Application of the API YeastIdent system in determining the enzymatic profiles of Nocardia asteroides isolates.

Glucose represses production of ammonium in many clinical isolates of Nocardia asteroides growing on bromcresolpurple casein glucose agar. Strains exhibiting this property are designated as group A, while group B represents isolates showing a high degree of proteolytic activity and a resulting rapid increase in pH. Twenty isolates of N. asteroides were characterized as group A or B. Enzymatic profiles obtained using the API YeastIdent system showed significant enzymatic variation between 12 group B and 8 group A isolates. Proteolytic enzymes which most varied in activity between groups were glycine aminopeptidase, histidine aminopeptidase and leucyl glycine aminopeptidase. As some of the N. asteroides isolates were isolated from asymptomatic patients, it is of interest to consider the possibility of one group being of low virulence while the other is more strongly associated with infection.

Rapid detection of infectious mononucleosis-associated heterophile antibodies by a novel immunochromatographic assay and a latex agglutination test.

A novel immunochromatographic assay, the CARDS O.S. MONO test (Pacific Biotech, San Diego, Calif.), and a latex agglutination test, the Infectious Mononucleosis Kit (Unipath Ltd., Hampshire, United Kingdom) were compared with the Paul-Bunnell-Davidsohn test. Of the 957 serum specimens studied, 78 were positive and 879 were negative by the Paul-Bunnell-Davidsohn test. After discrepancies were resolved by determining Epstein-Barr virus serology, the sensitivities of the CARDS O.S. MONO test and the Infectious Mononucleosis Kit were 91.0 and 96.2%, respectively, and both tests had a specificity and a positive predictive value of 100% and a negative predictive value and overall agreement of greater than 99%. The results show that both tests can accurately detect infectious mononucleosis-associated heterophile antibodies.

An autochthonous phaeohyphomycotic nail infection in Canada caused by Hendersonula toruloidea.

A case of an autochthonous toenail infection caused by Hendersonula toruloidea is presented. H. toruloidea was characterized by thick, branching septate brown-walled hyphae of a non-dermatophyte in microscopic tissue mounts in 25% Na0H with 5% glycerol. Cultures grew out on Littman's agar and Sabouraud peptone glucose agar plus chloramphenicol, but only if cycloheximide was not present. In vitro, the H. toruloidea isolate was characterized by copious 1-to 2-celled hyaline to dark-brown arthroconidia deriving from the fragmentation of aerial hyphae. It is likely that more infections due to H. toruloidea will be diagnosed if cycloheximide-free media are routinely used in the isolation of organisms from suspected dermatophyte infections.

Serotype distribution of meningococci isolated in South Australia 1971 through 1980.

The serotypes of meningococci isolated from 76 sporadic cases of meningococcal disease in South Australia during the years 1971 through 1980 were determined. Thirty-four (56%) of the 61 group B strains were nontypable; the remainder were of five serotypes namely 8 (16%), 1 (13%), 2 (2a and 2b) (9%), 12 (3%), and 15 (3%). Four of the five group B type 2 strains were serotype 2b. Serotype 2a accounted for four of seven group C strains and four of five group W135 strains. Overall serotypes 2 (2a and 2b) (17%), 8 (13%), and 1 (10%) occurred most frequently amongst the typable strains, whereas 40 (53%) of the 76 strains were nontypable. The results indicate that several serotypes and many nontypable strains were responsible for the sporadic disease occurring during a 10-year period in Australia.

Adjuvant and immunogenic properties of gonococcal R-type lipopolysaccharide in reaginic antibody formation in mice.

The ability of gonococcal R-type lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in the formation of IgE and IgG1 antibody responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG1 under a variety of experimental conditions. LPS was capable of enhancing IgE and IgG1 antibody responses to gonococcal protein (ZAB), but its adjuvant effect was weaker than that of A1(OH)3. The LPS-induced anti-ZAB IgE antibody titers showed a cycling phenomenon with time, but the IgG1 response was delayed and peaked only once.

Immunogenic and protective properties of meningococcal serotype 2a protein in the hen-embryo model.

The ability of serotype 2 outer membrane protein (SP-2) of Neisseria meningitidis strain M986 serogroup B, serotype 2a (B, 2a) to stimulate antibody formation in hens and to confer protection against meningococcal challenge to embryos from immunised hens was investigated. Hens, housed with roosters, were immunised with 100 micrograms of SP-2 once a week for 5 weeks to maintain consistent levels of serum antibody during the study. Antibodies in sera of hens, yolks and plasma of 13-day-old embryos reacted in enzyme-linked immunosorbent assays and immunodiffusion with outer membrane vesicles (OMV) from serotypes 2a, 2b and 2c and most of the other prototype group B strains of N. meningitidis. Cross-reactivity of hen sera with most OMV appeared after only one injection of SP-2. Embryos from immunised hens were protected against challenge with up to 10,000 LD50 doses of either the homologous strain M986 (B, 2a) or strain M1011 (B, 2a). Protection was also evident against strains 614 (W135, 2a), S5896 (Y, 2c) and 2241 (C, 2a), but not against strain 78704 (C, 2a) despite strong cross-reactivity of antibody with OMV of this strain. Embryos were only partially protected against strain 78069 (B, 2b) and were fully susceptible to strain 2996 (B, 2b). Some protection was also obtained against meningococcal strains M1080 (B, 1), M982 (B,9), S3032 (B, 12), 79001 (B, 12), 79694 (B, 15-related) but not strain 77252 (B, nontypable). These results suggest that proteins extracted from both serotype 2a and 2b meningococci would provide the broadest protection against infection with group B, serotype 2 meningococci and that antibody, presumably directed against common peptides in the major outer membrane proteins, can prevent infection by some other disease-associated serotypes of N. meningitidis.

A new serogroup (L) of Neisseria meningitidis.

A strain of neisseria meningitidis (LCDC 78189) isolated from the mother of a 3-year-old male with meningococcal meningitis was found to be antigenically distinct from the known serogroups A, B, C, D, H, I, K, X, Y, Z, 29E, and W135; it was designated serogroup L. Anti-78189 serum specifically agglutinated the homologous strain and three other strains which were isolated from the father and two other contacts of the child. Only those strains isolated from the contacts produced immunoprecipitates with the anti-78189 serum by the antiserum-agar method. A structurally unique capsular polysaccharide which was obtained from strain 78189 in a highly purified state was demonstrated to be the antigen responsible for the serological properties of the strain. The polysaccharide formed a precipitin band with the anti-78189 serum but not with the meningococcal grouping sera, and it was also able to absorb both the agglutinating and precipitating activity from the anti-78189 serum.

Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979.

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81%) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b, 77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS-PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

Evaluation of two Salmonella typhimurium hybrids as challenge organisms in a system for the assay of typhoid vaccines.

A mouse-virulent Salmonella typhimurium hybrid (H42), which expresses the Salmonella typhi Vi antigen in addition to S. typhi O antigens 9 and 12, and a mouse-virulent S. typhimurium hybrid (H1), which expresses only the 9 and 12 antigens of S. typhi, were compared in their behavior as challenge organisms in a system developed to assay the protective capabilities of typhoid vaccines. Swiss-Webster white mice, vaccinated intraperitoneally with live Escherichia coli hybrids expressing the S. typhi O antigens 9 and 12, were significantly protected against death from intraperitoneal challenge with each of the S. typhimurium hybrid strains. Vaccination with an E. coli hybrid expressing the S. typhi Vi antigen in addition to O antigens 9 and 12 was seen to confer no advantage in protection against either S. typhimurium hybrid challenge organism over that obtained by vaccination with an E. coli hybrid expressing only the O antigens of S. typhi. However, a notable difference in the behavior of the two S. typhimurium hybrids was seen in mice vaccinated with the parent of the E. coli hybrid vaccinating strains, E. coli F464, which expresses no surface antigens common to either of these S. typhimurium hybrid challenge organisms. A nonspecific (with respect to the vaccinating strain) protective effect, believed to be associated with Vi antigen expression by the challenge organism, was seen against the challenge with S. typhimurium hybrid H42 after F464 vaccination, whereas no protection was conferred by F464 vaccination against the challenge with Vi-nonexpressing S. typhimurium hybrid H1. Inasmuch as neither S. typhimurium hybrid discriminates between the expression or nonexpression of the Vi antigen in a vaccinating strain, it is concluded that the Vi-nonexpressing S. typhimurium hybrid H1, which more clearly indicates the vaccine-specific protective role of the S. typhi O antigens and does not exhibit the nonspecific protection response of hybrid H42, is the better choice as challenge organism for this vaccine assay system.

Enzyme-linked immunosorbent assay with polyvalent gonococcal antigen.

An indirect enzyme-linked immunosorbent assay (ELISA) with a mixture of eight different gonococcal outer-membrane proteins (OMP) as coating antigen was evaluated for detection of gonococcal antibody in 507 sera obtained from patients selected from high-risk and low-risk populations. The indirect ELISA method was more specific and sensitive when the polyvalent antigen was used than when OMP from only one serotype was used. Past episodes of the gonorrhoea had a significant influence on the seropositivity of the test. In a selected low-risk population the specificity of the assay was 94% and in a selected high-risk population the sensitivity was 78%. When sera from 24 asymptomatic individuals were tested the sensitivity was 83%. The ELISA polyvalent-antigen test should be useful as an aid for the detection of gonorrhoea in populations with a low prevalence.

Serotypes among Neisseria meningitidis serogroups B and C strains isolated in Canada.

Antisera made to prototype serogroup B strains of Neisseria meningitidis were used to serotype, by agar gel double diffusion, 262 meningococcal serogroups B and C strains isolated in Canada. The strains included 93 from patients and 169 from carriers. Serotype 2 was associated with 39 of 75 (52%) of group B strains and 14 of 18 (77.8%) of group C strains isolated from patients. The group B strains were mainly (87.2%) serotype 2b, while the majority (92.2%) of group C strains was serotype 2a. Other serotypes (including a new provisional serotype) represented 25.3 and 5.5% of groups B and C strains, respectively. The new serotype accounted for 13% of the group B strains. Approximately 23% of the strains isolated from patients were nontypable. The distribution of serotype 2, nontype 2 (other serotypes), and nontypable strains isolated from carriers was 2.1, 36.6, and 61.3%, respectively, for group B meningococci and 22.2, 29.6, and 48.25, respectively, for group C meningococci. Serotype 11 was the most prominent of the strains isolated from carriers. Approximately 7% of all the strains were multiple serotypes. Serotype 2 is an important virulence marker associated with meningococcal groups B and C disease in Canada, with serotypes 2a and 2b being markedly associated with groups C and B meningococcal disease, respectively.

Improved antiserum agar method for the serogroup differentiation of Neisseria meningitidis Y and W135.

Rabbit antisera prepared to meningococcal serogroups Y and W135 strains were compared with horse antisera using the antiserum agar method (ASA) for the serogroup identification of Neisseria meningitidis. Thirty-seven group Y strains formed immunoprecipitates with the Y rabbit serum only, whereas the same Y strains formed immunoprecipitates with both the Y and W135 horse antisera. Forty-seven W135 strains formed specific immunoprecipitates with both the rabbit and horse W135 antisera by ASA. None of the 166 meningococcal isolates, representative of other meningococcal serogroups, formed immunoprecipitates with the groups Y and W135 rabbit or horse antisera. Use of specific Y and W135 rabbit antisera in ASA provides an improved technique for the serogroup differentiation of groups Y and W135 meningococci.

Evaluation of the antiserum agar method for the serogroup identification of Neisseria meningitidis.

The antiserum agar method (ASA), which is based on the formation of immunoprecipitates around bacterial growth on agar containing meningococcal hyperimmune horse serum, was evaluated for serogroup identification of Neisseria meningitidis. Four hundred meningococcal stains were serogrouped by ASA employing horse antisera to serogroups A, B, C, Y, W135, Z, and 29E and compared to serogroup identification by bacterial slide agglutination (BA) employing rabbit antisera. Overall, there was 95% agreement between the two methods. The ASA proved to be more accurate than BA since 15 strains which cross-reacted with Y and W135 rabbit antisera by BA were specifically serogrouped as either Y or W135 by ASA. In addition, 5 out of 75 strains which were ungroupable by BA were serogrouped as either B or 29E by ASA. Repeat serogroup identification of 100 meningococcal strains by ASA provided identical results thus showing the reproducibility of the method. The ASA is advantageous to BA since it is more reliable, utilizes standard antisera which do not have to be absorbed to remove cross-reactions, does not require the preparation of standardized bacterial antigen, and is simple to perform.

Mouse protective capabilities of Escherichia coli hybrids expressing Salmonella typhi antigens.

An Escherichia coli hybrid, F1061, expressing Salmonella typhi somatic antigens 9 and 12, and a derivative of this hybrid, E. coli hybrid WR3078, expressing the S. typhi Vi antigen in addition to somatic antigens 9 and 12, were compared with S. typhi Ty2 in experiments to test their ability, as live vaccines, to protect Swiss white mice against death from challenge with a mouse-virulent Salmonella typhimurium hybrid expressing the S. typhi antigens 9, 12, Vi, and d. When the live, vaccinating organisms were administered intraperitoneally, 87.5% of the mice immunized with S. typhi Ty2 survived challenge, as compared with 62.5% of those immunized with E. coli hybrid F1061 and 55% of those inoculated with E. coli hybrid WR3078. When live organisms were administered orally at a dose of 10(9), 67.5% of the mice immunized with S. typhi Ty2 survived challenge as compared with 47.5% of those immunized with E. coli hybrid F1061 and 40% of those administered E. coli hybrid WR3078. Thus, the protection conferred by E. coli hybrid F1061 expressing only the S. typhi somatic antigens, although significant in this system, was inferior to that conferred by S. typhi Ty2 and the addition of the S. typhi Vi antigen to this hybrid (creating E. coli hybrid WR3078) did not enhance that protection.

Hen fluorescein-labeled gonococcal lipopolysaccharide antibody in the delayed fluorescent antibody technique for the confirmation of Neisseria gonorrhoeae.

A fluorescent antibody reagent (termed anti-LPS conjugate) was prepared from sera obtained from hens immunized with gonococcal R-type lipopolysaccharide. The reagent was absorbed with Formalin-treated cells of Neisseria meningitidis. The anti-LPS conjugate gave uniform brilliant staining of Neisseria gonorrhoeae with little background fluorescence, thus making interpretation and reading of fluorescence simple. The conjugate did not significantly stain cultures of N. meningitidis, Neisseria lactamica, nonpathogenic Neisseria species, or other gram-negative bacteria. Several preparations of the conjugate provided the same specificity and reproducibility of staining. The anti-LPS conjugate was compared with Difco Laboratories fluorescent antibody conjugate for staining of N. gonorrhoeae. Both conjugates stained cells of the light and dark variants of gonococcal colony types 1 and 2, as well as cells of colony types 3 and 4. When used for the confirmation of N. gonorrhoeae, the anti-LPS and Difco conjugates stained 426 of 431 (98.8%) and 210 of 213 (98.6%) of the gonococcal cultures, respectively. Absorption of the anti-LPS conjugate with R-type lipopolysaccharide removed the staining of gonococci. However, absorption of Difco conjugate with R-type lipopolysaccharide did not remove the staining of gonococci, suggesting that the majority of fluorescein-labeled antibody present in the Difco conjugate is directed to gonococcal cell surface components other than lipopolysaccharide. The results of this study indicate that fluorescein-labeled gonococcal lipopolysaccharide antibody should be a reliable fluorescent antibody reagent for the confirmation of N. gonorrhoeae.

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.

Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.

Identification of Neisseria gonorrhoeae from primary cultures by a slide agglutination test.

Hen antigonococcal lipopolysaccharide hen serum was used in a simple slide agglutination test for the identification of Neisseria gonorrhoeae from primary isolates.