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Colorimetric assay for the detection of benzimidazole resistance in trichostrongyles
Research in Veterinary Science /989. 46. 363 - 366
Colorimetric assay for the detection of benzimidazole resistance in trichostrongyles I. A. SUTHERLAND, D. L. LEE, Department of Pure and Applied Biology, University of Leeds, LS29JT, D. LEWIS, Pfizer Central Research, Sandwich, Kent, CTl39NJ
A modified version of the aphid tile-test, which is 1987), it seemed that a method similar to that used in used to detect insecticide resistance in single adult aphids may prove of value in the detection of aphids that are resistant or susceptible to organo- resistance to benzimidazole anthelmintics in trichophosphate or carbamate insecticides, was used to strongyles. compare the levels of non-specific esterases in strains of the trichostrongyle nematodes Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis which were known to be Materials and methods resistant or susceptible to benzimidazole (BZ) anthel- Nematodes mintics. This colorimetric assay has shown that there Both sz-resistant and sz-susceptible strains of H is significantly more non-specific esterase in the infective-stage larvae of sz-reslstant strains than in contortus, 0 circumcincta and T colubriformis were susceptible strains and this may prove to be of use in compared in this study. The countries of origin of the the detection of resistance to benzimidazole anthel- isolates are given in Tables I to 3. mintics. THE incidence of resistance in the trichostrongyle nematode: Haemonchus contort us, Ostertagia circumcincta and Trichostrongylus colubriformis to benzimidazole (HZ) anthelmintics has increased steadily in recent years (Waller 1985). The development of a reliable assay which will detect benzimidazole resistance in trichostrongyle infective-stage larvae would be of value in detecting resistant populations where fresh, unembryonated eggs are not available for use in an egg-hatch assay, such as that described by Le Jambre et al (1970), or where confirmation of a positive egg-hatch assay, using a different method, is required. A common method for the detection of resistance to organophosphate and carbamate insecticides in the aphid Myzus persicae is the rapid and simple aphid tile-test (Sawicki et al 1978). This involves crushing single adult aphids in a non-specific esterase stain in wells of a porcelain dish. As the levels of esterase are known to be elevated in organophosphate-resistant strains of this aphid (Sudderuddin 1973, Baker 1977, Beranek and Oppenoorth 1977), an elevation of intensity of colour change greater than that in known susceptible individuals is a good indicator of the resistance. As a study of esterase levels in the infective-stage larvae of trichostrongyles has shown that the enzyme content is greater in resistant than in susceptible strains of the species studied (Sutherland
Experimental procedure Samples of 30,000 infective-stage larvae were homogenised in I ml Jencons all-glass homogenisers in 10 mM Tris-HCI buffer containing I mM dithothreitol (pH 7· 5). Each homogenate was centrifuged at 2000 g for 10 minutes and the supernatant freezedried (Chern-Lab Model 6) at - 25°C. Samples were stored at - 70°C until required. Esterase activity was detected by reconstituting the samples with I ml of a non-specific esterase staining solution which consisted of: 100 mg a-naphthyl acetate (Sigma) in 10 ml 50 per cent aqueous acetone, 5 to 6 ml of which was added to 100 mg Fast Blue RR Salt (Sigma) in 38 ml distilled water. After conversion of the a-naphthyl acetate to anaphthol and acetic acid by the esterase, the anaphthol binds to the Fast Blue RR Salt to give a yellow-brown colour. After reconstitution with the enzyme stain, O' 25 ml of the larval sample (equivalent to 7500 larvae per well) were placed in each of four wells in columns on a 96-well microtest plate (up). In each assay, column I of the plate contained O' 25 ml of esterase stain, without larval samples, in each well. The samples were maintained at room temperature and the optical density (OD) of the samples was measured at 15 minute intervals on a Titertek Multiscan plate reader at a wavelength of 492 nm.
363
I. A. Sutherland, D. L. Lee, D. Lewis
364
TABLE 1: Increase in optical density (Multiscan units) with time of H contortus infective-stage larval homogenate, either resistant or susceptible to benzimidazole anthelmintics, after staining for non-specific esterase activity and measurement on a Titertek Multiscan plate reader at a wavelength of 492 nm
Minutes
2
10 ±
20 ±
30 ±
40 ±
50 ±
60 ± ± 1 2 3 4 5 6 7
0·25 0·03 0·426 0·039 0·543 0·092 0·631 0·098 0·713 0'091 0·783 0·106
± ± ± ± ± ±
3
0·204 0·029 0·330 0·027 0·449 0·051 0·532 0'045 0·598 0·067 0·646 0·081
± ± ± ± ± ±
Strain of H contortus 4
0·191 0·02 0·410 0·038 0·521 0·033 0·610 0·073 0·672 0·087 0·746 0·097
± ± ± ± ± ±
0·231 0·035 0·390 0'043 0'472 0·052 0·536 0·067 0·612 0·062 0·661 0·106
± ± ± ± ±
7
6 ± ± ± ± ± ±
0·161 0'02 0'203 0·03 0·260 0·045 0·305 0·05 0·260 0·054 0·220 0·051
± ± ± ± ± ±
0·126 0·024 0·192 0·041 0·248 0·049 0·230 0·061 0·180 0·065 0·175 0·083
standard deviation where n = 4 Weybridge CVL thiabendazole-resistant (UK) South African benzimidazole-resistant Australian cambendazole-resistant Pfizer thiabendazole-resistant (UK) Pfizer benzimidazole-susceptible (UK) Edinburgh benzimidazole-susceptible (UK) Australian benzimidazole-susceptible
Statistical analysis
Results
The readings taken immediately after addition of the stain were used as zero values. These values were subtracted from the mean of the following readings to give the increase in 00 from the origin with time (Tables I, 2 and 3), The change in 00 of each sample was then illustrated graphically. The significance of any differences found between the strains was tested by computing the 95 per cent confidence intervals of each sample reading. Where the confidence intervals do not overlap at a given time, the ODs in the strains examined are significantly different at that time.
H contortus
1
±
5 0·113 0·027 0·179 0·032 0·175 0·049 0·148 0·038 0'08 0·033 0·038 0·017
2
3
4
5
6
7
8
-r: '\.
t
\ ... FIG 1: Photograph of a microtest plate to show esterase activity of freeze-dried hornoqenates of benzimidazole-resistant and benzimidazole-susceptible H contortus infective-stage larvae after 60 minutes in the esterase stain. Column 1, esterase stain alone; 2, Weybridge CVL thiabendazole-resistant (UK); 3, South African benzimidazole-resistant; 4, Australian cambendazole-resistant; 5, Pfizer thiabendazole-resistant (Australia); 6, Pfizer benzimidazole-susceptible (UK); 7, Edinburgh benzimidazole-susceptible (UK); 8, Australian benzimidazole-susceptible
Each of the four sz-resistant strains of H contortus showed a more significant colour change than did the susceptible strains in this assay (Fig I). Each of the four resistant strains showed an increase in optical density (00) of over 0'7 Multiscan units after 60 minutes (Table 1) whereas the three anthelminticsusceptible strains showed much smaller degrees of colour change, between 0'3 and 0'013 units after 60 minutes (Table I).
1
2
3
4
5
6
.-•••• •••••• ••••;. • • • •1:• .
FIG 2: Photograph of a microtest plate to show esterase activity of freeze-dried homogenates of thiabendazole-resistant and thiabendazole-susceptible 0 circumcincta infective-stage larvae after 60 minutes in the esterase stain. Column 1, esterase stain alone; 2, Moredun thiabendazole-resistant (UK); 3, Britt thiabendazoleresistant (UK); 4, Weybridge CVL thiabendazole-resistant (UK); 5, Weybridge CVL thiabendazole-susceptible (UK); 6, ICI thiabendazolesusceptible (UK)
Diagnosis oj benzimidazole resistance
."••• ••••• ••••• •••• 1
TABLE 2: Increase in optical density (Multiscan units) with time of 0 circumcincta infective-stage larval homogenate, either resistant or susceptible to thiabendazole anthelmintics, after staining for non-specific esterase activity and measurement on tIJ Titertek Multiscan plate reader at a wavelength of 492 nm Strain of 0 circumcincta 4 2 3
Minutes
10
0·116 ± 0·03
20
0·187 ± 0·03
30
0·259 ± 0·024
40
50 60
0·13 ± 0·038
0,077 ± 0,027
0·206
0·210
0·113
0·119
± 0·038
± 0·034
± 0·029
0·252 ± 0·04
0·313
0·321 ± 0·044
0·356
0·052 ± 0·033
0·392 ± 0·06
0·419
0·440
± 0·056
± 0·065
0·264 ± 0·05
0·136
0·148
± 0·027
± 0·032
0·332
± 0,045
0·168 ± 0·022
0·370
± 0,05
0·184 ± 0·03
0·426 ± 0·05
0·175
± 0,006
0·212
0·232 ± 0·014
± standard deviation where n ; 4 1 Moredun thiabendazole-resistant (UK) 2 Britt thiabendazole-resistant (UK) 3 Weybridge CVLthiabendazole-resistant (UK) 4 Weybridge CVLthiabendazole-susceptible (UK) 5 ICI thiabendazole-susceptible (UK)
The significance of the difference in esterase activity between the resistant and susceptible populations was determined by fitting 95 per cent confidence limits to a graph comparing the resistant strain which showed the least increase in OD (South African BZresistant) with the susceptible strain which showed the greatest degree of colour change (Australian BZsusceptible).
5
+;'"
0·191 ± 0·0095
± 0·039
4
3
2
.,:
5
± 0·027
± 0·033 ± 0·05
0·108 ± 0·03
365
FIG 3: Photograph of a microtest plate to show esterase activity of freeze-dried homogenates of thiabendazole-resistant and thia-" bendazole-susceptible T colubriformis infective-stage larvae after 60 minutes in esterase stain. Column 1, esterase stain alone; 2, Pfizer thiabendazole-resistant (Australia); 3, VRSG thiabendazole-resistant (Australia); 4, German thiabendazole-resistant; 5, British thiabendazole-susceptible
(Australian 'rnz-resistant) with the susceptible strain was used to demonstrate the significance of the difference in esterase activity by fitting 95 per cent confidence limits to the data. This analysis showed that the raz-resistant strains had significantly greater esterase activity than the strains susceptible to the anthelmintic (Table 3).
o circumcincta The degree of colour change in the three thiabendazole (rszj-resistant strains of 0 circumcincta was much more pronounced than in the two TBZsusceptible strains (Fig 2), The resistant strains showed an increase in OD of about 0'45 Multiscan units compared to a rise of around O'25 in the two susceptible strains, after 60 minutes (Table 2). The resistant strains were shown to have significantly greater esterase activity than the susceptible strains by fitting 95 per cent confidence limits to a graph comparing the resistant strain which showed the lowest increase in OD with time (Moredun TBZresistant) against the most enzymically active susceptible strain (ICI rsz-susceptible).
TABLE 3: Increase in optical density (Multiscan units) with time of T colubriformis infective-stage larval homogenate. either resistant or susceptible to thiabendazole anthelmintics. after staining for non-specific esterase activity and measurement on a Titertek Multiscan plate reader at a wavelength of 492 nm Strain of T colubriformis 2 3
Minutes
10 ±
20 ±
30 ±
40 ±
50
T colubriformis The degree of colour change, after staining for nonspecific esterase activity in T colubrijormis, was greater in the three rnz-resistant strains than in the rnz-susceptible strain (Fig 3). Comparison of the resistant strain which showed the least colour change
60
± ±
0·085 0·014 0·130 0·022 0·175 0·027 0·237 0,033 0·274 0'04 0·316 0·049
± ± ± ± ± ±
0·062 0·017 0·116 0·025 0·187 0·024 0·244 0·037 0·310 0·045 0,332 0·048
± standard deviation where n ; 4 1 Pfizer thiabendazole-resistant (Australia) 2 VRSG thiabendazole-resistant (Australia) 3 German thiabendazole-resistant 4 British thiabendazole-susceptible
± ± ± ± ± ±
0·106 0·022 0·162 0·033 0·208 0·038 0·267 0'049 0·331 0·054 0·364 0·059
4 0·035 ± 0·0127 0,073 ± 0·022 0·108 ± 0·031 0·146 ± 0·035 0·178 ± 0·036 0·214 ± 0·033
366
I. A. Sutherland, D. L. Lee, D. Lewis
Discussion It has been established in this study that the principles of the aphid tile-test (Sawicki et a! 1978) c~n be applied to the detection of Bz-reSlStance. In infective-stage larvae of trichostrongyles. In the tiletest, the non-specific esterase content (~s revealed by intensity of staining) of single adult aphids crushed on a porcelain dish are compared with known standards after a period of time. As insecticide-resistant clones of Myzus persicae are known to contain elevated amounts of these enzymes (Sudderuddin 1973, Beranek and Oppenoorth 1977, Baker 1977), the degree of colour change is used to determi?e. the presence of reduced susceptibility. Using a similar, but quantitative, method the authors have shown that there is a similar association between resistance to the benzimidazole and elevation of esterase content in infective-stage larvae of trichostrongyles. Significantly greater degrees of colour change were detected in the az-resistant strains than in the corresponding susceptible strains of each species studied, suggesting that this method may prove of value in the detection of resistance to these anthelmintics. The process described here is an extension of the tile-test used to detect insecticide resistance in aphids, as it uses a significantly more sensitive approach in the interpretation of the results. In the aphid tile-test, no measurements are taken beyond that of visual estimation. While this may be suitable in rapid preliminary screening for highly resistant clones, aphids showing lower resistance factors, perhaps where resistance is at an early stage of development, will not necessarily be identified. Quantification of staining intensity with time, using densitometry, may allow the detection of relatively low-level resistance in strains of trichostrongyles. The diagnosis of low-level insecticide-resistance in aphids may also be affected by the size of the individual. Exceptionally small aphids with low-level resistance could conceivably have a similar esterase content to a normal sized susceptible individual. In screening for anthelmintic resistance in infective-stage larvae of trichostrongyles by this method, however, the use of relatively large numbers of larvae, although requiring greater preparation tha~ a. ~ingle .adult aphid, should negate the effects of l.ndlvldual ~Ize of subject, making the assay more reliable. Having to use about 7000 infective-stage larvae per well can, however, be a drawback if only small numbers of larvae are available. The versatility of this method of diagnosis appears to be an important factor in screening for resistance. Not only does the test give accurate measurements of
esterase activity through the use of a plate reader in the laboratory, the ease of operation and interpretation of results through direct visual examination only, at least in those strains with significant levels of resistance, also makes it suitable for use without a plate reader and in this respect it is very similar to the aphid tile-test. Freeze-dried material has been used in this study (for convenience of storage of many sa~ples), but there is no reason why homogenates of living larvae could not be substituted for the freeze-dried larvae. This would avoid the relatively lengthy process of freeze-drying and would speed up the testing of samples. If this method is to be used to detect resistance to anthelmintics it will be necessary to provide standard strains against which the colour change in the experimental samples can be compared. These would consist of known susceptible or resistant strains of the species investigated. As the ambient temperature will affect the enzyme kinetics, and thus the rate of colour change, these base values will ensure the reliable detection of anthelmintic resistance. Acknowledgements We thank Mr Peter Evans, University of Newcastle upon Tyne, Dr P. Andrews, Bayer Chemotherapie, Mr Alan Herbert and Mr Nick Leach-Bing, Pfizer Central Research, UK, and Mr Keith Hunt and Dr G. Cawthorne Central Veterinary Laboratory, Weybridge, for' the supply of resistant and susceptible strains of the parasites. This work was supported by SERC/CASE studentship (83507057) in conjunction with Pfizer Central Research, Sandwich, Kent. References BAKER, J. P. (1977) Annals of Applied Biology 86,1-9 BERANEK, A. P. & OPPENOORTH, F. J. (1977) Pesticide Biochemistry and Physiology 7, 16-20
LE JAMBRE, L. F., CROFTON, H. D. & WHITLOCK, J. H.
(1970) Transactions of the American Microscopical Society 89, 397-406 SAWICKI, R. M., DEVONSHIRE, A. L., RICE, A. D., MOORES, G. D., PETZING, S. M. & CAMERON, A. (1978) Pesticide Science 9, 189-201 SUDDERUDDIN, K. I. (1973) Comparative Biochemistry and Physiology 44, 923-929 SUTHERLAND, I. A. (1987) PhDthesis, University of Leeds WALLER, P. J. (1985) Resistance in Nematodes to Anthelmintic
Drugs. Eds N. Anderson & P. J. Waller. CSIRO/Australian Wool Corporation. pp 1-13
Received December 30, 1987 Accepted July 15. 1988
Colorimetric assay for the detection of benzimidazole resistance in trichostrongyles I. A. SUTHERLAND, D. L. LEE, Department of Pure and Applied Biology, University of Leeds, LS29JT, D. LEWIS, Pfizer Central Research, Sandwich, Kent, CTl39NJ
A modified version of the aphid tile-test, which is 1987), it seemed that a method similar to that used in used to detect insecticide resistance in single adult aphids may prove of value in the detection of aphids that are resistant or susceptible to organo- resistance to benzimidazole anthelmintics in trichophosphate or carbamate insecticides, was used to strongyles. compare the levels of non-specific esterases in strains of the trichostrongyle nematodes Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis which were known to be Materials and methods resistant or susceptible to benzimidazole (BZ) anthel- Nematodes mintics. This colorimetric assay has shown that there Both sz-resistant and sz-susceptible strains of H is significantly more non-specific esterase in the infective-stage larvae of sz-reslstant strains than in contortus, 0 circumcincta and T colubriformis were susceptible strains and this may prove to be of use in compared in this study. The countries of origin of the the detection of resistance to benzimidazole anthel- isolates are given in Tables I to 3. mintics. THE incidence of resistance in the trichostrongyle nematode: Haemonchus contort us, Ostertagia circumcincta and Trichostrongylus colubriformis to benzimidazole (HZ) anthelmintics has increased steadily in recent years (Waller 1985). The development of a reliable assay which will detect benzimidazole resistance in trichostrongyle infective-stage larvae would be of value in detecting resistant populations where fresh, unembryonated eggs are not available for use in an egg-hatch assay, such as that described by Le Jambre et al (1970), or where confirmation of a positive egg-hatch assay, using a different method, is required. A common method for the detection of resistance to organophosphate and carbamate insecticides in the aphid Myzus persicae is the rapid and simple aphid tile-test (Sawicki et al 1978). This involves crushing single adult aphids in a non-specific esterase stain in wells of a porcelain dish. As the levels of esterase are known to be elevated in organophosphate-resistant strains of this aphid (Sudderuddin 1973, Baker 1977, Beranek and Oppenoorth 1977), an elevation of intensity of colour change greater than that in known susceptible individuals is a good indicator of the resistance. As a study of esterase levels in the infective-stage larvae of trichostrongyles has shown that the enzyme content is greater in resistant than in susceptible strains of the species studied (Sutherland
Experimental procedure Samples of 30,000 infective-stage larvae were homogenised in I ml Jencons all-glass homogenisers in 10 mM Tris-HCI buffer containing I mM dithothreitol (pH 7· 5). Each homogenate was centrifuged at 2000 g for 10 minutes and the supernatant freezedried (Chern-Lab Model 6) at - 25°C. Samples were stored at - 70°C until required. Esterase activity was detected by reconstituting the samples with I ml of a non-specific esterase staining solution which consisted of: 100 mg a-naphthyl acetate (Sigma) in 10 ml 50 per cent aqueous acetone, 5 to 6 ml of which was added to 100 mg Fast Blue RR Salt (Sigma) in 38 ml distilled water. After conversion of the a-naphthyl acetate to anaphthol and acetic acid by the esterase, the anaphthol binds to the Fast Blue RR Salt to give a yellow-brown colour. After reconstitution with the enzyme stain, O' 25 ml of the larval sample (equivalent to 7500 larvae per well) were placed in each of four wells in columns on a 96-well microtest plate (up). In each assay, column I of the plate contained O' 25 ml of esterase stain, without larval samples, in each well. The samples were maintained at room temperature and the optical density (OD) of the samples was measured at 15 minute intervals on a Titertek Multiscan plate reader at a wavelength of 492 nm.
363
I. A. Sutherland, D. L. Lee, D. Lewis
364
TABLE 1: Increase in optical density (Multiscan units) with time of H contortus infective-stage larval homogenate, either resistant or susceptible to benzimidazole anthelmintics, after staining for non-specific esterase activity and measurement on a Titertek Multiscan plate reader at a wavelength of 492 nm
Minutes
2
10 ±
20 ±
30 ±
40 ±
50 ±
60 ± ± 1 2 3 4 5 6 7
0·25 0·03 0·426 0·039 0·543 0·092 0·631 0·098 0·713 0'091 0·783 0·106
± ± ± ± ± ±
3
0·204 0·029 0·330 0·027 0·449 0·051 0·532 0'045 0·598 0·067 0·646 0·081
± ± ± ± ± ±
Strain of H contortus 4
0·191 0·02 0·410 0·038 0·521 0·033 0·610 0·073 0·672 0·087 0·746 0·097
± ± ± ± ± ±
0·231 0·035 0·390 0'043 0'472 0·052 0·536 0·067 0·612 0·062 0·661 0·106
± ± ± ± ±
7
6 ± ± ± ± ± ±
0·161 0'02 0'203 0·03 0·260 0·045 0·305 0·05 0·260 0·054 0·220 0·051
± ± ± ± ± ±
0·126 0·024 0·192 0·041 0·248 0·049 0·230 0·061 0·180 0·065 0·175 0·083
standard deviation where n = 4 Weybridge CVL thiabendazole-resistant (UK) South African benzimidazole-resistant Australian cambendazole-resistant Pfizer thiabendazole-resistant (UK) Pfizer benzimidazole-susceptible (UK) Edinburgh benzimidazole-susceptible (UK) Australian benzimidazole-susceptible
Statistical analysis
Results
The readings taken immediately after addition of the stain were used as zero values. These values were subtracted from the mean of the following readings to give the increase in 00 from the origin with time (Tables I, 2 and 3), The change in 00 of each sample was then illustrated graphically. The significance of any differences found between the strains was tested by computing the 95 per cent confidence intervals of each sample reading. Where the confidence intervals do not overlap at a given time, the ODs in the strains examined are significantly different at that time.
H contortus
1
±
5 0·113 0·027 0·179 0·032 0·175 0·049 0·148 0·038 0'08 0·033 0·038 0·017
2
3
4
5
6
7
8
-r: '\.
t
\ ... FIG 1: Photograph of a microtest plate to show esterase activity of freeze-dried hornoqenates of benzimidazole-resistant and benzimidazole-susceptible H contortus infective-stage larvae after 60 minutes in the esterase stain. Column 1, esterase stain alone; 2, Weybridge CVL thiabendazole-resistant (UK); 3, South African benzimidazole-resistant; 4, Australian cambendazole-resistant; 5, Pfizer thiabendazole-resistant (Australia); 6, Pfizer benzimidazole-susceptible (UK); 7, Edinburgh benzimidazole-susceptible (UK); 8, Australian benzimidazole-susceptible
Each of the four sz-resistant strains of H contortus showed a more significant colour change than did the susceptible strains in this assay (Fig I). Each of the four resistant strains showed an increase in optical density (00) of over 0'7 Multiscan units after 60 minutes (Table 1) whereas the three anthelminticsusceptible strains showed much smaller degrees of colour change, between 0'3 and 0'013 units after 60 minutes (Table I).
1
2
3
4
5
6
.-•••• •••••• ••••;. • • • •1:• .
FIG 2: Photograph of a microtest plate to show esterase activity of freeze-dried homogenates of thiabendazole-resistant and thiabendazole-susceptible 0 circumcincta infective-stage larvae after 60 minutes in the esterase stain. Column 1, esterase stain alone; 2, Moredun thiabendazole-resistant (UK); 3, Britt thiabendazoleresistant (UK); 4, Weybridge CVL thiabendazole-resistant (UK); 5, Weybridge CVL thiabendazole-susceptible (UK); 6, ICI thiabendazolesusceptible (UK)
Diagnosis oj benzimidazole resistance
."••• ••••• ••••• •••• 1
TABLE 2: Increase in optical density (Multiscan units) with time of 0 circumcincta infective-stage larval homogenate, either resistant or susceptible to thiabendazole anthelmintics, after staining for non-specific esterase activity and measurement on tIJ Titertek Multiscan plate reader at a wavelength of 492 nm Strain of 0 circumcincta 4 2 3
Minutes
10
0·116 ± 0·03
20
0·187 ± 0·03
30
0·259 ± 0·024
40
50 60
0·13 ± 0·038
0,077 ± 0,027
0·206
0·210
0·113
0·119
± 0·038
± 0·034
± 0·029
0·252 ± 0·04
0·313
0·321 ± 0·044
0·356
0·052 ± 0·033
0·392 ± 0·06
0·419
0·440
± 0·056
± 0·065
0·264 ± 0·05
0·136
0·148
± 0·027
± 0·032
0·332
± 0,045
0·168 ± 0·022
0·370
± 0,05
0·184 ± 0·03
0·426 ± 0·05
0·175
± 0,006
0·212
0·232 ± 0·014
± standard deviation where n ; 4 1 Moredun thiabendazole-resistant (UK) 2 Britt thiabendazole-resistant (UK) 3 Weybridge CVLthiabendazole-resistant (UK) 4 Weybridge CVLthiabendazole-susceptible (UK) 5 ICI thiabendazole-susceptible (UK)
The significance of the difference in esterase activity between the resistant and susceptible populations was determined by fitting 95 per cent confidence limits to a graph comparing the resistant strain which showed the least increase in OD (South African BZresistant) with the susceptible strain which showed the greatest degree of colour change (Australian BZsusceptible).
5
+;'"
0·191 ± 0·0095
± 0·039
4
3
2
.,:
5
± 0·027
± 0·033 ± 0·05
0·108 ± 0·03
365
FIG 3: Photograph of a microtest plate to show esterase activity of freeze-dried homogenates of thiabendazole-resistant and thia-" bendazole-susceptible T colubriformis infective-stage larvae after 60 minutes in esterase stain. Column 1, esterase stain alone; 2, Pfizer thiabendazole-resistant (Australia); 3, VRSG thiabendazole-resistant (Australia); 4, German thiabendazole-resistant; 5, British thiabendazole-susceptible
(Australian 'rnz-resistant) with the susceptible strain was used to demonstrate the significance of the difference in esterase activity by fitting 95 per cent confidence limits to the data. This analysis showed that the raz-resistant strains had significantly greater esterase activity than the strains susceptible to the anthelmintic (Table 3).
o circumcincta The degree of colour change in the three thiabendazole (rszj-resistant strains of 0 circumcincta was much more pronounced than in the two TBZsusceptible strains (Fig 2), The resistant strains showed an increase in OD of about 0'45 Multiscan units compared to a rise of around O'25 in the two susceptible strains, after 60 minutes (Table 2). The resistant strains were shown to have significantly greater esterase activity than the susceptible strains by fitting 95 per cent confidence limits to a graph comparing the resistant strain which showed the lowest increase in OD with time (Moredun TBZresistant) against the most enzymically active susceptible strain (ICI rsz-susceptible).
TABLE 3: Increase in optical density (Multiscan units) with time of T colubriformis infective-stage larval homogenate. either resistant or susceptible to thiabendazole anthelmintics. after staining for non-specific esterase activity and measurement on a Titertek Multiscan plate reader at a wavelength of 492 nm Strain of T colubriformis 2 3
Minutes
10 ±
20 ±
30 ±
40 ±
50
T colubriformis The degree of colour change, after staining for nonspecific esterase activity in T colubrijormis, was greater in the three rnz-resistant strains than in the rnz-susceptible strain (Fig 3). Comparison of the resistant strain which showed the least colour change
60
± ±
0·085 0·014 0·130 0·022 0·175 0·027 0·237 0,033 0·274 0'04 0·316 0·049
± ± ± ± ± ±
0·062 0·017 0·116 0·025 0·187 0·024 0·244 0·037 0·310 0·045 0,332 0·048
± standard deviation where n ; 4 1 Pfizer thiabendazole-resistant (Australia) 2 VRSG thiabendazole-resistant (Australia) 3 German thiabendazole-resistant 4 British thiabendazole-susceptible
± ± ± ± ± ±
0·106 0·022 0·162 0·033 0·208 0·038 0·267 0'049 0·331 0·054 0·364 0·059
4 0·035 ± 0·0127 0,073 ± 0·022 0·108 ± 0·031 0·146 ± 0·035 0·178 ± 0·036 0·214 ± 0·033
366
I. A. Sutherland, D. L. Lee, D. Lewis
Discussion It has been established in this study that the principles of the aphid tile-test (Sawicki et a! 1978) c~n be applied to the detection of Bz-reSlStance. In infective-stage larvae of trichostrongyles. In the tiletest, the non-specific esterase content (~s revealed by intensity of staining) of single adult aphids crushed on a porcelain dish are compared with known standards after a period of time. As insecticide-resistant clones of Myzus persicae are known to contain elevated amounts of these enzymes (Sudderuddin 1973, Beranek and Oppenoorth 1977, Baker 1977), the degree of colour change is used to determi?e. the presence of reduced susceptibility. Using a similar, but quantitative, method the authors have shown that there is a similar association between resistance to the benzimidazole and elevation of esterase content in infective-stage larvae of trichostrongyles. Significantly greater degrees of colour change were detected in the az-resistant strains than in the corresponding susceptible strains of each species studied, suggesting that this method may prove of value in the detection of resistance to these anthelmintics. The process described here is an extension of the tile-test used to detect insecticide resistance in aphids, as it uses a significantly more sensitive approach in the interpretation of the results. In the aphid tile-test, no measurements are taken beyond that of visual estimation. While this may be suitable in rapid preliminary screening for highly resistant clones, aphids showing lower resistance factors, perhaps where resistance is at an early stage of development, will not necessarily be identified. Quantification of staining intensity with time, using densitometry, may allow the detection of relatively low-level resistance in strains of trichostrongyles. The diagnosis of low-level insecticide-resistance in aphids may also be affected by the size of the individual. Exceptionally small aphids with low-level resistance could conceivably have a similar esterase content to a normal sized susceptible individual. In screening for anthelmintic resistance in infective-stage larvae of trichostrongyles by this method, however, the use of relatively large numbers of larvae, although requiring greater preparation tha~ a. ~ingle .adult aphid, should negate the effects of l.ndlvldual ~Ize of subject, making the assay more reliable. Having to use about 7000 infective-stage larvae per well can, however, be a drawback if only small numbers of larvae are available. The versatility of this method of diagnosis appears to be an important factor in screening for resistance. Not only does the test give accurate measurements of
esterase activity through the use of a plate reader in the laboratory, the ease of operation and interpretation of results through direct visual examination only, at least in those strains with significant levels of resistance, also makes it suitable for use without a plate reader and in this respect it is very similar to the aphid tile-test. Freeze-dried material has been used in this study (for convenience of storage of many sa~ples), but there is no reason why homogenates of living larvae could not be substituted for the freeze-dried larvae. This would avoid the relatively lengthy process of freeze-drying and would speed up the testing of samples. If this method is to be used to detect resistance to anthelmintics it will be necessary to provide standard strains against which the colour change in the experimental samples can be compared. These would consist of known susceptible or resistant strains of the species investigated. As the ambient temperature will affect the enzyme kinetics, and thus the rate of colour change, these base values will ensure the reliable detection of anthelmintic resistance. Acknowledgements We thank Mr Peter Evans, University of Newcastle upon Tyne, Dr P. Andrews, Bayer Chemotherapie, Mr Alan Herbert and Mr Nick Leach-Bing, Pfizer Central Research, UK, and Mr Keith Hunt and Dr G. Cawthorne Central Veterinary Laboratory, Weybridge, for' the supply of resistant and susceptible strains of the parasites. This work was supported by SERC/CASE studentship (83507057) in conjunction with Pfizer Central Research, Sandwich, Kent. References BAKER, J. P. (1977) Annals of Applied Biology 86,1-9 BERANEK, A. P. & OPPENOORTH, F. J. (1977) Pesticide Biochemistry and Physiology 7, 16-20
LE JAMBRE, L. F., CROFTON, H. D. & WHITLOCK, J. H.
(1970) Transactions of the American Microscopical Society 89, 397-406 SAWICKI, R. M., DEVONSHIRE, A. L., RICE, A. D., MOORES, G. D., PETZING, S. M. & CAMERON, A. (1978) Pesticide Science 9, 189-201 SUDDERUDDIN, K. I. (1973) Comparative Biochemistry and Physiology 44, 923-929 SUTHERLAND, I. A. (1987) PhDthesis, University of Leeds WALLER, P. J. (1985) Resistance in Nematodes to Anthelmintic
Drugs. Eds N. Anderson & P. J. Waller. CSIRO/Australian Wool Corporation. pp 1-13
Received December 30, 1987 Accepted July 15. 1988