Strategies for preventing and managing botulism outbreaks
SYSTEMATICS
CLASSIFICATION
Clostridia can be categorized into four major groups based on the types of diseases they cause.
Histotoxic clostridia are responsible for a range of tissue (often muscle) infections that frequently occur after wounds or other trauma (e.g.,).
Hepatotoxic clostridia produce their toxins in the liver, leading to diseases such as bacillary haemoglobinuria and black disease (e.g.,).
Enterotoxigenic clostridium primarily cause enterotoxemia and food poisoning, though they can occasionally be histotoxic (e.g.,).
Neurotoxic clostridia cause diseases by producing potent exotoxins (neurotoxins) (e.g.,).
FAMILY CHARACTERS
Classification
Clostridia are pleomorphic, rod-shaped bacteria; long filaments and involution forms are common.
Spore formation varies in frequency across different species, and the shape and position of the spores also differ.
Most clostridia are motile with peritrichous flagella, except for C. welchii and C. tetani type VI. C. welchii is encapsulated, while others are not.
Clostridia are anaerobic bacteria. C. oedematiens are strict anaerobes and die upon exposure to oxygen.
C. histolyticum and C. welchii are aerotolerant and can even grow aerobically.
Clostridia are fermentative, oxidase-negative, and catalase-negative organisms.
A very useful medium for isolating clostridia is Robertson's cooked meat broth. Clostridia grow in this medium, making the broth turbid, and most species produce gas.
Saccharolytic species such as C. oedematiens, C. septicum, C. chauvoei, and C. welchii turn the meat pink.
Proteolytic species such as C. tetani, C. botulinum, and C. haemolyticum turn the meat black and produce a foul and pervasive odor.
In litmus milk medium, the production of acid, clot, and gas can be detected.
HISTORY
Tetanus has been recognized since ancient times, with Hippocrates describing it.
However, understanding of the disease was only achieved in 1884.
In 1886, Rosenbach demonstrated a slender bacillus with round terminal spores in a case of tetanus.
Kitasato, in 1889, isolated C. tetani in pure culture and successfully reproduced the disease in animals by inoculating them with this pure culture.
The term "tetanus," derived from the Greek word meaning "contracture," has roots in Latin medicine's "rigor."
HABITAT
Soil, particularly when contaminated by animal feces, serves as the natural habitat for C. tetani, which can transiently inhabit the intestines of horses and other animals.
The bacterium is widespread and has been found in various other sources, such as street and hospital dust, cotton wool, bandages, catgut, plaster of Paris, clothing, etc.
It may exist as a seemingly harmless contaminant in wounds.
MORPHOLOGY
C. tetani is a straight and slender Gram-positive rod. It typically forms terminal, spherical endospores that bulge the cell, resulting in a characteristic drumstick appearance (young spores may initially appear oval).
The bacterium is usually found singly but can occasionally form chains.
It lacks a capsule and moves via peritrichous flagella.
CULTURAL AND BIOCHEMICAL CHARACTERISTICS
Cultural characters
C. tetani is an obligate anaerobe that thrives at an optimal temperature of 37°C and pH 7.4.
It grows on standard media, and its growth is enhanced by blood and serum rather than glucose.
Obtaining surface colonies is challenging due to its tendency to swarm over agar surfaces, particularly in moist conditions.
The swarming behavior can be mitigated by increasing agar concentration up to 3% (stiff agar), resulting in the formation of individual rhizoid colonies.
On blood agar, it forms partially translucent, grayish colonies with filamentous edges, giving a fuzzy appearance.
Horse blood agar shows initial alpha hemolysis, progressing to beta hemolysis due to tetanolysin production.
In Robertson's cooked meat broth, C. tetani grows well, causing turbidity without digesting the meat, though prolonged incubation turns it black.
In gelatin stab cultures, it exhibits a fir tree-like growth pattern with slow liquefaction.
Media containing neutral red develop a greenish fluorescence in the presence of C. tetani.
Biochemical properties
C. tetani exhibits weak proteolytic activity and does not ferment sugars.
It produces indole and tests negative for both methyl red (MR) and Voges-Proskauer (VP).
It does not reduce nitrates.
RESISTANCE
The endospores of C. tetani are highly resistant; boiling can kill most strains' spores within 15 minutes.
Autoclaving at 121°C for 15 minutes or dry heat at 150°C for over an hour is completely sporicidal.
These spores can survive in soil for years and are resistant to most antiseptics.
They are not affected by 5% phenol or 0.1% mercuric chloride solutions even after two weeks.
Iodine (1% aqueous solution) and hydrogen peroxide (H2O2) can kill the spores within a few hours.
ANTIGENICITY
Ten serological types of C. tetani have been identified based on flagellar antigens (types I to X), with Type VI consisting of non-flagellated strains.
All types produce the same neurotoxin, tetanospasmin, which can be neutralized by a common antitoxin.
They share a common heat-stable somatic antigen across all types.
Additionally, types II, IV, V, and X share a second somatic antigen.
TOXINS
C. tetani produces at least two distinct toxins: tetanospasmin and tetanolysin.
These toxins are antigenically and pharmacologically different, and their production occurs independently of each other.
A third toxin, recently identified as a nonspasmogenic peripherally active neurotoxin, has been documented.
Its role in the pathogenesis of tetanus remains unclear.
Tetanolysin
It functions as a hemolysin or cytotoxin, capable of causing lysis of rabbit and horse red blood cells (RBCs).
This toxin is sensitive to heat and oxygen, similar to streptolysin O, d toxin of C. oedematiens, and j toxin of C. welchii.
Tetanospamin
It is an exceptionally potent neurotoxin responsible for the clinical symptoms of tetanus.
The toxin is stable in oxygen but relatively labile to heat, becoming inactive at 65°C within 5 minutes.
When highly purified, it spontaneously toxoids.
It serves as a robust antigen and is specifically neutralized by antitoxin. The toxin exists in two forms: a toxic monomer with a molecular weight of 68,000, and a non-toxic but antigenic dimer.
The purified toxin remains active in exceedingly small amounts, containing approximately 3 x 10^7 minimum lethal doses per milligram of protein.
Different animal species vary considerably in their susceptibility to the toxin.
Horses and humans are the most susceptible, followed by guinea pigs, mice, goats, and rabbits in descending order.
Birds and reptiles exhibit high resistance to the toxin.
PATHOGENESIS
C. tetani exhibits minimal invasive capabilities. Its endospores typically enter traumatized tissue or surgical wounds, especially post-castration or docking, through the umbilicus or uterus following dystocia in cattle and sheep.
Spores implanted in wounds germinate and multiply under favorable conditions. Factors like tissue destruction, necrosis, inadequate drainage, and soil contaminants create anaerobic environments that promote C. tetani spore germination.
Vegetative cells multiply locally, producing the potent neurotoxin tetanospasmin.
Tetanospasmin travels via peripheral nerves or bloodstream to ganglioside receptors on motor nerve terminals, eventually affecting cells in the spinal cord's ventral horn. This process leads to muscle spasms across various levels of the body.
The toxin acts presynaptically on motor neurons, blocking synaptic inhibition and causing spastic paralysis and characteristic tetanic spasms.
Tetanospasmin binds specifically to nerve tissue gangliosides and cannot be neutralized by antitoxin once bound.
In ascending tetanus, toxin ascends a regional motor nerve in a limb, first affecting muscles in that limb, then spreading to the opposite limb and moving upwards. This pattern is more common in less susceptible animals like dogs and cats.
Descending (generalized) tetanus is typical in more susceptible species such as humans and horses. Here, circulating toxin affects motor nerve centers serving the head and neck initially, then spreading to the limbs.
Once established, tetanus symptoms are similar across all affected animals.
PATHOGENICITY
Symptoms
Several factors influence tetanus, including the location and nature of the wound, as well as the dose and toxicity of the infecting organism.
The incubation period varies widely, ranging from 2 days to several weeks, but typically lasts 6-12 days.
Initial symptoms include mild stiffness and reluctance to move, progressing to rigidity in the head, neck, and tail.
Muscle twitching develops into pronounced spasms triggered by sudden noises or movements, causing the animal to fall and struggle to rise.
In the terminal stages, muscle rigidity spreads from the limbs to the trunk. Symptoms include dilated nostrils, erect ears, protruding nictitating membranes, and difficulty in chewing due to locked jaw (trismus).
Respiration becomes shallow and rapid before ultimately failing.
There are no specific lesions characteristic of tetanus, though superficial wounds from accidental injuries or surgeries may be present.
DIAGNOSIS
Direct microscopy
The presence of characteristic drumstick-shaped spores of C. tetani can be observed in Gram-stained smears of wound material. However, this observation alone is not confirmatory, as C. tetanomorphum and C. tetanoides also produce similar drumstick spores.
Isolation
Necrotic tissue or wound exudates are heated to 80°C for 20 minutes and used to inoculate two types of blood agar plates: one with stiff agar and another without.
Additionally, a tube of thioglycollate medium or cooked meat broth is inoculated and subcultured onto blood agar plates.
Plates are incubated anaerobically for 2-3 days. Growth, observed with a hand lens, appears as filamentous growth spreading throughout the medium.
Pure cultures are obtained from the edges of this growth upon subcultivation.
To confirm the presence of C. tetani, the toxin can be identified. This involves inoculating the toxin from animal serum or filtrate from cooked meat broth or thioglycollate medium into mice subcutaneously (S/C) or intramuscularly (I/M), and conducting neutralization or protection tests using specific antitoxin.
CONTROL AND PREVENTION
The disease results from the toxin's action, making active immunization the most reliable method of prevention.
Available prophylactic methods include:
Surgical intervention
Antibiotics
Immunization, which can be passive, active, or a combination of both.
Surgical attention focuses on removing foreign bodies, necrotic tissue, and blood clots to prevent creating an anaerobic environment favorable for the tetanus bacillus.
Flushing the wound area with hydrogen peroxide promotes aerobic conditions.
Antibiotics, such as large doses of penicillin, can prevent tetanus if administered within 4 hours of infection, but efficacy decreases after 8 hours, emphasizing the need for prompt treatment.
Local application of bacitracin or neomycin is recommended.
Penicillin can be administered via injections or orally until healing is achieved, though antibiotics do not affect the toxin itself.
Antitoxin should be promptly administered intravenously or into the subarachnoid space over three consecutive days to neutralize unbound toxin.
Tetanus toxoid may be administered subcutaneously to stimulate active immune response, even in animals previously treated with antitoxin.
Routine vaccination of farm animals with tetanus toxoid is crucial for prevention.
HISTORY
The term "botulism" originates from the Latin word "botulus," meaning sausage, due to its historical association with food poisoning linked to sausages.
C. botulinum was first isolated by Van Ermengam in 1896 from a piece of ham that caused a botulism outbreak.
C. botulinum refers to a group of bacteria that produce highly potent neurotoxins.
These toxins cause botulism, a condition characterized by flaccid paralysis in many animals and humans.
Botulism is most frequently observed in water birds, ruminants, horses, mink, and poultry.
The disease has been known by various names in different animals:
Horses: Spinal typhus or Shaker foal syndrome
Cattle: Lamsiekte, loin disease, and contagious bulbar paralysis
Waterfowl: Limber neck, alkali poisoning, and western duck sickness
Botulism is rare in domestic cats, while pigs and dogs exhibit relatively higher resistance.
HABITAT
Endospores are extensively found in soils and aquatic environments worldwide.
Botulism primarily results from ingesting preformed toxin.
Germination of endospores and growth of vegetative cells, leading to toxin production, occur in anaerobic conditions. These conditions include contaminated cans of meat, fish, or vegetables, carcasses of invertebrate and vertebrate animals, rotting vegetation, and baled silage.
MORPHOLOGY
These bacteria are Gram-positive, straight rods that can occur singly, in pairs, or occasionally in chains.
Their oval spores are wider than the bacilli and are located centrally, terminally, or subterminally.
They are non-capsulated and exhibit motility due to peritrichous flagella.
CULTURAL AND BIOCHEMICAL CHARACTERISTICS
It is an obligate anaerobe, thriving optimally at temperatures ranging from 30 to 37°C and pH levels between 7 and 7.6. It exhibits robust growth on standard media.
Surface colonies appear large, irregular, and semi-transparent with a fringed border.
Spores are consistently produced in alkaline glucose gelatin media at 20 to 25°C.
On horse blood agar, colonies are large, transparent, with irregular edges and a narrow zone of hemolysis.
On sheep blood agar, C. botulinum produces beta-hemolysis, with colonies slightly domed and ragged-edged.
In cooked meat medium, proteolytic strains (type A, B, and F) cause meat blackening, while non-proteolytic strains (type C, D, and E) do not.
Gelatin is rapidly liquefied by type A and B strains, whereas type C, D, and E liquefy it slowly or not at all.
RESISTANCE
Clostridium botulinum spores exhibit high resistance, surviving temperatures of 100°C for several hours and up to 120°C for up to 10 minutes.
However, spores can be effectively destroyed by autoclaving at 121°C for 15 minutes, whereas the toxins are rendered inactive at 100°C for 20 minutes.
ANTIGENS AND TOXINS
C.botulinum possesses a number of H, O and spore antigens
Eight types of C. botulinum have been classified (Types A to G) based on differences in the toxins they produce, which vary immunologically. These neurotoxins share identical pharmacological actions but differ in potency, distribution, and antigenicity. They can only be neutralized by their specific antiserum.
Unlike other exotoxins, the botulinum toxin is not released during the organism's life.
It only appears in the medium upon the death and autolysis of the cell.
Initially, it is believed to be synthesized as a nontoxic protoxin or progenitor toxin.
Progenitor toxin is activated to active toxin by enzymes like trypsin.
The toxin is sensitive to heat and has a molecular weight of 70,000.
One milligram of neurotoxin contains over 120 million mouse lethal doses.
The lethal dose for humans ranges from 1 to 2 micrograms. This toxin acts slowly, taking several hours to cause death.
Comparison of the toxins of C.tetani and C.botulinum
PATHOGENESIS
C. botulinum does not invade tissues and is considered virtually non-infectious. Botulism manifests in three types.
Food borne botulism
It occurs due to the ingestion of preformed toxin found in food.
The toxin is absorbed from the intestinal tract and travels through the bloodstream to peripheral nerve cells.
It binds to susceptible cells and inhibits the release of acetylcholine at the myoneural junctions.
This leads to flaccid paralysis, with death typically caused by circulatory failure and respiratory paralysis.
Wound botulism
Spores are introduced into wounds, where they germinate.
Toxin is produced at the site of germination and disseminates throughout the body.
This mechanism is believed to cause conditions like shaker foal syndrome in horses.
Infant botulism
This type of botulism occurs when spores are ingested through contaminated food and germinate in the intestines, especially when the normal flora has not fully developed.
It is observed in human infants (known as floppy baby syndrome) and occasionally in rare epidemics of type C botulism in broiler chickens and turkey poults.
PATHOGENICITY
Symptoms
In cattle, the incubation period ranges from 2 to 10 days depending on the dose of toxin ingested.
Initially, there is excitement, followed by incoordination and paralysis of the hind limbs.
Paralysis of muscles in the mouth, pharynx, and neck occurs, causing difficulty in swallowing and protrusion of the tongue. This ultimately leads to death.
In South Africa, this condition is known as lamsiekte in cattle, caused by type D botulinum toxin, especially in phosphorus-deficient animals.
In poultry, ingestion of type C toxin leads to a disease known as duck sickness or western duck disease, and as Limberneck in chickens.
Symptoms include paralysis of the wings, legs, and neck, protrusion of the nictitating membrane, diarrhea, and a comatose state before recovery occurs within 5-6 days.
Lesions
Pathological changes are observed in the central nervous system, particularly in the brainstem and third ventricle, as well as in the form of catarrhal gastroenteritis, hepatitis, and nephrosis.
DIAGNOSIS
Botulism diagnosis relies on history, clinical signs, and the detection and identification of toxin in the serum of critically ill or recently deceased animals. Additionally, testing suspected foodstuff for the presence of toxin and/or C. botulinum is essential.
Toxin demonstration
Serum or centrifuged serum exudates from animals can be directly injected intravenously (0.3ml) or intraperitoneally (0.5ml) into mice.
If toxin is present, mice will exhibit a characteristic "wasp waist" appearance within a few hours to up to 5 days.
This appearance is caused by abdominal breathing due to paralysis of respiratory muscles.
To extract toxin from foodstuffs, the material is ground in saline.
The resulting suspension is then centrifuged, and the supernatant is filtered through a 0.45μm filter.
Since the toxin may initially be in a protoxin form, nine parts of the filtrate are mixed with one part of 1% trypsin solution and incubated at 37°C for 45 minutes.
Mice or guinea pigs are then inoculated intraperitoneally.
Toxin identification
Mouse or guinea pig neutralization tests begin with the use of a polyvalent antitoxin initially, followed by a monovalent antitoxin.
Isolation of C. botulinum from foodstuffs
Several samples of the foodstuffs are blended with a small amount of physiological saline.
The suspension is heated at 65-80°C for 30 minutes to eliminate most contaminating organisms and induce germination of C. botulinum spores.
Blood agar plates are then inoculated with the suspension and placed in a CO2 environment at 35°C for up to 5 days.
To confirm if the isolate produces toxin, it is inoculated into cooked meat broth and incubated at 30°C for 5-10 days.
Filtrates are then prepared and used in laboratory animals to demonstrate and identify the toxin.
CONTROL AND PREVENTION
Polyvalent antiserum effectively neutralizes the toxin.
Therapeutic agents like tetraethylammonium and guanidine hydrochloride, which enhance transmitter release at neuromuscular junctions, may be beneficial when administered intravenously.
Immunization is not routinely practiced. In South Africa, efforts were made by administering two injections of types C and D toxoid several weeks apart.
Bivalent and trivalent antitoxins are also available.
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