Evaluation of the MicroScan enzyme-based system for the identification of foodborne yeasts

Eighty-nine strains representing 36 species of foodborne yeasts isolated from fruit juice concentrates were identified using the Baxter MicroScan enzyme-based kit, conventional tests according to a simplified identification method (SIM), and the API 20C kit. Of the 15 test species included in the MicroScan database, only 40% were correctly identified; 13% gave scores of unacceptably low probabilities, 20% were misidentified, and 27% could not be identified. Of the 21 test species not in the MicroScan database, 38% were misidentified and 62% produced biocodes with between-species differences not larger than differences between strains within species. The reliability of the MicroScan enzyme-based system is questioned, in that different results were sometimes obtained upon retesting the same strains. The MicroScan enzyme-based system is rapid, providing results within 4 h. However, because of its restricted and specific database and unreliability, the system appears to be unsuited for the identification of foodborne yeasts.     

Technical viability of the YF MAC-HD ELISA kit for use in yellow fever-endemic regions

Yellow fever (YF), an arboviral disease, affects an estimated 200,000 people and causes 30,000 deaths per year and recently has caused major epidemics in Africa and South America. Timely and accurate diagnosis of YF is critical for managing outbreaks and implementing vaccination campaigns. A YF immunoglobulin M (IgM) antibody-capture (MAC) enzyme-linked immunosorbent assay (ELISA) kit, the YF MAC-HD, was successfully introduced starting in 2018 to laboratories in Africa and South America. The YF MAC-HD kit can be performed in 3.5 hours, test up to 24 samples, and includes all reagents necessary to perform the test, except for water used to dilute wash buffer. In 2018 and 2019, a total of 56 laboratory personnel from 39 countries in Africa and South America were trained to use the kit during workshops, followed by take-home YF IgM proficiency testing (PT) exercises. Participants received either a 10- or 20-sample YF PT panel and performed testing using the YF MAC-HD kit. All countries obtained 90% or higher correct results. These results verified the technical viability and transferability of YF MAC-HD kit use for laboratories in YF-endemic countries.     

Land Use as a Driver of Patterns of Rodenticide Exposure in Modeled Kit Fox Populations

Although rodenticides are increasingly regulated, they nonetheless cause poisonings in many non-target wildlife species. Second-generation anticoagulant rodenticide use is common in agricultural and residential landscapes. Here, we use an individual-based population model to assess potential population-wide effects of rodenticide exposures on the endangered San Joaquin kit fox (Vulpes macrotis mutica). We estimate likelihood of rodenticide exposure across the species range for each land cover type based on a database of reported pesticide use and literature. Using a spatially-explicit population model, we find that 36% of modeled kit foxes are likely exposed, resulting in a 7-18% decline in the range-wide modeled kit fox population that can be linked to rodenticide use. Exposures of kit foxes in low-density developed areas accounted for 70% of the population-wide exposures to rodenticides. We conclude that exposures of non-target kit foxes could be greatly mitigated by reducing the use of second-generation anticoagulant rodenticides in low-density developed areas near vulnerable populations.     

Rapid and sensitive detection of DNA in Southern blots with chemiluminescence

A new chemiluminescent Southern blot procedure offers molecular biologists a safe, ultrasensitive and rapid alternative to conventional 32P-based systems. This new DNA detection system, SOUTHERN-LIGHT, has been developed by Tropix, Inc. The luminescent signal is produced from a direct chemiluminescent substrate, disodium 3-(4-methoxyspiro[1,2-dioxetane-3-2'-tricyclo-[3.3.1.1 .3,7]decan]-4-yl) phenyl phosphate (AMPPD), which decomposes upon dephosphorylation with alkaline phosphatase. SOUTHERN-LIGHT is an ultrasensitive, rapid detection kit for use with membrane-bound DNA. It is the first test kit to incorporate AMPPD. It requires no specialized equipment and results can be conveniently imaged on instant film or x-ray film within 5-60 min of exposure.     

Performance and behaviour of rabbit does in a group-housing system with natural mating or artificial insemination

This study compared reproductive performance and behaviour of does raised in a group-housing system and in a regular cage system. The group-housing pen was divided into different functional areas for suckling, resting, and eating and special hiding areas for kits when they had left the nest-boxes and does to favour the species specific behavioural traits. Does had access to their nest-box by means of an individual Electronic Nest-box Recognition System (ENRS) activated by a coded transponder placed in their eartags. Eight does were housed in each pen. Natural mating (NM, with a buck in the group) or artificial inseminations (AI) were applied. Litter size, kit mortality and kit weight at 14 d of age were similar for group-housing and cages when NM were applied. With a natural reproduction rhythm group-housing led to an increase of +38% of litters. However, from a management point of view, a cycled production system with AI is preferred. With AI and group-housing, a lower kindling rate and a lower kit weight at weaning were found. The lower kindling rate was partly caused by pseudo-pregnancies that were found in 23% (P < 0.01) of the does in the group-housing system against 0% in the control group. Sixteen to 20% of the does in the group-housing system had skin injuries, which is an indicator for aggression among does. Most of the injuries were seen on the body and most of them were superficial bites. Based on the results of this study, it can be concluded that group-housing of rabbit does seems possible, but more research is needed to solve the problems of the decreased kindling rate and occurrence of pseudo-pregnancies, the lower weight at weaning and aggressiveness among does.     

Decades-long social memory in bottlenose dolphins

Long-term social memory is important, because it is an ecologically relevant test of cognitive capacity, it helps us understand which social relationships are remembered and it relates two seemingly disparate disciplines: cognition and sociality. For dolphins, long-term memory for conspecifics could help assess social threats as well as potential social or hunting alliances in a very fluid and complex fission–fusion social system, yet we have no idea how long dolphins can remember each other. Through a playback study conducted within a multi-institution dolphin breeding consortium (where animals are moved between different facilities), recognition of unfamiliar versus familiar signature whistles of former tank mates was assessed. This research shows that dolphins have the potential for lifelong memory for each other regardless of relatedness, sex or duration of association. This is, to my knowledge, the first study to show that social recognition can last for at least 20 years in a non-human species and the first large-scale study to address long-term memory in a cetacean. These results, paired with evidence from elephants and humans, provide suggestive evidence that sociality and cognition could be related, as a good memory is necessary in a fluid social system.     

Memory carriers and stewardship of metropolitan landscapes

History matters, and can be an active and dynamic component in the present. We explore social-ecological memory as way to diagnose and engage with urban green space performance and resilience. Rapidly changing cities pose a threat and a challenge to the continuity that has helped to support biodiversity and ecological functions by upholding similar or only slowly changing adaptive cycles over time. Continuity is perpetuated through memory carriers, slowly changing variables and features that retain or make available information on how different situations have been dealt with before. Ecological memory carriers comprise memory banks, spatial connections and mobile link species. These can be supported by social memory carriers, represented by collectively created social features like habits, oral tradition, rules-in-use and artifacts, as well as media and external sources. Loss or lack of memory can be diagnoses by the absence or disconnect between memory carriers, as will be illustrated by several typical situations. Drawing on a set of example situations, we present an outline for a look-up table approach that connects ecological memory carriers to the social memory carriers that support them and use these connections to set diagnoses and indicate potential remedies. The inclusion of memory carriers in planning and management considerations may facilitate preservation of feedbacks and disturbance regimes as well as species and habitats, and the cultural values and meanings that go with them.     

一种优化的胡杨高效多重 (12重) SSR体系

微卫星多重PCR方法是一种非常经济并且高通量的基因分型技术。本研究在耐干旱、盐碱的胡杨（Populus euphratica）中开发出一套荧光标记的12重微卫星工作体系。该体系包含12条表达序列标签微卫星（EST SSR）引物,其中3条设计于NCBI,另外9条设计于二代的转录组序列。利用该多重微卫星体系可在单一的PCR反应体系中成功扩增出12条表达序列标签的微卫星短序列片段,并在胡杨的3个自然居群96个个体中对该体系进行了验证,结果显示该体系具有很高的稳定性及多样性。同时,在杨属的5个派7个种中对其通用性进行了检验,显示这些引物具有很高的通用性,成功扩增率为79%。本研究中提供的12重多重PCR结合本实验已经公开发表的2个8重体系对揭示胡杨及其他杨树的进化历史具有重要的作用。最后,本研究认为引物的选择,扩增效率,哑等位基因的检测是多重体系开发过程中最为关键的步骤。     

High Throughput SSR Multiplex Kits (12 plex) for Euphrates’ Poplar,Populus euphratica (Salicaceae)

Multiplex PCR of microsatellite is a cost effective and high throughput technique of genotyping. We developed a new 12 plex PCR kit for Populus euphratica, the only tree species in desert area ranging from Western China to Mediterranean coast. Three primers were designed for the expressed sequence tags (ESTs) sequences from the NCBI database and the other nine primers were designed based on the EST sequences of Peuphratica obtained by Solexa. The multiplex kit was tested by 96 samples from three natural populations. The results showed sufficient amplification stability and high polymorphism. All the 12 loci used in this kit showed a high transferability (79%) in other seven species from five sections of the genus. The new 12 plex kit combined with the two eight multiplex kits we had developed in previous studies, should be useful to reveal the genetic mechanism and evolution history of the Peuphratica and related species. During the research, we found that primers selection, amplification efficiency, null allele detection are the essential parts of the multiplex kit development.     

一种高效的哺乳动物粪便DNA提取通用方法

以粪便为材料提取动物DNA进行动物保护遗传学和分子生态学研究的关键是能否提取到高质量的粪便DNA.然而提取方法通用性不好和产物质量不高等问题阻碍了粪便DNA分析技术的推广.本文介绍的改进型十六烷基三甲基溴化铵提取法可广泛适用于各食性哺乳动物粪便DNA提取,在11种不同食性动物的粪便DNA提取实验中验证了它的可靠性和通用性.本方法成本低廉(3元/样),用实验室常规试剂即可完成粪便DNA提取,其产物纯度高于专用试剂盒QIAamp DNA Stool Kit,在拥有超过专业试剂盒提取效果的同时尽可能的降低了实验成本,有利于粪便DNA技术的推广.     

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.)

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref‐sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit‐SMsa1 and kit‐SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.     

A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung

Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412-bp fragment of the mitochondrial cytochrome- b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 'unknown' fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future.     

Optical immunoassay for snake venom detection

A sensitive and specific optical immunoassay (OIA) has been developed for snake venom detection. The assay is based on the principle of detection of physical changes in thickness of molecular thin film resulting from specific binding events on an optical silicon chip (SILAS-I, ThermoBioStar, Colorado, USA). The reflection of white light through the thin film results in destructive interference of a particular wavelength of the light from gold to purple-blue depending on the thickness of the thin film formed or the amount of venom in the test sample. A prototype test kit for the simultaneous identification of species and semi-quantitative detection of venoms from four medically important snakes of South Vietnam (Trimeresurus albolabris, Calloselasma rhodostoma, Naja kaouthia and Ophiophagus hannah) has been developed. The kit can detect venom analytes in blood, plasma, urine, wound exudates, blister fluid or tissue homogenates. The efficacy of the test kit in snakebite diagnosis has been demonstrated in experimental envenomations and sample analytes taken from snakebite victims in South Vietnam. This rapid snake venom detection kit based on OIA technique is potentially applicable in the clinics as well as in the field.     

Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq

The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.     

Statistical Computations Underlying the Dynamics of Memory Updating

Psychophysical and neurophysiological studies have suggested that memory is not simply a carbon copy of our experience: Memories are modified or new memories are formed depending on the dynamic structure of our experience, and specifically, on how gradually or abruptly the world changes. We present a statistical theory of memory formation in a dynamic environment, based on a nonparametric generalization of the switching Kalman filter. We show that this theory can qualitatively account for several psychophysical and neural phenomena, and present results of a new visual memory experiment aimed at testing the theory directly. Our experimental findings suggest that humans can use temporal discontinuities in the structure of the environment to determine when to form new memory traces. The statistical perspective we offer provides a coherent account of the conditions under which new experience is integrated into an old memory versus forming a new memory, and shows that memory formation depends on inferences about the underlying structure of our experience.     

A rapid and sensitive screening kit for the detection of anti-Toxocara larval ES antibodies

We developed a new screening kit for the detection of anti-Toxocara larval excretory-secretory antibodies. The test can be performed within 3 min for one sample and does not require a high-priced supplemental instrument. Moreover, results are easily and directly observed. Using this test kit, 22 sera taken from healthy subjects were negative for anti-Toxocara larval excretory-secretory antibodies at the serum dilution of 1:20. Of 14 proven cases of parasitic infections other than toxocariasis, one (gnathostomiasis) showed a positive result, but the others were negative. In serologically diagnosed toxocariasis, the test kit showed good correlation with ELISA, immunoblot and double gel diffusion tests. We designated this test kit as ToxocaraCHEK.     

Pathogen interactions, population cycles, and phase shifts

Interspecific pathogen interactions can profoundly affect pathogen population dynamics and the efficacy of control strategies. However, many pathogens exhibit cyclic abundance patterns (e.g., seasonality), and temporal asynchrony between interacting pathogens could reduce the impact of those interactions. Here we use an extension of our previously published model to investigate the effects of cycles on pathogen interaction. We demonstrate that host immune memory can maintain the impact of an interaction, even when the effector pathogen abundance is low or the pathogen is absent. Paradoxically, immune memory can result in pathogens interacting more strongly when temporally out of phase. We find that interactions between species can result in changes to the temporal pattern of the affected species. We further demonstrate that this may be observed in a natural host-pathogen system. Given the continuing debate regarding the relevance of pathogen interactions in natural systems and increasing concern about treatment strategies for coinfections, both the discovery of a shift in cycle in empirical data and the mechanism by which we identified it are important. Finally, because the model structure used here is analogous to models of a simple predator-prey system, we also consider the consequences of these findings in the context of that system.     

Alien Invasions, Ecological Restoration in Cities and the Loss of Ecological Memory

After a community or ecosystem is lost, it may leave behind an ecological memory. The site history, soil properties, spores, seeds, stem fragments, mycorrhizae, species, populations, and other remnants may influence the composition of the replacement community or ecosystem to varying degrees. The remnants may also hold the site to a trajectory that has implications for ecological restoration. This is true in urban situations in particular where repeated disturbance has masked the history of the site. The ecological memory remaining may be insufficient for a site to heal itself; restoration activities are required to direct the future of the site. Conversely, in light of climate change and other rapidly changing environments, the existing ecological memory may be poorly suited to the new conditions and restoration projects need to create new and perhaps novel ecosystems. The loss of ecological memory facilitates the establishment of foreign invasive species. These invasives may eventually create a new stability domain with its own ecological memory and degree of resilience. To be successful, invasive species control must address both internal within patch memory of invasives and external between patch memory. Further research is necessary to document and conserve ecological memory for ecological restoration in response to future ecosystem changes.     

以川陕哲罗鲑为目标物种的水样环境DNA分析流程的优化

水样环境DNA分析包括水样采集、DNA提取和分析等流程,已成为监测濒危水生生物种群分布调查的重要手段.为减少在监测目标物种尤其濒危物种中的不确定性,对水环境DNA分析流程的优化至关重要.本研究以川陕哲罗鲑为目标物种,采用滤膜法采集养殖池中的水样,设计了 250 mL、500 mL、1 L和2 L等4种水样采集量,分别采用 PoweWater DNA Isolation kit和DNeasy Tissue and Blood DNA extraction kit 提取水样环境DNA(eDNA),使用物种mtDNA D_loop区特异性引物进行PCR扩增,通过研究滤膜法、水样采集量和水样DNA提取方法对水样eDNA中目标基因检出率的影响,探索适宜的eDNA分析操作方案.结果表明: 使用DNeasy Tissue and Blood DNA extraction kit提取的水样DNA中目的基因的检出率为100%,效果明显优于PoweWater DNA Isolation kit(目标基因的检出率为0);目标基因扩增条带的亮度随水样采样量的增加而增加,其中2 L水样目标基因的扩增效果较理想;序列比对结果显示,本试验从水样DNA中成功扩增得到了川陕哲罗鲑mtDNA Dloop区部分序列.表明DNA提取方法和水样采集量对目标物种的检出率有显著的影响,滤膜法、2 L水样采集量、DNeasy Tissue and Blood DNA extraction kit更适宜进行水样的DNA分析,mtDNA D-loop区可作为川陕哲罗鲑识别的特异性分子标记.     

Where's my dinner? Adult neurogenesis in free‐living food‐storing rodents

Postnatal hippocampal neurogenesis in wild mammals may play an essential role in spatial memory. We compared two species that differ in their reliance on memory to locate stored food. Yellow-pine chipmunks use a single cache to store winter food; eastern gray squirrels use multiple storage sites. Gray squirrels had three times the density of proliferating cells in the dentate gyrus (determined by Ki-67 immunostaining) than that found in chipmunks, but similar density of young neurons (determined by doublecortin immunostaining). Three explanations may account for these results. First, the larger population of young cells in squirrels may increase the flexibility of the spatial memory system by providing a larger pool of cells from which new neurons can be recruited. Second, squirrels may have a more rapid cell turnover rate. Third, many young cells in the squirrels may mature into glia rather than neurons. The densities of young neurons were higher in juveniles than in adults of both species. The relationship between adult age and cell density was more complex than that has been found in captive populations. In adult squirrels, the density of proliferating cells decreased exponentially with age, whereas in adult chipmunks the density of young neurons decreased exponentially with age.