Dirofilaria immitis: fate and immunogenicity of irradiated infective stage larvae in beagles

Experiments were conducted on the fate of irradiated infective larvae of Dirofilaria immitis in dogs, and on the effect of these infections on a challenge dose of nonirradiated larvae administered at a later date. Six dogs were inoculated with 200 to 296 irradiated larvae; in no case was a patent infection established. No living worm was recovered beyond 66 days. Eight dogs inoculated with 200 to 2401 irradiated larvae over varying periods of time were exposed 57 to 190 days after the final inoculation of irradiated larvae, to a challenge infection of 200 to 250 nonirradiated (normal) larvae. The results showed that the number of worms which developed to maturity in these dogs was sharply reduced compared to that in the 5 controls (dogs inoculated with normal larvae only). The most striking effect was seen in “vaccinated” dogs which were challenged 3 months or more after the final administration of irradiated larvae.     

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.     

Dirofilaria immitis: molting process of third-stage larvae

The objective of this study was to determine the molting process of Dirofilaria immitis third-stage larvae (L-3) to fourth-stage larvae (L-4), as it occurred in vitro. After 48 hr in vitro, the L-4 epicuticle was completely formed, and by 72 hr there was a clear separation between the L-3 and L-4 cuticles. The thickness of the newly formed L-4 cuticle was significantly less than that which has been described for larvae recovered from dogs after a similar incubation time period. If culture conditions were lacking in bovine albumin or proper temperature, larvae successfully developed the L-4 epicuticle but did not complete ecdysis. The molting process of D. immitis L-3 was thus shown to be multistepped with different factors required to induce the various developmental phases.     

In vitro immune mechanisms associated with clearance of microfilariae of Dirofilaria immitis

An in vitro system has been developed to elucidate potential immune mechanisms associated with clearance of microfilariae (Mf) from the bloodstream in canine Dirofilaria immitis infection. Granulocytes as well as mononuclear cells adhere to Mf of Dirofilaria immitis in the presence of immune serum. Only granulocytes, however, were capable of killing Mf, whereas PBMC attach to but do not effectively kill Mf. In the presence of granulocytes 1% +/- 1, 10% +/- 2, and 12% +/- 3 of Mf were killed by heated normal (NDS), patent (PS), and occult serum (OS), respectively, after an 18-hr incubation. With the addition of fresh NDS there was an increase in killing to 5% +/- 1 (p less than 0.025) with heat-inactivated NDS, to 12% +/- 3 in the presence of PS and to 77% +/- 12 (p less than 0.005) in the presence of OS. On further purification of granulocyte cell populations with metrizamide gradients, neutrophils were found to be the predominant effector cells with 73% +/- 18 killing with neutrophils and 18% +/- 6 with eosinophils (p less than 0.0005). Only with neutrophils was a significant increase in killing of Mf observed when fresh NDS was added to delta OS. Fractionation of OS by gel filtration suggested that IgM was the opsonizing antibody in the occult serum. In addition, immunofluorescent studies showed only IgM bound to the surface on Mf on incubation in OS. The involvement of complement in the fresh serum enhancement of killing was supported by the finding, by immunofluorescence, of surface C3 on Mf after incubation in fresh OS.     

Dirofilaria immitis: diethylcarbamazine-induced anaphylactoid reactions in infected dogs

The immunopathogenesis of the anaphylactoid Mazzotti reactions has been studied by comparing physiologic and immunologic aspects of diethylcarbamazine-induced shock in Dirofilaria immitis infected dogs with antigen induced anaphylaxis in infected and uninfected controls. Filarial antigen, specific host IgG antibody, and C1 and C3 complement levels were quantitatively measured over time in relation to the levels of histamine and prostaglandin D2 in the blood and changes in mean blood pressure. D. immitis antigen injected into uninfected dogs having no detectable IgG antibody to D. immitis or Toxocara canis produced a rapid drop in blood pressure that paralleled a drop in C1 and C3 levels and an increase in prostaglandin D2. Antigen injected into infected dogs with IgG antibody produced a similar drop in blood pressure and complement and increase in prostaglandin D2 which differed from the uninfected group only in the slower clearance of antigen from the blood. Diethylcarbamazine alone produced no measurable changes in blood pressure or complement in uninfected hosts. Diethylcarbamazine, however, administered into skin test positive infected dogs, produced a temporally slower but quantitatively similar loss in blood pressure, drop in complement, and increase in prostaglandin D2 and histamine to that induced by antigen injection. Complement activation and immune complex formation are initiated by antigen release, and subsequent vasoactive mediator release leads to shock with prostaglandin D2 being quantitatively higher in blood than is histamine.     

Phosphatidylcholine synthesis in adult Dirofilaria immitis females

Crude homogenates of adult Dirofilaria immitis females were able to incorporate choline into phosphatidylcholine (PC) and also were able to methylate phosphatidyl (N,N-dimethyl)-ethanolamine, using S-adenosylmethionine as the methyl donor, to form PC. The finding of choline phosphotransferase (EC 2·7·8·2) and phosphatidyl (N,N-dimethyl) ethanolamine methyltransferase activity in the paniculate (mainly microsomal) fraction of the homogenates provided further evidence that adult D. immitis females can synthesize PC by way of choline and cytidine 5'-diphosphocholine (Kennedy pathway) and also by way of S-adenosylmethionine-mediated sequential methylation of phosphatidylethanolamine (Bremer-Greenberg pathway).     

Phosphatidylethanolamine synthesis in adult Dirofilaria immitis females

Srivastava Arvind K., Jaffe Julian J. and Lambert Roger A. 1985. Phosphatidylethanolamine synthesis in adult Dirofilaria immitis females. International Journal for Parasitology15: 429–433. Adult Dirofiliaria immitis females were found able to synthesize phosphatidylethanolamine (PE) by way of the following three pathways: (1) phosphorylethanolamine, cytidine diphosphoethanolamine and 1,2-diacylglycerol; (2) decarboxylation of phosphatidylserine (PS); and (3) direct exchange of ethanolamine for choline or serine in preformed phosphatidylcholine or PS. The latter two pathways were confined to the paniculate fraction of worm homogenates. Under stated assay conditions, the respective rates of PE formation by way of these pathways in the order given were around 250, 8500 and 2–3 pmol min^?1 mg^?1 protein.     

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.     

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.