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In vivo assays for drug resistance in Babesia divergens using the Mongolian gerbil, Meriones unguiculatus
Research in Veterinary Science 1992, 52, 126-128
In vivo assays for drug resistance in Babesia divergens using the Mongolian gerbil, Meriones unguiculatus J. S. GRAY, S. L. PARR, Department of Agricultural Zoology, UniversityCollege, Dublin, Ireland
Two in vivo drug resistance assays were developed using gerbils. Cross resistance, involving related babesicides as well as the chemically unrelated antibiotic, oxytetracycline, was demonstrated, but the suggestion that imidocarb may select for pathogenic strains of parasites was not supported. Limited tests of field strains did not detect resistance. It is suggested that an in vitro assay would be more appropriate for surveys through in vivo assays would be essential for confirmatory studies.
BOVINE babesiosis, caused by Babesia divergens, is a widespread and important disease in Ireland (Gray and Harte 1985) and surveys amongst farmers and veterinarians have suggested that drug resistance may play a part in its epidemiology (Gray and Looby 1984). Drug resistance in Babesia species has been reported before (Fulton and Yorke 1941, Dalgliesh and Stewart 1977, Yeruham et al 1985), but the high degree of host specificity shown by these parasites has made detailed studies using laboratory animals difficult. B divergens, however, can be cultured in the Mongolian gerbil (Meriones unguiculatus) and in this study gerbils were used to develop reference strains, develop resistance assays, isolate suspected resistant strains from the field and test some of these strains for drug resistance. The gerbils were obtained from an outbred colony maintained in the university animal facility. They were used when aged 12 to 16 weeks in groups of five and were matched for sex and weight. The laboratory reference strain of B divergensused was originally isolated from a drug susceptible case of babesiosis in Glencree, Co Wicklow, Ireland in 1976. After use in experimental cattle it was adapted to gerbils, in which it underwent 60 passages, and was then designated CE2/N. The babesicidal drugs used were quinuronium sulphate (Ludobal; Bayer and several generics) diminazene aceturate (Berenil; Hoescht), amicarbalide isethionate (Diampron; May & Baker) and imidocarb dipropionate (Imizol; Coopers Pitman-Moore). A fifth drug, long-acting oxytetracyline (Terramycin LA; Pfizer), has no therapeutic activity, but may have potential as a prophylactic (Taylor et al 1986). Gerbils were usually infected by intraperitoneal injections of infected red blood cells in phosphate buffered saline, though sub-
cutaneous and intracardiac infection routes were also used. Thin smears of tail blood were obtained daily from infected gerbils and parasitaemias determined after staining with Giemsa stain. Blood was collected by cardiac puncture into heparinised syringes under sodium pentobarbitone anaesthesia and when necessary was cryopreserved and frozen stabilates revived as described by Langley (1985). Attempts were made to produce drug-tolerant reference lines from CE2/N in two ways. Method A, designed for prophylactic drugs, involved treatment of pairs of gerbils with increasing subcurative doses of drug at the time of infection and subpassage once parasitaemias had risen above 5 per cent. Method B, designed for curative drugs, consisted of the treatment of pairs of infected gerbils with increasing curative doses at haemoglobinuria and the subpassage of surviving parasites at declining parasitaemias of not more than 5 per cent. Parasites were passaged at least 10 times and then stored in liquid nitrogen. Two lines were developed by method A, one with imidocarb (CE2/IMDP-A) and another with long-acting oxytetracycline (CE2/LAT-A)and three by method B with quinuronium (CEZ/QS-B),amicarbalide (CE2/AI-B) and imidocarb (CE2/IMDP-B). Several methods of in vivo resistance assessment, based initially on those described for resistance studies on malaria (Peters 1984), were investigated with the objective of obtaining as reproducible, rapid and cheap an assay as possible. Of these, only two proved to be sufficiently practicable and reproducible. These were a curative method (TM0 and a suppression method (TM2). In TM1, gerbils were infected with l06 infected red blood cells intraperitoneally and treated at the first sign of haemoglobinuria with minimum curative drug doses, based on those determined by Gray (1983) but modified to allow for continuing parasite adaptation to gerbils. This method detected increased drug tolerance in CE2/IMDP-A and CE2/QS-Busing the parameter of maximum parasitaemias, but it was at a very low level. Fulmination never occurred in these lines following treatment. There was no evidence of tolerance in CE2/AI-B or CE2/IMDP-B. In TM2, gerbils were infected with 106 infected red blood cells intraperitoneally and treated at the same time with the minimum 126
Babesia divergens drug resistance curative drug dose. The parameters used were maximum parasitaemias and time to 1 per cent parasitaemia expressed as the delay caused by drug treatment relative to controls. This method detected increased drug tolerance in CE2/IMDP-Aand CE2/QS-B, but not in CE2/LATA, CE2/IMDP-Aor CE2/AI-B.It had proved very difficult to increase drug doses when trying to induce tolerance in the latter three lines. TM1 required fewer gerbils than TM2, but needed more intensive monitoring and seemed less sensitive than TM2, though was arguably more relevant to the field situation. Problems arising from the comparison of gerbil-adapted parasites with bovine isolates in TM1 were overcome by pretreatment of gerbils with 5 mg kg-l dexamethasone (Dexafort; Intertvet), which also reduced variability. This procedure did not, however, improve TM2. The potential for cross resistance was studied using the TM1 assay in two experiments. First, involving CE2/QS-B and the drugs quinuronium, amicarbalide, diminazene and imidocarb and, secondly, involving CE2/IMDP-A and CE2/QS-B and long-acting oxytetracycline. The results of the first experiment showed that none of the drugs, which are all chemically related, were as effective against the drug tolerant QS-B as against the sensitive reference line CE2/N (Table 1). In the second series of experiments it was shown that the unrelated drug, long-acting oxytetracycline, was slightly less active against the drug tolerant reference lines, CE2/IMDP-A and CE2/QS-B, than against the sensitive line (Table 2).
127
Two experiments were conducted to investigate the suggestion that the prophylactic use ofimidocarb injections may select for pathogenic strains of parasites, resulting in apparent drug resistance. Infections of CE2/IMDP-A and CE2/N were initiated, first, by inoculations of 106 infected red blood cells intraperitoneally and, secondly, 108 infected red blood cells subcutaneously. Comparisons were made of prepatent periods, time to haemoglobinuria, packed cell volume changes, maximum parasitaemias and spleen weights. No significant differences were found for any of these parameters and it is concluded that virulence is not increased by the selection of imidocarb-tolerant parasites, at least in gerbils. A limited number of field isolates suspected of drug resistance were tested with the in vivo assays. These were obtained by post from veterinary practices as whole blood in EDTA. On receipt, 0.5 ml of washed packed cells were injected intraperitoneally into gerbils that had been pretreated with dexamethasone and the resulting infected gerbil blood was cryopreserved. A total of 32 isolates were received in this way and three of these were tested in the TM! and TM2 alongside drug tolerant and sensitive reference lines. However, none of these field strains showed any signs of resistance. It was evident from this that a large number of field isolates may have to be tested to identify drug resistance as a significant factor in cases that respond poorly to treatment. In vitro assays would probably be more appropriate as a primary screen for this purpose, though confirmatory in vivo assays would be essenti~tl.
TABLE 1 : Tolerance of CE2/QS-Bto quinuronium, amicarbalide, diminazene and imidocarb (test method 2) Quinuronium (1-5mg kg-1)
CE2/OS-B
Amicarbalide (7.0rag kg-1)
2'50 + 0"60 P>0"I
CE2/N
5"00 -- 0'27
Diminazene (5.0mg kg -1)
Patency delay (days + SE) 0"75 + 0'11 2'6 + 0'33 P<0"001 4"75 + 0"38 7"40 -+ 0"3
Imidocarb (1.5mg kg 1)
4"20 + 0"8 P<0"002 >10'0 -+ 0"0
Maximum parasitaemia CE2/QS~B
4"86
15"40 P<0"02
CE2/N
0"35
0'07
0"47
O'0
0"0
P<0"01 0'49
TABLE 2: Tolerance of CE2/QS-Band CE2/IMDP-Ato long-acting oxytetracycline (10 mg kg-1) (test method 2) Parasite line Experiment 1 CE2/QS-B
Patency delay (days _+sE)
Maximum parasitaemia
4"2 + 0"82
14"2 P>0'I
CE2/N Experiment 2 CE2/IMDP-A
4"8 + 0"9.8
0"56
3"17 + 1"17 8"5 + 0"62
3/5 P<0'05 5/5
3'9 P<0"002
CE/N
Survival
6/6 P>0"I
0"18
6/6
128
J. S. Gray, S. L. Parr
Acknowledgements We are grateful to G. Looby and N. O'Keefe for contributions in the initial stages of this study and to B. Kaye for occasional technical assistance. Thanks are also due to Bayer (Ireland), Hoechst Ireland, Rhone Poulenc Ireland and Coopers Pitman-Moore for supplying the drugs used in this study. References DALGLIESH, R. J. & STEWART, N. P. (1977) Tolerance to imidocarb induced experimentally in tick-transmitted Babesia argentina. Australian Veterinary Journal 53, 176-180 FULTON, J. D. & YORKE, W. (1941) Studies in chemotherapy. XXVIII Drug resistance in Babesia infections. Annals of Tropical Medicine and Parasitology 35, 229-235 GRAY, J. S. (1983) Chemotherapy of Babesia divergens in the gerbil, Meriones unguiculatus. Research in Veterinary Science 35, 318-324 GRAY, J. S. & HARTE, L. N. (1985) An estimation of the prevalence and economic importance of clinical bovine babesiosis in the
Republic of Ireland. Irish Veterinary Journal 39, 75-78 GRAY, J. S. & LOOBY, G. (1984) Studies on the epidemiology of acute babesiosis in Ireland. Proceedings of the Society of Veterinary Epidemiology and Preventive Medicine, University of Edinburgh, 10-11 July 1984. Ed M. V. Thrusfield. pp 140-147 LANGLEY, R. J. (1985) The host parasite relationship of the cattle piroplasm Babesia divergens in the Mongolian gerbil, Meriones unguiculatus. PhD thesis, National University of Ireland PETERS, W. (1984) Experimental production of drug resistance. In: Handbook of Experimental Pharmacology 68/1. Eds W. Peters and W. H. G. Richards, Berlin, Heidelberg, Springer-Verlag. pp 461-473 TAYLOR, S. M., ELLIOTT, C. T. & KENNY, J. (1986) Inhibition of Babesia divergens in cattle by oxytetracycline. Veterinary Reeord 118, 98-102 YERUHAM, I., PIPANO, E. & DAVIDSON, M. (1985) A field strain of Babesia boris apparently resistant to amicarbalide isethionate. Tropical Animal Health and Production 17, 29-30
Received June 26. 1991 Aeeepted Oeloher 10, 1991
In vivo assays for drug resistance in Babesia divergens using the Mongolian gerbil, Meriones unguiculatus J. S. GRAY, S. L. PARR, Department of Agricultural Zoology, UniversityCollege, Dublin, Ireland
Two in vivo drug resistance assays were developed using gerbils. Cross resistance, involving related babesicides as well as the chemically unrelated antibiotic, oxytetracycline, was demonstrated, but the suggestion that imidocarb may select for pathogenic strains of parasites was not supported. Limited tests of field strains did not detect resistance. It is suggested that an in vitro assay would be more appropriate for surveys through in vivo assays would be essential for confirmatory studies.
BOVINE babesiosis, caused by Babesia divergens, is a widespread and important disease in Ireland (Gray and Harte 1985) and surveys amongst farmers and veterinarians have suggested that drug resistance may play a part in its epidemiology (Gray and Looby 1984). Drug resistance in Babesia species has been reported before (Fulton and Yorke 1941, Dalgliesh and Stewart 1977, Yeruham et al 1985), but the high degree of host specificity shown by these parasites has made detailed studies using laboratory animals difficult. B divergens, however, can be cultured in the Mongolian gerbil (Meriones unguiculatus) and in this study gerbils were used to develop reference strains, develop resistance assays, isolate suspected resistant strains from the field and test some of these strains for drug resistance. The gerbils were obtained from an outbred colony maintained in the university animal facility. They were used when aged 12 to 16 weeks in groups of five and were matched for sex and weight. The laboratory reference strain of B divergensused was originally isolated from a drug susceptible case of babesiosis in Glencree, Co Wicklow, Ireland in 1976. After use in experimental cattle it was adapted to gerbils, in which it underwent 60 passages, and was then designated CE2/N. The babesicidal drugs used were quinuronium sulphate (Ludobal; Bayer and several generics) diminazene aceturate (Berenil; Hoescht), amicarbalide isethionate (Diampron; May & Baker) and imidocarb dipropionate (Imizol; Coopers Pitman-Moore). A fifth drug, long-acting oxytetracyline (Terramycin LA; Pfizer), has no therapeutic activity, but may have potential as a prophylactic (Taylor et al 1986). Gerbils were usually infected by intraperitoneal injections of infected red blood cells in phosphate buffered saline, though sub-
cutaneous and intracardiac infection routes were also used. Thin smears of tail blood were obtained daily from infected gerbils and parasitaemias determined after staining with Giemsa stain. Blood was collected by cardiac puncture into heparinised syringes under sodium pentobarbitone anaesthesia and when necessary was cryopreserved and frozen stabilates revived as described by Langley (1985). Attempts were made to produce drug-tolerant reference lines from CE2/N in two ways. Method A, designed for prophylactic drugs, involved treatment of pairs of gerbils with increasing subcurative doses of drug at the time of infection and subpassage once parasitaemias had risen above 5 per cent. Method B, designed for curative drugs, consisted of the treatment of pairs of infected gerbils with increasing curative doses at haemoglobinuria and the subpassage of surviving parasites at declining parasitaemias of not more than 5 per cent. Parasites were passaged at least 10 times and then stored in liquid nitrogen. Two lines were developed by method A, one with imidocarb (CE2/IMDP-A) and another with long-acting oxytetracycline (CE2/LAT-A)and three by method B with quinuronium (CEZ/QS-B),amicarbalide (CE2/AI-B) and imidocarb (CE2/IMDP-B). Several methods of in vivo resistance assessment, based initially on those described for resistance studies on malaria (Peters 1984), were investigated with the objective of obtaining as reproducible, rapid and cheap an assay as possible. Of these, only two proved to be sufficiently practicable and reproducible. These were a curative method (TM0 and a suppression method (TM2). In TM1, gerbils were infected with l06 infected red blood cells intraperitoneally and treated at the first sign of haemoglobinuria with minimum curative drug doses, based on those determined by Gray (1983) but modified to allow for continuing parasite adaptation to gerbils. This method detected increased drug tolerance in CE2/IMDP-A and CE2/QS-Busing the parameter of maximum parasitaemias, but it was at a very low level. Fulmination never occurred in these lines following treatment. There was no evidence of tolerance in CE2/AI-B or CE2/IMDP-B. In TM2, gerbils were infected with 106 infected red blood cells intraperitoneally and treated at the same time with the minimum 126
Babesia divergens drug resistance curative drug dose. The parameters used were maximum parasitaemias and time to 1 per cent parasitaemia expressed as the delay caused by drug treatment relative to controls. This method detected increased drug tolerance in CE2/IMDP-Aand CE2/QS-B, but not in CE2/LATA, CE2/IMDP-Aor CE2/AI-B.It had proved very difficult to increase drug doses when trying to induce tolerance in the latter three lines. TM1 required fewer gerbils than TM2, but needed more intensive monitoring and seemed less sensitive than TM2, though was arguably more relevant to the field situation. Problems arising from the comparison of gerbil-adapted parasites with bovine isolates in TM1 were overcome by pretreatment of gerbils with 5 mg kg-l dexamethasone (Dexafort; Intertvet), which also reduced variability. This procedure did not, however, improve TM2. The potential for cross resistance was studied using the TM1 assay in two experiments. First, involving CE2/QS-B and the drugs quinuronium, amicarbalide, diminazene and imidocarb and, secondly, involving CE2/IMDP-A and CE2/QS-B and long-acting oxytetracycline. The results of the first experiment showed that none of the drugs, which are all chemically related, were as effective against the drug tolerant QS-B as against the sensitive reference line CE2/N (Table 1). In the second series of experiments it was shown that the unrelated drug, long-acting oxytetracycline, was slightly less active against the drug tolerant reference lines, CE2/IMDP-A and CE2/QS-B, than against the sensitive line (Table 2).
127
Two experiments were conducted to investigate the suggestion that the prophylactic use ofimidocarb injections may select for pathogenic strains of parasites, resulting in apparent drug resistance. Infections of CE2/IMDP-A and CE2/N were initiated, first, by inoculations of 106 infected red blood cells intraperitoneally and, secondly, 108 infected red blood cells subcutaneously. Comparisons were made of prepatent periods, time to haemoglobinuria, packed cell volume changes, maximum parasitaemias and spleen weights. No significant differences were found for any of these parameters and it is concluded that virulence is not increased by the selection of imidocarb-tolerant parasites, at least in gerbils. A limited number of field isolates suspected of drug resistance were tested with the in vivo assays. These were obtained by post from veterinary practices as whole blood in EDTA. On receipt, 0.5 ml of washed packed cells were injected intraperitoneally into gerbils that had been pretreated with dexamethasone and the resulting infected gerbil blood was cryopreserved. A total of 32 isolates were received in this way and three of these were tested in the TM! and TM2 alongside drug tolerant and sensitive reference lines. However, none of these field strains showed any signs of resistance. It was evident from this that a large number of field isolates may have to be tested to identify drug resistance as a significant factor in cases that respond poorly to treatment. In vitro assays would probably be more appropriate as a primary screen for this purpose, though confirmatory in vivo assays would be essenti~tl.
TABLE 1 : Tolerance of CE2/QS-Bto quinuronium, amicarbalide, diminazene and imidocarb (test method 2) Quinuronium (1-5mg kg-1)
CE2/OS-B
Amicarbalide (7.0rag kg-1)
2'50 + 0"60 P>0"I
CE2/N
5"00 -- 0'27
Diminazene (5.0mg kg -1)
Patency delay (days + SE) 0"75 + 0'11 2'6 + 0'33 P<0"001 4"75 + 0"38 7"40 -+ 0"3
Imidocarb (1.5mg kg 1)
4"20 + 0"8 P<0"002 >10'0 -+ 0"0
Maximum parasitaemia CE2/QS~B
4"86
15"40 P<0"02
CE2/N
0"35
0'07
0"47
O'0
0"0
P<0"01 0'49
TABLE 2: Tolerance of CE2/QS-Band CE2/IMDP-Ato long-acting oxytetracycline (10 mg kg-1) (test method 2) Parasite line Experiment 1 CE2/QS-B
Patency delay (days _+sE)
Maximum parasitaemia
4"2 + 0"82
14"2 P>0'I
CE2/N Experiment 2 CE2/IMDP-A
4"8 + 0"9.8
0"56
3"17 + 1"17 8"5 + 0"62
3/5 P<0'05 5/5
3'9 P<0"002
CE/N
Survival
6/6 P>0"I
0"18
6/6
128
J. S. Gray, S. L. Parr
Acknowledgements We are grateful to G. Looby and N. O'Keefe for contributions in the initial stages of this study and to B. Kaye for occasional technical assistance. Thanks are also due to Bayer (Ireland), Hoechst Ireland, Rhone Poulenc Ireland and Coopers Pitman-Moore for supplying the drugs used in this study. References DALGLIESH, R. J. & STEWART, N. P. (1977) Tolerance to imidocarb induced experimentally in tick-transmitted Babesia argentina. Australian Veterinary Journal 53, 176-180 FULTON, J. D. & YORKE, W. (1941) Studies in chemotherapy. XXVIII Drug resistance in Babesia infections. Annals of Tropical Medicine and Parasitology 35, 229-235 GRAY, J. S. (1983) Chemotherapy of Babesia divergens in the gerbil, Meriones unguiculatus. Research in Veterinary Science 35, 318-324 GRAY, J. S. & HARTE, L. N. (1985) An estimation of the prevalence and economic importance of clinical bovine babesiosis in the
Republic of Ireland. Irish Veterinary Journal 39, 75-78 GRAY, J. S. & LOOBY, G. (1984) Studies on the epidemiology of acute babesiosis in Ireland. Proceedings of the Society of Veterinary Epidemiology and Preventive Medicine, University of Edinburgh, 10-11 July 1984. Ed M. V. Thrusfield. pp 140-147 LANGLEY, R. J. (1985) The host parasite relationship of the cattle piroplasm Babesia divergens in the Mongolian gerbil, Meriones unguiculatus. PhD thesis, National University of Ireland PETERS, W. (1984) Experimental production of drug resistance. In: Handbook of Experimental Pharmacology 68/1. Eds W. Peters and W. H. G. Richards, Berlin, Heidelberg, Springer-Verlag. pp 461-473 TAYLOR, S. M., ELLIOTT, C. T. & KENNY, J. (1986) Inhibition of Babesia divergens in cattle by oxytetracycline. Veterinary Reeord 118, 98-102 YERUHAM, I., PIPANO, E. & DAVIDSON, M. (1985) A field strain of Babesia boris apparently resistant to amicarbalide isethionate. Tropical Animal Health and Production 17, 29-30
Received June 26. 1991 Aeeepted Oeloher 10, 1991