bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of Superoxide Dismutases in the Response ofEscherichia colito Selenium Oxides
Authors: Bébien Magali , Lagniel Gilles , Garin Jérôme , Touati Danièle , Verméglio André , Labarre Jean
Published: July 2002

Selenium can provoke contrasting effects on living organisms. It is an essential trace element, and low concentrations have beneficial effects, such as the reduction of the incidence of cancer. However, higher concentrations of selenium salts can be toxic and mutagenic. The bases for both toxicity and protection are not clearly understood. To provide insights into these mechanisms, we analyzed the proteomic response ofEscherichia colicells to selenate and selenite treatment under aerobic conditions. We identified 23 proteins induced by both oxides and ca. 20 proteins specifically induced by each oxide. A striking result was the selenite induction of 8 enzymes with antioxidant properties, particularly the manganese and iron superoxide dismutases (SodA and SodB). The selenium inductions ofsodAandsodBwere controlled by the transcriptional regulators SoxRS and Fur, respectively. Strains with decreased superoxide dismutase activities were severely impaired in selenium oxide tolerance. Pretreatment with a sublethal selenite concentration triggered an adaptive response dependent upon SoxRS, conferring increased selenite tolerance. Altogether, our data indicate that superoxide dismutase activity is essential for the cellular defense against selenium salts, suggesting that superoxide production is a major mechanism of selenium toxicity under aerobic conditions.
Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease fromAlteromonassp. Strain O-7 and Its Role in the Chitinolytic System
Authors: Miyamoto Katsushiro , Nukui Eiji , Itoh Hiroyuki , Sato Takaji , Kobayashi Takeshi , Imada Chiaki , Watanabe Etsuo , Inamori Yoshihiko , Tsujibo Hiroshi
Published: July 2002

Alteromonassp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned inEscherichia coli. DNA sequencing analysis revealed that the open reading frame ofaprIVencoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to α-chitin and β-chitin. Western and Northern analyses demonstrated thataprIVwas induced in the presence ofN-acetylglucosamine,N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity fromAlteromonassp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.
Aortic Valve Endocarditis in a Dog Due toBartonella clarridgeiae
Authors: Chomel Bruno B. , Mac Donald Kristin A. , Kasten Rickie W. , Chang Chao-Chin , Wey Aaron C. , Foley Janet E. , Thomas William P. , Kittleson Mark D.
Published: July 2002

We report the first documented case of endocarditis associated withBartonella clarridgeiaein any species.B. clarridgeiaewas identified as a possible etiological agent of human cat scratch disease. Infective vegetative valvular aortic endocarditis was diagnosed in a 2.5-year-old male neutered boxer. Historically, the dog had been diagnosed with a systolic murmur at 16 months of age and underwent balloon valvuloplasty for severe valvular aortic stenosis. Six months later, the dog was brought to a veterinary hospital with an acute third-degree atrioventricular block and was diagnosed with infective endocarditis. The dog died of cardiopulmonary arrest prior to pacemaker implantation. Necropsy confirmed severe aortic vegetative endocarditis. Blood culture grew a fastidious, gram-negative organism 8 days after being plated. Phenotypic and genotypic characterization of the isolate, including partial sequencing of the citrate synthase (gltA) and 16S rRNA genes indicated that this organism wasB. clarridgeiae. DNA extraction from the deformed aortic valve and the healthy pulmonic valve revealed the presence ofB. clarridgeiaeDNA only from the diseased valve. NoBorrelia burgdorferiorEhrlichiasp. DNA could be identified. Using indirect immunofluorescence tests, the dog was seropositive forB. clarridgeiaeand had antibodies againstEhrlichia phagocytophilabut not againstEhrlichia canis, Ehrlichia ewingii,B. burgdorferi, orCoxiella burnetii.
Identification and Antimicrobial Susceptibility of
Alcaligenes xylosoxidans
Isolated from Patients with Cystic Fibrosis
Authors: Saiman Lisa , Chen Yunhua , Tabibi Setareh , San Gabriel Pablo , Zhou Juyan , Liu Zhenling , Lai Lena , Whittier Susan
Published: July 2002

In the past decade, potential pathogens, including
Alcaligenes
species, have been increasingly recovered from cystic fibrosis (CF) patients. Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF. We examined the frequency of correct identification of
Alcaligenes
spp. by microbiology laboratories affiliated with American CF patient care centers. Selective media, an
exotoxin A
probe for
Pseudomonas aeruginosa
, and a commercial identification assay, API 20 NE, were used for identification. The activity of antimicrobial agents against these clinical isolates was determined. A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied. Most (89%) were correctly identified by the referring laboratories as
Alcaligenes xylosoxidans
. However, 12 (11%) strains were misidentified; these were found to be
P. aeruginosa
(
n
= 10),
Stenotrophomonas maltophilia
(
n
= 1), and
Burkholderia cepacia
(
n
= 1). Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited. High concentrations of colistin (100 and 200 μg/ml) inhibited 92% of strains. Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains. Selective media and biochemical identification proved to be useful strategies for distinguishing
A. xylosoxidans
from other CF pathogens. Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of
A. xylosoxidans
should be determined.
Scedosporium prolificansKeratouveitis in Association with a Contact Lens Retained Intraocularly over a Long Term
Authors: Arthur Stella , Steed Lisa L. , Apple David J. , Peng Qun , Howard Gene , Escobar-Gomez Marcela
Published: July 2002

Scedosporium prolificansis a soil saprophyte that is associated with a large variety of infectious processes and with respiratory colonization in immunocompetent and immunocompromised patients. We report the first described case ofS. prolificanskeratouveitis associated with the intraocular long-term retention of a contact lens in a 76-year-old female patient.
Sensitive Detection ofEhrlichia chaffeensisin Cell Culture, Blood, and Tick Specimens by Reverse Transcription-PCR
Authors: Felek Suleyman , Unver Ahmet , Stich Roger W. , Rikihisa Yasuko
Published: July 2002

Ehrlichia chaffeensisis an obligatory intracellular bacterium of monocytes and macrophages and the etiologic agent of human monocytic ehrlichiosis, an emerging zoonosis. The Lone Star tick (Amblyomma americanum) has been implicated as the primary vector ofE. chaffeensis. The present study examined the sensitivity of the nested reverse transcription (RT)-PCR based on the 16S rRNA gene relative to that of the nested PCR for detection ofE. chaffeensisin infected DH82 cells, experimentally infected dog peripheral blood mononuclear cells, or experimentally infectedA. americanumtick samples. The RT-PCR was found to be approximately 100 times more sensitive than the PCR for detection ofE. chaffeensisregardless of the nature of the specimens. Thus, this RT-PCR is useful for detection ofE. chaffeensiswhen a high sensitivity is required. Positive results by RT-PCR also imply the presence of viable pathogens. This is the first demonstration of RNA ofE. chaffeensisin infected blood and acquisition-fed male, nymphal, and larvalA. americanumticks.
Immunodiagnostic Identification of Dairy Cows Infected withPrototheca zopfiiat Various Clinical Stages and Discrimination between Infected and Uninfected Cows
Authors: Roesler Uwe , Scholz Holger , Hensel Andreas
Published: July 2002

Protothecosis is a severe form of mastitis in cattle that is caused by colorless algae of the genusPrototheca. So far, no suitable serological test for the identification of infected animals is available for routine diagnosis. In this study an indirect enzyme-linked immunosorbent assay (ELISA) for the identification of infected cows and for discriminating among infected cows at various clinical stages was developed. Immunoglobulin G (IgG) in serum and IgA and IgG1 in whey were used as antibody isotypes. The ELISA was evaluated using serum and whey from animals at different clinical stages of infection. A total of 12 cows with acute clinical manifestation of protothecal mastitis, 22 cows with clinical signs of chronic mastitis, 40Prototheca zopfii-negative cows, and 18 cows with chronic clinical signs and earlier cultures positive forP. zopfiibut with presently negative culturing results were investigated. A sensitivity of 96% and a specificity of 94% were calculated for the ELISA based on IgA levels. Intra-assay and interassay variations were calculated to be 6.08 and 6.32%, respectively. Based on these data, this ELISA was found to be suitable for discrimination between infected and uninfected animals and might therefore be useful for screening affected herds.
Circulating Nucleic Acids of
Chlamydia pneumoniae
and Cytomegalovirus in Patients Undergoing Coronary Angiography
Authors: Smieja M. , Chong S. , Natarajan M. , Petrich A. , Rainen L. , Mahony J. B.
Published: July 2002

Peripheral blood mononuclear cells from 208 consecutive patients undergoing elective coronary angiography or angioplasty were collected before, immediately after, and 4 h after the procedure. Nucleic acids of
Chlamydia pneumoniae
and of cytomegalovirus (CMV) were detected by PCR and confirmed by hybridization. Circulating
C. pneumoniae
DNA was identified in 24 patients (11.5%) and was associated with current smoking (odds ratio [OR] = 4.5, 95% confidence interval [CI] = 1.6 to 12.2,
P
= 0.004) but not with arterial narrowing on coronary angiogram or with serological results positive for
C. pneumoniae
. Circulating CMV DNA was identified in 36 patients (17.3%) and was associated with anti-CMV immunoglobulin G (OR = 2.7, 95% CI = 1.2 to 6.3,
P
= 0.02) but not with angiographic arterial narrowing or with the need for revascularization. Neither
C. pneumoniae
nor CMV DNA detection increased after angioplasty, a procedure in which endothelium is disrupted. Larger prospective studies are needed to determine the prognostic significance of DNA detection.
Strain Composition of the EhrlichiaAnaplasma marginalewithin Persistently Infected Cattle, a Mammalian Reservoir for Tick Transmission
Authors: Palmer Guy H. , Rurangirwa Fred R. , McElwain Terry F.
Published: July 2002

Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure ofAnaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using themsp1α genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region whereA. marginaleis endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken byDermacentor andersoniticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneousA. marginaleand suggest that different genotypes are maintained by transmission within the reservoir population.
Isolation and Characterization of a Black-Pigmented
Corynebacterium
sp. from a Woman with Spontaneous Abortion
Authors: Shukla Sanjay K. , Vevea Dirk N. , Frank Daniel N. , Pace Norman R. , Reed Kurt D.
Published: July 2002

An unusual black-pigmented coryneform bacterium was isolated from the urogenital tract of a woman who experienced a spontaneous abortion during month 6 of pregnancy. Biochemical and chemotaxonomic analyses demonstrated that the unknown bacterium belonged to the genus
Corynebacterium
. Phylogenetic analysis based on 16S rRNA sequences (GenBank accession no.
AF220220
) revealed that the organism was a member of a distinct subline which includes uncultured
Corynebacterium
MTcory 1P (GenBank accession no.
AF115934
), derived from prostatic fluid, and
Corynebacterium
CDC B8037 (GenBank accession no.
AF033314
), an uncharacterized black-pigmented coryneform bacterium. On the basis of chemotaxonomic and phylogenetic evidence, this organism probably represents a new species and is most closely related to the uncharacterized Centers for Disease Control and Prevention group 4 coryneforms. Our strain is designated CN-1 (ATCC 700975).
Genotyping of EnterotoxigenicClostridium perfringensFecal Isolates Associated with Antibiotic-Associated Diarrhea and Food Poisoning in North America
Authors: Sparks Shauna G. , Carman Robert J. , Sarker Mahfuzur R. , McClane Bruce A.
Published: July 2002

Clostridium perfringenstype A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD). Recent studies suggest thatC. perfringenstype A food poisoning is caused byC. perfringensisolates carrying a chromosomalcpegene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmidcpeisolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34cpe-positive,C. perfringensfecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmidcpegene, but the North American food poisoning isolates all carry a chromosomalcpegene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmidcpeisolates cause most CPE-associated AAD cases and chromosomalcpeisolates cause mostC. perfringenstype A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.
Molecular Epidemiology ofStreptococcus uberisIsolates from Dairy Cows with Mastitis
Authors: Phuektes Patchara , Mansell Peter D. , Dyson Rodney S. , Hooper Narelle D. , Dick Jennifer S. , Browning Glenn F.
Published: July 2002

Pulsed-field gel electrophoresis and antimicrobial sensitivity testing were used as tools to investigate the epidemiology ofStreptococcus uberismastitis in dairy cows. A total of 62 different strains were found among 138 isolates from the four herds investigated, and between 10 and 26 different strains were found in each herd. There was no strain common to all four herds. Identical strains ofS. uberiswere detected from different quarters of individual cows and from cows within the same herd, suggesting that transmission from quarter to quarter and cow to cow had occurred. Despite the great variation inS. uberisstrains, persistent infection with the same strain within a lactation was observed in most cows. Predominant strains were present in two herds. Preliminary investigations could not clarify why these particular strains might predominate, but in one herd there was a significant difference between the prevalence of clinical mastitis in quarters infected with the predominant strain and that in quarters infected with other strains, suggesting the greater virulence of the predominant strain. The wide variety ofS. uberisstrains found is consistent with an environmental source ofS. uberis. However, evidence of direct transmission, the persistence of infection, and the predominance of particular strains in some herds indicate thatS. uberisinfections are epidemiologically complex and that the relative importance of these factors in the occurrence of mastitis may differ between herds.
Detection of Salmonellae in Chicken Feces by a Combination of Tetrathionate Broth Enrichment, Capillary PCR, and Capillary Gel Electrophoresis
Authors: Carli K. Tayfun , Unal Can Bora , Caner Vildan , Eyigor Aysegul
Published: July 2002

This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. PureSalmonella entericaserovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that aSalmonellagenus-specific 281-bp PCR product was detected whenSalmonellastrains but not non-Salmonellastrains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pureSalmonella entericaserovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml−1, respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml−1, respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples,Salmonellacould not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harborSalmonellaby both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detectSalmonellawith the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identifySalmonella-infected flocks.
Utility of Inoculum Counting (Walshe and English Criteria) in Clinical Diagnosis of Onychomycosis Caused by Nondermatophytic Filamentous Fungi
Authors: Gupta Aditya K. , Cooper Elizabeth A. , MacDonald Paul , Summerbell Richard C.
Published: July 2002

Opportunistic onychomycosis caused by nondermatophytic molds may differ in treatment from tinea unguium. Confirmed diagnosis of opportunistic onychomycosis classically requires more than one laboratory analysis to show consistency of fungal outgrowth. Walshe and English in 1966 proposed to extract sufficient diagnostic information from a single patient consultation by counting the number of nail fragments positive for inoculum of the suspected fungus. Twenty fragments were plated per patient, and each case in which five or more fragments grew the same mold was considered an infection by that mold, provided that compatible filaments were also seen invading the nail tissue by direct microscopy. This widely used and often recommended method has never been validated. Therefore, the validity of substituting any technique based on inoculum counting for conventional follow-up study in the diagnosis of opportunistic onychomycosis was investigated. Sampling of 473 patients was performed repeatedly. Nail specimens were examined by direct microscopy, and 15 pieces were plated on standard growth media. After 3 weeks, outgrowing dermatophytes were recorded, and pieces growing any nondermatophyte mold were counted. Patients returned on two to eight additional occasions over a 1- to 3-year period for similar examinations. Onychomycosis was etiologically classified based on long-term study. Opportunistic onychomycosis was definitively established for 86 patients. Counts of nondermatophyte molds in initial examinations were analyzed to determine if they successfully predicted both true cases of opportunistic onychomycosis and cases of insignificant mold contamination. There was a strong positive statistical association between mold colony counts and true opportunistic onychomycosis. Logistic regression analysis, however, determined that even the highest counts predicted true cases of opportunistic onychomycosis only 89.7% of the time. The counting criterion suggested by Walshe and English was correct only 23.2% of the time.
Acremonium
infections were especially likely to be correctly predicted by inoculum counting. Inoculum counting could be used to indicate a need for repeat studies in cases of false-negative results from laboratory direct microscopy. Inoculum counting cannot serve as a valid substitute for follow-up study in the diagnosis of opportunistic onychomycosis. It may, nonetheless, provide useful information both to the physician and to the laboratory, and it may be especially valuable when the patient does not present for follow-up sampling.
Recombinant Major Antigenic Protein 2 ofEhrlichia canis: a Potential Diagnostic Tool
Authors: Alleman A. Rick , McSherry Leo J. , Barbet Anthony F. , Breitschwerdt Edward B. , Sorenson Heather L. , Bowie Michael V. , Bélanger Myriam
Published: July 2002

The major antigenic protein 2 (MAP2) ofEhrlichia caniswas cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples fromE. canisimmunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected withE. canisand were previously demonstrated to contain antibodies reactive withE. canisby indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other thanE. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 ofE. canisin the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 ofE. caniscould be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis.
Genomic Heterogeneity and O-Antigenic Diversity ofCampylobacter upsaliensisandCampylobacter helveticusStrains Isolated from Dogs and Cats in Germany
Authors: Moser I. , Rieksneuwöhner B. , Lentzsch P. , Schwerk P. , Wieler L. H.
Published: July 2002

A serotyping scheme based on heat-stable surface antigens was established for 101Campylobacter upsaliensisand 10Campylobacter helveticusstrains isolated from 261 dogs and 46 cats of different ages originating from two geographically distinct regions in Germany. The prevalence ofC. upsaliensisvaried between 27.8% in juvenile dogs (<12 months of age) and 55.4% in adult dogs (P< 0.05). Of the cats, 19.6% harboredC. upsaliensis, whereas 21.7% carriedC. helveticus. Of theC. upsaliensisisolates from both host species, 93.1% belonged to five different serogroups, two of them being prevalent at rates of 47.5 and 27.7%, with different frequencies in both regions. Six (54.6%) of theC. helveticusisolates also belonged to serotypes found amongC. upsaliensisstrains, whereas five (45.4%) possessed an O antigen unique forC. helveticus. In contrast, a considerable degree of genomic diversity of the isolates was assessed by macrorestriction analyses with the endonucleasesSmaI andXhoI, using pulsed-field gel electrophoresis as well as enterobacterial repetitive intergenic consensus sequence PCR (ERIC PCR). Restriction withSmaI pointed towards the existence of clonal groups associated to some extent with serotypes, while restriction withXhoI disintegrated these groups to smaller noncoherent subgroups. Analysis of ERIC PCR profiles did not exhibit any associations with serotypes. In conclusion these data demonstrate the genomic heterogeneity amongC. upsaliensisstrains and indicate that the combination ofSmaI restriction with serotyping is a useful tool to investigate the expansion of clonal groups ofC. upsaliensis.
Rapid and Inexpensive Drug Susceptibility Testing of
Mycobacterium tuberculosis
with a Nitrate Reductase Assay
Authors: Ängeby K. A. Kristian , Klintz Lisbeth , Hoffner Sven E.
Published: July 2002

Multidrug-resistant tuberculosis is an increasing public health concern in many parts of the world, especially in low-income countries, where most cases occur. Traditional drug susceptibility testing is either time-consuming, such as the proportion method on solid media, or expensive, such as the BACTEC 460 system. We have evaluated a new nitrate reductase assay (NRA) that depends on the ability of
Mycobacterium tuberculosis
to reduce nitrate to nitrite. The reduction can be detected using specific reagents, which produce a color change. We tested a panel of 57
M. tuberculosis
strains with various resistance patterns. The bacteria were inoculated on Löwenstein-Jensen medium, either without drugs or with rifampin, isoniazid, streptomycin, or ethambutol and with potassium nitrate (KNO
3
) incorporated. After incubation for 7, 10, or 14 days, the reagents were added and nitrate reduction, indicating growth, could be detected by a color change. Sensitivities to and specificities for drugs as determined by the NRA method compared to those determined by the BACTEC 460 method were 100 and 100% for rifampin, 97 and 96% for isoniazid, 95 and 83% for streptomycin, and 75 and 98% for ethambutol, respectively. The results were in the majority of the cases available in 7 days. The evaluated method is rapid and inexpensive and could correctly identify most resistant and sensitive
M. tuberculosis
strains. It has the potential to become an interesting alternative to existing methods, such as the proportion and BACTEC methods, particularly in resource-poor settings.
Rapid Identification ofCandidaSpecies by Confocal Raman Microspectroscopy
Authors: Maquelin K. , Choo-Smith L.-P. , Endtz H. P. , Bruining H. A. , Puppels G. J.
Published: July 2002

Candidaspecies are important nosocomial pathogens associated with high mortality rates. Rapid detection and identification ofCandidaspecies can guide a clinician at an early stage to prescribe antifungal drugs or to adjust empirical therapy when resistant species are isolated. Confocal Raman microspectroscopy is highly suitable for the rapid identification ofCandidaspecies, since Raman spectra can be directly obtained from microcolonies on a solid culture medium after only 6 h of culturing. In this study, we have used a set of 42Candidastrains comprising five species that are frequently encountered in clinical microbiology to test the feasibility of the technique for the rapid identification ofCandidaspecies. The procedure was started either from a culture on Sabouraud medium or from a positive vial of an automated blood culture system. Prior to Raman measurements, strains were subcultured on Sabouraud medium for 6 h to form microcolonies. Using multivariate statistical analyses, a high prediction accuracy (97 to 100%) was obtained with the Raman method. Identification with Raman microspectroscopy may therefore be significantly faster than identification with commercial identification systems that allow various species to be identified and that often require 24 to 48 h before a reliable identification is obtained. We conclude that confocal Raman microspectroscopy offers a rapid, accurate, and easy-to-use alternative for the identification of clinically relevantCandidaspecies.
Frequency of Mutations Conferring Resistance to Nucleoside Reverse Transcriptase Inhibitors in Human Immunodeficiency Virus Type 1-Infected Patients in Korea
Authors: Cho Young Keol , Sung Heungsup , Ahn Sun Hee , Bae In Gyu , Woo Jun Hee , Won Young Ho , Kim Dae Ghon , Kang Moon Won
Published: July 2002

A nested PCR and direct sequencing methods were used to define human immunodeficiency virus type 1(HIV-1) reverse transcriptase codons 41 to 219 in DNA from 127 peripheral blood mononuclear cell samples obtained from 35 patients treated with nucleoside reverse transcriptase inhibitors (NRTI). The follow-up period after the initiation of NRTI therapy was 61.8 ± 31 months (mean and standard deviation). In addition to NRTI therapy, 32 of 35 patients were simultaneously treated with Korean red ginseng. The annual decrease in the CD4
+
T-cell count over 5 years was 13.2/μl. Twenty-eight (80%) of the 35 patients had mutations conferring resistance to NRTI. The frequencies of K70R, T215S/Y/F (i.e., mutation of T at codon 215 to S, Y, or F), D67N/E, K219Q, T69N/S/A, M41L, and L210W mutations conferring resistance to zidovudine were 57.6, 36.4, 36.4, 27.2, 24.2, 21.2, and 12.1%, respectively. Mutations conferring resistance to didanosine and lamivudine were detected in 2 (L74V and M184I; 14.2%) of 11 patients tested and in 4 (M184V; 57%) of 7 patients tested, respectively. In particular, the frequency of T69N/S/A increased sharply after more than 48 months of zidovudine monotherapy. However, Q151M was not detected. As the first report on the frequency of NRTI resistance mutations in Korea, our data suggest that genotypic antiretroviral drug testing should be considered for the design of better drug regimens to improve the management of HIV-1-infected patients.
Prevalent Bacterial Species and Novel Phylotypes in Advanced Noma Lesions
Authors: Paster B. J. , Falkler, W. A. , Enwonwu C. O. , Idigbe E. O. , Savage K. O. , Levanos V. A. , Tamer M. A. , Ericson R. L. , Lau C. N. , Dewhirst F. E.
Published: July 2002

The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods. 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned intoEscherichia coli. Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or closest relatives by comparison with sequences of known species or phylotypes. Nearly complete sequences of approximately 1,500 bases were obtained for most of the potentially novel species. A total of 67 bacterial species or phylotypes were detected, 25 of which have not yet been grown in vitro. Nineteen of the species or phylotypes, includingPropionibacterium acnes,Staphylococcusspp., and the opportunistic pathogensStenotrophomonas maltophiliaandOchrobactrum anthropiwere detected in more than one subject. Other known species that were detected includedAchromobacterspp.,Afipiaspp.,Brevundimonas diminuta,Capnocytophagaspp.,Cardiobacteriumsp.,Eikenella corrodens,Fusobacteriumspp.,Gemella haemoylsans, andNeisseriaspp. Phylotypes that were unique to noma infections included those in the generaEubacterium,Flavobacterium,Kocuria,Microbacterium, andPorphyromonasand the relatedStreptococcus salivariusand generaSphingomonasandTreponema. Since advanced noma lesions are infections open to the environment, it was not surprising to detect species not commonly associated with the oral cavity, e.g., from soil. Several species previously implicated as putative pathogens of noma, such as spirochetes andFusobacteriumspp., were detected in at least one subject. However, due to the limited number of available noma subjects, it was not possible at this time to associate specific species with the disease.
Dense Bodies of Human Cytomegalovirus Induce both Humoral and Cellular Immune Responses in the Absence of Viral Gene Expression
Authors: Pepperl Sandra , Münster Jürgen , Mach Michael , Harris J. Robin , Plachter Bodo
Published: July 2002

Infection of fibroblast cell cultures with human cytomegalovirus (HCMV) leads to the production of significant amounts of defective enveloped particles, termed dense bodies (DB). These noninfectious structures contain major antigenic determinants which are responsible for induction of both the humoral and the cellular immune response against HCMV. We tested the hypothesis that, by virtue of their unique antigenic and structural properties, DB could induce a significant immune response in the absence of infectious virus. Mice were immunized with gradient-purified DB, which were either left untreated or subjected to sequential rounds of sonication and freeze-thawing to prevent cellular entry. Titers of neutralizing antibodies induced by DB were in a range comparable to levels present in convalescent human sera. The virus-neutralizing antibody response was surprisingly durable, with neutralizing antibodies detected 12 months following primary immunization. The HCMV-specific major histocompatibility complex class I-restricted cytolytic T-cell (CTL) response was assayed using mice transgenic for the human HLA-A2 molecule. Immunization with DB led to high levels of HCMV-specific CTL in the absence of de novo viral protein synthesis. Maximal total cytolytic activity in mice immunized with DB was nearly as efficient as the cytolytic activity induced by a standard immunization with murine cytomegalovirus. Furthermore, DB induced a typical T-helper 1 (Th1)-dominated immune response in mice, as determined by cytokine and immunoglobulin G isotype analysis. Induction of humoral and cellular immune responses was achieved without the concomitant use of adjuvant. We thus propose that DB can serve as a basis for the future development of a recombinant nonreplicating vaccine against HCMV. Finally, such particles could be engineered for efficient delivery of antigens from other pathogens to the immune system.
Vaccination against Canine Distemper Virus Infection in Infant Ferrets with and without Maternal Antibody Protection, Using Recombinant Attenuated Poxvirus Vaccines
Authors: Welter Janet , Taylor Jill , Tartaglia James , Paoletti Enzo , Stephensen Charles B.
Published: July 2002

Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n= 5) or ALVAC-HF (n= 4) developed significant neutralizing titers (log10inverse mean titer ± standard deviation of 2.30 ± 0.12 and 2.20 ± 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 ± 0.57 versus 0.40 ± 0.37,P= 0.02) and a better survival rate (6/7 versus 0/5,P= 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n= 7) and ALVAC-HF (n= 7) developed significantly higher antibody titers (1.64 ± 0.54 and 1.28 ± 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 ± 0.59;n= 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 ± 0.32;n= 8,P= 7 × 10−6). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n= 9) produced higher titers (1.63 ± 0.25) than did i.n. immunization with NYVAC-HF (0.88 ± 0.36;n= 9) and ALVAC-HF (0.61 ± 0.43;n= 9,P= 3 × 10−7), and survival was also significantly better in the i.n.-parenteral group (3 of 9) than in the other HF-vaccinated animals (none of 18) or in controls immunized with RG (none of 5) (P= 0.0374). Multiple routes were not tested with the ALVAC vaccine. The results suggest that infant ferrets are less responsive to i.n. vaccination than are older ferrets and raises questions about the appropriateness of this route of immunization in infant ferrets or infants of other species.
CD4-Independent Infection of Two CD4−/CCR5−/CXCR4+Pre-T-Cell Lines by Human and Simian Immunodeficiency Viruses
Authors: Borsetti Alessandra , Parolin Cristina , Ridolfi Barbara , Sernicola Leonardo , Geraci Andrea , Ensoli Barbara , Titti Fausto
Published: July 2002

The infection of CD4-negative cells by variants of tissue culture-adapted human immunodeficiency virus type 1 (HIV-1) or HIV-2 strains has been shown to be mediated by the CXCR4 coreceptor. Here we show that two in vitro-established CD4−/CCR5−/CXCR4+human pre-T-cell lines (A3 and A5) can be productively infected by wild-type laboratory-adapted T-cell-tropic HIV-1 and HIV-2 strains in a CD4-independent, CXCR4-dependent fashion. Despite the absence of CCR5 expression, A3 and A5 cells were susceptible to infection by the simian immunodeficiency viruses SIVmac239 and SIVmac316. Thus, at least in A3 and A5 cells, one or more of the chemokine receptors can efficiently support the entry of HIV and SIV isolates in the absence of CD4. These findings suggest that to infect cells of different compartments, HIV and SIV could have evolved in vivo to bypass CD4 and to interact directly with an alternative receptor.
Infectivity of Moloney Murine Leukemia Virus Defective in Late Assembly Events Is Restored by Late Assembly Domains of Other Retroviruses
Authors: Yuan Bing , Campbell Stephen , Bacharach Eran , Rein Alan , Goff Stephen P.
Published: July 2002

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant
gag
gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.
Follicular Dendritic Cells and Dissemination of Creutzfeldt-Jakob Disease
Authors: Manuelidis Laura , Zaitsev Igor , Koni Pandelakis , Yun Lu Zhi , Flavell Richard A. , Fritch William
Published: July 2002

The contribution of immune system cells to the propagation of transmissible encephalopathies is not well understood. To determine how follicular dendritic cells (FDC) may act, we challenged lymphotoxin β null and wild-type (wt) controls with a Creutzfeldt-Jakob disease (CJD) agent. There was only a small difference in incubation time to clinical disease even after peripheral challenge with low infectious doses (31 in a total of 410 days). Brain pathology with extensive microglial infiltration, identified by keratan sulfate, as well as astrocytic hypertrophy, was also equivalent in all groups despite the fact that null mice had neither FDC nor splenic metallophilic macrophages that filter particulate antigen. Because FDC accumulate pathologic prion protein (PrP) in infected but not wt mice, we studied the cellular distribution of PrP by confocal microscopy. The majority of pathologic PrP collected on the plasma membrane of FDC, as identified by the Ca+2-binding protein S100A. This surface distribution suggested that agent aggregated with pathologic PrP might spread by cell-to-cell contacts. While several types of leukocytes may be involved in agent dissemination, cells of myeloid lineage were found to be infectious. Moreover, perivascular tracks of microglia and abnormal PrP after intraperitoneal inoculation were consistent with hematogenous spread. In summary, FDC are not required for CJD agent spread from the periphery, although FDC may enhance spread through surface accumulation of pathologic PrP. While it is still not clear where the infectious agent replicates, macrophages can sequester appreciable levels of infectivity and hence act as reservoirs for prolonged latent infection.
A Recombinant Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Complex Stabilized by an Intermolecular Disulfide Bond between the gp120 and gp41 Subunits Is an Antigenic Mimic of the Trimeric Virion-Associated Structure
Authors: Binley James M. , Sanders Rogier W. , Clas Brian , Schuelke Norbert , Master Aditi , Guo Yong , Kajumo Francis , Anselma Deborah J. , Maddon Paul J. , Olson William C. , Moore John P.
Published: July 2002

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.
Cytotoxic T-Lymphocyte Responses to a Polymorphic Epstein-Barr Virus Epitope Identify Healthy Carriers with Coresident Viral Strains
Authors: Brooks J. M. , Croom-Carter D. S. G. , Leese A. M. , Tierney R. J. , Habeshaw G. , Rickinson A. B.
Published: July 2002

Cytotoxic T-lymphocyte (CTL) responses to Epstein-Barr virus (EBV) tend to focus on a few immunodominant viral epitopes; where these epitope sequences are polymorphic between EBV strains, host CTL specificities should reflect the identity of the resident strain. In studying responses in HLA-B27-positive virus carriers, we identified 2 of 15 individuals who had strong CTL memory to the pan-B27 epitope RRIYDLIEL (RRIY) from nuclear antigen EBNA3C but whose endogenous EBV strain, isolated in vitro, encoded a variant sequence RKIYDLIEL (RKIY) which did not form stable complexes with B27 molecules and which was poorly recognized by RRIY-specific CTLs. To check if such individuals were also carrying an epitope-positive strain (either related to or distinct from the in vitro isolate), we screened DNA from freshly isolated peripheral blood mononuclear cells for amplifiable virus sequences across the EBNA3C epitope, across a different region of EBNA3C with type 1-type 2 sequence divergence, and across a polymorphic region of EBNA1. This showed that one of the unexplained RRIY responders carried two distinct type 1 strains, one with an RKIY and one with an RRIY epitope sequence. The other responder carried an RKIY-positive type 1 strain and a type 2 virus whose epitope sequence of RRIFDLIEL was antigenically cross-reactive with RRIY. Of 15 EBV-seropositive donors analyzed by such assays, 12 appeared to be carrying a single virus strain, one was coinfected with distinct type 1 strains, and two were carrying both type 1 and type 2 viruses. This implies that a small but significant percentage of healthy virus carriers harbor multiple, perhaps sequentially acquired, EBV strains.