Direct detection of Chlamydia trachomatis in clinical specimens by a dot-immunobinding technique using monoclonal antibody.

A dot-immunobinding technique (DIBT) has been developed to permit detection of Chlamydia trachomatis organisms or antigen in clinical specimens. This method was evaluated for the rapid diagnosis of chlamydia infections using monoclonal antibody. The membrane antigen extracted from reticulate bodies was used for the production of species-specific monoclonal antibodies by an in vitro immunization procedure. The DIBT involved spotting clinical specimen directly onto a nitrocellulose membrane followed by reaction with monoclonal antibody and a biotin-avidin-peroxidase indicator system. Specimens were tested for the presence of chlamydia by the cell culture method. Of these, 361 positives and 317 negatives were selected for detection of antigen using the DIBT method. Of 678 clinical specimens that were evaluated by DIBT, 654 (96.7%) gave identical results to the cell culture method, whereas 24 (3.5%) were positive by the DIBT but culture negative. The overall sensitivity was 100% with a specificity of 92.4%. The test could detect as little as 75 pg of chlamydial antigen.

Specificity of monoclonal antibodies reactive with Fusobacterium nucleatum: effect of formalin fixation.

Monoclonal antibody specific for Fusobacterium nucleatum was reacted with untreated and formalin fixed F. nucleatum cells by an enzyme-linked immunosorbent assay (ELISA) and by indirect immunofluorescence. Treatment of bacterial cells with formalin destroyed the antigenic determinant responsible for reactivity with this monoclonal antibody in both assays. Formalin fixation had no effect on hemagglutination activity (HA) of F. nucleatum cells or reactivity with polyvalent rabbit antiserum in double diffusion in agar. Scanning electron microscopy demonstrated that formalin fixation did not affect binding of F. nucleatum cells to microtiter plates. When developing monoclonal antibodies to be used as diagnostic reagents, the antigenic form utilized for immunization should be identical to the antigenic form which will eventually be used in the diagnostic assay.

Identification of an estrogen-binding protein in Pseudomonas aeruginosa.

A constitutive estrogen-binding protein (EBP) has been identified in the cytosol of Pseudomonas aeruginosa, a Gram-negative bacterium. All 14 strains tested contained the EBP. Estradiol binding was rapid and maximal binding occurred by 90 min at 0 degrees C. Dissociation of estradiol from the binding protein occurred at a rate of 4.6 fmol/min with a t1/2 of 42 min. EBP binding was destroyed by protease treatment and at high temperature. Sodium molybdate had no effect on binding. The Kd determined by Scatchard analysis was 3.9 nM and the Bmax was 323 fmol/mg protein. The EBP sedimented at 8.9 S on sucrose density gradients. The presence of 0.4 M KCl increased estradiol binding 6-fold but did not cause a shift in the sedimentation value. Gel filtration of the native protein gave an estimated molecular weight of 215,000 and a Stokes radius of 50.2 A. Steroid binding specificity, in order of decreasing affinity, was estradiol, estrone, dihydrotestosterone, estriol, testosterone, progesterone and promegestone. Other steroid hormones tested did not compete for estradiol binding. Identification of an EBP in a bacterium allows a comparative analysis of other steroid-binding proteins in unicellular microorganisms.

Isolation of Fusobacterium necrophorum from cancrum oris (noma).

A study of the predominant microflora in active sites of noma (cancrum oris) lesions was carried out in eight noma patients 3-15 years of age in Sokoto State in northwestern Nigeria. Paper point sampling and conventional anaerobic microbiologic techniques were used. Fusobacterium necrophorum was recovered from 87.5% of the noma lesions. Oral microorganisms included Prevotella intermedia, alpha-hemolytic streptococci, and Actinomyces spp. which were isolated from 75.0%, 50.0%, and 37.5% of the patients, respectively. Peptostreptococcus micros, Veillonella parvula, Staphylococcus aureus, and Pseudomonas spp. were each recovered from one lesion. The F. necrophorum and P. intermedia isolates were tested for antibiotic sensitivity to clindamycin, tetracycline, metronidazole, and penicillin using the E-test, and all strains were observed to be sensitive to all of the antibiotics tested with the exception of one strain of P. intermedia, which showed resistance to penicillin. The first reported isolation from human noma lesions of F. necrophorum, a pathogen primarily associated with animal diseases, may have important etiologic and animal transmission implications.

Isolation of Fusobacterium nucleatum and electron microscopic observations of spirochetes from tropical skin ulcers in Papua New Guinea.

Tropical ulcer is a disabling condition of the lower leg affecting mainly young adults and older children. Microscopic observations of lesion material have shown fusiform bacilli and spirochetes. We used anaerobic culture techniques to isolate and identify these fusiform bacilli. Electron microscopic (EM) studies were performed to characterize the spirochetes. Material collected on swabs was used to inoculate pre-reduced media and to prepare smears for gram staining; the swabs were placed in fixative for EM study. After incubation, colonies containing fusiform bacilli were subcultured. The anaerobic gram-negative fusiform isolates were identified as Fusobacterium nucleatum using biochemical reactions, hemagglutination testing, and reaction of antigen preparations of the isolates and ATCC strains in serological tests with rabbit antisera. EM observations of negatively stained spirochetes revealed an 8-16-8 periplasmic flagellar arrangement. F. nucleatum and spirochetes may participate in the pathogenesis of this polymicrobic infection.

Pathogenesis of cancrum oris (noma): confounding interactions of malnutrition with infection.

This study showed that impoverished Nigerian children at risk for cancrum oris (noma) had significantly reduced plasma concentrations of zinc (< 10.8 micromol/L), retinol (< 1.05 micromol/L), ascorbate (< 11 micromol/L), and the essential amino acids, with prominently increased plasma and saliva levels of free cortisol, compared with their healthy counterparts. The nutrient deficiencies, in concert with previously reported widespread viral infections (measles, herpesviruses) in the children, would impair oral mucosal immunity. We postulate, subject to additional studies, that evolution of the oral mucosal ulcers including acute necrotizing gingivitis to noma is triggered by a consortium of microorganisms of which Fusobacterium necrophorum is a key component. Fusobacterium necrophorum elaborates several dermonecrotic toxic metabolites and is acquired by the impoverished children via fecal contamination resulting from shared residential facilities with animals and very poor environmental sanitation.

Serologic reactions of oral gram negative anaerobic bacilli.

Serological reactions were performed using hyperimmune rabbit antisera and antigenic preparations of Leptotrichia buccalis, Fusobacterium fusiforme, and Fusobacterium polymorphum. All tests indicated that there was serologic cross reactivity between the two Fusobacterium species. No cross reactivity could be detected between the Fusobacterium species and L. buccalis. The findings suggest that F. fusiform and F. polymorphum are similar in their immunogenicity, and that the grouping of these two organisms as F. nucleatum may be justified.

Acinetobacter contamination of laboratory dental pumice.

Micro-organisms of the genus Acinetobacter, implicated as opportunistic pathogens, have been recovered from dentures after laboratory repair. A study was undertaken to determine if Acinetobacter could be isolated from used dental pumice. Cultural studies demonstrated that Acinetobacter calcoaceticus variety lwoffi was present in high numbers in used pumice and was a major gram-negative microbial contaminant.

Comparative microbiological and immunological studies of subgingival dental plaque from man and baboons.

Baboons may be useful as animal models for the study of human oral diseases and infections. They are closely related to man anatomically, physiologically, and phylogenetically. Plaque and gingival indices were relatively low in 18 baboons (Papio anubis). The mean scores ranged between 0.62 +/- 0.29 and 0.37 +/- 0.20, respectively. Gram-positive and Gram-negative cocci comprised 27.0 +/- 32.4 and 3.1 +/- 7.5% of the total viable counts in the dental plaque samples. Black-pigmented Bacteroides formed about 1.9 +/- 5.9% of the bacterial population in the samples. Anaerobic Gram-negative bacilli were found in 73.2% of the samples and averaged 19.2 +/- 26.3% of the total recovered flora. Species of the oral Actinomyces and other Gram-positive rods found in humans were not isolated. The composition of the oral flora in baboons appeared to be significantly different from that of man. Isolates of F. nucleatum, L. buccalis, and B. intermedius from the two mammals were biochemically similar, but were distinguishable by analysis with antibody, both by precipitin lines and/or differences between homologous and heterologous titers.

Serological studies of peptostreptococci using an indirect fluorescent antibody test.

Members of the genus Peptostreptococcus are frequent isolates from periodontal lesions. A study was undertaken for determination of the serological relationships of oral and non-oral strains of several species of this genus. Eighty-nine strains of peptostreptococci representing seven species were tested by means of an indirect fluorescent antibody technique (IFA), with rabbit antisera to Peptostreptococcus anaerobius ATCC 27337, Peptostreptococcus micros VPI 2618-A, and Peptostreptococcus productus ATCC 27340. Each antiserum showed positive fluorescence when reacted with homologous cells. When anti-P. micros VPI 2618-A serum was added to suspensions of P. micros ATCC 33270 and clinical isolates of P. micros in the IFA, positive fluorescence was observed to a titer of 1:64 with 26 out of 29 strains. Positive fluorescence was also seen when P. anaerobius VPI 5737 and clinical isolates of P. anaerobius were tested with anti-P. anaerobius ATCC 27337 serum; a titer of at least 1:64 was observed in 10 out of 14 strains. These results support the presence of two serological groups of P. anaerobius, rabbit anti-P. anaerobius ATCC 27337 serum reacting with Group II organisms. No interspecies cross-reactivity was observed except with four strains of Peptostreptococcus magnus, which reacted with several antisera as well as with normal rabbit serum and the saline controls. These results indicate that rabbit antisera and the use of an IFA are useful in the identification of P. anaerobius and P. micros from oral lesions.

A system to maximize the in vitro immunological stability of human Langerhans cells.

Langerhans cells (LC) are now known to be immunocompetent antigen-presenting cells (APC) that are involved with numerous immune reactions initiated in skin, including the induction of contact hypersensitivity and the rejection of skin grafts. Immortalized cells that closely resemble in vivo LC have not been developed and mature LC, in vitro, appear to only briefly pass through a stage of differentiation when immunological potential seems stable. The literature supports the concept that mature LC possess numerous ultrastructural and antigen (Ag) characteristics. These include suface ATPase activity, Fc receptors, MHC class II Ag, CD1a Ag and Birbeck granules. These studies were undertaken to determine the ideal conditions of LC isolation and enrichment for persistence of these characteristics, such that toxicological and immunoreactive studies could closely mimic the in vivo situation. Studies were carried out to evaluate sources of tissue, duration of manipulations, chemical perturbation, stored v. fresh tissue, methods of enrichment, donor serological information and cost. LC were evaluated for ultrastructural and Ag stability by light and electron microscopy utilizing monoclonal antibodies (Mab) to surface Ag and ultrastructural features. It was determined that the most critical conditions included duration of manipulations and method of enrichment. Differences between fresh-frozen and fresh tissue samples were negligible. Cells evaluated less than 18 hr post-collection and enriched by density gradient centrifugation consistently demonstrated stability while ATPase activity, MHC Ag and ultrastructural characteristics varied when other methods were used. Implications for immunological and toxicological testing of in vitro skin samples can be drawn.

Systemic antibody response of clinically characterized patients with antigens of Eubacterium brachy initially and following periodontal therapy.

Eubacterium brachy, a gram-positive anaerobic rod, has been implicated by cultural studies to be associated with the microflora of periodontal diseases. Serum samples from 184 clinically characterized patients were evaluated in a standardized enzyme-linked immunosorbent assay (ELISA) for reactivity to E. brachy antigens. Sera from clinically healthy subjects (HS) served as controls. Sera from rapidly progressive periodontitis (RP) patients demonstrated significantly greater reactivity by ELISA than did HS when reactivity with E. brachy antigens was determined (P less than 0.05). Juvenile periodontitis (JP) and adult periodontitis (AP) patients did not differ in reactivity by ELISA from HS (P greater than 0.05). Three to 4 years following successful periodontal therapy, reactivity was not significantly altered in any patient group (P greater than 0.05). The possible significance of these findings and the importance of an extracellular antigen of E. brachy in the immunopathology of periodontal diseases are discussed.

Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients.

B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.

Immunologic profile of juvenile periodontitis. I. Lymphocyte blastogenesis and the autologous mixed lymphocyte response.

Studies of blastogenesis of lymphocytes in culture from juvenile periodontitis (JP) patients have been inconclusive. Experiments demonstrating differences in lymphocyte blastogenesis to preparations of putative periodontopathogens in JP and phytohaemagglutinin (PHA) were performed. Variables in the blastogenesis assay system recently reported were controlled which included using a range of cell and activator concentrations, incubation times of 3, 5 and 7 days, conical-bottomed microtest wells and chemical inhibitors during labeling of DNA which permits accurate assessment of lymphocyte blastogenesis. Using these modified culture conditions, stimulated lymphocytes of localized (LJP) and generalized (GJP) forms of JP patients did not differ significantly from stimulated lymphocytes of healthy subjects in counts incorporated, stimulation index, incubation time, cell concentration or activator dose required for maximal blastogenesis to bacterial preparations of Actinobacillus actinomycetemcomitans (Y-4), Bacteroides gingivalis, Capnocytophaga ochraceus, Fusobacterium nucleatum, Streptococcus sanguis and Treponema denticola. Unstimulated lymphocyte cultures reflecting the autologous mixed lymphocyte response (AMLR) were increased for LJP compared to healthy subjects although not statistically significant. Unstimulated lymphocyte cultures of GJP were decreased compared to healthy subjects and LJP (P less than 0.05). These observations indicate that GJP patients may have abnormalities in T- and B-lymphocyte regulatory mechanisms.

Eubacterium brachy. Reactivity in in vitro bone resorptive bioassay.

Recent studies have demonstrated an association of Eubacterium sp. with the subgingival microflora of patients with chronic periodontitis. One species, Eubacterium brachy, was evaluated to determine the possible mechanisms by which this microorganism may contribute to this disease. An extracellular antigen was identified in the culture supernatant which reacted with antibodies in human sera. This antigen was isolated by methanol precipitation and purified by gel filtration. The purified extracellular antigen was reacted in vitro with 45CaCl2-labeled fetal rat bone in a bone resorptive bioassay. This antigen was shown to have a molecular weight of 170,000, to share a line of identity with a sonicated preparation of E. brachy whole cells and to result in increased 45CaCl2 release from fetal rat bones when cultures were exposed to the purified extracellular antigen at concentrations of 10 to 53 micrograms/ml.

The gingival immune response to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients.

Biologic activity of type I and type II Fusobacterium nucleatum isolates from clinically characterized sites.

Fusobacterium nucleatum is a microorganism commonly cultured from periodontal disease sites. F. nucleatum isolates (120) were obtained from subgingival plaque samples taken from 27 clinically characterized sites using a selective culture medium. All isolates were verified by morphology, Gram-stain reactions, oxygen tolerance and biochemical reactions. A total of eight clinical isolates and two typed strains were used for further evaluation. In this study, there was no relationship found between GI and probing depth or between probing depth and frequency of isolation of Type I or Type II F. nucleatum colonies. There was a significant increase in isolation of Type II colonies with a GI of 2 (P less than 0.0001). All isolates tested shared lines of identity by double diffusion in agar and displayed similar ability to hemagglutinate sheep erythrocytes and a reduction in this hemagglutination activity by previous exposure to 50 mM D-galactose. All isolates tested showed similar protein patterns as determined by polyacrylamide gel electrophoresis. By the methods used, no differences were detected between Type I and Type II F. nucleatum; however, there was a statistically significant increase in Type II isolates with increasing levels of gingivitis.

Reaction of human sera from juvenile periodontitis, rapidly progressive periodontitis, and adult periodontitis patients with selected periodontopathogens.

The levels of serum antibody reactive to selected periodontopathogens were determined in 182 clinically characterized patients: 35 healthy control, 50 juvenile periodontitis, 42 adult periodontitis and 55 rapidly progressive periodontitis. Reactive antibody levels were determined using an enzyme-linked immunosorbent assay with whole cell preparations of Bacteroides gingivalis, Capnocytophaga (Bacteroides) ochraceus, Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans (Y-4) serving as antigens. Increased reactivity to B. gingivalis and F. nucleatum was observed in all three disease groups studied while antibody reactive to A. actinomycetemcomitans was increased in juvenile and rapidly progressive periodontitis. Antibody levels reactive to C. ochraceus in healthy subjects did not differ from those observed in any disease patient groups. Possible implications in the etiology and progression of the diseases coupled with environmental changes which occur in the econiche of the periodontal pocket are described.

The ELISA system for measuring antibody reactive to Fusobacterium nucleatum in the sera of patients with chronic periodontitis.

Humoral IgG, IgA and IgM responses to three typed oral strains of F. nucleatum were examined in patients with chronic periodontitis and in individuals with clinically healthy gingiva. Attempts were made to isolate F. nucleatum from either the pathologic gingival crevices or healthy sulci in this study. Higher levels of IgG and IgA reactive to all three strains were observed in the chronic periodontitis patients; however, there were no differences between the IgM levels in the diseased and healthy individuals. Immunodiffusion studies with rabbit antiserum and adsorption studies of the human sera demonstrated a sharing of antigenic determinants by the F. nucleatum strains. F. nucleatum strains were isolated from 14 of the 15 individuals in the study.

A comparison of the reactivity of Eubacterium species with localized and serum immunoglobulins from rapidly progressive and adult periodontitis patients.

Immunoglobulins in sera and in supernatant fluids of explant cultures of diseased gingival tissues from 20 rapidly progressive and 20 adult periodontitis sites were tested by an ELISA assay for reactivity with typed strains of Eubacterium alactolyticum, E. brachy, E. limosum and E. nodatum. Immunoglobulins present in tissue culture fluids from both rapidly progressive and adult periodontitis samples reactive with E. brachy and E. nodatum were significantly greater (P less than 0.05) than those reactive with E. alactolyticum or E. limosum. The titers to E. brachy in tissue culture fluids from adult periodontitis were significantly greater (P less than 0.05) than those from rapidly progressive periodontitis; there was no difference in titers to the other three species. The only significant difference in serum titers was that sera from patients with rapidly progressive periodontitis had significantly greater reactivity to E. alactolyticum than did sera from adult periodontitis patients. These data indicate that immunoglobulins in the sera of rapidly progressive and adult periodontitis patients do not necessarily reflect the reactivity of localized immunoglobulins present in the diseased gingival tissue explant culture fluids from these patients.