Biology 216 - Lect 19
Cholera/ETEC
Nobel Prize in physiology/medicine this year?
Family: Vibrionaceae - Gm- curved rods, Ox+; 
	Members: Vibrio, Aeromonas, Plesiomonas
I. Non-cholera vibrios
	A. V. vulnificus - wound infections
	B. V. parahaemolyticus - grow in salt water 
		estuaries, halophilic.
		Contaminate foods and grow very quickly.
		#1 food illness in Japan - sushi
		Acute GI
II. Vibrio cholerae - explosive life-threatening diarrhea.
	Prototypic enteropathy - Other studies based on this 
	(ex. ETEC, Salmonella.....)
	A. Bacteriology - 
       Sequenced: http://www.tigr.org/tdb/CMR/gvc/htmls/SplashPage.html
       2 chromosomes 
		1. Serotypes based on O-Ag (O-1 agglutinable - 
			epidemic; NAGs most tox-; O-139)
			O-1 Ag - 3 serotypes (Inaba, Ogawa, Hikojima)
		2. Biotypes of O-1Ag - Different biochems (HA, VP, 
			phage sensitivity, polymyxin B)
			Classical and El Tor
	B. Cholera toxin - responsible for entire disease; 
		Prototype enterotoxin (E. coli, Campy...)
		Genes found in lysogenic phage in chromosome 
		(phage can be recovered)
		1. 1959 soluble toxin; Before i.p. in mouse gave 
			no response; ligated ileal loop
		2. Structure - 1A (enzymatic ADP-ribosylating),
			 5B (bind GM1 ganglioside - sugar/lipid)
			ctxA-ctxB single operon - differ in rbs efficiency
			toxR - transcriptional activator.
		3. MOA - modifies intestinal epithelial cell to excrete; 
			Does not kill; MOA OTB;Y-1 adrenal,CHO
	C. Pathogenesis - 7th great pandemic - 
			began 1961 (Asia-> Africa) - 
			40,000/yr, under reported probably 1 million.
			1991 Peru >300K. Now spread through latin and 
				south America (> 1 million)
				Began with Chinese frigate in Lima, sewage 
					dumped, fish -> sewage 
					Decreased chlorination of sewage.
		1. Transmission - 10(8) ie no direct transmission; 
				Ingestion of food, water, vegetable, 
				Found in shellfish and plankton beds. 
		2. Pass stomach (bolus); Gastric barrier; achlorhydria 
			and bicarb increase infection.
		3. Colonize and multiply in small intestine 
			(draw lumen, crypts, villi, microvilli, mucus)
			Role for motility, mucinase, attachment
		4. Cholera toxin - electrolyte efflux, water, diarrhea 
			(rice water stool), vomit, no damage
			May lose 20 liters/day for 3 days.
		5. Dehydration - sunken tissues (face, washer 
			woman hand), hypovolemic shock.
		6. 3 - 5 days vibrios detach. 60 % mortality 
			untreated; 1 % if treated.
		7. 1f - 20 % carriers after disease (carriage is 
			the rule/disease is a mistake)
	D. Lab diagnosis
		1. History, stool appearance, Fl-Ab of stool in epidemics.
		2. Isolation and ID - TCBS (thiosulfate citrate bile sucrose), 
			usually pure culture, biochem
	E. Treatment
		1. Replace fluid and electrolytes - iv (2 liter/hr);
			WHO  Oral rehydration pack (ORP)
			Gatorade; cholera cot
		2. Antibiotics - eliminate carrier state; 
			Some effect on course of disease.
		3. Exptl drugs based on MOA - chlorpromazine 
			(conc in sm intest; inhibit Ad cyclase)
	F. Prevention
		1. Sewage disposal - flush toilet; treat and 
			disinfect sewage (Peru); controllable
		2. Travelers to endemic areas - avoid vegetables; 
			raw seafood, tap water and ice, fruit
		3. Vaccines: Old vaccine is ip killed V. cholerae - ineffective.
			Need immune response at site of infection, 
				not circulating Ab.
			Live attenuated vaccines: M13 (reverted); 
				Texas star S/R; A-B+ recombinant
				Recombinant A-B+ (gene replacement HgR insert). 
				S. typhi carrying cloned genes.
                     New - oral rCTB + 4 killed strains
II. ETEC - First in swine piglets (plasmid, K88, LT, ST); 
		1/2 billion humans/year (CFA, LT, ST)
	Tijuana trots, Montezuma's revenge; mild to cholera-like.
	A. LT and CT - Ouchterlony
		MOA identical
		Clements purification of LT on A5M (galactose)
	B. ST - small MW non-immunogenic protein
		MOA - activates guanylate cyclase and 
			blocks absorption of electrolytes
		Now found in Yersinia and other bacteria
	C. Lab diagnosis - 
		Biochemically identical to other E. coli; 
			Certain serotypes; 
		Need for rapid determination of ETEC 
		(DNA hybridization to plasmid; ELISA toxin)
	D. Treat same as cholera
	E. Control - Diarrheal disease in WHO #1 target
		Exptl vaccines - LT/ST conjugates; 
		Cloned into S. typhi
		A subunit as mucosal adjuvant to stimulate 
			local immune response.