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Host effects on glutathione S-transferase activity in Fasciola hepatica
0
002~7519/93 %6.00 + 0.00 ~ergamon Press Ltd I993 Aurrralian Soaery for Parasiroloay
RESEARCHNOTE HOST EFFECTS
ON GLUTATHIONE S-TRANSFERASE FASCIOLA HEPATICA C. M.D.
*Division
of Biochemistry
TElizabeth
Macarthur
MILLER,* M.J.HowELL*
and J.C.
ACTIVITY
IN
BoRAYt
& Molecular
Agricultural
Biology. Faculty of Science, Australian National University, GPO Box 4, Canberra, A.C.T. 2601 Australia Institute, N.S.W. Department of Agriculture, Private Mail Bag 8, Camden N.S.W. 2570 Australia (Received 5 July 1993; accepted 12 July 1993)
Abstract-Mu.ER
C. M. D., HOWELL M. J. and BORAY J. C. 1993. Host effects on glutathione Stransferase activity in Fasciolu hepatica. International Journal for Parasitology 23: 1073-1076. Glntathione S-transferases (GST, E.C. 2.5.1.18) in Fasciola hepatira from sheep were previously found to be extremely variable with regard to specific GST activity and isoenzyme profile within and between parasite isolates. The effect of the host on GST activity and isoenzyme profile was examined by infecting mice, rats and cattle as well as sheep with one or the other of two isolates-ither salicylanilide-resistant or salicylanilide-susceptible F. hepatica. In the case of both isolates, GST activity in hosts relatively resistant to reinfection-rats and cattle-was lower and more restricted in range compared with hosts susceptible to multiple infection-mic;e and sheep. In the case of the rat flukes. there was little variation in isozyme profiles whereas cattle flukes appeared to exhibit more variation than sheep flukes. In mice, despite the apparent variability in GST activity, only one GST band was found in the isoenzyme profiles. Therefore, the host appears to exert a pronounced effect on the activity and expression of GSTs in F. heparica which may be related to variation in the immune responses of the different hosts during infection.
INDEX KEY WORDS: Fasciola hepatica; host effect; glutathione
GLUTATHIONES-transferases (GSTs) (E.C. 2.5.1.18) are a family of multifunctional proteins concerned and the detoxification of xenobiotics with endogenously derived toxic compounds using catalytic and binding mechanisms (Brophy, Papadopoulos, Touraki, Coles, Kiirting & Barrett, 1989). They appear to be ubiquitous throughout the plant and animal kingdoms and activity has been detected in a number of helminths where they appear to be the major detoxification enzymes since helminths apparently lack cytochrome P-450 (Precious & Barrett, 1989). GST activity has been correlated with drug resistance in Haemonchus conlortus (Kawalek, Rew & Heavner, 1984) and a connection has also been found between salicylanilidc resistance and GST activity in a number of isolates of FuscioIa hepaticu (C. M. D. Miller, M. J. Howell & J. C. Boray, submitted). However, activity levels and isoenzyme banding patterns in F. hepatica were found
to
be
extremely
variable
both
within
and
between different isolates. In an attempt to isolate factors involved in the modification of cxprcssion of
~To whom correspondence
S-transferase;
sheep: cattle: mice: rats.
GSTs in F. heppatica the effect of the species of host on activity and isoenzyme profile in particular isolates of F. hepatica was investigated. Two isolates of F. hepaticu with different susceptibilities to rafoxanide and closantel were used in this study. Both were originally derived from field infections and subsequently maintained in the laboratory (Boray & DeBono, 1989; 1990). Compton, originally isolated in the U.K., was fully susceptible to a range of anthelmintics while Jerangle K, originally isolated in N.S.W., exhibited resistance to rafoxanide and closantel (Boray, 1990). Flukes from both isolates were recovered 16 week post-infection (p.i.) from experimentally infected sheep, 21 days p.i. from Swiss mice or 49 days p.i. from Wistar rats. In addition, flukes from the Compton isolate were recovered from experimentally infected cattle at 16 week p.i. All flukes were allowed to disgorge caecal contents in H&don-Fleig saline (120 mh+NaCl, 4 mMKCI, 0.8 mM-Ca Cl,.2H,O, 2 mh+MgS0,.7H,O, 1.5% NaHCO,, 4% glucose) at 37°C before individuals were homogenised in 1 ml of 0.1% Triton X-100 (0.1 ml for flukes recovered from mice), briefly centrifuged at 12,OOOg and the supernatant stored at -20°C.
should be addressed.
1073
C. M. D. MILLER et al.
1074
A
2 A
n
A a
n
1 2 3 4 )_-.------___-__
A A
n
5
6
7
3
9
10 1112
13
n
n
A
b
FIG. 1. GST activity in crude homogenates of individual flukes of the Compton isolate (a) recovered from mice,
rats, cattle and sheep and the Jerangle K isolate (m) recovered from mice, rats and sheep. Each point represents specific GST activity
in an individual
fluke.
GST activity was assayed using CDNB as second substrate and isoenzyme profiles were examined using native PAGE and Western blotting as described elsewhere (C. M. D. Miller, M. J. Howell &J. C. Boray, submitted). The nature of expression of GST appears to be altered by the host environment as well as by the drug resistance status of the isolate. This is generally seen as a lower, narrower range of activity in mice, rats and cattle compared with sheep. The effect is more pronounced in the salicylanilide-susceptible isolate, Compton, than in the salicylanilide-resistant isolate, Jerangle K (Figure 1). Activities obtained for individual Compton flukes from sheep ranged from 0.27 to 6.52 units mg protein -’ (mean = 3.48 l 0.51, n = 12). By comparison, activities obtained from mice (1.11 f 0.14, n = 6), rats (0.27 f 0.05, 12 = 6) and cattle (0.34 f 0.05, n = 12) had a much narrower range and the mean level of activity was significantly lower than the level in flukes recovered from sheep (P ~0.05, Mann-Whitney U test).
FIG. 2. Diagram of Western blot of 12% native PAGE gel of crude homogenates of individual flukes from (a) the Compton isolate recovered from mice, rats, cattle and sheep and (b) the Jerangle K isolate recovered from mice, rats and sheep. Each blot was probed with serum from sheep injected with fluke GST (Howell et al., 1988). Negative results with control serum from a colostrum-deprived uninfected sheep confirmed the specificity of the anti-GST serum (data not shown). Approximately 40~1 was loaded for each sample; 2pg of purified GST (Howell et al., 1988) was loaded as a control. The homogenates of flukes from mice were combined to provide enough sample. All other samples were from individual flukes. Bands of darker intensity are shaded while fainter bands are open. (a) Compton flukes: Lane 1 purified GST; 2,3 flukes from mice; 4,5 flukes from rats; 69 flukes from cattle; l&l3 flukes from sheep. (b) Jerangle flukes: Lane 1 purified GST; 2,3 flukes from mice; 4-7 flukes from treated rats; 8-13 flukes from sheep.
Previous work has shown that a restriction in range and level of activity is observed with development of rafoxanide and closantel resistance (C. M. D. Miller, M. J. Howell & J. C. Boray, submitted). This could obscure effects seen due to the host and could explain why there was no significant difference between the level of activity in Jerangle K flukes from rats (0.60 f 0.11, n = 7) and sheep (1.15 * 0.43, n= 12). Contrary to what was observed with Compton flukes, Jerangle K flukes recovered from mice showed a significantly higher level of activity (2.73 + 0.37,
Research Note n = 6) although not a greater range than flukes recovered from sheep (P < 0.05, Mann-Whitney U test). Isoenzyme profiles in crude homogenates of Compton flukes recovered from rats, mice, sheep and cattle [Fig. 2(a)] and for Jerangle flukes recovered from rats, mice and sheep [Fig. 2(b)] were examined using native PAGE and Western blotting to see if particular isoenzyme profiles of the parasites could be related to the species of host animal. Homogenates of flukes from mice were pooled to provide enough sample. All other lanes were profiles obtained for individual flukes. The most striking observation is the single isoenzyme observed in flukes from mice in both Compton [Fig. 2(a), lanes 2,3] and Jerangle K [Fig. 2(b), Lanes 2, 31 despite the variability observed in activity amongst individual flukes from this host. Underlining the restriction in range and level of GST activity seen in Compton flukes from rats, the isoenzyme profiles [Fig. 2(a), Lanes 4, 51 showed a reduced number of bands and no apparent variation. In contrast, the isoenzyme profiles of flukes obtained from sheep were highly variable [Fig. 2(a), Lanes 10-131. Similarly, with Jerangle K rat flukes [Fig. 2(b), Lanes 4-71 no variation was seen in profiles between individuals although there were more bands present than observed in most sheep fluke profiles [Fig. 2(b), Lanes 8-131. Flukes from cattle [Fig. 2(a), Lanes 6 91, by contrast, showed a startling amount of variation, comparable with that observed in sheep flukes, despite an apparent restriction in range and level of GST activity. Examination of a larger sample size (data not shown) revealed that, although many of the bands were shared, most flukes had a unique profile. As was previously found (C. M. D. Miller, M. J. Howell & J. C. Boray, submitted), none of the profiles found in Compton or Jerangle K flukes in the various hosts corresponded to that found in the fluke GST purified by Howell, Board & Boray (1988) which was used as a positive control. Therefore, the host appears to have an effect on both the range and level of GST activity and the variability seen in isoenzyme profiles. This is not without precedent as host effects on metabolic enzymes of Hymenolepis diminuta have also been recorded. Examination of the levels of catabolic endproducts (lactate, succinate and acetate) of this parasite have revealed the presence of two types of worms. All individuals within one rat had the same metabolic profile but the profile varied between rats (Bennet, Behm & Bryant, 1990). The nature of the effects observed in H. diminuta has not been definitively established but they appear to be immunologically based since different inbred strains
1075
of rats affect the metabolic status of the worms in different ways and prior infection with intestinal nematodes, such as Nippostrongylus brasiliensis, that provoke an inflammatory response also cause profound metabolic changes in H. diminuta (Bennet et al., 1990). It is interesting to note that GST activity is lower in hosts that can develop resistance to reinfection (rats and cattle) compared with hosts that are susceptible to multiple infections (mice and sheep). Helminth GSTs are known to conjugate with other endogenous substances such as lipid hydroperoxides and are thought to have a role in the protection of the parasite from free radicals produced by the host (Smith, Davern, Board, Ti, Garcia & Mitchell, 1986; Brophy & Barrett, 1990). Rats have been found to produce 30 times more free radicals per animal than mice in response to F. hepatica challenge infection (Smith, Ovington & Boray, 1992) so perhaps the differences in GST activities and profiles between hosts are related to the operation of different immune mechanisms. There is some evidence that this may be occurring as rats can eliminate existing infections and show resistance to reinfection whereas sheep and mice can do neither (Hughes, 1987). Thus, rats may effectively (by as yet unknown means) restrict fluke activity, thus compromising the parasite’s ability to combat products of the oxidative burst. However, the immune response may not be involved at all as flukes from cattle show the same restricted range and level of GST activities as rats but immunological mechanisms are not considered to be involved in resistance to reinfection with F. hepatica in cattle. It appears that resistance in cattle may be nutritionally based i.e. adult flukes cannot survive in the bile ducts due to severe fibrosis and dystrophic calcification. In previously chronically infected cattle most immature flukes are eliminated within the liver parenchyma due to proliferating fibrosis (Ross, Todd & Dow, 1966; Boray, 1969; Doyle, 1973; Doy & Hughes, 1984). It may be of relevance that rat flukes all exhibited the same isoenzyme profile whereas profiles of cattle flukes exhibited a great variety. The reason for this is unclear and will remain so until the role of GSTs in the physiology of the liver fluke is better understood. Clearly, these results indicate that care should be taken when using different hosts (alternative species or strains for a particular parasite) for studies of parasite biochemistry as host effects can be profound. The plasticity in GST activity and isoenzyme profile also reinforces the notion that parasites are extremely adaptable to unusual or changed circumstances. Acknowledgements-Thanks technical
assistance
to Doreen DeBono for and to Dr N. C. Smith for many helpful
1076
C. M. D. MILLER et al.
comments. This work was funded, including the award of an Australian Wool Corporation Postgraduate Scholarship to C. M. D. Miller, by the Australian Wool Corporation. REFERENCES BENNET E. M., BEHM C. A. & BRYANT C. 1990. The role of
the host in the regulation of end-product formation in two strains of the rat tapeworm, Hymenolepis diminuta. International Journalfor Parasitology 20: 841-848. BORAY J. C. 1969. Experimental fascioliasis in Australia. Advances in Parasitology 7: 95-2 10. BORAY J. C. & DE BONO D. 1989. Drug resistance in Fasciola hepatica. In: Australian Advances in Veterinary Science (Edited by OUT-~ERIDGEP. M. & RICHARDS R. B.), pp. 166-169. Australian Veterinary Association, Sydney. BORAY J. C. 1990. Drug resistance in Fasciolu hepatica In: Resistance of Parasites to Antiparasitic Drugs. Round table conference at the VIIth International Conference of Parasitology Paris August 1990 (Edited by BORAYJ. C., MARTIN P. J. & ROUSH R. T.) pp. 51-60. MSD AGVET, Rahway, New Jersey. BROPHY P. M. & BARRETT J. 1990. Strategies for detoxification of aldehydic products of lipid peroxidation in helminths. Molecular and Biochemical Parasitology 42: 205-212. BROPHY P. M., PAPA~~ULOS A., TOURAKI M., COLES B., K~RTING W. & BARRETTJ. 1989. Purification of cytosolic glutathione transferases from Schistocephalus solidus (plerocercoid): interaction with anthelmintics and products of lipid peroxidation. Molecular and Biochemical Parasitology 36: 187-196. DOY T. G. & HUGHES D. L. 1984. Fasciola hepatica: site of resistance to reinfection in cattle. Experimental Parasitology 57: 247-278.
DOYLE J. J. 1973. The relationship between the duration of a primary infection and the subsequent development of any required resistance to experimental infections with Fasciola hepatica in calves. Research in Veterinary Science 14: 97-103. HOWELL M. J., BOARD P. G. & BORAY J. C. 1988. Glutathione S- transferases in Fusciolu hepatica. Journal of Parasitology 74: 7 15-7 18. HUGHES D. L. 1987. Fasciola and Fascioloides In: Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis Volume II: Trematodes and Cestodes (Edited by SOULSBYE. J. L.), pp. 91-114. CRC Press Inc., Boca Raton, Florida. KAWALEKJ. C., REW R. S. & HEAVNERJ. 1984. Glutathione S-transferase, a possible drug-metabolizing enzyme, in Haemonchus contortus: comparative activity of a cambenadazole-resistance and a susceptible strain. International Journalfor Parasitology 14: 173- 175. PRECIOUSW. Y. & BARRETTJ. 1989. Xenobiotic metabolism in helminths Parasitology Today 5: 156-160. Ross J. G., TODD J. R. & Dow C. 1966. Single experimental infections of calves with the liver fluke, Fasciola hepatica (Linnaeus 1758) Journal of Comparative Pathology 76: 67-8 1. SMITH D. B.,, DAVERN K. M., BOARD P. G., TI W. U., GARCIA E. G. & MITCHELLG. F. 1986. M, 26,000 antigen of Schistosoma japonicum recognised by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proceedings of the National Academy of Sciences of the United States of America 83: 8703-8707. SMITH N. C., OVINGTONK. S. & BORAY J. C. 1992. Fusciokz hepatica: free radical generation by peritoneal leukocytes in challenged rodents. International Journal for Parasitology 22: 281-286.
002~7519/93 %6.00 + 0.00 ~ergamon Press Ltd I993 Aurrralian Soaery for Parasiroloay
RESEARCHNOTE HOST EFFECTS
ON GLUTATHIONE S-TRANSFERASE FASCIOLA HEPATICA C. M.D.
*Division
of Biochemistry
TElizabeth
Macarthur
MILLER,* M.J.HowELL*
and J.C.
ACTIVITY
IN
BoRAYt
& Molecular
Agricultural
Biology. Faculty of Science, Australian National University, GPO Box 4, Canberra, A.C.T. 2601 Australia Institute, N.S.W. Department of Agriculture, Private Mail Bag 8, Camden N.S.W. 2570 Australia (Received 5 July 1993; accepted 12 July 1993)
Abstract-Mu.ER
C. M. D., HOWELL M. J. and BORAY J. C. 1993. Host effects on glutathione Stransferase activity in Fasciolu hepatica. International Journal for Parasitology 23: 1073-1076. Glntathione S-transferases (GST, E.C. 2.5.1.18) in Fasciola hepatira from sheep were previously found to be extremely variable with regard to specific GST activity and isoenzyme profile within and between parasite isolates. The effect of the host on GST activity and isoenzyme profile was examined by infecting mice, rats and cattle as well as sheep with one or the other of two isolates-ither salicylanilide-resistant or salicylanilide-susceptible F. hepatica. In the case of both isolates, GST activity in hosts relatively resistant to reinfection-rats and cattle-was lower and more restricted in range compared with hosts susceptible to multiple infection-mic;e and sheep. In the case of the rat flukes. there was little variation in isozyme profiles whereas cattle flukes appeared to exhibit more variation than sheep flukes. In mice, despite the apparent variability in GST activity, only one GST band was found in the isoenzyme profiles. Therefore, the host appears to exert a pronounced effect on the activity and expression of GSTs in F. heparica which may be related to variation in the immune responses of the different hosts during infection.
INDEX KEY WORDS: Fasciola hepatica; host effect; glutathione
GLUTATHIONES-transferases (GSTs) (E.C. 2.5.1.18) are a family of multifunctional proteins concerned and the detoxification of xenobiotics with endogenously derived toxic compounds using catalytic and binding mechanisms (Brophy, Papadopoulos, Touraki, Coles, Kiirting & Barrett, 1989). They appear to be ubiquitous throughout the plant and animal kingdoms and activity has been detected in a number of helminths where they appear to be the major detoxification enzymes since helminths apparently lack cytochrome P-450 (Precious & Barrett, 1989). GST activity has been correlated with drug resistance in Haemonchus conlortus (Kawalek, Rew & Heavner, 1984) and a connection has also been found between salicylanilidc resistance and GST activity in a number of isolates of FuscioIa hepaticu (C. M. D. Miller, M. J. Howell & J. C. Boray, submitted). However, activity levels and isoenzyme banding patterns in F. hepatica were found
to
be
extremely
variable
both
within
and
between different isolates. In an attempt to isolate factors involved in the modification of cxprcssion of
~To whom correspondence
S-transferase;
sheep: cattle: mice: rats.
GSTs in F. heppatica the effect of the species of host on activity and isoenzyme profile in particular isolates of F. hepatica was investigated. Two isolates of F. hepaticu with different susceptibilities to rafoxanide and closantel were used in this study. Both were originally derived from field infections and subsequently maintained in the laboratory (Boray & DeBono, 1989; 1990). Compton, originally isolated in the U.K., was fully susceptible to a range of anthelmintics while Jerangle K, originally isolated in N.S.W., exhibited resistance to rafoxanide and closantel (Boray, 1990). Flukes from both isolates were recovered 16 week post-infection (p.i.) from experimentally infected sheep, 21 days p.i. from Swiss mice or 49 days p.i. from Wistar rats. In addition, flukes from the Compton isolate were recovered from experimentally infected cattle at 16 week p.i. All flukes were allowed to disgorge caecal contents in H&don-Fleig saline (120 mh+NaCl, 4 mMKCI, 0.8 mM-Ca Cl,.2H,O, 2 mh+MgS0,.7H,O, 1.5% NaHCO,, 4% glucose) at 37°C before individuals were homogenised in 1 ml of 0.1% Triton X-100 (0.1 ml for flukes recovered from mice), briefly centrifuged at 12,OOOg and the supernatant stored at -20°C.
should be addressed.
1073
C. M. D. MILLER et al.
1074
A
2 A
n
A a
n
1 2 3 4 )_-.------___-__
A A
n
5
6
7
3
9
10 1112
13
n
n
A
b
FIG. 1. GST activity in crude homogenates of individual flukes of the Compton isolate (a) recovered from mice,
rats, cattle and sheep and the Jerangle K isolate (m) recovered from mice, rats and sheep. Each point represents specific GST activity
in an individual
fluke.
GST activity was assayed using CDNB as second substrate and isoenzyme profiles were examined using native PAGE and Western blotting as described elsewhere (C. M. D. Miller, M. J. Howell &J. C. Boray, submitted). The nature of expression of GST appears to be altered by the host environment as well as by the drug resistance status of the isolate. This is generally seen as a lower, narrower range of activity in mice, rats and cattle compared with sheep. The effect is more pronounced in the salicylanilide-susceptible isolate, Compton, than in the salicylanilide-resistant isolate, Jerangle K (Figure 1). Activities obtained for individual Compton flukes from sheep ranged from 0.27 to 6.52 units mg protein -’ (mean = 3.48 l 0.51, n = 12). By comparison, activities obtained from mice (1.11 f 0.14, n = 6), rats (0.27 f 0.05, 12 = 6) and cattle (0.34 f 0.05, n = 12) had a much narrower range and the mean level of activity was significantly lower than the level in flukes recovered from sheep (P ~0.05, Mann-Whitney U test).
FIG. 2. Diagram of Western blot of 12% native PAGE gel of crude homogenates of individual flukes from (a) the Compton isolate recovered from mice, rats, cattle and sheep and (b) the Jerangle K isolate recovered from mice, rats and sheep. Each blot was probed with serum from sheep injected with fluke GST (Howell et al., 1988). Negative results with control serum from a colostrum-deprived uninfected sheep confirmed the specificity of the anti-GST serum (data not shown). Approximately 40~1 was loaded for each sample; 2pg of purified GST (Howell et al., 1988) was loaded as a control. The homogenates of flukes from mice were combined to provide enough sample. All other samples were from individual flukes. Bands of darker intensity are shaded while fainter bands are open. (a) Compton flukes: Lane 1 purified GST; 2,3 flukes from mice; 4,5 flukes from rats; 69 flukes from cattle; l&l3 flukes from sheep. (b) Jerangle flukes: Lane 1 purified GST; 2,3 flukes from mice; 4-7 flukes from treated rats; 8-13 flukes from sheep.
Previous work has shown that a restriction in range and level of activity is observed with development of rafoxanide and closantel resistance (C. M. D. Miller, M. J. Howell & J. C. Boray, submitted). This could obscure effects seen due to the host and could explain why there was no significant difference between the level of activity in Jerangle K flukes from rats (0.60 f 0.11, n = 7) and sheep (1.15 * 0.43, n= 12). Contrary to what was observed with Compton flukes, Jerangle K flukes recovered from mice showed a significantly higher level of activity (2.73 + 0.37,
Research Note n = 6) although not a greater range than flukes recovered from sheep (P < 0.05, Mann-Whitney U test). Isoenzyme profiles in crude homogenates of Compton flukes recovered from rats, mice, sheep and cattle [Fig. 2(a)] and for Jerangle flukes recovered from rats, mice and sheep [Fig. 2(b)] were examined using native PAGE and Western blotting to see if particular isoenzyme profiles of the parasites could be related to the species of host animal. Homogenates of flukes from mice were pooled to provide enough sample. All other lanes were profiles obtained for individual flukes. The most striking observation is the single isoenzyme observed in flukes from mice in both Compton [Fig. 2(a), lanes 2,3] and Jerangle K [Fig. 2(b), Lanes 2, 31 despite the variability observed in activity amongst individual flukes from this host. Underlining the restriction in range and level of GST activity seen in Compton flukes from rats, the isoenzyme profiles [Fig. 2(a), Lanes 4, 51 showed a reduced number of bands and no apparent variation. In contrast, the isoenzyme profiles of flukes obtained from sheep were highly variable [Fig. 2(a), Lanes 10-131. Similarly, with Jerangle K rat flukes [Fig. 2(b), Lanes 4-71 no variation was seen in profiles between individuals although there were more bands present than observed in most sheep fluke profiles [Fig. 2(b), Lanes 8-131. Flukes from cattle [Fig. 2(a), Lanes 6 91, by contrast, showed a startling amount of variation, comparable with that observed in sheep flukes, despite an apparent restriction in range and level of GST activity. Examination of a larger sample size (data not shown) revealed that, although many of the bands were shared, most flukes had a unique profile. As was previously found (C. M. D. Miller, M. J. Howell & J. C. Boray, submitted), none of the profiles found in Compton or Jerangle K flukes in the various hosts corresponded to that found in the fluke GST purified by Howell, Board & Boray (1988) which was used as a positive control. Therefore, the host appears to have an effect on both the range and level of GST activity and the variability seen in isoenzyme profiles. This is not without precedent as host effects on metabolic enzymes of Hymenolepis diminuta have also been recorded. Examination of the levels of catabolic endproducts (lactate, succinate and acetate) of this parasite have revealed the presence of two types of worms. All individuals within one rat had the same metabolic profile but the profile varied between rats (Bennet, Behm & Bryant, 1990). The nature of the effects observed in H. diminuta has not been definitively established but they appear to be immunologically based since different inbred strains
1075
of rats affect the metabolic status of the worms in different ways and prior infection with intestinal nematodes, such as Nippostrongylus brasiliensis, that provoke an inflammatory response also cause profound metabolic changes in H. diminuta (Bennet et al., 1990). It is interesting to note that GST activity is lower in hosts that can develop resistance to reinfection (rats and cattle) compared with hosts that are susceptible to multiple infections (mice and sheep). Helminth GSTs are known to conjugate with other endogenous substances such as lipid hydroperoxides and are thought to have a role in the protection of the parasite from free radicals produced by the host (Smith, Davern, Board, Ti, Garcia & Mitchell, 1986; Brophy & Barrett, 1990). Rats have been found to produce 30 times more free radicals per animal than mice in response to F. hepatica challenge infection (Smith, Ovington & Boray, 1992) so perhaps the differences in GST activities and profiles between hosts are related to the operation of different immune mechanisms. There is some evidence that this may be occurring as rats can eliminate existing infections and show resistance to reinfection whereas sheep and mice can do neither (Hughes, 1987). Thus, rats may effectively (by as yet unknown means) restrict fluke activity, thus compromising the parasite’s ability to combat products of the oxidative burst. However, the immune response may not be involved at all as flukes from cattle show the same restricted range and level of GST activities as rats but immunological mechanisms are not considered to be involved in resistance to reinfection with F. hepatica in cattle. It appears that resistance in cattle may be nutritionally based i.e. adult flukes cannot survive in the bile ducts due to severe fibrosis and dystrophic calcification. In previously chronically infected cattle most immature flukes are eliminated within the liver parenchyma due to proliferating fibrosis (Ross, Todd & Dow, 1966; Boray, 1969; Doyle, 1973; Doy & Hughes, 1984). It may be of relevance that rat flukes all exhibited the same isoenzyme profile whereas profiles of cattle flukes exhibited a great variety. The reason for this is unclear and will remain so until the role of GSTs in the physiology of the liver fluke is better understood. Clearly, these results indicate that care should be taken when using different hosts (alternative species or strains for a particular parasite) for studies of parasite biochemistry as host effects can be profound. The plasticity in GST activity and isoenzyme profile also reinforces the notion that parasites are extremely adaptable to unusual or changed circumstances. Acknowledgements-Thanks technical
assistance
to Doreen DeBono for and to Dr N. C. Smith for many helpful
1076
C. M. D. MILLER et al.
comments. This work was funded, including the award of an Australian Wool Corporation Postgraduate Scholarship to C. M. D. Miller, by the Australian Wool Corporation. REFERENCES BENNET E. M., BEHM C. A. & BRYANT C. 1990. The role of
the host in the regulation of end-product formation in two strains of the rat tapeworm, Hymenolepis diminuta. International Journalfor Parasitology 20: 841-848. BORAY J. C. 1969. Experimental fascioliasis in Australia. Advances in Parasitology 7: 95-2 10. BORAY J. C. & DE BONO D. 1989. Drug resistance in Fasciola hepatica. In: Australian Advances in Veterinary Science (Edited by OUT-~ERIDGEP. M. & RICHARDS R. B.), pp. 166-169. Australian Veterinary Association, Sydney. BORAY J. C. 1990. Drug resistance in Fasciolu hepatica In: Resistance of Parasites to Antiparasitic Drugs. Round table conference at the VIIth International Conference of Parasitology Paris August 1990 (Edited by BORAYJ. C., MARTIN P. J. & ROUSH R. T.) pp. 51-60. MSD AGVET, Rahway, New Jersey. BROPHY P. M. & BARRETT J. 1990. Strategies for detoxification of aldehydic products of lipid peroxidation in helminths. Molecular and Biochemical Parasitology 42: 205-212. BROPHY P. M., PAPA~~ULOS A., TOURAKI M., COLES B., K~RTING W. & BARRETTJ. 1989. Purification of cytosolic glutathione transferases from Schistocephalus solidus (plerocercoid): interaction with anthelmintics and products of lipid peroxidation. Molecular and Biochemical Parasitology 36: 187-196. DOY T. G. & HUGHES D. L. 1984. Fasciola hepatica: site of resistance to reinfection in cattle. Experimental Parasitology 57: 247-278.
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