bluuebunny/crossref_metadata_2025 huggingface dataset:
Accumulation of Polyhydroxyalkanoic Acid Containing Large Amounts of Unsaturated Monomers in
Pseudomonas fluorescens
BM07 Utilizing Saccharides and Its Inhibition by 2-Bromooctanoic Acid
Authors: Lee Ho-Joo , Choi Mun Hwan , Kim Tae-Un , Yoon Sung Chul
Published: July 2002

A psychrotrophic bacterium,
Pseudomonas fluorescens
BM07, which is able to accumulate polyhydroxyalkanoic acid (PHA) containing large amounts of 3-hydroxy-
cis
-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. When it was grown on heptanoic acid (C
7
) to hexadecanoic acid (C
16
) as the sole carbon source, the monomer compositional characteristics of the synthesized PHA were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C
18
) led to no PHA accumulation, but instead free stearic acid was stored in the cell. The existence of the linkage between fatty acid de novo synthesis and PHA synthesis was confirmed by using inhibitors such as acrylic acid and two other compounds, 2-bromooctanoic acid and 4-pentenoic acid, which are known to inhibit β-oxidation enzymes in animal cells. Acrylic acid completely inhibited PHA synthesis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhibition of PHA synthesis occurred in 70 mM fructose-grown cells in the presence of 1 to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid were found to much inhibit PHA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from 1 to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cell growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA synthesis inhibition over a small range of concentrations below 100 μM, a finding indicative of a very specific inhibition, whereas 4-pentenoic acid showed a broad, featureless concentration dependence, suggesting a rather nonspecific inhibition. The apparent inhibition constant
K

i

(the concentration for 50% inhibition of PHA synthesis) for 2-bromooctanoic acid was determined to be 60 μM, assuming a single-site binding of the inhibitor at a specific inhibition site. Thus, it seems likely that a coenzyme A thioester derivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest that 2-bromooctanoic acid can substitute for the far more expensive (2,000 times) and cell-growth-inhibiting PHA synthesis inhibitor, cerulenin.
Enzyme-Linked Immunosorbent Assay Specific for (1→6) Branched, (1→3)-β-
d
-Glucan Detection in Environmental Samples
Authors: Milton Donald K. , Alwis K. Udeni , Fisette Leslie , Muilenberg Michael
Published: July 2002

(1→3)-β-
d
-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1→6) branched, (1→3)-β-
d
-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1→3)-β-
d
-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall β-
d
-glucans as a detector reagent. The assay was highly specific for (1→6) branched, (1→3)-β-
d
-glucans (such as that from
Saccharomyces
cerevisiae
) and did not show any response at 200 ng/ml to curdlan, laminarin, pustulan, dextran, mannan, carboxymethyl cellulose, and endotoxins. The detection level was 0.8 ng/ml for baker's yeast glucan and Betafectin. A coefficient of variation of 7.8% was obtained for (1→3)-β-
d
-glucans in house dust samples. Metal working fluids spiked with (1→3)-β-
d
-glucans inhibited the glucan assay. Because the assay is specific for (1→6) branched, (1→3)-β-
d
-glucans and is sensitive and reproducible, it will be useful for the investigation of health effects from exposure to this class of biologically active molecules.
Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of
Bacillus
sp. Strain GL1
Authors: Hashimoto Wataru , Miki Hikaru , Tsuchiya Noriaki , Nankai Hirokazu , Murata Kousaku
Published: July 2002

When grown on xanthan as a carbon source, the bacterium
Bacillus
sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520–2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of
Streptomyces griseus
(identity score, 37.7%).
Escherichia coli
transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4°C, the purified enzyme (97 kDa) from
E. coli
was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from
Bacillus
sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of
Bacillus
sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in
Bacillus
sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.
The
adnA
Transcriptional Factor Affects Persistence and Spread of
Pseudomonas fluorescens
under Natural Field Conditions
Authors: Marshall Bonnie , Robleto Eduardo A. , Wetzler Richard , Kulle Peter , Casaz Paul , Levy Stuart B.
Published: July 2002

A soil plot was inoculated with a mixture of
Pseudomonas fluorescens
Pf0-2, the wild type, and Pf0-5a, a Tn
5
insertion mutant in
adnA
, at 7.84 log CFU/g of soil. Over a period of 231 days, culturable populations of both strains were measured at selected times below and away from the point of inoculation. Pf0-5a did not spread as fast and attained significantly lower populations than Pf0-2. At sample depths below the inoculation site, the
adnA
mutant showed a significant decrease in CFU/g of soil as compared to Pf0-2. Pf0-2 was first detected at the 1.5-cm annular site at 3 days after inoculation, whereas Pf0-5a required 7 days to travel the same distance. At this distance, the wild-type strain could be detected at a 21.5- to 25-cm depth, whereas Pf0-5a could be detected only as deep as 15.5 to 18 cm. At 4.5 cm from the site of inoculation and in soil fractions corresponding to 13 to 18 cm, Pf0-2 was the only strain detected. These results suggest that the transcription factor AdnA provides a fitness advantage in
P. fluorescens
, allowing it to spread and survive in soil under field conditions.
Different Mechanisms of Resistance to
Bacillus thuringiensis
Toxins in the Indianmeal Moth
Authors: Herrero Salvador , Oppert Brenda , Ferré Juan
Published: July 2002

Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of
125
I-labeled Cry1Ab and Cry1Ac has been examined in three
Plodia interpunctella
colonies, one susceptible (688
s
) and two resistant (198
r
and Dpl
r
) to
Bacillus thuringiensis
. Toxicological studies showed that the 198
r
colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl
r
colony was 4-fold more resistant to protoxin versus toxin. Binding results with
125
I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl
r
colony showed a drastic reduction in binding affinity (60-fold higher

K
d

), although they had similar concentrations of binding sites. Insects from the 198
r
colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher

K
d

and ca. three-fold-lower

R
t

compared with the 688
s
colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198
r
colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473–23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of
B. thuringiensis
resistance genes occurring in natural populations.
Molecular Characterization of
Cryptosporidium
Oocysts in Samples of Raw Surface Water and Wastewater
Authors: Xiao Lihua , Singh Ajaib , Limor Josef , Graczyk Thaddeus K. , Gradus Steve , Lal Altaf
Published: July 2002

Recent molecular characterizations of
Cryptosporidium
parasites make it possible to differentiate the human-pathogenic
Cryptosporidium
parasites from those that do not infect humans and to track the source of
Cryptosporidium
oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize
Cryptosporidium
oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis.
Cryptosporidium
parasites were detected in 25 surface water samples and 12 raw wastewater samples.
C. parvum
human and bovine genotypes were the dominant
Cryptosporidium
parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas
C. andersoni
was the most common parasite in wastewater. There may be geographic differences in the distribution of
Cryptosporidium
genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of
Cryptosporidium
parasites in water.
Long-Range1H-15N Heteronuclear Shift Correlation at Natural Abundance: a Tool To Study Benzothiazole Biodegradation by TwoRhodococcusStrains
Authors: Besse Pascale , Combourieu Bruno , Boyse Gaëlle , Sancelme Martine , De Wever Heleen , Delort Anne-Marie
Published: July 2002

The biodegradation of benzothiazole and 2-hydroxybenzothiazole by two strains ofRhodococcuswas monitored by reversed phase high-pressure liquid chromatography and by1H nuclear magnetic resonance (NMR). Both xenobiotics were biotransformed into a hydroxylated derivative of 2-hydroxybenzothiazole by these two strains. The chemical structure of this metabolite was determined by a new NMR methodology: long-range1H-15N heteronuclear shift correlation without any previous15N enrichment of the compound. This powerful NMR tool allowed us to assign the metabolite structure to 2,6-dihydroxybenzothiazole.
Mutations in the
csgD
Promoter Associated with Variations in Curli Expression in Certain Strains of
Escherichia coli
O157:H7
Authors: Uhlich Gaylen A. , Keen James E. , Elder Robert O.
Published: July 2002

Single-base-pair
csgD
promoter mutations in human outbreak
Escherichia coli
O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression. Red phenotype
csgD
::
lacZ
promoter fusions had fourfold-greater expression than white promoter fusions. Cloning the red variant
csgDEFG
operon into white variants induced the red phenotype. Substrate utilization differed between red and white variants.
Attached and Unattached Microbial Communities in a Simulated Basalt Aquifer under Fracture- and Porous-Flow Conditions
Authors: Lehman R. Michael , Colwell Frederick S. , Bala Greg A.
Published: July 2002

Bench scale column studies were used to examine the partitioning of microorganisms between groundwater and a geologic medium and to examine the effect of hydrogeology (i.e., porous- versus fracture-flow) on organism partitioning. Replicated columns were constructed with intact basalt core segments that contained natural fractures and with the same basalt crushed into particles. The columns were perfused with groundwater, and upon reaching a steady state, the columns were sacrificed and the attached and unattached communities were analyzed by multiple approaches. The analyses included the total number of cells, the phylogenetic affiliation of the cells (i.e., the α, β, and γ subclasses of the class
Proteobacteria
and gram positives with high G+C DNA content) by fluorescent in situ hybridization (FISH), number and taxonomic affiliation by fatty acid methyl ester profiles of culturable heterotrophs, most-probable-number estimates of methanotrophs and phenol oxidizers, and whole-community sole carbon source utilization patterns from Biolog GN microplates. In the packed columns, about 99% of the total biomass (per cubic centimeter of porous medium) was attached to the geologic medium. Lack of equitable units precluded a comparison of attached and unattached biomasses in the fractured columns where the attached biomass was expressed per unit of surface area. Compositional differences in the attached and unattached communities were evidenced by (i) the recovery of
Pseudomonas stutzeri
, an
Enterococcus
sp., and
Bacillus psychrophilus
from the groundwater and not from the basalt, (ii) differences between community carbon source utilization patterns, and (iii) the relative abundances of different phylogenetic groups estimated by FISH in both column types. In the packed columns, attached communities were depleted of members of the α- and β-
Proteobacteria
subclasses in comparison to those in the corresponding groundwater. In the fractured columns, attached communities were enriched in gram-positive
Bacteria
and γ-
Proteobacteria
and depleted of β-
Proteobacteria
, in comparison to those in the corresponding groundwater. Segregation of populations and their activities, possibly modified by attachment to geologic media, may influence contaminant fate and transport in the subsurface and impact other in situ applications.
Incorporation of [
15
N]Ammonia by the Cellulolytic Ruminal Bacteria
Fibrobacter succinogenes
BL2,
Ruminococcus albus
SY3, and
Ruminococcus flavefaciens
17
Authors: Atasoglu Cengiz , Newbold C. James , Wallace R. John
Published: July 2002

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using
15
NH
3
. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of
Fibrobacter succinogenes
BL2 (51%),
Ruminococcus flavefaciens
17 (43%), and
Ruminococcus albus
SY3 (46%) were derived from non-NH
3
-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH
3
. More cell nitrogen was formed from NH
3
during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for
F. succinogenes
, and its
15
N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.
The Mannose-Sensitive Hemagglutinin ofVibrio choleraePromotes Adherence to Zooplankton
Authors: Chiavelli Deborah A. , Marsh Jane W. , Taylor Ronald K.
Published: July 2002

The bacteriumVibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats. TheV. choleraeO1 El Tor biotype andV. choleraeO139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereasV. choleraeO1 classical biotype strains do not. AlthoughV. choleraeO1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains. SinceV. choleraeO1 El Tor andV. choleraeO139 have displacedV. choleraeO1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton. We found that mutation ofmshAinV. choleraeO1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustaceanDaphnia pulex. The effect of the mutation was more pronounced forV. choleraeO139, essentially eliminating adherence. Adherence of theV. choleraeO1 classicalmshAmutant was unaffected. The results suggest that MSHA is a factor contributing to the ability ofV. choleraeto adhere to plankton. The results also showed that both biotypes ofV. choleraeO1 utilize factors in addition to MSHA for zooplankton adherence. The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner.
Distribution, Isolation, Host Specificity, and Diversity of Cyanophages Infecting Marine
Synechococcus
spp. in River Estuaries
Authors: Lu Jingrang , Chen Feng , Hodson Robert E.
Published: July 2002

The abundance of cyanophages infecting marine
Synechococcus
spp. increased with increasing salinity in three Georgia coastal rivers. About 80% of the cyanophage isolates were cyanomyoviruses. High cross-infectivity was found among the cyanophages infecting phycoerythrin-containing
Synechococcus
strains. Cyanophages in the river estuaries were diverse in terms of their morphotypes and genotypes.
Improved Properties of Baker's Yeast Mutants Resistant to 2-Deoxy-
d
-Glucose
Authors: Rincón Ana M. , Codón Antonio C. , Castrejón Francisco , Benı́tez Tahı́a
Published: July 2002

We isolated spontaneous mutants from
Saccharomyces cerevisiae
(baker's yeast V1) that were resistant to 2-deoxy-
d
-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-
d
-glucose-6-phosphate. Dough fermentation (CO
2
liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties.
Novel Alkylsulfatases Required for Biodegradation of the Branched Primary Alkyl Sulfate Surfactant 2-Butyloctyl Sulfate
Authors: Ellis Andrew J. , Hales Stephen G. , Ur-Rehman Naheed G. A. , White Graham F.
Published: July 2002

Recent reports show that contrary to common perception, branched alkyl sulfate surfactants are readily biodegradable in standard biodegradability tests. We report here the isolation of bacteria capable of biodegrading 2-butyloctyl sulfate and the identification of novel enzymes that initiate the process. Enrichment culturing from activated sewage sludge yielded several strains capable of growth on 2-butyloctyl sulfate. Of these, two were selected for further study and identified as members of the genus
Pseudomonas
. Strain AE-A was able to utilize either sodium dodecyl sulfate (SDS) or 2-butyloctyl sulfate as a carbon and energy source for growth, but strain AE-D utilized only the latter. Depending on growth conditions, strain AE-A produced up to three alkylsulfatases, as shown by polyacrylamide gel electrophoresis zymography. Growth on either SDS or 2-butyloctyl sulfate or in nutrient broth produced an apparently constitutive, nonspecific primary alkylsulfatase, AP1, weakly active on SDS and on 2-butyloctyl sulfate. Growth on 2-butyloctyl sulfate produced a second enzyme, AP2, active on 2-butyloctyl sulfate but not on SDS, and growth on SDS produced a third enzyme, AP3, active on SDS but not on 2-butyloctyl sulfate. In contrast, strain AE-D, when grown on 2-butyloctyl sulfate (no growth on SDS), produced a single enzyme, DP1, active on 2-butyloctyl sulfate but not on SDS. DP1 was not produced in broth cultures. DP1 was induced when residual 2-butyloctyl sulfate was present in the growth medium, but the enzyme disappeared when the substrate was exhausted. Gas chromatographic analysis of products of incubating 2-butyloctyl sulfate with DP1 in gels revealed the formation of 2-butyloctanol, showing the enzyme to be a true sulfatase. In contrast,
Pseudomonas
sp. strain C12B, well known for its ability to degrade linear SDS, was unable to grow on 2-butyloctyl sulfate, and its alkylsulfatases responsible for initiating the degradation of SDS by releasing the parent alcohol exhibited no hydrolytic activity on 2-butyloctyl sulfate. DP1 and the analogous AP2 are thus new alkylsulfatase enzymes with novel specificity toward 2-butyloctyl sulfate.
Effects of pH and Magnetic Material on Immunomagnetic Separation of
Cryptosporidium
Oocysts from Concentrated Water Samples
Authors: Kuhn Ryan C. , Rock Channah M. , Oshima Kevin H.
Published: July 2002

In this study, we examined the effect that magnetic materials and pH have on the recoveries of
Cryptosporidium
oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted.
Rapid PCR-Based Method Which Can Determine Both Phenotype and Genotype of
Lactococcus lactis
Subspecies
Authors: Nomura Masaru , Kobayashi Miho , Okamoto Takashi
Published: July 2002

A highly efficient, rapid, and reliable PCR-based method for distinguishing
Lactococcus lactis
subspecies (
L. lactis
subsp.
lactis
and
L. lactis
subsp.
cremoris
) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different
L. lactis
strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the
L. lactis
subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical
L. lactis
subsp.
cremoris
fragments.
Biotransformation of 2,7-Dichloro- and 1,2,3,4-Tetrachlorodibenzo-
p
-Dioxin by
Sphingomonas wittichii
RW1
Authors: Hong Hyo-Bong , Chang Yoon-Seok , Nam In-Hyun , Fortnagel Peter , Schmidt Stefan
Published: July 2002

Aerobic biotransformation of the diaryl ethers 2,7-dichlorodibenzo-
p
-dioxin and 1,2,3,4-tetrachlorodibenzo-
p
-dioxin by the dibenzo-
p
-dioxin-utilizing strain
Sphingomonas wittichii
RW1, producing corresponding metabolites, was demonstrated for the first time. Our strain transformed 2,7-dichlorodibenzo-
p
-dioxin, yielding 4-chlorocatechol, and 1,2,3,4-tetrachlorodibenzo-
p
-dioxin, producing 3,4,5,6-tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol; all of these compounds were unequivocally identified by mass spectrometry both before and after
N
,
O
-bis(trimethylsilyl)-trifluoroacetamide derivatization by comparison with authentic standards. Additional experiments showed that strain RW1 formed a second metabolite, 2-methoxy-3,4,5,6-tetrachlorophenol, from the original degradation product, 3,4,5,6-tetrachlorocatechol, by methylation of one of the two hydroxy substituents.
Development of a Procedure for Discriminating among
Escherichia coli
Isolates from Animal and Human Sources
Authors: Guan Shukui , Xu Renlin , Chen Shu , Odumeru Joseph , Gyles Carlton
Published: July 2002

Counts of
Escherichia coli
cells in water indicate the potential presence of pathogenic microbes of intestinal origin but give no indication of the sources of the microbial pollution. The objective of this research was to evaluate methods for differentiating
E. coli
isolates of livestock, wildlife, or human origin that might be used to predict the sources of fecal pollution of water. A collection of 319
E. coli
isolates from the feces of cattle, poultry, swine, deer, goose, and moose, as well as from human sewage, and clinical samples was used to evaluate three methods. One method was the multiple-antibiotic-resistance (MAR) profile using 14 antibiotics. Discriminant analysis revealed that 46% of the livestock isolates, 95% of the wildlife isolates, and 55% of the human isolates were assigned to the correct source groups by the MAR method. Amplified fragment length polymorphism (AFLP) analysis, the second test, was applied to 105 of the
E. coli
isolates. The AFLP results showed that 94% of the livestock isolates, 97% of the wildlife isolates, and 97% of the human isolates were correctly classified. The third method was analysis of the sequences of the16S rRNA genes of the
E. coli
isolates. Discriminant analysis of 105
E. coli
isolates indicated that 78% of the livestock isolates, 74% of the wildlife isolates, and 80% of the human isolates could be correctly classified into their host groups by this method. The results indicate that AFLP analysis was the most effective of the three methods that were evaluated.
Crescent Bodies of
Parachlamydia acanthamoeba
and Its Life Cycle within
Acanthamoeba polyphaga
: an Electron Micrograph Study
Authors: Greub Gilbert , Raoult Didier
Published: July 2002

Parachlamydiaceae
are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of
Parachlamydia acanthamoeba
within
Acanthamoeba polyphaga
was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. The multiplication of
Parachlamydia
bacteria by binary fission of reticulate bodies was independently associated with culture in PYG broth (odds ratio [OR] = 4.4; 95% confidence interval [CI], 1.55 to 12.46) and with the presence of reticulate bodies within the amoebae (OR = 2.10; 95% CI, 1.53 to 2.89). A third developmental stage was observed, the crescent body. Its presence outside and inside the amoebae was associated mainly with prolonged incubation time (OR = 3.98; 95% CI, 1.49 to 10.68, and OR = 5.98; 95% CI, 1.75 to 20.4, respectively). Elementary and crescent bodies were released into the extracellular medium within vesicles or after amoebal lysis. For both, phagocytosis was their mode of entry. This electron micrograph study revealed another infective developmental stage, the crescent body, and provided quantitative analysis of the life cycle of
P. acanthamoeba
within
A. polyphaga
.
Sequential Use of Paraformaldehyde and Methanol as Optimal Conditions for the Direct Quantification of ZEBRA and Rta Antigens by Flow Cytometry
Authors: Imbert-Marcille Berthe-Marie , Coste-Burel Marianne , Robillard Nelly , Foucaud-Gamen Jacqueline , Billaudel Sylviane , Drouet Emmanuel
Published: July 2002

A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.
Use of Monoclonal Antibodies for Rapid Diagnosis of Respiratory Viruses in a Community Hospital
Authors: Irmen Kenneth E. , Kelleher James J.
Published: July 2002

An indirect fluorescence antibody (IFA) procedure was used for the rapid detection of respiratory viruses in direct clinical specimens and for determining the epidemiology of viruses in a community hospital setting. Viral respiratory diseases were monitored for 10 consecutive respiratory seasons. The Bartels Viral Respiratory Screening and Identification Kit is an IFA method that contains pooled and individual monoclonal antibodies for seven common respiratory viruses. Compared with 8,670 conventional tube cell cultures, IFA staining of direct patient specimens had an overall sensitivity of 84.2% and a specificity of 87.7%. Yearly epidemics of respiratory syncytial virus were seen with alternating short and long intervals between successive periods when virus was isolated. Epidemics following short intervals were more severe. Influenza A virus epidemics occurred yearly, and influenza B virus activity was seen generally every other year. When influenza A and influenza B viruses were cocirculating in a given season, the months of peak activity of one virus were always within 1 month of the peak activity of the other virus. Parainfluenza virus type 1 was detected in the autumn of odd-numbered years, and parainfluenza type 2 virus was seen usually in the autumn of even-numbered years. Parainfluenza type 3 virus and adenovirus were the most ubiquitous agents, with peak incidence occurring in the late winter to spring.
A Latex Bead-Based Flow Cytometric Immunoassay Capable of Simultaneous Typing of Multiple Pneumococcal Serotypes (Multibead Assay)
Authors: Park M. K. , Briles D. E. , Nahm M. H.
Published: July 2002

A simple and rapid method of simultaneously determining 15
Streptococcus pneumoniae
serotypes was developed. Fifteen latex beads of different sizes and different red fluorescence levels were coated with 1 of 15 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, and 23F) of pneumococcal capsular polysaccharide (PS). The bead mixture was incubated with individual pneumococcal lysate, a pool of rabbit antisera capable of binding the 15 serotypes, and fluorescein (green fluorescence)-conjugated anti-rabbit antibody. Bead size, red fluorescence, and green fluorescence were measured in a single flow cytometer run. The green fluorescence of the beads was inhibited only when there was a serotypic match between PS on the bead and PS in the pneumococcal lysate. This method distinguished cross-reactive serotypes and correctly identified the serotypes in 100% of 86 pneumococcal isolates tested.
Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3
Authors: Martínez-Torrecuadrada Jorge L. , Lázaro Beatriz , Rodriguez José F. , Casal J. Ignacio
Published: July 2002

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions.
Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay
Authors: Tajima Tomoko , Zhi Ning , Lin Quan , Rikihisa Yasuko , Horowitz Harold W. , Ralfalli John , Wormser Gary P. , Hechemy Karim E.
Published: July 2002

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with
Escherichia coli
or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four
Ehrlichia chaffeensis
-positive and 10
Borrelia burgdorferi
-positive sera were negative by ELISA. However, two
Babesia microti
-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.
An IS
711
Element Downstream of the
bp26
Gene Is a Specific Marker of
Brucella
spp. Isolated from Marine Mammals
Authors: Cloeckaert Axel , Grayon Maggy , Grepinet Olivier
Published: July 2002

DNA polymorphism of the
bp26
gene, coding for a diagnostic protein antigen for brucellosis, was assessed by PCR and restriction fragment length polymorphism analysis using primers to amplify the
bp26
gene with its flanking regions. Surprisingly, whereas PCR performed on DNA of the reference strains of the six recognized
Brucella
species produced a product of the expected size (1,029 bp), PCR performed on DNA of three representative strains from marine mammals (from a seal, a dolphin, and a porpoise) produced a larger product, of about 1,900 bp. Nucleotide sequencing of the 1,900-bp PCR products revealed the presence of an insertion sequence, IS
711
, downstream of the
bp26
gene and adjacent to a Bru-RS1 element previously described as being a hot spot for IS
711
insertion. PCR performed on a large number of field strains from different geographic origins and from marine mammal isolates indicated that the occurrence of an IS
711
element downstream of the
bp26
gene was a feature specific to the marine mammal
Brucella
strains. Thus, this PCR assay is able to differentiate
Brucella
terrestrial isolates from marine mammal isolates and could be applied for diagnostic purposes.
Hyper-Immunoglobulin A in the Hyperimmunoglobulinemia D Syndrome
Authors: Klasen Ina S. , Göertz Joop H. C. , van de Wiel Gertrude A. S. , Weemaes Corry M. R. , van der Meer Jos W. M. , Drenth Joost P. H.
Published: July 2002

The hyperimmunoglobulinemia D syndrome (HIDS) is an autosomal recessive disorder characterized by recurrent febrile attacks with abdominal, articular, and skin manifestations. Apart from elevated immunoglobulin D (IgD) levels (>100 IU/ml), there are high IgA levels in the majority of cases. Mutations in the gene encoding mevalonate kinase constitute the molecular defect in HIDS. The cause of elevated IgA concentrations in HIDS patients remains to be elucidated. We studied the hyper-IgA response in serum of a group of HIDS patients. Elevated IgA concentrations result from increased IgA1 concentrations. IgA and IgA1 concentrations correlated significantly with IgD concentrations, and levels of IgA polymers were significantly higher than the levels in healthy donors. These results indicate a continuous, presumably systemic, stimulation of IgA in HIDS patients.
Canine Parvovirus (CPV) Vaccination: Comparison of Neutralizing Antibody Responses in Pups after Inoculation with CPV2 or CPV2b Modified Live Virus Vaccine
Authors: Pratelli Annamaria , Cavalli Alessandra , Martella Vito , Tempesta Maria , Decaro Nicola , Carmichael Leland Eugene , Buonavoglia Canio
Published: July 2002

Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population.
Seroprevalence of Canine Distemper Virus in Cats
Authors: Ikeda Yasuhiro , Nakamura Kazuya , Miyazawa Takayuki , Chen Ming-Chu , Kuo Tzong-Fu , Lin James A. , Mikami Takeshi , Kai Chieko , Takahashi Eiji
Published: July 2002

A seroepidemiological survey of canine distemper virus (CDV) infection in Asian felids revealed that the prevalence of antibodies varied depending on region and, in some cases, exposure to dogs. The serologic pattern in cats with antibodies indicated that they had likely been exposed to field strains rather than typical CDV vaccine strains.
Enhancement of Splenic-Macrophage Fcγ Receptor Expression by Treatment with Estrogens
Authors: Gomez F. , Ruiz P. , Bernal J. A. , Escobar M. , Garcia-Egido A. , Lopez-Saez J. J. B.
Published: July 2002

Splenic-macrophage Fcγ receptors (FcγRs) participate in the pathophysiologies of immune-complex diseases and in host defense against infection. Modulation of macrophage FcγR expression is an immuno-therapeutic target. Glucocorticoids, sex steroids, and dopaminergic drugs modulate macrophage FcγR expression. Previous data indicate that estradiol increases macrophage FcγR expression. Nevertheless, the effects of clinically used estrogens upon macrophage FcγR expression are unknown. We assessed the effects of treatment with commonly used estrogens on the expression of macrophage FcγRs using a guinea pig experimental model. Six estrogens have been studied: ethynylestradiol (Et), mestranol (M), chlortianisene (Ct), promestriene, 17-epiestriol, and 17β-estradiol. Following in vivo treatment of guinea pigs, we determined the clearance of immunoglobulin G (IgG)-sensitized erythrocytes in vivo, the binding of IgG-sensitized erythrocytes by isolated splenic macrophages, and splenic-macrophage FcγR cell surface expression. Estrogens enhance the clearance of IgG-sensitized erythrocytes by increasing splenic-macrophage FcγR expression. Et, M, and Ct were more effective than the other estrogens. Flow cytometry and fluorescence microscopy with monoclonal antibodies demonstrated that estrogens increase the cell surface expression of FcγR1 and -2 more than that of FcγR2. These data indicate that treatment with commonly used estrogens enhances the clearance of IgG-sensitized cells by improving splenic-macrophage FcγR expression.
Passive Immunization with Milk Produced from an Immunized Cow Prevents Oral Recolonization by
Streptococcus mutans
Authors: Shimazaki Yoshihiro , Mitoma Morihide , Oho Takahiko , Nakano Yoshio , Yamashita Yoshihisa , Okano Kaoru , Nakano Yutaka , Fukuyama Masataka , Fujihara Noboru , Nada Youichi , Koga Toshihiko
Published: July 2002

Cell surface protein antigen (PAc) and water-insoluble glucan-synthesizing enzyme (GTF-I) produced by cariogenic
Streptococcus mutans
are two major factors implicated in the colonization of the human oral cavity by this bacterium. We examined the effect of bovine milk, produced after immunization with a fusion protein of functional domains of these proteins, on the recolonization of
S. mutans
. To prepare immune milk, a pregnant Holstein cow was immunized with the fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I. After eight adult subjects received cetylpyridinium chloride (CPC) treatment, one subgroup (
n
= 4) rinsed their mouths with immune milk and a control group (
n
= 4) rinsed with nonimmune milk.
S. mutans
levels in saliva and dental plaque decreased after CPC treatment in both groups. Mouth rinsing with immune milk significantly inhibited recolonization of
S. mutans
in saliva and plaque. On the other hand, the numbers of
S. mutans
cells in saliva and plaque in the control group increased immediately after the CPC treatment and surpassed the baseline level 42 and 28 days, respectively, after the CPC treatment. The ratios of
S. mutans
to total streptococci in saliva and plaque in the group that received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling
S. mutans
in the human oral cavity.
Analogs of Eap Protein Are Conserved and Prevalent in Clinical
Staphylococcus aureus
Isolates
Authors: Hussain Muzaffar , Becker Karsten , von Eiff Christof , Peters Georg , Herrmann Mathias
Published: July 2002

Map and Eap are secreted
Staphylococcus aureus
proteins that interact with various extracellular matrix molecules. PCR analysis using
map
primers yielded positive reactions in 97.9% of
S. aureus
isolates but not in
Staphylococcus epidermidis
isolates. Cloning and sequencing of the conferring genes revealed a high degree of overall homology combined with size variability of the gene product due to various repeat numbers and early translation termination in a poly(A) region. Thus, Map and Eap may provide a potential novel tool for
S. aureus
identification and typing.
Bacterial Challenge Stimulates Formation of Arachidonic Acid Metabolites by Human Keratinocytes and Neutrophils In Vitro
Authors: Eberhard Jörg , Jepsen Søren , Pohl Lutz , Albers Hans Karl , Açil Yahya
Published: July 2002

Although the interactions of bacteria with keratinocytes induce the synthesis of various mediators, the capability of epithelial cells to form arachidonic acid mediators has not been studied, and therefore the first part of this study was initiated. The complex mixture of epithelium-derived mediators suggests that chemoattraction is not their only effect on neutrophils and that they may also affect neutrophil mediator synthesis. The effect of epithelium-derived mediators on neutrophil eicosanoide synthesis was evaluated in the second part of this study. We incubated human keratinocytes with human-pathogenic bacteria for 2 h and harvested the supernatants after 4, 6, 10, and 18 h of culture. Subsequently, the supernatants were coincubated for 5 min with human neutrophils with or without arachidonic acid. The formation of the arachidonic acid metabolites prostaglandin E2(PGE2), leukotriene B4(LTB4), 12-hydroxyeicosatetraenoic acid (12-HETE), and 15-HETE in keratinocytes and neutrophils was measured by reverse-phase high-pressure liquid chromatography. We demonstrated for the first time that keratinocytes produced significant amounts of LTB4and 12-HETE 4 to 6 h after bacterial challenge. Upon stimulation with epithelial supernatants, neutrophils produced significant amounts of PGE2, LTB4, 12-HETE, and 15-HETE throughout the observation period of 18 h, with a maximum synthesis by supernatants harvested 4 to 10 h after bacterial infection. The results of the study suggest that arachidonic acid mediator formation by epithelial cells following bacterial challenge may act as an early inflammatory signal for the initiation of the immune response. The epithelial supernatants were capable of inducing the formation of arachidonic acid mediators by neutrophils, which may have further regulatory effects on the immune response.
Infectious Disease Issues in Xenotransplantation
Authors: Boneva Roumiana S. , Folks Thomas M. , Chapman Louisa E.
Published: July 2002

Xenotransplantation, the transplantation of living organs, tissues, or cells from one species to another, is viewed as a potential solution to the existing shortage of human organs for transplantation. While whole-organ xenotransplantation is still in the preclinical stage, cellular xenotransplantation and extracorporeal perfusion applications are showing promise in early clinical trials. Advances in immunosuppressive therapy, gene engineering, and cloning of animals bring a broader array of xenotransplantation protocols closer to clinical trials. Despite several potential advantages over allotransplantation, xenotransplantation encompasses a number of problems. Immunologic rejection remains the primary hindrance. The potential to introduce infections across species barriers, another major concern, is the main focus of this review. Nonhuman primates are unlikely to be a main source for xenotransplantation products despite their phylogenetic proximity to humans. Genetically engineered pigs, bred under special conditions, are currently envisaged as the major source. Thus far, there has been no evidence for human infections caused by pig xenotransplantation products. However, the existence of xenotropic endogenous retroviruses and the clinical evidence of long-lasting porcine cell microchimerism indicate the potential for xenogeneic infections. Thus, further trials should continue under regulatory oversight, with close clinical and laboratory monitoring for potential xenogeneic infections.