Faecal sexual steroids in sex typing and endocrine status of great bustards

Faecal sexual steroids have been used in field studies evaluating the relationships between gender and the multiple factors influencing endocrine status of individuals. The determination of faecal steroids has been also proposed as an alternative, non-invasive sexing method when other methods were deemed impractical or risky for the health of birds. In this study, we quantified sexual steroid hormones in faeces of the great bustard (Otis tarda), a large and sexually dimorphic polyginic bird species that it is threatened and subjected to intense wildlife management. We evaluated differences between captivity and wild conditions, flocks and sexes, and used faecal steroids to develop sex determination procedures. We found similar steroid levels in captive and wild bustards, no differences between unisexual wild flocks and clear between-sexes differences in testosterone but not estradiol. Faecal steroids accurately discriminated gender in both captive and wild known-sex great bustards. Total testosterone concentration was always higher than estradiol concentration in faecal samples from males, but estradiol was not always higher than testosterone in females. Faecal steroids failed to reveal the presence of young males in female flocks during winter, despite faecal testosterone levels increased with age in a small sample of captive males. Our results show that faecal steroid measurement for both sexing and characterizing the endocrine status of great bustards is feasible, and therefore it should be valuable in wildlife management, especially in combination with additional information obtained from faeces as diet.     

Fecundity and egg output by Toxocara canis in the red fox, Vulpes vulpes

The role of the red fox Vulpes vulpes in the dissemination of eggs of Toxocara canis into the environment is considered with reference to female worm fecundity and egg output in the faeces of infected foxes collected from four localities in southern England. A significant positive correlation was found between female worm size and the number of eggs in the uterus but there was no significant relationship between T. canis worm numbers and egg output in fox faeces. Reliable estimates of worm burdens in foxes could not, therefore, be determined from faecal egg counts alone. The highest mean egg output of 2145.0 epg recorded from adult foxes indicated that fox cubs are not necessarily the main sources of environmental contamination with T. canis eggs. Saturated magnesium sulphate was found to be a more effective flotation solution than zinc sulphate and sodium chloride for recovering eggs from fox faecal samples.     

Molecular tracking of mountain lions in the Yosemite valley region in California: genetic analysis using microsatellites and faecal DNA

Twelve microsatellite loci were characterized in California mountain lions (Puma concolor) and sufficient polymorphism was found to uniquely genotype 62 animals sampled at necropsy. Microsatellite genotypes obtained using mountain lion faecal DNA matched those from muscle for all of 15 individuals examined. DNA from potential prey species and animals whose faeces could be misidentified as mountain lion faeces were reliably distinguished from mountain lions using this microsatellite panel. In a field application of this technique, 32 faecal samples were collected from hiking trails in the Yosemite Valley region where seven mountain lions previously had been captured, sampled, and released. Twelve samples yielded characteristic mountain lion genotypes, three displayed bobcat-type genotypes, and 17 did not amplify. The genotype of one of the 12 mountain lion faecal samples was identical to one of the mountain lions that previously had been captured. Three of the 12 faecal samples yielded identical genotypes, and eight new genotypes were detected in the remaining samples. This analysis provided a minimum estimate of 16 mountain lions (seven identified by capture and nine identified by faecal DNA) living in or travelling through Yosemite Valley from March 1997 to August 1998. Match probabilities (probabilities that identical DNA genotypes would be drawn at random a second time from the population) indicated that the samples with identical genotypes probably came from the same mountain lion. Our results demonstrate that faecal DNA analysis is an effective method for detecting and identifying individual mountain lions.     

A survey of Campylobacter species shed in faeces of beef cattle using polymerase chain reaction

A polymerase chain reaction (PCR)-based survey of campylobacters associated with faeces collected from 382 beef cattle was undertaken. To ensure the removal of PCR inhibitors present in faeces and determine if adequate extraction was achieved, faeces were seeded with internal control DNA (i.e., DNA designed to amplify with the Campylobacter genus primer set, but provide a smaller amplicon) before the extraction procedure. In only two samples (0.5%) were the internal control or Campylobacter genus amplicons not detected. In the remaining 380 faecal samples, Campylobacter DNA was detected in 83% of the faecal samples (80% of the faecal samples were positive for Campylobacter genus DNA, and 3% of the samples were negative for Campylobacter genus DNA but positive for DNA of individual species). The most frequently detected species was Campylobacter lanienae (49%), a species only recently connected to livestock hosts. Campylobacter jejuni DNA was detected in 38% of the faecal samples, and Campylobacter hyointestinalis and Campylobacter coli DNA were detected in 8% and 0.5% of the samples, respectively. Campylobacter fetus DNA was not detected. Twenty-four percent of the faecal samples contained DNA of at least two species of Campylobacter. Of these samples, the majority (81%) contained DNA of C. jejuni and C. lanienae. The results of this study indicate that beef cattle commonly release a variety of Campylobacter species into the environment and may contribute to the high prevalence of campylobacteriosis in humans inhabiting areas of intensive cattle production, such as southern Alberta. Furthermore, this study demonstrates the utility of using PCR as a rapid and accurate method for simultaneously detecting the DNA of a diverse number of Campylobacter species associated with bovine faeces.     

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/1 sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.     

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20 mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/l sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.     

Comparing plasma and faecal measures of steroid hormones in Adelie penguins Pygoscelis adeliae

Physiological measurements of both stress and sex hormones are often used to estimate the consequences of natural or human-induced change in ecological studies of various animals. Different methods of hormone measurement exist, potentially explaining variation in results across studies; methods should be cross-validated to ensure that they correlate. We directly compared faecal and plasma hormone measurements for the first time in a wild free-living species, the Adelie penguin (Pygoscelis adeliae). Blood and faecal samples were simultaneously collected from individual penguins for comparison and assayed for testosterone and corticosterone (or their metabolites). Sex differences and variability within each measure, and correlation of values across measures were compared. For both hormones, plasma samples showed greater variation than faecal samples. Males had higher mean corticosterone concentrations than females, but the difference was only statistically significant in faecal samples. Plasma testosterone, but not faecal testosterone, was significantly higher in males than females. Correlation between sample types was poor overall, and weaker in females than in males, perhaps because measures from plasma represent hormones that are both free and bound to globulins, whereas measures from faeces represent only the free portion. Faecal samples also represent a cumulative measure of hormones over time, as opposed to a plasma ‘snapshot’ concentration. Our data indicate that faecal sampling appears more suitable for assessing baseline hormone concentrations, whilst plasma sampling may best define immediate responses to environmental events. Consequently, future studies should ensure that they select the most appropriate matrix and method of hormone measurement to answer their research questions.     

Development and application of a spiral plating method for the enumeration of Escherichia coli O157 in bovine faeces

AIM: To develop and validate a direct plating method applicable to epidemiological studies for enumerating Escherichia coli O157 in cattle faeces. METHODS AND RESULTS: The spiral plate count method was used to enumerate E. coli O157 in faecal samples. The accuracy and variation of counts was then assessed using faecal samples inoculated with E. coli O157. There was good agreement between inoculated levels of E. coli O157 and those recovered from faeces, particularly when counts were > 10(2) CFU g(-1) of faeces. The method was applied to a small study assessing short-term survival of E. coli O157 in naturally infected cattle faeces. E. coli O157 was found to survive in faeces for over 10 days at concentrations above 10(3) CFU g(-1) of faeces. Populations of E. coli O157 were also found to increase 100-fold in the first few hours after defecation. CONCLUSIONS: The enumeration method is easy to implement and enables a quick throughput of large numbers of samples. The method is accurate and reliable and enables the inherent variation in count data to be explored but needs to be used in combination with a more sensitive method for samples containing < 10(2) CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is appropriate for enumeration of E. coli O157 in cattle faeces in large-scale epidemiological studies.     

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.     

Empirical evaluation of preservation methods for faecal DNA

We evaluate the relative effectiveness of four methods for preserving faecal samples for DNA analysis. PCR assays of fresh faecal samples collected from free-ranging baboons showed that amplification success was dependent on preservation method, PCR-product size, and whether nuclear or mitochondrial DNA was assayed. Storage in a DMSO/EDTA/Tris/salt solution (DETs) was most effective for preserving nuclear DNA, but storage in 70% ethanol, freezing at –20°C and drying performed approximately equally well for mitochondrial DNA and short (<200 bp) nuclear DNA fragments. Because faecal DNA is diluted and degraded, repeated extractions from faeces may be necessary and short nuclear markers should be employed for genotyping. A review of molecular scatology studies further suggests that three to six faeces per individual should be collected.     

Evaluation of a double centrifugation technique for the detection of Anoplocephala eggs in horse faeces

Faecal samples of 250 horses from farms with a known history of tapeworm infection were examined comparatively for cestode eggs using a double centrifugation/combined sedimentation-floatation technique. From each faecal sample, three 5?g and three 15?g subsamples were processed, each using either saturated NaCl solution, specific gravity (sp. g.) 1.2 [NaCl]; concentrated sugar solution, sp. g. 1.26 [sugar]; or concentrated ZnSO4 solution, sp. g. 1.3 [ZnSO4] for floatation. In total, faeces from 187 horses (?=?74.8%) tested 'positive' for Anoplocephala eggs. Percentages of samples testing 'positive' for Anoplocephala ova were: 57.2% for 5?g faeces/NaCl, 66% for 15?g faeces/NaCl, 66% for 5?g faeces/sugar, 72.8% for 15?g faeces/sugar, 55.6% for 5?g faeces/ZnSO4, and 61.2% for 15?g faeces/ZnSO4, respectively. Processing of 15?g faecal samples resulted in a significant (P?相似文献   

Development of Faecalibacterium 16S rRNA gene marker for identification of human faeces

Aims:  The focus of this study was to identify a bacterial 16S rRNA gene sequence, unique to microbiota in the human gut, for use in development of a dependable PCR assay to detect human faecal pollution in water.
Methods and Results:  Suppression subtractive hybridization (SSH) and bioinformatics were used to identify a genetic marker, within the 16S rRNA gene of Faecalibacterium , for the detection of human faeces. DNA sequencing analysis demonstrated that a majority (16) of 74 clones of the SSH library contained insertion sequences identified as Faecalibacterium 16S rRNA genes . Human faeces-specific sequences were derived and six PCR primer sets designed and tested against faecal DNA samples from human and nonhuman sources. One PCR primer set, HFB-F3 and HFB-R5, was exclusively associated with human faeces. These primers generated a human faeces-specific amplicon of 399 bp from 60·2% of human faecal samples and 100% of sewage samples.
Conclusions:  The subject Faecalibacterium marker is specific for sewage.
Significance and Impact of the Study:  This study represents the initial report of a Faecalibacterium marker for human faeces, which may prove useful for microbial source tracking.     

Detection of the nifH gene of Methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters

AIMS: The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage. METHODS AND RESULTS: PCR primers for the nifH gene of M. smithii were designed, tested and used to detect the presence or absence of this organism in faecal and environmental samples. Specificity analysis showed that the Mnif primers amplified products only in M. smithii pure culture strains (100%), human faeces (29%), human sewage samples (93%) and sewage-contaminated water samples (100%). No amplification was observed when primers were tested against 43 bacterial stock cultures, 204 animal faecal samples, 548 environmental bacterial isolates and water samples from a bovine waste lagoon and adjacent polluted creek. Sequencing of PCR products from sewers demonstrated that a 222-bp product was the nifH gene of M. smithii. The minimal amount of total DNA required for the detection of M. smithii was 10 ng for human faeces, 10 ng for faecally contaminated water and 5 ng for sewage. Recreational water seeded with M. smithii established a lower detection limit of 13 cells ml(-1). CONCLUSIONS: The Mnif assay developed during this investigation showed successful detection of M. smithii in individual human faecal samples, sewage and sewage-contaminated water but not in uncontaminated marine water or bovine-contaminated waters. The Mnif assay appears to be a potentially useful method to detect sewage-polluted coastal waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to utilize methanogens as an indicator of sewage pollution. Mnif PCR detection of M. smithii was shown to be a rapid, inexpensive and reliable test for determining the presence or absence of sewage pollution in coastal recreational waters.     

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.     

Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H2/CO2, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.     

Detection of Yersinia enterocolitica O:3 in faecal samples and tonsil swabs from pigs using IMS and PCR

Dilutions of faecal samples spiked with Yersinia enterocolitica O:3 were analysed using immunomagnetic separation (IMS) followed by PCR. In 10% faecal dilutions with added Y. enterocolitica cells; the limit of detection was 200 cells g^-1 faeces. Faecal samples from 38 pigs were analysed by IMS-PCR in parallel with detection and quantification of Y. enterocolitica O:3 using cold pre-enrichment culturing. Of the 15 culture-positive samples, only two were detected with IMS-PCR. These two samples contained 40–400 Y. enterocolitica O:3 cells g^-1 faeces; the highest level found in the investigation. This indicated that the low sensitivity of IMS-PCR was due to low amounts of cells in the faecal samples. Swab samples from 195 pig tonsils, taken on a slaughterline were examined using IMS-PCR and culture detection. Of 164 culture-positive samples, 60 were positive with IMS-PCR. In addition, IMS-PCR was positive for three culture-negative samples. Forty-five of the samples were further examined by IMS-PCR after 7–10 d of cold pre-enrichment. All 31 culture-positive samples as well as five culture-negative samples were detected by IMS-PCR. From these data it can be concluded that IMS-PCR can be used to detect Y. enterocolitica O:3 cells after pre-enrichment, but direct detection needs further optimization of the sample preparation procedures.     

Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H₂/CO₂, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurrries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.     

短尾猴陈旧粪便中DNA的提取

分子粪便学（Molecular scatology）是一门将传统粪便分析方法与分子生物学技术相结合,以动物粪便为实验材料进行多领域研究的学科（魏辅等,2001）。虽然该方法已在野生濒危动物保护遗传学和分子生态学研究中发挥了很大作用（Kohnand Wayne,1997）,但目前大多数分子粪便学研究中使用的材料是新鲜粪便,从保存时间很长的陈旧粪便中很难提取到高质量的DNA用于PCR扩增以及序列分析,严重制约了分子粪便学的广泛应用（Wasser et al．,1997;Constable et al．,2001;Murphy et al．．2002）。     

粪便收集方式对异育银鲫表观消化率测定的影响

消化率的高低是评价饲料营养价值的重要指标,但是粪便的不同收集方式会影响消化率的测定结果。研究观察了异育银鲫摄食后的排粪高峰以及排粪持续时间,探讨了粪便的不同收集方式对异育银鲫(Carassius auratus gibelio)(约100 g)表观消化率(Apparent digestibility coefficient,ADC)测定结果的影响。饲料中加入1%三氧化二铬(Cr2O3)作为指示剂,实验采用3种粪便收集方法(积粪器法、虹吸法和后肠取粪法),并且在积粪器法和虹吸法中设置了不同的收粪时间。结果表明,正常投喂的异育银鲫摄食后的排粪高峰出现在摄食结束后的5—10h;饥饿2d的异育银鲫恢复摄食后的排粪高峰出现在摄食结束后的7—9h;饲料的干物质表观消化率(ADC of dry matter,ADCd)和蛋白表观消化率(ADC of protein,ADCp)在排粪初始阶段较低,随着排粪高峰的出现而升高。粪便的不同收集方法和不同收集时间对ADCd和ADCp的测定结果均存在显著影响(P<0.05)。采用虹吸法收集粪便测得的ADCd与在排粪高峰时段内积粪器法的测定值相当;后肠取粪法收集粪便测得的ADCd低于积粪器法排粪高峰期的测定值。采用虹吸法收集粪便测得的ADCp略低于在排粪高峰时段内积粪器法的测定值;后肠取粪法收集粪便测得的ADCp低于积粪器法各时间段内的测定值。因此,在选择挑选新鲜完整粪便的前提下,在排粪高峰期采用虹吸法收集粪便用于ADC的测定是一种方便可靠的方法,反而后肠取粪法由于操作误差容易带来更大偏差。     

Comparison of culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry

Background

Performances of different salmonella detection methods were evaluated by applying them to of artificially contaminated faecal specimens from cattle, pigs and poultry. The NMKL71 method, being the standard reference method for detection of salmonella in the official Swedish control program, was compared with the proposed ISO method using MSRV-selective enrichment for culturing, and also with three commercial ELISA- based systems, Bioline Selecta, Bioline Optima and Vidas, a commercial PCR-based method, BAX^® system, and three different strategies using PCR detection using a non-commercial PCR system.

Results

Altogether, 391 samples were tested, and the overall results clearly indicate that, when faeces from all animal species and all serotypes were included, the MSRV performed best, with a calculated accuracy of 99% and a calculated sensitivity of 98%. The second most sensitive and specific method was the BAX^® system, using the modified enrichment protocol as recommended by the manufacturer for faecal samples. However, this protocol includes one additional day of work, as compared with the standard procedure for food sample analysis by the same method. The different strategies for salmonella detection using non-commercial PCR showed a sensitivity and specificity in the same range as the BAX^® method; furthermore, results were obtained more quickly. The various commercial ELISA methods and the NMKL method showed the poorest performance of the methods included in the study, and were closely dependent on the origin of the faeces used and on which salmonella strain was to be detected.

Conclusion

The study showed that the sensitivity of the different methods depended to a great extent on the origin of the faecal matrices and the salmonella strains used to "spike" the samples.