Protection induced in mice vaccinated with recombinant collagen-binding protein (CnBP) and alpha-toxoid against intramammary infection with Staphylococcus aureus.

Mice vaccinated with a combination of two Staphylococcus aureus antigens consisting of a recombinant collagen-binding protein (CnBP) and alpha-toxoid (alpha-toxoid) were significantly protected from intramammary challenge infection with S. aureus. The average number of bacteria recovered from the glands of mice vaccinated with the combination of CnBP/alpha-toxoid was significantly lower compared to the average number of bacteria recovered from the glands of mice vaccinated with only CnBP or alpha-toxoid or controls (P< or =0.01). Histopathological examination of mammary glands of mice vaccinated with CnBP together with alpha-toxoid showed no pathological changes, whereas glands of mice vaccinated with CnBP or alpha-toxoid alone developed severe mastitis and showed both focal and disseminated necrosis.

Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction.

A heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of the monoclonal antibodies F4, F5, and LT1 directed against mouse polyomavirus large T-antigen. Phage selected by biopanning was cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay. In phage reacting with the F5 antibody the deduced amino acid sequence of the displayed peptides corresponded to a segment of large T-antigen. In phage reacting with the antibodies F4 and LT1, no such similarity was observed. The kinetics of phage particle-monoclonal antibody complex formation and dissociation was analyzed in an optical biosensor instrument. Sensor chips of standard quality were useful for binding analysis of T7 phage in crude lysates of infected Escherichia coli. We synthesized peptides corresponding to selected consensus sequences and showed by biosensor analysis that these peptides (linear NH3-CPNSLTPADPTMDY-COOH and NH3-NSLTPCNNKPSNRC-COOH with an intramolecular S--S bridge) were able to compete with large T-antigen in binding to the corresponding antibodies (LT1 and F4). These synthetic peptides were also used for gentle and specific dissociation of large T-antigen-antibody complexes. The results demonstrate the potential of phage T7 for display of peptides and for rapid analysis of interactions of these peptides with ligands.

Binding of Galanthus nivalis lectin to Chlamydia trachomatis and inhibition of in vitro infection.

A glycoprotein present in Chlamydia trachomatis, serotype L1, elementary bodies (EBs) was earlier found to bind the lectin from Galanthus nivalis (GNA). In the present paper we investigate the interaction of GNA with chlamydial EBs and its effect on in vitro infectivity. The binding affinity was studied with 125I-GNA lectin. Within 15 min about 80% maximal binding was obtained. The chlamydia-GNA interaction was inhibited by alpha-methylmannoside, causing a decrease of about 50% at 1 mM. Curve fit analyses indicated two types of binding sites for GNA on the EBs. The affinity to these differed by a factor of 15. The influence of the lectin on the ability of C. trachomatis to infect McCoy cells was also investigated. There was a GNA-dependent inhibition with a 50% reduction in the number of intracellular inclusions at 0.2 microM of the lectin. The findings indicate the presence of terminal mannose structures on the chlamydial surface at or in the proximity of the cell-binding domains. Mannose-binding proteins of eukaryotic cells could be important for the initial uptake of EBs.

Vaginal microbiological flora and sexually transmitted diseases in women with recurrent or current vulvovaginal candidiasis.

A history of recurrent vulvovaginal candidiasis (RVVC) was reported by 102 women, while current vulvovaginal candidiasis (VVC) was diagnosed in 83 of the same 996 women. They had all attended two family planning and one youth clinic, respectively. Two women, without RVVC or VVC, matched for age for each case of RVVC, were selected as a comparison group (COMP). Recurrent, but not current VVC, was associated with a history of sexually transmitted disease. Those with current, but not with recurrent, VVC had significantly more often genital warts and bacteriuria (> 10(5) bacteria/ml), but significantly less often bacterial vaginosis than the COMP women. Both VVC and RVVC were inversely correlated to a vaginal flora change with a mixed anaerobic vaginal flora. Those with VVC had a greater number of lactobacilli on vaginal cultures, than those with RVVC and the women in the COMP group. VVC and a history of RVVC both occurred more frequently in women with a lactobacilli-predominated vaginal flora, as compared with those with a flora change with a mixture of anaerobic and facultative anaerobic bacteria.

Culture and wet smear microscopy in the diagnosis of low-symptomatic vulvovaginal candidosis.

OBJECTIVES: To compare the clinical usefulness of culture and wet smear microscopy in low-symptomatic vulvovaginal candidosis (VVC) diagnosis. STUDY DESIGN: Women attending for contraceptive advice were screened for vaginal yeast fungi by culture and wet smear microscopy. A positive culture was found in 130 (13.2%) of the 983 women studied, while a positive wet smear was found in 133 (13.9%). In 40 (30%) of these women both the culture and wet smear was positive. RESULTS: The methods were equally sensitive in predicting symptoms of VVC, such as pruritus, smarting and burning pain, as well as for dyspareunia (35% vs. 36%), but wet smear microscopy was more sensitive in predicting signs of VVC, such as erythema and abnormal discharge (52% vs. 34%). The highest sensitivity was reached when both methods were positive (60% for symptoms, 75% for signs). There was no quantitative correlation between number of Candida colonies on culture on the one hand and symptoms, signs or a positive wet smear on the other hand. Using four parameters as a diagnostic battery for VVC, the two methods complemented each other. The correlation between symptoms and/or signs for wet smear was high than for culture. CONCLUSION: Wet smear microscopy of vaginal secretion, along with signs found at examination, should be the first-line test in the diagnosis of VVC. Culture must, however, be used when there is a clinical suspicion of VVC and a negative wet smear, or when speciation or antibiotic susceptibility tests of isolates are required.

Comparison of polymerase chain reaction, direct immunofluorescence, cell culture and enzyme immunoassay for the detection of Chlamydia psittaci in bull semen.

The polymerase chain reaction (PCR), direct immunofluorescence (DIF; JMAGEN Chlamydia, DAKO Diagnostics, UK), cell culture (CC) and enzyme immunoassay (EIA; Syva Micro Trak) were evaluated for detection of Chlamydia psittaci in bull semen. Three specimens were collected from each of 47 bulls at 3-6 month intervals (134 samples). Judging by the number of samples tested (n = 134), PCR showed a sensitivity of 90.9%, DIF of 93.9%, CC of 72.7% and EIA of 81.8%. PCR, DIF, CC and EIA were 100% specific, respectively. Of the 47 bulls the maximum number of chlamydia-positive animals (n = 14) was revealed when repeated tests were made by PCR. PCR detected 21.4% more positives than DIF and CC and 35.7% more than EIA. Although CC was less sensitive judging by the number of samples tested, it was as sensitive as DIF (78.6%) when judged by the number of bulls investigated. All bulls found to be chlamydia-positive remained so throughout the investigation, which lasted 18 months.

Binding of type-I collagen to Chlamydia trachomatis.

Denatured type-I collagen was found to bind to Chlamydia trachomatis elementary bodies (EBs) in a time-dependent and specific manner. Specificity was tested by having a large excess of other proteins present in the binding mixture. Only denatured type-I collagen efficiently competed for binding. Radiolabelled fibronectin did not bind under the test conditions used. The binding was temperature-dependent and the interaction increased at the melting temperature of the collagen. Evidence was found for two binding sites: one with high affinity (Kd 3.3 x 10(-9)) and one with low affinity (Kd 1.7 x 10(-7)), with an estimated number of binding sites per EB of 590 and 2900, respectively. The interaction between C. trachomatis and collagen may also be relevant in vivo, since 50% binding occurred at 37 degrees C. The binding to denatured collagen may be of importance for the development of sexually acquired reactive arthritis.

Reactivity of antibodies to heteroclitic peptides based on the Chlamydia trachomatis major outer-membrane protein.

One problem of peptide vaccines is that antibodies generated against them react poorly with the target sequence on the native protein. Using monoclonal antibodies (mAbs) to the serovar L1 type-specific epitope on the major outer-membrane protein of Chlamydia trachomatis as our model in conjunction with the Pin Technology Epitope Scanning technique, we had previously identified the critical binding site at this epitope as DAVP. Amino acid substitution showed that AV were essential residues for binding. A series of structurally related (heteroclitic) peptides retaining AV were synthesized. Some of these were found to be much more reactive with the model mAb than peptides of cognate sequence. It was hypothesized that the DAVP peptide only approximated to the conformation of the homologous sequence in the native protein, whereas some of the flexible heteroclitic peptides produced conformations which more closely resembled the native constrained sequence. The key question was whether the most reactive heteroclitic peptide would also generate antibody capable of more efficient binding to the native protein. We therefore immunized mice with one of six heteroclitic peptides or one of two native sequence control peptides. The reactivity of these antisera with the peptide immunogens and with native chlamydial elementary bodies was then evaluated by enzyme immunoassay. Pooled antisera to two of the heteroclitic peptides reacted with significantly greater absorbance (P < 0.05) and at higher dilution with whole chlamydiae than did pooled antisera to the control peptides. This suggests that heteroclitic peptides may in some circumstances be useful to increase the reactivity of site-specific antibodies with epitopes on the native protein important for vaccine development or for serodiagnosis.

Antichlamydial activity of lacrimal fluid in patients with trachoma.

The effect of lacrimal fluid on the growth of Chlamydia trachomatis in cycloheximide-treated McCoy cells was studied in 172 persons with active trachoma (Group A), in 54 with scarring trachoma (Group B) and in 40 healthy subjects (Group C). The patients in groups A and B were treated with tetracycline eye ointment for 4-6 weeks after which tears were collected for retesting. Pooled lacrimal fluid from patients with active trachoma, collected before treatment, had a higher antichlamydial activity compared with healthy individuals. No reduction of the chlamydial inclusion count was seen with such fluid from patients with scarring trachoma. After tetracycline treatment, patients with active trachoma had a slight decrease in their inhibitory activity. In patients with scarring trachoma, the treatment did not significantly reduce the inclusion count. Antichlamydial antibodies were detected more often in patients with active trachoma than in patients with scarring trachoma, while the healthy individuals had no such antibodies. Ultrafiltered and nonfiltered lacrimal fluids were equally effective in inhibiting C. trachomatis inclusion-formation. The inhibitory principle had a molecular weight of less than 10,000 Da.

In vivo-like antigenic surface properties of Staphylococcus aureus from bovine mastitis induced upon growth in milk whey.

Antigenic surface properties of Staphylococcus aureus strains grown in milk whey were compared with TSB-grown bacteria using immuno-gold electron microscopy. It is shown that colloidal gold (CG) particles coated with polyclonal antibody raised against Staphylococcus aureus surface antigen expressed in vivo bound to the surface of S. aureus strain F1440 grown in milk whey, but not to homologous bacteria grown in TSB. S. aureus strains grown in milk whey agglutinated in the presence of the polyclonal antibody, whereas the corresponding bacteria grown in TSB did not agglutinate. Immuno-gold particles did not bind to milk whey-grown bacteria treated with periodate. Periodate-treated milk whey-grown bacteria did not agglutinate in the presence of the polyclonal antibody, whereas periodate treatment had no effect on TSB-grown bacteria.

Adhesion of Staphylococcus aureus to bovine mammary epithelial cells induced by growth in milk whey.

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey-grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB-grown bacteria had no effect on adhesion to the mammary gland epithelial cells.

Characterization of glycoproteins from Chlamydia trachomatis using lectins.

Glycoproteins in Chlamydia trachomatis, serotype L1, elementary bodies were studied by lectin blotting. A panel of 23 lectins representing a variety of sugar specificities was used. The pattern of lectin-binding specificities at a peptide band was studied in order to determine the type and structure of its glycoconjugate. To establish chlamydial origin of the glycopeptide bands in the blot, control samples from non-infected host cell membranes were run in parallel. Terminal mannosidic structures were demonstrated in a 72 kDa glycopeptide (gp72) by its selective binding of Galanthus nivalis lectin (GNA). Sialic acids were found in two chlamydial glycopeptides, gp40 and gp64, which appear to carry O-linked glycoconjugates as they bound the peanut agglutinin (PNA, both gp40 and gp64) and jackfruit lectin (Jac, only gp40). Such structures were also present in other chlamydial glycopeptides. Lectins with specificities for fucose in different links, galactose and N-acetyl glucosamine bound to several chlamydial peptides. On the basis of our results we suggest an alternative mechanism for uptake of chlamydial elementary bodies into host cells, namely phagocytosis mediated by eukaryotic cell surface lectins.

Age-dependent antichlamydial activity of human saliva. A study of infants, children and adults.

Saliva samples from 51 children ranging from 1 month to 12 years of age were studied for their effect on the capacity of Chlamydia trachomatis, serotypes I and L2 elementary bodies (EB), to form inclusions in cycloheximide-treated McCoy cell cultures. The results were compared to those of tests using saliva from adults. No antibodies against C. trachomatis or Chlamydia pneumoniae could be detected by microimmunofluorescence tests in either group. Saliva of children between 1-4 years of age showed an age-related decrease in the chlamydial inclusion count (i.c.). Saliva from children older than 4 years of age, like saliva from healthy adults, showed a pronounced reduction of the i.c. (up to 70%). The study indicates that children between 1-4 years gradually develop a natural antichlamydial activity against C. trachomatis, and above that age they exhibit the same level of antichlamydial activity as adults. The inhibitory activity was heat-resistant.

Fibronectin binding of Lactobacillus species isolated from women with and without bacterial vaginosis.

Lactobacilli isolated from the vaginas of healthy women (39 strains) and from the vaginal discharge of women with bacterial vaginosis (15 strains) were investigated for their binding to 125I-fibronectin. Nine of the 54 strains bound fibronectin at pH 7.2. The binding capacity of these nine strains was about the same as that observed with Staphylococcus aureas Cowan 1. The binding was specific; an excess of unlabelled fibronectin or its amino-terminal 29-kDa fragment effectively competed for binding, whereas bovine serum albumin, human IgG and orosomucoid did not. Incubation of lactobacilli with fibronectin for different periods revealed a time-dependent increase in binding. Lowering the pH to 4.0 increased the binding capacity of all of the lactobacilli tested; binding occurred with strains that had previously failed to bind at pH 7.2. The increased binding of lactobacilli to fibronectin at a low pH may play a role in the maintenance of the ecological balance of the vagina.

Influence of whole human milk, and fractions thereof, on inclusion-formation of Chlamydia trachomatis in McCoy cells.

A study of the effect of human breast milk, and components thereof, on the capacity of Chlamydia trachomatis to form inclusions in cycloheximide-treated McCoy cells, was undertaken. Pooled whole milk collected during the first week of breast feeding caused a concentration-dependent inhibition of the chlamydial inclusion-formation. The activity resided in the fat and fat globule membrane (FGM) components of the milk. The active principle in the FGM fraction is heat-stable and pronase-sensitive, but resistant to both neuraminidase and periodate. Immunoglobulins was not responsible for the inhibition. Whey and casein fractions of milk increased the chlamydial inclusion-formation. The activity of the whey was heat-stable, dose-related, and had a mol. wt. of greater than or equal to 12,000. The casein fraction was still active after heat treatment. Whey samples collected up to 28 days after delivery varied slightly in their stimulatory activity, with an optimum between the 7th and 14th days. The present study demonstrated a multieffect of breast milk on chlamydial inclusion-formation: an inhibitory activity due to a protein compound as well as another factor in the fat fraction and an enhancing effect due to a heat-stable factor(s).

Antichlamydial activity of saliva.

Saliva collected from adults with no antichlamydial antibodies in their serum or saliva, was tested for its capacity to inhibit the formation of inclusions of Chlamydia trachomatis in McCoy cell cultures. Pooled saliva, diluted in tissue culture medium and sterilized by filtration, was found to reduce the inclusion count by up to about 40%. However, the pretreatment of the chlamydial organisms for 2 hours with diluted saliva caused a 75% decrease in the number of inclusions. The inhibitory activity, which was concentration-dependent, seems to affect the attachment of the chlamydial elementary body to the host cell by acting on both the chlamydiae and the McCoy cells. Saliva did not seem to affect the intracellular development of the chlamydiae. The inhibitory activity was not affected by trypsin treatment, while absorbtion with a gel of a chelating agent caused total loss of the antichlamydial effect. The purpose of our study was to test saliva for its possible antichlamydial activity and to partially characterize the active principle.

Virulent Escherichia coli strains for chicks bind fibronectin and type II collagen.

125I-fibronectin and 125I-collagen (type II) binding was detected in Escherichia coli strains isolated from chickens and poults. High fibronectin binding-strains also bind the 29 kD aminoterminal fragment of fibronectin. Binding properties in strain CK28 were partially characterized. The highest binding of 125I-fibronectin and 125I-collagen for strain CK28 was obtained with bacteria grown at 33 degrees C. Binding of 125I-fibronectin, its 125I-29 kD fragment, and 125I-collagen, was very rapid, reaching a maximum in 5 min. Binding of 125I-fibronectin and 125I-collagen was considerably inhibited by preincubation of bacteria with unlabelled fibronectin and unlabelled type I collagen respectively, but not inhibited with human immunoglobulin G or bovine serum albumin. Inhibition experiments showed that the reversibility of 125I-fibronectin binding was estimated at approximately 50%, while reversibility for 125I-collagen binding was higher than 90%. Receptors for fibronectin, its 29 kD fragment, and collagen were released from the bacterial surface by treatment at different temperatures, and surface material released at 100 degrees C inhibited binding. There was cross-inhibition for both fibronectin and collagen binding when unlabelled fibronectin and unlabelled collagen were used as inhibitors, suggesting that binding receptors for both proteins may be closely located.

Antichlamydial activity of tear fluid.

Tear fluid collected from healthy children and adults, was tested for its capacity to inhibit Chlamydia trachomatis, serotype I, to form inclusions in McCoy cell cultures. Pooled tear fluid added to such cultures reduced the chlamydial inclusion count even at concentrations of 1%. The inhibitory activity was concentration-dependent. The chlamydial inhibitory factor has a molecular weight of less than 10,000 dalton and the principle is heat-stable. The antichlamydial factor seems to affect the attachment of the elementary body (EB) to the host cell surface, while no effect on the intracellular development and reproduction of the chlamydiae could be demonstrated. The activity could not be explained by the presence of antichlamydial antibodies.

Interaction of sub-epithelial connective tissue components with Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis.

Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis were examined for their ability to interact with 125I-labelled fibronectin, fibrinogen and type II collagen. Their relative surface hydrophobicity and production of extracellular capsule were also investigated. Almost all S. aureus strains bound fibronectin (mean value 23%), fibrinogen (mean value 12%) and type II collagen (mean value 16%). CNS bound fibronectin (mean value 6%) and type II collagen (mean value 7%), but not fibrinogen (mean value 2%). The specificity of binding of these proteins to S. aureus strain F1440 and to coagulase-negative Staphylococcus chromogenes strain BO52 was studied by adding an excess of unlabelled proteins. Fibronectin and collagen binding were observed to be specific, varying between 50 and 75%, whereas the specificity of fibrinogen binding to S. aureus strain F1440 was lower (26%). Most of the S. aureus strains (63%) showed very high surface hydrophobicity (autoaggregation) or lower hydrophobicity (29% of the strains) and the rest were hydrophilic. None of the CNS strains autoaggregated, 44% were classified as hydrophilic strains. Hydrophilic strains (except the reference strains) did not show extracellular capsule production. However, the encapsulated (reference) strains showed low binding to these proteins as compared to their unencapsulated variants. Pre-treatment of S. aureus strain F1440 and S. chromogenes strain BO52 with trypsin decreased their fibronectin binding capacity and surface hydrophobicity, whereas pre-treatment with bovine milk (except on collagen binding to strain F1440) did not significantly affect binding to these proteins. These data indicate that S. aureus and CNS isolated from bovine udder infection have the ability to bind to tissue matrix and plasma proteins which may be exposed in the traumatized or toxin-damaged udder epithelial lesions.

Adhesion of enterotoxigenic (ETEC) and bovine mastitis Escherichia coli strains to rat embryonic fibroblasts: role of amino-terminal domain of fibronectin in bacterial adhesion.

Adherence of human enterotoxigenic and bovine mastitis Escherichia coli to rat embryonic fibroblasts was studied. Adhesion of E. coli strains B34289c (human) and 1407 (bovine) was rapid and reached maximum after 30-40 min. Strain 1410 (bovine), which binds fibronectin but not its 29K amino-terminal fragment, did not adhere to the fibroblasts. Strain B34289c grown at 25 C or below and at 40 C or above lost its binding and adhesive properties simultaneously. Maximum binding and adhesion for this strain was achieved when it was grown at 33 C. Strains grown at this temperature adsorbed to fibronectin-, 29K fragment-, and Octyl Sepharose, with the exception of bovine strain 1410, which did not adsorb to 29K-Sepharose as expected. None of the strains adsorbed to cross-linked Sepharose 4B. 29K-IgG and Fab fragments thereof specifically blocked both binding (max 55%) and adhesion (greater than 95%). Sonicated and trypsin-treated bacteria were no longer able to bind or adhere. The supernatant of sonicated bacteria inhibited both binding and adhesion. Penicillin G at 0.5 micrograms/ml (1/5 minimal inhibitory concentration: MIC) and tetracycline at 0.2 micrograms/ml (1/5 MIC), when included in the growth medium, suppressed the cell surface components responsible for fibronectin binding and fibroblast adhesion. The presence of fibronectin was demonstrated in the fibroblast extracellular matrix by immunofluorescens with 29K-IgG antibodies.