Efficacy of laboratory tests for the detection of enterotoxigenic Clostridium perfringens.

Nineteen Clostridium perfringens strains with positive erythemal and ligated intestinal loop reactions, and 22 strains with negative reactions, originating from food-poisoning cases, were tested comparatively using the fluorescent antibody (FA), reversed passive hemagglutination (RPHA), and immunodiffusion (ID) tests. All the biologically positive strains were detected by the three immunological tests used. The FA test detected five additional strains among the biologically negative group which did not react in RPHA or ID tests. Sporulating culture supernatant fluids, after 13 to 17h of growth, were satisfactory for testing for the presence of enterotoxin by the RPHA and ID tests. The FA test was used on cell smears.     

Comparison of Indirect Hemagglutination and Immunodiffusion Tests for Detecting Type II Leukosis (Marek's) Infection in S- and K-Line Chickens

The indirect hemagglutination and immunodiffusion tests were compared for detection of antigen and antibody to JM strain of leukosis virus infection between S- and K-line chickens. The indirect hemagglutination test was more sensitive than the immunodiffusion test for detecting the smallest amount of viral antigen and corresponding antibody in the plasma of infected chickens. The Cornell S-line had higher levels of antigen and antibody as compared with the Cornell K-line during the 20-week experimental period.     

Comparison of Indirect Hemagglutination and Immunodiffusion Tests for Detecting Type II Leukosis (Marek''s) Infection in S- and K-Line Chickens

The indirect hemagglutination and immunodiffusion tests were compared for detection of antigen and antibody to JM strain of leukosis virus infection between S- and K-line chickens. The indirect hemagglutination test was more sensitive than the immunodiffusion test for detecting the smallest amount of viral antigen and corresponding antibody in the plasma of infected chickens. The Cornell S-line had higher levels of antigen and antibody as compared with the Cornell K-line during the 20-week experimental period.     

Experimental diarrhea in cynomolgus monkeys by oral administration with Clostridium perfringens type A viable cells or enterotoxin.

Purified C. perfringens type A enterotoxin fed orally in an amount of 5 mg caused both vomiting and diarrhea in the monkey only when the gastric juice had been neutralized. Exposure of enterotoxin to pH 4.0 or below rapidly destroyed the activity. All three monkeys receiving sodium bicarbonate and 2.4 X 10(10) viable cells grown in DS medium developed diarrhea, and only one of them vomited once. The diarrhea lasted for 13, 18 and 19 hr. The symptoms were similar to those reported in human cases of C. perfringens food poisoning. These results have verified the general notion that C. perfringens food poisoning should be categorized as a true "intravital intoxication". The reversed passive hemagglutination test detected enterotoxin directly in most fecal samples. This method may be applicable for diagnosis of human cases of C. perfringens food poisoning. Neither enterotoxin nor anti-enterotoxin was detected in serum samples taken from any monkey up to 21 days after the challenge. We are tempted to conclude, therefore, that no significant amount of C. perfringens enterotoxin is absorbed from the intestine.     

Enterotoxin B: Serological Assay in Cultures by Passive Hemagglutination

Bis-diazotized benzidine hemagglutination with Formalinized sheep erythrocytes was adapted to the rapid and specific detection of enterotoxin B in staphylococcal culture fluids. There was complete agreement between hemagglutination inhibition (HI) and gel diffusion in detecting 8 enterotoxin B-positive cultures from a total of 68 staphylococcal cultures tested. The sensitivity of HI equals or exceeds that of gel diffusion. Also, results can be obtained in several hours, even with extremely low concentrations of enterotoxin, whereas it may require 24 hr to 1 week to obtain comparable results with gel diffusion. Problems associated with the presence of potent hemagglutinins for sheep erythrocytes in several staphylococcal culture fluids are discussed.     

A survey of Clostridium perfringens enterotoxin antibody in human and animal sera in western Canada

Sera from human, cattle, sheep, swine, and horse populations in western Canada were tested for the presence of Clostridium perfringens enterotoxin antibody by the passive hemagglutination (PHA) test, supplemented by an immunodiffusion test and by counterimmunoelectrophoresis. A total of 224 human, 345 cattle, 165 sheep, 620 swine, and 768 horse serum samples were examined. Low-titer reactions in the PHA test were detected in human, cattle, horse, and swine sera, in that order, with no titers demonstrated in sheep. The titers in human sera ranged up to 1:128 and three of these samples were also positive in the other two serological tests. The significance of this antibody is not clear, but it is suggested that the low prevalence of the antibody may reflect a low prevalence of enterotoxigenic C. perfringens strains in western Canada. Such serological surveys may be applicable to epidemiological studies involving enterotoxigenic C perfringens.     

Serological studies with the ficin-antificin system.

Rabbit antisera prepared against ficin reacted with it in a series of serologic tests. Upon immunodiffusion analysis, ficin was found to consist of at least nine antigenic components. Ficin will adsorb spontaneously to erythrocytes which can be used in passive hemagglutination tests. The enzymatic activity of ficin was not abolished by antificin sera.     

Assay Methods for Clostridium perfringens Type A Enterotoxin

Enterotoxin produced by a sporulating culture of Clostridium perfringens type A NCTC 8798 was purified to a level of 3,500 mouse mean lethal doses per mg of nitrogen. High-titer sera were obtained from rabbits injected with enterotoxin and used to compare the sensitivity of serological tests and bioassays for C. perfringens enterotoxin. Reversed passive hemagglutination was by far the most sensitive test, followed by microslide diffusion, single gel diffusion and electroimmunodiffusion, guinea pig skin test, mouse test, and rabbit ileal loop test.     

A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin

Fourteen strains of Bacillus cereus isolated from different sources were examined for their ability to produce diarrhoeal enterotoxin by two commercial immunoassay kits (Oxoid BCET-RPLA and Tecra ELISA) and the microslide immunodiffusion assay. One strain that was positive in monkey feedings, as well as a number of other strains isolated from diarrhoeal outbreaks, gave positive results in the ELISA and negative results in the RPLA test systems. When tested in the microslide assay, these strains produced only one antigen which formed a line of identity with the reference toxin. The results of the control toxins provided with the kits substantiated that the two commercial assays did not detect the same antigen. Cultures positive with both assay kits were shown to produce diarrhoeal enterotoxin (by a line of identity) and other antigens in the microslide immunodiffusion assay.     

Isolation of influenza A virus from budgerigars and its serological characterization

A hemagglutinating agent was isolated from the respiratory organs of budgerigars suffering from diarrhea and malnutrition. This agent, possessing neuraminidase activity, was identified as influenza A virus by the double immunodiffusion test. The results of hemagglutination and neuraminidase-inhibition tests with monospecific antisera to the isolated surface antigens showed that the isolates possessed Hav4 hemagglutinin and Nav1 neuraminidase subunits both of which were closely related to the corresponding antigens of A/duck/Czech/56 (Hav4 Nav1).     

Enterotoxigenicity and genetic relatedness of Clostridium perfringens isolates from retail foods in the United States

Clostridium perfringens is a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high. For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness. Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic. It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness. We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States. Forty isolates were obtained by using the iron milk method at 45 degrees C, with confirmation by use of motility nitrate and lactose gelatin media. The presence of the C. perfringens enterotoxin (cpe) and alpha toxin (cpa) genes was determined by PCR using previously published primer sequences. All isolates possessed cpa. None of the isolates were identified as carrying the cpe gene by this method or by another method using a digoxigenin-labeled gene probe. Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination. Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates. About 5% of the isolates were considered to be closely related (2- to 3-band difference). The others were considered to be unrelated to one another. The results demonstrate the rarity of cpe(+) strains in retail foods and the genetic diversity among nonoutbreak strains.     

Echinococcus granulosus: comparison between antigens in scolices and hydatid fluid.

The sheep hydatid fluid and scolex antigens of Echinococcus granulosus were precipitated by increasing ammonium sulphate concentrations. The antigenic profiles, obtained by complement fixation and indirect hemagglutination inhibition tests on the ammonium sulphate precipitates after linear sucrose gradient ultracentrifugation, were different comparing the hydatid fluid and the scolex extracts. Antigenic non-identity was found between sheep hydatid fluid and scolex extracts by immunodiffusion and indirect hemagglutination inhibition tests. The ammonium sulphate precipitates of hydatid fluid and scolex extracts revealed several different bands by slab-gel examination.     

Hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

The hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin (LTc-B) produced by chicken enterotoxigenic Escherichia coli was studied by hemagglutination and hemagglutination inhibition. No or weak hemagglutination of intact human erythrocytes was found by the LTc-B at the highest concentration used, whereas strong hemagglutination of both neuraminidase- and pronase-treated human erythrocytes was found. Enhancement in hemagglutination of treated human erythrocytes induced by the LTc-B was over 2 to 120-fold for type A and B erythrocytes and over 8-fold for type O erythrocytes, respectively. With intact and treated sheep erythrocytes, on the other hand, no hemagglutination was found by the LTc-B at the highest concentration used. Hemagglutination of pronase-treated human type B erythrocytes by the LTc-B was inhibited by methyl-alpha-D-galactopyranoside, galactose, melibiose, hog A + H, asialo-bovine salivary mucin and asialo-thyroglobulin among mono-, di- and polysaccharides and glycoproteins used as inhibitors. These results suggest that the LTc-B is a galactose-specific bacterial lectin.     

Detection of Staphylococcal Enterotoxin in Foods

A short procedure for the extraction of staphylococcal enterotoxins from food materials has been developed. The procedure involves extraction of the food at pH 4.5, centrifugation, extraction of the supernatant with CHCl(3) (pH 7.5), extraction of the enterotoxin from the water layer with CG-50 ion exchange resin (pH 5.4 to 5.9), and treatment of the eluate with agar and concentration with Carbowax 20-M. The concentrate was extracted with CHCl(3), and the water layer was lyophilized. The dried material was dissolved in a 1% trypsin solution and placed on microslides, which were incubated 24 h at 37 C. The time required for enterotoxin analysis was 3 days with microslides and 1 day with the reversed passive hemagglutination technique.     

A simple procedure for isolation and purification of A-type staphylococcus enterotoxin

A new procedure is presented for the isolation and purification of A-type staphylococcus enterotoxin. Homogeneous enterotoxin preparation was obtained by purification in 2 phases. In radial double agar-gel immunodiffusion the smallest precipitating dose of the isolated and purified enterotoxin was found to be 1.4-0.7 micrograms protein and 0.4-0.1 micrograms nitrogen. In cat experiments the dose giving a positive reaction was 2 micrograms protein or 0.5 micrograms nitrogen calculated for kg body weight.     

Improved Reversed Passive Hemagglutination for Simple and Rapid Detection of Staphylococcal Enterotoxins A~E in Food

Detection and identification of staphylococcal enterotoxins in food or culture filtrates were performed using the reversed passive hemagglutination (RPHA) technique, with formalized sheep red blood cells (FSRBC) sensitized with immunoglobulins of anti-A, B, C, D, and E rabbit hyperimmune sera fractionated by affinity chromatography. The FSRBC sensitized with anti-A~E immunoglobulins showed a high level of reactivity and specificity in RPHA, against homologous types of purified enterotoxins and culture filtrates of toxin-producing strains. No non-specific reactions with various ingredients in foods nor cross-reactions among enterotoxin types were observed. The minimum amount of enterotoxins in foods detected by RPHA was calculated to be 0.01 μg/g without concentration, and the recovery rate of experimentally added toxins was calculated to be about 80%. Under routine laboratory practice, detection and identification of enterotoxins from incriminated foods of five food poisoning outbreaks were performed by RPHA within 3 hr after reception of the specimens. Among them, three were determined to be enterotoxin A food poisoning, one to be toxin C and the rest to be intoxication of A and D. The concentration of the toxins was between 0.014 and 3.65 μg per gram of food.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.     

Production and Evaluation of a Purified Adenovirus Group-Specific (Hexon) Antigen for Use in the Diagnostic Complement Fixation Test

A simple procedure for the production of large volumes of purified adenovirus group-specific complement-fixing (CF) (hexon) antigen by selective adsorption to and elution from CaHPO(4) is described. Results of immunodiffusion tests, electrophoresis, electron microscopy, and tests for hemagglutination and infectivity indicate that the purified antigen consisted of a single virus component (hexon). The purified product contained little host materials. Unlike the crude virus harvest usually employed for serodiagnostic CF tests, the purified antigen demonstrated no anticomplementary activity and did not develop such activity during storage. The purified antigen was equal to or slightly more sensitive than crude virus harvests for serodiagnosis of adenovirus infections.     

New Method for the Isolation of Membranes from Mycoplasma gallisepticum

Mycoplasma gallisepticum lysed readily in carbonate bicarbonate buffer at pH 9.2 to 10.5. The hemagglutination titer of the lysates was 2- to 16-fold greater than a cell suspension at the same protein concentration in buffered saline. Membranes prepared from cells lysed by this method at pH 10 were relatively free from cytoplasmic contaminants as shown by electron microscopy of thin sections. The membranes retained their hemagglutination activity, gave reactions in immunodiffusion tests identical to those obtained by osmotic lysis and sonic treatment, and showed a similar pattern of protein bands by polyacrylamide disk electrophoresis. When inoculated into rabbits, the membranes gave rise to antibodies active in growth-, metabolic- and hemagglutination-inhibition tests. On the average, membranes obtained by lysis at pH 10 contained 44% of the original cell protein. The method is simple, giving high yields of membranes, and may be adaptable to other mycoplasmas.     

The application of QAE-Sephadex for the purification of two staphylococcal enterotoxins. I. Purification of enterotoxin C2.

A new method developed for purification of enterotoxin C2 from Staphylococcus aureus strain 361 consisted of four steps: batchwise adsorption from culture supernatant on QAE-Sephadex; gel filtration on Sephadex G-100; chromatography on QAE-Sephadex using a buffer of constant pH and molarity; and gel filtration using a volatile buffer of constant pH and molarity; and gel filtration using a volatile buffer as the eluting solvent. The purified enterotoxin appeared homogeneous by gel immunodiffusion, gel chromatography and in the analytical ultracentrifuge, although an apparent heterogeneity was noted on QAE-Sephadex chromatography and polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.04 mug/kg of animal weight. Upon treatment with sodium dodecylsulfate, beta-mercaptoethanol and urea, enterotoxin C2 separated into 3 bands in sodium dodecylsulfate-electrophoresis. One band mol. wt 29000, and two bands of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight oligopeptides emerged as a single peak at the same position as untreated enterotoxin C2 during gel filtration with buffer lacking thiol and denaturant, and gave a reaction of complete identify to enterotoxin C2 in Ouchterlony immunodiffusion. The results suggest that enterotoxin C2 is a mixture composed of intact polypeptide chains, mol. wt 29000, and two fragments cleaved in the disulfide region of molecular weight of approx. 15400 and 12800 linked by the single disulfide bond in the toxin molecule. Amino acid analysis indicates that enterotoxin C2 consists of 255 amino acid residues.