Botulinum Toxin and Its Biological Significance: A Review

Abstract

Botulism is a severe neuroparalytic bacterial disease caused by gram positive, anaerobic, spore-forming microorganisms, Clostridium botulinum, referred to as Botulinum Neurotoxin (BoNT) producing bacteria. The objective of this paper is to review botulinum toxin and its biological significance. This bacteria can produce seven types of toxins (A - G) known as BoNT. BoNT is highly potent preformed toxin that affects humans, all warm-blooded animals, and fishes due to consumption of contaminated silage, carcass, water, industrial by product, and canned foods like meat, milk, fruits and vegetables. Botulism is an important disease in the world, particularly where stock graze under range conditions and are subject to periods of protein and phosphorous deficiency. There is no geographical limitation for botulism because sporadic outbreaks occur in the most countries. The main route of transmission of botulism is by oral ingestion and wound infection with spore. The clinical signs occur within 24 hour up to 17 days. BoNT contain zinc endopeptidase that blocks vesicle of acetylcholine binding with the terminal membrane of the motor neuron and causes flaccid muscle paralysis with lateral recumbency, generalized muscle weakness and dysphagia. Finally results in death due to respiratory arrest, paralysis of pharyngeal and diaphragmatic muscles. There is no specific lesion during postmortem examination but may be seen in chronic case. Diagnosis is based on clinical sign and laboratory examination like, ELISA, MPT and culture for isolation of bacteria. Although this toxin in animals represents a serious environmental and economic concern because of the high mortality during the outbreak, it provides some biological significance after extraction by genetic recombination like in cosmetics, chemotherapy, and biological weapons. There is no effective treatment but ampicillin used as antibiotics. Prevention by polyvalent vaccine and proper feeding management is better than treatment. Therefore it recommended that the farmers should store animals feed with proper ventilation to avoid multiplication of bacterial spore.

Keywords: Botulism; Bacteria; Neurotoxin; Zinc endopeptidase; Paralysis

Introduction

Botulism is a severe neuroparalytic bacterial disease affects humans, all warm-blooded animals, and fishes [1]. It is caused by exposure to botulinum neurotoxins (BoNTs), which are produced by gram positive, anaerobic, spore-forming microorganism’s genus Clostridium, referred to as BoNT-producing clostridia [2]. BoNTs act on nerve endings to block acetylcholine release. Their potency depends on two factors: their enzymatic activity and their selective affinity for binding neurons. It is also an exotoxin-induced flaccid paralysis in animals and human. Because of its bio-warfare agent and potent toxin BoNT causes disorder in living beings [3].

The word ‘botulism’ is derived from the Latin word ‘botulus’ meaning ‘sausage’. Hence, the name ‘botulism’ refers to the poisoning caused due to consumption of sausages. In 1869, Muller report that eating fish cause food poisoning. He was the first person to call the term ‘Botulism’ in his report. The bacteria are found in the intestinal tracts of some healthy fish, birds and mammals, and the gills and viscera of shellfish (crabs) [4].

The causative agent, Clostridium botulinum is appear as soil- borne pathogen, prefers to grow in decaying organic matter [5]. This toxin is a protein in nature and heat labile neurotoxin which affects acetylcholine release at the neuromuscular junction and causes botulism [6]. It is fatal bacterial disease because of neuronal paralysis which cannot be reversed by therapeutic options. However administration of antitoxin was recommended as the first line of management. Human botulism treated effectively with antitoxin, mechanical ventilation, and other symptomatic therapeutic measures [7]. But, the availability of antitoxin in developing countries is limited. However, antitoxin therapy would be effective if it is injected before the toxin reaches motor-end plate [1].

An important source of intoxication is contaminated silage, canned foods, either from consumption of food that has not been heated properly before canning or from food that was not properly cooked from the can before consuming. Three important types of botulism are identified, namely food botulism, infant botulism and wound botulism [8].

In the recent year, sporadic incidences and high amount of poultry outbreak with bovine botulism have been reported from different parts of the world [9,10]. This disease in animals represents a serious environmental and economic concern because of the high mortality during the outbreak and used as biological weapons. More than a million deaths have been reported during outbreaks in particular area in a year with losses of 50,000 birds [1]. Therefore the objective of this paper is to review botulinum toxin and its biological significance.

Literature Review

Etiology

Botulism is disease caused by a toxin produced by the bacterium Clostridium botulinum which is a spore forming, gram positive, an anaerobic bacteria. This bacteria multiplies in an oxygen deficient (oxygen-free environment). In this condition with warmth and moisture, C. botulinum multiplies fast and produce highly lethal toxin. All warm-blooded animals can be paralyzed because the toxin blokes nerve function. As result this toxin is known as neurotoxin [11].

Based on their genotypic, phenotypic, and biochemical characteristics, these strains of microorganism can be divided into six groups: C. botulinum (groups’ I-IV), C. butyricum, and C. baratii. Groups I and II, C. butyricum, and C. baratii are associated with human botulism, and group III causes animal botulism [3]. Group IV organisms (C. argentinense), are associated with wound botulism [12] .This bacteria can produce seven types of toxins (A, B, C, D, E, F and G). Most clostridial strains produce only one toxin type. All of the botulinum toxins cause the same clinical signs but different in severity of the disease. Knowing the type of toxin is important in selecting an antiserum for treatment because antiserum produced against one type is not effective for others. In people, botulism is caused by types A, B and E. Types C and D are the most common causes of disease in other mammals and birds. Type C is especially common in birds, mink and horse. Cattle that fed poultry litter and dogs that eat contaminated bird carcasses are also affected by type C. Types A and B affect horses in the U.S. Type E toxin is found in aquatic environments, and can cause botulism in fish and fisheating birds. In addition C. botulinum type C also produce C2 toxin, which causes an enterotoxin with gastrointestinal signs. In humans, botulism is caused by group I or group II organisms [13].

Group I and II organisms can producing type A, B, E, and F toxins while group III organisms produce type C, D, and their mosaic C/D and D/C toxins [14]. Group I contains proteolytic strains and group IV, C. butyricum, and C. baratii can produce type G, E, and F toxins, respectively [15]. And group II consists of nonproteolytic strains that form B, E or F toxins. Group I and II C. botulinum strains differ in heat resistance. Spores from group I organisms are more resistant to heat, growth temperatures, and other characteristics that inhibit the types of foods where they tend to grow. Group III strains produce toxins C or D that cause botulism in animals. Group IV produces the type G toxin which reclassified as C. argentinense. This species cause outbreak of human botulism in Switzerland [13].

Epidemiology

Mammals are susceptible to botulinum neurotoxin and develop botulism with the same clinical sign to humans. Most of the cases are caused by C. botulinum group III, even if groups I and II are also reported in animal botulism. Horses are very sensitive to BoNTs and equine botulism occurs sporadically worldwide, both as feed poisoning and as toxico-infectious forms. Avian botulism is usually caused by BoNT type C1, to which most birds seem to be susceptible. It is also very serious problem in fish farming. Contaminated silage becomes major cause for outbreak of botulism in cattle [2]. There is no geographical limitation for botulism because sporadic outbreaks occur in the most countries. The source of the toxin and risk for the disease varies from regions to region due to food storage, feeding and management practices. Outbreaks of disease occur with ingestion of toxin in feeds that is common in northern USA and Europe. Additionally outbreaks in animals on pasture are reported from South Africa, Australia and Gulf coast of USA [16].

Geographic distribution: The geographical distribution of bacterial strain that conducted in the USA indicates, type A was found in the neutral and alkaline soil in the west whereas type B and C in damp or wet soil all over, except that B was not found in south. Type C was found in soils in Gulf coast and type D in alkaline soil in west. The prevalence of the disease is high in area where, canning fruits and vegetables is more common like tropical countries [17].

Botulism is an important disease in the world, particularly where stock graze under range conditions and are subject to periods of protein and phosphorous deficiency. It has been reported in feedlots and in dairy cattle under intensive feeding systems. There are seven recognized types of botulism organisms but only two, types C and D, are important in cattle [11]. The distribution of the organism is not the same and more common in certain geographical areas because the environmental factors can influence the occurrence of botulism For instance, it is common in cattle from areas with phosphorus-poor soils, like in southern Africa [13]. The toxin does not affect Fly larvae and other invertebrates. However, feeding on toxigenic carcasses make this organism victim of the toxin. Ingestion of a single toxigenic maggot could be lethal. This is described as the carcass-maggot cycle of botulism [18].

Animal botulism: The primary contamination route for either animal botulism or human botulism is the ingestion of preformed toxins in foods or feeds. Raw material, such as grass, hay, rotting vegetation, and slaughterhouse waste, decay of vertebrate carcasses, invertebrates, and sewage may support BoNT-producing clostridia growth and toxin production. Animals may directly ingest decaying organic matter containing toxin, or from the consumption of zooplankton or invertebrates, such as larva that carry toxin. A second form of animal botulism is due to absorption of BoNTs produced in vivo in the intestinal tract. This form of botulism, seen in chickens and horses is known as toxicoinfection. A third form of animal botulism is caused by the germination and production of toxin by C. botulinum spores in infected wounds. The last 2 forms are often referred to as toxicoinfectious form of botulism [19].

Susceptibility: The susceptibility of cattle to botulinum poisoning is depends on presence of the following factors: phosphorous and protein deficiency, carcass and bone chewing, bacterial distribution, toxin, unvaccination and improper vaccination [11]. The exposure to poultry litter as feed or bedding may be risk factor in the occurrence of cattle botulism [9]. Phosphorus deficiency in cattle may result in pica that tend to chew on cadavers and bones to balance their mineral deficiency which means a high risk of BoNT ingestion [20- 22].

Transmission: The main route of transmission of botulism is by oral ingestion and wound infection with spore. Because all species of Clostridium can produce spores that make them dormant and highly resistant to disinfectants, heat and environmental conditions that destroy vegetative cells. These spores can survive for many years until favorable conditions allow them to germinate and grow. C. botulinum spores are common in the soils, in sediments in lakes, streams and coastal waters. Also found in the intestinal tracts of some healthy fish, birds and mammals, and the gills and viscera of shellfish (crabs). Also the toxin has been detected in snails, earthworms, maggots feeding on contaminated carcasses, and nematodes. Because invertebrates are not affected by the toxin, they are involved in transmitting it to species such as birds [13].

Pathogenesis

Botulinum toxin is a dichain polypeptide: a heavy chain of 100 KDa is attached by a single disulfide bond with 50 KD of light chain, which contain zinc endopeptidase that blocks vesicle of acetylcholine binding with the terminal membrane of the motor neuron and causes flaccid muscle paralysis. This toxin is the most lethal toxin and all seven types act in similar ways. Thus results in death due to respiratory arrest, paralysis of pharyngeal and diaphragmatic muscles [23]. Mental functioning is not impaired by BoNTs, so the patient remains alert and conscious throughout the disease [2].

It produced during bacterial vegetative growth as inactive single-chain polypeptides then activated by bacterial or tissue protease. Naturally this toxin found as progenitor toxins containing the neurotoxin and nontoxic associated proteins which protect neurotoxin from environmental factors [1]. The genes for encoding BoNTs found in the chromosome or on extrachromosomal elements, such as plasmids or bacteriophages [24]. But toxin genes for group III organisms are carried by bacteriophages that exert rapid change on lysogenic cycle. Molecular and genomic analysis of the bacteriophage genome describes that this phage exists as a circular plasmid prophage in the lysogenic state but does not accepted by host chromosome [25].

The mechanism of action of Botox follows steps in the system of the body. Frist, active toxin is absorbed in the small intestine by binding to the receptors on the apical surface of gut epithelial cells. Second, released into the systemic circulation, reaching all peripheral cholinergic nerve endings. Third, in these sites, the toxin binds to specific receptors. Then internalized into the cytosol of the nerve terminus, where it blocks the release of acetylcholine, finally results in characteristic paralysis [26].

Post-mortem examinations

Postmortem examination is not usually evident but may be seen in chronic case [27]. Diffuse intestinal hemorrhage may be observed as some strains of C. botulinum type C and D because of an enterotoxin they produce. But these changes are not sufficient to specifiy confirmatory diagnosis. However examination of stomach contents like decayed carcass material, bones, maggots may be consistent [28,29].

Clinical signs

There are three forms of botulism, food-borne, wound and intestinal form. Those cause disease by absorption of botulinum toxin into the circulation. The incubation period on the mucosal surface of the wound depends on the rate and amount of toxin absorbed [30].

Food-borne botulism (classical botulism) was the first form of the disease reported in literature. Food poisoning due to this toxin was emerged as a problem when food preservation became a common practice in the world. Infant botulism, recognized as a clinical disease over three decades ago in USA. The initial neurological symptoms of infant botulism are largely the same as in other forms of botulism, but these are usually missed by parents and doctors because the infant cannot verbalize them. The most common sources of infection for infants appear to be honey and environmental exposure. Another form of botulism is analogous to tetanus, in that BoNT is determined from C. botulinum growing in vivo in abscessed wounds called wound botulism. Most cases occur in physically active young males who are presumable at higher risk of traumatic injuries. Wound botulism has emerged following subcutaneous injection of spore [2].

All forms of botulism results in progressive, symmetrical, flaccid paralysis that starting from the hindquarters with weakness, muscle tremors, stumbling, ataxia, eyes appear closed, pupils dilated, papillary reflexes sluggish and recumbency, finally results in death. It may be in peracute, acute, and chronic forms. In general, the clinical signs occur within 24 hours up to 17 days. The incubation period of food borne botulism in monogastric animal is shorter than in ruminants which show clinical signs after a week. The incubation period for toxicoinfectious botulism is longer (4 to 14 days) [31].

Diagnosis

Clinical diagnosis: Clinical signs of animal botulism are not specific but indicative. Confident diagnosis is based on signs observed in sick animals, the duration of the outbreak, the postmortem findings and by ruling out other differential diagnoses. In cattle, flaccid paralysis, the epidemiology of the outbreak, the clinical chemistry like hyperglycemia and neutrophilia support the diagnosis [1].

Laboratory diagnosis: Laboratory confirmation can be done by following methods; first, by examination of BoNTs in serum, gastrointestinal content, liver, and wound; second by BoNTproducing clostridia in gastrointestinal content, liver, and wound; third by BoNTs or BoNT-producing clostridia in feed or the close environment of the sick animal and by antibody response in an animal with symptoms of botulism [27,32]. Detection of toxin by protecting with monovalent toxin allows diagnosis of botulism with testing of toxin in plasma or tissue before death of animals. In addition to this the toxin can be demonstrated by ELISA technique, injecting intra peritoneal the extract of food or culture into mice or guinea pig and isolation of bacteria by culturing [33].

Enzyme Linked Immuno Sorbant Assey (ELISA): This test is used to show that an animal has antibodies to against toxin in its blood serum. Antibodies arise from either natural exposure to a toxin or from vaccination. The test can identify the type of toxin involved (type C or D) with natural infection, and the level of antibodies in the animal. Because of cross reactions following vaccination, it is not possible to differentiate between type C and D vaccination titres. This test is useful for assessing the success of a vaccination program. In unvaccinated herds the ELISA test is very useful as a positive result shows natural exposure. However, it is an expensive test. It can be used together with the fecal culture test to confirm that animals have been exposed to botulism. A repeat sample taken from survivors two weeks following the outbreak should indicate rising levels of antibodies if botulism infection has occurred [11]. However toxin detection by ELISA test appears less sensitive than mouse bioassay [33].

Mouse protection test (MPT) (Toxin neutralization test): Detection of toxin using bioassay in mice coupled with toxin neutralization with polyvalent antitoxins used but the sensitivity is low in both ruminants and horse because they are more sensitive than mice to botulism toxin. The test results in paralysing mice with an injection of a toxic bacterial or toxic serum from an affected animals and then protecting them with specific type C or D botulism antiserum. It is good for identifying the presence of toxic botulism bacteria and is used with the ELISA test. However, it is not so useful in proving that a paralyzed beast has botulism. This is because only very low doses of toxin are present for short periods in the bovine serum and the mouse is relatively resistant to the toxin compared to cattle [11]. After demonstration of BoNTs in serum, feed material, or intestinal content inject into the mouse and taking bioassay is the gold standard for laboratory confirmation of botulism. But negative mouse bioassay does not always mean no botulism, because the toxin may be present at a level below the limit of detection or may have been biodegraded by microbes in the intestinal tract of the animals [33].

Culture for isolation of bacteria: Examination of the toxin in feed stuff, fresh stomach content or vomitus assists diagnosis of botulism. The spoilage of food or swelling of cans or presence of bubbles inside the can indicate clostridial growth. Food is homogenized in broth and incubated in Robertson cooked meat medium and blood agar or egg yolk agar, which are incubated anaerobically for 3-5 days at 37° [16]. The botulism organism can be grown from any gut contents or even carcass material. Once the organism is grown in the laboratory, tests are carried out to show that it is C. botulinum, and to identify the type. This test will show that a toxic bacterium may be present but it does not prove that it was the cause of death. It may have been present without ill effect [11].

Biological significance of botulinum toxin

Preparations of botulinum toxin: Serotype A (BTX-A) is the most commercially available toxin for clinical use .Also the efforts have been made for the commercial production of serotypes B, C, and F. The two available market preparations of BTX-A are by the trade names Dysport and Botox BTX-A is prepared by laboratory fermentation of Clostridium botulinum cultures. Crude botulinum toxin is a protein with a molecular weight of about 190, KDa. After purification, the toxin is diluted with human serum albumin, bottled in vials, lyophilized (freeze-dried), and sealed. Each freeze-dried vial contains 100 units (U) of BTX-A which is reconstituted with preservative-free normal saline (1-5 ml) just before use. The toxin should be used within 4 hours of reconstitution. Within these four hours; reconstituted botulinum should be clear, colourless and free of particulate matter. The shelf life of the packaged product is 36 months when stored at 2°C to 8°. The potency of BTX-A is measured in mouse units (MU). One MU of BTX-A is equal to the amount of toxin that kills 50% of a group of 20g Swiss Webster mice within 3 days of intraperitoneal injection(LD50) [39].

Importance of botulinum toxin in cosmetics: Today, BoNT is the most commonly performed cosmetic procedure in the world. The main significance of botulinum toxin in cosmetic use is on the muscles of facial expression gives beautiful apperance. Because this toxin reduces hyperfunctional muscles and eliminating the overlying skin line or ridge [35]. It is also used in treatment of glabellar lines, horizontal forehead lines, wrinkles correction, brow lift, nasal scrunch, rejuvenation of mouth and mandibular contouring [36]. Now days, glabella is the only FDA-approved site for cosmetic injection of BOTOX -A in the USA. Because it is the most common site for patients and physicians to begin treatment with BoNT-A. Injections of the small muscles in this area are technically simple to perform and they result in a high degree of patient satisfaction. Close attention should be paid to the eyelid and eyebrow for possible ptosis and redundant eyelid skin that made patient dissatisfayed following treatment Stretching the skin in this area will form creases and repeated treatment should be given within 3-4 month intervals to reduce wrinkles in the area where treated [38]. One recent study has publicized that glabellar treatment may help convey positive and relaxed emotions correctly and that BoNT-A injections of the glabella can be beneficial for patients, who believe their faces are not communicating their emotions properly [36,38]. Botulinum neurotoxin type A injection is a simple, safe, and very effective treatment of the aging face, reducing wrinkles through the temporary and reversible paralysis of treated muscles [35,36].

Importance of botulinum toxin as therapeutic agent: The first batch of botulinum toxin type A manufactured by Scott and Schantz was named Oculinum. Later by 1991, the manufacturing facility and license were turned over to Allergan and got a new name Botox. The clinical use of botulinum toxin is to change extra ocular muscle to different position during surgical treatment of strabismus (heterotropia). In animal botulinum toxin produced long lasting, localized, dose dependant muscle weakness with no systemic toxicity and necrotizing side effects. This toxin is used in humans according to Investigational New Drug (IND) license for the treatment of strabismus, blepharospasm, hemifacial spasm, cervical dystonia (torticollis), thigh adductor spasm and, hyperhidrosis. At now a day number of label used botulinum toxin. Such as in tremor, spasticity, over active bladder, anal fissure, achalasia, various pain disorders including headache. The most recent indication of botulinum toxin (botox) is used for wrinkles and various cosmetic activities [4].

Currently available pharmaceutical preparations of botulinum toxins for the treatment of human diseases in ophthalmology, neurology and dermatology are marketed under the trade names Botox®(USA), Dysport® (United kingdom)and Xeomin® (South America and Asia) (based on botulinum neurotoxin A), Neuronox (South korea) and Myoblock® /Neuroblock® (based on botulinum neurotoxin B). With the exception of Xeomin, this is practically devoid of complexing protein [2] (Table 1).

  

    
NO 
    
Properties 
    
Preparations 
  
  

    
 
    
 
    
Botox 
    
Dysport 
    
XEOMIN 
    
NEUROBLOCK 
  
  

    
1
    
Formulation
    
Powder
    
Powder
    
Powder
    
Ready to use    solution
  
  

    
2
    
Storage condition
    
< 8°C
    
< 8°C
    
< 25°C
    
< 8°C
  
  

    
3
    
Self life
    
36 months
    
24 months
    
36 months
    
24 months
  
  

    
4
    
Botulinum toxin    type
    
A

    
A
    
A
    
B
  
  

    
5
    
SNARE target
    
SNAP25
    
SNAP25
    
SNAP25
    
VAMP
  
  

    
6
    
Purification    process
    
Precipitation and
      
chromatography
    
Precipitation and
      
chromatography
    
Precipitation and
      
chromatography
    
Precipitation and
      
chromatography
  
  

    
7
    
pH value
    
7.4
    
7.4
    
7.4
    
5.6
  
  

    
8
    
Stabilization
    
Vacuum drying
    
Freeze drying
    
Vacuum drying
    
pH reduction
  
  

    
9
    
Excipients
    
Human serum
      
albumin, NaCl
    
Human serum
      
albumin, lactose
    
Human serum
      
albumin, sucrose
    
Human serum    albumin,
      
disodium    succinate, NaCl,
      
H2O, hydrochloric    acid
  
  

    
10
    
Biological    activity
    
100 MU-A/vial
    
500 MU-I/ vial
    
100 MU-M/vial
    
1.0-2.5/10 k    MU-E/ vial
  
  

    
Source: Amarnath et    al., [4].
      
MU-A:    Mouse unit in the Allergan mouse lethality assay; MU-I: Mouse unit in the    Ipsen mouse lethality assay; MU-M: Mouse unit in the Merz mouse lethality    assay; MU-E: Mouse unit in the Solstice mouse lethality assay [4].
      
 
      
 
  

Table 1:  Comparison of different botulinum toxin drugs.

  

    
NO
    
Indication
    
Example
  
  

    
1
    
Dystonias
    
Cervical dystonia, Oromandibular dystonia, Pharyngolaryngial dystonias, Jaw closure/opening dystonias, Occupational cramps, Limb and axial dystonias
  
  

    
2
    
Spasticity
    
Cerebral palsy, Brain injury, Spinal cord injury
  
  

    
3
    
Eyelid spasm
    
Blepharospasm, Hemifacial spasm, Eyelid twitch
  
  

    
4
    
Exocrine gland hyperactivity
    
Focal hyperhidrosis, Relative sialorrohoea, Crocodile tears syndrome
  
  

    
5
    
Movement disorders
    
Tremors, Bruxism, Tic
  
  

    
6
    
Pain syndromes
    
Migraine, Back spasm
  
  

    
7
    
Urinary bladder dysfunction
    
Sphincter- detrusor dyssenergia, detrusor hyperreflexia
  
  

    
8
    
Opthalmology
    
Strabismus, Entropion, Protective ptosis
  
  

    
9
    
Cosmetology
    
Hyperactive facial lines-brow lines, Frown lines,
  
  

    
10
    
Gastroenterology
    
Achalasia, Anal fissures, Anismus
  
  

    
11
    
Gynecology
    
Vaginismus
  
  

    
12
    
Urology
    
Sterile prostatitis
  
  

    
13
    
Dentistry
    
Muscle spasm associated with temporomandibular    joint pathology
  
  

    
14
    
Veterinary
    
Barking dogs
  
  

    
Source:    Dhaked et al., [2].
      
 
  

Table 2:  Clinical use of botulinum neurotoxin.

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Citation: Desta S, Melaku M and Abdela N. Botulinum Toxin and Its Biological Significance: A Review. Austin J Vet Sci & Anim Husb. 2016; 3(1): 1021. ISSN: 2472-3371

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