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At least 577 records · Page 32Linked to original sources
PubMed1983

Brugia malayi and B. pahangi: cultivation in vitro of infective larvae to the fourth and fifth stages.

Cultivation of fourth stage Brugia pahangi and B. malayi larvae from infective larvae (stage 3) were obtained in culture medium RPMI 1640 supplemented with 10% human AB serum and an LCC-MK2 rhesus monkey kidney continuous cell line feeder layer. This culture system kept larvae alive in excess of 7 weeks, and served as a source for collection of the worms' secretory, excretory, and moulting antigens.

Animals↗
PubMed1983

Dirofilaria immitis: performance and standardization of specific antibody immunoassays for filariasis.

The factors associated with the development, optimization, and validation of immunoassays for the detection of parasite-specific antibody in filariasis infections were investigated using the dog heartworm, Dirofilaria immitis as a model. We examined two assays, the Protein A solid-phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent assay (ELISA), for quantitation of specific antibody to the parasite in canine serum. Precision, reproducibility, and parallelism were examined using response-error relationships and precision profile analyses. A staphylococcal Protein A saturation analysis was applied to the standardization of IgG anti-parasite antibody reference sera in weight per volume units (microgram/ml). Using the mean minimal detectable dose + 3 SD and an intraassay precision profile less than 10% coefficient of variation (CV) as criteria for assay sensitivity, the SPRIA and ELISA displayed comparable positive thresholds of 1 microgram/ml IgG anti-parasite antibody/ml of serum. Both assays demonstrated good reproducibility (less than 15% interassay CV) and parallelism (less than 20% interdilutional CV) over their working ranges (SPRIA: 1-40 micrograms/ml; ELISA: 1-5 micrograms/ml). Specificity of each assay was enhanced by preadsorption of cross-reacting antibodies in canine serum (i.e., specific for Toxocara canis antigens) with solid-phase antigen prior to assay. Methods for comparing different immunoassay designs are considered in relation to the variables that influence the assays' performance characteristics.

Adsorption↗
PubMed1983

Brugia pahangi: antibodies and microfilaremias in Lewis rats.

Male and female Lewis rats were inoculated subcutaneously in the left groin with 75 infective larvae of Brugia pahangi and microfilaremias were followed for as long as 420 days postinoculation. Patent infections developed in 64% of the female rats and 95% of the male rats. Mean prepatent periods were similar (65.9 and 63.9 days, respectively), but mean microfilaremias in males rose much higher, to a mean of 218 mf/0.25 ml blood at 270 days postinoculation. IgG titers, as measured by enzyme-linked immunosorbent assay (ELISA), to adult worm somatic antigen were higher than those to microfilariae in almost all rats. For both sexes, the most consistently microfilaremia rats had highest titers to these antigens. Granulomas with degenerating microfilaria were present in the spleen of male rats with high microfilaremias (greater than 100-300 mf/0.25 ml blood). Ouchterlony precipitin reactions suggested that most rats with spleen granulomas responded to microfilarial antigen components to which most rats without granulomas did not. Neither spleen granulomas nor antibody responses measured in this study appeared to have protective (microfi8laremia-lowering) value. As measured by microfilaremias, the male Lewis rat is not as susceptible as some conventional hosts of B. pahangi, but it does consistently become infected and remains microfilaremic for more than a year. Preferential male susceptibility indicates that this model may be useful for studying this aspect of human lymphatic filariasis.

Animals↗
PubMed1984

Brugia malayi: stage-specific expression of carbohydrates containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae.

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.

Acetylglucosamine↗
PubMed1986

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 less than 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 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM 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 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Dirofilaria immitis: identification and partial characterization of parasite antigens in the serum of infected dogs.

We have recently developed a sensitive and specific immunodiagnostic test for canine Dirofilaria immitis infection based on detection of soluble parasite antigens in dog sera by monoclonal antibody-based enzyme immunoassay. In addition to their importance as markers of infection, these antigens may contribute to the pathogenesis of heartworm disease in dogs. In the present study, a variety of methods were used to identify and characterize circulating D. immitis antigens. Two antigens were identified in infected dog sera that formed lines of identity in rocket-line immunoelectrophoresis with soluble antigens extracted from adult D. immitis. Circulating D. immitis antigens were also demonstrated in infected dog sera by immunoblot analysis with polyclonal and monoclonal antibodies. These antigens had apparent molecular weights that ranged from 50 to 250 kDa. Most of the circulating D. immitis antigens contained the epitope defined by monoclonal antibody 1418BF2.1 which is used in our enzyme immunoassay for circulating D. immitis antigen. Studies of parasite antigens released during in vitro culture indicated that the circulating D. immitis antigens in dog sera that are detected by our enzyme immunoassay are primarily derived from adult female worms.

Animals↗
PubMed1987

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.

Animals↗
PubMed1988

Further characterization of refractoriness in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy).

Factors which control the expression of the refractory or susceptible condition to infection with Dirofilaria immitis in the mosquito. Aedes aegypti, were investigated using three protocols. (1) Microfilariae and prelarvae were injected into the hemocoel of susceptible A. aegypti. Some microfilariae and prelarvae developed to the L1 larval stage but they failed to complete development to the infective stage. (2) Enema of microfilariae and prelarvae from infected susceptible and refractory donor females were given into the midgut of uninfected susceptible and refractory recipient females. The results indicate that the conditions which inhibit the initiation of development are present in the Malpighian tubules and not in the midgut of the refractory mosquitoes. (3) Transplants of infected Malpighian tubules from susceptible and refractory donor females were made into the abdominal hemocoel of uninfected susceptible and refractory recipient females. The results showed that the refractory condition depends on the genetic makeup of the donor, not the recipient, mosquito. The above results taken as a whole indicate that the factors which control refractoriness are not present in the midgut but are present in the Malpighian tubule cells of refractory A. aegypti.

Aedes↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Animals↗