Analysis of Wuchereria bancrofti infections in a village community in northern Nigeria: increased prevalence in individuals infected with Onchocerca volvulus

Infections with Wuchereria bancrofti causing lymphatic filariasis still represent one of the major health problems in the tropics, with 120 million people infected and over 750 million exposed to this filarial parasite. We have studied lymphatic filariasis infections as part of a multi-parasite survey in a village community in the savannah of northern Nigeria. We analysed serum samples from 341 individuals aged 5-70 years, detecting a W. bancrofti circulating antigen using the commercially available ICT Filariasis card test. The prevalence of infections was 10% and clearly age-dependent, increasing from below 2% in children to over 20% in subjects older than 40 years. Measuring IgG4 antibodies against the recombinant W. bancrofti antigen SXP1 showed that 36% of all tested individuals had been at least exposed to the parasite. Antibody levels also increased very significantly with age. A further analysis measuring Onchocerca volvulus-specific IgG4 antibodies showed a very significant association between infections with O. volvulus and those with W. bancrofti. Our data show that infections with W. bancrofti in Nigeria are still a frequently occurring health problem, since they are more prevalent than previously reported, and that individuals with an O. volvulus infection are more often infected with W. bancrofti than expected statistically.     

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.     

Transmission intensity determines lymphocyte responsiveness and cytokine bias in human lymphatic filariasis

Humans living in areas where filariasis is endemic vary greatly in their exposure to mosquito-borne infective third-stage larvae (L3) of these parasitic helminths. Because the intensity of exposure to Ags affects T cell differentiation and susceptibility to parasitic infections in murine models, we compared T cell and cytokine responses in 97 residents of two villages in Papua New Guinea, where transmission intensity of Wuchereria bancrofti differed by 63-fold (37 vs 2355 L3 per person per year). Residents of the high transmission village had 4- to 11-fold lower proliferation and IFN-gamma responses to filarial Ags, nonparasite Ag, and PHA by PBMC compared with the low transmission village (p < 0.01) even when subjects were matched for intensity of infection. In contrast, filarial Ag-driven IL-5 production was 5.5-fold greater (p < 0.001), and plasma IL-4 and TGF-beta levels were 4-fold and 34% higher, respectively, in residents of the high transmission village. IL-4 and IL-10 responses by PBMC differed little according to village, and increased production of the counterregulatory cytokines IL-10 or TGF-beta by PBMC did not correlate with weak proliferation and IFN-gamma responses. Plasma IL-5, IFN-gamma, and IL-10 levels were similar in the two villages. These data demonstrate that the intensity of exposure to L3 affects lymphocyte responsiveness and cytokine bias possibly by a mechanism that alters APC function.     

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freund's incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.     

Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses

BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

Antibody responses of Wuchereria bancrofti patients to recombinant Brugia pahangi superoxide dismutase

Lymphatic filarial parasite Brugia malayi contains significant amount of Cu/Zn superoxide dismutase (SOD) activity in the extract of different life stages and in the excretory-secretory product of adults. In the present study recombinant SOD from B. pahangi has been used to see the antibody response in Wuchereria bancrofti infected patients. The recombinant SOD from B. pahangi reacted specifically with W. bancrofti infected sera in ELISA and immunoblotting. The reactivity of IgM subclass was more as compared to IgG subclass both in the asymptomatic microfilaraemic and symptomatic amicrofilaraemic when tested by ELISA. Serum from other helminthic infection was very low and found to be insignificant. The antibody response to rec SOD was directly proportional to the number of microfilariae in infected patients. The circulating filarial SOD was detected in filarial patients using polyclonal antibodies raised against recombinant Cu/Zn SOD in rabbits. The apparent molecular masses as determined by immunoblotting were 29 and 22 kDa. The specificity of recombinant SOD could be explored for its use in immunodiagnosis of lymphatic filariasis.     

Immunological aspects of murine infection with the rat nematode Strongyloides ratti Sandground, 1925

In a study of the immune response of the rat to infection with the nematode Strongyloidis ratti, the antigens of the infective larval stage (L3) and of the parasitic, parthenogenetic female (Fp) were investigated. From both the larvae and the adult females, one metabolic (exoantigen) and two somatic antigens were extracted. Of the two somatic antigens, one was soluble and obtainable by physical means while the other was separated by chemical means from the tegument of the parasite. Humoral responses to the various antigens were evaluated by immunodiffusion and ELISA techniques, while the overall immune response was assayed by the worm burden in the immunized and subsequently infected rats. Agar-gel double diffusion yielded precipitin bands only with larval somatic antigens. ELISA proved positive at a titer of 20,000 with larval metabolic antigen and sera of rats immunized against either larval metabolic or somatic antigens. By 20 days post challenge infection, however, this titer diminished to 4000. In vivo studies of worm burden in rats immunized with the various antigens and then exposed to the live L3 of the nematode showed that there were significantly fewer adult worms in the rats immunized with larval somatic antigen and adult metabolic antigen than in those immunized with adult somatic antigen or larval metabolic antigen.     

MHC restriction of the antibody repertoire to secretory antigens, and a major allergen, of the nematode parasite Ascaris

Humans vary considerably in the antigen specificity of their immune responses to parasitic nematodes, and in the infection loads of individuals living in the same environment. The possibility that the former has a genetic basis operating through repertoire control of the immune system was investigated using infection of mice with the nematode Ascaris. The specificity of the antibody response was examined using excretory/secretory (ES) materials of the parasite as target Ag. No strain of mouse was found to recognize all of the potentially antigenic components of ES, and the Ag recognition patterns varied considerably from strain to strain. Using H-2 congenic mice on both the BALB and B10 backgrounds, it was established that the antigen recognition patterns were MHC-determined. Focusing on one particular component of ES, of Mr 14,000, only H-2s strains responded in IgG. This MHC restriction of the repertoire was confined to infection, and broke down under adjuvant-assisted immunization with the purified protein. The Mr 14,000 molecule was also found to be a potent allergen in a passive cutaneous anaphylaxis assay, and the IgE response to it was also restricted to H-2s. This haplotype was, however, a low IgE responder on the SJL background. There is, therefore, MHC control of the specificity of the immune response to this molecule, but non-MHC control of the amplitude of the IgE antibody response to it. Hybrids between responder and nonresponder strains (BALB/c x SJL)F1, responded to the Mr 14,000, but their responses to other ES components could not be predicted from the response patterns of parental strains. For example, the BALB/c parent responded to a 118-kDa component, but the SJL parent and the F1 progeny did not. Moreover, the response to a 41-kDa Ag was substantially down-regulated in the F1, whereas both parental strains responded vigorously. This new model system, therefore, has implications for MHC control of responses to the allergens of pathogens, and for the complex immunoregulation in heterozygotes in the context of infection.     

Distribution of filarial elephantiasis and hydrocele in Matara district, Sri Lanka, as reported by local leaders, and an immunological survey in areas with relatively high clinical rates

To eliminate lymphatic filariasis by means of mass drug administration, it is essential to have reliable data on the disease distribution and prevalence in targeted areas. In Matara district, Sri Lanka, self-administered questionnaires were mailed to 2105 local leaders questioning the presence and the numbers of elephantiasis and hydrocele cases. The information provided by them revealed that elephantiasis was clearly aggregated in the southern part of the district along the coast, while hydrocele was distributed rather evenly in the whole district, including Deniyaya region where no endemic filariasis had been known. To confirm active transmission of filariasis in Deniyaya, Wuchereria bancrofti antigen and filaria-specific urinary IgG4 antibody were measured with 2436 subjects. The positive rates for antigen and antibody were 0.6% and 4.3%, respectively. The titer analysis of IgG4 according to age revealed that the youngest IgG4 positive was 3 years old, and that in 10 years old or less, there were 16 positives out of 607 children examined (2.6%). It was concluded that filarial transmission at a low level was going on in the region.     

Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretory-secretory antigens of Anisakis simplex in patients with gastric anisakiasis

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1-2 proteins in the 65-95 kDa region. The antigenicity of the excretory-secretory products was lost following treatment by 0.2% trypsin, but not by 0.2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.     

The effect of HIV on filarial-specific antibody response before and after treatment with diethylcarbamazine in Wuchereria bancrofti infected individuals

The effect of HIV on filarial-specific antibody response before and after treatment with diethylcarbamazine (DEC) was analysed by comparing two groups of Wuchereria bancrofti-infected adult individuals (positive for circulating filarial antigen) who were positive (n = 15) or negative (n = 21) for HIV co-infection. Prior to DEC treatment there was no significant difference in filarial-specific IgG1, IgG2, IgG4 and IgE antibody response between the HIV negative and the HIV positive group, while a five times (statistically significant) higher filarial-specific IgG3 response was observed in the HIV positive than in the HIV negative group. At 12 weeks after treatment with DEC, a significant decrease in filarial-specific IgG4 was observed in the HIV positive but not in the HIV negative group, indicating that DEC treatment had a stronger antifilarial effect in individuals co-infected with HIV. DEC treatment had no significant effect on the other classes of filarial specific antibodies, neither in the HIV negative or the HIV positive group.     

Early events of electroporation-mediated intramuscular DNA vaccination potentiate Th1-directed immune responses

BACKGROUND: Application of electrical pulses after DNA injection into muscle increases expression of the encoded genes, and is shown to improve antigen-specific immune responses when used for DNA vaccination. In addition, electroporation causes tissue injury and inflammatory reactions. Together with immune stimulatory motifs in the injected DNA these factors may potentiate the immune response by acting as adjuvants for the antigen. Here, we have examined the role of these factors in promoting the efficiency of DNA vaccination. METHODS: We injected a plasmid DNA vector containing the gene Ag85B from M. tuberculosis into mouse quadriceps muscles followed by electroporation. Ag85B was under control of a Tet-responsive promoter, and was expressed either immediately or up to 28 days later by administrating doxycycline to the mice. Delayed expression was combined with injection of non-coding DNA or saline with or without electroporation to examine the ability of these factors to enhance the Ag85B-specific antibody response in the blood and cellular responses in the spleen. Blood samples were analysed with ELISA, while the number of Ag85B-specific IFN-gamma- and IL-4-producing spleenocytes was analysed with ELISpot. RESULTS: Delaying Ag85B expression by 5 or 28 days caused lower anti-Ag85B-specific IgG2a levels. In contrast, the IgG1 antibody response was not significantly affected. Injection of non-coding DNA followed by electroporation moderately increased the IgG2a response. Delaying the Ag85B expression by 28 days reduced the average number of Ag85B-specific IFN-gamma-producing spleenocytes by over 60%. No significant change in the number of IL-4-producing Ag85B-specific spleenocytes was observed. CONCLUSIONS: These results suggest that DNA and electroporation per se may act as good adjuvants in promoting efficient Th1-directed responses during DNA vaccination.     

Elevated adaptive immune responses are associated with latent infections of Wuchereria bancrofti

In order to guarantee the fulfillment of their complex lifecycle, adult filarial nematodes release millions of microfilariae (MF), which are taken up by mosquito vectors. The current strategy to eliminate lymphatic filariasis as a public health problem focuses upon interrupting this transmission through annual mass drug administration (MDA). It remains unclear however, how many rounds of MDA are required to achieve low enough levels of MF to cease transmission. Interestingly, with the development of further diagnostic tools a relatively neglected cohort of asymptomatic (non-lymphedema) amicrofilaremic (latent) individuals has become apparent. Indeed, epidemiological studies have suggested that there are equal numbers of patent (MF⁺) and latent individuals. Since the latter represent a roadblock for transmission, we studied differences in immune responses of infected asymptomatic male individuals (n = 159) presenting either patent (n = 92 MF⁺) or latent (n = 67 MF⁻) manifestations of Wuchereria bancrofti. These individuals were selected on the basis of MF, circulating filarial antigen in plasma and detectable worm nests. Immunological profiles of either Th1/Th17, Th2, regulatory or innate responses were determined after stimulation of freshly isolated PBMCs with either filarial-specific extract or bystander stimuli. In addition, levels of total and filarial-specific antibodies, both IgG subclasses and IgE, were ascertained from plasma. Results from these individuals were compared with those from 22 healthy volunteers from the same endemic area. Interestingly, we observed that in contrast to MF⁺ patients, latent infected individuals had lower numbers of worm nests and increased adaptive immune responses including antigen-specific IL-5. These data highlight the immunosuppressive status of MF⁺ individuals, regardless of age or clinical hydrocele and reveal immunological profiles associated with latency and immune-mediated suppression of parasite transmission.     

The mouse antibody response to Trichinella spiralis defines a single, immunodominant epitope shared by multiple antigens.

Immunoblot analysis was used to characterize the Trichinella spiralis L1 larval Ag recognized by antisera from T. spiralis-infected AKR/J mice. Antisera were analyzed for reactivity with crude worm extract, excretory/secretory proteins and cuticle proteins from L1 larvae. The response was biphasic; antibodies against one set of Ag were detected 13 days after infection (group I Ag), and antibodies against a different set of Ag were detected 35 days after infection (group II Ag). Excretory/secretory and cuticle proteins were recognized only by antibodies produced late in infection. The predominant isotype in day 42 antiserum was IgG1, and 80% of these IgG1 antibodies reacted with a stage-specific determinant shared by virtually all group II Ag. A mAb reactive with the shared determinant was used to purify the group II Ag from L1 larval extract. Such affinity-purified Ag were protective when used to immunize mice against subsequent infection, and T cells from infected mice proliferated when cultured with these Ag in vitro. Other mouse strains also made a strong serum antibody response to the group II Ag. We hypothesize that immune responses to the shared epitope play an important role in determining the outcome of the host-parasite interaction during infection.     

Th1-type immune response to a Coccidioides immitis antigen delivered by an attenuated strain of the non-invasive enteropathogen Vibrio cholerae

The antigen-2 or proline rich antigen (Ag2/PRA) from Coccidioides immitis, known to protect mice against experimental Coccidioidomycosis, was expressed in the genetically attenuated cholera vaccine candidate Vibrio cholerae 638 and its thymine auxotrophic derivative 638T. Intranasal immunization of mice with strains producing Ag2/PRA induced serum vibriocidal antibody and Ag2/PRA-specific total IgG responses in outbred Swiss Webster and inbred BALB/c mice. Analysis of IgG subclasses showed a predominance of IgG2a subclass antibodies. Lymphocytes from immunized mice stimulated with pure Ag2/PRA showed a significant proliferative response with production of interferon-gamma. Positive selection for plasmid maintenance in vivo did not enhance immune response to Ag2/PRA. These results demonstrate that genetically attenuated strains of the non-invasive pathogen V. cholerae can be used to express and deliver foreign antigens to stimulate a Th1 type of immune response.     

PCR and Mosquito dissection as tools to monitor filarial infection levels following mass treatment

BACKGROUND: Entomological methods may provide important tools for monitoring the progress of lymphatic filariasis elimination programs. In this study, we compared dissection of the vector, Culex quinquefasciatus, with the polymerase chain reaction (PCR) to assess filarial infection levels in mosquitoes in the context of a lymphatic filariasis elimination program in Leogane, Haiti. METHODS: Mosquitoes were collected using gravid traps located in 4 sentinel communities with Wuchereria bancrofti microfilaria prevalence that ranged from 0.8% to 15.9%. Captured mosquitoes were divided between dissection, to enumerate W. bancrofti larvae (L1, L2, L3) and desiccation for later analysis by PCR. PCR was conducted on DNA extracts from pooled mosquitoes (1-15 pooled females) utilizing a competitive PCR system with primers specific for the Ssp I repeat. PCR products were analyzed with a hybridization ELISA using probes specific for a control sequence and the Ssp I repeat. RESULTS: The prevalence of mosquito infection with W. bancrofti ranged from 0%-3.66% by dissection (L1-L3) and point estimates of infection prevalence, as assayed by PCR, ranged from 0.25% - 9.16%. Following mass treatment, W. bancrofti infection prevalence dropped significantly as determined by PCR and dissection in 2 of the 4 sentinel sites (Leogane and Barrier Jeudi, P = 0.04 and P = 0.005, respectively). Although transmission declined in the other two sites, larval recoveries were low and these changes were not statistically significant. DISCUSSION: Our results suggest that a single round of mass treatment can have an impact on transmission of lymphatic filariasis. The use of entomologic methods as a tool to monitor filariasis programs and the statistical limitations of mosquito trapping are discussed.     

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels of microfilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in a microfilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.