Jon A. Yates

Differential recognition of two cloned Brugia malayi antigens by antibody class

The humoral and cellular immune response to filarial parasites is complex. Numerous studies have shown that antibodies to a large number of protein and non-protein antigens may be produced over the course of infection and that immune recognition of any given antigen may vary by disease manifestation and by immunoglobulin class. We have used the techniques of molecular cloning to attempt to dissect this complex interaction, and describe here two clones, isolated from an expression library constructed from Brugia malayi genomic DNA, whose products are recognized by distinct immunoglobulin classes. A lambda gt11 fusion protein containing part of the B. malayi myosin tail region is recognized by antibodies of the IgG class from a high percentage of bancroftian filariasis patients. A fusion protein containing a collagen-like sequence is less frequently and weakly recognized under the same experimental conditions, but is almost universally recognized when the developing reagent is specific for IgE. We thus identify specific filarial proteins against which the infected human host responds preferentially with antibodies of a specific immunoglobulin class.

Influence of gonadal steroids on susceptibility to Brugia malayi in scid mice.

In the present study, we demonstrate that male scid/scid mice have higher adult worm burdens than do female scid/scid mice following equal challenge doses of infective Brugia malayi L3 larvae. Gonadectomy of four week old immature mice has no effect on worm burden in either sex, suggesting that the gender dichotomy between males and females does not depend on continued presence of endogenous gonadal steroids. The worm yield from female, but not male, mice can be increased by prepubertal oophorectomy combined with administration of either estradiol or testosterone in depot form. Our results raise the possibility that prepubertal steroid pulse(s) result(s) in physiological changes in male scid/scid mice that enhance worm growth. These studies confirm earlier reports of epidemiological data in humans suggesting a sexual dimorphism in susceptibility to filarial infection. Our data suggest that this gender difference is not due simply to the presence of adult gonadal steroids, but rather to ontogenic differentiative actions of sex steroids in the host.

Infectivity and normal development of third stage Brugia malayi maintained in vitro.

Shipment of infective-stage filarial larvae (L3s) usually has been accomplished by transporting living infected vectors or L3s cryopreserved in liquid nitrogen. Our objective was to find culture conditions for transporting L3s that would promote survival of Brugia malayi larvae without altering their capacity to infect susceptible animals. In preliminary studies we observed that Hams nutrient mixture F-12, with antibiotics and 1% fetal calf serum, could support L3s without apparent development for at least 10 days. In order to evaluate the effect of culture temperatures on infectivity, fresh L3s were divided into groups that were either immediately injected into jirds (infectivity control) or incubated for 24, 48, or 120 hr in tightly sealed tubes maintained horizontally at either 0 C, 20 C, or 37 C, before they were injected into jirds. Necropsies were performed on the jirds 120-130 days after injection to recover and count adult worms. Levels of microfilaremia were also determined. We found that L3s held overnight at 0 C, although apparently viable, were unable to survive in jirds. However, larvae kept at 20 C and 37 C produced patent infections with adult worms in normal locations even after 120 hr of in vitro cultivation. There was no statistical difference in mean worm recovery or size of worms from jirds infected with freshly harvested L3s and jirds injected with larvae that were maintained overnight at 20 C or 37 C. When cultured L3s were shipped from Michigan to Connecticut by overnight air courier, along with infected living mosquitos, the L3s appeared to be 99% viable upon arrival. L3s shipped in F-12 produced patent infections in C.B.-17 scid/scid mice with worm recoveries comparable to those observed in mice injected with L3s freshly obtained from shipped mosquitos.

Localization of Paramyosin, Myosin, and a Heat Shock Protein 70 in Larval and Adult Brugia malayi

Recombinant filarial proteins are of interest as potentially protective immunogens for lymphatic filariasis. We have previously identified paramyosin, myosin, and a heat shock protein 70 (HSP) 70 as prominent immunogens in individuals residing in an area endemic for lymphatic filariasis. Our goal in the present work was to identify the Brugia malayi tissues that contain these proteins. Polyclonal rabbit antisera with high levels of immunoglobulins to each of these proteins were prepared for use in indirect immunofluorescence microscopy studies of third-and fourth-stage larvae (L3s and L4s) and adult worms. Myosin and paramyosin were found within the longitudinal somatic musculature in all of these life stages. In L4s and adult worms, myosin and paramyosin were also detected within the walls of the reproductive and alimentary tracts of male and female worms. HSP 70 was evident within the somatic musculature, hypodermis, lateral chords, alimentary tract, and reproductive structures in L4s and adult worms. HSP 70 was not detected in sections of freshly obtained L3s. However, L3s cultured at 37 C for 24 hr before fixation demonstrated a classic heat shock response. In these larvae, intracellular HSP 70 was observed in all tissues. None of the antigens studied appeared to be located on cuticular surfaces.

Dipetalonema (Alafilaria) hydrochoerus subgen. et sp. n. (Nematoda: Filarioidea) from Colombian Capybaras

Dipetalonema (Alafilaria) hydrochoerus subgen. et sp. n. is described from specimens recovered from skeletal muscle fascia of the capybara, Hydrochoerus hydrochacris, from several localities in Colombia, South America. The microfilaria, which is found in the skin of the host, is also described for the first time. The monotypic species of the subgenus Alafilaria can be distinguished from existing Dipetalonema subgenera and all filariae known to us, on the basis of numerous preanal caudal papillae in males, small size of petals on the caudal extremity of each sex, and low, bluntly-rounded lateral alae in the cuticle of adult worms of both sexes. Unusual and distinctive features of the microfilaria include conspicuous lateral cuticular alae and a caudal extremity devoid of nuclei.

Use of parasite antigen detection to monitor macrofilaricidal therapy in Brugia malayi-infected jirds.

Improved methods are needed to evaluate new treatments for filarial infections. We have recently developed a monoclonal antibody-based enzyme immunoassay to detect circulating parasite antigen in sera from Brugia malayi-infected jirds. In the present study, parasite antigen levels were compared to parasitological parameters after treatment of B. malayi-infected jirds with CGP 20376 that has been reported to be active against both microfilariae and adult worms of this parasite. Microfilariae were cleared promptly and permanently after CGP 20376 treatment, and no adult worm was recovered in jirds at necropsy 20 wk after treatment. In contrast, untreated animals had sustained microfilaremia throughout the course of the study, and adult worms were recovered in all control animals (mean worm recovery; 24.3 +/- 7.8 SE). Parasite antigen was present in sera from all infected animals before treatment. Parasite antigen titers in sera were unchanged 5 wk after treatment but fell to undetectable levels in 4 of 6 animals by 20 wk after treatment. Low-level antigenemia was detected in 2 of 6 animals at 20 wk, perhaps suggesting incomplete killing of parasites or incomplete clearance of antigen. Parasite antigen levels were stable throughout the study in control animals. These preliminary results suggest that parasite antigen detection is useful as a means of noninvasively monitoring the efficacy of anti-filarial drug therapy.

DEVELOPMENT OF TETRAPETALONEMA LLEWELLYNI TO THE INFECTIVE STAGE IN CULICOIDES HOLLENSIS

The vector requirements and course of larval development for tetrapetalonema llewellyni Price, 1962, a common filaria of raccoons in Louisiana, are described for the first time. Development (from microfilaria to infective stage) took place in the thoracic muscles of the biting midge, Culicoides hollensis (Melander and Brues, 1902) Foote and Pratt, 1954, and required 9 days under laboratory conditions. The findings of this study suggest that species of Culicoides may serve as natural vectors of T. llewellyni.