Veterinary Parasitology, 44 ( 1992 ) 87-95 Elsevier Science Publishers B.V., Amsterdam
87
Isoenzyme analysis of Haemonchus contortus resistant or susceptible to ivermectin F.A.M. Echevarria, S.M. Gennari I and A. Tait Department of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK (Accepted 25 February 1992)
ABSTRACT Echevarria, F.A.M., Gennari, S.M. and Tait, A., 1992. Isoenzyme analysis ofHaemonchus contortus resistant or susceptible to ivermectin. Vet. Parasitol., 44: 87-95. Three different strains of Haemonchus contortus (susceptible to ivermectin, S-IVM; selected for resistance to ivermectin, R-IVM; a multiple resistant strain, i.e. resistant to benzimidazole and ivermectin, R-IVM/SA) were examined for isoenzyme variation by starch gel electrophoresis. Using stains for seven enzymes separated in five different buffer systems, no differences in the electrophoretic mobility could be detected between any of the strains. Results demonstrate a low level of enzyme variation in H. contortus and no differences in enzyme electrophoretic profile between IVM-sensitive and IVM-resistant parasites. Differences between the ivermectin-sensitive and both ivermectin-resistant strains were observed with the propionyl esterases and although some of the differences are probably associated with benzimidazole resistance, others are associated with resistance to ivermectin. The three strains of H. contortus are generally identical; however, differences between all strains of H. contortus and a strain of Dictyocaulus viviparus were detected.
INTRODUCTION
As resistance in sheep nematodes to all broad spectrum anthelmintics (benzimidazoles, levamisole, morantel and ivermectin) continues to evolve, in vitro tests have been developed to differentiate resistant strains of parasites from susceptible strains (Le Jambre, 1976; Coles and Simpkin, 1977; Hall et al., 1978; Whitlock et al., 1980; Dobson et al., 1986; Lacey and Snowdon, 1988). Unfortunately these tests only detect resistance when it has reached high levels and there is considerable variability in these measurements (Borgsteede and Couwenberg, 1987; Martin et al., 1990 ). A technique which would detect resistance prior to its clinical appearance would be extremely Correspondence to: F.A.M. Echevarria, Department of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK. ~Present address: ICB, Universidade de Sao Paulo, Sao Paulo, 01.051, Brazil.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
88
F.A.M. ECHEVARRIAET AL.
valuable as it would allow other control measures to be introduced before widespread anthelmintic failures occurred with resultant economic losses. Recently, genetic techniques have been used in an attempt to provide an alternative method for the characterisation of nematode species and strains (Duncan, 1990; Roos et al., 1990). A number of different approaches to the measurement of genetic variation are available involving either the direct analysis of gene polymorphism by using restriction fragment length polymorphism or analysis of electrophoretic variation in gene products by a wide range of electrophoretic techniques. The analysis of the electrophoretic mobility of a range of enzymes by starch gel electrophoresis can provide a genetic profile which can be used to identify strains within a species (Nomura, 1984 ). Such analysis has given useful information on the characterisation of strains and species of a large number of parasites, for example, the identification of zoonotic trypanosome subspecies (Tait et al., 1985 ) and the identification of benzimidazole -resistant strains of nematodes (Sutherland et al., 1988 ). The latter authors showed differences in the esterase patterns between benzimidazole-susceptible and benzimidazole-resistant strains of Haemonchus COtltOrllgS.
Benzimidazole resistance in H. contortus is widespread and the development of ivermectin (IVM), an unrelated c o m p o u n d (Burg et al., 1979) has provided an alternative for use in nematode control. Nevertheless, resistance has developed against this drug under field conditions in South Africa (Van Wyk and Malan, 1988 ) and in Brazil (Echevarria and Trindade, 1989 ). This study was carried out to examine whether isoenzyme analysis could be used as a test to demonstrate differences between a susceptible strain ofH. contortus and a derivative strain which was selected for resistance to ivermectin. MATERIALS AND METHODS
Parasite strains I VM-sensitive stra in (S-1VM) An ivermectin-susceptible strain of H. contortus obtained from The Moredun Research Institute, Edinburgh, was maintained for more than 5 years by continuous passages through housed, worm-naive lambs. This strain was susceptible to ivermectin. IVM-resistant strain (R-IVM) The susceptible strain of H. contortus ( S - I V M ) was selected for resistance by treatment of infected lambs with ivermectin at 0.02 mg kg-~ over nine passages. Treatment successfully reduced the faecal egg counts by more than 99% up to the seventh treatment. Following the eighth treatment, there was no reduction in worm egg counts, the larvae were then exposed to a full ther-
ISOENZYME ANALYSIS OF HAEMONCHUS CONTORTUS
89
apeutic dose of IVM and the resulting larvae used to generate the resistant strain.
Multiple resistant strain (R-IVM/SA) A multiple resistant strain, i.e. resistant to ivermectin, benzimidazole and closantel (Van Wyk and Malan, 1988 ) was imported from South Africa. This strain was maintained at Glasgow University Veterinary School (under isolation conditions) by four passages in parasite-naive lambs using ivermectin treatment at 0.2mg kg- 1 during each passage. Dictyocaulus viviparus Third-stage larvae were obtained from Intervet, Cambridge, UK.
Sample preparation Third- stage larvae (L3) were exsheathed with 0.1% w/v sodium hypochlorite and washed five times with 0.85% sodium chloride. Exsheathed larvae were then homogenised using a glass homogeniser in 0.2 mM EDTA (pH 8) containing the protease inhibitors N-o~-p-tosyl-~_-lysine chloromethyl ketone (TLCK, 50 ~tg m1-1 , N-1-tosyl-I.-phenylalanine chloromethyl ketone (TPCK, 25/~g m l - 1), phenyl methylsulphonylfluoride (PMSF, 1 mM), 1,10 phenanthroline ( 1 mM) and antipain (4 ~tM). The homogenate was centrifuged at 10 0 0 0 × g for 10 min and the supernatant removed, filtered and stored at - 2 0 ° C. Measurement of the protein concentration in the samples from the different strains was carried out using the Bradford Protein Assay (Bradford, 1976).
Horizontal starch gel electrophoresis The techniques used were as described by Smith William ( 1968 ) and Brewer (1970). Briefly, hydrolysed starch (Connaught Laboratories, Canada) and the appropriate gel buffer were mixed, boiled with swirling over a bunsen, degassed and poured into a gel mould. The gel was left to set on a level surface for 1 h at room temperature followed by 1 h at 4°C; sample slots (0.3 cm deep) were cut in a horizontal line across the gel. The methods for electrophoresis are those described by Tait et al. ( 1985 ) using four buffer systems (Tris-citrate, pH 7.0; Tris-citrate, pH 8.6; Trisphosphate, pH 7.0; Tris-phosphate, pH 9.3). The staining protocols employed were adapted from Harris and Hopkinson (1976) and are described by Tait et al. (1985). Enzyme staining systems for glucose-6-phosphate dehydrogenase (G-6-PDH, malate dehydrogenase ( M D H ) , malic enzyme (ME), phosphoglucomutase (PGM), propionate esterase (EstP) and three
90
F.A.M. ECHEVARRIA ET AL.
peptidases, using either L-alanyl-tyrosine (Ala-Tyr), L-leucycl-leucine (LeuLeu) or L-Leucyl-alanine (Leu-Ala) as substrates were used. RESULTS
Initially a series of different enzymes was assayed using the four different buffer systems (see Materials and Methods) in order to determine which enzymes were active and which buffer system (s) gave the sharpest resolution and maximal electrophoretic mobility. Based on this initial analysis, specific combinations of buffer system/enzyme were chosen for the comparisons between the different strains of H. contortus. Extracts of D. viviparus were used as a control to demonstrate that electrophoretic variation could be detected. Glucose-6-phosphate dehydrogenase was one of the enzymes which produced high resolution with most of the buffers used; however, there were no differences in band patterns between the three strains of H. contortus tested. The best resolution was achieved with either Tris-citrate buffer pH 8.6 or Tris-potassium phosphate pH 9.3, where a band of activity was detected at 3.5 cm from the origin (Fig. 1 ). Malate dehydrogenase had a low mobility in all four buffer systems. The results with three buffer systems (Tris-potassium pH 9.3, Tris-phosphate pH 7.0 and Tris-phosphate pH 8.6 ) are shown in Fig. 2. Single identical bands of activity were found in comparisons between S-IVM and R-IVM strains in the I*~ 6.0cm->
3.5cm->
®
iiiii
2.5¢m-> 2.0cm->
,@@ ,®,.
iiirl
I .Scm-O.5cm[I
:!F~
:IF~
R
S
@
:ii~ SA
D
TC pH 8.5
H. contortus
R
S
TK-PO 4 pH 9.3
R= resistant S= susceptible SA= South Africa
,lllf~
@
R
S
(-slot
TP pH 7.0 |120
D= D. viviparus
Fig. 2. Malate dehydrogenase enzyme pattern by starch gel electrophoresis.
first two buffer systems while an additional cathodal band was observed using Tris-citrate pH 8.6, which was also identical between all strains. A sample of D. viviparus L3 was included for comparison and it was found that for G-6-PDH with Tris-citrate pH 8.6 no activity could be detected while for M D H with Tris-citrate pH 8.6 a clear band of different mobility to the H. contortus samples was observed (Fig. 2 ). Malic enzyme showed very low levels of activity in all the systems tested and could not be used for a comparative analysis. Similar problems were experienced with PGM with all the buffer systems examined. Peptidase activity was analysed using all four buffer systems and three dipeptide substrates (Leu-Leu, Leu-Ala and Ala-Tyr). The best resolution and activity was achieved with Tris-phosphate pH 7.0 and Tris-citrate pH 8.6 (Fig. 3 ). The substrate Ala-Tyr with Tris-citrate pH 8.6 showed two faint bands for D. viviparus at 1 and 2 cm (Fig. 3). Based on electrophoretic mobility these substrates detect two different peptidases; one, using either leucyl-alanine or leucyl-leucine, shows a single band of activity in either Tris-phosphate pH 7.0 or Tris-citrate pH 8.6 at 2.5 cm or 7.5 cm from the slot, respectively. The second peptidase uses alanyl-tyrosine and gave a single band of activity 1.0 cm from the slot in the tris-citrate pH 8.6 system. No differences between the strains were observed. The three strains of H. contortus (S-IVM, R-IVM and R-IVM/SA) were analysed for the electrophoretic patterns ofpropionyl esterases. It was found, using the tris-citrate pH 7.0 buffer system, that the R-IVM/SA strain showed a faint band at 1.5 cm from the slot with most of the activity remaining in the
F.A.M.ECHEVARRIAET AL.
92 Leu-Ala
Leu-Ala
Leu-Leu
Leu-Leu
Ala-Tyt
(+) 7.5cm->
2.5cm-> 1.Ocm->
,I i~
R
S
87
Isoenzyme analysis of Haemonchus contortus resistant or susceptible to ivermectin F.A.M. Echevarria, S.M. Gennari I and A. Tait Department of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK (Accepted 25 February 1992)
ABSTRACT Echevarria, F.A.M., Gennari, S.M. and Tait, A., 1992. Isoenzyme analysis ofHaemonchus contortus resistant or susceptible to ivermectin. Vet. Parasitol., 44: 87-95. Three different strains of Haemonchus contortus (susceptible to ivermectin, S-IVM; selected for resistance to ivermectin, R-IVM; a multiple resistant strain, i.e. resistant to benzimidazole and ivermectin, R-IVM/SA) were examined for isoenzyme variation by starch gel electrophoresis. Using stains for seven enzymes separated in five different buffer systems, no differences in the electrophoretic mobility could be detected between any of the strains. Results demonstrate a low level of enzyme variation in H. contortus and no differences in enzyme electrophoretic profile between IVM-sensitive and IVM-resistant parasites. Differences between the ivermectin-sensitive and both ivermectin-resistant strains were observed with the propionyl esterases and although some of the differences are probably associated with benzimidazole resistance, others are associated with resistance to ivermectin. The three strains of H. contortus are generally identical; however, differences between all strains of H. contortus and a strain of Dictyocaulus viviparus were detected.
INTRODUCTION
As resistance in sheep nematodes to all broad spectrum anthelmintics (benzimidazoles, levamisole, morantel and ivermectin) continues to evolve, in vitro tests have been developed to differentiate resistant strains of parasites from susceptible strains (Le Jambre, 1976; Coles and Simpkin, 1977; Hall et al., 1978; Whitlock et al., 1980; Dobson et al., 1986; Lacey and Snowdon, 1988). Unfortunately these tests only detect resistance when it has reached high levels and there is considerable variability in these measurements (Borgsteede and Couwenberg, 1987; Martin et al., 1990 ). A technique which would detect resistance prior to its clinical appearance would be extremely Correspondence to: F.A.M. Echevarria, Department of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK. ~Present address: ICB, Universidade de Sao Paulo, Sao Paulo, 01.051, Brazil.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
88
F.A.M. ECHEVARRIAET AL.
valuable as it would allow other control measures to be introduced before widespread anthelmintic failures occurred with resultant economic losses. Recently, genetic techniques have been used in an attempt to provide an alternative method for the characterisation of nematode species and strains (Duncan, 1990; Roos et al., 1990). A number of different approaches to the measurement of genetic variation are available involving either the direct analysis of gene polymorphism by using restriction fragment length polymorphism or analysis of electrophoretic variation in gene products by a wide range of electrophoretic techniques. The analysis of the electrophoretic mobility of a range of enzymes by starch gel electrophoresis can provide a genetic profile which can be used to identify strains within a species (Nomura, 1984 ). Such analysis has given useful information on the characterisation of strains and species of a large number of parasites, for example, the identification of zoonotic trypanosome subspecies (Tait et al., 1985 ) and the identification of benzimidazole -resistant strains of nematodes (Sutherland et al., 1988 ). The latter authors showed differences in the esterase patterns between benzimidazole-susceptible and benzimidazole-resistant strains of Haemonchus COtltOrllgS.
Benzimidazole resistance in H. contortus is widespread and the development of ivermectin (IVM), an unrelated c o m p o u n d (Burg et al., 1979) has provided an alternative for use in nematode control. Nevertheless, resistance has developed against this drug under field conditions in South Africa (Van Wyk and Malan, 1988 ) and in Brazil (Echevarria and Trindade, 1989 ). This study was carried out to examine whether isoenzyme analysis could be used as a test to demonstrate differences between a susceptible strain ofH. contortus and a derivative strain which was selected for resistance to ivermectin. MATERIALS AND METHODS
Parasite strains I VM-sensitive stra in (S-1VM) An ivermectin-susceptible strain of H. contortus obtained from The Moredun Research Institute, Edinburgh, was maintained for more than 5 years by continuous passages through housed, worm-naive lambs. This strain was susceptible to ivermectin. IVM-resistant strain (R-IVM) The susceptible strain of H. contortus ( S - I V M ) was selected for resistance by treatment of infected lambs with ivermectin at 0.02 mg kg-~ over nine passages. Treatment successfully reduced the faecal egg counts by more than 99% up to the seventh treatment. Following the eighth treatment, there was no reduction in worm egg counts, the larvae were then exposed to a full ther-
ISOENZYME ANALYSIS OF HAEMONCHUS CONTORTUS
89
apeutic dose of IVM and the resulting larvae used to generate the resistant strain.
Multiple resistant strain (R-IVM/SA) A multiple resistant strain, i.e. resistant to ivermectin, benzimidazole and closantel (Van Wyk and Malan, 1988 ) was imported from South Africa. This strain was maintained at Glasgow University Veterinary School (under isolation conditions) by four passages in parasite-naive lambs using ivermectin treatment at 0.2mg kg- 1 during each passage. Dictyocaulus viviparus Third-stage larvae were obtained from Intervet, Cambridge, UK.
Sample preparation Third- stage larvae (L3) were exsheathed with 0.1% w/v sodium hypochlorite and washed five times with 0.85% sodium chloride. Exsheathed larvae were then homogenised using a glass homogeniser in 0.2 mM EDTA (pH 8) containing the protease inhibitors N-o~-p-tosyl-~_-lysine chloromethyl ketone (TLCK, 50 ~tg m1-1 , N-1-tosyl-I.-phenylalanine chloromethyl ketone (TPCK, 25/~g m l - 1), phenyl methylsulphonylfluoride (PMSF, 1 mM), 1,10 phenanthroline ( 1 mM) and antipain (4 ~tM). The homogenate was centrifuged at 10 0 0 0 × g for 10 min and the supernatant removed, filtered and stored at - 2 0 ° C. Measurement of the protein concentration in the samples from the different strains was carried out using the Bradford Protein Assay (Bradford, 1976).
Horizontal starch gel electrophoresis The techniques used were as described by Smith William ( 1968 ) and Brewer (1970). Briefly, hydrolysed starch (Connaught Laboratories, Canada) and the appropriate gel buffer were mixed, boiled with swirling over a bunsen, degassed and poured into a gel mould. The gel was left to set on a level surface for 1 h at room temperature followed by 1 h at 4°C; sample slots (0.3 cm deep) were cut in a horizontal line across the gel. The methods for electrophoresis are those described by Tait et al. ( 1985 ) using four buffer systems (Tris-citrate, pH 7.0; Tris-citrate, pH 8.6; Trisphosphate, pH 7.0; Tris-phosphate, pH 9.3). The staining protocols employed were adapted from Harris and Hopkinson (1976) and are described by Tait et al. (1985). Enzyme staining systems for glucose-6-phosphate dehydrogenase (G-6-PDH, malate dehydrogenase ( M D H ) , malic enzyme (ME), phosphoglucomutase (PGM), propionate esterase (EstP) and three
90
F.A.M. ECHEVARRIA ET AL.
peptidases, using either L-alanyl-tyrosine (Ala-Tyr), L-leucycl-leucine (LeuLeu) or L-Leucyl-alanine (Leu-Ala) as substrates were used. RESULTS
Initially a series of different enzymes was assayed using the four different buffer systems (see Materials and Methods) in order to determine which enzymes were active and which buffer system (s) gave the sharpest resolution and maximal electrophoretic mobility. Based on this initial analysis, specific combinations of buffer system/enzyme were chosen for the comparisons between the different strains of H. contortus. Extracts of D. viviparus were used as a control to demonstrate that electrophoretic variation could be detected. Glucose-6-phosphate dehydrogenase was one of the enzymes which produced high resolution with most of the buffers used; however, there were no differences in band patterns between the three strains of H. contortus tested. The best resolution was achieved with either Tris-citrate buffer pH 8.6 or Tris-potassium phosphate pH 9.3, where a band of activity was detected at 3.5 cm from the origin (Fig. 1 ). Malate dehydrogenase had a low mobility in all four buffer systems. The results with three buffer systems (Tris-potassium pH 9.3, Tris-phosphate pH 7.0 and Tris-phosphate pH 8.6 ) are shown in Fig. 2. Single identical bands of activity were found in comparisons between S-IVM and R-IVM strains in the I*~ 6.0cm->
3.5cm->
®
iiiii
2.5¢m-> 2.0cm->
,@@ ,®,.
iiirl
I .Scm-O.5cm[I
:!F~
:IF~
R
S
@
:ii~ SA
D
TC pH 8.5
H. contortus
R
S
TK-PO 4 pH 9.3
R= resistant S= susceptible SA= South Africa
,lllf~
@
R
S
(-slot
TP pH 7.0 |120
D= D. viviparus
Fig. 2. Malate dehydrogenase enzyme pattern by starch gel electrophoresis.
first two buffer systems while an additional cathodal band was observed using Tris-citrate pH 8.6, which was also identical between all strains. A sample of D. viviparus L3 was included for comparison and it was found that for G-6-PDH with Tris-citrate pH 8.6 no activity could be detected while for M D H with Tris-citrate pH 8.6 a clear band of different mobility to the H. contortus samples was observed (Fig. 2 ). Malic enzyme showed very low levels of activity in all the systems tested and could not be used for a comparative analysis. Similar problems were experienced with PGM with all the buffer systems examined. Peptidase activity was analysed using all four buffer systems and three dipeptide substrates (Leu-Leu, Leu-Ala and Ala-Tyr). The best resolution and activity was achieved with Tris-phosphate pH 7.0 and Tris-citrate pH 8.6 (Fig. 3 ). The substrate Ala-Tyr with Tris-citrate pH 8.6 showed two faint bands for D. viviparus at 1 and 2 cm (Fig. 3). Based on electrophoretic mobility these substrates detect two different peptidases; one, using either leucyl-alanine or leucyl-leucine, shows a single band of activity in either Tris-phosphate pH 7.0 or Tris-citrate pH 8.6 at 2.5 cm or 7.5 cm from the slot, respectively. The second peptidase uses alanyl-tyrosine and gave a single band of activity 1.0 cm from the slot in the tris-citrate pH 8.6 system. No differences between the strains were observed. The three strains of H. contortus (S-IVM, R-IVM and R-IVM/SA) were analysed for the electrophoretic patterns ofpropionyl esterases. It was found, using the tris-citrate pH 7.0 buffer system, that the R-IVM/SA strain showed a faint band at 1.5 cm from the slot with most of the activity remaining in the
F.A.M.ECHEVARRIAET AL.
92 Leu-Ala
Leu-Ala
Leu-Leu
Leu-Leu
Ala-Tyt
(+) 7.5cm->
2.5cm-> 1.Ocm->
,I i~
R
S
