Treponema pallidum organisms suspended in equal parts of Nelson's survival medium alone and with addition of various sera were counted in 5 microliters samples before and after 18 hours' anaerobic incubation at 35 degrees C. Samples in which the treponemes were not Poisson-distributed were excluded from the study. A loss of greater than 30% occurred in 230 out of 391 samples. The disappearance of treponemes did not seem to be caused by treponemal agglutination during incubation. There was no adherence of treponemes to the formed elements remaining in the suspension. The loss was not dependent on the concentration of syphilitic serum, nor was it influenced by the presence of immobilizing antibody in the serum. Lack of rigorous anaerobiosis during the manipulations did not influence the treponemal loss. The loss was independent of the density of the treponemal suspensions which did not contain unheated guinea-pig serum (GPS). The relative frequency of the loss exceeding 30% was highest in suspensions containing unheated GPS. However, the extent of loss in individual samples was haphazardly distributed among suspensions of all the compositions examined. The loss of treponemes seems to depend on at least two factors: a) a factor which is eluted together with the treponemes from the syphilitic orchitis and b) the presence of active guinea-pig complement. It is conjectured that the complement is non-immunologically activated and acts through the alternative pathway.
The Ibc protein fraction of group B streptococci was prepared by HCL extraction of the type Ic strains A909 and 335. The fraction from strain A909 contained two protein antigens (alpha A 909 and beta A909) that could be separated by ion-exchange chromatography and isoelectric focusing. The 335 extract contained the alpha (alpha 335)-but not the beta antigen. The alpha 335 antigen was purified by similar procedures. The beta A909 antigen had a molecular weight of several hundred thousands, was immunogenic in rabbits and dissociated into several polypeptides on SDS-PAGE. Polypeptides with sub-unit molecular weights corresponding to 70,000 daltons showed antigenic activity. The alpha 335 antigen had a molecular weight of approximately 75,000 daltons as judged from gel filtration and SDS-PAGE. The antigen was immunogenic in rabbits. In contrast, the alpha A909 antigen showed neither protein lines on SDS-PAGE, nor immunogenicity in rabbits. However, the two alpha antigens showed serological crossreactivity in tests with the anti-alpha 335 serum.
Microsporidian organisms causing great losses among young blue foxes (alopex lagopus) were isolated and propagated in monolayer cell cultures and examined by transmission- and scanning electron microscopy. The parasite was found to fulfil the criteria set up for the genus Encephalitozoon and the ultrastructural findings indicated that the parasite was morphologically identical to Encephalitozoon cuniculi previously isolated from some other mammalian species. Terminal vesicles on the extruded polar filaments seen on scanning electron micrographs were discussed to be similar formation as described for the microsporidian Nosema michaelis.
Uptake of non-opsonized Escherichia coli by mouse peritoneal macrophages (MPM) is influenced by pre-treatment of MPM with homologous interferon preparations. Incubation of MPM with 10(2)-10(3) units of interferon per ml for 18 hours resulted in a 30-70 per cent increase in the phagocytosis rate as well as maximal phagocytic capacity. This effect was time dependent. At least seven hours of interferon pre-treatment and a phagocytosis period of 90 minutes were necessary for a statistically significant increase of the phagocytosis. Treatment of MPM with high concentrations of interferon (i.e. more than 10(4) units per ml) had a depressive effect on the phagocytosis. Both the enhancing and the depressive effects were characterized by the standard physico-chemical properties of interferons. Both effects were species specific. The phagocytosis enhancing effect was neutralized by anit-interferon serum.
Clumping of different staphylococci by fibronectin and other purified plasma proteins has been investigated. Purified fibronectin was capable of clumping Staphylococcus aureus strains in concentrations identical with concentrations of fibronectin in human plasma. S. epidermidis and S saprophyticus were not clumped by fibronectin. The binding of fibronectin to S. aureus was not mediated by protein-A, as a strain lacking protein-A clumped in the presence of fibronectin, and the presence of IgG could not inhibit the clumping of S. aureus strains. The fibronectin-binding component on the staphylococcal cell wall seems to be unrelated to the fibrinogen-binding clumping factor.
A purified polypeptide antigen from Staphylococcus simulans CCM 2705 produced one precipitation line by double diffusion in agar with rabbit antiserum against homologous whole bacteria. The purified antigen did not induce antibody production in rabbits. However, when the antigen was complexed with methylated bovine serum albumin, antibodies with specificity against the polypeptide were produced. The antigen did not sensitize normal or tanned erythrocytes for agglutination in antiserum. The polypeptide antigen induced a primary skin reaction and was toxic for mice. It also induced production of MIF as demonstrated by the migration inhibition test. The polypeptide was found to be a leukotaxigen. No difference between C4 normal and C4 deficient serum was noted. C5 was found to be necessary for the induction of chemotaxis.
The Diffusion‐In‐Gel Enzyme Linked Immunosorbent Assay (DIG‐ELISA) is a new and simple method for the quantitation of antibodies. The prodedure is based on the ability of the antibodies to diffuse from wells in gel in petri dishes and adsorb to an antigen coated to the plastic surface prior to testing. The antigen‐antibody reaction is visualized with horseradish‐peroxidase conjugated class‐specific anti‐immunoglobulins. The DIG‐ELISA permits detection of 0.1 μg/ml of specific antibodies. This method was used to determine the IgG, IgA and IgM antibody levels to Yersinia enterocolitica O:3 in sera from patients with antibody titres to this microorganism, as determined by direct agglutination and complement‐fixation tests. The DIG‐ELISA IgG antibody values, in contrast to IgA and IgM, correlated well to titres obtained by the direct agglutination method. Low degrees of correlations were found for all three immunoglobulin classes when compared to the complement fixation test.
Through hydrophobic interaction, sphingomyelin was adsorbed to agarose beads containing octyl groups by a stepwise dilution procedure. This immobilized lipid was used as a substrate for three bacterial phospholipases C (E.C. 3.1.4.3.). The degradation with time of this substrate showed two different fractions of the substrate according to hydrolysing velocity in the early part of the time-curve when phospholipases C from Bacillus cereus and Clostridium perfringens were used. The early fractions could be predigested by the enzymes, a procedure which resulted in linear time-curves. The corresponding early part of the time-curve for phospholipase C from Staphylococcus aureus was linear, indicating a comparatively large early fraction of the substrate for this enzyme. The stock gel of the immobilized lipid substrate could be stored for months. It was easily and reproducibly handled as a water suspension. After enzymatic hydrolysis the substrate was rapidly separated from enzyme and product by filtration. The enzyme assay presented thus represents a convenient way to avoid the difficulties connected with the use of temporary sonicated suspensions as substrate for bacterial phospholipases C.
Towels from private homes and public offices and underwear contaminated by being used by employees at a public health laboratory were examined for occurrence of radiation-resistant bacteria and fungi. Three different methods were used for isolation of the most resistant organisms, one with multiplication of the microbial population prior to an irradiation used for selection, and two without this multiplication and with the organisms placed on membrane filters or in situ on the textiles, respectively. A total of 44 different strains were isolated. Differences in the three methods used for selection of the most radiation-resistant microorganisms were not reflected in the results. 16 pigment-producing Gram-positive cocci, tentatively classified as Micrococcus radiodurans, were the most radiation-resistant and were isolated in about half of the examinations. Other Gram-positive cocci, non-spore forming rods, some Nocardia and Candida parapsilosis strains and two Bacillus strains constituted the rest of the collection. Wtih few exceptions dose-response curves for the strains were upward convex. D-6 values were determined to be between 1.5 megarad for the most radiation sensitive, a Candida, and 5.7 megarad for the most resistant,, tentatively classified as M. radiodurans. The D-6 values for the Bacillus strains were in both cases 1.8 megarad, consistent with a D-value of 0.3 megarad. The same resistance is reported to be the maximum resistance for B pumilus, strain E601, commonly used as reference strain in the literature on radiation sterilization of medical devices and supplies.
Antimicrobial susceptibility of 180 recent isolates of Streptococcus pneumoniae was determined by microdilution technic. There was a high degree of susceptibility to both penicillin G and cefuroxime, except for one strain which required 0.25 microgram/ml. All strains were inhibited by 0.06 microgram/ml of ampicillin, clindamycin and erythromycin. When tested against doxycycline 97.2% of the strains were inhibited by 1.0 microgram/ml. 8 microgram/ml inhibited all strains. Three of the strains were chloramphenicol-resistant with MIC more than 8 microgram/ml. These strains could be shown to inactivate chloramphenicol. All strains but three were susceptible to 20/l microgram/ml of sulfamethoxazole/trimethoprim.
In this work we have analyzed the bacterial composition of anaerobically cultured cecal contents (mixed broth culture) of adult fowls previously shown able to protect 1 day old chickens from oral Salmonella infections. All four cultures studied in this paper gave complete protection against Salmonella infantis when used undiluted or in the 10(-2) dilution and at least some protection in the 10(-4) dilution. The total aerobic as well as anaerobic counts on the nonselective medium used (VLMH) were of the order of 10(8) viable organisms/ml indicating that the mixed broth culture consisted predominantly of facultative organisms. From a total of 239 colonies isolated on the basis of colony morphology 66 were obligate anaerobes and 173 facultative anaerobic or microaerophilic species. Isolates were selected from each medium. They were grouped and tentatively classified on the basis of their ability to grow on selective media, their colony and cell morphology. Gram stain and products formed from glucose fermentation (obligate anaerobes). Further characterization was performed using conventional carbohydrate fermentation and biochemical tests. However only a minor fraction of the anaerobic isolates could be identified as being identical to known species. The most numerous species were E. coli and various Lactobacilli which were still found in the highest dilution (10(-8)) of the broths. Fecal streptococci were the next most frequent, isolated at the 10(-7) dilution. The obligate anaerobes isolated included Gram-positive cocci and Gram-positive and -negative rods tentatively classified as Eubacteria, Propionibacteria, Clostridia, Fusobacteria and Bacteroides. A large number of them were isolated from the 10(-6) dilution and some species of Eubacteria and Clostridia could only be recovered from the 10(-3) or 10(-4) dilutions.
Various auxiliary treatments of L-cells employed for the isolation and cultivation of C. psittaci were investigated in order to develop an improved method for the detection of the agent, in addition to the aid obtained by centrifugation and cycloheximide treatment. Glucocorticoid treatment increased the observed number of inclusions considerably through a preservative effect on host cells and enhanced an spontaneous re-infection. Besides, the hormone made the scanning of cell culture for inclusions more convenient through an altered cell morphology. This method was tested with two extreme species types that differed as regards cytopathogenicity and growth rate. The length of the cultivation period was of great importance for the diagnostic result. Especially the cytopathogenic agent-type influenced the optimal time of cell culture fixation, which was situated around 48 or 88 hours post infection (h p.i.). Owing to the cytotoxicity of field samples (milk secretion), the cell culture technique (48 h p.i.) was less sensitive compared to the conventional isolation method in embryonated eggs. However, a different sampling technique improved the result, and simultaneous use of the secondary multiplication cycle of chlamydia (88 h p.i.) makes the less cumbersome cell culture technic recommendable.
Minimum inhibitory concentrations (MIC) were determined against 662 recent clinical isolates for eight cephalosporins representing first, second and third generation compounds. All four third-generation cephalosporins tested (cefoperaxone, cefotaxime, ceftazidime and moxalactam) were significantly more active against aerobic gram-negative bacteria than the older compounds (cephalothin, cefamandole, cefoxitin, and cefuroxime). Cefotaxime and moxalactam were most active against Enterobacteriaceae with extremely low MIC-values. Ceftazidime was definitely most active against Pseudomonas aeruginosa with more than 90% of strains inhibited at 4 micro g/ml. MIC-values for cefotaxime against Staphylococcus aureus were for all strains 1-2 micro g/ml, slightly higher for cefoperazone, while the effect of ceftazidime and moxalactam was more limited. All third generation cephalosporins demonstrated efficiency against Streptococcus pyogenes, cefotaxime being most active and moxalactam least active, but were essentially ineffective against Streptococcus faecalis. Moxalactam demonstrated higher activity against Bacteroides fragilis than other second and third generation cephalosporins including cefoxitin. Previous studies have demonstrated a very high activity of all third generation cephalosporins against Haemophilus influenzae and Neisseria gonorrhoeae, including beta-lactamase producing strains.
The virus‐specific protein synthesis of the hamster brain cells persistently infected with a wild‐type measles virus (M‐HB/MVB) was investigated in vivo and in vitro and compared to the protein synthesis of the Vero‐cells lytically infected with the wild‐type measles virus and the SSPE‐measles virus strain LEC. The virus‐specific protein synthesis in the M‐HB/MVB‐cells in vivo was weak. Only two virus‐specific polypeptides, corresponding to polypeptide G and nucleocapsid polypeptide NP, were detected. On the other hand, the in vitro translation products, coded by the RNA extracted from the actinomycin D‐treated M‐HB/MVB‐cells, were similar compared to those products coded by the RNAs from the lytically infected Vero‐cells. The polypeptides with MWs of 75000, 61000, 40000, 37000 and 18000 were coded with all three RNA‐extracts. So the similar mRNAs were induced in the actinomycin D‐treated M‐HB/MVB‐cells, but their translation in vivo was limited. In addition to those polypeptides mentioned above the polypeptide with a MW of 42000 was detected among the in vitro translation products of the M‐HB/MVB‐cells.
Sixty-six strains of R, S, and RS streptococci could be distinguished from other species of streptococci by their ability to ferment inulin and glycogen and to hydrolyze arginine, combined with resistance to optochin. Likewise, they were easily recognizable serologically. The capsular reaction test is the test of choice.
Binding of fibrinogen degradation products was measured to Gram-positive cocci known to carry receptors for human fibrinogen. Forty-one strains of S. aureus and group A, C and G streptococci were studied. The largest plasmin produced fragment (X-fragment, HMWDP) showed higher binding levels to the bacterial receptors in all four species as compared to intact fibrinogens. The enzymatically produced C-terminal fragment D was strongly reactive, whereas the N-terminal fragment E was non-reactive. Inhibition experiments showed that reactive fragments were bound to the same receptors as fibrinogen. Three chemically-produced and well-characterized fragments from the terminal and the middle portions of the fibrinogen molecule were all negative in binding assays. The binding patterns for fibrinogen and the fragments tested were identical in the four bacterial species tested, S. aureus and group A, C and G streptococci. The results confirm and extend earlier data on similarities between fibrinogen receptors on these Gram-positive bacterial species.
A method was developed for the characterization of proteins non-covalently bound to the cell wall of Gram-positive cocci. The method employs radioactive labelling of cell wall proteins followed by solubilisation and analysis on polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Using this experimental procedure, protein patterns obtained from group A, C and G streptococcal strains showed marked similarities within each group. Protein peaks were also found to be shared between group C and G strains. Two major peaks with molecular weights of about 30 000 and 70 000 characterized group A strains, whereas group C and G strains showed one consistent peak of about 45 000, thus reflecting the closer relationship between these two groups as compared to group A streptococci. By incubating bacteria with human serum proteins before labelling, solubilisation and electrophoretic analysis, it was also possible to study external proteins specifically bound to the bacterial surface. A group G streptococcus, strain G 148, showed protein peaks corresponding to its known specific binding of human albumin and immunoglobulin G, but also additional protein peaks. When Staphylococcus aureus, strain Cowan I, was pre-incubated with human serum in excess, protein peaks corresponding to heavy and light chains of immunoglobulins were seen. Three more protein peaks of serum origin were also detected, indicating binding of proteins other than Ig to S. aureus. Experiments with protein A-coated Sepharose beads resulted in the same protein pattern, suggesting that binding of these different polypeptides is indeed mediated by protein A.
Sera taken from a well-mapped epidemic of Salmonella enteritidis gastro-enteritis among school children and teachers have been tested for antibodies of the IgG, IgM and IgA classes by MRPAH (mixed reverse passive antiglobulin haemagglutination) and for IgG and IgM classes by ELISA. When suitable threshold titres were chosen all sera taken up to 10 days after infection were negative by MRPAH, whereas all samples but one taken between day 16 and 48 showed the presence of antibodies of each three immunoglobulin classes. IgG antibody titres often remained elevated one year after infection whereas IgM and particularly IgA antibodies returned to negative. Up to the 28th day the IgG antibody titre showed a logarithmic increase with time, whereas no simple relationship was observed for the IgM and IgA antibody responses. Estimation of the regression equations for the titres measured by ELISA and MRPAH shows agreement between the two methods.
The effect of ampicillin on gonococci was investigated in chambers subcutaneously implanted in rabbits. An intramuscular injection of ampicillin resulted in a rapid increase of the ampicillin concentration in serum, whereas the diffusion of ampicillin into the fluid of a non-infected chamber was comparatively slow. The ampicillin concentration was, however, maintained in the chamber fluid during a prolonged period of time as compared to ampicillin in serum. The concentration profile of ampicillin in the infected chamber was similar to that of the non-infected chamber, though at a lower level. No viable gonococci were detected 120 minutes after the injection of ampicillin. In contrast, the same concentration of ampicillin in a liquid culture resulted in slower reduction in the viability of the gonococcal strain. Even after 300 minutes a small population of gonococci was viable. Thus, a difference in the activity of ampicillin could be observed between the in vitro and the in vivo test situation.
An antigen fraction isolated by various forms of chromatography from phenol/water extracts of Bacteroides fragilis NCTC 9343 was devoid of phosphorus, fatty acids and amino acids. Rhamnose, fucose, galactose, glucose and a neutral sugar tentatively identified as arabinose were present at molar ratios of 1:1:1.2:0.9:0.7. The fraction contained an unidentified reducing and ninhydrin positive substance and trace amounts of glucosamine and galactosamine. The antigen fraction produced three immunoelectrophoretic bands against NCTC 9343 antiserum, and reacted in complement fixation tests.
