bluuebunny/crossref_metadata_2025 huggingface dataset:
Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine
Authors: Gerritsen M J , Olyhoek T , Smits M A , Bokhout B A
Published: January 2020

An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitive detection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction (PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows were used. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 10(7) to 10(8) Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelleted leptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation method enabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the need for dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by experimentally infected cows.
Use of monoclonal antibodies with neutralizing effects on toxic antigens from human bacterial plaque to detect specific bacteria by colony blotting
Authors: Levine M , Miller F C
Published: January 2020

Inflammatory periodontal diseases are provoked by bacteria which adhere to teeth at the gingival margin and form plaques containing toxins detectable by their effect on mammalian cells in culture. The aim of this study was to make toxin-neutralizing monoclonal antibodies and determine whether they detect antigen in specific oral bacteria. Bacterial plaque was collected from teeth and homogenized, and the fluid phase (plaque extract) was boiled or first fractionated over Sephacryl S-300. Hybridomas from immunized mice secreted immunoglobulin M (IgM) antibodies which reacted to plaque antigens. Neutralization was detected by an increase in the growth of HL60 cells which were exposed to plaque toxins in the presence of IgM from hybridoma culture or ascitic fluids. However, the neutralization was obvious only when the plaque toxins reduced growth by 50% or less. Plaque toxin preparations were found to contain proteases which hydrolyzed all of the IgM in ascitic fluids within 24 h. Replenishing the IgM daily preserved protection compared with protection from IgM from other hybridomas or saline only. The decrease in the specific activity of plaque proteins caused by replenishing one such antibody (3hE5) was 2.5-fold compared with activity with unreplenished 3hE5, 3.8-fold compared with activity with saline only, and 10.7-fold compared with activity with replenished, unrelated antibody. The neutralizing IgM detected an array of 14,000- to 22,000-molecular-weight antigens. The native toxins may be aggregates of these antigens, or the array may indicate fragments of an undetected, larger antigen or a common, nonpeptide adduct. Only 0.5 to 0.8% of the bacteria from sites with periodontitis and grown on blood agar contained antigen. One group of reactive bacteria was identified as Actinomyces odontolyticus serotype I. Other isolates were identified as Staphylococcus epidermidis, but antigen disappeared from the these isolates within 6 weeks of subculture. Epitope-containing antigens were also found in streptococcal and Eikenella isolates, and it is likely that the antigens from only some of these bacteria are toxic.
Phage typing of Salmonella enteritidis in the United States
Authors: Hickman-Brenner F W , Stubbs A D , Farmer J J
Published: January 2020

The number of reported isolates of Salmonella enteritidis has increased dramatically in the last 10 years. For many years phage typing has been a useful epidemiologic tool for studying outbreaks of S. typhi and S. typhimurium. In 1987, Ward et al. (L. R. Ward, J. De Sa, and B. Rowe, Epidemiol. Infect. 99:291-294, 1987) described a phage typing scheme for S. enteritidis. This system differentiated 27 phage types by use of 10 typing phages. With these phages, we typed 573 strains of S. enteritidis from humans (42 outbreaks), animals, food, and the environment. Ninety-six percent of the strains were typeable. The most common phage types were 8 (48.2%), 13a (20.1%), 13 (7.8%), and 14b (7.8%). Most of the strains were specifically collected from egg-related outbreaks in the northeastern United States in 1988 and 1989, probably accounting for the distribution of the four most common types in this sample. This system was particularly useful for differentiating a group of animal strains that had a number of diverse phage types. For 49 animal strains typed, 16 different patterns were obtained. Phage type 8 represented 32% of these strains, but no other phage type represented more than 8% of these strains. One-half of the 16 animal strains that were phage type 8 were from poultry. This phage typing system will be useful for comparing phage types found in the United States with those types encountered worldwide and for determining whether virulent strains of phage type 4 are entering the United States. Additional phage typing systems as well as molecular techniques are being studied to determine whether they can differentiate strains of phage types 8 and 13a.
Fibronectin binding by Salmonella strains: evaluation of a particle agglutination assay
Authors: Baloda S B , Dyal R , González E A , Blanco J , Hajdu L , Månsson I
Published: January 2020

Thirty-five Salmonella strains isolated from human cases of salmonellosis were tested and compared for their fibronectin (fn) binding capacities by using two fn-particle agglutination assays (fn-PAAs) prepared by coating with human fn either (i) latex beads (Difco; 0.81-micron diameter) (L-fn-PAA) or (ii) heat-killed formalin-treated Staphylococcus aureus Cowan 1 cells (C-fn-PAA). Six S. aureus strains were also included in this study as controls. The strains were cultured on colonization factor antigen agar and blood agar and in tryptic soy broth and brain heart infusion broth. The Salmonella and S. aureus strains were cultured at 33 and 37 degrees C, respectively, for optimal expression of fn-binding proteins. Bacterial cells (approximately 10(10) cells per ml) harvested from growth in various culture media and suspended in 0.02 M potassium phosphate buffer (pH 6.8) agglutinated the fn-PAA reagents. These reactions were scored semiquantitatively from + to + depending on the speed or intensity of the reactions within 2 min. Maximum agglutination in fn-PAA systems was observed when the cells were grown in brain heart infusion broth, while tryptic soy broth proved to be least suitable media for culturing cells for fn-PAAS. Although a statistically highly significant correlation was obtained between results of assays of radiolabeled fn and 29-kDa fragment binding, no significant correlation was observed (i) between the results of strains cultured in different media or (ii) when semiquantitative score results of the two fn-PAA systems were compared with those of the conventional radiolabeled fn assay. To enhance the efficiency of the test system, the C-fn-PAA reagent was stained with methylene blue (2% in 0.17 M glycine-NaOH buffer [pH 6.8]). This facilitated easy interpretation of results, which could be performed on hydrophobic paper instead of glass slides. The results obtained with both unstained C-fn-PAA and stained C-fn-PAA were comparable to each other and reproducible. Although the fn-PAAs are simple and easy to perform, the results did not differentiate between negative, low, moderate, and high binding abilities when Salmonella strains were evaluated for fn binding, and the results were not comparable to those obtained by the conventional radiolabeling method.
Detection of Helicobacter pylori in stomach tissue by use of a monoclonal antibody
Authors: Husson M O , Leclerc H
Published: January 2020

Monoclonal antibodies were produced against an acid glycine extract of Helicobacter pylori ATCC 43504T. One of these appeared to be specific for H. pylori; it recognized all H. pylori isolates by an indirect immunofluorescence assay (IIF) but it did not cross-react with the other strains tested, including different species of the genera Helicobacter, Campylobacter, and Wolinella. Different strains of members of the families Enterobacteriaceae and Pseudomonadaceae or other gram-negative bacteria tested also gave negative reactions. Indirect immunofluorescence assay of antral biopsy specimens identified 54 of 56 infected patients (96.4%), and it may be able to detect nonviable organisms after antibiotic therapy.
Evaluation of the Vitek GPS-TA card for laboratory detection of high-level gentamicin and streptomycin resistance in enterococci
Authors: Metchock B , McGowan J E
Published: January 2020

The Vitek GPS-TA card (Vitek Systems, Hazelwood, Mo.) was compared with single-concentration broth microdilution and disk diffusion methods using high-content disks for the detection of high-level resistance to gentamicin and streptomycin in 99 isolates of enterococci (81 Enterococcus faecalis isolates and 18 Enterococcus faecium isolates). The GPS-TA card accurately detected high-level resistance to gentamicin, but not streptomycin, in E. faecalis. When streptomycin is being considered for therapy, either disk diffusion or time-kill studies should be used to confirm susceptible results obtained by Vitek testing. Additional studies are needed to determine the best method for testing E. faecium isolates.
Glycosidase profiles of members of the family Enterobacteriaceae
Authors: Kämpfer P , Rauhoff O , Dott W
Published: January 2020

A total of 712 strains representing 47 taxa of the family Enterobacteriaceae were tested for the ability to hydrolyze 14 4-methylumbelliferyl (4-MU)-linked substrates within 3 h of incubation. In addition to the well-known differentiation potential of the hydrolysis of 4-MU-beta-D-galactopyranoside, 4-MU-beta-D-glucuronide, and 4-MU-beta-D-xylopyranoside, the hydrolysis of some other fluorogenic substrates (e.g., 4-MU-beta-D-fucopyranoside, 4-MU-N-acetyl-beta-D-galactosaminide, and 4-MU-alpha-D-galactopyranoside) can also be used for species differentiation within the family Enterobacteriaceae.
Emergence of homogeneously methicillin-resistant Staphylococcus aureus
Authors: Fung-Tomc J , Huczko E , Gradelski E , Denbleyker K , Bonner D P , Kessler R E
Published: January 2020

Forty-seven clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), collected between 1986 and 1990 from 29 institutions, were analyzed for susceptibility to various antibiotics. Twenty-six strains were homogeneously methicillin resistant (i.e., greater than or equal to 10% of the cells in these strains were able to grow on Mueller-Hinton agar containing 50 micrograms of methicillin per ml). The MICs of gentamicin, clindamycin, trimethoprimsulfamethoxazole, methicillin, and imipenem for homogeneous MRSA strains were higher than those for heterogeneously resistant strains. Both types of strains were, for the most part, susceptible to vancomycin and trimethoprim-sulfamethoxazole. Ciprofloxacin-resistant MRSA strains were not isolated prior to 1988 but made up 40% of the post-1987 strains. The level of methicillin resistance correlated well with the imipenem MIC, suggesting that susceptibility to imipenem may serve as a marker to identify and monitor the prevalence of homogeneous MRSA strains.
Prevalence of virulent Rhodococcus equi in isolates from soil and feces of horses from horse-breeding farms with and without endemic infections
Authors: Takai S , Ohbushi S , Koike K , Tsubaki S , Oishi H , Kamada M
Published: January 2020

The prevalence of virulent Rhodococcus equi in isolates from soil and feces of foals on a farm with endemic R. equi infections was significantly higher than that of a farm with no history of the disease. Foals bred on a farm with the endemic disease might be constantly exposed to virulent R. equi in their environment.
Restriction enzyme and Southern hybridization analyses of Pseudomonas aeruginosa strains from patients with cystic fibrosis
Authors: Loutit J S , Tompkins L S
Published: January 2020

Conventional typing schemes for Pseudomonas aeruginosa may not discriminate strains. P. aeruginosa isolates from patients with cystic fibrosis were examined by restriction enzyme analysis and Southern hybridization. There was marked diversity of restriction enzyme analysis patterns among P. aeruginosa DNAs from cystic fibrosis isolates; however, sequential isolates obtained from individual patients showed very little variation over an 8-year period. DNA fragments from the alginate biosynthesis gene complex, the exotoxin A gene, and the elastase gene of P. aeruginosa were used in Southern hybridization analysis. The patterns of hybridization to the elastase and algD gene probes were highly conserved in all isolates, therefore, these DNA fragments are not useful in discriminating strains, in contrast to the exotoxin A gene probe.
Detection of antibodies to Mycoplasma gallisepticum in egg yolk versus serum samples
Authors: Brown M B , Stoll M L , Scasserra A E , Butcher G D
Published: January 2020

Serum (n = 1,636) and egg yolk (n = 802) samples collected from hens on four commercial egg farms in Florida were tested for the presence of specific antibodies to Mycoplasma gallisepticum in a commercially available enzyme-linked immunosorbent assay. No significant differences were noted between serum and egg yolk samples with respect to distribution of positive, suspect, and negative test results or for the mean sample/positive control ratio values of positive, suspect, and negative test results. A linear relationship between the distribution of positive and negative results and the age of the birds was observed for results obtained with both serum and egg yolk samples. On the basis of the results of this study, egg yolk samples can be used in lieu of serum samples to screen flocks for antibodies to Mycoplasma gallisepticum.
Evaluation of the Roche Septi-Chek AFB system for recovery of mycobacteria
Authors: D'Amato R F , Isenberg H D , Hochstein L , Mastellone A J , Alperstein P
Published: January 2020

The Septi-Chek AFB system for the recovery of mycobacteria from clinical specimens was compared with a conventional approach using Lowenstein-Jensen and Middlebrook 7H11 agars. A total of 1,532 clinical specimens were analyzed; 132 yielded mycobacteria. Mycobacterium tuberculosis and Mycobacterium avium complexes were the predominant isolates. With the conventional combination of Lowenstein-Jensen and 7H11 agars, 75.8% of the isolates were recovered; the Septi-Chek AFB allowed recovery of 100% of the isolates. Septi-Chek AFB required less time for the detection of mycobacteria than did the conventional media.
Role of cattle in the epidemiology of tick-bite fever in Zimbabwe
Authors: Kelly P J , Mason P R , Manning T , Slater S
Published: January 2020

Almost 100% of 52 cattle tested from the southern areas of Zimbabwe were found to have antibodies reactive with Rickettsia conorii compared with less than 30% of 120 cattle from the north. Steers artificially infected with R. conorii isolated from Amblyomma hebraeum were found to show no hematological or biochemical signs of disease but did seroconvert. Clinical signs of infection were restricted to regional lymphadenopathy and dermal erythema, edema, and tenderness at the inoculation site. Rickettsemia was detectable for at least 32 days postinfection. Our findings indicate that cattle could be involved in the transmission of rickettsias by A. hebraeum and may serve as a reservoir of human tick-bite fever in southern Africa.
Multilocus enzyme electrophoresis analysis of the Mycobacterium avium complex and other mycobacteria
Authors: Wasem C F , McCarthy C M , Murray L W
Published: January 2020

Multilocus enzyme electrophoresis analysis was used to evaluate the Mycobacterium avium complex (MAC), M. paratuberculosis, and nine other mycobacterial species. The average number of alleles per locus was 2.8 for the 35 MAC and 2 M. paratuberculosis strains which represented 24 electrophoretic types (ETs) and two distinct groups. The M. avium group was resolved into 17 ETs and contained the M. paratuberculosis ET. The M. intracellulare group consisted of six ETs. There was complete agreement between Gen-Probe identification and group placement by multilocus enzyme electrophoresis. The mean genetic diversity per locus for the 24 MAC ETs was 0.38. This procedure subdivided some serovars and, if implemented, should prove to be a powerful epidemiologic tool for the MAC. Eleven additional ETs were formed after the data for the other mycobacterial species were pooled with those for the MAC.
Evaluation of a commercial enzyme-linked immunosorbent assay for Johne's disease
Authors: Collins M T , Sockett D C , Ridge S , Cox J C
Published: January 2020

A new commercial kit for diagnosis of bovine paratuberculosis (Johne's disease), called the Johne's Absorbed EIA (enzyme immunoassay; Commonwealth Serum Laboratories, Parkville, Victoria, Australia), was evaluated by using serum specimens from the National Repository for Paratuberculosis Specimens. The evaluation was specifically designed to measure test sensitivity and specificity for detection of dairy cattle with subclinical paratuberculosis. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis from fecal samples or internal organs of cattle without diarrhea or chronic weight loss. Animals designed as free of the disease originated exclusively from four herds in Wisconsin that were certified to be free of disease. The kit had a sensitivity of 47.3% for serum specimens from 150 infected cattle. The test detected 59.7% of animals that shed M. paratuberculosis in their feces, as defined by conventional fecal culture, at the time of serum collection. Testing of 196 serum specimens from cattle without paratuberculosis yielded two false-positive results; the test specificity was thus 99.0%. Decision analysis procedures on the economics of using the kit in a test-and-cull disease control program indicated it would be cost-effective in any herd with a true paratuberculosis prevalence of greater than or equal to 3%. Comparison of the sensitivity and specificity of the Johne's Absorbed EIA with those of other tests for detection of subclinical paratuberculosis indicated that it may be the most accurate commercially available test at present and better than standard complement fixation test used in the United States.
Outbreak of Brucella melitensis among microbiology laboratory workers in a community hospital
Authors: Staszkiewicz J , Lewis C M , Colville J , Zervos M , Band J
Published: January 2020

From May to September 1988, eight employees of a microbiology laboratory developed acute brucellosis (attack rate, 31%). Seven of the eight affected employees had clinical illness ranging from a nonspecific, flulike illness to severe hepatitis. Blood cultures obtained from five of the affected employees (63%) were positive for Brucella melitensis, biotype 3. Comparison of cases and controls showed that there were no risk factors besides employment in the laboratory. Based on work locations, assignments, and interviews, it was found that person-to-person, droplet, food-borne, and waterborne spread were unlikely. Our investigation disclosed that 6 weeks before the outbreak began, a frozen brucella isolate from a patient hospitalized 3 years earlier had been thawed and subcultured without the use of a biologic safety cabinet. This clinical isolate was subsequently identified as B. melitensis, biotype 3, identical to the employee isolates. It is presumed that transmission occurred via the airborne route. This outbreak reemphasized that all work on Brucella species, an established biosafety level 3 organism, must be conducted under a biologic safety hood. Furthermore, it might be prudent to perform all clinical "setups" under a safety hood since aerosolization commonly occurs during the initial processing of specimens and the majority of these specimens are from patients with uncertain diagnoses.
Characterization of noncapsulate Haemophilus influenzae by whole-cell polypeptide profiles, restriction endonuclease analysis, and rRNA gene restriction patterns
Authors: Bruce K D , Jordens J Z
Published: January 2020

Thirty-four clinical isolates of noncapsulate Haemophilus influenzae representing isolates with either related or dissimilar patterns of whole-cell polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were further characterized by restriction enzyme analysis (REA) and rRNA gene restriction patterns. Total cellular DNA was extracted by a rapid, microcentrifuge-scale method and digested with BamHI, which gave a pattern of about 18 discrete bands. This confirmed the five closely related groupings suggested by SDS-PAGE. Isolates dissimilar by SDS-PAGE were also distinguishable by REA. However, there was no correlation between the degrees of similarity estimated from whole-cell polypeptide profiles and those obtained from REA for the dissimilar isolates. Therefore, inferences of genetic relatedness made on the basis of these data should be interpreted with caution. rRNA gene restriction patterns also confirmed the groupings suggested by the other two techniques. We conclude that the three methods were highly discriminatory and that whole-cell polypeptide patterns or REA with BamHI would be appropriate techniques for epidemiological studies of noncapsulate H. influenzae.
Chemotypes of Mycobacterium malmoense based on glycolipid profiles
Authors: Katila M L , Brander E , Jantzen E , Huttunen R , Linkosalo L
Published: January 2020

Thin-layer chromatographic analysis of 72 Finnish clinical mycobacterial isolates presumptively identified as Mycobacterium malmoense revealed four major glycolipid profiles with two minor variations. An additional glycolipid profile was found in three British M. malmoense-like strains. No clear distinction between the strains could be made by means of gas chromatography of cellular fatty acids. The two M. malmoense-specific constituents, 2-methyleicosanoate and 2,4,6-trimethyltetracosanoate, were detected in all strains. The frequency of chemotypes other than that of the type strain was 8% among the Finnish isolates. This variation should be recognized when confirmative identification of mycobacteria is based on thin-layer chromatography of glycolipid extracts.
Performance of a BACTEC nonradiometric medium for pediatric blood cultures
Authors: Morello J A , Matushek S M , Dunne W M , Hinds D B
Published: January 2020

A BACTEC aerobic nonradiometric medium, PEDS Plus, designed for diagnosis of pediatric bacteremia was evaluated in three hospital centers. Equivalent blood volumes (up to 5 ml) were inoculated into and incubated in BACTEC NR-6A (6A) and PEDS Plus broths. Among 4,581 compliant sets, 289 clinically significant organisms, representing more than 20 bacterial and two Candida species, were isolated. One hundred eighty-one isolates were recovered in both bottles, 75 in PEDS Plus only, and 33 in 6A only (P less than 0.001). Time to detection when both bottles were positive was the same for 129 isolates, detection with PEDS Plus was earlier for 39, and detection with 6A was earlier for 13 (P less than 0.005). Staphylococcus aureus was recovered significantly more often in PEDS Plus than in 6A (P less than 0.01), and more coagulase-negative staphylococci and pediatric pathogens (pneumococci, Haemophilus influenzae, and Streptococcus agalactiae) were recovered in PEDS Plus than in 6A. Coagulase-negative staphylococci and H. influenzae were detected significantly earlier in PEDS Plus (P less than 0.05 and less than 0.01, respectively). When the eight species of the family Enterobacteriaceae isolated were considered together, recovery in PEDS Plus was better than in 6A (P less than 0.05). For 66 of the 143 isolates from patients known to be on antimicrobial therapy at the time blood was drawn, PEDS Plus was superior to 6A. In 45 cases, organisms were isolated from PEDS Plus only (P less than 0.001) and in 21 cases they were isolated from PEDS Plus before 6A (P less than 0.01). PEDS Plus broth aids diagnosis of pediatric bacteremia by increasing recovery of etiologic agents and decreasing the time required for detection.
Campylobacter butzleri sp. nov. isolated from humans and animals with diarrheal illness
Authors: Kiehlbauch J A , Brenner D J , Nicholson M A , Baker C N , Patton C M , Steigerwalt A G , Wachsmuth I K
Published: January 2020

Seventy-eight aerotolerant Campylobacter isolates were characterized phenotypically and by DNA hybridization (hydroxyapatite method at 50 and 65 degrees C). Two DNA relatedness groups were found. (i) Sixty-four strains belonged to aerotolerant Campylobacter DNA hybridization group 2. These organisms were isolated from humans, primarily with diarrheal illness, and animals on several continents. Strains were aerotolerant at 30 and 36 degrees C and catalase negative or weakly catalase positive, grew in media containing glycine and on MacConkey agar, were susceptible to nalidixic acid, and were resistant to cephalothin. The name Campylobacter butzleri sp. nov. is proposed for this group. (ii) DNA hybridization group 1 consisted of the type strain of Campylobacter cryaerophila and 13 additional strains isolated from 10 animals outside the United States and from three humans within the United States. This group was genetically diverse; five strains were closely related to the type strain of C. cryaerophila (DNA hybridization group 1A), and eight strains were more closely related to one another (DNA hybridization group 1B). Strains in DNA hybridization group 1B were phenotypically diverse, with two of eight strains resembling C. cryaerophila. The seven strains from DNA hybridization groups 1A and 1B which resembled C. cryaerophila and the C. cryaerophila type strain were aerotolerant only at 30 degrees C and catalase positive, did not grow in glycine or on MacConkey agar, were generally susceptible to nalidixic acid, and were resistant to cephalothin. The remaining six strains of DNA hybridization group 1B phenotypically resembled C. butzleri; however, they were generally catalase positive and susceptible to nalidixic acid and cephalothin. DNA hybridization group 1B is not designated as a separate species at this time since it cannot, with certainty, be separated genetically from C. cryaerophila or phenotypically from C. butzleri.
Detection of bacteria in blood by centrifugation and filtration
Authors: Bernhardt M , Pennell D R , Almer L S , Schell R F
Published: January 2020

Culture of blood is the most frequent means of diagnosing bacteremia. However, conventional blood culturing methods are slow in isolating bacteria. We developed a method for isolation of bacteria by centrifugation and filtration. Fresh human whole blood was inoculated with facultatively anaerobic and aerobic microorganisms (3 to 172 microorganisms per 5 ml). Seeded blood was then mixed with Ficoll-Hypaque (density, 1.149 +/- 0.002 g/ml) and centrifuged (386 x g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micron membrane filter (Millipore). The filters were then placed on chocolate agar plates and incubated at 35 degrees C in a humidified atmosphere containing 5% CO2. For each bacterium tested, approximately 35 to 100% of the viable microorganisms were recovered when compared with control cultures (pour plates of seeded blood). All bacteria produced isolated colonies on filters after overnight incubation (18 h). This procedure may prove to be a more rapid method for isolating bacteria from clinical blood samples than the blood culture bottle technique.
Identification of 15- to 17-kilodalton antigens associated with virulent Rhodococcus equi
Authors: Takai S , Koike K , Ohbushi S , Izumi C , Tsubaki S
Published: January 2020

Antigens of Rhodococcus equi were analyzed by immunoblotting with naturally infected foal sera. Immunoblots of whole-cell antigen preparations of clinical isolates of R. equi revealed that major protein bands with molecular masses of 15 to 17 kDa were present in all clinical isolates tested and all isolates virulent for mice. In contrast, the 15- to 17-kDa antigens were not identified by immunoblotting in ATCC 6939, a type strain of R. equi that was avirulent for mice. Whole-cell antigens of 102 environmental isolates were investigated by immunoblotting and the mouse pathogenicity test. Twenty-five of these isolates were demonstrated to contain the 15- to 17-kDa antigens by immunoblotting and were virulent for mice. The remaining 77 environmental isolates lacked the 15- to 17-kDa antigens and were avirulent for mice. These data suggest that the diffuse 15- to 17-kDa proteins are virulence-associated antigens with immunogenicity in foals and that they may be useful in marking virulent R. equi contamination in the environment of a horse-breeding farm.
Legionella fairfieldensis sp. nov. isolated from cooling tower waters in Australia
Authors: Thacker W L , Benson R F , Hawes L , Gidding H , Dwyer B , Mayberry W R , Brenner D J
Published: January 2020

Three Legionella-like organisms were isolated from water from the cooling towers of two Australian institutions. The strains grew on buffered charcoal-yeast extract (BCYE) agar but not on BCYE agar in the absence of L-cysteine. Gas-liquid chromatography profiles of the isolates were consistent with those for Legionella spp. They were serologically distinct from other legionellae in a slide agglutination test. DNA hybridization studies showed that the three isolates belong to a new species of Legionella, Legionella fairfieldensis (ATCC 49588).
In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory
Authors: Powers D C , Fries L F , Murphy B R , Thumar B , Clements M L
Published: January 2020

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.
Polymerase chain reaction analyses identify two distinct classes of Borrelia burgdorferi
Authors: Rosa P A , Hogan D , Schwan T G
Published: January 2020

We sequenced homologous chromosomal loci from several North American and European isolates of the Lyme disease spirochete Borrelia burgdorferi, as well as from the relapsing fever spirochete Borrelia hermsii. Inter- and intraspecies sequence comparisons permitted the design of B. burgdorferi-specific polymerase chain reaction primers that detected all strains tested (n = 31) from diverse geographical and biological origins. Polymerase chain reaction "typing" with other unique sets of primers subdivided B. burgdorferi isolates into two groups: all North American isolates and a few European isolates made up one group, while the majority of the European and Asian isolates made up the second group. This classification may have a clinical correlate reflected in differences between "typical" Lyme borreliosis in North America and Europe.
Comparison of the E Test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria
Authors: Baker C N , Stocker S A , Culver D H , Thornsberry C
Published: January 2020

The E Test (AB Biodisk, Solna, Sweden) is a new method for performing antimicrobial susceptibility tests. It consists of an impervious carrier (5- by 50-mm strip) with a predefined antimicrobic gradient which is placed on an inoculated agar plate and processed like a disk diffusion test. Results are generated directly as MICs from a continuous concentration gradient covering 15 twofold dilutions, and MICs are read where the edge of the inhibition zone intersects the strip. We compared the E Test with disk diffusion, broth microdilution, and agar dilution tests by using a challenge set of 195 gram-positive and gram-negative bacteria for 14 antimicrobial agents. Also, disk diffusion, broth microdilution, and agar dilution tests were compared with each other. All test method comparisons gave greater than 94% agreement for the category of susceptibility. The E Test category agreement with disk diffusion and broth microdilution was 95.1%, and with agar dilution it was 95.2%. The E Test results were as reliable as the results obtained by the standard antimicrobial susceptibility testing methods.
Aeromonas jandaei (formerly genospecies DNA group 9 A. sobria), a new sucrose-negative species isolated from clinical specimens
Authors: Carnahan A , Fanning G R , Joseph S W
Published: January 2020

A large numerical taxonomy study conducted in 1988 of 165 mostly clinical Aeromonas strains from diverse geographic sources produced a cluster (S = 84%, SSM) of four sucrose-negative strains that included the DNA definition strain for DNA group 9 A. sobria (CDC 0787-80). These four strains, together with five additional strains received in 1989, were subjected to DNA-DNA hybridization (hydroxyapatite, 32P, 60 and 75 degrees C), and all eight strains were closely related to the ninth labeled DNA group 9 definition strain CDC 0787-80 (73 to 86% relatedness at 60 degrees C and 68 to 80% relatedness at 75 degrees C; percent divergence, 2.0 to 3.5). Type strains and DNA definition strains for all other established Aeromonas species were only 35 to 72% related (60 degrees C) to CDC 0787-80. We propose the name Aeromonas jandaei for this highly related group of nine strains, formerly known as DNA group 9 A. sobria. The type strain was designated ATCC 49568 (CDC 0787-80). The nine strains were examined at 36 degrees C and were found to be resistant to 0/129 (vibriostatic agent) and uniformly positive for oxidase, gas production from glucose, indole, lysine decarboxylase, arginine dihydrolase, o-nitrophenyl-beta-D-galactopyranoside, motility (25 degrees C), nitrate reduction, citrate utilization, hemolysis on sheep blood agar, and growth in Trypticase soy broth with no added NaCl. They all fermented D-glucose, D-mannitol, and mannose but did not ferment sucrose, cellobiose, L-arabinose, inositol, salicin, or D-sorbitol. They were uniformly negative for esculin and urea hydrolysis, elastase production, ornithine decarboxylation, and the string test. The antibiogram of A. jandaei resembled that of other aeromonads (resistance to ampicillin and cephalothin), but it differed from most other aeromonads because of resistance to single dilution of colistin and differed from clinical A. veronii biogroup sorbria (formerly A. sobria) by its nearly uniform resistance to cephalothin. The esculin-, sucrose-, and cellobiose-negative and colistin-resistant profile distinguished A. jandaei from other Aeromonas species. These A. jandaei strains were isolated from blood (two strains), wounds (two strains), diarrheal stools (four strains), and a prawn (one strain). The blood and wound isolates, in particular, suggest that there is a possible clinical significance for this species and justify identification of and further research on this group of motile aeromonads.
Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus DNA
Authors: Schiffman M H , Bauer H M , Lorincz A T , Manos M M , Byrne J C , Glass A G , Cadell D M , Howley P M
Published: January 2020

A methodologic study was performed to compare the polymerase chain reaction (PCR) and Southern blot hybridization, two commonly used testing strategies for the detection of human papillomavirus (HPV) infection. Three laboratories tested masked aliquots of exfoliated cervical cell specimens obtained from 120 women by cervicovaginal lavage. The study population included 32 women with condylomatous atypia or cervical intraepithelial neoplasia and 88 control women with no known history of cervical neoplasia. Two laboratories used PCR with different sets of consensus primers for HPV detection. The third laboratory used low-stringency Southern blot hybridization to identify all HPV types, followed by high-stringency Southern and/or dot blot hybridization to confirm specific HPV types. One of the PCR primer sets detected HPV types with a differential efficiency that was not predicted by analysis of DNA sequences or direct testing of HPV-containing plasmids. In contrast, the second PCR primer set was shown to be a much broader consensus system, detecting the same HPV types as Southern blotting, though requiring much less clinical specimen. Over 80% of women with cervical intraepithelial neoplasia or condylomatous atypia were found to be HPV infected both by Southern blotting and by the second PCR primer set. Among the control women, 11% were HPV positive by Southern blotting, while 31% were positive with the second set of primers. Most of the HPV infections found only by PCR were not due to HPV type 6, 11, 16, 18, 31, 33, or 45. These known HPV types were uncommon among normal women in the study population, even as determined by the PCR method.
Comparison of biotinylated DNA and RNA probes for rapid detection of varicella-zoster virus genome by in situ hybridization
Authors: Forghani B , Yu G J , Hurst J W
Published: January 2020

We describe a general method for the production of nonisotopic DNA and RNA probes for the detection of the varicella-zoster virus (VZV) genome by in situ hybridization. VZV DNA was extracted from purified viral nucleocapsids, cleaved with restriction enzyme (RE) BamHI, and cloned into plasmid pBR322 by the standard vector insert procedure. We cloned over 85% of the VZV genome and obtained 18 recombinants. Plasmids containing the B, F, G, H, and J fragments of VZV DNA were labeled by the nick translation method with biotin-11-dUTP as the dTTP analog. Additionally, the B fragment was cleaved with RE AvaI, subcloned into the plasmid pGEM-4 transcription vector, and subsequently linearized with REs PstI and EcoRI. RNA was transcribed with T7 or SP6 polymerase, with a substitution of allylamine-UTP as the UTP analog, and labeled with epsilon-caproylamidobiotin-N-hydroxysuccinimide ester. The DNA and RNA probes were used under full-stringency conditions for in situ hybridization with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate-Nitro Blue Tetrazolium as the substrate. When tested under comparable conditions, the RNA probe was slightly more sensitive than was the DNA probe: both probes showed homology only with VZV-infected cells and clinical tissues and not with the other herpesviruses. Probes prepared from variable regions of the genome (fragments F and J) performed as well as did those from conserved regions (fragments B. G. and H). Biotinylated probes have distinct advantages over isotopic probes and retain their full potency for more than 2 years when stored properly.
Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen
Authors: Weare J A , Robertson E F , Madsen G , Hu R , Decker R H
Published: January 2020

Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a negative population (n = 160) from assay cutoffs. With a selected population (n = 307), inclusion of reductant eliminated apparent anti-HBc activity in 54 of 81 samples in the 30 to 70% inhibition range. Reductant-stable anti-HBc samples were strongly associated with the presence of antibody to hepatitis B surface antigen (21 of 27). The association persisted below the detection limits of current assays to 0.3 to 0.4 Paul Ehrlich Institute units per ml. Only 1 of 54 reduction-sensitive borderline samples was confirmed to be positive for antibody to hepatitis B surface antigen. The modified procedures had unchanged or slightly improved sensitivity for immunoglobulin G (IgG)-associated anti-HBc activity. Although IgM anti-HBc detection was reduced from four- to eightfold in the presence of reductants, sensitivities remained at least twofold greater than tha of an enzyme immunoassay (Corzyme M; Abbott) designed to detect acute-phase levels of IgM anti-HBc. The use of reducing agents should significantly improve the reliability of anti-HBc testing, especially near assay cutoffs.
Improvements in detection of antibody to hepatitis B core antigen by treating specimens with reducing agent in an automated microparticle enzyme immunoassay
Authors: Spronk A M , Schmidt L , Krenc C , Pavlis-Jenkins L , Brady J , Taskar S , Angus-Finn L , Mimms L
Published: January 2020

A fully automated microparticle enzyme immunoassay (EIA), IMx Core, was developed for the detection of antibody against hepatitis B core antigen (anti-HBc). IMx Core sensitivity was less than 0.5 Paul Ehrlich Institut units per ml and was greater than that of the commercial radioimmunoassay (RIA) or EIA, Corab and Corzyme, respectively. Specimens from blood donors and diagnostic and hospital patients, which included individuals with a variety of infectious and immune diseases, were tested in parallel by the IMx Core and EIA. Overall agreement of 99.1% (4,797 of 4,841) was obtained. Prevalence of anti-HBc tested by IMx Core ranged from 1.2% in volunteer blood donors to 9.1% in hospital laboratories. Discordant specimens reactive by IMx Core but negative by Corzyme or Corab resulted from the increased sensitivity of the IMx Core assay, since other hepatitis B markers were usually present. However, most discordant specimens were positive by the EIA or RIA but negative by IMx Core. No other hepatitis B markers could be detected in these discordants, and after addition of reducing agent, these specimens also became negative by EIA or RIA. In clinical trials, 30% (14 of 47) of volunteer blood donors and 8% (9 of 119) of hospital patients testing repeatedly reactive by the EIA had reduction-sensitive (unspecific) anti-HBc reactivity. The reducing agent, dithiothreitol, was added to each specimen automatically in the IMx assay to eliminate these unspecific reactions without significantly affecting anti-HBc reactivity resulting from hepatitis B virus infection as judged by the correlation with other hepatitis B markers.
Gen-Probe Rapid Diagnostic System for the Mycobacterium avium complex does not distinguish between Mycobacterium avium and Mycobacterium paratuberculosis
Authors: Thoresen O F , Saxegaard F
Published: January 2020

Three reference and 16 field strains of Mycobacterium paratuberculosis were tested with the Gen-Probe Mycobacterium avium complex DNA probe (Gen-Probe Inc., San Diego, Calif.). All reference strains and 12 of 16 field strains gave positive hybridization results with the probe. This study shows that the M. avium complex probe does not distinguish between M. avium and M. paratuberculosis and indicates heterogeneity in the 16S rRNA gene of M. paratuberculosis.
Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells
Authors: Keller G H , Huang D P , Manak M M
Published: January 2020

We developed an improved microtiter-based assay for the detection of polymerase chain reaction (PCR)-amplified DNA sequences. The synthetic DNA sequences used to prime the PCR were labeled with biotin at their 5' ends so that the specific PCR product was labeled with biotin. Following amplification, an aliquot of the PCR product was denatured and hybridized to a capture DNA sequence immobilized in a microtiter well. The capture sequence was complementary to a portion of the sequence between the primers, so that only extended primers were captured. The captured PCR product was detected colorimetrically by using a streptavidin-peroxidase conjugate and tetramethylbenzidine substrate.
Use of dried blood spot specimens in the detection of human immunodeficiency virus type 1 by the polymerase chain reaction
Authors: Cassol S , Salas T , Arella M , Neumann P , Schechter M T , O'Shaughnessy M
Published: January 2020

Dried blood spots (DBSs) constitute a potentially valuable source of material for human immunodeficiency virus (HIV) serologic and molecular testing. To facilitate molecular testing, we have adapted the polymerase chain reaction (PCR) to the detection of HIV proviral DNA in DBS samples. The method is highly reproducible, with 75 microliters of whole dried blood providing sufficient DNA for duplicate testing with three primer sets. By using DBS PCR, 66 of 69 (95.6%) seropositive at-risk individuals tested positive by at least two primer sets and 85 of 85 (100%) low-risk seronegative blood donors tested negative by all three sets of primers. The frequency of HIV DNA detection in seronegative at-risk individuals was low, with only 1 of 58 (1.7%) individuals testing positive. These results show that in a clinical environment, HIV PCR analysis of DBS specimens is specific and sensitive. The method is cost effective and presents a useful alternative to the isolation of HIV from seropositive babies with an undefined infection status.