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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 4  |  Page : 323-329

Species-specific and cross-reactive antigen of filarial worm: Brugia malayi and Setaria digitata


1 Department of Immunology, Regional Medical Research Centre, Bhubaneswar, India
2 Department of Virology, Regional Medical Research Centre, Bhubaneswar, India

Date of Submission16-Mar-2018
Date of Acceptance16-Jul-2018
Date of Web Publication30-Nov-2020

Correspondence Address:
Dr. Prakash Kumar Sahoo
Department of Virology, ICMR-Regional Medical Research Centre, Chandrasekharpur, Bhubaneswar–571 023
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.s302035

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  Abstract 

Background & objectives: Generally filarial antigens have been found to be cross-reactive in nature. Identification of genus and species-specific antigens has not been successful so far. Due to lack of human adult filarial parasite, researchers have been using other adult worms like Setaria digitata, a cattle parasite or Brugia malayi, a rodent model for their research work. In this situation, specificity of the prepared antigen (S. digitata or B. malayi) to detect the antibodies to Wuchereria bancrofti is questionable.
Methods: In the present investigation, we have tested a panel of human sera (collected from the areas, endemic for bancroftian filariasis) to correlate the immune reactivity against somatic antigens of adult stages and microfilarial stages of S. digitata and B. malayi. Further, using intact microfilariae (mf) from the above two parasites along with W. bancrofti, we have analyzed the antibody response to the sheath antigens. A panel of infected human and cattle sera was tested by immunoperoxidase assay using intact mf of three different parasites, viz. W. bancrofti, B. malayi, and S. digitata.
Results: A very significant positive correlation in filarial Igs (polyvalent), IgG, IgM, IgE and IgG4 levels were found between the two adult somatic antigens of B. malayi and S. digitata when tested against human filarial sera. However, such a correlation was not found when mf antigens of B. malayi and S. digitata were tested against a panel of W. bancrofti sera indicating that antigens present in mf could be far less cross-reactive in comparison to those in adult stage parasites.
Interpretation & conclusion: The results indicated the differential cross-reactivity of antisheath antibodies to the mf sheath of three different filarial parasites. Soluble antigens of S. digitata could inhibit antisheath antibody reactivity to only S. digitata mf sheath and not to mf sheath of W. bancrofti further confirming the specificity of sheath antigen.

Keywords: Antisheath antibodies; Brugia malayi; cross-reactive antigen; IgG4; IPA; Setaria digitata; species-specific


How to cite this article:
Satapathy AK, Sahoo PK. Species-specific and cross-reactive antigen of filarial worm: Brugia malayi and Setaria digitata. J Vector Borne Dis 2019;56:323-9

How to cite this URL:
Satapathy AK, Sahoo PK. Species-specific and cross-reactive antigen of filarial worm: Brugia malayi and Setaria digitata. J Vector Borne Dis [serial online] 2019 [cited 2021 Jan 25];56:323-9. Available from: https://www.jvbd.org/text.asp?2019/56/4/323/302035


  Introduction Top


Lymphatic filariasis caused by filarial parasites, Wuchereria bancrofti, Brugia malayi and B. timori are transmitted by mosquitoes[1]. Wuchereria bancrofti infects only humans, B. malayi infects both the man and animal whereas S. digitata infects cattle. Cross-reaction has been reported between the antigens of Setaria spp. with filarial infected human sera[2],[3],[4]. Further, antigen of Dirofilaria immitis and B. pahangi has been found to have cross-reaction with human sera from filarial endemic area[5],[6]. Filarial antigens have been generally found to be mostly common between several parasites, viz. W. bancrofti, B. malayi, S. digitata, D. immitis, Onchocerca volvulus, etc. Almost every known cloned of filarial antigen from one filarial parasite has been demonstrated to be present in other filarial parasites. There has been successful demonstration of stage-specific filarial antigens (viz. larval, adult stage and microfilarial specific) at least in B. malayi. Attempts to demonstrate species/genus-specific antigens of filarial parasites have been largely unsuccessful[7].

The extensive cross-reactive nature of different filarial parasites has been used for studying immune response in human filariasis. Wuchereria bancrofti, the parasite responsible for the widely prevalent bancroftian filariasis has not been adopted in laboratory animals and consequently the developmental stages of the parasites are not extensively available for use in immunological studies. As far as our knowledge goes most of the immunological studies (including B & T lymphocyte functions, antibody and cytokine responses etc.) in bancroftian filariasis have been performed using antigen prepared from B. malayi, another human filarial parasite[8],[9].

In the present study, we have evaluated the degree of cross-reactivity of two filarial parasites B. malayi and S. digitata (adult as well as microfilarial antigens) against a panel of bancroftian filarial sera. While the adult antigens were found to be widely cross-reactive, the microfilarial soluble antigens and surface (sheath) antigens of the two parasites appeared to be different further confirming the specificity of sheath antigen.


  Material & Methods Top


Patients and samples

A mass night blood survey for filariasis was conducted in coastal village of Jatni area approximately 40 km from Bhubaneswar, India. This area was reported as filarial endemic area[10]. About 2 ml of venous blood samples were collected from individuals for plasma and microfilaria. Thick blood smear was prepared with 20 μl and stained with Giemsa stain for microscopic examination of microfilaria. One ml of whole blood was passed through 5 mm polycarbonate membrane (Nuclopore Corporation, CA, USA) for purification of mf by membrane filtration method. Plasma were separated from the blood and stored at –20 °C for further use.

Parasite and antigens

Setaria digitata and B. malayi adult parasites were collected from peritoneal cavity of infected cattle and birds, respectively. For preparations of mf slides, mature mf was harvested from the circulation of infected cattle by filtration of infected blood through 5 mM polycarbonate membranes (Nucleopore, USA) as described earlier[11]. Brugia malayi mf and adult worm were collected from the peritoneum of infected birds by membrane filtration. About 25 μl of mf suspension (1500 mf/ml) were spotted onto microscopic slides, air-dried, fixed with acetone and stored at 4 °C for further use as ‘antigen slides’ in immuno-peroxidase assay (IPA).

Preparation of antigens

Antibodies to soluble antigens of adult and micro-filariae antigens of S. digitata were quantified by ELISA. For preparation of antigens, worms (adults and mf) were extensively washed and homogenized in PBS (Polytron homogenizer, USA) and micro centrifuged at 10,000 g for 5 min and the clear supernatant was stored at –20 °C for further use.

Antibodies detection

ELISA was performed with human sera. Briefly, 2–3 μg/well of antigen was coated in an ELISA plate. The plates were incubated for 4 h at 37 °C and shifted to 4 °C for overnight incubation. After three washes, the wells were blocked with 1% skim milk for 1h to avoid nonspecific binding. After three washes, the plates were incubated with 500-fold diluted sera (serum titration indicated 1/500 to be optimum for maximum sensitivity of ELISA values) and the bound antibodies were detected using sequentially 500-fold diluted rabbit antihuman Igs and antibovine Igs peroxides conjugate (Dakopatt, Denmark). After incubation, the plates were washed with PBS-T 20 and enzyme activity was measured using O-phenylenediamine. The absorbance at 492 nm was measured by an EIA reader (Bio Rad, USA). The absorbance values were expressed as optical density (OD).

Immunoperoxidase assay (IPA)

Antibodies to mf sheath in sera were detected by IPA as described elsewhere[11]. Briefly, acetone-fixed mf on microscopic slides were treated with 1% H2O2 in methanol to remove endogenous peroxidase activity and incubated with 5-fold diluted human or cattle sera for 2 h at 37 °C. After washing, the slides were incubated with 100-fold diluted antihuman Igs conjugated with peroxidase (Dakopatt, Denmark). The slides were stained with diaminobenzidine (0.5 mg/ml in Tris-HCl buffer with H2O2), dehydrated in ethanol and observed using a light microscope to score reactivity.

Immunofluorescent assay (IFA)

Immunofluorescent assay using W. bancrofti, S. digitata or B. malayi was performed essentially as described previously[12]. Briefly 1/5 diluted sera in PBS were added onto mf spotted slide and incubated for 2 h at 37 °C. After extensive washing in PBS, slides were probed with 100-fold diluted rabbit antihuman Ig FITC conjugate (Dakopatt, Denmark) for 2 h at 37 °C. Washed slides were mounted with 50% glycerol and intensity of the reactive to sheath fluorescent was scored under fluorescent microscope.

Inhibition of antibodies

For inhibition test, samples were co-incubated with S. digitata soluble antigen for 1 h at 37 °C and the immunoperoxidase assay was performed with the pre-incubated sera as described above.

Statistical analysis

Statistical analysis was performed by using Pearson’s correlation coefficient between appropriate parameters. Graph Pad prism Version 4 software was used for statistical analysis.

Ethical statement

Before collection of blood samples, people of the villages were properly informed about the study by the local health workers by our group. The samples were collected after taking informed consent from each individual.


  Results Top


Adult soluble antigen of S. digitata cross-reacts with B. malayi

Human serum samples collected from bancrofian filariasis endemic area were tested against somatic antigens of adult S. digitata and B. malayi to evaluate the cross-reactivity between them. About 40 serum samples were tested for qualitative analysis of Ig antibodies against S. digitata and B. malayi adult antigens. The IgG antibodies to human filarial sera reacted with adult antigen of both B. malayi and S. digitata. There was a statistically significant positive correlation (Pearson’s, r = 0.6501, p <0.001) between the two antigens [Figure 1]a indicating that both antigens have great similarity. IgG antibodies of same filarial sera were also tested against other animal parasite B. pahangi. The reactivity of human IgG antibodies to adult antigen of S. digitata was compared with B. pahangi. The result is shown in [Figure 1]b. A significantly positive correlation of the antibodies between S. digitata and B. pahangi (Pearson’s, r = 0.5591, p < 0.0001) was observed indicating the presence of cross-reactive antigen in adult worm of S. digitata, B. malayi and B. pahangi.
Figure 1:

Click here to view


Similarly, qualitative analyses of antibodies to human IgM and IgE were also evaluated. A panel of 39 human sera collected from filarial endemic area was tested to know the qualitative difference of IgM antibodies reactivity to both B. malayi and S. digitata adult antigens. The levels of IgM antibodies were compared between them. The antibody titres were significantly correlated between the two parasite antigens (Pearson’s, r = 0.4232, p <0.0073) as shown in [Figure 1]c. The same human filarial sera were tested to evaluate the antibodies to human IgE as shown in [Figure 1]d. The antibodies levels were compared between adult antigens of B. malayi and S.digitata. The antibodies to human IgE levels were significantly positively correlated between the two different filarial parasites (Pearson’s, r = 0.6643, p <0.0019) indicating the presence of similar antigens between the two parasites. Interestingly, the subtype, IgG4 antibodies, a marker for human active filarial infection was also tested between adult antigens of B. malayi and S. digitata. Relationship between IgG4 antibodies to adult antigens of B. malayi and S. digitata are shown in [Figure 1]e. A significant positive correlation was found between B. malayi and S. digitata adult soluble antigens (Pearson’s, r = 0.4294, p <0.0057) indicating a significant relation between them.

Crude extract of microfilaria (mf) antigen of S. digitata did not cross-react with B. malayi

Apart from testing the human filarial sera against soluble adult antigens of two filarial worms, we also tested the same filarial sera against soluble microfilarial antigens of both parasites to evaluate the cross-reactivity between them. Soluble mf antigens of both B. malayi and S. digitata were prepared and tested with 40 human filarial sera to know the qualitative relationship between antigens of two species. Antibodies to human IgG levels were compared using these two mf antigens. The results are shown in [Figure 2]. IgG antibodies of filarial sera did not recognize soluble antigen of S .digitata as well as B. malayi mf antigens. There was no significant correlation (Pearson's, r = – 0.1443, p <0.3745) between these two mf antigens suggesting that there is no significant relation between them.
Figure 2: Relationship between IgG antibodies to microfilaria antigens of B. malayi and S. digitata. A panel of 40 human sera were tested (r = –0.1443 and p = 0.3745). The coefficient value suggested that there was no significant relation between them.

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Microfilaria surface antigen of S. digitata differentially cross-reacts with B. malayi

Microfilaria of B. malayi, W. bancrofti and S. digitata were purified and antigen spots were prepared for IPA and immunofluorescent assay. Human filarial sera were tested against microfilaria surface antigen to know the specificity. It was found that human sera were reacted differentially with this three mf surface antigen [Figure 3]. One of the sera (P-1) strongly reacted with W. bancrofti than with S. digitata and B. malayi. At the same time, four sera (P-4, P-8, P-65 and P-105) reacted with one mf and not with others when tested by IPA as shown in [Figure 3]a. Further, to confirm the differential reactivity, another group of 12 human filarial sera were tested by other immunological technique like immunofluorescent assay (IFA) with these three different mfs. The results of this experiment are shown in [Figure 3]b. Four of these sera (G-102, G-140, G-129 and G -131) strongly reacted with mf of W. bancrofti than S. digitata or B. malayi. Likewise, three sera (G-114, G-120 and G-135) had a strong reactivity with mf of S. digitata than W. bancrofti or B. malayi. The above results indicate that the surface antigens are more specific because of their differential reactivity. Differential reactivity to mf sheath antigens was observed in human filarial sera as shown in [Figure 3]a & [Figure 3]b. This was further tested by pre-incubation of soluble S. digitata antigens with human or cattle filarial sera and subsequently tested against surface antigens of W.
Figure 3: Diagrammatic representation of immunoperoxidase assay (IPA)/immune fluorescent assay (IFA) for antisheath antibodies performed with 1/5 diluted sera : (a) Sixteen human filarial sera were tested against microfilaria of W. bancrofti, S. digitata and B. malayi by IPA; (b) Twelve human filarial sera were tested against microfilaria of W. bancrofti, S. digitata and B. malayi by IFA; and (c) Four of human sera and three of cattle sera were incubated with soluble Setaria antigen and tested against microfilaria of W. bancrofti, and S. digitata for reactivity and scored as follows:
◻ No reactivity; ◼4+; •3+; ▴2+ ♦1+


Click here to view


bancrofti and S. digitata [Figure 3]c. Since S. digitata is a common filarial parasite of cattle, we used cattle sera as a control in this inhibition test. All the samples pre-incubated with soluble mf antigen of S. digitata lost their reactivity to mf sheath of S. digitata. Soluble antigens of S. digitata could inhibit antisheath antibody reactivity to only S. digitata mf sheath, but not to mf sheath of W. bancrofti indicating the specificity of mf antigen.


  Discussion Top


Filarial antigens have been generally found to be extensively common between several parasites, viz. W. bancrofti, B. malayi, S. digitata, D. immitis, O. volvulus, etc. The extensive cross-reactive nature of different filarial parasites has been used for studying immune response in human filariasis. Due to non-vailability of the W. bancrofti parasite, almost all immunological studies in bancroftian filariasis have been performed using antigen prepared from B. malayi, another animal filarial parasite. In this study, we attempted to determine the cross-reactivity and species-specific antigens derived from adult and microfilaria stages of parasite. The homology of the antigens between the two parasites S. digitata and B. malayi was compared by using human filarial sera. The reactivity of human sera to both the adult parasite antigens was significantly correlated. IgG, IgM, IgE and IgG4 antibodies in human sera were found to recognize both S. digitata and B. malayi adult stages soluble antigens indicating the antigenic similarity or homology between the two parasites. The cross-reactivity of human sera to animal parasite has been demonstrated earlier by using soluble antigen of S. equine[13], D. immitis[5], S. digitata[7] and B. malayi[6]. The cross-reaction between S. cervi and human filariasis has been reported earlier[14],[15]. The parasite-specific and cross-reactive antigen of Onchocerciasis of cattle and man has also been reported earlier[16]. Antigenically, S. digitata has been demonstrated to be closer to W. bancrofti[17],[18]. Our findings add to the existing evidence that the soluble adult antigen of S. digitata cross-reacts with B. pahangi.

In the present study, we have evaluated the degree of cross-reactivity of two filarial parasites B. malayi and S. digitata (adult as well as microfilarial antigens) against a panel of Bancroftian filariasis sera. Setaria digitata is a common filarial parasite of cattle. Due to the easy availability of large quantities of parasites from slaughtered animals, the parasite has been increasingly used in recent years for immunological, chemotherapeutic and other biological studies[19],[20]. Setaria digitata microfilariae are morphologically very similar to mf of human filarial parasite W. bancrofti and B. malayi in terms of shape, size, presence of sheath, etc. Therefore, we evaluated the cross-reactivates between the two parasites S. digitata and B. malayi. Interestingly, no cross-reactivity was found between two parasites’ mf antigens when the same set of filarial sera was tested against soluble mf antigen. Further, IPA and IFA tests were performed by using intact mf to know the cross-reactivity/specificity of mf surface (sheath) antigen of three different species W. bancrofti, B. malayi and S. digitata. In both IFA and IPA experiments, a differently relativities to microfilarial sheath antigens of different species by human filarial sera has been observed. Since antisheath antibodies recognize mostly carbohydrate determinants on the microfilarial sheath[21], it is possible that the microfilariae sheath might represent different carbohydrates antigens. Soluble antigens of S. digitata could inhibit antisheath antibody reactivity to only S. digitata mf sheath but not to mf sheath of W. bancrofti indicating the specificity of mf antigen. The results of the present investigation indicate the adult antigens were found to be widely cross-reactive at the same time there was a differential cross-reactivity of antisheath antibodies to the mf sheath of the three different filarial parasites. Further, analysis of sheath antigen could provide the molecule(s) responsible for the specificity. Currently, the work is in progress to identify such species-specific determinants.


  Conclusions Top


The present study concluded that the somatic adult antigens of S. digitata, B. malayi and W. bancrofti were found to be widely cross-reactive. Due to the extensive cross-reactive nature of different filarial parasites S. digitata adult antigen can be used for studying immune response in the people residing in either B. malayi or W. bancrofti endemic area. Further, all immunological studies (including B & T lymphocyte functions, antibody and cytokine responses etc.) in filariasis can be performed by using any of the three filarial antigens. On the other hand there was a differential cross-reactivity of antisheath antibodies to the mf sheath of three different filarial parasites. The soluble as well as surface (sheath) antigens of mf appeared to be different confirming the specificity of sheath antigen.

Conflict of interest

The authors declare no conflict of interest.


  Acknowledgements Top


The authors express their sincere thanks to the patients who were voluntarily participant in the study. The authors are also grateful to the Director of ICMR—Regional Medical Research Centre, Bhubaneswar for her constant support and providing the infrastructures and facilities for the study.

 
  References Top

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