Multiplex PCRs for the specific identification of marsupial and deer species from faecal samples as a basis for non-invasive epidemiological studies of parasites

Helicobacter pyloriinfection, often acquired in early childhood, is a global cause of undernutrition, gastritis, peptic ulcer disease and gastric carcinoma. This study tested the feasibility of usingH. pylorished in the faeces as a source of DNA for non-invasive epidemiological studies.H. pyloriDNA was chemically recovered and isolated using a specific biotinylated oligonucleotide probe with magnetic capture from 28H. pyloripositive faecal samples obtained from children attending hospital for the investigation of suspectedH. pyloriinfection, together with close family members. Random amplification of polymorphic DNA (RAPD) was subsequently used to discriminate each isolate. 93% of stool samples selected were typeable. Parent, child and sibling samples were compared and similarities determined. Phylogenetic analysis showed thatH. pyloriDNA obtained from the faeces can be used to genotype individual strains, offering a means of studying intrafamilial transfer of this microorganism.

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Non-Invasive Molecular Survey of Sarcoptic Mange in Wildlife: Diagnostic Performance in Wolf Faecal Samples Evaluated by Multi-Event Capture–Recapture Models

Pathogens ◽

10.3390/pathogens10020243 ◽

2021 ◽

Vol 10 (2) ◽

pp. 243

Author(s):

Julieta Rousseau ◽

Mónia Nakamura ◽

Helena Rio-Maior ◽

Francisco Álvares ◽

Rémi Choquet ◽

...

Keyword(s):

Molecular Detection ◽

High Specificity ◽

Sarcoptes Scabiei ◽

Sarcoptic Mange ◽

Diagnostic Specificity ◽

Non Invasive ◽

Faecal Samples ◽

Capture Recapture ◽

The Mean ◽

Event Capture

Sarcoptic mange is globally enzootic, and non-invasive methods with high diagnostic specificity for its surveillance in wildlife are lacking. We describe the molecular detection of Sarcoptes scabiei in non-invasively collected faecal samples, targeting the 16S rDNA gene. We applied this method to 843 Iberian wolf Canis lupus signatus faecal samples collected in north-western Portugal (2006–2018). We further integrated this with serological data (61 samples from wolf and 20 from red fox Vulpes vulpes, 1997–2019) in multi-event capture–recapture models. The mean predicted prevalence by the molecular analysis of wolf faecal samples from 2006–2018 was 7.2% (CI95 5.0–9.4%; range: 2.6–11.7%), highest in 2009. The mean predicted seroprevalence in wolves was 24.5% (CI95 18.5–30.6%; range: 13.0–55.0%), peaking in 2006–2009. Multi-event capture–recapture models estimated 100% diagnostic specificity and moderate diagnostic sensitivity (30.0%, CI95 14.0–53.0%) for the molecular method. Mange-infected individually identified wolves showed a tendency for higher mortality versus uninfected wolves (ΔMortality 0.150, CI95 −0.165–0.458). Long-term serology data highlights the endemicity of sarcoptic mange in wild canids but uncovers multi-year epidemics. This study developed and evaluated a novel method for surveying sarcoptic mange in wildlife populations by the molecular detection of S. scabiei in faecal samples, which stands out for its high specificity and non-invasive character.

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Molecular Characterisation of Cryptosporidium spp. in Mozambican Children Younger than 5 Years Enrolled in a Matched Case-Control Study on the Aetiology of Diarrhoeal Disease

Pathogens ◽

10.3390/pathogens10040452 ◽

2021 ◽

Vol 10 (4) ◽

pp. 452

Author(s):

Augusto Messa, Jr. ◽

Pamela C. Köster ◽

Marcelino Garrine ◽

Tacilta Nhampossa ◽

Sérgio Massora ◽

...

Keyword(s):

Diarrhoeal Disease ◽

Epidemiological Studies ◽

Domestic Animal ◽

Cryptosporidium Species ◽

Older Children ◽

Species Determination ◽

Cryptosporidium Spp ◽

Faecal Samples ◽

Matched Case

Cryptosporidium is a leading cause of childhood diarrhoea and associated physical and cognitive impairment in low-resource settings. Cryptosporidium-positive faecal samples (n = 190) from children aged ≤ 5 years enrolled in the Global Enteric Multicenter Study (GEMS) in Mozambique detected by ELISA (11.5%, 430/3754) were successfully PCR-amplified and sequenced at the gp60 or ssu rRNA loci for species determination and genotyping. Three Cryptosporidium species including C. hominis (72.6%, 138/190), C. parvum (22.6%, 43/190), and C. meleagridis (4.2%, 8/190) were detected. Children ≤ 23 months were more exposed to Cryptosporidium spp. infections than older children. Both C. hominis and C. parvum were more prevalent among children with diarrhoeal disease compared to those children without it (47.6% vs. 33.3%, p = 0.007 and 23.7% vs. 11.8%, p = 0.014, respectively). A high intra-species genetic variability was observed within C. hominis (subtype families Ia, Ib, Id, Ie, and If) and C. parvum (subtype families IIb, IIc, IIe, and IIi) but not within C. meleagridis (subtype family IIIb). No association between Cryptosporidium species/genotypes and child’s age was demonstrated. The predominance of C. hominis and C. parvum IIc suggests that most of the Cryptosporidium infections were anthroponotically transmitted, although zoonotic transmission events also occurred at an unknown rate. The role of livestock, poultry, and other domestic animal species as sources of environmental contamination and human cryptosporidiosis should be investigated in further molecular epidemiological studies in Mozambique.

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Using reliable predator identification to investigate feeding habits of Neotropical carnivores (Mammalia, Carnivora) in a deforestation frontier of the Brazilian Amazon

Mammalia ◽

10.1515/mammalia-2018-0106 ◽

2019 ◽

Vol 83 (5) ◽

pp. 415-427 ◽

Cited By ~ 1

Author(s):

Flávia P. Tirelli ◽

Thales R.O. de Freitas ◽

Fernanda Michalski ◽

Alexandre R. Percequillo ◽

Eduardo Eizirik

Keyword(s):

Feeding Habits ◽

Brazilian Amazon ◽

Ecological Studies ◽

Accurate Identification ◽

Predator Species ◽

Non Invasive ◽

Carnivore Species ◽

Faecal Samples ◽

Critical Requirement ◽

Better Than

Abstract Accurate identification of predator species is a critical requirement to investigate their diet using faecal samples. We used non-invasive sampling and two methods of predator identification to investigate the diets of sympatric carnivores in a highly deforested region of the Brazilian Amazon. Of 108 scats, 81 could be identified at the species level using DNA sequencing and/or trichology. The former performed better than the latter (81.5% vs. 54.3% of the identified samples), and results were quite congruent (89.7% concordance in the 29 samples that could be assessed with both approaches). Nine species were identified, out of which four (crab-eating fox, ocelot, puma and jaguar) presented a sufficient number of samples to allow dietary analyses. The crab-eating fox was the most generalist (BA=0.92); ocelots focused on small- to medium-sized prey; pumas fed mostly on medium-sized items; and jaguars mostly targeted large-sized prey. A considerable overlap was observed between ocelots and pumas in all estimations (O=0.47–0.83). The presence of jaguars in the same region could be driving pumas to select medium- and small-sized prey. The results of this study highlight the importance of reliable predator identification and the need for in-depth ecological studies in areas where carnivore species are sympatric.

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Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples

Veterinary Microbiology ◽

10.1016/j.vetmic.2008.09.087 ◽

2009 ◽

Vol 136 (1-2) ◽

pp. 166-172 ◽

Cited By ~ 30

Author(s):

Léonid M. Irenge ◽

Karl Walravens ◽

Marc Govaerts ◽

Jacques Godfroid ◽

Valérie Rosseels ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Specific Identification ◽

Faecal Samples ◽

Development And Validation

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Sex determination in gibbons of genus Nomascus using non-invasive method

Acta Veterinaria Brno ◽

10.2754/avb201685040363 ◽

2016 ◽

Vol 85 (4) ◽

pp. 363-366 ◽

Cited By ~ 1

Author(s):

Petra Bolechová ◽

Kateřina Ječmínková ◽

Michal Hradec ◽

Tomáš Kott ◽

Jana Doležalová

Keyword(s):

Sex Determination ◽

Direct Examination ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Non Invasive ◽

Faecal Samples ◽

Invasive Method ◽

Polymerase Chain ◽

Template Dna

Gibbons of the genus Nomascus have a strong sexual dimorphism and dichromatism. As they mature, both sexes develop sex-specific pelage colour. In combination with physical similarities in the genitalia with both sexes, there are problems with determining the sex of young individuals compared to other genus of gibbons. This is a pilot study applying a multiplex polymerase chain reactions based on a non-invasive method for sex determination of gibbons. The study was conducted on 22 faecal samples from gibbons of the genus Nomascus. The animals were monitored by staff so that the samples were identified correctly and each sample was collected immediately after the defecation. Results confirmed the sex in all adult and juvenile animals with known sex; and 2 females and 5 males in juveniles were determined with unknown sex. The results of direct examination completely corresponded with the PCR results. The PCR reaction with template DNA isolated from faecal material required BSA usage, however, we observed the occurrence of nonspecific fragments. This did not affect the reliability of our results and we confirmed the usability of this method for this genus.

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Genetic diversity of African clawless otters (Aonyx capensis) occurring in urbanised areas of Gauteng, South Africa

South African Journal of Science ◽

10.17159/sajs.2019/4889 ◽

2019 ◽

Vol 115 (7/8) ◽

Author(s):

Damian W. Ponsonby ◽

M. Thabang Madisha ◽

Schwaibold Schwaibold ◽

Desiré L. Dalton

Keyword(s):

South Africa ◽

Genetic Diversity ◽

Gene Flow ◽

Environmental Changes ◽

Urban Expansion ◽

Evolutionary Potential ◽

Non Invasive ◽

Faecal Samples ◽

Physical Barriers ◽

High Gene

Genetic diversity is the basis of the evolutionary potential of species to respond to environmental changes. However, restricting the movement of species can result in populations becoming less connected which can reduce gene flow and can subsequently result in a loss of genetic diversity. Urban expansion can lead to the fragmentation of habitats which affects the ability of species to move freely between areas. In this study, the genetic diversity of the African clawless otter (Aonyx capensis) in Gauteng (South Africa) was assessed using non-invasive sampling techniques. DNA was extracted from spraint (faecal) samples collected along nine rivers and genotyped using 10 microsatellites to assess population structure and genetic diversity. Samples were grouped based on locality and by catchment to determine whether isolated subpopulations exist. Genetic diversity of A. capensis in Gauteng was found to be low (mean observed heterozygosity (Ho)=0.309). Analysis of genetic structure provides support for the otter populations being panmictic with high gene flow between populations from different rivers. Results from the study indicate that the movement of A. capensis is not affected by physical barriers in urbanised areas. However, because the genetic diversity of the species in the study area is low, these animals may not be able to cope with future environmental changes.

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Factors influencing genotyping success and genotyping error rate of Eurasian otter (Lutra lutra) faeces collected in temperate Central Europe

European Journal of Wildlife Research ◽

10.1007/s10344-020-01444-4 ◽

2020 ◽

Vol 67 (1) ◽

Author(s):

Marcia Sittenthaler ◽

Eva Maria Schöll ◽

Christoph Leeb ◽

Elisabeth Haring ◽

Rosemarie Parz-Gollner ◽

...

Keyword(s):

Influential Factors ◽

Lutra Lutra ◽

Sample Treatment ◽

Success Rates ◽

Sample Collection ◽

Allelic Dropout ◽

Non Invasive ◽

Eurasian Otter ◽

Faecal Samples ◽

Elusive Species

AbstractThe use of non-invasively collected DNA source material for genetic and genomic applications is usually characterized by low target DNA concentration and quality, genotyping errors and cost-intensive lab procedures. However, for otters (Lutrinae) as elusive species of conservation concern, genetic non-invasive sampling has become an important tool to study their ecology and demography. To increase cost-efficiency of monitoring programmes and to promote the expansion of genomic approaches to non-invasive samples, we aimed to refine sample collection and preparation. Therefore, we examined the effects of intrinsic sample characteristics (including diet), environmental conditions in the field and sample treatment in the molecular laboratory on the success of genotyping and allelic dropout (ADO) rates using microsatellite markers in 1970 fresh Eurasian otter (Lutra lutra) scats. Using fresh samples only, we probably eliminated one of the most important impediments of genotyping DNA from otter faecal samples beforehand. But, we observed higher genotyping success and lower ADO rates for anal glad secretions and faecal samples containing high proportions of mucus. Moist conditions during sample collection may promote DNA degradation and PCR inhibition, leading to decreased genotyping success rates. ADO was further affected by the type of extraction kit. However, a high proportion of variance remaining unexplained by our models implied that additional parameters were acting (amount of PCR inhibitors, non-uniform distribution of intestinal cells, efficiency of PCRs, specific microclimate at marking sites). We summarized influential factors maximizing genotyping quality of otter scats and give recommendations for sample collection, storage and DNA extraction based on our results and current literature.

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Gut microbiome analysis as a tool towards targeted non-invasive biomarkers for early hepatocellular carcinoma

Gut ◽

10.1136/gutjnl-2017-315084 ◽

2018 ◽

Vol 68 (6) ◽

pp. 1014-1023 ◽

Cited By ~ 102

Author(s):

Zhigang Ren ◽

Ang Li ◽

Jianwen Jiang ◽

Lin Zhou ◽

Zujiang Yu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Gut Microbiome ◽

Cross Validation ◽

Area Under The Curve ◽

Northwest China ◽

Central China ◽

Advanced Hcc ◽

Non Invasive ◽

Faecal Samples ◽

Early Hcc

ObjectiveTo characterise gut microbiome in patients with hepatocellular carcinoma (HCC) and evaluate the potential of microbiome as non-invasive biomarkers for HCC.DesignWe collected 486 faecal samples from East China, Central China and Northwest China prospectively and finally 419 samples completed Miseq sequencing. We characterised gut microbiome, identified microbial markers and constructed HCC classifier in 75 early HCC, 40 cirrhosis and 75 healthy controls. We validated the results in 56 controls, 30 early HCC and 45 advanced HCC. We further verified diagnosis potential in 18 HCC from Xinjiang and 80 HCC from Zhengzhou.ResultsFaecal microbial diversity was increased from cirrhosis to early HCC with cirrhosis. Phylum Actinobacteria was increased in early HCC versus cirrhosis. Correspondingly, 13 genera including Gemmiger and Parabacteroides were enriched in early HCC versus cirrhosis. Butyrate-producing genera were decreased, while genera producing-lipopolysaccharide were increased in early HCC versus controls. The optimal 30 microbial markers were identified through a fivefold cross-validation on a random forest model and achieved an area under the curve of 80.64% between 75 early HCC and 105 non-HCC samples. Notably, gut microbial markers validated strong diagnosis potential for early HCC and even advanced HCC. Importantly, microbial markers successfully achieved a cross-region validation of HCC from Northwest China and Central China.ConclusionsThis study is the first to characterise gut microbiome in patients with HCC and to report the successful diagnosis model establishment and cross-region validation of microbial markers for HCC. Gut microbiota-targeted biomarkers represent potential non-invasive tools for early diagnosis of HCC.

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Sapogenol is a Major Microbial Metabolite in Human Plasma Associated with High Protein Soy-Based Diets: The Relevance for Functional Food Formulations

Foods ◽

10.3390/foods9040422 ◽

2020 ◽

Vol 9 (4) ◽

pp. 422

Author(s):

Madalina Neacsu ◽

Vassilios Raikos ◽

Yara Benavides-Paz ◽

Sylvia H. Duncan ◽

Gary J. Duncan ◽

...

Keyword(s):

Functional Foods ◽

Biological Activities ◽

Epidemiological Studies ◽

High Protein ◽

Biologically Active ◽

Soya Meal ◽

Human Volunteers ◽

Faecal Samples ◽

Health Promoting

Legumes are a source of health-promoting macro- and micronutrients, but also contain numerous phytochemicals with useful biological activities, an example of which are saponins. Epidemiological studies suggest that saponins may play a role in protection from cancer and benefit human health by lowering cholesterol. Therefore, they could represent good candidates for specialised functional foods. Following the consumption of a soya-rich high-protein weight-loss diet (SOYA HP WL), the concentrations of Soyasaponin I (SSI) and soyasapogenol B (SSB) were determined in faecal samples from human volunteers (n = 10) and found to be between 1.4 and 17.5 mg per 100 g fresh faecal sample. SSB was the major metabolite identified in volunteers’ plasma (n = 10) after consumption of the soya test meal (SOYA MEAL); the postprandial (3 h after meal) plasma concentration for SSB varied between 48.5 ng/mL to 103.2 ng/mL. The metabolism of SSI by the gut microbiota (in vitro) was also confirmed. This study shows that the main systemic metabolites of soyasaponin are absorbed from the gut and that they are bioavailable in plasma predominantly as conjugates of sapogenol. The metabolism and bioavailability of biologically active molecules represent key information necessary for the efficient development of functional foods.

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