were interested in whether genetic engineering could be
used to improve autocidal control,”
Unhealthy exposure on the reservation
Apperson, a professor in the Entomology Department of the College of Agriculture and Life Sciences, is investigating La Crosse encephalitis, or LACE, a leading cause of pediatric encephalitis in the United States. The disease is restricted in North Carolina to the western mountains, where there are large populations of Eastern tree-hole mosquitoes and Asian tiger mosquitoes that carry the virus.
Physicians often confuse LACE with other ailments, and therefore cases are underreported, Apperson said. He hopes that documenting the costs of the illness, monetary as well as physical and emotional, will lead to greater awareness and greater prevention efforts.
Symptoms of the disease include headache, fever and disorientation. Extreme cases can cause seizures, swelling of the brain and even death. The disease may be mistaken for meningitis or other types of encephalitis, characterized by brain inflammation.
In the late 1980s and early ’90s, Apperson was involved in monitoring mosquitoes found at the homes of LACE patients. “We found that there were large numbers of mosquitoes produced in discarded containers around the home. Hence, we feel pretty strongly that’s why there are so many LACE cases reported from the reservation itself,” he said.
“Plus the living style there is very conducive to exposure. They live in wooded areas, and the kids, by and large, play outside where they are exposed to mosquitoes.”
Those living on the reservation are four times more likely to have antibodies to the LACE virus, indicating more exposure to the virus, than people living elsewhere. While about 4.5 percent of the general population outside the reservation has antibodies to the virus, 20 percent of those living on the reservation have those antibodies.
And, for those living on the reservation, the chances of exposure climb with age. Half of those age 75 and older have been exposed to the virus sometime during their lives. But while adults may develop antibodies to the LACE virus, only children actually become sick from it, and researchers aren’t sure why.
“How do you solve this problem? There is no vaccine for it, so really the only way to avoid becoming ill or getting sick is to avoid mosquito bites. And that involves better mosquito control, public education, increased awareness and better reporting, so we know the true number of cases,” Apperson said.
“Our thinking is that we need to increase public awareness and physician awareness, and the way to do that is to really quantify the costs of the disease. I think the disease is really underrecognized and underappreciated because no one has bothered to document the residual effects.
“Nobody’s ever documented the costs associated with the disease, which are considerable. These patients spend a week to several weeks in the hospital in intensive care, so you’re not talking about a trivial sum of money, you’re talking about a pretty substantial sum of money,” Apperson said.
Most patients recover from the disease, but many report residual effects, including learning disabilities, recurring seizures and other physical and mental problems because the virus invades nervous tissue. A recent study published in the New England Journal of Medicine confirmed Apperson’s suspicions that LACE may be more common and produce more severe, on-going symptoms than previously believed.
At a meeting on the reservation, Apperson heard health officials and mothers of LACE victims tell stories of the devastating and ongoing effects that the disease had on their children and families.
“We met with Indian health services officials and tribal health delivery personnel. Mothers spoke of the grief and pain and suffering they had experienced from this disease and the fact that many of their kids were still suffering. And we hope that will be a stepping stone for what we want to do and a stepping stone for developing a more comprehensive and sustainable education program,” he said.
“Parents told of more devastating effects than those in the literature,” said Todd Utz, a master’s degree student in entomology, who is working with Apperson on the project. This summer, Utz conducted 21 interviews on the reservation and in other parts of North Carolina to gather data on disease costs, both medical and nonmedical.
Medical costs include hospitalization, doctor visits and therapeutic drugs. Nonmedical costs include things like mileage to the doctor’s office, time lost from work to care for a sick child and time lost from school for the patient.
To measure loss of life-quality for the patient, Utz is using a World Health Organization index called Disability Adjusted Life Years, or DALY. The index uses information about residual effects from the disease, their duration and severity, to determine how many productive life years a community may lose as a result of the disease. DALY has been used to measure the impact of diseases ranging from malaria to alcoholism.
In addition, Utz will measure the emotional costs of the disease and how it has disrupted families and communities. Through the interviews, he identifies stressors associated with the disease and rates those by stress level. Stressors include logistical problems such as transportation to the doctor for those without a car, a lack of understanding about the disease or anxiety over a parent’s missed work days. Utz is also using an “impact-of-La-Crosse-encephalitis scale” to measure the impact of the illness on the life of the patient.
“What I feel is important is looking at the situation and finding out the true cost, both economic and human, and getting a number that people can look at to help them appreciate the disease. Hopefully, they will then say, ‘O.K. this is an important disease. How can we now take steps to prevent this and educate the public about this?’ ” Utz said.
In June, Apperson received permission from the Cherokee tribal council to conduct the study, which involves contacting and interviewing families of LACE victims. The university’s committee on research in human subjects also has endorsed the project.
Though Utz found LACE patients across the state, he reports almost all of the patients contracted the disease while visiting the North Carolina mountains west of Asheville. The one-to-two-hour interviews were mostly with the parents or guardians of children who had tested positive for the LACE virus.
Spraying is ineffective at killing mosquito species that carry La Crosse encephalitis. “The mosquitoes in the mountains that carry this virus are active during the day, and they breed in small, discreet containers. They don’t breed in ponds or lakes or puddles, ” Apperson said.
Apperson recommends the practice of “tip-and-toss” prevention, regularly dumping water that collects in containers such as buckets, old tires and even saucers beneath potted plants.
“The whole problem is a result of what we call KAP, which stands for knowledge, attitude and practice. Homeowners have very little knowledge of mosquitoes. They have a very passive attitude toward mosquito production, mosquito breeding and the disease itself. And their practices are conducive to mosquito production. They leave containers out, which mosquitoes will breed in.
“This is a social, cultural problem. That’s why we need to do an economic analysis,” Apperson said. “We need to document all the pain and suffering associated with the disease so that we can increase the awareness of it and encourage people to be vigilant about cleaning up around the home.”
Infant formula like mother's milk
The research of Drs. Jack Odle and Bob Harrell, nutrition professors in the Department of Animal Science, and graduate student Susan Matthews has helped lead to federal acceptance of two oils for infant formula that provide fatty acids commonly found in mothers’ milk. The oils are sources of two long-chain polyunsaturated fatty acids (LCPUFA) that are important in the brain and visual development of infants.
Odle’s research area, nutritional biochemistry of the neonate, focuses on animal and human infant nutrition, using piglets as a model. Piglets were used to study two different sources of the LCPUFA and the benefits that each provided to infant nutrition.
The pigs were chosen to test safety and delivery aspects of LCPUFA from two sources produced by Martek Biosciences Corp. Odle’s work has long involved using piglets as a model for studying infant and piglet nutrition. Piglets are good research models because of their size — three to four pounds at birth — and because their gastrointestinal tract is similar to that of humans.
“The young pig, in many ways, is an excellent animal pediatric model, especially if you compare it to rodents, which are used frequently to model human nutrition,” Odle said. “The infant formula companies have really liked the piglet model and have employed it increasingly to examine various aspects of pediatric nutrition and metabolism.
“The piglet also is a good bridge to a very nice pre-clinical model where we can feed the pigs and measure their growth, and we can also closely examine various tissues and organ systems to ensure health and safety.”
As a result of the team’s work, the U.S. Food and Drug Administration has issued notification that the two oils developed by Martek are generally recognized as safe for inclusion in U.S. infant formula. More than 60 countries throughout the world already use these oils in infant formula.
“Breast milk is the gold standard that the infant formula companies try to mimic or emulate as best they can,” Odle said. “Of course, over the years, infant formula has been refined to be more and more similar to breast milk. In this research, we are striving to better align the fat profile of infant formula to that of breast milk.”
Although fat often gets a bad name in the nutrition world, certain types of fat are necessary in the diet, especially for infants, Odle said. For many years, infant formula contained two essential fatty acids — linoleic and linolenic acid. Nutritionists believed that infants could metabolize these fatty acids, by adding carbons to each, to create arachidonic acid and docosahexaenoic acid, the LCPUFA that are found naturally in breast milk.
“These converted fats show up in mothers’ milk in small amounts,” Odle said. “If a mother’s diet is adequate, she will make these LCPUFA and pass them along in her milk.”
In recent years, researchers have come to believe that infants are not capable of metabolizing sufficient amounts of the essential fatty acids in infant formula to provide the same nutrition as when the LCPUFA are fed directly. Many nutritionists now believe the LCPUFA should be provided directly to the infant’s diet to enhance growth and development.
“These LCPUFA are primarily used in neural and visual development,” said graduate student Susan Matthews. “Most of the trials that have been done with infants (who consume formula with LCPUFA) show these infants have better visual acuity. Interestingly, other studies have shown that children who were breast-fed as infants have higher IQs than children who were formula-fed. We speculate that these differences could be due, in part, to differences in fatty acid composition.”
“In model species, where you can get in and see where these fatty acids end up in the body, they concentrate in the retina and in neural tissue in the brain,” Odle said. “It’s not really understood their precise involvement, but these studies that show differences in IQ are pretty remarkable.”
An efficient use of genetic engineering
That is why scientists in the College of Agriculture and Life Sciences at North Carolina State University are thinking today about how best to use this powerful new technology to control insect pests tomorrow.
Dr. Fred Gould, a William Neal Reynolds professor of entomology, and two colleagues, Dr. Paul Schliekelman and Dr. Stephen Ellner, have tried to consider all the variables that may affect what Gould calls “autocidal” control of insect pests using genetic engineering techniques.
While the genetic manipulations Gould has been considering were not possible two years ago, breakthroughs in the genetic manipulation of insects have recently opened the door for such approaches. Molecular geneticists, who are just now beginning to develop autocidal pest control strategies, will be able to use what Gould has done to perform their work more efficiently.
The lure of autocidal pest control strategies that employ genetic engineering is that they have the potential to be, in Gould’s words, “almost surgically precise. The only thing harmed is the pest species.” Indeed, even without the advantage of genetic engineering, this type of pest control is very specific and has little environmental impact. Only the insect pest is affected, and no insecticides are used.
Autocidal techniques have been used in the past with considerable success, Gould said. Perhaps the best known example of autocidal pest control is the release into the environment of sterile insects raised in captivity. The sterile insects mate with wild insects but don’t produce offspring. Eventually, the wild population is eradicated.
Gould said this technique worked particularly well in the 1950s and ’60s to control a pest of cattle in Texas called the screwworm fly. Male screwworm flies were raised in captivity, then subjected to radiation to sterilize them. The sterile flies were then released among wild flies. The screwworm fly was eventually eradicated.
A similar technique is used today to try to ensure that the Mediterranean fruit fly does not gain a foothold in the United States. Traps are used to monitor for the presence of fruit flies. Whenever a fruit fly turns up in a trap, millions of sterile fruit flies are released in the area, ensuring that a fruit fly population does not develop.
But this type of autocidal control has its drawbacks. Gould pointed out that it’s not very efficient. If it is to be effective, sterile insects must outnumber fertile insects by a wide margin, sometimes as much as 1,000 to one. Multiple releases of sterile insects are usually required to eradicate an insect population, and the technique doesn’t work well if the wild insect population is spread over a wide geographic area.
Genetic engineering could make autocidal insect control more effective; Gould has been considering how to use genetic engineering most efficiently.
Working primarily with Ellner, an expert in biomathematics, and Schliekelman, who was then a graduate student, Gould assessed autocidal techniques he thinks will be used in the future. As part of Schliekelman’s doctoral thesis, he developed mathematical models the scientists used to evaluate different autocidal strategies. Schliekelman and Ellner have since left N.C. State. Schliekelman is now a postdoctoral fellow at the University of California, Berkeley, while Ellner is on the faculty at Cornell University.
“We were interested in whether genetic engineering could be used to improve autocidal control,” Gould said.
The research team considered two autocidal techniques. They looked first at using genetic engineering techniques to insert into insects what Gould calls a “dominant conditional lethal gene.”
It should be possible, Gould explained, to add to an insect’s genetic makeup a genetic package containing a switch called a promoter that turns on a gene that kills the insect. The promoter would respond to a cue, probably environmental, such as the shorter day length as seasons change. Insects with this genetic package would die at a certain time of year.
Genetically modified insects would be released into a wild population of insects. As the genetically modified insects bred with their wild cousins, the lethal gene would spread among the population. Then, when the gene was triggered, most of the target population would die. But how much of the target population would be killed? And what factors would influence the success of the strategy? Those are some of the questions Gould, Schliekelman and Ellner tried to answer.
Gould said advances in genetic engineering now make it feasible to insert such a genetic package at multiple locations within an insect’s genome, or genetic makeup. One of the many variables his team looked at was how many insertions would be most efficient. The number of generations the target population goes through from the release of genetically engineered insects until the lethal gene triggered would also affect efficiency, as would factors such as mating behavior and whether male or female only or male and female insects are released. The model showed that at least four generations from the time of release are necessary for maximum effectiveness.
The reproductive fitness of released insects in relation to wild insects would also affect efficiency, Gould said. He explained that genetic manipulation and rearing insects in captivity both tend to have a cost in terms of decreased reproductive efficiency. Genetically engineered and laboratory raised insects usually do not reproduce as successfully as wild insects. All these factors are considered in the model Gould’s team developed.
A similar model was developed for the release of insects carrying a dominant gene that kills only female insects, leaving a male-dominated population that cannot reproduce. The advantage of this technique is that the pest population is reduced while the introduced gene remains in the population.
The models showed that the success of releasing insects carrying a female-killing gene is not as sensitive to the number of generations the target population goes through following the release as is releasing insects carrying a conditional lethal gene. The models showed that both control strategies are more effective when more copies of the lethal gene are inserted into the insect’s genome. In some cases, these techniques are expected to be 100 times more efficient than the traditional sterile insect approach. However, effectiveness can decrease rapidly if genetic manipulation decreases reproductive fitness.
Gould said the models show that if molecular geneticists can develop techniques that allow them to produce insect strains that carry multiple copies of a lethal gene without degrading reproductive fitness, then autocidal strategies could be a powerful method of insect control. The models should be valuable to molecular geneticists, for they balance the various factors that are likely to affect the efficiency of genetic engineering techniques. The models point up the need for molecular geneticists to work with entomologists who understand insect ecology and pest management specialists in order to make the best use of genetic engineering technology.
Gould is currently working with Dr. Trudy Mackay, a genetics professor at N.C. State, and Dr. Luke Alphey, a geneticist at Oxford University in Great Britain, to test his models with genetically altered fruit flies. At the same time, the federal Centers for Disease Control is interested in Gould’s work and how it may apply to efforts to genetically engineer mosquitos to limit the spread of malaria.