Comparison of gull feces-specific assays targeting the 16S rRNA genes of Catellicoccus marimammalium and Streptococcus spp

Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.     

Tracking the Primary Sources of Fecal Pollution in a Tropical Watershed in a One-Year Study

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n = 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n = 340) and wastewater treatment samples (n = 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of host-specific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings.     

New molecular quantitative PCR assay for detection of host-specific Bifidobacteriaceae suitable for microbial source tracking

Bifidobacterium spp. belong to the commensal intestinal microbiota of warm-blooded animals. Some strains of Bifidobacterium show host specificity and have thus been proposed as host-specific targets to determine the origin of fecal pollution. Most strains have been used in microbial-source-tracking (MST) studies based on culture-dependent methods. Although some of these approaches have proved very useful, the low prevalence of culturable Bifidobacterium strains in the environment means that molecular culture-independent procedures could provide practical applications for MST. Reported here is a set of common primers and four Bifidobacterium sp. host-associated (human, cattle, pig, and poultry) probes for quantitative-PCR (qPCR) assessment of fecal source tracking. This set was tested using 25 water samples of diverse origin: urban sewage samples, wastewater from four abattoirs (porcine, bovine, and poultry), and water from a river with a low pollution load. The selected sequences showed a high degree of host specificity. There were no cross-reactions between the qPCR assays specific for each origin and samples from different fecal origins. On the basis of the findings, it was concluded that the host-specific qPCRs are sufficiently robust to be applied in environmental MST studies.     

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.     

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.     

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.     

Phylogenetic analysis of Bacteroidales 16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real‐time PCR

Aims: The aims of this study were to evaluate the host‐specific distribution of Bacteroidales 16S rRNA gene sequences from human‐ and animal‐related effluents and faeces, and to define a ruminant‐specific marker. Methods and Results: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human‐, bovine‐, pig‐ and wild bird‐specific clusters. From the bovine‐specific cluster II, we designed a ruminant‐specific marker, Rum‐2‐Bac, and this showed 97% sensitivity (n = 30) and 100% specificity (n = 40) when tested by TaqMan^® real‐time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8·2 ± 0·5, 8·4 ± 1·3 and 7 ± 0·5 log₁₀ copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5·1 ± 0·3 log₁₀ copies per millilitre water. Conclusions: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host‐specific distributions. Identification of a bovine‐specific cluster made it possible to design a reliable ruminant‐specific marker. Significance and Impact of the Study: The host‐specific distribution of Bacteroidales sequences from effluents mirrored the host‐specific distribution of sequences observed in individual faeces. This efficient new ruminant‐specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.     

七种圈养鹤的部分血液生化指标分析

为探索圈养鹤类的血液生化指标差异,本研究对白鹤(Grus leucogeranus)、丹顶鹤(G.japonensis)、黑冠鹤(Balearica pavonina)、黑颈鹤(G.nigricollis)、灰冠鹤(B.regulorum)、蓝鹤(Anthropoides paradiseus)和白枕鹤(G.vipio)的10项血液生化指标(总胆固醇、甘油三酯、总蛋白、白蛋白、球蛋白等)进行统计分析,并与国际物种信息系统(ISIS)提供的数据进行比较。结果表明,1)不同鹤类之间总胆固醇差异显著,而甘油三酯和血糖没有显著性差异,除了黑冠鹤和灰冠鹤的总胆固醇以及蓝鹤的血糖外,其余鹤类的常值均高于ISIS的平均值。2)不同鹤类之间的总蛋白、球蛋白和白蛋白的浓度差异显著,但总蛋白、球蛋白和白蛋白均在ISIS的正常范围内。3)不同鹤类间肌酐和尿酸不具有显著性差异,其中尿酸低于ISIS平均值,但仅尿素氮差异显著。不同鹤类之间血液生化指标存在差异,与圈养鹤的营养水平和环境有关。因此血液生化指标对于圈养鹤类营养状态和健康情况的判断具有重要参考价值,对圈养动物饲养管理具有重要的指导作用。     

Coccidia of whooping cranes

Coccidial oocysts were observed in 6 of 19 fecal samples from free-ranging whooping cranes (Grus americana) and 4 of 16 samples from captive whooping cranes. Eimeria gruis occurred in four free-ranging whooping cranes and E. reichenowi in two free-ranging and two captive whooping cranes. Fecal samples from two captive cranes contained oocysts of Isospora lacazei which was considered a spurious parasite. Oocysts of both species of Eimeria were prevalent in fecal samples collected from three free-ranging Canadian sandhill cranes (G. canadensis rowani) from whooping crane wintering grounds in Texas. These coccidia were prevalent also in fecal samples from 14 sandhill cranes (of 4 subspecies) maintained in captivity at the Patuxent Wildlife Research Center in Maryland.     

Microbial source tracking by DNA sequence analysis of the Escherichia coli malate dehydrogenase gene

Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.     

内蒙古胡列也吐的鹤类及保护

胡列也吐位于内蒙古自治区呼伦贝尔盟陈巴尔虎旗境内 ,地理位置在东经 4 9°4 8′,北纬 1 1 8°2 7′ ,总面积为 4 0km2 。 1 998年笔者对本区的鹤类资源进行了为期一年的调查。本区共有 5种鹤栖息 ,本次共记录到 4种鹤计 78只。其中白头鹤 52只 ,丹顶鹤 1 1只 ,白枕鹤 4只 ,蓑羽鹤 1 1只。灰鹤在本区有分布 ,但本次调查未见。此外还记录到我国最大的白头鹤夏季集群。     

应用肠道微生物示踪地表水粪便污染的研究进展

人和动物的粪便已成为水污染的重要污染源, 严重威胁着饮水安全和经济发展。水质污染微生物的传统检测指示菌是总大肠菌群、粪大肠菌群、埃希大肠菌、肠球菌和梭菌属。经过调查发现, 上述指示菌由于在体外能存活并繁殖, 并且不同宿主之间没有差异性, 不能准确用于追踪污染粪便的来源, 因此该指标难以直接说明粪便污染源和污染程度。最近的研究表明, Faecalibaterium作为水体粪便污染来源追踪的指示微生物具有很多优点。本文综述了粪便污染指示菌以及其相关替代方法在水质检测中的研究进展, 对各种指示菌进行了优、缺点比较, 展望了Faecalibaterium的应用前景。     

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.     

A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area

AIMS: The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes. METHODS AND RESULTS: The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. CONCLUSIONS: The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. Significance AND IMPACT OF THE STUDY: The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.     

中国鹤类现状及其保护对策

中国有9种鹤类,其中灰鹤（Grus grus）、黑颈鹤（Grus nigricollis）、丹顶鹤（Grus japonensis）、白枕鹤（Grus vipio）、赤颈鹤（Grus antigone）、蓑羽鹤（Anthropoides virgo）在中国进行繁殖。中国政府为保护鹤类采取了大量有效措施。本文在结合当前鹤类数量和保护现状的基础上,对中国鹤类的保护对策进行探讨。     

Use of Bacteroidales Microbial Source Tracking To Monitor Fecal Contamination in Fresh Produce Production

In recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based on Bacteroidales 16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universal Bacteroidales marker (AllBac), including 66% of samples from cantaloupe farms (3.6 log₁₀ genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log₁₀ GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log₁₀ GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation between Bacteroidales and generic Escherichia coli across all samples. This study provides evidence that Bacteroidales markers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.     

Coccidian Parasites and Conservation Implications for the Endangered Whooping Crane (Grus americana)

While the population of endangered whooping cranes (Grus americana) has grown from 15 individuals in 1941 to an estimated 304 birds today, the population growth is not sufficient to support a down-listing of the species to threatened status. The degree to which disease may be limiting the population growth of whooping cranes is unknown. One disease of potential concern is caused by two crane-associated Eimeria species: Eimeria gruis and E. reichenowi. Unlike most species of Eimeria, which are localized to the intestinal tract, these crane-associated species may multiply systemically and cause a potentially fatal disease. Using a non-invasive sampling approach, we assessed the prevalence and phenology of Eimeria oocysts in whooping crane fecal samples collected across two winter seasons (November 2012–April 2014) at the Aransas National Wildlife Refuge along the Texas Gulf coast. We also compared the ability of microscopy and PCR to detect Eimeria in fecal samples. Across both years, 26.5% (n = 328) of fecal samples were positive for Eimeria based on microscopy. Although the sensitivity of PCR for detecting Eimeria infections seemed to be less than that of microscopy in the first year of the study (8.9% vs. 29.3%, respectively), an improved DNA extraction protocol resulted in increased sensitivity of PCR relative to microscopy in the second year of the study (27.6% and 20.8%, respectively). The proportion of positive samples did not vary significantly between years or among sampling sites. The proportion of Eimeria positive fecal samples varied with date of collection, but there was no consistent pattern of parasite shedding between the two years. We demonstrate that non-invasive fecal collections combined with PCR and DNA sequencing techniques provides a useful tool for monitoring Eimeria infection in cranes. Understanding the epidemiology of coccidiosis is important for management efforts to increase population growth of the endangered whooping crane.     

Temporal Assessment of the Impact of Exposure to Cow Feces in Two Watersheds by Multiple Host-Specific PCR Assays

Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.     

Accurate analysis of prevalence of coccidiosis in individually identified wild cranes in inhabiting and migrating populations in Japan

Eimeria gruis and E. reichenowi cause coccidiosis, a major parasitic disease of cranes. By non-invasive molecular approaches, we investigated the prevalence and genetic characterization of pathogens in two Japanese crane habitats; one is Hokkaido inhabited by the endangered red-crowned crane, and the other is Izumi in Kyushu where populations that consist mainly of vulnerable hooded and white-naped cranes migrate in winter. The non-invasively collected faecal samples from each wintering population were first subjected to host genomic DNA-targeted analyses to determine the sample origin and avoid sample redundancy. Extremely high prevalence was observed in the Izumi populations (> 90%) compared with the Hokkaido population (18-30%) by examining 470 specimens by microscopy and PCR-based capillary electrophoresis (PCR-CE), using genetic markers in the second internal transcribed spacer (ITS2). Correspondence analysis of PCR-CE data revealed differences in community composition of coccidia between hooded and white-naped cranes. 18S rRNA and ITS2 sequences were determined from single oocysts excreted by red-crowned and hooded cranes. Phylogenetic analysis of 18S rRNA suggested that E. reichenowi was polyphyletic while E. gruis was monophyletic. Together with PCR-CE data, these results indicate different host specificity among the E. reichenowi type. Our data suggest that E. reichenowi comprises multiple species.     

Phylogenetic diversity and molecular detection of bacteria in gull feces

In spite of increasing public health concerns about the potential risks associated with swimming in waters contaminated with waterfowl feces, little is known about the composition of the gut microbial community of aquatic birds. To address this, a gull 16S rRNA gene clone library was developed and analyzed to determine the identities of fecal bacteria. Analysis of 282 16S rRNA gene clones demonstrated that the gull gut bacterial community is mostly composed of populations closely related to Bacilli (37%), Clostridia (17%), Gammaproteobacteria (11%), and Bacteriodetes (1%). Interestingly, a considerable number of sequences (i.e., 26%) were closely related to Catellicoccus marimammalium, a gram-positive, catalase-negative bacterium. To determine the occurrence of C. marimammalium in waterfowl, species-specific 16S rRNA gene PCR and real-time assays were developed and used to test fecal DNA extracts from different bird (n = 13) and mammal (n = 26) species. The results showed that both assays were specific to gull fecal DNA and that C. marimammalium was present in gull fecal samples collected from the five locations in North America (California, Georgia, Ohio, Wisconsin, and Toronto, Canada) tested. Additionally, 48 DNA extracts from waters collected from six sites in southern California, Great Lakes in Michigan, Lake Erie in Ohio, and Lake Ontario in Canada presumed to be impacted with gull feces were positive by the C. marimammalium assay. Due to the widespread presence of this species in gulls and environmental waters contaminated with gull feces, targeting this bacterial species might be useful for detecting gull fecal contamination in waterfowl-impacted waters.