7

Vector Biology, Ecology, and Control

WHERE WE WANT TO BE IN THE YEAR 2010

Vector biology will play a major role in the battle against malaria. Improved vector surveillance networks will allow most countries, particularly those in Africa, to mount effective control efforts and to predict outbreaks of disease. Researchers will be able to conduct epidemiologic surveys and track drug resistance simply by analyzing mosquito populations. Simple techniques will be used in the field to identify morphologically indistinguishable mosquitoes that have different capabilities to transmit malaria parasites, leading to more effective application of vector control measures. The entomological risk factors for severe disease and death will be identified, and interventions will be implemented. The development of environmentally safe antimosquito compounds will complement traditional residual insecticide spraying, and genetically engineered microbial agents will be used to kill mosquito larvae. An antimosquito vaccine will add to the growing arsenal of malaria control weapons. Feasibility studies will be carried out to replace populations of malaria vectors with natural or genetically altered forms that cannot transmit human malaria.



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MALARIA: Obstacles and Opportunities 7 Vector Biology, Ecology, and Control WHERE WE WANT TO BE IN THE YEAR 2010 Vector biology will play a major role in the battle against malaria. Improved vector surveillance networks will allow most countries, particularly those in Africa, to mount effective control efforts and to predict outbreaks of disease. Researchers will be able to conduct epidemiologic surveys and track drug resistance simply by analyzing mosquito populations. Simple techniques will be used in the field to identify morphologically indistinguishable mosquitoes that have different capabilities to transmit malaria parasites, leading to more effective application of vector control measures. The entomological risk factors for severe disease and death will be identified, and interventions will be implemented. The development of environmentally safe antimosquito compounds will complement traditional residual insecticide spraying, and genetically engineered microbial agents will be used to kill mosquito larvae. An antimosquito vaccine will add to the growing arsenal of malaria control weapons. Feasibility studies will be carried out to replace populations of malaria vectors with natural or genetically altered forms that cannot transmit human malaria.

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MALARIA: Obstacles and Opportunities WHERE WE ARE TODAY Vector biology, broadly defined, is the science devoted to studying insects that transmit pathogens, their contact with humans, and their interaction with the disease-causing organisms. In the case of malaria, the vector is the anopheline mosquito and the disease-causing organism is the malaria parasite. Humans and anopheline mosquitoes are both considered to be the parasite's hosts. One of the primary goals of vector biology in malaria research is to promote a better understanding of the disease cycle that will facilitate more effectively targeted control strategies. The vast majority of successful antimalaria campaigns have relied heavily on vector control. The distribution of malaria within human populations is linked closely to site-specific characteristics of vector populations. Within any given area, there are usually fewer than five vector species, although the biology of each species is unique in many respects, including the sites where larvae develop, adult mosquito behavior (especially human-biting behavior), susceptibility to Plasmodium parasites, and the ability to transmit these parasites. Not all mosquitoes can transmit human malarial parasites. Of the thousands of described mosquito species, only a fraction of those in the genus Anopheles serve as vectors. Some anopheline species do not feed on humans, others are not susceptible to human malaria parasites, and a number have life spans too short to allow the parasite to fully mature. Vector species that pose the greatest threat are abundant, long-lived, commonly feed on humans, and typically dwell in proximity to people. Their role in malaria transmission depends largely on the presence of a favorable environment for larval development and adult survival, and the ability to feed on humans. Transmission also depends significantly on human habits that promote host-vector contact. Perhaps the least understood process in malaria transmission is the development of the parasite in the vector. To transmit malaria, vectors must be able to support parasite development through several key stages over 8 to 15 days. Only then are the sporozoite-stage parasites present and ready for transmission to new human hosts. Thus, from the standpoint of vector biology, there are three main points of attack for controlling malaria: the environment, human habits, and parasite development in the vector. In cases in which the impact and feasibility of vector control are questioned, the result is often an overwhelming reliance on chemotherapy-based measures for reducing malaria-related mortality and morbidity. In countries with the most severe malaria problems, there are seldom funds for anything but antimalarial drugs and, in some cases, for limited vector control activities (mostly in urban areas). Such approaches usually do little

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MALARIA: Obstacles and Opportunities to prevent malaria transmission, however. The continuous need for adequate drug supplies to treat clinically ill residents of endemic areas severely limits progress toward malaria prevention. In most malarious regions of the world, there is little baseline information on vector populations and variation in the intensity of malaria transmission. Thus, it is exceedingly difficult, and often unrealistic, for developing countries to formulate malaria control strategies aimed at prevention. As in other areas of tropical health, distinctions between field and laboratory research in vector biology are sometimes blurred, since basic research problems often require use of field-collected specimens to explore natural phenomena. Similarly, even the least sophisticated laboratories are now using modern techniques. The distinction between basic and applied research in vector biology is difficult to make, because most research topics have long-term applied or operational applications. Throughout the world, vector biology field studies generally use a common set of techniques for collecting vectors and processing field-collected specimens. The same general methods used to study malaria transmission and vector behavior are used to evaluate new vector control strategies. As new vector-related techniques are developed for investigating the biology of anopheline mosquitoes, they are quickly adopted by field-based malaria control programs. Thus, developments in malaria vector control are highly dependent on basic research. Vector-Parasite Interactions Sporogonic Development in Anopheles Mosquitoes The four human malarial parasites—Plasmodium falciparum, P. vivax, P. malariae, and P. ovale—all undergo a similar process of sporogonic development in the mosquito host (Garnham, 1966). Development begins when a susceptible female mosquito ingests microgametocytes (male forms) and macrogametocytes (female forms) during blood feeding on an infected human. Sexual reproduction (and, importantly, genetic recombination) occurs in the mosquito host as microgametocytes quickly exflagellate, producing microgametes that fuse with macrogametes to form zygotes. Zygotes develop into ookinetes, which penetrate the midgut epithelial cells and mature into oocysts. These in turn mature and release thousands of sporozoites into the mosquito hemolymph system. A mosquito is considered infective as soon as sporozoites invade the salivary glands. Transmission to humans occurs when sporozoites are injected with salivary fluids during a blood meal. The time needed for sporozoites to reach the salivary glands of the mosquito depends on both the species of parasite and the ambient temperature. For example, P. falciparum takes 9 days at 30°C, 10 days at 25°C,

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MALARIA: Obstacles and Opportunities 11 days at 24°C, and 23 days at 20°C, a difference of 14 days over a range of 10°C. At 25°C, the process is completed in 9 days for P. vivax, compared with 15 to 20 days for P. malariae and 16 days for P. ovale. The relatively short extrinsic incubation periods of P. falciparum and P. vivax are among the several reasons why these parasite species are more common than either P. malariae or P. ovale. Once a female mosquito is infective, she remains so for life. Generally, mosquitoes are capable of transmitting sporozoites during each blood-feeding episode, sometimes to multiple individuals during each feeding cycle. Boyd and Stratman-Thomas (1934) demonstrated that P. vivax-infected mosquitoes could infect 90 percent of patients during the first three weeks, 66 percent by the fifth week, and only 20 percent by the seventh week. Although old infective mosquitoes that have fed 5 to 10 times can still transmit malaria sporozoites, over time these sporozoites tend to lose infectivity. Factors Affecting Susceptibility Factors that affect the susceptibility of anopheline mosquitoes to human malaria parasites are poorly understood. Mosquitoes of the genera Culex and Aedes contain numerous species that feed on humans and transmit a number of infectious diseases. However, none of these species transmit human malarias. The physiological and genetic basis of this insusceptibility to the human malaria parasite is unknown, just as are the differences in susceptibility among various Anopheles species. The inability of malaria parasites to develop in some mosquito species may be due to the absence of some critical factor in the mosquito required for normal parasite development, or it could be due to the presence of a toxin that actively inhibits or aborts parasite development (Weathersby, 1952). One mechanism that may make mosquitoes susceptible to parasites is species-specific stimulation of exflagellation (Micks, 1949; Nijhout, 1979), while encapsulation of ookinetes and oocysts (Collins et al., 1986) and the failure of sporozoites to penetrate salivary glands (Rosenberg, 1985) may help explain mosquito resistance to malaria parasites. The genetic basis for mosquito susceptibility or refractoriness to malaria is extremely complex (Curtis and Graves, 1983). Using laboratory-reared vectors and malaria parasites from animals, it is possible to select for highly susceptible and highly refractory strains of mosquitoes. In most cases, several genes and often complicated modes of inheritance appear to be involved. Factors Affecting Transmission The basic process of sporogonic development in susceptible vector species is poorly understood. The numbers of gametocytes ingested, ookinetes

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MALARIA: Obstacles and Opportunities and oocysts that develop, sporozoites in the hemolymph and in the salivary glands, and sporozoites transmitted during a blood meal have not been well quantified. Most studies of vector competence count only oocysts on the midgut wall and crudely estimate salivary gland sporozoites. Thus, there is little information on this very important process for any vector species, and there is no basis for comparison among vector species. Studies of sporogonic development in the vector and vector-parasite relationships for human malaria parasites are largely restricted to P. falciparum, the only species that can be grown in vitro. The extent to which similar vector-related studies, using animal model systems (Mons and Sinden, 1990), are relevant to human malaria is unknown. Malaria Transmission Most vector biology field studies focus on determining human-vector contact, feeding and resting habits, survival, and other life history parameters of vector populations. Usually, the vector status of populations is defined by determining sporozoite and oocyst rates (the proportion of infective mosquitoes in a vector population and the proportion of mosquitoes with oocysts, respectively). This approach provides essential but not sufficient information about vectorial systems (all anopheline species in a given area that transmit malaria). Field studies of malaria transmission need to be reoriented toward quantifying other important epidemiologic parameters of anopheline populations. For example, little is known about the variation in the number of sporozoites in mosquito salivary glands (sporozoite loads), nor is there much information on the numbers of sporozoites transmitted per feeding and whether this parameter is affected by sporozoite loads. Globally, the diversity of vectorial systems should allow for great heterogeneity in the ability of vectors to transmit sporozoites; this has significant implications for malaria control. For example, a sporozoite vaccine may be effective in one country where a certain Anopheles species transmits an average of 5 sporozoites per bite, but not in another country where a different Anopheles species transmits 500 sporozoites per bite. Factors influencing variation in sporozoite rates and in sporozoite loads, within geographic zones, are equally important. Life stages of Plasmodium in the vector, other than oocysts and sporozoites, have never been studied in nature. Lack of information about the early stages of sporogonic development, from the point of ingestion of gametocytes to ookinetes to the appearance of oocysts, is critical because these stages influence the development of sporozoites. It is also likely that the life history parameters of vector populations, such as vector size, feeding habits, frequency of feeding, age, and reproductive state, can influence the mosquito's susceptibil-

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MALARIA: Obstacles and Opportunities ity to parasites and the probability it will survive long enough for the parasite to fully develop. Vector biologists know very little about vector-related factors that affect sporozoite viability in nature. Epidemiologic studies indicate that, at most, between 1 and 20 percent of sporozoite inoculations produce infections in nature (Pull and Grab, 1974). Effective, direct assays for determining sporozoite viability for individual, field-collected mosquitoes do not exist. Human antibodies ingested by mosquitoes may play some role in regulating sporozoite infectivity. In one study, human immunoglobulin G antibody was found on sporozoites in over 80 percent of infected mosquitoes sampled in Kenya (Beier et al., 1989); the significance in terms of sporozoite infectivity is unknown. Regulation of Vector Populations—Larval Ecology The mechanisms that regulate vector populations are poorly understood but are of great importance for malaria control (Molineaux, 1988). For example, there is limited information on the biology of aquatic stages of malaria vectors. The factors affecting larval survival and the mechanisms controlling adult production are largely unknown for even the most important vector species. The basic concept of density-dependent regulation has never been studied for populations in nature. It is extremely important to know whether populations are regulated through competition (intra-and/or interspecific) and predation in the aquatic habitat. Furthermore, there is no baseline information on the foraging habits and strategies of larval-stage vector populations. The study of larval biology is complicated further by inadequate techniques for the identification of larvae belonging to species complexes. Consequently, few entomologists seek to tackle this important area of anopheline biology. A basic understanding of the aquatic stages of vectors is extremely relevant to malaria control. Source reduction through the modification of larval habitats was the key to malaria eradication efforts in the United States, Israel, and Italy (Kitron and Spielman, 1989). In these countries, a variety of measures directed against the aquatic stages of important vectors reduced cases of malaria and eliminated parasite transmission. Vector Incrimination The identification of anopheline mosquitoes responsible for malaria transmission is known as vector incrimination, and the approach is the same for any given area. Mosquitoes, preferably those coming to feed on humans, are collected, identified, and dissected to determine the presence of sporozoites in the salivary glands. Immunological techniques can be used to

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MALARIA: Obstacles and Opportunities identify particular Plasmodium species. This is important since sporozoites of all Plasmodium species that infect humans are morphologically similar, and sporozoites of most animal malarias cannot be distinguished morphologically from those that infect humans. Species are sometimes referred to as primary and secondary vectors. The incrimination of primary vectors is usually clear-cut; they are often abundant, commonly feed on humans, and have measurable sporozoite rates. Incrimination of secondary vectors is more complicated, because these species may be uncommon and have low sporozoite rates. However, they may be seasonally abundant and, at times, play a major role in transmission. Adult mosquito behavior and larval ecology may be significantly different in primary versus secondary vectors, and measures taken to control primary vectors may not have an impact on secondary vectors. Gathering site-specific information about vectors is an important first step in planning vector control measures. It is sometimes necessary to extrapolate vector-related data from areas where actual vector identification has been performed. Such an approach is not without problems, since epidemiologically significant shifts in primary vector species can occur due to changes in the environment, such as urbanization, deforestation, and irrigation. Vector Species and Distributions Haworth (1988) provides a detailed review of the global distribution of human malaria and, for each geographic zone, lists the primary and secondary vectors. In general, malaria in each zone is transmitted by a specific set of Anopheles species. Distribution patterns for mosquito species are fairly stable. Vector species rarely completely disappear from a region, and in no case have indigenous vectors been deliberately eradicated. The introduction of malaria vectors into nonindigenous areas is a serious public health concern. For example, the introduction of Anopheles gambiae to Brazil and Egypt in the 1940s caused devastating epidemics and required unparalleled efforts to eliminate the newly arrived vector (Duffy, 1977). The natural distribution patterns of anophelines are largely determined by environmental conditions. Each species has unique environmental tolerance limits. The same is true for malarial parasites. For example, the distributions of P. vivax and P. falciparum are theoretically limited by summer isotherms of 15°C and 18°C, respectively, the temperatures required for the completion of the sporogonic cycle in the mosquito host (Boyd, 1949).

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MALARIA: Obstacles and Opportunities Mosquito Taxonomy and Species Complexes In malaria entomology, anopheline species are grouped according to morphological criteria and related taxonomic information. Much of modern-day taxonomy addresses problems associated with species complexes, that is groups of morphologically indistinguishable species that are genetically different and that may differ greatly in vectorial potential. The limits of the traditional morphological approaches to species identification became apparent in Europe with the puzzling observation of “anophelism without malaria”: certain areas with low densities of An. maculipennis had malaria transmission, while other areas with an abundance of An. maculipennis had no malaria transmission at all. This observation subsequently led to the discovery of the An. maculipennis complex. There are more than 20 recognized species complexes in the genus Anopheles, many of which include malaria vectors (Coluzzi, 1988). A variety of methods are available for discriminating among species—the gold standard relies on cytogenetics—but none are simple or as yet, practical for routine use in the field. The identification of vectors that belong to species complexes has long been a stumbling block in malaria epidemiology and control. The failure to recognize sibling species can mean that vector species are mistaken for nonvector species, and vice versa. The results of field studies that evaluate larval ecology, seasonal biting rates, host preference, infection rates, resting habits, and malaria control efforts may be misleading if morphologically defined “species” actually are a mixture of two or three species. In Africa, for example, sibling mosquito species living in the same area respond differently to insecticides, presenting formidable obstacles to vector control operations. Parasite Transmission There are distinct groups of mosquito vectors associated with almost every major type of malaria (see Chapter 10). Very often, the single greatest source of variation within these regions is the mosquito itself. The variation among species, feeding habits, seasonality, abundance, and vectorial capacity all help determine how malaria is transmitted to and expressed in individuals and populations. Field Studies The principles and methods used for sampling anopheline mosquitoes have been the same for the past 25 years, and are described in detail by the

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MALARIA: Obstacles and Opportunities World Health Organization (1975). Methods include collecting mosquitoes that come to feed on humans or animals and collecting resting vectors in houses, in animal shelters, outdoors, or in traps. In general, these methods can be adapted to any geographic location or malaria situation. Once collected, mosquitoes can be identified by external morphology and dissected for malaria oocysts and salivary gland sporozoites, and ovaries can be examined to determine parity and egg-stage development. Further analysis can reveal blood meal contents, insecticide susceptibility, chromosomal patterns for species identification, and the presence of nonmalaria parasites (World Health Organization, 1975). Immunological methods for detecting species-specific malaria sporozoites in mosquitoes have been developed (Zavala et al., 1982; Wirtz et al., 1987). In malaria field studies, these methods are useful for estimating sporozoite rates and for identifying the species of Plasmodium detected by dissection (Beier et al., 1990a). Advances in methods used to analyze blood meals (Beier et al., 1988; Tempelis, 1989) and to determine insecticide resistance (Brown and Brogdon, 1987) have been made. Major advances in the genetic analysis of vector populations using DNA-based technologies also have been made (Collins et al., 1990). A combination of traditional methods and a battery of newer immunological or molecular assays can now be performed on single specimens of anopheline mosquitoes to yield critical biological and epidemiologic information. Indices of Malaria Transmission Determining the intensity of malaria transmission by mosquito populations is a key component of epidemiologic studies of malaria. Two important dimensions of malaria transmission are the entomological inoculation rate (EIR) and vectorial capacity (VC). The EIR is a measure of the number of infective bites each person receives per night, and is a direct measure of the risk of human exposure to the bites of infective mosquitoes. The VC measures the potential for malaria transmission, based on several key parameters of vector populations. It is important to realize how these measurements differ and how each is important for describing malaria situations. The EIR is the product of the human-biting rate and the sporozoite rate. Human-biting rates are best estimated by all-night collections of mosquitoes that come to feed on humans. The sporozoite rate is determined by dissection and examination of mosquito salivary glands or by immunological methods. Measurement of EIRs during longitudinal studies provides information on seasonal variations in transmission. The EIR is the only direct measure of malaria transmission and the only useful index for predicting malaria epidemics (Onori and Grab, 1980). Vectorial capacity is based on several key parameters of vector popula-

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MALARIA: Obstacles and Opportunities tions. The equation used to calculate VC is C = ma2pn/−logcp, where C = vectorial capacity, m = density of vectors in relation to humans, a = number of blood meals taken on humans per vector per day, p = daily survival probability of vectors (measured in days), and n = incubation period in the vector (measured in days). The formula expresses the capacity of a vector population to transmit malaria based on the potential number of secondary inoculations originating per day from an infective person. The formula is specific for a given species of vector, because different species vary with respect to m, a, and p. If several vector species coexist, the VC is the sum of the vectorial capacities of each of the individual vector species. Vectorial capacity is an essential component of mathematical models of malaria transmission. There are a number of assumptions that must be taken into account when VC is used to either assess the status of a malarious situation or predict its evolution (Molineaux, 1988). Theoretically, VC can predict the extent to which mosquito populations must be reduced to affect the intensity of malaria transmission. For example, according to the formula, the mosquito population would have to be reduced by 99 percent in a holoendemic area before any change in transmission would occur. Such predictions are difficult to verify in natural situations, however. Indeed, there is a need to determine how reductions in vectorial capacity affect patterns of disease in human populations. Surveillance Strategies Malaria surveillance may be the most important first step for endemic countries hoping to understand and manage their malaria problems. Surveillance networks must be able to monitor the disease in human populations, track patterns of parasite drug resistance, and monitor transmission by vector populations. The geographic variation in the intensity of malaria transmission is of prime importance for development of appropriate control measures. Few endemic countries have useful information on the patterns and intensities of transmission. This is in marked contrast to small, size-limited studies, in which information has been collected over many years. For example, investigators have studied malaria in the Kisumu area of Kenya for 60 years, but relatively little is known about vectors and transmission anywhere else in the country. Recently developed immunoassays that detect sporozoites in mosquitoes make vector surveillance more feasible, especially in areas where vectors prefer indoor environments and can be easily sampled, as in parts of Africa. The ability to dry and store mosquitoes for later processing by enzyme-linked immunosorbent assay will facilitate the collection of mosquitoes from multiple sites for testing in central facilities. Indices of trans-

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MALARIA: Obstacles and Opportunities mission intensity, derived from measurements of vector densities and sporozoite rates (analogous to the EIR), can provide useful information about patterns of endemicity in human populations. For the purposes of malaria management, it is useful to know whether individuals receive 5, 50, or 500 infective bites per year, as well as when the transmission occurs. Vector-derived EIR estimates are highly correlated with measurements of malaria incidence. In this respect, EIRs provide stronger predictive capabilities than do estimates of prevalence. Analysis of fresh blood meals of human-fed anophelines for the presence of antimalarial antibodies represents a possible alternative approach to serosurveys. Human antiparasite antibodies remain intact, undigested, for about 24 hours. Even though anophelines ingest only 1 to 2 microliters of blood, stage-specific antibodies can be detected by simple immunoassays (Beier et al., 1989). Although molecular probes do not appear to be sensitive enough to detect the presence of parasites in this small volume of blood, polymerase chain reaction methods could be used in the future to detect low numbers of parasites in mosquito blood meals. If sufficient epidemiologic information can be extracted from analyzing the blood meals of mosquitoes, there may be less need to draw blood from residents of endemic communities. As a replacement for traditional surveys, then, the effectiveness of such an approach deserves consideration. Entomological Components of Malaria Vaccine Development The development and testing of P. falciparum sporozoite vaccines depend on the availability of sporozoites from experimentally infected mosquitoes. Sporozoites are in great demand for use in antibody assays and in the characterization and evaluation of candidate vaccines. Studies testing the efficacy of vaccines against sporozoite challenge have used infective mosquitoes to feed on volunteers. This methodology raises a number of vector-related questions. For example, since the number of sporozoites transmitted by infective mosquitoes is unknown, vaccines are being tested without reference to levels of sporozoite inocula. There is also the concern that laboratory infected mosquitoes may have a greater sporozoite transmission potential than do those in nature, since sporozoite loads are generally 10 to 200 times higher in the former (Davis et al., 1989). These entomological concerns are of equal importance when one is testing vaccines against blood stages of the malaria parasites. Vector-related issues will become even more important as vaccines move from laboratory testing to field evaluation. Some of the concerns have already been outlined (World Health Organization, 1986). A prerequisite for vaccine field trials is the long-term characterization of malaria transmis-

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MALARIA: Obstacles and Opportunities use in indoor residual spraying, provided that the vector is susceptible. This chlorinated hydrocarbon remains one of the most effective insecticides for malaria control efforts in endemic countries. Compared with other available insecticides, it is inexpensive and, importantly, is nontoxic to humans. Few of the accidental poisonings each year can be attributed to DDT use in public health disease control programs. Parenthetically, the use of DDT as a residual indoor spray does not introduce DDT into the environment in amounts sufficient to enter the food chain, and thus this usage does not have adverse ecological consequences. Innovative Vector Control Measures Antimosquito Vaccines The idea that blood-feeding vectors may be damaged by mammalian antibodies directed against insect tissues is not new. Recently, scientists developed an antitick vaccine that protects cattle against tick infestations (Willadsen et al., 1989). The antibodies induced by this vaccine are directed against “concealed antigens” on tick gut cells that are not exposed during the blood-feeding process. Preliminary studies using homogenized mosquito tissues or whole mosquitoes as the immunogen have demonstrated significant effects on mosquito survival, fecundity, and egg viability (see Chapter 9). The direct effects of antibodies produced by these methods on the sporogonic development of P. falciparum and other human malaria parasites are unknown, however, as are the mosquito antigens that might elicit the most effective human antibody response. Modern tools of immunology and molecular biology offer hope for the development of mosquito-derived antigens that could serve as candidate vaccines for malaria control. Genetic Modification of Mosquito Vectors It is theoretically possible to replace populations of malaria-transmitting mosquitoes with genetically altered forms that cannot transmit the malaria parasite. The foundation for vector replacement strategies is based on continued progress and major advances in critical areas of vector biology. Already there have been successful attempts to insert foreign genes into the mosquito genome (Miller et al., 1987), and molecular probes for species identification have been developed and field tested (Collins et al., 1988). Much research remains to be done before genetically engineered mosquitoes can be used for malaria control. Regardless of the long-term outcome of such research, however, valuable new techniques and approaches for identifying the genetic basis of malaria transmission will be developed along the way. Environmentally Safe Biological Control Agents Natural predators and pathogens, ranging from viruses to nematodes, help regulate populations of both

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MALARIA: Obstacles and Opportunities immature and adult-stage mosquitoes. A number of studies are looking at candidate biological control agents. Although some appear to have an impact on mosquito populations, none have a primary role in current antimalaria control operations (Service, 1983). The most promising avenue in biological control is focusing on the larval ecology of major vector species to determine which organisms serve as food resources in the aquatic environment. Modern genetic engineering methods may eventually be used to insert genes that would promote the release of substances that would kill or disable mosquito larvae. Training in Vector Biology The contributions of vector biology to infectious disease research are diminishing around the world because of a lack of trained personnel, training resources, career opportunities in academia and the government, and financial support (Reeves, 1989; Moore, 1990). From 1970 to 1982, at 24 leading training institutions in the United States, 144 doctoral degrees were awarded in vector biology and 88 postdoctoral fellows were trained in the field (National Research Council, 1983). Slightly more than a fifth of these doctoral and postdoctoral scientists received their training at medical schools or schools of public health. Each year, only 10 new doctoral candidates can be expected to enter vector biology training programs in the United States. A workshop sponsored by the National Research Council developed recommendations to prevent or delay the expected national shortage of vector biologists, but these recommendations have so far not been implemented. Although there have been no assessments since 1982, there appear to be fewer and fewer students receiving training in vector biology. Many of the outstanding educators in the field have retired and have not been replaced. Several vector biology training programs have simply closed down. None of the six textbooks on medical entomology published since 1970 are still in print, and there are no replacements in sight (Reeves, 1989). The declining number of students trained in vector biology is related to a general trend in infectious disease research. The emphasis on biochemical- and molecular-level investigations of pathogens has superseded the biological studies of vectors, vector-pathogen interactions, and the specialty areas of medical entomology and disease ecology. The vector biologist in academia has limited access to research grant funding; work in this area is generally not considered to be at the cutting edge of science. In the immediate future, the need for vector biologists will be tied closely to increases in malaria and other vector-borne diseases expected to result from the repercussions of population growth over the next 25 years. It may

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MALARIA: Obstacles and Opportunities be that special programs will need to be developed to fund those scientists willing to devote years to working in relative isolation in the field. RESEARCH AGENDA Over the next 15 to 20 years, malaria research in the area of vector biology should focus concurrently on four areas: field investigations, laboratory-based research in support of field investigations, innovative methods for malaria control, and vector control evaluation in endemic areas. Field Investigations Patterns of Transmission Each type of malaria (see Chapter 10) is associated with a distinct group of mosquito vectors that vary greatly in their potential to transmit malaria parasites. An understanding of patterns of malaria transmission depends on vector field studies focused on defining ecological interactions among mosquito vector populations, malaria parasites, and humans. The identification of key points at which transmission can be interrupted will provide clues for malaria control in areas of high transmission and will lead to new strategies for malaria eradication in areas of low transmission. RESEARCH FOCUS: The dynamics of malaria transmission by vector populations and the risk of exposure for human populations in various ecosystems. Microepidemiology A critical question is why some individuals living in areas of stable malaria transmission develop severe and life-threatening disease. The incidence of severe malaria may depend, among other things, on the local patterns (microdistribution) of malaria transmission and on the intensity of sporozoite inoculation. Identifying vector-related determinants of severe malaria requires new approaches for measuring house-by-house variations in malaria transmission. RESEARCH FOCUS: Characterization of “microepidemiologic” patterns of malaria transmission, including the identification of vector-related and environmental risk factors for the development of severe malaria.

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MALARIA: Obstacles and Opportunities Regulation of Anopheline Populations Factors affecting larval survival and mechanisms controlling adult production in aquatic habitats are largely unknown, even for the most important vector species. There is limited information on the foraging habits of larvae (what they eat), and the extent to which natural populations are limited by intra- and/or interspecific competition and predation is unknown. The failure to understand population survival strategies and natural mechanisms of competitive exclusion among Anopheles taxa presents obstacles for the development of population replacement strategies, whereby attempts could be made to replace a vector species with introduced non-vector mosquitoes. Another obstacle involves difficulties in identifying larvae belonging to species complexes. Lack of knowledge in all of these areas hinders malaria control efforts. RESEARCH FOCUS: The foraging habits of larvae and the role of intra- and/or interspecific competition and predation in regulating vector populations. Country-Wide Vector Surveillance Geographic variation in the intensity of malaria transmission is of prime importance for the development and stratification of control measures. In most endemic countries, there is little or no information on patterns and intensities of transmission on a national scale. Efficient new surveillance techniques are needed to establish country-wide networks for monitoring the disease in human populations, patterns of drug resistance in parasites, and transmission intensity by vector populations. In areas of tropical Africa, where vectors are highly anthropophilic and endophilic, vector sampling inside houses and corresponding evaluation of sporozoite rates by immunoassay would provide sensitive indicators of transmission intensity (estimates of EIR). There are unexplored possibilities for assaying human blood meals of mosquitoes for the presence of stage-specific malaria antibodies (objective: analogous to serosurveys), malaria parasite antigens (objective: analogous to prevalence surveys), antimalarial drugs (objective: monitor drug use by humans), and even drug-resistant parasites (objective: monitor drug resistance). Progress in the area of vector surveillance for monitoring malaria on a national scale will minimize the need of traditional mass blood surveys and would promote more effective measures for predicting epidemics and allocating resources for malaria control. RESEARCH FOCUS: Innovative vector surveillance strategies for assessing malaria transmission by vector popu-

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MALARIA: Obstacles and Opportunities lations, for measuring the risk of exposure for human populations, and for monitoring epidemiologic parameters in human populations (e.g., malaria antibodies, malaria antigens, and drug use) in country-wide efforts to improve malaria management capabilities. Vaccine Field Trials Vector-related issues assume great importance as candidate vaccines move from laboratory testing to field evaluation. Baseline information on both the intensity and seasonality of transmission is necessary for planning vaccine trials. During vaccine field trials, concurrent measurements of sporozoite challenge (EIR) will be useful for gauging vaccine efficacy, especially in large trials conducted at sites with different transmission intensities. RESEARCH FOCUS: Site-specific characterization of malaria transmission by vector populations before vaccine field trials and concurrent efforts to monitor transmission intensity during field trials. Transmission-blocking vaccines are being developed to elicit human antigametocyte antibodies that block early stages of parasite development in the mosquito. Such vaccines, when used in a mixture with other vaccines, may extend their effective life by reducing the rate at which variant forms of parasites appear. Both naturally occurring and laboratory-generated transmission-blocking antibodies are usually tested by mixing sera containing antibodies with malaria parasites and assessing the development of oocysts in the vector. It is difficult to predict the potential efficacy of transmission-blocking vaccines because the extent to which antigametocyte antibodies regulate vector infectivity in nature has never been determined in focused studies on vector populations. RESEARCH FOCUS: Field studies to assess natural effects of transmission-blocking antibodies on the early stages of parasite development in vector populations. Laboratory-Based Research Malaria Parasite Development in Mosquitoes Malaria parasite development in anopheline vectors is a complex process that takes 8 to 15 days from the ingestion of gametocytes to the appearance of sporozoites in the salivary glands of the mosquito. Regula-

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MALARIA: Obstacles and Opportunities tory mechanisms affecting sporogonic development in susceptible anopheline species are largely unknown. A greater understanding of the differences in sporogonic development and transmission potential among vector species may provide important clues about malaria epidemiology and establish new directions for blocking the transmission cycle in nature. In marked contrast to parasite stages in the human host (see Chapter 6), there have been relatively few attempts to understand the behavior, physiology, and bio-chemistry of parasite stages in the definitive host, the mosquito. RESEARCH FOCUS: Basic mechanisms of malaria parasite development in anopheline species and vector-parasite interactions that affect sporozoite transmission potential. RESEARCH FOCUS: Development of in vitro culture systems for stages of the malaria parasite that occur in the mosquito host. The fact that most anopheline mosquitoes cannot transmit malaria indicates the presence of one or more refractory mechanisms that inhibit parasite development. The problems of maintaining colonies of anopheline species and cultures of human malaria parasites in the laboratory make the study of these mechanisms difficult. RESEARCH FOCUS: The genetic, physiological, and bio-chemical basis for mechanisms of mosquito refractoriness to malaria parasites, with the goal of developing novel approaches for blocking or interrupting sporogonic development in the vectors. Technique Development for Field Studies Given advances in biotechnology, there are a number of field-test systems that would be extremely useful to vector biologists. The most immediate need is for simple and inexpensive methods to differentiate among anopheline species in areas where morphologically indistinguishable vector and nonvector species exist. Field researchers also require better methods to identify genetic variants within anopheline species, as well as new techniques for assessing the chronological age of vectors and for characterizing parasites in vectors according to drug resistance and genetic diversity. The ideal assay system would be capable of simultaneously analyzing individual mosquitoes for multiple epidemiologic variables, including sporozoites, blood meals, insecticide resistance, and species identification.

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MALARIA: Obstacles and Opportunities RESEARCH FOCUS: Development and field testing of immunological and molecular assay systems, in conjunction with studies of natural malaria transmission and malaria control efforts for determining vector-related parameters. Innovative Methods for Malaria Control Antimosquito Vaccines Unlike vaccines directed against the malaria parasite, an antimosquito vaccine would be effective against all Plasmodium species carried by a common vector. A successful vaccine would not necessarily have to kill the mosquito immediately after it feeds on humans to be effective. Vaccine-induced effects on mosquito survival, blood meal digestion, feeding frequency, reproduction, or physiological processes may have profound effects on the sporogonic development of the parasites and, ultimately, vector transmission potential. RESEARCH FOCUS: Development of candidate anti-mosquito vaccines that produce host antibodies with an immunopathological impact on the vector or that disrupt parasite development in the vector. Genetic Modification of the Mosquito Vector An interesting future possibility for malaria control involves the replacement of vector mosquitoes by genetically altered mosquitoes that cannot transmit malaria. To progress beyond the theoretical, simultaneous advances are needed in key areas of vector biology, including the identification of factors regulating parasite development in vectors; the identification, genetics, and movement of refractory genes; the development of molecular probes for identifying members of species complexes; and various other aspects of mosquito molecular biology. A long-term goal would be to use techniques and findings from molecular-level studies to explore mechanisms operating in nature, with the ultimate hope of genetically engineering mosquitoes for release in endemic areas for malaria control. RESEARCH FOCUS: Further development of molecular-level approaches for understanding the genetics of vector populations and their natural abilities to transmit malaria parasites.

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MALARIA: Obstacles and Opportunities Drug Development A potentially promising area for malaria prevention is the use of antimalarial drugs to reduce the intensity of parasite transmission by vectors. There is a continuing need for new drugs that kill or reduce the infectivity of gametocytes in the human bloodstream. At the same time, a number of already available drugs may prove effective against parasite stages that occur in the vector. The identification and evaluation of these compounds may be one of the best hopes for preventing malaria in endemic countries. Determining the ability of these drugs to affect malaria transmission will require field studies in endemic areas, probably in conjunction with trials of schizonticidal drugs. RESEARCH FOCUS: New or existing drugs that kill or reduce the infectivity of gametocytes or reduce the transmission potential of vectors through disrupting normal sporogonic development. RESEARCH FOCUS: Vector-based field evaluation of transmission-blocking drugs in endemic areas. Evaluation of Vector Control Methods Every effort at malaria control should be evaluated for its impact on the intensity of transmission and the incidence of disease. It is equally important to assess how the control measures are received at the community level and whether they have any adverse effects on humans or the environment. Continuous evaluations of control activities will ensure that the methods used are appropriate for the particular epidemiologic situation. RESEARCH FOCUS: The impact of existing and future vector control interventions on the intensity of malaria transmission and patterns of disease. REFERENCES Beier, J. C., P. V. Perkins, R. A. Wirtz, J. Koros, D. Diggs, T. P. Gargan II, and D. K. Koech. 1988. Bloodmeal identification by direct enzyme-linked immunosorbent assay (ELISA), tested on Anopheles (Diptera: Culicidae) in Kenya. Journal of Medical Entomology 25:9-16. Beier, J. C., C. N. Oster, J. K. Koros, F. K. Onyango, A. K. Githeko, E. Rowton, D. K. Keoch, and C. R. Roberts. 1989. Effect of human circumsporozoite antibodies in Plasmodium-infected Anopheles (Diptera: Culicidae). Journal of Medical Entomology 26:547-553.

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