Adult Dirofilaria Immitis Research Articles

Dirofilaria immitis: Biochemical and immunological characterization of the surface antigens from adult parasites

The molecules associated with the surface of adult Dirofilaria immitis were identified and characterized employing IODO-GEN-mediated surface labeling methods. D. immitis female and male parasites were found to have a limited number of surface-associated proteins (17.5,16, and 14.5 kDa) and glycoproteins (49 and 20 kDa) which were readily extracted from parasite homogenates in the absence of detergent. The major surface labeled proteins and glycoproteins were antigenic in rabbits, but appeared to elicit only a weak humoral response in dogs with patent dirofilariasis. In addition, a 10- to 6-kDa surface-associated glycolipid was identified which may form a coat on the outside of the parasite and play a role in immune evasion. In immunoprecipitation experiments, the glycolipid was not recognized by the antibodies from rabbits exposed to the glycolipid or by antibodies in the sera of patently infected animals. The glycolipid and the 14.5-kDa surface protein were selectively released by the adult parasite during in vitro culture.

Parasite excretory-secretory antigen and antibody to excretory-secretory antigen in body fluids and kidney tissue of Dirofilaria immitis infected dogs.

Excretory-secretory (ES) antigens of adult Dirofilaria immitis were detected by enzyme-linked immunosorbent assay (ELISA) in serum and urine from each of 12 experimentally infected dogs. Excretory-secretory antigens in serum were first detected 154 days postinfection. Serum antibodies directed against parasite ES antigens were detected by ELISA in all dogs. Kidney tissue elution studies were performed in 10 dogs, and antibody and parasite ES antigens were demonstrated in each case. Antibody or parasite antigen was not detected in serum, urine, or kidney eluates from uninfected dogs. At peak concentrations of the ES antigens in serum, there were correlations with the number of adult D. immitis present in the dogs (r2 = 23.8, P less than 0.05) and with the antigen concentration in kidney eluates (r2 = 73.5, P less than 0.001). Peak serum antibody concentrations were not correlated with either the number of adult worms or the antibody concentrations in kidney eluates. This study suggests that detection of parasite antigens in urine may be an important diagnostic aid. In addition, the correlation between the concentration of D. immitis ES antigens in kidney tissue and in serum without a similar correlation between serum and kidney antibody concentrations suggests that D. immitis ES antigens adhere to kidney tissue.

Evidence of Keratin and Peroxidase Activity in Adult Dirofilaria immitis

FIGURE 1. A. Cross section of an body wall stained with Gomori's tric like fibers (small arrow) surrounded t arrow) in the cuticle. B. Anti-keratin dase staining (small arrow) of fibers section of the cuticle. Bars = 6 am. tis century, the this material has been given the name cuticubrates has been lin (Fugimoto and Shigenori, 1973, Archives of , keratin is not Biochemistry and Biophysics 157: 1-6). Keratinimals produce like materials have also been described in the that covalently nematode cuticle (Anya, 1966, Nature 209: 827, Experimental 828). 355; Lee, 1966, The specific origin of the nematode cuticle re-250; McBride mains unclear. Some authors have concluded that tistry 6: 1484the cuticle is formed exclusively by secreted coles not digest a lagens (Bird, 1957, Experimental Parasitology 6: 2ris cuticle, and 383-403; Lee, 1972, Advances in Parasitology 10: 347-369). However, the discovery of RNA _ and ATPase in the nematode cuticle indicated cellular characteristics associated with this structure (Anya, 1966, Parasitology 56: 179-198). Because keratin is an intracellular protein, the presence of keratin in the nematode cuticle would support the theory that the cuticle is at least partly cellular. We have examined cross sections of Dirofilaria immitis stained with Gomori's one-step trichrome method (Sheehan, 1980, Theory and practice of histotechnology, C. V. Mosby Company, St. Louis, Missouri, pp. 191-192). Frozen A, d sections were stained with an immunohistology kit for keratin (Miles Scientific, Naperville, Illinois, #8631). The antiserum in the immunohistology kit was prepared in rabbits challenged with cytokeratins isolated from the stratum corneum of the human foot. Negative control serum from nonimmunized animals of the same species was supplied by the manufacturer. Adult heartworms were harvested from a pound dog; samples of footpad from the dog were also collected for a positive keratin control. Some worms and footpad samples were fixed in 10% neutral buffered t __ j | formalin for 48 hr, dehydrated in an alcohol seB ries, and embedded in paraffin. Other worms and footpad samples were frozen in liquid nitrogen adult heartworm for cryostat sectioning. Jhrome. KeratinA heartworm section stained with Gomori's immunoperoxitrichrome is shown in Figure 1A. Fibers in the in frozen cross cuticle stained red and showed strong birefringence similar to keratin in the footpad control.

Phosphatidylinositol, phosphatidylglycerol and cardiolipin synthesis in adult Dirofilaria immitis females

Srivastava A. K. and Jaffe J. J. 1987. Phosphatidylinositol, phosphatidylglycerol, and cardiolipin synthesis in adult Dirofilaria immitis females. International Journal for Parasitology 17:917–920. The pathways leading to the formation of phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL) in adult Dirofilaria immitis females were investigated. PI was synthesized by both de novo as well as via base exchange pathway in the worms. Under specified assay conditions, the respective rates of PI formation by way of these pathways in crude homogenates of the worms in the order given were around 3.0 and 0.75 nmol min −1 mg −1 protein. PG synthesizing activity in the worms was mainly associated with the particulate fractions and the rate of formation by these fractions was around 1.5 nmol min −1mg −1 protein. The worms were unable to synthesize CL by the pathway found in mammals.

Glutathione S-transferase in adult Dirofilaria immitis and Brugia pahangi

Glutathione S-transferase (EC 2.5.1.18) was detected in the cytosolic and microsomal fractions of adult Dirofilaria immitis females at respective levels of 30 nmol and 3 nmol min −1 (mg protein) −1 activity with the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The transferase activity in the cytosolic fraction of adult Brugia pahangi females was 10 nmol min −1 mg −1 with CDNB; determination of its activity in the microsomal fraction of this filariid was not attempted. These filarial glutathione S-transferases were further characterized after their purification by glutathione-affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytosolic transferase from D. immitis, molecular weight 47 000, yielded a single subunit of around 28 kDa. The cytosolic and microsomal transferases from D. immitis differed in their activity with CDNB, 1,2-dichloro-4-nitrobenzene, 4-benzylchloride and ethacrynic acid. The cytosolic transferase from B. pahangi was distinguished by its high activity with ethacrynic acid. Both glutathione S-transferases from D. immitis also functioned as a glutathione peroxidase, strongly preferring cumene hydroperoxide as a substrate over hydrogen peroxide. Both were equiactive inhibitors of malonaldehyde formation in the NADPH-microsomal lipid peroxidation system. Thus, in addition to the ability of filarial glutathione S-transferases to detoxify electrophilic xenobiotics, at least those from D. immitis also exhibited selenium-independent glutathione peroxidase activity. Their glutathione S-transferase function suggests a potential role for these enzymes in the leukotriene synthetic pathway, if filariae can form such eicosanoids from arachidonate. Functioning as a glutathione peroxidase, they could serve to protect filarial membrane lipids from peroxidation.

Dirofilaria immitis: Comparison of cytosolic and mitochondrial glutamate dehydrogenases

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8–8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and <100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 μ M NADH, the K m for the substrate, α-ketoglutarate, was equivalent for the two enzymes, but the K m for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 μ M NADH, 1 m M ADP approximately doubled and 1 m M ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 m M glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate K m of M-GDH. However, addition of as little as 3m M glutamate reduced M-GDH velocity about 20%. The two glutamate dehydrogenases of D. immitis were found to differ in almost every respect.

Phosphatidylserine synthesis in adult dirofilaria immitis females

Phosphatidylserine synthesis in adult Dirofilria immitis females. International Journal for Parasitology 16: 9–11. Phosphatidylserine synthesis in extracts of Dirofilaria immitis females was found to occur exclusively by way of a calcium-stimulated enzyme-catalyzed exchange of l-serine for the base components of preformed phospholipids. Under specified conditions the rate of serine incorporation into phospholipid(s) was around 44 pmol min −1 mg −1 protein.

Effects of diethylcarbamazine on the motility of Angiostrongylus cantonensis and Dirofilaria immitis.

Effects of piperazine derivatives, especially of diethylcarbamazine (DEC) on adult Angiostrongylus cantonensis and Dirofilaria immitis were examined. Piperazine (3 X 10(-5)-10(-4) M) paralyzed A. cantonensis and the action was antagonized by picrotoxin. 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (10(-5)-10(-4) M) caused contraction but little effect was produced by strychnine. An inhibitory effect on untreated preparations was caused by lower concentrations (3 X 10(-6)-10(-5) M) of diethylcarbamazine citrate (DEC) and also on the preparations contracted by eserine. A stimulatory effect was also seen when higher concentrations (10(-4)-3 X 10(-4) M) of this drug were applied to both preparations. The inhibitory action of DEC was antagonized by gabergic antagonists such as picrotoxin and bicuculline, but not by alpha-adrenergic antagonists like dibenamine and phentolamine. When the worm preparation was paralyzed by strychnine or hexylresorcinol (inhibitors of the release of acetylcholine in this worm), the stimulatory effect of DEC was blocked, but pyrantel (a nicotinic cholinergic agonist) contracted the paralyzed preparation. However, the effect of DEC on D. immitis (10(-7)-3 X 10(-4) M) was inhibitory, and this action was also antagonized by picrotoxin. These results suggest that the DEC inhibitory and stimulatory action is through the gabergic and cholinergic mechanisms in adult A. cantonensis and D. immitis.

Dirofilaria immits in a portacaval shunt

The fifth known case of intravascular human infection with an adult Dirofilaria immitis (canine heartworm) is reported. This mature, but nongravid, female nematode was recovered at autopsy from a prosthetic portacaval shunt. A brief review of human dirofilariasis is presented.

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 Parasitology 15: 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.

Transplantation of Adult Dirofilaria immitis into Lewis Rats: Parasitologic and Serologic Findings

porarily sterilized), fecal examinations made weekly for 6 mo after deworming were negative. When cultures were maintained at 15 C, those containing the mixed infection and those monospecific for A. caninum exhibited a rise in larval numbers on day 12 and were identified as a mixed population of U. stenocephala and A. caninum larvae in the former and a homogenous population of A. caninum in the latter. Results indicate that a monospecific isolate of U. stenocephala larvae can be separated from a mixed infection with A. caninum by culturing at 15 C and recovering larvae on the eighth day.

Experimental production of lesions in canine pulmonary arteries similar to those produced by Dirofilaria immitis infection.

Pulmonary arterial disease was produced by the placement of flexible polyvinyl chloride threads (similar in size, shape and flexibility to adult female Dirofilaria immitis) in the pulmonary arteries of dogs. Several different types of pathological change were apparent ranging from subendothelial oedema and endothelial loss, mild intimal proliferation and thrombus formation to organised fibrous encasement of the threads in areas where they had become impacted in the lumen of the artery. This range of pathological changes is very similar to that produced by D immitis and suggests that the intima of the canine pulmonary artery may react to different insults in a similar way.

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).

A highly purified allergen from excretory and secretory products of Dirofilaria immitis

Preceding data revealed that the allergen concentrated mainly in excretory and secretory (ES) products exhausted by adult Dirofilaria immitis. The present paper reported that a highly purified allergen was obtainable from ES products more easily and effectively. An allergen in ES products was purified by a combination of DEAE-Sephadex A-50 chromatography, Sephadex G-200 and Sephadex G-100 gel filtration. The purified preparation was proved to be one protein by sodium dodecyl sulfate (SDS) gel electrophoresis and to be exact the same allergen with the one obtained from the crude extract of adult Dirofilaria worms. The molecular weight of the purified allergen was estimated to be 15,000, and the allergen was inclined to aggregate in the buffered solution.

Isolation, partial purification and some properties of two acid proteases from adult Dirofilaria immitis

Two acid proteases were isolated from the soluble extracts of adult Dirofilaria immitis, the filarial heartworm of canines. Activity of these proteases was detected using 3H-labeled bovine α-casein as substrate, and they were designated Fp-I and Fp-II in order of their elution from a CM-cellulose column. The molecular weight of partially purified Fp-I was approximately 170000, and it was active between pH 4.6–5.8. The activity of Fp-I doubled in the presence of various sulfhydryl reagents at 5 mM, and it was inhibited 50–60% by the sulfhydryl inhibitors p-hydroxymercuribenzoate and iodoacetate at 1 mM, the heavy metal chelating agent o-phenanthroline at 1 mM and the peptide aldehyde protease inhibitors pepstatin (10 μM), leupeptin, antipain and chymostatin (50 μM). The molecular weight of the more extensively purified Fp-II is approximately 48000. This protease was active between pH 2.6–3.4 and was highly sensitive to inhibition by pepstatin (80% inhibition at 10 nM). Fp-II was not significantly affected by sulfhydryl reagents, sulfhydryl inhibitors, metal chelating agents or peptide aldehyde protease inhibitors other than pepstatin. These properties of dirofilarial Fp-II resemble those of mammalian cathepsin D.

Antigenicity of microfilarial an adult Dirofilaria immitis in indirect fluorescent antibody test.

Antigenicity of microfilarial an adult Dirofilaria immitis in indirect fluorescent antibody test.

Thymidine kinase activity and thymidine salvage in adult Brugia pahangi and Dirofilaria immitis

Cytosolic thymidine kinase (EC 2.7.1.75), the initial enzyme in the thymidine salvage pathway, was detected in crude homogenates of adult female Brugia pahangi and Dirofilaria immitis, with respective specific activities of 100 and 460 nmol/h/mg protein. Partially purified filarial thymidine kinases were found to have molecular weights of approximately 180 000, to be most active in the presence of Mg 2+ and ATP, to have a sharp pH optimum (pH 7.0) and to be heat-labile in the absence of added thymidine. For both, the respective K m values for thymidine and ATP were 60 μM and 1.6 mM, and 5-iodo-2′-deoxyuridine was as good a substrate as thymidine. A distinguishing property was the 3-fold higher sensitivity of the B. pahangi enzyme to feedback inhibition by thymidine 5′-triphosphate. Adult female B. pahangi took up and incorporated [ methyl- 3H] thymidine into DNA when they were exposed to this radiolabeled deoxynucleoside in vivo, but the thymidine salvage pathway in these worms was essentially nonfunctional in vitro.

Glycosyl transferase activity in homogenates of adult Dirofilaria immitis

Homogenates of adult Dirofilaria immitis possess a microsomal enzyme system able to transfer mannose from GDP-mannose to endogenous lipid intermediate(s) and exogenous dolichol monophosphate. A divalent metal was required with Mn 2+ being the most effective; other requirements for optimal activity included Triton X-100, EDTA and either ATP or NaF. The maximal rate of mannose transfer to the lipid acceptor by the filarial system, 1.6 pmol · min −1 · mg −1 protein, occurred at 37°C and pH 7.0, and this was inhibited 50% by 8 μM diumycin and not at all by 100 μM tunicamycin. D. immitis microsomes also were shown to promote the transfer of mannose to derivatives of α-lactalbumin, resulting in the synthesis of a mannose-labeled glycoprotein.

Inhibition of NADP-linked malic enzyme from Onchocerca volvulus and Dirofilaria immitis by suramin

NADP-linked malic enzyme (malate dehydrogenase (oxaloacetate-decarboxylating) NADP +, EC 1.1.1.40) has been partially purified from adult Onchocerca volvulus and Dirofilaria immitis. Suramin was found to inhibit the activity of malic enzyme from both filarial worms. The inhibition constants for suramin were calculated to be 0.011 μM and 0.015 μM for the enzymes from O. volvulus and D. immitis, respectively. In the case of NADP-linked malic enzyme from Trypanosoma brucei and chicken liver the inhibition by suramin was less pronounced. The inhibition constants were found to be 0.8 μM and 2.5 μM for the protozoan and vertebrate enzymes, respectively. The type of inhibition was competitive with respect to malate. The Michaelis constants for malate and pyruvate were determined to be 0.9 and 4.5 mM for O. volvulus and 0.85 and 5.0 mM for D. immitis, respectively. The low K m values for malate compared to those for pyruvate and the about 15-fold greater turnover in the direction of decarboxylation compared to carboxylation indicate that malic enzyme from both filarial sources might be involved in an alternative pathway leading from phosphoenolpyruvate via oxaloacetate, malate and pyruvate to lactate. It is suggested, that the inhibition of malic enzyme activity from O. volvulus by suramin might interfere with the generation of NADPH for biosynthetic reactions.

Synthesis of ubiquinone 9 by adult Brugia pahangi and Dirofilaria immitis: evidence against its involvement in the oxidation of 5-methyltetrahydrofolate.

Among various ubiquinone (Q) isoprenologues tested, only Q7 was more efficient than menadione in promoting the oxidation of 5-methyltetrahydrofolate (CH3FH4) by 5,10-methylenetetrahydrofolate reductase isolated from adult Brugia pahangi, whereas Q10 was the best cofactor in the same reaction catalysed by the analogous enzyme from adult Dirofilaria immitis. Menoctone (3-[1-cyclohexyloctyl] -2-hydroxy-1,4-naphthoquinone) was a strong competitive inhibitor of both these ubiquinone isoprenologues in the respective reactions. When incubated in the presence of D,L-[14C]-mevalonate, adult B. pahangi and D. immitis synthesized radiolabelled Q9 only, in addition to other isoprenoid derivatives in the neutral lipid fraction. In view of the inability of Q9 to promote the oxidation of CH3FH4 by 5,10-methylenetetrahydrofolate reductase from B. pahangi, it seems unlikely that this filaria uses Q9 as a cofactor in this reaction. Conceivably, D. immitis could use Q9 as a cofactor in its enzymatic oxidation of CH3FH4, since in this circumstance, it was a better cofactor than menadione.