Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen.

Dirofilaria immitis, a filarial nematode, is the causative agent of canine and feline heartworm disease. Previous research has demonstrated that immunity to D. immitis can be induced in dogs by repeated chemical abbreviation of infections while the parasite is a fourth-stage larva. Sera obtained from dogs immunized in this manner has been effective in passively transferring larval killing and stunting. These immune sera, by comparison to nonimmune sera from infected cohorts, recognize a number of unique D. immitis antigens, some of which are larval specific. In this study immune dog sera were used to screen a D. immitis larval cDNA expression library. Three overlapping cDNA clones, Di22, Di18 and Di16, were obtained that encode a portion of a large molecule, greater than 150 kDa, that is composed of multiples of a 399-bp repeat. This protein when immunoblotted with antibody against a recombinant expressed Di22 fusion protein is found in larval as well as adult extracts and excretory-secretory products, and is seen as a series of ascending subunits, each approximately 15 kDa larger than the previous one. This antigen is highly immunogenic, as evidenced by the strong reactivity of the recombinant expressed Di22 fusion protein with sera from immune dogs, microfilaremic dogs and infected amicrofilaremic dogs. While the function of this antigen is unknown it has significant sequence similarity with an allergen found in Ascaris.

Circulating excretory-secretory antigen levels and specific antibody responses in mice infected with Toxocara canis.

Circulatory excretory-secretory antigen levels and IgM and IgG responses to larval antigens were monitored in the serum of 20 BALB/c mice that had been given approximately 500 infective eggs of Toxocara canis by stomach tube. Other groups of mice received different doses of infective eggs, ranging from 5 to 1,250 eggs. Excretory-secretory antigens were collected from culture fluid in which mechanically hatched larvae of T. canis were maintained. An indirect enzyme-linked immunosorbent assay was used to monitor specific antibody responses. Circulating antigen levels were monitored using a direct ELISA which incorporated an IgG fraction of a rabbit antiserum to the excretory-secretory antigens as a capture antibody and a biotin-conjugated form of the same rabbit IgG as the second antibody. The antigen-specific IgM response was evident the first week of infection and peaked 3 to 6 weeks post-infection. The antigen-specific IgG response first appeared the second week of infection and peaked at 6 to 8 weeks post-infection. Both isotype levels stayed near their peak values for the remainder of the study. In the untreated sera, circulating antigen was initially evident and highest the first week of infection; the antigen concentrations dropped by the third month of infection to low, but significant, levels that persisted for the duration of the study. The administration of greater than 25 eggs produced antigenemias. There appeared to be a positive linear trend between the number of eggs given and the amount of antigen in the circulation.

Induction of protective immunity in dogs to infection with Dirofilaria immitis using chemically-abbreviated infections.

Four dogs were immunized against Dirofilaria immitis infection by a series of 3 larval infections which were each subsequently terminated by ivermectin treatment. Two control dogs received ivermectin treatment alone. Following the final ivermectin treatment, dogs were challenged with infective larvae by subcutaneous inoculation, both free and contained within diffusion chambers. Three weeks after larval challenge the chambers were removed and live larvae were enumerated. Seven months after challenge dogs were killed and necropsied to collect and count adult D. immitis. Chambers recovered from immunized dogs had 63% fewer larvae than chambers from control dogs. At necropsy, control dogs had a mean of 28.5 adult worms whereas the immunized animals had an average of 0.5 worms (range 0-2). Sera collected from immune dogs throughout the study had elevated antibody levels to third- and fourth-larval stage antigens. Significant levels of immune protection were achieved with this immunization regimen. The data suggest that a multiple-stage parasite killing occurs in immune animals. It was not possible to associate immune protection with any of the 5 antigen subsets.

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.

In vitro development of third- and fourth-stage larvae of Dirofilaria immitis: comparison of basal culture media, serum levels and possible serum substitutes.

In vitro development and survival of third-stage larvae of Dirofilaria immitis were compared in four different culture media and in the presence of varying concentrations of four different medium supplements. Motility and the incidence of third- to fourth-stage molting were used as criteria for evaluating different culture conditions. No significant differences in either motility or molting response were detected between larvae cultured in NCTC-135, F12(K), CMRL 1066 or Dulbecco's Modified Eagle's Medium. Fetal calf serum enhanced development and survival of the cultured larvae in dose-dependent fashion. Its effects were maximal at a concentration of 20 percent of the total medium volume. Addition of a commercial medium supplement, NuSerum, also gave a dose-related increase in larval development and viability. The activity of NuSerum in this respect was comparable to that of fetal calf serum. The tripeptide glycylhistidyllysine and bovine serum albumin, fraction V both failed to stimulate development of third-stage D. immitis larvae in vitro.

Recovery and viability of Dirofilaria immitis microfilariae.

The viability of Dirofilaria immitis microfilariae recovered from canine blood by different methods was determined. Microfilaria recovery techniques included saponin lysis, saponin lysis with a trypsin treatment, dextran sedimentation and phytohemagglutinin treatment. Criteria for evaluating viability were microfilarial motility in vitro at 37 degrees C, microfilarial development in mosquitoes and the ability of microfilariae to circulate in mice. Although each method produced motile microfilariae, differences among groups of microfilariae recovered by different techniques were apparent by each of the criteria for viability. Saponin lysis gave superior yields of viable microfilariae.

In vitro culture of Dirofilaria immitis third- and fourth-stage larvae under defined conditions.

The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.

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.

Canine Dirofilaria immitis infection in a hyperenzootic area: examination by parasitologic findings at necropsy and by two serodiagnostic methods.

A total of 174 dogs from an area hyperenzootic for Dirofilaria immitis were grouped into 4 age categories and necropsied; information was obtained on adult D immitis infections and on the presence of microfilariae. Serum samples from these dogs were examined by an enzyme-linked immunosorbent assay (ELISA) for antibody to adult D immitis and by an indirect fluorescent antibody test (IFAT) for antibody to microfilarial surface antigens. In dogs less than or equal to 5 months of age, necropsy demonstrated no evidence of infection; however, positive serologic results indicated that some of these dogs had prepatent infections. The percentage of dogs with ELISA titers (positive) increased with age, as did the percentage of dogs with adult D immitis infections. The IFAT results were positive in some dogs in each age category. Sera from all 29 dogs with occult infections were positive by ELISA. Sera from 6 of 20 dogs with occult dual-sex heartworm infections and 1 of 9 dogs with occult single-sex heartworm infections were positive by IFAT. For diagnosing occult dirofilariasis, the ELISA had a positive predictive value which increased with age of the dog to a maximum of 65.0% in dogs greater than or equal to 12 months of age; ELISA had a negative predictive value of 100% in all age groups. In contrast, positive and negative predictive values for the IFAT decreased with age of the dog to 60% and 37.5%, respectively, in dogs greater than or equal to 12 months of age.

Serologic pattern of canine heartworm (Dirofilaria immitis) infection.

A total of 602 dogs at the Louisiana State University Veterinary Teaching Hospital were tested for antibodies to Dirofilaria immitis by enzyme-linked immunosorbent assay (ELISA), using a purified adult dirofilaria-derived antigen. Most dogs also were evaluated for heartworm infection by a complete WBC count and a Knott test for circulating microfilariae. The serologic prevalence of heartworm infection was 34.7%; prevalence increased significantly (P = 0.0003) with age up to 8 years and then decreased. Dogs usually kept indoors were significantly (P = 0.005) less likely to be infected, as were dogs given diethylcarbamazine therapy (P = 0.0004). Coat length, sex, breed, and presence of intestinal parasites were not associated (P greater than 0.05) with a positive heartworm ELISA result. The ELISA titers showed a positive relationship with both eosinophil and basophil counts. A total of 99 dogs evaluated radiographically were grouped according to results of the Knott test and radiographic examination as follows: (i) negative Knott test and negative radiographic examination (14 dogs), (ii) negative Knott test and positive radiographic examination (57 dogs), and (iii) positive Knott test and positive radiographic examination (28 dogs). The serologic prevalences of D immitis infection in each of these groups were 35.7%, 56.1%, and 85.7% for groups (i), (ii), and (iii), respectively. The ELISA, when used in conjunction with the Knott test results, record of exposure, clinical signs, laboratory results, and radiographic changes, was found to be useful for studying serologic patterns and identifying risk factors for canine heartworm infection.

Cryopreservation of Dirofilaria immitis microfilariae and third-stage larvae.

Microfilariae of Dirofilaria immitis retained their infectivity for susceptible mosquitoes after cooling to -196 degrees C in the presence of 5% dimethylsulphoxide (Me2SO) using a two-step cooling sequence. Motility and in vitro development of cryopreserved microfilariae also compared favourably with unfrozen controls. Third-stage larvae frozen by the same cooling sequence in the presence of either 5% Me2SO or 16% hydroxyethyl starch were motile upon thawing. Thawed larvae completed the third- to fourth-stage moult in vitro at a frequency approximately 5 to 10% of that seen in unfrozen controls.

Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae.

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.

Dirofilaria immitis: surface properties of third- and fourth-stage larvae.

The objective of this study was to analyze surface properties of larval Dirofilaria immitis with potential relevance to protective immunity. Comparisons were made between third (L3)- and fourth-stage larvae (L4) based on their net surface charge, surface carbohydrate and antigen composition, ability to nonspecifically absorb host proteins, complement activation, and nonspecific cellular adherence. It was determined that L3 had a net negative surface charge, whereas L4 had either a neutral or weakly positive surface charge. The lectin Con A, but not any of the other lectins tested, bound only to the surface of L4, and not to that of L3. Monoclonal antibodies were prepared which reacted with the surface of L3 or with the surface of L4, but never both. L4 were found to nonspecifically adsorb host protein to their surfaces, whereas L3 did not. Both L3 and L4 were found to activate complement through the alternate pathway. Finally, nonspecific cellular adherence was found on L3 both in vitro and in vivo but not on L4. The surfaces of L3 and L4 were thus shown to be significantly different and, potentially, in ways which would have great impact in the generation and effectiveness of a protective immune response.

Development and dynamics of regional specialization within the syncytial epidermis of the rat tapeworm, Hymenolepis diminuta.

The epidermis of the tapeworm Hymenolepis diminuta is a highly organized syncytium, composed of an outer layer of continuous cytoplasm, or ectocytoplasm, and an inner layer of nucleated cell bodies, or perikarya. The perikarya are in direct cytoplasmic continuity with the ectocytoplasm via narrow plasmalemma-bound bridges called internuncial process. Although distinct structural and functional differences are apparent between ectocytoplasm and perikarya, all of the perikarya along the body of the cestode are morphologically similar, as are all regions of ectocytoplasm. However, immunocytochemically distinct subpopulations of perikarya and regionally defined areas of ectocytoplasm were identified along the tapeworm strobila by the use of monoclonal antibodies raised against a preparation of isolated tegument. The different types of perikarya and the regionally specialized areas of ectocytoplasm were organized in a topographically precise manner along the body of the parasite. Examination of labeling patterns after colchicine treatment suggests that different types of perikarya are specialized for biosynthesis of specific tegumental molecules and for turnover or recycling of tegumental material. Furthermore, it appears that a 52 kDa polypeptide synthesized by one population of perikarya is transported through the syncytium and ultimately resorbed by a different population of tegumental perikarya. These data suggest that the syncytial epidermis of parasitic platyhelminthes exhibits a more complex organization of function than previously appreciated.

Identification of Dirofilaria immitis larval antigens with immunoprophylactic potential using sera from immune dogs.

Previous research has demonstrated that dogs that received chemically abbreviated Dirofilaria immitis larval infections were significantly immune to challenge infections. Sera from those immune animals have been effective in passively transferring larval killing and stunting. In the present study, sera from immune and control animals were used to screen various Ag subsets for unique Ag. Through Western blot analysis of larval extracts and excretory-secretory products, and immunoprecipitation of metabolically labeled proteins and larval surface Ag, it was determined that as many as 12 molecules were uniquely recognized by protective immune sera. A 39-kDa molecule was present in both soluble lysates of third- and fourth-stage larvae and larval excretory-secretory products; it was recognized by each of the immune dogs and by none of the infected or uninfected control animals. The 39-kDa molecule appeared to be absent from adults and microfilariae of the parasite. In addition to the unique recognition by immune dog sera, larval stage specificity of this molecule suggests that it may be useful as a vaccine candidate.