"Changing Perspectives on the Ecology of Infectious Disease: The Case of Cholera"
Dr. Rita R. Colwell
Director
National Science Foundation
U.S.-Japan Cooperative Medical Sciences Program
8th International Conference on Emerging Infections in the Pacific Rim
Dhaka, Bangladesh
December 11, 2003
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[Title Slide]
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Thank you the opportunity to speak to you today. This promises to be a successful conference on emerging infections.
As we speak of emerging and re-emerging infectious disease, we must contemplate the disturbing facts. HIV/AIDS, cholera, and other bacterial enteric diseases continue to devastate societies around the world, despite continuing advancements in scientific discovery and understanding. This is very sobering. Progress thus far, as well as current work on these infections, is promising. But it is obvious that so much more needs to be done.
This conference brings together working scientists from several countries to initiate further and, hopefully, more rapid progress. This international audience represents a mix of unique scientific perspectives. However, in addition to a fluent exchange of ideas, there is a perspective that I would like to share with you that I believe will be integral to success in understanding and controlling infectious disease.
[Slide 2: new biocomplexity slide]
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I use the term biocomplexity to describe this mode of envisioning. It is a way of thinking that has emerged from the most recent research examining the complex interactions between climate and health, as well as my own work.
Biocomplexity denotes the study of complex interactions in biological systems, including humans, and their physical environments. Ecosystems do not respond linearly to environmental change, nor do the pathogens that live in them. Here, I use the form of a spiral, symbolic of life at every level, to underscore the point that understanding demands observing and integrating at multiple scales, from the nano to the global. From reductionism we need to move toward an holistic conceptualization of infectious disease.
The spiral of complexity begins at the scale of the atom, and curves up through successive levels of life, through the cell, the organism, the community, the ecosystem. Complexity principles emerge at each level. With the perspective of biocomplexity, disciplinary worlds, formerly discrete, intersect to form fuller, more nuanced viewpoints.
Most of science, up to now, has followed a reductionist approach. We have sought understanding by taking things apart. This has been a dominant trend in medicine and health as in many disciplines. This approach has given us the lion's share of scientific knowledge to date. Now we're ready for a new perspective that integrates across disciplines and scales, a perspective that roots epidemiology firmly in ecology and takes advantage of simulation science. The spiral of complexity curves both ways - outward, integrating the levels of life, and inward, back to the center.
Biocomplexity reaches beyond the scope of ecology, linking the molecular level to forces operating at the planetary scale, such as climate.
Biocomplexity as we define it, from atoms to the planetary level, is now a priority research area at the National Science Foundation.
The lens of biocomplexity helps to focus on the links between climate and health. As a lead-in to cholera, I'll begin by applying the biocomplexity perspective to another disease. In this case it was critical to solving a mystery that began in 1993 in the Four Corners area of the United States, when young and otherwise healthy people began dying from what was then an unknown disease.
[Slide 3: Not available]
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The culprit turned out to be a Hantavirus unknown in the New World until the outbreak, and now familiar to all of us. The mortality rate of those infected with the virus - more than 50% -- is second only to Ebola. Was the new virus a mutant, or had the environment been harboring it all the time?
[Slide 4: Not available]
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The carrier turned out to be a rodent, the deer mouse. Biologists working at an NSF-sponsored Long-Term Ecological Research site, led by Terry Yates at the University of New Mexico and his team, were able to detect the deadly virus in mouse tissue that had been archived years before. As it happened, Native American legends corroborated a history of outbreaks.
[Slide 5: Not available]
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In addition, the investigators showed a link between climate and the outbreak of disease. Mild and wet winters associated with a periodic climate pattern, El Nino-Southern Oscillation, had provided more food for the rodents, whose populations had increased dramatically in 1993.
The Hantavirus story is just one compelling chapter in the saga of ecology and infectious disease and exemplifies the need to be able to trace complexity in order to predict, to treat, and to prevent.
[Slide 6: 3 types of networks]
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Biocomplexity employs the connection between mathematics and epidemiology. In 1998, Strogatz and Watts presented the first mathematical model of so-called "small-world networks." Small-world networks result when even a very small number of short cuts are added to a regular network.
[Slide 7: Not available]
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Mathematicians further specified a type of small-world network called a scale-free network. As you can see in this figure, scale-free networks contain nodes that are more connected throughout the network than are others.
[Slide 8: image of "human" network]
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Why is this important to epidemiology? Because it turns out that small-world networks model the human societal network quite well. Human interactions are not completely regular, nor are they completely random. Short cuts in the societal network are created by air-travel, linking corners of the world in a matter of hours. The highly connected nodes of a scale-free societal network are representative of individuals who interact with a large number of other individuals. In the case of HIV/AIDS, these nodes can represent promiscuous individuals, potentially escalating the rate of disease spread throughout a society.
The ability to mathematically model a system allows us to understand better the processes and interactions implicit in the system's multiple components. When it comes to infectious disease, knowing how disease travels through societal networks can give us a significantly improved perspective into how we can contain disease epidemics, and how we can prevent the worst possible scenario.
[Slide 9: global spread of cholera-recent]
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My own research on cholera has focused on the interrelationship between climate and health.
Cholera is an excellent example of how investigation of environmental factors - climate foremost among them - gives a clearer picture of a disease, from virulence to transmission to epidemiology.
Although not all members of the genus Vibrio produce toxins, many do. Twelve of the 30 known species of this bacterial genus cause human diseases.
[Slide 10: Biocomplexity spiral with molecular level highlighted]
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As we begin to trace the spiral of complexity that surrounds the mystery of cholera, let us begin with insights from the smallest scale.
[Slide 11: picture: life at undersea vent]
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Where did cholera come from? Once again we turn to the environment. The requirement of Vibrio cholerae for salt for growth led us to suggest that its ancestral home is the sea. Genomics helped substantiate this theory.
Here is a community of life around a deep-sea vent. The isolation of the genus Vibrio from deep-sea hydrothermal vents was first reported in 1981. Vibrio species have also been found in a variety of deep-sea habitats.
[Slide 12: East Pacific Rise: map]
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In 1999, during dives by the submersibles Alvin and Nautile, sulfide chimneys were collected from undersea hydrothermal vents on the East Pacific Rise. Vibrio species isolated from the chimneys were identified that bore significant similarity to Vibrio cholerae, suggesting that it is autochthonous, or native, to the deep sea. V. cholerae serves important functions in ocean and estuarine ecosystems, such as chitin digestion and nitrogen cycling.
[Slide 13: Small chromosome and Large chromosome]
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In 2000, the genomes of the two chromosomes possessed by V. cholerae were sequenced. The toxin genes, of which there are about fifty, reside on the large chromosome. The sequencing data confirm that V. cholerae is a versatile organism, able to live in several habitat types, as well as to infect the human gastrointestinal tract.
Vibrio cholerae has a gene acquisition system located on its small chromosome as well as hot spots for DNA rearrangement. Lateral transfer of genetic material is clearly occurring in this organism.
[Slide 14: spiral with organism level highlighted]
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Ascending a level of biocomplexity to that of the organism lends insight into the remarkable capacity of V. cholerae to evolve.
Virulence genes are distributed in environmental strains of V. cholerae from various serogroups - apparently an environmental reservoir of such genes. 0139 acquired novel DNA. Serotypes that do not cause epidemic cholera can pick up toxin genes from other cholera bacteria in its environment. This underscores the versatility of cholera, and gives even greater credence to the significance of the environment in understanding cholera's complexity. We also know, however, that it is difficult to isolate V. cholerae 01 from the environment, where it is competing with some 250 other serotypes.
[Slide 15: List of VBNC organisms]
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A major insight from the past few decades is the discovery that pathogens can exist in a viable state even though they cannot be cultured. This list gives a quick glimpse of some of the pathogens in which the "viable but non-culturable" phenomenon has been studied, from E. coli to Helicobacter pylori (the cause of ulcers), to Legionella and Salmonella.
[Slide 16: Chesapeake Bay]
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In the late 1960s and early 1970s, my students and I discovered that the ocean and estuaries are a reservoir for V. cholerae, when we identified the organism in water samples from the Chesapeake Bay and off the coast of Maryland and Delaware. Cholera's interactions with its environment are a major line of inquiry. In the Chesapeake Bay, native V. cholerae populations fluctuate with the seasons, just as they do in Bangladesh.
My student, Valerie Louis, recently completed her Ph.D. thesis investigating how salinity affects cholera concentrations in the water. V. cholerae is more common in the northern part of the bay where salinity is low and when the weather is warmer.
In fact, temperature and salinity combined predict the presence of V. cholerae with an accuracy of between 76 and 90 percent.
Furthermore, changes in salinity from year to year, due to the influx of freshwater from the Susquehanna River at the head of the Bay, may cause V. cholerae populations to fluctuate greatly. We are building a computer model to be able to predict occurrences of V. cholerae.
With climate change - carbon dioxide increasing in the atmosphere, global warming, and more rainfall - river flow would be expected to increase, altering salinity and affecting V. cholerae population distribution in the Bay. There is a similarity here with the Bay of Bengal, monsoons, and cholera.
[Slide 17: Not available]
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The ecological relationship of cholera and planktonic copepods was first established in 1983 when we showed that V. cholerae attached to live copepods in the Chesapeake Bay and around Bangladesh, following on the relationship between Vibrio parahaemolyticus and the blue crab in the Chesapeake Bay, the work of my student Dr. Tatsuo Kaneko in 1970-1974. The conclusion is that cholera is not an eradicable disease but a controllable disease, because the causative organism lives naturally in riverine, brackish and estuarine ecosystems. Preventing its ingestion by humans can be fully achieved by provision of filtered and chlorinated drinking water and proper sanitation.
Currently, my student, Tonya Rawlings at the University of Maryland, is investigating whether the seasonality of cholera in endemic areas is associated not only with temperature and salinity, but also interaction of V. cholerae with given species of hosts - namely these copepods, perhaps explaining, in part, the seasonality of cholera epidemics. At the present time, Tonya is investigating whether 01 and 0139 have preference for attaching to specific genera of copepods. Three candidate copepod genera in Chesapeake Bay are: Oithona, Eurythemora, and Acartia. Not all bacteria compete well for space on surfaces. The pathogenic species of V. cholerae attach best, another example of cholera's environmental capabilities and further evidence of its autochthonous aquatic nature.
[Slide 18: copepod with egg case]
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This copepod is a female whose egg case, on the left, is covered with vibrios. V. cholerae colonizes the oral region and egg sacs of copepods. The oral location probably reflects the normal ingestion of bacteria as a food source, but the egg sac substrate is more attractive to vibrios.
Our hypothesis, in fact, is that cholera originally evolved commensally with marine animals such as copepods, which provided them a surface to grow, nutrition and perhaps other mutual benefits.
[Slide 19: spiral with habitat and population highlighted]
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Moving up the complexity spiral to the levels of population and habitat, we trace the ecological interactions of V. cholerae populations in two distinct habitats: the human intestine and the aquatic environment.
We are finding that Eurythemora is more prevalent in the upper Chesapeake Bay, with more Acartia in the lower Bay. We have also noted seasonal fluctuations in the prevalence of these species. These results underscore the need to do intensive work to see if copepod species selectivity - for example, the ability of the 01 to attach better to a particular copepod species - might be linked to a more severe cholera epidemic when a particular copepod species is more abundant.
Indeed, many factors govern the adhesive ability of V. cholerae, whether in the environment or in the human gut. The "environmental" capabilities of V. cholerae include its ability to secrete a powerful chitinase, which assists its growth on chitin surfaces. Besides colonizing copepods, V. cholerae is also present in shellfish. In the ocean ecosystem generally, the breakdown of chitin is an important ecological function. Another interesting capability is V. cholerae's production of a protease, which enables it to penetrate the mucus barrier that covers the gastrointestinal epithelium of humans and the egg sac of copepods.
Carla Pruzzo of the Universita Politecnica delle Marche has reported preliminary data that serum from Mytilus (mussel) hemolymph increases attachment to intestinal epithelial cells. The upshot is that when Vibrio is ingested with seafood, cholera acquires "bridging molecules" which make cholera very adhesive in the intestine. As Pruzzo explains, "Both virulence and infectivity depend on both bacterial properties and environmental factors."
[Slide 20: spiral: ecosystem level]
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Let's move up the spiral to take in a broader view, looking at cholera from the perspective of the ecosystem.
We know that cholera epidemics are seasonal. Using remote sensing imagery, we discovered that, in areas of Bangladesh, cholera outbreaks occur shortly after sea surface temperature and sea surface height are at their zenith. This usually occurs twice a year, in spring and fall.
Recently we have used remote sensing to track sea surface temperature and sea surface height. We discovered that temperature patterns were clearly linked to the pattern of cholera outbreaks in Bangladesh and in South America. Simply stated, we have found a positive correlation between increased sea surface temperature and sea surface height and subsequent outbreaks of cholera.
Heating of surface waters, especially off a tropical or subtropical coast, results in an increase in phytoplankton. Through remote sensing, we can now determine when that bloom is occurring. Phytoplankton, in turn, provide food for zooplankton, including the copepods, which then increase, followed by an abundance of vibrios in the water when the zooplankton bloom "crashes."
[Slide 21: Heidelberg: Choptank map]
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Populations of bacterial species such as Vibrio cholerae are very patchy in distribution--they vary over both time and space. Vibrio species vary seasonally from spring to winter in the Choptank River, a tributary of the Chesapeake, and even markedly from one week to the next.
Work on this question by my student, John Heidelberg, now at The Institute for Genomic Research, is important for decisions based on environmental sampling, when samples at two nearby locations, or a few days apart, may differ greatly.
[Slide 22: Heidelberg: graphs]
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The upper graph shows how numbers of vibrios, measured by FISH -- fluorescent DNA probes -- vary over spring to winter, from one week to the next. The lower graph shows the variation from week to week of the number of V. cholerae cells found on just one zooplankter such as a copepod.
The highest measurement was 7,100 (7 x 103) vibrios on a single copepod. Ingestion of a couple of copepods would approximate an infectious dose -- a dose, based on human volunteer studies, that could cause cholera in a human being. We see how factors at scales large and small, seasonal and microscopic, might interact to shape populations of cholera bacteria.
[Slide 23: Bangladesh picture: woman straining water]
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In cholera-endemic areas, human beings are part of the cholera bacteria's ecosystem. Yet there is a simple and inexpensive tool available to thwart its progress.
A sari cloth such as this, available even in the poorest household, can be folded eight to ten times. This creates a 20-micron mesh filter, as we determined by electron microscopy. As we saw in the film excerpt from Bangladesh, we have found that straining water through several layers of sari cloth may be enough to prevent ingestion of infectious levels of cholera bacteria.
[Slide 24: micrographs of old and new sari cloth]
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The pictures show why old sari cloth, not new, is preferred - because its holes are smaller (ca. 20 µ) and better able to trap the plankton. Laboratory studies show that old sari cloth folded at least eight times filtered out more than 99% of the V. cholerae attached to plankton.
[Slide 25: Not available]
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We published in Proceedings of the National Academy of Sciences results of a three-year study carried out in 65 villages in Matlab, Bangladesh, comprising a total study population of about 133,000 people. You can see the result here for filters made of sari cloth and nylon net versus the control group.
The incidence of cholera was reduced more than 50% in villages that used sari filters. The severity of disease also appears to have been reduced in villages that filtered, but this will need confirmation, which we hope to achieve in continuing studies.
[Slide 26: ballast ship]
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Another way human beings interact with V. cholerae in the ecosystem is through discharge of ship ballast.
[Slide 27: Not available]
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The student we see here is inside a ballast tank of a ship collecting water samples.
Cholera bacteria have been detected in ballast water of ships entering the Chesapeake Bay and the U.S. Great Lakes after an ocean crossing. It has not been shown that cholera can colonize a new geographical area permanently this way. Nonetheless, if coastal environments continue to warm, an organism adapted to warmer temperatures and frequently transported in ballast water may be deposited in a local site, transiently at least temporarily, and cause a localized outbreak from ingestion of polluted shellfish.
[Slide 28: spiral: global level]
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From a global perspective, the largest epidemic of cholera to strike the Americas occurred toward the close of the last century.
[Slide 29: Peruvian coast]
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The coast of Peru has added new insights to the cholera story. Here, cholera surfaced in 1991 after a century of absence in Latin America. Cholera has recurred in Peru since then, following a seasonal pattern, with the greatest number of cases in the summer (June-March) in Lima and other major cities along the coast.
[Slide 30: Table 4 - Lipp et al.]
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Erin Lipp and others studied the seasonal distribution in coastal Peru of total V. cholerae, V. cholerae 01, and ctxA. The percentages for each are shown in this table. V. cholerae detection followed ambient temperature increases and coincided with or preceded annual outbreaks of cholera in summer.
[Slide 31: Not available]
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Again off the Peruvian coast, we have reported a significant correlation between cholera incidence and elevated sea surface temperature. This study by Ana Gil and others covered October 1997 to June 2000 and included the 1997-98 El Nino event. The lines show sea surface temperature at different study sites, while the bars give rates of cholera at the same four sites.
We hypothesize that toxigenic V. cholerae 01 El Tor was present along the coast at least since October 1990, causing sporadic cases until the epidemic began full force in January 1991 as shown by Dr. Brad Sack and co-workers, including members of my laboratory. Continuing work during and after the 1997-98 El Nino showed that, indeed, the coastal waters of Peru harbor V. cholerae 01 in seawater and associated with zooplankton.
We hypothesize that the El Nino event triggered the resurgence of cholera in Peru. Warm water along the coast, coupled with plankton blooms fostered by El Nino rains, may have helped amplify the population of cholera bacteria already in the environment.
Work by Mercedes Pascal, myself and my students suggests that the ENSO pattern also affects atmospheric circulation in the Indian Ocean and South Asia. El Nino may influence regional climate in Bangladesh-- affecting the prevalence of cholera.
[Slide 32: Not available]
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The spiral of biocomplexity comes full circle, connecting climate patterns to cholera in Latin America, Bangladesh, and even its presence (without epidemics) in the Chesapeake Bay.
Now we are poised on the threshold of prediction for complex systems such as cholera ecology.
For example, satellite data suggest that it is to the north of Bangladesh, over the Himalayas, that certain temperature patterns unfold six months before the incidence of cholera rises in Bangladesh.
We want to be able to incorporate such climate patterns into an early warning system for cholera, especially for "good" versus "bad" cholera years, and this is now feasible. As science moves from reduction to integration, so we move from reaction to prediction.
Connecting cholera to climate has required insights from sociologists, physicians, field extension agents, microbiologists, epidemiologists, ecologists, statisticians, remote sensing scientists, and environmental scientists.
The spiraling perspective of biocomplexity allows us to blend insights from the smallest to the largest scales. Biocomplexity shows how cholera and climate entangle, and how epidemiology and ecology intertwine.
Only now, armed with this new perspective, with the integration across disciplines, and the explosion of opportunities to share scientific insights around the world, can we unravel the mysteries and assemble the puzzle pieces of emerging and re-emerging infection, paving the way to an even greater understanding of the ecology of infectious disease.
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