Microbiota Analysis of an Environmental Slurry and Its Potential Role as a Reservoir of Bovine Digital Dermatitis Pathogens

ABSTRACT At present, very little information exists regarding what role the environmental slurry may play as an infection reservoir and/or route of transmission for bovine digital dermatitis (DD), a disease which is a global problem in dairy herds. To investigate whether DD-related bacteria belong to the indigenous microbiota of the dairy herd environment, we used deep amplicon sequencing of the 16S rRNA gene in 135 slurry samples collected from different sites in 22 dairy farms, with and without DD-infected cows. Both the general bacterial populations and digital dermatitis-associated Treponema were targeted in this study. The results revealed significant differences in the bacterial communities between the herds, with only 12 bacterial taxa shared across at least 80% of all the individual samples. These differences in the herd microbiota appeared to reflect mainly between-herd variation. Not surprisingly, the slurry was dominated by ubiquitous gastrointestinal bacteria, such as Ruminococcaceae and Lachnospiraceae. Despite the low relative abundance of spirochetes, which ranged from 0 to 0.6%, we were able to detect small amounts of bacterial DNA from DD-associated treponemes in the slurry. However, the DD-associated Treponema spp. were detected only in samples from herds with reported DD problems. These data indicate that treponemes involved in the pathogenesis of DD are not part of the normal environmental microflora in dairy herds without clinical DD and, consequently, that slurry is not a primary reservoir of infection. IMPORTANCE Bovine digital dermatitis (DD), a dermal disease which causes lameness in dairy cattle, is a serious problem worldwide. To control this disease, the infection reservoirs and transmission routes of DD pathogens need to be clarified. The dairy herd slurry may be a pathogen reservoir of DD-associated bacteria. The rationale for the present study was, therefore, to examine whether DD-associated bacteria are always present in slurry or if they are found only in DD-afflicted herds. The results strongly indicated that DD Treponema spp. are not part of the indigenous slurry and, therefore, do not comprise an infection reservoir in healthy herds. This study applied next-generation sequencing technology to decipher the microbial compositions of environmental slurry of dairy herds with and without digital dermatitis.

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Discovery of Bovine Digital Dermatitis-Associated Treponema spp. in the Dairy Herd Environment by a Targeted Deep-Sequencing Approach

Applied and Environmental Microbiology ◽

10.1128/aem.00873-14 ◽

2014 ◽

Vol 80 (14) ◽

pp. 4427-4432 ◽

Cited By ~ 31

Author(s):

Kirstine Klitgaard ◽

Martin W. Nielsen ◽

Hans-Christian Ingerslev ◽

Mette Boye ◽

Tim K. Jensen

Keyword(s):

High Throughput ◽

Deep Sequencing ◽

Dairy Herd ◽

Operational Taxonomic Unit ◽

Rrna Gene ◽

Digital Dermatitis ◽

Content Type ◽

Phylogenetic Characterization ◽

Geographically Dispersed ◽

Environmental Reservoir

ABSTRACTThe bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genusTreponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of rRNA gene sequences from DD-associated treponemes in the dairy herd environment. Although the spread of DD appears to be facilitated by wet floors covered with slurry, no DD-associated treponemes have been isolated from this environment previously. Consequently, there is a lack of knowledge about the spread of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six geographically dispersed dairy herds. Species belonging to theTreponema denticola/Treponema pedis-like andTreponema phagedenis-like phylogenetic clusters were among the most prevalent treponemes in both the dairy herd environment and the DD lesions. By the high-throughput approach presented here, we have demonstrated that cow feces and environmental slurry are possible reservoirs of DD-associated treponemes. This method should enable further clarification of the etiopathogenesis of DD.

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The dysbiosis of ovine foot microbiome during the development and treatment of contagious ovine digital dermatitis

Animal Microbiome ◽

10.1186/s42523-021-00078-4 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

J. S. Duncan ◽

J. W. Angell ◽

P. Richards ◽

L. Lenzi ◽

G. J. Staton ◽

...

Keyword(s):

Antibiotic Treatment ◽

Amplicon Sequencing ◽

Strong Tendency ◽

Rrna Gene ◽

Digital Dermatitis ◽

Healthy State ◽

Foot Disease ◽

End Stage ◽

And Control ◽

Interdigital Dermatitis

Abstract Background Contagious Ovine Digital Dermatitis (CODD) is an emerging and common infectious foot disease of sheep which causes severe welfare and economic problems for the sheep industry. The aetiology of the disease is not fully understood and control of the disease is problematic. The aim of this study was to investigate the polybacterial aetiopathogenesis of CODD and the effects of antibiotic treatment, in a longitudinal study of an experimentally induced disease outbreak using a 16S rRNA gene amplicon sequencing approach. Results CODD was induced in 15/30 experimental sheep. During the development of CODD three distinct phenotypic lesion stages were observed. These were an initial interdigital dermatitis (ID) lesion, followed by a footrot (FR) lesion, then finally a CODD lesion. Distinct microbiota were observed for each lesion in terms of microbial diversity, clustering and composition. Porphyromonadaceae, Family XI, Veillonellaceae and Fusobacteriaceae were significantly associated with the diseased feet. Veillonellaceae and Fusobacteriaceae were most associated with the earlier stages of ID and footrot rather than CODD. Following antibiotic treatment of the sheep, the foot microbiota showed a strong tendency to return to the composition of the healthy state. The microbiota composition of CODD lesions collected by swab and biopsy methods were different. In particular, the Spirochaetaceae family were more abundant in samples collected by the biopsy method, suggesting that these bacteria are present in deeper tissues of the diseased foot. Conclusion In this study, CODD presented as part of a spectrum of poly-bacterial foot disease strongly associated with bacterial families Porphyromonadaceae, Family XI (a family in Clostridiales also known as Clostridium cluster XI), Veillonellaceae and Fusobacteriaceae which are predominately Gram-negative anaerobes. Following antibiotic treatment, the microbiome showed a strong tendency to return to the composition of the healthy state. The composition of the healthy foot microbiome does not influence susceptibility to CODD. Based on the data presented here and that CODD appears to be the severest end stage of sheep infectious foot disease lesions, better control of the initial ID and FR lesions would enable better control of CODD and enable better animal welfare.

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Sponge-Associated Bacteria Are Strictly Maintained in Two Closely Related but Geographically Distant Sponge Hosts

Applied and Environmental Microbiology ◽

10.1128/aem.05285-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7207-7216 ◽

Cited By ~ 76

Author(s):

Naomi F. Montalvo ◽

Russell T. Hill

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Communities ◽

Sponge Species ◽

Rrna Gene ◽

Gene Sequences ◽

Associated Bacteria ◽

Content Type ◽

Key Species ◽

Sponge Associated Bacteria

ABSTRACTThe giant barrel spongesXestospongiamutaandXestospongiatestudinariaare ubiquitous in tropical reefs of the Atlantic and Pacific Oceans, respectively. They are key species in their respective environments and are hosts to diverse assemblages of bacteria. These two closely related sponges from different oceans provide a unique opportunity to examine the evolution of sponge-associated bacterial communities. Mitochondrial cytochrome oxidase subunit I gene sequences fromX.mutaandX.testudinariashowed little divergence between the two species. A detailed analysis of the bacterial communities associated with these sponges, comprising over 900 full-length 16S rRNA gene sequences, revealed remarkable similarity in the bacterial communities of the two species. Both sponge-associated communities include sequences found only in the twoXestospongiaspecies, as well as sequences found also in other sponge species and are dominated by three bacterial groups,Chloroflexi,Acidobacteria, andActinobacteria. While these groups consistently dominate the bacterial communities revealed by 16S rRNA gene-based analysis of sponge-associated bacteria, the depth of sequencing undertaken in this study revealed clades of bacteria specifically associated with each of the twoXestospongiaspecies, and also with the genusXestospongia, that have not been found associated with other sponge species or other ecosystems. This study, comparing the bacterial communities associated with closely related but geographically distant sponge hosts, gives new insight into the intimate relationships between marine sponges and some of their bacterial symbionts.

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Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants

Applied and Environmental Microbiology ◽

10.1128/aem.03044-19 ◽

2020 ◽

Vol 86 (9) ◽

Cited By ~ 5

Author(s):

Jannie Munk Kristensen ◽

Marta Nierychlo ◽

Mads Albertsen ◽

Per Halkjær Nielsen

Keyword(s):

Wastewater Treatment ◽

Pathogenic Bacteria ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Content Type ◽

Effluent Quality ◽

Culture Independent ◽

Influent Wastewater

ABSTRACT Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the “clean” effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock. IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri. Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

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Endophytic bacterial communities and spatiotemporal variations in cotton roots in Xinjiang, China

Canadian Journal of Microbiology ◽

10.1139/cjm-2020-0249 ◽

2020 ◽

Author(s):

Yingwu Shi ◽

Hongmei Yang ◽

Min Chu ◽

Xinxiang Niu ◽

Xiangdong Huo ◽

...

Keyword(s):

Community Structure ◽

Endophytic Bacteria ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Bacterial Populations ◽

Operational Taxonomic Units ◽

Cotton Sample ◽

Endophytic Bacterial Community ◽

V3 Region ◽

Illumina Amplicon Sequencing

Endophytic bacteria may be important for plant health and other ecologically relevant functions of cotton. However, the endophytic bacterial community structure and diversity in cotton is still poorly characterized. We investigated the community structure of endophytic bacteria in cotton roots growing in Xinjiang, China, using the Illumina amplicon sequencing. A total of 60.84 M effective sequences of 16S rRNA gene V3 region were obtained from cotton samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in cotton, that is, 81-338 OTUs in a cotton sample, at 3% cutoff level and sequencing depth of 50000 sequences. We identified 23 classes from the resulting 2,723,384 sequences. Gammaproteobacteria were the dominant class in all cottons, followed by Alphaproteobacteria, Actinobacteria and Bacilli. A marked difference in the diversity of endophytic bacteria in cotton for different growth periods was evident. The greatest number of OTUs was detected during seedling (654 OTUs) and budding (381 OTUs). Endophytic bacteria diversity was reduced during flowering (350 OTUs) and boll-opening (351 OTUs). 217 OTUs were common to all four periods. There were more tags of Pantoea in Shihezi than other locations. While there were more tags of Erwinia in Hami than other locations. The dynamics of endophytic bacteria communities were influenced by plant growth stage. These results show the complexity of the bacterial populations present in inner tissues of cotton.

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Spatial and Temporal Variation in Enterococcal Abundance and Its Relationship to the Microbial Community in Hawaii Beach Sand and Water

Applied and Environmental Microbiology ◽

10.1128/aem.00135-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3601-3609 ◽

Cited By ~ 28

Author(s):

Henglin Cui ◽

Kun Yang ◽

Eulyn Pagaling ◽

Tao Yan

Keyword(s):

Microbial Community ◽

Temporal Variation ◽

Beach Sand ◽

Spatial And Temporal Variation ◽

Rrna Gene ◽

Fecal Indicator ◽

Bacterial Populations ◽

Mass Unit ◽

Content Type ◽

Beach Water

ABSTRACTRecent studies have reported high levels of fecal indicator enterococci in marine beach sand. This study aimed to determine the spatial and temporal variation of enterococcal abundance and to evaluate its relationships with microbial community parameters in Hawaii beach sand and water. Sampling at 23 beaches on the Island of Oahu detected higher levels of enterococci in beach foreshore sand than in beach water on a mass unit basis. Subsequent 8-week consecutive samplings at two selected beaches (Waialae and Kualoa) consistently detected significantly higher levels of enterococci in backshore sand than in foreshore/nearshore sand and beach water. Comparison between the abundance of enterococci and the microbial communities showed that enterococci correlated significantly with totalVibrioin all beach zones but less significantly with total bacterial density andEscherichia coli. Samples from the different zones of Waialae beach were sequenced by 16S rRNA gene pyrosequencing to determine the microbial community structure and diversity. The backshore sand had a significantly more diverse community and contained different major bacterial populations than the other beach zones, which corresponded to the spatial distribution pattern of enterococcal abundance. Taken together, multiple lines of evidence support the possibility of enterococci as autochthonous members of the microbial community in Hawaii beach sand.

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Growth and Population Dynamics of the Anaerobic Fe(II)-Oxidizing and Nitrate-Reducing Enrichment Culture KS

Applied and Environmental Microbiology ◽

10.1128/aem.02173-17 ◽

2018 ◽

Vol 84 (9) ◽

Cited By ~ 15

Author(s):

Claudia Tominski ◽

Helene Heyer ◽

Tina Lösekann-Behrens ◽

Sebastian Behrens ◽

Andreas Kappler

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Community Composition ◽

Enrichment Culture ◽

Operational Taxonomic Unit ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Organic Substrates ◽

Content Type ◽

Heterotrophic Denitrification

ABSTRACTMost isolated nitrate-reducing Fe(II)-oxidizing microorganisms are mixotrophic, meaning that Fe(II) is chemically oxidized by nitrite that forms during heterotrophic denitrification, and it is debated to which extent Fe(II) is enzymatically oxidized. One exception is the chemolithoautotrophic enrichment culture KS, a consortium consisting of a dominant Fe(II) oxidizer,Gallionellaceaesp., and less abundant heterotrophic strains (e.g.,Bradyrhizobiumsp.,Nocardioidessp.). Currently, this is the only nitrate-reducing Fe(II)-oxidizing culture for which autotrophic growth has been demonstrated convincingly for many transfers over more than 2 decades. We used 16S rRNA gene amplicon sequencing and physiological growth experiments to analyze the community composition and dynamics of culture KS with various electron donors and acceptors. Under autotrophic conditions, an operational taxonomic unit (OTU) related to known microaerophilic Fe(II) oxidizers within the familyGallionellaceaedominated culture KS. With acetate as an electron donor, most 16S rRNA gene sequences were affiliated withBradyrhizobiumsp.Gallionellaceaesp. not only was able to oxidize Fe(II) under autotrophic and mixotrophic conditions but also survived over several transfers of the culture on only acetate, although it then lost the ability to oxidize Fe(II).Bradyrhizobiumspp. became and remained dominant when culture KS was cultivated for only one transfer under heterotrophic conditions, even when conditions were reverted back to autotrophic in the next transfer. This study showed a dynamic microbial community in culture KS that responded to changing substrate conditions, opening up questions regarding carbon cross-feeding, metabolic flexibility of the individual strains in KS, and the mechanism of Fe(II) oxidation by a microaerophile in the absence of O2.IMPORTANCENitrate-reducing Fe(II)-oxidizing microorganisms are present in aquifers, soils, and marine and freshwater sediments. Most nitrate-reducing Fe(II) oxidizers known are mixotrophic, meaning that they need organic carbon to continuously oxidize Fe(II) and grow. In these microbes, Fe(II) was suggested to be chemically oxidized by nitrite that forms during heterotrophic denitrification, and it remains unclear whether or to what extent Fe(II) is enzymatically oxidized. In contrast, the enrichment culture KS was shown to oxidize Fe(II) autotrophically coupled to nitrate reduction. This culture contains the designated Fe(II) oxidizerGallionellaceaesp. and several heterotrophic strains (e.g.,Bradyrhizobiumsp.). We showed that culture KS is able to metabolize Fe(II) and a variety of organic substrates and is able to adapt to dynamic environmental conditions. When the community composition changed andBradyrhizobiumbecame the dominant community member, Fe(II) was still oxidized byGallionellaceaesp., even when culture KS was cultivated with acetate/nitrate [Fe(II) free] before being switched back to Fe(II)/nitrate.

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Analysis of Bacterial Communities during Clostridium difficile Infection in the Mouse

Infection and Immunity ◽

10.1128/iai.00145-15 ◽

2015 ◽

Vol 83 (11) ◽

pp. 4383-4391 ◽

Cited By ~ 28

Author(s):

Ekaterina G. Semenyuk ◽

Valeriy A. Poroyko ◽

Pehga F. Johnston ◽

Sara E. Jones ◽

Katherine L. Knight ◽

...

Keyword(s):

Clostridium Difficile ◽

16S Rrna ◽

Bacterial Species ◽

Intestinal Content ◽

Rrna Gene ◽

Gi Tract ◽

Bacterial Populations ◽

Content Type ◽

Minority Member ◽

Gene Sequence Analysis

ABSTRACTClostridium difficileinfection (CDI) is a major cause of health care-associated disease. CDI initiates with ingestion ofC. difficilespores, germination in the gastrointestinal (GI) tract, and then colonization of the large intestine. The interactions betweenC. difficilecells and other bacteria and with host mucosa during CDI remain poorly understood. Here, we addressed the hypothesis that, in a mouse model of CDI,C. difficileresides in multicellular communities (biofilms) in association with host mucosa. To do this, we paraffin embedded and then sectioned the GI tracts of infected mice at various days postinfection (p.i.). We then used fluorescentin situhybridization (FISH) with 16S rRNA probes targeting most bacteria as well asC. difficilespecifically. The results revealed thatC. difficileis present as a minority member of communities in the outer (loose) mucus layer, in the cecum and colon, starting at day 1 p.i. To generate FISH probes that identify bacteria within mucus-associated communities harboringC. difficile, we characterized bacterial populations in the infected mouse GI tract using 16S rRNA gene sequence analysis of bacterial DNA prepared from intestinal content. This analysis revealed the presence of genera of several families belonging toBacteroidetesandFirmicutes. These data suggest that formation of multispecies communities associated with the mucus of the cecum and colon is an important early step in GI tract colonization. They raise the possibility that other bacterial species in these communities modulate the ability ofC. difficileto successfully colonize and, thereby, cause disease.

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16S rRNA Gene Amplicon Sequencing Data of Tailing and Nontailing Rhizosphere Soils of Mimosa pudica from a Heavy Metal-Contaminated Ex-Tin Mining Area

Microbiology Resource Announcements ◽

10.1128/mra.00761-20 ◽

2020 ◽

Vol 9 (42) ◽

Author(s):

Saidu Abdullahi ◽

Hazzeman Haris ◽

Kamarul Z. Zarkasi ◽

Hamzah G. Amir

Keyword(s):

Heavy Metal ◽

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Mimosa Pudica ◽

Sequencing Data ◽

Content Type ◽

Rhizosphere Soils

ABSTRACT The 16S rRNA gene amplicon sequence data from tailing and nontailing rhizosphere soils of Mimosa pudica from a heavy metal-contaminated area are reported here. Diverse bacterial taxa were represented in the results, and the most dominant phyla were Proteobacteria (41.2%), Acidobacteria (17.1%), and Actinobacteria (14.4%).

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Most of the Dominant Members of Amphibian Skin Bacterial Communities Can Be Readily Cultured

Applied and Environmental Microbiology ◽

10.1128/aem.01486-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6589-6600 ◽

Cited By ~ 25

Author(s):

Jenifer B. Walke ◽

Matthew H. Becker ◽

Myra C. Hughey ◽

Meredith C. Swartwout ◽

Roderick V. Jensen ◽

...

Keyword(s):

Bacterial Community ◽

Batrachochytrium Dendrobatidis ◽

Amplicon Sequencing ◽

Reference Database ◽

Rrna Gene ◽

Amphibian Skin ◽

Amphibian Species ◽

Content Type ◽

Average Percentage ◽

Culture Independent

ABSTRACTCurrently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogenBatrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened byB. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individual's bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health.

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