"Wonders unconceived": reflections on the birth of medical entomology

Prior to Patrick Manson's discovery in 1877 that the mosquito Culex fatigans was the intermediate host of filariasis, the association of insects with disease and the nature of disease transmission was almost entirely speculation. Manson's work was incomplete, however, because it showed the manner in which the mosquito acquired the infection from humans, but failed to show the way in which the mosquito passed the infection to humans. That pathogens were transmitted by the bite of an infected female mosquito was later proven experimentally with bird malaria by Manson's protégé, Ronald Ross. In 1898 Ross demonstrated that the infective stage of the malarial parasite was injected into the host when the mosquito released saliva into the wound prior to injesting blood. Insects were suspected as carriers of disease for centuries, yet it was not until the late 1870s that the uncritical acceptance of folk beliefs was supplanted by research-based scientific medicine. Why did it take so long? The answer lies in the fact that early medicine itself was imprecise and could not have pursued the subject with any hope of useful results until the last quarter of the 19th century. A better understanding of the nature of the disease process (germ theory of disease) and improved technology (microscopes and oil-immersion lenses with greater resolving power, and synthetic tissue stains) were indispensable for revealing the nexus between those partners in crime: insects and parasites.     

Changes in the haemocyte population of the mosquito, Culex quinquefasciatus, following infection with the filarial parasite, Wuchereria bancrofti

The mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is the vector of the filarial parasite Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), which causes human bancroftian filariasis. Information on the mosquito humoral response against the filarial parasite during the process of its infection and development is important, as it decides the vector competence of the mosquito. Visible changes in the haemocyte population of mosquito, if any, will be an indicator of the possible humoral factors. The present study was aimed at investigating changes in the populations of various types of haemocytes of Cx. quinquefasciatus following infection with W. bancrofti. On day 2 post-feeding on microfilaraemic blood, the haemolymph perfusate of infected mosquitoes with L1 stage of the parasite showed 44.1% granulocytes, 42% prohaemocytes and 13.9% plasmatocytes, whereas that of the control mosquitoes fed on amicrofilaraemic blood showed 63.4% plasmatocytes, 22.2% prohaemocytes and 14.4% granulocytes. Differences in the population numbers of haemocyte types between the infected and control were significant (P > 0.05). However, the mosquitoes examined on day 6 post-feeding, when the parasite was in L2 stage, did not show any such changes. But, similar changes reappeared on day 12 in mosquitoes with L3 stage of the parasite. The observed haemocyte population changes indicate the possibility of some amount of humoral immune response, through the production of certain immune molecules, in Cx. quinquefasciatus infected with W. bancrofti. The nature and exact role of such a response on the filarial parasite development need further investigation.     

Manson's triple error

The author discusses the significance, implications and limitations of Manson's work. How did Patrick Manson resolve some of the major problems raised by the filarial worm life cycle? The Amoy physician showed that circulating embryos could only leave the blood via the percutaneous route, thereby requiring a bloodsucking insect. The discovery of a new autonomous, airborne, active host undoubtedly had a considerable impact on the history of parasitology, but the way in which Manson formulated and solved the problem of the transfer of filarial worms from the body of the mosquito to man resulted in failure. This article shows how the epistemological transformation operated by Manson was indissociably related to a series of errors and how a major breakthrough can be the result of a series of false proposals and, consequently, that the history of truth often involves a history of error.     

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.     

Incorporation of radioactive precursors into filarial larvae of Brugia developing in susceptible and refractory mosquitoes.

The incorporation of tritiated precursors injected into mosquito hosts parasitized by developing filarial larvae of Brugia patei has been studied by autoradiography in 2 species of mosquito, Aedes togoi in which filarial development was normal and Anopheles labranchiae atroparvus in which filarial development was abnormal. In both mosquito hosts there was significant incorporation into 4--5-day-old developing larvae of uridine and amino acids (isoleucine, leucine, valine, arginine, lysine, cystine, methionine, phenylalanine, tyrosine, tryptophan, histidine, and proline), although lower incorporation of methionine, tyrosine, and tryptophan was found during abnormal development. No incorporation of thymidine, hydroxytryptophan, dopa, or carbohydrate was found at this stage of larval development. Some incorporation of glucose and dopa was found in or around earlier stages of development in An. l. atroparvus. Mosquito flight muscle showed lower incorporation of glucose, but not of amino acids, around the site of filarial parasite development. The flight muscle of An. l. atroparvus showed a higher level of incorporation of lysine compared to that in A. togoi and higher levels of lysine and valine were found in the abnormally developing filarial larvae in the refractory mosquito.     

Chyluria with proteinuria or filarial nephropathy? An enigma

Lymphatic filariasis is endemic in India. Out of 128 million infected individuals worldwide, India accounts for 48 million cases [Manson's Tropical Diseases, 21st Ed. p 1488]. Filariasis can have protean manifestations, but Tropical pulmonary eosinophilia and chyluria are unusual manifestations reported mainly from South Asian countries [Manson's Tropical Diseases, 21st Ed. p 1494]. Chyluria occurs only in 2% of filarial afflicted patients in the filarial belt [Diamond E, Schapira HE. Chyluria--a review of literature. Urology 1985;26(5): 427-31]. Lymphatic filariasis presenting as chyluria may be equally rare. Predominant chyluria with no overt lymphatic filariasis remains an enigma.     

Dirofilaria immitis: effect on the longevity of Aedes trivittatus

Laboratory studies on vector mortality as related to parasite burden revealed that the mosquito Aedes trivittatus showed considerable tolerance to infection with the filarial nematode Dirofilaria immitis. Although mosquitoes exposed to a dog with a high microfilaremia (347 microfilariae/20 mm³) had a significant increase in mortality during the first 16 days postexposure, 66% of the mosquitoes lived long enough for complete parasite development to occur. Those mosquitoes exposed to a dog with a low microfilaremia (62 microfilariae/20 mm³) had no significant increase in mortality. There was a strong negative correlation between parasite burden and mosquito survival, but only mosquitoes harboring more than 15 juveniles had an increased chance of dying before D. immitis could develop to the infective stage. The retention of microfilariae within the blood clot and peritrophic membrane of A. trivittatus seems beneficial to this vector-parasite system by reducing the parasite burden and increasing mosquito longevity.     

Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship

Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasite's vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite.     

Reproductive aspects of the mosquito Culex quinquefasciatus (Diptera:Culicidae) infected with Wuchereria bancrofti (Spirurida: Onchocercidae)

This study reports on the relationship between Wuchereria bancrofti infection and female body size, intake of blood and fecundity in the mosquito Culex quinquefasciatus, vector of this filarial parasite in Recife (Brazil). Adults from field collected larvae were infected via a membrane feeding procedure, using blood with parasitaemia ranging from 724-6,000 mf/ml. A positive correlation was observed between mosquito size (measured by wing length) and egg production in uninfected females. However, this relationship did not exist in W. bancrofti infected mosquitoes. This change is unlikely to be the result of changes in blood ingestion as no significant difference was found when infected and uninfected females were compared. Variation in egg production observed between trials could not be associated with parasite density in the blood. These results suggest infection with W. bancrofti may disrupt the relationship between mosquito size and egg production during the first gonotrophic cycle of C. quinquefasciatus such that fecundity is sometimes reduced. However, this overall affect is variable and many groups of mosquitoes do not respond in this way.     

Plasmodium differentiation in the mosquito

The essential passage of the malarial parasite through a mosquito vector results in major population bottlenecks in parasite numbers. The volume of the bloodmeal ingested by the female mosquito is 1-2 microliters. This may contain from 1 to 10(5) gametocytes. Of these, it is normal for just 12 to become macrogametes; 5-6 become ookinetes, and 2 develop into oocysts 2-7 days later. Of the 16,000 sporozoites produced from these two oocysts just 10-20 are inoculated by the malaria-infected female mosquito each time she probes when taking a subsequent bloodmeal. These significant population bottlenecks suggest that parasite differentiation is severely constrained by the environment in the mosquito, and therefore by the interactions between the parasite and the vector. This review will describe parasite differentiation in the mosquito and try to highlight the more important interactions between the parasite, the bloodmeal and the mosquito, attempting to identify those interactions which are essential to parasite differentiation, and those where the mosquito may be mounting effective strategies against this important pathogen. The potential exploitation of these interactions as possible mechanisms for intervention will be discussed.     

A new insight into the pathogenesis of filarial disease

Filariasis is a major public health problem throughout many regions of the tropics. The disease is caused by several species of filarial nematode including Wuchereria bancrofti and Brugia malayi, the agents of lymphatic filariasis, and Onchocerca volvulus, the cause of 'riverblindness'. Disease caused by these worms varies depending on the tissue location of the parasite, and is associated with episodes of acute and chronic inflammation. These pathologies, including elephantiasis and blindness, rank among the most disabling in the world. Studies aimed at characterizing the molecular nature of the inflammatory stimuli derived from filarial nematodes uncovered a long forgotten secret, their symbiont Wolbachia. LPS-like molecules from these intracellular bacteria are responsible for potent inflammatory responses from macrophages and in animal models of filarial disease. Wolbachia has also been associated with severe inflammatory reactions to filarial chemotherapy, being released into the blood following the death of the parasite. Recent studies in animal models even implicate Wolbachia in the onset of lymphodema and blindness. Taken together these studies suggest a major role for Wolbachia in the pathogenesis of filarial disease. It may be possible, through the use of antibiotic therapy, to clear worms of their bacteria, in the hope that this will prevent the onset and development of filarial pathology.     

Filarial worms reduce Plasmodium infectivity in mosquitoes

Background

Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG). Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness).

Methodology/Principal Findings

Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis) but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria parasite infections.

Conclusions/Significance

These results could have an impact on vector infection and transmission dynamics in areas where Anopheles transmit both parasites, i.e., the elimination of filarial worms in a co-endemic locale could enhance malaria transmission.     

Brugia pahangi: effects upon the flight capability of Aedes aegypti.

Aedes aegypti mosquitoes were adversely affected by infections of the filarial worm Brugia pahangi. Infected mosquitoes flew significantly shorter distances and showed marked reductions in total flight time during 24-hr flight mill tests compared to uninfected controls. Total flight range and duration flown by infected mosquitoes remained relatively constant throughout the infection process, while control mosquitoes flew further and longer with increasing time after their blood meal. Furthermore, a significantly greater number of infected mosquitoes either died or were rendered incapable of flight. Of flying and nonflying mosquitoes with 6-day-old or older infections dissected for parasite burdens, the nonflying group contained significantly more worms. Results of this study indicate that developing filarial larvae within this mosquito vector reduce its ability to survive and to transmit its infection by reducing its flight capabilities. Conclusions from this study relate only to A. aegypti homozygous for the gene fm which is fully susceptible to this filarial parasite.     

High prevalence and lineage diversity of avian malaria in wild populations of great tits (Parus major) and mosquitoes (Culex pipiens)

Avian malaria studies have taken a prominent place in different aspects of evolutionary ecology. Despite a recent interest in the role of vectors within the complex interaction system of the malaria parasite, they have largely been ignored in most epidemiological studies. Epidemiology of the disease is however strongly related to the vector's ecology and behaviour, and there is a need for basic investigations to obtain a better picture of the natural associations between Plasmodium lineages, vector species and bird hosts. The aim of the present study was to identify the mosquito species involved in the transmission of the haemosporidian parasites Plasmodium spp. in two wild populations of breeding great tits (Parus major) in western Switzerland. Additionally, we compared Plasmodium lineages, based on mitochondrial DNA cytochrome b sequences, between the vertebrate and dipteran hosts, and evaluated the prevalence of the parasite in the mosquito populations. Plasmodium spp. were detected in Culex pipiens only, with an overall 6.6% prevalence. Among the six cytochrome b lineages of Plasmodium identified in the mosquitoes, three were also present in great tits. The results provide evidence for the first time that C. pipiens can act as a natural vector of avian malaria in Europe and yield baseline data for future research on the epidemiology of avian malaria in European countries.     

Geographic and ecologic heterogeneity in elimination thresholds for the major vector-borne helminthic disease,lymphatic filariasis

Background

Large-scale intervention programmes to control or eliminate several infectious diseases are currently underway worldwide. However, a major unresolved question remains: what are reasonable stopping points for these programmes? Recent theoretical work has highlighted how the ecological complexity and heterogeneity inherent in the transmission dynamics of macroparasites can result in elimination thresholds that vary between local communities. Here, we examine the empirical evidence for this hypothesis and its implications for the global elimination of the major macroparasitic disease, lymphatic filariasis, by applying a novel Bayesian computer simulation procedure to fit a dynamic model of the transmission of this parasitic disease to field data from nine villages with different ecological and geographical characteristics. Baseline lymphatic filariasis microfilarial age-prevalence data from three geographically distinct endemic regions, across which the major vector populations implicated in parasite transmission also differed, were used to fit and calibrate the relevant vector-specific filariasis transmission models. Ensembles of parasite elimination thresholds, generated using the Bayesian fitting procedure, were then examined in order to evaluate site-specific heterogeneity in the values of these thresholds and investigate the ecological factors that may underlie such variability

Results

We show that parameters of density-dependent functions relating to immunity, parasite establishment, as well as parasite aggregation, varied significantly between the nine different settings, contributing to locally varying filarial elimination thresholds. Parasite elimination thresholds predicted for the settings in which the mosquito vector is anopheline were, however, found to be higher than those in which the mosquito is culicine, substantiating our previous theoretical findings. The results also indicate that the probability that the parasite will be eliminated following six rounds of Mass Drug Administration with diethylcarbamazine and albendazole decreases markedly but non-linearly as the annual biting rate and parasite reproduction number increases.

Conclusions

This paper shows that specific ecological conditions in a community can lead to significant local differences in population dynamics and, consequently, elimination threshold estimates for lymphatic filariasis. These findings, and the difficulty of measuring the key local parameters (infection aggregation and acquired immunity) governing differences in transmission thresholds between communities, mean that it is necessary for us to rethink the utility of the current anticipatory approaches for achieving the elimination of filariasis both locally and globally.