Rapid, Direct Tissue Culture Test for Toxigenicity of Corynebacterium Diphtheriae

A method for testing toxigenicity of Corynebacterium diphtheriae in tissue culture is described. The technique, called the colony overlay test (COT), involves inoculating material from an isolated colony of C. diphtheriae to a small area on the surface of an agar medium which overlays a monolayer of toxin-susceptible HeLa cells. If toxin is produced during incubation at 37 C, it diffuses to the tissue monolayer and destroys the cells below the inoculation site. Twenty-four hours after inoculation, organisms are killed and tissue cells are fixed with formaldehyde. The agar overlay is then removed, and the monolayer is stained with crystal violet. Toxin-affected areas fail to stain or stain poorly. A second plate with antitoxin incorporated in the overlay serves as a control for specificity. Forty-eight strains of C. diphtheriae were tested by the COT, guinea pig, and in vitro, gel diffusion tests. The COT is as specific as the other two tests, is easy to read, and can be used to test large numbers of isolates for toxin action more conveniently than by animal inoculation.     

Determination of the toxigenicity of Corynebacterium diphtheriae by using paper indicator disks

The method for the determination of the toxigenicity of C. diphtheriae by means of discs impregnated with diphtheria antitoxin is proposed. The discs can be prepared long before use and stored for a year. The determination of toxigenicity with these discs is not inferior in its sensitivity to the routine procedure of the immunoprecipitation test. The disc method decreases the consumption of unavailable diphtheria antitoxin by 10 times, that of culture media by 2-2.5 times: besides, it is less labor consuming.     

The use of polymerase chain reaction in the diagnosis of diphtheria infection

624 Corynebacterium diphtheriae strains, newly isolated from patients and carriers, were studied with the use of the methods of gel immunodiffusion (Elek's test) and polymerase chain reaction (PCR). In the evaluation of 388 C. diptheriae strains, found to be toxigenic in PCR, the results of Elek's test coincided with those of PCR on 98% of cases. In 38 out of 143 strains (26.5%), nontoxigenic according to the results of Elek's test, the presence of the A-fragment of the tox-gene was established. Subculturing in nutrient media made it possible to determine the presence of toxin in 19 out of 38 of these strains; the remaining strains, isolated mainly from carriers, were found to have the "silent" gene. The advantage of using PCR for the detection of toxigenic and nontoxigenic C. diphtheriae strains of different origin was shown.     

Relative sensitivity of various cell cultures to the thermostable exotoxin of B. thuringiensis]

The "thermostable" B. thuringiensis exotoxin is active on cell cultures of Mammals "in vitro", except on the KB strain from a human tumor. The primary cultures are the most sensitive: first, with monkey kidney cells, the growth is inhibited by 0.1 mg of toxin per ml; next, the young rabbit kidney cells react to 0.25 mg of toxin per ml. The established lines of cells come last: human diploid cells (Lyon 4) and heteroploid cells (BHK21C13), with the same active dose of 1 mg of toxin per ml. No protection is obtained by adding ATP to monkey kidney cells at the same time as the exotoxin.     

Detection of Nuclease Activity in Semisolid and Broth Cultures

A method for the detection of deoxyribonuclease activity in semisolid agar cultures was investigated. When cultures were overlaid with an acridine orangedeoxyribonucleate-agar (ADA) mixture, incubated for 1 to 3 hr, and observed under ultraviolet light, clear halos developed around colonies that produced deoxyribonuclease. A variation of the method for use with broth cultures involved impregnation of filter-paper discs with a small portion of the culture and overlaying the discs with the ADA mixture. This alteration has the advantage that the test tube cultures are easily heat-treated prior to assay to determine the heat resistance of the enzyme.     

Detection of toxin and evaluation of the degree of toxin-formation of Corynebacterium diphtheriae using immunoenzyme analysis

Toxin production and the intensity of toxin formation in 265 C. diphtheriae strains circulating in different areas of the USSR have been studied by the method of the enzyme-linked immunosorbent assay (ELISA). This study has been carried out with the use of the assay system consisting of monoclonal antibodies to the COOH-area of the B-fragment of the toxin molecule adsorbed onto the surface of polystyrene plates, affinity-purified polyclonal antidiphtheria antibodies labeled with horse-radish peroxidase and substrate indicator mixture (5-aminosalicylic acid and hydrogen peroxide). Some specific features of using ELISA for the detection of C. diphtheriae toxin directly in liquid culture medium are presented. High sensitivity, specificity and good reproducibility of this method permitting the detection of C. diphtheriae toxin and the determination of the intensity of toxin formation in the C. diphtheriae strains under study are shown. The method may be recommended for practical use at health institutions.     

Kinetics of the Vaccinia Virus Plaque Neutralization Test

Neutralization of vaccinia virus with immune rabbit serum occurred optimally when incubated at 37 C for 16 to 24 hr in the plaque neutralization test employing the MA-104 embryonic rhesus monkey kidney cell line.     

Pathogenicity of Corynebacterium diphtheriae circulating in Rostov-on-Don City and Rostov region during interepidemic period

Main pathogenic characteristics (toxin production, tox-gene detection, adhesiveness) of 59 strains of C. diphtheriae circulating in Rostov-on-Don city and Rostov region in 2004-2005 were studied. Study of toxigenicity of 15 tox+ C.di phtheriae strains showed full coincidence of Elek immunoprecipitation test and polymerase chain reaction (PCR). Presence of part of A-fragment of tox-gene was detected in 5 (11.4%) of 44 C. diphtheriae strains that were negative in Elek test. Hemagglutinating activity of toxin producing strains was intermediate (40%) or high (60%). Among non-toxigenic strains those with intermediate adhesiveness were predominated (45,5%), the intermediate or high adhesiveness was detected in strains positive in PCR. Obtained characteristics of C. diphtheriae can be useful for surveillance for diphtheria infection during interepidemic period.     

Interaction of Staphylococcal Alpha Toxin and the Estrogenic Hormone Diethylstilbestrol

Previous work in this laboratory showed that diethylstilbestrol was capable of suppressing induced furunculosis in rabbits. The present study indicates that the synthetic estrogenic hormone diethylstilbestrol which is used for acne, estrogen deficiency, cancer, and other disorders, can reduce the cytolytic action of staphylococcal alpha toxin. The cytotoxic action of purified alpha toxin for tissue cultures was evaluated by use of such parameters as total and viable cell counts, glucose, and protein determination, and cytopathic effects (CPE) in the presence and absence of steroids. To 3-day-old primary rabbit baby kidney tissue cultures, 1 to 5 μg of diethylstilbestrol per ml was added; growth of tissue cultures in Eagles medium was continued till the 6th day, and then one tissue cytopathic dose per milliliter of alpha toxin was added, and the subsequent fate of tissue cultures was assayed. Such cultures yielded higher total and viable cell counts, utilized more glucose, and contained more protein than the control cultures. In control cultures, CPE was observed on the 3rd hr after the addition of alpha toxin, and it was complete in 24 hr, whereas in tissue cultures treated with diethylstilbestrol, the CPE was significantly reduced. The data presented in this study made possible the availability of a suppressor of the cytolytic action of alpha toxin and might be useful in assaying the action of alpha toxin in an in vitro inexpensive test system.     

Rubratoxin production by penicillium rubrum when grown in a synthetic medium containing different sources of carbon and nitrogen

A sterile mineral salts broth was fortified with different additives, inoculated with conidia ofPenicillium rubrum P-13, and incubated quiescently for 14 days or with shaking for 3 to 5 days. Maximal fungal growth and rubratoxin production occurred when the broth contained 20% sucrose. Broth with 10% glucose, 10% fructose, 5% maltose, or 1% asparagine supported formation of substantial amounts of rubratoxin (52.9–78.5 mg/100 ml). When the broth was fortified with glucose plus lysine, arginine aspartic acid, cystine, ammonium citrate, or ammonium phosphate, moderate amounts (27.5–39.5 mg/100 ml) of rubratoxin and mycelium (0.1–1.5 g/100 ml) were produced. Presence in the broth of 5% galactose or starch resulted in accumulation of small amounts (22.2 and 24.6 mg/100 ml, respectively) of rubratoxin and mold tissue (0.70 and 0.5 g/ 100 ml, respectively). Whereas some toxin was recovered from mineral salts broth fortified with lactose or ribose, toxin was not recovered when the mold grew in broth containing mannitol or fumarate. With the exception of gluconate which supported some growth and toxin formation and ethanol which permitted formation of small amounts of toxin, other carbon sources resulted in little or no fungal growth and no toxin formation. Yields of rubratoxin decreased with an increase in amount of agitation or length of incubation ofP. rubrum cultures. Mold growth increased and toxin formation decreased with an increase in volume of culture.     

CULTURE OF HUMAN GENITAL "T-STRAIN" PLEUROPNEUMONIA-LIKE ORGANISMS.

Ford, Denys K. (University of British Columbia, Vancouver, Canada). Culture of human genital "T-strain" pleuropneumonia-like organisms. J. Bacteriol. 84:1028-1034. 1962.-The conditions under which "T-strain" pleuropneumonia-like organisms, as described by Shepard, are best cultured were investigated. The organisms were found to grow on several types of nutrient agar and broth, of which PPLO medium supplemented with yeast extract and horse serum was the simplest. Subculture was possible through broth cultures, provided the broths were not incubated longer than 16 hr. The organisms on agar required either Fortner's anaerobic atmosphere or 10% CO(2), but broth cultures grew aerobically. "T-strains" grew over a pH range of 6.8 to 7.8, and a temperature range of 30 to 36 C. They were viable after storage for 16 days at 4 C and for 90 days at -20 C, and they resisted lyophilization. They were sensitive to 1.5 mug per ml of tetracycline and streptomycin, but were resistant to ampicillin and penicillin. Quantitative studies showed maximal concentration in broth of 10(6) to 10(7) organisms per ml, and logarithmic multiplication for the first 12 hr of broth culture, with a subsequent rapid decline in number. Colonial morphology was maintained after numerous subcultures.     

Microtissue Culture Test for the Titration of Low Concentrations of Diphtheria Antitoxin in Minimal Amounts of Human Sera

A microtissue culture method for the assay of low concentrations of diphtheria antitoxin in human sera has been developed, using a monkey kidney cell (VERO) culture technique. Results obtained with sera from nonvaccinated children and with immune sera from children vaccinated with three and four injections of diphtheria pertussis tetanus vaccine were in agreement with antitoxin levels considered necessary to denote immunity to diphtheria. The use of microplates and organic buffer for culturing the animal cells improved the stability of the tissue culture system. The described method is sensitive, economical, and applicable for the titration of antitoxin in human sera particularly from infants and children from whom a minimum amount of serum is available.     

Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production

Mutants of Corynebacterium diphtheriae C7(beta) that are resistant to the inhibitory effects of iron on toxinogenesis were identified by their ability to form colonies surrounded by toxin-antitoxin halos on agar medium containing both antitoxin and a high concentration of iron. Chromosomal mutations were essential for the altered phenotypes of four independently isolated mutant strains. During growth in deferrated liquid medium containing various amounts of added iron, these mutants differed from wild-type C. diphtheriae C7(beta) in several ways. Their growth rates were slower under low-iron conditions and were stimulated to various degrees under high-iron conditions. The concentrations of iron at which optimal toxin production occurred were higher for the mutants than for wild-type C. diphtheriae C7(beta). Toxin production by the mutants during growth in low-iron medium occurred throughout the period of exponential growth at nearly constant rates that were proportional to the bacterial growth rates. In contrast, toxin production by wild-type C. diphtheriae C7(beta) in similar low-iron cultures occurred predominantly during the late exponential phase, when iron was a growth-limiting nutrient. Additional studies demonstrated that these mutants had severe defects in their transport systems for ferric iron. We propose that the altered regulation of toxinogenesis by iron in our mutants was caused by the severe defects in their iron transport systems. As a consequence, the mutants exhibited a low-iron phenotype during growth under conditions that permitted wild-type C. diphtheriae C7(beta) to exhibit a high-iron phenotype.     

Bacteriophage and the Toxigenicity of <Emphasis Type="Italic">Clostridium botulinum</Emphasis> Type D

THE data of Inoue and Iida^1,2 strongly suggest that bacteriophage is involved in the toxigenicity of Clostridium botulinum types C and D. They were able to recover toxigenic isolates from nontoxigenic cultures incubated in broth containing filtrates of the toxigenic strains (Stockholm strain of type C and strain 1873 of type D). Lysis was observed in the cultures, but plaques were not demonstrated on solid medium. The change from hontoxigenicity to toxigenicity in C. botulinum type C strain 468C has been shown³ to require the active and continued participation of a specific bacteriophage designated CEβ.     

Pathogenic properties of Corynebacterium diphtheriae

The main pathogenic properties of 73 C. diphtheriae strains (their adhesive, invasive and cytotoxic activity) were characterized in the cultures of cells HEp-2 and Vero. The quantitative determination of the toxigenicity of 381 strains in the indirect hemagglutination test was made, and the strains were distributed by the degree of their toxigenicity. The characteristics of C. diphtheriae obtained with the use of in vitro experimental models, coincided with the severity of clinical manifestations of the diphtheria in humans, which made it possible to regard the models used in this study as adequate. On the basis of the chosen criteria the characterized strains could be subdivided into highly, moderately and low virulent and the degree of their potential epidemic danger could be determined.     

Role of complement in immune inactivation of Mycoplasma gallisepticum

Studies were undertaken to evaluate the role of complement in the interaction between mycoplasmas and antiserum. A suspension of the A-1 strain of Mycoplasma gallisepticum in PPLO broth was incubated at 37 C with rabbit immune serum which had been heated for 30 min at 56 C. Samples were removed from the mixture at timed intervals for 1 hr for titration of the mycoplasmas in broth. When normal guinea pig serum was included in the mixture at a final dilution of 1:40, the titer fell rapidly from 10(6) to 10(2) organisms per 0.2 ml. When the guinea pig serum was heated for 30 min at 56 C or was omitted from the mixture, the immune serum did not reduce the titer. The rate of inactivation was related to the final concentration of antiserum and to the incubation temperature. The effect of the guinea pig serum was eliminated by the addition of 0.01 m sodium ethylenediaminetetraacetate or by prior absorption with an unrelated antigen-antibody complex. It was concluded that complement-like substances play an important role in immune inactivation of M. gallisepticum.     

New Method of Isolating Salmonellae from Milk

The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations.     

Effects on Growth and Toxin Production of Exposure of Spores of Clostridium botulinum Type F to Sublethal Doses of Gamma Irradiation

Spores of the Langeland strain of Clostridium botulinum type F were grown at 30 or 10 C after exposure to 0.0, 0.1, or 0.2 megarad of cesium-137 gamma irradiation. When incubated at 30 C, cultures irradiated at the 0.2-megarad level reached the stationary growth phase 15 hr earlier than the 0.0 or 0.1 megarad-irradiated cultures. This was not the result of earlier or more frequent germination of the irradiated spores, the formation of larger individual cells, filament formation, or cell clumping. It appeared to result from elimination of a lytic phenomenon noted in 0.0 and 0.1 megarad-irradiated cultures after 26 and 29 hr of incubation, respectively, which was followed by a second exponential-growth response 5 hr later in these cultures. The time of toxin appearance in culture supernatant fractions was independent of prior irradiation treatment and occurred after 36 hr of incubation. Toxin release was essentially logarithmic until maximal titers were reached and maximal toxin titers were higher in irradiated than in unirradiated cultures. The higher toxin level was sustained over a period of 23 days of 30 C. Toxin produced in the 30 C cultures could not be trypsin-activated. An incubation temperature of 10 C resulted in no outgrowth of spores subjected to 0.2 megarad of irradiation, although spore germination did occur. At 10 C, outgrowth of the 0.1-megarad culture was faster with slightly higher quantities of a more stable toxin than was seen in the unirradiated control. At 10 C, trypsinization was necessary to demonstrate the toxin present in the cultures.     

Cytotoxin Production by Clostridium sordellii Strains

A total of 55 strains of Clostridium sordellii, 21 lethal toxin-positive and 34 lethal toxin-negative, were tested for cytotoxin production in brain heart infusion medium supplemented with 0.2% Na₂HPO₄ (m-BHI) and cooked-meat-glucose (CMG) medium using baby hamster kidney (BHK-21/WI-2) cells as indicator cells. The m-BHI medium was preferred to CMG medium and 24 hr of incubation was sufficient for cytotoxin production. Nineteen of the 21 toxigenic strains were also cytotoxigenic, and the strength of the cytotoxigenicity was approximately parallel with that of the lethal toxigenicity. Clostridium difficile antitoxin neutralized C. sordellii cytotoxin and also C. sordellii antitoxin neutralized C. difficile cytotoxin.     

Use of Antibiotics in the Preparation of Canine Kidney Tissue Culture Vaccines to Eliminate Leptospiral Infection Hazards

The potential leptospiral infection hazard in the use of vaccines prepared from canine kidney monolayer cultures was studied. Cell cultures were prepared from kidneys of dogs experimentally infected with Leptospira serotype canicola. Viable leptospires were found in kidney cell suspensions at the time of seeding, surviving trypsinization either at room temperature for approximately 2 hr or overnight at 4 C, even in the presence of antibiotics. In tissue cultures maintained without antibiotics, leptospires were cultured up to the time of involution of cells at 25 to 34 days of incubation. Cytopathogenic effects of leptospires on cultured kidney cells were not noted; neither was growth of leptospires remarkable. Generally, the leptospire culture titer decreased to 10^-4 or 10^-5 at the 4th hr or 1st day of incubation to 10^-1 or negative by the 30th or 34th day of incubation. The addition of either a combination of penicillin (100 units per ml) plus streptomycin (100 μg/ml) or polymyxin B (50 units per ml) plus dihydrostreptomycin (100 μg/ml) to seeding cell suspensions resulted in the elimination of viable leptospires by the 4th hr of incubation. From cell cultures treated with neomycin (100 μg/ml) or chloramphenicol (100 μg/ml), leptospires were recovered, respectively, after 24 and 48 hr, but not thereafter. It was apparent that antibiotics, particularly the combination of polymyxin B and dihydrostreptomycin, could be effectively used to eliminate leptospires in tissue culture. Other antibiotics with known antileptospiral activities probably would be effective also. If antibiotics are not used in canine kidney tissue culture employed for viral vaccine preparations, rigid testing for the presence of leptospires in donor dogs and tissue-culture vaccine is indicated.