Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Immunodiffusion method for detection of type A Clostridium botulinum

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Large-Plate Method for the Assay of Neomycin in Serum

A large-plate method employing radial diffusion from small paper discs for assaying serum levels of neomycin is described. More than 120 discs placed on a loading plate and loaded with 20 muliters of sample could all be brought into contact with the agar plate at one time. The requirement for elaborate statistical design to compensate for time-dependent bias was thus eliminated. The dose-response curve was linear for a range of at least 0.5 to 2.5 mug/ml. The experimental limits for the actual zone width (distance from the edge of the paper disc to the outer edge of the inhibition zone) for 120 repetitions of the assay of neomycin in one and the same serum, carried out simultaneously on one large plate, were about +/- 6% (95% confidence interval). The 95% confidence interval for the distribution of difference between duplicate zones obtained for the assay of neomycin in 34 different sera, also carried out on one plate, was about +/- 10%. The dose-response lines for a standard and for three unknown sera, when carried out together on one plate, were parallel within the variability (+/- SD one) of the zone width. The large-plate method is considered to be more efficient than the use of the smaller petri dishes. The method is suitable for the assay of penicillin in serum and can most probably be used for the assay of a wide variety of substances for which radial diffusion from paper discs into agar is feasible.     

A microbiological test system for determining dioxidine levels in biological fluids

A microbiological procedure for determining dioxidine concentrations in biological fluids with using E. coli AB 2472 rec A 16, a reparation deficient strain as a test organism is described. Cell suspension of the strain 24-hour culture is added to 1.2 per cent agar with Hottinger digest (140 mg per cent of amine nitrogen), 3 g/l of disubstituted sodium phosphate and 0.4 per cent of glucose cooled to 50 degrees C. 10 ml of the medium are added to every Petri dish with metallic cylinders put on the agar. After the medium solidification the cylinders are removed and 0.1 ml of the solution being tested is added to every well. The dishes are incubated for 24 hours under anaerobic conditions. The test system sensitivity is 0.2 microgram/ml of dioxidine. The relationship between the growth inhibition zone and the drug concentration is linear within dioxidine concentrations of 0.2 to 3.2 micrograms/ml.     

Production of a monovalent staphylococcal enterotoxic antiserum type A and its use for typing staphylococcal enterotoxins

A monovalent specific staphylococcal antiserum, type A, was obtained by means of the isolated and purified preparation of type A staphylococcal enterotoxin. This antiserum was proved to be identical to antiserum of the same type, manufactured by Serva Feinbiochemica GmbH & Co. (West Germany). The titer of the newly obtained antiserum in the precipitation test was 1 : 16, and its use allowed one to detect enterotoxin of the above-mentioned type at a concentration of 0.004 mg/ml. The study of 320 staphylococcal strains with the use of this antiserum revealed that 25 strains (7.8%) produced type A enterotoxin.     

DNA-binding proteins specified by bacteriophage T1

Abstract A type A Clostridium perfringens enterotoxin was chially purified by ammonium sulfate precipitation (0 to 15%) and was submitted to polyacrylamide gel electrophoresis (7%). A specific enterotoxin antiserum was obtained by inoculating a rabbit with the polyacrylamide gel strip containing the enterotoxin. This serum gave only one precipitin line with purified enterotoxin and cellular extract in immunodiffusion and immunoelectrophoresis. The titer (1:8) in counter-immunoelectrophoresis was sufficient to detect 0.39 μg/ml enterotoxin by this technique. This serum neutralized the mouse lethality, cytotoxicity and plating efficiency of Vero cells.     

Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.     

Purification of pig renin

1. A new method of purification of renin is described. This method employs the following procedures: ethanol precipitation; saline extraction; precipitation of renin with 40% ammonium sulphate; precipitation of impurities with 3% ammonium sulphate at pH2.5; chromatography on DEAE-cellulose and CM-Sephadex; gel filtration on Sephadex G-100 (both normal and superfine grade); finally, starch-gel electrophoresis. 2. The final renin preparation had a specific activity 10(4) times that of the initial saline extract. 3. A single band of stained protein corresponding to the renin activity was present on starch-gel electrophoresis in the final step and a single precipitin line was obtained to this material with rabbit anti-(pig renin) serum. 4. Double diffusion in agar with rabbit anti-(pig renin) serum showed one major precipitin line, probably due to renin-anti-renin complex, and in addition two minor components.     

Monoclonal antibodies with specificity for three different serotypes of Marek's disease viruses in chickens

A total of 50 antibody-secreting hybridoma cells against Marek's disease virus (MDV) and turkey herpesvirus (HVT) have been produced. Eleven hybridomas were used for serotyping a panel of 15 pathogenic and nonpathogenic strains of MDV and HVT, representing three serotypes. The antibodies from the culture medium have fluorescence antibody (FA) titers of up to 100 and those from mouse ascitic fluid have titers ranging from 10(4) to 10(6). Monoclonal antibody T81 is type-common, i.e., it reacts at equal titer with all MDV and HVT tested. Of the remaining 10 antibodies, eight react only with pathogenic and attenuated strains of MDV (presumably serotype 1), one reacts only with nonpathogenic MDV (presumably) serotype 2), and one reacts only with strains of HVT (presumably serotype 3). Two hybridomas belong to IgG2a and IgG2b subclasses, respectively, and the remaining nine belong to IgG1 subclass. None of the antibodies specific for MDV strains reacted with homologous viruses in serum neutralization (SN), agar gel precipitin (AGP), or membrane immunofluorescence tests. Antibody L78, which is specific for HVT, was reactive with its homologous virus in the SN test; antibody from the culture medium showed an SN titer of 10 and that from mouse ascites a titer of 10,000. None of the antibodies specific for MDV or HVT reacted with other avian or mammalian herpesviruses, avian leukosis viruses (ALV), reticuloendotheliosis viruses (REV), or Marek's disease tumor-associated surface antigen (MATSA) expressed in a lymphoblastoid cell line, MDCC-MSB-1.     

Selective and differential medium for detecting Clostridium botulinum.

A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F. The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction. Growth of proteolytic types of C. botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed. Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer. Optimum antibody titer varied with toxin type. The proposed selective differential medium should be valuable in isolating and typing of proteolytic C. botulinum types A, B, and F from samples containing mixed microbial populations.     

Selective and differential medium for detecting Clostridium botulinum

A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F. The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction. Growth of proteolytic types of C. botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed. Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer. Optimum antibody titer varied with toxin type. The proposed selective differential medium should be valuable in isolating and typing of proteolytic C. botulinum types A, B, and F from samples containing mixed microbial populations.     

A consideration to immune doses of staphlococcal enterotoxin B to rabbits

Immune doses of staphylococcal enterotoxin B to rabbits were studied by comparing the efficiency of various immunization schedules. It was experienced that even two subcutaneous injections each with such a small dose as 10 mug of enterotoxin B, the primary one with Freund's complete adjuvant and a booster one without adjuvant, could stimulate a rabbit to develop the antibody to a satisfactorily high titer determined by agar gel diffusion and passive hemagglutination.     

Radial diffusion in gel for micro determination of enzymes. I. Muramidase, alpha-amylase, DNase 1, RNase A, acid phosphatase, and alkaline phosphatase

The principle of radial diffusion in substrate containing agar gel has been applied for the quantitative assessment of several enzymes. Muramidase, alpha-amylase, DNase I, RNase A, acid phosphatase, and alkaline phosphatase have been investigated. Clearing zones in the opalescent agar, staining of the substrate incorporated in the agar, or a colored insoluble hydrolysis product indicate the diffusion zone of the enzyme. A linear relationship was found between the logarithm of the enzyme concentration and the corresponding diameters of the diffusion zones over a wide range. Standard dilutions of the different enzymes are used as reference.     

Immunochemical analysis of a Smith-like antigen isolated from two human strains of Staphylococcus aureus.

A surface antigen consisting of aminoglucuronic acid and N-acetyl-L-alanine was isolated from the culture filtrates of two human strains of Staphylococcus aureus. Double diffusion analysis in agar suggested that the antigen is immunologically similar to the alanyl-aminoglucuronic acid capsule of the Smith strain of S. aureus. Quantitative precipitin inhibition studies indicated that N-acetyl-L-alanine is the immunodominant determinant of the acidic antigen. In addition, conjugates consisting of N-acetyl-L-alanine coupled to bovine serum albumin gave a significant precipitin reaction with anti-staphylococcal serum which is rich in alanyl-aminoglucuronic acid polymer antibodies. Antibodies with N-acetyl-L-alanine specificity were isolated from N-acetyl-L-alanine-Sepharose immunoabsorbent columns. Double diffusion analysis in agar indicated that the eluted antibodies were serologically reactive and belonged to the IgG class of immunoglobulins.     

Immunodiffusion test in avian encephalomyelitis. I. Standardization of procedure and detection of antigen in infected chickens and embryos.

The double immunodiffusion technique was applied to avian encephalomyelitis virus (AEV). Agar gel medium containing such a high concentration of NaCl as 15% was more preferable for highly diluted quantities of reactants than any other NaCl-containing medium. A single precipitin line appeared on the 1st to 7th days of diffusion at room temperature. The specificity of reaction between AEV antigen and homologous immune chicken serum has been demonstrated by no cross reaction between heterologous viruses and specific absorption by homologous virus. The antigen was produced in the brain, viscera, eyeball, whole body and yolk sac of chick embryos inoculated via yolk sac, as well as in the thigh muscles of chicks subcutaneously inoculated at 2 days of age. Antigenicity was detectable in 50% emulsion of these organs with a virus titer more than 10(5.0) per 0.1 g of tissue weight.     

Nonspecific Precipitation of Serum Proteins by Sodium Lauryl Sulfate in Agar Diffusion and Immunoelectrophoresis

The anionic detergent sodium lauryl sulfate (SLS), in a final concentration of 0.1% and greater, reacted with whole serum in agar diffusion and immunoelectrophoresis to form artifactual precipitin lines. These lines occurred when either Ionagar or agarose was used as the supporting gel and were not affected by the presence of urea and 2-mercaptoethanol. Analytic chemical tests confirmed that the precipitating agent is SLS, and staining techniques showed that the detergent precipitates both protein and lipoprotein components of whole serum. Multiple artifactual precipitin lines occurred with a wide variety of animal sera, and a single line formed with human 7S immunoglobulin. Hence, in agar diffusion studies in which SLS is present in the test system, these artifactual lines may be easily misinterpreted as true antigen-antibody precipitin reactions.     

Serum 25-Hydroxyvitamin D Level and Influenza Vaccine Immunogenicity in Children and Adolescents

Background

Vaccination is an important strategy in the prevention of influenza, but immunologic response to vaccination can vary widely. Recent studies have shown an association between serum 25-hydroxyvitamin D (25[OH]D) levels and immune function. The purpose of this study was to determine if serum 25(OH)D level correlates with influenza vaccine immunogenicity in children and adolescents.

Methods

We conducted a prospective cohort study of children age 3 to 15 years of age vaccinated with trivalent influenza vaccine (A/Brisbane/59/2007[H1N1]-like virus, A/Brisbane/10/2007 [H3N2]-like virus and B/Florida/4/2006-like virus) in Hutterite communities in Alberta, Saskatchewan and Manitoba. Serum 25(OH)D levels were measured at baseline and immunogenicity was assessed using hemagluttination inhibition (HAI) titers done at baseline and 3–5 weeks post vaccination. Logistic regression was used to assess the relationship between serum 25(OH)D level as both a continuous and dichotomous variable and seroprotection, seroconversion, fold increase in geometric mean titer (GMT) and post vaccination titer.

Results

A total of 391 children and adolescents were included in the study and 221 (57% had post-vaccination HAI titers. The median serum 25(OH)D level was 61.0 nmol/L (Interquartile range [IQR] 50.0, 71.0). No relationship was found between serum 25(OH)D level and seroprotection (post-vaccination titer ≥40 and ≥320) or seroconversion (post-vaccination titer ≥40 for participants with pre-vaccine titer <10 or four-fold rise in post-vaccination titer for those with a pre-vaccine titer ≥10).

Conclusion

Serum 25(OH)D level was not associated with influenza vaccine immunogenicity in otherwise healthy children and adolescents. Other strategies to enhance influenza vaccine response should continue to be evaluated in this population.The role of serum 25(OH)D level in vaccine responsiveness in other populations, especially those hyporesponsive to influenza vaccination, requires further study.     

Transfection of Lysostaphin-treated Cells of Staphylococcus aureus

After treatment with 1 unit of lysostaphin per ml for 3 min, two strains of Staphylococcus aureus, 233 and PS 44A HJD, were transfected with phenol-extracted deoxyribonucleic acid (DNA) from the staphylococcal bacteriophages, 53 and 44A HJD, respectively. The number of transfected cells was low in both systems, approximately two in 10(7) enzyme-treated cells. There was a saturation effect at high concentrations of DNA; optimal results were obtained at concentrations between 10 to 25 mug/ml. Growth curves and fluctuation tests indicated that cells of strain 44A HJD infected with phage, then converted to protoplasts by a 10-min treatment with lysostaphin, produce only one phage particle and lose their ability to lyse spontaneously in hypertonic media.     

Enhancement of antitoxic immunity in newborn infants by peroral administration of donor antistaphylococcal immunoglobulin

The possibility of enhancing specific immunity by the oral administration of homologous antistaphylococcal immunoglobulin in a dose of 50 I. U./kg b. w. before the first feeding was shown in 75 newborn infants with a high risk of staphylococcal infection. 24 hours after the first administration of Ig the titer of staphylococcal anti-alpha toxin in the blood rose from 0.68 +/- 0.05 I. U./ml to 2.9 +/- 0.14 I. U/ml, on day 7 this titer persisted at the level of 2.86 +/- 0.12 I. U./ml, and 3 months later the titer was 1.5 +/- 0.05 I. U./ml. No side effects were observed. In the reference group (50 infants) antitoxic titers remained low. No suppurative-septic diseases were observed in the test group within 3 months, while in the controls, focal forms of staphylococcal infection (12 cases) and sepsis (1 case) were registered.     

The propagation of a porcine epidemic diarrhea virus in swine cell lines

A strain of porcine epidemic diarrhea virus (PEDV), P-5V, utilized as a live virus vaccine in Japan was infected to a swine cell lines, KSEK6 and IB-RS-2 cells. Clear CPE, characterized by cellular destruction, started to appear in the infected cells on 2-3 days post infection (DPI) and affected cells was completely degenerated on 4 DPI. The virus was serially passaged in the cells even without addition of trypsin. Small but clear plaques were formed under an agar overlay medium on the cells. The infective titer in the order of 10(7.00-7.50) TCID50 per ml was obtained at usual incubation temperature.