bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical properties of the outer membrane of Treponema denticola
Authors: Yotis W W , Sharma V K , Gopalsami C , Chegini S , McNulty J , Hoerman K , Keene J , Simonson L G
Published: January 2020

The outer membranes (OMs) from serovars a, b, and c of Treponema denticola, originally isolated from periodontal patients, were prepared. Dialysis of the OMs against 20 mM MgCl2 yielded the aggregable (A) and the nonaggregable (NA) moieties of the OMs. The absence of muramic acid, adenosine triphosphatase, hexokinase, and nucleic acid as well as electron microscopy indicated that the OM preparations were homogeneous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the A and NA moieties of the OMs showed approximately 25 Coomassie brilliant blue R-250 stain-positive bands or 47 silver-stained polypeptides. The relative molecular masses ranged between 14 and 97 kDa. The electrophoretic polypeptide profiles of the A and NA moieties shared many similarities among serovars a, b, and c. However, they exhibited variation in the overall pattern, intensity, or location of the polypeptide stained zones. This was especially true for serovar b. Two-dimensional electrophoretic studies showed an excess of 100 silver-stained spots with isoelectric points of 4.6 to 7.0 and relative molecular masses in the 14- to 97-kDa range. The OMs contained simple proteins, glycoproteins, and lipoproteins. The NA moieties of the OMs contained 4 to 6, 10 to 12, and 4 to 6 glycopeptides as well as two, seven, and two lipoprotein bands for serovars a, b, and c, respectively. The A moieties of the OMs showed 7 to 9, 11 to 13 and 5 to 6 glycopeptides as well as four, five, and three lipoprotein bands for serovars a, b, and c, respectively. Lipopolysaccharide was detected in the OMs of the three serovars following removal of proteins with proteinase K, pronase and silver staining of sodium dodecyl sulfate-polyacrylamide gels, or removal of lipopolysaccharide from the OMs by hot phenol extraction. The 66- and 53-kDa bands were present in serovars b and c, while a band with a relative molecular mass of 45 kDa was present only in serovar c. Endotoxin-like activity was also shown in the OMs of the three serovars by the Limulus amebocyte clotting assay and the chick embryo lethality test. This is the first report on selected biochemical properties of the OM macromolecules of three known serovars of T. denticola.
Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis
Authors: Fulop M J , Webber T , Manchee R J , Kelly D C
Published: January 2020

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.
Enzyme immunoassay for detection of antibodies against eastern equine encephalomyelitis virus in sentinel chickens
Authors: Olson J G , Scott T W , Lorenz L H , Hubbard J L
Published: January 2020

We developed an enzyme immunoassay (EIA) for the detection of immunoglobulin M (IgM) and IgG subclass antibodies directed against eastern equine encephalomyelitis (EEE) virus in chickens. The assays were compared with the serum plaque reduction neutralization test (PRNT) and the hemagglutination inhibition (HI) test for ability to detect antibodies against EEE virus in laboratory-infected birds. No cross-reactivity was detected in serum from chickens inoculated with St. Louis encephalitis or Highlands J virus. The interval after infection when EEE virus-specific antibodies were first detected by IgM and IgG EIAs was found to be similar to that determined by the PRNT and HI tests: 2 to 4 days. The IgG EIA, PRNT, and HI test detected antibodies to EEE virus for at least 27 to 30 days after inoculation. In contrast, serum from five of seven chickens did not contain detectable IgM 30 days after infection. Similarly, in all three naturally infected sentinel chickens from Maryland, IgM class antibody was undetectable 1 to 5 weeks after IgM was initially detected. EIAs provide simple and rapid alternatives to traditional tests for monitoring EEE virus infections in sentinel chicken flocks. Moreover, the IgM EIA provides a means to separate recently infected chickens from those infected greater than or equal to 1 month earlier.
Effects of incubation time and temperature on microbiologic sampling procedures for hemodialysis fluids
Authors: Arduino M J , Bland L A , Aguero S M , Favero M S
Published: January 2020

To prevent pyrogenic reactions and bacteremia in hemodialysis patients, the Association for the Advancement of Medical Instrumentation and the Centers for Disease Control recommend culturing of hemodialysis fluids (water and dialysate) at least once a month. The recommendations for total microbial counts are (i) less than or equal to 200 CFU/ml in water used to prepare dialysate or reprocess hemodialyzers and (ii) less than or equal to 2,000 CFU/ml for the dialysate. In accordance with the Association for the Advancement of Medical Instrumentation recommendations all cultures should be incubated at 37 degrees C for 48 h on suitable culture media, such as Trypticase soy agar, standard methods agar, or one of several commercially available assay systems. There have been suggestions that lower temperatures and longer incubation might improve the recovery of bacteria from water and dialysate. In this study bacterial recovery from various dialysis fluids (water, bicarbonate dialysate, and bicarbonate concentrate) at 30 and 37 degrees C was compared. Duplicate sets of samples were membrane filtered (pore size, 0.45 microns); one set was incubated at 30 degrees C and the other was incubated at 37 degrees C for 72 h. The number of visible colonies was counted every 24 h by using a dissecting microscope. No significant difference was observed in specimens incubated at 37 degrees C for 48 h compared with those incubated at 30 degrees C for 72 h. Also, bacterial recovery was significantly better when samples of bicarbonate dialysate or bicarbonate concentrate were plated on Trypticase soy agar as opposed to standard methods agar.
Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates
Authors: Marcom K A , Pearson L D , Chung C S , Poulson J M , DeMartini J C
Published: January 2020

Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats.
Detection of tetanus antitoxin using Eu(3+)-labeled anti-human immunoglobulin G monoclonal antibodies in a time-resolved fluorescence immunoassay
Authors: Schröder J P , Kuhlmann W D
Published: January 2020

Tetanus antitoxin in human sera was detected with solid-phase immunoassays in microtitration modules coated with tetanus toxoid by using Eu(3+)-labeled anti-human monoclonal antibodies on the basis of an exactly calibrated antibody standard. The use of a time-resolved fluorescence immunoassay (TR-FIA) significantly improved the quantitative detection of tetanus antitoxin over that of the enzyme-linked immunosorbent assay (ELISA) technique because of its high sensitivity and its wide measurement range, detecting antibody levels between 0.001 and 12.5 IU/ml with a single serum dilution of 1:100. For the same purpose, two different serum dilutions (1:100 and 1:1,000) were needed in the ELISA technique. TR-FIA is reproducible and can be performed in 3.5 h. A study of 2,630 serum samples was undertaken to examine the age-dependent distribution of titer levels, indicating the decline of sufficient protection in patients older than 60 years. The wide measurement range of TR-FIA enabled fast examination of large numbers of serum samples without the need for repetition, with further sample dilution, as was often necessary in the ELISA procedure.
Differentiation of Bacillus anthracis from Bacillus cereus by gas chromatographic whole-cell fatty acid analysis
Authors: Lawrence D , Heitefuss S , Seifert H S
Published: January 2020

Three strains of Bacillus anthracis and seven strains of Bacillus cereus were grown on complex medium and on synthetic medium. Gas chromatographic analysis of whole-cell fatty acids of strains grown on complex medium gave nearly identical fatty acid patterns. Fatty acid patterns of strains grown on synthetic medium showed a high content of branched-chain fatty acids. Significant differences between the fatty acid patterns of the two species were found. Odd iso/anteiso fatty acid ratios were about equal in B. anthracis strains, whereas in B. cereus strains the fractions of iso acids were at least twice as high as the fractions of anteiso acids. The method described herein is used in our diagnostic laboratory to help differentiate between these two species.
Isolation and identification of a variant of bluetongue virus serotype 11 from a ram in a bluetongue outbreak in western Texas
Authors: Chung S I , Billingsley P , Livingston C W , Collisson E W
Published: January 2020

A field strain of bluetongue virus was isolated from a blood sample of a ram during an outbreak of bluetongue in November 1985 in western Texas. In this bluetongue outbreak at least 25 of the 2,000 sheep were infected. Isolation was made by intravenous inoculation of 11-day-old embryonated chicken eggs. The serotype was identified as serotype 11 by serum neutralization tests. The genomic pattern on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of the new isolate is similar to that of bluetongue virus prototype 11. Comparisons were also made with proteins labeled in vivo with [3H]leucine and separated by SDS-PAGE. We conclude that this virus belongs to serotype 11, with slight differences in both genome and protein electrophoretic patterns.
Suitability of infection of cells in suspension for detection of herpes simplex virus
Authors: Luker G , Chow C , Richards D F , Johnson F B
Published: January 2020

Detection of herpes simplex virus in clinical specimens by the suspension-infection technique was compared with detection by conventional cell culture. The sensitivity and specificity of the suspension-infection technique compared with those of conventional culture were 95.9 to 98.2% and 97.5 to 100%, respectively, depending on the cell line used in the tests. The mean time to diagnosis by the suspension-infection technique was 1 day, compared with 4.8 days by conventional culture. Comparable detection of low-level positive specimens was observed with the methods. In a clinical setting, the isolation rates obtained by suspension-infection and conventional culture were indistinguishable. These results indicate that the suspension-infection method can be used for the detection of herpes simplex virus and can yield rapid diagnostic results without a time-consuming centrifugation step.
Human papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients
Authors: Bleul C , Müller M , Frank R , Gausepohl H , Koldovsky U , Mgaya H N , Luande J , Pawlita M , ter Meulen J , Viscidi R
Published: January 2020

Antibody-reactive regions on the human papillomavirus type 18 (HPV-18) E6 and E7 proteins were identified with rabbit polyclonal anti-fusion protein sera by screening of an fd phage expression library containing subgenomic HPV-18 DNA fragments and by testing of overlapping decapeptides representing the E6 and E7 open reading frames. Peptides comprising the delineated regions (designated E6/1 to E6/4 and E7/1) were synthesized and used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-HPV-18 antibodies in human sera. A total of 232 human serum samples (identical numbers of cervical cancer patients and age-matched controls) collected in Tanzania were tested. Similar prevalences (between 0.8 and 4.3%) of antibodies recognizing the different E6 peptides were found in the sera from tumor patients and controls. With a synthetic 28-mer peptide (designated pepE701) comprising the E7/1 region, a significant difference was found: 10 of 116 tumor serum samples but 0 of 116 control serum samples showed a specific reaction (P less than 0.001). This observation confirms earlier results with HPV-16 E7 fusion proteins (I. Jochmus-Kudielka, A. Schneider, R. Braun, R. Kimmig, U. Koldovsky, K. E. Schneweis, K. Seedorf, and L. Gissmann, J. Natl. Cancer Inst. 81:1698-1704, 1989). A lower prevalence of anti-HPV-18 E7 antibodies was observed when 188 human serum samples collected in Germany from tumor patients and controls were tested (3 of 94 positive in the cancer group; 0 of 94 positive in the control group). The type specificity of anti-HPV-18 E7 antibodies was demonstrated when the HPV type found by Southern hybridization in the cervical cancer biopsies was compared with seroreactivity: 4 of 8 serum samples obtained from HPV-18 DNA-positive but 0 of 16 serum samples from HPV-18 DNA-negative tumor patients reacted in the HPV-18 E7 ELISA. In addition, HPV-18-positive sera failed to react in a peptide ELISA with the homologous HPV-16 E7 region (M. Müller, H. Gausepohl, G. de Martinoff, R. Frank, R. Brasseur, and L. Gissmann, J. Gen. Virol. 71:2709-2717, 1990) and vice versa.
43-kilodalton glycoprotein from Paracoccidioides brasiliensis: immunochemical reactions with sera from patients with paracoccidioidomycosis, histoplasmosis, or Jorge Lobo's disease
Authors: Puccia R , Travassos L R
Published: January 2020

Sera from patients with paracoccidioidomycosis (PCM), histoplasmosis (HP), or Jorge Lobo's disease (JL) were titrated against purified gp43 from Paracoccidioides brasiliensis by using both enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IPP) reactions with 125I-labeled antigens. In IPP, PCM sera and other sera could be distinguished on the basis of serum titers, whereas in ELISA, 53% of the HP sera and 29% of the JL sera reacted similarly to the PCM sera. To investigate the possible role of the carbohydrate epitopes in these reactions, we compared the reactivities of sera from several patients with native and deglycosylated gp43. Competition experiments were carried out with monosaccharides as inhibitors. The results suggest that greater than 85% of the reactions of the PCM sera with gp43 involved peptide epitopes. Cross-reactions with HP and JL sera in ELISA were predominantly attributed to periodate-sensitive carbohydrate epitopes containing galactosyl residues. HP and JL sera which reacted strongly with gp43 in ELISA were only weakly reactive or did not react in IPP with labeled antigens in solution. Moreover, ELISA reactions could be significantly inhibited either by monosaccharides or by periodate treatment. Apparently, carbohydrate epitopes in gp43 are more accessible to the antibodies when the molecule is bound to a plastic substrate than when it is in solution. Structural changes in the gp43 antigen arising by N deglycosylation abolish reactivity with PCM sera and support the existence of conformational peptide epitopes.
Comparison of the PREMIER cryptococcal antigen enzyme immunoassay and the latex agglutination assay for detection of cryptococcal antigens
Authors: Gade W , Hinnefeld S W , Babcock L S , Gilligan P , Kelly W , Wait K , Greer D , Pinilla M , Kaplan R L
Published: January 2020

A new enzyme immunoassay (EIA), PREMIER Cryptococcal Antigen, was compared with latex agglutination (LA) for the detection and quantitation of circulating capsular polysaccharide antigen from Cryptococcus neoformans. The clinical evaluation of PREMIER EIA as a screening assay, including 475 specimens with 120 LA and EIA positives, resulted in 99% sensitivity and 97% specificity. The clinical specimens included sera and cerebrospinal fluids as well as 10 rheumatoid factor-positive and 20 anti-nuclear antibody-positive serum samples. This monoclonal antibody-based assay detects serotypes A to D at 0.63, 0.63, 7.8, and 62 ng/ml, respectively. With three different known positive specimens, the assay was found to yield coefficients of variation of 2 to 12% for intra- and interassay comparisons of precision and reproducibility. The primary use for semiquantitative values derived with the LA or EIA is to follow the course of disease and monitor drug therapies. The present data suggest that the PREMIER EIA will be a valuable method for this purpose. We conclude that the PREMIER Cryptococcal Antigen EIA provides a rapid, convenient, and reliable antigen detection method for screening and semiquantitative determination of antigen levels.
Identification of Plasmodium falciparum histidine-rich protein 2 in the plasma of humans with malaria
Authors: Parra M E , Evans C B , Taylor D W
Published: January 2020

Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) is a water-soluble protein released from parasitized erythrocytes into in vitro culture supernatants. This study sought to determine whether PfHRP-2 could be detected in the plasma of humans with P. falciparum malaria. A monoclonal antibody (1E1) is described that binds to PfHRP-2. By using monoclonal antibody 1E1, PfHRP-2 was identified by Western blot (immunoblot) analysis in the plasma of 37 of 39 (95%) patients experiencing either a first or repeat episodes of P. falciparum malaria and by dot blot analysis in the plasma of 40 of 41 patients tested. PfHRP-2 was not detected in 30 control, uninfected subjects. The current demonstration of PfHRP-2 in plasma, plus the fact that it is a structurally well-characterized molecule present in all natural isolates of P. falciparum tested, makes PfHRP-2 of interest for its potential effects on the host immune system and as an antigen for specific diagnosis of malaria.
Fluorescence immunoassay for detecting periodontal bacterial pathogens in plaque
Authors: Wolff L F , Anderson L , Sandberg G P , Aeppli D M , Shelburne C E
Published: January 2020

A particle concentration fluorescence immunoassay has been modified into a bacterial concentration fluorescence immunoassay (BCFIA) to rapidly detect periodontopathic bacteria in human plaque samples. The BCFIA utilizes fluorescently tagged monoclonal antibodies (MAbs) directed against the lipopolysaccharide of selected gram-negative plaque bacteria. Microorganisms closely associated with periodontal disease that can be identified in plaque with the BCFIA include Porphyromonas gingivalis, Bacteroides intermedius, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Eikenella corrodens. Briefly, the procedure involved mixing a patient's plaque sample or other bacterial preparation with a species-specific fluorescein isothiocyanate-labeled MAb in a specialized microtiter plate. This mixture was incubated to allow binding of the MAb to its homologous bacteria. The bound and unbound fluorescent tagged MAbs were separated by filtration in the modified microtiter plate, and the total bacterial bound fluorescence was determined with a fluorimeter. The number of a specific bacterial species in a given plaque sample or other bacterial suspension was estimated by reference to a primary standard carried through the BCFIA. The lower detection limit of the BCFIA was 10(3) to 10(4) bacterial cells from single cultures of bacteria or 10(4) bacterial cells in mixed cultures. The coefficient of variation within and between plates for each of the five bacterium-specific MAbs in screening plaque for the periodontal pathogens was less than 10%. These results demonstrate that microbes in plaque can be used as the solid phase in the BCFIA to detect and quantitate MAbs associated with specific bacteria quickly and reliably.
Mycobacterium paratuberculosis antigen D: characterization and evidence that it is a bacterioferritin
Authors: Brooks B W , Young N M , Watson D C , Robertson R H , Sugden E A , Nielsen K H , Becker S A
Published: January 2020

By using a combination of agarose and polyacrylamide gel electrophoresis, Mycobacterium paratuberculosis antigen D was resolved from a crude sonicated preparation of the organism and characterized as a component with a molecular mass of approximately 400,000 Da. While this component was composed mainly of protein, with unusually high proportions of glutamic acid and leucine, it was resistant to digestion with a number of proteolytic enzymes. Structural detail revealed by electron microscopy, amino acid sequence data, and the demonstration of a Soret band in its absorption spectrum indicated that antigen D was similar to an Escherichia coli bacterioferritin.
Restriction fragment length polymorphisms in the ribosomal genes for species identification and subtyping of aerotolerant Campylobacter species
Authors: Kiehlbauch J A , Plikaytis B D , Swaminathan B , Cameron D N , Wachsmuth I K
Published: January 2020

Whole-cell chromosomal digests of 84 strains of aerotolerant Campylobacter (AC) were examined by using PvuII restriction fragment length polymorphisms of rRNA genes followed by hybridization with Escherichia coli 16S and 23S rRNA (ribotyping). The AC strains belonged to Campylobacter cryaerophila (n = 13) and a newly defined species, "C. butzleri" (n = 64). Strains of C. cryaerophila belonged to two hybridization groups: DNA group 1A (including the type strain of C. cryaerophila) and DNA group 1B (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). Six AC strains not classified as C. cryaerophila or "C. butzleri" were also included. All 35 sporadic human and animal isolates of "C. butzleri" sent to the Centers for Disease Control for identification showed different ribotype patterns. However, most "C. butzleri" strains contained common bands at approximately 3.0, 6.2, 12.0, and 15.0 kb; the 3.0-kb band was present in all but four strains. An additional 23 strains of "C. butzleri," isolated as part of special studies, contained the 3.0-kb band. Thus, on the basis of visual identification of the 3.0-kb band, 94% of available strains were correctly identified as "C. butzleri." Ribotyping demonstrated that C. cryaerophila strains (DNA groups 1A and 1B) were different from C. butzleri strains. All C. cryaerophila strains demonstrated a common ribosomal DNA restriction fragment of 3.2 kb; DNA group 1B strains contained an additional common band at 2.6 kb. Ribotyping patterns of AC species were easily distinguished from patterns of other Campylobacter, Helicobacter, and Wolinella species.(ABSTRACT TRUNCATED AT 250 WORDS)
Genetic dissimilarity of commensal strains of Candida spp. carried in different anatomical locations of the same healthy women
Authors: Soll D R , Galask R , Schmid J , Hanna C , Mac K , Morrow B
Published: January 2020

Candida spp. carriage and strain relatedness were assessed in 52 healthy women at 17 anatomical locations by using an isolation procedure which assesses carriage intensity and by using a computer-assisted DNA fingerprinting system which computes genetic similarity between strains on the basis of the patterns of Southern blots probed with the moderately repetitive sequence Ca3. Candida spp. were cultured from 73% of the test individuals, most frequently from the oral (56%), vulvovaginal (40%), and anorectal (24%) regions. Half of the test individuals with Candida spp. carried the organism simultaneously in more than one of the three general areas of carriage. Isolates from different body locations of the same individual were either completely unrelated, identical, or highly similar but nonidentical. In 11 cases in which Candida spp. were simultaneously isolated from the oral cavity and vaginal canal, seven pairs of isolates were genetically unrelated and four pairs were similar but nonidentical. In the latter cases, the isolate pairs each appear to have arisen by genetic divergence from a single progenitor. A comparison of the genetic relatedness of isolates from different individuals further uncovered a single strain which was vaginospecific in the Iowa City, Iowa area and reduced genetic diversity among vulvovaginal strains compared with those isolated from other body locations. These results suggest that strains adapt to different anatomical locations and, conversely, that in a healthy individual there is anatomical selection of vaginotropic, anotropic and orotropic strains of Candida spp.
Collaborative feasibility study of a biphasic system (Roche Septi-Chek AFB) for rapid detection and isolation of mycobacteria
Authors: Isenberg H D , D'Amato R F , Heifets L , Murray P R , Scardamaglia M , Jacobs M C , Alperstein P , Niles A
Published: January 2020

A study to delineate the feasibility of a biphasic-culture approach for detection and isolation of mycobacteria from clinical specimens except blood was conducted in four medical centers. The biphasic system (Septi-Chek AFB, Roche Diagnostic Systems, Nutley, N.J.) was compared with conventional mycobacterial isolation media and the BACTEC system. Septi-Chek AFB showed the highest degree of mycobacterial recovery. In addition, Septi-Chek AFB consistently shortened the time required for recovery of mycobacteria from clinical specimens and supported the growth of small inoculum numbers of stock cultures of 14 mycobacterial species. The study indicates the feasibility and potential advantages of the biphasic approach for detection and isolation of mycobacteria.
Use of pooled formalin-preserved fecal specimens to detect Giardia lamblia
Authors: Wahlquist S P , Williams R M , Bishop H , Addiss D G , Stewart J M , Finton R J , Juranek D D , Sullivan J J
Published: January 2020

Three formalin-preserved fecal specimens from the same child attending a child-care center were pooled and compared with the three separate individual specimens by a single microscopic examination of concentration sediment for Giardia lamblia. The sensitivity of the pooled system was 100% when two or more individual specimens were positive and 88% when only one individual specimen was positive. The organism density in a single specimen was not a factor of whether the pool of specimens was positive or negative. Nearly half of the pools that contained positive specimens had only one of three specimens with positive results, reinforcing the need for multiple stool examinations when diagnosing G. lamblia infections.
Evaluation of the An-Ident system and an indole spot test for the rapid differentiation of porcine treponemes
Authors: Bélanger M , Jacques M
Published: January 2020

The An-Ident strip system (Analytab Products Inc., St-Laurent, Quebec, Canada) was evaluated for its ability to differentiate Treponema hyodysenteriae from Treponema innocens. Of the 20 tests included on this strip, 15 yielded identical results for the two species. Among the other five assays, none could be considered as a positive discriminator for the two species. However, when an indole spot test with 1% p-dimethylaminocinnamaldehyde was used in parallel, all reference strains and 97% of the isolates of T. hyodysenteriae were positive, whereas all isolates of T. innocens were negative. Our results indicate that An-Indent is of little value for the differentiation of the two species. Our results also suggest that a rapid and simple differentiation between T. hyodysenteriae and T. innocens can be achieved by using the hemolysis and ring phenomenon tests in conjunction with an indole spot test.
Reproducibility of a scoring system for gram stain diagnosis of bacterial vaginosis
Authors: Joesoef M R , Hillier S L , Josodiwondo S , Linnan M
Published: January 2020

A total of 225 pairs of duplicate Gram-stained slides from three hospitals in Jakarta were evaluated independently by a local (University of Indonesia, Jakarta) and a referral (University of Washington, Seattle) laboratory by the new scoring criteria proposed by Nugent et al. The correlation coefficients of the duplicate Gram stain scores ranged from 0.65 to 0.83. The kappa statistics for the bacterial vaginosis category (no, score of 0 to 6; yes, score of 7 to 10) ranged from 0.62 to 0.77. These findings confirm the good to excellent interobserver reliability of the new scoring system and the importance of slide preparation.
Changes in subgenome types of adenovirus type 4 isolated from patients with ocular disease between 1985 and 1989 in Sapporo, Japan
Authors: Itakura S , Aoki K , Sawada H , Ishiguro N , Shinagawa M
Published: January 2020

A total of 122 adenovirus type 4 (Ad4) strains isolated from patients with epidemic keratoconjunctivitis at an eye clinic in Sapporo, Japan, from 1985 to 1989 were identified as Ad4a. By the use of HinfI and TaqI restriction endonucleases, these Ad4a strains were classified into 12 subgenome types. During this observation period, two epidemics of Ad4 infection relating to epidemic keratoconjunctivitis occurred. During the Ad4a epidemic in 1985, three subgenome types became predominant successively. However, except for one strain, the prevalent strains isolated during the epidemic of Ad4a in 1988 all belonged to a single subgenome type. In the years when an Ad4a epidemic was not observed, such Ad4a strains were new or previous subgenome types. The prevalent subgenome types tended to change within several years.
Detection of untreated mycobacteria by using polymerase chain reaction and specific DNA probes
Authors: Fries J W , Patel R J , Piessens W F , Wirth D F
Published: January 2020

A method for specific identification of mycobacteria by using the polymerase chain reaction on organisms taken from liquid cultures, frozen suspensions, or colonies grown on Lowenstein-Jensen slants is presented. This direct detection of mycobacterial organisms has important implications for strain typing and diagnosis.
Protective value of elevated levels of gamma interferon in serum against exoerythrocytic stages of Plasmodium falciparum
Authors: Deloron P , Chougnet C , Lepers J P , Tallet S , Coulanges P
Published: January 2020

In vitro experiments suggested that gamma interferon, CD8+ T cells, and anticircumsporozoite antibodies inhibited the exoerythrocytic stages of malaria parasites. To assess the role played in vivo by these factors, we conducted a prospective study in Madagascar. Forty individuals with a negative blood smear were followed for 8 weeks to detect the appearance of parasites in blood. Nineteen subjects remained negative for malaria, whereas 21 individuals became positive during follow-up. Among these, seven presented with blood parasites within the first 2 weeks and were excluded, as they probably were infected by sporozoites at enrollment. When measured at enrollment, antibodies to the synthetic peptide (NANP)5, lymphocyte proliferation with (NANP)5, and various lymphocytes subsets were similar among individuals that later presented with a Plasmodium falciparum blood infection or were not infected. Conversely, the level of gamma interferon in serum was higher in individuals that did not present with a P. falciparum infection during follow-up. These data suggest that gamma interferon may inhibit the malaria exoerythrocytic stages of development under in vivo conditions, as it does in vitro.
Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences
Authors: Wesley I V , Wesley R D , Cardella M , Dewhirst F E , Paster B J
Published: January 2020

Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA.
Identification of clinical isolates of gram-negative nonfermentative bacteria by an automated cellular fatty acid identification system
Authors: Osterhout G J , Shull V H , Dick J D
Published: January 2020

An automated cellular fatty acid (CFA) bacterial identification system, Microbial Identification System (MIS; Microbial ID, Newark, Del.), was compared with a conventional system for the identification of 573 strains of gram-negative nonfermentative bacteria. MIS identifications were based exclusively on the CFA composition following 22 to 26 h of growth at 28 degrees C on Trypticase soy agar. MIS identifications were listed with a confidence measurement (similarity index [SI]) on a scale of 0 to 1.0. A value of greater than or equal to 0.5 was considered a good match. The MIS correctly listed as the first choice 478 of 532 (90%) strains contained in the data base. However, only 314 (59%) had SI values of greater than or equal to 0.5. Of the 54 strains in which there was not agreement, 37 belonged to the genera Acinetobacter, Moraxella, or Alcaligenes or were Pseudomonas pickettii. Reproducibility studies suggest that SI variation is most likely a function of a difference in culture age at the time of analysis, which is due to the relatively low temperature and time of incubation. Other discrepancies were attributable to insufficiently characterized library entries or an inability to differentiate chemotaxonomically closely related species. The MIS, as the first automated CFA identification system, is an accurate, efficient, and relatively rapid method for the identification of gram-negative nonfermentative bacteria. The development of a CFA library with the media and incubation conditions routinely used for the isolation of clinical pathogens could further decrease the identification time and provide an increase in accuracy.
Protein X of Streptococcus agalactiae induces opsonic antibodies in cows
Authors: Rainard P , Lautrou Y , Sarradin P , Poutrel B
Published: January 2020

Protein X of Streptococcus agalactiae is a surface protein frequently associated with strains isolated from cases of mastitis of dairy cows. By immunizing cows with purified protein X, we obtained an antibody response which was restricted to X-bearing strains of S. agalactiae in a whole-cell enzyme-linked immunosorbent assay. This response resulted in an increase in the opsonic activity of serum for strains bearing protein X, as assessed through the augmentation of the chemiluminescence response of phagocytosing polymorphonuclear cells and through an increased ingestion of bacteria, although the proportion of ingested bacteria which were killed (about 60%) remained unchanged. Protein X behaved as a target of opsonins and, as such, could be a protective antigen worth incorporating in a vaccine against S. agalactiae mastitis.
Evidence for the genetic unrelatedness of nosocomial vancomycin-resistant Enterococcus faecium strains in a pediatric hospital
Authors: Bingen E H , Denamur E , Lambert-Zechovsky N Y , Elion J
Published: January 2020

Vancomycin-resistant enterococci are now found with increasing frequency. Up to now, epidemiological studies of enterococci have been limited by the lack of convenient and accessible methods for comparing strains. In this study, we report an epidemiological investigation on 16 nosocomial vancomycin-resistant Enterococcus faecium strains isolated from 15 patients in four different wards of a children's hospital over a period of 17 months. Analysis of restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) was used as a typing approach. Each strain produced a different total DNA RFLP pattern after HindIII and PvuII digestion, except for two strains that were isolated from a single patient and that gave indistinguishable patterns. In our system, ribotyping was less discriminative than RFLP of total DNA. This approach, therefore, shows the genetic unrelatedness of the nosocomial strains studied and excludes patient-to-patient strain transmission either in the same ward or between wards.
Isolation and continuous culture of Neorickettsia helminthoeca in a macrophage cell line
Authors: Rikihisa Y , Stills H , Zimmerman G
Published: January 2020

Experimental evidence is presented supporting the development of a system for the isolation and propagation of a Neorickettsia sp. in a continuous canine macrophage cell line (DH82). To isolate a Neorickettsia sp. pathogenic to the canine species, three naive dogs were fed metacercaria-encysted kidneys of salmon caught in a river where infection of metacercariae with Neorickettsia helminthoeca has been circumstantially known for decades. Clinically, the classic course of salmon poisoning disease developed in all of the dogs. Parasitemia began on day 8 to 11 postinfection, when the dogs developed a febrile peak, and continued until euthanasia. At necropsy, characteristic gross and microscopic lesions of the disease were present. A Neorickettsia sp. was also isolated from liver and spleen samples of these animals. The isolates have been continuously propagated and passed in DH82 cells for more than 6 months. Electron microscopic examination confirmed that the rickettsial organisms multiplied in the membrane-bound compartment of DH82 cells and that they morphologically closely resembled rickettsia belonging to the genus Ehrlichia. An indirect fluorescent antibody test using Neorickettsia organisms cultured in DH82 cells showed that all dogs seroconverted 13 to 15 days postinfection. Finally, inoculation of the cell-cultured Neorickettsia organisms into a naive dog reproduced clinically typical salmon poisoning disease which was of greater severity and had a more rapid time course than that in the dogs from which the original isolation was made. On the basis of the clinical and pathologic responses of the dogs in our study, we believe that virulent N. helminthoeca was isolated and cultured in a continuous cell line.
Molecular and immunological analysis of a polymorphic periplasmic protein of Borrelia burgdorferi
Authors: Simpson W J , Schrumpf M E , Hayes S F , Schwan T G
Published: January 2020

Borrelia burgdorferi is the causative agent of Lyme disease, a tick-borne spirochetosis with a worldwide prevalence. To assist the categorization and typing of fresh isolates from global foci, we have identified a unique species-specific periplasmic protein (P22-A) conserved among all North American and European isolates examined. The gene encoding this antigen was cloned, and the recombinant was used to screen serum collected from experimentally infected animals. Although antibodies were detected in all infected animals at 21 days after inoculation with live, low-passage spirochetes, the response was stronger in other animals that were inoculated with inactivated and lysed bacteria. This result, along with the immune electron microscopy data, suggests P22-A is concentrated in the periplasmic space. The P22-A antigens exhibited size heterogeneity among different isolates, ranging between 20 and 23 kDa, but as a group the P22-A antigens appeared to retain antigenic homogeneity. Thus, P22-A can serve as a structural marker for characterizing new isolates of B. burgdorferi and may prove useful in future serological assays with a mixture of B. burgdorferi-specific antigens.
Molecular characterization of Haemophilus ducreyi by ribosomal DNA fingerprinting
Authors: Sarafian S K , Woods T C , Knapp J S , Swaminathan B , Morse S A
Published: January 2020

Intraspecies genotypic heterogeneity among Haemophilus ducreyi isolates was examined by using genomic fingerprints with rRNA from Escherichia coli as a probe. DNA from 44 isolates of H. ducreyi was digested by restriction endonuclease HincII or HindIII, separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with 32P-labeled 16S and 23S rRNA. HincII digests yielded four hybridization patterns (ribotypes), whereas HindIII digests yielded eight ribotypes. Four HincII and five HindIII ribotypes were observed among 14 H. ducreyi isolates collected within a period of 1 month in Kenya, where chancroid is endemic. In contrast, one HincII and two HindIII ribotypes were observed among 28 isolates collected during the Orange County, Calif., chancroid epidemic that occurred in 1981 and 1982. The plasmid content, in conjunction with ribotyping, provided additional differentiation among some isolates of H. ducreyi. This study demonstrates that ribotyping of H. ducreyi may be used to study the epidemiology of chancroid.
Rapid presumptive identification of black-pigmented gram-negative anaerobic bacteria by using 4-methylumbelliferone derivatives
Authors: Moncla B J , Braham P , Rabe L K , Hillier S L
Published: January 2020

A rapid method for presumptive identification of black-pigmented gram-negative anaerobic rods was developed. Using filter paper spot tests for indole production, sialidase, alpha-glucosidase, beta-glucosidase, alpha-fucosidase, and trypsinlike enzyme activities, 100% of Porphyromonas gingivalis, Prevotella intermedia, and Bacteroides levii and 89% of Prevotella corporis isolates were correctly identified to the species level. Porphyromonas asaccharolytica and Porphyromonas endodontalis could not be differentiated from each other but could be distinguished from all other species tested. Similarly, Prevotella denticola, Prevotella loescheii, and Prevotella melaninogenica could not be differentiated from each other. The methods described are based on 4-methylumbelliferone derivatives of the various substrates and are simple to perform, rapid (less than 15 min), and applicable to difficult-to-cultivate anaerobic rods.
Monoclonal antibodies specific for the Escherichia coli heat-stable enterotoxin STb
Authors: Urban R G , Pipper E M , Dreyfus L A
Published: January 2020

Protein A-STb and STb-alkaline phosphatase protein fusions were used as immunogen and antigen, respectively, for the generation and screening of monoclonal antibodies to the Escherichia coli heat-stable enterotoxin STb. Following immunization with immunoglobulin G-Sepharose-purified protein A-STb and hybridoma construction, STb-alkaline phosphatase hybrid protein was used in a labeled antigen capture assay to detect the production of STb-specific monoclonal antibody. STb-specific monoclonal antibodies were characterized by using a combination of immunoblotting and synthetic-peptide-based enzyme immunoassay techniques. Four distinct anti-STb antibodies were identified and characterized.
Detection and strain differentiation of Chlamydia psittaci mediated by a two-step polymerase chain reaction
Authors: Kaltenboeck B , Kousoulas K G , Storz J
Published: January 2020

Specific and sensitive amplification of major outer membrane protein (MOMP) gene DNA sequences of Chlamydia psittaci was achieved in a two-step polymerase chain reaction. First, oligonucleotide primers specific for 5' and 3' nontranslated regulatory regions of the MOMP gene were used in a polymerase chain reaction to amplify a DNA fragment of approximately 1,400 bp. A portion of this DNA fragment was amplified in a second reaction using a degenerate oligonucleotide primer specific for a DNA sequence contained within the 1,400-bp DNA fragment and one of the first-step primers. This method detected 10 cognate chlamydial genomes. C. psittaci MOMP genes from two avian strains and from mammalian serovars 1, 7, and 8 were amplified and analyzed by restriction endonuclease digestion. MOMP genes from mammalian serovars 2 through 6 and 9 and from strains of C. trachomatis and C. pneumoniae could not be amplified. Restriction endonuclease analysis with HaeIII indicated a close relationship between C. psittaci strains of avian and mammalian serovar 1 lineage, while those of mammalian serovars 7 and 8 exhibited distinct restriction patterns. DNA sequences corresponding to the mammalian serovar 1-wild type parakeet MOMP genotype of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.
Prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray
Authors: Ward R L , Bernstein D I , Knowlton D R , Sherwood J R , Young E C , Cusack T M , Rubino J R , Schiff G M
Published: January 2020

A model was developed to examine the effects of disinfectants on the transmission of infectious rotavirus from a dried surface to humans. The initial experiments were designed to find a method of preserving rotavirus infectivity during drying. Culture-adapted human rotavirus (CJN strain) was dried at room temperature in different organic suspensions, including fecal matter, several laboratory media, and nonfat dry milk (NDM). Recoveries of infectious virus were then compared. Fecal matter provided little protection in this study relative to distilled water, but the other suspensions were quite protective, especially NDM, which consistently allowed recoveries of greater than 50%. When 10(3) focus-forming units of unpassaged CJN virus were dried in NDM and administered to subjects who licked the dried material, 100% (8 of 8) became infected. The effect of Lysol brand disinfectant spray (LDS) was next examined. Although NDM provided some protection against inactivation by LDS, spraying under conditions recommended by the manufacturer consistently caused the CJN virus titer to decrease greater than 5 log10. Consumption of CJN virus (10(3) focus-forming units) sprayed with LDS caused no infection in 14 subjects, whereas 13 of 14 subjects who consumed the unsprayed virus became infected (P less than 0.00001). The methods developed in this study could be used to test the effects of other disinfectants on the spread of infectious rotavirus from inanimate surfaces to humans.