Acta Tropica, 51(1992)167 171
167
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0001-706X/92/$05.00 ACTROP 00217 Short Communication
The isolation of the sheath/epicuticle of Brugia pahangi microfilariae B. R a v i n d r a n a a n d E. D e v a n e y b aDepartment of Applied Immunology, Regional Medical Research Centre, Chandrasekharpur, Bhubaneswar 751 016, India and ULiverpoolSchool of Tropical Medicine, Pembroke Place, Liverpool, UK
(Received 27 September 1991; Accepted 5 March 1992) Key words: Brugiapahangi; Microfilariae;Sheath; Epicuticle; Purification
Microfilariae of each of the species of lymphatic filarial nematodes are bound by a sheath, an elongate structure derived from the embryonic eggshell (Rogers et al., 1976). In some species of filarial parasite, e.g., Onchocerca volvulus, the sheath is cast while the microfilariae are in the uterus of the adult female and the microfilariae in the skin are unsheathed. In parasites such as Brugia and Wuchereria species the sheath is retained until the microfilariae are ingested by a suitable mosquito vector and exsheathment usually occurs in the mosquito mid gut. The microfilarial sheath has elicited a great deal of interest, largely because of the observation that in natural infections, the appearance of antibodies to the sheath is often correlated with the disappearance of microfilariae from the circulation (McGreevy et al., 1980). A number of monoclonal antibodies have also been described which appear to recognize surface components of the microfilariae and which clear microfilariae from the circulation when administered to experimental hosts (Canlas et al., 1984; Aggarwal et al., 1985). The immunochemical and biochemical analysis of the microfilarial sheath has been greatly hampered by the inability to obtain large quantities of pure material. Microfilariae of B. pahangi can be exsheathed by exposure to increased concentrations of Ca 2 + or to a variety of proteolytic enzymes (Devaney and Howells, 1979; Devaney, 1985). After Ca2+-induced exsheathment it is sometimes possible to observe large clumps of empty sheaths (see Fig. 3 in Devaney and Howells, 1979), but it has not proved possible to routinely use this technique to obtain purified sheaths. After enzyme exsheathment, no intact sheaths have been observed, presumably due to their proteolytic degradation, although Srivastava (1985) used this method followed by density gradient centrifugation to isolate sheaths of B. malayi. Purified sheaths of L. carinii can be obtained by a combination of freeze-thawing and filtration, but although the resulting preparation of sheaths is clean, the yield is low, approximately 15% (Bardehle et al., 1987). This method has also been adapted to Correspondenceaddress: E. Devaney,LiverpoolSchoolof Tropical Medicine,Pembroke Place, Liverpool,
L3 5QA, UK.
168
the purification of sheaths of Brugia species (Klonisch et al., 1991). In this communication we describe a simple and reproducible method for obtaining large quantities of microfilarial sheaths largely free from somatic material; while the yield from this method is high, the purified sheaths still contain the remnants of the insoluble epicuticle and are consequently not as pure as those obtained using the method of Bardehele et al. (1987). The purified sheaths/epicuticles have been subjected to a variety of physiochemical analyses and have been characterised with respect to their antigenicity. Microfilariae were harvested from the peritoneal cavity of infected jirds and separated from contaminating host cells by centrifugation on a cushion of Lymphoprep (Flow). If necessary, contaminating erythrocytes were lysed with sterile distilled water prior to the Lymphoprep step. The pellet of microfilariae was washed twice with Hanks' balanced salts solution (HBSS) and either used immediately or stored in liquid nitrogen until required. 50 gl of a microfilarial suspension (approximately 2 x 106 organisms per ml) was pelleted and resuspended in 200 gl of 10 mM Tris (pH 6.8) containing 1% sodium dodecyl sulphate (SDS) and 50 mM dithiothreitol (DTT). The microfilariae were incubated at room temperature on a rotator and examined by phase contrast microscopy after 30, 60 and 120 min of incubation. Incubation of microfilariae in 1% SDS, 50 mM DTT for 60 min resulted in the solubilization of the somatic structure of the mf (Fig. 1, panel 1) with the exception of the epicuticle, which, as in the adult parasite (Betschart et al., 1985), is resistant to SDS/DTT. Incubation of microfilariae in SDS alone or in 50 mM DTT alone was insufficient to digest the soma, with most microfilariae remaining structurally intact. Although resonably clean sheaths were obtained in 0.5% SDS and 10-50 mM DTT, the best results were obtained in 1% SDS and 10-50 mM DTT. This method has proved reproducible in many separate experiments and where the recoveries have been quantified, the yield has been in the order of 70% (63-74%). The only problem encountered has been in the extraction of a very large pellet of microfilariae ( > 1 X 106) and the subsequent lysis of the nuclei, which can lead to the trapping of cytoplasmic components in the insoluble pellet. This problem can be overcome by treatment with DNase. Microfilarial sheaths isolated by incubation for 2 h in 1% SDS, 50 mM DTT were fixed for ultrastructural examination using standard methods. Ultrastructural examination confirmed the light level observations, demonstrating the apparent structural integrity of the sheath (Fig. 1, panel 2A and B). The conditions required to produce clean sheaths are rather harsh and may result in the extraction of integral components of the sheath. The 'antigenic intactness' of the isolated sheaths was assessed by binding FITC labelled wheat-germ agglutin (Devaney, 1985) or using a cat antiserum (a kind gift from Dr. D. Denham,) which binds to the sheath of live microfilariae of B. pahangi. SDS/DTT isolated sheaths bound both WGA and the immune cat serum (Fig. 1, panel 3) as strongly as intact microfilariae. No fluoresence was observed with control cat serum. In order to better characterise the isolated sheaths/epicuticles, intact microfilariae were labelled with lzsI via the Iodogen method and the sheaths purified as described above. The conditions used for iodination were 100 gg Iodogen, 400 gCi 125I for 10 min at room temperature. Following the iodination, the parasites were washed and then extracted for 2 h at room temperature in 1% SDS, 100 mM DTT. In two separate experiments, the proportion of radioactivity associated with the SDS/DTT-
169
A
B O.51 ~
Fig 1. Panel (1) Population of isolated sheaths after incubation for 60 min in 1% SDS, 50 mM DTT. × 700. (2A) Isolated sheaths were fixed for 60 rain in 3% glutaraldehyde in cacodylate buffer and post-fixed in osmium, embedded in Epon/araldite and stained en bloc with uranyl acetate. Sections were stained with uranyl acetate and lead citrate following standard methods. (2B) Higher magnification to show structure of cuticle and sheath. (3) Isolated sheath of B. pahangi mf reacted with 1/20 dilution of immune cat serum, followed by 1/60 FITC anti-cat IgG. x 200.
soluble s u p e r n a t a n t a n d insoluble sheath/epicuticle fraction was quantified. Th e c o u n t s associated with the S D S / D T T - s o l u b l e s u p e r n a t a n t were e s t i m a t e d by precipitation o f d u p li c a te a l i q u o t s with ice-cold t r i ch l o r o acet i c acid; the counts associated
170 with the insoluble fraction were estimated by counting duplicate aliquots in the gamma counter. After SDS-DTT extraction of 125I labelled mf, 13-16% of the counts were associated with the soluble supernatant and 84 87% of the counts with the insoluble sheath/epicuticle fraction. Whether the radioactivity in the sheath pellet was associated with the sheath itself or the epicuticle is more difficult to define. A previous study using the same labelling method (Philipp et al., 1986) had shown by ultrastructural autoradiography that most of the silver grains appeared to be over the sheath itself. In an attempt to address this question, the susceptibility of the isolated sheaths/ epicuticles to various enzymes was investigated. 125I-labelled sheaths were incubated with elastase (Sigma type IV, 200 ~tg), pronase E (Sigma proteinase type XXV, 50 p.g) or chitinase (Sigma from Streptomyces gr&eus, 10 units). Pronase and elastase treatments were carried out at 37°C for 60 min, and treatment with chitinase overnight at 28°C in the presence of a cocktail of proteinase inhibitors (2 mM PMSF, 200 p.M TPCK, 200 ~tM TLCK; Devaney, 1988). Control tubes contained sheaths and buffer alone. Following the incubation, the sheaths were spun through a Spinnex 0.45 lam spin filter. The supernatant and the retained sheaths were counted in the gamma counter. The susceptibility of unlabelled purified sheaths to these enzymes was followed in parallel by phase contrast microscopy. Treatment of the insoluble sheath fraction with either pronase or elastase released approx. 80% of the counts to the supernatant. Microscopic observations of purified sheaths exposed to elastase demonstrated that, as in the adult parasite (Selkirk et al., 1989), the epicuticle was degraded. The sheaths appeared morphologically intact, but the pellet of sheaths was greatly reduced in size, suggesting that the sheaths were partially digested by elastase. Incubation in pronase digested the sheath into small pieces leaving only traces of the epicuticle from the tail-end of the microfilariae intact. Thus using these enzymes it was not possible to distinguish whether the radioactivity was associated with the sheath or epicuticle. Treatment of isolated B. pahangi sheaths with chitinase in the presence of proteinase inhibitors had no effect on the structure of the sheath; equivalent numbers of counts were released to the supernatant with chitinase or control buffer when 12SI-labelled sheaths were used. In conclusion, we have described a fast and simple method for obtaining resonably clean sheaths of microfilariae of B. pahangi. The purified sheaths appear to be antigenically intact and would therefore be a useful source of antigen for the preparation of sheath-specific antibodies.
Acknowledgements B.R. was supported by a W H O Training Fellowship. E.D. is a Medical Research Council Senior Fellow (Non-Clinical). We thank Dr. D.A. Denham, of the London School of Hygiene and Tropical Medicine, for supplying the immune cat serum.
References Aggarwal, A., Cuna, W., Haque, A., Dissous, C. and Capron, A. (1985)Resistanceagainst Brugiamalayi microfilariaeinduced by a monoclonalantibody which promotes killing by macrophagesand recognizes surface antigen(s). Immunology 54, 655-663.
171 Bardehle, G., Klonisch, Th., Schott, H.-H., Stirm, S. and Zahner, H. (1987) Isolation of pure sheaths of Lmitomosoides carinii microfilariae. Parasitol. Res. 74, 188-190. Betschart, B., Rudin, W. and Weiss, N. (1985) The isolation and immunogenicity of the cuticle of Dipetalonema vitae (Filarioidea). Z. Parasitenkd. 71, 87-95. Canlas, M., Wadee, A., Lamontagne, L. and Piessens, W.F. (1984) A monoclonal antibody to surface antigens on microfilariae of Brugia malayi reduces microfilaremia in infected jirds. Am. J. Trop. Med. Hyg. 33, 420-424. Devaney, E. (1985) Lecitin-binding characteristics of Brugia pahangi microfilariae. Trop. Med. Parasit. 36, 25-28. Devaney, E. (1988) The biochemical and immunochemical characterisation of the 30 kilodalton surface antigen of Brugia pahangi. Mol. Biochem. Parasitol. 27, 83-92. Devaney, E. and Howells, R.E. (1979) The exsheathment of Brugia pahangi microfilariae under control conditions in vitro. Ann. Trop. Med. Parasitol. 73, 227-233~ Klonisch, T., Bardehle, G., Linder, D., Boschek, B., Schott, H.-H., Zahner, H. and Stirm, S. (1991) The sheaths of Brugia microfilariae: isolation and composition. Parasitol. Res. 77, 448-451. McGreevy, P.B., Ratiwayanto, S., Sekar Tuti, McGreevy, M.M. and Dennis, D.T. (1980) Brugia malayi: relationship between anti-sheath antibodies and amicrofilaremia in natives living in an endemic area of South Kalimantan, Borneo. Am. J. Trop. Med. Hyg. 29, 553-562. Philipp, M., Maizels, R.M., McLaren, D.J., Davies, M.W., Suswillo, R. and Denham, D.A. (1986) Expression of cross-reactive surface antigens by microfilariae and adult worms of Brugia pahangi during infections in cats. Trans. R. Soe. Trop. Med. Hyg. 80, 385-393. Rogers, R., Ellis, D.S. and Denham, D.A. (1976) Studies with Brugia pahangi 14. Intrauterine development of the microfilaria and a comparison with other filarial species. J. Helminth. 50, 251-257. Selkirk, M.E., Nielsen, L., Kelly, C., Partono, F., Sayers, G. and Maizels, R.M. (1989) Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and Brugia pahangi. Mol. Biochem. Parasitol. 32, 229-246. Srivastava, A.K. (1985) A method for isolation and purification of the sheath of microfilariae of Brugia malavi. J. Parasitol. 71,257-258.
167
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0001-706X/92/$05.00 ACTROP 00217 Short Communication
The isolation of the sheath/epicuticle of Brugia pahangi microfilariae B. R a v i n d r a n a a n d E. D e v a n e y b aDepartment of Applied Immunology, Regional Medical Research Centre, Chandrasekharpur, Bhubaneswar 751 016, India and ULiverpoolSchool of Tropical Medicine, Pembroke Place, Liverpool, UK
(Received 27 September 1991; Accepted 5 March 1992) Key words: Brugiapahangi; Microfilariae;Sheath; Epicuticle; Purification
Microfilariae of each of the species of lymphatic filarial nematodes are bound by a sheath, an elongate structure derived from the embryonic eggshell (Rogers et al., 1976). In some species of filarial parasite, e.g., Onchocerca volvulus, the sheath is cast while the microfilariae are in the uterus of the adult female and the microfilariae in the skin are unsheathed. In parasites such as Brugia and Wuchereria species the sheath is retained until the microfilariae are ingested by a suitable mosquito vector and exsheathment usually occurs in the mosquito mid gut. The microfilarial sheath has elicited a great deal of interest, largely because of the observation that in natural infections, the appearance of antibodies to the sheath is often correlated with the disappearance of microfilariae from the circulation (McGreevy et al., 1980). A number of monoclonal antibodies have also been described which appear to recognize surface components of the microfilariae and which clear microfilariae from the circulation when administered to experimental hosts (Canlas et al., 1984; Aggarwal et al., 1985). The immunochemical and biochemical analysis of the microfilarial sheath has been greatly hampered by the inability to obtain large quantities of pure material. Microfilariae of B. pahangi can be exsheathed by exposure to increased concentrations of Ca 2 + or to a variety of proteolytic enzymes (Devaney and Howells, 1979; Devaney, 1985). After Ca2+-induced exsheathment it is sometimes possible to observe large clumps of empty sheaths (see Fig. 3 in Devaney and Howells, 1979), but it has not proved possible to routinely use this technique to obtain purified sheaths. After enzyme exsheathment, no intact sheaths have been observed, presumably due to their proteolytic degradation, although Srivastava (1985) used this method followed by density gradient centrifugation to isolate sheaths of B. malayi. Purified sheaths of L. carinii can be obtained by a combination of freeze-thawing and filtration, but although the resulting preparation of sheaths is clean, the yield is low, approximately 15% (Bardehle et al., 1987). This method has also been adapted to Correspondenceaddress: E. Devaney,LiverpoolSchoolof Tropical Medicine,Pembroke Place, Liverpool,
L3 5QA, UK.
168
the purification of sheaths of Brugia species (Klonisch et al., 1991). In this communication we describe a simple and reproducible method for obtaining large quantities of microfilarial sheaths largely free from somatic material; while the yield from this method is high, the purified sheaths still contain the remnants of the insoluble epicuticle and are consequently not as pure as those obtained using the method of Bardehele et al. (1987). The purified sheaths/epicuticles have been subjected to a variety of physiochemical analyses and have been characterised with respect to their antigenicity. Microfilariae were harvested from the peritoneal cavity of infected jirds and separated from contaminating host cells by centrifugation on a cushion of Lymphoprep (Flow). If necessary, contaminating erythrocytes were lysed with sterile distilled water prior to the Lymphoprep step. The pellet of microfilariae was washed twice with Hanks' balanced salts solution (HBSS) and either used immediately or stored in liquid nitrogen until required. 50 gl of a microfilarial suspension (approximately 2 x 106 organisms per ml) was pelleted and resuspended in 200 gl of 10 mM Tris (pH 6.8) containing 1% sodium dodecyl sulphate (SDS) and 50 mM dithiothreitol (DTT). The microfilariae were incubated at room temperature on a rotator and examined by phase contrast microscopy after 30, 60 and 120 min of incubation. Incubation of microfilariae in 1% SDS, 50 mM DTT for 60 min resulted in the solubilization of the somatic structure of the mf (Fig. 1, panel 1) with the exception of the epicuticle, which, as in the adult parasite (Betschart et al., 1985), is resistant to SDS/DTT. Incubation of microfilariae in SDS alone or in 50 mM DTT alone was insufficient to digest the soma, with most microfilariae remaining structurally intact. Although resonably clean sheaths were obtained in 0.5% SDS and 10-50 mM DTT, the best results were obtained in 1% SDS and 10-50 mM DTT. This method has proved reproducible in many separate experiments and where the recoveries have been quantified, the yield has been in the order of 70% (63-74%). The only problem encountered has been in the extraction of a very large pellet of microfilariae ( > 1 X 106) and the subsequent lysis of the nuclei, which can lead to the trapping of cytoplasmic components in the insoluble pellet. This problem can be overcome by treatment with DNase. Microfilarial sheaths isolated by incubation for 2 h in 1% SDS, 50 mM DTT were fixed for ultrastructural examination using standard methods. Ultrastructural examination confirmed the light level observations, demonstrating the apparent structural integrity of the sheath (Fig. 1, panel 2A and B). The conditions required to produce clean sheaths are rather harsh and may result in the extraction of integral components of the sheath. The 'antigenic intactness' of the isolated sheaths was assessed by binding FITC labelled wheat-germ agglutin (Devaney, 1985) or using a cat antiserum (a kind gift from Dr. D. Denham,) which binds to the sheath of live microfilariae of B. pahangi. SDS/DTT isolated sheaths bound both WGA and the immune cat serum (Fig. 1, panel 3) as strongly as intact microfilariae. No fluoresence was observed with control cat serum. In order to better characterise the isolated sheaths/epicuticles, intact microfilariae were labelled with lzsI via the Iodogen method and the sheaths purified as described above. The conditions used for iodination were 100 gg Iodogen, 400 gCi 125I for 10 min at room temperature. Following the iodination, the parasites were washed and then extracted for 2 h at room temperature in 1% SDS, 100 mM DTT. In two separate experiments, the proportion of radioactivity associated with the SDS/DTT-
169
A
B O.51 ~
Fig 1. Panel (1) Population of isolated sheaths after incubation for 60 min in 1% SDS, 50 mM DTT. × 700. (2A) Isolated sheaths were fixed for 60 rain in 3% glutaraldehyde in cacodylate buffer and post-fixed in osmium, embedded in Epon/araldite and stained en bloc with uranyl acetate. Sections were stained with uranyl acetate and lead citrate following standard methods. (2B) Higher magnification to show structure of cuticle and sheath. (3) Isolated sheath of B. pahangi mf reacted with 1/20 dilution of immune cat serum, followed by 1/60 FITC anti-cat IgG. x 200.
soluble s u p e r n a t a n t a n d insoluble sheath/epicuticle fraction was quantified. Th e c o u n t s associated with the S D S / D T T - s o l u b l e s u p e r n a t a n t were e s t i m a t e d by precipitation o f d u p li c a te a l i q u o t s with ice-cold t r i ch l o r o acet i c acid; the counts associated
170 with the insoluble fraction were estimated by counting duplicate aliquots in the gamma counter. After SDS-DTT extraction of 125I labelled mf, 13-16% of the counts were associated with the soluble supernatant and 84 87% of the counts with the insoluble sheath/epicuticle fraction. Whether the radioactivity in the sheath pellet was associated with the sheath itself or the epicuticle is more difficult to define. A previous study using the same labelling method (Philipp et al., 1986) had shown by ultrastructural autoradiography that most of the silver grains appeared to be over the sheath itself. In an attempt to address this question, the susceptibility of the isolated sheaths/ epicuticles to various enzymes was investigated. 125I-labelled sheaths were incubated with elastase (Sigma type IV, 200 ~tg), pronase E (Sigma proteinase type XXV, 50 p.g) or chitinase (Sigma from Streptomyces gr&eus, 10 units). Pronase and elastase treatments were carried out at 37°C for 60 min, and treatment with chitinase overnight at 28°C in the presence of a cocktail of proteinase inhibitors (2 mM PMSF, 200 p.M TPCK, 200 ~tM TLCK; Devaney, 1988). Control tubes contained sheaths and buffer alone. Following the incubation, the sheaths were spun through a Spinnex 0.45 lam spin filter. The supernatant and the retained sheaths were counted in the gamma counter. The susceptibility of unlabelled purified sheaths to these enzymes was followed in parallel by phase contrast microscopy. Treatment of the insoluble sheath fraction with either pronase or elastase released approx. 80% of the counts to the supernatant. Microscopic observations of purified sheaths exposed to elastase demonstrated that, as in the adult parasite (Selkirk et al., 1989), the epicuticle was degraded. The sheaths appeared morphologically intact, but the pellet of sheaths was greatly reduced in size, suggesting that the sheaths were partially digested by elastase. Incubation in pronase digested the sheath into small pieces leaving only traces of the epicuticle from the tail-end of the microfilariae intact. Thus using these enzymes it was not possible to distinguish whether the radioactivity was associated with the sheath or epicuticle. Treatment of isolated B. pahangi sheaths with chitinase in the presence of proteinase inhibitors had no effect on the structure of the sheath; equivalent numbers of counts were released to the supernatant with chitinase or control buffer when 12SI-labelled sheaths were used. In conclusion, we have described a fast and simple method for obtaining resonably clean sheaths of microfilariae of B. pahangi. The purified sheaths appear to be antigenically intact and would therefore be a useful source of antigen for the preparation of sheath-specific antibodies.
Acknowledgements B.R. was supported by a W H O Training Fellowship. E.D. is a Medical Research Council Senior Fellow (Non-Clinical). We thank Dr. D.A. Denham, of the London School of Hygiene and Tropical Medicine, for supplying the immune cat serum.
References Aggarwal, A., Cuna, W., Haque, A., Dissous, C. and Capron, A. (1985)Resistanceagainst Brugiamalayi microfilariaeinduced by a monoclonalantibody which promotes killing by macrophagesand recognizes surface antigen(s). Immunology 54, 655-663.
171 Bardehle, G., Klonisch, Th., Schott, H.-H., Stirm, S. and Zahner, H. (1987) Isolation of pure sheaths of Lmitomosoides carinii microfilariae. Parasitol. Res. 74, 188-190. Betschart, B., Rudin, W. and Weiss, N. (1985) The isolation and immunogenicity of the cuticle of Dipetalonema vitae (Filarioidea). Z. Parasitenkd. 71, 87-95. Canlas, M., Wadee, A., Lamontagne, L. and Piessens, W.F. (1984) A monoclonal antibody to surface antigens on microfilariae of Brugia malayi reduces microfilaremia in infected jirds. Am. J. Trop. Med. Hyg. 33, 420-424. Devaney, E. (1985) Lecitin-binding characteristics of Brugia pahangi microfilariae. Trop. Med. Parasit. 36, 25-28. Devaney, E. (1988) The biochemical and immunochemical characterisation of the 30 kilodalton surface antigen of Brugia pahangi. Mol. Biochem. Parasitol. 27, 83-92. Devaney, E. and Howells, R.E. (1979) The exsheathment of Brugia pahangi microfilariae under control conditions in vitro. Ann. Trop. Med. Parasitol. 73, 227-233~ Klonisch, T., Bardehle, G., Linder, D., Boschek, B., Schott, H.-H., Zahner, H. and Stirm, S. (1991) The sheaths of Brugia microfilariae: isolation and composition. Parasitol. Res. 77, 448-451. McGreevy, P.B., Ratiwayanto, S., Sekar Tuti, McGreevy, M.M. and Dennis, D.T. (1980) Brugia malayi: relationship between anti-sheath antibodies and amicrofilaremia in natives living in an endemic area of South Kalimantan, Borneo. Am. J. Trop. Med. Hyg. 29, 553-562. Philipp, M., Maizels, R.M., McLaren, D.J., Davies, M.W., Suswillo, R. and Denham, D.A. (1986) Expression of cross-reactive surface antigens by microfilariae and adult worms of Brugia pahangi during infections in cats. Trans. R. Soe. Trop. Med. Hyg. 80, 385-393. Rogers, R., Ellis, D.S. and Denham, D.A. (1976) Studies with Brugia pahangi 14. Intrauterine development of the microfilaria and a comparison with other filarial species. J. Helminth. 50, 251-257. Selkirk, M.E., Nielsen, L., Kelly, C., Partono, F., Sayers, G. and Maizels, R.M. (1989) Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and Brugia pahangi. Mol. Biochem. Parasitol. 32, 229-246. Srivastava, A.K. (1985) A method for isolation and purification of the sheath of microfilariae of Brugia malavi. J. Parasitol. 71,257-258.
