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Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use
fnternorionnlJournolfor Primed in Great Brrrain
Parnsimlogy
002~7519/89 53.00 + 0.00 Pergamon Press p/c % 1989 Ausrrolian Socieryfor Porosirology
Vol. 19, No. 8. pp. 957-959, 1989
RESEARCHNOTE ISOLATION
OF ONCHOCERCA GIBSONITISSUE FROM NODULES FOR BIOCHEMICAL USE
FROZEN
L. H. SEMPREVIVO and M. D. MALONEY Department
of Zoology,
University
of Massachusetts,
Amherst,
(Received 29 June 1989; accepted 29 Augur
MA 01003, U.S.A.
1989)
L. H. and MALONEY M. D. 1989. Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use. International Journal for Parasitology 19: 957-959. A new procedure is described which enables gram quantities of adult Onchocerca tissue to be isolated from frozen connective tissue nodules, thus minimizing the risk of enzymatic degradation. Bovine connective tissue nodules containing adult Onchocerca gibsoni worms were obtained from Australia frozen at - 70°C and sectioned while still frozen into 3 mm thick slabs. The sections were thawed immediately before use, worm segments removed, rinsed, pelleted, and flash frozen in liquid nitrogen. Quality of the isolated material was demonstrated by the presence of an intact adult epicuticle as determined by electron microscopy, and by the presence ofviable uterine larvae and cells. This procedure is applicable to other nodule-forming worms such as Onchocerca volvulus and is suitable for investigations which require the isolation of labile molecules or those present in minute quantities, AbStraCt-SEMPREVIVO
INDEX
KEY WORDS:
Glycolipids;
Onchocerca gibsoni; epicuticle;
tissue isolation.
being cut. The frozen slabs were cleaned of kerf by gentle scraping with a single-edged razor blade and separated into individual nodule sections which were kept frozen on solid CO, until processed. The nodule sections were then placed under a dissecting microscope, thawed, and the worm segments rapidly harvested (within 30 min) using fine-tipped forceps. The segments were collected on a 30-mesh stainless steel screen in ice cold Hanks’ balanced salt solution (HBSS), pH 7.4 (without glucose, phenol red or sodium bicarbonate), and rinsed free of host material by passing excess ice cold HBSS over them. The worm material was then transferred to a Petri dish containing ice cold HBSS and inspected under a dissecting microscope for the presence of host tissue or abnormal worm material. The worm segments were transferred to a pre-weighed microcentrifuge tube and centrifuged at 11,600 gfor 3 min at 4°C (Eppendorf Centrifuge No. 5412). The supernatant was removed, pellet weight calculated, and the tube with pellet flash frozen in liquid nitrogen and stored at - 100°C until used. To check the quality of the isolated material, samples were examined for cell viability, motility of uterine larvae, and the integrity of the worm surface. Parasite components escaping from the cut ends of the adult worm pieces were examined for viability using erythrocin B (Hodgkinson & Herman, 1980). In all samples inspected, more than 80% of the cells examined, including those of the intra-uterine larvae and eggs, did not absorb dye thus indicating their
THE objective of our research is to investigate the glycolipids of Unchocerca gibsoni adults as a possible
source of vaccine components. The quantity of glycolipids needed for this experimentation requires that gram amounts of pristine adult worm tissue routinely be available for extraction. Nodules containing 0. gibsoni adults are available from Australia but for practical reasons of preservation are distributed frozen. Freezing and thawing of such material may be expected to result in some cellular damage and release of host and parasite enzymes. Methods available to isolate adult worms from encapsulating host connective tissue, including simple dissection or collagenase digestion, either could not produce the quantity of worm tissue needed or required prolonged periods for isolation during which there was a substantial risk of enzymatic degradation. The technique described below was developed to permit efficient isolation of gram quantities of adult worm tissue from frozen nodules with minimal chance for enzymatic degradation. Bovine connective tissue nodules containing 0. gibsoni adults were collected at abattoirs in Australia, shipped to us on solid CO, frozen in aggregates of approximately 1 dozen, and stored at - 100°C until processed for adult worm tissue. To collect worm material, an aggregate of nodules was sectioned into slabs 3 mm thick using an electric scroll saw (Sears) equipped with a 0.5 mm thick blade oscillating at 1700 strokes min-‘. All slabs were processed within 8 h of 957
L. H. SEMPREVWO
and M. D. MALONEY
FIG. 1. Electron micrographs of outer surface of 0. gibsoni adult segments. (A) Section of outer cuticle of 0. gibsoni segment isolated as described in text. Scale bar = 1.5 pm. (B) Micrograph of the outer surface. Scale bar = 0.2 pm. (C) Higher magnification micrograph of outer surface showing the surface folds and trilaminate structure of the epicuticle. Scale bar = 0.1 pm.
Research Note viability. It appears that minimal during the isolation procedure.
tissue damage occurs
The epicuticular surface of the isolated worm segments was examined by electron microscopy. of 2% Segments were fixed in a solution glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, at 4°C for 2 h. The tissue was then washed in the cacodylate buffer and post-fixed in 1% osmium tetroxide in the same buffer at 4°C for 4 h. Dehydration was performed in ethanol with embedment in Epon-Araldite. Thin sections were cut with diamond knives on a Reichert Ultracut, mounted on naked 2~i3~ mesh copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 100 S electron microscope operating at 80 kv. Electron micrographs of the adult surface are shown in Fig. 1. The epicuticle with its characteristic sinuous folds (Franz, Schulz-Key & Copeman, 1987) appears to be intact and free of host material contamination. The epicuticular structure observed is very similar to that reported for Onchocercu volvulus in which living worms were fixed for electron microscopy in situ within a nodule (Deas, Aguilar & Miller, 1974). These results indicate that the procedure described here for isolating adult worm material results in removal of superficial host material associated in the nodule with the worm surface but preserves the worm surface itself. A third check of the quality of the isolated material was made by incubating the uterine larvae which spilled from the cut worms in HBSS. It was found that after a few minutes to an hour at room temperature a high percentage of the larvae became active attesting to the biological integrity of the isolated material. In cases where nodules had thawed and were refrozen, the associated uterine larvae did not become active.
959
Because thawing and refreezing of nodules decreases the quality of worm tissues, such nodules were deemed unsuitable for our procedure and were not used as a source of worm material. Collection followed by immediate freezing of connective tissue nodules is a convenient means by which adult worms of nodule-forming Onchocerca species may be preserved for later use or distribution (Greene, 1988). The procedure described here provides a means by which gram quantities of worm tissue may be harvested efficiently from these frozen nodules with minimal risk of degradation. The procedure may be of value to investigations requiring the isolation of labile molecules or those present in minute quantities. Acknowledgements-We thank Dr D. B. Copeman for supplying the 0. gibsoni nodules and for generously sharing his knowledge of Onchocerca. We also thank Lucy Yin, Microscopy Center, University of Massachusetts, and Judy Semprevivo for technical assistance. This work was supported by a grant from the Edna McConnell Clark Foundation.
REFERENCES DEASJ. E., AGUILARF. J. & MILLERJ. H. 1974. Fine structure of the cuticle of female Onchocerca volvulus. Journal of
Parasitology 60: 1006-1012. FRANZM., SCHULZ-KEYH. & COPEMAND. B. 1987. Electron-
microscopic observations on the female worms of six Onchocercu species from cattle and red deer. Parasitology Research 74: 73-83. GREENE B. M. 1988. Handling
of Onehocereu VOICES nodules and isolated worms for immunologic studies.
Tropical Medicine and Parasitology 39: 464. HODGKINSONV. H. & HERMAN R. 1980. in vivo assay of viability of amastigotes of Leishmania donovani. Journal of Parasitology 66: 245-249.
Parnsimlogy
002~7519/89 53.00 + 0.00 Pergamon Press p/c % 1989 Ausrrolian Socieryfor Porosirology
Vol. 19, No. 8. pp. 957-959, 1989
RESEARCHNOTE ISOLATION
OF ONCHOCERCA GIBSONITISSUE FROM NODULES FOR BIOCHEMICAL USE
FROZEN
L. H. SEMPREVIVO and M. D. MALONEY Department
of Zoology,
University
of Massachusetts,
Amherst,
(Received 29 June 1989; accepted 29 Augur
MA 01003, U.S.A.
1989)
L. H. and MALONEY M. D. 1989. Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use. International Journal for Parasitology 19: 957-959. A new procedure is described which enables gram quantities of adult Onchocerca tissue to be isolated from frozen connective tissue nodules, thus minimizing the risk of enzymatic degradation. Bovine connective tissue nodules containing adult Onchocerca gibsoni worms were obtained from Australia frozen at - 70°C and sectioned while still frozen into 3 mm thick slabs. The sections were thawed immediately before use, worm segments removed, rinsed, pelleted, and flash frozen in liquid nitrogen. Quality of the isolated material was demonstrated by the presence of an intact adult epicuticle as determined by electron microscopy, and by the presence ofviable uterine larvae and cells. This procedure is applicable to other nodule-forming worms such as Onchocerca volvulus and is suitable for investigations which require the isolation of labile molecules or those present in minute quantities, AbStraCt-SEMPREVIVO
INDEX
KEY WORDS:
Glycolipids;
Onchocerca gibsoni; epicuticle;
tissue isolation.
being cut. The frozen slabs were cleaned of kerf by gentle scraping with a single-edged razor blade and separated into individual nodule sections which were kept frozen on solid CO, until processed. The nodule sections were then placed under a dissecting microscope, thawed, and the worm segments rapidly harvested (within 30 min) using fine-tipped forceps. The segments were collected on a 30-mesh stainless steel screen in ice cold Hanks’ balanced salt solution (HBSS), pH 7.4 (without glucose, phenol red or sodium bicarbonate), and rinsed free of host material by passing excess ice cold HBSS over them. The worm material was then transferred to a Petri dish containing ice cold HBSS and inspected under a dissecting microscope for the presence of host tissue or abnormal worm material. The worm segments were transferred to a pre-weighed microcentrifuge tube and centrifuged at 11,600 gfor 3 min at 4°C (Eppendorf Centrifuge No. 5412). The supernatant was removed, pellet weight calculated, and the tube with pellet flash frozen in liquid nitrogen and stored at - 100°C until used. To check the quality of the isolated material, samples were examined for cell viability, motility of uterine larvae, and the integrity of the worm surface. Parasite components escaping from the cut ends of the adult worm pieces were examined for viability using erythrocin B (Hodgkinson & Herman, 1980). In all samples inspected, more than 80% of the cells examined, including those of the intra-uterine larvae and eggs, did not absorb dye thus indicating their
THE objective of our research is to investigate the glycolipids of Unchocerca gibsoni adults as a possible
source of vaccine components. The quantity of glycolipids needed for this experimentation requires that gram amounts of pristine adult worm tissue routinely be available for extraction. Nodules containing 0. gibsoni adults are available from Australia but for practical reasons of preservation are distributed frozen. Freezing and thawing of such material may be expected to result in some cellular damage and release of host and parasite enzymes. Methods available to isolate adult worms from encapsulating host connective tissue, including simple dissection or collagenase digestion, either could not produce the quantity of worm tissue needed or required prolonged periods for isolation during which there was a substantial risk of enzymatic degradation. The technique described below was developed to permit efficient isolation of gram quantities of adult worm tissue from frozen nodules with minimal chance for enzymatic degradation. Bovine connective tissue nodules containing 0. gibsoni adults were collected at abattoirs in Australia, shipped to us on solid CO, frozen in aggregates of approximately 1 dozen, and stored at - 100°C until processed for adult worm tissue. To collect worm material, an aggregate of nodules was sectioned into slabs 3 mm thick using an electric scroll saw (Sears) equipped with a 0.5 mm thick blade oscillating at 1700 strokes min-‘. All slabs were processed within 8 h of 957
L. H. SEMPREVWO
and M. D. MALONEY
FIG. 1. Electron micrographs of outer surface of 0. gibsoni adult segments. (A) Section of outer cuticle of 0. gibsoni segment isolated as described in text. Scale bar = 1.5 pm. (B) Micrograph of the outer surface. Scale bar = 0.2 pm. (C) Higher magnification micrograph of outer surface showing the surface folds and trilaminate structure of the epicuticle. Scale bar = 0.1 pm.
Research Note viability. It appears that minimal during the isolation procedure.
tissue damage occurs
The epicuticular surface of the isolated worm segments was examined by electron microscopy. of 2% Segments were fixed in a solution glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, at 4°C for 2 h. The tissue was then washed in the cacodylate buffer and post-fixed in 1% osmium tetroxide in the same buffer at 4°C for 4 h. Dehydration was performed in ethanol with embedment in Epon-Araldite. Thin sections were cut with diamond knives on a Reichert Ultracut, mounted on naked 2~i3~ mesh copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 100 S electron microscope operating at 80 kv. Electron micrographs of the adult surface are shown in Fig. 1. The epicuticle with its characteristic sinuous folds (Franz, Schulz-Key & Copeman, 1987) appears to be intact and free of host material contamination. The epicuticular structure observed is very similar to that reported for Onchocercu volvulus in which living worms were fixed for electron microscopy in situ within a nodule (Deas, Aguilar & Miller, 1974). These results indicate that the procedure described here for isolating adult worm material results in removal of superficial host material associated in the nodule with the worm surface but preserves the worm surface itself. A third check of the quality of the isolated material was made by incubating the uterine larvae which spilled from the cut worms in HBSS. It was found that after a few minutes to an hour at room temperature a high percentage of the larvae became active attesting to the biological integrity of the isolated material. In cases where nodules had thawed and were refrozen, the associated uterine larvae did not become active.
959
Because thawing and refreezing of nodules decreases the quality of worm tissues, such nodules were deemed unsuitable for our procedure and were not used as a source of worm material. Collection followed by immediate freezing of connective tissue nodules is a convenient means by which adult worms of nodule-forming Onchocerca species may be preserved for later use or distribution (Greene, 1988). The procedure described here provides a means by which gram quantities of worm tissue may be harvested efficiently from these frozen nodules with minimal risk of degradation. The procedure may be of value to investigations requiring the isolation of labile molecules or those present in minute quantities. Acknowledgements-We thank Dr D. B. Copeman for supplying the 0. gibsoni nodules and for generously sharing his knowledge of Onchocerca. We also thank Lucy Yin, Microscopy Center, University of Massachusetts, and Judy Semprevivo for technical assistance. This work was supported by a grant from the Edna McConnell Clark Foundation.
REFERENCES DEASJ. E., AGUILARF. J. & MILLERJ. H. 1974. Fine structure of the cuticle of female Onchocerca volvulus. Journal of
Parasitology 60: 1006-1012. FRANZM., SCHULZ-KEYH. & COPEMAND. B. 1987. Electron-
microscopic observations on the female worms of six Onchocercu species from cattle and red deer. Parasitology Research 74: 73-83. GREENE B. M. 1988. Handling
of Onehocereu VOICES nodules and isolated worms for immunologic studies.
Tropical Medicine and Parasitology 39: 464. HODGKINSONV. H. & HERMAN R. 1980. in vivo assay of viability of amastigotes of Leishmania donovani. Journal of Parasitology 66: 245-249.