1. Bacterial toxin Clostridium botulinum The most potent biological toxin 10 ng can kill a human 1 g can kill 100 million people Preserved foods such as sausage (botulus) C. botulinum was first recognized and isolated in 1897 by Emile van Ermengem from home cured ham and victim's liver in Belgian. Inject the isolated culture into animals and the same symptoms of human were obtained.

2. 11 people died after eating wax-bean in Darmstadt, Germany. – Not only meat but also vegetables Antiserum of German and Belgian could not neutralize each other. – Classified as A and B Home and commercial canned foods in early 20 century. Autoclave was invented to control bolutlism.

3. types Neurotoxin types, A – G Food-borne – Type A, B – Type E in seafoods, F is rare Wound bolutism – Deep infection in wounds – 華陀刻骨療傷關公 though symptoms described in the novel were close to anthrax infection

4. Infant botulism – Less than one year old – Ingestion of C. botulinum spores – Colonization on intestinal tract, and produce toxin – Infants do not possess normal intestinal flora which can suppress C. botulinum – Type A and B – Honey – Sudden infact death syndrome (SIDS)

6. animal Undetermined C, D, G,

7. The organism Strict anaerobic, gram-positive, spore-forming (spores live forever and happily ever after germination) Four groups Group I: proteolytic, sacchrolytic, heat resistance Group II: non-proteolytic but sacchrolytic, psychrotrophic, heat-liable Group III: mildly or non-proteolytic but saccharolytic, intermediate heat resistant Group IV: requires truptic to full toxicity (type G) pH, temperature, NaCl, heat resistant

11. Neurotoxin Similar to PSP, puffer fish, …etc Toxin is produced during the exponential phase of growth As cells cease to grow and divide, toxin is release as a result of cell lysis. (group I) Group II doesn’t autolysis and do not efficiently release toxin.

12. Toxin classification Type A to G Immunological response One strain only produce one type of toxin C1 and C2: one of them is dominant in one strain

14. Role of bacteriophage Some strain of C. botulinum need to be infected with certain types of phage to produce toxin Toxigenic strains can reserve to non-toxigenic strains when inducing phage is lost or vice versa. Toxin production is under control of viral gene Non-toxigenic strain could convert into toxigenic ones after infected with phage

15. properties 12S or larger size S: 7S M: 12S L : 16S XL: 19S Type A: 19, 16, 12S Group II: trypsin inactivate toxicity

16. Heavy (H) and light (L) chain connected with disulfide bond Individual antibodies can neutralize the complete toxin

18. stability Unstable in alkaline pH – Denaturing agents: alcohols – Amino-reacting agents – Sulfhydryl-reacting – Irradiation to destroy histidine – Oxidizing agent Heat liable Acid food is more sentative

19. toxicity Mouse bioassay – The only assay accepted by FDA – I. p. injection May God bless thee and tender thee May God protect thee and give thee strength to walk through valley of death May thee have better next life

20. Oral toxicity Lethal dose decrease as the size of toxin complex increase (in other word, larger protein has higher toxicity) Oral lethal dose of type A toxin 50,000 to 250,000 times that of the intraperitoneal lethal dose Large mice are more sensitive than smaller mice because of large surface area of intestine Toxicity of progenitor toxin is much higher than that of the derivative toxin

21. Vegetable support the production of L and LL toxin Meat supported production of M toxin of type A and B Boiling vegetable has more fetal cases than meat

22. Intraperitoneal toxicity Smaller size, the smaller the quantity of nontoxic protein and therefore the higher its specific toxicity. Nontoxic protein offer protection during ingestion for oral toxicity but not for i. p. Minimum 0.3 ng/kg

23. Intestinal absorption Upper intestines and appears in the lymphatics Transport of the toxin to target cells is unknown

24. Mode of action Three steps – Binding – Internalization – Intracelar action H chain is responsible for selective binding of the toxin to neurons, internalization of the entire neurotoxin, intraneuronal sorting, and translocation of the L chain into cytosol.

25. L chain block exocytosis as soon as they are release into the cytoplasm. Small number of high affinity binding sites and a large number of sites with much lower affinity on the synaptosomes ( 神經突觸體 ). Double receptor – Toxin first bind to the negatively charged surface of membrane which contains high amount of acid lipid – After binging C-terminal half of the H-chain to acid lipid, toxin diffuse laterally in the mebrane to bind to a protein receptor

26. Neuromuscular junction, entre neurotoxin is internalized by receptor-mediated endocytosis. Can not be neutralized by anti-toxin after endocytosis. H chain aggregates and forms channels to allow L chain into cytoplasm. L chain inhibits neurotransmitter (acetylcholine) release independently. Zinc dependent Degraded synaptosome- associated protein and cause docking and fusion of synaptic vesicle block and neuotransmitter release. C2 toxin causes respiratory difficulty through hypotension.

27. Genetic regulation Chromosome C1 and D are encoded by bacteriophage G on plasmid

28. Symptoms and diagnosis Serious disease – Symptom appear within 24 h – Nausea and vomiting, – Visual impairment – Loss of normal mouth and throat function (speaking) – Fatigue and lack of muscle coordination – Respiratory impairment – Fatality rates was about 50% in early 20 century – Current 10%

29. treatment 1.Neutralization of circulating toxin with antiserum (antidote); the most effective; 50% to 8% in China, 0% in US and Canada 2.Use of enema (vomiting enhancer) to remove residual 3.Gastric lavage or treatment with emetics

30. Mouse assay – Ethically undesirable and time-consuming (4 days) Replacement – Sensitivity, specificity, reliability – 1 ng/mL = 100 mouse LD 50 dose – False positive and negative – Unable to detect all types of toxins

31. Foods Type A and B: meat, vegetables, – East of Rocky Mountains usually contain type B and type A is predominate in the western States. Type E: seafood Eviscerate (remove inner organs and clean)

39. cases Sausage – Nitrate is added to inhibit growth of C. botulinum Under heated cans Fermented fishes – Fermented salmons for native Alaskan – Fermented fish case in Austria – A mother was prosecuted for murder after all her family member was dead after consuming home- made fermented fish.

40. Pickle eggs – A man used toothpicks to penetrate fermented eggs Potato mash – Whole pile of mashed potato create anaerobic condition inside the pile Baked potatoes

41. Vibrio toxin All Vibrios are water-associated – V. cholerae – fresh water – V. parahaemolyticus and vulnificus – salted water (see water is the only one kind of salted water)

42. Vibrio parahaemolyticus Hemolyticus toxin – Kanagawa ( 神奈川、金川 ) phenomenon – gene: tdh, tlh, trh – Proein: TDH, TLH, TRH

43. Thermostable Direct Hemolysin (TDH)Clinical strains of V. parahaemolyticus are capable of producing thermostable direct hemolysin (TDH). An enzyme that can lyse red blood cells on Wagatsuma blood agar plates named the Kanagawa phenomenon (KP). Hemolytic activity of TDH has been reported to be commonly associated with strains isolated from humans with gastroenteritis but were rarely observed in environmental isolates. TDH has been recognized the major virulence factor of V. parahaemolyticus.

44. EHEC Produce toxin which can kills African green monkey kidney (Vero) cells during cell culture – VTEC Shiga-like toxin – SLTEC Virulence – A/E lesion enterocyte effacemetn (LEE) – Shiga toxin – 60-Mda plasmid

45. enterocyte effacemetn (LEE) – Bacterial surface or interact with host cells – Tir (EspE) protein – Primary determinant of bacterial adherence to epithelial cells – Intimin is a virulence factor (adhesin) of EPEC (e.g. E. coli O127:H6) and EHEC (e.g. E. coli O157:H7) E. coli strains. It is an attaching and effacing (A/E) protein which with other virulence factors is responsible for enteropathogenic and enterohaemorrhagic diarrhea.

46. Shiga like toxin – S. dysenteriae (meaning bloody diarrhea) Stx1 and Stx2 Subunit A (32 Kda) and B (7.7 Kda) – AB 5 (one A and five B subunits per toxin protein) A nicked into 2 products B binds to the receptor on the cell membrane Endocytosis

47. Vesicle Golgi apparatus Endoplasmic reticulum Release A1 subunit 28S rRNA Inhibition of protein synthesis and cell death

48. After epithelial cells die, toxin goes into blood stream and circulates to kidney tissue. Causing death of kidney cells and malfunction of kidney – HUS (hemolytic-uremic syndrome)

49. plamsid Hemolysin (HlyA) – Stimulate bacterial growth in the gut by releasing hemoglobin from read blood cells. – increase iron source Catalase-peroxidase (KatP) EspP – Surface-associated protein – Serine protease cleaves the coagulation factor V and cytotoxic for Vero cells