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A new simplified assay for larval migration inhibition
hrernarional Journalfor Printed in Great Britain
Parasitology
002~7519/92 $5.00 + 0.00 Pergamon Press Lid Socieryfor Pmo~irologv
Vol. 22, No. 8, pp. I183-1185, 1992
CQ 1992 Awrralian
~~~EA~C~~~~~ A NEW SIMPLIFIED
ASSAY FOR LARVAL MIGRATION
INHIBITION
B. M. WAGLAND, W. 0. JONES,L. HRIBAR, T. BENDIXSENand D. L. EMERY CSIRO Division of Animal Health, McMaster Laboratory, Private Bag No. 1, Glebe, New South Wales 2037, Australia (Received 9 March 1992; accepted 4 September 1992)
AIrstract-WAoLANo B. M., JONESW. O., HR~BARL., BENDIXSEN T. and EMERYD. L. 1992. A new simplified assay for larval migration inhibition. International Journal for Parasitology 22: 1183-I 185. A simple method is described for the in vitro detection of substances that impair the motility of third-stage larvae of gastro-int~tinal nematodes. The test is based on the ability of larvae to freely migrate through selected mesh sizes of nylon sieves and the reduced ability of larvae to migrate after preincubation with, and in the presence of, substances that inhibit or reduce larval motility. INDEX KEY WORDS: Trichosfrongylus calubriformis; sheep; larval migration inhibition assay; in vitro.
A number of in vitro assays have been described for screening potential anthelmintics with toxic or paralysing properties and for the detection of resistance to these anthelmintics in parasites. These assays
generally depend on subjective assessment of paralysis, inhibited development or quantitative measurement by photodetectors of reduced movement (Martin & Le Jambre, 1979; Boisvenue, Brand& Galloway & Hendrix, 1983; Dobson, Donald, Waller & Snowdon, 1986; Gill, Redwin, Van Wyk & Lacey, 1991; Folz, Pax, Thomas, Bennett, Lee & Conder, 1987). Unfortunately, these assays are not readily adaptable for detecting antiparasitic activity in complex biological samples such as mucus or intestinal contents. However, Douch, Harrison, Buchanan & Greer (1983) developed an in vitro assay for the presence of ‘antiparasitic’ substances in intestinal mucus while investigating rejection of Trichostrongylas coi~~rl~r~~s larvae from immune sheep. In their assay, mucus from immune sheep was mixed with exsheathed larvae, incorporated in agar blocks and the proportion of larvae that migrated out of the agar blocks was counted. This larval migration inhibition (LMI) assay has been used by Kimambo & MacRae (1988) to confirm that intestinal mucus and digesta from immune sheep contained substance(s) capable of immobilizing parasitic third-stage T. coEubrt$ormis larvae. However, attempts to use the LMI assay at this laboratory have been frustrated by difficulties in the preparation of the agar gel blocks containing viable larvae and the putative inhibitory substances. A modi~~ation of the method of Douch et al. (1983) was described by Gamble & Zajac (1992) to measure the factors in abomasal mucosa with
paralysing
against exsheathed larvae of but this modification also depended on the migration of larvae out of agar/ agarose blocks. The published assays also depend on the inhibitory substance acting immediately and irreversibly on larvae, the dist~bution of larvae within the agar block arid whether all larvae had an equal opportunity to migrate out of the agar block. This communication describes an alternative and simpler technique for performance of LMI assays and is based on the ability of larvae to migrate through fine nylon mesh after preincubation and while in continuous contact with substances affecting larval motility. The modified test is easier to perform than the Douch et al. (1983) and Gamble & Zajac (1992) assays, and is amenable for testing large numbers of samples. In the modified LMI assay, infective larvae of T. ~olubrt~orm~ were exsheathed as described by O’Donnell, Dineen, Wagland, Letho, Dopheide, Grant & Ward (1989), and the exsheathed larvae were separated from the sheaths by allowing the exsheathed larvae, but not sheaths, to pass through a 20 pm mesh sieve. Exsheathed larvae remained viable and suitable for use in LMI assays if stored at a concentration of 2000 larvae ml-’ in flat containers at 4°C. The modified LMI assay was performed using small sieves made from 35 mm lengths of glass tubing (7 mm OD, 6 mm ID) rather than agar blocks. The tubes were constructed by grinding one end of the tube with abrasive paper and attaching a 10 mm circle of nylon mesh (Swiss Screens, Australia) with epoxy resin. Excess mesh was Haemonchus
1183
activity
contortus,
1184
B. M. WAGLAND,
W. 0.
JONES, L. HRIBAR, T. BENDIXSEN and D. L. EMERY
100
Lrl 0
60
k
2 A
60
40 20
a
00.0I
09
Levomisole
1.0
IO
concentration
(pg
FIG. 1. Percentage of T. colubriformis migrating
loo
ml?
larvae inhibited from
in the presence of increasing concentrations
of
levamisole.
trimmed and the sieve examined microscopically to ensure there was a complete seal between glass and mesh. The LMI tubes were placed in the wells of a 48well tissue culture plate (Costar, Cambridge, MA). Rubber ‘0’ rings were placed around the LMI tubes so that the ‘0’ rings rested on the rims of the wells and supported the LMI tubes to ensure the mesh on the bottom of the LMI tubes was just above the bottom of the well. The standardized LMI assay was performed by preincubation of 100 ~1 of larval suspension (2000 larvae ml-‘) with 750 ~1 of test solution at room temperature (approximately 22°C) for 2 h. Then the larvae, suspended in the test solution, were transferred by Pasteur pipette into the LMI tubes. Care was taken to insure that the test solution passed freely through the mesh and there were no air-bubbles beneath the mesh to prevent the larvae from migrating through the mesh in a column of liquid and falling to the bottom of the well. Usually 16 h were allowed for the larvae to migrate before the LMI tubes were removed and the numbers of larvae in the wells were counted. Unless > 80% of larvae in control tubes [water, phosphate buffered saline (PBS)] had migrated, the larvae were not considered sufficiently viable for use in LMI assays. All tests were performed in triplicate and included a negative control (water, PBS) and a positive inhibition control-usually levamisole at 20 pg ml-‘. The percentage of larvae inhibited was calculated from the formulae: % inhibited
= NS - NE NS
where tube
NS = number
migrated
NE = number migrated
in negative in test solution.
control
Preliminary studies showed that > 90% of viable exsheathed T. colubriformis larvae migrated through meshes ranging in size from 20 to 100 pm, but < 10% migrated through the 10 ,um mesh. Furthermore, as less than 5% of larvae killed either by heat or by repeated freezing and thawing passed through 20 pm mesh, it was decided that the 20 pm mesh was the most appropriate size mesh for LMI assays with T. colubriformis. An experiment to observe the effect of number of larvae per tube on migration in either PBS or levamisole (20 pg ml-‘) showed that the % inhibited was independent of the number of larvae per tube for the range of SO-300 larvae per tube. However, as the coefficient of variation for counts of larvae was usually less than 10% when the tubes contained 150 or more larvae, but was greater than 10% when the tubes contained < 150 larvae, it was decided to standardize the number of larvae per tube at 200. Sheathed larvae were also suitable for the LMI assay as it was found that the percentage of exsheathed larvae inhibited by levamisole (20 pg ml-‘) was only slightly lower than for sheathed larvae. Temperature affected larval migration only slightly in PBS but significantly in levamisole and probably reflects the greater motility of larvae at warmer than at colder temperatures. When the preincubation and migration were carried out at 4,24 or 37°C the percentage of larvae inhibited in the presence of levamisole at 1 pg ml-’ was 34, 66 and 88%, respectively. Omission of the preincubation step resulted in an increase in the proportion of larvae migrating in the presence of levamisole. The results of an LMI assay using concentrations of levamisole ranging from 0.1 to 50 /lg ml-’ are illustrated in Fig. 1. Inhibition of migration occurred at concentrations as low as 0.5 pg ml-’ and maximum inhibition occurred at concentrations > 5 c(g ml-‘. LMI assays have also been performed using T. cohbriformis larvae suspended in thiabendazole or ivermectin as well as in levamisole to observe whether the results of the LMI assay correlated with the known modes of action of these anthelmintics. According to Behm & Bryant (1985) thiabendazole does not cause paralysis but acts by binding to tubulin and having an antimitate effect, whereas levamisole and ivermectin cause paralysis by interfering with the parasite’s nervous system. After dissolving thiabendazole and ivermectin in dimethyl sulphoxide (DMSO) and dilution in PBS and using equivalent concentrations of DMSO in PBS as controls, it was found that thiabendazole had minimal effect on the migration of larvae at concentrations of less than 100 pg ml-‘. In contrast, ivermectin at concentrations ranging from 0.1 to 100 fig ml-’
Researc:h Note
resulted in 840% inhibition of migration. These observations indicate that the LMI assay may be used to screen substances for anthelmintic activity which interferes with the neurophysiology or neuromuscular co-ordination of the parasitic stages used in LMI assay. Similarly, the assay could be used to detect resistance in parasites against anthelmintics which affect the nervous system. These applications are additional to previously reported studies on mechanisms of rejection of nematodes from the intestinal tract of immune hosts (Douch et al., 1983; Kimambo & MacRae, 1988). Acknowledgemenr-This work was supported by a grant (No. CSOSP) from the Australian Wool Corporation. REFERENCES BEHMC. A. & BRYANTC. 1985. The modes of action of some modem anthelmintics. In: Resistance in Nematodes to Anthelmintic Drugs (Edited by ANDERSON N. & WALLER P. J.), pp. 5767. Commonwealth Scientific and Industrial Research Organization and Australian Wool Corporation, Sydney. B~ISVENUER. J., BRANDT M. C., GALLOWAYR. B. & HENDIUX J. C. 1983. In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae. Veterinary Parasitology 13: 341-347. DOBSONR. J., DONALDA. D., WALLERP. J. & SNOWDON K. L. 1986. An egg-hatch assay for resistance to levamisole in trichostrongyloid nematode parasites. Veterinary Parasitology 21: 77-84.
1185
DOUCHP. G. C., HARRISONG. B. L., BUCHANAN L. L. & GREER K. S. 1983. In vitro bioassay of sheep gastrointestinal mucus for nematode paralysing activity mediated by substances with some properties characteristic of SRS-A. International Journal for Parasitology 13: 207-212. FOLZ S. D., PAX R. A., THOMASE. M., BENNETT J. L., LEEB. L. & CONDERG. A. 1987. Development and validation of an in vitro Trichostrongylus colubrlformis motility assay. International Journalfor Parasitology 17: 144-1444. GAMBLEH. R. & ZAJACA. M. 1992. Resistance of St. Croix lambs to Haemonchus contortus in experimentally and naturally acquired infections. Veterinary Parasitology 41: 21 l-225. GILL J. H., REDWINJ. M., VAN WYK J. A. & LACEY E. 1991. Detection of resistance to ivermectin in Haemonchus contortus. International Journal for Parasitology 21: 771776. KIMAMBO A. E. & MACRAEJ. C. 1988. Measurement in vitro of a larval migration inhibitory factor in gastrointestinal mucus of sheep made resistant to the roundworm Trichostrongylus colubriformis. Veterinary Parasitology 28: 213-222. MARTINP. J. & LE JAMBREL. F. 1979. Larval paralysis as an in vitro assay of levamisole and morantel tartrate resistance in Ostertagia. Veterinary Science Communications 3: 159-164. O’DONNELLI. J., DINEENJ. K., WAGLANDB. M., LETHOS., DOPHEIDE T. A. A., GRANTW. N. & WARD C. W. 1989. Characterization of the major immunogen in the excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis. International Journalfor Parasitology 19: 793-802.
Parasitology
002~7519/92 $5.00 + 0.00 Pergamon Press Lid Socieryfor Pmo~irologv
Vol. 22, No. 8, pp. I183-1185, 1992
CQ 1992 Awrralian
~~~EA~C~~~~~ A NEW SIMPLIFIED
ASSAY FOR LARVAL MIGRATION
INHIBITION
B. M. WAGLAND, W. 0. JONES,L. HRIBAR, T. BENDIXSENand D. L. EMERY CSIRO Division of Animal Health, McMaster Laboratory, Private Bag No. 1, Glebe, New South Wales 2037, Australia (Received 9 March 1992; accepted 4 September 1992)
AIrstract-WAoLANo B. M., JONESW. O., HR~BARL., BENDIXSEN T. and EMERYD. L. 1992. A new simplified assay for larval migration inhibition. International Journal for Parasitology 22: 1183-I 185. A simple method is described for the in vitro detection of substances that impair the motility of third-stage larvae of gastro-int~tinal nematodes. The test is based on the ability of larvae to freely migrate through selected mesh sizes of nylon sieves and the reduced ability of larvae to migrate after preincubation with, and in the presence of, substances that inhibit or reduce larval motility. INDEX KEY WORDS: Trichosfrongylus calubriformis; sheep; larval migration inhibition assay; in vitro.
A number of in vitro assays have been described for screening potential anthelmintics with toxic or paralysing properties and for the detection of resistance to these anthelmintics in parasites. These assays
generally depend on subjective assessment of paralysis, inhibited development or quantitative measurement by photodetectors of reduced movement (Martin & Le Jambre, 1979; Boisvenue, Brand& Galloway & Hendrix, 1983; Dobson, Donald, Waller & Snowdon, 1986; Gill, Redwin, Van Wyk & Lacey, 1991; Folz, Pax, Thomas, Bennett, Lee & Conder, 1987). Unfortunately, these assays are not readily adaptable for detecting antiparasitic activity in complex biological samples such as mucus or intestinal contents. However, Douch, Harrison, Buchanan & Greer (1983) developed an in vitro assay for the presence of ‘antiparasitic’ substances in intestinal mucus while investigating rejection of Trichostrongylas coi~~rl~r~~s larvae from immune sheep. In their assay, mucus from immune sheep was mixed with exsheathed larvae, incorporated in agar blocks and the proportion of larvae that migrated out of the agar blocks was counted. This larval migration inhibition (LMI) assay has been used by Kimambo & MacRae (1988) to confirm that intestinal mucus and digesta from immune sheep contained substance(s) capable of immobilizing parasitic third-stage T. coEubrt$ormis larvae. However, attempts to use the LMI assay at this laboratory have been frustrated by difficulties in the preparation of the agar gel blocks containing viable larvae and the putative inhibitory substances. A modi~~ation of the method of Douch et al. (1983) was described by Gamble & Zajac (1992) to measure the factors in abomasal mucosa with
paralysing
against exsheathed larvae of but this modification also depended on the migration of larvae out of agar/ agarose blocks. The published assays also depend on the inhibitory substance acting immediately and irreversibly on larvae, the dist~bution of larvae within the agar block arid whether all larvae had an equal opportunity to migrate out of the agar block. This communication describes an alternative and simpler technique for performance of LMI assays and is based on the ability of larvae to migrate through fine nylon mesh after preincubation and while in continuous contact with substances affecting larval motility. The modified test is easier to perform than the Douch et al. (1983) and Gamble & Zajac (1992) assays, and is amenable for testing large numbers of samples. In the modified LMI assay, infective larvae of T. ~olubrt~orm~ were exsheathed as described by O’Donnell, Dineen, Wagland, Letho, Dopheide, Grant & Ward (1989), and the exsheathed larvae were separated from the sheaths by allowing the exsheathed larvae, but not sheaths, to pass through a 20 pm mesh sieve. Exsheathed larvae remained viable and suitable for use in LMI assays if stored at a concentration of 2000 larvae ml-’ in flat containers at 4°C. The modified LMI assay was performed using small sieves made from 35 mm lengths of glass tubing (7 mm OD, 6 mm ID) rather than agar blocks. The tubes were constructed by grinding one end of the tube with abrasive paper and attaching a 10 mm circle of nylon mesh (Swiss Screens, Australia) with epoxy resin. Excess mesh was Haemonchus
1183
activity
contortus,
1184
B. M. WAGLAND,
W. 0.
JONES, L. HRIBAR, T. BENDIXSEN and D. L. EMERY
100
Lrl 0
60
k
2 A
60
40 20
a
00.0I
09
Levomisole
1.0
IO
concentration
(pg
FIG. 1. Percentage of T. colubriformis migrating
loo
ml?
larvae inhibited from
in the presence of increasing concentrations
of
levamisole.
trimmed and the sieve examined microscopically to ensure there was a complete seal between glass and mesh. The LMI tubes were placed in the wells of a 48well tissue culture plate (Costar, Cambridge, MA). Rubber ‘0’ rings were placed around the LMI tubes so that the ‘0’ rings rested on the rims of the wells and supported the LMI tubes to ensure the mesh on the bottom of the LMI tubes was just above the bottom of the well. The standardized LMI assay was performed by preincubation of 100 ~1 of larval suspension (2000 larvae ml-‘) with 750 ~1 of test solution at room temperature (approximately 22°C) for 2 h. Then the larvae, suspended in the test solution, were transferred by Pasteur pipette into the LMI tubes. Care was taken to insure that the test solution passed freely through the mesh and there were no air-bubbles beneath the mesh to prevent the larvae from migrating through the mesh in a column of liquid and falling to the bottom of the well. Usually 16 h were allowed for the larvae to migrate before the LMI tubes were removed and the numbers of larvae in the wells were counted. Unless > 80% of larvae in control tubes [water, phosphate buffered saline (PBS)] had migrated, the larvae were not considered sufficiently viable for use in LMI assays. All tests were performed in triplicate and included a negative control (water, PBS) and a positive inhibition control-usually levamisole at 20 pg ml-‘. The percentage of larvae inhibited was calculated from the formulae: % inhibited
= NS - NE NS
where tube
NS = number
migrated
NE = number migrated
in negative in test solution.
control
Preliminary studies showed that > 90% of viable exsheathed T. colubriformis larvae migrated through meshes ranging in size from 20 to 100 pm, but < 10% migrated through the 10 ,um mesh. Furthermore, as less than 5% of larvae killed either by heat or by repeated freezing and thawing passed through 20 pm mesh, it was decided that the 20 pm mesh was the most appropriate size mesh for LMI assays with T. colubriformis. An experiment to observe the effect of number of larvae per tube on migration in either PBS or levamisole (20 pg ml-‘) showed that the % inhibited was independent of the number of larvae per tube for the range of SO-300 larvae per tube. However, as the coefficient of variation for counts of larvae was usually less than 10% when the tubes contained 150 or more larvae, but was greater than 10% when the tubes contained < 150 larvae, it was decided to standardize the number of larvae per tube at 200. Sheathed larvae were also suitable for the LMI assay as it was found that the percentage of exsheathed larvae inhibited by levamisole (20 pg ml-‘) was only slightly lower than for sheathed larvae. Temperature affected larval migration only slightly in PBS but significantly in levamisole and probably reflects the greater motility of larvae at warmer than at colder temperatures. When the preincubation and migration were carried out at 4,24 or 37°C the percentage of larvae inhibited in the presence of levamisole at 1 pg ml-’ was 34, 66 and 88%, respectively. Omission of the preincubation step resulted in an increase in the proportion of larvae migrating in the presence of levamisole. The results of an LMI assay using concentrations of levamisole ranging from 0.1 to 50 /lg ml-’ are illustrated in Fig. 1. Inhibition of migration occurred at concentrations as low as 0.5 pg ml-’ and maximum inhibition occurred at concentrations > 5 c(g ml-‘. LMI assays have also been performed using T. cohbriformis larvae suspended in thiabendazole or ivermectin as well as in levamisole to observe whether the results of the LMI assay correlated with the known modes of action of these anthelmintics. According to Behm & Bryant (1985) thiabendazole does not cause paralysis but acts by binding to tubulin and having an antimitate effect, whereas levamisole and ivermectin cause paralysis by interfering with the parasite’s nervous system. After dissolving thiabendazole and ivermectin in dimethyl sulphoxide (DMSO) and dilution in PBS and using equivalent concentrations of DMSO in PBS as controls, it was found that thiabendazole had minimal effect on the migration of larvae at concentrations of less than 100 pg ml-‘. In contrast, ivermectin at concentrations ranging from 0.1 to 100 fig ml-’
Researc:h Note
resulted in 840% inhibition of migration. These observations indicate that the LMI assay may be used to screen substances for anthelmintic activity which interferes with the neurophysiology or neuromuscular co-ordination of the parasitic stages used in LMI assay. Similarly, the assay could be used to detect resistance in parasites against anthelmintics which affect the nervous system. These applications are additional to previously reported studies on mechanisms of rejection of nematodes from the intestinal tract of immune hosts (Douch et al., 1983; Kimambo & MacRae, 1988). Acknowledgemenr-This work was supported by a grant (No. CSOSP) from the Australian Wool Corporation. REFERENCES BEHMC. A. & BRYANTC. 1985. The modes of action of some modem anthelmintics. In: Resistance in Nematodes to Anthelmintic Drugs (Edited by ANDERSON N. & WALLER P. J.), pp. 5767. Commonwealth Scientific and Industrial Research Organization and Australian Wool Corporation, Sydney. B~ISVENUER. J., BRANDT M. C., GALLOWAYR. B. & HENDIUX J. C. 1983. In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae. Veterinary Parasitology 13: 341-347. DOBSONR. J., DONALDA. D., WALLERP. J. & SNOWDON K. L. 1986. An egg-hatch assay for resistance to levamisole in trichostrongyloid nematode parasites. Veterinary Parasitology 21: 77-84.
1185
DOUCHP. G. C., HARRISONG. B. L., BUCHANAN L. L. & GREER K. S. 1983. In vitro bioassay of sheep gastrointestinal mucus for nematode paralysing activity mediated by substances with some properties characteristic of SRS-A. International Journal for Parasitology 13: 207-212. FOLZ S. D., PAX R. A., THOMASE. M., BENNETT J. L., LEEB. L. & CONDERG. A. 1987. Development and validation of an in vitro Trichostrongylus colubrlformis motility assay. International Journalfor Parasitology 17: 144-1444. GAMBLEH. R. & ZAJACA. M. 1992. Resistance of St. Croix lambs to Haemonchus contortus in experimentally and naturally acquired infections. Veterinary Parasitology 41: 21 l-225. GILL J. H., REDWINJ. M., VAN WYK J. A. & LACEY E. 1991. Detection of resistance to ivermectin in Haemonchus contortus. International Journal for Parasitology 21: 771776. KIMAMBO A. E. & MACRAEJ. C. 1988. Measurement in vitro of a larval migration inhibitory factor in gastrointestinal mucus of sheep made resistant to the roundworm Trichostrongylus colubriformis. Veterinary Parasitology 28: 213-222. MARTINP. J. & LE JAMBREL. F. 1979. Larval paralysis as an in vitro assay of levamisole and morantel tartrate resistance in Ostertagia. Veterinary Science Communications 3: 159-164. O’DONNELLI. J., DINEENJ. K., WAGLANDB. M., LETHOS., DOPHEIDE T. A. A., GRANTW. N. & WARD C. W. 1989. Characterization of the major immunogen in the excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis. International Journalfor Parasitology 19: 793-802.