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  • 1. FOOD BORNE BACTERIAL TOXINS
  • 2.  INTRODUCTION  CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN  PATHOGENESIS BASED EXOTOXINS CLASSIFICATION  EVENTS OF FOOD BORNE DISEASES  FOOD BORNE ILLNESS  ENTEROTOXINS  PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS  HEMOLYTIC EXOTOXINS  NEUROTOXIN  METHODS FOR DETECTION OF TOXINS.  SUMMARY OVERVIEW OF PRESENTATION
  • 3.  Toxins are the toxicants or poisonous substance liberated or produced by living organism and generally not well defined chemically. INTRODUCTION Depending on origin Bacterial toxins Mycotoxins Zootoxins Phytotoxins
  • 4. BACTERIAL TOXIN ENDOTOXIN EXOTOXIN Cont…
  • 5. ENDOTOXIN Cont…
  • 6. PROPERTY ENDOTOXIN EXOTOXIN CHEMICAL NATURE LPS (mw = 10kDa) Protein (mw = 50-1000kDa) RELATIONSHIP TO CELL Part of outer membrane Extracellular, Diffusible HEAT STABILITY Yes (600 C) Relatively No ANTIGENIC Yes Yes FORM TOXOID No Yes POTENCY Relatively low (100ug) Relatively high (1 ug) SPECIFICITY Low degree High degree ENZYMATIC ACTIVITY No Often PYROGENICITY Yes Occasionally SYNTHESIS CONTROL Chromosomal gene Extrachromosomal gene CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN
  • 7. ENTEROTOXINS V. cholerae, E. coli, B. cereus, Cl. perfringens, Salmonella, Staph. aureus, Shigella, NAG vibrios, Aeromonas. HEMOLYTIC EXOTOXINS Staph. aureus, Streptococcus, Cl.perfringens, V. parahaemolyticus, B. cereus, Aeromonas hydrophila. NEUROTOXIN Cl. botulinum, Cl. tetani, Shigella dysenteriae. CYTOTOXIC –CYTOLYTIC EXOTOXIN Strept. Staph. aureus, Shigella , Aeromonas, V. parahaemolyticus, Cl. difficile. DIRECT MACROMOLECULAR SYNTHESIS INHIBITOR Corynebacterium diphtheriae, E. coli B.thuringensis, Yersinia pestis, Pseudomonas, Vibrio cholerae. PATHOGENESIS BASED EXOTOXINS CLASSIFICATION
  • 8. Reservoir of Pathogen Contamination of food Viral or Parasitic infection Growth of pathogenic bacteria Infection Intoxication Toxicoinfection Food+ Live cells Food +Toxin Invasive Infection EVENTS OF FOOD BORNE DISEASES Mycotoxin
  • 9. 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…
  • 10. BACTERIAL FOOD BORNE INFECTIONFOOD BORNE BACTERIAL ILLNESS
  • 11. FOOD BORNE BACTERIAL ILLNESSCont…
  • 12. FOOD BORNE BACTERIAL ILLNESSCont…
  • 13.  Bacterial toxins that exert some deleterious effect and host response exclusively in the small or large intestine.  Alteration in intestinal cell structure or function by - o A diversity of mode of action, o Target cell types, o Receptors.  Protein associated with cell wall- o Produces fluid loss in infant mice.  Heat stable and rapid in action.  Heat labile and delayed response :- o Mol. Wt. of 25 kDa, o Increases in cAMP and PGE2 levels. o Stimulate secretion in 18-h Salmonella ENTEROTOXINS
  • 14.  Cholera is caused by V. cholerae of the O1 and O139 serogroups.  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 holotoxin to its GM1 receptor o A is the subunit responsible for the intracellular changes in cyclic AMP levels. V. cholerae C T Zot Ace Cont…
  • 15. Mechanism of Action of Cholera Toxin Cont… GM1
  • 16.  If a strain is CT positive, it is almost always zot positive (48 KDa peptide) .  The onset of action of crude Zot is immediate and reversible.  Zot may contribute to diarrhea in cholera by altering the permeability of intestinal tissue.  Ace 11.3-kDa protein. stimulate electrogenic chloride secretion & contribute, to the pathogenicity of V. cholerae O1.  Produce a 17-a.a. NAG-ST that shares 50% sequence homology with the STa of ETEC .  This toxin is found only in a minority of non-O1/non-O139 V. cholerae strains. NAG Vibrio Cont…
  • 17.  EAEC-  EIEC –  EHEC - E. coli Stx1, SLT-I or VTI ShET2 or EIEC enterotoxin EAST 1 4.1 kDa protein Heat-labile protein 108 kDa ( Pet) S. flexneri Stx2, SLT-II or VT2 (Stx2c,2d,2e) Cont… Chrom.encoded 110 kDa (Pic) STx, SLT or VT S.dysenteriae Shiga toxin
  • 18. ST LT STbSTa LTI LTII o Cysteine-rich, o 18 / 19-a.a. peptide o Mol wt 2 kDa o Bind to G C C- Increase cGMP- PKA- CFTR- Chloride sec.- o Diarrhea o Children -more effected o Human & Animal o Trypsin-sensitive protein o No toxic domain o Nor Intestinal receptor o Sulfatid receptor o No effect on cAMP or cGMP. o Not Stimulate chloride sec. o Bicarbonate sec. o Piglets o Mol wt 80,000 o Bind to GM 1 o Activate Adenylate cyclase o Increase cAMP o Increase PG o Stimulate GIT Nervous system o Human & Animal o Absent of B subunit o 2 sub unit LTIIa &LTIIb o Bind to- Gangliosid GD1b or GD1a o Increase cAMP o Animal disease  ETEC Cont…
  • 19. Shigella S. flexneri 2a. S. dysenteriae I Shiga toxin ShET2 or EIEC enterotoxinShET1 Cont… o Chomo. Encoded o Iron dependent o 55 KDa o Plasmid encoded protein o 63 KDa
  • 20. Y. enterocolitica C. jejuni o Heat-labile enterotoxin (LT-like) o 60 to 70 kDa o Iron regulated. o Yst o Produced at - o 30o C o Increased osmolarity & pH at 370 C favour Yst. o Guanylate cyclase activation o Increase cGMP levels o Yst-II o 10- to 30-kDa o Protein toxin o Similar to E. coli STa Cont…
  • 21. Staph. aureus Exotoxins PTS Ags Hemolysins SEsTSST-1 o 18 toxins- o Classical enterotoxins - SEA to SEE o Recently discovered – o True SEs-- SEG, SEH & SEI o SEls- ( SElJ to SElP and SElU ). Cont…
  • 22.  SEs - thermostable , resistant to GIT enzymes. Heat stability depend on the environmental factors - aw & pH . (Balaban et al., 2000)  SEA and SED, the most frequently involved in food poisoning . (Balaban et al., 2000 ; Le et al ., 2003 )  5% cases attributed to new enterotoxins & SEH most commonly reported . (Jorgensen et al ., 2005)  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 of the individual. Cont…
  • 23. 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 DIARRHEAGENIC toxin EMETIC toxin 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…
  • 24. o Wide-range of foods. o Large no. of the m.o. o IP 8–16.5 hr. o Diarrhea, abd. cramps & tenesmus. o Long-incubation form. o Clostridium perfringens Diarrheal type Toxicoinfection Cont… o Cooked rice & improperly refrigerated foods . o Preformed toxin o I P 1–5 hours o Nausea & Vomiting o Recovery within 24 hr o Staphylococcus aureus Emetic Form Intoxication
  • 25.  Several cytotonic enterotoxins o 15-20 kDa heat stable protein o 44 kDa heat labile protein o CT-like enterotoxin . Aeromonas Cont…
  • 26. PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS Heat Labile Heat Stable Salmonella, E. coli, Campylobacter , Vibrio cholerae, Aeromonas, B.cereus E.coli, Y.enterocolitica, Staph.aureus, B.cereus. o PRODUCTION : o In host o During vegetative growth o Secreted in Ileum o Sub unit 6 subunits (1 A & 5 B ) o TRYPSIN o Proteolysis o Heat stability o 60 0 C(650 C -30 min.) o ENZYMATIC ACTIVITY o ADP ribosylate o AFFECT o Bind to Ganglioside receptor (epithelialCell) o Alteration of trans membrane signal transduction o Diarrhoea 1000 C -30 min. No effect.
  • 27. Staph.aureus α β γ δ Streptococcus SLS SLO Cl.perfringens α δ θ V. parahaemolyticus TDH(1000 C -10 min) B. cereus Primary hemolysin – H-1, Cereolysin & Mouse lethal factor 2ndry – H -II A. hydrophila α hemolysin β hemolysin (Aerolysin) HEMOLYTIC EXOTOXINS
  • 28. :-  BoNT also known as “Botox”.  150-kDa zn-binding metallo protease (holotoxin) 100-kDa heavy & 50- kDa light chain connected by a reducible disulphide bond . (Schiavo et al., 2000)  100,000 times more toxic than sarin . (Shapiro et al., 1998).  Estimated human lethal i.v. dose 1-2 ng/kg body weight . ( Arnon et al., 2001)  Botulin toxin - potential bioweapon, 75 nanograms to kill a person (LD50- 1ng/kg). (Fleming., 2000)  500 grams is enough to kill half of the entire human population  Shiga toxin. NEUROTOXIN Clostridium botulinum Shigella dysenteriae
  • 29.  BIOASSAY METHOD.  IMMUNOLOGICAL ASSAYS.  MOLECULAR TECHNIQUES.  CELL CULTURES. METHODS FOR DETECTION OF TOXINS
  • 30. MOUSE LETHALITY o Botulinum toxin in foods o Cl.perfringens enterotoxin o Listeria virulence assesment WHOLE ANIMAL ASSAYS BIOASSAY METHOD
  • 31. FERRETS o Staphylococcal enterotoxin B Cont… MONKEY FEEDING TEST o Staph enterotoxins ( Most sensitive )
  • 32. SUNCUS MURINUS EMESIS MODEL o Cereulide – B. cereus Cont… KITTEN / CAT TEST o Staph enterotoxins
  • 33. RABBIT & MOUSE DIARRHOEA o E.coli o Y.enterocolitica o Vibrio cholerae (enterotoxins) Cont… SUCKLING / INFANT MOUSE o E. Coli enterotoxins ( STa, STb ) o Y. enterocolitica (Yst)
  • 34. RABBIT SKIN TEST Vascular permeability- o E. Coli (diarrheagenic enterotoxin) Cont… GUINEA PIG SKIN TEST Erythemal activity- o Cl. perfringens enterotoxin
  • 35. SERENY & ANTON TEST Guinea pig, Mouse & Rabbit o Shigella o EIEC o Listeria Cont…
  • 36. LIGATED LOOP TECHNIQUE o B.cereus, o Cl perfringens, o E. coli, o V. parahaemolyticus RITARD MODEL o ETEC o V.Cholerae o C. Jejuni o Aeromonas ANIMAL MODELS REQUIRE SURGICAL PROCEDURES Cont…
  • 37. ELISA Double antibody "sandwich" ELISA Bennett,.et al; 1994 IMMUNOLOGICAL ASSAYS  Staph enterotoxins A - E
  • 38.  Solid phase RIA : Staph. ET -: A to E  Sensitivity - 1-5 ng range  E.coli STa Radioimmunoassay Cont…
  • 39.  Antibody coupled to sheep RBCs  Agglutination if toxin present  Sensitivity for SE–B : 1.5 ng /ml Reverse passive haemagglutination VIDAS SET Assay Immunoenzymatic test.  Specific for Staph enterotoxin A - E.  Staph. enterotoxins A - E  Sensitivity of at least 1 ng/ml. Cont…
  • 40.  Microslide gel double diffusion test:  B.cereus diarrhoeal enterotoxins  Cl. perfringens enterotoxins  Staph. B enterotoxin  Simple gel ID tech. (Biken’s test):  E. coli  Sensitivity : 98%  Time: 3-4 days Gel diffusion assay Cont…
  • 41.  SET-A  B. cereus enterotoxin  Cl.botulinum enterotoxin A to E. Electroimmunodiffusion Cont…
  • 42.  Detection of ST- A,B  Sensitivity – 0.3mcg /ml Radial immunodiffusion assay Cont…
  • 43.  Detection of soluble antigen  Antibody + latex particle  Presence of toxin : diffuse layer at base  Negative : button formation  Simple, rapid  B. cereus emetic toxin. Reverse passive latex agglutination Cont…
  • 44.  Assay with a large immuno-sorbent surface area.  Captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate.  An inexpensive, simple and robust procedure that ensures high analytical specificity and attomolar sensitivity for the detection of BoNT/A in complex biological samples BoNT/A ALISSA Cont…
  • 45.  ELECTRO CHEMI LUMINESCENCE  To detect Clostridium botulinum neurotoxins serotypes A, B, E, and F  Similar to the gold standard mouse bioassay. ECL ASSAYS Cont…
  • 46.  Duplex SYBR green I real-time PCR assay- for one-step differentiation between emetic B. cereus and S. aureus. (Mackay., 2004)  Real-time PCR-based assays - BoNT A, B, E and F gene fragments (Akbulut ., 2005) PCR MOLECULAR TECHNIQUES
  • 47. TaqMan-based, real-time PCR assay –  Provide a rapid and sensitive method for the specific detection of emetic B. cereus in food NUCLEIC ACID PROBES  Cl.perfringens enterotoxin  Staphylococcal enterotoxin Cont…
  • 48.  E.coli LT  V.cholerae ET  Salmonella ET CELL CULTURES Human foetal intestine CHO monolayer  V.parahaemolyticus
  • 49. Vero cells  E.coli LT  Cl.perfringens ET  Salmonella cytotoxin  A.hydrophilla toxin Y-1 adrenal cells  E.coli ET  V.cholerae toxin Cont…
  • 50. 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 cultures are used to detect the bacterial toxin.