food borne food borne bacterial toxins. introduction characteristics of bacterial endotoxins and...
TRANSCRIPT
FOOD BORNE FOOD BORNE
BACTERIAL TOXINSBACTERIAL TOXINS
INTRODUCTION INTRODUCTION
CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN
PATHOGENESIS BASED EXOTOXINS CLASSIFICATION PATHOGENESIS BASED EXOTOXINS CLASSIFICATION
EVENTS OF FOOD BORNE DISEASES
FOOD BORNE ILLNESS
ENTEROTOXINS
PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS
HEMOLYTIC EXOTOXINSHEMOLYTIC EXOTOXINS
NEUROTOXIN
METHODS FOR DETECTION OF TOXINSMETHODS FOR DETECTION OF TOXINS..
SUMMARY
OVERVIEW OF PRESENTATION
Toxins are the toxicants or poisonous substance liberated or produced by Toxins are the toxicants or poisonous substance liberated or produced by living organism and generally not well defined chemically. living organism and generally not well defined chemically.
INTRODUCTIONINTRODUCTION
Depending on origin
Bacterial toxins Mycotoxins Zootoxins Phytotoxins
BACTERIAL TOXIN
ENDOTOXIN EXOTOXIN
Cont…Cont…
ENDOTOXINCont…Cont…
PROPERTYPROPERTY ENDOTOXINENDOTOXIN EXOTOXINEXOTOXIN CHEMICAL NATURECHEMICAL NATURE LPS (mw = 10kDa)LPS (mw = 10kDa) Protein (mw = 50-1000kDa)Protein (mw = 50-1000kDa)
RELATIONSHIP TO CELLRELATIONSHIP TO CELL Part of outer membranePart of outer membrane Extracellular, DiffusibleExtracellular, Diffusible
HEAT STABILITYHEAT STABILITY Yes (60Yes (6000 C) C) Relatively NoRelatively No
ANTIGENICANTIGENIC YesYes YesYes
FORM TOXOIDFORM TOXOID NoNo YesYes
POTENCYPOTENCY Relatively low (100ug)Relatively low (100ug) Relatively high (1 ug)Relatively high (1 ug)
SPECIFICITYSPECIFICITY Low degreeLow degree High degreeHigh degree
ENZYMATIC ACTIVITYENZYMATIC ACTIVITY NoNo OftenOften
PYROGENICITYPYROGENICITY YesYes OccasionallyOccasionally
SYNTHESIS CONTROLSYNTHESIS CONTROL Chromosomal geneChromosomal gene Extrachromosomal geneExtrachromosomal gene
CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN
ENTEROTOXINSENTEROTOXINS V. cholerae, E. coli, B. cereus, V. cholerae, E. coli, B. cereus, Cl. perfringens, Salmonella, Staph. aureus, Cl. perfringens, Salmonella, Staph. aureus, Shigella, NAG vibrios, Aeromonas.Shigella, NAG vibrios, Aeromonas.
HEMOLYTIC EXOTOXINSHEMOLYTIC EXOTOXINS Staph. aureus, Streptococcus, Staph. aureus, Streptococcus, Cl.perfringens, V. parahaemolyticus, Cl.perfringens, V. parahaemolyticus, B. cereus, Aeromonas hydrophila. B. cereus, Aeromonas hydrophila.
NEUROTOXINNEUROTOXIN Cl. botulinum, Cl. tetani, Cl. botulinum, Cl. tetani,
Shigella dysenteriae.Shigella dysenteriae.
CYTOTOXIC –CYTOLYTICCYTOTOXIC –CYTOLYTIC
EXOTOXINEXOTOXIN
Strept. Staph. aureus, Shigella , Strept. Staph. aureus, Shigella , Aeromonas, V. parahaemolyticus, Aeromonas, V. parahaemolyticus, Cl. difficile.Cl. difficile.
DIRECT MACROMOLECULAR DIRECT MACROMOLECULAR
SYNTHESIS INHIBITORSYNTHESIS INHIBITOR
Corynebacterium diphtheriae, E. coliCorynebacterium diphtheriae, E. coli
B.thuringensis, Yersinia pestis, B.thuringensis, Yersinia pestis, Pseudomonas, Vibrio cholerae. Pseudomonas, Vibrio cholerae.
PATHOGENESIS PATHOGENESIS BASEDBASED EXOTOXINS CLASSIFICATION EXOTOXINS CLASSIFICATION
Reservoir of Pathogen Contamination of foodViral or Parasitic
infection
Growth of pathogenic bacteria
Infection Intoxication
Toxicoinfection
Food+ Live cells Food +Toxin
Invasive Infection
EVENTS OF FOOD BORNE DISEASES
Mycotoxin
Invasive Infection IntoxicationToxicoinfection
o Salmonella
o L. monocytogenes
o E. coli ( enteric type)
o Shigella
o Campylobacter
o Yersinia
o Vibrio parahaemolyticus
o Aeromonas
o Staph. Aureus
o B.Cereus (Emetic type)
o Cl. botulinum
o Bacillus cereus (Diarrhoel)
o Cl.botulinum ( Infant) o Cl . perfringens
o Vibrio cholarae
o E. Coli (Enterotoxigenic)
Cont…Cont…
BACTERIAL FOOD BORNE INFECTIONFOOD BORNE BACTERIAL ILLNESS
FOOD BORNE BACTERIAL ILLNESSCont…Cont…
FOOD BORNE BACTERIAL ILLNESSCont…Cont…
Bacterial toxins that exert some deleterious effect and host response exclusively in Bacterial toxins that exert some deleterious effect and host response exclusively in the small or large intestine.the small or large intestine.
Alteration in intestinal cell structure or function byAlteration in intestinal cell structure or function by - -
o A diversity of mode of action,A diversity of mode of action,o Target cell types, Target cell types, o Receptors.Receptors.
Protein associated with cell wall-Protein associated with cell wall-o Produces fluid loss in infant mice.Produces fluid loss in infant mice.
Heat stable and rapid in action.Heat stable and rapid in action.
Heat labile and delayed response :-Heat labile and delayed response :-o Mol. Wt. of 25 kDa,Mol. Wt. of 25 kDa,o Increases in cAMP and PGE2 levels. Increases in cAMP and PGE2 levels. o Stimulate secretion in 18-hStimulate secretion in 18-h
Salmonella
ENTEROTOXINS
Cholera is caused by Cholera is caused by V. cholerae V. cholerae of the O1 and O139 serogroups. of the O1 and O139 serogroups.
CT is the prototypic A-B subunit toxin (A/B ratio, 1:5), CT is the prototypic A-B subunit toxin (A/B ratio, 1:5),
o B is the subunit (11.6 kDa) responsible for binding of the B is the subunit (11.6 kDa) responsible for binding of the holotoxin to its GM1 receptorholotoxin to its GM1 receptor
o A is the subunit responsible for the intracellular A is the subunit responsible for the intracellular changes in cyclic AMP levels.changes in cyclic AMP levels.
V. cholerae
C TC T ZotZot AceAce
Cont…Cont…
Mechanism of Action of Cholera Toxin
Cont…Cont…
GM1
If a strain is CT positive, it is almost always If a strain is CT positive, it is almost always zot zot positive (48 KDa peptide) . positive (48 KDa peptide) .
The onset of action of crude Zot is immediate and reversible. The onset of action of crude Zot is immediate and reversible.
Zot may contribute to diarrhea in cholera by altering the permeability of Zot may contribute to diarrhea in cholera by altering the permeability of intestinal tissue. intestinal tissue.
Ace 11.3-kDa protein. stimulate electrogenic chloride secretion & contribute, Ace 11.3-kDa protein. stimulate electrogenic chloride secretion & contribute, to the pathogenicity of to the pathogenicity of V. cholerae V. cholerae O1.O1.
Produce a 17-a.a. NAG-ST that shares 50% sequence homology with the STa Produce a 17-a.a. NAG-ST that shares 50% sequence homology with the STa of ETEC . of ETEC .
This toxin is found only in a minority of non-O1/non-O139 This toxin is found only in a minority of non-O1/non-O139 V. cholerae V. cholerae strains.strains.
NAG Vibrio
Cont…Cont…
EAEC- EAEC-
EIEC –EIEC –
EHEC -EHEC -
E. coli
Stx1, SLT-I or VTIStx1, SLT-I or VTI
ShET2 or ShET2 or EIEC enterotoxinEIEC enterotoxin
EAST 1 EAST 1 4.1 kDa protein4.1 kDa protein
Heat-labile proteinHeat-labile protein108 kDa ( Pet)108 kDa ( Pet)
S. flexneriS. flexneri
Stx2, SLT-II or VT2Stx2, SLT-II or VT2(Stx2c,2d,2e)(Stx2c,2d,2e)
Cont…Cont…
Chrom.encodedChrom.encoded110 kDa (Pic)110 kDa (Pic)
STx, SLT or VTSTx, SLT or VT
S.dysenteriaeShiga toxin
ST ST LT LT
STbSTbSTaSTa LTILTI LTIILTII
o Cysteine-rich, Cysteine-rich, o 18 / 19-a.a. peptide18 / 19-a.a. peptideo Mol wt 2 kDaMol wt 2 kDao Bind to G C C-Bind to G C C- Increase cGMP-Increase cGMP- PKA- PKA- CFTR-CFTR- Chloride sec.-Chloride sec.-o DiarrheaDiarrheao Children -more Children -more effectedeffectedo Human & Animal
o Trypsin-sensitive Trypsin-sensitive proteinproteino No toxic domainNo toxic domaino Nor Intestinal Nor Intestinal receptorreceptoro Sulfatid receptorSulfatid receptoro No effect on cAMPNo effect on cAMP or cGMP.or cGMP.o Not Stimulate Not Stimulate chloride sec.chloride sec.o Bicarbonate sec.Bicarbonate sec.o Piglets Piglets
o Mol wt 80,000Mol wt 80,000o Bind to GM 1 Bind to GM 1 o Activate Activate Adenylate cyclaseAdenylate cyclaseo Increase cAMPIncrease cAMPo Increase PGIncrease PGo Stimulate GIT Stimulate GIT Nervous systemNervous systemo Human Human & Animal& Animal
o Absent of B Absent of B subunitsubunito 2 sub unit 2 sub unit LTIIa <IIb LTIIa <IIb o Bind to-Bind to- Gangliosid Gangliosid GD1b or GD1aGD1b or GD1ao Increase cAMPIncrease cAMPo Animal diseaseAnimal disease
ETECETEC
Cont…Cont…
Shigella
S. flexneri S. flexneri 2a. 2a.
S. dysenteriae S. dysenteriae II Shiga toxinShiga toxin
ShET2 or ShET2 or EIEC enterotoxinEIEC enterotoxinShET1ShET1
Cont…Cont…
o Chomo. Encodedo Iron dependento 55 KDa
o Plasmid encoded proteino 63 KDa
Y. enterocolitica
C. jejuni
o Heat-labile enterotoxin (LT-like) Heat-labile enterotoxin (LT-like) o 60 to 70 kDa 60 to 70 kDa o Iron regulated. Iron regulated.
o YstYsto Produced at -Produced at -
o 3030oo C Co Increased osmolarity Increased osmolarity & pH at 37& pH at 3700 C favour Yst. C favour Yst.
o Guanylate cyclase activationGuanylate cyclase activationo Increase Increase cGMP levels cGMP levels
o Yst-IIYst-IIo 10- to 30-kDa10- to 30-kDao Protein toxin Protein toxin o Similar to Similar to E. coli E. coli STaSTa
Cont…Cont…
Staph. aureus
ExotoxinsExotoxins
PTS AgsPTS Ags HemolysinsHemolysins
SEsSEsTSST-1TSST-1
o 18 toxins- 18 toxins- o Classical enterotoxins -Classical enterotoxins -
SEA to SEESEA to SEEo Recently discovered – Recently discovered –
o True SEs-- SEG, SEH & SEI True SEs-- SEG, SEH & SEI
o SEls-SEls- ( SElJ to SElP and SElU ).( SElJ to SElP and SElU ).
Cont…Cont…
SEs - thermostable , resistant to GIT enzymes. Heat stability depend on SEs - thermostable , resistant to GIT enzymes. Heat stability depend on the environmental factors - athe environmental factors - aww & pH . & pH . (Balaban (Balaban et alet al., 2000)., 2000)
SEA and SED, the most frequently involved in food poisoning .SEA and SED, the most frequently involved in food poisoning . (Balaban (Balaban et alet al., 2000 ; Le ., 2000 ; Le et alet al ., 2003 ) ., 2003 )
5% cases attributed to new enterotoxins & SEH most commonly 5% cases attributed to new enterotoxins & SEH most commonly reported . reported . (Jorgensen (Jorgensen et alet al ., 2005) ., 2005)
SEs are potent emetic agents whereas the other PTSAgs are not . SEs are potent emetic agents whereas the other PTSAgs are not .
The conc. of SE from 0.5 to 1.0 ng/ml and depends on the susceptibility The conc. of SE from 0.5 to 1.0 ng/ml and depends on the susceptibility of the individual. of the individual.
Cont…Cont…
B. cereus
o Thermolabile antigenic protein
o Mol wt. 38,000 - 57,000 Da
o Susceptible to – o 560 C - 30 min.
o Trypsin & Pronase digestion
DIARRHEAGENICtoxin
EMETICtoxin
o Highly stable
o Probably a peptide
o Mol. Wt. < 10,000
o Withstand –o 1260C - 1.5 hr,
o Extreme pH,
o Typsin & Pepsin exposure
Cont…Cont…
o Wide-range of foods.Wide-range of foods.
o Large no. of the m.o.Large no. of the m.o. o IP 8–16.5 hr. IP 8–16.5 hr.
o Diarrhea, abd. cramps Diarrhea, abd. cramps & tenesmus.& tenesmus. o Long-incubation form.Long-incubation form. o Clostridium perfringensClostridium perfringens
Diarrheal typeDiarrheal type
Toxicoinfection
Cont…Cont…
o Cooked rice & improperlyCooked rice & improperly refrigerated foods .refrigerated foods .
o Preformed toxinPreformed toxin o I P 1–5 hoursI P 1–5 hours
o Nausea & VomitingNausea & Vomiting o Recovery within 24 hrRecovery within 24 hr o Staphylococcus aureusStaphylococcus aureus
Emetic FormEmetic Form
Intoxication
Several cytotonic enterotoxinsSeveral cytotonic enterotoxins
o 15-20 kDa heat stable protein15-20 kDa heat stable protein
o 44 kDa heat labile protein44 kDa heat labile protein
o CT-like enterotoxinCT-like enterotoxin
.
Aeromonas
Cont…Cont…
PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS
Heat Labile Heat StableSalmonella, E. coli, Campylobacter ,
Vibrio cholerae, Aeromonas, B.cereus E.coli, Y.enterocolitica, Staph.aureus, B.cereus.
o PRODUCTION : o In host o During vegetative growtho Secreted in Ileumo Sub unit 6 subunits (1 A & 5 B )
o TRYPSINo Proteolysis
o Heat stabilityo 60 0 C(650 C -30 min.)
o ENZYMATIC ACTIVITY o ADP ribosylate
o AFFECTo Bind to Ganglioside receptor (epithelialCell) o Alteration of trans membrane signal transductiono Diarrhoea
1000 C -30 min.
No effect.
Staph.aureus Staph.aureus
αα
ββ γγ δδ
StreptococcusStreptococcus SLSSLS SLOSLO
Cl.perfringensCl.perfringens
αα δδ θ
V. parahaemolyticusV. parahaemolyticus
TDH(100TDH(1000 0 C -10 min)C -10 min)
B. cereusB. cereus
Primary hemolysin –Primary hemolysin –
H-1, Cereolysin & H-1, Cereolysin &
Mouse lethal factorMouse lethal factor
2ndry –2ndry –
H -IIH -II
A. hydrophilaA. hydrophila
αα hemolysin hemolysin
β β hemolysinhemolysin
(Aerolysin)(Aerolysin)
HEMOLYTIC EXOTOXINSHEMOLYTIC EXOTOXINS
:-:-
BoNT also known as “Botox”.BoNT also known as “Botox”.
150-kDa zn-binding metallo protease (holotoxin) 100-kDa heavy & 50-150-kDa zn-binding metallo protease (holotoxin) 100-kDa heavy & 50-kDa light chain connected by a reducible disulphide bond . kDa light chain connected by a reducible disulphide bond . (Schiavo (Schiavo et alet al., 2000)., 2000)
100,000 times more toxic than sarin . (Shapiro 100,000 times more toxic than sarin . (Shapiro et alet al., 1998).., 1998).
Estimated human lethal Estimated human lethal i.v.i.v. dose 1-2 ng/kg body weight . dose 1-2 ng/kg body weight . ( Arnon ( Arnon et alet al., 2001)., 2001)
Botulin toxin - potential bioweapon, 75 nanograms to kill a person Botulin toxin - potential bioweapon, 75 nanograms to kill a person (LD50- 1ng/kg). (Fleming(LD50- 1ng/kg). (Fleming., 2000)., 2000)
500 grams is enough to kill half of the entire human population 500 grams is enough to kill half of the entire human population
Shiga toxinShiga toxin. .
NEUROTOXIN
Clostridium botulinumClostridium botulinum
Shigella dysenteriaeShigella dysenteriae
BIOASSAY METHOD. BIOASSAY METHOD.
IMMUNOLOGICAL ASSAYS. IMMUNOLOGICAL ASSAYS.
MOLECULAR TECHNIQUES. MOLECULAR TECHNIQUES.
CELL CULTURES.CELL CULTURES.
METHODS FOR DETECTION OF TOXINSMETHODS FOR DETECTION OF TOXINS
MOUSE LETHALITY
o Botulinum toxin in foods
o Cl.perfringens enterotoxin o Listeria virulence assesment
WHOLE ANIMAL ASSAYS
BIOASSAY METHODBIOASSAY METHOD
FERRETS
o Staphylococcal enterotoxin B
Cont…Cont…
MONKEY FEEDING TEST
o Staph enterotoxins ( Most sensitive )
SUNCUS MURINUS EMESIS MODEL
o Cereulide – B. cereus
Cont…Cont…
KITTEN / CAT TEST
o Staph enterotoxins
RABBIT & MOUSE DIARRHOEA
o E.colio Y.enterocoliticao Vibrio cholerae (enterotoxins)
Cont…Cont…
SUCKLING / INFANT MOUSE o E. Coli enterotoxins ( STa, STb )
o Y. enterocolitica (Yst)
RABBIT SKIN TEST
Vascular permeability-o E. Coli (diarrheagenic enterotoxin)
Cont…Cont…
GUINEA PIG SKIN TEST
Erythemal activity- o Cl. perfringens enterotoxin
SERENY & ANTON TEST
Guinea pig, Mouse & Rabbit
o Shigella
o EIEC o Listeria
Cont…Cont…
LIGATED LOOP TECHNIQUE
o B.cereus, o Cl perfringens, o E. coli, o V. parahaemolyticus
RITARD MODEL
o ETECo V.Choleraeo C. Jejunio Aeromonas
ANIMAL MODELS REQUIRE SURGICAL PROCEDURES
Cont…Cont…
ELISAELISA
Double antibody "sandwich" ELISA
Bennett,.et al; 1994
IMMUNOLOGICAL ASSAYSIMMUNOLOGICAL ASSAYS
Staph enterotoxins A - E
Solid phase RIA : Solid phase RIA :
Staph. ETStaph. ET -: A to E -: A to E Sensitivity - 1-5 ng Sensitivity - 1-5 ng
rangerange E.coliE.coli STa STa
RadioimmunoassayRadioimmunoassay
Cont…Cont…
Antibody coupled to sheep RBCsAntibody coupled to sheep RBCs
Agglutination if toxin presentAgglutination if toxin present
Sensitivity forSensitivity for SE–BSE–B : 1.5 ng /ml: 1.5 ng /ml
Reverse passive haemagglutinationReverse passive haemagglutination
VIDAS SET AssayVIDAS SET Assay
Immunoenzymatic testImmunoenzymatic test..
Specific for Staph enterotoxin A - E.
Staph. enterotoxins A - E Staph. enterotoxins A - E
Sensitivity of at least 1 ng/ml.Sensitivity of at least 1 ng/ml.
Cont…Cont…
Microslide gel double diffusion test:Microslide gel double diffusion test:
B.cereus diarrhoeal enterotoxinsB.cereus diarrhoeal enterotoxins
Cl. perfringens enterotoxinsCl. perfringens enterotoxins
Staph. B enterotoxinStaph. B enterotoxin
Simple gel ID tech. (Biken’s test):Simple gel ID tech. (Biken’s test):
E. coliE. coli
Sensitivity : 98%Sensitivity : 98%
Time: 3-4 daysTime: 3-4 days
Gel diffusion assayGel diffusion assay
Cont…Cont…
SET-ASET-A
B. cereus B. cereus enterotoxinenterotoxin
Cl.botulinum Cl.botulinum enterotoxin A to E.enterotoxin A to E.
ElectroimmunodiffusionElectroimmunodiffusion
Cont…Cont…
Detection of ST- A,BDetection of ST- A,B
Sensitivity – Sensitivity –
0.3mcg /ml0.3mcg /ml
Radial immunodiffusion assayRadial immunodiffusion assay
Cont…Cont…
Detection of soluble antigenDetection of soluble antigen
Antibody + latex particleAntibody + latex particle
Presence of toxin : diffuse layer Presence of toxin : diffuse layer at baseat base
Negative : button formationNegative : button formation
Simple, rapidSimple, rapid
B. cereus emetic toxin.B. cereus emetic toxin.
Reverse passive latex agglutinationReverse passive latex agglutination
Cont…Cont…
Assay with a large immuno-sorbent surface area. Assay with a large immuno-sorbent surface area.
Captures a low number of toxin molecules and measures their intrinsicCaptures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate.metalloprotease activity with a fluorogenic substrate.
An inexpensive, simple and robust procedure that ensures high analytical An inexpensive, simple and robust procedure that ensures high analytical specificity and attomolar sensitivity for the detection of BoNT/A in complexspecificity and attomolar sensitivity for the detection of BoNT/A in complex biological samplesbiological samples
BoNT/A ALISSABoNT/A ALISSA
Cont…Cont…
ELECTRO CHEMI LUMINESCENCE
To detect Clostridium botulinum neurotoxins serotypes A, B, E, and F
Similar to the gold standard mouse bioassay.
ECL ASSAYS
Cont…Cont…
Duplex SYBR green I real-time Duplex SYBR green I real-time PCR assay-PCR assay-
for one-step differentiation for one-step differentiation between emetic B. cereus and S. between emetic B. cereus and S. aureus. (Mackay., 2004) aureus. (Mackay., 2004)
Real-time PCR-based assaysReal-time PCR-based assays - - BoNT A, B, E BoNT A, B, E and F gene fragments and F gene fragments
(Akbulut ., 2005)(Akbulut ., 2005)
PCRPCR
MOLECULAR TECHNIQUESMOLECULAR TECHNIQUES
TaqMan-based, real-time TaqMan-based, real-time PCR assayPCR assay – –
Provide a rapid and Provide a rapid and sensitive method for the sensitive method for the specific detection of emetic B. specific detection of emetic B. cereus in foodcereus in food
NUCLEIC ACID PROBESNUCLEIC ACID PROBES
Cl.perfringensCl.perfringens enterotoxin enterotoxin
Staphylococcal enterotoxinStaphylococcal enterotoxin
Cont…Cont…
E.coli E.coli LTLT V.choleraeV.cholerae ET ET SalmonellaSalmonella ET ET
CELL CULTURESCELL CULTURES
Human foetal intestineHuman foetal intestine
CHO monolayerCHO monolayer
V.parahaemolyticusV.parahaemolyticus
Vero cellsVero cells
E.coli E.coli LTLT
Cl.perfringensCl.perfringens ET ET
SalmonellaSalmonella cytotoxin cytotoxin A.hydrophillaA.hydrophilla toxin toxin
Y-1 adrenal cellsY-1 adrenal cells
E.coli E.coli ETET
V.choleraeV.cholerae toxin toxin
Cont…Cont…
SUMMARY
Both Gram positive and Gram negative bactera are resposible for most of the outbreak of the food poisining.
Bacterial exotoxins have enterotoxic, cytotoxic, hemolytic and neurotoxic effect .
It is the bacterial enterotoxins which are responsible for production of various type of gastrointestinal manifestations like diarrhea and vomiting during different food borne bacterial illness.
Some bacterial toxins are very potent and relatively easy to produce and classified As bio-threat agents. Eg.. Botulinum neurotoxins.
Bioassay method, Immunological assays, Molecular techniques & Cell Bioassay method, Immunological assays, Molecular techniques & Cell cultures are used to detect the bacterial toxin.cultures are used to detect the bacterial toxin.
REFERENCES:www.Slideshare.com