bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Bacterial Community Composition and Dynamics and Viral Mortality Rates Associated with Enhanced Flagellate Grazing in a Mesoeutrophic Reservoir
Authors: Šimek Karel , Pernthaler Jakob , Weinbauer Markus G. , Hornák Karel , Dolan John R. , Nedoma Jirı́ , Mašı́n Michal , Amann Rudolf
Published: July 2002

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-μm treatment) or enhance (<5-μm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the “grazer-free” (0.8-μm-filtered) treatment, subject only to a relatively low mortality rate (∼17% day
−1
) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-μm filtrate), grazing mortality was ∼200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the
Flectobacillus
lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the
Cytophaga-Flavobacterium
cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.
Detection of Toxigenicity by a Probe for the Microcystin Synthetase A Gene (mcyA)of the Cyanobacterial GenusMicrocystis: Comparison of Toxicities with 16S rRNA and Phycocyanin Operon (Phycocyanin Intergenic Spacer) Phylogenies
Authors: Tillett Daniel , Parker Dorothy L. , Neilan Brett A.
Published: July 2002

The relationship between toxigenicity and phylogeny within the cyanobacterial genusMicrocystisis unclear. To investigate this issue, we have designed PCR primers for theN-methyltransferase (NMT) domain of the microcystin synthetase genemcyAand have probed 37Microcystissp. cultures as well as several field samples. The NMT region was present in all 18 laboratory strains that gave positive reactions in the protein phosphatase inhibition assay for microcystin but was absent in 17 nontoxic strains. Two other nontoxic strains, one of which had previously been reported to produce microcystin, possessed the NMT region. Detection of NMT-specific DNA in field samples corresponded to periods of toxicity as assessed by protein phosphatase inhibition. TheMicrocystisstrains formed a monophyletic cluster based on 16S rRNA gene sequences but comprised two groups with respect to phycocyanin intergenic spacer (PC-IGS) sequences. Toxic and nontoxic strains appeared to be erratically distributed within the PC-IGS and 16S rRNA trees. Sequence analysis of the NMT domain revealed two coherent groups. The genomic region immediately downstream of themcyABCcluster in all 20 NMT-positive strains contained an open reading frame of unknown function (uma1) at a conserved distance frommcyC. All nontoxic strains also containeduma1, which is not cotranscribed withmcyABC. The consistent linkage ofmcyCtouma1suggests thatmcyChas not been frequently transferred into nontoxic strains via any mechanism involving insertion at random chromosomal locations. These results are discussed with respect to various mechanisms that could explain the patchy distribution of toxigenicity among the variousMicrocystisclades.
Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials
Authors: Borchardt S. A. , Allain E. J. , Michels J. J. , Stearns G. W. , Kelly R. F. , McCoy W. F.
Published: July 2002

Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation inPseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. TheChromobacterium violaceumCV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine algaLaminaria digitatademonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. TheChromobacterium violaceumactivity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling.
Biotransformation of Various Substituted Aromatic Compounds to Chiral Dihydrodihydroxy Derivatives
Authors: Raschke Henning , Meier Michael , Burken Joel G. , Hany Roland , Müller Markus D. , Van Der Meer Jan Roelof , Kohler Hans-Peter E.
Published: July 2002

The biotransformation of four different classes of aromatic compounds by the
Escherichia coli
strain DH5α(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) from
Pseudomonas
sp. strain P51, was examined. CDO oxidized biphenyl as well as monochlorobiphenyls to the corresponding
cis
-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring. No higher substituted biphenyls were oxidized. The absolute configurations of several monosubstituted
cis
-benzene dihydrodiols formed by CDO were determined. All had an
S
configuration at the carbon atom in
meta
position to the substituent on the benzene nucleus. With one exception, the enantiomeric excess of several 1,4-disubstituted
cis
-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases. Naphthalene was oxidized to enantiomerically pure (+)-
cis
-(1
R
,2
S
)-dihydroxy-1,2-dihydronaphthalene. All absolute configurations were identical to those of the products formed by toluene dioxygenases of
Pseudomonas putida
UV4 and
P. putida
F39/D. The formation rate of (+)-
cis
-(1
R
,2
S
)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted
cis
-benzene dihydrodiols and more than four times higher than the formation rate of
cis
-benzene dihydrodiol. A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation products.
Differentiation of
Lactobacillus plantarum,
L. pentosus,
and
L. paraplantarum
by
recA
Gene Sequence Analysis and Multiplex PCR Assay with
recA
Gene-Derived Primers
Authors: Torriani Sandra , Felis Giovanna E. , Dellaglio Franco
Published: July 2002

In this study, we succeeded in differentiating
Lactobacillus plantarum,
Lactobacillus pentosus,
and
Lactobacillus paraplantarum
by means of
recA
gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the
recA
gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for
L. plantarum
, 218 bp for
L. pentosus,
and 107 bp for
L. paraplantarum
. This strategy permitted the unambiguous identification of strains belonging to
L. plantarum,
L. pentosus,
and
L. paraplantarum
in a single reaction, indicating its applicability to the speciation of isolates of the
L. plantarum
group.
Presence of Nitrate-Accumulating Sulfur Bacteria and Their Influence on Nitrogen Cycling in a Shallow Coastal Marine Sediment
Authors: Sayama Mikio
Published: July 2002

Nitrate flux between sediment and water, nitrate concentration profile at the sediment-water interface, and in situ sediment denitrification activity were measured seasonally at the innermost part of Tokyo Bay, Japan. For the determination of sediment nitrate concentration, undisturbed sediment cores were sectioned into 5-mm depth intervals and each segment was stored frozen at −30°C. The nitrate concentration was determined for the supernatants after centrifuging the frozen and thawed sediments. Nitrate in the uppermost sediment showed a remarkable seasonal change, and its seasonal maximum of up to 400 μM was found in October. The directions of the diffusive nitrate fluxes predicted from the interfacial concentration gradients were out of the sediment throughout the year. In contrast, the directions of the total nitrate fluxes measured by the whole-core incubation were into the sediment at all seasons. This contradiction between directions indicates that a large part of the nitrate pool extracted from the frozen surface sediments is not a pore water constituent, and preliminary examinations demonstrated that the nitrate was contained in the intracellular vacuoles of filamentous sulfur bacteria dwelling on or in the surface sediment. Based on the comparison between in situ sediment denitrification activity and total nitrate flux, it is suggested that intracellular nitrate cannot be directly utilized by sediment denitrification, and the probable fate of the intracellular nitrate is hypothesized to be dissimilatory reduction to ammonium. The presence of nitrate-accumulating sulfur bacteria therefore may lower nature's self-purification capacity (denitrification) and exacerbate eutrophication in shallow coastal marine environments.
Isolation of
Gemmata
-Like and
Isosphaera
-Like Planctomycete Bacteria from Soil and Freshwater
Authors: Wang Jenny , Jenkins Cheryl , Webb Richard I. , Fuerst John A.
Published: July 2002

New cultured strains of the planctomycete division (order
Planctomycetales
) of the domain
Bacteria
related to species in the genera
Gemmata
and
Isosphaera
were isolated from soil, freshwater, and a laboratory ampicillin solution. Phylogenetic analysis of the 16S rRNA gene from eight representative isolates showed that all the isolates were members of the planctomycete division. Six isolates clustered with
Gemmata obscuriglobus
and related strains, while two isolates clustered with
Isosphaera pallida
. A double-membrane-bounded nucleoid was observed in
Gemmata
-related isolates but not in
Isosphaera
-related isolates, consistent with the ultrastructures of existing species of each genus. Two isolates from this study represent the first planctomycetes successfully cultivated from soil.
Identification ofCryptosporidium parvumOocysts by an Artificial Neural Network Approach
Authors: Widmer Kenneth W. , Oshima Kevin H. , Pillai Suresh D.
Published: July 2002

Microscopic detection ofCryptosporidium parvumoocysts is time-consuming, requires trained analysts, and is frequently subject to significant human errors. Artificial neural networks (ANN) were developed to help identify immunofluorescently labeledC. parvumoocysts. A total of 525 digitized images of immunofluorescently labeled oocysts, fluorescent microspheres, and other miscellaneous nonoocyst images were employed in the training of the ANN. The images were cropped to a 36- by 36-pixel image, and the cropped images were placed into two categories, oocyst and nonoocyst images. The images were converted to grayscale and processed into a histogram of gray color pixel intensity. Commercially available software was used to develop and train the ANN. The networks were optimized by varying the number of training images, number of hidden neurons, and a combination of these two parameters. The network performance was then evaluated using a set of 362 unique testing images which the network had never “seen” before. Under optimized conditions, the correct identification of authentic oocyst images ranged from 81 to 97%, and the correct identification of nonoocyst images ranged from 78 to 82%, depending on the type of fluorescent antibody that was employed. The results indicate that the ANN developed were able to generalize the training images and subsequently discern previously unseen oocyst images efficiently and reproducibly. Thus, ANN can be used to reduce human errors associated with the microscopic detection ofCryptosporidiumoocysts.
Novel
Bacillus thuringiensis
Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm,
Diabrotica virgifera virgifera
LeConte
Authors: Ellis R. Tracy , Stockhoff Brian A. , Stamp Lisa , Schnepf H. Ernest , Schwab George E. , Knuth Mark , Russell Josh , Cardineau Guy A. , Narva Kenneth E.
Published: July 2002

A new family of insecticidal crystal proteins was discovered by screening sporulated
Bacillus thuringiensis
cultures for oral activity against western corn rootworm (WCR) larvae.
B. thuringiensis
isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant
B. thuringiensis
strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known
B. thuringiensis
insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from
Bacillus sphaericus
are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.
Induction, Isolation, and Characterization of Two Laccases from the White Rot BasidiomyceteCoriolopsis rigida
Authors: Saparrat Mario C. N. , Guillén Francisco , Arambarri Angélica M. , Martínez Angel T. , Martínez María Jesús
Published: July 2002

Previous work has shown that the white rot fungusCoriolopsis rigidadegraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH2). The presence of ABTS in the laccase reaction expanded the substrate range ofC. rigidalaccases to nonphenolic compounds and that of MBQH2extended the reactions catalyzed by these enzymes to the production of H2O2, the oxidation of Mn2+, the reduction of Fe3+, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.
Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems
Authors: Kulakov Leonid A. , McAlister Morven B. , Ogden Kimberly L. , Larkin Michael J. , O'Hanlon John F.
Published: July 2002

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4′,6′-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included
Ralstonia pickettii
,
Bradyrhizobium
sp.,
Pseudomonas saccharophilia
, and
Stenotrophomonas
strains. These bacteria constituted a significant part of the total number of isolated strains (≥20%). Two sets of primers specific to
R. pickettii
and
Bradyrhizobium
sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly,
nifH
gene sequences were found in
Bradyrhizobium
sp. and some
P. saccharophilia
strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.
Responses of
Listeria monocytogenes
to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques
Authors: Shabala Lana , Budde Birgitte , Ross Tom , Siegumfeldt Henrik , Jakobsen Mogens , McMeekin Tom
Published: July 2002

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in
Listeria monocytogenes
subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H
+
at either side of the bacterial membrane in real time and therefore to evaluate the rate of H
+
flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH
o
) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH
i
) and net H
+
fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH
i
remained above 7.0 within a pH
o
range of 4 to 6 and decreased below pH
o
4. Above pH
o
4, H
+
extrusion increased correspondingly, with the maximum value at pH
o
5.5, and below pH
o
4, a net H
+
influx was observed. Without glucose in the medium, the pH
i
decreased, and a net H
+
influx was observed below pH
o
5.5. A high correlation (
R
= 0.75 to 0.92) between the pH
i
and net H
+
flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH
i
homeostasis in
L. monocytogenes
subjected to acid stress.
Towards Growth of Arbuscular Mycorrhizal Fungi Independent of a Plant Host
Authors: Hildebrandt Ulrich , Janetta Katharina , Bothe Hermann
Published: July 2002

When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF)Glomus intraradicesSy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified asPaenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence ofP. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA fromAgrobacterium rhizogenes. The presence ofP. validusalone also enabledG. intraradicesto form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experimentsP. validuscould not be replaced by bacteria such asEscherichia coliK-12 orAzospirillum brasilenseSp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) fromP. validuswhich stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.
Bradyrhizobia from Wild
Phaseolus
,
Desmodium
, and
Macroptilium
Species in Northern Mexico
Authors: Parker Matthew A.
Published: July 2002

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (
Amphicarpaea
) that are closely related to
Bradyrhizobium elkanii.
An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete
Gloeophyllum trabeum
Authors: Jensen, Kenneth A. , Ryan Zachary C. , Vanden Wymelenberg Amber , Cullen Daniel , Hammel Kenneth E.
Published: July 2002

The brown-rot basidiomycete
Gloeophyllum trabeum
uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by
G. trabeum
under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by
G. trabeum
(2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that
k
cat
/

K
m

for the quinones is greater than 10
8
M
−1
s
−1
. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The
G. trabeum
quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.
Metabolic Engineering of Glycerol Production inSaccharomyces cerevisiae
Authors: Overkamp Karin M. , Bakker Barbara M. , Kötter Peter , Luttik Marijke A. H. , van Dijken Johannes P. , Pronk Jack T.
Published: July 2002

Inactivation ofTPI1, theSaccharomyces cerevisiaestructural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. Intpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect oftpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, atpi1Δnde1Δnde2Δgut2Δ quadruple mutant was constructed.NDE1andNDE2encode isoenzymes of mitochondrial external NADH dehydrogenase;GUT2encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH inS. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h−1at 100 g of glucose · liter−1). In aerated batch cultures grown on 400 g of glucose · liter−1, this engineeredS. cerevisiaestrain produced over 200 g of glycerol · liter−1, corresponding to a molar yield of glycerol on glucose close to unity.
Relationship between IS901in theMycobacterium aviumComplex Strains Isolated from Birds, Animals, Humans, and the Environment and Virulence for Poultry
Authors: Pavlik Ivo , Svastova Petra , Bartl Jiri , Dvorska Lenka , Rychlik Ivan
Published: July 2002

A total of 738 strains ofMycobacterium aviumcomplex (MAC) were examined in biological experiments on poultry by use of PCR methods with primers for detection of the insertion sequence IS901. Serotype strains of MAC from all known 28 serotypes were examined. Further strains were isolated from human immunodeficiency virus (HIV)-negative and HIV-positive patients, 6 animal species, 17 bird species, and the environment. Of 165 strains virulent for poultry, characterized by generalized tuberculosis, 164 strains contained IS901, a result which is statistically highly significant (P, 0.01). The remaining 573 strains were nonvirulent; however, IS901was present in 24 strains. From among 20 strains of serotypes 1, 2, and 3, IS901was found in 15 strains, only 5 of which were virulent for poultry. The remaining 111 strains, of serotypes 4 to 28, were nonvirulent and did not incorporate IS901. None of the 152 strains isolated from humans was virulent for poultry, including 12 strains which were IS901positive.
Primary Structure of the Sialodacryoadenitis Virus Genome: Sequence of the Structural-Protein Region and Its Application for Differential Diagnosis
Authors: Yoo Dongwan , Pei Yanlong , Christie Natasha , Cooper Melissa
Published: July 2002

Sialodacryoadenitis virus (SDAV) is a coronavirus that is commonly found in laboratory rats and that causes sialodacryoadenitis and respiratory illness. We cloned and sequenced the 3′ terminal 9.8 kb of the genomic RNA and analyzed the structure of the viral genome. As with mouse hepatitis coronaviruses (MHVs), the SDAV genome was able to code for a spike protein, a small membrane protein, a membrane-associated protein, and a nucleocapsid protein. In addition, the hemagglutinin-esterase gene capable of encoding a protein of 439 amino acids (aa) was identified. The putative functional site for acetylesterase activity was present in the HE protein as Phe-Gly-Asp-Ser (FGDS), suggesting that the SDAV HE protein might have retained the esterase activity. Immediately upstream of the HE gene and downstream of the polymerase 1b gene, the NS2 nonstructural-protein gene was identified with a coding capacity of 274 aa. A motif of UCUAAAC was identified as a potential transcription signal for subgenomic mRNA synthesis. Large insertions of 172, 127, and 44 aa were detected in the N-terminal half of the predicted S protein of SDAV when its sequence was compared to the sequences of MHV 2, MHV JHM, and MHV A59, respectively. The sequence information on the SDAV S-protein gene was applied to a differential diagnostic PCR to detect and distinguish the rat coronavirus from mouse coronaviruses. This is the first report on the comprehensive genetic information of any rat coronavirus.
A Single Amino Acid Change within Antigenic Domain II of the Spike Protein of Bovine Coronavirus Confers Resistance to Virus Neutralization
Authors: Yoo Dongwan , Deregt Dirk
Published: July 2002

The spike glycoprotein is a major neutralizing antigen of bovine coronavirus (BCV). Conformational neutralizing epitopes of group A and group B monoclonal antibodies (MAbs) have previously been mapped to two domains at amino acids 351 to 403 (domain I) and amino acids 517 to 621 (domain II). To further map antigenic sites, neutralization escape mutants of BCV were selected with a group A MAb which has both in vitro and in vivo virus-neutralizing ability. The escape mutants were demonstrated to be neutralization resistant to the selecting group A MAb and remained sensitive to neutralization by a group B MAb. In radioimmunoprecipitation assays, the spike proteins of neutralization escape mutants were shown to have lost their reactivities with the selecting group A MAb. Sequence analysis of the spike protein genes of the escape mutants identified a single nucleotide substitution of C to T at position 1583, resulting in the change of alanine to valine at amino acid position 528 (A528V). The mutation occurs in domain II and in a location which corresponds to the hypervariable region of the spike protein of the coronavirus mouse hepatitis virus. Experimental introduction of the A528V mutation into the wild-type spike protein resulted in the loss of MAb binding of the mutant protein, confirming that the single point mutation was responsible for the escape of BCV from immunological selective pressure.
Infectivity-Neutralizing and Hemagglutinin-Inhibiting Antibody Responses to Respiratory Coronavirus Infections of Cattle in Pathogenesis of Shipping Fever Pneumonia
Authors: Lin Xiaoqing , O'Reilly Kathy L. , Burrell Mamie L. , Storz Johannes
Published: July 2002

Respiratory bovine coronaviruses (RBCV) emerged as an infectious agent most frequently isolated from respiratory tract samples of cattle with acute respiratory tract diseases. Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) antibodies induced by RBCV infections were monitored in sequential serum samples collected from cattle during a naturally evolving and experimentally monitored epizootic of shipping fever pneumonia (SFP). Cattle nasally shedding RBCV at the beginning of the epizootic started with low levels of serum IN and HAI antibodies. An increase in serum IN antibody after day 7 led to reduction of virus shedding in nasal secretions by the majority of the cattle between days 7 and 14. A substantial rise in the serum HAI antibody was observed during the initial phase among the sick but not the clinically normal cattle which were infected with RBCV. The RBCV isolation-positive cattle that developed fatal SFP had minimal serum IN and HAI antibodies during the course of disease development. Cattle that remained negative in RBCV isolation tests entered this epizootic with high levels of serum IN and HAI antibodies, which dramatically increased during the next two weeks. Protection against SFP was apparently associated with significantly higher levels of serum IN antibodies at the beginning of the epizootic. The RBCV-neutralizing activity is associated with serum immunoglobulin G (IgG), particularly the IgG2 subclass, while RBCV-specific HAI antibody is related to both serum IgG and IgM fractions.
Field Validation of the Use of RB51 as Antigen in a Complement Fixation Test To Identify Calves Vaccinated withBrucella abortusRB51
Authors: Adone Rosanna , Ciuchini Franco , Olsen Steven
Published: July 2002

In order to confirm the efficiency of an experimental RB51-based complement fixation (CF) test in identifying cattle vaccinated withBrucella abortusstrain RB51, 831 sera from 110 vaccinated and 48 unvaccinated Hereford heifers of Iowa, collected for studies conducted in different years, were sent to Italy without coding to be tested in a CF test using RB51 as antigen. Most of the calves, aged from 3 to 10 months, were vaccinated subcutaneously with the recommended dosage of 1010CFU of RB51 commercial vaccine, while only six calves received 109CFU of the same vaccine. Serum samples for serologic testing, collected until 16 postinoculation weeks (PIW), were also tested by routine surveillance tests for brucellosis such as rose bengal plate and CF tests performed withB. abortussmooth strain 99 as control antigen. RB51 CF test results obtained by testing sera from cattle vaccinated in 1999 indicate that the sensitivity of the reaction is 97% at 2 to 3 PIW and 90% until 8 PIW and decreases to 65% at 12 PIW, the specificity remaining at 100%. Collectively, the results of this study confirm that serologic standard tests fail to detect antibodies to RB51 while the RB51-based CF test is able to monitor antibody responses to RB51 until 15 to 16 PIW with a specificity of 100%. In addition, unlike the RB51-based dot blot assay, which is the only test currently used to monitor antibody responses to RB51, the CF test also detected specific responses following vaccination with 109CFU of RB51, although seroconversion was only 50% at 8 PIW. In conclusion, because of high specificity and sensitivity, the CF test described here can be used to efficaciously monitor serologic responses following RB51 vaccination in cattle and could also be employed to detect RB51 infection in humans exposed to this strain.
Evaluation of a Latex Agglutination Kit (Virogen Rotatest) for Detection of Bovine Rotavirus in Fecal Samples
Authors: Al-Yousif Yousif , Anderson Joe , Chard-Bergstrom Cindy , Bustamante Adrian , Muenzenberger Margaret , Austin Kimberly , Kapil Sanjay
Published: July 2002

The performance of the Virogen Rotatest latex agglutination test (LAT) was evaluated for detection of bovine rotavirus antigen. Sixty-three fecal samples from diarrheic calves were collected from November 1999 to May 2000 and screened by LAT, the Rotazyme II enzyme-linked immunosorbent assay (ELISA), and virus isolation (VI) followed by an anti-rotavirus fluorescent-antibody (FA) test to detect the presence of group A rotavirus antigen. Of the 63 samples screened by VI-FA, 33 (58%) tested positive for rotavirus antigen. When the results from the LAT were compared to those from VI-FA, the “gold standard” for detection of bovine rotavirus in fecal samples, the sensitivity and specificity were found to be 87.8 and 73.3%, respectively. Latex agglutination compared with ELISA (the reference method) showed 100% sensitivity and 96.3% specificity, and when ELISA was compared with VI, the sensitivity was 84.8% and the specificity was 73.3%. Latex agglutination is easy to perform in a short time and does not require expensive equipment or skilled personnel, and the reagents have long shelf lives. These factors make the LAT suitable and highly efficient for use in a clinical laboratory as a rapid screening test for bovine rotavirus.
Oral Administration of Influenza Vaccine in Combination with the Adjuvants LT-K63 and LT-R72 Induces Potent Immune Responses Comparable to or Stronger than Traditional Intramuscular Immunization
Authors: Barackman John D. , Ott Gary , Pine Samuel , O'Hagan Derek T.
Published: July 2002

Mucosal immunization strategies are actively being pursued in the hopes of improving the efficacy of vaccines against the influenza virus. Our group investigated the oral immunization of mice via intragastric gavage with influenza hemagglutinin (HA) combined with mutantEscherichia coliheat-labile enterotoxins K63 (LT-K63) and R72 (LT-R72). These oral immunizations resulted in potent serum antibody and HA inhibition titers, in some cases stronger than those obtained with traditional intramuscular administration, in addition to HA-specific immunoglobulin A in the saliva and nasal secretions. This study demonstrates that it may be possible to develop effective oral influenza vaccines.
Anti-CD20 Monoclonal Antibody Treatment of Human Herpesvirus 8-Associated, Body Cavity-Based Lymphoma with an Unusual Phenotype in a Human Immunodeficiency Virus-Negative Patient
Authors: Pérez Celeste L. , Rudoy Silvia
Published: July 2002

Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus first detected in Kaposi's sarcoma tumor cells and subsequently in primary effusion lymphoma (PEL) tumor cells and peripheral blood mononuclear cells from PEL patients. PEL has been recognized as an individual nosologic entity based on its distinctive features and consistent association with HHV-8 infection. PEL is an unusual form of body cavity-based B-cell lymphoma (BCBL). It occurs predominantly in human immunodeficiency virus (HIV)-positive patients but occasionally also in elderly HIV-negative patients. We describe a case of PEL, with ascites, bilateral pleural effusions, and a small axillary lymphadenopathy, in a 72-year-old HIV-negative man. PCR performed on a lymph node specimen and in liquid effusion was positive for HHV-8 and negative for Epstein-Barr virus. The immunophenotype of the neoplastic cells was B CD19
+
CD20
+
CD22
+
with coexpression of CD10 and CD23 and with clonal kappa light chain rearrangement. The patient was treated with Rituximab, a chimeric (human-mouse) anti-CD20 monoclonal antibody. Thirteen months later, the patient continued in clinical remission. This is the first report of an HHV-8-associated BCBL in an HIV-negative patient in Argentina.
Antibodies to an Epitope from the Cha Human Autoantigen Are Markers of Chagas' Disease
Authors: Gironès Núria , Rodrı́guez Clara I. , Basso Beatriz , Bellon José M. , Resino Salvador , Muñoz-Fernández M. Angeles , Gea Susana , Moretti Edgardo , Fresno Manuel
Published: July 2002

Chagas' disease is a prevalent disease in South America that is thought to have an autoimmune etiology. We previously identified human Cha as a new autoantigen recognized by chagasic sera. Those sera recognized an epitope spanning amino acids 120 to 129 of Cha, named R3. In the present study we have used the synthetic R3 peptide for the detection of serum immunoglobulin G antibodies from patients at different stages of Chagas' disease, including a therapeutically treated group. The immunoreactivity with R3 by enzyme-linked immunosorbent assay (ELISA) showed 92.4% sensitivity and 100% specificity for Chagas' disease sera. This sensitivity and specificity were higher than for any other autoantigen described to date. No anti-R3 antibodies were detected in sera from
Leishmania
-infected or idiopathic dilated cardiomyopathy patients or healthy controls from the same areas. Moreover, anti-R3 antibody reactivity detected by ELISA correlated with conventional serological tests as indirect immunofluorescence and ELISA assays with
Trypanosoma cruzi
extracts and other diagnostic tests as indirect hemagglutination. The levels of anti-R3 antibodies increased with progression and symptomatology of Chagas' disease. More interestingly, a statistically significant fall in anti-R3 antibody titer was observed in patients treated with antiparasitic drugs. Those results suggest that the presence of anti-R3 antibodies is a highly specific marker of Chagas' disease and that R3 ELISA could be helpful in the diagnosis and monitoring of this disease.
Modified Indirect Porcine Circovirus (PCV) Type 2-Based and Recombinant Capsid Protein (ORF2)-Based Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to PCV
Authors: Nawagitgul Porntippa , Harms Perry A. , Morozov Igor , Thacker Brad J. , Sorden Steven D. , Lekcharoensuk Chalermpol , Paul Prem S.
Published: July 2002

Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations.
Lymphocyte Depletion in Ileal Peyer’s Patch Follicles in Lambs Infected withEimeria ovinoidalis
Authors: Aleksandersen Mona , Lie Kai-Inge , Gjerde Bjørn , Landsverk Thor
Published: July 2002

A total of 14 lambs were experimentally infected withEimeria ovinoidalisin two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer’s patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5′- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severeE. ovinoidalisinfection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer’s patch follicles.
Impact of Hepatitis B and Hepatitis C Virus Infections in a Hematology-Oncology Unit at a Children's Hospital in Nicaragua, 1997 to 1999
Authors: Visoná Kirsten , Baez Fulgencio , Taylor Lizeth , Berríos René , León Bernal , Pacheco Carlos , Jirón Roberto , Luftig Ronald B. , Somarriba M. Mercedes
Published: July 2002

The risk of acquiring both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in patients with hematological-oncological disorders has been documented. However, the impact and risk factors for such infections from different geographical areas vary, and the use of both immunological and molecular assays to determine HCV infections has been our approach. Children from a hematology-oncology unit (HOU) in Nicaragua were studied for both HBV and HCV serological markers; studies for the latter used both immunological (anti-HCV) and molecular (HCV RNA) assays. The children from the HOU included patients with leukemia, lymphoma, other neoplasias, and anemia and a smaller group with other hematological diseases. As a control group, children from other units at the same hospital were enrolled, as well as health care workers attending both patient populations. Pertinent clinical and personal data for each child at the HOU were obtained for statistical analysis. Of the 625 children from the HOU enrolled in this study 53.3% were infected with HCV and 29.4% had a prior or present HBV infection. In the child patient control group 3.2% had HBV markers and all were negative for HCV. The group of children with leukemia had the highest infection rate for both HBV and HCV. However, the determination of anti-HCV was found to have an overall low sensitivity in children from HOU, and a retest consisting of a molecular assay to determine HCV RNA was performed to better establish the total number of HCV-infected subjects in this group. The highest independent risk factor for infection was hospitalization. The very high prevalence rates for both HBV and HCV infection in this patient group indicate an urgent need to implement better control of known risk factors and to consider the use of both immunological and molecular assays for HCV diagnostic purposes.
Bioterrorism: Implications for the Clinical Microbiologist
Authors: Klietmann Wolfgang F. , Ruoff Kathryn L.
Published: July 2002

The specter of bioterrorism has captured the attention of government and military officials, scientists, and the general public. Compared to other sectors of the population, clinical microbiologists are more directly impacted by concerns about bioterrorism. This review focuses on the role envisioned for clinical laboratories in response to a bioterrorist event. The microbiology and clinical aspects of the biological agents thought to be the most likely tools of bioterrorists are presented. The historical background of the problem of bioterrorism and an overview of current U.S. preparedness planning, with an emphasis on the roles of health care professionals, are also included.