Heat resistance of Pectinatus sp., a beer spoilage anaerobic bacterium

D. WATIER, I. LEGUERINEL, J.P. HORNEZ, I. CHOWDHURY AND H.C. DUBOURGUIER. 1995. The heat resistance of three strains of Pectinatus (Pectinatus cerevisiiphilus DSM 20466, Pectinatus sp. DSM 20465 and Pectinatus frisingensis ATCC 33332) were measured at different temperatures (50–60°C), different pH (4–6·3), in two media (MRS and wort). Similar D ₆₀ values were obtained for DSM 20465 and ATCC 33332, but DSM 20466 was more resistant in MRS. Moreover the z values were higher in MRS than in wort for the three strains. Thermal destruction at different pH was variable. Results were discussed in relation to factors affecting heat resistance.     

Identification of Streptococcus Agalactiae by the Fluorescent Antibody

A fluorescent antibody technic for identification of Streptococcus agalactiae is described. Smears from colonies on blood agar plates were tested. A pool of conjugates to four different Streptococcus agalactiae antisera stained all the 80 Streptococcus agalactiae strains investigated. The pool proved superior to individual conjugates. Also strains of groups C and G were stained by the Streptococcus agalactiae conjugates. These, however, could be differentiated from Streptococcus agalactiae strains by examination of the controls because the conjugates of antisera to some group C strains stained group C and G strains but not Streptococcus agalactiae strains.     

Antigenic Relationships Among the Proteolytic and Nonproteolytic Strains of Clostridium botulinum

Relationships of the somatic antigens among Clostridium botulinum strains have been investigated by tube agglutination and agglutinin absorption tests. Results revealed a relationship by which strains of C. botulinum are grouped by their proteolytic capacity rather than by the type of specific toxin produced. Thus, C. botulinum type E and its nontoxigenic variants, which are nonproteolytic, share common somatic antigens with the nonproteolytic strains of types B and F. Absorption of antiserum of a strain of any one type with antigen of any of the others removes the antibody to all three types. In the same manner, C. botulinum type A shares somatic antigens with the proteolytic strains of types B and F, and absorption of any one antiserum with an antigen of either of the other two types removes the antibody to all three types. Partial cross-agglutination of C. sporogenes, C. tetani, and C. histolyticum with the somatic antisera of the proteolytic group was also observed.     

The Use of Standardized Reference Antigens in the Serogrouping of Streptococci

Streptococci isolated from the dental plaque of five animal species were identified by physiological and serological methods. Twenty-nine strains of streptococci considered to resemble Streptococcus mitior or Strep. bovis on the basis of physiological data reacted with Lancefield group B or group K antisera respectively in tube precipitation tests. Further serological studies with standardized antigens from known serogroup B and K streptococci revealed that only three of these 29 isolates had been serogrouped accurately and carried the appropriate group antigen. Comparisons were made between the reactivity of the antisera produced by Difco and Wellcome Reagents with acid and autoclaved extracts of the strains. It was shown that the accuracy of serogrouping such isolates could be improved if the tests were made in a gel diffusion system that included a reference antigen.     

Genetic Analysis of Adult-Specific Surface Antigenic Differences between Varieties of the Nematode Caenorhabditis elegans

We have studied developmental stage-specificity and genetic specification of surface antigens in the nematode Caenorhabditis elegans. Rabbit antisera directed against the adult C. elegans cuticle were used in conjunction with antiserum adsorption experiments to obtain antibody reagents with specificity for the adult surface. Adult-specific antibodies were used to identify several varietal strains of C. elegans that display antigen-negative phenotypes as adults. Genetic mapping results using the surface antigen phenotype as a marker indicated that a single gene (designated srf-1) or cluster of genes on linkage group II determines the adult surface antigen phenotype.     

Solid Phase Radioimmunoassay for Identification of Herpesvirus hominis Types 1 and 2 from Clinical Materials

A solid phase radioimmunoassay (RIA) was developed for typing Herpesvirus hominis (HVH) strains isolated from clinical materials, and it also proved to be applicable to the direct detection and typing of HVH antigen in human and animal brain tissue. The procedure utilized virus-infected human fetal diploid cells or brain tissue smears in the bottom of 1-dram glass vials, antigen was detected through the use of intermediate HVH antisera produced in rabbits or hamsters and cross-absorbed with the HVH heterotype, and (125)I-labeled anti-species (rabbit or hamster) globulins produced in goats were used for detection of immune complexes. The cross-absorbed HVH antisera could be used at high dilutions in the RIA test, and they reacted with marked type-specificity in the RIA system. Specificity of the test was also improved by determining and using optimal concentrations of intermediate sera and of (125)I-labeled anti-species globulins. Results of typing HVH isolates by the RIA procedure agreed in all instances with those obtained by direct fluorescent antibody staining with cross-absorbed conjugates. The RIA procedure was effective and more sensitive than direct fluorescent antibody for demonstrating and typing HVH antigen directly in smears of infected human brain tissue.     

Extended serotyping scheme forVibrio cholerae

Fifty-seven new O serogroups have been added to the existing serotyping scheme ofVibrio cholerae to extend the scheme from O84 to O140. Prominent new additions were serogroups O139 and O140. The reference strain of O139 was isolated from a patient from an epidemic of cholera-like diarrhea in Madras, Southern India. Serogroup O140 was assigned to a group ofV. cholerae strains which were tentatively named as the Hakata serogroup and which possessed the C (Inaba) factor but not the B (Ogawa) nor the A (major specific antigen of O1 serogroup ofV. cholerae). As all antisera against reference strains ofV. cholerae contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera must be absorbed with the reference rough strain, CA385.     

Serological properties of Abiotrophia and Granulicatella species (nutritionally variant streptococci)

Serological variations were examined among 12 type or reference strains and 91 oral isolates of vitamin B6-dependent Abiotrophia and Granulicatella spp. Rabbits were immunized with whole cells of 12 selected strains and 10 typing antisera were obtained, which were unreactive with the Lancefield group A to G antigen preparations. The reactivity of the antisera and autoclaved cell surface antigen extracts was tested by double diffusion in agar gel and a capillary precipitin test. These typing antisera categorized all Abiotrophia defectiva strains, all except one Granulicatella elegans strain, three-quarters of the Granulicatella adiacens, and half of the Granulicatella paraadiacens into 8 serotypes and 2 subserotypes. The Granulicatella balaenopterae type strain was unserotypable. All A. defectiva strains were serotype I, some of which were divided into subserotype I-1 and/or I-5. The G. adiacens strains generally belonged to serotype II or III, and the G. paraadiacens strains to serotype IV, V or VI. All G. adiacens or G. paraadiacens serotype II strains were also subserotype I-5. The G. elegans strains were serotype VII or VIII. These Abiotrophia and Granulicatella serotypes were undetectable among 33 strains of the other 11 species including the bacteriolytic enzyme-producing but vitamin B6-independent strains of Streptococcus, Enterococcus, Dolosigranulum and Aerococcus. The proposed serotyping system for Abiotrophia and Granulicatella spp. would be helpful in the identification and classification of these unique coccal isolates in ecological and epidemiological studies.     

Production of Multivalent Fluorescent Antisera for Identification of Organisms in the Mycobacterium avium-Mycobacterium intracellulare Complex

Antisera to ten strains of mycobacteria in the Mycobacterium avium-Mycobacterium intracellulare group were obtained by injecting rabbits with ultraviolet light-killed cells. The antisera were conjugated with fluorescein isothiocyanate and used in the direct fluorescent antibody test. Individual antisera reacted specifically with the mycobacterial serotype used to produce them. The antisera were then combined in two multivalent pools. Each multivalent pool reacted specifically with its corresponding antigens. The multivalent antisera were thus found to provide a rapid identification method for the mycobacteria studied.     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serogrouping of Rhodococcus equi

The serological relationships among 27 isolates of Rhodococcus equi selected from a total of 1,195 isolates were investigated by cross-agglutination and absorption tests. The presence of capsular material was demonstrated in all the 27 isolates by electron microscopic observation. Antisera were prepared by employing formalized antigen of each isolate. In the cross-agglutination test with formalized antigen, 13 antisera reacted with homologous antigens alone, but the remaining 14 antisera reacted not only with homologous antigens but also with one to four heterologous antigens. When these 14 antisera possessing heterologous agglutinins were absorbed with each of the cross-reacting antigens, 14 specific antisera were obtained. The cross-agglutination test with these 27 antisera proved the 27 strains examined to be serologically distinct from one another. These strains were designated serogroups 1 to 27. Thus the same number of grouping antisera were prepared. The distribution of each serogroup among the 1,195 isolates and 15 reference strains was investigated by the slide agglutination test. All the strains were found to be groupable. Most of them belonged to serogroups 1 to 4, 7 to 9, 11, 14, and 15. Of the serogroups designated, 4, 16, 2, 12, 21, 1, and 9 were identical with Prescott's serovars 1, 2, 3, 4, 5, 6, and 7, respectively.     

Evidence for the presence of a K antigen in strains ofVibrio anguillarum

ElevenVibrio anguillarum O group 1 strains, isolated from different species of diseased fish, were selected for an immunoelectrophoretic study. Antigenic preparations for immunoelectrophoresis were simple water extracts boiled for 1 h. Using O and OK antisera, immunoelectrophoretic patterns suggested the presence of a K antigen; there is evidence that examined strains possess a common K antigen.     

Isolation and partial characterization of a genus common antigen and species specific antigen of Capnocytophaga

Sixteen strains of Capnocytophaga were isolated from the pocket of a localized juvenile periodontitis patient. These strains were divided into four groups on the basis of morphological and physiological traits. Strains from group I and group III were identified as C. ochracea and group II as C. sputigena. An antigen common to genus Capnocytophaga was purified utilizing immunoabsorbent chromatography from lysates obtained by sodium dodecyl sulfate treatment of C. ochracea strain S1. An antigen specific to C. ochracea was prepared by sequential gel filtration and preparative isoelectric focusing. The genus common and species specific antigens isolated were immunologically unique and pure when tested by immunoelectrophoresis and immunodiffusion against rabbit antisera prepared to Capnocytophaga and other gram-negative rods. The genus common antigen was susceptible to trypsin and pronase digestion, was soluble in chloroform-methanol, but was unaltered by ribonuclease and deoxyribonuclease treatments and periodate oxidation. Antigenicity of the species specific antigen was destroyed by periodate oxidation. The genus common antigen appeared to be lipid-associated protein, while the species specific antigen consisted mainly of carbohydrate. These specific immunological reagents would be valuable in diagnosing and monitoring diseases.     

Indirect Fluorescent-Antibody Method for the Identification of Corynebacterium vaginale

The indirect fluorescent-antibody technique was employed in an attempt to develop a rapid method of identification of Corynebacterium vaginale. Six reference strains and ten clinical isolates selected on the basis of morphology and conventional biochemical tests were compared. Antisera were prepared in rabbits against the six reference strains. The most satisfactory antiserum was that prepared using strain 14018 grown diphasically (14018 Di) as the antigen. Certain of the antisera did exhibit a cross-reacting titer when reacted against Corynebacterium diptheriae, Corynebacterium xerosis, or Lactobacillus acidophilus. However, antisera adsorbed with these bacteria did not exhibit a significant decrease in titer when reacted against the homologous strain. Various other species of Corynebacterium as well as species of Nocardia, Actinomyces, Hemophilus, and Streptococcus did not fluoresce with the antisera. A specific antiserum was prepared by adsorbing anti-14018 Di with L. acidophilus. The adsorption removed the cross-reacting antibody but did not affect the staining reaction with C. vaginale strains. All reference strains and clinical isolates characterized as C. vaginale gave a definite positive reaction with the adsorbed anti-14018 Di. The specificity of the reactions was assessed by adsorbing the antiserum with the homologous strain. The data suggest that the indirect staining method will be of value in the rapid presumptive identification of C. vaginale.     

Protection against Pseudomonas aeruginosa infection by passive transfer of anti-flagellar serum

The specificity of adsorbed flagellar antisera for H-antigen was demonstrated in vitro by cross-agglutination assays, motility inhibition, and an ELISA. The specific flagellar antibody was determined to be an IgG. Complete protection against burn wound sepsis was achieved with flagellar antisera. Cross-protection experiments revealed that protection was not only H-antigen dependent, but specific for the flagella antigen type. Antiserum raised against b-type flagella would only protect against homologous bacterial challenge and not against a-type flagellated strains. Results using a-type antisera were consistent, giving protection only against the homologous strain. In contrast, protective capacity was selectively removed from antisera by adsorbing with Fla+ cells. Bacteria colonized the burn wounds of passively protected mice to similar levels as seen in nonprotected animals, but the colonization remained localized and did not result in systemic infection, a pattern similar to infections with motility mutants observed in other studies. Animals rendered neutropenic prior to burning were not protected with flagellar antisera. These data suggested a role for phagocytic cells in protection. Immobilization by flagellar antiserum was observed both by microscopic studies and by inhibition of colony spreading. Antiflagellar antibody is hypothesized as exerting its protective capacity possibly in two ways; first by inhibiting the motility of invading bacteria by binding to the flagellum and immobilizing the bacteria, and secondly by acting as an opsonin, targeting either immobilized or mobile cells for phagocytosis.     

Common Antigenic Determinant in a Rumen Organism and in Salmonellae Containing the Antigen O4

Nineteen of 28 strains of rumen organisms isolated from a cow on a high roughage diet and identified morphologically as butyrivibrios, reacted to a low agglutinin titer with salmonella antisera, forming five groups. However only one strain reacted with polyvalent O salmonella antiserum. This strain reacted with O4 factor serum and with antisera to Salmonella strains containing the antigen O4, and agglutinin absorption tests showed the presence of an antigen identical to O4. When 16 further strains of butyrivibrio-like rumen organisms isolated from three cows and one steer were examined, one possessed an antigen similar to but not identical with the antigen O9, and two strains reacted with specific O6,7 factor serum but were not examined further. These four strains were presumptively identified by physiological tests as butyrivibrios. The possible site of antigenic stimulation by such organisms is discussed.     

Beta haemolytic streptococci of the Lancefield groups E. P and U:Streptococcus infrequens

The physiological and biochemical characteristics of isolates from swine in Sweden and The Netherlands were compared with those of strains from several culture collections. These characteristics were found to be similar for all three Lancefield groups and they form a well defined pattern distinct from other known streptococcal species. It is suggested that these streptococci be classified together in one species:Streptococcus infrequens.Group and type sera of Group E streptococci have no affinity to Group P and Group U streptococci, and vice versa. None of the sera prepared against group E type strains contains the group antibody. Group P and Group U streptococci have an antigen in common. This common antigen is present in formamide extracts. It is not demonstrable in acid extracts. None of the group P sera tested contains the common antibody. Group P serum has to be considered as a type serum. Group U sera contain the common antibody, and when absorbed with group P cells prove to contain another type antibody, which reacts with extracts of most group U strains.Isolates of all three Lancefield groups were obtained from a variety of pathological conditions in swine.     

Serologic differences in strains of Sporothrix schenckii.

To obtain evidence that Sporothrix scheneckii enters the body by contact with contaminated materials, the antigenic property of strains from different sources was investigated. The reciprocal absorption test of the antisera against a soil isolate and a human isolate (KO 4606) showed that the absorbed antisera against KO 4606 possessed unique antigen(s) in addition to the common antigen of both strains. Twenty-three clinical isolates were tested with absorbed antisera. Not all of them possessed the unique antigen(s), but there were serologic varieties among S. schenckii strains, regardless of their sources, clinical type of the disease and the morphology of the yeast phase cells.     

Molecular size variation of the hemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis

The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.