Lecture 32

Question 1

1. What is cholera?
   
   1. How many cases per year?
   2. How fatal is diarrhoeal diseases in youth?
2. What are the symptoms of cholera?

Answer 1

1. One of the most important bacterial diseases transmitted by water. A “water-borne” infectous gastroenteritis. An intestinal infection causing a life-threatening diarrhea.
   
   1. 3 to 5 million cases and 100,000 to 120,000 deaths each year.
   2. 2nd most leading cause of death among children under 5 years of age.
2. Cramps, vomiting, voluminous and nearly continuous but painless watery “rice water” diarrhea.

Question 2

1. How many liters of fluid can you lose per day?
2. What is the mortality rate without treatment?
3. What is the cause of death?

Answer 2

1. Over a four-day period, circa 30 liters of fluid can be lost.
2. Without treatment (rehydration therapy, below), death occurs in 50-60% of cases.
3. Death is due to profound dehydration and severely altered levels of electrolytes. In sever cases, death can occur in a few hours.

Question 3

1. How old is cholera?
2. Where is it uncommon, and common today?

Answer 3

1. Reported from several centuries BC. First European description, 1502. In recent times, cholera has been a common disease in Europe and North America.
2. Uncommon now in U.S. and Europe, but the Gulf Coast of Louisiana and Texas may be an endemic focus. Still common in Asia, Africa, and some areas of Central and South America.

Question 4

1. What is the causative agent of Cholera?
2. Who discovered it?
3. Who was the first to isolate the bacterium?
4. What is its natural habitat?

Answer 4

1. Vibrio cholerae. Gram-negative, facultatively aerobic, curved rod, single polar flagellum, family Vibrionaceae. A member of Gammaproteobacteria.
2. Isolated by Robert Koch (German), 1883. In 1883, Koch worked with a French research team in Egypt, studying cholera. Koch identified the bacterium that caused cholera, but was not able to prove it in experiments.
3. Italian anatomist Filippo Pacini in 1854, but his work had been ignored due to the predominance of the miasma theory of disease. Koch was unaware of Pacini’s work and made an independent discovery, and his greater preeminence allowed the discovery to be widely spread for the benefit of others. In 1965, however, the bacterium was formally renamed Vibrio cholera Pacini 1854.
4. Bays, estuaries, lakes; can tolerate brackish water also, common in coastal seawater. Commonly associated with the surface of algae and metazoans.

Question 5

1. How many cases are reported in the U.S.?
2. How is the disease transmitted?

Answer 5

1. 5 to 10 cases are reported in the U.S. each year. Most are linked to eating undercooked seafood (oysters, crabs), some cases result from foreign travel (foods eaten abroad). No deaths in the U.S. due to cholera in the past 15 years. Some strains are luminous, but they are non-toxigenic (lack the ctxAB genes).
2. The main route of transmission, historically - drinking water contaminated by human sewage. Also, consumption of raw shellfish (beds in raw sewage) and raw vegetables are sources of the disease.

Question 6

1. Who is John Snow?
2. What did he compare?

Answer 6

1. A British physician working in London during an outbreak of cholera, 1850s.
2. Snow compared the distribution of houses of cholera patients with the sources of water different households recieved. At that time in London, water was supplied to households by private water companies that delivered the water.
   
   1. In the same neighborhood, different companies, competing for business, would deliver water to different houses.
   2. Snow compared rates of cholera with which company provided the water to different households. All of London - 59 cases per 10,000.
   3. Southwark and Vauxhall - 350 cases per 10,000.
   4. Lambeth - 37 cases per 10,000.

Question 7

1. What did Snow find during his studies?

Answer 7

1. Found that both Southwark and Vauxhall Company pumped water from the Thames River, below where Snow noticed that raw human sewage was released into the river.
2. Found that Lambeth Company, however, pumped its water upstream from where the city released its sewage.
3. Results were then published in 1855, led Snow to hypothesize that cholera was caused by drinking “fecalized water”.
4. This was before the germ theory of disease had been established and when many scientists believed that disease was caused by miasma (bad air). Study disproved this theory.

Question 8

1. What else is Snow known for?
2. How did he go about indentifing the cause of cholera on Broad Street?

Answer 8

1. Well known for providing an early example of enlightened health care, in 1854. In some neighborhoods, households took their water from neighborhood pumps that were supplied by different sources of water.
2. Snow plotted the incidence of Cholera in neighborhoods in London and the locations of the pumps where people from those neighborhoods got their water. He identified a high incidence of cholera with a specific pump, in the Broad Street area. He then ordered the removal of the handle of the Broad Street pump, forcing those households to use other pumps for their water.
   
   1. Stopped the cholera outbreak in the Broad Street neighborhood. And, this established the link between this infection and water as its vector.

Question 9

1. How many pandemics of cholera has their been since 1817?
   
   1. How many people have been killed since?
   2. Is there a pandemic currently?

Answer 9

1. There has been 7 major pandemics, affecting primarily Asia, the Middle East, and Africa.
   
   1. Killed more than 50,000,000 people since 1817.
   2. Currently in the 7th pandemic, began in 1961 in the Celebes, Indonesia. Then spread to eastern Asia and reached Bangladesh in 1963, India in 1964, the USSR, Iran , Iraq, and South America. More than 5 million cases and more than 250,000 deaths reported.

Question 10

1. How do we type the bacterium?
2. What are the serogroups of V. cholera?

Answer 10

1. Typing of a bacterium by agglutination reactions using serotyping-specific antiserum. Serum containing antibodies against specific antigens. The “0” side chain of lipopolysaccharide of the outer membrane.
2. Six total, two of which are able to produce enterotoxin and cause the disease.
   
   1. 01 - two biotypes (strains). Classic, caused epidemics until the middle of the 20th Century. El Tor, causative agent of most current cholera outbreaks.
   2. 0139 - a newer serogroup, emerged in the Bay of Bengal area in 1992. Not so common, but might be starting an 8th outbreak.
   3. non-01/0139 - very common, but not associated with disease.

Question 11

1. What does V. cholera colonize?
2. What does it require?
3. How many cells does it take to cause the disease?
   
   1. How can number be lowered?
   2. How many for ingestion with food? What is the incubation time?

Answer 11

1. Colonizes the small intestine.
2. A large inoculum, for some cells to survive and get past the killing effect of stomach acidity.
3. Ingestion of 10⁶ to 10⁸ cells.
   
   1. Humans volunteers given bicarbonate to neutralize gastric acidity developed the disease when only 10⁴ cells were administered.
   2. Far lower cell numbers required to initiate infection if V. cholerae is ingested with food (protection). 24-72 hr incubation period follows before disease symptoms begin.

Question 12

1. Where do the bacteria adhere to? How do they do this?
   
   1. What do they do at this point?
2. What kind of toxin is Cholera’s?
   
   1. What does the B subunit do?
   2. What does the A subunit do?
3. What does the addition of a ADP-ribosyl group do?
4. Whats the result of the increased levels of cAMP?

Answer 12

1. Adhere to the intestinal mucosa of the small intestine, using a mucinase to get into the mucosal layer, and specific mucosal cell receptors for attachment.
   
   1. They grow and produce an enterotoxin (exotoxin active in the small intestine).
2. Cholera toxin is an A-B toxin; two functional units, an enzymatic A subunit and an intestinal receptor-binding B subunit.
   
   1. Binds specifically to ganglioside GM1, a complex glycolipid on the mucosal cell surface. The binding triggers endocytosis of the A subunit by intestinal epithelial cells.
   2. Activates the host cell enzyme adenylate cyclase by the addition of an ADP-ribosyl group (from NAD⁺​). Process called ADP-ribosylation.
3. Blocks the ability of the enzyme to shut itself off. Host cell adenylate cyclase synthesized cAMP, which plays a major role in eukaryotic membrane permeability. So, the enzyme is “locked” in the active position, causing an overproduction of cAMP.
4. Cause a change in membrane permeability, resulting in the hypersecretion of water and chloride ions out, into the gut lumen, while inhibiting absorption of sodium ions.

Question 13

1. What two genes encode the toxin?
   
   1. What carries these genes?
2. How does a phage get into the bacterium?
3. What controls the expression of the genes?

Answer 13

1. ctxA and ctxB
   
   1. A bacteriophage, called CTX.
2. Phage binds to the pilus used by the bacterium to colonize the host’s gut, enters the bacterium, and incorporates its genes into the bacterial chromosome.
3. The chromosomal toxR gene product, ToxR protein, controls expression of the ctxAB genes.

Question 14

1. What are some treatments?

Answer 14

1. Oral rehydration therapy, reduces mortality to less than 1%.
2. Salts (NaCl, NaHCO₃, KCl) plus glucose (or sucrose) to stimulate water uptake by the intestine.
   
   1. 1/2 Teaspoon of salt
   2. 1/4 Teaspoon of sodium bicarbonate (trisodium citrate dihydrate)
   3. 1/4 Teaspoon of potassium chloride
   4. 4 Tablespoons of glucose (sucrose)

Question 15

1. Antibiotic Therapy
2. Prevention
3. Control
4. Note: Look at Haiti Slide

Answer 15

1. Tetracycline, trimethoprim-sulfamethoxazole, or ciprofloxacin.
2. Vaccination - available, but immunity is not attained by all individuals and it does not last long.
3. Proper sanitation to avoid fecal contamination, especially of water supplies.