Validating gut flushing as a non-lethal sampling technique for diet monitoring in agastric fish.

The acquisition of tissue samples from wild populations is a constant challenge in conservation biology, especially for endangered species and protected species where nonlethal sampling is the only option. Whole blood has been suggested as a nonlethal sample type that contains a high percentage of bodywide and genomewide transcripts and therefore can be used to assess the transcriptional status of an individual, and to infer a high percentage of the genome. However, only limited quantities of blood can be nonlethally sampled from small species and it is not known if enough genetic material is contained in only a few drops of blood, which represents the upper limit of sample collection for some small species. In this study, we developed a nonlethal sampling method, the laboratory protocols and a bioinformatic pipeline to sequence and assemble the whole blood transcriptome, using Illumina RNA-Seq, from wild greater mouse-eared bats (Myotis myotis). For optimal results, both ribosomal and globin RNAs must be removed before library construction. Treatment of DNase is recommended but not required enabling the use of smaller amounts of starting RNA. A large proportion of protein-coding genes (61%) in the genome were expressed in the blood transcriptome, comparable to brain (65%), kidney (63%) and liver (58%) transcriptomes, and up to 99% of the mitogenome (excluding D-loop) was recovered in the RNA-Seq data. In conclusion, this nonlethal blood sampling method provides an opportunity for a genomewide transcriptomic study of small, endangered or critically protected species, without sacrificing any individuals.

Detection accuracy of Renibacterium salmoninarum in Chinook salmon, Oncorhynchus tshawytscha (Walbaum) from non-lethally collected samples: Effects of exposure route and disease severity

Nonlethal sampling techniques have previously been evaluated for detection of a variety of viral salmonid pathogens. However, many of these studies have used molecular assays in lieu of widely accepted cell culture techniques to evaluate the sampled tissues. Samples were collected from female steelhead Oncorhynchus mykiss broodstock using three potential nonlethal sampling methods (mucus/skin scrape, pectoral fin clip, and gill tissue biopsy) and evaluated for the presence of infectious hematopoietic necrosis virus (IHNV) via cell culture techniques. The results were compared with those from samples collected using a standard lethal sampling method (pooled anterior kidney and spleen tissues) applied to the same fish. Of the three nonlethal sampling techniques that were evaluated, fin clipping was the easiest and least invasive method. Furthermore, fin tissue was as sensitive as or more sensitive than kidney/spleen tissue for detecting IHNV in this population of fish. However, with the exception of gill tissue, the nonlethal samples did not appear to be appropriate surrogates for lethally collected tissues with regard to identifying an active infection in a particular fish. Nevertheless, nonlethal sampling coupled with cell culture appears to be suitable for helping to define the IHNV status of a steelhead population. Received July 27, 2016; accepted December 11, 2016.

As top consumers and generalist predators, spiders are ideal organisms to study food webs and complex ecological functions using stable isotopes. Most researchers use whole‐body samples to analyze stable isotope ratios in spiders. Spiders can regrow lost legs and produce multiple molts during a life cycle, and nonlethal sampling utilizing legs and molts may provide a useful alternative to whole‐body sampling especially in larger bodied or threatened species. Furthermore, removing spider abdomens and thus leftover prey in the gut contents may provide a more accurate isotopic value. We tested the hypothesis that the δ15N, δ13C, or δ2H isotopic values in spider legs are reliable proxies for spider prosomas, abdomens, or whole bodies. We used laboratory‐reared large‐bodied spiders (Pterinochilus murinus) and field‐collected Lycosidae to compare lethal and nonlethal tissue isotopic values. We found that nonlethal samples of spider legs and molts are acceptable alternatives to lethal whole‐body samples to determine δ13C and δ15N stable isotope signatures. Nonlethal samples are not suitable proxies for whole‐body samples to determine δ2H isotopic values. Using nonlethal leg or molts samples in stable isotope investigations of spiders will allow researchers to promote conservation efforts and study threatened species while ensuring accurate and repeatable results.

Monitoring of contaminants in fish generally involves lethal sampling, but public scrutiny and increased pressure on fisheries have driven the need to develop nonlethal sampling methods. We examined the ability of adipose, anal, and caudal fin tissues to serve as nonlethal surrogates for lethal muscle tissue samples in the analysis of metals (elements). First, we evaluated the use of biopsies by examining relationships between concentrations of 39 elements in low-volume dorsal muscle biopsies and high-volume muscle samples from Arctic Grayling Thymallus arcticus and Bull Trout Salvelinus confluentus collected in the South Nahanni River watershed, Northwest Territories, Canada. Low-volume dorsal biopsy samples in this study served to most closely model the concentrations of elements found in high-volume dorsal samples; caudal and anal fins were more representative of high-volume dorsal samples than were adipose fins. Regressions between high- and low-volume dorsal muscle samples were significant for 12 elements/species, with Cs, Rb, and Tl having the strongest relationships in both species. Regression analyses comparing low-volume muscle samples and fin samples revealed variation between Arctic Grayling and Bull Trout, but Co, Hg, and Tl concentrations among samples were strongly related for both species. Addition of fish length or age as a covariate did not greatly improve the predictive power of calculated regressions. For future monitoring, selection of a nonlethal sampling strategy (e.g., use of dorsal biopsy or adipose fin samples) will require consideration of the element of interest, the primary route of exposure, interaction with other elements, and the basic biology and ecology of the fish species. Ideally, nonlethal sampling tools can be further developed for the two species to promote inclusion of community partners; these tools offer sustainable, long-term approaches for monitoring sensitive fish populations in northern Canadian habitats. Received February 29, 2016; accepted September 9, 2016Published online December 20, 2016

The Wanshan mercury mining area (WMMA) in Guizhou Province, China, has been identified as a region at high ecological risk owing to heavy metal contamination. This study employed non-lethal sampling methods, using the phalanges of Pelophylax nigromaculatus in the WMMA as analytical material. Ten heavy metal (metalloid) elements were selected for analysis, including Hg, Cr, Mn, Ni, Cu, Zn, Cd, Pb, As, and Se. These elements were measured using inductively coupled plasma mass spectrometry (ICP-MS), while Hg was determined using atomic fluorescence spectrometry (AFS). The bioaccumulation characteristics of heavy metals in P. nigromaculatus were analyzed through the bioaccumulation factor (BAF), and the extent of heavy metal pollution along with potential ecological risk was assessed using the potential ecological risk index (RI). The scaled mass index (SMI) was utilized to evaluate the body condition of P. nigromaculatus. Additionally, a combination of Spearman correlation analysis and non-negative matrix factorization (NMF) was employed to identify the sources and contributions of heavy metal elements. The results indicated that the distribution characteristics of heavy metal elements in the phalanges of P. nigromaculatus varied. Except for Pb, the concentrations of essential trace elements were significantly higher than those of non-essential elements. The bioaccumulation potential of P. nigromaculatus for heavy metal elements in the soil was found to be low, whereas the exposure risk from heavy metals in the water was high. The presence of heavy metal elements poses a significant potential ecological risk to P. nigromaculatus, with Hg, Cd, and As identified as the primary contributors to this risk. The environmental sources of heavy metals were identified as follows: Hg, Pb, Zn, and Se mainly originated from mining pollution sources; As and Cd primarily came from atmospheric pollution sources; Cr and Cu were mainly from natural activities sources; and Mn and Ni had multiple composite sources. Although heavy metal pollution negatively impacted the physical condition of P. nigromaculatus, no significant differences in SMI were observed across different regions. We believe that this is primarily attributable to the high levels of Se, which effectively mitigate the toxicity of heavy metals. The study demonstrated that using frog phalanges for heavy metal bioaccumulation analysis and SMI for physical condition assessment are simple, safe, and non-lethal methods, which can serve as useful indicators of environmental pollution and help trace the sources of environmental pollutants.

Pediatric submersion victims often require admission. We wanted to identify a cohort of children at low risk for submersion-related injury who can be safely discharged from the emergency department (ED) after a period of observation. This was a single-center retrospective derivation/validation cross-sectional study of children (0-18 years) who presented postsubmersion to a tertiary care, children's hospital ED from 2008 to 2015. We reviewed demographics, comorbidities, and prehospital and ED course. Primary outcome was safe discharge at 8 hours postsubmersion: normal mentation and vital signs. To identify potential scoring factors, any p-value of ≤0.25 was included in binary logistic regression; p-values < 0.05 were included in the final score. In the validation data set, we generated a one-point scoring system for each normal ED item. Receiver operating characteristic curves with area under the curve (AUC) were generated to test sensitivity and specificity. The derivation data set consisted of 356 patients and validation data set of 89 patients. Five factors generated a safe discharge score at 8 hours: normal ED mentation, normal ED respiratory rate, absence of ED dyspnea, absence of need for airway support (bag-valve mask ventilation, intubation, and CPAP), absence of ED systolic hypotension (maximum score = 5; range = 0-5). Only the 80 patients with values for all five factors were included in the sensitivity/specificity analysis. This resulted in an AUC of 0.81 (95% confidence interval [CI] = 0.71-0.91; p < 0.001). Based on the sensitivity/specificity analysis, the discriminative ability peaks at 75% with a score of ≥3.5. A score of 4 or higher in the ED would suggest a safe discharge at 8 hours (sensitivity = 88.2% [95% CI = 72.5%-96.7%]; specificity = 62.9% [95% CI = 44.9%-78.5%]; positive predictive value = 69.8% [95% CI = 53.9%-82.8%]; negative predictive value = 84.6% [95% CI = 65.1%-95.6%]). A risk score can identify children at low risk for submersion-related injury who can be safely discharged from the ED after observation.

Nonlethal sampling methods for diagnosis of Streptococcus agalactiae infection in Nile tilapia, Oreochromis niloticus (L.)

AEI Aquaculture Environment Interactions Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AEI 4:251-261 (2013) - DOI: https://doi.org/10.3354/aei00087 Improved method for triacylglycerol-derived fatty acid profiling by various non-lethal and lethal sampling techniques in Atlantic salmon Rolf Erik Olsen1,*, Geir Lasse Taranger2, Terje Svåsand2, Ove Tommy Skilbrei2 1Institute of Marine Research, Matre Research Station, 5984 Matredal, Norway 2Institute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway *Email: rolf.erik.olsen@imr.no ABSTRACT: The current paper compares the use of adipose fin and muscle biopsies as non-lethal sampling procedures, and the suitability of triacylglycerol (TAG) versus total lipid (TOT) fatty acid composition (fatty acid methyl esters, FAME) to estimate dietary history in farmed and wild maturing Atlantic salmon Salmo salar. TAG FAME gives best resemblance to dietary history. Fatty acid composition differs between tissues, and sample series should preferably be from 1 tissue only. TOT FAME supplies informative data on dietary history in fatty tissues, but differs from TAG FAME when total lipid levels are low. The reason is a larger contribution of phospholipid FAME. In wild maturing salmon, lipid content in adipose fin and muscle is low. TAG provides good data on dietary history, while TOT is less informative. Escapees are well identified analysing 18:2n-6 and 18:3n-3 fatty acids, which are high in commercial feed. For estimating a wider range of dietary history after escape (switch to wild prey), or feed preferences in wild fish, other fatty acids have to be taken into account. This requires the use of multivariate methods, like principal component analysis. Adipose fin and muscle biopsies are good alternatives for non-lethal sampling using the TAG method. The minimum amounts of samples to be used are proposed to be 0.5 to 1 g of adipose fin and 60 to 100 mg of muscle. The method of storage (liquid nitrogen/dry ice) does not affect fatty acid compositions. Other tissues can also be used for fatty acid profiling. Snout is a good alternative, being lipid-rich even in wild fish with low muscle lipid. KEY WORDS: Fatty acid composition · Fatty acid profiling · Adipose fin · Triacylglycerol · Muscle · Snout · Escaped farmed salmon Full text in pdf format PreviousNextCite this article as: Olsen RE, Taranger GL, Svåsand T, Skilbrei OT (2013) Improved method for triacylglycerol-derived fatty acid profiling by various non-lethal and lethal sampling techniques in Atlantic salmon. Aquacult Environ Interact 4:251-261. https://doi.org/10.3354/aei00087 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AEI Vol. 4, No. 3. Online publication date: November 14, 2013 Print ISSN: 1869-215X; Online ISSN: 1869-7534 Copyright © 2013 Inter-Research.

Traditional methodologies to identify fish pathogens require euthanasia before the collection of tissue samples. While these methods are standardized and proven, there are instances where nonlethal alternatives would be preferred. Despite the need to develop nonlethal sampling techniques, few publications have focused on them and even fewer have used these approaches to identify viruses from infections occurring in wild fish populations. In this study, we compared the ability of nonlethal sampling techniques with traditional methods for the detection of Largemouth Bass virus (LMBV) from a wild population of Largemouth Bass Micropterus salmoides from the upper Mississippi River. Largemouth bass virus was isolated from 30% of the Largemouth Bass sampled using traditional methods where tissue samples were inoculated on Bluegill fry (BF-2) cells. Furthermore, when using tissue cell culture to isolate LMBV, there was no significant difference observed in the overall proportion that was positive between the mucus samples and the kidney and spleen samples. Mucus swabs analyzed with molecular methods (conventional PCR and quantitative PCR) were more sensitive than traditional tissue cell culture-based methods as they detected LMBV from >70% of the samples; limitations to these methods (i.e., carryover contamination) were also identified. The results of this study suggest that nonlethal sampling may be a useful option for detecting LMBV from fish populations.

The influence of gait stance on pedestrian lower limb injury risk

Koi herpesvirus (KHV) causes an economically important, highly infectious disease in common carp and koi, Cyprinus carpio L. Since the occurrence of mass mortalities worldwide, highly specific and sensitive molecular diagnostic methods have been developed for KHV detection. The sensitivity and reliability of these assays have essentially focused at the detection of low viral DNA copy numbers during latent or persistent infections. However, the efficacy of these assays has not been investigated with regard to low-level viraemia during acute infection stages. This study was conducted to compare the sensitivity of seven different polymerase chain reaction (PCR) assays to detect KHV during the first hours and days post-infection (hpi; dpi), using lethal and non-lethal sampling methods. The results highlight the limitations of the assays for detecting virus during the first 4dpi despite rapid mortality in experimentally infected carp. False-negative results were associated with time post-infection and the tissue sampled. Non-lethal sampling appears effective for KHV screening, with efficient detection in mucus samples obtained from external swabs during this early infection period (<5dpi), while biopsies from gills and kidney were negative using the same PCR assays. Non-lethal sampling may improve the reliability of KHV detection in subclinical, acutely infected carp.

The dose-response relationship of alcohol and injury and the effects of country-level detrimental drinking pattern (DDP) and alcohol control policy on this relationship are examined for specific causes of injury. The dose-response risk of injury is analyzed on 18,627 injured patients in 22 countries included in the International Collaborative Alcohol and Injury Study, using case-crossover analysis by cause of injury (traffic, violence, falls, other), DDP, and the International Alcohol Policy and Injury Index. Risk of all injury was higher at all volume levels in higher DDP countries compared to lower DDP countries and for each cause of injury. Risk of injury from traffic was significantly greater in higher DDP than lower DDP countries at 3.1 to 6 drinks (odds ratio (OR)=2.64, confidence interval (CI)=1.17 to 5.97) and at ≤3 drinks for falls (OR=2.51, CI=1.52 to 4.16) and injuries from other causes (OR=1.72, CI=1.10 to 2.69). Countries with higher restrictive alcohol policy were at a lower risk of injury at lower levels of consumption (≤3 drinks) for all injuries (OR=0.72, CI=0.56 to 0.92) and for injuries from other causes (OR=0.46, CI=0.29 to 0.73) and at a lower risk of traffic injuries at higher levels of consumption (≥10 drinks). At higher levels of consumption (≥10 drinks), countries with higher alcohol policy restrictiveness were at greater risk of all injuries (OR=2.03, CI=1.29 to 3.20) and those from violence (OR=9.02, CI=3.00 to 27.13) and falls (OR=4.29, CI=1.86 to 9.91). Countries with high DDP are at higher risk of injury from most causes at a given level of consumption, while countries with low restrictiveness of alcohol policy are at higher risk of injury at lower levels of consumption and at higher risk of traffic injuries at high levels of consumption. These findings underscore the importance of aggregate-level factors which need to be considered in developing effective intervention and prevention strategies for reducing alcohol-related injury.

The objective of this study was to determine the effects of teaching the scooping-resheathing method on the incidence of needle-stick injuries in medical students. Before starting their first clerkship, 81 medical students were given a 15-min lecture on the high incidence and dangers of needle-stick injuries and a demonstration of the scooping-resheathing method. The number of needle-stick injuries that occurred during the 3-month clerkship was compared with the number reported by 86 medical students who had completed their first clerkship 1 year previously and had not been given such instruction. Compared with controls, the study group had a 3.8-fold lower risk of needle-stick injury (95% confidence interval, 2.0-7.4, P < 0.0001) and a 8.3-fold lower risk of multiple needle-stick injuries (95% confidence interval, 2.0-35.0, P < 0.001). Those in the study group, who consistently used the scooping method had a much lower risk of injury than those who did not (1 of 36 [2.8%] vs. 8 of 45 [17.4%], P = 0.039). We conclude that a lecture recommending the scooping-resheathing method is effective in reducing the risk of needle-stick injuries in medical students during their first rotation. Because this is the first time that an intervention not requiring change in equipment has been successful, further studies are warranted to substantiate our findings and for extrapolation to other medical personnel in other cultural settings.

Noninvasive sampling methods for studying intestinal microbiomes are widely applied in studies of endangered species and in those conducting temporal monitoring during manipulative experiments. Although existing studies show that noninvasive sampling methods among different taxa vary in their accuracy, no studies have yet been published comparing nonlethal sampling methods in adult amphibians. In this study, we compare microbiomes from two noninvasive sample types (faeces and cloacal swabs) to that of the large intestine in adult cane toads, Rhinella marina. We use 16S rRNA gene sequencing to investigate how microbial communities change along the digestive tract and which nonlethal sampling method better represents large intestinal microbiota. We found that cane toads' intestinal microbiota was dominated by Bacteroidetes, Proteobacteria and Firmicutes and, interestingly, we also saw a high proportion of Fusobacteria, which has previously been associated with marine species and changes in frog immunity. The large and small intestine of cane toads had a similar microbial composition, but the large intestine showed higher diversity. Our results indicate that cloacal swabs were more similar to large intestine samples than were faecal samples, and small intestine samples were significantly different from both nonlethal sample types. Our study provides valuable information for future investigations of the cane toad gut microbiome and validates the use of cloacal swabs as a nonlethal method to study changes in the large intestine microbiome. These data provide insights for future studies requiring nonlethal sampling of amphibian gut microbiota.