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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.

Aedes↗

Nutritional factors and antimicrobials on development of infective larvae of subperiodic Brugia malayi (Nematoda: Filarioidea) in Anopheles quadrimaculatus and Aedes aegypti (Diptera: Culicidae).

The effects of nutritional factors and antimicrobials on the development of infective larvae of subperiodic Brugia malayi in susceptible Anopheles quadrimaculatus Say and Aedes aegypti (L.) were investigated. Larvae of both species of mosquitoes were reared on brewers yeast or a 1:1 brewers yeast-liver powder mixture. After emergence, one-half of the adults from each rearing condition were maintained on a 10% sucrose solution and the other half on a 10% sucrose solution containing 0.1% p-aminobenzoic acid (PABA). Females of both species of mosquitoes that fed on B. malayi-infected jirds showed a significant increase in the ineffective rate and intensity of infectiveness when the mosquito larvae were reared on the brewers yeast-liver powder diet. The addition of 0.1% PABA to the adult diet increased numbers of infective larvae of B. malayi that developed in Ae. aegypti but not in An. quadrimaculatus. The intensity of infectiveness of B. malayi was significantly greater when An. quadrimaculatus females were provided with a second blood meal from an uninfected jird and when females of both species were maintained on different concentrations of an antimicrobial solution.

4-Aminobenzoic Acid↗

Variation of mosquito (Diptera: Culicidae) relative abundance and Dirofilaria immitis (Nematoda: Filarioidea) vector potential in coastal North Carolina.

At an enzootic focus of Dirofilaria immitis in coastal North Carolina, mosquito populations were sampled June-September 1985 and on several occasions during August-October 1986 and June-August 1987, to identify local vectors and to determine relative abundance and D. immitis infection rates. Predominant species collected were Anopheles bradleyi King (66.6%), Culex salinarius Coquillett (15.9%), Aedes taeniorhynchus (Wiedemann) (8.2%), and Aedes sollicitans (Walker) (4.9%). Population abundance varied within and among seasons. D. immitis infection was found in An. bradleyi (1.2%), Ae. taeniorhynchus (0.9%), and Ae. sollicitans (0.9%). Of infected An. bradleyi, 88% were collected June-2 July, whereas the highest number of infected Ae. taeniorhynchus and Ae. sollicitans were found during mid-July 1985. Ae. sollicitans and Ae. taeniorhynchus were the only infected species found during 1986 and 1987. However, based on overall relative abundance and infective rate, An. bradleyi appeared to be the primary vector of D. immitis in the study area.

Aedes↗

Salivarectomy effects on the ingestion of Brugia pahangi (Nematoda: Filarioidea) microfilariae by susceptible and refractory strains of Aedes aegypti (Diptera: Culicidae).

Ingestion of Brugia pahangi Buckley & Edeson microfilariae by susceptible (Liverpool) and refractory (Bora-Bora) strains of salivary duct-severed Aedes aegypti (L.) was investigated. The transection at either the salivary common duct or the two lateral ducts of mosquitoes did not affect the mean number of microfilariae nor the mean amount of blood ingested per mosquito. These results indicated that mosquito salivary secretion did not have a chemotropic effect on microfilariae.

Aedes↗

Polymerase chain reaction-based method for the detection of canine heartworm (Filarioidea: Onchocercidae) in mosquitoes (Diptera: Culicidae) and vertebrate hosts.

A polymerase chain reaction (PCR)-based test was developed to aid in identification of Dirofilaria sp. and for use in surveys for infected vectors of Dirofilaria immitis (Leidy). A set of PCR primers was designed based on the DNA sequence of a D. immitis surface antigen gene. The predicted product was a 378 base-pair DNA fragment. The target fragment was amplified from free D. immitis larvae (both L3 and microfilariae), individual infected mosquitoes, pools of 30 mosquitoes (including a single infected mosquito), infected mosquitoes stored desiccated at room temperature for 7 mo, and whole blood from a dog infected with D. immitis. These primers did not amplify a homologous fragment from a mermithid or from 4 other species of filarioid nematodes (including 1 other Dirofilaria species). Third-stage larvae from field-collected mosquitoes also were tested. Field-collected L3, identified tentatively as D. immitis, were confirmed by PCR. There was no amplification using the PCR test for L3 identified tentatively as Dirofilaria sp. (possibly D. tenuis Chandler). These data provide a strong indication of the specificity of these primers. The potential utility of this technique for detecting the presence of D. immitis in field populations of mosquitoes is discussed.

Animals↗

Cellular hemolymph response of Simulium vittatum (Diptera:Simuliidae) to intrathoracic injection of Onchocerca lienalis (Filarioidea:Onchocercidae) microfilariae.

Hemolymph cellular changes in Simulium vittatum Zetterstedt in response to intrathoracic injection with Onchocerca lienalis Stiles were characterized by increased numbers of tissue fragments that contained cells embedded within a noncellular matrix. Cell numbers within the matrix fragments increased both for sham and microfilariae-injected files, indicating a blastogenic response to injection alone. Morphological characteristics of fixed, Giemsa-stained cells within these tissue fragments were most similar to those previously described for prohemocytes. The total population of freely circulating hemocytes collected 24 h after injection also responded to injection alone. Differential cell counts showed a complex pattern of changes that was influenced strongly by increased numbers of prohemocytes at 24 h in microfilariae-injected flies. Fat body fragments collected in hemocoel perfusates at 24 h were fewer when flies were maintained at 27 degrees C than at 21 degrees C. More fat body fragments were collected from microfilariae-injected flies than from control and sham-injected flies held at 27 degrees C. Injection of 1.25-5 micrograms of lipopolysaccharide per fly did not elicit similar hemolymph-associated matrix tissue and fat body changes, indicating that S. vittatum response to microfilaria infection and bacteria infection are likely to involve different mechanisms.

Animals↗

Multiplex polymerase chain reaction for detection of Dirofilaria immitis (Filariidea: Onchocercidae) and Wuchereria bancrofti (Filarioidea: Dipetalonematidae) in their common vector Aedes polynesiensis (Diptera: Culicidae).

In French Polynesia, Aedes polynesiensis (Marks) is the vector of the human filarial parasite Wuchereria bancrofti (Cobbold) and dog heartworm, Dirofilaria immitis (Leidy). A multiplex polymerase chain reaction (PCR) assay was designed to screen pools of field-collected Ae. polynesiensis for the presence of both parasites simultaneously using primers specific for each parasite. The sensitivity of detection on purified DNA was 1 and 10 pg, equivalent to 0.1 and 1 L3 larva per pool for W. bancrofti and D. immitis, respectively. Codetection was performed at an hybridization temperature of 58 degrees C to avoid competition between heterologous DNA and primers that was observed at 55 degrees C. In addition, D. immitis was detected by PCR in the blood of infected dogs.

Aedes↗

Determination of sample sizes for the estimation of Onchocerca volvulus (Filarioidea: Onchocercidae) infection rates in biting populations of Simulium ochraceum s.l. (Diptera: Simuliidae) and its application to ivermectin control programs.

Monthly samples of biting Simulium ochraceum s.l. Walker were collected before and after ivermectin treatment in southern Mexico and analyzed for Onchocerca volvulus Leuckart infection rates, infection intensity, and the characteristics of larval distribution among parous flies. The variance over mean ratio (VMR) indicated that in all cases this distribution departed from Poisson and was strongly aggregated (VMR > 1). The negative binomial was found to be an adequate model with a small value of the aggregation parameter k, but the degree of larval overdispersion increased as the mean larval load decreased, invalidating the use of a common kc value. A linear relationship between k and the mean (mu) was established, k(mu) = k1 mu, which permitted exploration of the relationship between the observed proportion of infected flies, p, and the estimated mean larval burden per fly, m (all larval stages in parous flies). This would allow mean numbers of larvae per parous fly to be predicted from presence-absence data (e.g., from infection rates provided by polymerase chain reaction methods applied to pools of flies), assuming that k1 is a known parameter. Given that both p and m are naturally low in S. ochraceum, their relationship was practically linear within the range of observed values. Predictions were tested with the Mexican data from which the clumping parameter was estimated as well as for Guatemalan data for which this information was not available. Results showed a highly satisfactory degree of agreement between predictions and observations. The sample sizes required to estimate mean larval loads from prevalence data for fixed levels of precision (defined as the ratio between SE[m] and m) were calculated for realistic S. ochraceum infection rates (those found in published pre- and postcontrol field surveys as well as in this work). For the special case in which the relationship between k and the mean is linear and goes through the origin, k(mu) = k1 mu, the number of flies to be examined for O. volvulus infections does not explicitly depend on the aggregation parameter, but rather on the unknown proportion of infected flies. Practical recommendations for the calculation of sample sizes are discussed. For infection levels < 0.2%, a minimum number between 6,000 and 13,000 parous flies would have to be examined to estimate the mean larval load with a precision between 0.20 and 0.30. The linearity between onchocercal infection rate and infection intensity in the fly population indicates that relationships between the former and onchocerciasis patterns in the human population should be further explored for the purposes of monitoring the impact of ivermectin control programs through entomological evaluations.

Animals↗

Effect of nutrient levels and Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) infections on the vector competence of Aedes albopictus (Diptera: Culicidae) for Dirofilaria immitis (Filarioidea: Onchocercidae).

The effect of habitat nutrients and Ascogregarina taiwanensis (Lein & Levine) infection on the vector competence of a New Orleans strain of Aedes albopictus (Skuse) for Dirofilaria immitis (Leidy) were evaluated. Larvae were infected with A. taiwanensis oocysts and reared under high and deficient nutrient conditions using leaf litter as a food source. Ascogregarine-infected and uninfected females were fed on D. immitis-infected blood and examined after 15 d for filariae. Susceptibility to infection with filaria was 60-70% for all females. In groups reared under high nutrients, the infective rate (3rd-stage larvae present) of coinfected females (18%) was significantly greater than females infected only with heartworm (6%). Host mortality following blood meals was significantly less in coinfected (22%) than in heartworm-infected females (37%). Under deficient nutrient conditions, there was no significant difference between the infective rate (8%) or post-blood meal mortality (5%) of coinfected females compared with heartworm-infected females. Prevalence of melanization reactions in coinfected females was significantly higher (31%) than in females infected only with heartworm (6%) at both nutrient levels. It is concluded that high nutrient levels and ascogregarine infection increase the vector competence of Ae. albopictus for D. immitis by enhancing the immune response so that fewer filariae develop, causing lower host mortality. Under low nutrient conditions, the smaller host contains less food reserves for filariae development, and in coinfected females melanization reactions and damage to the Malpighian tubules may reduce vector survival.

Aedes↗

Aedes albopictus (Diptera: Culicidae): an experimental and natural host of Dirofilaria immitis (Filarioidea: Onchocercidae) in Florida, U.S.A.

Females of an Aedes albopictus (Skuse) colony from southeastern Florida, U.S.A., ingested low (22.9 +/- 3.2 mg/Female) and high (243.2 +/- 37.6 mf/Female) numbers of microfilariae from a dog infected with Dirofilaria immitis (Leidy). High mortality of females occurred during the first 4 d after infection regardless of the number of microfilariae ingested; daily mortality was almost negligible during 5-15 d after infection. Percentage of survival 15 d after infection was higher (63%) in females that ingested low numbers of microfilariae than those (15%) that ingested high numbers of microfilariae. The development of most of the D. immitis larvae was arrested in late L1 stage with some of the L1 stage larvae becoming melanized intracellularly in the Malpighian tubule cells of Ae. albopictus. Fifteen days after infection, development of D. immitis to the infective L3 stage occurred in only 10.9% of the surviving F1 and F2 Ae. albopictus that ingested low numbers of microfilariae, but in 94% of Anopheles quadrimaculatus Say ingesting similar numbers of microfilariae, as a control. Females of Ae. albopictus ingesting high numbers of microfilariae had more surviving females with L3 than those ingesting low numbers of microfilariae. The number of L3 larvae in the Malpighian tubules, hemocoel, head capsule and proboscis ranged from 1 to 37 per female, indicating the potential of Ae. albopictus to transmit D. immitis. Development of D. immitis larvae was not affected by co-infection with Ascogregarina taiwanensis (Lien & Levine), although both parasites infect the Malpighian tubules, the first intracellularly and the second extracellularly. After one generation of selection, a strain of Ae. albopictus susceptible to D. immitis developed 2.5 times more L3 than the parent strain. These results show that a small portion of the natural population of Ae. albopictus is susceptible to infection with D. immitis and that susceptibility may be increased rapidly by selection. The presence of developing Dirofilaria sp. larvae in the Malpighian tubules of field-caught females indicated that Ae. albopictus may be infected naturally with D. immitis in Florida.

Aedes↗

Natural infection of Culex theileri (Diptera: Culicidae) with Dirofilaria immitis (Nematoda: Filarioidea) on Madeira Island, Portugal.

Field and laboratory studies were performed to verify whether Culex theileri Theobald functions as a natural vector of Dirofilaria immitis (Leidy) on Madeira Island, Portugal. CO2-baited light traps (EVS traps) were use to sample mosquitoes monthly basis between February 2002 and February 2003 in the area of Quebradas (Funchal). Three mosquito species were captured, including 58 Culex pipiens L., 790 Cx. theileri, and three Culiseta longiareolata (Macquart). Only C. theileri tested positive for D. immitis. The presence of this filarial worm was detected by direct observation, infectivity assay dissection technique, and polymerase chain reaction methods. Infected mosquitoes were recovered in October and December 2002 and January 2003. These data provide evidence that Cx. theileri could be the main vector of D. immitis in Funchal, Madeira.

Animals↗

Density and distribution of Dirofilaria immitis (Nematoda: Filarioidea) third-stage larvae in Aedes sollicitans and Aedes taeniorhynchus (Diptera: Culicidae).

Numbers and the distribution of third-stage larvae (L3) were investigated in Aedes sollicitans (Walker) and Aedes taeniorhynchus (Wiedemann) female mosquitoes fed Dirofilaria immitis (Leidy) infectious-blood with densities of microfilariae (mf) ranging from approximately 7,100-43,400 mf/ml. At each microfilarial density, a maximum of 63-66 infective larvae were recovered from an Ae. sollicitans alive on day 15 after infection. In comparison with Ae. taeniorhynchus, Ae. sollicitans averaged greater numbers of L3 and from 1.4 to 2.4 times more L3 in the head and labium per infected female. The trend was for greater numbers of L3 to be found in the labium than in the head of Ae. sollicitans, but there were no significant differences between numbers of L3 recovered from these sites in differentially infected females. However, numbers of L3 recovered from the head versus the labium of differentially infected Ae. taeniorhynchus varied significantly with the infectious blood microfilarial density. At the two lowest and highest microfilarial densities, greater numbers of L3 were recovered from the head and labium, respectively. Variations among species in the female body size, blood meal size, and retention of L3 may be factors responsible for differences observed between the total numbers and percentage distribution of D. immitis L3 recovered from the simultaneous blood-fed Ae. sollicitans and Ae. taeniorhynchus.

Aedes↗

Development of Onchocerca volvulus (Filarioidea: Onchocercidae) in the West African black fly Simulium yahense (Diptera: Simuliidae) in Liberia.

Simulium yahense black flies infected with microfilaria of Onchocerca volvulus were kept in a defined insectary environment in Liberia, West Africa. A daily sample of infected flies was dissected for larvae developing in the thoracic muscles and examined for growth in stadial development. Microfilariae ingested by black flies transformed to the L1 larval stage without molting. Successive larval development included molting to the L2 stage and, finally, to the L3 stage, which was infective in humans. The cephalic cap, consisting of a laterally located hook and central stoma, occurs in the first larval stage. The caudal appendix and the laterally located anal opening are apparent in the L1 larva. In the L2 stage, the cephalic cap is lost and the large circular stoma becomes surrounded with elevated flaps. The caudal appendix was lost after larvae molted to the L3 stage, and in its place, 3 terminal papillae developed. Sense organs, such as 2 opposing phasmids and 8 papillae that were arranged into 2 circles, developed in the cephalic region of the L3 larva. The evidence of pathological consequences due to the presence of the L3 larva in the fly host are illustrated and discussed.

Animals↗

Further studies on the development of onchocerca spp. (Nematoda: Filarioidea) in Nearctic black flies (Diptera: Simuliidae).

Of three species of Nearctic black flies tested, Simulium pictipes Hagen proved the most efficient laboratory vector of the bovine parasite Onchocerca lienalis. Among flies inoculated intrathoracially with 40 microfilariae, numbers of 3rd-stage larvae per fly were 7.63 for S. pictipes, 7.54 for S. vittatum Zetterstedt, and 0.83 for S. decorum Walker. S. pictipes survived the longest under laboratory conditions, with 83.3% of the females remaining alive 10 days after inoculation with 40 microfilariae of O. lienalis. Using an artificial membrane feeding system, S. pictipes could be routinely infected with O. lienalis by mouth. This black fly was also susceptible to infection with the Guatemalan strain of O. volvulus. Among flies injected with 10 microfilariae the rate of infection with 3rd-stage larvae was 93%, with a mean of three 3rd-stage larvae per fly. Successful techniques for the large-scale recovery and cryopreservation of 3rd-stage larvae of O. lienalis were also developed. A motility rate of 92.7% was observed in larvae cryopreserved within vector black flies.

Animals↗