Resistance to fascioliasis — A review

Veterinary Parasitology, 20 (1986) 63--93 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

63

R E S I S T A N C E T O F A S C I O L I A S I S -- A R E V I E W

EL TAHIR M. HAROUN and GEORGE V. HILLYER 1. USPHS-NIH Fogarty International Fellow, Department, of Pathology, Faculty of Veterinary Sciences, University of Khartoum, P.O. Box 32, Khartoum North (Sudan) z Laboratory of Parasite Immunology, Department of Biology, University of Puerto Rico, Rio Piedras, PR 00931 (U.S.A.) ABSTRACT

Haroun, E1 Tahir M. and Hillyer, G.V., 1986. Resistance to fascioliasis -- a review. Vet. Parasitol., 20: 63--93. Attempts to actively stimulate or passively transfer resistance to Fasciola hepatica or F. gigantica in various laboratory and farm animals including mice, rats, rabbits, sheep, goats and cattle have been reviewed. These attempts comprised sensitization by primary homologous or heterologous normal or irradiated infections per os, sensitization by subcutaneous, intramuscular or intraperitoneal implantation with the various fluke stages, sensitization by somatic extracts or metabolic products of mature or immature flukes and passive transfer of resistance by immune serum or sensitized lymphocytes. INTRODUCTION

Fascioliasis is an e c o n o m i c a l l y i m p o r t a n t disease caused b y digenetic t r e m a t o d e s o f t h e genus Fasciola, and its o c c u r r e n c e is d e p e n d e n t o n t h e presence o f b i o t o p e s suitable f o r the d e v e l o p m e n t o f t h e snail i n t e r m e d i a t e h o s t o f t h e parasite. F. hepatica and F. gigantica are t h e t w o species m o s t l y i n c r i m i n a t e d f o r causing the disease and b o t h are t r a n s m i t t e d b y snails o f the family L y m n a e s i d a e . F. hepatica prevails in t e m p e r a t e zones while F. gigantica is p r e d o m i n a n t in t r o p i c a l regions (Over, 1982). Fasciolasis is e c o n o m i c a l l y m o s t i m p o r t a n t in sheep and cattle, b u t it can also a f f e c t a wide range o f susceptible hosts including goats, camels, buffalo, swine and Equidae. Wild m a m m a l s m a y also c o n t r a c t t h e disease and some o f these act as reservoir hosts (Dinnik and Dinnik, 1959; H a m m o n d , 1972). H u m a n fascioliasis has also b e e n r e p o r t e d f r o m m a n y parts o f the w o r l d (Dawes and Hughes, 1 9 6 4 ; B o r a y 1969; H a r d m a n et al., 1 9 7 0 ; H a m m o n d , 1974). T h e e c o n o m i c losses caused b y fascioliasis can be v e r y serious, particularly w h e n t h e local e n v i r o n m e n t a l c o n d i t i o n s are c o n d u c i v e f o r high and persiste n t rates o f transmission. In such situations animals are o f t e n e x p o s e d t o large doses o f i n f e c t i o n , s o m e t i m e s taking the f o r m o f o u t b r e a k s o f an acute disease with high mortalities, particularly in sheep (Karib, 1 9 6 2 ; H a r o u n , * Author to w h o m correspondence should be addressed.

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64 1975). However, of even greater overall economic importance are the less dramatic but insidious, long term and deleterious effects of the chronic wasting condition. The sequelae of such types of chronic disease may include inappetence, poor food utilization, loss in body weight, reduced milk and wool production and stillbirth (Pantelouris, 1965; Sinclair, 1967; Roseby, 1970; Crossland et al., 1977. In addition, condemnation of infected livers at meat inspection results in a considerable loss of revenue. Conventional methods for the control of fascioliasis include snail eradication by molluscicides, use of fasciolicides to reduce contamination and improved drainage systems to adversely influence the snail habitat (Armour, 1975). The use of molluscicides, however, involves certain hazards such as accumulation of toxic residues in water and soil which may have immediate or long-term effects on the surrounding fauna (Amin and Fe~wick, 1977). Furthermore, resistant strains of the target snail may develop. Regular application of molluscicides into extensive breeding areas does not result in complete eradication; the survivors are necessarily the resistant members of the snail population which may quickly repopulate the treated areas with resistant progeny. Moreover, the need for repeated applications of molluscicides to maintain the optimum concentration in contaminated water bodies makes it an expensive means of control. In spite of their wide use against fascioliasis, anthelmintics have their drawbacks. Many drugs are ineffective at the recommended dose against the immature stages of the liver fluke. Incomplete elimination of the infection results in subclinical fascioliasis with continuous contamination of pasture (Ollerenshaw, 1971). This is most importan~ in sheep, which can harbour the subclinical form of the disease for a long time. Therapeutic and toxic doses of anthelmintics are very close (Gibson, 1964), so employing skilled labor for careful administration adds to the high cost of these chemicals, particularly in endemic areas where mass chemotherapy is necessary. Thus it appears that there is a need for an alternative and more effective means of control, such as vaccination. Various attempts have been made to stimulate resistance against fascioliasis in different hosts and to understand the mechanism of this resistance with the ultimate goal of producing a vaccine. These attempts are reviewed in the following pages. STIMULATION OF RESISTANCE TO FASCIOLA HEPATICA OR F. GIGANTICA BY PRIMARY HOMOLOGOUS INFECTION Mammalian hosts seem to vary in their ability to acquire resistance against challenge infection with F. hepatica or F. gigantica following primary homologous sensitization. Mice

Lang (1967) reported that mice sensitized with two infections o f F . hepatica before challenge showed an earlier migration of the worms from the hepatic

65 parenchyma to the bile ducts 20 days after, challenge; however, 40 days after challenge they harboured significantly fewer worms than the controls. According to this worker, the nature and timing of l y m p h o c y t e infiltrations and the histopathology of the liver indicated that the delayed (cellular) hypersensitivity might be responsible for the resistance manifested by the sensitized mice. This conclusion was corroborated by the results of another experiment (Lang et al., 1967) in which the transfer of peritoneal exudate cells from isologous donors caused more rapid response in the recipients and a significant reduction in worms. In contrast to these results, Chapman and Mitchell (1982) reported the failure of several strains of mice to develop resistance to challenge with F. hepatica following one or two primary infections for 4--8 weeks. A primary infection with large doses of cysts (10--30) followed by chemotherapy also failed to stimulate resistance to challenge infection. Additional work to resolve this apparent discrepancy in mice is clearly warranted.

Rats Rats consistently exhibit significant resistance to reinfection with F. hepatica. Hayes et al. (1973) reported 76% fewer flukes from a challenge infection in rats with a primary infection of F. hepatica than in rats n o t previously exposed. At 7 weeks post-infection the livers of rats infected with one fluke showed little damage excluding any important role for non-specific liver inflammation and damage existing at the time of challenge in the resistance to F. hepatica in rats. Hayes et al. (1974a) also reported significant resistance to superinfection with F. hepatica in rats harbouring chronic primary infections of 7 or 12 months' duration. Pathological changes in the livers of those rats were minimal and it was unlikely that they had an effect on resistance. Furthermore, Hayes and Mitrovic (1977) and Rajasekariah and Howell (1977) reported evidence that the gut acts as an effective barrier to the freshly-excysted juveniles of the challenge infection with F. hepatica. Besides reductions in worm recovery from previously infected rats, livers of rats orally challenged after a previous infection appeared to be free from secondary damage while considerable damage was evident in rates which received an intraperitoneal challenge. An immunological basis for the resistance against challenge in sensitized hosts was further indicated by the repeated finding that resistance stimulated by primary infection persists in rats even after the removal of the infection by anthelmintic treatment. Goose and MacGregor (1973) showed that in resistant superinfected rats the flukes of the immunizing primary infection in the bile ducts are not affected by the immunological process which acts against the worms of the challenge infection. They suggested that either the flukes in the bile ducts are not accessible to the host defense mechanisms or that host mimicry is operating in this host-parasite relationship.

66

Rabbits The protective effect of primary infections with F. hepatica against challenge seems to be controversial in rabbits. Ross (1966) reported a reduction in the size and number of worms recovered from a challenge infection following primary sensitization but there was considerable individual variation and the reduction was not statistically significant. Kendall et al. (1967) also found a reduction in the number of worms recovered from a challenge infection following sensitization, but they attributed this to an inhibition of growth due to overcrowding rendering the worms difficult to find rather than to real resistance. Significant resistance was, however, reported in rabbits with a primary infection which had been terminated by hexachloroethane 2 days before challenge (Kendall and Sinclair, 1971). This resistance was considered to be only apparent since no protection could be found when the primary infection was removed 4 days before challenge. The apparent resistance was attributed to the interaction of the sensitizing infection and the drug resulting in a transient release of antigen which initiates a delayed hypersensitivity in the liver and leads to non-specific allergic inflammation, thus creating an environment adverse to the challenge infection. On the other hand, F o r t m e y e r (1973) found significant resistance to oral challenge in repeatedly infected rabbits, but not to intraperitoneal challenge. He concluded that t w o kinds of immune mechanism operate, one in the intestinal wall and the other during the migration phase. Bolbol et al. {1978) and Haroun et al. (1980a) did n o t find resistance in rabbits with a single primary mature or immature infection which had been eliminated by rafoxanide at least 1 week before challenge. However, following sensitization with t w o curtailed immature infections, Bolbol et al. (1978) reported a significant resistance (45%) after challenge. In contrast, Haroun et al. (1980a) found a non-significant reduction of 33% in a similar experiment.

Sheep It appears that there is no evidence to indicate that primary sensitization of sheep with F. hepatica stimulates any resistance to challenge in terms of reduction in the number of worms recovered from challenge infection. However, other manifestations such as retarded worm growth, decrease in worm size, reduced egg production of adult worms, delay of onset of anemia and elevated antibody titers can be seen as effects of a primary sensitizing infection. Thus, in sheep infected with 150 metacercariae of F. hepatica on four occasions before challenge, Sinclair (1962) could not find any resistance to challenge, although he observed delayed and reduced egg production by the adult flukes. Boray (1967) infected sheep with large doses (1000 cysts) of normal F. hepatica, terminated the infections with an anthelmintic and then challenged these sheep and controls with 4000 cysts each. He found no appreciable difference in the number of flukes recovered from the challenge

67 infections compared with the controls. However, the previously infected sheep lived longer and developed anemia later than the controls. He concluded that hepatic fibrosis in repeatedly infected sheep acts as a mechanical barrier which does not reduce the number and size but may reduce the mobility of the migrating flukes of the challenge infection. Sinclair (1970) reported that following a primary infection in sheep, the juvenile flukes of the challenge infection migrate more rapidly to the bile ducts due to an earlier local reaction to the parasites. The protective effect of immature flukes was investigated by Sinclair (1971a) when he compared t w o groups of sheep, one previously uninfected and one which had been treated to terminate an 8-week primary infection. The challenged sheep showed a marked temporary retardation of fluke development 8 weeks after infection as well as fibroblastic activity together with lymphocytic infiltration which ocurred earlier than in the livers of the controls. Although fluke recoveries were similar, unlike his results in 1970, there was evidence of delayed entry to the bile ducts in the challenged group. The retardation of fluke development was related to the earlier occurrence of fibroblastic activity and lymphocytic infiltration in the livers of the challenged group of sheep. Sinclair (1971a) suggested that the lymphocytes, which are the antigen-sensitive cells directly involved in antib o d y production and the carriage of immunological memory, are probably concerned in the retarded development of the flukes in the challenged group. Differences between the degree of eosinophilia in the blood and tissues of the livers of the challenged and control sheep were also reported in this study, and it was suggested that this eosinophilia is correlated with the retardation of the flukes. These results were confirmed by the author in 1973 when he compared three groups of sheep, one previously uninfected, one which had been treated to eliminate a 9-week old primary infection and one which had received five previous infections each terminated after 1 week. In the two challenged groups there was an earlier and more marked eosinophilia and a temporary retardation of fluke development, but fluke recoveries from the challenged and control groups were similar. Flukes recovered from sheep exposed to a primary infection of 9 weeks were smaller in size than those recovered from the challenged controls. Findings similar to those of Sinclair (1971a, 1973) were also reported by Rushton (1977). This investigator superinfected one group of sheep 11 weeks after a primary infection with F. hepatica, challenged another group one week after the elimination of a 10-week primary infection and compared worm recoveries with those from a control group. No protective effect was found. Knight {1980) also studied the effect of primary sensitization of sheep with repeated low levels of infection (20 cysts each weekday for 5 weeks) followed by treatment with albendazole and challenge with 500 cysts. Smaller worms were recovered from the sensitized sheep but there was no redution in worm numbers as compared with the challenge controls.

68 Failure of mature sensitizing infections to stimulate resistance against challenge with F. hepatica in sheep has also been reported by Sandeman and Howell (1981). They were unable to detect an anamnastic response after challenge and .suggested that the antibody response during this period was suppressed. According to the author.s, this suppression could be due to an immunosuppressive factor released from adult flukes or, alternatively, immature flukes of the challenge infection or mature flukes from the primary infection may secrete enough antigen to cause a persistent fall in the level of free antibody, thus leading to the formation of immune complexes that suppress T- and B-cell responses.

Goats Experiments in goats have shown that a primary infection with F. gigantica stimulates resistance to homologous challenge (Haroun and E1 Sanhouri, 1985). This resistance persists even after the elimination of the sensitizing primary infection and it may be initiated by one immature (4-week), one mature (8-week) or two mature (8 + 8-week) primary infections. The resistance is manifested by significant reductions in the numbers of flukes recovered from challenge infections. Worms recovered from the goats which had mature, treated infections were also smaller in size than those from the respective controls; however, worms recovered from the group which had an immature primary infection were similar in size to those from their respective controls, probably because recovery was carried out 4 weeks after challenge.

Cattle It has been established that cattle acquire resistance to challenge infection with F. hepatica or F. gigantica when they are sensitized by primary homologous patent or drug-abbreviated infections. This resistance is usually manifested by a decrease in the size and number of flukes recovered from challenge. Ross (1967) and Boray (1967) indicated that hepatic fibrosis due to primary infection is probably an important contributory factor in this resistance; a positive correlation between the duration of primary infection and resistance to challenge has also been indicated by Doyle (1973). Kendall et al. (1978) showed that the resistance stimulated by primary infection with F. hepatica may persist for a long period after the removal of the sensitizing infection by anthelmintic treatment. They terminated a long duration infection (32 weeks) in calves, then challenged them 3 weeks or 22 weeks later. A similar degree of resistance was obtained in both cases. D o y and Hughes (1984) found 56% resistance following primary sensitization of 18 weeks. When they extended the period of sensitization to 26 weeks, they obtained a higher level of resistance (94%) and thus suggested that the extra few weeks of exposure were necessary for the full development of resistance.

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Haroun and Yagi (1985) also found significant resistance to F. gigantica in zebu calves sensitized with a curtailed homologous primary infection for 8 weeks. In summary, then, resistance to challenge infection of primary sensitizing doses of Fasciola are clearly host-dependent. Cattle, rats and goats clearly develop significant levels of resistance, and mice probably fall into this category although additional work in this area is warranted. Sheep and rabbits are susceptible hosts and develop little resistance. STIMULATION OF HOMOLOGOUS FASCIOLA HEPATICA

RESISTANCE

BY

IMPLANTATION

WITH

The involvement of immunological factors in the development of resistance to challenge infections with F. hepatica was indicated by experiments in which the hepatic migration of a primary sensitizing infection is by-passed by implanting different stages of flukes before challenge. Such implantations were also used to study the protective ability of the various fluke stages. Thus, Lang and Dronen (1972) and Lang (1974) implanted mice intraperitoneally with juvenile flukes of different ages (8, 12, 14, 16, 18, 20 or 24 days) and challenged them orally when the implanted worms were 40 days old. Except for 20 and 24-day-old flukes, worms of the other ages stimulated significant resistance to oral challenge. They concluded that a duration of hepatic migration of at least 10--11 days by juvenile flukes is responsible for the stimulation of resistance and not the particular age of the fluke. While significant resistance to oral challenge was reported in albino rats implanted with adult flukes by Eriksen and Flagstad (1974), in inbred Piebald Viral Glaxo rats by Anderson et al. (1975) and in Porton Wistar rats by Haroun et al. (1980b), such resistance could not be found in Buffalo inbred rats by Rajasekariah and Howell (1978), although they found that subcutaneous implantation of the latter strain of rats with eggs of F. hepatica, metacercariae or 4-week-old flukes stimulated significant resistance to challenge. They attributed the discrepancy in their results with implantations of adult flukes with results reported by previous workers to differences in rat strains. Haroun et al. (1980b) found a protective immune response in rats implanted with mature living flukes in diffusion chambers but not in those with similarly implanted dead flukes, and suggested that this response was initiated by soluble metabolic products released from the living flukes within the chambers. They also found that the serological response induced by such metabolic products diminishes with time and may no longer be detectable 12 weeks after implantation, by which time the flukes will be dead. It can, however, be restored by restimulation with the same diffusible antigens, Hughes et al. (1976b) found that subcutaneous implantation of rats with one mature fluke did not stimulate resistance to intraperitoneal or subcutaneous challenge with mature flukes. However, oral sensitization stimulated significant resistance and this led to the suggestion that sensitization with all

70 stages of the fluke (i.e. oral challenge) may be necessary for the development of resistance against adult challenge. Later Hughes et al (1981a) found that Piebald Viral Glaxo rats subjected to intraperitoneal sensitization for 3 weeks by immature flukes 2 h, 6 days or 14 days old were protected against challenge with adult flukes. According to the authors the stimulation of resistance by implantation of newly-excysted flukes excluded any important role for the gut penetration stage as well as the cyst wall or any of the products associated with excystment in the initiation of resistance. Curiously, Ross et al. (1967) were able to induce resistance in sheep by intramuscular injection of 4- or 6-week-old F. hepatica worms. Additional work on this model is clearly warranted. STIMULATION OF RESISTANCE TO F. HEPATICA OR F. GIGANTICA BY IRRADIATED HOMOLOGOUS CYSTS It has been shown that ionizing radiation reduces the pathogenicity of parasitic larvae w i t h o u t affecting their antigenic potential (Jarret et al., 1958; Dawes, 1964; Sokolic, 1971). Sokolic (1968) suggested that irradiated parasite vaccines initiate a greater antigenic stimulation than normal infections because of the larger dose of the former which a host can withstand. Moreover, it has been reported that irradiated schistosomal vaccines produce more resistance than can be induced by non-irradiated cercarial infection in sheep (Taylor et al., 1976; Bickle et al., 1979). Studies on stimulation of resistance to F. hepatica or F. gigantica utilizing irradiated metacercariae are listed in Table I. Irradiated cycsts of Fasciola species, like normal cysts, clearly stimulate strong protective responses against homologous challenge in cattle and rats. However, this appears to be controversial in sheep, rabbits and mice. The few experiments carried o u t in rabbits showed that irradiated cysts of F. hepatica do n o t stimulate resistance against homologous challenge (Hughes, 1962), and most experiments in mice also gave negative results (Hughes, 1962; Dawes, 1964; Sokolic, 1968). However, Harness et al. (1976) reported significant resistance in mice sensitized by irradiated cysts, with worms recovered 2 days after challenge. In a later experiment, however, Harness et al. (1977) found that the immature flukes of the challenge infection in the sensitized mice migrate more rapidly from the peritoneum to the liver than in the controls and that there was no real resistance. Recovery of worms 2 weeks after challenge revealed no difference between the sensitized and control mice. Hughes et al. (1981a) found that metacercariae of F. hepatica irradiated at 3.8 Kr, and so prevented from developing b e y o n d the 8--10 day stage, did not stimulate resistance against challenge with adult flukes. Thus they suggested the the ability to produce immunity in rats, as demonstrated by others, m a y be dependent on the level of irradiation and fluke development b e y o n d the 8--10 day stage.

71 TABLE I Stimulation of resistance to Fasciola hepatica or F. gigantica by irradiated homologous metacercariae Host

Experimental procedure

Resistance

Reference

A. Mice and rabbits

Sensitization by m/c of

n.s. a

F. hepatica irradiated

Hughes (1962)

Sensitization for 22 days by m/c of F. hepatica irradiated at 3 Kr before oral challenge

n.s.

Dawes (1964)

Sensitization by irradiated m/c of F. hepatica before challenge

n.s.

Sokolic (1968)

36%

Harness et al. (1976)

at 2--4 Kr before oral challenge B. Mice

Sensitization for 3 weeks by a single dose of F. hepatica m/e irradiated at 3.8 Kr before oral challenge with 50 normal m/c, with necropsy 2 days after challenge SensitizaLion by 2 doses of F. hepatica m/c irradiated at 3.8 Kr (1 week intervals) and orally challenged with 100 m/c 3 weeks after the 2nd immunizing dose, with necropsy 2 days after challenge Sensitization for 3 weeks by a single dose of F. hepatica m/c irradiated at 3.8 Kr before oral challenge with 100 m/c: a) in mice killed 2 days after challenge b) in mice killed 12 days after challenge c) in mice killed 14 days after challenge C. Rats

(P ~ 0.001)

39% (P <~ 0.01)

Harness et al. (1976)

Harness et al. (1977)

38% (P ~ 0.001) 17% 0

Sensitization by F. hepatica m/c irradiated at 2.5 Kr before challenge

50%

Thorpe and Broome (1962)

Sensitization by 3 weekly doses of F. hepatica m/c irradiated at 2.5 Kr

(P ~ 0.001)

Corba et al. (1971)

Sensitization by 3 doses of

56% (P ~ 0.001)

Armour and Dargie (1974)

F. hepatica m/c irradiated at 3 Kr before challenge

72 TABLE I (continued) Sensitization for 3 weeks by a single dose of F. hepatica m/c irradiated at 3.8 Kr before i/p challenge with 3 adult flukes Sensitization for 3 weeks by a single dose of F. hepatica m/c irradiated at: a) 3 Kr or b) 4 Kr with necropsy 48 h after oral challenge: a) irradiation at 3 Kr b) irradiation at 4 Kr

n.8.

Hughes et al. (1981a) Hughes et al. (1982a)

sig. n.s.

Sensitization by 3 doses of 20-Kr-irradiated m/c of F. hepatica given at intervals of 6 weeks before challenge

n.s.

Boray (1967)

Sensitization by 6 doses of 3-Kr-irradiated m/c of F. hepatica before challenge

64% (n.s.)

Armour et al. (1974)

Sensitization by m/c of F. hepotico irradiated at 2-5 Kr before challenge

n.s.

Campbell et al. (1978)

Sensitization for 8 weeks by m/c of F. gigantica irradiated at 3 Kr before challenge, with necropsy 8 weeks later

80% (P < 0.005)

A/Gadir et al. (1985)

E. Goats

Sensitization for 3 weeks by m/c of F. gigantica irradiated at 3 Kr before challenge, with necropsy 8 weeks later

43% (P < 0.01)

El Sanhouri et al. (1985)

F. Cattle

Sensitization by 3 doses of 20-Kr-irradiated m/c of F. hepatica given at intervals of 6 weeks before challenge

n.s.

Boray (1967)

Sensitization by 2 doses of 3-Kr-irradiated mlcof F. gigantica given at intervals of 6 weeks followed by challenge after 14 weeks

98%

Bitakaramire (1973)

Sensitization by 2 doses of 3 l/2-Kr-irradiated m/c of F. hepatica at intervals of 4 weeks followed by challenge after 8 weeks

70%

Armour et al. (1974)

D. Sheep

73 TABLE

I (continued) Sensitization by 2 doses of 3 1/2-Kr-irradiated m/c F. hepatica at intervals of 4 weeks followed by challenge after 4 weeks

30%

Armour et al. (1974)

Sensitization by 3 doses of 3-Kr-irradiated m/c of F. hepatica at intervals of 4 weeks, followed by challenge 4 weeks later by allowing to graze in infected field for 24 weeks

71%

Nansen (1975)

Sensitization for 8 weeks by a single dose of m/c of F. gigantica irradiated at 3 Kr followed by challenge

65%

Younis et al. (1985)

Sensitization for 4 weeks by a single dose of m/c of F. gigantica irradiated at 3 Kr before challenge

18%

Younis et al. (1985)

Sensitization for 8 weeks by a single dose of F. gigantiea m/c irradiated at 20 Kr followed by challenge

80%

Younis et al. (1985)

Sensitization by 2 doses of F. gigantica m / c irradiated at 3 Kr (at intervals of 4 weeks) followed by challenge

63%

Younis et al. (1985)

a n.s. = not significant.

In sheep, Boray (1967) found that sensitization with three doses of metacercariae of F. hepatica X-irradiated at 20 Kr did not protect animals against challenge with 4000 normal cysts, although a delay of onset of anemia was observed. Campbell et al. (1978) also found that sensitization with 100 or 1000 metacercariae irradiated at 2--5 Kr produced no resistance against challenge infection. On the other hand, Dargie et al. (1974) sensitized sheep with six doses of 100 metacercariae irradiated at 3 Kr before challenge with 75 normal cysts. Although the reduction in fluke recovery was considerable (64%), it was statistically insignificant. The authors, however, considered it to be partial evidence for the ability of sheep to acquire resistance against challenge with

F. hepatica. Metacercariae of F. gigantica gamma-irradiated at 3 Kr were capable of inducing significant resistance, as indicated by reductions in the size and

74 number (80%) of worms recovered from homologous challenge in Sudanese desert sheep (A/Gadir et al., 1985). The sensitized lambs also showed less hepatic damage compared with the controls, as indicated by lower levels of the serum enzymes glutamate dehydrogenase and sorbitol dehydrogenase. In contrast to the controls, the sensitized lambs also showed insignificant reductions in the erythrocyte count, packed cell volume and hemoglobin values. E1 Sanhouri et al. (1985) found that sensitization of goats for 8 weeks with metacercariae of F. gigantica which had been irradiated at 3 Kr stimulated resistance which resulted in a significant reduction in the size and number (43%) of worms recovered from a challenge infection. Vaccinated goats showed less severe changes in hemoglobin concentration after challenge than controls, where a progressive decline was observed. The protective effect of irradiated cysts of F. hepatica or F. gigantica against homologous challenge seems to be less controversial in cattle than in sheep. Boray (1967) did not find resistance to challenge with 5000 cysts of F. hepatica in three calves sensitized with three doses of 3000 X-irradiated metacercariae (20 Kr) given at intervals of 6 weeks. On the other hand, Bitakaramire (1973) reported a highly significant degree of protection (98%) in seven zebu calves sensitized with two dose of F. gigantica cysts irradiated at 3 Kr and administered at intervals of 6 weeks. The livers of the sensitized calves were apparently normal while those of the challenge controls had marked gross lesions. Significant resistance against challenge with F. hepatica was also reported by Armour et al. (1974) in calves sensitized with two doses of cysts irradiated at 3.5 Kr and administered at an interval of 4 weeks. A reduction in fluke burden of 30% was obtained when the sensitized calves were challenged 4 weeks after the second dose, whereas a reduction of 70% was found when the sensitized calves were challenged 8 weeks after the second sensitizing dose. Nansen (1975) also immunized calves at intervals of 4 weeks with three doses of F. hepatica metacercariae irradiated at 3 Kr. Four weeks after the last dose the sensitized animals, together with controls, were allowed to graze in fluke-infected fields for a period of 24 weeks. A resistance of 71% was recorded at necropsy. The author indicated, however, that some metacercariae of the immunizing infection escaped the irradiated effects and developed to maturity. Recently, Younis et al. (1985) reported significant resistance as indicated by a reduction in the size and number of worms {65%) recovered after challenge with F. gigantica in zebu calves sensitized for 8 weeks with homologous cysts irradiated at 3 Kr. Although a mean of 5.6% of the irradiated worms reached maturity, the serum enzymes sorbitol dehydrogenase and glutamate dehydrogenase and the liver histopathology indicated less hepatic damage in the sensitized animals than in the controls. When the period of sensitization with irradiated cysts (3 Kr) of F. gigantica was reduced to 4 weeks before challenge an insignificant reduction (18%) in parasitic burden was obtained. However when a 'booster' dose was adminis-

75

tered 4 weeks after the first dose of irradiated cysts, a reduction of 62% in parasitic burden was obtained. Sensitization of calves for 8 weeks with F. gigantica metacercariae irradiated at 20 Kr also resulted in a significant resistance (80%) against challenge. A mean of 2.8% stunted, irradiated metacercariae-derived flukes was recovered. The liver of the sensitized animals showed much less damage than those of the controls, as indicated by the histopathological picture and by low levels of the serum enzymes glutamate dehydrogenase and sorbitol dehydrogenase. The manner in which irradiation activates the resistance-inducing potential of the larvae is not yet elucidated. Hanna (1980a) indicated that certain secretion granules in the tegument (To, T1, and T2 ) contain antigens which are sequentially produced with the development of the flukes. According to Burden et al. {1983), metacercarial irradiation results in slower development and thus the release of the various juvenile antigens continues for longer in irradiated than in normal infections. Irradiated cysts therefore produce more antigenic stimulation than normal. The latter authors also pointed out the differences in the growth rates of F. hepatica in different hosts (rats and mice}, with the consequent differences in the rates of development of the tegument in each host and differences in the expression of tegumental antigens. They suggested that for immunity to develop in the rat the sensitizing infection must develop to the stage when TI and/or T2 granules are functional as surface antigens. They suggested that gamma-irradiation at 46 Kr prevented the development of T1 granules in rats but not in mice, although it delayed their production in the latter host, and the To granules were not affected by irradiation. STIMULATION OF RESISTANCE TO F. H E P A T I C A BY SOMATIC F L U K E EXTRACTS

Attempts at active immunization to stimulate resistance to F. hepatica utilizing somatic fluke extracts are summarized in Table II. Sensitization by somatic antigens generally failed in stimulating resistance against F. hepatica in rabbits (Urquhart et al., 1954; Healy, 1955; Ross, 1967), sheep {Ross, 1967) and sometimes in mice {Burden et al., 1982; Chapman and Mitchell, 1982) and rats {Hughes et al., 1981a; Oldham and Hughes 1982; Burden et al. 1982). However, Lang and Hall (1977) and Hall and Lang (1978) succeeded in inducing significant resistance to challenge in mice and cattle by sensitization with sonicated 16-day-old fluke antigen. In contrast to Burden et al. (1982), Oldham and Hughes {1982) obtained 80% resistance in rats vaccinated intraperitoneally with adult fluke antigen in Freund's incomplete adjuvant and Bordetella pertussis. They suggested that the intraperitoneal route of sensitization may have induced a type of immune response not produced by the subcutaneous or intramuscular sensitizations which failed to stimulate resistance. Alternatively, the adjuvant

76 TABLE II Stimulation of resistance to F. hepatica by somatic fluke extracts Host

Experimental procedure

Resistance

Reference

A. Mice

Sensitization for 6 weeks by a single i/p dose of sonicated 16-day-old F. hepatica followed by challenge

86%

Lang and Hall (1977)

Sensitization by 2 i/p doses of sonicated 16-dayold fluke antigen (at 3-week intervals)followed by challenge

82%

Lang and Hall (1977)

Sensitization by 3 s/c doses of sonicated newly-excysted flukes before challenge

10% (n.s.)a

Chapman and Mitchell

Sensitization by 2 s/c doses of sonicated 16-day-old flukes before challenge

0--4%

Sensitization by i/m or s/c injections with adult fluke extract in Freund's complete adjuvant on 2 occasions 3 weeks apart before challenge with adult flukes

n.s.

Hughes et al. (1981a)

Sensitization by 3 s/c injections of adult fluke extract in Freund's complete adjuvant followed by oral challenge

0

Oldham Hughes

Sensitization by 3 i/m injections of adult fluke extract in Freund's incomplete adjuvant followed by oral challenge

0

Oldham and Hughes (1982)

Sensitization by 3 i/p injections of adult fluke extract (4 ml) in Freund's incomplete adjuvant followed by challenge

48%

Oldham and Hughes (1982)

B. rats

(1982) Chapman and Mitchell

(1982)

and

(1982)

(P < 0.05)

77 TABLE II (continued) Sensitization by 3 i/p injections of adult fluke extract in Freund’s incomplete adjuvant + Bordetella pertussis followed by oral challenge

67% (P < 0.001)

Oldham and Hughes (1982)

Sensitization by 3 i/p injections of adult fluke extract in Freund’s incomplete adjuvant -I- Bordetella pertussis followed by oral challenge

80%

Oldham and Hughes (1982)

Sensitization by i/p injection of adult fluke extract from different batches in Freund’s incomplete adjuvant: 2 mg injected 3 times 2 mg injected 3 times 2 mg injected 3 times 2 mg injected 3 times 10 mg injected twice 10 mg injected twice 10 mg injected twice 10 mg injected 3 times 10 mg injected 3 times Sensitization by sonicated 14- or 16-day-old flukes before challenge Sensitization by 2 s/c injections of 14-day-old flukes in: a) Freund’s adjuvant b) alum followed by challenge C. Rabbits

Oldham (1983)

40% 29% 41% 55% 84% 36% 86% 65% 70% 0 and 13%

50% 49% (P < 0.05)

Burden et al. (1982) Chapman and Mitchell (1982)

Sensitization by adult fluke extract before challenge

0

Healy (1955)

Sensitization by 3 i/m injections of adult fluke extract at intervals of 8-10 days before challenge

32% (n.6.)

Urquhart et al (1954)

Sensitization by 6 i/m injections of adult fluke extract at intervals of B-10 days before challenge

1%

Urquhart et al. (1954)

78 TABLE II (continued) Sensitization by 2 s/c injections of 6-week-old F. hepatica homogenate at intervals of 2 weeks followed by challenge 3 weeks later

n.s.

~oss (1967)

Sensitization by primary infection and i/v injection of adult fluke extract followed by oral challenge

54--75%

Sinclair and Joyner (1974)

D. Sheep

Sensitization for 2 weeks by adult fluke extract followed by challenge

n.s.

Ross (1967)

E. Cattle

Sensitization for 2 weeks by adult fluke extract followed by challenge

n.s.

Ross (1967)

Sensitization for 100 days by a single s/c dose of sonicated 16-day-old flukes before challenge

98%

Hall and Lang (1978)

Sensitization for 38 days by a single s/c dose of sonicated 16-day-old flukes before challenge

90.6%

Hall and Lang (1978)

Sensitization for 200 days by a single s/e dose of sonieated 16-day-old flukes before challenge

99.6%

Hall and Lang (1978)

a n.s. = not significant.

(alone o r w i t h antigen), m a y have a t t r a c t e d a higher c o n c e n t r a t i o n o f e f f e c t o r cells i n t o t h e p e r i t o n e u m w h i c h w e r e able t o kill t h e m i g r a t i n g flukes. O l d h a m ( 1 9 8 3 ) f o u n d t h a t increasing t h e a m o u n t o f sensitizing antigen i m p r o v e d t h e degree o f p r o t e c t i o n against challenge a n d t h a t at least t w o sensitizing doses o f a d u l t f l u k e a n t i g e n w e r e r e q u i r e d t o s t i m u l a t e resistance in rats. T h i s i n d i c a t e d a n e e d f o r a s e c o n d a r y i m m u n e r e s p o n s e t y p e o f react i o n , r e q u i r i n g an i n c r e a s e d a n t i b o d y t i t e r or a switch f r o m an IgM to an I g G r e s p o n s e . H o w e v e r , n e i t h e r t h e age o f t h e i n f e c t i o n at t h e t i m e o f a d u l t f l u k e r e c o v e r y , n o r t h e species o f t h e a n i m a l f r o m w h i c h t h e f l u k e s w e r e c o l l e c t e d h a d a n y e f f e c t o n t h e resistance. T h e a u t h o r p o i n t e d o u t t h a t a l t h o u g h a d u l t flukes' antigens m a y be c o n v e n i e n t to o b t a i n , t h e y m a y n o t be t h e r i c h e s t s o u r c e o f p r o t e c t i v e antigens. O l d h a m ( 1 9 8 3 ) c o n f i r m e d p r e v i o u s results ( O l d h a m a n d Hughes, 1 9 8 2 ) i n d i c a t i n g t h e i m p o r t a n c e o f t h e r o u t e o f sensitization; he f o u n d t h a t while

79 subcutaneous sensitization with adult fluke antigen in Freund's adjuvant did not stimulate resistance in rats, it was successfully initiated when the latter procedure was coupled with intraperitoneal injection of saline in Freund's adjuvant. Thus, he indicated the probable operation of two mechanisms, one which requires priming by intraperitoneal or subcutaneous sensitization and another that requires the action of adjuvant in the peritoneal cavity. Sinclair and Joyner (1974) found 54--75% resistance in rabbits sensitized by a primary infection and intravenous injection of adult fluke antigen before challenge. They attributed this resistance to an immunological reaction which initiated an anaphylactic response that damaged the hepatic cells and formed a barrier to the migration of the juvenile flukes. These studies suggest that active immunization with somatic extracts may be possible in mice and rats, although work by other investigators utilizing the mouse model are clearly desirable. The limited experience with cattle also requires additional work. Bennett and Threadgold (1973, 1975) described the ultrastructure of the development of the F. hepatica tegument and indicated the possibility of surface antigenic variation during fluke development. Different antigens were associated with the appearance of different granules (To, T1 and T2 ) in the tegument: To granules were associated with metacercariae and early juvenile stages, T, predominantly occurred in the migratory stages in the hepatic parenchyma and T2 granules were mainly associated with the tegument of mature flukes. This hypothesis has been further supported by Bennett (1978) and by Hanna (1980a,b,c). Specific antibodies associated with T1 and T2 antigens in cattle and sheep have been demonstrated by Hanna (1980a) and by Hughes et al. (1981b), and compared with antibody profiles from animals infected with irradiated flukes (Hughes et al., 1982b). Irradiated metacercariae were shown to stimulate a normal humoral response to To, T1 and gut antigens in cattle, sheep and rats. The occurrence of antibodies against T2 antigens probably depends on 'breakthrough' and maturation of a few irradiated larvae. Identification and characterization of these tegumental antigens is an important step towards employing them in immunization trials (Bennet et al., 1982; Hanna and Trudgett, 1983; Hanna and HiUyer, 1984). Hillyer (1984) thoroughly reviewed the use of heterologus trematode antigens in the immunity to schistosomes. The clearest evidence of crossresistance to S. mansoni was found with F. hepatica, and the separation and use of Fasciola antigens in this cross-resistance were discussed. The author pointed out that higher degrees of antigen purification allowed higher levels of resistance against S. mansoni to be obtained with smaller amounts of antigen, indicating the operation of immunological mechanisms in this resistance. A certain Fasciola/Schistosoma-defined immunity cross-reactive antigen was isolated from F. hepatica and designated FhSmIII(M) (Hillyer, 1979). It

80

was shown that this antigen or some of its determinants were present on (or in) S. mansoni, S. bovis and Paragonimus westermani and a c o m m o n link of cross-resistance shared between these trematodes was indicated. S T I M U L A T I O N O F R E S I S T A N C E T O F. H E P A T I C A BY M E T A B O L I C P R O D U C T S O F FLUKES

Attempts to stimulate resistance to F. hepatica by sensitization with metabolic products (secretory/excretory p r o d u c t s ) o f flukes were consistently unsuccessful in stimulating resistance in rabbits and sheep (Healy, 1955; Lalic et al., 1976; Lehner and Sewell, r1979; Sandeman et al., 1980). Metabolic products of mature flukes also failed to stimulate resistance in mice (Rajasekariah et al., 1979a) and rats {Burden and Hammet, 1980; Burden et al., 1982). However, while sensitization by metabolic products of immature flukes was successful in stimulating resistance against challenge with F. hepatica in some experiments in mice (Lang, 1976; Lang and Hall, 1977) and rats (Rajasekariah et al., 1979a), no such resistance was seen in other experiments in mice (Rajasekariah et al., 1979a; Lehner and Sewell, 1979; Burden et al., 1982; Chapman and Mitchell, 1982} and rats (Lehner and Sewell, 1979; Davies et al., 1979; Burden et al., 1982). The antigen-antibody complexes which form around immature flukes cultured in vitro in immune serum, first observed by Wikerhauser (1961), were found to stimulate resistance to challenge with F. hepatica in rats (Howell, 1979; Howell and Sandeman, 1979) but not in sheep (Sandeman et al., 1980) although high levels of antibody wdre stimulated by the complex and were present at the time of infection. Sandeman and Howell (1982) examined the role of ovine antibodies which precipitate with surface antigens of F. hepatica and concluded that some effector mechanism other than antibody, such as T-cell-mediated reactions, is defective in sheep. P A S S I V E I M M U N I Z A T I O N A G A I N S T F. H E P A T I C A O R F. G I G A N T I C A BY TRANSFER OF IMMUNE SERUM

Although early attempts to passively transfer resistance by immune serum were unsuccessful (see reviews by Dawes and Hughes, 1964; Sinclair, 1967, Geyer, 1967; Sokolic, 1968), evidence for the involvement of humoral components in the resistance to fasciolasis has been obtained by the successful transfer of resistance by immune serum in rats, rabbits, sheep and cattle. Armour and Dargie (1974) observed that passive resistance positively correlates with large volumes of immune sera. Passive resistance also seems to be expressed against juvenile flukes during their migration in the peritoneum. Hayes et al. (1974b) and Chapman and Mitchell (1982) found flukes older than 4 or 14 days, respectively, to be refractory to immunization by immune serum. Hayes et al. (1974c) also found that surviving flukes recovered from

81

rats treated with immune serum were similar in size or even larger than those recovered from controls and suggested that immune serum has an all-ornothing effect against juvenile flukes. According to these authors, the protective effect of immune serum could be absorbed by live or dead flukes and it may also be eliminated by heat (56°C). Haroun et al. (1981) confirmed the ability of metabolic products from living mature flukes to absorb out the protective effect from immune serum. However, Chapman and Mitchell (1982) found that heating at 56°C had no effect on the protective ability of immune serum and concluded that the heat labile components of serum (such as complement and IgE) are not responsible for the transfer of resistance. Passive resistance appears also to be influenced by the species of the donor and recipient. Baalawy (1975) reported a low level of resistance by transfer of immune homologous serum to rabbits but a high level of protection was conferred in rabbits by immune serum from goats. Haroun et al. (1981) also f o u n d that resistance to challenge with F. hepatica can be conferred on rats by immune serum from rats, rabbits or cattle and that although immune sera from rats or cattle protect rabbits, immune rabbit serum was ineffective in the homologous host. Thus it appears that immune rabbit serum is less protective than immune rat, goat or cattle serum. Haroun et al. (1981) suggested that this may be due to the relative concentration of the protective antibodies in the respective sera, or it may indicate that various hosts form a different spectrum of antibodies following infection with F. hepatica. Chapman and Mitchell (1982) also found that immune mouse serum is n o t protective to mice or rats, and that mice do n o t even develop resistance to reinfection. However, Armour and Dargie (1974) and Mitchell et al. (1981) reported that immune ovine serum is protective to rats and that immune serum from donors which do not develop resistance to reinfection {like sheep) is still capable of conferring resistance to species which can become resistant, such as rats. I M M U N I Z A T I O N A G A I N S T F. H E P A T I C A BY T R A N S F E R O F S E N S I T I Z E D LYMPHOCYTES

Sinclair (1971b) found that homogenates of lymph nodes and spleen from donor sheep infected with F. hepatica for 8 weeks did not confer resistance to homologous challenge, although retardation in fluke development was observed in the recipient sheep. On the other hand, Lang et al. (1967) were able to confer significant resistance against F. hepatica in mice by intraperitoneal transfer of peritoneal exudate cells from donor mice with 35-weekold homologous infection. Corba et al. (1971) also f o u n d that lymphoid cells from rats or cattle infected with F. hepatica were able to confer a high degree of resistance against challenge infection to homologous recipients. Similar results were also reported in rats by Armour and Dargie (1974) and by Rajasekariah and Howell (1979).

82 L y m p h o c y t e s from donors with immature infections failed to confer protection on recipient rats, although lymphocytes from donors infected with irradiated cysts did so, thus excluding the need to obtain l y m p h o c y t e s from donors with mature infections as a prerequisite for the successful transfer of resistance (Corba et al., 1971). The degree of antigenic stimulation in the donors also seems to be important; Armour and Dargie (1974) found that the adoptive transfer of lymphoid cells from rats with high parasitic burdens conferred significantly more resistance to recipients than cells obtained from donors harboring few flukes. Lang (1967, 1968) suggested that sensitized lymphocytes may be attracted to sites of antigen depositon, where they initiate a delayed-type hypersensitivity reaction which renders the environment unsuitable for the young parasites. However, Armour and Dargie (1974) maintained that an antibodymediated immunity is probably implicated together with cell-mediated mechanisms in the resistance to F. hepatica, with the latter functioning as a second line of defense. CROSS-RESISTANCE BETWEEN LIVER FLUKES AND OTHER HELMINTHS It has been established that cross-resistance exists between Fasciola and Schistosoma species in many hosts. Hillyer (1976) and Christensen et al. (1978, 1980) showed that adult F. hepatica infections induce significant resistance to subsequent challenge with S. mansoni in mice. Similarly, mice with mature primary infections of S. mansoni were found to show significant resistance to challenge with F. hepatica (Christensen et al., 1978, Hillyer, 1981). Christensen et al. (1980) also showed that mice with immature infections ( 9 4 weeks) with F. hepatica developed resistance to challenge with S. mansoni. Single-sex infections of S. mansoni, however, did not stimulate resistance to F. hepatica. Evidence for the involvement of immunological factors (rather than mere physical changes due to primary sensitizing infection) was indicated by the successful use of crude or purified antigens of F. hepatica to stimulate resistance to challenge with S. mansoni in mice and hamsters (Hillyer et al., 1975, 1977; Hillyer and Sagramoso de Ateca, 1979; Hillyer, 1979, 1981; Hillyer and Serrano, 1982). Furthermore, the protective F. hepatica worm antigens were those which b o u n d to antibodies to S. mansoni, and, as antigen purifications proceeded, smaller amounts were required to obtain significant levels of protection. These t w o factors, cross-reactivity and improved protection with increasing antigen purity, are both supportive of an immunological basis for protection against S. mansoni. Cross-resistance between schistosomes and liver flukes has also been demonstrated in farm animals. Monrad et al. (1981) found significant resistance to F. hepatica, as indicated by less hepatic damage and reduced worm burden, in sheep harboring 2--3-week-old (70%) and 7--8-week-old S. bovis infections (93%); although 16--17-week-old infections with S. bovis were not protective.

83 Calves harboring mature primary infection with S. bovis also showed significant resistance (30%) to challenge with F. hepatica (Sirag et al., 1981). Moreover, the resistance was indicated by less hepatic damage and lower serum gammaglutamyl-transpeptidase values in sensitized calves compared with the controls. These authors did not attribute the resistance to hepatic damage because of the low density of schistosomal eggs in the liver and the absence of liver fibrosis. Heterologous resistance between F. gigantica and S. bovis has also been demonstrated in cattle (Yagi et al., 1985}. These workers found significant resistance (94%) to challenge with S. bovis in zebu calves primarily infected with F. gigantica for 8 weeks and a reduction of 84% in a challenge infection with F. gigantica in calves primarily infected with S. bovis for a similar period. The level of heterologous resistance stimulated by S. bovis against F. gigantica was markedly higher (84%) than that stimulated by S. bovis against challenge with F. hepatica (30%} in Jersey calves (Sirag et al., 1981), but was comparable with that induced by a patent infection in European breeds of sheep (93%) against challenge with F. hepatica (Monrad et al., 1981). Although sensitization with normal cercariae of S. bovis engendered resistance to heterologous challenge with F. gigantica, such resistance was not induced in cattle or goats when the cercariae were irradiated at 3 Kr (Yagi, et al., 1985; E1 Sanhouri, et al., 1985). Cercariae of S. bovis irradiated at 3 Kr were, however, found to stimulate significant resistance against homologous S. bovis challenge (Bushara et al., 1978). Thus Yagi et al. (1985) suggested that heterologous resistance may be stimulated by stage-specific adult worm antigens which would not be expressed by irradiated infections, or that a degree of hepatic damage due to primary infection is important in the stimulation of heterologous resistance between these trematodes. Experiments were recently carried out by Haroun and Hillyer (unpublished data) to investigate cross-resistance between F. hepatica and S. mansoni in sheep and to see whether shared antigens, and in particular a certain antigen isolated from F. hepatica (Hillyer, 1979) and designated FhSmIII(M) can be used for immunization against these trematodes in farm animals. This antigen stimulated significant resistance to S. mansoni and F. hepatica in mice (Hillyer, 1979, 1985), and induced high antibody titers (as shown by ELISA) and a reduction of 55% in the worm burden of calves challenged with F. hepatica, when compared with controls (Hillyer et al., unpublished data). Sheep are partially susceptible to primary infection with S. mansoni and they show great individual variations in their pathophysiological responses. S. mansoni eggs were first seen in feces 9 weeks after infection and no eggs could be detected after 14 weeks. Tissue eggs counts were also low. Following infection with 5000 cercariae of S. mansoni, egg counts ranged from 0 to 133 in the liver and from 0 to 257 in the intestine. Primary infection with S. mansoni resulted in a reduction of 51% in worm recovery following a challenge infection with F. hepatica (Table III). There

84 TABLE III Recovery of F. hepatica Group

Sheep no.

No. ofF.

Percent

hepatica

reduction

recovered at necropsy A (5000 c of S. mansoni for 10w + 400m/c of F. hepatica) Mean + SD

415 416 417 418 419

27 71 47 43 69 51 + 19

B (400 m/c of

445 446 447 448 449

50 27 132 113 197 104 + 68

F. hepatica) Mean + SD

51

was a clear t e n d e n c y towards n o r m o c y t i c n o r m o c h r o n i c anemia following primary infection with S. mansoni; however, blood values were m ore reduced in the challenge controls than in the animals which received primary infection with S. mansoni. Primary infection of sheep with F. hepatica for 10 weeks followed by challenge with S. mansoni resulted in a reduction of 22% and 16% in S. mansoni egg counts in the liver and intestines, respectively, but only dead S. mansoni were recovered at perfusion (Haroun and Hillyer, unpublished data). Heterologous resistance against F. hepatica has also been d e m o n s t r a t e d in sheep harboring a primary infection with the metacestode of Taenia hydatigena (Cysticerus tenuicollis) for 12 weeks (Campbell et al., 1977). Before sensitization, sheep were treated with levamisole to eliminate nemat o de infections. Sensitized sheep were resistant to F. hepatica w het her challenge was superimposed u p o n the cestode infection or after the removal o f the cestodes with mebendazole. There was, however, no evidence of resistance in sheep sensitized with the cestode for only 3 weeks. F u r t h e r studies by Dineen et al. (1978) showed t hat sheep which resisted challenge with F. hepatica after a primary infection with Taenia hydatigena for 12 weeks maintained this resistance against a second challenge 9 m o n t h s later. However, sheep in which the cestode infection had been eliminated after 12 weeks were fully susceptible to the second challenge with F. hepatica after 9 months, even though t h e y were resistant after 12 weeks. Sheep which were susceptible to challenge with F. hepatica after a 3-week primary infection with T. hydatigena, were also susceptible to the second challenge with F. hepatica after 9 months. This correlation between the maintenance of resistance against F. hepatica

85 and the persistence of infection with the metacestodes in a site remote from the reactive tissue led the authors to suggest that infection with T. hydatigena stimulates an immunological mechanism rather than a physical barrier against F. hepatica. The finding that sheep which had been susceptible to challenge with F. hepatica after a 3-week infection with the cysticerci were also susceptible to the second challenge 9 months later, led to the suggestion that the first challenge with F. hepatica prevented the development of crossresistance because it destroyed the cysticerci before they were able to stimulate resistance against F. hepatica. The authors, however, did not exclude the possibility that competitive interaction between the two parasites could be the cause of cross-resistance. Hughes et al. (1978) infected sheep with Taenia hydatigena and challenged them with F. hepatica after 12 weeks. No evidence of resistance was found, and this was attributed to the fact that levamisole, which has immunostimulating properties, had not been used in the experiment. Failure to stimulate resistance against F. hepatica, by primary infections with C. tenuicollis in sheep has also been reported by Campbell et al. (1979} and Mitchell and Armour (1981). The latter workers also reported the failure of primary infection with Ascaris suum, Ostertagia circumcincta and Trichostrongylus colubriformis to initiate any degree of resistance against challenge with F. hepatica in sheep. However, they found that combined levamisole treatment and primary infection with the former helminth species stimulate significant resistance against challenge with F. hepatica in sheep and suggested that levamisole corrects the immunosuppression induced by the interaction of primary nematode and subsequent fluke infections. Rajasekariah et al. (1979b) also reported the failure of attempts to immunize rats and mice against F. hepatica by oral dosing with T. hydatigena eggs, extracts from cysticerci and cyst fluid or culture incubate of larvae for 48 hours or 14 days. In view of the significant resistance stimulated in sheep against F. hepatica by 12 weeks' infection with T. hydatigena, previously reported by Campbell et al. (1977), the former workers suggested that the resistance against F. hepatica might be stimulated by antigens from mature T. hydatigena rather than immature worms. Alternatively, they suggested that antigens from T. hydatigena may be effective in stimulating resistance against F. hepatica in sheep but not in rats or mice, the latter being unnatural hosts for T.

hydatigena. On the other hand, Goose (1977) reported that rats primarily infected with F. hepatica showed resistance to challenge with Nippostrongylus brasiliensis and D o y et al. (1981) found that primary infection with N. brasiliensis initiates significant resistance to F. hepatica in rats. CONCLUDING REMARKS Although resistance to fascioliasis can be acquired in some hosts, such as rats and cattle, this seems to be doubtful or controversial in other hosts like

86

mice, rabbits and sheep. The mechanism of this resistance has n o t been clearly elucidated, but important steps have been made and it is now evident that the resistance to fascioliasis is immunologically-mediated through humoral and/or cellular reactions. Various means have been tried to stimulate these immunological responses against F. hepatica or F. gigantica with the ultimate objective of producing a vaccine. In view of the significant resistance stimulated by irradiated metacercariae in cattle and the successful use of irradiation-attenuated larvae of a number of other parasites in stimulating resistance against diseases such as parasitic bronchitis (Jarrett et al., 1958), canine ancylostomiasis (Miller, 1965); ovine schistosomiasis (Taylor et al., 1976; Bickle et al., 1979) and bovine schistosomiasis (Bushara et al., 1978; Majid et al., 1980), as well as other parasitic infections (Taylor, 1980, 1981), irradiated cysts of liver flukes seem to constitute a promising tool for vaccination against bovine fascioliasis. However, the optimum vaccination dose, best irradiation level, optimum number of immunizations and o p t i m u m interval between successive immunizations which stimulate maximum resistance must be determined. More importantly, the logistic problems of preserving irradiated vaccines and employing them for practical purposes agains fascioliasis in the field need to be solved. The alternative practical tool for vaccination against fascioliasis would appear to be the use of specific antigens obtained from flukes. With advancing means of isolation, purification and characterization of antigens, an effective vaccine against fascioliasis may be available for use in the not-too-distant future. ACKNOWLEDGEMENTS

These investigations were supported primarily by USPHS Grant No. R22 AI 20974 administered by the U.S.--Japan Cooperative Medical Science Program and by USPHS Grant No. R R 8102.

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