Studies with Brugia pahangi. II. The effect of repeated infection on parasite levels in cats

Denham D. A., Ponnudurai T., Nelson G. S., Rogers Rosemary and Guy Frances 1972. Studies with Brugia pahangi—II. The effect of repeated infection on parasite levels in cats. International Journal for Parasitology2: 401–407. 21 cats were given a primary infection of 100–200 infective larvae of Brugia pahangi followed, some time later, by repeated challenge with 50 larvae per time at 10-day intervals. In most cats the microfilarial levels increased considerably but in a minority the levels remained the same as those seen in cats given only one infection. Adult worm recoveries were very much higher than after a single infection but after about 20 challenges there was no further increase in the number of worms establishing an infection. After a long series of challenge infections, the microfilarial counts of some cats suddenly fell and the blood became free of microfilariae.     

In vitro study on humoral encapsulation of microfilariae: effects of diethyldithiocarbamate and dopachrome on the reaction

The effects of a phenoloxidase inhibitor, diethyldithiocarbamate, and an intermediate of the melanin biosynthetic pathway, dopachrome, on the humoral encapsulation of microfilariae were studied in vitro. Diethyldithiocarbamate inhibited the melanization but did not prevent the humoral encapsulation of microfilariae. In the presence of diethyldithiocarbamate in the in vitro system, transparent material in the form of numerous granules were deposited on the surface of the microfilariae and coalesced to form a consolidated transparent capsule around microfilariae. The thickness of transparent capsule was significantly greater than that of normal melanotic capsule. The transparent capsule material, which was probably the precursor of the melanotic capsule, was demonstrated histochemically as a protein-carbohydrate complex. The ultrastructure of transparent capsule showed that the transparent capsule material was very different from that of melanotic capsule material, being more flocculent and less granular in appearance. When dopachrome was concurrently present with the inhibitor in the in vitro encapsulation system, it failed to restore normal melanotic encapsulation. The data presented lead to the following conclusions: 1. The melanin biosynthetic pathway in the humoral encapsulation of microfilariae is similar to that found in the synthesis of mammalian melanin. 2. The humoral encapsulation of the microfilariae in vitro is a two-step process. The protein-carbohydrate complex is deposited on the surface of microfilariae. Then the tyrosine group of the complex is catalysed by phenoloxidase to melanin.     

Brugia pahangi: Immunization with early L3 ES alters parasite migration,and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus)

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24 h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.     

Ultrastructure of the intracellular melanization of Brugia malayi (Buckley) (Nematoda : Filarioidea) in the thoracic muscles of Anopheles quadrimaculatus (Say) (Diptera : Culicidae)

Ultrastructural details of the melanization of the filarial parasite Brugia malayi (Nematoda : Filarioidea) in the thoracic muscle cell of selected susceptible and refractory strains of Anopheles quadrimaculatus (Diptera : Culicidae) were examined. In both susceptible and refractory strains, following infection of the muscle cells, there is a dissolution of the sarcoplasmic cytoskeletal matrix surrounding the myofibrils, causing the myofibrils and mitochondria to drift apart and lose their regular spacing. This is coupled with a reorganization of the cytoplasm near the normally developing intracellular larva. Masses of vesicles and vesiculated cytoplasmic bodies accumulate near the larva. Eventually, these form a dense layer of cytoplasm around the parasite. Fine grains of melanin begin to appear in this dense cytoplasmic layer, increasing gradually until a nearly complete capsule of melanin surrounds the larva. The time course of melanization was found to be quite variable among and even within mosquitoes. Strains of A. quadrimaculatus selected to be refractory or susceptible to B. malayi, were found to vary substantially in the frequency of intracellular melanization, but not in the mechanism.     

Observations on the basal follicle numbers developed per female of two strains of Aedes aegypti after being fed on hosts with different levels of microfilariae of Brugia pahangi

Results indicate that there is a significant correlation between the density of Brugia pahangi microfilariae on which Aedes aegypti feed, the mean number of infective larvae produced per mosquito, and the mean number of basal follicles developed per female. When the density of microfilariae in cat's blood was high, the mean number of basal follicles developed per female was proportionally lower. It is concluded that the infected mosquitos show a significant drop in numbers of basal follicles produced per female. Both in the susceptible and refractory mosquito strains infected with B. pahangi, there was a negative linear correlation between parasite load and the number of basal follicles developed per female following their infecting blood meal.     

Acknowledgment

The uptake and incorporation in vitro of various nucleic acid precursors by microfilariae, third-stage infective larvae, 10-day-old juveniles and adult worms of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. A significant uptake of uracil and of purines, including adenine, hypoxanthine, and guanine was demonstrated in this study. No evidence was obtained for the uptake and incorporation of thymine, cytosine, orotate, formate, folate or p-aminobenzoic acid by either micro- or macrofilariae of B. pahangi.     

Defense reactions of mosquitoes to filarial worms: potential mechanism for avoidance of the response by Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and the black-eyed Liverpool (LVP) and Rocke-feller (RKF) strains of Aedes aegypti against intrathoracically inoculated Brugia pahangi microfilariae (mff) that previously had penetrated LVP, RKF, or A. trivittatus midguts in vitro was assessed at 1, 3, and 5 days postinoculation (PI). LVP and RKF midgut-derived mff almost totally avoided the melanization response and developed normally in LVP strain A. aegypti, and although over 90% of these mff died by 5 days PI in RKF mosquitoes, the majority of these were not melanized. A. aegypti midgut-derived mff also were able to avoid the response of A. trivittatus in 33–43% of the cases. Penetration through A. trivittatus midguts, however, did not significantly affect the ability of mff to avoid the melanization response in any of the mosquitoes examined. Allogeneic and xenogeneic tissue inplants were accepted by all three mosquito species examined. Data presented support the hypothesis that mff avoid immune recognition in compatible mosquitoes by coating themselves with midgut material(s) during penetration of the midgut in their migration to the hemocoel.     

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.     

Defense reactions of mosquitoes to filarial worms: comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and Dirofilaria immitis microfilariae

The melanization response of Aedes trivittatus and the Rockefeller (RKF) and black-eyed Liverpool (LVP) strains of Aedes aegypti against intrathoracically inoculated Dirofilaria immitis and Brugia pahangi microfilariae (mff) was investigated. All mff of either species were melanized in A. trivittatus following Day 2 postinoculation, and the response of this species was significantly more rapid and effective than either strain of A. aegypti. The refractory RKF strain had a significantly greater response against both D. immitis and B. pahangi than the highly susceptible LVP strain, but data suggest that the increased responsiveness was due to a physiologic incompatibility in RKF A. aegypti, thereby resulting in a greater mortality and subsequent melanization of inoculated mff. Inoculation of large numbers of mff overloaded the defense capabilities of A. aegypti (LVP), but not those of A. trivittatus. The melanization response against D. immitis mff was effectively reduced for up to 4 days in A. aegypti (LVP), but for only 1 day in A. trivittatus, when mosquitoes were maintained on a 0.3 m sucrose diet containing from 0.1 to 1.0% (w/v) phenylthiourea.     

Brugia pahangi and Dirofilaria immitis: experimental infections in the ferret, Mustela putorius furo.

Ferrets were inoculated with 160 third-stage larvae of the filarial nematode Brugia pahangi, followed 23 days later by 15 larvae of another filarial nematode, Dirofilaria immitis. Other ferrets received only one of these species. Microfilaremia developed in some ferrets with single infections of each species and in some ferrets with dual infections. The nature of the experiment did not permit a thorough study of microfilaremia, but B. pahangi microfilariae were found in numbers as high as 15,650/ml. At necropsy, approximately 8 months after inoculation, adult B. pahangi were recovered from the lymphatic vessels of all 8 ferrets inoculated only with that species, the recovery rate (based on 6 animals only) varying from 2 to 50% of the inoculum (mean 25%). Adult D. immitis were recovered from the heart of all three ferrets inoculated only with that species, the recovery rate being 7, 47, and 60% (mean 38%) of the inoculum. All 5 ferrets inoculated with both species yielded both adult B. pahangi (6 to 23%, mean 16% of inoculum) and adult D. immitis (13 to 67%, mean 37% of inoculum). It is concluded that the ferret is highly susceptible to both species and that concurrent infections with both species may readily be established.     

Successful Treatment of Brugia pahangi in Naturally Infected Cats with Ivermectin

Lymphatic filariasis is a common parasitic disease of cats in tropical regions including Thailand. The objective of this study was to determine the efficacy of ivermectin against microfilariae of Brugia pahangi in naturally infected cats. Eight cats naturally infected with B. pahangi were divided into control (untreated) and treated groups. Cats in the latter group were given ivermectin injection at 400 µg/kg weekly for 2 months. Microfilariae were counted every week until 48 weeks. Microfilaremia was significantly decreased in the treated group 4 weeks after starting the treatment and become zero at week 9 and afterwards. On the other hand, cats in the control group had high microfilaremia throughout the study. It was successful to treat and control B. pahangi infection in naturally infected cats using ivermectin.     

Cytochemical localization of lectin-binding to intracellularly melanized first stage larvae of Brugia malayi (Buckley) (Nematoda: Filarioidea) in Anopheles quadrimaculatus (Say) (Diptera: Culicidae)

Cytochemical localization of Concanavalin A (Con A) lectin followed by gold-labeled horseradish peroxidase binding to carbohydrate moieties on intracellularly developing normal and melanized first stage (L₁) larvae of Brugia malayi (Nematoda: Filarioidea) was investigated in the thoracic muscles of Anopheles quadrimaculatus (Diptera: Culicidae) females. Con A did not bind to the carbohydrate moieties on the larval cuticle or in tissues around the normally developing L₁, but bound moderately to the carbohydrate moieties in the cellular matrix of the larva. It bound intensely to the carbohydrate moieties of the dense cytoplasmic material and melanin deposits in the melanized capsule surrounding the melanized L₁. The results suggest that the dense cytoplasmic material in the melanized capsule surrounding the L₁ contains materials with exposed carbohydrate moieties specific for Con A.     

What does not kill them makes them stronger: larval environment and infectious dose alter mosquito potential to transmit filarial worms

For organisms with complex life cycles, larval environments can modify adult phenotypes. For mosquitoes and other vectors, when physiological impacts of stressors acting on larvae carry over into the adult stage they may interact with infectious dose of a vector-borne pathogen, producing a range of phenotypes for vector potential. Investigation of impacts of a common source of stress, larval crowding and intraspecific competition, on adult vector interactions with pathogens may increase our understanding of the dynamics of pathogen transmission by mosquito vectors. Using Aedes aegypti and the nematode parasite Brugia pahangi, we demonstrate dose dependency of fitness effects of B. pahangi infection on the mosquito, as well as interactions between competitive stress among larvae and infectious dose for resulting adults that affect the physiological and functional ability of mosquitoes to act as vectors. Contrary to results from studies on mosquito–arbovirus interactions, our results suggest that adults from crowded larvae may limit infection better than do adults from uncrowded controls, and that mosquitoes from high-quality larval environments are more physiologically and functionally capable vectors of B. pahangi. Our results provide another example of how the larval environment can have profound effects on vector potential of resulting adults.     

Studies with Brugia pahangi. I. Parasitological observations on primary infections of cats (Felis catus)

The large majority of cats given a single inoculation of third stage larvae of Brugia pahangi became microfilaraemic. Some cats had microfilariae in their blood 53 or 54 days after infection and most had become positive before 72 days after infection. In the majority of cats microfilarial counts remained very steady between 2 and 10 microfilariae per mm³ for long periods. At autopsy 10·7% of the infective larvae injected were recovered as adult worms. The recovery of adult worms was directly related to the number of larvae injected. The microfilarial level did not increase significantly with an increase in the number of adult worms.     

Carbohydrate metabolism in Brugia pahangi (Nematoda: Filarioidea)

Carbohydrate metabolism in Brugia pahangi (Nematoda:Filarioidea). International Journal for Parasitology16: 465–469. Adult B. pahangi have a complete glycolytic sequence and tricarboxylic acid cycle. The activities of the glycolytic enzymes are extremely high, but their relative activities are similar to those found in other glycolytic tissues. A comparison of the mass action ratios of the glycolytic enzymes with their apparent equilibrium constants indicates that phosphorylase, hexokinase, phosphofructokinase and pyruvate kinase are non-equilibrium regulatory enzymes. There is also some evidence that aldolase may be pseudoregulatory. Adult B. pahangi have measurable amounts of fumarate reductase as well as phosphoenolpyruvate carboxykinase and NADP-linked malic enzyme. Apart from lactate. the only other acidic end-product detected was traces of alanine; lactate production was little affected by the presence or absence of oxygen, unless exogenous glucose was present.     

A Repurposing Strategy for Hsp90 Inhibitors Demonstrates Their Potency against Filarial Nematodes

Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties.     

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.     

PHENOL OXIDASE ACTIVITY IN ANOPHELES SINENSIS AND AEDES ALBOPZCTUS EXPOSED TO MICROFILARIAE OF BRUGIA MALAYI

Abstract Phenol oxidase (PO activity was investigated in cell-free hemolymph collected from Anopheles sinensis and Aedes albopictlcs female adults intrathoracically inoculated with Brugia dayi microfilariae (mff), inoculated with saline alone, or from uninoculated mosquitoes. The activity of PO from uninoculated 1-day-old mosquitoes was significantly greater than those of mosquitoes on 3, 12 and 16 day. PO activity in mff-inoculated A. sinensis at 24 h postinoculation (PI) was 2–3 times higher as compared with uninoculated or saline-inoculated mosquitoes. Inoculation of B. malayi mff into A. albopictus elicited ?-fold increase in PO activity at 24 h PI as compared with uninoculated mosquitoes. Immune-activated levels of PO activity in A. alboplctus was significantly higher as compared with those seen in A. sinensis. The relationship of observed differences in PO activity to differences in immunological capability between A. sinensis and A. albopictus is discussed.     

Brugia pahangi: infections and their effect on the lymphatic system of Mongolian jirds (Meriones unguiculatus)

Brugia pahangi has been found to be primarily a lymphatic-dwelling parasite in jirds when infections are induced by the subcutaneous injection of infective larvae or by allowing infected Aedes aegypti to feed.Migration to the regional lymphatics occurred as early as 1–4 days. Although some injected larvae remained in the skin for as long as 30 days and some became localized in the heart, lungs, pleural cavity, or peritoneal cavity, about three-fourths of the recovered filariae were found in the regional lymphatics. In contrast, when larvae were injected peritoneally they remained largely in the peritoneal cavity for at least 30 days.The relevant lymphatics and their drainage patterns in jirds have been described.The major pathological changes noted in jirds involved the regional lymphatic vessels and nodes, which were severely affected when they contained dead worms. Pulmonary granulomas due to dead microfilariae and occasionally to dead larvae or adult worms were noted.Observations are included on the susceptibility and course of B. pahangi infections in jirds.