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.

Antigen shedding from the surface of the infective stage larvae of Dirofilaria immitis.

A qualitative and quantitative analysis was made of the release of surface-associated molecules from developing Dirofilaria immitis infective-stage larvae (L3). D. immitis L3s were labelled with 125I using an Iodogen catalysed reaction and either maintained in culture or placed in chambers that were implanted into Lewis rats. The larvae released 10-20% of the labelled material each day during the first 4 days of in vitro and in vivo development. The loss of surface-labelled peptides from developing larvae corresponded with an increase in the amount of trichloroacetic acid-precipitable radioactivity found in the culture medium. SDS-PAGE analysis of the labelled material showed that the same 35 and 6 kDa components found in larval extracts were shed into culture medium by the developing parasites. Metabolic labelling studies and experiments in which larvae were labelled after different times in culture indicated that, once released, the surface-associated molecules were not replaced, and that this net loss of surface peptides resulted in a reduction in the antigenic potential of the cuticular surface. Antibodies from both immunized rabbits and naturally infected dogs immunoprecipitated the 35 kDa component. In contrast, the 6 kDa molecule was not recognized by the antibodies in any of the sera tested. Shedding of surface peptides and reducing surface antigenicity may represent mechanisms by which D. immitis infective-stage larvae evade immune attack.

Dirofilaria immitis: studies on anti-microfilarial immunity in Lewis rats.

The effect of vaccination on rates of microfilarial clearance using Dirofilaria immitis in male Lewis rats was examined. Animals were immunized with whole, dead microfilariae or a PBS extract of microfilariae in Freund's adjuvant. The immunized animals, as well as untreated and adjuvant controls, were challenged intravenously with 4 x 10(5) viable microfilariae (mf). The duration of microfilaremia was 15.5 days in rats vaccinated with whole mf, 17.7 days in those vaccinated with a PBS extract, 36.3 days for those vaccinated with adjuvant alone, and greater than 70 days for the untreated group. Analysis of the anti-microfilarial IgG response by ELISA and Western blots demonstrated that immunization induced significant amounts of antibody against high molecular weight peptides, particularly a peptide located at 105 kDa. Antibody levels in both groups of immunized animals continued to rise following challenge, reaching peak levels of 78-80 micrograms/ml on the day of microfilarial clearance. Decreasing microfilaremia following challenge was associated with an enhanced recognition of low molecular weight peptides.

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.

Dirofilaria immitis: identification and characterization of circulating parasite antigens.

A method for the identification of circulating parasite antigens in filarial nematode infections was developed using canine infections with Dirofilaria immitis as a model. Filarial antigens ranging in molecular weight from 211 to 13 kDa were extracted from the sera of microfilaremic dogs by a solid phase immunobinding procedure and identified by immunostaining of Western blots. A major antigen of 104 kDa was selected for further characterization. The 104 kDa circulating antigen showed antigenic and biochemical identity with 104 kDa peptides found in extracts of adult male and microfilarial stages of the parasite. The 104 kDa peptide was antigenically stable under a variety of storage conditions. Its potential as a diagnostic target is discussed.

Proteolytic cleavage of IgG and other protein substrates by Dirofilaria immitis microfilarial enzymes.

Proteases were detected in aqueous extracts of Dirofilaria immitis microfilariae. Enzymes within the extract were capable of hydrolyzing Azocoll, a general protease substrate, at pH's 7, 8, and 9. Sensitivities to a variety of protease inhibitors indicated that multiple azocollytic enzymes were present in the extract, most prominent of which appear to belong to the serine class of proteases. By incorporating various substrates into the matrices of polyacrylamide gels, 2 SDS-resistant, mercaptoethanol-sensitive proteases in the MF extract were identified at 22 and 76 kDa. These proteases showed differential abilities to digest casein, fibrinogen, hemoglobin, and IgG. The MF extract hydrolyzed radiolabeled IgG into 8-10-kDa fragments following a 20-hr incubation. A similar degree of digestion was observed in 2 hr when viable microfilariae were used. The potential significance of these proteases in the evasion of host effector mechanisms is discussed.

Antigenic peptides on the surface of Dirofilaria immitis microfilariae.

Various methods of radioiodination were employed to identify peptides on the surface of Dirofilaria immitis microfilariae. Optimum surface radiolabelling occurred with the lactoperoxidase-catalyzed reaction. Two major peptides of 16 and 14 kDa were labelled by this method. These peptides were soluble in Nonidet P-40, were not glycosylated, and showed no signs of disulfide linkages. These peptides were immunoprecipitated by sera from D. immitis-infected dogs, but not by sera from uninfected dogs or sera from dogs with potentially cross-reactive nematode infections. Analysis of the 14 and 16 kDa peptides by two-dimensional gel electrophoresis revealed that the 16 kDa peptide was a single unit with a pI of 5.25 whereas the 14 kDa band was composed of three individual peptides with pI values ranging from 5.6 to 6.1. Iodination by chloramine T resulted in the same panel of labelled peptides but suffered from poor efficiency of 125I incorporation. The viability of microfilariae labelled by the standard Bolton-Hunter method decreased by 50% following the reaction which resulted in the labelling of a variety of internal components.

Quantitative and qualitative changes in the humoral response of dogs through the course of infection with Dirofilaria immitis.

The present study compared the humoral response of dogs which developed microfilaremic or occult forms of dirofilariasis following experimental infection with Dirofilaria immitis L3 larvae. Quantitative analysis by ELISA revealed that antibody levels to adult somatic (AS), excretory-secretory (ES), and microfilarial (MF) antigens were highest during the patent phase of infection in dogs with either form of dirofilariasis. Patent sera from dogs destined for occult infections contained anti-AS and anti-MF antibody concentrations of 1,572 and 1,004 micrograms/ml, respectively, while microfilaremic-bound dogs contained 1,044 and 906 micrograms/ml, respectively. Chronic sera (430 days post infection) from occult dogs contained anti-AS and anti-MF antibody levels of 982 and 600 micrograms/ml, respectively, which were higher than in microfilaremic dogs. The antibody response to ES antigen was generally 10-fold less in absolute antibody concentrations at all time points tested. Immunoperoxidase staining of antigens transferred to nitrocellulose revealed the presence of several antigenic proteins which were recognized by occult, and to a lesser extent or not at all, by microfilaremic dogs. Sera drawn from occult-bound dogs 280 days post-infection, a time corresponding to microfilarial clearance (transition phase), contained higher antibody activity to microfilarial proteins weighing 47.5, 42.0, 34.2, and 22.4 kilodaltons compared to the microfilaremic dogs. This difference in antigen recognition became more apparent during the chronic phase of infection.

Inhibition of the adverse reaction to diethylcarbamazine in Dirofilaria immitis-infected dogs by diazepam.

Eight dogs with a Dirofilaria immitis microfilaraemia predictive for a moderately severe post-diethylcarbamazine (DEC) reaction, and 6 dogs with a microfilarial density predictive for a severe, fatal or near-fatal, reaction were given 5 mg diazepam (Valium) by intramuscular injection followed, 30 minutes later, by an intramuscular injection of 5 mg/kg DEC. Diazepam completely, or almost completely, blocked the post-DEC adverse reaction in all dogs of both groups. However, 5 D. immitis-infected dogs given DEC immediately following the injection of diazepam all developed an adverse reaction of a degree of severity, as measured by clinical and haematological parameters, customary for their level of microfilaraemia.