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)     

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)     

Brugia pahangi: effects of maternal filariasis on the responses of their progeny to homologous challenge infection.

Granulomatous lesion formation and immune responses to Brugia pahangi infections were compared in age-matched male progeny of homologously infected and uninfected female jirds. Infections initiated in 2-week-old offspring yielded mean +/- SD adult worm recoveries of 6.0 +/- 5.7 and 4.2 +/- 5.4 in offspring from infected or uninfected mothers, respectively. Infections initiated in 4-week-old offspring resulted in an mean +/- SD recovery of adult worms of 11.3 +/- 11.3 and 10.2 +/- 5.8 in offspring from infected and uninfected mothers, respectively. The ratio of intralymphatic thrombi per intralymphatic worm was similar between infected offspring from infected or uninfected mothers within experiments. Areas of granulomas around B. pahangi antigen-coated beads embolized in the lungs were not significantly affected by maternal origin in infected or uninfected progeny. Offspring infected at 2 or 4 weeks of age from infected mothers exhibited significantly reduced titers of serum IgG antibodies to Brugia antigens at 5-8 weeks postinfection compared to infected offspring of uninfected mothers. Infected offspring from infected mothers also had significantly fewer splenic IgG plaque-forming cells to B. pahangi antigens at 5 weeks postinfection than similarly infected offspring from uninfected mothers. Western immunoblot analysis indicated qualitative and quantitative reductions in serum antibody reactivity to adult B. pahangi antigens in infected progeny of infected females compared to age-matched infected controls. Reduced homologous serum antibody responses in progeny exposed to maternal B. pahangi infection suggest that maternal immunoregulation to filarial antigens may occur. Reduced antibody responsiveness to B. pahangi antigens observed in infected offspring from infected mothers, however, had no demonstrable effect on adult worm burdens, microfilaremias, lymphatic lesion formation, or antigen-specific granulomatous inflammatory responses compared to infected progeny of uninfected mothers.     

Brugia pahangi: circulating antibodies to adult worm antigens in uninfected progeny of homologously infected female jirds

Serum IgG antibody levels to adult Brugia pahangi antigens were measured in uninfected offspring from uninfected and B. pahangi-infected female jirds. Antibody titers to B. pahangi antigens in sera of offspring from infected females mimicked the maternal titer during the suckling period. Neonate titers peaked at 2 weeks of age at levels as high as 1:4100, then decreased to levels well below maternal titers by 8-12 weeks of age. Concurrent maternal and 2-week-old neonate sera recognized identical B. pahangi antigens in Western blots. Spleen cells from 2-week-old filariae-exposed and unexposed offspring failed to produce measurable antibody to B. pahangi in vitro. Progeny of uninfected mothers nursed by B. pahangi-infected females showed circulating IgG antibody titers to adult worm antigens similar to those of homologously reared offspring. Conversely, offspring born to B. pahangi-infected females and nursed by an uninfected female had no serum antibodies to B. pahangi antigens. Blastogenic responses of spleen cells to the mitogens phytohemagglutinin and pokeweed mitogen, and adult B. pahangi antigens, were not different between offspring groups. Mean areas of pulmonary granulomas induced by the intravenous inoculation of B. pahangi antigen-coated beads also did not differ between 4- and 8-week-old progeny of uninfected or infected females. These results suggest that the circulating IgG antibodies to adult B. pahangi antigens demonstrated in offspring of infected female jirds are maternally derived via the milk and do not alter the cellular responses of uninfected offspring to B. pahangi antigens as measured by antigen-stimulated blastogenesis or pulmonary granulomatous inflammatory response.     

Brugia pahangi: comparative susceptibility of the Mongolian jird, Meriones unguiculatus, and the PD4 inbred hamster, Mesocricetus auratus

The susceptibility of Mongolian jirds, Meriones unguiculatus, and PD4 hamsters, Mesocricetus auratus, to Brugia pahangi was compared based on the percentage adult worm recoveries, mean microfilaremia levels, and adult worm lengths. Fourteen male jirds and seventeen male PD4 hamsters were each inoculated subcutaneously in the left inguinal region with 90-100 L3 of B. pahangi and necropsied 130-150 days after inoculation. There were no significant differences between jirds and hamsters in mean adult worm recoveries (24.7 vs 25.4%) and prepatent periods (69.9 vs 77 days after inoculation). In hamsters, 85% of recovered worms were found in the heart and lungs and 15% were found in genital lymphatic vessels. In jirds, distribution of recovered worms was 66% in genital lymphatics, 23% in the heart and lungs, 8% in the peritoneal cavity, and 3% in lymphatic vessels in other sites. The mean microfilaremia level in jirds (16.5/20 microliter) was significantly higher than in hamsters (8.7/20 microliter. Female worms in the genital lymphatics of jirds were significantly longer than female worms in the genital lymphatics of PD4 hamsters (33.5 vs 27.3 mm). Lengths of worms in other locations were similar between the two species.     

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.     

Pulmonary granuloma formation in murine toxocariasis: transfer of granulomatous hypersensitivity using bronchoalveolar lavage cells

A major drawback to studying granuloma formation in murine toxocariasis is the ability of the second-stage larva of Toxocara canis to escape from a developing granuloma, migrate elsewhere, and initiate granuloma formation anew. In an attempt to circumvent this difficulty, 2 different T. canis-derived antigenic preparations were covalently attached to Sepharose 4B beads and embolized into the microvasculature of the lungs of CBA/J mice that had been infected 10 days previously with 25 T. canis ova. Both T. canis egg extract (TEE) and T. canis exoantigens (TEX) were able to elicit antigen-specific granulomas 6 days postembolization as determined by both histologic and morphologic criteria. Histologically, the eosinophil-rich granulomas forming around antigen-coated beads embolized into infected mice resembled the developing granuloma previously described forming around the second-stage larva. Attempts to transfer granulomatous reactivity in this model using either immune spleen cells or immune serum were unsuccessful. Successful transfer of granulomatous hypersensitivity was achieved using cells obtained by bronchoalveolar lavage of previously infected mice. The results suggest the feasibility of using this embolic model of granuloma formation in murine toxocariasis.     

Role of interleukin 1 in experimental pulmonary granuloma in mice

Pulmonary granulomas were induced in immunized BALB/c mice by the intratracheal injection of antigen-coated and plain agarose beads. Prominent lesions developed within 24 hr, reached peak intensity within 3 days, and gradually declined in size thereafter. The hypersensitivity granulomas induced in sensitized mice by antigen-coated beads were much larger than the lesions induced by plain beads. Minimal inflammation was produced in unsensitized mice injected with antigen-coated or plain beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in sensitized mice by antigen-coated beads contained high levels of interleukin 1 (IL 1) and migration inhibition factor (MIF) activities. The kinetics of appearance of these mediators were similar. Lower but detectable activity of both mediators was detected in extracts prepared from sensitized mice injected with plain beads. Neither interleukin 2 (IL 2) nor IL 2 neutralizing activities were detected in the extracts. The presence of IL 1 and MIF in extracts prepared from early and peak pulmonary granulomatous lesions suggests that these soluble factors are produced by cells within the lesions, and that they are involved in mediating the expression and/or maintenance of the granulomas.     

Induction of lymphatic lesions by Brugia pahangi in jirds with large and small preexisting homologous intraperitoneal infections

The effect of intraperitoneal (i.p.) infections induced by inoculations of 30 or 150 Brugia pahangi third-stage larvae (L3) on the development of infections and lymphatic lesions induced by subsequent homologous subcutaneous (s.c.) inoculations were compared in the present study. Lymphatic lesion severity, as judged by the numbers of lymph thrombi present, and lymphatic lesion scores were significantly reduced in both groups of jirds with existing i.p. infections. The numbers of adult worms that developed, locations of these worms, and the subsequent microfilaremias did not differ significantly between groups. All jirds with i.p. infections developed similar antibody titers to crude somatic adult antigen as measured by ELISA. These levels did not change following s.c. infections. Immediate and delayed footpad swelling responses were also similar in all groups. Results of these experiments support and extend previous studies indicating that i.p. infections of B. pahangi induce a hyporesponsive state in jirds to subsequent s.c. infections without significantly affecting the subsequent parasite burden. This effect appears to be independent of the numbers of L3 inoculated i.p. prior to lymphatic-induced infection. Circulating antibody titers and footpad swelling responses to B. pahangi antigen were not reduced in jirds with the hyporesponsive lymphatic inflammatory response and do not correlate with this condition.     

Brugia pahangi: infections and their effect on the lymphatic system of Mongolian jirds (Meriones unguiculatus)

Brugia pahangi has been found to be primarily a lymphatic-dwelling parasite in jirds when infections are induced by the subcutaneous injection of infective larvae or by allowing infected Aedes aegypti to feed.Migration to the regional lymphatics occurred as early as 1–4 days. Although some injected larvae remained in the skin for as long as 30 days and some became localized in the heart, lungs, pleural cavity, or peritoneal cavity, about three-fourths of the recovered filariae were found in the regional lymphatics. In contrast, when larvae were injected peritoneally they remained largely in the peritoneal cavity for at least 30 days.The relevant lymphatics and their drainage patterns in jirds have been described.The major pathological changes noted in jirds involved the regional lymphatic vessels and nodes, which were severely affected when they contained dead worms. Pulmonary granulomas due to dead microfilariae and occasionally to dead larvae or adult worms were noted.Observations are included on the susceptibility and course of B. pahangi infections in jirds.     

Profiling the cellular immune response to multiple Brugia pahangi infections in a susceptible host

Human lymphatic filariasis is caused primarily by Brugia malayi and Wuchereria bancroffi. Unraveling this disease is complex, as people living in endemic areas exhibit a vast array of clinical states and immune responses. The Mongolian gerbil (Meriones unguiculatus)-B. pahangi model of human lymphatic filariasis has provided much information on immune parameters associated with filarial infection. Prior investigations in our laboratory have shown that gerbils closely mimic a subset of patients classified as microfilaremic but asymptomatic, a group that comprises the majority of people living in endemic areas. Worm recovery data suggest that gerbils carrying current B. pahangi infections do not show any resistance to subsequent subcutaneous B. pahangi infections. The aim of the present studies was to investigate the T cell cytokine response in gerbils receiving multiple infections of B. pahangi as a means of mimicking the conditions experienced by people in endemic areas. The T cell cytokine profile generated by multiply infected gerbils was not different from that previously generated by gerbils infected only once with B. pahangi. Gerbils infected multiple times with B. pahangi showed a transient increase in IL-5, which corresponded to the increased eosinophil levels previously reported from multiply infected gerbils. Chronically infected gerbils showed elevated IL-4 mRNA levels, as has been reported from gerbils infected only once with B. pahangi. Chronic infections were also associated with a state of immune hyporesponsiveness, as determined by the characterization of lymphatic thrombi and lymphoproliferation of spleen and renal lymph node cells to worm antigen.     

Differences in adult egg output of Schistocephalus solidus from singly- and multiply-infected sticklebacks

In this study, we recovered Schistocephalus solidus plerocercoids from singly and multiply infected three-spine sticklebacks, Gasterosteus aculeatus, transferred them individually to in vitro culture conditions, and quantified their lifetime egg output. We found a significant difference in the relationships between plerocercoid mass and lifetime egg output for parasites recovered from singly and multiply infected sticklebacks. Although egg output was strongly and positively related to plerocercoid mass amongst worms from singly infected fish, for those recovered from multiply infected sticklebacks the relationship was marginally nonsignificant and negative, with small worms achieving high levels of egg production. We suggest 2 hypotheses that may explain differences in the egg production of plerocercoids from singly and multiply infected fish. One possibility is that smaller plerocercoids in asymmetric multiple infections develop precocially, in response to host manipulation strategies of larger worms that decrease survival prospects of the host. Alternatively, small worms in singly infected sticklebacks may be prevented from becoming sexually mature because they face energetic constraints associated with having to overcome the host's immune response alone. We discuss our results in terms of recent studies examining strategic egg production in helminths.     

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.     

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.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.     

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.     

Schistosoma japonicum: A comparison of the development of the parasite and associated pathological changes in mice and jirds (Meriones unguiculatus)

In this paper an attempt is made to compare the development of Schistosoma japonicum in Meriones unguiculatus and in mice, as well as the associated pathological changes. Total worm recovery was lower and worm development more rapid in jirds than in mice but there was no obvious difference in the copulation rate of worms in the two hosts. Differences observed in the pathological lesions indicate that those which develop in jirds bear a stronger resemblance to those seen in man than do the corresponding changes in mice, and that the immune reactions elicited against egg antigens is stronger in jirds. It is concluded that M. unguiculatus may serve as a useful experimental model for studies on the immunopathology of Schistosomiasis japonicum.     

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.     

Production and characterization of monoclonal antibodies against excretory/secretory products of adult Echinococcus granulosus, and their application to coproantigen detection

Two IgM murine monoclonal antibodies (MAbs), EgC1 and EgC3, were produced against the excretory/secretory (E/S) products of Echinococcus granulosus adult worms. Immunoblotting revealed that both predominantly recognized a 50 kDa antigen in the somatic extract and an 85 kDa component in the E/S products. Immunolocalization showed that both MAbs reacted with the tegument of the parasite, and additionally EgC3 reacted with parenchyma and the tegument lining the external surface of the reproductive organs. A coproantigen capture ELISA was developed using a rabbit polyclonal antibody against E/S products from adult tapeworms as catching antibodies, and each one of MAbs as detecting antibody. The assays detected seven out of eight (EgC1), and eight out of eight (EgC3) experimentally infected dogs (worm burdens ranging from 61 to 57,500), using heat-treated samples obtained at prepatent period, and none (n=8) of helminth-free samples. Time course analysis showed that, after a 12-25 days lag, coproantigen levels rose above cut off O.D. values and typically peaked around 30 days post-infection (DPI) at the end of the experiment. One dog experimentally infected with Taenia hydatigena metacestodes was slightly detected as positive at different time points after 30 DPI. Both MAbs showed a similar pattern of recognition, but T. hydatigena antigens were undetectable for a longer period, and reached lower O.D. values with EgC1. Interestingly, fecal samples from two experimentally infected dogs with Echinococcus multilocularis were not recognized by the EgC1 assay, suggesting a potential value as species-specific diagnostic tool.