risk sites for sea otter infection with T. gondii, with seroprevalence rates of 79% in otters sampled in this area (35). To date, the reasons for the high sea otter prevalence of infections with T. gondii at this site remain unknown. This estuarine habitat has been extensively altered by human activities and is listed as an impaired body of water by the State of California (9). Specifically, extensive degradation has been observed in the slough, with over one-third of vegetated wetlands converted to mudflats due to erosion (49). While the effect of this landscape alteration on the transport of waterborne pathogens is not currently known, such degradation may facilitate contamination of nearshore waters with T. gondii.Wetland habitats provide valuable ecosystem services, including improvement of effluent water quality characteristics through removal of a variety of pollutants (28, 50, 57). Artificially constructed wetlands are now used globally in water treatment facilities to remove nutrients, chemical pollutants, and fecal pathogens from contaminated waters before discharge into receiving water bodies (8, 17, 21, 26, 27). However, compared with freshwater and constructed wetlands, significantly less research has focused on the effects of natural, estuarine wetlands on water quality. In the few studies that investigated the impact of saltwater marshes on marine water quality, these habitats were shown to reduce concentrations of chemicals and nutrients that reach coastal waters in contaminated overland runoff (5, 51). In addition, the percentage of watershed-impervious surface coverage and reduction of natural coastal habitats due to anthropogenic changes has been associated with increased coastal water pollution (33, 34). Despite previous research suggesting a link between wetland degradation and coastal pathogen pollution (5, 33, 34, 51), the role estuarine wetlands play in the transport of terrestrial pathogens from land to sea has not been previously investigated.The overall goal of our research was to evaluate the effect of coastal wetland degradation on contamination of estuarine and coastal waters with terrestrially derived, zoonotic pathogens. Specifically, the objective of this study was to measure T. gondii oocyst transport through vegetated estuarine wetlands and nonvegetated mudflats to quantify the effect of vegetation loss on the flux of this zoonotic pathogen to coastal waters. Due to the biohazard risks associated with the release of environmentally resistant oocysts, experiments used previously validated surrogate microspheres and a specially designed flume that was deployed in vegetated and mudflat (nonvegetated) estuarine wetland habitats. The flume-in-field study design allowed for replication of experiments using specific hydrological parameters while conducting the study within a natural estuarine environment with in situ vegetation, substrate, and water. The two autofluorescent microspheres used in this study have similar physical and surface chemistry properties to T. gondii oocysts and have been previously evaluated as surrogate particles for this protozoan parasite (44). Our results provide novel insights into the consequences of changes in coastal habitat on the ecology of zoonotic infectious disease organisms in coastal marine ecosystems.     

Health assessment of free-ranging alligator snapping turtles (Macrochelys temminckii) in Georgia and Florida

The Alligator Snapping Turtle (Macrochelys temminckii) is a large freshwater turtle endemic to river systems that drain into the Gulf of Mexico. Turtle populations were sharply reduced by commercial harvest in the 1970s and 1980s; however, the species has yet to be protected under the Endangered Species Act. While anthropogenic stressors such as habitat fragmentation and degradation and illegal capture continue to threaten populations, the degree to which disease may be contributing to any decline of the Alligator Snapping Turtle is unknown. Data were collected from 97 free-ranging Alligator Snapping Turtles in nine waterways in Florida and Georgia from 2001 to 2006. Eleven turtles were captured more than once, resulting in a total sample pool of 123. Reference ranges were established for complete blood count, plasma biochemistry values, trace metals (mercury, zinc, copper, lead, and arsenic), and nutrient parameters (vitamins A, E, D, and selenium). Variations by capture location, sex, and season were detected and likely resulted from external factors such as habitat and diet. Turtles sampled in one location were positive for tortoise herpesviral antibodies. Blood mercury values also differed among populations. This study provides justification for the use of these long-lived aquatic turtles as biologic monitors of the health of local freshwater ecosystems.     

Structural asymmetry in a trimeric Na+/betaine symporter, BetP, from Corynebacterium glutamicum

The Na⁺-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.     

Comparative health assessment of Western Pacific leatherback turtles (Dermochelys coriacea) foraging off the coast of California, 2005-2007

Leatherback turtles (Dermochelys coriacea) are critically endangered, primarily threatened by the overharvesting of eggs, fisheries entanglement, and coastal development. The Pacific leatherback population has experienced a catastrophic decline over the past two decades. Leatherbacks foraging off the coast of California are part of a distinct Western Pacific breeding stock that nests on beaches in Indonesia, Papua New Guinea, and the Solomon Islands. Although it has been proposed that the rapid decline of Pacific leatherback turtles is due to increased adult mortality, little is known about the health of this population. Health assessments in leatherbacks have examined females on nesting beaches, which provides valuable biological information, but might have limited applicability to the population as a whole. During September 2005 and 2007, we conducted physical examinations on 19 foraging Pacific leatherback turtles and measured normal physiologic parameters, baseline hematologic and plasma biochemistry values, and exposure to heavy metals (cadmium, lead, and mercury), organochlorine contaminants, and domoic acid. We compared hematologic values of foraging Pacific leatherbacks with their nesting counterparts in Papua New Guinea (n=11) and with other nesting populations in the Eastern Pacific in Costa Rica (n=8) and in the Atlantic in St. Croix (n=12). This study provides the most comprehensive assessment to date of the health status of leatherbacks in the Pacific. We found significant differences in blood values between foraging and nesting leatherbacks, which suggests that health assessment studies conducted only on nesting females might not accurately represent the whole population. The establishment of baseline physiologic data and blood values for healthy foraging leatherback turtles, including males, provides valuable data for long-term health monitoring and comparative studies of this endangered population.     

Role of Mitochondria in Parvovirus Pathology

Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV) associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association.     

Efficacy and Safety of Vitamin K-Antagonists (VKA) for Atrial Fibrillation in Non-Dialysis Dependent Chronic Kidney Disease

Background

Essential information regarding efficacy and safety of vitamin K-antagonists (VKA) treatment for atrial fibrillation (AF) in non-dialysis dependent chronic kidney disease (CKD) is still lacking in current literature. The aim of our study was to compare the risks of stroke or transient ischemic attack (TIA) and major bleeds between patients without CKD (eGFR >60 ml/min), and those with moderate (eGFR 30–60 ml/min), or severe non-dialysis dependent CKD (eGFR <30 ml/min).

Methods

We included 300 patients without CKD, 294 with moderate, and 130 with severe non-dialysis dependent CKD, who were matched for age and sex. Uni- and multivariate Cox regression analyses were performed reporting hazard ratios (HRs) for the endpoint of stroke or TIA and the endpoint of major bleeds as crude values and adjusted for comorbidity and platelet-inhibitor use.

Results

Overall, 6.2% (45/724, 1.7/100 patient years) of patients developed stroke or TIA and 15.6% (113/724, 4.8/100 patient years) a major bleeding event. Patients with severe CKD were at high risk of stroke or TIA and major bleeds during VKA treatment compared with those without renal impairment, HR 2.75 (95%CI 1.25–6.05) and 1.66 (95%CI 0.97–2.86), or with moderate CKD, HR 3.93(1.71–9.00) and 1.86 (95%CI 1.08–3.21), respectively. These risks were similar for patients without and with moderate CKD. Importantly, both less time spent within therapeutic range and high INR-variability were associated with increased risks of stroke or TIA and major bleeds in severe CKD patients.

Conclusions

VKA treatment for AF in patients with severe CKD has a poor safety and efficacy profile, likely related to suboptimal anticoagulation control. Our study findings stress the need for better tailored individualised anticoagulant treatment approaches for patients with AF and severe CKD.     

Using Molecular Epidemiology to Track Toxoplasma gondii from Terrestrial Carnivores to Marine Hosts: Implications for Public Health and Conservation

Background

Environmental transmission of the zoonotic parasite Toxoplasma gondii, which is shed only by felids, poses risks to human and animal health in temperate and tropical ecosystems. Atypical T. gondii genotypes have been linked to severe disease in people and the threatened population of California sea otters. To investigate land-to-sea parasite transmission, we screened 373 carnivores (feral domestic cats, mountain lions, bobcats, foxes, and coyotes) for T. gondii infection and examined the distribution of genotypes in 85 infected animals sampled near the sea otter range.

Methodology/Principal Findings

Nested PCR-RFLP analyses and direct DNA sequencing at six independent polymorphic genetic loci (B1, SAG1, SAG3, GRA6, L358, and Apico) were used to characterize T. gondii strains in infected animals. Strains consistent with Type X, a novel genotype previously identified in over 70% of infected sea otters and four terrestrial wild carnivores along the California coast, were detected in all sampled species, including domestic cats. However, odds of Type X infection were 14 times higher (95% CI: 1.3–148.6) for wild felids than feral domestic cats. Type X infection was also linked to undeveloped lands (OR = 22, 95% CI: 2.3–250.7). A spatial cluster of terrestrial Type II infection (P = 0.04) was identified in developed lands bordering an area of increased risk for sea otter Type II infection. Two spatial clusters of animals infected with strains consistent with Type X (P≤0.01) were detected in less developed landscapes.

Conclusions

Differences in T. gondii genotype prevalence among domestic and wild felids, as well as the spatial distribution of genotypes, suggest co-existing domestic and wild T. gondii transmission cycles that likely overlap at the interface of developed and undeveloped lands. Anthropogenic development driving contact between these cycles may increase atypical T. gondii genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife.     

A strong decline of the endangered Apollo butterfly over 20 years in the archipelago of southern Finland

Journal of Insect Conservation - Insect groups are declining worldwide; Lepidoptera are among the taxa most affected in terrestrial ecosystems. The main drivers of these declines are a diverse set...     

Cholesterol supports the retinoic acid-induced synaptic vesicle formation in differentiating human SH-SY5Y neuroblastoma cells

Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.