Instituto Valenciano de Microbiología
(IVAMI)

Masía El Romeral
Ctra. de Bétera a San Antonio Km. 0.3
46117 Bétera (Valencia)
Phone. 96 169 17 02
Fax 96 169 16 37
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www.ivami.com
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Clostridium botulinum - Botulism: Diagnostic tests; recommended tests; detection of toxin; C. botulinum culture; botulinum toxin typing; Molecular diagnosis (PCR).

Clostridium botulinum - Botulism (Clinical forms). Tests for diagnosis of botulism. Detection of toxin in patients, food, feed, sludge or aquatic sediments; waterfowl, other samples. Recommended tests: detection toxin, C. botulinum culture, Typed botulinum toxin; Molecular diagnosis (PCR).

 

Characteristics of Clostridium botulinum and botulinum toxin/s

 

Clostridium botulinum is an strict anaerobic grampositive bacillus, genus Clostridium, family Clostridiaceae, spore-forming, that produces a neurotoxic toxin. This bacterium is usually found in soil and untreated fresh water and sediments (oceans, lakes), with a worldwide distribution. In some circumstances this organism can contaminate food and grow in them to produce their toxin/s. Botulism, a serious form of food poisoning, results from ingestion of food containing the toxin. Although this disease is rare, their mortality rate is high. When the toxin type was determined among 1036 cases detected in US between 1899 and 1990, 384 were for A toxin, 106 due to B toxin, 105 to E toxin and 3 to F toxin. Sometimes may be cases due to two toxins, for example A and B.

All forms of human or animal botulism, are caused by absorption of botulinum toxin formed during the multiplication of the bacterium Clostridium botulinum. The toxin has a toxicity (neurotoxicity) very high, so that exerts its action at extremely low levels, is thermolabile, while spores of the bacteria are heat resistant and survive in foods heated to over 100ºC, such as canned under thermal treatment. Besides, some strains of C. botulinum, C. butyricum, C. baratii and C. argentinense can produce botulinum neurotoxins.

There are seven toxin types (A - G) differentiable by neutralization tests, useful for clinical and epidemiology. Types A, B, E and F are the main causes of human botulism, while types C and D are found in cases of animal botulism, the most affected being wild birds and poultry, cattle, horses and some species of fishes. Types A and B are the most common in men, and are mainly related to contamination of home-prepared canned vegetables, but in Europe these types have also been found in relation to meat products. The E-type (fish) found in aquatic environments and correlates with E botulism cases concerning contaminated fish or shellfish, and is increasing. The F type is exceptional. Type C is subdivided into C1 (neurotoxin) and C2 (not neurotoxin affects vascular permeability and enterotoxigenic). The G type is produced by C. argentiniense (isolated from ground in Argentina, serum of deceased patients, although it is unclear involvement).

Toxins are synthesized during the growth of the bacterium as an inactive protein (150 kDa), which is released from the bacteria during lysis. To activate the toxin formed should degrade into two polypeptide chains (50 and 100 kDa).            

C. botulinum, can differentiate into groups according to their culture characteristics, biochemical and physiological. All cultures type A and some of the type B and F are proteolytic. Cultures of C. botulinum toxins produced by C and D are not proteolytic, when cultured in a medium with coagulated egg white or flesh. All types E and some of the type B and F are non-proteolytic, but have characteristics of carbohydrate metabolism that differ from non-proteolitics groups types C and D. The strains of type G not have It has been studied in sufficient detail for effective and satisfactory characterization.

The optimum temperature for growth and toxin production is about 35ºC for the proteolytic strains; for non-proteolytic strains is 26-28ºC. Non-proteolytic types B, E and F strains can produce toxin at refrigeration temperatures (3-4ºC). Toxins of non-proteolytic strains do not show maximum toxicity until activated toxin with trypsin. Toxins from proteolytic strains are generally produced in its activated form.

Clinical forms of botulism

There are four clinical forms of human botulism:

In any of the clinical forms of human botulism, and equal in the animal botulism, toxin penetrates the blood from the gastrointestinal tract when ingested preformed with a food, or when produced by the bacterium that colonizes the digestive tract (young children or adults), or in exceptional cases children from an infected wound with the bacteria. There are some foods that are more likely than others to contain botulinum toxin. Foods with a pH less than 4.5 are more difficult to be cause of botulism since at this pH C. botulinum is unable to multiply and produce toxin (this is the case of fruit juices, vinegar marinated food, etc.). Conversely, foods with pH equal or higher than 4.5 can cause botulism, since in them the multiplication and toxin production is possible (this is the case of meat, fish, vegetables, prepared dishes, etc.), on all those foods oxygen-free exposure, as with canned or vacuum-packed food, and having a pH greater than 4.6. Examples are dangerous food: cured ham, smoked, canned fish or vegetable (subjected to heat treatment insufficient to kill spores), etc. The cans contaminated with C. botulinum are usually curved, though this does not occur with type E.

The toxin absorbed irreversibly binds to the neuromuscular junctions of motor neurons, preventing the release of acetylcholine and causing flaccid paralysis or muscle weakness.

Clinically is characterized by acute flaccid paralysis, which usually begins with bilateral involvement of the cranial nerves, affecting the muscles of the face, head and pharynx, and then descends symmetrically to affect the chest muscles and limbs. Death, when it occurs, it is due to respiratory failure by paralysis of the tongue and pharyngeal muscles occluding the upper airway, or by paralysis of the diaphragm and the intercostal muscles. For this reason, patients should receive botulinum antitoxin and respiratory intensive care needed.

Diagnostic tests for botulism

 

Detection of botulinum toxin in samples or food without culture (see recommended sample in the test section "Test offered by IVAMI and samples required").

This is recommended in a patient with clinical evidence of botulism.

Detecting botulinum toxin can be performed in a liquid such as serum obtained from blood. It can also be detected from the remains of food eaten that has caused a case or an outbreak of botulism. To perform the test using remains of food it is necessary to obtain an extract filtrate from it. In cases of infant botulism or intestinal botulism can be detected the presence of toxin in the feces of children or patients, but consider more advisable to perform a preliminary culture of faeces (see below ). Botulism toxin detection in any of the cases is performed by inoculation in micethat develop paralytic symptoms, followed by death, if inoculated with botulinum toxin. To confirm that the mice have died from the inoculated botulinum toxin, it is necessary a neutralization test, which confirms that there botulinum toxin was present and identifies the type of botulinum toxin present. This neutralization test is performed facing the inoculated product (person or animal serum, extract from ingested food, ...) to specific antisera of each type of toxin.

Prior to the neutralization test step it is necessary to calculate the minimum lethal dose (MLD). Thus we calculate the maximum (highest) dilution which causes the death of the infected animals, and the inoculated volume has caused the death of the animals contain a lethal minimum dose (LMD).

To identify the type of toxin, once known the "Minimum Letal dose", this is faced with different anti-toxin botulinum type antisera. These mixtures will be inoculated in equal volume to experimental mice and those which survive, have been inoculated with the mixture of "minimum lethal dose" plus antiserum to a type that has been able to neutralize.

Detection of toxin in samples / products after preculture (see Recommended sample after in the the test  offered by IVAMI and samples required).

When the bacteria Clostridium botulinum, which produces the toxin, can be found in the sample it is recommended prior cultivation, to investigate the toxin after having cultivated the sample. This is the case of feces of a patient with infant botulism or intestinal botulism, the remains of food eaten in which had proliferated bacteria (e.gr., can prerserves), a suspected food that might contain the bacteria (e.gr., a bulged preserves), a food under control to exclude the presence of this bacterium (e.gr., sausages, hams, cans), or other samples such as fresh water sludges or aquatic marine sediments, etc., from areas where mortality has been observed in waterfowl.

During culture in the laboratory, in suitable culture media, the toxin is produced if there is a bacteria and obtaining a culture filtrate can be useful to investigate its presence, inoculating the filtrate to mice. If inoculated mice are affected indicate the likely presence of Clostridium botulinum in the sample o growth in culture. However, mice may have died from another cause, so necessary, before issuing the report, must be check that they have really died for having been inoculated with botulinum toxin.

To confirm the presence of botulinum toxin in the culture neutralization tests using specific antisera for each type of botulinum toxin (neutralization test) can be performed, or detection for the presence of Clostridium botulinum genes and its type in the culture medium (molecular detection by PCR).

Neutralization test is performed facing the inoculated sample (culture filtrate) to specific antisera for each type of toxin.

Prior to the neutralization test step it is necessary to calculate the minimum lethal dose (MLD). Thus the (highest) maximum dilution which causes the death of the inoculated animals is calculated, and the inoculated volume has caused the death of the animals contain a minimum lethal dose.

To identify the type of toxin, once known the "minimum lethal dose", confrontation the minimum lethal dose with different types of anti-botulinum sera is performed. These mixtures will be inoculated in equal volume to experimental mice and those who survive, have been inoculated with the mixture of "minimum lethal dose" plus antiserum to a type that has been able to neutralize.

The molecular detection by PCR avoids the time required for calculating the minimum lethal dose (MLD) and neutralization test.

Detection of anti-botulinum toxin antibodies (see recommended sample after in the section Test offered by IVAMI and samples required)

Detection of anti-botulinum toxin antibodies have interest in the following cases:

Clostridium botulinum toxin, at very high dilutions, has been used by local administration to treat spastic processes. These processes have been shown to be a useful remedy. These processes are usually chronic spastic thus requiring that the toxin is administered permanently. Therefore, resistance can arise during treatment due to progressive immunization of the patient throughout the treatment, in which case the effect would be limited. To detect this immunization is required measure accurately and sensitively the existence of antibodies against botulinum toxin A and/or B.

The accepted reference method for detecting and quantifying antibodies to botulinum toxin is the neutralization test in mice (Mouse Neutralization Assay), in which a dilution of botulinum toxin, quantified by the Lethal Dose 50% for mouse (DL50), mixed with various base 2 or base 4 dilutions of the serum/plasma, and after incubation each are inoculated intraperitoneally to groups of mice. The highest dilution of test serum that reduces toxicity correspond to the antibody titer against the corresponding botulinum toxin. This dilution, compared to an international standard allows obtaining results in international units (IU/mL) (1 IU is defined as the amount of antibody that neutralizes 10,000 LD50 of toxins A or B, or 1000 LD50 type E). The amount of toxin used in the tests is one that is neutralized by 0.02; 0.005 and 0.0125 IU/mL of antitoxin for types A, B and E, respectively (Hatheway et al. 1984). Sera that protect the mice titer of 1: 4 are reported as <0.08 IU / mL for type A, or <0.02 IU / mL for type B. The test is laborious, expensive and long duration performing, so alternatives have been sought enmzimoinmunoanálisis based methods (ELISA) using microplates coated with botulinum toxin. However, the values obtained by ELISA sometimes not fully correlate with neutralization test in mice.

Prior to the neutralization test step it is necessary to calculate the minimum lethal dose (DLM) of toxin. The calculation of the minimum lethal dose is performed by base 10 dilutions of the culture filtrate, diluting half with physiological saline to obtain the same dilution as the toxin mixed with serum or plasma from patient, and inoculating each dilution mixture to laboratory mice. Thus the (highest) maximum dilution which causes the death of the infected animals is calculated. The inoculated volume has caused the death of the animals contain a minimum lethal dose (MLD).

Once known the minimum lethal dose, calculate the minimum non-lethal dose (dmnm) corresponding to the minimum amount of toxin in the presence of a constant amount of antitoxin, that donot kill inoculated mice. This amount of toxin is being neutralized by the corresponding units of anti-A antiotoxina or anti-B antitoxin. Minimum non-lethal dose is called because it is the minimum amount of toxin that causes no death of the mice in the presence of antitoxin .

Tests offered by IVAMI and samples required:

Time for report of results (TAT)

Form with product characteristics and test/s chosen

Sample volume

Storage conditions and shipping of samples

Cost of testing