Vibrios are one of the most common organisms in surface waters of the
world. They occur in both marine and freshwater habitats and in associations
with aquatic animals. Some species are bioluminescent and live in mutualistic
associations with fish and other marine life. Other species are pathogenic
for fish, eels, and frogs, as well as other vertebrates and invertebrates.
V. cholerae and V. parahaemolyticus are pathogens of humans. Both produce diarrhea, but in ways that are entirely different. V. parahaemolyticus is an invasive organism affecting primarily the colon; V. cholerae is noninvasive, affecting the small intestine through secretion of an enterotoxin.
V. cholerae produces cholera toxin, the model for enterotoxins, whose action on the mucosal epithelium is responsible for the characteristic diarrhea of the disease cholera. In its extreme manifestation, cholera is one of the most rapidly fatal illnesses known. A healthy person may become hypotensive within an hour of the onset of symptoms and may die within 2-3 hours if no treatment is provided. More commonly, the disease progresses from the first liquid stool to shock in 4-12 hours, with death following in 18 hours to several days.
The clinical description of cholera begins with sudden onset of massive diarrhea. The patient may lose gallons of protein-free fluid and associated electrolytes, bicarbonates and ions within a day or two. This results from the activity of the cholera enterotoxin which activates the adenylate cyclase enzyme in the intestinal cells, converting them into pumps which extract water and electrolytes from blood and tissues and pump it into the lumen of the intestine. This loss of fluid leads to dehydration, anuria, acidosis and shock. The watery diarrhea is speckled with flakes of mucus and epithelial cells ("rice-water stool") and contains enormous numbers of vibrios. The loss of potassium ions may result in cardiac complications and circulatory failure. Untreated cholera frequently results in high (50-60%) mortality rates.
Treatment of cholera involves the rapid intravenous replacement of the lost fluid and ions. Following this replacement, administration of isotonic maintenance solution should continue until the diarrhea ceases. If glucose is added to the maintenance solution it may be administered orally, thereby eliminating the need for sterility and iv. administration. By this simple treatment regimen, patients on the brink of death seem to be miraculously cured and the mortality rate of cholera can be reduced more than ten-fold. Most antibiotics and chemotherapeutic agents have no value in cholera therapy, although a few (e.g. tetracyclines) may shorten the duration of diarrhea and reduce fluid loss.
History and spread of epidemic cholera
Cholera has smoldered in an endemic fashion on the Indian subcontinent for centuries. There are references to deaths due to dehydrating diarrhea dating back to Hippocrates and Sanskrit writings. Epidemic cholera was described in 1563 by Garcia del Huerto, a Portuguese physician at Goa, India. The mode of transmission of cholera by water was proven in 1849 by John Snow, a London physician. In 1883, Robert Koch successfully isolated the cholera vibrio from the intestinal discharges of cholera patients and proved conclusively that it was the agent of the disease.
The first long-distance spread of cholera to Europe and the Americas began in 1817 and by the early 20th century, six waves of cholera had spread across the world in devastating epidemic fashion. Since then, until the 1960s, the disease contracted, remaining present only in southern Asia. In 1961, the "El Tor" biotype (distinguished from classic biotypes by the production of hemolysins) reemerged to produce a major epidemic in the Philippines and to initiate a seventh global pandemic (See map below). Since then this biotype has spread across Asia, the Middle East, Africa, and more recently, parts of Europe.
There are several characteristics of the El Tor strain that confer upon it a high degree of "epidemic virulence" allowing it to spread across the world as previous strains have done. First, the ratio of cases to carriers is much less than in cholera due to classic biotypes (1: 30-100 for El Tor vs. 1: 2 - 4 for "classic" biotypes). Second, the duration of carriage after infection is longer for the El Tor strain than the classic strains. Third, the El Tor strain survives for longer periods in the extraintestinal environment. Between 1969 and 1974, El Tor replaced the classic strains in the heartland of endemic cholera, the Ganges River Delta of India.
The global spread of cholera during the seventh pandemic, 1961-1971
El Tor broke out explosively in Peru in 1991 (after an absence of cholera there for 100 years), and spread rapidly in Central and South America, with recurrent epidemics in 1992 and 1993. From the onset of the epidemic in January 1991 through September 1, 1994, a total of 1,041,422 cases and 9642 deaths (overall case-fatality rate: 0.9%) were reported from countries in the Western Hemisphere to the Pan American Health Organization. In 1993, the numbers of reported cases and deaths were 204,543 and 2362, respectively.
So far, the United States has been spared except for imported cases, or clusters of infections from imported food. In the United States during 1993 and 1994, 22 and 47 cholera cases were reported to CDC, respectively. Of these, 65 (94%) were associated with foreign travel.
In 1982, in Bangladesh, a classic biotype resurfaced with a new capacity to produce more severe illness, and it rapidly replaced the El Tor strain which was thought to be well-entrenched. This classic strain has not yet produced a major outbreak in any other country.
In December, 1992, a large epidemic of cholera began in Bangladesh,
and large numbers of people have been involved. The organism has been characterized
as V. cholerae O139 "Bengal". It is derived genetically from the
El Tor pandemic strain but it has changed its antigenic structure such
that there is no existing immunity and all ages, even in endemic areas,
are susceptible. The epidemic has continued to spread. and V. cholerae
O139 has affected at least 11 countries in southern Asia. Specific totals
for numbers of V. cholerae O139 cases are unknown because affected
countries do not report infections caused by O1 and O139 separately.
The toxin has been characterized and contains 5 binding (B) subunits of 11,500 daltons, an active (A1) subunit of 23,500 daltons, and a bridging piece (A2) of 5,500 daltons that links A1 to the 5B subunits. Once it has entered the cell, the A1 subunit enzymatically transfers ADP ribose from NAD to a protein (called Gs or Ns), that regulates the adenylate cyclase system which is located on the inside of the plasma membrane of mammalian cells.
Enzymatically, fragment A1 catalyzes the transfer of the ADP-ribosyl
moiety of NAD to a component of the adenylate cyclase system. The process
is complex. 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. The normal situation is described
The 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.
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. 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.
After natural infection by V. cholerae, circulating antibodies can be detected against several cholera antigens including the toxin, somatic (O) antigens, and flagellar (H) antigens. All these antibodies can also be raised by parenteral injection of antigens as vaccine components. Antibodies directed against Vibrio O antigens are considered "vibriocidal" antibodies because they will lyse V. cholerae cells in the presence of complement and serum components. Vibriocidal antibodies reach a peak 8-10 days after the onset of clinical illness, and then decrease, returning to the baseline 2 - 7 months later. Their presence correlates with resistance to infection, but they may not be the mediators of this protection and the role of circulating antibodies in natural infection is unclear.
After natural infection, patients also develop toxin-neutralizing antibodies. In a natural setting for cholera, there is no correlation between antitoxic antibody levels and the incidence of disease.
Since cholera is essentially a topical disease of the small intestine, it would seem that topical defense might be a main determinant of protection against infection by V. cholerae. Recurrent infections of cholera are in fact, rare, and this is probably due to local immune defense mediated by antibodies secreted onto the surfaces of the intestinal mucosa.
Secretory IgA, as well as IgG and IgM in serum exudate, can be detected in the intestinal mucosa of immune individuals. Although these antibodies presumably have to function in the absence of complement they still bring about protective immunity. Motility is important in pathogenesis, and antibodies against flagella could immobilize the vibrios. Antibodies against flagella or somatic O antigens could cause clumping and arrested motion of cells. Antitoxic antibodies could react with toxin at the epithelial cell surface and block binding of the toxin. The process to which the vibrios attach to the intestinal epithelium is highly specific and antibodies against Vibrio fimbriae or other surface components (LPS?) could block attachment.
The observation that natural infection confers effective and long-lasting immunity against cholera has led to efforts to develop a vaccine which will elicit protective immunity. The first attempts at a vaccine in 1960s were directed at whole cell preparations injected parenterally. At best, 90% protection was achieved and this immunity waned rapidly to the baseline within one year. Purified LPS fractions from different biotypes have also been given as vaccines with variable success. The cholera toxin can be converted to toxoid in the presence of formalin and glutaraldehyde. The toxoid is a poor antigen, however, and it elicits a very low level of protection. At the present, a good vaccine for cholera does not exist.
Attempts are underway to develop an oral vaccine from a live attenuated strain of V. cholerae. The ideal properties for such a strain would be to have all the pathogenicity factors required for colonization of the small intestine (motility, fimbriae, neuraminidase, etc.) but not to produce a complete toxin molecule. Ideally it should produce only the B subunit of the toxin which would stimulate formation of antibodies that could neutralize the binding of the native toxin molecule to epithelial cells.
A new vaccine has been developed to combat the Vibrio cholerae Bengal strain that has started spreading in epidemic fashion in the Indian subcontinent and Southeast Asia. The Bengal strain differs from previously isolated epidemic strains in that it is serogroup 0139 rather than 01, and it expresses a distinct polysaccharide capsule. Since previous exposure to 01 Vibrio cholerae does not provide protective immunity against 0139, there is no residual immunity in the indigenous population to the Bengal form of cholera.
The noncellular vaccine is relatively nontoxic and contains little or no LPS and other impurities. The vaccine will be used for active immunization against Vibrio cholerae Bengal and other bacterial species expressing similar surface polysaccharides. In addition, human or other antibodies induced by this vaccine could be used to identify Vibrio cholerae Bengal for the diagnosis of the infection and for environmental monitoring of the bacterium.
Enterotoxins, toxins which act in the GI tract, are produced by a wide
variety of bacteria. The family of heat-stable (ST) enterotoxins of E.
coli, which activate guanylate cyclase, are unrelated to LT toxin or
cholera toxin. Other enterotoxins, which elicit cytotoxic effects on intestinal
epithelial cells, have been described from Escherichia, Klebsiella,
and Staphylococcus aureus.