Zeitschrift for
Z. Parasitenkd. 58,175-180 (1979)
ParasllBRkIHId8 Parasitology Research
9 by Springer-Verlag 1979
Attempts to Immunise Rats and Mice Against Infection With Fasciola hepatica Using Antigens Prepared From Taenia hydatigena G.R. Rajasekariah 1, M.D. Rickard 2, P.E. Montague 1, and G.F. Mitchell 3 1 ICI Research Laboratories, Ascot Vale, Victoria, 3032, Australia 2 Universityof Melbourne, Veterinary Clinical Centre, Princes Highway,Werribee, Victoria 3030, Australia 3 Walter and Eliza Hall Institute, Royal Melbourne Hospital P.O., Victoria 3050, Australia
Summary. Attempts were made to immunise rats and mice against infection with F. hepatica by oral dosing with T. hydatigena eggs, or by vaccination with various T. hydatigena antigen preparations. These antigens included extracts from 1". hydatigena cysticerci and cyst fluid, and antigens collected during short-term (48 h) and long-term (14 days) in vitro cultivation of larvae. hnmunity was assessed by the numbers of F. hepatica recovered from the challenge infection in rats, and the mortality rates of infected mice. None of the immunisation regimes with T. hydatigena antigens induced consistent, significant immunity. This was in contrast to the high level of immunity shown by rats dosed orally with F. hepatica metacercariae four weeks prior to challenge infection.
Introduction In a recent experiment Campbell et al. (1977) demonstrated a high level of resistance to Fasciola hepatica infection in sheep which had been infected with the larval stages of Taenia hydatigena 12 weeks previously. Prior infection with T. hydatigena not only significantly reduced the numbers of F. hepatica recovered from the challenge infection, but also protected against the pathogenic effects of the liver fluke. There have been a number of experiments which suggest that rats and mice are useful models for studying aspects of the biology and immunology of F. hepatica infection (Hayes et al., 1972; Armour and Dargie, 1974; Lang, 1968, 1976; Howell et al., 1977; Rajasekariah and Howell, 1977 b; 1978). The present experiments were designed to investigate the efficiency of various T. hydatigena antigen preparations in protecting rats and mice against a challenge F. hepatica infection. To whom offprint requestsshould be sent: Dr. G.R. Rajasekariah, Universityof Melbourne, Veterinary Clinical Centre, Princes Highway,Werribee, Victoria 3030, Australia
0044-3255/79/0058/0175/$01.20
176
G.R. Rajasekariah et al.
Rickard and Coman (1977) showed that rabbits dosed orally with eggs of T. hydatigena developed significant immunity against a subsequent challenge infection with eggs of the homologous parasite T. pisiformis; immunity was stimulated despite the fact that only a limited degree of migration and development of the heterologous T. hydatigena oncospheres had occurred in the rabbits. These experiments suggested that intestinal activation of, and penetration by, taeniid oncospheres can occur in other than their natural hosts. Therefore in the present experiments oral administration of T. hydatigena eggs was chosen as one possible means of immunisation. Antigens prepared from somatic tissues and cyst fluid of mature T. hydatigena cysticerci were also tested; although such preparations have given little protection against larval cestode infections in sheep (Gemmell and Soulsby, 1968), nevertheless, comparable somatic antigen preparations have shown some effect against T. pisiformis infection of rabbits (Miller and Kerr, 1932; Kerr, 1935), T. taeniaeformis in rats (Miller, 1932), and T. saginata in cattle (Gallie and Sewell, 1976). Antigens collected during in vitro cultivation of larval T. ovis, T. saginata, and T. pisiformis have been highly effective in immunising the host against homologous challenge infection (Rickard and Bell, 1971 ; Rickard and Outteridge, 1974; Heath, 1976; Rickard and Adolph, 1976, 1977; Rickard et al., 1976). Furthermore, antigens collected during short-term in vitro maintenance of T. ovis oncospheres stimulated high levels of resistance in lambs (Rickard and Adolph, 1977). Therefore, antigens collected during both long- and short-term in vitro culture of T. hydatigena were tested in the present experiments.
Materials and Methods Experimental Animals. Beagle puppies used for stock infections with T. hydatigena were reared cestode-free and fed on proprietary-line dog feed (Puppy Chow; Harper and Co., Mulgrave, Victoria: Lucky Dog Pellets; I.G.Y. Veterinary Products, Marrackville, N.S.W.). Rats were male Wistar breed and maintained at the I.C.I. Central Research Laboratories, Melbourne in conventional plastic cages at 22_+1~ and fed mouse breeding cubes (Barastoc; Melbourne) and water ad libitum. Rats were used in experiment when five weeks of age because resistance to Fasciola hepatica is known to increase with age in this animal (Rajasekariah and Howell, 1977a). Mice were SPF male C57B1/6 bred in an SPF facility but maintained conventionally at the Walter and Eliza Hall Institutel Melbourne in plastic cages between 20 and 25 ~ C and fed on Barastoc cubes and water ad libitum; the mice were 13 weeks of age at the time of immunisation. Parasites. Adult T. hydatigena were obtained from dogs experimentally infected with cysticerci recovered from naturally infected sheep slaughtered at the abattoir. Methods for infecting dogs, collecting tapeworms and their eggs, and counting and storing the eggs have been described previousIy (Coman and Rickard, 1975). Metaeercariae (mc) of F. hepatica were obtained from experimentally infected Lymnaea tomentosa snails. They were checked microscopically to assess their viability (Rajasekariah and Howell, 1977b) before administration into rats and mice. Somatic and Cyst-fluid Antigens of T. hydatigena. Cysticerci of T. hydatigena were freshly collected from sheep slaughtered at the abattoir and removed from their host capsules. Twenty-five cysts (total volume 250 ml) were washed several times in cold 0.01 M phosphate buffered saline (PBS) pH 7.2 and homogenised at 4 ~ C in a Sorvall Omnimixer (Sorvall, Connecticut, USA). The homogenate was frozen overnight at - 2 0 ~ C, thawed, and re-homogenised at 4 ~ C. After lyophilisation
Immunisation Against Fasciola hepatica in Rats and Mice
177
the antigen was reconstituted to 60 ml with PBS, centrifuged at 10,000 xg for 30 min and the snpernatant removed. Merthiolate was added to a concentration of 1/10,000 and the antigen stored at 4~ C before use. The protein concentration in the antigen was I0 mg/ml (biuret method).
In Vitro Culture Antigens. Methods for hatching and activating the T. hydatigena eggs and techniques for culture were as described previously for 7". ovis (Rickard and Bell, 1971 ; Rickard and Adolph, 1977). Antigens were collected during a 48 h maintenance period in vitro in Medium 858 (Common; wealth Serum Laboratories (CSL), Melbourne) containing 20% inactivated (56~ C for 30 rain) foetal calf serum (FCS) (CSL, Melbourne) or during 14-day in vitro cultivation in Medium 858 supplemented with 20% of inactivated colostrum-deprived lambs' serum. In vitro culture antigens were concentrated by dialysis against polyethylene glycol to a concentration such that 1 ml of antigen was equivalent to culture products from I0,000 oncospheres. Vaccination of Rats and Mice. All antigen preparations were emulsified in an equal volume of Freund's Complete Adjuvant (FCA) (Difco) and both rats and mice were injected subcutaneously. Rats received a total volume of 0.2 ml of antigen-FCA homogenate (1 mg protein of somatic antigen preparation; antigen from 1,000 oncospheres in 48 h and 14 day culture) and mice were injected with 0.1 ml each. Sham vaccination consisted of injection of equivalent volumes of a 50/50 mixture of FCS and colostrum-deprived lambs' serum homogenised in an equal volume of FCA. Challenge Infection of Rats and Mice and Measurement of their Immune Status. Rats and mice were challenged orally with 20 and 5 mc respectively. Immunity in rats was measured by the numbers of F. hepatica recovered at necropsy from the peritoneal cavity, liver parenchyma, and the bile ducts (Rajasekariah and Howell, 1977b). In mice the mortality of animals was noted, and any mice surviving until eight weeks after infection were killed and examined for F. hepatica infection (Lang, 1968).
Experiments and Results Immunisation o f Rats against F. hepatica Infection. N i n e t y rats were divided into six groups of 12 each a n d one g r o u p of 18. The group of 18 rats were the i m m u n e controls a n d at the time of first v a c c i n a t i o n of the other groups each rat received 5 mc orally. Six of these a n i m a l s were killed at the time of challenge of the other rats to ascertain that F. hepatica had established f r o m the i m m u n i s i n g infection. O n e g r o u p of rats served as the u n t r e a t e d controls a n d a further g r o u p was s h a m - v a c c i n a t e d four a n d two weeks before challenge. T e n t h o u s a n d T. hydatigena eggs were a d m i n i s t e r e d orally to a n o t h e r g r o u p at four a n d two weeks prior to challenge infection. The r e m a i n i n g three groups were vaccinated with either the somatic a n d cyst fluid antigen p r e p a r a t i o n or the 48 h or 14-day culture antigens at four weeks a n d two weeks before challenge infection. Of each g r o u p of rats, six were killed a n d e x a m i n e d at four weeks a n d six at eight weeks after challenge infection. The results of the e x p e r i m e n t are s h o w n in T a b l e 1. The rats i m m u n i s e d by prior infection with F. hepatica h a d significantly fewer parasites from the challenge infection at b o t h four a n d eight weeks post-challenge. F u r t h e r m o r e , n o m a r k e d degree of gross p a t h o l o g y was evident in the liver p a r e n c h y m a w h e n c o m p a r e d to n o t r e a t m e n t controls. However, there was n o significantly consistent effect o n fluke recovery of a n y i m m u n i s i n g schedule using cestode antigens, n o r any a p p a r e n t difference in gross p a t h o l o g y of the liver, a l t h o u g h
178
G.R. Rajasekariah et al.
Table 1. Recovery of F. hepatica (mean_+SD) from challenge infection (20 mc) of groups of six rats immunised with various antigens prepared from T. hydatigena Weeks after challenge
4 8
T. hydatigena antigen used for immunisation Eggs orally (a)
Somatic and cyst fluid (b)
48 h culture (c)
14 day culture (d)
Sham Immune vaccinated control a (e) (f)
No treatment (g)
2.0_+0.6 3.6_+1.6
3.5_+1.3 2.8_+1.4
3.5_+1.3 3.7_+1.8
4.2_+2.1 4.0_+0.8
2.3_+1.9 5.0_+1.1
5.0_+1.7 3.0_+1.4
1.0-+2.2 0.1_+0.4
Statistical analyses (Mann-Whitney U-test) P < 0.05: Four weeks after challenge; a < g, f< g, f
Z. Parasitenkd. 58,175-180 (1979)
ParasllBRkIHId8 Parasitology Research
9 by Springer-Verlag 1979
Attempts to Immunise Rats and Mice Against Infection With Fasciola hepatica Using Antigens Prepared From Taenia hydatigena G.R. Rajasekariah 1, M.D. Rickard 2, P.E. Montague 1, and G.F. Mitchell 3 1 ICI Research Laboratories, Ascot Vale, Victoria, 3032, Australia 2 Universityof Melbourne, Veterinary Clinical Centre, Princes Highway,Werribee, Victoria 3030, Australia 3 Walter and Eliza Hall Institute, Royal Melbourne Hospital P.O., Victoria 3050, Australia
Summary. Attempts were made to immunise rats and mice against infection with F. hepatica by oral dosing with T. hydatigena eggs, or by vaccination with various T. hydatigena antigen preparations. These antigens included extracts from 1". hydatigena cysticerci and cyst fluid, and antigens collected during short-term (48 h) and long-term (14 days) in vitro cultivation of larvae. hnmunity was assessed by the numbers of F. hepatica recovered from the challenge infection in rats, and the mortality rates of infected mice. None of the immunisation regimes with T. hydatigena antigens induced consistent, significant immunity. This was in contrast to the high level of immunity shown by rats dosed orally with F. hepatica metacercariae four weeks prior to challenge infection.
Introduction In a recent experiment Campbell et al. (1977) demonstrated a high level of resistance to Fasciola hepatica infection in sheep which had been infected with the larval stages of Taenia hydatigena 12 weeks previously. Prior infection with T. hydatigena not only significantly reduced the numbers of F. hepatica recovered from the challenge infection, but also protected against the pathogenic effects of the liver fluke. There have been a number of experiments which suggest that rats and mice are useful models for studying aspects of the biology and immunology of F. hepatica infection (Hayes et al., 1972; Armour and Dargie, 1974; Lang, 1968, 1976; Howell et al., 1977; Rajasekariah and Howell, 1977 b; 1978). The present experiments were designed to investigate the efficiency of various T. hydatigena antigen preparations in protecting rats and mice against a challenge F. hepatica infection. To whom offprint requestsshould be sent: Dr. G.R. Rajasekariah, Universityof Melbourne, Veterinary Clinical Centre, Princes Highway,Werribee, Victoria 3030, Australia
0044-3255/79/0058/0175/$01.20
176
G.R. Rajasekariah et al.
Rickard and Coman (1977) showed that rabbits dosed orally with eggs of T. hydatigena developed significant immunity against a subsequent challenge infection with eggs of the homologous parasite T. pisiformis; immunity was stimulated despite the fact that only a limited degree of migration and development of the heterologous T. hydatigena oncospheres had occurred in the rabbits. These experiments suggested that intestinal activation of, and penetration by, taeniid oncospheres can occur in other than their natural hosts. Therefore in the present experiments oral administration of T. hydatigena eggs was chosen as one possible means of immunisation. Antigens prepared from somatic tissues and cyst fluid of mature T. hydatigena cysticerci were also tested; although such preparations have given little protection against larval cestode infections in sheep (Gemmell and Soulsby, 1968), nevertheless, comparable somatic antigen preparations have shown some effect against T. pisiformis infection of rabbits (Miller and Kerr, 1932; Kerr, 1935), T. taeniaeformis in rats (Miller, 1932), and T. saginata in cattle (Gallie and Sewell, 1976). Antigens collected during in vitro cultivation of larval T. ovis, T. saginata, and T. pisiformis have been highly effective in immunising the host against homologous challenge infection (Rickard and Bell, 1971 ; Rickard and Outteridge, 1974; Heath, 1976; Rickard and Adolph, 1976, 1977; Rickard et al., 1976). Furthermore, antigens collected during short-term in vitro maintenance of T. ovis oncospheres stimulated high levels of resistance in lambs (Rickard and Adolph, 1977). Therefore, antigens collected during both long- and short-term in vitro culture of T. hydatigena were tested in the present experiments.
Materials and Methods Experimental Animals. Beagle puppies used for stock infections with T. hydatigena were reared cestode-free and fed on proprietary-line dog feed (Puppy Chow; Harper and Co., Mulgrave, Victoria: Lucky Dog Pellets; I.G.Y. Veterinary Products, Marrackville, N.S.W.). Rats were male Wistar breed and maintained at the I.C.I. Central Research Laboratories, Melbourne in conventional plastic cages at 22_+1~ and fed mouse breeding cubes (Barastoc; Melbourne) and water ad libitum. Rats were used in experiment when five weeks of age because resistance to Fasciola hepatica is known to increase with age in this animal (Rajasekariah and Howell, 1977a). Mice were SPF male C57B1/6 bred in an SPF facility but maintained conventionally at the Walter and Eliza Hall Institutel Melbourne in plastic cages between 20 and 25 ~ C and fed on Barastoc cubes and water ad libitum; the mice were 13 weeks of age at the time of immunisation. Parasites. Adult T. hydatigena were obtained from dogs experimentally infected with cysticerci recovered from naturally infected sheep slaughtered at the abattoir. Methods for infecting dogs, collecting tapeworms and their eggs, and counting and storing the eggs have been described previousIy (Coman and Rickard, 1975). Metaeercariae (mc) of F. hepatica were obtained from experimentally infected Lymnaea tomentosa snails. They were checked microscopically to assess their viability (Rajasekariah and Howell, 1977b) before administration into rats and mice. Somatic and Cyst-fluid Antigens of T. hydatigena. Cysticerci of T. hydatigena were freshly collected from sheep slaughtered at the abattoir and removed from their host capsules. Twenty-five cysts (total volume 250 ml) were washed several times in cold 0.01 M phosphate buffered saline (PBS) pH 7.2 and homogenised at 4 ~ C in a Sorvall Omnimixer (Sorvall, Connecticut, USA). The homogenate was frozen overnight at - 2 0 ~ C, thawed, and re-homogenised at 4 ~ C. After lyophilisation
Immunisation Against Fasciola hepatica in Rats and Mice
177
the antigen was reconstituted to 60 ml with PBS, centrifuged at 10,000 xg for 30 min and the snpernatant removed. Merthiolate was added to a concentration of 1/10,000 and the antigen stored at 4~ C before use. The protein concentration in the antigen was I0 mg/ml (biuret method).
In Vitro Culture Antigens. Methods for hatching and activating the T. hydatigena eggs and techniques for culture were as described previously for 7". ovis (Rickard and Bell, 1971 ; Rickard and Adolph, 1977). Antigens were collected during a 48 h maintenance period in vitro in Medium 858 (Common; wealth Serum Laboratories (CSL), Melbourne) containing 20% inactivated (56~ C for 30 rain) foetal calf serum (FCS) (CSL, Melbourne) or during 14-day in vitro cultivation in Medium 858 supplemented with 20% of inactivated colostrum-deprived lambs' serum. In vitro culture antigens were concentrated by dialysis against polyethylene glycol to a concentration such that 1 ml of antigen was equivalent to culture products from I0,000 oncospheres. Vaccination of Rats and Mice. All antigen preparations were emulsified in an equal volume of Freund's Complete Adjuvant (FCA) (Difco) and both rats and mice were injected subcutaneously. Rats received a total volume of 0.2 ml of antigen-FCA homogenate (1 mg protein of somatic antigen preparation; antigen from 1,000 oncospheres in 48 h and 14 day culture) and mice were injected with 0.1 ml each. Sham vaccination consisted of injection of equivalent volumes of a 50/50 mixture of FCS and colostrum-deprived lambs' serum homogenised in an equal volume of FCA. Challenge Infection of Rats and Mice and Measurement of their Immune Status. Rats and mice were challenged orally with 20 and 5 mc respectively. Immunity in rats was measured by the numbers of F. hepatica recovered at necropsy from the peritoneal cavity, liver parenchyma, and the bile ducts (Rajasekariah and Howell, 1977b). In mice the mortality of animals was noted, and any mice surviving until eight weeks after infection were killed and examined for F. hepatica infection (Lang, 1968).
Experiments and Results Immunisation o f Rats against F. hepatica Infection. N i n e t y rats were divided into six groups of 12 each a n d one g r o u p of 18. The group of 18 rats were the i m m u n e controls a n d at the time of first v a c c i n a t i o n of the other groups each rat received 5 mc orally. Six of these a n i m a l s were killed at the time of challenge of the other rats to ascertain that F. hepatica had established f r o m the i m m u n i s i n g infection. O n e g r o u p of rats served as the u n t r e a t e d controls a n d a further g r o u p was s h a m - v a c c i n a t e d four a n d two weeks before challenge. T e n t h o u s a n d T. hydatigena eggs were a d m i n i s t e r e d orally to a n o t h e r g r o u p at four a n d two weeks prior to challenge infection. The r e m a i n i n g three groups were vaccinated with either the somatic a n d cyst fluid antigen p r e p a r a t i o n or the 48 h or 14-day culture antigens at four weeks a n d two weeks before challenge infection. Of each g r o u p of rats, six were killed a n d e x a m i n e d at four weeks a n d six at eight weeks after challenge infection. The results of the e x p e r i m e n t are s h o w n in T a b l e 1. The rats i m m u n i s e d by prior infection with F. hepatica h a d significantly fewer parasites from the challenge infection at b o t h four a n d eight weeks post-challenge. F u r t h e r m o r e , n o m a r k e d degree of gross p a t h o l o g y was evident in the liver p a r e n c h y m a w h e n c o m p a r e d to n o t r e a t m e n t controls. However, there was n o significantly consistent effect o n fluke recovery of a n y i m m u n i s i n g schedule using cestode antigens, n o r any a p p a r e n t difference in gross p a t h o l o g y of the liver, a l t h o u g h
178
G.R. Rajasekariah et al.
Table 1. Recovery of F. hepatica (mean_+SD) from challenge infection (20 mc) of groups of six rats immunised with various antigens prepared from T. hydatigena Weeks after challenge
4 8
T. hydatigena antigen used for immunisation Eggs orally (a)
Somatic and cyst fluid (b)
48 h culture (c)
14 day culture (d)
Sham Immune vaccinated control a (e) (f)
No treatment (g)
2.0_+0.6 3.6_+1.6
3.5_+1.3 2.8_+1.4
3.5_+1.3 3.7_+1.8
4.2_+2.1 4.0_+0.8
2.3_+1.9 5.0_+1.1
5.0_+1.7 3.0_+1.4
1.0-+2.2 0.1_+0.4
Statistical analyses (Mann-Whitney U-test) P < 0.05: Four weeks after challenge; a < g, f< g, f
