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Human serum albumin and immunoglobulin on Dracunculus medinensis
Acta Tropica 73 (1999) 135 – 141 www.elsevier.com/locate/actatropica
Human serum albumin and immunoglobulin on Dracunculus medinensis Paul Bloch *, Mette Lund, Birgitte J. Vennervald, Paul E. Simonsen Danish Bilharziasis Laboratory, Jaegersborg Alle 1 D, DK-2920 Charlottenlund, Denmark Received 31 July 1998; received in revised form 11 December 1998; accepted 17 February 1999
Abstract Dracunculus medinensis recovered from infected humans were examined for the occurrence and localization of human serum albumin and immunoglobulins. Immunoelectrophoretic examination of homogenates prepared from adult female worms (ADGW) and first stage larvae (LVGW) showed that anti-human albumin antibodies reacted to both stages of the parasite. By direct fluorescence antibody technique, antigens resembling human albumin and human immunoglobulins (isotype IgG) were identified on the surface of adult female worms. The occurrence of host-like compounds on the parasites may be an adaptation for survival of the parasites. A possible interference of these compounds should be considered in attempts to develop methods for immunological diagnosis of D. medinensis infections. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Human albumin; Human immunoglobulin; Dracunculus medinensis; Dracunculiasis
1. Introduction Antigens with epitopes resembling those of host proteins have been identified on the surface of different filarial parasites (Behnke et al., 1992). Thus, for example antigens resembling bovine albumin have been detected on skin microfilariae of Onchocerca gibsoni from cattle (Forsyth et al., 1984), antigens resembling dog albumin, dog IgG and dog complement factor C3 have been demonstrated on adult * Corresponding author. Tel.: +45-77-327732; fax: +45-77-327733. E-mail address: [email protected] (P. Bloch) 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 9 ) 0 0 0 2 4 - 8
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P. Bloch et al. / Acta Tropica 73 (1999) 135–141
Dirofilaria immitis from dogs (Kadipasaoglu and Bilge, 1989), and antigens resembling human albumin and human IgG have been found on Wuchereria bancrofti (Maizels et al., 1984; Kar et al., 1993) and Brugia malayi microfilariae (Piessens et al., 1980; Mania and Kar, 1994) recovered from humans. The detection of specific markers of infection is crucial in immunodiagnostic assays, and information on the occurrence of host-like antigens on parasites of medical importance is of key importance when developing immunodiagnostic assays and when attempting to prepare parasite specific polyclonal antibodies by immunizing experimental animals with antigen material. In an attempt to develop a method for immunological diagnosis of Dracunculus medinensis infection, we have performed detailed analyses of the humoral responsiveness to this parasite in endemic populations (Bloch et al., 1993; Bloch and Simonsen, 1998a,b Bloch and Simonsen, 1998c). The present study searched for antigenic human components (albumin and immunoglobulin) in D. medinensis parasite material recovered from humans, by immunoelectrophoretic examination of crude homogenates prepared from adult female worms and first stage larvae and by direct fluorescence antibody examination of intact first stage larvae and sections of adult female worms.
2. Subjects and methods
2.1. Parasite material D. medinensis adult females and first stage larvae, obtained from individuals from northern Ghana, were analyzed in intact (larvae only), sectioned (adult worms only) and homogenized form (larvae and adult worms). The adult worms were extracted surgically just prior to emergence (Rohde et al., 1993). They were thoroughly washed in PBS (phosphate buffered saline, pH 7.4) and frozen at − 20°C in PBS containing sodium azide (NaN3, 15 mM). First stage larvae were gently milked into distilled water from the uterus of live adult females. The larvae were thoroughly washed in distilled water and either frozen at−20°C in distilled water containing NaN3 (15 mM), or preserved in 1.0% formalin at 4°C. Crude homogenates of D. medinensis adult worms (ADGW) and first stage larvae (LVGW) were prepared from the frozen parasite material as described previously (Bloch and Simonsen, 1998a). Thin cross-sections were prepared from the mid-body region of adult D. medinensis females. Briefly, two non-fixed worms were embedded in Tissue-Tech (O.C.T. 4583, Miles Scientific Lab, USA) according to procedure from the manufacturer, and sectioned by cryostat microtomy. Sections were kept frozen on microscope slides at−20°C until use. Larger pieces of approximately 1 mm in thickness were manually cut with a scalpel from the mid-body region of two adult female worms. These pieces were either used without further treatment or after removal of the internal parts (primarily muscles, uterus and larvae) by dissection.
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2.2. Crossed immunoelectrophoresis ADGW and LVGW were analyzed by crossed immunoelectrophoresis (CIE) by applying previously described procedures (Grubb, 1983; Krøll, 1983; Johnstone and Thorpe, 1987). First dimension electrophoresis was carried out with 50 m1 ADGW or LVGW diluted to a protein concentration of 2 mg/ml or with 50 ml dissolved human albumin (A-7030, Sigma, St. Louis, USA) diluted to a concentration of 200 mg/ml. In the second dimension, 200–400 ml undiluted antiserum (rabbit-anti-human albumin or rabbit-anti-human IgG; both from Dako, Denmark) was added to the gel. The CIE plates were photographed for presentation following protein staining using coomasie brilliant blue (R-250, Bio-Rad, USA).
2.3. Direct fluorescence antibody test The direct fluorescence antibody test was applied according to procedure described by Hudson and Hay (1989). Approximately 100 1.0% formalin fixed first stage larvae were washed three times in PBS (pH 7.4). After the last centrifugation (at 2000 rpm for 5 min), the supernatant was carefully removed until 300 ml remained. To reduce the amount of autofluorescence, 90 ml Evans blue counterstain (Sigma E0133) diluted 1:100 in PBS was added together with optimally diluted fluorescein-isothiocyanate (FITC) labelled antibody (1:40 for rabbit-anti-human albumin, Dako, Denmark; 1:40 for rabbit-anti-human IgG, Dako; 1:10 for goat anti-human IgE, Sigma, USA). After incubation for 1 h at 20°C and three times washing, the larvae were transferred to a microscope slide. One drop of buffered glycerol (i.e. a mixture in the proportion 1:3 of PBS and glycerol, Merck 4093, USA) was added. The preparation was examined by reflected light fluorescence microscopy (mercury lamp, 490 nm excitation filter, 515 nm barrier filter). Thin cross-sections of adult D. medinensis females remained on the microscope slides during processing and the same incubation steps and dilutions described above were followed. The specimens were washed by gently rinsing the slides three times in PBS, and incubations were carried out by adding a 50 m1 drop of FITC-labelled antibody diluted in PBS (containing diluted Evans blue) on top of each specimen. The larger hand-cut pieces of adult worms were tested with FITC-labelled conjugates according to the above standard procedure with the modification that incubations and washings were carried out in 200 ml buffer volumes in wells of Nunclon D multidish plates (Nunc 143982, Life technologies, Denmark). Specimens processed without FITC-labelled conjugates were included as controls in all tests.
3. Results Anti-human albumin antibodies reacted with both ADGW and LVGW in CIE as reflected by the appearance of distinct precipitation curves (Fig. 1A and 1B). For
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Fig. 1. Reactivity of rabbit-anti-human albumin with (A) LVGW; (B) ADGW; (C) LVGW and dilute human albumin applied tandemly; and (D) ADGW and dilute human albumin applied tandemly, in crossed immunoelectrophoresis. Fig. 2. Pieces of adult female D. medinensis examined by direct fluorescence microscopy. FITC-conjugated rabbit-anti-human albumin diluted 1:40 applied to a worm from which no structures had been removed (A; 100 × magnification) and to a worm from which the intemal parts (e.g. muscles, uterus and larvae) had been removed (B; 200 × magnification).
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each antigen homogenate, the tail of the most distinct precipitation curve was linked to the precipitation curve formed by the human albumin control (Fig. 1C and 1D), as an additional indication of the presence of antigenic epitopes resembling human albumin in the parasite homogenates. In contrast, no reaction was observed between anti-human IgG antibodies and LVGW or ADGW in CIE (data not shown). The CIE experiments were repeated three times, and similar results were obtained each time. Batches of formalin fixed D. medinensis first stage larvae were examined by direct fluorescence antibody test. When treating first stage larvae with FITC-labelled anti-human IgG, FITC-labelled anti-human IgE or FITC-labelled anti-human albumin, no emission of green fluorescence was observed (data not shown). Binding of FITC-labelled anti-human albumin to the surface (but not to the internal parts) of adult female D. medinensis was observed on the thin worm cross-sections. In such sections FITC-labelled anti-human IgG (but not IgE) was also shown to bind to the surface (but not to the internal parts) of the worm. These frozen tissue sections had a faint emission of fluorescent light and are thus not shown. Attempts were made to obtain a brighter emission of fluorescent light by examining numerous larger hand-cut sections of adult female D. medinensis in the direct fluorescence antibody test after treatment with FITC-labelled anti-human albumin. Emission of bright green fluorescence from the worm surface was observed on all sections. When removing the major internal parts, which emitted autofluorescence (Fig. 2A), the emission of green fluorescence from the surface of the worms became more distinct (Fig. 2B).
4. Discussion D. medinensis first stage larvae and adult female worms recovered from humans were analyzed for the occurrence of antigens resembling human albumin or immunoglobulin. Crossed immunoelectrophoresis identified antigens in both ADGW and LVGW which reacted with anti-human albumin antibodies. The albumin-like antigenic determinants in ADGW and LVGW were closely related to antigenic determinants in dissolved human albumin, as reflected by the formation of connected CIE precipitation curves when the antigen suspensions were applied tandemly, i.e. beside each other in paired wells (Krøll, 1983). Direct fluorescence antibody examination furthermore identified albumin-like antigens on the surface of adult female worms. In contrast, this examination technique did not identify such antigens on the surface of first stage larvae, perhaps because the antigens were present in low amounts, or because they were damaged or displaced by the light formalin fixation. Kar et al. (1993) reported that after fixation with acetone or methanol, host albumin and immunoglobulin could no longer be detected on the surface of W. bancrofti microfilariae. The origin of the albumin-like antigens is unclear. They may be components of human albumin adsorbed to the cuticle, metabolic waste products
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from nutritional utilization of human albumin or parasite antigens to which anti-human albumin antibodies cross-react. It has been suggested for other parasitic nematodes that host antigens adsorbed to the surface may be of importance to parasite survival by masking the worms (causing molecular mimicry) and thereby protecting them from the hostile environment (Behnke et al., 1992). Epitopes similar to human IgG (but not IgE) were identified on the surface of adult female D. medinensis by direct fluorescence antibody examination. This reflects that the surface of the worms is exposed to antibodies in the human host. Similar antibody-like epitopes were not identified on the surface of D. medinensis first stage larvae, probably because the uterus dwelling larvae are well protected from the hosts immune responses. No evidence was obtained by CIE for the occurrence of human immunoglobulins in ADGW, probably for technical reasons (migration of immunoglobulins is not favored at the pH normally applied in CIE). Nevertheless, the observation that the human immunoglobulin-like epitopes appeared to be present on adult worms only, combined with the observed extensive sharing of antigenic determinants between the different developmental stages of the parasite (Bloch and Simonsen, 1998a) points to an advantage of using first stage larvae for experiments in which contamination of the parasite material with human products should be avoided, such as the preparation of a specific polyclonal anti-D. medinensis antiserum for use in diagnostic assays based on detection of circulating antigens. Cuticle associated canine IgG-like epitopes have previously been observed on adult D. immitis from dogs (Kadipasaoglu and Bilge, 1989), and human IgG-like epitopes have been observed on the sheath of W. bancrofti (Kar et al., 1993) and B. malayi (Mania and Kar, 1994) microfilariae recovered from humans. The conjugates used for detection of immunoglobulins on the surface of D. medinensis were specific for the Fc-portion of IgG, and it appears likely that the detected host immunoglobulins bound to the parasites as part of a specific anti-parasitic immune response. Nevertheless, having failed to kill the parasite, these immunoglobulins may be involved in protection of the parasite from further host immune responses, similar to the function of cuticle associated host albumin-like epitopes described above. The present study has provided information about the occurrence and localization of human-like antigen components of D. medinensis adult females and first stage larvae. This information may be used to assess antigenic characteristics of adult female worms and first stage larvae as a basis for selecting an appropriate parasite material for use in immunodiagnostic and immunoepidemiological studies.
Acknowledgements The study was carried out as part of an agreement between the Danish Bilharziasis Laboratory and the Ministry of Health of Ghana on the strengthening of the Guinea Worm Eradication Program in the Northern Region of Ghana. We thank the Ministry of Health of Ghana for its logistic and technical support, especially Dr Sam Bugri, Von Asigri, Lawrence Yelifari, Albano Bayitaa and Abdul Rahman
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Yakubu; Professor Peter Lind and technician Anni Ravn, Danish Veterinary Laboratory, Copenhagen, Denmark, for their efforts with the preparation of cross-sections of frozen guinea worms. The study was sponsored by the Danish Bilharziasis Laboratory and the Danish International Development Agency (Danida).
References Behnke, J.M., Barnard, C.J., Wakelin, D., 1992. Understanding chronic nematode infections: evolutionary considerations, current hypotheses and the way forward. Int. J. Parasitol. 22, 861 – 907. Bloch, P., Simonsen, P.E., Vennervald, B.J., 1993. The antibody response to Dracunculus medinensis in an endemic human population of northern Ghana. J. Helminthol. 67, 37 – 48. Bloch, P., Simonsen, P.E., 1998a. Studies on immunodiagnosis of dracunculiasis. I. Detection of specific serum antibodies. Acta Tropica 70, 73 – 86. Bloch, P., Simonsen, P.E., 1998b. Immunoepidemiology of Dracunculus medinensis infections. I. Antibody responses in relation to infection status. Am. J. Trop. Med. Hyg. 59, 978 – 984. Bloch, P., Simonsen, P.E., 1998c. Immunoepidemiology of Dracunculus medinensis infections. II. Variation in antibody responses in relation to transmission season and patency. Am. J. Trop. Med. Hyg. 59, 985–990. Forsyth, K.P., Copeman, D.B., Mitchell, G.F., 1984. Differences in the surface radioiodinated proteins of skin and uterine microfilariae of Onchocerca gibsoni. Mol. Biochem. Parasitol. 10, 217 – 229. Grubb, A.O., 1983. Crossed immunoelectrophoresis. Scand. J. Immunol. 17 (Suppl. 10), 113 – 124. Johnstone, A., Thorpe, R., 1987. Immunochemistry in Practice. Blackwell, Oxford. Hudson, L., Hay, F.C., 1989. Practical Immunology. Blackwell, Oxford. Kadipasaoglu, A.K., Bilge, F.H., 1989. Partial characterization of the adsorbed protein layer on Dirofilaria immitis (Nematode) cuticle. Parasitol. Res. 75, 554 – 558. Kar, S.K., Mania, J., Baldwin, C.I., Denham, D.A., 1993. The sheath of the microfilaria of Wuchereria bancrofti has albumin and immunoglobulin on its surface. Parasite Immunol. 15, 297 – 300. Krøll, J., 1983. Tandem crossed immunoelectrophoresis. Scand. J. Immunol. 17 (Suppl. 10), 135 – 139. Maizels, R.M., Philipp, M., Dasgupta, A., Partono, F., 1984. Human serum albumin is a major component on the surface of microfilariae of Wuchereria bancrofti. Parasite Immunol. 6, 185 – 190. Mania, J., Kar, S.K., 1994. Detection of albumin and immunoglobulin on the surface of Brugia malayi mf by immunofluorescence. Indian J. Med. Res. 99, 65 – 67. Piessens, W.F., McGreevy, P.B., Ratiwayanto, S., McGreevy, M., Piessens, P.W., Koiman, I., Saroso, J.S., Dennis, D.T., 1980. Immune responses in human infections with Brugia malayi: correlation of cellular and humoral reactions to microfilarial antigens with clinical status. Am. J. Trop. Med. Hyg. 29, 563–570. Rohde, J.E., Sharma, B.L., Patton, H., Deegan, C., Sherry, J.M., 1993. Surgical extraction of guinea worm: disability reduction and contribution to disease control. Am. J. Trop. Med. Hyg. 48, 71 – 76.
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Human serum albumin and immunoglobulin on Dracunculus medinensis Paul Bloch *, Mette Lund, Birgitte J. Vennervald, Paul E. Simonsen Danish Bilharziasis Laboratory, Jaegersborg Alle 1 D, DK-2920 Charlottenlund, Denmark Received 31 July 1998; received in revised form 11 December 1998; accepted 17 February 1999
Abstract Dracunculus medinensis recovered from infected humans were examined for the occurrence and localization of human serum albumin and immunoglobulins. Immunoelectrophoretic examination of homogenates prepared from adult female worms (ADGW) and first stage larvae (LVGW) showed that anti-human albumin antibodies reacted to both stages of the parasite. By direct fluorescence antibody technique, antigens resembling human albumin and human immunoglobulins (isotype IgG) were identified on the surface of adult female worms. The occurrence of host-like compounds on the parasites may be an adaptation for survival of the parasites. A possible interference of these compounds should be considered in attempts to develop methods for immunological diagnosis of D. medinensis infections. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Human albumin; Human immunoglobulin; Dracunculus medinensis; Dracunculiasis
1. Introduction Antigens with epitopes resembling those of host proteins have been identified on the surface of different filarial parasites (Behnke et al., 1992). Thus, for example antigens resembling bovine albumin have been detected on skin microfilariae of Onchocerca gibsoni from cattle (Forsyth et al., 1984), antigens resembling dog albumin, dog IgG and dog complement factor C3 have been demonstrated on adult * Corresponding author. Tel.: +45-77-327732; fax: +45-77-327733. E-mail address: [email protected] (P. Bloch) 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 9 ) 0 0 0 2 4 - 8
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P. Bloch et al. / Acta Tropica 73 (1999) 135–141
Dirofilaria immitis from dogs (Kadipasaoglu and Bilge, 1989), and antigens resembling human albumin and human IgG have been found on Wuchereria bancrofti (Maizels et al., 1984; Kar et al., 1993) and Brugia malayi microfilariae (Piessens et al., 1980; Mania and Kar, 1994) recovered from humans. The detection of specific markers of infection is crucial in immunodiagnostic assays, and information on the occurrence of host-like antigens on parasites of medical importance is of key importance when developing immunodiagnostic assays and when attempting to prepare parasite specific polyclonal antibodies by immunizing experimental animals with antigen material. In an attempt to develop a method for immunological diagnosis of Dracunculus medinensis infection, we have performed detailed analyses of the humoral responsiveness to this parasite in endemic populations (Bloch et al., 1993; Bloch and Simonsen, 1998a,b Bloch and Simonsen, 1998c). The present study searched for antigenic human components (albumin and immunoglobulin) in D. medinensis parasite material recovered from humans, by immunoelectrophoretic examination of crude homogenates prepared from adult female worms and first stage larvae and by direct fluorescence antibody examination of intact first stage larvae and sections of adult female worms.
2. Subjects and methods
2.1. Parasite material D. medinensis adult females and first stage larvae, obtained from individuals from northern Ghana, were analyzed in intact (larvae only), sectioned (adult worms only) and homogenized form (larvae and adult worms). The adult worms were extracted surgically just prior to emergence (Rohde et al., 1993). They were thoroughly washed in PBS (phosphate buffered saline, pH 7.4) and frozen at − 20°C in PBS containing sodium azide (NaN3, 15 mM). First stage larvae were gently milked into distilled water from the uterus of live adult females. The larvae were thoroughly washed in distilled water and either frozen at−20°C in distilled water containing NaN3 (15 mM), or preserved in 1.0% formalin at 4°C. Crude homogenates of D. medinensis adult worms (ADGW) and first stage larvae (LVGW) were prepared from the frozen parasite material as described previously (Bloch and Simonsen, 1998a). Thin cross-sections were prepared from the mid-body region of adult D. medinensis females. Briefly, two non-fixed worms were embedded in Tissue-Tech (O.C.T. 4583, Miles Scientific Lab, USA) according to procedure from the manufacturer, and sectioned by cryostat microtomy. Sections were kept frozen on microscope slides at−20°C until use. Larger pieces of approximately 1 mm in thickness were manually cut with a scalpel from the mid-body region of two adult female worms. These pieces were either used without further treatment or after removal of the internal parts (primarily muscles, uterus and larvae) by dissection.
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2.2. Crossed immunoelectrophoresis ADGW and LVGW were analyzed by crossed immunoelectrophoresis (CIE) by applying previously described procedures (Grubb, 1983; Krøll, 1983; Johnstone and Thorpe, 1987). First dimension electrophoresis was carried out with 50 m1 ADGW or LVGW diluted to a protein concentration of 2 mg/ml or with 50 ml dissolved human albumin (A-7030, Sigma, St. Louis, USA) diluted to a concentration of 200 mg/ml. In the second dimension, 200–400 ml undiluted antiserum (rabbit-anti-human albumin or rabbit-anti-human IgG; both from Dako, Denmark) was added to the gel. The CIE plates were photographed for presentation following protein staining using coomasie brilliant blue (R-250, Bio-Rad, USA).
2.3. Direct fluorescence antibody test The direct fluorescence antibody test was applied according to procedure described by Hudson and Hay (1989). Approximately 100 1.0% formalin fixed first stage larvae were washed three times in PBS (pH 7.4). After the last centrifugation (at 2000 rpm for 5 min), the supernatant was carefully removed until 300 ml remained. To reduce the amount of autofluorescence, 90 ml Evans blue counterstain (Sigma E0133) diluted 1:100 in PBS was added together with optimally diluted fluorescein-isothiocyanate (FITC) labelled antibody (1:40 for rabbit-anti-human albumin, Dako, Denmark; 1:40 for rabbit-anti-human IgG, Dako; 1:10 for goat anti-human IgE, Sigma, USA). After incubation for 1 h at 20°C and three times washing, the larvae were transferred to a microscope slide. One drop of buffered glycerol (i.e. a mixture in the proportion 1:3 of PBS and glycerol, Merck 4093, USA) was added. The preparation was examined by reflected light fluorescence microscopy (mercury lamp, 490 nm excitation filter, 515 nm barrier filter). Thin cross-sections of adult D. medinensis females remained on the microscope slides during processing and the same incubation steps and dilutions described above were followed. The specimens were washed by gently rinsing the slides three times in PBS, and incubations were carried out by adding a 50 m1 drop of FITC-labelled antibody diluted in PBS (containing diluted Evans blue) on top of each specimen. The larger hand-cut pieces of adult worms were tested with FITC-labelled conjugates according to the above standard procedure with the modification that incubations and washings were carried out in 200 ml buffer volumes in wells of Nunclon D multidish plates (Nunc 143982, Life technologies, Denmark). Specimens processed without FITC-labelled conjugates were included as controls in all tests.
3. Results Anti-human albumin antibodies reacted with both ADGW and LVGW in CIE as reflected by the appearance of distinct precipitation curves (Fig. 1A and 1B). For
138
P. Bloch et al. / Acta Tropica 73 (1999) 135–141
Fig. 1. Reactivity of rabbit-anti-human albumin with (A) LVGW; (B) ADGW; (C) LVGW and dilute human albumin applied tandemly; and (D) ADGW and dilute human albumin applied tandemly, in crossed immunoelectrophoresis. Fig. 2. Pieces of adult female D. medinensis examined by direct fluorescence microscopy. FITC-conjugated rabbit-anti-human albumin diluted 1:40 applied to a worm from which no structures had been removed (A; 100 × magnification) and to a worm from which the intemal parts (e.g. muscles, uterus and larvae) had been removed (B; 200 × magnification).
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each antigen homogenate, the tail of the most distinct precipitation curve was linked to the precipitation curve formed by the human albumin control (Fig. 1C and 1D), as an additional indication of the presence of antigenic epitopes resembling human albumin in the parasite homogenates. In contrast, no reaction was observed between anti-human IgG antibodies and LVGW or ADGW in CIE (data not shown). The CIE experiments were repeated three times, and similar results were obtained each time. Batches of formalin fixed D. medinensis first stage larvae were examined by direct fluorescence antibody test. When treating first stage larvae with FITC-labelled anti-human IgG, FITC-labelled anti-human IgE or FITC-labelled anti-human albumin, no emission of green fluorescence was observed (data not shown). Binding of FITC-labelled anti-human albumin to the surface (but not to the internal parts) of adult female D. medinensis was observed on the thin worm cross-sections. In such sections FITC-labelled anti-human IgG (but not IgE) was also shown to bind to the surface (but not to the internal parts) of the worm. These frozen tissue sections had a faint emission of fluorescent light and are thus not shown. Attempts were made to obtain a brighter emission of fluorescent light by examining numerous larger hand-cut sections of adult female D. medinensis in the direct fluorescence antibody test after treatment with FITC-labelled anti-human albumin. Emission of bright green fluorescence from the worm surface was observed on all sections. When removing the major internal parts, which emitted autofluorescence (Fig. 2A), the emission of green fluorescence from the surface of the worms became more distinct (Fig. 2B).
4. Discussion D. medinensis first stage larvae and adult female worms recovered from humans were analyzed for the occurrence of antigens resembling human albumin or immunoglobulin. Crossed immunoelectrophoresis identified antigens in both ADGW and LVGW which reacted with anti-human albumin antibodies. The albumin-like antigenic determinants in ADGW and LVGW were closely related to antigenic determinants in dissolved human albumin, as reflected by the formation of connected CIE precipitation curves when the antigen suspensions were applied tandemly, i.e. beside each other in paired wells (Krøll, 1983). Direct fluorescence antibody examination furthermore identified albumin-like antigens on the surface of adult female worms. In contrast, this examination technique did not identify such antigens on the surface of first stage larvae, perhaps because the antigens were present in low amounts, or because they were damaged or displaced by the light formalin fixation. Kar et al. (1993) reported that after fixation with acetone or methanol, host albumin and immunoglobulin could no longer be detected on the surface of W. bancrofti microfilariae. The origin of the albumin-like antigens is unclear. They may be components of human albumin adsorbed to the cuticle, metabolic waste products
140
P. Bloch et al. / Acta Tropica 73 (1999) 135–141
from nutritional utilization of human albumin or parasite antigens to which anti-human albumin antibodies cross-react. It has been suggested for other parasitic nematodes that host antigens adsorbed to the surface may be of importance to parasite survival by masking the worms (causing molecular mimicry) and thereby protecting them from the hostile environment (Behnke et al., 1992). Epitopes similar to human IgG (but not IgE) were identified on the surface of adult female D. medinensis by direct fluorescence antibody examination. This reflects that the surface of the worms is exposed to antibodies in the human host. Similar antibody-like epitopes were not identified on the surface of D. medinensis first stage larvae, probably because the uterus dwelling larvae are well protected from the hosts immune responses. No evidence was obtained by CIE for the occurrence of human immunoglobulins in ADGW, probably for technical reasons (migration of immunoglobulins is not favored at the pH normally applied in CIE). Nevertheless, the observation that the human immunoglobulin-like epitopes appeared to be present on adult worms only, combined with the observed extensive sharing of antigenic determinants between the different developmental stages of the parasite (Bloch and Simonsen, 1998a) points to an advantage of using first stage larvae for experiments in which contamination of the parasite material with human products should be avoided, such as the preparation of a specific polyclonal anti-D. medinensis antiserum for use in diagnostic assays based on detection of circulating antigens. Cuticle associated canine IgG-like epitopes have previously been observed on adult D. immitis from dogs (Kadipasaoglu and Bilge, 1989), and human IgG-like epitopes have been observed on the sheath of W. bancrofti (Kar et al., 1993) and B. malayi (Mania and Kar, 1994) microfilariae recovered from humans. The conjugates used for detection of immunoglobulins on the surface of D. medinensis were specific for the Fc-portion of IgG, and it appears likely that the detected host immunoglobulins bound to the parasites as part of a specific anti-parasitic immune response. Nevertheless, having failed to kill the parasite, these immunoglobulins may be involved in protection of the parasite from further host immune responses, similar to the function of cuticle associated host albumin-like epitopes described above. The present study has provided information about the occurrence and localization of human-like antigen components of D. medinensis adult females and first stage larvae. This information may be used to assess antigenic characteristics of adult female worms and first stage larvae as a basis for selecting an appropriate parasite material for use in immunodiagnostic and immunoepidemiological studies.
Acknowledgements The study was carried out as part of an agreement between the Danish Bilharziasis Laboratory and the Ministry of Health of Ghana on the strengthening of the Guinea Worm Eradication Program in the Northern Region of Ghana. We thank the Ministry of Health of Ghana for its logistic and technical support, especially Dr Sam Bugri, Von Asigri, Lawrence Yelifari, Albano Bayitaa and Abdul Rahman
P. Bloch et al. / Acta Tropica 73 (1999) 135–141
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Yakubu; Professor Peter Lind and technician Anni Ravn, Danish Veterinary Laboratory, Copenhagen, Denmark, for their efforts with the preparation of cross-sections of frozen guinea worms. The study was sponsored by the Danish Bilharziasis Laboratory and the Danish International Development Agency (Danida).
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