Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bloodmeal microfilariae density and the uptake and establishment of Wuchereria bancrofti infections in Culex quinquefasciatus and Aedes aegypti.

The relationship between ingestion of microfilariae (mf), production of infective larvae (L3) and mf density in human blood has been suggested as an important determinant in the transmission dynamics of lymphatic filariasis. Here we assess the role of these factors in determining the competence of a natural vector Culex quinquefasciatus and a non vector Aedes aegypti to transmit Wuchereria bancrofti. Mosquitoes were infected via a membrane feeding procedure. Both mosquito species ingested more than the expected number of microfilariae (concentrating factor was 1.28 and 1.81 for Cx. quinquefasciatus and Ae. aegypti, respectively) but Cx. quinquefasciatus ingested around twice as many mf as Ae. aegypti because its larger blood meal size. Ae. aegypti showed a faster mf migration capacity compared to Cx. quinquefasciatus but did not allow parasite maturation under our experimental conditions. Similar proportions of melanized parasites were observed in Ae. aegypti (2. 4%) and Cx. quinquefasciatus (2.1%). However, no relationship between rate of infection and melanization was observed. We conclude that in these conditions physiological factors governing parasite development in the thorax may be more important in limiting vectorial competence than the density of mf ingested.     

Susceptibility of mosquitoes in central Taiwan to natural infections of Dirofilaria immitis

From October 1997 to September 1998, 3085 Culex quinquefasciatus (Say) (Diptera: Culicidae), 584 Cx. tritaeniorhynchus (Giles) (Diptera: Culicidae), 392 Cx. annulus (Theobald) (Diptera: Culicidae), 374 Aedes albopictus (Skuse) (Diptera: Culicidae) and 102 Armigeres subalbatus (Coquillet) (Diptera: Culicidae) were collected and examined for Dirofilaria immitis (Leidy) (Spirurida: Filariidae) infection. However, only Cx. quinquefasciatus and Ae. albopictus were infected, with a prevalence of 4.28% and 3.74%, respectively. The intensity of D. immitis found in Ae. albopictus (3.43 larvae/mosquito) was higher than that found in Cx. quinquefasciatus (2.89 larvae/mosquito). After being fed with canine blood containing 7500 microfilariae (mf) per mL, Cx. quinquefasciatus ingested approximately two times as many mf as Ae. albopictus (mean of 31.73 in comparison to 16.47). However, almost three times as many third-stage infective larvae developed in Ae. albopictus as in Cx. quinquefasciatus (mean of 3.25 as compared with 1.10), with a vector efficiency index (VEI) of 19.73 and 3.47, respectively. The results showed that Cx. quinquefasciatus and Ae. albopictus served as natural vectors of D. immitis in central Taiwan. Although Ae. albopictus was more efficient for heartworm transmission, Cx. quinquefasciatus may play a more prominent role on the transmission of dirofilariasis in central Taiwan.     

Synergistic efficacy of botanical blends with and without synthetic insecticides against Aedes aegypti and Culex annulirostris mosquitoes.

Increasing insecticide resistance requires strategies to prolong the use of highly effective vector control compounds. The use of combinations of insecticides with other insecticides and phytochemicals is one such strategy that is suitable for mosquito control. In bioassays with Aedes aegypti and Culex annulirostris mosquitoes, binary mixtures of phytochemicals with or without synthetic insecticides produced promising results when each was applied at a LC25 dose. All mixtures resulted in 100% mortality against Cx. annulirostris larvae within 24 h rather than the expected mortality of 50%. All mixtures acted synergistically against Ae. aegypti larvae within the first 24 h except for one mixture that showed an additive effect. We conclude that mixtures are more effective than insecticides or phytochemicals alone and that they enable a reduced dose to be applied for vector control potentially leading to improved resistance management and reduced costs.     

Vector competence of Culex quinquefasciatus say from different regions of Brazil to Dirofilaria immitis

The vector competence of Culex quinquefasciatus from five localities in Brazil to Dirofilaria immitis was evaluated experimentally. Females from each locality were fed on an infected dog ( approximately 6 microfilariae/microl blood). A sample of blood fed mosquitoes were dissected approximately 1 h after blood meal. These results demonstrated that all had ingested microfilariae (mean, 4.8 to 24.6 microfilariae/mosquito). Fifteen days after the infected blood meal, the infection and infective rates were low in all populations of Cx. quinquefasciatus. The mean number of infective larvae detected in the head and proboscis of these mosquitoes was 1-1.5. The vector efficiency, the number of microfilariae ingested/number of infective larvae, was low for all populations of Cx. quinquefasciatus. However, the survival rate for all populations was high (range 50-75%). The survival rate of Aedes aegypti assayed simultaneously for comparison was low (24.7%), while the vector efficiency was much higher than for Cx. quinquefasciatus. These data suggest that the vector competence of all assayed populations of Cx. quinquefasciatus to D. immitis in Brazil is similar and that this species is a secondary vector due to its low susceptibility. Nevertheless, vector capacity may vary between populations due to differences in biting frequency on dogs that has been reported in Brazil.     

Biological control of Culicidae with the copepod Mesocyclops aspericornis and larvivorous fish (Poeciliidae) in a village of French Polynesia

The copepod Mesocyclops aspericornis Daday and the larvivorous fishes Gambusia affinis (B. & G.) and Poecilia reticulata R. & B., were released into mosquito breeding sites in Tuherahera village, Tikehau atoll, French Polynesia, to control larvae of Aedes aegypti (L.), Ae.polynesiensis Marks, Culex annulirostris Skuse and Cx quinquefasciatus Say. Treatments were completed within a week, in January 1990. Fish quickly eliminated mosquito larvae from the open breeding sites (ponds, wells). The impact of copepods in water tanks, drums and covered wells was inconsistent, apparently depending on the availability of microfaunal diet for growth of copepod nauplii. As the biting rate of adult Ae.aegypti seemed to be unaffected by the biological control of larvae, this village-scale experiment was judged to be unsuccessful as a means of vector control.     

Four species of mosquito as possible vectors for Dirofilaria immitis piedmont rice-fields

Wild mosquitoes were allowed to feed, during two nights, on a heartworm-infected dog with a very high microfilaraemia (72,000 and 78,000 microfilariae/ml just prior to be allocated in the live trap). A heartworm-free dog was used as control bait in the second night. Engorged mosquitoes (Aedes caspius, Anopheles maculipennis s.l., Culex modestus, and Cx pipiens) were kept under laboratory conditions, artificially fed until day 10 post-infection (PI) and then dissected for the presence of Dirofilaria immitis larvae. Mortality of Ae. caspius and Cx modestus was significantly higher than controls on day 3 PI (89.4 and 80.3%, respectively), but survival rates were similar in the following period. Third-stage larvae were observed from day 12 to 17 PI in the four mosquito species studied. However, vector efficiency was significantly higher in Ae. caspius which produced 102 (73.9%) of the 138 infective larvae found. Although less efficient vectors, the other 3 species may contribute to D. immitis transmission in the study area due to their abundance (Cx modestus) or higher resistance to the negative effects of infection (An. maculipennis s.l., Cx pipiens). As far as Cx pipiens is concerned, this mosquito, which luckily fed the least frequently on the dog, confirmed to act as powerful vector in iperendemic areas. The risks for veterinary and medical health, related to the zoo-antropophylic host-feeding pattern of the studied species, are discussed.     

Distribution and habitats of mosquito larvae in the Kingdom of Tonga

Abstract  Mosquitoes are a significant pest and human health issue in the Kingdom of Tonga. The occurrence of species and habitats used by mosquito larvae were investigated to determine the potential for control through larval habitat management. Forty-two sites, including 22 villages and 20 farm plantations on the six islands of Tongatapu, Pangaimotu, Vava'u, Pangaimotu (Vava'u group), 'Utungake and Nuku, were surveyed in April 2006. A total of eight mosquito species were collected: Aedes aegypti (Linnaeus), Ae. horrescens (Edwards), Ae. nocturnus (Theobold), Ae. tongae (Edwards), Culex albinervis (Edwards), Cx. annulirostris (Skuse), Cx. quinquefasciatus (Say) and Cx. sitiens (Wiedemann). Several species were widespread, particularly Ae. aegypti and Ae. nocturnus on the main island of Tongatapu, whereas Ae. aegypti dominated sites on islands of the Vava'u group. Comparative sampling of 17 village and 17 rural sites showed that larval habitat was more abundant in towns than in rural areas. Larvae were found in a wide range of habitats but were particularly abundant in artificial water bodies (e.g. disused concrete water tanks, 44-gallon drums and used car tyres). In rural sites, habitats were generally sparse except in rain-filled branch stems of giant taro plants. Mosquito populations in artificial habitats could be markedly reduced by seeding disused water tanks with aquatic predators already present in Tonga, using mesh-net covers over 44-gallon drums, and drilling holes in used car tyres.     

The vector competence of Culex annulirostris, Aedes sagax and Aedes alboannulatus for Murray Valley encephalitis virus at different temperatures

Culex annulirostris Skuse, colonized from Brisbane, Queensland, and Mildura, Victoria, Australia, were effective vectors of Murray Valley encephalitis virus at 20, 27 and 32-35 degrees C with full extrinsic incubation periods of 15, 10 and 4 days respectively. At 20 degrees C, 7-11 days post-infection, transmission by the Mildura colony (0-20%) was less efficient than the Brisbane colony (30-70%) but both were capable of 75-100% transmission after longer extrinsic incubation periods. Discriminant analysis of body and salivary gland titres showed that these were not satisfactory indicators of transmission. Wild-caught Aedes sagax (Skuse) and Cx annulirostris from the Murray Valley showed equal competence, but Aedes alboannulatus (Macquart) was a poor vector. The results provide data on rural amplification of Murray Valley encephalitis virus during spring and suggest that further work on the potential of Ae. sagax as a natural vector is warranted.     

Seasonal abundance of Culex annulirostris and other mosquitoes at Kowanyama, north Queensland, and Charleville, south west Queensland.

At Kowanyama, 104,006 adult female mosquitoes of a probable 47 species were collected on twelve occasions from April, 1972, to April, 1976. At Charleville, 71,791 adult females of 20 taxa were taken during thirteen periods from February, 1974, to February, 1976. Culex annulirostris was predominant at Kowanyama and especially at Charleville where it comprised up to 99% of dry season collections. Culex fatigans, Ae normanensis, An annulipes and, at Kowanyama only, An bancroftii were the other major species collected. Several bait trapping methods were suitable for monitoring densities of Cx annulirostris and other species but mammalian baits were best. Because Cx annulirostris activity continued throughout winter even at Charleville, continuous cycles of arbovirus transmission are possible, although the potential of dormant Aedes eggs also bears investigation as a method of arbovirus survival.     

Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae

A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.     

Vector competence of Australian Culex gelidus Theobald (Diptera: Culicidae) for endemic and exotic arboviruses

The recent recognition of established populations of the mosquito, Culex gelidus Theobald, in Australia has raised concerns about local transmission of arboviruses. The vector competence of a mainland population of Cx. gelidus was investigated for two local alphaviruses, Ross River (RRV) and Barmah Forest (BFV) viruses, and three flaviviruses, Japanese encephalitis (JEV), Kunjin (KUNV) and Murray Valley encephalitis (MVEV) viruses. Colonised mosquitoes were exposed to virus via blood-soaked pledgets and transmission was tested using a capillary-tube method. The important Australian vectors, Aedes vigilax (Skuse) and Culex annulirostris Skuse, were used as internal controls for the alphaviruses and flaviviruses, respectively. Overall, Cx. gelidus was a more efficient vector of flaviviruses than alphaviruses. Culex gelidus was refractory to infection with BFV, and nearly 25% transmitted RRV, which was comparable to Ae. vigilax . Culex gelidus was susceptible to all three flaviviruses, with transmission rates of 96%, 95% and 41% for JEV, KUNV and MVEV, respectively. JEV transmission rates in Cx. annulirostris were unexpectedly low and this was possibly due to differences in susceptibility to JEV genotypes I and II. Considering the high susceptibility to the flaviviruses demonstrated here, and the natural infections with RRV and JEV that have been detected from northern Australian populations, the establishment of the exotic mosquito, Cx. gelidus , in Australia is potentially a significant public health concern.     

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.     

Effect of N-acetyl-D-glucosamine on the migration of Brugia pahangi microfilariae into the haemocoel of Aedes aegypti

Two strains of Aedes aegypti (L.), differing in their susceptibility to Brugia pahangi (Buckley & Edeson), were examined with regard to the effect on the proportion of microfilariae migrating from the mid-gut, of specific carbohydrate supplements in the infecting bloodmeal. N-Acetyl-D-glucosamine (GlcNAc), a sugar also present on the microfilarial sheath, significantly increased the migration rate. This enhancement is greater for the refractory strain of Ae.aegypti. The use of sucrose as a control sugar results in no enhancement of microfilariae migration. It is postulated that the GlcNAc is acting by blocking endogenous gut/peritrophic membrane carbohydrate binding proteins, which would normally inhibit microfilariae migration. Furthermore, there is a significant correlation whereby increasing loads of microfilariae ingested result in decreasing proportions migrating across the mid-gut.     

A comparison of mosquito predation by the fish Pseudomugil signifier Kner and Gambusia holbrooki (Girard) in laboratory trials.

Using larval Cx. annulirostris mosquitoes, this study compared the predation rate of Pseudomugil signifer and Gambusia holbrooki for four larval instars, three prey densities, and three vegetation densities. There was no significant difference in the quantity of first, second, and third instar larvae consumed by the two fish species after 24 h. There was a significant negative relationship between the predation rate and both larval instar and prey density for both fish species. In simulated vegetation trials, P. signifer performed marginally better than G. holbrooki in medium to high density vegetation (0.3 stems/cm 2 and 0.6 stems/cm2, respectively).     

Antifilarial effect of Zingiber officinale on Dirofilaria immitis

Dogs, naturally infected with Dirofilaria immitis, were treated with the residues of the alcoholic extracts of the rhizomes of Zingiber officinale (ginger). Twelve subcutaneous injections of the extract given at 100 mg/kg reduced microfilarial concentration in blood by a maximum of 98%. Fifty five days after the last injection there was 83% reduction in microfilarial concentration suggesting partial destruction of adult worms. Half of the treated dogs showed some lethargy at the beginning of treatment possibly due to the mass annihilation of microfilariae in blood.