Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets

Non-invasive wildlife research using DNA from feces has become increasingly popular. Recent studies have attempted to solve problems associated with recovering DNA from feces by investigating the influence of factors such as season, diet, collection method, preservation method, extraction protocol, and time. To our knowledge, studies of this nature have not addressed DNA degradation over time in wet environments, and have not been performed on fecal pellets of ungulates. Therefore, our objective was to determine the length of time a fecal pellet from a Sitka black-tailed deer (Odocoileus hemionus sitkensis) could remain in the field in a temperate rainforest environment before the DNA became too degraded for individual identification. Pellets were extracted from the rectum of recently killed deer and placed in an environment protected from rainfall and in an environment exposed to rainfall. Pellets from each treatment group were sampled at intervals of 2, 7, 14, 21, and 28 days after deer harvest. DNA was extracted from sampled pellets and individual samples were genotyped using microsatellite markers. Amplification failure and errors (dropout and false alleles) were recorded to determine extent of DNA degradation. Eighty percent of samples in the protected environment and 22% of samples in the exposed environment were successfully genotyped during the 28-day experiment. With no samples being successfully genotyped in the exposed environment after 7 days, our study showed that rainfall significantly increases degradation rates of DNA from ungulate pellets.     

Estimating abundance of Sitka black-tailed deer using DNA from fecal pellets

Densely vegetated environments have hindered collection of basic population parameters on forest-dwelling ungulates. Our objective was to develop a mark–recapture technique that used DNA from fecal pellets to overcome constraints associated with estimating abundance of ungulates in landscapes where direct observation is difficult. We tested our technique on Sitka black-tailed deer (Odocoileus hemionus sitkensis) in the temperate coastal rainforest of Southeast Alaska. During 2006–2008, we sampled fecal pellets of deer along trail transects in 3 intensively logged watersheds on Prince of Wales Island, Alaska. We extracted DNA from the surface of fecal pellets and used microsatellite markers to identify individual deer. With genotypes of individual deer, we estimated abundance of deer with moderate precision (±20%) using mark–recapture models. Combining all study sites, we identified a 30% (SE = 5.1%) decline in abundance during our 3-year study, which we attributed to 3 consecutive severe winters. We determined that deer densities in managed land logged >30 years ago (7 deer/km², SE = 1.3) supported fewer deer compared to both managed land logged <30 years ago (10 deer/km², SE = 1.5) and unmanaged land (12 deer/km², SE = 1.4). Our study provides the first estimates of abundance (based on individually identified deer) for Sitka black-tailed deer and the first estimates of abundance of an unenclosed ungulate population using DNA from fecal pellets. Our tool enables managers to accurately and precisely estimate the abundance of deer in densely vegetated habitats using a non-invasive approach. © 2011 The Wildlife Society.     

Non-invasive genotyping of transgenic animals using fecal DNA

For genotyping of transgenic animals, many IACUC guidelines recommend the use of fecal DNA when possible because this approach is non-invasive. Existing methods for extracting fecal DNA may be costly or involve the use of toxic organic solvents. Furthermore, feces contain an abundance of PCR inhibitors that may hinder DNA amplification when they are co-purified with fecal DNA. Here the authors describe a cost-effective, non-toxic method for genotyping transgenic animals by using the reagent AquaStool to extract fecal DNA and remove PCR inhibitors. Genotyping results obtained from fecal DNA samples extracted using AquaStool were reliably accurate when compared with results obtained from tail DNA samples. Because it is non-invasive, the authors believe that use of this method for genotyping transgenic animals using fecal DNA samples may improve animal welfare.     

Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses

In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3 years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3 years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458 pg/μl, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25 pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.     

Quantification and genetic profiling of DNA isolated from free-floating feces of the North Atlantic right whale (Eubalaena glacialis)

Fecal analysis from the highly endangered North Atlantic right whale provides valuable information about health and reproductive parameters of individual animals. Genetically profiling the feces facilitates this connection when the sample originator is unknown. Although genetic analysis of feces collected in terrestrial systems has become well established, genetic studies of cetacean DNA are rare. Here, the use of free‐floating feces as a source of right whale DNA and the reliability of the genotypes produced are examined. On average, fecal extracts yielded 25 ng of DNA/mg dry weight, but less than 1% was right whale DNA. Although all samples were amplified using genus‐specific mitochondrial control region primers, the quantity of right whale DNA present was over estimated when compared to amplifications using nuclear primers. No correlation was found between the quantity of right whale DNA recovered and the duration the sample sat in the water. Composite microsatellite profiles from multiple amplifications of 28 fecal samples of known origin were consistent with profiles of the same individuals obtained from skin biopsies, however, the rate of allelic dropout varied depending on the amount of right whale DNA added. A screening and genotyping protocol for reliable genetic profiling based on fecal DNA quantification is presented.     

Morphometric analyses of non-invasive fecal samples of the Korean long-tailed goral (Naemorhedus caudatus) for species and age identification

The Korean long-tailed goral (Naemorhedus caudatus) is at risk of population decline due to habitat loss and fragmentation. Therefore, it is essential to ascertain its presence and/or the identity of individuals of the goral using non-invasive fecal samples for its conservation and management. In this study, we examined the morphology of fecal samples to provide the baseline data that can be used to distinguish species and age of goral individuals. We detected a significant difference in the length-to-width ratios of feces among the five ungulate species found in Korea. Also, we detected a significant difference in the length-to-width ratios of feces of gorals depending on the age groups. To assess the accuracy of species and age identification based on the fecal morphology, we conducted a series of blind comparison between the mean length-to-width ratios of the fecal pellets and the reference mean ratio values of the fecal pellets. Using 20 fecal pellets, our results showed 73%–86% probability of correct identification of the three species (gorals, goats, and roe deer), and 83%–90% probability of correct identification of >5 year-old goral individuals. The use of fecal morphometric analyses will be useful for the studies of Korean ungulate species, particularly the endangered gorals.     

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis)

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as “fecal hairs”. In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I—the highest-quality DNA, grade II—high-quality DNA, and grade III—poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis. Zoo Biol 28:49–58, 2009. © 2008 Wiley-Liss, Inc.     

Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method

Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition.     

Escherichia coli Survival in,and Release from,White-Tailed Deer Feces

White-tailed deer are an important reservoir for pathogens that can contribute a large portion of microbial pollution in fragmented agricultural and forest landscapes. The scarcity of experimental data on survival of microorganisms in and release from deer feces makes prediction of their fate and transport less reliable and development of efficient strategies for environment protection more difficult. The goal of this study was to estimate parameters for modeling Escherichia coli survival in and release from deer (Odocoileus virginianus) feces. Our objectives were as follows: (i) to measure survival of E. coli in deer pellets at different temperatures, (ii) to measure kinetics of E. coli release from deer pellets at different rainfall intensities, and (iii) to estimate parameters of models describing survival and release of microorganisms from deer feces. Laboratory experiments were conducted to study E. coli survival in deer pellets at three temperatures and to estimate parameters of Chick''s exponential model with temperature correction based on the Arrhenius equation. Kinetics of E. coli release from deer pellets were measured at two rainfall intensities and used to derive the parameters of Bradford-Schijven model of bacterial release. The results showed that parameters of the survival and release models obtained for E. coli in this study substantially differed from those obtained by using other source materials, e.g., feces of domestic animals and manures. This emphasizes the necessity of comprehensive studies of survival of naturally occurring populations of microorganisms in and release from wildlife animal feces in order to achieve better predictions of microbial fate and transport in fragmented agricultural and forest landscapes.     

Testing the reliability of noninvasive genetic sampling by comparing analyses of blood and fecal samples in Barbary macaques (Macaca sylvanus).

Genetic studies of wild animal populations are often hindered by difficulties in obtaining blood samples. Recent advances in molecular biology have allowed the use of noninvasive samples as sources of DNA (e.g., hair or feces), but such samples may provide low-quality DNA and prevent the determination of true genotypes in subsequent DNA analysis. We present a preliminary study aimed at assessing the reliability of using fecal samples for genotyping in Barbary macaques (Macaca sylvanus). The test was performed on samples of blood and feces from 11 captive animals, using three dinucleotide microsatellites. The CTAB DNA extraction method was found to be the most relevant for Barbary macaque feces, yielding successful amplification at all loci for 70% of PCRs. All the fecal samples tested gave correct genotypes at least once for each locus when referenced against blood-derived genotypes. An average of 18.3% of PCRs displayed spurious genotypes (false homozygous or false allele). The minimum theoretical probability required to obtain a 100% accurate genotype is 0.74, based on the criterion that a correct genotype is assessed only if it was observed at least twice. The observed probability of obtaining a correct genotype from three PCRs, based on our genotyping results, was greater (0.81 on average) than the minimum threshold. In conclusion, our comparison of blood and fecal samples showed that fecal sampling is a reliable tool for the further study of wild Barbary macaque populations.     

Single-copy nuclear DNA sequences obtained from noninvasively collected primate feces

Noninvasively collected primate feces have been shown to provide a useful source of mitochondrial DNA for sequencing and nuclear microsatellite DNA for size analysis. In this study, single-copy nuclear DNA sequences were obtained from noninvasively collected fecal samples of two species of wild tamarins, Saguinus fuscicollis and S. mystax, in the context of a project on the functional utility of color vision. Noninvasive genotyping of the X-linked opsin gene is important for future studies of selection and adaptation at this locus in a number of primate species. The wide range of techniques that can now be applied successfully to DNA extracted from feces introduces a broad spectrum of potential genetic studies that can be undertaken on primates, without the need for intrusive or invasive methods.     

原麝和林麝冬夏两季粪便真菌菌群多样性

肠道微生物群对宿主健康的影响取决于饮食环境和宿主等因素.本研究通过Illumina MiSeq平台对冬、夏两季林麝和原麝粪便真菌ITS1区段进行测序,探究原麝和林麝肠道真菌菌群结构特性及季节因素对其多样性的影响.结果 表明,林麝和原麝肠道真菌组成中子囊菌门Ascomycota均为优势菌门,它们的相对丰度随物种和季节因素...     

通过粪球形态快速鉴定马鹿性别的方法与分子鉴定

通过SRY 基因扩增,对76 份野生东北马鹿粪便DNA 进行了性别鉴定,其中雄性49 份,雌性27 份。观察发现粪球形态可分两类:子弹状、枣核状。子弹状呈短粗型,长宽比较小;枣核状呈细长型,长宽比较大。对76 份样品进行分类:子弹状51 份、枣核状25 份。并以样品长、宽平均值的比值(R)为指标快速聚类,并建立了判别方程。统计指出判别结果与实际性别吻合率79.71% ;形态分类与实际性别吻合率90.56% ,即子弹状为雄性,枣核状为雌性。结果提示今后的野外研究可直接利用粪球形态判定粪样性别,长宽比判别方程可作为辅助。     

Evaluation of two library-independent microbial source tracking methods to identify sources of fecal contamination in French estuaries

In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193'), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.     

使用粪堆数量估计日本奄美群岛上琉球兔的种群数量

为了测量奄美岛上濒危物种琉球兔 (Pentalagusfurnessi)的丰盛度 ,我们计数了森林中道路、溪流和森林地被物中的粪堆数量。计算用的模型包括新粪堆的数量及其年龄、总粪堆数量、每天产生的平均粪堆数 ,以及在森林地被物和溪边的粪堆数量的回归方程。估计在 1 993- 1 994年期间该岛上有 2 5 0 0 - 6 1 0 0只琉球兔 ,在 2 0 0 2 - 2 0 0 3年间有 2 0 0 0 - 4 80 0只。通过比较粪便丰盛度和在森林道路旁观察到的琉球兔数量 ,我们考察了该模型的有效性 ,最后将以前调查的数据应用于该模型以确定种群下降的程度     

Ectomycorrhizal ecology under primary succession on coastal sand dunes: interactions involving Pinus contorta, suilloid fungi and deer

Ectomycorrhizal fungi (EMF) are critical for pine establishment under primary succession. The species of EMF supporting primary successional pine seedlings on coastal sand dunes and mechanisms for their establishment were investigated. Fungi were identified from ectomycorrhizal roots using molecular techniques. Field seedlings were collected from forested and nonforested zones. Laboratory seedlings were grown in soils collected from the same zones, and in sterile soils inoculated with fresh and 1-yr-old dry deer fecal pellets. Suilloid fungi were frequently observed on all seedlings. A diverse group of fungi was available to seedlings in forested zones. A less diverse group of fungi was available to field seedlings in nonforested zones and all laboratory bioassay seedlings. Deer fecal inoculant yielded an average of two EMF per seedling. Both Suillus and Rhizopogon species dominated seedlings inoculated with fresh deer feces, but only Rhizopogon species dominated seedlings inoculated with 1-yr-old feces. Suilloid fungi are dispersed by deer, produce resistant spore banks and are the principle fungi supporting seedlings on the sand dunes.     

Escherichia coli O157:H7 in free-ranging deer in Nebraska.

In order to determine the prevalence and distribution of the human pathogen, Escherichia coli O157:H7, in free-ranging deer, hunters were asked to collect and submit fecal samples from deer harvested during a regular firearm season (14-22 November 1998). Prior to the season, 47% of the hunters with permits in the southeastern Nebraska (USA) study area indicated a willingness to participate in the study. Approximately 25% of successful hunters in the area submitted deer fecal samples. Escherichia coli O157:H7 was cultured from four (0.25%) of 1,608 total samples submitted. All of the fecal samples that were properly identified (1,426) and all that were positive for E. coli O157:H7 were from white-tailed deer (Odocoileus virginianus). We were unable to detect a statistically significant geographic distribution pattern of E. coli O157:H7. The presence of E. coli O157:H7 in the feces of free-ranging deer has implications not only for hunters, consumers of venison, and others in contact with deer or deer feces, but also for the development of strategies aimed at reducing and/or controlling this pathogen in water sources and domestic livestock.     

Low genotyping error rates in non-invasively collected samples from roe deer of the Bavarian Forest National Park

Genetic wildlife monitoring is increasingly carried out on the basis of non-invasively collected samples, whereby the most commonly used DNA sources are skin appendages (hairs, feathers) and faeces. In order to guide decisions regarding future adequate ways to monitor the roe deer (Capreolus capreolus) population of the Bavarian Forest National Park in Germany, we tested these two different types of DNA source materials to compare their suitability for genetic monitoring. We determined the haplotypes (d-loop) of 19 roe deer and genotyped each individual (tissue, hairs, faeces) across 12 microsatellite loci. The amount of missing and erroneous microsatellite alleles obtained from hair and faeces samples, respectively, was estimated based on comparisons with the corresponding tissue sample control. We observed no missing alleles in hair samples, but in fecal samples PCR failed in 30 out of 228 instances (19 individuals x 12 loci), corresponding to a frequency of missing alleles of 13.2% across all loci and individuals. In genotypes generated from hairs erroneous alleles were detected in 2 out of 228 instances (0.9%), while genotypes retrieved from fecal samples displayed erroneous alleles in 6 out of 198 remaining instances (3%). We conclude that both hair and fecal samples are generally well suited for genetic roe deer monitoring, but that fecal sample based analyses require a larger sample size to account for higher PCR failure rates.     

Distribution of Mycobacterium avium subspecies paratuberculosis in the lower Florida Keys

Johne's disease, a fatal and contagious gastrointestinal infection caused by Mycobacterium avium subsp. paratuberculosis (Map), was first diagnosed in an endangered Florida Key deer (Odocoileus virginianus clavium) in 1996 and later in six additional Key deer deaths from 1998 to 2004. We investigated the geographic distribution of Map in the Lower Florida Keys from February 2005 through May 2006 via collection of blood and fecal pellets from 51 live-captured deer, collection of 550 fecal samples from the ground, and by necropsies of 90 carcasses. Tissue and fecal samples also were submitted from 30 raccoons (Procyon lotor), three feral cats (Felis catus), an opossum (Didelphis virginiana), and a Lower Keys marsh rabbit (Sylvilagus palustris hefneri). Mycobacterium avium subsp. paratuberculosis was identified in 23 Key deer fecal samples collected from the ground, tissue samples from two clinically ill Key deer, and from the mesenteric lymph node of a raccoon. The results of this study indicate that Map persists in the Key deer population and environment at a low prevalence, but its distribution currently is limited to a relatively small geographic area within the range of Key deer.     

Feasibility and Recommendations for Swift Fox Fecal DNA Profiling

Abstract: Genetic profiling using fecal samples collected noninvasively in the wild can provide managers with an alternative to live-trapping. However, before embarking on a large-scale survey, feasibility of this methodology should be assessed for the focal species. Costs associated with fecal genotyping can be high because of the need for multiple amplifications to prevent and detect errors. Assessing the relative amount of target DNA before genotyping means samples can be eliminated where error rates will be high or amplification success will be low, thereby reducing costs. We collected fecal samples from an endangered population of swift fox (Vulpes velox) and employed target-DNA quantification and a screening protocol to assess sample quality before genetic profiling. Quantification was critical in identifying samples of low quality (68%, <0.2 ng/μL). Comparison of the amplification, from a subset of loci in 25 samples that did not meet the screening criteria, confirmed the effectiveness of this method. The protocol, however, used a considerable amount of DNA, and an assessment of the locus and sample variability allowed us to refine it for future population surveys. Although we did not use <50% of the samples we collected, the remaining samples provided 36 unique genotypes, which corresponded to approximately 70% of animals estimated to be present in the study area. Although obtaining fecal DNA from small carnivores is challenging, our protocol, including the quantification and qualification of DNA, the selection of markers, and the use of postgenotyping analyses, such as DROPOUT, CAPWIRE, and geographic information, provides a more cost-effective way to produce reliable results. The method we have developed is an informative approach that wildlife managers can use to conduct population surveys where the collection of feces is possible without the need for live-trapping.