scholarly journals Molecular Characterization of Bacteriophages for Microbial Source Tracking in Korea

2009 ◽  
Vol 75 (22) ◽  
pp. 7107-7114 ◽  
Author(s):  
Jung Eun Lee ◽  
Mi Young Lim ◽  
Sei Yoon Kim ◽  
Sunghee Lee ◽  
Heetae Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Phylogenetic Analyses ◽  
Microbial Source Tracking ◽  
Group Iv ◽  
Source Tracking ◽  
Fecal Samples ◽  
Human Waste ◽  
Reverse Transcription Pcr ◽  
Group I

ABSTRACT We investigated coliphages from various fecal sources, including humans and animals, for microbial source tracking in South Korea. Both somatic and F+-specific coliphages were isolated from 43 fecal samples from farms, wild animal habitats, and human wastewater plants. Somatic coliphages were more prevalent and abundant than F+ coliphages in all of the tested fecal samples. We further characterized 311 F+ coliphage isolates using RNase sensitivity assays, PCR and reverse transcription-PCR, and nucleic acid sequencing. Phylogenetic analyses were performed based on the partial nucleic acid sequences of 311 F+ coliphages from various sources. F+ RNA coliphages were most prevalent among geese (95%) and were least prevalent in cows (5%). Among the genogroups of F+ RNA coliphages, most F+ coliphages isolated from animal fecal sources belonged to either group I or group IV, and most from human wastewater sources were in group II or III. Some of the group I coliphages were present in both human and animal source samples. F+ RNA coliphages isolated from various sources were divided into two main clusters. All F+ RNA coliphages isolated from human wastewater were grouped with Qβ-like phages, while phages isolated from most animal sources were grouped with MS2-like phages. UniFrac significance statistical analyses revealed significant differences between human and animal bacteriophages. In the principal coordinate analysis (PCoA), F+ RNA coliphages isolated from human waste were distinctively separate from those isolated from other animal sources. However, F+ DNA coliphages were not significantly different or separate in the PCoA. These results demonstrate that proper analysis of F+ RNA coliphages can effectively distinguish fecal sources.

Application of mussels as biosamplers for characterization of faecal pollution in coastal recreational waters

2010 ◽  
Vol 62 (3) ◽  
pp. 586-593 ◽  
Author(s):  
P. Roslev ◽  
A. S. Bukh ◽  
L. Iversen ◽  
H. Sønderbo ◽  
N. Iversen
Keyword(s):  
Molecular Markers ◽  
Blue Mussel ◽  
Unknown Origin ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Recreational Waters ◽  
Human Waste ◽  
Faecal Pollution ◽  
Faecal Bacteria ◽  
Seawater Samples

Sources of faecal pollution in coastal recreational waters may be identified by analysing different host associated microorganisms or molecular markers. However, the microbial targets are often present at low numbers in moderately impacted waters, and often exhibit significant temporal and spatial variability in waters with fluctuating faecal loads. This patchy occurrence can limit successful detection of relevant targets in microbial source tracking studies. In this study, we explored the possibility for using the blue mussel (Mytilus edulis) as a biosampler for accumulation of faecal bacteria relevant for microbial source tracking. Non-contaminated blue mussels were transferred to three coastal recreational waters affected by faecal pollution of unknown origin. Molecular markers associated with animal and human waste were targeted by PCR and compared in seawater and mussel samples. The results demonstrated that transplanted mussels in simple enclosures accumulated and retained elevated levels of molecular markers associated with different types of faecal pollution. The targets included a novel putative human associated E. coli subgroup B2 VIII clone, and animal and human associated markers in enterococci (esp, M19, M66, M90, and M91). Human (sewage) associated markers including esp and M66 were sometimes not detectable in seawater samples despite known wastewater contamination, whereas the markers were detectable in mussels. We suggest that transplanted mussels should be considered as potential biosamplers in studies focusing on identifying source of faecal pollution in low or moderately impacted recreational waters. Bioaccumulation of molecular markers in mussels for several days may represent the water quality better than traditional grab samples from the water column.

Molecular and phylogenetic analyses of the liver amphistome Explanatum explanatum (Creplin, 1847) Fukui, 1929 in ruminants from Bangladesh and Nepal based on nuclear ribosomal ITS2 and mitochondrial nad1 sequences

2016 ◽  
Vol 91 (4) ◽  
pp. 497-503 ◽  
Author(s):  
U.K. Mohanta ◽  
H.B. Rana ◽  
B. Devkota ◽  
T. Itagaki
Keyword(s):  
Molecular Characterization ◽  
Phylogenetic Analyses ◽  
Economic Loss ◽  
Fixation Index ◽  
Its2 Region ◽  
Group I ◽  
Severe Liver Damage ◽  
Second Internal Transcribed Spacer ◽  
First Time

AbstractExplanatum explanatum flukes, liver amphistomes of ruminants, cause significant economic loss in the livestock industry by inducing severe liver damage. A total of 66 flukes from 26 buffaloes and 7 cattle in four different geographic areas of Bangladesh and 20 flukes from 10 buffaloes in the Chitwan district of Nepal were subjected for analysis. The sequences (442 bp) of the second internal transcribed spacer (ITS2) of ribosomal DNA and the variable fragments (657 bp) of mitochondrial nicotinamide dehydrogenase subunit 1 (nad1) of E. explanatum flukes from Bangladesh and Nepal were analysed. The aim of this study was molecular characterization of the flukes and to elucidate their origin and biogeography. In the ITS2 region, two genotypes were detected among the flukes from Bangladesh, while flukes from Nepal were of only one genotype. Phylogenetic analyses inferred from the nad1 gene revealed that at least four divergent populations (groups I–IV) are distributed in Bangladesh, whereas two divergent populations were found to be distributed in Nepal. Fst values (pairwise fixation index) suggest that Bangladeshi and Nepalese populations of group I to IV are significantly different from each other; but within groups III and IV, the populations from Bangladesh and Nepal were genetically close. This divergence in the nad1 gene indicates that each lineage of E. explanatum from diverse geography was co-adapted during the multiple domestication events of ruminants. This study, for the first time, provides molecular characterization of E. explanatum in Bangladesh and Nepal, and may provide useful information for elucidating its origin and dispersal route in Asia.

Hybridisation of F+ RNA coliphages detected in shellfish samples with oligonucleotide probes to assess the origin of microbiological pollution of shellfish

2006 ◽  
Vol 54 (3) ◽  
pp. 219-223 ◽  
Author(s):  
A. Vantarakis ◽  
D. Venieri ◽  
G. Komninou ◽  
M. Papapetropoulou
Keyword(s):  
Group Iv ◽  
Specific Information ◽  
Faecal Material ◽  
Human Waste ◽  
The Public ◽  
E Coli ◽  
Group I ◽  
Viral Indicators ◽  
Time Of Year ◽  
Male Specific

Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator, male-specific B+ coliphages, have been investigated as viral indicators of faecal contamination that may provide source-specific information for impacted environmental waters. This study compared the distribution of E. coli and F+ RNA bacteriophages in shellfish grown in harvesting areas of Greece and also examined the presence and proportions of the different subgroups of F+ RNA coliphages in shellfish. F+ RNA bacteriophages were present in shellfish at higher concentrations than E. coli. Elevated numbers of F+ RNA bacteriophages observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in Greece. The majority of F+ RNA coliphages detected in shellfish samples belonged to group IV which indicated the possible presence of animal faecal material in sample harvesting areas. Phages of groups II and III (human waste and human faecal material, respectively) were present at low levels. Finally, 8% of the phages hybridised were found to belong to group I. The presence of group IV showed seasonal distribution (more in winter, less in summer) whereas the other groups did not show any difference. Monitoring of F+ coliphage subgroups may indicate the presence and major sources of microbial inputs to surface waters; however, environmental effects on the relative occurrence of different groups need to be considered.

Revisiting theAcanthamoebaspecies that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences

Parasitology ◽  
2011 ◽  
Vol 139 (1) ◽  
pp. 45-52 ◽  
Author(s):  
ANA C. M. MAGLIANO ◽  
MARTA M. G. TEIXEIRA ◽  
SILVIA C. ALFIERI
Keyword(s):  
Opportunistic Infections ◽  
Phylogenetic Analyses ◽  
Sequence Divergence ◽  
Rrna Gene ◽  
Pathogenic Potential ◽  
Morphological Group ◽  
Isolation And Characterization ◽  
Group I ◽  
Group 2

SUMMARYFree-living amoebae of the genusAcanthamoebaare the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1–T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmentalAcanthamoebaisolates, all assigned to group I. Phylogenetic analyses based on partial (∼1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related toA. comandoni(genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2 0% sequence divergence) that likely represents a newAcanthamoebaspecies. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the newAcanthamoebaisolates induced the disruption of SIRC and HeLa cell monolayers.

scholarly journals Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization

2004 ◽  
Vol 70 (10) ◽  
pp. 5996-6004 ◽  
Author(s):  
Jan Vinjé ◽  
Sjon J. G. Oudejans ◽  
Jill R. Stewart ◽  
Mark D. Sobsey ◽  
Sharon C. Long
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Phylogenetic Analyses ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Reverse Line Blot ◽  
Source Tracking ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Male Specific

ABSTRACT In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qβ, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

scholarly journals Evolution and Characterization of Tetraonine Endogenous Retrovirus: a New Virus Related to Avian Sarcoma and Leukosis Viruses

2001 ◽  
Vol 75 (4) ◽  
pp. 2002-2009 ◽  
Author(s):  
Derek E. Dimcheff ◽  
Mallika Krishnan ◽  
David P. Mindell
Keyword(s):  
Southern Blotting ◽  
Phylogenetic Analyses ◽  
Endogenous Retrovirus ◽  
Pcr Analysis ◽  
Bonasa Umbellus ◽  
Long Terminal Repeats ◽  
Proviral Sequence ◽  
Reverse Transcription Pcr ◽  
Avian Sarcoma

ABSTRACT In a previous study, we found avian sarcoma and leukosis virus (ASLV) gag genes in 19 species of birds in the order Galliformes including all grouse and ptarmigan (Tetraoninae) surveyed. Our data suggested that retroviruses had been transmitted horizontally among some host species. To further investigate these elements, we sequenced a replication-defective retrovirus, here named tetraonine endogenous retrovirus (TERV), from Bonasa umbellus (ruffed grouse). This is the first report of a complete, replication-defective ASLV provirus sequence from any bird other than the domestic chicken. We found a replication-defective proviral sequence consisting of putative Gag and Env proteins flanked by long terminal repeats. Reverse transcription-PCR analysis showed that retroviral gagsequences closely related to TERV are transcribed, supporting the hypothesis that TERV is an active endogenous retrovirus. Phylogenetic analyses suggest that TERV may have arisen via recombination between different retroviral lineages infecting birds. Southern blotting usinggag probes showed that TERV occurs in tetraonines but not in chickens or ducks, suggesting that integration occurred after the earliest phasianid divergences but prior to the radiation of tetraonine birds.

scholarly journals Phylogenetic Analysis of Deformed Wing Virus Genotypes from Diverse Geographic Origins Indicates Recent Global Distribution of the Virus

2007 ◽  
Vol 73 (11) ◽  
pp. 3605-3611 ◽  
Author(s):  
Olga Berényi ◽  
Tamás Bakonyi ◽  
Irmgard Derakhshifar ◽  
Hemma Köglberger ◽  
Gražyna Topolska ◽  
...  
Keyword(s):  
New Zealand ◽  
Nucleic Acid ◽  
United Arab Emirates ◽  
Phylogenetic Trees ◽  
Phylogenetic Analyses ◽  
Pcr Amplification ◽  
Structural Protein ◽  
Deformed Wing Virus ◽  
Reverse Transcription Pcr ◽  
Geographic Origins

ABSTRACT Honeybees originating from 10 different countries (Austria, Poland, Germany, Hungary, Slovenia, Nepal, Sri Lanka, the United Arab Emirates, Canada, and New Zealand) located on four continents were analyzed for the presence of deformed wing virus (DWV) nucleic acid by reverse transcription-PCR. Two target regions within the DWV genome were selected for PCR amplification and subsequent sequencing, i.e., a region within the putative VP2 and VP4 structural-protein genes and a region within the RNA helicase enzyme gene. DWV nucleic acid was amplified from 34 honeybee samples representing all the above-mentioned countries with the notable exception of New Zealand. The amplification products were sequenced, and phylogenetic analyses of both genomic regions were performed independently. The phylogenetic analyses included all sequences determined in this study as well as previously published DWV sequences and the sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus 1 (VDV-1). In the sequenced regions, the DWV genome turned out to be highly conserved, independent of the geographic origins of the honeybee samples: the partial sequences exhibited 98 to 99% nucleotide sequence identity. Substitutions were most frequently observed at the same positions in the various DWV sequences. Due to the high level of sequence conservation, no significant clustering of the samples in the phylogenetic trees could be identified. On the other hand, the phylogenetic analyses support a genetic segregation of KGV and VDV-1 from DWV.

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