Thermotaxis in third and fourth-stage Dirofilaria immitis larvae

Third-stage and fourth-stage Dirofilaria immitis larvae exhibited positive thermotaxis when placed in a thermal gradient. Negative thermotaxis was not observed. Positive thermotaxis may be important for the successful transmission and for directing third and fourth-stage larval migration toward predilection sites in the host.     

Active and passive immunization of mice against larval Dirofilaria immitis

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.     

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.     

Dirofilaria immitis: effect on the longevity of Aedes trivittatus

Laboratory studies on vector mortality as related to parasite burden revealed that the mosquito Aedes trivittatus showed considerable tolerance to infection with the filarial nematode Dirofilaria immitis. Although mosquitoes exposed to a dog with a high microfilaremia (347 microfilariae/20 mm³) had a significant increase in mortality during the first 16 days postexposure, 66% of the mosquitoes lived long enough for complete parasite development to occur. Those mosquitoes exposed to a dog with a low microfilaremia (62 microfilariae/20 mm³) had no significant increase in mortality. There was a strong negative correlation between parasite burden and mosquito survival, but only mosquitoes harboring more than 15 juveniles had an increased chance of dying before D. immitis could develop to the infective stage. The retention of microfilariae within the blood clot and peritrophic membrane of A. trivittatus seems beneficial to this vector-parasite system by reducing the parasite burden and increasing mosquito longevity.     

Dirofilaria immitis: the moulting of the infective larva in vitro

A number of in vitro culture systems were tested for their ability to support the development of Dirofilaria immitis infective larvae to the fourth larval stage. In cultures of medium ML-15 containing a feeder layer of Dog Sarcoma (DS) cells larvae successfully moulted and showed a small but significant increase in length. Ultrastructural observations demonstrated that the fourth-stage cuticle was synthesized in vitro and in some larvae was fully formed by 60 hours of culture. The hypodermis of moulting larvae contained numerous multi-vesicular bodies. It is concluded that the moult in vitro is a true moult and not an atypical response of the larvae to the conditions of culture.     

Identification of Dirofilaria immitis larval antigens with immunoprophylactic potential using sera from immune dogs.

Previous research has demonstrated that dogs that received chemically abbreviated Dirofilaria immitis larval infections were significantly immune to challenge infections. Sera from those immune animals have been effective in passively transferring larval killing and stunting. In the present study, sera from immune and control animals were used to screen various Ag subsets for unique Ag. Through Western blot analysis of larval extracts and excretory-secretory products, and immunoprecipitation of metabolically labeled proteins and larval surface Ag, it was determined that as many as 12 molecules were uniquely recognized by protective immune sera. A 39-kDa molecule was present in both soluble lysates of third- and fourth-stage larvae and larval excretory-secretory products; it was recognized by each of the immune dogs and by none of the infected or uninfected control animals. The 39-kDa molecule appeared to be absent from adults and microfilariae of the parasite. In addition to the unique recognition by immune dog sera, larval stage specificity of this molecule suggests that it may be useful as a vaccine candidate.     

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.     

Lipid synthesis in the adult dog heartworm, Dirofilaria immitis.

1. Incorporation studies with three labelled substrates--[14C]2-glycerol, [14C]1-acetate and [14C]1-oleic acid--demonstrated that adult dog heartworms can synthesize all classes of complex lipids present, including free cholesterol. 2. Diacylglycerols and phosphoglycerides were most rapidly labelled regardless of the precursor employed. 3. 14C from glycerol was found in the aqueous phase of saponified lipids, whereas that from oleic acid was in the fatty acid portion. 4. Tag from acetate was predominantly in the fatty acid portion of saponified lipids and also occurred in the unesterified fatty acids. 5. Acetate and unesterified fatty acids, as represented by oleic acid, were more readily used for lipid synthesis than was glycerol.     

Glycerol metabolism in the adult dog heartworm, Dirofilaria immitis.

1. When adult Dirofilaria immitis were incubated up to 60 min in a medium containing physiological concentrations of glycerol and glucose, the mean amount of free glycerol present did not change significantly. 2. [14C]2-glycerol disappeared from media in which 1 g heartworms were incubated at a linear rate of about 0.7 mumol/hr/g during the first hour. 3. 85-95% of the radioactivity in the Folch extract remained in the aqueous phase. 4. Phosphoglycerides and diacylglycerols accounted for better than 90% of the 14C in the lipids. 5. The rate of glycerol conversion to lactate was only about 0.02% of the rate of glucose conversion under experimental conditions. 6. The data indicate that, although dog heartworms do utilize glycerol as a substrate in glycolysis, it is probably of more importance in lipid synthesis.     

In vitro culture of Dirofilaria immitis third- and fourth-stage larvae under defined conditions

The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.     

The effects of various carbohydrate diets on Aedes aegypti infected with Dirofilaria immitis.

Aedes aegypti infected with Dirofilaria immitis and uninfected mosquitoes were maintained on various carbohydrate diets (glucose, galactose, fructose, sucrose, trehalose, maltose, and melibiose). The value of each of these sugars in supporting survival of adult A. aegypti, and in supporting egg production, viability of eggs, and development of third-stage larvae of D. immitis in A. aegypti was analyzed. Fructose, glucose, maltose, sucrose, and trehalose provided the strongest support for survival of adult male, and infected and uninfected adult female A. aegypti. Galactose and melibiose provided the least support for survival of all groups of mosquitoes. The mean number of eggs laid per uninfected adult female A. aegypti was greatest when mosquitoes were maintained on glucose, melibiose, maltose, fructose, sucrose, and trehalose. The same was true for female mosquitoes infected with D. immitis; except for melibiose which provided poor support for egg production. In both Dirofilaria-infected and in uninfected mosquitoes, galactose supported the production of low mean numbers of eggs per adult female A. aegypti. High percentages of eggs laid by uninfected and by infected female mosquitoes fed glucose, melibiose, maltose, sucrose, and trehalose hatched. While galactose supported a high percentage of hatching in eggs laid by uninfected A. aegypti, a much lower percentage of eggs laid by infected female mosquitoes maintained on this same carbohydrate hatched. The lowest percentages of eggs that hatched were from among those laid by infected and by uninfected females fed fructose. The highest mean number of D. immitis larvae (L₃) were recovered from adult A. aegypti fed glucose, maltose, fructose, and sucrose; the second best sugar in this regard was trehalose. The lowest mean number of D. immitis larvae were isolated from female A. aegypti fed galactose and melibiose.     

Reconstructing the spread of Dirofilaria immitis in California coyotes

Dirofilaria immitis is a filarial nematode parasite that is currently widely enzootic in dog and coyote (Canis latrans) populations of California. Weak historical evidence suggests that the initial focus of D. immitis in California occurred 3 decades ago in the Sierra Nevada foothills (SNF) and spread to other parts of California thereafter. However, this hypothesis is difficult to evaluate because of the lack of epidemiological studies on heartworm in California before 1970. We investigated this hypothesis by comparing D. immitis prevalence in coyotes between initial (1975-1985) and current (2000-2002) surveys in the SNF and 2 coastal regions. In the SNF, prevalence of heartworm was not significantly different between initial (35%, n = 169) and current (42%, n = 60) surveys (P = 0.17), suggesting the existence of a stable enzootic focus in the initial survey period. In contrast, current prevalence was 4 times higher than initial prevalence in the northern Coast Range foothills (44 vs. 10%; n = 119, 107; P < 0.001) and in the south San Francisco Bay foothills (32 vs. 8%; n = 31, 59; P = 0.005), suggesting that initial surveys were made during the early stages of colonization. Dirofilaria immitis prevalence, intensity, and abundance was similar in a coastal location in Mendocino County between 1994-1996 and 1999-2002, suggesting some degree of stability in this enzootic focus. Collectively, these findings support the hypothesis that D. immitis established itself initially in California coyotes living in the SNF and subsequently expanded its range of enzootic foci in central California.