Tetanus Toxin

1. Clostridium tetani: Tetanus Toxin
Dr Ravi Kant Agrawal, MVSc, PhD,
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. What is Tetanus?
 An infectious disease caused by contamination
of wounds from the bacteria Clostridium tetani
or the spores they produce that live in the soil,
and animal feces.
 Greek words -“tetanos and teinein”, meaning
rigid and stretched, which describe the
condition of the muscles affected by the toxin,
tetanospasmin, produced by Clostridium tetani.
 The estimated minimum human lethal dose is
2.5 nanograms per kilogram of body weight, or
175 nanograms in a 70 kg (154 lb) human.
• Also known as lockjaw, it is characterised by
muscular spasms.
• In the most common type the spasms begin in
the jaw and then progresses to the rest of the
body.
• These spasms usually last a few minutes each
time and occur frequently for three to four
weeks.

3. Epidemiology
 Tetanus is one of the most basic fatal diseases in existence.
 Clostridium tetani is considered to be the deadliest
bacteriological pathogen in existence, second only to
Clostridium botulinum.
 Tetanus is an international health problem, as spores are
ubiquitous.
 The disease occurs almost exclusively in persons who are
unvaccinated or inadequately immunized.
 Tetanus occurs worldwide but is more common in hot, damp
climates with soil rich in organic matter.
 More common in developing and under developing countries.
 More prevalent in industrial establishment, where agriculture
workers are employed.
 Tetanus neonatorum is common due to lack of MCH care.
 The more frequent cases like neonatal tetanus resulted in over
59000 infant mortalities in 2008 alone.
 The United States alone reports over 30 cases of infection each
year.
 Lack of a proper diagnostic test makes Tetanus all the more
potent a killer.

4. Route of Entry
 Apparently trivial injuries- Usually a puncture
wound - injury by rusty nails/splinters or less
frequently by laceration
 Open fractures
 Burns
 Gangrene or Dead tissue
 Abscess
 Animal bites/human bites
 In neonates: usually via infected umbilical stumps
 Woman during childbirth, through the injured
uterine mucosa
 Parenteral drug abuse
Tetanus prone wound
 A wound sustained more than 6 hr before surgical
treatment.
 A wound sustained at any interval after injury
which is puncture type or shows much devitalized
tissue or is septic or is contaminated with soil or
manure.

5. Source of Infection
 The spores are widely distributed in soil and in the intestines
and feces of horses, sheep, cattle, dogs, cats, rats, guinea pigs,
and chickens.
 Manure-treated soil may contain large numbers of spores.
 In agricultural areas, a significant number of human adults may
harbor the organism.
 The spores can also be found on skin surfaces and in
contaminated heroin.

6. Mechanism of Infection
 C. tetani spores are found throughout the environment, usually in
soil, dust, and animal waste.
 Tetanus is acquired through contact with the environment; it is not
transmitted from person to person.
 C. tetani usually enter the body through an open wound, leading to
spore germination under anaerobic conditions.
 Once spore germination has occurred, toxins are released into the
bloodstream and lymphatic system.
 These toxins act at several locations within the central nervous
system, interfering with neurotransmitter release and blocking
inhibitor impulses.
 Such disruptions lead to un-controllable muscle contractions.
Mechanism of Infection:
 Entry wound -> germinate -> Produce toxin -> Spread through blood
stream -> moves up nerves
 Results in: Spastic paralysis/Rigid Paralysis, cardiac failure, respiratory
failure, trismus, risus sardonicus, opisthotonos.
Cephalic -- poor prognosis
Localized -- favourable prognosis
Prevention: toxoid, 3% formaldehyde

7. Incubation Period
 Varies from 1 day to several months.
 The incubation period is usually 7-10 days.
 A period of onset of less than 48 hr is associated with the
development of severe tetanus.
 In theory, the farther the injury site is from the central nervous
system, the longer the incubation period, and the less severe
are the symptoms experienced.

8. C. tetani: Historical aspects
Tetanus was known to ancient people, who recognized the
relationship between wounds and fatal muscle spasms.
In 1884, Arthur Nicolaier isolated the strychnine-like toxin of
tetanus from free-living, anaerobic soil bacteria.
The etiology of the disease was further elucidated in 1890 by
Antonie Carl and Giorgio Rattone, who demonstrated the
transmissibility of tetanus for the first time. They produced
tetanus in rabbits by injecting their sciatic nerve with pus from a
fatal human tetanus case in that same year.
In 1889, C. tetani was isolated from a human victim, by
Kitasato Shibasaburō, who later showed that the organism
could produce disease when injected into animals, and that the
toxin could be neutralized by specific antibodies.
In 1897, Edmond Nocard showed that tetanus antitoxin induced
passive immunity in humans, and could be used for prophylaxis
and treatment.
Tetanus toxoid vaccine was developed by P. Descombey in 1924,
and was widely used to prevent tetanus induced by battle
wounds during World War II.

9.  Clostridium tetani is a Gram-positive, thin
rod, 2.4-5 μm x 0.5-1.1 μm.
 It is motile by peritrichous flagella.
 Contains granular inclusions which occur
centrally and at the ends of the cell.
 The organism produces round terminal
spores which give it the appearance of a
drumstick or tennis racket.
 Found in soil, carried by horses.
 Electron microscopy shows that the cell wall
is composed of five layers and the
cytoplasmatic membrane of three layers.
 The cytoplasm is dense, granular and
contains ribosomes and polysomes.
 The nucleoid is compact and occupies a
small part of the cell.
 The spores are enclosed by a sporangium.
 The G+C content in DNA is 25%.
 During maximum liberation of the exotoxin,
the cytoplasmatic membrane draws away
from the cell wall and the main bulk of the cell
is lysed.
Clostridium tetani
Round terminal spores give
cells a “drumstick” or “tennis
racket” appearance.

10. Sporulated Vegetative

11. Clostridium tetani with terminal spores

12.  Cl. tetani produces an extremely potent exotoxin
tetanospasmin, A NEUROTOXIN, which causes muscle contraction,
and tetanolysin, A HEMOLYSIN which haemolyses erythrocytes.
 Tetanolysin is related functionally and serologically to
streptolysin O and belongs to a large group of OXYGEN SENSITIVE
HEMOLYSINS from a variety of bacteria.
 In addition to erythrocytes, tetanolysin lyses a variety of cells
such as polymorphonuclear neutrophils, macrophages,
fibroblasts, ascites tumor cells, and platelets.
 It is unknown, however, whether it plays any significant role in
infections by C. tetani.
Toxin production

13.  Tetanospasmin is synthesized in the bacterium as a single
polypeptide chain, but after its release by lysis of the organism, a
bacterial protease cleaves one peptide bond to yield two chains that
remain linked together through a disulfide bond.
 The larger chain (H chain) has a molecular weight of 100,000
daltons, and it possesses the specific receptors that bind the toxin
to the neuronal gangliosides.
 The smaller peptide (L chain) has a molecular weight of 50,000
daltons and is thought to exert the biologic effect of the toxin.
 Tox
 Ne
gangl
Move
transp
Tetanospasmin

14.  The mechanism of action of the toxin is not fully understood,
but it is known that the toxin is first bound to neuronal cells at
the neuromuscular junction.
 The complete toxin then crosses the nerve cell membrane and
is transported retrogradely to the inhibitory inter-neurons.
 There, by an as yet unknown mechanism, the toxin enters the
interneurons and blocks the exocytosis of inhibitory
transmitters, namely, glycine and gamma-aminobutyric acid
(GABA).
 The final effect is a SPASTIC PARALYSIS characterized by the
convulsive contractions of voluntary muscles.
 Because the spasms frequently involve the neck and jaws, the
disease had been referred to as LOCKJAW.
 Death ordinarily results from muscular spasms affecting the
mechanics of respiration.
 In an analogous situation,

15. Toxins: Plasmid coded
 Interestingly, all toxin-producing strains of C. tetani possess a
large plasmid, which encodes for the synthesis of the toxin.
 Loss of the plasmid converts the cell to an avirulent, non-toxin-
producing organism.
 Tetanus toxin is generated in living bacteria, and is released
when the bacteria lyse, such as during spore germination or
vegetative growth.
 A minimal amount of spore germination and vegetative cell
growth are required for toxin production.

16. Tetanus toxin: MOA
Tetanospasmin released in the wound is absorbed into the
circulation and reaches the ends of motor neurons all over the
body.
 The toxin acts at several sites within the central nervous system,
including nerve terminals, the spinal cord, and brain, and within the
sympathetic nervous system.
 By binding to peripheral motor neuron terminals, the toxin enters
the nerve axons, and is transported across synaptic junctions to the
nerve-cell body in the brain stem and spinal cord by
retrograde intraneuronal transport, until it reaches the central
nervous system, where it rapidly binds to gangliosides at the pre-
synaptic membrane of inhibitory motor nerve endings.
The clinical manifestations of tetanus are caused when tetanus
toxin blocks inhibitory impulses, by interfering with the release of
neurotransmitters, including glycine and gamma-aminobutyric acid.
These inhibitory neurotransmitters inhibit the alpha motor neurons.
With diminished inhibition, the resting firing rate of the alpha
motor neuron increases, producing rigidity, unopposed muscle
contraction and spasm.

17.  TeNT is a neuro-specific toxin that binds to Motor Neurons at the
Neuromuscular Junction, where it is internalized and undergoes
axonal retrograde transport to the cell body.
 It is then secreted and taken up by adjacent inhibitory inter-
neurons, where it blocks neurotransmitter release by cleaving
VAMP/synaptobrevin.
 It’s course of action involves:
1. Specific binding in the periphery neurons.
2. Retrograde axonal transport to the central nervous system (CNS)
inhibitory inter-neurons (Movement toward the cell body is
called retrograde transport and movement toward the synapse is
called anterograde transport)
3. Transcytosis from the axon into the inhibitory inter-neurons.
4. Reduction of the disulphide bond between the light and heavy
chain
5. Temperature and pH mediated translocation of the light chain
into the cytosol.
6. Cleavage of synaptobrevin / VAMP (Vesicle associated membrane
proteins (VAMP) (VAMP are a family of SNARE proteins with
similar structure, and are mostly involved in vesicle fusion).

Tetanus toxin: MOA……

18. Tetanus toxin: MOA….
 Mechanism of tetanus toxin similar
to botulinum toxin.
 Difference: CAUSE SPASTIC
INSTEAD OF FLACCID PARALYSIS
 Delivery of toxin to inhibitory
neurons
 GABA and glycine not produced
 Excitatory signal overwhelms
 Muscles go to extreme spasm
 A 0.0000005 ml (0.5nL) dose of
tetanospasmin obtained from a
broth culture filtrate kills a white
mouse which weighs 20 g; and
0.000000005 g (5ng) of dry toxin
obtained by ammonium sulphate
precipitation is fatal to the mouse.
 Several million lethal mouse doses
are contained in 1 mg of crystalline
toxin.

20. Course of Action
• Tetanus toxin is composed of a heavy chain and light chain, which are
attached by a disulphide bond.
• Tetanus toxin fragment C (TeNT-FC) is a 47-kDa fragment on the heavy
chain molecule that contains the ganglioside-binding domain.
• TTFC attaches to gangliosides on the peripheral nerves, and as a
result, the toxin is internalized.
• Through trans-synaptic spread, the toxin can spread to the central
nervous system.
• The light chain contains a ZINC METALLOPROTEASE DOMAIN which
can cleave proteins that facilitate synaptic vessel fusion with the
plasma membrane of the neuron – namely, the integral protein
SYNAPTOBREVIN.
• As a result, the neurotransmitter glycine and γ-aminobutyric acid
(GABA) is blocked from reaching the synaptic cleft, and the excitation
of motor neurons persists.
• Persistent neuron signalling leads to the motor spasms seen in a
typical tetanus patient

28. TOXINA TETANOESPASMINA
Cadena A (ligera 50 KD)Cadena A (ligera 50 KD)
Cadena B (Pesada 100 KD)Cadena B (Pesada 100 KD)
Puentes disulfuroPuentes disulfuro

36.  Cl. tetani is not serologically homogeneous and 11 serological
variants have been recognized.
 Eleven strains of C. tetani differ primarily in flagellar antigens
and in their ability to produce tetanospasmin.
 Variant specificity is associated with the H-antigen and group
specificity with the O-antigen.
 The genes for toxin production are encoded on a plasmid which
is present in all toxigenic strains.
 All strains that are capable of producing toxin produce identical
toxins.
 The I, III, VI, and VII types exhibit a manifest specificity.
 The motile strains contain the H-antigen, and the non-motile
strains contain only the O-antigen.
Antigenic structure

37.  Vegetative cells of the tetanus organism withstand a
temperature of 60-70° C for 30 minutes and are destroyed quite
rapidly by all commonly used disinfectants.
 The spores are very resistant, and survive in soil and on various
objects over a long period of time and with-stand boiling for 10-90
minutes or even, as with spores of certain strains, for 1-3 hours.
 The spores are killed by exposure to a 5% phenol solution for 8-
10 hours, and by a 1% formalin solution, for 6 hours.
 Direct sunlight destroys them in 3-5 days.
Resistance

38. Clinical features: Symptoms
 Risus sardonicus: (a rigid smile) Contraction of the muscles at
the angle of mouth and frontalis
 Trismus (Lock Jaw): Spasm of Masseter muscles.
 Opisthotonus: (Arched back) Spasm of extensor of the neck,
back and legs to form a backward curvature.
 Muscle spasticity
 Prolonged muscular action causes sudden, powerful, and
painful contractions of muscle groups. This is called tetany.
 These episodes can cause fractures and muscle tears.
 Seizures may occur, and the autonomic nervous system may
also be affected.
 Loss of inhibition also affects preganglionic sympathetic
neurons in the lateral gray matter of the spinal cord and
produces sympathetic hyperactivity and high circulating
catecholamine levels.
 Hypertension and tachycardia alternating
with hypotension and bradycardia may develop.
 If respiratory muscle is involved – apnoea.

39. Other Symptoms
Other symptoms include:
 Drooling
 Excessive sweating
 Fever
 Hand or foot spasms
 Irritability
 Swallowing difficulty
 Uncontrolled urination or defecation
 Headache, high blood pressure, and a fast heart rate.

40. Lockjaw

41. Risus sardonicus

42. Opisthotonos

43. Tetanus Neonatorum

44.  Immunity following tetanus infection is mainly antitoxic in
character and of low grade.
 Re-infections may occur.
Immunity

45.  There are currently no blood tests that can be used to diagnose
tetanus - Diagnosis is done clinically.
Laboratory diagnosis is usually not carried out because clinical
symptoms of the disease are self-evident.
 Objects of epidemiological significance (soil, dust, dressings,
preparations used for parenteral injections) are examined
systematically.
 Wounds, dressings, and medicaments used for parenteral injections
are examined for the presence of Cl. tetani and their spores by the
following procedures.
 Specimens are inoculated into flasks or test tubes.
 The sowings are kept at a temperature of 80° C for 20 minutes to
suppress the growth of any non-spore forming microflora which may
be present.
 After ‘2-10 days’ incubation at 35° C, the culture is studied
microscopically and tested for the presence of toxin by injection into
mice.
 If Cl. tetani is present, tetanus of the tail develops during 24-48 hours,
followed by tetanus of the body and death (mouse inoculation test).
 The disease does not occur in mice which have been inoculated with
anti-tetanus serum (mouse protection test).
Laboratory diagnosis

46.  If no tetanus toxin is detected in the first inoculation but
microscopic examination reveals the presence of organisms
morphologically identical with Cl. tetani, the initial culture is
inoculated into a condensated water of coagulated blood
serum.
 A thin film will appear over the entire surface of the medium
after 24 hours' growth in anaerobic conditions.
 Experimental animals are infected with a culture grown on
liquid nutrient medium and kept for 4-5 days at 35° C.
 A biological test is employed for detecting the exotoxin in the
test material extract.
 Two white mice are given intramuscular injections of 0.5-1.0 ml
of a centrifuged precipitate or filtrate of the extract. An equal
amount of the filtrate is mixed with antitoxic serum, left to
stand for 40 minutes at room temperature, and then injected
into another two mice in a dose of 0.75 or 1.5 ml per mouse. If
the toxin is present in the filtrate, the First two mice will die in
2-4 days while the other two (control mice) will survive.

47. Differential Diagnosis
 Rabies
 Masseter muscle spasm due to dental abscess
 Dystonic reaction to phenothiazine
 Hysteria

48.  The organisms are obligate anaerobes.
 They grow on sugar and blood agar at pH 7.0-7.9 and at a
temperature of 38 °C (no growth occurs below 14 and above 45 °C).
 Sometimes a zone of haemolysis is produced around the colonies.
 Brain medium and bismuth-sulphite agar are blackened by Cl.
tetani.
1. Robertson’s cooked meat medium: turbidity & some gas
formation. The meat is not digested but turns black on prolonged
incubation.
2. Blood agar: fine translucent film of growth. α hemolysis is
produced, which later develops into β hemolysis, due to the
production of hemolysin (tetanolysin)
3. Deep agar shake cultures: spherical fluffy balls, 1-3mm in diameter,
made of filaments with radial arrangement.
4. Gelatin stab culture: FIR TREE APPEARANCE with slow liquefaction.
 A uniform turbidity is produced on Kitt-Tarozzi medium with
liberation of gas and a peculiar odour as a result of proteolysis.
Cultivation

49. Clostridium tetani: Colonies in
stab agar culture

50.  No carbohydrates are usually fermented.
 Cl. tetani causes slow gelatin liquefaction and
produces no indole.
 Nitrates are rapidly reduced to nitrites.
 The organisms coagulate milk slowly, forming small
flakes.
Fermentative properties

51. Principle of Treatment
1. Neutralization of unbound toxin with Human tetanus immuno-
globulin
2. Prevention of further toxin production by
- Wound debridement
- Antibiotics (Metronidazole)
3. Control of spasm
- Nursing in quiet environment
- avoid unnecessary stimuli
- Protecting the airway
4. Supportive care
- Adequate hydration
- Nutrition
- Treatment of secondary infection
- prevention of bed sores.

52.  Intramuscular injections of large doses of antitoxic anti-tetanus serum are
employed. The best result is produced by gamma-globulin obtained from the
blood of humans immunized against tetanus.
 Anti-convulsant therapy includes intramuscular injections of 25% solutions of
magnesium sulphate, administration of diplacine (A nicotinic cholinoreceptor
antagonist at the neuromuscular junction; curare-like ganglioblocker),
condelphine (Nicotinic acetylcholine receptor antagonist.), aminazine
(Chlorpromazine) (Antipsychotic (neuroleptic) from the group of
phenothiazine derivatives. Has expressed an antipsychotic, sedative,
antiemetic effect. Attenuates or eliminates the delusions and hallucinations,
relieves agitation reduces affective reactions, anxiety, restlessness, reduces
locomotor
activity.),pipolphen(a PHENOTHIAZINE derivative having marked antihistaminic
activity as well as sedative andantiemetic actions; used to provide bedtime, sur
gical, and obstetrical sedation, to treat allergic
conditions including rhinitis, conjunctivitis, and itching, to manage nausea, vom
iting, and vertigo associated with surgery, pregnancy, or motion
sickness, and as an ingredient in cough and coldpreparations; administered oral
ly, rectally, intramuscularly, and intravenously as the hydrochloridesalt.) or
andaxine and chloral hydrate introduced in enemas.
Treatment

53.  Un-inoculated persons are subjected to active and passive
immunization.
 This is achieved by injecting 0.5 ml of toxoid and 3.000 units of
antitoxic serum and then 5 days later, another 0.5 ml of toxoid.
 The tetanus antitoxin is also introduced into previously
inoculated individuals suffering from a severe wound.
 Injection of the total dose of antitoxin is preceded by an intra-
cutaneous test for body sensitivity to horse protein.
 This is carried out by introducing 0.1 ml of antitoxin,
previously diluted 1:100, into the front part of the forearm.
 If the intra-cutaneous test proves negative, 0.1 ml of whole
antitoxin is injected subcutaneously and if no reaction is
produced in 30 minutes, the total immunization dose is
introduced.

54. The complex of prophylactic measures includes adequate
surgical treatment of wounds.
The organisms are sensitive to penicillin, but the antibiotic
has no effect on the neutralization of the toxin.
However, after surgical cleansing of the wound, antibiotic
therapy can be helpful in preventing any additional growth of the
organisms.

55. Prevention
 Tetanus is completely preventable by active
immunization against tetanus .
 Immunization is thought to provide
protection for 10 years.
 Begins in infancy with the DTP series of
shots. The DTP vaccine is a "3-in-1" vaccine
that protects against diphtheria, pertussis,
and tetanus.
 Can be achieved by active immunization by
tetanus toxoid (5 doses – 0 day, 1 month, 6
month, 1 year, 1 year).
 Older teenagers and adults who have
sustained injuries, especially puncture-type
wounds, should receive booster
immunization for tetanus if more than 10
years have passed since the last booster.
 Clinical tetanus does not produce immunity
to further attacks. Therefore, even after
recovery patients must receive a full course
of tetanus toxoid.

56. Prophylaxis is ensured by prevention of occupational
injuries and traumas in everyday life.
Active immunization is achieved with tetanus toxoid.
It is injected together with a tetravalent or polyvalent
vaccine or maybe a component of an associated adsorbed
vaccine.
The pertussis-diphtheria-tetanus vaccine and associated
diphtheria-tetanus toxoid are employed for specific tetanus
prophylaxis in children.
Immunization is carried out among all children from 5-6
months to 12 years of age, individuals living in certain rural
regions (in the presence of epidemiological indications),
construction workers, persons working at timber, water-supply,
cleansing and sanitation, and peat enterprises, and railway
transport workers.
Prophylaxis

57. Immunization with tetanus toxoid stimulates the
production of sufficient amounts of antitoxin.
Immunity lasts for a period of 2 or 3 years.
The effectiveness of the immunization has made tetanus a
relatively rare disease in the developed countries (36 cases in the
United States during 1994).
In Third World countries, however, most persons are not
immunized, and infections of the umbilical cord give rise to large
numbers of neonatal tetanus. For example, special surveillance
programs indicate death rates from neonatal tetanus to be 2% of
all live births in Bangladesh, 6% in Papua, New Guinea, and 14.5%
in Haiti.
One study relating the proportion of pregnant females with
tetanus antitoxin titres adequate to provide protection for the
newborn found 96% protected in New Haven, Connecticut, and
only 19% in Santiago, Chile.

58. Oral immunization may become possible using a live
attenuated strain of Salmonella typhimurium that was transfected
with a plasmid engineered to express a 50–kd fragment of the
tetanus toxin. Given orally, this strain provided protective
immunity in mice.
After an injury, human tetanus immune globulins should be
administered to those who have never been immunized with
tetanus toxoid or to those who did not receive the full three doses
of toxoid.
Booster injections of toxoid also are given if the immune
status of the patient is unknown, or if it has been over 5 years
since the last dose of toxoid.

59. Toxin similarity
 Botulinum and Tetanospasmin are Zn requiring Endopeptidases
that cleave a set of proteins. Synaptobrevins found in synaptic
vesicles of neurons.
 Interfere with release of neurotransmitters and the normal
inhibitory function.
BUT
Binding regions of tetanus toxin and botulinum toxin are different
in terms of cell specificity.

60. Thanks
Acknowledged: The presentations available online on the subject
are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the
source and authenticity of the content.