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.     

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.     

Opisthorchis viverrini: influence of maternal infection in hamsters on offspring infected with homologous parasite and their IgG antibody response

We investigated the influence in hamsters of a maternal Opisthorchis viverrini infection on their offspring infected with homologous parasites and the kinetics of the O. viverrini-specific IgG antibody responses. No significant difference (P > 0.05) was found in the specific IgG antibody response and the number of O. viverrini eggs per gram feces (EPG) between infected offspring from infected mothers and infected offspring from uninfected mothers. A significant difference (P < 0.05) of EPG per worm was found between infected offspring from infected mothers and infected offspring from uninfected mothers only when the offspring were infected with O. viverrini after weaning at 5 weeks of age. The worm loads in infected offspring from infected mothers were significantly less than that in infected offspring from uninfected mothers. This study demonstrated that maternal infection effects worm fecundity and the worm load in an infected offspring.     

Studies with Brugia pahangi. 16. Precipitation antibody in infected cats detected by counterimmunoelectrophoresis

In cats infected with normal, or irradiated, infective (L3) larvae of Brugia pahangi counterimmunoelectrophoresis revealed the presence of antibody to soluble antigens derived from microfilariae, adults and infective larvae of the same parasite. Infected cats with a persistently high to moderate microfilaraemia gave positive precipitin reactions to L3, microfilarial and adult worm antigens. Cats which had become amicrofilaraemic had antibody to L3 and microfilarial antigens but not to adult worm antigen. Serum from cats inoculated with irradiated L3 larvae produced a precipitin reaction only to the L3 antigen.     

Inhibition of antigen-driven proliferative responses and enhancement of antibody production during infection with Brugia pahangi

Long standing Brugia pahangi infections in seven dogs, restricted to one rear limb popliteal lymph node and its afferent ducts, were monitored with regard to proliferative responses and antibody production specific for a PBS extract of B. pahangi (BpA) by cells from infected and uninfected lymph nodes and by PBL. Five of 10 dogs were negative for proliferative responses to BpA in node cells from infected limbs, yet they had positive PBL responses, and another was negative in both node cells and PBL. Production of BpA-specific antibody was detected in cultures of node cells from infected limbs of 9 of 10 dogs, but only in two cultures of node cells from uninfected limbs and not at all in PBL cultures. Three dogs with responsive node cells produced the least amount of anti-BpA antibody in culture. Injections of B. pahangi adult worm excretory/secretory products (ES), totaling 1 mg over 48 h, into the limb of the original infections in seven dogs, resulted in inhibition of Ag-driven proliferation by cell populations previously responsive to BpA. There was a loss of PBL responsiveness by all but one infected dog and a loss of node cell response by the two dogs previously responsive in infected and uninfected nodes. This loss of responsiveness lasted at least 28 days in three dogs. There was no evidence of suppression of responses to mitogens either before or after ES injection. In contrast, BpA-specific antibody production was greatly increased in node cells from infected limbs injected with ES. Similar injections into the uninfected limbs of two infected dogs produced no change of proliferative responses or of antibody production in the uninfected node. These results indicate that ES can modulate immune cell, Ag-driven proliferation, and simultaneously enhance antibody production in previously infected nodes. This may promote parasite survival by inhibiting cellular attack based on delayed-type hypersensitivity while directing immune responses toward production of antibodies that are less damaging to the adult helminth.     

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).     

Circulating parasite antigen in Brugia pahangi-infected jirds

The Mongolian jird is used widely in filariasis research for studies of protective immunity, pathogenesis, and therapy. The purpose of this study was to evaluate parasite antigen detection as a means of noninvasively monitoring Brugia pahangi infection in jirds. A parasite antigen with Mr of 105-110 kDa was identified in sera from i.p.- and s.c.-infected jirds by immunoblot with a monoclonal antibody to phosphorylcholine. The same antibody was used in a direct sandwich enzyme immunoassay to measure antigen in jird sera. Parasite antigen was detectable as early as 2 wk after i.p. or s.c. injection of L3. Antigen titers increased between 2 and 12 wk and stabilized between 12 and 36 wk after infection in s.c.-infected animals. A different pattern was seen in i.p.-infected jirds with antigen titers peaking at 16 wk and falling significantly between 16 and 32 wk after infection. Parasite antigen titers correlated significantly with adult worm infection intensities in jirds with mature i.p. and s.c. infections. Antigenemia was also detectable in sera from jirds after i.p. implantation of adult parasites of either sex. However, antigen was not detected in sera from infant offspring of antigenemic infected mothers. We conclude that parasite antigen detection allows B. pahangi development and survival as well as infection intensity to be monitored in living animals with unprecedented sensitivity and accuracy. This technique should facilitate drug and vaccine studies in this important experimental filariasis model.     

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.     

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: 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)     

Parasite-induced suppression of the immune response in Aedes aegypti by Brugia pahangi

The melanization response against intrathoracically inoculated Brugia pahangi and Dirofilaria immitis microfilariae (mff) isolated from vertebrate host blood was evaluated in both uninfected Aedes aegypti black-eyed Liverpool strain and in mosquitoes harboring a developing B. pahangi infection. The immune response against inoculated mff of either species was significantly reduced by 28-47% in infected as compared with uninfected mosquitoes. Attempts to passively transfer this suppression factor(s) by inoculating naive mosquitoes with 0.1-0.2 microliter of hemolymph from B. pahangi-infected mosquitoes produced equivocal results. The role this parasite-induced immune suppression might play in aiding parasite survival in compatible vectors is discussed.     

Detection of antibodies in Brugia pahangi-infected cats by counter immunoelectrophoresis, indirect fluorescent antibody test and enzyme-linked immunosorbent assay

In cats infected with Brugia pahangi, antibodies first appeared against the larvae (L3), then against the adults (L5) and the microfilariae (mf). Homologous antigens were better than antigens prepared from heterologous species (Dirofilaria immitis, Dipetalonema viteae, Litomosoides carinii and Onchocerca gutturosa) in detecting antibodies to B. pahangi in the infected cats by indirect fluorescent antibody test (IFAT). Metabolic products of L5, but not L3 or mf, of B. pahangi were antigenic and were used in the enzyme-linked immunosorbent assay (ELISA) for detection of antibodies. Using various homologous antigens, IFAT was found to be more sensitive than counter immunoelectrophoresis and ELISA in the detection of antibodies in the infected cats. The best antigen was cryosections of L3, with a positivity rate of 81%. However, using L3, L5 and mf antigens in IFAT, a total positivity of 97% was obtained.     

Studies with Brugia pahangi 10. An attempt to demonstrate the sharing of antigenic determinants between the worm and its hosts.

Infective stage Brugia pahangi that were reared in Aedes aegypti survived equally well in cats that had previously been immunized against mosquito tissue and in a normal cat. The survival of third, fourth, juvenile, adult and microfilarial stages of B. pahangi that were recovered from cats was similar in jirds that had been immunized against cat antigens and in normal jirds. Host antigenic determinants were not detected on the surface of larvae in substantial amounts using fluorescent antibody techniques. It is unlikely that B. pahangi evades the immune response of its vertebrate hosts by masquerading as "self" behind host antigens.     

Zoonotic Brugia pahangi filariasis in a suburbia of Kuala Lumpur City, Malaysia

Five local Malaysian patients with clinical manifestations consistent with lymphatic filariasis were referred to our medical centre between 2003 and 2006. Although no microfilariae (mf) were detected in their nocturnal blood samples, all were diagnosed to have lymphatic filariasis on the basis of clinical findings and positive serology results. PCR on their blood samples revealed that two of the patients were infected with Brugia pahangi, an animal filarial worm hitherto not known to cause human disease in the natural environment. All the patients were successfully treated with anti-filarial drugs: four patients were treated with a combination of diethylcarbamazine (DEC) and albendazole, and one with doxycycline. Four of them were residents of Petaling Jaya, a residential suburbia located 10 km southwest of Kuala Lumpur city, Malaysia. The fifth patient was a frequent visitor of the suburbia. This suburbia has no history or record of B. malayi infection. The most likely vector of the worm was Armigeres subalbatus as extensive entomological surveys within the suburbia revealed only adult females of this mosquito species were infected with B. pahangi larvae. Wild monkeys caught in the suburbia were free from B. pahangi mf, but domestic cats were mf positive. This suggests that infected cats might be the source of the zoonotic infection in the suburbia.     

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.     

Brugia pahangi and Wolbachia: the kinetics of bacteria elimination,worm viability,and host responses following tetracycline treatment

Wolbachia spp., first reported from filariae nearly 30 years ago, have been suggested to contribute to the pathogenesis associated with human filarial infection. Tetracycline has been used to cure filariae of Wolbachia, as a novel means of chemotherapeutic treatment for both ocular and lymphatic filariasis. Tetracycline treatment of L4 or adult Brugia pahangi in vivo resulted in Wolbachia clearance. Less tetracycline was required to clear Wolbachia when treatment began at the L4 stage, compared with adults. Female worms died earlier than male worms when tetracycline was administered at the L4 stage. In all cases, Wolbachia clearance was closely associated with worm death. Worm recoveries decreased following the L4-L5 molt, suggesting tetracycline does not interrupt molting in this model system. Despite worm death and the assumed release of both bacterial- and worm-derived molecules, differences in inflammatory cell population and T cell cytokine mRNA profiles were negligible between tetracycline-treated and non-treated B. pahangi infected gerbils. These data suggest the contribution of Wolbachia to the in vivo induction of the gerbil immune response to B. pahangi may be small.     

Differentiation of species and life cycle stages of Brugia spp. by isoenzyme analysis

The isoenzyme patterns of glucose phosphate isomerase and phosphoglucomutase of 3 species of Brugia, B. pahangi, subperiodic B. malayi, and B. patei, and 3 life cycle stages, adult, third-stage larva, and microfilaria were compared using the technique of isoelectricfocusing on polyacrylamide gels. The results demonstrated that the adults of all 3 species could be identified from one another and that differences existed between the sexes of any one species. Hybridization between B. pahangi and B. patei could be detected in the progeny of the cross. Both the third-stage larvae and microfilariae of B. malayi and B. pahangi were differentiated and the epidemiological significance and the application of these findings to arthropod-borne filarial infections were discussed.     

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.     

Flight muscle ultrastructure of susceptible and refractory mosquitoes parasitized by larval Brugia pahangi.

On parasitization with larval Brugia pahangi the infected flight muscle fibres of "resistant" Anopheles labranchiae atroparvus undergo the following ultrastructural changes. The fibres become almost totally devoid of glycogen, their sarcoplasmic reticulum becomes elongate and closely associated with muscle fibrils. These fibrils degenerate and vesicles appear both within the degenerate fibril and within elements of the sarcoplasmic reticulum. Vesicles accumulate around the worm and degenerate to a uniform mass which eventually becomes melanized from its inner edge (next to the parasite) outwards. The infected flight muscle fibres of both "resistant" Aedes aegypti and "susceptible" Aedes togoi are almost totally devoid of glycogen granules, but show no other ultrastructural change from the uninfected state.     

Effect of immunostimulatory oligodeoxynucleotides on host responses and the establishment of Brugia pahangi in Mongolian gerbils (Meriones unguiculatus)

Infection of humans with filarial parasites has long been associated with the maintenance of a dominant Th2-type host immune response. This is reflected by increases in interleukin (IL)-4- and IL-5-producing T cells, elevated immunoglobulin (Ig)E and IgG4 levels, and a pronounced eosinophilia. The Mongolian gerbil (Meriones unguiculatus) is permissive for the filarial nematodes Brugia malayi and B. pahangi. As in humans, persistent microfilaremic infections of gerbils with Brugia spp. results in increases in Th2 cytokines such as IL-4 and IL-5. The association of dominant Th2 cytokine profiles with the maintenance of infection suggests that the introduction of Brugia spp. into a strongly Th1-biased environment may adversely affect parasite establishment. Indeed, studies conducted in mice with B. malayi suggest that depleting Th1 effectors such as interferon (IFN)-gamma and nitric oxide results in increased worm recoveries. In the present studies, the Mongolian gerbil was used as a model to investigate the effect of a dominant Th1 cytokine environment on the establishment of B. pahangi. Intraperitoneal (i.p.) administration of immunostimulatory oligodeoxynucleotide (IS ODN) induced the production of IFN-gamma in the peritoneal exudate cells and spleen of gerbils. The presence of IFN-gamma at the time of B. pahangi infection did result in an altered host immune response to B. pahangi. Gerbils that received IS ODN before i.p. B. pahangi infections showed lower levels of the Th2-type cytokines IL-4 and IL-5, compared with animals that received B. pahangi alone (0 + Bp). This alteration in cytokine profile, however, did not alter the establishment or development of B. pahangi in the peritoneal cavity. Furthermore, there was no difference in the granulomatous response of gerbils to soluble adult B. pahangi antigen bound to beads embolized in their lungs, regardless of treatment group, suggesting that IL-4 and IL-5 are not essential contributors to the systemic host inflammatory response to B. pahangi in this model.