- Email: [email protected]
Changes in lc50 in an in vitro egg development assay during the patent period of Haemonchus contortus in sheep
Research in Veterinary Science /987, 42, 4/3-4/4
Changes in LCSO in an in vitro egg development assay during the patent period of Haemonchus contortus in sheep F. H. M. BORGSTEEDE, T. COUWENBERG, Central Veterinary Institute, PO Box 65, 8200 AB Lelystad, The Netherlands
Using an in vitro egg development assay with thiabendazole as anthelmintic the LCSO of eggs produced by two sheep infected with either a susceptible or a resistant strain of Haemonchus contortus was followed during the patent period from 22 days to 77 days after infection, In both sheep the LCSO followed the same pattern. At the start of the patent period the average values for the susceptible and the resistant strain were, respectively, 0·05 and 0·21 /olg ml- I thiabendazole, after 40 to 60 days 0·09 and 0·35 /olg ml- I , while after 77 days 0·07 and 0·24 /olg ml- I were recorded. SINCE the introduction of the egg hatch assay for the detection of anthelmintic resistance of nematodes against benzimidazoles (LeJambre 1976, Coles and Simpkin 1977), many investigators have used this technique. However, some limitations were described, for instance the recommendation to carry out the test within four hours after faeces collection. Smith-Buijs and Borgsteede (1986) demonstrated that faeces containing eggs of Haemonchus contortus stored at 4°C, still yielded suitable eggs for some days to run a reliable test. During the present resistance studies in which the egg development assay was used it was observed that there was a variation in the LC50 which could not be ascribed' to the usual sources of variance such as animals differing with regard to age or parasitological history, different stock solutions or other people running the test. Therefore, it was decided to follow the pattern of the LC50 during the patent period of a resistant and susceptible strain of Haemonchus contortus in sheep.
Two sheep were used, born in April 1983 and reared indoors free of nematodes. On December 19, 1983 one sheep (I) was infected with 20,000 larvae of a strain of Haemonchus contortus, kept at the institute since 1971 and passed 32 times through sheep without any contact with anthelmintics. This strain is designated susceptible. On the same day the other sheep (2) was infected with 20,000 larvae of a Haemonchus contortus strain isolated from the field in 1981 and resistant to fenbendazole, and since then passaged five times always after treatment with 5 mg kg - I. Daily egg counts were made starting 18 days after infection. From 22 days (sheep I) and 23 days (sheep 2) after infection an egg development assay with thiabendazole was carried out three times each week in triplicate according to the methods described by Coles and Simpkin (1977) and modified by Boersema et al (1982). The results of the eggs per gram counts are presented in Fig I. Although both sheep were infected with the same dose of larvae the egg output of the resistant strain was considerably higher during the whole patent period. Towards 80 days after infection there was a rapid decline in egg output in both strains and no more egg development assays were performed. The results of the in vitro egg development tests are given in Fig 2 for the susceptible strain and in Fig 3 for the resistant strain. In both figures the maximum and minimum values of each triple test are indicated. The solid line represents the
0'09
40 35
I
0'08
30
N
~ 0'07
"'0 25
I
E
x 20
~ 0'06
Cl
g- 15
0'05
10
5
0·04
o +-,I~...-rr-,-,--.---,r--.--.---.--,-,---'<-" o 22 27 32 37 42 47 52 57 62 67 72 77 82
20
Days after infection FIG 1: Eggs per 9ram pattern of the susceptible strain of Haemonchus contorlus (0---0 sheep 11 and of the resistant strain (. - - . sheep 2)
I 30
50 40 60 Days after infection
70
80
FIG 2: Patterns of Le50 of the susceptible strain of Haemonchus contorCus (sheep 1) with indication of the maximum and minimum values in each triple test. TBZ thiabendazole
413
F. H. M. Borgsteede, T. Couwenberg
414
0'35
I NO·3 III
...
E Cl
"-0·25
0·2 20
I
30
40
50
60
70
80
Days after infection FIG 3: Pattern of LC50 of the resistant strain of Haemonchus contortus (sheep 21 with indication of the maximum and minimum values in each triple test. TBZ thiabendazole
equation of the third degree y=(0'4549x 1O- 6)x 3 + (0'997 X 1O- 4)x2 + (0'6583 x 1O- 2)x -0'04843 for the susceptible strain and y = (I . 663 x 10- 6)x 3 - (0' 3815 x 10- 3)x 2 + 0'02532x - 0'1829 for the resistant strain. These were the best fitted curves after multiple regression analysis of the results. From the results it is clear that in both strains the pattt'rn of the LC50 followed the same course. After the start of the patent period the values were the lowest. In the suseptible strain the lowest value of the triple test on day 22 was O' 044 J.lg ml- I and the rounded off average O' 05 J.lg ml- I. In the resistant strain the lowest value of the triple test on day 23 was 0·205 J.lg ml- I and theaverageO·21. After the start of egg production an increase in LC50 was seen in both strains. Between 40 to 60 days after infection the highest values were found. In the susceptible strain this was O' 09 for the average and 0'103 for an individual test and in the resistant strain 0·35 as the average and 0·370 for an individual test. After 60 days a steady decrease was observed although the low values of the first part of the patent period were not reached. After 77 days the average values in both strains were O' 078 and O' 24, respectively. It is not easy to find an explanation for this phenomenon which was recently also observed by others (D. Kerboeuf, personal communication). One has to consider that the anthelmintic activity of benzimidazoles in the host has not necessarily a linear relationship with the ovicidal effect of these drugs as measured in vitro in the egg development assay. This means that an LC50 might not be suitable for indicating the level of resistance of the adult worm. This can be illustrated by the author's resistant strain. After isolation from the field an LC50 of 0·22 J.lg ml- I thiabendazole was found (Boersema et al 1982). At the present moment the adult worms in sheep can even survive a dose of 20 mg kg - I fenbendazole, but the LC50 of the eggs produced thereafter is not much higher compared with the results in the present experiment. In vivo selection was also not reflected in in
vitro results. Apart from this observation one has to take into account that the strain was selected .with fenbendazole while the tests were run with thiabendazole. The present results might be explained by assuming that both strains were not fully homozygous for the resistance genes although the susceptible strain was passaged so many times that this seems unlikely (Anderson 1983). If one accepts that the populations were not homozygous one can imagine that the most susceptible females reached patency first. Thereafter their egg production was surpassed by that of the majority of the female worms which were more resistant. At the end of the patent period the influence of the susceptible females might become greater because of a longer lifespan. The same reasoning might be applied for males and for the combination of both. The decrease in LC50 towards the end of the patent period might also be influenced by a certain senility of the worms leading to a lower LC50, although the mechanisms are not clear. Maybe the most interesting and promising explanation could be given by the phenomena of maternal inheritance and endomitosis mentioned by LeJambre (1985). In strongylids selective division of the chromosomes which are carrying the resistance genes might increase the level of resistance observed as an increase of the LC50, although it is still not clear how this has to be interpreted, for instance in terms of reduced tubulin binding, the major mechanism of resistant worms to escape the effects of benzimidazole drugs (Lacey and Prichard 1986). The practical consequences of the observed changes in LC50 during a patent period are great. Many conclusions drawn on single observations have to be interpreted in another way if one takes into account a variation of a factor of about two in LC50 as found in the present experiment (0'04 toO'IO in the susceptible strain and 0·21 to O' 37 in the resistant strain). References ANDERSON, R. M. (1983) Facts and ~eflections IV. Resistance of Animals to Anthelmintics. Eds F. H. M. Borgsteede, Sv.Aa. Henriksen and H. J. Over. Lelystad, CEC Workshop, Central Veterinary Institute. pp 51-58 BOERSEMA, J. H., LEWING-VAN DER WIEL, P. J. & BORGSTEEDE, F. H. M. (1982) Veterinary Record 110, 203-204 COLES, G. C. & SIMPKIN, K. G. (1977) Research in Veterinary Science 22. 386-387 LACEY, E. & PRICHARD, R. K. (1986) Molecular and Biochemical Parasitology 19,171-181 LEJAMBRE, L. F. (1976) Veterinary Parasitology 2,385-391 LEJAMBRE, L. F. (1985) Resistance in Nematodes to Anthelmintic Drugs. Eds N. Anderson and P. J. Waller. CSIRO Division of Animal Health, Australian Wool Corporation. pp 97-106 SMITH-BUIJS, C. M. C. & BORGSTEEDE, F. H. M. (1986) Research in Veterinary Science 40, 4-7
Received for publication September 19, 1986 Accepted December 12, 1986
Changes in LCSO in an in vitro egg development assay during the patent period of Haemonchus contortus in sheep F. H. M. BORGSTEEDE, T. COUWENBERG, Central Veterinary Institute, PO Box 65, 8200 AB Lelystad, The Netherlands
Using an in vitro egg development assay with thiabendazole as anthelmintic the LCSO of eggs produced by two sheep infected with either a susceptible or a resistant strain of Haemonchus contortus was followed during the patent period from 22 days to 77 days after infection, In both sheep the LCSO followed the same pattern. At the start of the patent period the average values for the susceptible and the resistant strain were, respectively, 0·05 and 0·21 /olg ml- I thiabendazole, after 40 to 60 days 0·09 and 0·35 /olg ml- I , while after 77 days 0·07 and 0·24 /olg ml- I were recorded. SINCE the introduction of the egg hatch assay for the detection of anthelmintic resistance of nematodes against benzimidazoles (LeJambre 1976, Coles and Simpkin 1977), many investigators have used this technique. However, some limitations were described, for instance the recommendation to carry out the test within four hours after faeces collection. Smith-Buijs and Borgsteede (1986) demonstrated that faeces containing eggs of Haemonchus contortus stored at 4°C, still yielded suitable eggs for some days to run a reliable test. During the present resistance studies in which the egg development assay was used it was observed that there was a variation in the LC50 which could not be ascribed' to the usual sources of variance such as animals differing with regard to age or parasitological history, different stock solutions or other people running the test. Therefore, it was decided to follow the pattern of the LC50 during the patent period of a resistant and susceptible strain of Haemonchus contortus in sheep.
Two sheep were used, born in April 1983 and reared indoors free of nematodes. On December 19, 1983 one sheep (I) was infected with 20,000 larvae of a strain of Haemonchus contortus, kept at the institute since 1971 and passed 32 times through sheep without any contact with anthelmintics. This strain is designated susceptible. On the same day the other sheep (2) was infected with 20,000 larvae of a Haemonchus contortus strain isolated from the field in 1981 and resistant to fenbendazole, and since then passaged five times always after treatment with 5 mg kg - I. Daily egg counts were made starting 18 days after infection. From 22 days (sheep I) and 23 days (sheep 2) after infection an egg development assay with thiabendazole was carried out three times each week in triplicate according to the methods described by Coles and Simpkin (1977) and modified by Boersema et al (1982). The results of the eggs per gram counts are presented in Fig I. Although both sheep were infected with the same dose of larvae the egg output of the resistant strain was considerably higher during the whole patent period. Towards 80 days after infection there was a rapid decline in egg output in both strains and no more egg development assays were performed. The results of the in vitro egg development tests are given in Fig 2 for the susceptible strain and in Fig 3 for the resistant strain. In both figures the maximum and minimum values of each triple test are indicated. The solid line represents the
0'09
40 35
I
0'08
30
N
~ 0'07
"'0 25
I
E
x 20
~ 0'06
Cl
g- 15
0'05
10
5
0·04
o +-,I~...-rr-,-,--.---,r--.--.---.--,-,---'<-" o 22 27 32 37 42 47 52 57 62 67 72 77 82
20
Days after infection FIG 1: Eggs per 9ram pattern of the susceptible strain of Haemonchus contorlus (0---0 sheep 11 and of the resistant strain (. - - . sheep 2)
I 30
50 40 60 Days after infection
70
80
FIG 2: Patterns of Le50 of the susceptible strain of Haemonchus contorCus (sheep 1) with indication of the maximum and minimum values in each triple test. TBZ thiabendazole
413
F. H. M. Borgsteede, T. Couwenberg
414
0'35
I NO·3 III
...
E Cl
"-0·25
0·2 20
I
30
40
50
60
70
80
Days after infection FIG 3: Pattern of LC50 of the resistant strain of Haemonchus contortus (sheep 21 with indication of the maximum and minimum values in each triple test. TBZ thiabendazole
equation of the third degree y=(0'4549x 1O- 6)x 3 + (0'997 X 1O- 4)x2 + (0'6583 x 1O- 2)x -0'04843 for the susceptible strain and y = (I . 663 x 10- 6)x 3 - (0' 3815 x 10- 3)x 2 + 0'02532x - 0'1829 for the resistant strain. These were the best fitted curves after multiple regression analysis of the results. From the results it is clear that in both strains the pattt'rn of the LC50 followed the same course. After the start of the patent period the values were the lowest. In the suseptible strain the lowest value of the triple test on day 22 was O' 044 J.lg ml- I and the rounded off average O' 05 J.lg ml- I. In the resistant strain the lowest value of the triple test on day 23 was 0·205 J.lg ml- I and theaverageO·21. After the start of egg production an increase in LC50 was seen in both strains. Between 40 to 60 days after infection the highest values were found. In the susceptible strain this was O' 09 for the average and 0'103 for an individual test and in the resistant strain 0·35 as the average and 0·370 for an individual test. After 60 days a steady decrease was observed although the low values of the first part of the patent period were not reached. After 77 days the average values in both strains were O' 078 and O' 24, respectively. It is not easy to find an explanation for this phenomenon which was recently also observed by others (D. Kerboeuf, personal communication). One has to consider that the anthelmintic activity of benzimidazoles in the host has not necessarily a linear relationship with the ovicidal effect of these drugs as measured in vitro in the egg development assay. This means that an LC50 might not be suitable for indicating the level of resistance of the adult worm. This can be illustrated by the author's resistant strain. After isolation from the field an LC50 of 0·22 J.lg ml- I thiabendazole was found (Boersema et al 1982). At the present moment the adult worms in sheep can even survive a dose of 20 mg kg - I fenbendazole, but the LC50 of the eggs produced thereafter is not much higher compared with the results in the present experiment. In vivo selection was also not reflected in in
vitro results. Apart from this observation one has to take into account that the strain was selected .with fenbendazole while the tests were run with thiabendazole. The present results might be explained by assuming that both strains were not fully homozygous for the resistance genes although the susceptible strain was passaged so many times that this seems unlikely (Anderson 1983). If one accepts that the populations were not homozygous one can imagine that the most susceptible females reached patency first. Thereafter their egg production was surpassed by that of the majority of the female worms which were more resistant. At the end of the patent period the influence of the susceptible females might become greater because of a longer lifespan. The same reasoning might be applied for males and for the combination of both. The decrease in LC50 towards the end of the patent period might also be influenced by a certain senility of the worms leading to a lower LC50, although the mechanisms are not clear. Maybe the most interesting and promising explanation could be given by the phenomena of maternal inheritance and endomitosis mentioned by LeJambre (1985). In strongylids selective division of the chromosomes which are carrying the resistance genes might increase the level of resistance observed as an increase of the LC50, although it is still not clear how this has to be interpreted, for instance in terms of reduced tubulin binding, the major mechanism of resistant worms to escape the effects of benzimidazole drugs (Lacey and Prichard 1986). The practical consequences of the observed changes in LC50 during a patent period are great. Many conclusions drawn on single observations have to be interpreted in another way if one takes into account a variation of a factor of about two in LC50 as found in the present experiment (0'04 toO'IO in the susceptible strain and 0·21 to O' 37 in the resistant strain). References ANDERSON, R. M. (1983) Facts and ~eflections IV. Resistance of Animals to Anthelmintics. Eds F. H. M. Borgsteede, Sv.Aa. Henriksen and H. J. Over. Lelystad, CEC Workshop, Central Veterinary Institute. pp 51-58 BOERSEMA, J. H., LEWING-VAN DER WIEL, P. J. & BORGSTEEDE, F. H. M. (1982) Veterinary Record 110, 203-204 COLES, G. C. & SIMPKIN, K. G. (1977) Research in Veterinary Science 22. 386-387 LACEY, E. & PRICHARD, R. K. (1986) Molecular and Biochemical Parasitology 19,171-181 LEJAMBRE, L. F. (1976) Veterinary Parasitology 2,385-391 LEJAMBRE, L. F. (1985) Resistance in Nematodes to Anthelmintic Drugs. Eds N. Anderson and P. J. Waller. CSIRO Division of Animal Health, Australian Wool Corporation. pp 97-106 SMITH-BUIJS, C. M. C. & BORGSTEEDE, F. H. M. (1986) Research in Veterinary Science 40, 4-7
Received for publication September 19, 1986 Accepted December 12, 1986