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Histochemical differentiation of Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit, Leucognost-SP.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.

Acid Phosphatase↗

An ivermectin-sensitive glutamate-gated chloride channel subunit from Dirofilaria immitis.

Dirofilaria immitis is a filarial nematode that infects dogs and causes cardiopulmonary disease. The most effective way of controlling the infection is by chemoprophylaxis, using members of the avermectin/milbemycin (A/M) class of anthelmintics, which includes ivermectin; these drugs act at invertebrate glutamate-gated chloride channels (GluCl). We have cloned two cDNAs encoding D. immitis GluCl subunits and demonstrated that at least one may be an important molecular target for the A/Ms in vivo. The subunits are orthologues of the alternatively spliced GluClalpha3A and alpha3B subunits (encoded by the avr-14 gene) previously identified in Caenorhabditis elegans and in Haemonchus contortus. Although the alternative splicing of avr-14 is conserved across the species, the processing of the mature GluClalpha3A mRNA differs in D. immitis compared to C. elegans and H. contortus. Two-electrode voltage clamp recordings were made from Xenopus oocytes injected with subunit-specific cRNAs. The DiGluClalpha3B subunit formed channels that were gated by L-glutamate (1-100 mM) and ivermectin (1 microM). Oocytes injected with DiGluClalpha3A cRNA failed to respond to L-glutamate. The qualitative responses obtained were consistent with the pharmacology observed for the GluClalpha3 subunits from C. elegans and H. contortus.

Alternative Splicing↗

Susceptibility of Aedes aegypti to infections with Dirofilaria immitis and Dirofilaria repens.

Eight stocks of Aedes aegypti-4 geographic stocks and 4 selected stocks-were tested for their susceptibility to infection with Dirofilaria immitis and D. repens. Four of the stocks were found to be susceptible and the other 4 were refractory of infection with D. immitis. All the stocks of Ae. aegypti that were susceptible to D. immitis were also susceptible to D. repens but the stocks that were refractory to D. immitis were all partially susceptible to D. repens.

Aedes↗

[Human dirofilariasis: identification of a 44kD antigen recognized by IgM against Dirofilaria immitis].

BACKGROUND: Dirofilaria immitis is a nematode parasite of canids that, in endemic areas, can infect humans. The use of adult somatic antigens for the diagnosis of human dirofilariasis has the disadvantage of a low sensibility because of cross-reactivity with other helminths, specially with IgM antibodies. We describe in this work proteins of the adult somatic antigenic complex that are specifically recognized by human IgM. METHODS: Human sera with different characteristics were analyzed by an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-D. immitis IgM seropositive individuals. Moreover, an enzyme-linked immuno-electro-transfer blot (EITB) was performed for the detection of polypeptides that specifically react with IgM anti-D. immitis. RESULTS: EITB analysis shows that 2 proteins of 44 and 18 kD are recognized by IgM in human sera from an endemic area with a high antibody titer against D. immitis measured by ELISA. Sera from a non-endemic area and from patients suffering from other parasitic and non-parasitic diseases did not recognize these antigens. CONCLUSIONS: We believe that the 44 kD protein could be a good marker of recent D. immitis infection.

Animals↗

Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis.

A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.

Amino Acid Sequence↗

Hexokinase of the adult dog heartworm, Dirofilaria immitis.

1. Dirofilaria immitis hexokinase was relatively heat stable and had a pH optimum range between 7.8 and 8.2. 2. Mean Vmax was 0.40 +/- 0.10 (S.D.) mumole/min/100 mg of worm. 3. Mean Km values were 0.32 mM for glucose, 0.86 mM for fructose and 0.39 mM for ATP. 4. Glucose-6-phosphate was not a strong product inhibitor. 5. Starch gel electrophoresis demonstrated at least three isozymes.

Animals↗

Vectorial competence of Aedes aegypti (Linnaeus 1762) Rio de Janeiro strain, to Dirofilaria immitis (Leidy 1856).

Dirofilaria immitis (Leidy 1856), a nematode parasite, is the etiologic agent of canine heartworm disease and mosquitoes are essential intermediate hosts. Mosquito susceptibility to the worms differ with species, strains and also among individuals of the same strain. To evaluate the degree of susceptibility of Rio de Janeiro laboratory raised strain of Aedes aegypti, we fed mosquitoes on canine blood with different densities of microfilariae (mf). There was no significant difference in the rate of development among the three different densities of mf. Infective larvae were found in the head and proboscis of all mosquitoes provided bloodmeals with different densities of mf after the 11th day post-infection. The infection rate of mosquitoes after ingestion of blood containing 3,000 mf/ml, 5,000 mf/ml and 7,000 mf/ml were 55.3%, 66.7% and 100%, respectively. The vector efficiency indices ranged from 1.6 to 9.3. The finding of L3 stage larvae, high infection rates and vector efficiency indices suggest that Ae. aegypti, Rio de Janeiro laboratory strain, is a potential vector of D. immitis, although of low efficiency.

Aedes↗

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.

Animals↗

Identification and analysis of genes expressed in the adult filarial parasitic nematode Dirofilaria immitis.

The heartworm Dirofilaria immitis is a filarial parasitic nematode infecting dogs and other mammals worldwide causing fatal complications. Here, we present the first large-scale survey of the adult heartworm transcriptome by generation and analysis of 4005 expressed sequence tags, identifying about 1800 genes and expanding the available sequence information for the parasite significantly. Brugia malayi genomic data offered the most valuable information to interpret heartworm genes, with about 70% of D. immitis genes showing significant similarities to the assembly. Comparative genomic analyses revealed both genes common to metazoans or nematodes and genes specific to filarial parasites that may relate to parasitism. Characterization of abundant transcripts suggested important roles for genes involved in energy generation and antioxidant defense in adults. In particular, we proposed that adult heartworm likely adopted an anaerobic electron transfer-based energy generation system distinct from the aerobic pathway utilized by its mammalian host, making it a promising target in developing next generation macrofilaricides and other treatments. Our survey provided novel insights into the D. immitis transcriptome and laid a foundation for further comparative studies on biology, parasitism and evolution within the phylum Nematoda.

Animals↗

Activation in vitro of some biological systems by extracts of adult worms and microfilariae of Dirofilaria immitis.

Extracts of Dirofilaria immitis adults and microfilariae cause the degranulation of mast cells in rat mesentery when applied in vitro and also cause increased vascular permeability in guinea-pigs and rabbits. Adult worms but not microfilariae are able to activate complement in vitro and both stages contain a plasminogen activator. No proteolytic activity or haemolysins could be detected in either stage. The possibility that these factors may be important in the pathogenesis of dirofilariasis and in the adverse drug reactions that may follow treatment of microfilaraemic animals with diethylcarbamazine is discussed.

Animals↗

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.

Animals↗

Glycosyl transferase activity in homogenates of adult Dirofilaria immitis.

Homogenates of adult Dirofilaria immitis possess a microsomal enzyme system able to transfer mannose from GDPmannose to endogenous lipid intermediate(s) and exogenous dolichol monophosphate. A divalent metal was required with Mn2+ 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 degrees C and pH 7.0, and this was inhibited 50% by 8 microM diumycin and not at all by 100 microM tunicamycin. D. immitis microsomes also were shown to promote the transfer of mannose to derivatives of alpha-lactalbumin, resulting in the synthesis of a mannose-labeled glycoprotein.

Animals↗

Increased rat alveolar macrophage expression of functional iNOS induced by a Dirofilaria immitis immunoglobulin superfamily protein.

Dirofilaria immitis is a worldwide filarial nematode causing heartworm disease in dogs and cats. Several mosquito species, which are able to feed both on humans and animals, can transmit this parasite. Inflammatory progression of host tissues induced by parasites are mediated by several molecules, including nitric oxide (NO), which usually exerts deleterious effects on parasites and occasionally on the host. We analyze the in vitro effect of total D. immitis adult worm somatic antigens on naïve rat alveolar macrophage NO production and further separation of parasite proteins to define specific D. immitis somatic molecules influencing host cell NO secretion. Additionally, we address the possible influence of Wolbachia spp. on the in vitro production of NO by macrophages. Our results demonstrate that D. immitis adult worm soluble antigens are able to specifically induce NO production from host macrophages. Furthermore, we demonstrated that this effect is due to nematode antigens rather than to defined components (LPS and metabolic molecules) derived from its endosymbiont, Wolbachia spp. In addition, we were able to isolate and identify one of the parasite specific components from the DiSo extract, denominated DiID35.3 and putatively belonging to the Immunoglobulin Superfamily Protein (ISP) group, triggering NO release from macrophages in a dose-dependent and specific manner.

Animals↗

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.

Animals↗

Pulmonary presentation of Dirofilaria immitis (canine heartworm) in man.

Canine heartworm or Dirofilaria immitis can occasionally infect man. We present the case of a 36-year-old Balkan woman referred to us for a thoracoscopic biopsy of a well defined pulmonary mass. The latter was thoracoscopically resected and proved to be due to Dirofilaria immitis on histopathological examination. To our knowledge this is the second reported case in Western Australia, the first being that reported by Brine et al. The prevalence of the disease in Australia was recognized in 1969 in one study in the state of Queensland, where 12% of 761 dogs at one veterinary clinic and 20% of 296 dogs at another were shown to be infected. Dirofilaria immitis resides in the right ventricle and pulmonary arteries of dogs. The microfilariae are released into the blood stream and are transmitted to secondary hosts by mosquitoes.

Adult↗

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.

Animals↗

Pulmonary coin lesions caused by Dirofilaria immitis.

The dog heartworm Dirofilaria immitis has been diagnosed by thoracotomy as the etiology of neoplastic-appearing nodules in two patients in the Peoria, Illinois area. This brings the total number of reported cases of human pulmonary dirofilariasis to approximately 81 in the United States. The major concern of this benign disease is that in making the diagnosis the patients undergo the risk of surgery because of the presumed preoperative diagnosis of cancer. Greater awareness of this disease is needed as the geographic distribution of human pulmonary dirofilariasis expands in this country.

Adult↗