Seasonal prevalence of third-stage larvae of Dirofilaria immitis in mosquitoes from Florida and Louisiana

Heads of 109,597 mosquitoes collected during 1996 and 1997 from Gainesville, Florida (1996, n = 39,131; 1997, = 34,209), Bartow, Florida (1996, n = 12,000; 1997, n = 12,000), and Baton Rouge, Louisiana (1996, n = 12,257) were tested by a polymerase chain reaction and Southern hybridization-based test for the presence of third-stage larvae of the canine heartworm Dirofilaria immitis. Mosquito heads were pooled (1-200 heads) by month, locality, and species for testing. The test used was species specific for D. immitis and was capable of detecting DNA from a single larva in a pool of 200 mosquito heads. Specificity for the third larval stage was achieved by probing only mosquito heads. One or more D. immitis-infected mosquito heads were detected in each month of the year from Barrow in both 1996 and 1997. No infected mosquito heads were detected from Gainesville or Baton Rouge in December, January, February, or March. These results are in general agreement with previous sentinel dog and model prediction studies that showed heartworm transmission in the warm temperate Gulf coast region of the United States to be seasonal rather than continuous as previously believed.     

Hemocyte monophenol oxidase activity in mosquitoes exposed to microfilariae of Dirofilaria immitis

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.     

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.     

Defense reactions of mosquitoes to filarial worms: effect of host age on the immune response to Dirofilaria immitis microfilariae

The melanization response of Aedes aegypti black-eyed Liverpool strain (LVP) and Aedes trivittatus against intrathoracically inoculated Dirofilaria immitis microfilariae (mff) was assessed in mosquitoes less than 1, 14, 21, and 28 days after adult ecdysis. There was a significant decrease in the melanization response of A. aegypti 14 days of age and older at 1, 3, and 5 days postinoculation (PI) compared to less than 1-day-old mosquitoes. The response also was reduced significantly in 14- to 28-day-old A. trivittatus on days 1 and 3 PI. Although essentially 100% of recovered mff were melanized by day 5 PI in A. trivittatus, the amount of melanin deposited was much less than that seen in 0-day-old mosquitoes. Potential mechanisms responsible for a reduced immune competence in older mosquitoes and the possible relationship to vector potential are discussed.     

Cell-mediated immunity to Dirofilaria immitis.

Cell-mediated immunity to Dirofilaria immitis (DI) in guinea pigs was confirmed by the migration inhibition test (MIT), the blast transformation test (BTT), the delayed skin reaction, and the skin reaction by passive transfer with sensitized peritoneal exudate (PE) cells. All migration inhibition (MI) positive cases were always associated with positive skin reactions and two cases showed positive skin reactions without MI. The cellular antibody confirmed by MIT first appeared on the 4th day after single sensitization, but DNA synthesis in splenic lymphocytes had already started on the 3rd day in the absence of delayed skin reaction and MI. Then, the role of this cellular antibody in the immune mechanism against DI infection was investigated by the in vitro and in vivo cytotoxicity test using microfilariae (Mf) of this species as a target. The cytotoxic activity significantly increased in the sensitized splenic and PE cells, and in vivo normal PE cells implanted into sensitized animals.     

Experimental infection of cats with Dirofilaria immitis.

Natural and experimental infections of cats with Dirofilaria immitis have been reported. Experimental infections of D. immitis in cats with the subsequent detection of microfilaremia and immediate skin hypersensitivity to antigen to D. immitis were produced. Cutaneous nodules and chylothorax were also detected in some infected cats. Adult worm recoveries were low and dead worms were found in some cats indicating the unsuitability of the cat as a host for D. immitis. However, one successful mosquito passager of D. immitis from a cat to a dog was accomplished.     

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.     

Separation of Dirofilaria immitis allergen from the IgG-inducing antigens.

Allergen in crude extract of Dirofilaria immitis was purified and separated from the IgG-inducing antigens by a combination of DEAE-Sephadex A-50 chromatography and Sephadex G-200 gel filtration. The molecular weight of the purified preparation was estimated to be approximately 20,000 by gel filtration. The carbohydrate content of the preparation was apparently low, about 2%. Rats immunized with the allergenic fraction developed only a homocytotropic antibody and no hemagglutination antibody.     

Laboratory observations on the insusceptibility of raccoons to Dirofilaria immitis

Two raccoons, Procyon lotor, were exposed to Dirofilaria immitis by subcutaneous injection of infective third stage larvae obtained from experimentally-infected Aedes trivittatus. Nematodes were not recovered from either raccoon when examined at necropsy 223 and 254 days postexposure. Large numbers of adult D. immitis were found in six dogs used as controls. These data indicate that raccoons cannot support the development of D. immitis.     

Genetic control of murine immune responses to larval Dirofilaria immitis

Previous studies have demonstrated that BALB/c mice, immunized against infection with Dirofilaria immitis, were capable of killing a significant percentage of challenge larvae found within diffusion chambers. The percentage of larvae killed by immunized mice was, however, less than in immunized dogs and unlike immunized dogs, mice were unable to retard the development of the surviving larvae. The objective of the present study was to test 3 inbred strains of mice to determine whether a higher level of protective immunity would develop in these hosts and if larval growth retardation would occur. DBA/2J and C57BL/6J mice and their F1 hybrids B6D2F1/J were used in these studies; it was determined that there were differences in susceptibility among the 3 strains but no difference in ability to eliminate larvae from challenge infections. Growth retardation was seen in larvae recovered from immunized DBA/2J and C57BL/6J mice but not in B6D2F1/J. No difference was noted between immune and control mice in the cell types found in the diffusion chambers. The predominant cell types seen were mononuclear macrophages, multinucleate syncytial cells, and neutrophils. Antibody responses to soluble third- and fourth-stage larval antigens and larval excretory/secretory antigens were measured. Although antibodies to all 3 antigen groups were found in higher concentrations in immunized mice than in their respective controls, only antibody responses to soluble L-3 antigens provided a clear correlation with protective immunity.     

Immunoblotting analysis of somatic components of Dirofilaria immitis

SDS-PAGE analysis of Dirofilaria immitis extracts demonstrated the complexity of somatic protein component of adult male similar to that of adult female worm. Western blot analysis showed six major peptide bands of 85, 66, 42, 20, 16.2 and 14.5 kDa recognized in the sera of infected dogs. Western blotting sera from dogs with Dirofilaria immitis infection suggest that antigenic components in the low molecular weight region may be related to the anti-parasitic mechanism of the host.     

Cryopreservation of Dirofilaria immitis microfilariae and third-stage larvae

Microfilariae of Dirofilaria immitis retained their infectivity for susceptible mosquitoes after cooling to -196 degrees C in the presence of 5% dimethylsulphoxide (Me2SO) using a two-step cooling sequence. Motility and in vitro development of cryopreserved microfilariae also compared favourably with unfrozen controls. Third-stage larvae frozen by the same cooling sequence in the presence of either 5% Me2SO or 16% hydroxyethyl starch were motile upon thawing. Thawed larvae completed the third- to fourth-stage moult in vitro at a frequency approximately 5 to 10% of that seen in unfrozen controls.