bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimization of a Reusable Hollow-Fiber Ultrafilter for Simultaneous Concentration of Enteric Bacteria, Protozoa, and Viruses from Water
Authors: Morales-Morales Hugo A. , Vidal Guadalupe , Olszewski John , Rock Channah M. , Dasgupta Debanjana , Oshima Kevin H. , Smith Geoffrey B. 
Published: July 2003

ABSTRACT

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10
5
to 10
6
CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased
Escherichia coli
strain K-12 recoveries from 70 to 84% and
Salmonella enterica
serovar Enteritidis recoveries from 36 to 72%. An
E. coli
antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When
E. coli
XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 × 10
5
and 2.4 × 10
3
per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of
E. coli
XL1-Blue (9 × 10
5
CFU/liter),
Cryptosporidium parvum
oocysts (10 oocysts/liter), phage T1 (10
5
PFU/liter), and phage PP7 (10
5
PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of
Cryptosporidium
oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.
Ingestion of
Salmonella enterica
Serotype Poona by a Free-Living Nematode,
Caenorhabditis elegans
, and Protection against Inactivation by Produce Sanitizers
Authors: Caldwell Krishaun N. , Adler Barbara B. , Anderson Gary L. , Williams Phillip L. , Beuchat Larry R. 
Published: July 2003

ABSTRACT

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables.
Caenorhabditis elegans
was selected as a model to study the effectiveness of sanitizers in killing
Salmonella enterica
serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on
S. enterica
serotype Poona were treated with produce sanitizers. Treatment with 20 μg of free chlorine/ml significantly (α = 0.05) reduced the population of
S. enterica
serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of
S. enterica
serotype Poona cells surviving treatments with 20 to 500 μg of chlorine/ml, suggesting that reductions caused by treatment with 20 μg of chlorine/ml resulted from inactivation of
S. enterica
serotype Poona on the surface of
C. elegans
but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 μg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log
10
CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 μg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log
10
CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing
S. enterica
serotype Poona and lactic acid was the most effective. Treatment with up to 500 μg of chlorine/ml, 1% hydrogen peroxide, 2,550 μg of Sanova/ml, 40 μg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of
C. elegans
. Treatments were also applied to cantaloupe rind and lettuce inoculated with
S. enterica
serotype Poona or
C. elegan
s that had ingested
S. enterica
serotype Poona. Protection of ingested
S. enterica
serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that
S. enterica
serotype Poona ingested by
C. elegans
may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.
Use of Pulsed-Field Gel Electrophoresis To Characterize the Heterogeneity and Clonality ofSalmonellaIsolates Obtained from the Carcasses and Feces of Swine at Slaughter
Authors: Wonderling Laura , Pearce Rachel , Wallace F. Morgan , Call Jeffrey E. , Feder Ingrid , Tamplin Mark , Luchansky John B. 
Published: July 2003

ABSTRACTSalmonella entericaisolates were recovered from swine at a collaborating processing plant over a 2-month period in the spring of 2000. In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmedSalmonellaisolates from the 84Salmonella-positive samples obtained from the previous study. A total of 32 different PFGE pulsotypes were observed visually, and a BioNumerics software analysis clustered those pulsotypes into 12 PFGE groups. The B, F, and G groups predominated throughout the sampling period and were isolated from 39, 22, and 13% of the swine, respectively. In addition, multiple isolates were obtained from 67 of the 84Salmonella-positive samples, and subtyping revealed multiple PFGE profiles in 35 of these 67 (62%) samples. Both carcass and fecal isolates ofSalmonellawere recovered from 13 swine, resulting in “matched” samples. Molecular typing of the 252 isolates recovered from the matched samples revealed that 7 (54%) of the 13 carcasses were contaminated withSalmonellapulsotypes that were not isolated from the feces of the same animal. Conversely, from 6 of the 13 (46%) matched animals,Salmonellaclonal types were isolated from the feces that were not isolated from the carcass of the same animal. These data establish that each lot of swine introduces new contaminants into the plant environment and that swine feces from one animal can contaminate many carcasses. In addition, these results indicate that the examination of multipleSalmonellaisolates from positive samples is necessary to determine the variety of potential contaminants of swine carcasses during slaughter and processing.
Identification ofCryptosporidiumspp. Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay
Authors: Nichols R. A. B. , Campbell B. M. , Smith H. V. 
Published: July 2003

ABSTRACTWe describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities ofCryptosporidiumspp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted fromCryptosporidium parvumoocysts,C. muris(RN 66),C. baileyi(Belgium strain, LB 19), human-derivedC. meleagridis,C. felis(DNA from oocysts isolated from a cat), andC. andersoniwas used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymesDraI andVspI. Discrimination betweenC. andersoniandC. murisisolates was confirmed by a separate, subsequent digestion withDdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. TheCryptosporidiumspecies detected in these finished water samples wasC. parvumgenotype 1. This method consistently and routinely detected >5 oocysts per sample.
A Selective Medium for Quantitative Reisolation of
Trichoderma harzianum
from
Agaricus bisporus
Compost
Authors: Williams Josie , Clarkson John M. , Mills Peter R. , Cooper Richard M. 
Published: July 2003

ABSTRACT

We adapted a selective medium, previously developed for reisolation of
Trichoderma
spp. from soil, for quantitative determination of growth of
T. harzianum
from commercial
Agaricus bisporus
composts. This medium enables comparisons of aggressive (sensu inhibition of
A. bisporus
yield) with nonaggressive
T. harzianum
groups. The resulting medium contains the antimicrobials chloramphenicol, streptomycin, quintozene, and propamocarb and was highly selective, allowing the recovery of
T. harzianum
, as viable conidia and hyphal fragments, in compact colonies with the absence of visible microbial contaminants.
Detection and Diversity Assessment of
Xylella fastidiosa
in Field-Collected Plant and Insect Samples by Using 16S rRNA and
gyrB
Sequences
Authors: Rodrigues Jorge L. M. , Silva-Stenico M. E. , Gomes J. E. , Lopes J. R. S. , Tsai S. M. 
Published: July 2003

ABSTRACT

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium
Xylella fastidiosa.
The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the
gyrB
gene were determined for 18 strains of
X. fastidiosa
isolated from different plant hosts. A phylogenetic analysis, based on
gyrB
, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and
gyrB
genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR.
Xylella fastidiosa
was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of
X. fastidiosa
may facilitate the understanding of its ecological significance and prevention of spread of the disease.
Mode of Bactericidal Action of Silver Zeolite and Its Comparison with That of Silver Nitrate
Authors: Matsumura Yoshinobu , Yoshikata Kuniaki , Kunisaki Shin-ichi , Tsuchido Tetsuaki 
Published: July 2003

ABSTRACT
The properties of the bactericidal action of silver zeolite as affected by inorganic salts and ion chelators were similar to those of silver nitrate. The results suggest that the contact of the bacterial cell with silver zeolite, the consequent transfer of silver ion to the cell, and the generation of reactive oxygen species in the cell are involved in the bactericidal activity of silver zeolite.
Motility of
Colwellia psychrerythraea
Strain 34H at Subzero Temperatures
Authors: Junge Karen , Eicken Hajo , Deming Jody W. 
Published: July 2003

ABSTRACT

We examined the Arctic bacterium
Colwellia psychrerythraea
strain 34H for motility at temperatures from −1 to −15°C by using transmitted-light microscopy in a temperature-controlled laboratory. The results, showing motility to −10°C, indicate much lower temperatures to be permissive of motility than previously reported (5°C), with implications for microbial activity in frozen environments.
Identification of Novel
Cryptosporidium
Genotypes from the Czech Republic
Authors: Ryan Una , Xiao Lihua , Read Carolyn , Zhou Ling , Lal Altaf A. , Pavlasek Ivan 
Published: July 2003

ABSTRACT

Isolates of
Cryptosporidium
from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.
Inhibitory Effect of Maillard Reaction Products on Growth of the Aerobic Marine Hyperthermophilic Archaeon
Aeropyrum pernix
Authors: Kim Kee Woung , Lee Sun Bok 
Published: July 2003

ABSTRACT

It was found that the growth of
Aeropyrum pernix
was severely inhibited in a medium containing reducing sugars and tryptone due to the formation of Maillard reaction products. The rate of the Maillard browning reaction was markedly enhanced under aerobic conditions, and the addition of Maillard reaction products to the culture medium caused fatal growth inhibition.
Molecular Phylogenetic Exploration of Bacterial Diversity in a Bakreshwar (India) Hot Spring and Culture of
Shewanella
-Related Thermophiles
Authors: Ghosh Dhritiman , Bal Bijay , Kashyap V. K. , Pal Subrata 
Published: July 2003

ABSTRACT

The bacterial diversity of a hot spring in Bakreshwar, India, was investigated by a culture-independent approach. 16S ribosomal DNA clones derived from the sediment samples were found to be associated with gamma-
Proteobacteria
, cyanobacteria, and green nonsulfur and low-GC gram-positive bacteria. The first of the above phylotypes cobranches with
Shewanella
, a well-known iron reducer. This phylogenetic correlation has been exploited to develop culture conditions for thermophilic iron-reducing microorganisms.
Large-Restriction-Fragment Polymorphism Analysis of
Mycobacterium chelonae
and
Mycobacterium terrae
Isolates
Authors: Vanitha J. Daisy , Venkatasubramani R. , Dharmalingam K. , Paramasivan C. N. 
Published: July 2003

ABSTRACT

Mycobacterium chelonae
and
Mycobacterium terrae
were reported to be frequently present in the environment of the
Mycobacterium bovis
BCG trial area in south India. Six isolates of
M. chelonae
and four isolates of
M. terrae
obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with
Dra
I and
Xba
I restriction enzymes. With the exception of one isolate of
M. terrae
, DNA from all other isolates could be digested with
Dra
I and
Xba
I and resulted in separable fragments. Visual comparison of the LRFs showed a unique pattern for each of the isolates tested. A computer-assisted dendrogram of the percent similarity demonstrated a high degree of genetic diversity in this group of isolates. This study demonstrates that species of nontuberculous mycobacteria, particularly
M. chelonae
and
M. terrae
, can be successfully typed by their LRF pattern using PFGE, which does not require species-specific DNA probes.
The Human Cytomegalovirus UL44 Protein Is a Substrate for the UL97 Protein Kinase
Authors: Krosky Paula M. , Baek Moon-Chang , Jahng Wan Jin , Barrera Imma , Harvey Robert J. , Biron Karen K. , Coen Donald M. , Sethna Phiroze B. 
Published: June 2003

ABSTRACT

The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [
32
P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a
UL97
null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.
Murine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame M35 Is Defective in Growth In Vivo
Authors: Tam Ada , Zhu Jiaming , Hai Rong , Haghjoo Erik , Tong Tuong , Zhan Xiaoyan , Lu Sangwei , Liu Fenyong 
Published: June 2003

ABSTRACT

We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn
3
-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.
Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein
Authors: Song Chisu , Hunter Eric 
Published: June 2003

ABSTRACT
The transmembrane protein of Mason-Pfizer monkey virus contains two heptad repeats that are predicted to form amphipathic α-helices that mediate the conformational change necessary for membrane fusion. To analyze the relative sensitivity of the predicted hydrophobic face of the N-terminal heptad repeat to the insertion of uncharged, polar, and charged substitutions, mutations that introduced alanine, serine, or glutamic acid into positions 436, 443, 450, and 457 of the envelope protein were examined. Novel systems using Tat protein and the GHOST cell line were developed to test and quantitate the effects of the mutations on Env-mediated fusion and infectivity of the virus. While no single amino acid change at any of the positions interfered significantly with the synthesis, processing, or transport to the plasma membrane of glycoprotein complexes, 9 of the 12 nonconservative mutations in these residues completely abolished fusion activity and virus infectivity. Mutations in the central positions (443 and 450) of the heptad repeat region were the most detrimental to Env function, and even single alanine substitutions in these positions dramatically altered the fusogenicity of the protein. These results demonstrate that this N-terminal heptad repeat plays a critical role in Env-mediated membrane fusion and highlight the key function of central hydrophobic residues in this process and the sensitivity of all positions to charge substitutions.
Differential Inhibition of RNA Editing in Hepatitis Delta Virus Genotype III by the Short and Long Forms of Hepatitis Delta Antigen
Authors: Cheng Qiufang , Jayan Geetha C. , Casey John L. 
Published: June 2003

ABSTRACTHepatitis delta virus (HDV) produces two essential forms of the sole viral protein from the same open reading frame by using host RNA editing activity at the amber/W site in the antigenomic RNA. The roles of these two forms, HDAg-S and HDAg-L, are opposed. HDAg-S is required for viral RNA replication, whereas HDAg-L, which is produced as a result of editing, inhibits viral RNA replication and is required for virion packaging. Both the rate and amount of editing are important because excessive editing will inhibit viral RNA replication, whereas insufficient editing will reduce virus secretion. Here we show that for HDV genotype III, which is associated with severe HDV disease, HDAg-L strongly inhibits editing of a nonreplicating genotype III reporter RNA, while HDAg-S inhibits only when expressed at much higher levels. The different inhibitory efficiencies are due to RNA structural elements located ca. 25 bp 3′ of the editing site in the double-hairpin RNA structure required for editing at the amber/W site in HDV genotype III RNA. These results are consistent with regulation of amber/W editing in HDV genotype III by a negative-feedback mechanism due to differential interactions between structural elements in the HDV genotype III RNA and the two forms of HDAg.
Application of Chimeric Feline Foamy Virus-Based Retroviral Vectors for the Induction of Antiviral Immunity in Cats
Authors: Schwantes Astrid , Truyen Uwe , Weikel Joachim , Weiss Christian , Löchelt Martin 
Published: June 2003

ABSTRACT
In order to define the potential and applicability of replication-competent foamy virus-based vaccine vectors, recombinant feline foamy virus (FFV) vectors encoding defined segments of the feline calicivirus (FCV) capsid protein E domain were constructed. In cell cultures, these FFV-FCV vectors efficiently transduced and expressed a hybrid fusion protein consisting of the essential FFV Bet protein and the attached FCV E domains. The stability of the vectors in vitro was inversely correlated to the size of the heterologous insert. The deletion of a part of the FFV U3 sequence in these FFV-FCV vectors did not interfere with replication and titer in cell cultures but increased the genetic stability of the hybrid vectors. Selected chimeric vectors were injected into immunocompetent cats and persisted in the transduced host concomitant with a strong and specific humoral immune response against vector components. In a substantial number of cats, antibodies directed against the FCV E domain were induced by the FFV-FCV vectors, but no FCV-neutralizing activities were detectable in vitro. When the vaccinated cats were challenged with a high-titer FCV dose, sterile immunity was not induced by any of the hybrid FFV-FCV vectors. However, the FFV-FCV vector with a truncated U3 region of the long terminal repeat promoter significantly reduced the duration of FCV shedding after challenge and suppressed the appearance of FCV-specific ulcers. Possible mechanisms contributing to the partial protection will be discussed.