scholarly journals Microbial Signatures of Oral Dysbiosis, Periodontitis and Edentulism Revealed by Gene Meter Methodology

2016 ◽  
Author(s):  
M. Colby Hunter ◽  
Alex E. Pozhitkov ◽  
Peter A. Noble
Keyword(s):  
Clinical Status ◽  
Clinical Signs ◽  
Rrna Gene ◽  
False Negatives ◽  
Gene Target ◽  
Adsorption Models ◽  
Highly Correlated ◽  
Oral Conditions ◽  
Specific Disease State ◽  
Generation Sequencing

ABSTRACTConceptual models suggest certain microorganisms (e.g., the red complex) are indicative of a specific disease state (e.g., periodontitis); however, recent studies have questioned the validity of these models. Here, the abundances of 500+ microbial species were determined in 16 patients with clinical signs of one of the following oral conditions: periodontitis, established caries, edentulism, and oral health. Our goal was to determine if the abundances of certain microorganisms reflect dysbiosis or a specific clinical condition that could be used as a signature for dental research. Microbial abundances were determined by the analysis of 138,718 calibrated probes using Gene Meter methodology. Each 16S rRNA gene was targeted by an average of 194 unique probes (n=25 nt). The calibration involved diluting pooled gene target samples, hybridizing each dilution to a DNA microarray, and fitting the probe intensities to adsorption models. The fit of the model to the experimental data was used to assess individual and aggregate probe behavior; good fits (R2>0.90) were retained for back-calculating microbial abundances from patient samples. The abundance of a gene was determined from the median of all calibrated individual probes or from the calibrated abundance of all aggregated probes. With the exception of genes with low abundances (< 2 arbitrary units), the abundances determined by the different calibrations were highly correlated (r ∼1.0). Seventeen genera were classified as signatures of dysbiosis because they had significantly higher abundances in patients with periodontitis and edentulism when contrasted with health. Similarly, 13 genera were classified as signatures of periodontitis, and 14 genera were classified as signatures of edentulism. The signatures could be used, individually or in combination, to assess the clinical status of a patient (e.g., evaluating treatments such as antibiotic therapies). Comparisons of the same patient samples revealed high false negatives (45%) for next-generation-sequencing results and low false positives (7%) for Gene Meter results.

scholarly journals Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Elton J. R. Vasconcelos ◽  
Chayan Roy ◽  
Joseph A. Geiger ◽  
Kristina M. Oney ◽  
Melody Koo ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Next Generation Sequencing ◽  
Complete Analysis ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Spotted Fever Group ◽  
Next Generation ◽  
Vector Borne ◽  
16 S Rrna ◽  
Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

scholarly journals Comparative analysis of bacterioplankton assemblages from two subtropical karst reservoirs of southwestern China with contrasting trophic status

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiang Li ◽  
Yadan Huang ◽  
Shenglin Xin ◽  
Zhongyi Li
Keyword(s):  
Trophic Status ◽  
Alpha Diversity ◽  
Decisive Role ◽  
Sensu Stricto ◽  
Karst Area ◽  
Southwestern China ◽  
Rrna Gene ◽  
Karst Water ◽  
Bacterioplankton Community ◽  
Generation Sequencing

AbstractAlthough bacterioplankton play an important role in aquatic ecosystems, less is known about bacterioplankton assemblages from subtropical karst reservoirs of southwestern China with contrasting trophic status. Here, 16S rRNA gene next-generation sequencing coupled with water chemistry analysis was applied to compare the bacterioplankton communities from a light eutrophic reservoir, DL Reservoir, and a mesotrophic reservoir, WL Reservoir, in subtropical karst area of southwestern China. Our findings indicated that Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria and Verrucomicrobia dominated bacterioplankton community with contrasting relative frequency in the two subtropical karst reservoirs. Proteobacteria and Bacteroidetes were the core communities, which played important roles in karst biogeochemical cycles. Though WT, TN and DOC play the decisive role in assembling karst aquatic bacterioplankton, trophic status exerted significantly negative direct effects on bacterioplankton community composition and alpha diversity. Due to contrasting trophic status in the two reservoirs, the dominant taxa such as Enterobacter, Clostridium sensu stricto, Candidatus Methylacidiphilum and Flavobacteriia, that harbor potential functions as valuable and natural indicators of karst water health status, differed in DL Reservoir and WL Reservoir.

scholarly journals Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

2020 ◽  
Vol 9 (1) ◽  
pp. 21
Author(s):  
Abdul Ghafar ◽  
Anson V. Koehler ◽  
Ross S. Hall ◽  
Charles G. Gauci ◽  
Robin B. Gasser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Water Buffalo ◽  
Hypervariable Region ◽  
Small Subunit ◽  
Rrna Gene ◽  
Next Generation ◽  
Small Subunit Rrna ◽  
Tropical Theileriosis ◽  
Ecological Zones ◽  
Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

scholarly journals The application of a new laminitis scoring method to model the rate and pattern of improvement from equine endocrinopathic laminitis in a clinical setting

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
A. Meier ◽  
J. McGree ◽  
R. Klee ◽  
J. Preuß ◽  
D. Reiche ◽  
...  
Keyword(s):  
Best Practice ◽  
Clinical Status ◽  
Clinical Signs ◽  
Scoring Method ◽  
Pain Medications ◽  
Exponential Decay Model ◽  
History Of ◽  
Consistent Method ◽  
Foot Disease ◽  
New Treatments

Abstract Background Endocrinopathic, or hyperinsulinaemia-associated laminitis (HAL) is a common and debilitating equine foot disease, and although no pharmacological treatments are registered, several are under development. To evaluate the effect of such treatments, an accurate and consistent method is needed to track the clinical signs of laminitis over time, and the natural history of the disease, in terms of a ‘normal’ pattern of improvement, needs to be understood. This study examined the improvement pattern in clinical cases of naturally-occurring HAL subjected to a range of best-practice interventions, using two different scoring methods. Eighty horses and ponies with suspected HAL were enrolled in a study conducted at 16 veterinary practices across Germany. The severity of laminitis was assessed by independent veterinarians using both the traditional Obel method and a modified Obel method developed by Meier and colleagues. Assessments were made on the day of diagnosis (d 0), then on days 4, 9, 14, 25 and 42 during the intervention period. Pain medications were withheld for 24 h prior to clinical examination in all cases. Results Time to marked improvement from laminitis varied between individuals, but was difficult to monitor accurately using the Obel method, with the median grade being 2/4 on days 0 and 4, then 0/4 from d 9 onwards. More subtle changes could be identified using the Meier method, however, and the median scores were seen to follow the form of an exponential decay model in most horses, improving from 8/12 on d 0, to 0/12 on d 25. Within this composite scoring method, considerable variation was observed in the rate of improvement of individual clinical signs, with the average time taken for each sign to reach a median score of 0 ranging from 4 days (foot lift and weight shifting) to 25 days (gait when turned in a circle) across all 80 horses. Conclusions The Meier method provides a reliable and consistent method for monitoring the clinical status of horses with HAL, and despite the variability, the pattern of improvement described here should provide a useful benchmark against which individual cases and new treatments can be assessed.

scholarly journals Structure of Microbial Communities When Complementary Effluents Are Anaerobically Digested

2021 ◽  
Vol 11 (3) ◽  
pp. 1293
Author(s):  
Ana Eusébio ◽  
André Neves ◽  
Isabel Paula Marques
Keyword(s):  
Anaerobic Digestion ◽  
Microbial Communities ◽  
Volatile Fatty Acids ◽  
Olive Mill Wastewater ◽  
Organic Load ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Olive Mill

Olive oil and pig productions are important industries in Portugal that generate large volumes of wastewater with high organic load and toxicity, raising environmental concerns. The principal objective of this study is to energetically valorize these organic effluents—piggery effluent and olive mill wastewater—through the anaerobic digestion to the biogas/methane production, by means of the effluent complementarity concept. Several mixtures of piggery effluent were tested, with an increasing percentage of olive mill wastewater. The best performance was obtained for samples of piggery effluent alone and in admixture with 30% of OMW, which provided the same volume of biogas (0.8 L, 70% CH4), 63/75% COD removal, and 434/489 L CH4/kg SVin, respectively. The validation of the process was assessed by molecular evaluation through Next Generation Sequencing (NGS) of the 16S rRNA gene. The structure of the microbial communities for both samples, throughout the anaerobic process, was characterized by the predominance of bacterial populations belonging to the phylum Firmicutes, mainly Clostridiales, with Bacteroidetes being the subdominant populations. Archaea populations belonging to the genus Methanosarcina became predominant throughout anaerobic digestion, confirming the formation of methane mainly from acetate, in line with the greatest removal of volatile fatty acids (VFAs) in these samples.

scholarly journals Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

2020 ◽  
Vol 5 (4) ◽  
pp. 2473011420S0002
Author(s):  
Yoonjung Choi ◽  
Irvin Oh
Keyword(s):  
Next Generation Sequencing ◽  
False Positive ◽  
Bacterial Genome ◽  
Rrna Gene ◽  
Next Generation ◽  
Antibiotic Resistant ◽  
Streptococcus Species ◽  
Resistant Genes ◽  
Standard Culture ◽  
Generation Sequencing

Category: Other Introduction/Purpose: Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method. Methods: We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed. Results: In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens. Conclusion: NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy. [Table: see text]

scholarly journals The role of ultrasound imaging in vascular compression syndromes

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Renato Farina ◽  
Pietro Valerio Foti ◽  
Andrea Conti ◽  
Francesco Aldo Iannace ◽  
Isabella Pennisi ◽  
...  
Keyword(s):  
Thoracic Outlet Syndrome ◽  
Clinical Signs ◽  
Vascular Compression ◽  
Nutcracker Syndrome ◽  
Vascular Changes ◽  
False Negatives ◽  
Dunbar Syndrome ◽  
Level Method ◽  
Compression Syndromes

AbstractVascular compression syndromes are rare alterations that have in common the compression of an arterial and/or venous vessel by contiguous structures and can be congenital or acquired. The best known are the Thoracic Outlet Syndrome, Nutcracker Syndrome, May–Thurner Syndrome, and Dunbar Syndrome. The incidence of these pathologies is certainly underestimated due to the non-specific clinical signs and their frequent asymptomaticity. Being a first-level method, Ultrasound plays a very important role in identifying these alterations, almost always allowing a complete diagnostic classification. If in expert hands, this method can significantly contribute to the reduction of false negatives, especially in the asymptomatic population, where the finding of the aforementioned pathologies often happens randomly following routine checks. In this review, we briefly discuss the best known vascular changes, the corresponding ultrasound anatomy, and typical ultrasound patterns.

scholarly journals Serra da Estrela PDO Cheese Microbiome as Revealed by Next Generation Sequencing

2021 ◽  
Vol 9 (10) ◽  
pp. 2007
Author(s):  
Rui Rocha ◽  
Manuela Vaz Velho ◽  
Joana Santos ◽  
Paulo Fernandes
Keyword(s):  
Next Generation Sequencing ◽  
Relative Abundance ◽  
Raw Materials ◽  
Rrna Gene ◽  
Next Generation ◽  
Candida Spp ◽  
Characterization Study ◽  
Materials Used ◽  
Serra Da Estrela ◽  
Generation Sequencing

Serra da Estrela PDO cheese is the oldest traditional cheese manufactured in Portugal. In this work, its microbiome as well as the main raw materials used in cheese production, raw ewes’ milk and thistle flowers (Cynara cardunculus L.), were characterized using next generation sequencing. Samples were accordingly retrieved from a local producer over two consecutive production campaigns and at different time periods within each campaign. The bacterial and fungi communities associated with each matrix were accessed through sequencing of V3−V4 and Internal Transcribed Spacer 2 regions of rRNA gene amplicons, respectively. A high microbial diversity was found associated to each matrix, differing significantly (p < 0.05) from each other. Over 500 taxa were identified in each analyzed matrix, ranging from dominant (relative abundance > 1%), sub-dominant (0.01−1%) and rare taxa (<0.01%). Specifically, in cheese, 30 taxa were present in all analyzed samples (core taxa), including species of Leuconostoc spp. and Lactococcus spp. for bacteria and Candida spp., Debaryomyces spp. and Yarrowia spp. for fungi, that were cumulatively the most prevalent genera in Serra da Estrela PDO cheese (average relative abundance ≥10%). Ultimately, this characterization study may contribute to a better understanding of the microbial dynamics of this traditional PDO product, namely the influence of raw materials on cheese microbiome, and could assist producers interested in preserving the identity, quality and safety of Serra da Estrela PDO cheese.

scholarly journals Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

2021 ◽  
Vol 15 (10) ◽  
pp. e0009779
Author(s):  
Fakhriddin Sarzhanov ◽  
Funda Dogruman-Al ◽  
Monica Santin ◽  
Jenny G. Maloney ◽  
Ayse Semra Gureser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gastrointestinal Symptoms ◽  
Molecular Methods ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Next Generation ◽  
Dientamoeba Fragilis ◽  
Significant Difference ◽  
Generation Sequencing ◽  
Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

scholarly journals Bacterial community associated with worker honeybees (Apis mellifera) affected by European foulbrood

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3816 ◽  
Author(s):  
Tomas Erban ◽  
Ondrej Ledvinka ◽  
Martin Kamler ◽  
Bronislava Hortova ◽  
Marta Nesvorna ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Clinical Symptoms ◽  
Clinical Signs ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Melissococcus Plutonius ◽  
European Foulbrood ◽  
Bacterial Groups ◽  
V3 Region

BackgroundMelissococcus plutoniusis an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis melliferaL.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies.MethodsThe study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1–V3 region, as performed through Illumina MiSeq amplicon sequencing.ResultsThe bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence ofM. plutoniusthan those from EFB1 asymptomatic colonies.Melissococcus plutoniuswas identified in all EFB1 colonies as well as in some of the control colonies. The proportions ofFructobacillus fructosus,Lactobacillus kunkeei,Gilliamella apicola,Frischella perrara, andBifidobacterium coryneformewere higher in EFB2 than in EFB1, whereasLactobacillus melliswas significantly higher in EFB2 than in EFB0.Snodgrassella alviandL. melliventris,L. helsingborgensisand,L. kullabergensisexhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence ofBartonella apisandCommensalibacter intestiniwere higher in EFB0 than in EFB2 and EFB1.Enterococcus faecalisincidence was highest in EFB2.ConclusionsHigh-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence ofM. plutoniuswithin the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmittingM. plutoniusdue to the greatly increased incidence of the pathogen. The presence ofM. plutoniussequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms thatE. faecalisis a secondary invader toM. plutonius; however, other putative secondary invaders were not identified in this study.

Sign in / Sign up

Export Citation Format

Share Document