Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies.

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. Potency testing of toxin and antitoxin therapies is entirely dependent on mouse lethality bioassay which is associated with extreme suffering of large numbers of animals to ensure high precision. The mouse phrenic nerve-diaphragm assay is an ex vivo assay that closely mimics in vivo respiratory paralysis offering substantial refinement and reduction in the number of animals used. A range of botulinum antitoxin standards, one licenced product and experimental antitoxins were tested for neutralising potency using ex vivo hemidiaphragm assay and compared with in vivo determined activities. Overall, there was an excellent agreement between neutralising activity detected by the two assay systems and for each toxin serotype using only 4-7 replicates for each product (almost perfect concordance for type A antitoxins: ρ = 0.997, and substantial concordance for type B antitoxins: ρ = 0.991 and type E antitoxins: ρ = 0.964, respectively). These findings confirm that the mouse nerve-diaphragm preparation can provide a functional ex vivo replacement assay for specific, sensitive and precise assessment of toxin and antitoxin activity.

Attempts to quantity Clostridium botulinum type A toxin and antitoxin in serum of two cases of infant botulism in Japan.

Serum samples taken from two infant botulism cases during hospitalization were titrated for botulinum toxin by both the intraperitoneal (ip) injection method and the score method in mice. By the ip method, in which death is the only parameter, such low levels of toxin as lower than 4 ip LD50/ml may not be titrated even though the surviving mice show abdominal palsy. By the score method based on the degree of abdominal palsy, such low levels of toxin as 1.1 and 0.8 ip LD50/ml were detected in specimens of one of the patient's serum. No antitoxin was demonstrated in either case of infant botulism by applying the score method. It is not known whether spontaneous recovery from infant botulism is due to the antitoxin production.

Titration of botulinum antitoxin of low levels by the score method.

Botulinum antitoxin is commonly titrated by injecting a mixture of toxin and antitoxin into mice and by utilizing deaths as a marker to measure the amount of unneutralized toxin. We attempted to titrate antitoxin by converting the severity of symptoms (notably palsy) and time-to-death in days into scores. In neutralization tests with toxin levels at 5.9 LD50 and 23.5 LD50, a linear relationship was obtained for antitoxin dose in a range between 0.03 to 0.003 IU/ml. Statistical analysis showed that homogeneity of variance or slope was not denied for the scores obtained on any day from the first to the fourth days after injection, demonstrating that this method can titrate accurately antitoxin of such a low level as 0.003 IU/ml within 4 days after injection.

Kinetics study of immunological response to Clostridium botulinum toxin.

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of humoral antibody to type A botulinal toxin was developed. This assay was used to study the kinetics of antibody response of a volunteer to botulinal toxoid. The circulating type A antitoxin was first detected by the ELISA 2 weeks after the first booster injection of the toxoid. The antibody titer stayed level until the second booster at 12 weeks. The titer then continued to rise throughout the remaining study period. The neutralizing antibody to type A toxin was detected by mouse assay 15 weeks after detection of antitoxin by the ELISA.

In vitro assays for botulinum toxin and antitoxins.

Clostridium botulinum produces several powerful neuroparalytic toxins which, although rare in food-poisoning instances, are generally fatal. A considerable amount of effort has therefore been made by the food industry to ensure that food treatment processes adequate to prevent growth and toxin production by Cl. botulinum. Laboratory mice and guinea-pigs are presently used extensively both for the assay of botulinum toxins and for the development and assessment of vaccines used to protect laboratory workers. An amplified ELISA, using a monoclonal antibody, has been developed for botulinum type A toxin with a sensitivity similar to that of the mouse acute toxicity test. The immunoassay has been found to be applicable to the detection of toxin in foodstuffs and could replace the currently used mouse bioassay in many laboratories. Immunoassays have also been developed for the detection of antibodies to botulinum toxins. A preliminary study has shown that antibody titres to botulinum types A and B toxins in sera taken from immunised personnel, as measured by ELISA, showed limited correlation with those measured by the toxin neutralisation test in mice. A more extensive study should determine whether the latter test can be replaced by the ELISA.

Antitoxin levels in botulism patients treated with trivalent equine botulism antitoxin to toxin types A, B, and E.

Serum levels of equine-botulism antitoxin to toxin types A, B, and E were measured in four type-A botulism patients who had received equine-botulism antitoxin. High circulating levels capable of neutralizing in excess of 1 X 10(8), 9 X 10(7), and 6 X 10(6) 50% mouse lethal doses of toxin of types A, B, and E, respectively, were detected. There was little depletion of type-A antitoxin even though two of the patients had circulating type-A toxin before treatment. The half-life for antitoxin persistence for one patient was calculated as being 6.5, 7.6, and 5.3 days for antitoxin types A, B, and E, respectively. Antitoxin levels were not proportionate to the amount (range, 2-4 vials) injected and did not appear to be affected by whether the route of administration was iv or im. Peak serum levels of antitoxin were 10-1,000 times higher than amounts needed to neutralize the toxin measured in the serum of these and other patients with botulism.

An outbreak of type C botulism in captive monkeys.

In an outbreak of type C botulism in a group of captive primates, six squirrel monkeys, six white throated capuchin monkeys and two weeper capuchin monkeys succumbed rapidly and died. Clostridium botulinum type C toxin was detected in the remains of the chopped chicken feed and in nine of 11 blood samples and one of three stomach contents samples from the affected animals.

Experimental study of skin-sensitizing antibodies after aerosol and subcutaneous immunization.

The reaction of passive skin anaphylaxis was used to investigate the accumulation and the dynamics of skin-sensitizing antibodies (SSA) in the sera of guinea pigs immunized with botulin anatoxin by means of different methods: the aerosol and the subcutaneous method. In the aerosol-immunized animals (unlike the subcutaneously immunized ones) SSA appeared in a negligible amount only after the fourth immunization and disappeared rapidly.

The antibody response of cattle to Clostridium botulinum types C and D toxoids.

The resistance of cattle with varying serum-antitoxin titres was determined by per os challenge. The results proved that a solid immunity can be produced against C. botulinum toxins C1 and D. The immune response of cattle to various quantities of C. botulinum C1 and D toxoids, aluminium-phosphate-adsorbed and in water-in-oil emulsion was investigated. The response to antigen in water-in-oil emulsion was far superior to the other when they were used for primary and secondary stimuli. When cattle had been given a solid basic immunity with 2 injections of antigen in water-in-oil emulsion, essentially the same booster effect was obtained with antigen in water-in-oil emulsion and in aqueous solution. Only some of the animals injected intramuscularly with antigens in water-in-oil emulsion developed local lesions. These lesions were not large and their histological picture indicated a noticeable decline in severity within 20 weeks. A case is thus made out for the use of C. botulinum C1 and D toxoids in water-in-oil emulsion for the primary and secondary stimuli and an aqueous solution of these antigens for any booster stimulus as an improved method of protecting cattle against botulism.

Botulinum antitoxins and antibacterial IgM and IgG antibodies in sera of persons immunized with botulinum polytoxoid combined with cholera vaccine. I. Response to botulinum toxoid.

Two groups of subjects were immunized with combined vaccine containing aluminum hydroxide-adsorbed botulinum toxoids, types A, B, and E, and 4 mld of formaldehyde-inactivated V. cholerae Inaba and Ogawa organisms. The first group included laboratory workers who were previously immunized against cholera and had professional contact with botulinum toxins and viable V. cholerae organisms. The second group included young men who were never vaccinated against botulism or cholera. The three-dose immunization schedule with combined vaccine resulted in clear-cut antitoxin response; after the third dose, the A, B, and E antitoxin level ranged from 0-2 to 10 IU/ml. Immunity against botulinum toxins lasted at least one year. Distribution of the antitoxins among IgM and IgG globulin classes resembled that in the case of response to other toxoids; 21 days after the third immunization antitoxin activity was found in IgG globulins.

Antitoxin responses to Clostridium botulinum vaccines types C and D in guinea pigs.

In guinea pigs, the type C and/or type D antitoxin responses to a single dose of a bivalent or monovalent Cl. botulinum vaccine increase markedly between the fourth and ninth week after injection and still increase markedly by the ninth week. For type C, a similar pattern has been found in cattle. Antigens of types C and D mutually interfere with the antitoxin responses in guinea pigs. Graded doses of vaccine arouse graded antitoxin responses in guinea pigs. Stability trials of vaccines have emphasized the unsatisfactory nature of an absolute-response type of assay, rather than revealing any loss of potency during storage. Attention is drawn to the need for a graded-response type of assay in which vaccines under test are compared with a reference vaccine.

Botulinum antitoxins and antibacterial IgM and IgG antibodies in sera of persons immunized with botulinum polytoxoid combined with cholera vaccine. II. Response to cholera vaccine.

The response of humans to cholera vaccine was very heterogeneous. The proportion of IgG vibriocidal antibodies was high in persons having previous natural or artificial contact with V. cholerae antigens. Predominance of IgM antibody response was seen in persons vaccinated for the first time. This type of response was sometimes evoked by unspecific stimuli such as botulinum polytoxoid without cholera vaccine. Antibodies passively protecting mice were found both in IgM and IgG globulins but the activity of these antibodies was higher in IgG than in IgM globulins.