What is Cholera?

2. What is Cholera?

3. Cholera • A life-threatening secretory diarrhea induced by enterotoxin secreted by V. cholerae • Water-borne illness caused by ingesting water/food contaminated by copepods infected by V. cholerae • An enterotoxic enteropathy (a non-invasive diarrheal disease) • A major epidemic disease

4. Recent Cholera Pandemics • 1-6th pandemic: • V. cholerae O1 biotype classical • 1817-1923, Asia, Africa, Europe, America and Australia • 7th pandemic: • V. cholerae O1 biotype El Tor • Began in Asia in 1961 • Spread to other continents in 1970s and 1980s • Spread to Peru in 1991 and then to most of South & Central America and to U.S. & Canada • By 1995 in the Americas, >106 cases; 104 dead • 1993: Cholera in Bengal caused by O139 may be cause of 8th pandemic

6. Vibrio • Vibrio cholerae--gastroenteritis • Vibrio parahaemolyticus -- gastroenteritis, wound infection, bacteremia • Vibrio vulnificus -- wound infection, bacteremia

7. V. cholerae • Grows in salt and fresh water • Endemic in areas of poor sanitation (India and Bangladesh ), transmitted by fecal-oral route • Can survive and multiply in brackish water by infecting copepods • Has over 150 identified serotypes based on O-antigen • Only O1 and O139 are toxigenic and cause Cholera disease

8. 小川型 稻叶型 彦岛型 Classification: O1 Antigen

9. O139 Serogroup In 1993, an entirely new serogroup (O139) cause an epidemic in Bangladesh. O139 organisms produce a polysaccharide capsule but do not produce O1 LPS or O1 antigen. Non-O1, Non-O139 Serogroup Most are CT (cholera toxin) negative and are not associated with epidemic disease. Classification: Other antigens

10. Profile of vibrio cholerae • G-, curved or comma-shaped rods • Highly motile; polar flagellum • Sensitive to low pH and die rapidly in solutions below pH 6 • Proliferate in summers • Cholera toxin • Pathogenic and nonpathogenic strains • 206 serogroups

11. Profile of vibrio cholerae • Similarities to Enterobacteriaceae • G-, Facultative anaerobes • Fermentative bacilli • Differences from Enterobacteriaceae • Polar flagella • Oxidase positive • Formerly classified together as Vibrionaceae • Primarily found in water sources • Cause gastrointestinal disease • Shown not closely related by molecular methods

12. Physiology of Vibrio • Broad temperature & pH range for growth on media • 18-37C • pH 7.0 - 9.0 (useful for enrichment) • Grow on variety of simple media including: • MacConkey’s agar • TCBS (Thiosulfate Citrate Bile salts Sucrose) agar • V. cholerae grow without salt • Most other vibrios are halophilic

13. People Most at Risk People with low gastric acid levels (103 -105 CFU ) Children: 10 × more susceptible than adults Elderly Blood types O>> B > A > AB

14. Period of Communicability During acute stage A few days after recovery By end of week, 70% of patients non-infectious By end of third week, 98% non-infectious

15. Symptoms • Occur 2-3 days after consumption of contaminated food/water • Usually mild, or no symptoms at all • 75% asymptomatic • 20% mild disease • 2-5% severe • Vomiting • Cramps • Watery diarrhea (1L/h) • Without treatment, death in 18 h-several days

16. Cholera Gravis (霍乱肌无力) More severe symptoms Rapid loss of body fluids 6 liters/hour 107 vibrios/mL Rapidly lose more than 10% of bodyweight Dehydration and shock Death within 12 hours or less Death can occur within 2-3 hours

17. Pathogenesis of V. cholerae Virulent factors • Toxins and enzymes - heat stable endotoxin - enterotoxin (exotoxin [cholera toxin CT]) CT is antigenically and pharmacologically identical in all sero and bio types

18. Cholera Toxin (CT) Structure • The A subunit contains an intracellular ADP-ribosyltransferase activity. • The mature A subunit is proteolytically cleaved to produce an A1 polypeptide, which contains the intracellular enzymatic activity, and an A2 polypeptide. • CT is a prototype A/B subunit toxin, 1A+5B • The B subunit form a pentameric ring, which binds the holotoxin to a eukaryotic cell surface receptor.

19. Cholera Toxin (CT) Structure • After cleavage, the A1 and A2 polypeptides remain linked by a disulphide bond. • A and B subunits are connected through the C-terminus of the A2 subunit, which is inserted through the central pore of the B pentamer.

20. How Does Cholera Toxin Work? • The biological activity of CT is dependent on binding of B pentamer to specific receptors GM1 ganglioside. • Internalization is initiated once CT-GM1 complexes cluster which then invaginate to form apical endocytic vesicles.

21. How Does Cholera Toxin Work? • These vesicles enter cellular trafficking pathways leading to the trans-Golgi network (TGN). • The toxin then moves retrograde via the Golgi cistern to the ER. • Once in the ER, CT is processed to activate the A1 peptide, which then targets the basolateral membrane (heterotrimeric GTPase and adenylate cyclase (AC)).

22. How Does Cholera Toxin Work? • Adenylate cyclase (AC) is activated normally by a regulatory protein (GS) and GTP; however activation is normally brief because another regulatory protein (Gi), hydrolyzes GTP. NORMAL CONDITION

23. CHOLERA How Does Cholera Toxin Work? • A1 fragment catalyzes the attachment of ADP-Ribose (ADPR) to the regulatory protein forming Gs-ADPR from which GTP cannot be hydrolyzed. • Since GTP hydrolysis is the event that inactivates the adenylate cyclase, the enzyme remains continually activated.

24. How Does Cholera Toxin Work? • Thus, the net effect of the toxin is to cause cAMP to be produced at an abnormally high rate which stimulates mucosal cells to pump large amounts of Cl- into the intestinal contents.

25. H2O, Na+ and other electrolytes follow due to the osmotic and electrical gradients caused by the loss of Cl-. The lost H2O and electrolytes in mucosal cells are replaced from the blood. Thus, the toxin-damaged cells become pumps for water and electrolytes causing the diarrhea, loss of electrolytes, and dehydration that are characteristic of cholera.  How Does Cholera Toxin Work?

26. How Does Cholera Toxin Work? • Inactivates GTPase function of G-protein coupled receptors in intestinal cells • G proteins stuck in “On” position • 100 fold increase in cAMP • Activation of ion channels • Ions flow out and water follows

29. Diagnosis • Cholera should be suspected when patients present with watery diarrhea, severe dehydration • Based on clinical presentation and confirmed by isolation of vibrio cholera from stool • No clinical manifestations help distinguish cholera from other causes of severe diarrhea: Enterotoxigenic E. coli Viral gastroenteritis Bacterial food poisoning

30. Diagnosis: Visible Symptoms • Decreased skin turgor • Sunken eyes, cheeks • Almost no urine production • Dry mucous membranes • Watery diarrhea consists of: • fluid without RBC, proteins • electrolytes • enormous numbers of vibrio cholera (107 vibrios/mL)

31. Laboratory Diagnosis • Visualization by dark field or phase microscopy • Look like “shooting stars” • Gram Stain • Red, curved rods of bacteria • Isolate V. cholerae from patient’s stool • Plate on sucrose agar • Yellow colonies form

33. VibrioPrevention & Control • Disrupt fecal-oral transmission Improved sanitation • Fluid and electrolyte replacement • Antibiotic prophylaxis • Improved food handling

34. Vibrio parahemolyticus • One kind of halophilic vibrios; • optimal NaCl concentration contained in culture media is 3.5%; • hemolysin related to its pathogenicity, can be detected by human or rabbit RBC test (Kanagawa test); • cause food poisoning in human beings. • raw sea-food

35. Vibrio parahemolyticus • Clinical manifestations • Self-limiting diarrhea to mild cholera-like illness • 24 hours after ingestion-explosive water diarrhea • Headache, abdominal cramps, nausea, vomiting, low grade fever for 72 hours or more • Uneventful recovery • Wound infections in people exposed to seawater-containing vibrios

36. Helicobacter pylori

37. Helicobacter pylori • G- with S or spiral-shaped • Very Motile ---corkscrew motion • 2~6 flagella at one end of HP • 5% O2+10% CO2 at 37 oC • Reaction of urea hydrolysis and creates an ammonia cloud

38. Pathogenesis of Hp CagA (Cytotoxin associated) VacA (vacuolationg associated) LPS also play great importance Flagellum and urease is necessary for its adhesion and inhabitation Adhesin

39. Duodenal Ulcer (DU) Gastric Ulcer (GU)

41. Campylobacter jejuni • G- rods with comma, S, or “gull-wing” shapes. • Motive, with a single polar flagellum • No spore & no capsule • 5% O2+10% CO2 • Two types of colonies:  watery and spreading round and convex

42. Pathogenesis of Campylobacter • Produces a toxin called Cytolethal Distending Toxin (CDT). • CDT activity requires activation of three genes: cdtA, cdtB, and cdtC. • CdtB is nuclease that damages DNA and causes cell cycle arrest. • Causes cell death.

43. What Are the Symptoms? • Diarrhea • Usually watery and sticky • Can contain blood and fecal leucocytes • Fever • Abdominal pain • Nausea and vomiting • Headache • Muscle pain

44. Who is affected? • All warm-blooded animals can become affected. Some animals carry the disease without exhibiting symptoms. • Any person can become infected. • Children under 5 and young adults ages 15-29 are most often affected. • Most deaths occur among the elderly and the immune-suppressed.

45. Summary • Properties of Vibrio (stain, culture, biochemical reaction, antigens and virulence factors) • Pathogenesis of V. parahemolyticus, H. pylori, C. jejuni