Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop‐mediated isothermal amplification (LAMP) reaction

Aims

To develop two assays based on the loop‐mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes.

Methods and Results

Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction.

Conclusions

The two LAMP assays permitted to quickly and specifically identify DNA from OTA‐producing black aspergilli, as well as isolates grown in pure culture.

Significance and Impact of the Study

Monitoring vineyards for the presence of OTA‐producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA‐producing black aspergilli.     

Early-life exposure to a herbicide has enduring effects on pathogen-induced mortality

Exposure to stressors at formative stages in the development of wildlife and humans can have enduring effects on health. Understanding which, when and how stressors cause enduring health effects is crucial because these stressors might then be avoided or mitigated during formative stages to prevent lasting increases in disease susceptibility. Nevertheless, the impact of early-life exposure to stressors on the ability of hosts to resist and tolerate infections has yet to be thoroughly investigated. Here, we show that early-life, 6-day exposure to the herbicide atrazine (mean ± s.e.: 65.9±3.48 µg l⁻¹) increased frog mortality 46 days after atrazine exposure (post-metamorphosis), but only when frogs were challenged with a chytrid fungus implicated in global amphibian declines. Previous atrazine exposure did not affect resistance of infection (fungal load). Rather, early-life exposure to atrazine altered growth and development, which resulted in exposure to chytrid at more susceptible developmental stages and sizes, and reduced tolerance of infection, elevating mortality risk at an equivalent fungal burden to frogs unexposed to atrazine. Moreover, there was no evidence of recovery from atrazine exposure. Hence, reducing early-life exposure of amphibians to atrazine could reduce lasting increases in the risk of mortality from a disease associated with worldwide amphibian declines. More generally, these findings highlight that a better understanding of how stressors cause enduring effects on disease susceptibility could facilitate disease prevention in wildlife and humans, an approach that is often more cost-effective and efficient than reactive medicine.     

The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report

BackgroundIn vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE).AimTo investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers.MethodsPast and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed.ResultsIVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001).ConclusionsOur results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.     

Combined Effects of Pesticides and Trematode Infections on Hourglass Tree Frog <Emphasis Type="Italic">Polypedates cruciger</Emphasis>

The impact of widespread and common environmental factors, such as chemical contaminants, on infectious disease risk in amphibians is particularly important because both chemical contaminants and infectious disease have been implicated in worldwide amphibian declines. Here we report on the lone and combined effects of exposure to parasitic cercariae (larval stage) of the digenetic trematode, Acanthostomum burminis, and four commonly used pesticides (insecticides: chlorpyrifos, dimethoate; herbicides: glyphosate, propanil) at ecologically relevant concentrations on the survival, growth, and development of the common hourglass tree frog, Polypedates cruciger Blyth 1852. There was no evidence of any pesticide-induced mortality on cercariae because all the cercariae successfully penetrated each tadpole host regardless of pesticide treatment. In isolation, both cercarial and pesticide exposure significantly decreased frog survival, development, and growth, and increased developmental malformations, such as scoliosis, kyphosis, and also edema and skin ulcers. The combination of cercariae and pesticides generally posed greater risk to frogs than either factor alone by decreasing survival or growth or increasing time to metamorphosis or malformations. The exception was that lone exposure to chlorpyrifos had higher mortality without than with cercariae. Consistent with mathematical models that suggest that stress should increase the impact of generalist parasites, the weight of the evidence from the field and laboratory suggests that ecologically relevant concentrations of agrochemicals generally increase the threat that trematodes pose to amphibians, highlighting the importance of elucidating interactions between anthropogenic activities and infectious disease in taxa of conservation concern.     

An interaction between climate change and infectious disease drove widespread amphibian declines

Climate change might drive species declines by altering species interactions, such as host–parasite interactions. However, few studies have combined experiments, field data, and historical climate records to provide evidence that an interaction between climate change and disease caused any host declines. A recently proposed hypothesis, the thermal mismatch hypothesis, could identify host species that are vulnerable to disease under climate change because it predicts that cool‐ and warm‐adapted hosts should be vulnerable to disease at unusually warm and cool temperatures, respectively. Here, we conduct experiments on Atelopus zeteki, a critically endangered, captively bred frog that prefers relatively cool temperatures, and show that frogs have high pathogen loads and high mortality rates only when exposed to a combination of the pathogenic chytrid fungus (Batrachochytrium dendrobatidis) and high temperatures, as predicted by the thermal mismatch hypothesis. Further, we tested various hypotheses to explain recent declines experienced by species in the amphibian genus Atelopus that are thought to be associated with B. dendrobatidis and reveal that these declines are best explained by the thermal mismatch hypothesis. As in our experiments, only the combination of rapid increases in temperature and infectious disease could account for the patterns of declines, especially in species adapted to relatively cool environments. After combining experiments on declining hosts with spatiotemporal patterns in the field, our findings are consistent with the hypothesis that widespread species declines, including possible extinctions, have been driven by an interaction between increasing temperatures and infectious disease. Moreover, our findings suggest that hosts adapted to relatively cool conditions will be most vulnerable to the combination of increases in mean temperature and emerging infectious diseases.     

Impacts of thermal mismatches on chytrid fungus Batrachochytrium dendrobatidis prevalence are moderated by life stage,body size,elevation and latitude

Global climate change is increasing the frequency of unpredictable weather conditions; however, it remains unclear how species‐level and geographic factors, including body size and latitude, moderate impacts of unusually warm or cool temperatures on disease. Because larger and lower‐latitude hosts generally have slower acclimation times than smaller and higher‐latitude hosts, we hypothesised that their disease susceptibility increases under ‘thermal mismatches’ or differences between baseline climate and the temperature during surveying for disease. Here, we examined how thermal mismatches interact with body size, life stage, habitat, latitude, elevation, phylogeny and International Union for Conservation of Nature (IUCN) conservation status to predict infection prevalence of the chytrid fungus Batrachochytrium dendrobatidis (Bd) in a global analysis of 32 291 amphibian hosts. As hypothesised, we found that the susceptibility of larger hosts and hosts from lower latitudes to Bd was influenced by thermal mismatches. Furthermore, hosts of conservation concern were more susceptible than others following thermal mismatches, suggesting that thermal mismatches might have contributed to recent amphibian declines.