[Pulmonary dirofilariasis].

A 70-year-old man was admitted to our department for pulmonary nodule of 15 mm in diameter in the left lower lobe detected by chest computed tomography (CT). A possibility of malignant tumor could not be ruled out, and lung partial resection was performed. Pathological examination during operation revealed a coagulation necrosis and the lesion was finally diagnosed as pulmonary dirofilariasis.

Morphological redescription of Dirofilaria immitis.

Morphological descriptions of Dirofilaria immitis are scarce. For this reason, we carried out morphological studies using both light and scanning electron microscopy for this filaroid species. Morphometric and morphological data were compatible with previous descriptions of D. immitis , but several anatomical structures are described by scanning electron microscopy for the first time, such as details of the cuticular striations, positioning of amphids, visualization of anal and vulvar opening, descriptions of deirids, lateral line, the pair of phasmids in the posterior end in females, and visualization of a small pair of latero-terminal papillae in the posterior end in males.

Scanning electron microscopic observation of ultrastructure of Dirofilaria immitis microfilaria.

Ultrastructure of Dirofilaria immitis microfilaria(Mf) was imaged through scanning and transmission electron microscopies. Transverse annular striations covered all over the surface of the whole body. Two small pores on the cephalic disk and the mouth-like cavity at a ventral side of the first striation next to the cephalic disk were observed. A single triangular hook was projected from the upper palate. The excretory pore was observed at the 80th annulus from the anterior end, and the anal pore at the 90th annulus from the posterior end. The both pores were located at the ventral side of the body. This study first demonstrated that a large number of nuclear column cells were distributed in the body cavity. These cells were spherical and about 1 microm in diameter. Each of the cells contained a spherical nucleus and was connected to each other by micro-strings that were running radially. Many flattened muscle cells were located at the inside of the hypodermis of the whole body. The tail contained only a single longitudinal muscle cell.

Kidney ultrastructural lesions in dogs experimentally infected with Dirofilaria immitis (Leidy, 1856).

Kidneys of 16 beagles with experimentally induced heartworm (Dirofilaria immitis) infections and 4 heartworm-nai;ve dogs were studied by light and electron microscopy. The infections were induced either by subcutaneous injection of infective larvae or by the transplantation of adult parasites, and infection periods varied from 111 to 818 days and 365 to 923 days, respectively. One control group of heartworm-naïve dogs and four groups of heartworm-infected dogs, which were divided according to the type and the length of infection, were used. In the infected dogs, thickening of the glomerular basement membrane (GBM), the presence of dense deposits in the GBM, and foot process effacement were the most frequent lesions observed. In some dogs, electron dense deposits were seen in the GBM and the mesangium and/or enlargement of the mesangial matrix could be characterized. The longer the infection period, the thicker the GBM and the more common the occurrence of foot process effacement. In general, these alterations were more evident in animals that had been infected for more than 1 year, had high microfilaremia, and had 14 or more parasites in the main pulmonary artery and its branches. The presence of dense deposits suggests that the pathogenesis of kidney disease in dirofilariasis is associated with deposits of immune complexes in the membrane. The finding of ultrastructural changes in dogs with early prepatent infections suggests that immature heartworms, as well as microfilariae and possibly adult worms, contribute to the glomerulonephropathy.

Dirofilaria immitis: further characterization of the transglutaminase enzyme and its role in larval molting.

We recently reported the cDNA cloning and functional characterization of a novel transglutaminase (TGase) from the dog filarial parasite Dirofilaria immitis. D. immitis TGase (DiTG) has no sequence similarity with any other known TGase, but has significant similarity to protein disulfide isomerase (PDI)-related endoplasmic reticulum protein ERp60. In the present study. we further characterized the recombinant DiTG (rDiTG) and studied its role in the molting process of third-stage larvae. The enzymatic activity of rDiTG requires Ca2+, and the maximum activity was observed at a calcium concentration of 4 mM. Interestingly, the rDiTG was highly thermostable, with optimal activity observed at 55 degrees C, similar to that seen with the native enzyme. Dithiothreitol (DTT) was not essential for enzyme activity. In fact, rDiTG was more active in the absence of DTT. The known inhibitors of TGase, such as monodansylcadaverine (MDC), cystamine and iodoacetamide, inhibited the TGase activity, but not the PDI activity, of rDiTG, demonstrating the dual activity of rDiTG. The TGase-specific pseudosubstrate, MDC, completely inhibited the molting of D. immitis L3 to L4 if present during the first 24-48 h of the molting process. Electron microscopic studies revealed that MDC-treated infective larvae failed to show separation between the L3 and L4 cuticles. The L4 cuticle and accompanying hypodermis were much thinner in MDC-treated worms than in controls. Using anti-rDiTG antiserum, the native DiTG antigen was localized in the hypodermis, afibrillar muscle cells and gut epithelium in adult male and female worms as well as developing embryos in the females.

Does fertilization in the filarial nematode Dirofilaria immitis occur through endocytosis of spermatozoa?

Information on the ultrastructural details of fertilization in filarial nematodes are still unavailable. Here we report new data on this process in Dirofilaria immits, the heartworm of dogs and cats. Electron microscopy allowed us to observe oocytes engulfing spermatozoa through an endocytosis-like process. We also observed spermatozoa inside the oocytes which still possessed their plasma membrane and which were clearly enveloped by a further membrane, likely derived from the endocytosis process. At this stage, at the interface between the sperm membrane and the endocytotic membrane (vacuolar space), we observed flocculent material in the proximity of the membranous organelles (MOs) of the sperm. In the proximity of the MOs, we also observed the enlargement of the vacuolar space. Other images showed the dissolution of the sperm membrane, and the release of nuclear masses and organelles in the egg cytoplasm. We did not observe the fusion of lysosomes to the endocytotic vacuoles. In addition, the lysis of the sperm organelles has never been observed inside the vacuoles containing the whole sperm. Thus we suggest that the degradation of the endocytotic and sperm plasma membranes is determined by material released by the MOs. Since we did not observe the entry of sperm into the oocytes by other mechanisms, we also suggest that endocytosis is the normal process used by the spermatozoon to get into the egg cytoplasm in D. immitis. Finally, during our observations of the seminal receptacle we did not observe any structure in the spermatozoa which could be interpreted as an intracellular bacterium. This is consistent with previous results indicating that the bacterium Wolbachia in filarial nematodes is not transmitted through the sperm.

Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

On the rugose area and caudal papillae distribution in Dirofilaria immitis (Leidy, 1856) Railliet & Henry, 1911, by scanning electron microscopy.

The objective of this study was to obtain a better characterization of Dirofilaria immitis (Leidy, 1856) Railliet & Henry, 1911, in Brazil, ratifying previous accounts and adding new data on the rugose area on the ventral surface of the spiralled posterior portion of males observed by scanning electron microscopy, which presents a different arrangement compared to other species of the genus, and differs also from the genera Litomosoides Chandler, 1931, and Wuchereria Silva Araújo, 1877, adding another taxonomic character to Dirofilariinae and Onchocercinae. Scanning electron microscopy studies also showed some aspects of the distribution of caudal papillae in D. immitis that have not been described before.

Dirofilaria immitis: ultrastructural localization, molecular characterization, and analysis of the expression of p27, a small heat shock protein homolog of nematodes.

A cDNA fragment encoding the complete coding region of a 27-kDa protein (p27) of Dirofilaria immitis was cloned. Antibody to the recombinant p27 bound to hypodermal tissues of third (L3) and fourth stage larvae (L4) of D. immitis and to both the hypodermis and the cuticle of L3s of Onchocerca volvulus, as visualized by immunoelectronmicroscopy. The deduced amino acid sequence of the central and C-terminal regions of p27 (amino acids S83 to H222) is 18-36% identical to members of the sHsp/alpha-crystallin family of proteins. The homologous region is thought to be responsible for the molecular chaperone activity of members of this family. The p27 cDNA does not encode a hydrophobic signal peptide. At least two homologous yet distinct p27 genes were identified in the D. immitis genome by Southern hybridization using the p27 cDNA as a probe. The p27 transcript was 0.9 kb in length on Northern blots. The expression of p27 in L3s of D. immitis was neither upregulated by heat shock (43 degrees C) nor by incubation at the physiologic temperature of 37 degrees C. Pulse-labeling experiments of both D. immitis and Brugia malayi L3s during the L3-L4 molt in vitro showed that synthesis of p27 is also not upregulated during this developmental phase. However, p27 is expressed constitutively throughout the D. immitis L3-L4 molt and therefore by both larval stages. In addition, both female and male adult worms of this species express p27 constitutively. P27, or an allomorph thereof, was detected in each of nine species representing four nematode superfamilies, thus indicating that this molecule is ubiquitous within the phylum Nematoda. In view of the hypodermal localization of p27, its constitutive expression, and its retention among nematodes, the function of this protein in essential housekeeping roles such as that of molecular chaperone during the molting process is discussed.

Mechanism of increased dissemination of chikungunya virus in Aedes albopictus mosquitoes concurrently ingesting microfilariae of Dirofilaria immitis.

We investigated whether concurrent ingestion of chikungunya virus and microfilariae of Dirofilaria immitis increases viral dissemination and multiplication in a mosquito vector. The increased rate of dissemination of this virus in mosquitoes concurrently ingesting both agents was found when homogenates of bodies and those of legs only were examined. It was significantly higher than that of controls ingesting the virus alone through the end of the experiment on day 14 after infection. We next studied the mechanism by which the presence of microfilariae enabled the virus to enter into the hemocoel and to reach the salivary glands. We checked our results using histopathologic procedures and electron microscopy by identifying holes produced by the microfilariae that penetrated the midgut epithelial layer. When the midgut of mosquitoes was punctured with a thin needle immediately after the mosquitoes ingested viruses, higher infection rates were observed than in mosquitoes without such punctures.

Dirofilaria immitis: effect of fluoromethyl ketone cysteine protease inhibitors on the third- to fourth-stage molt.

D. immitis third-stage larvae (L3) were cultured with fluoromethyl ketone cysteine protease inhibitors. By Day 5 in culture, none of the larvae cultured with 0.1, 0.2, 0.6, or 1.0 mM benzyloxycarbonyl-Phe-Ala-CH2F (Z-Phe-Ala-CH2F) has molted, while 63.2% of larvae in media without inhibitor had molted. At the two lower concentrations of inhibitor more larvae had initiated, but not completed, the molt. In addition to Z-Phe-Ala-CH2F, four other fluoromethyl ketone derivatives, Z-Phe-Arg-CH2F, amorpholine urea-(Mu)-Leu-Phe-CH2F, Mu-Tyr-Phe-CH2F, and Mu-Phe-Phe-CH2F, were tested to determine their effects on L3 in culture. All fluoromethyl ketones tested except Z-Phe-Arg-CH2F inhibited molting. Larvae cultured in inhibitors were determined to be alive as judged qualitatively by motility and quantitatively by reduction of 3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium. Electron microscopy demonstrated that L3 which were unable to molt after being cultured in a fluoromethyl ketone derivative had synthesized the new fourth-stage (L4) cuticle but had not shed the L3 cuticle. The same fluoromethyl ketone derivative that did not inhibit molting, Z-Phe-Arg-CH2F, was a slightly less effective inhibitor of larval extract-initiated hydrolysis of the synthetic peptide substrate, Z-Val-Leu-Arg-7-amino-4-methyl coumarin. L3 were also cultured through the molt in media containing the synthetic peptide substrate Z-Val-Leu-Arg-4- methoxy-B-naphthylamide to examine cysteine protease activity in situ. Fluorescence as seen on Days 0-4 during the molting process was first observed on the anterior tip of the larvae, and subsequently in the pharynx, with progression down the L4 as it shed the L3 cuticle.

Determination of the role of cuticular carbohydrates in the hemocompatibility of Dirofilaria immitis (Nematoda).

We have partially characterized surface glycoproteins of the canine heartworm, Dirofilaria immitis. Histochemical studies indicated the presence of neutral and acidic mucopolysaccharides at the blood-cuticle interface. Fluorescein isothiocyanate-conjugated lectin binding patterns suggested the presence of alpha-D-glucosyl and/or alpha-D-mannosyl, beta-galactosyl, N-acetylneuraminyl and N-acetylated-D-hexosaminyl (sialic and glucuronic acids, respectively) terminal residues among the constituent sugars of the glycocalyx. An additional goal of this study was to assess the significance of each carbohydrate in parasite hemocompatibility by using scanning electron microscopy, internal reflection infrared spectroscopy, and comprehensive contact angle measurements. Each carbohydrate identified in the glycocalyx was selectively cleaved with the appropriate exoglycosidase. Heart-worms bearing native and enzyme-altered surfaces were exposed to platelet-rich canine plasma. Activation and aggregation of platelets were significantly increased on enzyme-treated surfaces as compared with native surfaces. Enzyme-induced cleavage of carbohydrate residues was associated with an increase in critical surface tension or a loss in cuticular structural integrity or both. Hemocompatibility of the heartworm cuticle depends on the retention of a stable saccharide-rich layer that minimizes interaction with plasma proteins and platelets; thus, carbohydrate residues on the glycocalyx may contribute to parasite hemocompatibility. The presence of similar low-critical-surface-tension coatings with high mechanical integrity may impart thromboresistance to other polyphenolic or chitinous substances.

Cloning of a cuticular antigen that contains multiple tandem repeats from the filarial parasite Dirofilaria immitis.

An unusual antigen composed of tandemly repeated protein units was cloned from the filarial parasite Dirofilaria immitis. The antigen was initially identified by screening a lambda gt11 cDNA library with serum from dogs immunized with irradiated D. immitis third-stage larvae. DNA sequence analysis of the cDNA clone, Di5, revealed a continuous open reading frame composed of two 399-base-pair repeats arranged in tandem. Southern blot analysis of genomic D. immitis DNA showed that the gene coding for Di5 is composed of a tandem array of 25-50 copies of this same 399-base-pair repeat. Antiserum raised against recombinant Di5 protein detected a protein "ladder," from about 14 to greater than 200 kDa with steps approximately 15 kDa apart, on immunoblots of D. immitis extract. Metabolic labeling of adult parasites with [35S]methionine showed that Di5 is synthesized as a large precursor that is subsequently cleaved to produce the ladder-like array. These results suggest that the characteristic ladder is created by proteolytic cleavage of the precursor at the same site in each monomer. The Di5 antigen was localized to the cuticle and hypodermis of adult D. immitis by immunoelectron microscopy. Both male and female parasites were found to release Di5 when cultured in vitro. DNA hybridization analysis demonstrated that Di5 is a member of a gene family present in many filarial parasites that infect both animal and human populations.

Ultrastructural localization of Dirofilaria immitis antigen in canine lung tissue using the protein A-gold labelling technique.

Dirofilaria immitis antigens were demonstrated in the pulmonary tissues of dogs, with natural and experimental infection (D. immitis) in dogs using an immunostaining colloidal gold technique. Electron microscopy of thin sections treated with anti-heartworm serum revealed antigen deposition within the pulmonary microvasculatur and perivascular areas.

Surface-associated antigens of second, third and fourth stage larvae of Dirofilaria immitis.

The surface-associated molecules of the second (L2), third (L3) and fourth (L4) larval stages of Dirofilaria immitis were characterized employing radiolabeling techniques and SDS-PAGE analysis. Major labeled components of 35 kDa and 6 kDa were present in extracts from IODO-GEN-labeled L2 and L3 parasites. The results of lactoperoxidase-catalyzed reactions also demonstrated that L2 and L3 stages of D. immitis have a similar repertoire of surface-associated antigens. However, in contrast to the results obtained with IODO-GEN, lactoperoxidase reactions labeled components with apparent molecular weights of 66, 48, 25, 16.5 and 12 kDa. The similarities in the molecular weights of the L2 and L3 surface-associated components and the results of immunoprecipitation experiments which demonstrated that antibodies produced against the 35 kDa molecule from D. immitis L3s also recognize the 35 kDa component from L2 parasites suggest that synthesis of the molecules found at the surface of mature infective larvae begins as early as day 6 of development in the mosquito, D. immitis L4s emerged from the molting process with a repertoire of surface-associated antigens distinct from those found on L2s and L3s. IODO-GEN labeling of D. immitis L4s showed major surface-associated molecules with apparent molecular weights of 57, 40, 25, 12 and 10 kDa when analyzed under non-reducing conditions. In addition to molecules of 57, 40, 25, 12 and 10 kDa, extracts of D. immitis L4s labeled with lactoperoxidase contained additional major bands at 45, 43 and 8 kDa. Metabolic labeling experiments demonstrated a shift in the amount and complexity of the excretory/secretory products released by D. immitis during L3 to L4 development.

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.

Surface characterization of the cuticle of Dirofilaria immitis.

The surface reactivity of the dog heartworm (D. immitis) was evaluated by comprehensive contact angle measurements and a platelet retention test. Contact angle data yielded calculated surface energy terms very similar to those previously reported for intact vascular endothelium. The platelet test revealed the native worm surface to be nonreactive, retaining fewer platelets than glass or worms whose surfaces had been modified by extraction with acid and high salt solutions. The cuticular morphology of the heartworm was studied with both light and electron microscopy, the latter coupled with ferritin-conjugated double-layer immunolabeling to reveal adsorbed host protein on the cuticle surfaces. Multiple attenuated internal reflection (MAIR) IR spectroscopy confirmed the general composition of this surface layer to be glycoproteinaceous. Morphological and histochemical studies confirmed and extended previous descriptions of nematode cuticle, adding ultrastructural detail on cortical, medial, and basal layers. A trilaminar membrane, apparently corresponding to a mammalian cell membrane (plasmalemma), constituted the external cortical layer as observed in high magnifications. The existence of a glycocalyx of varying thickness was demonstrated in ruthenium red-stained sections. MAIR IR spectra showed this glycoproteinaceous film to appear, in fully hydrated samples, as a loose biological gel. Ferritin-antibody conjugate labeling confirmed the presence of adsorbed dog albumin, dog immunoglobulin class G (IgG) and dog complement fraction 3 (C3) in the cuticular surface layer. It is likely, therefore, that D. immitis heartworms demonstrate long-term thromboresistance at least in part due to their passive low-surface-energy overcoating with host proteins.

Partial characterization of the adsorbed protein layer on Dirofilaria immitis (Nematoda) cuticle.

Electron microscopy coupled with ferritin-conjugated indirect immunolabeling was used to locate sites of adsorbed host protein on cuticular surfaces of the adult canine heartworm, Dirofilaria immitis. The epicuticle appeared as a trilaminated structure. At high magnifications, the outermost layer of this structure was resolved into a trilaminar layer, which might correspond to the plasma membrane of animal cells. A ruthenium red-positive layer was external to the epicuticle. Ferritin-antibody conjugates showed evidence of adsorbed dog albumin, dog immunoglobulin class G (IgG), and dog complement fraction 3 (C3) on the surface. Ferritin adsorption to control surfaces was minimal. Possible causes and effects of interfacial host-protein adsorption are discussed in an attempt to bring insight to the hemocompatible nature of the parasitic cuticle.