To the vector go the spoils

By breaking down the complex cycle that allows mosquitoes, tsetse flies, ticks and other arthropods to transmit fatal disease, Yale scientists are providing new ammunition in the fight against malaria and other vector-borne illnesses.

Last July, something went very wrong in New York City’s crow population. Signs of trouble appeared first in the Bronx, where birds were observed flying erratically, staggering on the ground and suffering convulsions. Before long, crow carcasses began to dot the landscape near the 265-acre Bronx Zoo, much to the alarm of its veterinarians. They had good reason to worry.

Around the same time that birds were dying in the Bronx, a disease with similar neurological symptoms was affecting humans in New York. The Centers for Disease Control and Prevention (CDC) identified it at first as St. Louis encephalitis, a viral infection carried by mosquitoes. The CDC suggested that the deaths of birds, including flamingos, pheasants, cormorants and a bald eagle at the zoo, had been caused by mosquito-borne Eastern equine encephalitis, a disease that also affects horses and people. But Tracey McNamara, D.V.M., the zoo’s chief of pathology, had her doubts. Although Eastern equine encephalitis does kill birds, the birds at the zoo that would have been most susceptible were all healthy. Australian emus, considered “sentinel birds” for the disease, showed no signs of illness.

By late September the CDC had arrived at a new diagnosis: Both the human and the avian deaths had been caused by West Nile virus, a mosquito-borne disease never before seen in the Western Hemisphere. By the end of summer, West Nile encephalitis would kill not only the zoo’s birds and thousands of crows, but humans as well. Six New Yorkers and a Canadian tourist were dead, and 52 others had fallen ill.

The sudden appearance of West Nile in New York is yet another indication that the battle against infectious disease is far from over, says Durland Fish, Ph.D., an associate professor of epidemiology who has been a frequent commentator on the West Nile outbreak. A generation ago, as medicine marveled at the dramatic successes of antibiotics and the first effective vaccines, it seemed that communicable disease was on the way out. But in the last decade, the emergence of new maladies in the industrialized world, along with new strains of bacteria resistant to antibiotics, has changed that picture.

Many of the emerging diseases are vector-borne, which is to say that they rely on a complex cycle of transmission enabled by intermediaries. These “vectors” include mosquitoes (which carry the West Nile virus and the protozoan responsible for malaria), sand flies, ticks and other arthropods. Vector-borne illnesses from Lyme disease to African sleeping sickness have been on the rise around the globe and resurgent in places where they had once been controlled.

“The wealthiest people in the world, living near New York City, and amongst the poorest people in the world, living in rural communities in sub-Saharan Africa, all have to deal with vector-borne disease,” says Scott L. O’Neill, Ph.D., associate professor and head of the vector biology section at the School of Public Health. “It’s a universal problem.”

Vector-borne diseases are re-emerging, not only because insects have developed resistance to pesticides, and pathogens to medication, but also because war, human migration, poverty and complacency have undermined many of the disease-control strategies that were once effective. In addition, diseases are spreading to new places through international trade, travel and refugee resettlement. Patterns of disease may also be altering because of global warming, deforestation and even reforestation (as in New England, where the growth of new forest brings deer hosts for the tick responsible for Lyme disease into close contact with people).

The human cost of West Nile is dwarfed by the devastation caused by other vector-borne diseases, major killers. Malaria sickens 300 to 500 million people each year, according to the World Health Organization, and kills an estimated 2 million people annually—roughly the population of metropolitan Denver. Half of those who die are children. Other serious diseases, including yellow fever and dengue, are transmitted by mosquitoes. Tsetse flies spread fatal African sleeping sickness, and sand flies carry the protozoan responsible for two forms of leishmaniasis; the less deadly form, causing disfiguring skin infections, was contracted by some Gulf War veterans. A triatomid commonly called the “kissing bug” transmits the pathogen for Chagas’ disease, which can lead to encephalitis and heart disorders. Ticks carry not only the Lyme disease spirochete (which causes chronic neurological and joint problems in some patients), but also pathogens for two diseases newly discovered in the northeastern U.S., ehrlichiosis and babesiosis.

To counter these threats, vector biologists at Yale are developing novel strategies, drawing on fundamental new information about the genetic and molecular bases of these disease-transmission systems. It’s a strategy that looks at disease from many angles and incorporates entomology, molecular biology, field ecology, parasitology, population genetics and taxonomy.

Research into vector-borne disease calls for this broad approach because it requires a multidisciplinary understanding of how vector and pathogen interact with each other as well as the environment. That is because vectors transmit disease biologically rather than mechanically. As O’Neill explains it, “Vectors do not act as simple syringes injecting pathogens from one human to another the way a hypodermic needle might pass on AIDS or hepatitis.” Instead, they are the vital biological links that allow the pathogens to spread. Bacteria and viruses proliferate within the shelter of the vector’s body. Protozoa use the vector as a nursery where they multiply by completing a multistage life cycle. For example, the life cycle of the Plasmodium protozoan that causes malaria begins in the mosquito’s stomach, where the protozoa multiply, and culminates in the mosquito’s salivary gland. From there, Plasmodium parasites are passed on to a new host through the mosquito’s bite. In the human, these malaria pathogens then invade the liver, causing spleen enlargement, kidney disease or malignancy.

In recent years there has been a growing awareness of the importance of the vector-pathogen interaction. Yale investigators are looking for vulnerable points in the insect at which the chain of disease transmission can be interrupted. They are looking for ways to make the vector unfriendly to the disease pathogen or, alternatively, to prevent the pathogen from maturing or multiplying within the vector. One strategy is to make use of symbiotic bacteria that normally live in a mosquito or fly. The aim is to use the bacteria to “hitchhike” in new genes that interfere with a disease pathogen’s ability to multiply or to complete its life cycle within the vector. If it can’t mature or multiply within a vector, the pathogen cannot infect a new host.

Although vector-borne diseases have plagued humans throughout history and, indeed, affected the fate of civilizations, the notion that they were spread by insects did not even arise until a century ago. The discoveries by Ronald Ross and Giovanni Battista Grassi in the late 1890s that malaria was transmitted by Anopheles mosquitoes, rather than “mal air,” revolutionized epidemiology. Since then, however, the complexity of the interplay between vector and pathogen has hindered researchers seeking to block disease transmission. Humans fighting vector-borne diseases still do pretty much what they have been doing for generations: attempt to kill the vector. Spraying DDT on mosquitoes in the Solomon Islands, spraying deltamethrin on tsetse flies in Zaire or spraying malathion on mosquitoes in Queens are all based on the same strategy.

“You don’t have to know much about disease-transmission mechanisms to do that,” says O’Neill. The good news, he says, is that “we are at a turning point. We’re understanding on a more fundamental level how these organisms interact. This information will open up new approaches to disease control.”

Associate Professor Serap Aksoy, Ph.D., provides a dramatic picture of how the pace of research has accelerated since 1982, when she wrote her doctoral thesis. Her research, which looked at a single gene in an E. coli bacterium, took her three years to complete. Today it would require a week. “I wouldn’t even give it as a project to an undergraduate in my lab. It would be too easy,” says Aksoy, a medical entomologist. “If somebody told me 20 years ago what I’d be doing now—trying to change the genome of an insect so it doesn’t transmit parasites—I’d have told them it was science fiction.”

The new insectary at Yale is central to the work of the vector biology group. The year-old, $700,000 facility at the School of Public Health provides a controlled environment for the insect colonies maintained by the five principal investigators (who have come to Yale from four continents) and the 35 postdoctoral fellows, lab assistants and students who compose the vector biology group. Each chamber of the insectary is entered through a heavy, locked door and a thick screen zipped in place to contain the insects. Scientists entering and leaving the anteroom of the locked facility walk under a powerful fan designed to blow off any clinging insects.

The four-chamber, 1,200-square-foot containment laboratory is tightly monitored to maintain correct temperature and humidity; its lighting simulates the diurnal cycle. The insects here are mostly laboratory colonies whose ancestors were collected in Kenya, Mali, Thailand and India, now living in small mesh cages. The colonies in residence recently included two species of mosquitoes that can serve as malaria vectors; tsetse flies, which can carry the pathogen for African sleeping sickness; mosquitoes trapped at the Bronx Zoo to be tested for the West Nile virus; and fruit flies and moths that serve as experimental models. (Ticks, which are arthropods but not insects, live down the hall.) As a rule, the insects here are not carriers of disease, although the laboratory does have a separate chamber and a higher-level safety protocol for carriers, if needed.

In one of the humid insectary chambers on a recent morning, white-coated research associate Irene Kasumba, M.Sc., monitored a mating cage for Glossina palpalis palpalis, or tsetse flies. She pointed out two plump fly couples mating in the little mesh enclosure, members of the only colony of tsetse flies in North America and one of only a handful of lab colonies in the world. In the tray below them were a number of small, black pupae, the size and shape of apple seeds. Kasumba moved them to closed, aerated dishes, where they would remain until emerging a month later. In the adjacent chamber housing mosquitoes, larvae and pupae wriggled in pans of water. Mosquito pupae ready to emerge as winged adults had graduated to little bowls of water within mesh cages.

O’Neill tells the story of a fellow graduate student back in Australia who coddled her mosquitoes by feeding them the very best blood: her own. Since some mosquitoes feed at night and are adapted to thrive on human blood, she would take the mosquitoes home, strap little mesh boxes on her arms and let them bite her while she slept. At Yale, researchers depend on hamster collaborators to supply the blood. The hamsters lie supine, anesthetized, on mesh hammocks on top of the insect cages, providing meals to the insects flying around below.

Other insects are stored in freezers upstairs. Research Scientist Leonard E. Munstermann, Ph.D., keeps frozen (and sometimes live) sand flies, the vector for the Leishmania protozoan. Associate Professor Fish has frozen 500 more Bronx Zoo mosquitoes that he is screening for West Nile infection. Knowing the prevalence of West Nile in last summer’s mosquitoes will give public health officials some idea of what to expect this summer. In May, Fish, Munstermann and a group of public health students will collect more mosquitoes at the zoo, identifying the species and testing them for West Nile virus.

“We’ll know pretty early whether the virus is going to re-emerge or not,” says Fish. “We’re going to have a very tight surveillance program there.” If the virus does reappear, Fish recommends “source control,” using fish, bacteria or insecticide to kill mosquito larvae in places like storm drains and catch basins. He has been vocal in urging lawmakers to pay attention to the dangers posed by mosquitoes, attending legislative hearings and appearing on television news programs.

Vector biologists acknowledge that eradicating vector-borne diseases may prove impossible. Each time human beings make a new attempt to defeat them, vectors and pathogens contrive to survive the assault. “I think you can expect them to adapt to most things,” says O’Neill. “Evolution’s a pretty powerful thing. When you’re faced with extinction, it’s amazing how quickly a population will respond or change for survival. It’s a continual arms race with biological organisms.” YM

Related People

Leonard Munstermann

Senior Research Scientist in Epidemiology (Microbial Diseases)

Durland Fish

Professor Emeritus of Epidemiology (Microbial Diseases)

Serap Aksoy

Professor of Epidemiology (Microbial Diseases)