"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.

Vector transmission heterogeneity and the population dynamics and control of lymphatic filariasis.

A long-standing gap in lymphatic filariasis epidemiology is quantifying the potential effect that heterogeneous infection processes occurring in the major mosquito vector genera may have on parasite transmission and control. Although previous studies have focussed on examining the forms of the density dependent mechanisms regulating larval infection in various mosquito genera, there has been little work done thus far in investigating how such differential processes might interact with density-dependent processes occurring in other stages of the parasite life cycle to influence overall transmission dynamics between areas exposed to different transmitting vector populations. Here, we explore the impact that differences in vector genus-related larval infection dynamics may have on filariasis transmission and control using newly derived parasite transmission models incorporating the forms of the density-dependent processes regulating larval infection in the two major vectors transmitting filariasis, viz. culicine and anopheline mosquitoes. The key finding in this work is that filarial infection thresholds, system resilience, transmission dynamics and parasite response to control efforts, can all be influenced by the prevailing transmitting mosquito genus. In particular, we show that infection thresholds may be raised, system resilience to perturbations lowered and effects of repeated mass treatments in eliminating infection enhanced in anopheline filariasis compared to culicine filariasis, as a direct result of the occurrence and action of multiple positive density-dependent mechanisms influencing infection in this vector-parasite system, such as the "facilitation" function regulating larval infection dynamics in the vector and the inverse probability function governing adult worm mating in the host. These findings indicate that anopheline filariasis may be easier to eradicate than culicine filariasis for a given precontrol infection level, although the actual intensity of interventions required to achieve eradication may in fact be similar to that for culicine filariasis because of the higher infection levels generated as a result of the "facilitation" process in Anopheles transmission areas.

Comparison of the Impact of Annual and Semiannual Mass Drug Administration on Lymphatic Filariasis Prevalence in Flores Island, Indonesia.

We compared the impact of annual and semiannual mass drug administration (MDA) on the prevalence of Brugia timori and Wuchereria bancrofti in Flores Island. Two villages (Paga, B. timori only; Lewomada, co-endemic) received annual MDA with diethylcarbamazine/albendazole and a larger village (Pruda, co-endemic) received semiannual MDA. Infection parameters (microfilariae [Mf], antibodies to recombinant filarial antigen BmR1 [Brugia Rapid (BR)], and a test for W. bancrofti antigenemia [immunochromatographic test (ICT)]) were assessed before and after treatment. The crude Mf prevalence in Pruda decreased after five semiannual treatments from 14.2% to 1.2%, whereas the Mf prevalence in the other two villages decreased after three annual treatments from 3.9% to 0% and from 5% to 0.3%, respectively. ICT positivity prevalence in Pruda and Lewomada decreased from 22.9% and 6.5% to 7% and 0.8%, respectively, whereas BR antibody prevalence in Pruda, Lewomada, and Paga decreased from 28.9%, 31.7%, and 12.5% to 3.6%, 4.1%, and 1.8%, respectively. Logistic regression analysis indicated that that Mf, BR, and ICT prevalence decreased significantly over time and that for the Mf and ICT outcomes the semiannual treatment had higher odds of positivity. Model-adjusted prevalence estimates revealed that apparent differences in treatment effectiveness were driven by differences in baseline prevalence and that adjusted prevalence declined more rapidly in the semiannual treatment group. We conclude that in this setting, annual MDA was sufficient to reduce Mf prevalence to less than 1% in areas with low to moderate baseline prevalence. Semiannual MDA was useful for rapidly reducing Mf prevalence in an area with higher baseline endemicity.

Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids.

The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids.

Lymphatic filariasis: new insights into an old disease.

Lymphatic filariasis has afflicted people in the tropical areas of the world for thousands of years but even up to comparatively recent times it has been poorly understood and its importance under recognised. In the last 2 decades or so there has been a flurry of activity in filariasis research, which has provided new insights into the global problem of filariasis, the pathogenesis of filarial disease, diagnosis and control.

Oral transmission of Brugia pahangi to dogs.

Oral transmission of Brugia pahangi, already demonstrated in jirds, has now been accomplished in dogs. Beagle puppies, four anesthetized and two unanesthetized, were exposed to B. pahangi by instilling third-stage larvae (L-3s) into the mouth. Infections matured in all the dogs, and adult worms were recovered mainly from the mandibular, retropharyngeal, and axillary lymphatics. Worms were relatively numerous and peripheral microfilaremia developed in the dogs exposed under anesthetic, while worms were infrequent and microfilariae were found only intracardially in the dogs exposed without anesthetic. It appears that in orally exposed dogs, as in jirds studied earlier, the successful L-3s probably penetrated mucosa in or near the mouth.

Infective larvae of Brugia: escape from mosquitoes into water and subsequent oral infectivity in jirds.

Published work showed that third-stage larvae (L-3s) escape into water from dead or dying, Brugia pahangi-infected, Aedes aegypti. The present study revealed the same escape phenomenon among B. pahangi-infected Armigeres subalbatus, Anopheles quadrimaculatus, and Aedes togoi, and among Brugia malayi-infected Ae. aegypti and Ae. togoi. L-3s maintained in water or in Lum's solution for 3 hours retained infectivity when tested in orally or subcutaneously exposed jirds; furthermore, L-3s recovered from mosquitoes dead for 24 to 48 hours were also infective by either portal of entry in jirds. Since L-3s may escape and remain infective in the field, it is conceivable that natural filarial infections might thus be acquired orally by definitive hosts.

Differential pathogenicity of Brugia malayi, B. patei and B. pahangi in immunodeficient nude mice.

Immunodeficient nude mice chronically parasitized by subperiodic Brugia malayi developed an elephantoid appearance with persistent lymphoedema of limbs and massive lymphangiectasis of subcutaneous vessels containing viable adult worms. Removal of worms reversed the process. The syndrome was not caused by B. patei or B. pahangi and was not correlated with the presence or absence of microfilaremia. Histologic examination of elephantoid mice revealed dilated and tortuous lymphatics containing small nonobstructive lymph thrombi composed of small mononuclear cells and multinucleate giant cells. Draining lymph nodes were not enlarged or congested and mast cells in oedematous tissue were not degranulated. Analysis of lymph aspirated from dilated lymphatics showed increased total protein content: bacterial sepsis was not detected. This work suggests that viable adult B. malayi exert direct pathologic effects upon lymphatics and that this parasite is more pathogenic than related Brugia spp.

The effect of p-aminobenzoic acid and folic acid on the development of infective larvae of Brugia malayi in Aedes aegypti.

Adult Aedes aegypti mosquitoes, infected with the subperiodic Brugia malayi, were found to enhance the development of the filarial parasites to the infective stage when they were exposed to a cotton pad soaked in 10% sucrose solution containing p-aminobenzoic acid (PABA) in 0.001, 0.005, 0.01, 0.05 and 0.1% concentrations. Similarly, larval development increased when the mosquitoes were fed with folic acid at 0.001, 0.01 and 0.1% concentrations. This stimulation was more when PABA or folic acid was given prior to the infected blood meal through the developmental period of the larvae. The data thus suggest that PABA and folic acid are nutrients for the development of B. malayi-microfilariae to the infective stage in A. aegypti.

Susceptibility of Anopheles quadrimaculatus (Diptera: Culicidae) to subperiodic Brugia malayi and Brugia pahangi (Nematoda: Filarioidea) adapted to nude mice and jirds.

Anopheles quadrimaculatus and Aedes aegypti (Black-eyed Liverpool strain) were fed on jirds and nude mice (jird-jird infection, jird-mouse infection, and mouse-jird infection) infected with subperiodic Brugia malayi and B. pahangi. Microfilariae of B. malayi from jird-mouse and mouse-jird infections developed normally in An. quadrimaculatus, whereas those from jird-jird infections did not develop. Microfilariae of both species from jirds and nude mice developed normally in Ae. aegypti and those of B. pahangi developed normally in An. quadrimaculatus. It is suggested that microfilariae from nude mice are modified physiologically, immunologically, or both so that they can develop in refractory An. quadrimaculatus, thus indicating that susceptibility and refractoriness of An. quadrimaculatus to B. malayi also is influenced by factors relating to the vertebrate host in addition to mosquito genetic factors.

Parameters influencing the susceptibility of neonate mice to infection with Brugia pahangi.

A total of 307 young mice between less than 1 day (neonate) and 4 wk after birth were inoculated either subcutaneously or intraperitoneally with infective larvae of Brugia pahangi to determine the best protocol for the establishment of patent infections. For both male and female neonates, i.p. infection produced higher adult worm burdens than did s.c. infection. Although the numbers of adult worms harbored by male and female mice were not statistically different, male mice were more prone to develop a patent infection; no neonate female mice became microfilaremic , whereas seven of 113 i.p.-infected male mice developed microfilaremia. More female adult worms were recovered, on the average, than were male worms, regardless of the age of mice used for infection. However, the younger the mice were at infection, the higher were the numbers of male worms recovered. A high number of gravid female worms were recovered from amicrofilaremic mice. Adult female and male worms harvested from amicrofilaremic mice, implanted into the peritoneal cavity of jirds (Meriones unguiculatus), did not produce microfilariae although approximately 50% of the jirds contained gravid female parasites. Jirds implanted with worms from microfilaremic mice did, however, contain peritoneal microfilariae. It appeared that amicrofilaremic mice irreversibly damaged female worms to the extent that worms could survive and appear healthy, but could not release microfilariae.

Altered adult worm location in young male jirds infected with Brugia pahangi.

Male jirds (Meriones unguiculatus) were inoculated subcutaneously with 100 Brugia pahangi L3 each at 2, 6, 10, and 15 wk of age to compare their susceptibility and pathologic reactivity to infection. Adult worm recoveries (mean +/- SD) ranged from 24.1 +/- 15.1 to 36.4 +/- 13.9 at 60 days postinfection. No significant difference in susceptibility was measured among the 4 age groups. Jirds infected at 2 wk of age had significantly fewer (alpha less than or equal to 0.025) testicular and intralymphatic worms than all other age groups. Numbers of intralymphatic thrombi were significantly lower (alpha less than or equal to 0.01) in jirds infected at 2 wk of age. Lymphatic lesion severity, expressed as the number of intralymphatic thrombi per intralymphatic worm, was similar between age groups. These data indicate no differences in susceptibility or lymphatic lesion formation following B. pahangi infection in 2-wk-old male jirds, despite altered adult worm location.

Brugia timori: experimental infection in some laboratory animals.

Experimental infection with Brugia timori of 7 jirds (Merionesunguiculatus), 4 cats and 2 monkeys (Macaca fascicularis) is described. Although no microfilariae were detected by examining 20 microliter samples of tail blood of jirds, adult worms were recovered from 6 of the 7 jirds at autopsy 69-141 days following infection. Some worms were gravid and microfilariae were found in visceral blood of 2 animals. The adult recovery rate in jirds was 16%; the male to female ratio was 1:3. In cats patent infection developed in 95-105 days but microfilaraemias were of low level and transient. No parasites were recovered from monkeys.

Laboratory transmission of lymphatic filariasis by vector mosquitoes.

Aedes togoi and Ae. aegypti were used to examine the transmission potential of Brugia pahangi to one of its natural hosts, the domestic cat. Although a larger proportion of microfilariae taken in by Ae. togoi developed into infective larvae, the total number of B. pahangi larvae recovered from a cat exposed to Ae. aegypti was larger than from a cat exposed to Ae. togoi. Factors influencing the transmission dynamics included: development of microfilariae to infective larvae; survival of mosquitoes; willingness to take repeated blood meals; and proportion of infective larvae that egress from mosquitoes during the feeding process. From 19 to 25% of infective larvae were transferred to a susceptible host. The feasibility of using a Brugia-cat model to do comparative vector efficiency studies was demonstrated.