bluuebunny/crossref_metadata_2025 huggingface dataset:
Exposure to low oxygen tension and increased osmolarity enhance the ability of Mycobacterium avium to enter intestinal epithelial (HT-29) cells
Authors: Bermudez L E , Petrofsky M , Goodman J
Published: January 2020

Current evidence indicates that Mycobacterium avium infection in patients with AIDS is acquired mostly through the gastrointestinal (GI) tract and that M. avium binds to and invades GI mucosal cells in vitro. Since M. avium is exposed to specific environmental conditions in the GI tract such as changes in pH, low oxygen (O2) tension, increased osmolarity, and low concentration of iron, we investigated the effects of these conditions on the bacterium's ability to enter HT-29 intestinal cells. M. avium 101 (serovar 1) was cultured in 7H9 broth and then exposed to pH 4.5 to 8.0, low O2 tension, 0.1 to 0.3 M dextrose, and absence of iron for 2 h. After washing, bacteria (10(7)/ml) were used in the invasion assay. Confluent HT-29 cells were exposed to 10(6) bacteria for 1 h and then treated with amikacin (200 microg/ml) for 2 h to selectively kill extracellular but not intracellular M. avium. The supernatant was then removed, the monolayer was lysed, and the lysate was plated onto 7H10 agar plates. While exposure to acidic and basic pHs, as well as iron-free medium, had no significant effect on M. avium invasion of intestinal epithelial cells, low O2 tension and increased osmolarity enhanced invasion 11- and 9-fold, respectively, compared with the control. Exposure of M. avium to the combination of low O2 concentration and hyperosmolarity resulted in an approximate 10- to 15-fold increase in penetration of HT-29 cells. Hyperosmolarity and low O2 tension induced the invasive M. avium phenotype and can be useful for the identification of genes associated with M. avium invasion of intestinal mucosa.
Heterogeneity of antibodies reactive with the dominant antigen of Actinobacillus actinomycetemcomitans
Authors: Nakashima K , Schenkein H A , Califano J V , Tew J G
Published: January 2020

The serotype b-specific carbohydrate antigen (SbAg) of Actinobacillus actinomycetemcomitans Y4 is reported to be the O antigen of lipopolysaccharide, and the highest titers of serum antibody reactive with A. actinomycetemcomitans in early-onset periodontitis (EOP) patients bind SbAg. These high titers of serum antibody reactive with SbAg are associated with a lesser extent and severity of periodontal disease. The aim of this study was to determine if a limited number of genes code for anti-SbAg antibodies as has been shown for immunoglobulin G (IgG) reactive with the type b polysaccharide from Haemophilus influenzae. Serum IgG reactive with the SbAg was prepared from 20 high-titer EOP patients by affinity chromatography. The IgG subclass concentrations were determined, and heterogeneity was analyzed by isoelectric focusing (IEF). IgG2 was the dominant subclass (83% of total IgG) in the anti-SbAg IgG fraction and represented an average of 1.33% of total serum IgG2. The IgG2 reactive with SbAg was isolated from the affinity-purified IgG fraction by affinity chromatography with protein A and subclass-specific monoclonal antibodies. On IEF gels, only 4 to 20 bands were observed in the anti-SbAg IgG fractions, indicating limited heterogeneity. N-terminal amino acid sequence analysis of eight representative anti-SbAg IgG2 preparations indicated that variable heavy and light chains consisted largely of V(H)III and V(kappa)II, respectively. However, a significant fraction of anti-SbAg may use V(H) and V(lambda) genes with blocked N termini. In short, these findings indicate that IgG reactive with SbAg is very much like the antibody reactive with H. influenzae type b polysaccharide. Similarities include IgG2 dominance, limited bands on IEF gels, supporting an oligoclonal response, and use of genes from V(H)III and V(kappa)II regions.
In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies
Authors: Elliot B C , Wisnewski A V , Johnson J , Fenwick-Smith D , Wiest P , Hamer D , Kresina T , Flanigan T P
Published: January 2020

Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection.
Neutrophils play a critical role in early resistance to amebic liver abscesses in severe combined immunodeficient mice
Authors: Seydel K B , Zhang T , Stanley S L
Published: January 2020

Animal models of liver abscess formation with Entamoeba histolytica suggest that the neutrophil is the first cell of the host immune system to interact with the invading ameba. In vitro studies have suggested that lysis of neutrophils by virulent amebae may exacerbate the damage seen in amebic liver abscesses. To investigate the role of neutrophils in vivo, we used the severe combined immunodeficient (SCID) mouse model of amebic liver abscess formation and compared liver damage in neutrophil-depleted and control mice. We found that neutrophil-depleted animals have significantly larger amebic liver abscesses at early stages of infection and that abscesses in neutrophil-depleted SCID mice lack the prominent inflammatory cell ring seen in amebic liver abscesses in control SCID mice. These data suggest that neutrophils play a protective role in the early host response to amebic infection of the liver.
Polymorphism in ospC gene of Borrelia burgdorferi and immunoreactivity of OspC protein: implications for taxonomy and for use of OspC protein as a diagnostic antigen
Authors: Theisen M , Frederiksen B , Lebech A M , Vuust J , Hansen K
Published: January 2020

The nucleotide sequences of the ospC gene from five Danish human Borrelia burgdorferi isolates representing all three B. burgdorferi genospecies (B. burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461) and from the American type strain B31 were determined and compared with the published ospC sequence from the German B. burgdorferi isolate PKo (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). The ospC gene was present in all isolates, regardless of the presence or absence of its product, OspC. The deduced amino acid sequences of OspC from the seven isolates were aligned and revealed pairwise sequence identities ranging from 60.5 to 100%. Differences were scattered throughout the amino acid sequences. A phylogenetic tree was constructed and revealed three distinct phenotypic groups OspCI to OspCIII corresponding to the three delineated genospecies. Immunoblot analysis revealed that the seven OspC proteins tested have both common and specific epitopes. There is significant epitope diversity, since even polyclonal antisera showed serotype-restricted specificity. Therefore, a serodiagnostic assay for Lyme borreliosis utilizing OspC as a test antigen should include all three OspC phenotypes in order to obtain a species-wide sensitivity.
Use of multiple molecular subtyping techniques to investigate a Legionnaires' disease outbreak due to identical strains at two tourist lodges
Authors: Mamolen M , Breiman R F , Barbaree J M , Gunn R A , Stone K M , Spika J S , Dennis D T , Mao S H , Vogt R L
Published: January 2020

A multistate outbreak of Legionnaires' disease occurred among nine tour groups of senior citizens returning from stays at one of two lodges in a Vermont resort in October 1987. Interviews and serologic studies of 383 (85%) of the tour members revealed 17 individuals (attack rate, 4.4%) with radiologically documented pneumonia and laboratory evidence of legionellosis. A survey of tour groups staying at four nearby lodges and of Vermont-area medical facilities revealed no additional cases. Environmental investigation of common tour stops revealed no likely aerosol source of Legionella infection outside the lodges. Legionella pneumophila serogroup 1 was isolated from water sources at both implicated lodges, and the monoclonal antibody subtype matched those of the isolates from six patients from whom clinical isolates were obtained. The cultures reacted with monoclonal antibodies MAB1, MAB2, 33G2, and 144C2 to yield a 1,2,5,7 or a Benidorm 030E pattern. The strains were also identical by alloenzyme electrophoresis and DNA ribotyping techniques. The epidemiologic and laboratory data suggest that concurrent outbreaks occurred following exposures to the same L. pneumophila serogroup 1 strain at two separate lodges. Multiple molecular subtyping techniques can provide essential information for epidemiologic investigations of Legionnaires' disease.
Enhanced detection of intracellular organism of swine proliferative enteritis, ileal symbiont intracellularis, in feces by polymerase chain reaction
Authors: Jones G F , Ward G E , Murtaugh M P , Lin G , Gebhart C J
Published: January 2020

A sensitive assay based on amplification of a 319-bp DNA fragment of the intracellular bacterium of swine proliferative enteritis was developed for the detection of the organism in the feces of swine. A vernacular name, ileal symbiont intracellularis (IS-intracellularis), has recently been published for the intracellular bacterium, which was formerly known as a Campylobacter-like organism (C.J. Gebhart, S.M. Barnes, S. McOrist, G.F. Lin, and G.H.K. Larson, Int. J. Syst. Bacteriol. 43:533-538, 1993). As few as 10(1) IS-intracellularis organisms purified from intestinal mucosa, or 10(3) IS-intracellularis per g of feces, were detected. No amplification product was produced from a polymerase chain reaction performed on DNA extracted from the feces of healthy pigs. A 319-bp DNA fragment specific for IS-intracellularis was produced on amplification of DNA from the feces of pigs with experimental and naturally occurring proliferative enteritis.
Quantitation of enteroviral RNA by competitive polymerase chain reaction
Authors: Martino T A , Sole M J , Penn L Z , Liew C C , Liu P
Published: January 2020

The polymerase chain reaction (PCR) is a new diagnostic technique for the detection of enteroviral infection; however, it currently provides only qualitative results. The aim of this study was to adapt PCR for the accurate quantitation of enteroviral RNA in clinical specimens. For this purpose, we designed a standard RNA which was homologous to sequences at the 5' end of the coxsackie B3 enterovirus genome but contained a single-base-pair mutation which created a novel internal restriction site. Serial dilutions of this standard template RNA were mixed with a fixed concentration of coxsackie B3 enterovirus RNA. The viral and standard templates were reversed transcribed to cDNA and coamplified by PCR, and a comparison of the radioactive PCR products was made. Since the templates were both present in a single reaction tube and competed for the same primers, the ratio of products remained proportional throughout the amplification process. By this approach, a fourfold-difference in viral titer was clearly distinguishable. Moreover, we were able to accurately quantitate as few as 15 50% tissue culture infectious doses, which reflects common clinical viral titers. This study lays the foundation for quantitation of enteroviral RNA in clinical specimens and establishes a technique that can readily be applied to the diagnosis of enteroviral infection.
An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera
Authors: Mahan S M , Tebele N , Mukwedeya D , Semu S , Nyathi C B , Wassink L A , Kelly P J , Peter T , Barbet A F
Published: January 2020

Heartwater, a major constraint to improved livestock production in Zimbabwe, threatens to invade areas which have been previously unaffected. To monitor its spread in Zimbabwe, an immunoblotting diagnostic assay based on the responses of animals to the immunodominant, conserved 32-kDa protein of Cowdria ruminantium was evaluated. In this assay, no false reactions were detected with sera known to be positive and negative, but sera from some cattle, sheep, and goats from heartwater-free areas of Zimbabwe reacted strongly with the 32-kDa protein, suggesting that either these animals had previous exposure to heartwater or they were false positives. To investigate the possibility of previous exposure to heartwater, 11 immunoblot-positive and 6 immunoblot-negative sheep from heartwater-free areas of Zimbabwe were compared regarding their susceptibilities to challenge with C. ruminantium. Prior to challenge, C. ruminantium could not be detected in any sheep by transmission to Amblyomma hebraeum ticks or by the polymerase chain reaction (PCR) conducted with plasma samples. All sheep were equally susceptible to the challenge, and infection was confirmed by brain biopsy, necropsy, PCR, and transmission of C. ruminantium to ticks. Our data suggest that the immunoblot-positive reactions of sera from heartwater-free areas were due not to previous C. ruminantium infection but rather to antigenic cross-reactivity between C. ruminantium and another agent(s) such as Ehrlichia species. In conclusion, the immunodominant 32-kDa protein is not antigenically specific to C. ruminantium and its use in serological diagnosis of heartwater requires reevaluation.
Monoclonal antibody specific to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi
Authors: Takai S , Iie M , Kobayashi C , Morishita T , Nishio T , Ishida T , Fujimura T , Sasaki Y , Tsubaki S
Published: January 2020

Virulent Rhodococcus equi produces 15- to 17-kDa surface protein antigens. These antigens are used as markers to identify virulent R. equi isolates from foals and their environment by Western blot (immunoblot) analysis with naturally infected foal serum. In the present study, a monoclonal antibody (MAb; 10G5) was generated against the 15- to 17-kDa antigens excised from sodium dodecyl sulfate-polyacrylamide gels to develop sensitive and specific immunoblot assays for the identification of virulent R. equi. MAb 10G5 strongly reacted with R. equi ATCC 33701 and L1, which expressed 15- to 17-kDa antigens by Western blot, colony blot, and dot immunobinding assays, but it did not react with strains ATCC 33701P- and L1P-, which lacked the antigens. For identification of virulent R. equi, clinical and environmental isolates were tested by these assays with the MAb, and all virulent strains were successfully identified; these strains possessed virulence plasmids. These results suggest that the MAb is a useful reagent for the identification of virulent R. equi.
Evaluation of MicroScan rapid panels for detection of high-level aminoglycoside resistance in enterococci
Authors: Woods G L , DiGiovanni B , Levison M , Pitsakis P , LaTemple D
Published: January 2020

The ability of MicroScan rapid panels to detect high-level aminoglycoside-resistant enterococci was evaluated. By agar dilution, 46 of 139 isolates were susceptible to gentamicin (GNT) and streptomycin (STRP); the rest were highly resistant to one or both agents. Rapid panels detected 97.5% of STRP- and GNT-resistant isolates and had a specificity of 95.6%. Detection of resistance by conventional panels at 18 h was 64.6% for STRP and 90.2% for GNT.
Evaluation of a genetically engineered cell line and a histochemical beta-galactosidase assay to detect herpes simplex virus in clinical specimens
Authors: Stabell E C , O'Rourke S R , Storch G A , Olivo P D
Published: January 2020

A novel histochemical method was compared with a cytopathic effect (CPE) assay for the identification of herpes simplex virus (HSV) in clinical specimens. The method utilizes a stably transformed cell line (BHKICP6LacZ-5) that expresses beta-galactosidase only after infection with HSV. A total of 96 specimens submitted to our diagnostic virology laboratory were analyzed. Thirty-one specimens contained HSV as evidenced by positive CPE, and all were positive for beta-galactosidase staining. CPE were not evident for 2 or more days in 15 of the 31 positive specimens, whereas the histochemical stain was positive in all 31 positives by 16 to 24 h. This preliminary study shows that the BHKICP6LacZ-5 cell line can be used in a rapid, sensitive, and specific assay for the detection of HSV in clinical specimens.
Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases
Authors: Whittier S , Shapiro D S , Kelly W F , Walden T P , Wait K J , McMillon L T , Gilligan P H
Published: January 2020

Four commercial enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxin A have recently been developed and marketed (Premier, Meridian Diagnostics, Cincinnati, Ohio; VIDAS, bioMerierux Vitek, Inc., Hazelwood, Mo.; Tox-A-Test, TechLab, Blacksburg, Va.; and Bartels, Baxter Diagnostics, McGaw Park, Ill.). The performances of these EIAs were compared with those of the tissue culture cytotoxicity assay and a definition of C. difficile-associated disease based on both laboratory and clinical criteria for 329 clinical specimens. Two EIAs (Premier and VIDAS) showed good overall agreement (96 and 95%, respectively) with the cytotoxicity assay. However, they were less sensitive (84 and 71%, respectively) than the Bartels (94%) or Tox-A-Test (93%) EIAs. The Bartels and Tox-A-Test assays were much less specific, resulting in poor positive predictive values (56%) of the two assays when compared with that of the cytotoxicity assay. Tox-A-Test had the added drawback of having a significant number of indeterminate results (6.4%). These data indicate that the four EIAs all have specific shortcomings. When using these EIAs, testing strategies that take these shortcomings into consideration should be developed.
Role of specific immunoglobulin E in diagnosis of acute toxoplasma infection and toxoplasmosis
Authors: Wong S Y , Hajdu M P , Ramirez R , Thulliez P , McLeod R , Remington J S
Published: January 2020

Toxoplasma immunoglobulin E (IgE) antibodies were evaluated in an immunosorbent agglutination assay (ISAGA) and an enzyme-linked immunosorbent assay (ELISA) to determine their usefulness in the diagnosis of acute infection with Toxoplasma gondii. IgE antibodies were not detected in serum specimens from otherwise seronegative individuals, individuals with chronic toxoplasma infection, or infants without congenital toxoplasmosis. In contrast, they were detected in pregnant women who seroconverted during gestation (100% by ELISA, 63% by ISAGA), patients with toxoplasmic lymphadenopathy (96% by ELISA, 88% by ISAGA), infants with signs of congenital toxoplasmosis which prompted serologic testing in the postnatal period (92% by ELISA, 67% by ISAGA), children and adults with toxoplasmic chorioretinitis (36% by ELISA, 18% by ISAGA), and adult patients with AIDS and toxoplasmic encephalitis (33% by ELISA, 25% by ISAGA). In many of the serum specimens, the titer of IgE antibodies detected by the ISAGA were close to or at the positive cutoff value. The duration of detectable IgE antibodies in patients with acute infections varied considerably among individuals but showed a trend toward a briefer duration by the ISAGA than by the ELISA. These results reveal that recrudescence of IgE antibodies in patients with reactivated chronic infection (toxoplasmic chorioretinitis and toxoplasmic encephalitis) may be useful diagnostically and that demonstration of toxoplasma IgE antibodies is a useful adjunct to currently available serologic tests for the diagnosis of acute toxoplasma infection and toxoplasmosis.
Analysis of discordant test results among five second-generation assays for anti-hepatitis C virus antibodies also tested by polymerase chain reaction-RNA assay and other laboratory and clinical tests for hepatitis
Authors: Nakagiri I , Ichihara K , Ohmoto K , Hirokawa M , Matsuda N
Published: January 2020

The diagnostic performances of five commercially available second-generation assays for anti-hepatitis C virus antibody, two enzyme-linked immunosorbent assays, one enzyme immunoassay, and two particle agglutination assays (passive hemagglutination assay and particle agglutination assay), were evaluated. Among 104 samples from healthy subjects and 300 consecutive samples from patient ordered for routine determinations of anti-hepatitis C virus antibody in serum, assay results showed variable degrees of discordance for 17 samples (4.2%). These 17 samples were further tested by an immunoblot assay, the polymerase chain reaction-RNA assay, and the hemagglutination inhibition assay. Four of the 17 samples were regarded as true positive, since all supplementary assays and clinical data indicated active hepatitis C virus infection. Another five samples were considered false positive because no confirmatory evidence was obtained from the laboratory analysis or clinical data. The remaining eight samples were negative for hepatitis C virus RNA, but the results of the other supplementary tests were indeterminate. Some of these samples with indeterminate results may have been from patients with subclinical cases of disease who spontaneously recovered from hepatitis with persistent anti-hepatitis C virus antibody in their sera.
Epidemic of Pseudomonas cepacia in an adult cystic fibrosis unit: evidence of person-to-person transmission
Authors: Smith D L , Gumery L B , Smith E G , Stableforth D E , Kaufmann M E , Pitt T L
Published: January 2020

An epidemic of Pseudomonas cepacia occurred in an adult cystic fibrosis center in the United Kingdom, despite a policy of segregation of infected and noninfected patients within the hospital. Investigation of the outbreak by ribotyping and pulsed-field gel electrophoresis to characterize P. cepacia strain genomes together with inquiry into social contacts between patients revealed evidence of person-to-person transmission outside the hospital environment. Segregation policies aimed at reducing the spread of this infection in the cystic fibrosis community need to encompass patient contacts outside the hospital environment.
Invasive strains of Escherichia coli belonging to serotype O121:NM
Authors: Matsushita S , Yamada S , Kai A , Kudoh Y
Published: January 2020

Ten strains of Escherichia coli isolated from 10 travellers with sporadic diarrhea who were returning to Tokyo, Japan, from abroad were found to be of serotype O121:NM and were positive in the Serény test for invasiveness; this suggests that this serotype can cause a shigellosis-like illness in humans. The E. coli O121:NM strains were positive in the cell invasion test in HeLa cells. Analysis of the plasmid content of these strains showed that they contained a high-molecular-mass plasmid of 120 to 140 MDa which has been associated with invasiveness and were positive in an enzyme-linked immunosorbent assay for detection of the virulence plasmid-encoded proteins of Shigella spp. and enteroinvasive E. coli.
Multiple stool examinations for ova and parasites and rate of false-negative results
Authors: Marti H , Koella J C
Published: January 2020

A mathematical model that shows the relationship between the number of stool examinations for ova and parasites and the rate of false-negative results is described. An analysis of 1,869 patients with three stool examinations revealed various sensitivities for different parasites.
Relative frequencies of G (VP7) and P (VP4) serotypes determined by polymerase chain reaction assays among Japanese bovine rotaviruses isolated in cell culture
Authors: Suzuki Y , Sanekata T , Sato M , Tajima K , Matsuda Y , Nakagomi O
Published: January 2020

The relative frequencies of both the G (VP7) and P (VP4) serotypes of 40 bovine rotaviruses isolated in cell culture from diarrheic calves in Japan between January 1983 and February 1991 were determined by recently developed polymerase chain reaction assays. Isolates with G serotype 6 and P serotype 5 (UK-like strains) were most frequently found (42.5%) followed by isolates with G6P11 (17.5%), G6P1 (10%), or G10P5 (10%). Isolates with G10P11 (B223-like strains) were least frequently found (7.5%). The presence of various combinations of G and P serotypes suggests frequent reassortment in nature among bovine rotaviruses.
Ehrlichia chaffeensis and Rochalimaea antibodies in Kawasaki disease
Authors: Rathore M H , Barton L L , Dawson J E , Regnery R L , Ayoub E M
Published: January 2020

Sera from 38 patients with Kawasaki disease were tested for immunofluorescent antibodies to Ehrlichia chaffeensis, Rochalimaea henselae, and R. quintana Oklahoma. Only 2.5% of the patients tested positive for E. chaffeensis, and 5% were positive for R. henselae and R. quintana Oklahoma. Our data suggest that Ehrlichia and Rochalimaea spp. do not play a unique role in the etiology of Kawasaki disease.
Mycobacterium interjectum, a new species isolated from a patient with chronic lymphadenitis
Authors: Springer B , Kirschner P , Rost-Meyer G , Schröder K H , Kroppenstedt R M , Böttger E C
Published: January 2020

Mycobacterium-like organisms, isolates 2081/92 and 4185/92, were recovered from a lymph node of a child with chronic lymphadenitis. The growth characteristics, acid-fastness, and mycolic acids of the isolate were consistent with those for Mycobacterium species. The isolates were biochemically distinct from described Mycobacterium species, although they most closely resembled M. scrofulaceum. Comparative 16S rDNA sequencing showed that these isolates represent a new slow-growing Mycobacterium species which is named M. interjectum. Our results demonstrate the importance of 16S rDNA sequencing for recognizing the diversity of species within the genus Mycobacterium.
Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies
Authors: Nelson E A , Christopher-Hennings J , Drew T , Wensvoort G , Collins J E , Benfield D A
Published: January 2020

Monoclonal antibodies (MAbs) to two U.S. isolates of porcine reproductive and respiratory syndrome (PRRS) virus were prepared. Two MAbs specifically recognized a conserved epitope on the putative 15-kDa nucleocapsid protein of U.S. and European isolates of PRRS virus. Four other MAbs recognized epitopes on the 15-kDa protein of U.S. but not European isolates of PRRS virus. Collectively, this indicates that PRRS viruses contain both conserved and divergent epitopes on the 15-kDa viral protein.
Identification of mycobacteria from animals by restriction enzyme analysis and direct DNA cycle sequencing of polymerase chain reaction-amplified 16S rRNA gene sequences
Authors: Hughes M S , Skuce R A , Beck L A , Neill S D
Published: January 2020

Two methods, based on analysis of the polymerase chain reaction-amplified 16S rRNA gene by restriction enzyme analysis (REA) or direct cycle sequencing, were developed for rapid identification of mycobacteria isolated from animals and were compared to traditional phenotypic typing. BACTEC 7H12 cultures of the specimens were examined for "cording," and specific polymerase chain reaction amplification was performed to identify the presence of tubercle complex mycobacteria. Combined results of separate REAs with HhaI, MspI, MboI, and ThaI differentiated 12 of 15 mycobacterial species tested. HhaI, MspI, and ThaI restriction enzyme profiles differentiated Actinobacillus species from mycobacterial species. Mycobacterium bovis could not be differentiated from M. bovis BCG or Mycobacterium tuberculosis. Similarly, Mycobacterium avium and Mycobacterium paratuberculosis could not be distinguished from each other by REA but were differentiated by cycle sequencing. Compared with traditional typing, both methods allowed rapid and more accurate identification of acid-fast organisms recovered from 21 specimens of bovine and badger origin. Two groups of isolates were not typed definitively by either molecular method. One group of four isolates may constitute a new species phylogenetically very closely related to Mycobacterium simiae. The remaining unidentified isolates (three badger and one bovine) had identical restriction enzyme profiles and shared 100% nucleotide identify over the sequenced signature region. This nucleotide sequence most closely resembled the data base sequence of Mycobacterium senegalense.
Demonstration of circulating pneumococcal immunoglobulin G immune complexes in patients with community-acquired pneumonia by means of an enzyme-linked immunosorbent assay
Authors: Holloway Y , Snijder J A , Boersma W G
Published: January 2020

An enzyme-linked immunosorbent assay was developed for quantitation of circulating immune complexes (CICs) containing specific antipneumococcal immunoglobulin G (IgG). These CICs were detected in 17 (85%) of 20 patients with bacteremic pneumococcal pneumonia, 4 (36.4%) of 11 patients with probable pneumococcal pneumonia, 3 (16.7%) of 18 patients with pneumonia of other (nonpneumococcal) etiology, and 13 (41.9%) of 31 patients with pneumonia of unknown etiology. There was no correlation between CICs and serum IgG antibody levels. Pneumococcal capsular antigen was demonstrated in dissociated CICs by latex agglutination.
Roseomonas, a new genus associated with bacteremia and other human infections
Authors: Rihs J D , Brenner D J , Weaver R E , Steigerwalt A G , Hollis D G , Yu V L
Published: January 2020

In the 1980s, a pink bacterium different from species of the genus Methylobacterium was implicated in human infection. Using biochemical tests and DNA hybridization, we examined 42 strains of pink-pigmented, gram-negative bacteria that were not members of the genus Methylobacterium. The isolates included 6 strains each of CDC "pink coccoid" groups I, II, III, and IV; 10 isolates from Gilardi's "unnamed taxon"; and 8 blood isolates from ill, debilitated, or immunosuppressed patients. The DNA hybridization studies supported the creation of six genomospecies encompassing the 42 strains. Reactions for esculin hydrolysis, glycerol oxidation, and D-mannose oxidation enabled separation of genomospecies 1 through 4. These tests, as well as motility, nitrate reduction, citrate utilization, and oxidation of L-arabinose, D-galactose, and D-xylose, differentiated genomospecies 5 and 6 from each other and from genomospecies 1 through 4. These organisms were susceptible in vitro to the aminoglycosides, tetracycline, and imipenem and generally susceptible to the quinolones. We propose the new genus, Roseomonas, for these bacteria to include three named species, Roseomonas gilardii sp. nov., Roseomonas cervicalis sp. nov., and Roseomonas fauriae sp. nov., and three unnamed genomospecies.
Conservation of a protective surface antigen of Tritrichomonas foetus
Authors: Ikeda J S , BonDurant R H , Campero C M , Corbeil L B
Published: January 2020

Bovine trichomoniasis is a sexually transmitted disease caused by the flagellated protozoan Tritrichomonas foetus. A protective surface antigen was previously identified and immunoaffinity purified from T. foetus isolate D1 with cross-reactive monoclonal antibodies (MAbs) TF1.15 and TF1.17 (BonDurant, R. H., R. R. Corbeil, and L. B. Corbeil, Infect. Immun. 61:1385-1394, 1993). This antigen elicited antibody responses in the serum and cervicovaginal mucus of heifers. Thus, it may be useful as an immunodiagnostic reagent as well as a subunit vaccine. Conservation of the antigen in all strains would be crucial for either application. We investigated the conservation of this antigen among 36 isolates of T. foetus from Argentina, Costa Rica, and the United States using MAbs TF1.15 and TF1.17 in an enzyme-linked immunosorbent assay. MAb TF1.17 reacted with 32 of the 36 isolates, whereas MAb TF1.15 reacted with all of the isolates tested. One of the isolates which did not react with MAb TF1.17 (i.e., D1#3) was investigated further by Western blotting (immunoblotting) to determine the reason for the lack of reactivity with one of the two cross-reactive MAbs. The antigenic band that was reactive with MAb TF1.15 had a molecular mass slightly lower than that of the corresponding band from isolate D1, which reacted with both MAbs TF1.15 and TF1.17. Thus, at least a major portion of the antigen appeared to be conserved. This was confirmed in a study of heifers infected with isolate D1#3. The vaginal immunoglobulin A antibodies of these infected heifers reacted with the antigen of isolate D1 that was immunoaffinity purified with MAb TF1.17. Therefore, even though the epitope recognized by MAb TF1.17 was missing in the challenge isolate (D1#3), the heifers developed an immune response to the rest of the molecule. These results indicate that the major portion of the previously described protective antigen is conserved in different isolates of T. foetus. This portion contains the epitope that reacts with MAb TF1.15. Most isolates express the whole antigen, which possesses both TFl.15 and TF1.17 epitopes, but the few isolates that are missing the portion containing the TF1.17 epitope may still elicit an immune reponse to the conserved portion. Thus, the protective surface antigen is promising for use in immunodiagnosis or vaccination against bovine trichomoniasis.
Rapid method for presumptive identification of Corynebacterium jeikeium
Authors: Cartwright C P , Stock F , Kruczak-Filipov P M , Gill V J
Published: January 2020

Corynebacterium jeikeium causes systemic infections, particularly in immunocompromised hosts. A minitube assay has been developed for the presumptive identification of C. jeikeium. With our rapid sucrose-urea test and conventional biochemical tests, sixty isolates of gram-positive, catalase-positive bacilli were identified in our laboratory. Results indicated that our assay has a sensitivity of 100% and a specificity of 90%.
Prosthetic hip joint infection due to Campylobacter fetus
Authors: Yao J D , Ng H M , Campbell I
Published: January 2020

A case of postoperative prosthetic hip joint infection due to Campylobacter fetus subsp. fetus is described. Difficulties in isolation and antimicrobial susceptibility testing of this organism are discussed.
New method for detection of Candida albicans in human blood by polymerase chain reaction
Authors: Miyakawa Y , Mabuchi T , Fukazawa Y
Published: January 2020

We developed a new method for detection of Candida albicans cells in human blood by the polymerase chain reaction that amplifies a 125-bp region within a species-specific, multiple-copy DNA fragment. In preparing template DNA from C. albicans-containing blood, the addition of C. tropicalis cells, together with antibody which reacts with both cells, raised the sensitivity of detection of C. albicans to about 3 cells per 0.1 ml.
Application of Lior biotyping by use of genetically identified Campylobacter strains
Authors: Nicholson M A , Patton C M
Published: January 2020

We used the scheme of Lior to biotype 140 genetically identified Campylobacter strains. Our results confirmed previous studies and extended Lior biotyping to show that nine C. jejuni subsp. doylei strains (100%) were one biotype and nine C. jejuni subsp. jejuni nalidixic acid-resistant strains (100%) were C. jejuni biotype I or II. All C. jejuni subsp. jejuni hippurate-negative strains studied and 6 of 35 C. lari strains (17%) were grouped with C. coli biotypes. These findings may be useful in epidemiologic investigations.
Comparison of the Gen-Probe Group A streptococcus Direct Test with culture and a rapid streptococcal antigen detection assay for diagnosis of streptococcal pharyngitis
Authors: Heiter B J , Bourbeau P P
Published: January 2020

The Gen-Probe Group A Streptococcus Direct Test (GP-ST) is a new assay which utilizes a nucleic acid probe to detect group A streptococci directly from pharyngeal swabs. In this study, 1,103 specimens were cultured and tested by GP-ST. The sensitivities and specificities were as follows: culture, 98.8 and 100%; GP-ST, 92.4 and 99.6%. Of the 1,103 specimens, 808 were also tested with the TestPack Strep A assay. For the specimens tested by all three methods, the sensitivities and specificities were as follows: culture, 99.5 and 100%; TestPack Strep A assay, 76.3 and 99.7%; GP-ST, 93.5 and 99.7%. The GP-ST is a very user-friendly assay which has the potential to replace culture for the diagnosis of streptococcal pharyngitis.
Typing of pneumococci by using 12 pooled antisera
Authors: Sørensen U B
Published: January 2020

A new simplified chessboard system for typing of Streptococcus pneumoniae is described. It is intended for typing or grouping of 90 to 95% of the pneumococcal strains most commonly isolated from blood or cerebrospinal fluid and is based on 12 pooled diagnostic antisera, each reacting with 7 to 11 single types, together covering the 23 different vaccine-related types as well as 25 other cross-reacting types. Worldwide surveillance of the type distribution is important in order to ensure an optimal formulation of pneumococcal polysaccharide vaccines and, in the future, of polysaccharide-protein conjugate vaccines. The simplified typing system described in this paper makes it easier to carry out surveillance in other than specialized reference laboratories. Finally, it takes advantage of the fact that some types cause disease more often in children--as opposed to adults--than do others.
Relative frequency of VP4 gene alleles among human rotaviruses recovered over a 10-year period (1982-1991) from Japanese children with diarrhea
Authors: Gunasena S , Nakagomi O , Isegawa Y , Kaga E , Nakagomi T , Steele A D , Flores J , Ueda S
Published: January 2020

The relative frequencies of the Wa (corresponding to serotype P1A), DS-1(P1B), M37(P2), and AU-1(P3) alleles of the VP4 gene from rotaviruses collected from the stools of individuals in Japan between 1982 and 1991 were determined to be 83.1, 15.6, 0, and 1.3%, respectively, by a polymerase chain reaction-based typing assay.
Ceftizoxime interpretive criteria for in vitro susceptibility tests with Neisseria gonorrhoeae
Authors: Barrett M S , Jones R N , Erwin M E
Published: January 2020

Ceftizoxime was tested in triplicate against 100 clinical strains of Neisseria gonorrhoeae in accordance with National Committee for Clinical Laboratory Standards guidelines to establish susceptibility testing interpretive criteria. The MICs for 50 and 90% of the strains tested were 0.008 and 0.03 microgram/ml, respectively. These results confirm those of other studies reporting ceftizoxime's excellent activity against gonococci. Because no resistant strains were identified, a breakpoint MIC of < or = 0.5 microgram/ml was selected, with a correlate zone diameter of > or = 32 mm. Ceftizoxime appears to represent an alternative to other beta-lactamase-stable beta-lactams for cost-effective therapy of uncomplicated gonorrhea.
Recovery of a strain of Agrobacterium radiobacter with a mucoid phenotype from an immunocompromised child with bacteremia
Authors: Dunne W M , Tillman J , Murray J C
Published: January 2020

Agrobacteria are associated more commonly with plant than with human disease. The isolation of Agrobacterium radiobacter from blood cultures of an immunocompromised child with a transcutaneous catheter prompted a review of human infections caused by Agrobacterium species. Only 12 reports describing 19 cases of Agrobacterium infections in humans have appeared in the literature. Sixteen of the patients (84%) were equipped with implantable or transcutaneous medical devices at the time of infection, and 14 of the 19 (80%) patients could be considered immunocompromised because of underlying disease processes. Unlike those in previous reports, however, this patient was infected with a novel mucoid phenotype of A. radiobacter. Because of the significant relationship between infection and biomedical implants, we evaluated the adhesion of this mucoid strain and a nonmucoid strain of A. radiobacter to plastic by using two in vitro assays. No adhesion or biofilm formation was detected for either strain, but nonetheless it is clear from this review that the isolation of Agrobacterium spp. from patients with indwelling medical appliances should not be dismissed as an environmental contaminant.
Epidemiological study of Norwalk virus infections in Japan and Southeast Asia by enzyme-linked immunosorbent assays with Norwalk virus capsid protein produced by the baculovirus expression system
Authors: Numata K , Nakata S , Jiang X , Estes M K , Chiba S
Published: January 2020

In this study, we investigated Norwalk virus (NV) antigen and antibody to recombinant NV (rNV) in human populations in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs). Baculovirus-expressed recombinant NV (rNV) capsid protein was used for preparing antisera to rNV or used as an antigen for detecting antibody to rNV. The ELISAs were specific for NV and had sensitivities equivalent to or higher than those of the previously developed radioimmunoassays. In 159 stool samples obtained from children, mainly younger than 10 years old, with acute gastroenteritis due to small round structured viruses in Japan, only 1 was positive for NV antigen. The pattern of acquisition of antibody to rNV was quite different from those of antibodies to group A rotavirus and human calicivirus Sapporo (HuCV-Sa) strain. The prevalence of antibody to rNV remained at a low level throughout childhood and then showed a steep rise during school age and early adulthood in Japan. A high prevalence of antibody was observed in samples collected from healthy adults in Japan and Southeast Asia. These results suggested that NV infection is common in adults in Japan and Southeast Asia but may be rare in infants in Japan. The HuCV-Sa strain was negative by the ELISA, and no serological relationship between NV and the HuCV-Sa strain was found. NV may be quite different from the HuCV-Sa strain, although both viruses are classified in the family Caliciviridae.
Cultural, molecular, and immunological characterization of the etiologic agent for atypical canine ehrlichiosis
Authors: Kakoma I , Hansen R D , Anderson B E , Hanley T A , Sims K G , Liu L , Bellamy C , Long M T , Baek B K
Published: January 2020

More than 100 cases of canine ehrlichiosis, with three fatalities, were serologically negative by the indirect immunofluorescent antibody (IFA) test with Ehrlichia canis or E. sennetsu antigen but were reactive at titers of 10 to 640 with E. risticii. Ehrlichia-like agents were isolated from three such cases. The agents isolated from those cases were morphologically indistinguishable from each other and from a prototype, E. risticii, the etiologic agent of equine monocytic ehrlichiosis, in terms of growth characteristics and by light or electron microscopy. The patterns of and products from PCR were identical to those of E. risticii. The 16S rRNA sequences were distinct from those of E. canis and E. ewingii but were identical to those of E. risticii. A PCR product corresponding to the 5' half of the 16S rRNA gene was obtained from amplification of DNA from E. risticii and both sources of the atypical canine ehrlichiosis agent but was not obtained from uninfected host cells. The entire sequence of 719 nucleotides was identical for all three sources. The percentages of relatedness of the partial 16S rRNA gene of the atypical canine ehrlichiosis agent to E. risticii, E. sennetsu, E. platys, E. equi, E. phagocytophila, E. canis, E. chaffeensis, and E. ewingii were 100.0, 98.9, 83.7, 83.0, 83.0, 82.2, 81.8, and 81.5, respectively. These data are consistent with the identity of these isolates as E. risticii. The caninotropic characteristics of naturally acquired infections due to E. risticii are herein described for the first time, and the epizootiological implications are discussed in relation to the host range of E. risticii, which may include dogs as reservoirs.
Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis
Authors: Espino A M , Finlay C M
Published: January 2020

A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive.
Differentiation between wild and vaccine-derived strains of poliovirus by stringent microplate hybridization of PCR products
Authors: Takeda N , Sakae K , Agboatwalla M , Isomura S , Hondo R , Inouye S
Published: January 2020

Procedures for differentiation between wild and vaccine-derived strains of poliovirus are required, particularly in countries where wild and vaccine-related strains coexist. For this differentiation, we tested the method of Inouye and Hondo (S. Inouye and R. Hondo, Arch. Virol. 129:311-316, 1993) for discrimination of closely related viruses by using stringent microplate hybridization of PCR products. We used a pair of primers with enterovirus common sequences (between these primers there is a variable region for capsid proteins) for PCR using templates from wild and vaccine-derived poliovirus strains which were isolated in tissue culture and serotyped by neutralization assay. We also used the same primers for preparation of probes, which were labelled by incorporation of biotin-dUTP in the PCR, with the three original Sabin vaccine virus strains used as templates. The amplified DNAs from the isolates were immobilized on microplate wells and were then hybridized with the labelled probes. We found that, under the usual hybridization conditions, the Sabin vaccine virus strain probes hybridized with both wild and vaccine-derived viruses, but under stringent conditions, they reacted only with vaccine-derived viruses of the same serotype, clearly differentiating these from wild-type viruses.