Regulation of parasite antigen-induced T cell growth factor activity and proliferative responsiveness in Brugia pahangi-infected jirds

Previous studies have demonstrated that the induction of immunoregulatory mechanisms in the spleens of Brugia pahangi-infected jirds is correlated with the onset of microfilaremia. This study investigated the relationship between production of a factor with IL-2-like activity and the regulation of T cell-mediated responses in jirds experimentally infected with B. pahangi. A factor present in culture supernatants of mitogen-stimulated jird lymphocytes supported the proliferation of murine CTLL cells and provided the basis for an IL-2 assay. Mitogen induced proliferative responses and IL-2 production of spleen cells but not lymph node cells from pre-patent and microfilaremic jirds were suppressed. Both B. pahangi Ag-induced proliferative responsiveness and IL-2 production of spleen cells from microfilaremic jirds were also suppressed relative to lymph node cells from the same animals or spleen cells from B. pahangi immunized or prepatent jirds. Depletion of histamine receptor-bearing cells restored the ability of spleen cells from microfilaremic jirds to produce significant levels of IL-2. In addition, in add-mixture experiments, spleen cells from microfilaremic jirds suppressed Ag-induced IL-2 production by cells from either B. pahangi- or KHL-immunized jirds. Exogenous IL-2 failed to reconstitute the suppressed Ag-induced proliferative response of spleen cells from microfilaremic jirds. This study demonstrates that the down-regulation of immune responses in B. pahangi infection is a cell-mediated event and is associated with an inability to produce IL-2.     

Brugia malayi: detection of parasite antigen in sera from infected jirds

Sera from Brugia malayi-infected jirds were demonstrated to contain a heat-stable, 95- to 105-kDa parasite antigen by immunoblot with rabbit antibody to the parasite and with a monoclonal antibody that binds to phosphorylcholine. This antigen is a major component of B. malayi adult worm excretory/secretory antigen, and it is present in lavage fluid obtained from ip-infected animals. The antigen was detected by enzyme immunoassay in all sera collected from jirds 9-54 weeks after sc injection with 100 or 300 infective larvae (L3). Parasite antigen titers were higher in animals infected with the higher L3 dose. Antiphosphorylcholine antibodies were present in jird sera for the first 12 weeks after larval injection, but thereafter, antibody titers decreased to undetectable levels. Parasite antigen was not detected by immunoblot or enzyme immunoassay in sera from 21 human subjects with B. malayi microfilaremia. Antigen may be cleared from human sera by antiphosphorylcholine antibodies, which were present in all sera tested. The practical significance of B. malayi antigen detection in the jird is that it provides a sensitive means of noninvasively monitoring the status of infection in this important experimental filariasis model.     

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.     

Effect of CGP 20376 on Brugia malayi and parasite antigenemia in jirds

This study was designed to investigate the activity of CGP 20376, a benzothiazole derivative, against Brugia malayi in jirds and to illustrate the utility of parasite antigen detection as a means of monitoring drug efficacy in filariasis. Drug treatment was 100% effective in jirds treated 3 or 24 days after infection. Microfilaria and adult worm counts were reduced (relative to counts in sham-treated control animals) by 96% and 95%, respectively, in animals treated 153 days after infection. Four of 6 animals in this treatment group cleared their microfilaremias and were free of adult worms 5 mo after treatment. Thus, CGP 20376 was effective against all life cycle stages of B. malayi in jirds. Parasite antigen levels in jird sera were consistent with parasitological results in all treatment groups, but antigen clearance was incomplete in some cases after apparently successful treatment of mature and immature infections.     

Adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi in jirds

A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adult Setaria cervi females using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity against Brugia malayi (a human filarial parasite) in jirds (Meriones unguiculatus) in vitro and in situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells to B. malayi microfilariae (mf) and infective larvae (L3) in vitro and induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi 175 kDa antigen serum was more effective in inducing cytotoxicity to B. malayi L3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.     

Immunoregulation in experimental filariasis. II. Responses to parasite and nonparasite antigens in jirds with Brugia pahangi

Chronic B. pahangi infection (greater than or equal to 5 mo) in the jird, Meriones unguiculatus, leads to the induction of adherent nonspecific suppressor cells that are capable of modulating the in vitro mitogen responsiveness of spleen cells. In the present studies, a correlation between suppression of mitogen responsiveness and lack of reactivity to B. pahangi antigens was observed in vitro with splenic lymphocytes from chronically infected animals. However, the ability of jirds with a chronic B. pahangi infection to develop in vivo humoral responsiveness to SRBC and DTH to DNFB was comparable to that of uninfected controls. Analysis of the relationship between the development of antigen-specific and nonspecific immunoregulatory activity over the course of the infection was undertaken, too. Altered in vitro responsiveness of spleen cells from infected jirds to mitogens and B. pahangi antigens was associated with the onset of microfilaremia (8 wk post-infection). A transient lack of reactivity to SRBC was observed after the development of a patent infection in jirds. However, nonspecific suppressor cells capable of modifying the in vitro mitogen responsiveness of normal lymphocytes were not observed in the spleens of B. pahangi-infected animals exhibiting a lack of reactivity to SRBC. The relationship of antigen-specific suppressor cells to immunoregulation in experimental filariasis is discussed.     

A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds

A 43 kiloDaltan (kDa) antigen fraction (CFA₂-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory–secretory products. Further analysis was done on the immunoprophylactic potential of CFA₂-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA₂-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA₂-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA₂-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA₂-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.     

Non-specific immune suppression by CD8+ T cells in Brugia pahangi-infected rats

Non-specific suppression of the immune response was investigated in Brugia pahangi-infected Lewis rats. The proliferative response of peripheral blood lymphocytes or splenic non-adherent cells to mitogens was significantly reduced by B. pahangi infection. The degree of hyporesponsiveness of splenic non-adherent cells to mitogens was comparable between microfilaremic and non-microfilaremic animals. The suppressed proliferative response of splenic non-adherent cells was restored by blocking with anti-CD8 monoclonal antibody. After separation of T cells into CD4+ and CD8+ subpopulations, only CD8+ T cells from B. pahangi-infected rats suppressed the proliferative response of normal spleen cells to concanavalin A. CD8+ T cells from normal rats had no suppressive effect. On the other hand, the proliferative response of CD4+ T cells to concanavalin A was comparable between normal and infected rats. These results suggest that CD8+ T cells participate in the non-specific suppression of immune response in experimental filariasis.     

Degenerative changes in lymphatic endothelium of jirds infected with Brugia pahangi

The quantitative changes of cytoplasmic vesicles and vacuoles in lymphatic endothelial cells of the mongolian jirds associated with Brugia pahangi infections were observed by transmission electron microscopy. The present study revealed a decrease in the proportion of cytoplasm occupied by vesicles and in the number of cytoplasmic vesicles in endothelial cells from lymphatic vessels harboring B. pahangi at 3, 4, and 10 mo after infection (3.55, 3.36, and 2.55 vesicles/micron 2, respectively) when compared with cells from uninfected control vessels (7.03 vesicles/micron 2). On the contrary, there was an increase in the area of vacuoles in endothelial cells of jirds at 3, 4, and 10 mo postinfection. The mean +/- SD diameter of vesicles in cells from lymphatic vessels at 10 mo after infection was significantly smaller (78.6 +/- 5.6 nm) compared to vesicles in uninfected vessels (87.5 +/- 9.7 nm).     

Delayed macrofilaricidal activity of diethylcarbamazine against Brugia pahangi in Mongolian jirds

The macrofilaricidal activity of diethylcarbamazine (DEC) was confirmed in jirds infected with Brugia pahangi. Seventy jirds were inoculated subcutaneously with 100 infective larvae. At 20 weeks post-infection, the microfilaraemic jirds were divided into two groups, untreated and treated. For the treated group, 200 mg kg(-1) of DEC was injected intraperitoneally for 5 consecutive days. One, 4, 8, 12, 16 and 27 weeks after the final treatment, 4-7 jirds in each group were sacrificed to measure adult worm burdens. The number of adult worms recovered from treated jirds was comparable to controls at earlier necropsy (1 and 4 weeks post-treatment). However, at late necropsy (8 weeks and later) the recovery rate of adult worms in treated jirds was significantly lower than that in untreated controls, indicating an adultcidal effect of DEC. The present study demonstrates that DEC requires 8 weeks to kill B. pahangi adult worms in jirds and that the Mongolian jird is a useful model for screening antifilarial activity.     

Altered adult worm location in young male jirds infected with Brugia pahangi

Male jirds (Meriones unguiculatus) were inoculated subcutaneously with 100 Brugia pahangi L3 each at 2, 6, 10, and 15 wk of age to compare their susceptibility and pathologic reactivity to infection. Adult worm recoveries (mean +/- SD) ranged from 24.1 +/- 15.1 to 36.4 +/- 13.9 at 60 days postinfection. No significant difference in susceptibility was measured among the 4 age groups. Jirds infected at 2 wk of age had significantly fewer (alpha less than or equal to 0.025) testicular and intralymphatic worms than all other age groups. Numbers of intralymphatic thrombi were significantly lower (alpha less than or equal to 0.01) in jirds infected at 2 wk of age. Lymphatic lesion severity, expressed as the number of intralymphatic thrombi per intralymphatic worm, was similar between age groups. These data indicate no differences in susceptibility or lymphatic lesion formation following B. pahangi infection in 2-wk-old male jirds, despite altered adult worm location.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Comparative susceptibility to anthelmintics of Brugia pahangi in jirds infected by different methods

It is possible to infect jirds with Brugia pahangi by three methods. Infective larvae (L3) can be injected either intraperitoneally (ip), when adults develop in the peritoneal cavity, or sub-cutaneously (sc), when they develop in the lymphatics or the heart and blood vessels associated with the lungs. Alternatively adult worms which have been grown in the peritoneal cavities of jirds can be implanted into the peritoneal cavities of other jirds. This latter system has been widely used for screening for new filaricides. We have compared the activity of 9 macrofilaricidal compounds against these 3 types of infection. Mebendazole and albendazole were more active against implanted adults than against L3 induced adults in the peritoneal cavity. Oxibendazole, flubendazole, CGP24588A and oxfendazole were equally active against both types of worm. CGP20376, Mel Ga and Mel Ni were more active against adult worms derived from inoculated L3 than implanted worms. When comparing intra-lymphatic and ip adults (both derived from L3 infections and in the same jirds) albendazole and CGP20376 were active at the same levels against both types of infection. Mebendazole, flubendazole, oxfendazole, CGP24588A, Mel Ga and Mel Ni were more active against ip adults than intra-lymphatic adults. No drug was more active against intra-lymphatic adults than against adults.     

Induction of protective immunity to Brugia pahangi in jirds by drug-abbreviated infection.

Protective immunity of homologous challenge infection was examined in jirds after drug-abbreviated infection with Brugia pahangi. Mebendazole (MBZ) treatment at the early prepatent (5-7 weeks of post infection) or the late prepatent (7-9 weeks of post infection) period was highly effective in causing almost complete eradication of the primary infection. After challenge infection, the worm burden was significantly reduced 19% (31.1 in average) and 77% (9.5) to that of the controls (38.8 and 41.7), respectively. The magnitude of eosinophil response paralleled the degree of protection. No or only a few microfilariae were seen after challenge infection in jirds treated during the prepatent periods. They were also resistant to intravenous challenge with the microfilariae of B. pahangi. MBZ treatment at the patent period was, on the contrary, incomplete against primarily infected adult worms, and was not able to induce either significant protection (30.1 vs 33.1 in control) or eosinophil response to the challenge infection.     

Brugia pahangi: granulomatous lesion development in jirds following single and multiple infections

The development of adult worm burdens and microfilaremias were determined in jirds which received 2, 3, or 4 subcutaneous inoculations of 50 Brugia pahangi infective larvae. Parasite burdens in multiply inoculated jirds were compared to those in four different groups of jirds which received single inoculations of 50 infective larvae. One of each of these singly inoculated groups was infected on the same day that one of the inoculations was given to the multiply infected jirds. Thus, the duration of the infections in the four groups of jirds receiving one inoculation was 54, 118, 189, and 254 days. The development of lymphatic lesions and granulomatous hypersensitivity to B. pahangi antigen was assessed in all jirds at necropsy. The percentage recoveries of adult worms and their locations did not differ in the singly inoculated jirds with infections of different durations. A protective resistance to reinfection, as measured by adult worm recovery in multiply infected jirds, did not occur. The lymphatic lesion scores and numbers of intralymphatic thrombi was greatest in singly inoculated jirds examined 54 days after infection. Pulmonary granuloma areas around adult filarial antigen coated beads embolized in the lungs of jirds 3 days prior to necropsy were also greatest in singly inoculated jirds examined 54 days after infection. Using criteria of lesion scores and lymph thrombi numbers to assess lymphatic lesion severity, a decrease in lesion severity as well as pulmonary granuloma size around antigen coupled beads was seen by 118 days after infection in singly inoculated jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.     

Qualitative characterization of antibody responses to single and multiple Brugia pahangi infections in jirds

Antibody responses of jirds, singly and multiply inoculated with Brugia pahangi infective larvae (L3), to soluble somatic extracts of adult parasites were characterized by western blot analysis. Forty-two protein bands ranging in molecular weight from 12 to 160 kDa were recognized by sera from infected jirds. Antibody recognition of individual B. pahangi antigen bands in this assay appears to be independent of antibody enzyme-linked immunosorbent assay (ELISA) titers to crude parasite extract, severity of lymphatic lesions, levels of microfilaremia, numbers of L3 inoculated, or numbers of adult parasites in individual jirds. Antibody recognition of protein bands with molecular weights of 37 kDa, 21 kDa, and 17 kDa, however, did temporally correspond with certain parasitological and pathologic events. Antibody against the 37-kDa protein band first was identified at the onset of patency, reaching a 90% prevalence rate by 90 days postinfection (DPI). The prevalence of this antibody remained high. Antibody recognition of the 21-kDa protein band first occurred at 90 DPI and gradually increased in prevalence during the course of infection temporally similar to the increase in microfilaremia. Recognition of the 17-kDa protein band first occurred at 48 DPI, reached a maximum prevalence of 80% at 90 DPI, and decreased to a minimal prevalence by 160 DPI. Prevalence of antibody responses to the 17-kDa protein band corresponded temporally with the kinetics of the rise and fall of numbers of intralymphatic thrombi. The patterns of antibody response to these 3 bands were similar in both singly and multiply inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds

Sullivan J. J. and Chernin E. 1976. Oral transmission of Brugia pahangi and Dipetalonema viteae to adult and neonatal jirds. International Journal for Parasitology6: 75–78. Confirming previous studies, anaesthetized adult jirds became infected after oral doses of 100 infective larvae of B. pahangi; 4 of 5 jirds became microfilaria-positive and all 5 harbored adult worms. Among 7 unanaesthetized adult jirds similarly exposed, none developed microfilaraemia although 5 each harbored a few adult worms. In these unanaesthetized jirds, presumably, rapid passage of the inoculum through the mouth permitted fewer larvae to penetrate the mucosa, and the rest were probably killed in the stomach. Unanaesthetized 4-day old jirds proved highly and equally susceptible to oral or subcutaneous infection with B. pahangi as indicated by microfilaraemias and large worm-burdens. Direct and indirect evidence suggest that the baby's stomach and small intestine are not inimical to swallowed larvae, thus accounting for the relatively numerous mature worms in the peritoneal cavity. Third-stage larvae of D. viteae, readily infective subcutaneously, succeeded relatively infrequently in maturing when given orally to anaesthetized adult or to unanaesthetized baby jirds. Consistent oral infectivity may thus be a feature of filariae more closely related to B. pahangi.