Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 × 10⁻⁵ to 2.8 × 10⁻⁷ g (dry weight) of feces/liter and 6.8 × 10⁻⁷ g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.     

Microplate Subtractive Hybridization To Enrich for Bacteroidales Genetic Markers for Fecal Source Identification

The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).     

Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species

To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.     

Quantitative PCR for Genetic Markers of Human Fecal Pollution

Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 × 10⁶ copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.Waterborne diseases that originate from human fecal pollution remain a significant public health issue. As a result, a large number of methods have been developed to detect and quantify human fecal pollution (10, 12, 18, 20). The majority of these methods are based on real-time quantitative PCR (qPCR) assays designed to estimate the concentrations of 16S rRNA gene sequences from various subpopulations within the order Bacteroidales. This bacterial order constitutes a large proportion of the normal gut microbiota of most animals, including humans (3, 15, 27). Bacterial 16S rRNA genes are useful as markers because they have relatively low mutation rates (7) and are typically present in multiple operons, increasing template DNA levels available for detection (2, 11, 17, 29). While several studies have demonstrated the value of Bacteroides 16S rRNA gene-based qPCR assays, currently available assays cannot discriminate between several animal sources closely associated with humans, including cats, dogs, and/or swine (10, 12, 18, 20). Alternative qPCR assays targeting genes directly involved in host-specific interactions may be capable of increased discrimination of fecal pollution sources (22, 23) and are needed to complement existing qPCR-based approaches used to identify sources of human fecal pollution.A recent metagenomic survey of a human fecal bacterial community using genome fragment enrichment has led to the identification of hundreds of candidate human fecal bacterium-specific DNA sequences (23). PCR assays targeting two gene sequences encoding a hypothetical protein potentially involved in remodeling of bacterial surface polysaccharides and lipopolysaccharides (assay 19) and a putative RNA polymerase extracytoplasmic function sigma factor (assay 22) from Bacteroidales-like microorganisms exhibited a high level of specificity (100%) for human fecal material (23). However, it remained to be determined whether these reported chromosomal DNA sequences are abundant and uniform enough within human populations to be detected once diluted in the environment. On the basis of these considerations, the next steps toward the application of these gene sequences for water quality monitoring applications were to design qPCR assays for their detection and then to use these assays to evaluate the overall abundance and distribution of these sequences in human populations relative to those of rRNA gene sequences from different currently recognized fecal indicator bacterial groups.Here, we report the development of two qPCR assays for quantification of the human-specific DNA sequences targeted by previously reported PCR assays 19 and 22 (23). Method performance characteristics, including specificity, range of quantification (ROQ), limit of quantification, amplification efficiency, and analytical precision, were defined for each assay. An internal amplification control (IAC) was designed to monitor for the presence of inhibitors commonly associated with environmental sampling that can confound DNA target copy number estimations. Finally, the abundance of each DNA target in primary effluent wastewater samples representative of 20 geographically distinct human populations was measured by qPCR analysis. In addition, the abundances of these human-specific DNA genes in wastewater were compared to those of rRNA genes of Bacteroidales and enterococci, two general fecal indicator bacterial groups that have been widely used for water quality testing.     

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased,and cost-effective method for the isolation of high molecular weight(23 kb) metagenomic DNA(260/280 ratio 1.8) with a good yield(55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination(eubacterial: human genomic DNA marker genes = 2~(27.9):1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16 S r RNA gene sequencing using 454 FLX+.Moreover, 16 S r RNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richnessand does not show microbial diversity biases, which is further confirmed by q PCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality,amount, user-friendliness, and cost effectiveness for its suitability toward usage for cultureindependent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.     

Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats

Understanding the survival of fecal indicator bacteria (FIB) and microbial source-tracking (MST) markers is critical to developing pathogen fate and transport models. Although pathogen survival in water microcosms and manure-amended soils is well documented, little is known about their survival in intact cow pats deposited on pastures. We conducted a study to determine decay rates of fecal indicator bacteria (Escherichia coli and enterococci) and bovine-associated MST markers (CowM3, Rum-2-bac, and GenBac) in 18 freshly deposited cattle feces from three farms in northern Georgia. Samples were randomly assigned to shaded or unshaded treatment in order to determine the effects of sunlight, moisture, and temperature on decay rates. A general linear model (GLM) framework was used to determine decay rates. Shading significantly decreased the decay rate of the E. coli population (P < 0.0001), with a rate of −0.176 day⁻¹ for the shaded treatment and −0.297 day⁻¹ for the unshaded treatment. Shading had no significant effect on decay rates of enterococci, CowM3, Rum-2-bac, and GenBac (P > 0.05). In addition, E. coli populations showed a significant growth rate (0.881 day⁻¹) in the unshaded samples during the first 5 days after deposition. UV-B was the most important parameter explaining the decay rate of E. coli populations. A comparison of the decay behaviors among all markers indicated that enterococcus concentrations exhibit a better correlation with the MST markers than E. coli concentrations. Our results indicate that bovine-associated MST markers can survive in cow pats for at least 1 month after excretion, and although their decay dynamic differs from the decay dynamic of E. coli populations, they seem to be reliable markers to use in combination with enterococci to monitor fecal pollution from pasture lands.     

Host Distributions of Uncultivated Fecal Bacteroidales Bacteria Reveal Genetic Markers for Fecal Source Identification

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.     

Biotic Interactions and Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the Upper Mississippi River

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r², 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.     

A Widely Applicable Protocol for DNA Isolation from Fecal Samples

Feces are increasingly used as sources of DNA for genetic and ecological research. This paper describes a new method for isolation of DNA from animal feces. This method combines multiple purification steps, including pretreatment with ethanol and TE, an inhibitor-absorber made of starch, the CTAB method, the phenol-chloroform extraction method, and the guanidinium thiocyanate-silica method. The new method is efficient according to PCR results of 585 fecal samples from 23 species and costs much less than the commercial kits. The protocol can be tailored to the specific purpose of examining different diets of animals and can be performed with routine laboratory reagents.     

GPA: A Microbial Genetic Polymorphisms Assignments Tool in Metagenomic Analysis by Bayesian Estimation

Identifying antimicrobial resistant(AMR) bacteria in metagenomics samples is essential for public health and food safety. Next-generation sequencing(NGS) technology has provided a powerful tool in identifying the genetic variation and constructing the correlations between genotype and phenotype in humans and other species. However, for complex bacterial samples, there lacks a powerful bioinformatic tool to identify genetic polymorphisms or copy number variations(CNVs) for given genes. Here we provide a Bayesian framework for genotype estimation for mixtures of multiple bacteria, named as Genetic Polymorphisms Assignments(GPA). Simulation results showed that GPA has reduced the false discovery rate(FDR) and mean absolute error(MAE) in CNV and single nucleotide variant(SNV) identification. This framework was validated by whole-genome sequencing and Pool-seq data from Klebsiella pneumoniae with multiple bacteria mixture models, and showed the high accuracy in the allele fraction detections of CNVs and SNVs in AMR genes between two populations. The quantitative study on the changes of AMR genes fraction between two samples showed a good consistency with the AMR pattern observed in the individual strains. Also, the framework together with the genome annotation and population comparison tools has been integrated into an application, which could provide a complete solution for AMR gene identification and quantification in unculturable clinical samples. The GPA package is available at https://github.com/IID-DTH/GPA-package.     

Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.     

Quantification of the Flavonoid-Degrading Bacterium Eubacterium ramulus in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe

To investigate the occurrence of the flavonoid-degrading bacterium Eubacterium ramulus in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E.ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of E. ramulus in each individual tested in numbers from 4.4 × 10⁷ to 2.0 × 10⁹ cells/g of fecal dry mass. Six fecal E. ramulus isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA.     

Natural Hybridization between Diploid Crucian Carp Species and Genetic Independence of Triploid Crucian Carp Elucidated by DNA Markers

The population structure of genus Carassius in Lake Koyama, southeast Japan, was analyzed by genetic markers as microsatellite DNA, mtDNA RFLP and isozymes. Based on the ploidy level and morphological analysis, four Carassius groups were detected. The triploid group was identified as Ginbuna (C. langsdorfii). In the diploid group, Nagabuna (C. burugeri sp) and Gengoroubuna, (C. cuvieri) were identified. Remaining diploid individuals had morphological traits that were intermediate between Nagabuna and Gengoroubuna. These were considered as hybrids and their descendants. From the results of mtDNA RFLP and isozyme patterns, the triploid population was considered to be independent from the gene pools of diploid. The hybrids had the mtDNA haplotypes which were common to Gengoroubuna and Nagabuna populations. Based on the three microsatellite loci, Ginbuna was classified into six clonal lines. In the diploid population, substitution of the major alleles of Nagabuna and Gengoroubuna were found. The hybrids had alleles that were common in Nagabuna and Gengoroubuna. The values of the hybrid index (IH) which are ranged from 0.771 to 0.964 in Nagabuna, from 0.102 to 0.806 in the hybrids and from 0.068 to 0.157 in Gengoroubuna. The hybrid population was verified to be derived from crossbreeding between the Gengoroubuna and Nagabuna populations. Evidence of backcrossing in nature by microsatellite DNA markers was also obtained in the diploid populations.     

Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.The ability to discriminate between bovine and other sources of fecal contamination is necessary for the accurate evaluation of human health risks associated with agricultural runoff and focused water quality management to make waters safe for human use. Many methods have been proposed to identify bovine fecal pollution using a variety of different microbiology and molecular techniques. One of the most widely used approaches utilizes a PCR to amplify a gene target that is specifically found in a host population. Currently, there are numerous PCR-based assays for the detection and/or quantitative assessment of bovine fecal pollution available for microbial source-tracking (MST) applications (1, 5-7, 11, 14, 17, 18, 21, 23). These assays target genes ranging from mitochondrial DNA to ribosomal rRNA to other functional genes involved in microorganism-host interactions.The majority of the reported bovine-associated PCR assays target 16S rRNA genes from the order Bacteroidales. This bacterial group constitutes a large proportion of the normal gut microbiota of most animals, including bovines (28), and contains subpopulations closely associated with other animal hosts such as swine, horse, and human (1, 3, 6, 18, 24). Host-associated PCR-based assays targeting Bacteroidales genetic markers have been used to investigate the sources and levels of fecal pollution at a number of beaches and inland watersheds, with variable levels of success (10, 13, 22, 27). Researchers have postulated that differences in host animal age, health, diet, and geographic location may influence bacterial community structures in the bovine gastrointestinal tract (2, 9, 26). Without a priori knowledge of the potential representational bias introduced by such factors, it may be difficult to use these assays with confidence as indicators of bovine fecal pollution.Assay specificity and sensitivity and the prevalence and abundance of genetic marker determinations are typically estimated from the systematic testing of a collection of reference fecal sources collected from known animal sources. However, the characterization of assay performance has been limited, in most cases, to animal sources originating from a particular geographic region or industry, such as dairy or beef. The determination of assay performance across a range of different host populations is essential as the field moves toward the implementation of PCR-based host-associated fecal pollution assessment approaches.We report a performance study of seven PCR and quantitative PCR (qPCR) assays targeting Bacteroidales genes reported to be associated with either ruminant (e.g., bovine, goat, sheep, deer, and others) or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations. Assay specificity was determined by testing 175 fecal DNA extracts from 24 different animal species. For qPCR assays, the abundance of each genetic marker was measured within each bovine population and compared to quantities of Bacteroidales 16S rRNA genetic markers. These analyses indicated large discrepancies in assay performance across different bovine populations.     

Identification of 13 Human Microsatellite Markers via Cross-species Amplification of Fecal Samples from Rhinopithecus bieti

Yunnan snub-nosed monkeys (Rhinopithecus bieti) are 1 of 3 snub-nosed monkey species endemic to China. Only ca. 1500 individuals remain in high-altitude forests 3000–4500 m above sea level on the Tibetan Plateau, making them the nonhuman primate living at the highest known elevation. It is one of the most endangered 25 primate species in the world. Proper knowledge of the population genetics and social system of Rhinopithecus bieti will contribute to more appropriate conservation management decisions. Cross-species amplification of human microsatellite loci has facilitated analysis of the population genetics and reproductive strategies of various primate species. We screened 72 human-derived markers to assess their utility in Yunnan snub-nosed monkeys. Thirteen of them produced reliable results and exhibited moderate levels of polymorphism.     

Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 10⁶ copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.     

Identification of Bacterial DNA Markers for the Detection of Human Fecal Pollution in Water

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.     

Feral Horse Space Use and Genetic Characteristics from Fecal DNA

Feral horses (Equus ferus caballus) in the western United States are managed by the Bureau of Land Management (BLM) and United States Forest Service in designated areas on public lands with a goal of maintaining populations in balance with multiple uses of the landscape. Small, isolated populations can be at risk of extirpation from stochastic events and deleterious genetic effects resulting from inbreeding and reduced heterozygosity. The genetic diversity of feral horse herds is periodically monitored using blood or hair samples collected during management gathers (i.e., occasions when the herd is rounded up). We conducted a study to examine genetic characteristics of the feral horse population at the BLM Little Book Cliffs Herd Management Area (HMA) in Colorado, USA, using non-invasively collected fecal samples. Additionally, we explored whether genotypes could be used to document space use and potential sub-population development. We used a random sampling scheme, walking transects in sampling areas covering most of the HMA to find and collect fecal samples of all ages, except those that were deteriorating. We collected >1,800 fecal samples from across the study area in May, August, and October 2014. We then identified unique individuals using a suite of microsatellite loci. Our estimates of genetic diversity from fecal samples were higher than those reported from blood and hair samples taken during recent horse gathers, likely because our sample size and spatial distribution was larger. Genotypes revealed that some individuals were found only in certain parts of the study area and at a higher proportion than random; thus, they could be considered residents in those sampling areas. Using discriminant function analyses, we detected 5 genetic groups in the sample population, but these did not correspond to individuals in specific parts of the study area. Our results support the use of fecal DNA to augment direct observations of horse presence and could be used to detect habitat use and areas of high density. Non-invasive techniques such as fecal DNA sampling can help managers decide whether new individuals need to be translocated to a closed population to maintain genetic diversity without the human safety and animal welfare concerns associated with gathers and invasive techniques. © 2021 The Wildlife Society.     

DNase SISPA-Next Generation Sequencing Confirms Schmallenberg Virus in Belgian Field Samples and Identifies Genetic Variation in Europe

In 2011, a novel Orthobunyavirus was identified in cattle and sheep in Germany and the Netherlands. This virus was named Schmallenberg virus (SBV). Later, presence of the virus was confirmed using real time RT-PCR in cases of congenital malformations of bovines and ovines in several European countries, including Belgium. In the absence of specific sequencing protocols for this novel virus we confirmed its presence in RT-qPCR positive field samples using DNase SISPA-next generation sequencing (NGS), a virus discovery method based on random amplification and next generation sequencing. An in vitro transcribed RNA was used to construct a standard curve allowing the quantification of viral RNA in the field samples. Two field samples of aborted lambs containing 7.66 and 7.64 log(10) RNA copies per μL total RNA allowed unambiguous identification of SBV. One sample yielded 192 SBV reads covering about 81% of the L segment, 56% of the M segment and 13% of the S segment. The other sample resulted in 8 reads distributed over the L and M segments. Three weak positive field samples (one from an aborted calf, two from aborted lambs) containing virus quantities equivalent to 4.27-4.89 log(10) RNA copies per μL did not allow identification using DNase SISPA-NGS. This partial sequence information was compared to the whole genome sequence of SBV isolated from bovines in Germany, identifying several sequence differences. The applied viral discovery method allowed the confirmation of SBV in RT-qPCR positive brain samples. However, the failure to confirm SBV in weak PCR-positive samples illustrates the importance of the selection of properly targeted and fresh field samples in any virus discovery method. The partial sequences derived from the field samples showed several differences compared to the sequences from bovines in Germany, indicating sequence divergence within the epidemic.     

DNA分子标记在实验动物遗传分析中的应用

DNA分子标记技术很多,基本都是建立在RFLP、PCR和重复顺序的基础上的。本文重点介绍了限制性片段长度多态性（RFLP）标记、随机扩增多态性DNA（RAPD）标记、微卫星DNA（STR）标记、DNA指纹（DFP）标记、扩增片段长度多态性（AFLP）标记等几种重要的DNA分子标记技术的定义、结构、分布、组成、保守性、优点及丰富的多态性等。并重点介绍了微卫星DNA（STR）标记在分子遗传监测、遗传多样性分析和遗传血缘关系及个体识别等领域的应用。