THE FOLLOWING ARTICLE APPEARED IN THE

SEPTEMBER 2000 ISSUE

THE POISON SLEUTHS

DEATH BY BOTULINUM TOXIN

-Dr. Anil Aggrawal

“Good morning doctor. Oh, my God, what are you doing today? You have the dead body of a young man today. What happened to him? Please tell me.”

“Good morning Tarun. The name of this young man is Badal and he died in the hospital today morning, i.e. on 16th September at about 9 am. The doctors could not find out the cause of his death. He is only 25 years old, and everybody is concerned why he died. That is why the police has brought his body to me. My job of course is to find out how he died.”

“Please explain the circumstances of his death doctor.”

“Badal and Shatru were friends and they both loved a 21 year old girl Sudha. Both wanted to marry her, but Shatru could go to any length to get her. It so happened that Sudha showed more interest in Badal. Many times Shatru had asked Badal to get out of his way, otherwise the consequences would not be good for him. But Badal ignored his threats taking them to be just friendly pranks. On the outside both of them remained as friends, although it appeared to many that Shatru held a grudge against Badal. Badal was working as a clerk in a local private company and was living alone on a rented flat. On 12th September Shatru came to his flat in the evening with a bottle of whiskey apparently to celebrate his birthday. They both had whiskey together, and had dinner together. On 13th Badal was fine. On 14th morning, Badal started having some strange symptoms. First he had nausea, vomiting, diarrhea, abdominal distension and abdominal pain. He thought he was having an ordinary bout of diarrhea and contacted a local doctor for this. He too thought that he was having an attack of gastroenteritis, and prescribed some standard medicines. But they could not prove beneficial. On 15th morning, Badal developed double vision, technically known as diplopia, difficulty in speaking (dysarthria and dysphonia) and difficulty in swallowing (dysphagia). He became alarmed, informed one of his neighbors who immediately took him to a nearby hospital, where the doctors started investigating his illness. At the hospital, he reported some additional symptoms. He had dizziness, very dry and sore throat, blurred vision and drooping of eyelids. But the most alarming symptom of all was a paralysis of the muscles of his face and neck, which gradually moved downwards. That means that after the development of paralysis of muscles of his face and neck, first his arm muscles became paralysed, and then his leg muscles. Quite remarkably there was no fever. I have talked to the doctors who were treating him, and I have been told that his pupils were dilated. The doctors tried their level best to save him, but he died today morning, i.e. after about 48 hours of having those strange symptoms. On hearing his death, Badal’s family members have arrived here from the capital. They are blaming Shatru for his death. They think that when Shatru came to Badal’s house on 12th, he probably gave him some poison mixed in whiskey. They say that Shatru could do anything to get him out of his way. They are obviously very well connected. There was tremendous pressure on police to catch Shatru, and they have caught him today. But Shatru says he is innocent, and that he has done nothing.”

“Doesn’t look like Shatru killed him. After all, Badal started having those symptoms almost 36 hours after he had that meal with Shatru. I have been discussing poisons with you for quite some time now. I don’t know of any poison which starts its action so late after being administered. To me it looks like he had a terrible disease. What disease did he die of doctor? Tell me.”

“Tarun, I can say anything definitely only after a thorough post-mortem examination, but from the symptoms which have been told to me by the police and by the treating doctors at the hospital, I am of the opinion that Badal died of Botulism.”

“Botulism? As far as I know this is a disease connected with food poisoning. Then why have the police apprehended Shatru? They must immediately release him.”

“Tarun, I have asked them to continue keeping him in custody. I learnt that Shatru had studied microbiology at M.Sc. level. So I don’t want to take chances. I want to exclude everything, before I can say anything with confidence. In one of my earlier discussions with you (See Science Reporter May 1998 issue), I told you that the symptoms of botulism may resemble that of barium poisoning . In fact, so similar may be the symptoms that according to most doctors, barium poisoning should always be considered as a possible diagnosis in all cases of food poisoning accompanied by neurological complications. So the first thing I did was to exclude barium poisoning in this case, by checking for barium levels in various organs of Badal. And I could safely exclude barium poisoning, as I did not find any barium there.”

“But if it is not barium, how can it be botulism? I mean how could Shatru give him Botulism even if he wanted to?”

“Tarun, do you know that Botulin toxin is now used as a medicine too? And that medicine is available in vials. A certain number of vials, if mixed in one’s food, can definitely kill a person.”

“A toxin being used as a medicine? Never heard of that. Looks like we are on to one more of your fantastic poison stories. Well, why don’t we begin from the beginning?”

“Tarun, Botulism is a disease caused by the gram positive bacterium Clostridium botulinum. Before proceeding further I may tell you that the term gram positive refers to certain bacteria which take up a violet stain when stained with a special staining technique called gram staining. Gram staining was developed in 1884 by a Danish physician Hans Christian Joachim Gram (1853-1938). Bacteria which take up a pink color by this staining method are referred to as gram negative bacteria. So much so about gram staining and its nomenclature. So I was telling you about the Clostridium botulinum bacterium. I may tell you that its name comes from Latin botulus, meaning sausage. Indeed consumption of contaminated sausage was one of the major causes of this condition, when it was first recognized. It can however be caused by contamination of any type of foodstuff - vegetarian and non vegetarian. This bacterium is present in soil and infected water. Improper canning of foods at home may cause some contamination of food with soil or infected water during the process of canning. When canning is complete, the contents inside are devoid of air, a condition which is very much loved by these bacteria. In fact in technical terms Clostridium botulinum is known as a strict anaerobe, which means that a lack of oxygen is necessary for its growth; it gets killed in the presence of oxygen. This might appear paradoxical to you, since we are so used to talking about organisms which need oxygen for growth, but this is a fact. In an environment, which lacks oxygen, i.e. the closed can, these organism keep on multiplying. They produce a toxin known as botulin, which is supposed to be the most toxic substance found in nature. When someone consumes this food, he unwittingly consumes the toxin, and immediately becomes sick. He is said to be suffering from botulism. And he depicts almost the same symptoms, which were shown by Badal before his death. To summarize, we can say that Botulism is caused when food contaminated with Clostridium botulinum is canned or otherwise stored for long periods in anaerobic conditions.”

“Doctor, many bacteria have different strains. Are there different strains of these bacteria too?”

“Yes, there are eight different types of botulinum toxins have been described, and all of them are released by different strains of bacteria. They are all Clostridium botulinum, but differ in very subtle ways, so they are known as different strains of Clostridium botulinum. These different toxins are designated as A, B, C1, C2, D, E, F, and G. All are neurotoxins (poisons affecting the nerves), except for C2, which is a cytotoxin (poison having a direct effect on cells). Its significance is not clear. All these toxins are proteinaceous in nature, and thus antibodies against them may be prepared. However antibodies against any one of these toxins is effective only against that particular toxin and not against any other. Human poisoning mostly occurs due to A, B, and E. Type E poisoning is frequently associated with fish products. Rarely F and G can also cause human poisoning. C1, C2, and D cause poisoning in some mammals and birds. The toxin is not destroyed by acid or proteolytic (protein splitting) enzymes, but is heat labile and is destroyed when heated to 800C for 30 minutes, or 1000C for 10 minutes as during routine home cooking. It is thus a good practice to heat the canned food properly before cooking. Botulinum Toxin A (also known as BTX-A) has been isolated as a pure crystalline protein and is the most potent toxin known to man. You may be surprised at the fatal dose of this toxin. It is just 1 pg (10-12 g)/kg of body weight. This means that just about 10-10 g of toxin is required to kill a 100 Kg man! Even lesser doses would be required for ordinary human beings weighing 60-70 kg. Another figure may give you some idea of its lethality. One g of BTX-A toxin would kill 30,000 million mice! About 3 million molecules injected in the abdomen of a mouse would kill it. That would sound like lot of molecules, but the very fact that its lethal dose can be described in terms of molecules rather than in milligrams or grams speaks volumes about its lethality! So less is the fatal dose of BTX-A that it is often measured in a special unit called a mouse unit. It is a very small quantity. One Mouse Unit of BTX-A is equal to just 3 x 107 molecules. Again because of its such high lethality, many nations have seriously thought about using it as a biological weapon. As a biowarfare or terrorist agent, exposure is likely to occur following inhalation of aerosolized toxin or ingestion of food contaminated with the preformed toxin or microbial spores. Recently, Iraq admitted to active research on the offensive use of botulinum toxins and that they weaponized and deployed over 100 munitions with botulinum toxin in 1995.”

“Oh, that is really interesting. Doctor, does this bacterium forms spores too?”

“Oh, yes. The bacteria does form spores, when it finds that the conditions have become difficult for it to live. These spores (in contrast to toxin) are highly heat resistant, requiring exposure to moist heat at 1200C for 30 minutes for inactivation, as in steam sterilizers or pressure cookers. The spores are ubiquitous. They are present in soil, sea water and even air. Earlier I was telling about the fatal dose of BTX-A. I must tell you that its fatal period is just about 24 hours. I may also tell you that the toxins formed by Clostridium botulinum are known as exotoxins. Very broadly speaking exotoxins are those toxins which are produced by bacteria while they are still alive. On the contrary, there are certain other bacteria which release toxins when they get killed. Their cell walls rupture and the toxins are released. Those toxins are known as endotoxins. Thus the correct term for Botulinum toxin type A is BTX-A exotoxin.”

“That is a lot of information doctor. You told that these bacteria make toxins while remaining in the can. Then the food inside must get spoiled too. Then why at all does someone consume such food?”

“Unfortunately that is not the case. The food may or may not appear spoiled, depending on what strain of bacteria attacked it. If it released proteolytic enzymes, (the enzymes which break down proteins) along with the toxin, the food would spoil, otherwise not. Food contaminated by type A and B bacteria often appear putrefied, because they release proteolytic enzymes too. In contrast food infected with type E bacteria may look and taste normal, as they do not produce proteolytic enzymes. There are however ways to prevent botulism. First of all, if you are buying cans of food from a supermarket, never buy cans which are leaking, spouting, dented or otherwise show any other sign of abnormality. This is because the bacteria may release gases during their activity. And this build up of gas pressure inside the can may cause all these effects. Secondly all canned items should be cooked thoroughly by boiling and stirring for 15 minutes. This destroys any toxin if it is present. Commercial canners usually heat food to at least 1200C to guarantee the destruction of both toxins and spores. If you don’t want to use the food cans immediately, the cans should be frozen or refrigerated at very low temperatures. This is because some strains, especially type E, can produce toxin at temperatures as low as 50C. So a temperature lower than this must be achieved. There are some precautions suggested for those who do canning of food at home. One is the use of phosphoric and citric acids during canning. This is because the optimum pH for the growth of these bacteria is between 4.6 to 7.0. pH above and below this range are not conducive to their growth. Acid environment obviously is not conducive to their growth, and the use of phosphoric and citric acids provide a sound acidic environment. This is also the reason why acidic fruits may generally be safely canned. Many other facts seem to prove that an acidic environment prevents the growth of C. botulinum. Infants below the age of one year seem to become victims of infant botulism for the reason that their gastro-intestinal tract (GIT) is deficient in bile acids and gastric acid, which normally prevent the growth of C. botulinum. For preventing botulism, it has also been suggested that if you are canning meat at home, it should be cured with sodium nitrite (3.5% to 6%). This is said to prevent botulism, as sodium nitrite has an antimicrobial, especially antibotulinal activity. There is however a controversy that when such cured meats are fried, the nitrites may get converted to nitrosamines, a known carcinogen. But this is a different controversy and we would not concentrate it here now.”

“Doctor, how does Botulinum A Exotoxin work?

“BTX-A is an enzyme. It produces its effects by preventing release of acetylcholine (ACh) from the nerve endings. This chemical is very necessary for the transmission of impulses across nerves. Thus in a way the connections between nerves become ‘dead’, and the activities of the whole organism come to a stand still. Do you remember how Badal had a paralysis of his arms and legs? This is typical of botulism. Actually it was this symptom, which partly alerted me and made me think in this direction. I may tell you that the symptoms of food borne botulism start within about 18-36 hours of ingestion of contaminated food. After having food with Shatru, Badal also developed his symptoms roughly in the same time period. This period has often been referred to as the incubation period, but it is a actually a misnomer. The illness may be very mild for which no medical advice may be sought, or it may be so fulminant as to cause death within 24 hours. Badal died within three and a half days.”

“Doctor, you have just mentioned a term “food borne botulism”. Does this mean there are other forms of botulism too?”

“Oh yes. In fact the Center for Disease Control in Atlanta, USA (often referred to as CDC) currently classifies four different types of botulism. The first is the food borne botulism, which occurs from ingestion of preformed toxins. This is the best known form, and I have been talking about this form till now. The second form is infant botulism, which occurs mostly in infants below 6 months of age (90% cases). Rest of the cases occur between the ages of 6 months to 1 year. It occurs not from the ingestion of preformed toxins, but from ingestion of spores, which germinate in the mildly alkaline environment of the intestines, and produce toxin in-vivo, which gets absorbed. In adults the bile acids and the gastric acid tend to restrict the growth of C. botulinum. But the gastrointestinal tract of infants lacks these acids, and this factor may be responsible for their growth. Since the toxin is formed within the intestine and is absorbed gradually, the symptoms and signs are less dramatic, compared to adult type food-borne botulism, where preformed toxin is ingested all at once. The first signs to toxicity in infant botulism are constipation, feeding difficulty, feeble crying, and a “floppy” baby with diffuse decreased muscle tone, particularly apparent in the limbs and neck. Infant botulism is the most common form of botulism. Certain infants are more susceptible to infant botulism, while others seem resistant. The reason for this strange phenomenon is not clear. It is believed that giving of honey to infants may be a source of infection, as it contains spores of C. botulinum. Giving of honey to young infants is thus not advised now. The third form is Wound botulism, which occurs due to contamination of wounds with spores of C. botulinum. This usually occurs when they get soiled with dirt and nothing is done about it. The condition is also seen in chronic drug abusers and after caesarian delivery. This entity was first noted in California in 1976. This has now become the most common form of botulism. The symptoms and signs are quite similar to those seen in food borne botulism, except for the fact that gastric symptoms like nausea and vomiting are absent in wound botulism. Finally there is the Indeterminate type, which occurs in patients over 1 year of age with no recognized source for disease. Some authorities suggest that a mechanism similar to infant botulism may be at work here. Their studies suggest that the adults may ingest spores of C. botulinum, which may germinate in the gut, much like they do in infants. Several factors such as achlorhydria in these people, i.e. lack of acid in the stomach may be responsible for their germination. You may recall that I told you strongly acidic environments restrict the growth of this bacteria.”

“Yes, I do. Doctor, in the beginning you told that this toxin is also used as a medicine. How can this toxin - which you say is the most dangerous toxin in the world - be used as a medicine? To me this sounds like a paradox.”

“Tarun, now it is known that wrinkles in old people are caused by sustained contraction of facial muscles. If somehow these muscles could be paralysed, these wrinkles would go away too. BTX-A does this remarkably well. This toxin in nanogram doses is used for removal of wrinkles. Injection of BTX-A directly in these muscles causes their paralysis, which are then allowed to weaken by atrophy over a 3-week period. Similarly the toxin has been used in the treatment of certain eye disorders such as blepharospasm (abnormal contraction of eye lids) and in squint surgery. Commercially BTX-A is available under two different trade names - BOTOX and Dysport (produced by Speywood, Maidenhead, United Kingdom). BOTOX is prepared by a multiple precipitation technique, whereas Dysport is produced by column-based methods. These are technical terms and you need not bother about them. Suffice it to say, that this difference in production methods produces a differential between the effects on humans and mice. In particular, the dose (in mouse units) of Dysport required to produce a given effect is 3 to 4 times greater than the dose of BOTOX. Now I will come to the most important point. Work in primates, in conjunction with estimates derived from accidental botulism, suggests a human fatal dose of approximately 40 U/Kg if the toxin is delivered systemically. The BOTOX vial contains 100 units; the Dysport vial, 500 units. Thus a few vials if mixed in someone’s whiskey would be enough to kill him. This is actually what Shatru had done. I suspected him the moment I came to know that he was a brilliant student of microbiology and also when I heard the typical symptoms of Badal. I have chemically examined the blood of Badal and it shows BTX-A. I did not stop at that. I also did bioassays which proved that the blood of Badal contained something which paralyses muscles.”

“What is a bioassay doctor?

“Bioassay is a term which refers to determining a chemical by biological means. I prepared an extract from Badal’s serum, and injected it in mice. By doing this, I could produce paralysis in mice, which was a positive indication that some abnormal substance was there in Badal’s blood. I have lab reports to show to the court that this abnormal substance was in fact BTX-A. Now I will tell you one more secret. I sent policemen to Shatru’s house and they have recovered some empty BOTOX vials from his house. These vials had no business being present in his house, which tells us that he is the one who mixed those vials surreptitiously in Badal’s whiskey. Come let us tell the police to put him under more sustained questioning, and he would surely spill the beans.”

“That is very clever of you doctor. Without your clever deduction it would have been impossible to say how Badal died and Shatru may have gone scot-free. What are you going to tell me next time?”

“Tarun, next time, I would tell you about a very interesting poison - Lithium.”