Induction of Protective Immunity in Cattle ... - Infection and Immunity

7 downloads 136 Views 309KB Size Report
JOHN P. DALTON,1* SHARON MCGONIGLE,1 TIMOTHY P. ROLPH,2† AND ...... Smith, A. M., A. J. Dowd, M. Heffernan, C. D. Robertson, and J. P. Dalton. 1993.
INFECTION AND IMMUNITY, Dec. 1996, p. 5066–5074 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 64, No. 12

Induction of Protective Immunity in Cattle against Infection with Fasciola hepatica by Vaccination with Cathepsin L Proteinases and with Hemoglobin JOHN P. DALTON,1* SHARON MCGONIGLE,1 TIMOTHY P. ROLPH,2†

AND

STUART J. ANDREWS2

School of Biological Sciences, Dublin City University, Dublin 9, Republic of Ireland,1 and Mallinckrodt Veterinary Ltd., Harefield, Uxbridge, Middlesex UB9 6LS, United Kingdom2 Received 8 July 1996/Returned for modification 9 September 1996/Accepted 2 October 1996

Two cathepsin L proteinases, cathepsin L1 and cathepsin L2, secreted by liver flukes may be involved in tissue penetration, nutrition, and protection from immune attack. To ascertain the immunoprophylactic potential of these proteinases, and of another molecule, liver fluke hemoglobin (Hb), we performed vaccine trials in cattle. In the first vaccine trial various doses of cathepsin L1 were tested. The mean protection level obtained was 53.7%. In a second vaccine trial cathepsin L1 and Hb elicited 42.5 and 43.8% protection levels, respectively, while a combination of the two molecules induced a significantly higher level of protection (51.9%). Cathepsin L2 was not examined alone; however, vaccination of cattle with a combination of cathepsin L2 and Hb elicited the highest level of protection (72.4%). The animals that received cathepsin L1-Hb or cathepsin L2-Hb showed reduced liver damage as assessed by serum glutamic dehydrogenase and gamma-glutamyl transferase levels. Furthermore, a reduced viability was observed for fluke eggs recovered from all vaccine groups. This anti-embryonation effect of vaccination was particularly evident in the group that received cathepsin L2-Hb where >98% of the eggs recovered did not embryonate to miracidia. Although all vaccine preparations induced high antibody titers which were boosted following the challenge infection, there was no correlation between antibody titers and protection. The results of these trials demonstrate that cathepsin Ls and Hb could form the basis of a molecular vaccine that would not only reduce parasite burden but would also prevent transmission of liver fluke disease. showed that the two enzymes differed in their specificities for hydrolyzing peptide bonds (12). Therefore, the enzymes were termed cathepsin L1 (9, 27) and cathepsin L2 (9, 12). Another antigen secreted by adult flukes into culture medium was isolated by McGonigle and Dalton (18). Spectrometric studies revealed that this molecule contained a heme group and was a liver fluke hemoglobin (Hb) (10, 18). Hb is important in the aerobic respiration of immature flukes within the liver mass. However, in adult flukes, which have a largely anaerobic metabolism, it is more essential for oxygen-dependent functions such as egg production (3, 17, 30). As each of the above-described molecules may be involved in processes that are crucial to the development and survival of the parasite, we considered them potential targets at which a molecular vaccine could be directed. In the present study we report the results of vaccine trials carried out in cattle to test the immunoprophylactic potential of cathepsin L1, cathepsin L2, and Hb, either alone or in combinations. The first trial demonstrated that vaccination of cattle with cathepsin L1 can induce high levels of protection (mean, 53.7%) against a heterologous challenge infection of metacercariae of F. hepatica. A subsequent vaccine trial confirmed the protective nature of cathepsin L1 and showed that Hb could also elicit protective immune responses. Vaccination with a combination of these molecules elicited a higher level of protection than did vaccination with either alone. Most significantly, a 72.4% protection against challenge was achieved by vaccination with cathepsin L2 and Hb. The surviving flukes in this vaccinated group were more stunted in their growth than those recovered from the unvaccinated control animals. Consequently, these vaccinated animals showed little liver damage, as assessed by their serum glutamic dehydrogenase (GDH) and gamma-glutamyl transferase (GGT) levels. Furthermore, .98% of the eggs pro-

The trematode Fasciola hepatica is a causative agent of liver fluke disease, or fascioliasis, in mammals. Liver fluke disease of agricultural animals, such as cattle and sheep, has a worldwide distribution and results in large economic losses in many agriculture-dependent countries. Recent reports indicate that F. hepatica is also a major human pathogen (14). Infection is primarily acquired by the ingestion of vegetation on which metacercariae are encysted. Within the duodenum the metacercariae excyst, penetrate the intestinal wall, and then migrate via the peritoneal cavity to the liver. Here, the immature flukes spend 7 to 12 weeks migrating through the tissue causing extensive hemorrhage and fibrosis before they move into the bile ducts and mature to adults. Mature flukes produce numerous eggs which are deposited on pastures in the feces. Miracidia hatch from the eggs and penetrate an intermediate snail host, from which the infective metacercariae erupt after a developmental period. Dalton and Heffernan (11) showed, using gelatin substrate polyacrylamide gel electrophoresis (PAGE), that when immature and mature flukes are maintained in vitro they secrete cysteine endoproteinases into the culture medium. Several functions were suggested for these enzymes, including facilitation of migration through host tissue (11), the acquisition of nutrient (11, 27), and evasion of host immunity (8, 11, 26). Two cysteine proteinases were isolated and both were characterized as having physicochemical properties in common with mammalian lysosomal cathepsin L proteinases (12, 27). Kinetic studies with specific fluorogenic peptide substrates, however, * Corresponding author. Phone: 353-1-7045407. Fax: 353-1-7045412. Electronic mail address: [email protected]. † Present address: Animal Health Discovery, Pfizer Central Research, Pfizer Ltd., Sandwich, Kent CT13 9NJ, United Kingdom. 5066

VACCINATION OF CATTLE AGAINST F. HEPATICA INFECTION

VOL. 64, 1996

5067

duced by the flukes that did mature in these vaccinated animals did not embryonate to miracidia. MATERIALS AND METHODS Source and purification of parasite antigens. Mature F. hepatica flukes were removed from the bile ducts of infected bovine livers obtained from an abattoir in Ireland. The flukes were washed six times in phosphate-buffered saline (PBS), pH 7.3, and incubated for 16 h at 378C in RPMI 1640, pH 7.3, containing 2% glucose, 30 mM HEPES (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid), and 25 mg of gentamycin per ml. Following incubation, the medium was removed and centrifuged at 14,900 3 g for 30 min, and the supernatant, termed excretorysecretory (ES) products, was then collected and stored at 2208C (11). F. hepatica cathepsin L1 and cathepsin L2 were isolated from the ES products and purified by gel filtration chromatography on a Sephacryl S200HR column and ion exchange chromatography on a QAE Sephadex column as previously detailed (12, 27). F. hepatica Hb was eluted in a high-molecular-sized protein peak by gel filtration on Sephacryl S200HR as described before (18). Protein concentrations were determined by the MicroBCA protein assay (Pierce, Rockford, Ill.). Formulation, preparation, and administration of vaccines. The purified antigens were dialyzed against distilled water, freeze-dried, and stored at 2208C. On the day before vaccine administration the powder was reconstituted in PBS, and the vaccines were formulated by mixing 1 ml of the reconstituted antigen with an equal volume of Freund’s complete adjuvant (FCA) or Freund’s incomplete adjuvant (FIA) (Sigma Chemical Co., Poole, United Kingdom). The mixture was emulsified by sonication (7 3 30 s bursts, duty cycle 0.7). Vaccination of cattle. Two vaccine trials were performed with cattle. All animals were fluke free and housed indoors to preclude helminth infection. In the first trial 18 female Holstein-Friesian calves, aged 4 to 6 months, were randomly allocated into five groups of similar mean weight. Group 1 animals (n 5 4) received 10 mg of cathepsin L1 per immunization, Group 2 animals (n 5 4) received 50 mg of cathepsin L1 per immunization, Group 3 animals (n 5 3) received 200 mg of cathepsin L1 per immunization, Group 4 animals (n 5 3) received 500 mg of cathepsin L1 per immunization, and Group 5 control animals (n 5 4) received 150 mg of horse spleen ferritin (HSF). Each animal received a first injection of 1 ml of the vaccine preparation formulated in FCA into the biceps femoris of each hind leg on day 0. A second injection on day 28 (week 4) and a third on day 56 (week 7), both formulated in FIA, were given into each side of the back between the biceps femoris and gluteus medius. In the second trial, the effects of F. hepatica cathepsin L1, cathepsin L2 and Hb, and other combinations of these, were compared. Thirty-five male (castrated or entire) Holstein-Freisian calves, aged 1 year, were randomly allocated into five groups, each comprising seven calves of similar weights. Calves in Group 1 were vaccinated with 200 mg of cathepsin L1 per injection, calves in Group 2 received 200 mg of Hb per injection, calves in Group 3 received 200 mg of both cathepsin L1 and Hb per injection, calves in Group 4 received 200 mg of both cathepsin L2 and Hb, and calves in Group 5 (control) received 200 mg of HSF per injection. The first injection of 2 ml of antigen preparation, formulated in an equal volume of FCA, was injected into the biceps femoris of the left hind leg on day 0. Booster injections into the biceps femoris of the right hind leg on day 35 (week 5) and into the gluteus medius on day 63 (week 9) were formulated in an equal volume of FIA. Parasite challenge. Metacercariae of F. hepatica were purchased from Compton Paddock Laboratories, Newbury, United Kingdom, and stored at 48C. Gelatin capsules containing metacercariae (less than 4 months old) were inserted into a gelatin-agar bolus (2) and administered per os to each animal with an esophageal balling gun. In the first trial each animal received ca. 500 metacercariae 28 days after the third injection. In the second trial each animal received ca. 600 metacercariae 16 days after the third injection. Assessment of protection. All cattle were killed 11 (first trial) or 13 (second trial) weeks after the challenge. The duodenum was ligated 6 in. (1 in. 5 2.54 cm) each side of the common bile duct and removed with the liver and gall bladder. The number of flukes in the liver was estimated by standard techniques (20). In the second trial the numbers of flukes less than and greater than 6-mm long were also recorded. Total worm burdens were summarized for each vaccinated and control group by arithmetic means. Statistical differences between vaccinated and control groups were tested by analysis of variance. Any differences found to be significant at a 5% level (P 5 0.05) or less were further investigated by a multiple-range test. Liver enzyme analysis. Liver damage in all vaccinated and control animals was assessed by measuring the levels of the liver enzymes GDH (EC 1.4.1.3.) and GGT (EC 2.3.2.2.) in blood samples taken weekly from the time of challenge infection to the day of slaughter (1). Estimation of egg viability. The gall bladders were separated from the livers and sliced open, and the bile was decanted into a conical vessel. After 1 h the supernatant was siphoned off and the deposit, containing the F. hepatica eggs, was washed five times with water. The eggs were then incubated in darkness at 228C for 14 days. After incubation they were examined daily for miracidial development. Viability was assessed by estimating the percentage of eggs that had embryonated and developed to miracidia. In cases where low numbers of

FIG. 1. Polyacrylamide gel analysis of isolated F. hepatica ES molecules used in vaccine trials. (A) SDS reducing 10% PAGE analysis of total adult fluke ES products (lane 1), purified 27-kDa cathepsin L1 (CL1) (lane 2), and purified 29.5-kDa cathepsin L2 (CL2) (lane 3); (B) non-SDS native 10% PAGE analysis of total adult fluke ES (lane 1) and purified hemoglobin (lane 2).

eggs were recovered from the gall bladder, all eggs were considered in the calculation, and in cases where many eggs were recovered, 300 eggs were taken as representative. Analysis of the antibody responses of cattle by immunoblotting. Mature F. hepatica ES products were separated on sodium dodecyl sulfate (SDS) reducing 10% polyacrylamide gels for analysis of anti-cathepsin L1 and anti-cathepsin L2 responses (12, 27) or on non-SDS native 10% polyacrylamide gels for analysis of Hb responses (18) and transferred to nitrocellulose filters (0.45-mm pore size, BA85; Schleicher & Schuell) by using an Atto semidry blotting system. Nonspecific binding sites were blocked with PBS containing 1% fetal calf serum and 0.5% Tween 20. The nitrocellulose filters were used to analyze the specificity of the immune responses of vaccinated and control cattle. Filters were probed with serum (1:500 dilution), and bound immunoglobulin (Ig) was visualized by using alkaline phosphatase-conjugated anti-bovine Ig. Nitroblue tetrazolium and 5-bromo-5-chloro-3-indolylphosphate prepared in dimethylformamide were used as a substrate (Sigma Chemical Co.). Analysis of antibody responses by ELISA. Antibody responses were also examined by enzyme-linked immunosorbent assay (ELISA). Wells of a 96-well tissue culture plate were each coated with 1 mg of purified cathepsin L1 (first trial) or 2 mg of adult F. hepatica ES products (second trial) by adding the antigen in PBS to the wells and incubating overnight at 378C. The excess binding sites were blocked with 2% bovine serum albumin–1% Tween 20 in PBS at 378C for 1 h. After being washed three times in blocking buffer, the bovine sera (100 ml, 1:8,000 dilution) were added to the wells and the plates were incubated at 378C for 1 h. Bound antibody was detected by using alkaline phosphatase-conjugated rabbit anti-bovine IgG and the substrate p-nitrophenol phosphate as described by the manufacturer (Sigma Chemical Co.).

RESULTS Purification of vaccine components. SDS-reducing PAGE analysis of the medium in which adult liver flukes were maintained (ES products) showed that the major proteins secreted by these parasites were the 27-kDa cathepsin L1 and 29.5-kDa cathepsin L2 proteinases (Fig. 1A, lane 1) (11, 12, 27). Both of these enzymes were isolated by gel filtration which was followed by ion exchange chromatography as previously described (Fig. 1A, lanes 2 and 3) (12, 27). The cathepsin L1 is heterogeneous in its migration, whereas the cathepsin L2 migrates as a single band. Enzymatic assays performed with specific substrates for each enzyme demonstrated that there is no cross contamination between the two proteinases (data not shown).

5068

DALTON ET AL.

INFECT. IMMUN.

FIG. 2. Analysis of antibody responses to cathepsin L1 in vaccinated and control cattle by ELISA. Sera were taken during the course of vaccination (A) and following the challenge infection (B). Cathepsin L1 was used as antigen and was probed with sera from each animal (1:8,000 dilution). Each absorbance (Abs) represents the mean response in animals immunized with 10 mg (Group 1, large open squares), 50 mg (Group 2, small open squares), 200 mg (Group 3, large solid squares), or 500 mg (Group 4, small solid squares) of cathepsin L1 or with 150 mg of HSF (Group 5, controls, open triangles) per immunization.

Another molecule secreted by adult flukes was identified by McGonigle and Dalton (18) in the ES products when they were analyzed by non-SDS–native PAGE (Fig. 1B, lane 1 and 2). This molecule, which was characterized as fluke Hb, was not observed in SDS-reducing PAGE (Fig. 1A, lane 1) (18). The Hb molecule is susceptible to proteolytic cleavage and therefore migrates as a single band preceded by a smear (Fig. 1B, lane 2) (18). Antibody responses of cattle vaccinated with various doses of F. hepatica cathepsin L1. The first trial was carried out to determine the dose of cathepsin L1 required to elicit an antibody response in cattle. In addition, we examined whether the induction of immune responses to cathepsin L1 could confer protection against a subsequent heterologous challenge with metacercariae of F. hepatica. All animals immunized with cathepsin L1 produced an antibody response, including those in Group 1, which received only 10 mg of cathepsin L1 per injection, as judged by ELISA using purified cathepsin L1 as the antigen (Fig. 2A). Antibodies appeared in the sera of animals vaccinated with 200 and 500 mg of cathepsin L1 (Groups 3 and 4) within 4 weeks after the first injection, whereas a significant rise in antibodies was not observed in those receiving 10 and 50 mg (Groups 1 and 2) until 6 weeks after the first injection. The second and third injections, formulated in FIA, resulted in a boosting of the antibody responses in all groups, with the exception of the third immunization of Group 4 (500 mg per injection). The sera of animals immunized with HSF (Group 5) did not contain antibodies reactive with cathepsin L1 (Fig. 2A). The antibody responses of each animal to cathepsin L1 were monitored weekly following challenge. Antibodies to cathepsin L1 were detected in the sera of animals in the control group (Group 5) within 3 weeks after challenge, indicating that this molecule is immunogenic during the early stages of infection (Fig. 2B). The anti-cathepsin L1 responses in these animals slowly increased over the first 4 weeks of infection and then remained constant for the subsequent 7 weeks of the study. In

all the vaccinated groups (Groups 1 to 4) anti-cathepsin L1 antibody responses were boosted following challenge. Anticathepsin L1 antibody responses began to rise in these animals within 2 weeks of the infection and continued to rise over the following 7 weeks. There were no significant differences in the anti-cathepsin L1 antibody levels among the vaccinated groups before or during infection. However, throughout the entire course of infection the anti-cathepsin L1 levels in the vaccinated animals were always in the range of 10 to 15 times greater than those in the control animals (Fig. 2B). Adult F. hepatica ES products were electrophoretically separated by SDS-reducing PAGE, transferred to nitrocellulose, and probed with sera from both vaccinated and control animals. Sera of all vaccinated animals (Groups 1 to 4), taken in the week of challenge, contained antibodies that were reactive with two molecules in the adult fluke ES products (Fig. 3A, representatives from each group shown). These molecules, of 27 and 29.5 kDa, have been previously characterized by our laboratory as cathepsin L1 (27) and cathepsin L2 (12), respectively. Dowd et al. (12) showed by immunoblotting experiments that antisera prepared against homogeneous cathepsin L1 or cathepsin L2 was reactive with both cathepsin L1 and cathepsin L2; hence, both molecules share antibody epitopes. It is therefore not surprising to observe the reactivity of antibodies in the sera of the cathepsin L1-vaccinated animals with both cathepsin L1 and cathepsin L2. The intensities of the cathepsin L bands detected by the animal sera were highly variable even within groups (Fig. 3A and data not shown). Sera taken before challenge from animals in the control group (Group 5) did not contain antibodies reactive with liver fluke antigens (Fig. 3A). Similar immunoblot analyses were carried out with sera from the vaccinated animals following challenge. The cathepsin L1 and cathepsin L2 were the major components in the adult liver fluke ES products reactive with antibodies in these sera. The intensities of the cathepsin L1 and cathepsin L2

VOL. 64, 1996

VACCINATION OF CATTLE AGAINST F. HEPATICA INFECTION

FIG. 3. Immunoblot analysis of sera taken from cathepsin L1-vaccinated and control cattle in the first vaccine trial. Adult liver fluke ES products were separated by SDS reducing 10% PAGE and transferred to nitrocellulose. Sera obtained from animals in each group at the time of challenge (A) and 6 weeks following infection (B) were used to probe the nitrocellulose filters (a representative from each group is shown). Groups 1 to 4 received 10, 50, 200, and 500 mg of cathepsin L1 per immunization, respectively, and Group 5 (controls) received 150 mg of HSF per immunization. Grp, group; CL1, cathepsin L1; CL2, cathepsin L2.

bands were greater in the blots probed with postchallenge sera those probed with prechallenge sera, indicating increases in antibody titers to these molecules following challenge. The only other immunogen detected with these antisera was at .200 kDa (Fig. 3B; only data obtained by using sera taken at 6 weeks after infection are shown). These three molecules were also the only components reactive with antibodies in sera taken from the nonvaccinated control animals after challenge (Fig. 3B). Parasite burdens in cattle vaccinated with F. hepatica cathepsin L1. Eleven weeks after challenge the animals were killed and the parasite burdens were estimated (Table 1). In the control group (Group 5) the mean fluke infection level was 30.4%, which is consistent with other liver fluke infection studies in bovines (5). All groups vaccinated with cathepsin L1 showed a reduction in fluke burden, with the level of protection ranging from 38.2 to 69.5%. Group 1 (10 mg per injection) and Group 2 (50 mg per injection) showed significant levels of protection compared with the control group (Group 5) (P , 0.05). A significant level of protection was also induced in Group 3 (200 mg of injection) (P , 0.01). However, Group 4 (500 mg) was not significantly different from the control group. Although the level of protection increased with increased doses of antigen in the 10 to 200 mg range, statistical analysis revealed no significant differences among Groups 1, 2, and 3. When all the vaccinated animals (Groups 1 to 4) were treated as a single group and compared with the control group (Group 5), a significant difference was observed (P , 0.05) and the mean level of protection was 53.7%. Antibody responses of animals vaccinated with cathepsin L1, cathepsin L2, and Hb. A second trial was performed in which the immunoprotective potential of F. hepatica cathepsin L1 was further examined. The cathepsin L1 was tested at a dose which showed maximum protection in the first trial, that is, 200 mg per injection. In addition, the vaccine potentials of cathepsin L2 cysteine proteinase (12) and Hb (18) were also examined either alone (Hb) or in combinations. The antibody responses of vaccinated and control animals were analyzed by ELISA using total adult F. hepatica ES products as antigens. Figure 4 shows the mean antibody levels of each group obtained with serum dilutions of 1:8,000. Antibodies were observed in all vaccinated animals 2 weeks after the first immunization (data not shown). A boosting of the anti-

5069

body responses was observed in all groups following both the second and third immunizations (Fig. 4A). The group that received cathepsin L1 alone (Group 1) showed the lowest mean antibody levels of all the vaccinated groups. The sera taken before challenge from the control animals (Group 5) did not contain antibodies reactive with antigen in the liver fluke ES products (Fig. 4A). An increase in the antibody titers in all vaccinated animals was observed within 1 week following challenge (Fig. 4B); since in this vaccine trial the animals were challenged 2 weeks after the final immunization, this observed increase in antibodies may be due to a maturation of the immune response to the vaccination and/or to a boosting by invading parasites. Antibodies in all vaccinated groups remained high throughout the infection but decreased at approximately 6 weeks after infection, which may coincide with the beginning of parasite entry into the bile ducts. Antibodies to adult fluke ES products in the sera of the control group (Group 5) increased within the first 2 to 3 weeks after infection and then remained constant throughout the subsequent weeks (Fig. 4B). The specificities of the antibody responses of all animals, before and after the challenge, were examined by immunoblotting with adult F. hepatica ES products as the antigen. The sera obtained at the time of challenge and at 6 weeks after challenge from animals in the group vaccinated with cathepsin L1, either alone (Group 1) or in combination with Hb (Group 3), contained antibodies reactive with the 27-kDa cathepsin L1 and 29.5-kDa cathepsin L2. In addition, sera taken at the same times from animals that received cathepsin L2 with Hb (Group 4) also contained antibodies to both cathepsin L molecules. These immunoblots are not shown, as they were similar to those presented in Fig. 3. Antibody responses to Hb were examined with non-SDS native 10% polyacrylamide gels (18). Immunoblots showed that the sera taken from animals vaccinated with Hb at the time of challenge and 6 weeks later (Groups 2, 3, and 4) contained antibodies reactive with Hb (Fig. 5A). In similar immunoblot analyses the sera taken from animals vaccinated with cathepsin L1 alone (Group 1) or from control animals (Group 5) at the same times were not reactive with Hb (Fig. 5A and B) (18). Parasite burdens in animals vaccinated with cathepsin L1, cathepsin L2, and Hb. In the second trial fluke burdens were assessed in all animals 13 weeks after the challenge (Table 2). The mean recovery of liver flukes from the controls, Group 5, was 152.1, which represents 25.4% of the infection dose. This level of infection was lower than that observed in the first trial and may reflect differences in the infectivity of the metacercariae used in each trial. However, the animals used in this trial were older by 6 to 8 months. Earlier studies have shown that as cattle age they become more resistant to fluke infection (5). All

TABLE 1. Vaccination of cattle with F. hepatica cathepsin L1a Group (dose [mg]/injection)

Group Group Group Group Group

1 2 3 4 5

(10) (50) (200) (500) (ctlb)

Recovered flukes Mean 6 SD

Flukes/animal

72.25 6 26.2 68.5 6 34.4 46.3 6 15.5 94.0 6 30.3 152.0 6 9.3

70, 67, 12, 140 147, 105, 5, 17 17, 52, 70 91, 148, 43 142, 145, 180, 141

% Reduction vs control

52.5 54.9 69.5 38.2

a Groups of cattle were vaccinated with purified cathepsin L1 formulated in FCA and in FIA. b ctl, control.

5070

DALTON ET AL.

INFECT. IMMUN.

FIG. 4. Analysis of antibody responses of vaccinated and control cattle in the second vaccine trial by ELISA. Sera were taken during the course of vaccination (A) and following the challenge infection (B). Adult liver fluke ES products were used as antigens and were probed with sera from each animal (1:8,000 dilution). Each absorbance (Abs) represents the mean response in animals immunized with 200 mg of cathepsin L1 (Group 1, large open squares), 200 mg of Hb (Group 2, large solid squares), 200 mg of both cathepsin L1 and Hb (Group 3, small open squares), or 200 mg of both cathepsin L2 and Hb (Group 4, small solid squares) or 150 mg of HSF (Group 5, controls, open triangles) per immunization.

vaccinated groups (Groups 1 to 4) exhibited fluke burdens which were significantly lower than that of the control group (P , 0.05). The mean protection level obtained with vaccination with cathepsin L1 alone (Group 1) was 42.5%, which confirmed the protective nature of this molecule. Vaccination with Hb (Group 2) induced a similar level of protection (43.8%). Vaccination with the combination of cathepsin L1 and Hb (Group 3) induced a protection level of 51.9%, which

was significantly higher (P , 0.05) than that obtained with cathepsin L1 (Group 1) or Hb (Group 2) alone. The highest level of protection was achieved with a combination of cathepsin L2 and Hb (Group 4, 72.4%); this protection level was significantly higher (P , 0.05) than that observed in all other groups (Table 2). In the first trial it was noted that flukes recovered from the vaccinated animals tended to be shorter and narrower than those recovered from the control animals, which indicated that vaccination adversely affected the normal development of the parasites. In the second trial the recovered parasites were separated into size groups of less than and greater than 6 mm (Table 2). This size was chosen as an indicator of development since liver fluke parasites reach approximately 6 mm before migrating into the bile duct and developing to full maturity. However, flukes of less than 6 mm may not necessarily have been recovered from the liver parenchyma nor have undevel-

TABLE 2. Vaccination of cattle against F. hepatica infection with cathepsin L1, cathepsin L2, and Hba Group (antigen[s])

FIG. 5. Immunoblot analysis of sera taken from vaccinated and control cattle in the second trial. Purified Hb was separated by native 10% PAGE and transferred to nitrocellulose. Pools of sera obtained from animals in each group at the time of challenge (A) and 6 weeks following infection (B) were used to probe the nitrocellulose filters. Animals were immunized with 200 mg of cathepsin L1 (Group 1), 200 mg of Hb (Group 2), 200 mg each of cathepsin L1 and Hb (Group 3), 200 mg each of cathepsin L2 and Hb (Group 4), or 150 mg of HSF (Group 5, controls) per immunization. Grp, group.

Group 1 (cathepsin L1) Group 2 (Hb) Group 3 (cathepsin L1 plus Hb) Group 4 (cathepsin L2 plus Hb) Group 5 (HSF, control)

No. of flukes .6 mm

No. of flukes ,6 mm

Total no. of flukes

% Reduction vs control

27.4 6 3.7

60.1 6 9.5

87.5 6 12

42.5

26.4 6 3.2 25.8 6 9.3

59.1 6 8 53.1 6 6.8

85.5 6 8.2 79.0 6 8.2

43.8 51.9

8.0 6 4.8

34.0 6 11.4

42.0 6 16

72.4

78.4 6 15.9

73.7 6 13.8

152.1 6 20.3

a Groups of seven cattle were vaccinated with liver fluke antigens formulated in FCA and in FIA.

VOL. 64, 1996

VACCINATION OF CATTLE AGAINST F. HEPATICA INFECTION

5071

FIG. 6. Analysis of the levels of GDH (A) and GGT (B) in the sera of vaccinated and control cattle in the second trial. Sera were obtained thrice weekly, before and after challenge, from all animals immunized with 200 mg of cathepsin L1 (Group 1, open bars), 200 mg of Hb (Group 2, bars with horizontal lines), 200 mg each of cathepsin L1 and Hb (Group 3, bars with vertical lines), 200 mg each of cathepsin L2 and Hb (Group 4, bars with diagonal lines), or 150 mg of HSF (Group 5, controls, hatched bars) per immunization. Normal levels of GDH and GGT range between 5 and 20 U/liter (12). Bars represent the mean enzyme levels within each group 6 standard deviation.

oped internal organs. In the control animals the mean number of recovered flukes that were ,6 mm was approximately equal to the number that were .6 mm. However, parasites recovered from animals that were vaccinated with cathepsin L1 alone (Group 1), Hb alone (Group 2), or a combination of these (Group 3) tended to be smaller than those recovered from the control animals; in each group only approximately 30% of the parasites reached a size of .6 mm. In the animals vaccinated with a combination of cathepsin L2 and Hb (Group 4) the mean number of recovered parasites that developed beyond 6 mm was only approximately 20% of the total. Furthermore, the number of recovered flukes .6 mm in this group was only 10% of the number recovered from the control animals (Group 5) (Table 2). Serum GDH and GGT levels in animals vaccinated with cathepsin L1, cathepsin L2, and Hb. Since extensive liver damage is a pathological consequence of liver fluke infection, in the second trial we examined the degree of liver damage in the vaccinated and control animals by measuring their serum GDH and GGT levels throughout the course of infection (1). Increased GDH levels in serum are indicative of liver damage, particularly to the liver parenchyma cells. In liver fluke infections, this enzyme is observed in the serum as immature flukes enter the liver tissue, and its concentration rises as damage ensues. In all vaccinated and the control animals a rise in serum GDH level was observed within 6 weeks after the challenge infection; at this time there were no significant differences between any vaccine group and the control group (Fig. 6A). As the infection progressed the mean serum GDH levels increased in the vaccinated animals in groups 1, 2, and 3 and in the control group animals. However, from 6 weeks after infection to the day of slaughter the mean serum GDH levels in the animals that were vaccinated with cathepsin L2 and Hb (Group 4) did not increase significantly. The mean serum GDH levels in this group from 6 weeks after infection to the

week of slaughter were significantly lower (P , 0.05) than in all other vaccinated groups (Groups 1, 2, and 3) and the control group (Group 5) (Fig. 6A). In liver fluke infection an increased serum GGT level is indicative of hyperplasia in the bile ducts and is associated with the entrance and residence of the flukes in the bile ducts, which begins at approximately 9 weeks after infection. The mean serum GGT levels in the control group animals (Group 5) increased sharply from 9 to 12 weeks after infection (Fig. 6B). The mean serum GGT levels in the animals vaccinated with cathepsin L1 alone (Group 2) or Hb alone (Group 2) showed similar increases relative to the control group. The mean serum GGT level in the animals vaccinated with a combination of cathepsin L1 and Hb (Group 3) was significantly lower than in the control group at week 12 after infection (Fig. 6B). The serum GGT levels in the animals vaccinated with a combination of cathepsin L2 and Hb (Group 4) did not increase above preinfection levels at 9 weeks after infection and were only slightly increased at 12 weeks after infection; the GGT levels of this group were significantly lower than those of all other vaccinated groups and the control group (P , 0.05) (Fig. 6B). Viability of eggs recovered from animals vaccinated with cathepsin L1, cathepsin L2, and Hb. We examined whether our vaccinations had an effect on the viability of eggs produced by flukes that remained in the vaccinated animals. The viability data for eggs recovered from each animal are presented in Table 3. Eggs with a viability of 96 to 100% were recovered from the gall bladders of all animals in the control group (Group 5). The mean viabilities of eggs recovered from animals vaccinated with cathepsin L1 alone (Group 1), Hb alone (Group 2), and with a combination of cathepsin L1 and Hb (Group 3) were 40.6, 35, and 38.5%, respectively, taking into account that eggs recovered from some animals partially developed but did not embryonate to miracidia. Eggs were not recovered from three of the seven animals vaccinated with

5072

DALTON ET AL.

INFECT. IMMUN.

TABLE 3. Viability of F. hepatica eggs recovered from gall bladders of cattle in the second vaccine trial Animal no.

Egg viability (%)a

Group 1 (cathepsin L1)

4117 4141 4129 4122 4074 4051 4078

40 65 nd nd Partial 40 58

Group 1 (Hb)

4053 4080 4108 4071 4109 4138 4105

75 35 30 nd 42 Partial 30

Group 3 (cathepsin L1 1 Hb)

4119 4079 4052 4139 4107 4055 4128

0 20 65 80 55 Partial 50

Group 4 (cathepsin L2 1 Hb)

4058 4124 4076 4068 4125 4136 4110

7 No eggs Partial Partial Partial No eggs No eggs

Group 5 (HSF, control)

4142 4113 4143 4147 4137 4057 4114

98 98 100 100 96 100 100

Group (antigen)

a Partially embryonated, containing a blastocyst but no miracidium. nd, not determined.

cathepsin L2 and Hb protein (Group 4), presumably because flukes in these animals did not develop to maturity. Eggs recovered from three other animals in this group did not embryonate to miracidia, and only 7% of the eggs from the remaining animal embryonated. Therefore, vaccination with a combination of cathepsin L2 and Hb elicited an overall anti-embryonation effect of .98% (Table 3). DISCUSSION Proteinases are involved in many crucial processes of parasitic helminths, including tissue invasion, feeding, and immune evasion (11, 19). Accordingly, they have created much interest as targets for immunoprophylaxis. In the present report we have demonstrated that we can induce high levels of protection in cattle against a heterologous challenge of F. hepatica by immunization with cathepsin L cysteine proteinases. Additionally, we have shown that another molecule, liver fluke Hb, can also elicit significant levels of protection in cattle. Besides eliciting immunoprotection and a decrease in pathology associated with liver fluke infection, vaccination with these antigens adversely affects fluke growth and egg viability.

The liver fluke cathepsin Ls are packaged in vesicles in the epithelial cells lining the alimentary tract (27). These cells secrete the vesicles into the gut lumen where the proteinases are released and most probably degrade ingested host proteins (11, 27). Since the gut contents of the parasite’s blind-ended digestive tract are voided regularly by regurgitation, these enzymes must reach the surrounding tissues (13). Therefore, in addition to the digestive function in the fluke gut, cathepsin Ls may play some extracorporeal role that would benefit the parasite. The most likely extracorporeal function of the cathepsin Ls would be the degradation of host tissue, which would facilitate parasite migration and at the same time provide peptides that, following further hydrolysis, could be utilized as nutrients (7, 11, 12). In addition, the cathepsin L proteinases could be involved in protecting the parasite against immune attack (7, 11). We have shown that cathepsin L1 cleaved Ig at the hinge region and could prevent the antibody-mediated attachment of eosinophils to juvenile flukes (7, 26). We speculated that vaccination with fluke cathepsin L would induce antibodies that could interfere with the activity of these enzymes and thereby block some or all of the above-described crucial processes. In vitro studies showed that the binding of rabbit anti-cathepsin L1 IgG to the enzyme resulted in its inactivation and inhibited the ability of cathepsin L1 to prevent antibody-mediated eosinophil attachment to flukes (25). In the present study we show that immunization of cattle with cathepsin L1 can elicit high levels of protection against infection. In the first vaccine trial the protection level ranged from 38.2 to 69.5%. Although statistical analysis revealed that the level of protection obtained in the animals receiving 10, 50, or 200 mg of cathepsin L1 per immunization was dose related, this correlation may have occurred by chance since those animals receiving 500 mg per immunization exhibited the least protection. Nevertheless, in the first trial the average level of protection obtained by vaccination with cathepsin L1 was 53.7%. The ability of cathepsin L1 to elicit protective immune responses was confirmed in the second trial in which a mean protection level of 42.5% was obtained. There were several variations between the two trials which may have contributed to the difference in mean levels of protection; these included the age of animals, vaccine regime, and infection dose. The immunoprotective potential of a second cysteine proteinase, cathepsin L2 (12), was examined but only in combination with Hb. Insufficient quantities of purified cathepsin L2 were available to test its vaccine efficacy alone. Surprisingly, the combination of cathepsin L2 and Hb induced a significantly higher level of protection and had greater effects on fluke growth and egg viability than the combination of cathepsin L1 and Hb. Although both cathepsin molecules have common epitopes and induce cross-reactive humoral immune responses, cathepsin L2 may have a better immunoprotective property than cathepsin L1 by stimulating cell-mediated immune responses in cattle more effectively. Since the two molecules migrate differently in native and reducing SDS-PAGE, a structural difference clearly exists between them. Additionally or alternatively, the blocking of cathepsin L2 activity by immune effector mechanisms may have a more profound effect on parasite survival than the blocking of cathepsin L1 activity. An examination of the substrate specificities of the two proteinases revealed that while cathepsin L1 and cathepsin L2 have similar specificities for peptide bonds, cathepsin L2 cleaved these bonds with a greater efficiency. In particular, cathepsin L2 cleaved several peptide bonds with a proline in the P-2 position that were poorly cleaved by cathepsin L1 (12). This difference in substrate specificity indicates that the two

VOL. 64, 1996

VACCINATION OF CATTLE AGAINST F. HEPATICA INFECTION

cysteine proteinases may have distinct roles in digestion, tissue penetration, or immune evasion. Immunization with Hb also elicited protective immune responses in cattle against a liver fluke infection. Furthermore, when used in combination with cathepsin L1 the protection level was significantly higher, albeit not additive, than that achieved with each preparation alone. Little is known about hemoglobins of helminths, although besides being involved in the movement of oxygen through tissues, they are believed to function as myoglobin-like oxygen stores (4, 16). Migrating liver flukes have a predominantly aerobic energy metabolism and a functioning Krebs cycle which is almost exclusive to the tegument (29, 31). However, both the growth of the parasite, which limits oxygen diffusion to the tissues, and the anaerobic environment of the bile duct induce a switch to anaerobic respiration (30). It is possible, therefore, that immunization with Hb induced an immune response that is capable of interfering with the oxygen metabolism in the tegument of the migrating parasite. Antibodies prepared against the Hb were reactive with the immature fluke tegument (31a). All vaccine preparations used in this study exerted their effects on the migratory fluke stages, before the flukes entered the bile ducts. Flukes recovered from vaccinated animals tended to be smaller than those recovered from control animals, and in particular, in those animals vaccinated with a combination of cathepsin L2-Hb (Group 4) the number of flukes of .6 mm was only 10% of that recovered from the control animals. Serum enzyme analysis revealed that serum GGT levels, an indicator of bile duct damage, were significantly lower in the animals in the groups that received the most effective preparations (cathepsin L1-Hb and cathepsin L2-Hb). Animals vaccinated with cathepsin L2-Hb also had reduced serum GDH levels, indicating that they suffered little damage to the liver parenchyma. In an earlier study we showed that cathepsin L proteinases are expressed by all fluke stages that parasitize the mammalian host, including the juveniles that excyst in the intestines (6). In addition, antibodies to the Hb can be detected by ELISA as early as 1 week after infection (18). Therefore, it is possible that both cathepsin L and Hb are accessible to vaccine-induced immune responses at the early stages of infection. Since the mortality and morbidity associated with liver fluke infection is a direct consequence of liver pathology caused by migrating flukes, a vaccine will be effective only if it prevents the development of these early stages. An important feature of these vaccines was their effect on the viability of eggs recovered from the gall bladders of the vaccinated animals. Most remarkable, however, was the almost complete anti-embryonation effect of vaccination with a combination of cathepsin L2 and Hb. Anti-embryonation effects mediated by immune responses are well established in schistosomiasis, a disease caused by another trematode, Schistosoma spp. The pathology associated with Schistosoma mansoni and Schistosoma japonicum infections is a direct consequence of inflammatory responses to eggs trapped in the liver. Granulomata form around the eggs, but as infection proceeds the size of these decrease with a concurrent abatement of the disease (21). Granuloma modulation is believed to involve anti-egg antibodies that have an anti-embryonation effect, although the antigens responsible for inducing these antibodies have not been identified (21). However, passive immunization of S. mansoni-infected mice with anti-glutathione S-transferase (Sm28GST) monoclonal antibody significantly reduced the production and viability of eggs without having an effect on worm burdens (33). While eggs are not associated with pathology in liver fluke disease caused by Fasciola spp., blocking of miracidial development and hatching would have a profound

5073

effect on the extent of pasture contamination and hence disease transmission. The mechanism(s) by which immune responses to cathepsin L and Hb exert the anti-embryonation effects is not understood. In one study the presence of cathepsin L proteinases in the oocytes, Mehlis’ gland, and vitelline glands of flukes was demonstrated by immunohistochemistry (32). Furthermore, although adult flukes have an anaerobic respiration, oxygen is required for egg production and phenolase tanning of eggshell proteins (3, 17), and Hb may be involved in transporting oxygen to this tissue. Possibly, antibodies directly interact with cathepsin Ls and Hb in the vitelline glands or Mehlis’ glands. Alternatively, the production of nonviable eggs may be a favorable, but indirect, consequence of the effect of the vaccines on the parasite’s nutrition and development. High antibody titers were induced by all vaccine preparations, and these were boosted following the challenge infection. Therefore, these vaccines formulated in Freund’s adjuvant could induce memory B cells that are stimulated by antigens released from the parasite. No correlation was found between total antibody responses, assessed by ELISA and immunoblotting, and the level of protection obtained in both trials. Sexton et al. (24) found no correlation between antibody (IgG1 and IgG2) responses to linear peptide epitopes and the level of protection induced in sheep by vaccination with F. hepatica GST. At present, we have no information regarding cellular immune responses; however, studies are under way to assess both B- and T-cell responses to our vaccines formulated in Freund’s adjuvant and in some commercially approved adjuvants. Serious economic losses are caused by infection of both cattle and sheep by liver flukes. While we obtained high levels of protection in cattle, it still has to be determined whether these vaccine candidates will induce similar responses in sheep. Cattle show a high level of natural resistance to infection, whereas sheep do not (1, 14). Furthermore, vaccination of sheep with crude antigen preparations has been generally unsuccessful (24). Vaccination of sheep with purified liver fluke GST induced highly variable results with protection levels ranging from 0 to 57% (23, 24, 28). Wijffels et al. (32) also reported that vaccination of sheep with a mixture of F. hepatica cysteine proteinases had no effect on the parasite burden following a challenge infection but did reduce the egg output of the parasites. In this study we have demonstrated that a molecular vaccine against bovine fascioliasis is feasible. The efficacy of one of the vaccine preparations used in this study, cathepsin L2 and Hb, is close to that obtained by some flukicides. A comparative study of triclabendazole and nitroxynil showed that these drugs have efficacies against fluke infection of 96.9 and 76.4%, respectively (22). The reduction of worm burdens and development observed with this vaccine was reflected in a reduction of the severity of liver damage. Of particular significance, however, were the observed anti-embryonation effects. A commercial vaccine capable of inducing an anti-embryonation effect of .98% would undoubtedly have major epidemiological implications since pasture contamination, and hence the transmission of disease, would be negligible. Research toward the testing of recombinant cathepsin Ls and Hb as immunoprophylactic reagents is in progress and will form the basis of a commercial antiparasite and anti-disease transmission vaccine. ACKNOWLEDGMENTS We thank the Teagasc, Grange, Dunsany, Co. Meath, Ireland, for their animal husbandry expertise and Grace Mulcahy, Diane Clery, and Paul Torgenson, Department of Veterinary Microbiology and

5074

DALTON ET AL.

Parasitology, University College, Dublin, Ireland, for some of the postmortem analysis of fluke infection. This work was supported by grants received from Mallinckrodt Veterinary Ltd. and Dublin City University. REFERENCES 1. Anderson, P. H., S. Berrett, P. J. Brush, C. N. Hebert, J. W. Parfitt, and D. S. P. Patterson. 1977. Biochemical indicators of liver injury in calves with experimental fascioliasis. Vet. Rec. 100:43. 2. Andrews, S. J., S. McGonigle, A. M. Smith, J. P. Dalton, D. Clery, and G. Mulcahy. 1995. A bolus for the administration to cattle of metacercariae of the liver fluke Fasciola hepatica. J. Helminthol. 69:1–3. 3. Bjorkman, N., and W. Thorsell. 1963. On the fine morphology of the eggshell globules in the vitelline glands of the liver fluke (Fasciola hepatica). Exp. Cell. Res. 32:153–156. 4. Blaxter, M. L. 1993. Nemoglobins: divergent nematode globin. Parasitol. Today 9:353–360. 5. Boray, J. C. 1969. Experimental fascioliasis in Australia. Adv. Parasitol. 7:95–210. 6. Carmona, C., A. J. Dowd, A. M. Smith, and J. P. Dalton. 1993. Fasciola hepatica: cathepsin L proteinase secreted in vitro prevents the antibody mediated eosinophil attachment to newly excysted juveniles. Mol. Biochem. Parasitol. 62:9–18. 7. Carmona, C., S. McGonigle, A. J. Dowd, A. M. Smith, S. Coughlan, E. McGowan, and J. P. Dalton. 1994. A dipeptidylpeptidase secreted by Fasciola hepatica. Parasitology 109:113–118. 8. Chapman, C. B., and G. F. Mitchell. 1982. Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica. Vet. Parasitol. 11:165–178. 9. Dalton, J. P., and S. J. Andrews. October 1992. Vaccine containing a thiol proteinase. Patent application PCT/GB93/02172. 10. Dalton, J. P., and S. J. Andrews. October 1994. Vaccine containing a haembinding protein. Patent application PCT/GB95/02350. 11. Dalton, J. P., and M. Heffernan. 1989. Thiol proteases released in vitro by Fasciola hepatica. Mol. Biochem. Parasitol. 35:161–166. 12. Dowd, A. J., A. M. Smith, S. McGonigle, and J. P. Dalton. 1994. Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica. Eur. J. Biochem. 223:91–98. 13. Halton, D. W. 1967. Observations on the nutrition of digenetic trematodes. Parasitology 57:639–660. 14. Haroun, E. M., and G. V. Hillyer. 1986. Resistance to fascioliasis: a review. Vet. Parasitol. 20:63–70. 15. Hillyer, G. V., M. S. De Galanes, J. Rodriguez-Perez, J. Bjorland, M. S. De Lagrava, S. R. Guzman, and R. T. Bryan. 1992. Use of the Falcon assay screening test-enzyme-linked assay (Fast-ELISA) and the enzyme-linked immunoelectron transfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian Altiplano. Am. J. Trop. Med. Hyg. 46:603–609. 16. Lee, D. L., and M. H. Smith. 1965. Haemoglobins of parasitic animals. Exp. Parasitol. 16:393–424. 17. Mansour, T. E. 1958. The effect of serotonin on the phenol oxidase from the liver fluke, Fasciola hepatica, and from other sources. Biochem. Biophys. Acta 30:492–500. 18. McGonigle, S., and J. P. Dalton. 1995. Isolation of a Fasciola hepatica

Editor: J. M. Mansfield

INFECT. IMMUN. haemoglobin. Parasitology 111:209–215. 19. McKerrow, J. H., and M. J. Doenhoff. 1988. Schistosome proteases. Parasitol. Today 4:334–340. 20. Ministry of Agriculture, Fisheries and Food. Manual of veterinary parasitological laboratory techniques, reference book 418. United Kingdom Ministry of Agriculture, Fisheries and Food, London. 21. Mitchell, G. F. 1991. Infection characteristics of Schistosoma japonicum in mice and relevance to the assessment of schistosome vaccines. Adv. Parasitol. 30:167–200. 22. Rapic, D., N. Dzakula, D. Sakar, and R. J. Richards. 1988. Comparative efficacy of triclabendazole, nitroxynil and rafoxanide against immature and mature Fasciola hepatica in naturally infected cattle. Vet. Rec. 122:59– 62. 23. Sexton, J. L., A. R. Milner, M. Panaccio, J. Waddington, G. Wijffels, D. Chandler, C. Thompson, L. Wilson, T. W. Spithill, G. F. Mitchell, and N. J. Campbell. 1990. Glutathione S-transferase: novel vaccine against Fasciola hepatica infection in sheep. J. Immunol. 145:3905–3910. 24. Sexton, J. L., M. C. J. Wilce, T. Colin, G. Wijffels, L. Salvatore, S. Feil, M. W. Parker, T. W. Spithill, and C. Morrison. 1994. Vaccination of sheep against Fasciola hepatica with glutathione S-transferase: identification and mapping of antibody epitopes on a three-dimensional model of the antigen. J. Immunol. 152:1861–1872. 25. Smith, A., A. Dowd, C. S. McGonigle, C. Carmona, and J. P. Dalton. 1994. Neutralisation of the activity of a Fasciola hepatica cathepsin L proteinase by anti-cathepsin L antibodies. Parasite Immunol. 16:325–328. 26. Smith, A. M., A. J. Dowd, M. Heffernan, C. D. Robertson, and J. P. Dalton. 1993. Fasciola hepatica: a secreted cathepsin L-like proteinase cleaves host immunoglobulin. Int. J. Parasitol. 23:977–983. 27. Smith, A. M., A. J. Dowd, S. McGonigle, P. S. Keegan, G. Brennan, A. Trudgett, and J. P. Dalton. 1993. Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica. Mol. Biochem. Parasitol. 62:1–8. 28. Spithill, T., and C. A. Morrison. 1995. Molecular vaccines for the control of Fasciola hepatica infection in ruminants. In J. Boray (ed.), Immunology, pathophysiology and control of fascioliasis. MSD AgVET, Rahway, N.J. 29. Tielens, A. G. M., J. M. Van Den Heuvel, and S. G. Van Den Bergh. 1982. Changes in energy of the juvenile Fasciola hepatica during its development in the liver parenchyma. Mol. Biochem. Parasitol. 6:277–286. 30. Tielens, A. G. M., J. M. Van Den Heuvel, and S. G. Van Den Bergh. 1984. The energy metabolism of Fasciola hepatica during its development in the final host. Mol. Biochem. Parasitol. 13:301–307. 31. Tielens, A. G. M., P. Van Der Meer, and S. G. Van Den Bergh. 1981. The aerobic energy metabolism of the juvenile Fasciola hepatica. Mol. Biochem. Parasitol. 3:205–214. 31a.Trudgett, A., and J. P. Dalton. Unpublished data. 32. Wijffels, G. L., L. Salvatore, M. Dosen, J. Waddington, L. Wilson, C. Thompson, N. Campbell, J. Sexton, J. Wicker, F. Bowen, T. Friedel, and T. W. Spithill. 1994. Vaccination of sheep with purified cysteine proteinases of Fasciola hepatica decreases worm fecundity. Exp. Parasitol. 78:132–148. 33. Xu, C. B., C. Verwaerde, J. M. Grych, J. Fontaine, and A. Capron. 1991. A monoclonal antibody blocking the Schistosoma mansoni 28-kDa glutathione S-transferase activity reduces female worm fecundity and egg viability. Eur. J. Immunol. 21:1801–1807.