ELSEVIER Veterinary Parasitology 56 (1995) 57-66

veterinary parasitology

Detection of excretory-secretory circulating antigens in sheep infected with Fasciola hepatica and with

Schistosoma mansoni and F. hepatica

Jos6 Rodriguez-P6rez l, George V. Hillyer* Laboratory of Parasite Immunology and Pathology, Department of Pathology and Laboratory Medicine, University of Puerto Rico School of Medicine, San Juan 00936-5067, Puerto Rico

Accepted 22 February 1994

Abstract

Two groups of sheep were exposed to Fasciola hepatica and Schistosoma mansoni/F. hepatica infections, respectively, to determine the time of appearance of F. hepatica cir- culating excretory-secretory (FhES) antigens. Five sheep were infected orally with 400 F. hepatica metacercariae. Five additional sheep were infected first percutaneously with 5000 S. mansoni cercariae, and l0 weeks later orally with 400 F. hepatica metacercariae. Anti- gen detection was performed in the two groups by a double antibody microELISA. In the group infected with F. hepatica, circulating FhES antigens were detected in all five ani- mals. In the group infected first with S. mansoni none of the sera had positive optical density values for FhES antigens throughout the 10 week period following infection. After F. hepatica challenge, FhES antigens were detected in all animals. Thus, sheep infected with S. mansoni did not develop detectable F. hepatica circulating antigens during the 10 week period following Schistosoma infection. Anti-FhES antibodies were detected by FAST- ELISA, and all animals infected with F. hepatica were positive. In the group infected with S. mansoni, cross-reactive antibodies to F. hepatica were detected by 4-6 weeks post infec- tion. All sheep with double infections showed high antibody levels by 2 weeks after infec- tion with F. hepatica.

Keywords: Fasciola hepatica; Schistosoma mansoni; Sheep-Trematoda; Immune response-Trematoda

* Corresponding author. Tel.: (809) 756-7700. Fax: (809) 751-9210. Present address: Abbott Diagnostics Division, P.O. Box 278, Barceloneta 00617, Puerto Rico.

0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0304-4017 (94) 00666-Z

58 J. Rodrlguez-P~rez, G. V. Hillyer / Veterinary Parasitology 56 (1995) 57-66

1. Introduction

Fasciola hepatica is a common parasite of domestic livestock (cattle, sheep and goats) and causes severe disease and important economic losses in the animal husbandry industry. An important feature of this infection is that the pathology and disease occur as early as 3 weeks after infection, yet parasitologic diagnosis can only be confirmed by coprology at 2 months post infection (p.i.) in cattle (Hillyer et al., 1985) or 3 months p.i. in sheep (Zimmerman et al., 1982). For this reason, detection of faseioliosis early in infection, using antibody detection tests, makes immunodiagnosis an important adjunct for determining infection status and to prevent economic loss. These immunological tests have recently focused primarily on antibody detection by ELISA (Hillyer et al., 1985; Zimmer- man et al., 1985; Santiago and Hillyer, 1988; Hillyer and Soler de Galanes, 1991 ). Although ELISA-based antibody detection systems are excellent for the early de- termination of infection with F. hepatica in cattle and sheep, persistence of anti- body for long periods after cure makes it unsuitable for the prediction of success of chemotherapy.

The current study was designed to determine the appearance of circulating ex- cretion-secretion F. hepatica antigens in sheep with fascioliosis. Moreover, the effect of primary infection with Schistosoma mansoni in sheep, followed by infec- tion with F. hepatica, on the appearance of circulating antigens was also studied.

2. Materials and methods

2.1. F. hepatica metacercariae

F. hepatica metacercariae were purchased from Baldwin Aquatics, Inc. (Mon- mouth, OR, USA).

2.2. Animal infections and sera

Five 3-month-old lambs were infected orally with 400 F. hepatica metacercar- iae. Five additional lambs were infected first percutaneously with 5000 S. man- soni cercariae, and 10 weeks later orally with 400 F. hepatica metacercariae. The sheep were bled every 2 weeks from the jugular vein, and all sera were stored at - 7 0 ° C until used. Animals were necropsied 10 weeks after being infected with F. hepatica, and adult flukes were recovered from the bile ducts and liver and counted (Haroun and Hillyer, 1988). All of the sheep involved in this study were healthy and from a farm that had no history of fascioliosis. Monieza expansa eggs were found in the feces of some lambs and thiabendazole was given for treatment before the experiment commenced. Afterwards they were kept in tiled enclosures, and maintained on concentrate feed with free access to water (Haroun and HiUyer, 1988).

J. Rodriguez-Pbrez, G. V. Hillyer / Veterinary Parasitology 56 (I 995) 5 7-66 59

2.3. Antigen preparation

F. hepatica excretory-secretory antigens (FbES) were prepared as follows. Living intact adult flukes were obtained from bovine livers at a local slaughter- house and rinsed with phosphate-buffered saline (PBS), pH 7.2, to remove all traces of blood and bile. Flukes were incubated for 3 h in PBS containing 1 mM phenylmethyl sulfonyl fluoride (PMSF) at 37°C. The medium was collected, concentrated 20-fold by polyethylene glycol 15 000 mw and centrifuged at 20 000g for 20 min at 4 ° C. After protein measurement (Bradford, 1976 ), the supernatant was stored in aliquots at - 7 0 °C until used (Hillyer, 1993 ).

2.4. Antibody preparation

Polyclonal anti-FhES sera were developed in New Zealand white rabbits hyper- immunized subcutaneously with FhES. The initial immunization ( 100/zg FLIES) was in Freund's complete adjuvant and all subsequent ones were in Freund's in- complete adjuvant. Anti-FhES globulins were obtained by precipitation with 50% ammonium sulfate and then dialyzed exhaustively against 10 mM PBS (pH 7.2). Anti-F. hepatica sheep globulins containing IgG were obtained in the same way from sheep infected orally with F. hepatica for 10 weeks. After protein measure- ment, rabbit and sheep immunoglobulins were aliquoted and stored at - 7 0 ° C until used.

2.5. Antigen detection by a double antibody ELISA

The rabbit anti-FhES globulins were plated in wells (2.5 gg ml -~ in 10 mM PBS, pH 7.2, 100 #1 per well) at 4°C and incubated overnight. Plates were then washed three times each with 10 mM PBS containing 0.05% Tween 20 (PBST) for 5 rain. Blocking of excess-binding sites was performed by incubation with 200 #1 of PBST containing 4% bovine serum albumin (BSA) for 1 h at 37°C. After a new wash cycle as above, 100/A of each serum was added undiluted and incu- bated for 3 h at 37°C. All sera were tested in triplicate. Plates were then washed as above and 0.25 gg of anti-Fh sheep globulins in 100 gl of PBST containing 4% BSA were added to each well and incubated for 1 h at 37°C. Plates were then washed as above and 0.25 gg of anti-Fh sheep globulins in 100 gl of PBST con- taining 4% BSA were added to each well and incubated for 1 h at 37°C. After washing, plates were then washed and 100 gl of peroxidase-conjugated anti-sheep IgG (KPL, Gaithersburg, MD, USA) were added to each well ( 1 / 1500 dilution in 4% BSA-PBST) and incubated for 1 h at 37°C. Finally, 100 gl of substrate solution consisting of 10 mg of ortho-phenylenediamine, 1 ml of methanol, 99 ml of distilled water and 10/d of 30% hydrogen peroxide were added to each well and the plate was incubated in the dark for 30 min at room temperature. The color reaction was stopped with the addition of 50 gl of 4 N sulfuric acid, and absorbances were read at 490 nm using a Bio-Rad Microplate reader Model 3550

60 J. Rodriguez-P~rez, G. V. Hillyer / Veterinary Parasitology 56 (1995) 57-66

(Bio-Rad Laboratories, Life Sciences Group, Hercules, CA, USA). The result of each serum was expressed as the mean of the optical density (OD) reading of triplicate wells. The negative control OD values were the preinfection serum of each animal as well as a pool of the preinfection sera. A positive was defined as the mean OD value of the negative control sera plus three standard deviations (SD) of that mean (Hillyer et al., 1992).

2.6. Antibody detection by the FAST-ELISA

The Falcon assay screening test-enzyme-linked immunosorbent assay (FAST- ELISA) was performed as described by Hancock and Tsang (1986) for the de- tection of anti-S, mansoni antibodies, with modifications for fascioliosis using FhES antigens (Hillyer and Soler de Galanes, 1988, 1991 ). FhES was used at 4 /zg ml-l; horseradish peroxidase-labeled rabbit anti-sheep IgG (KPL, Gaithers- burg, Maryland) was used as conjugate antibody at a 1:300 dilution. Absor- bances were read at 655 nm using the Bio-Rad Microplate reader. A pool of nor- mal, preinfection sera was used as a negative control. A positive OD was determined as above (Hillyer, et al., 1992 ).

2. 7. Statistical analysis

Results were analyzed statistically using the Student's t-test.

3. Results

3. I. F. hepatica worm burden recoveries

Sheep infected with F. hepatica alone had a mean of 104 F. hepatica worms recovered from the liver and gall bladder of each animal. Those first infected with S. mansoni and then with a similar number of F. hepatica metacercariae to the above group had almost half (n= 51 ) the F. hepatica worm burden recovery of the singly infected group. No S. mansoni worms were recovered from this second group (at 20 weeks of S. mansoni infection), although there had been evidence of infection, as S. mansoni eggs were detected in feces by 9-11 weeks of S. man- soni infection (Haroun and Hillyer, 1988 ).

3.2. Antigen detection

Positive values were the mean OD of all preinfection sera (x= 0.261 ) plus three standard deviations. Mean preinfection, normal serum OD values were 0.261 with a standard deviation of 0.045. Thus, positive F. hepatica ES antigen values were 0.396 or higher with a confidence interval of 99%.

In the group of sheep which were infected only with F. hepatica, circulating FhES antigens were detected in all five animals. The mean values of all sheep per week of infection are shown in Fig. 1. In three sheep, antigens were detected as

J. Rodriguez-P~rez, G. F.. Hillyer / Veterinary Parasitology 56 (1995) 5 7-66 61

E C 0 O~

t~ J~

0 . 7 '

0.6 ¸

0.5 ~

0.4-

0.3

0.2

0.1

0.0 1'o

Weeks of Infection Fig. 1. Double antibody enzyme-linked immunosorbent assay (ELISA) of sera from sheep infected with Fasciola hepatica. OD value for each date is the mean of the five individual sera. Standard error bars are shown.

E C 0

¢/1 en <¢

S.

0.5

0.4

0.3'~

0.2"

0.1

0.0 2 4 6 8 1'0 1'2 14 1'6 1'8 2'0

t Weeks F. het~atlca Infection mansoni Infection

Fig. 2. Double antibody enzyme-linked immunosorbent assay (ELISA) of sera from sheep infected with Fasciola hepatica and from sheep infected with Schistosoma mansoni and challenged with F. hepatica. OD value for each date is the mean of the five individual sera. Standard error bars are shown.

early as 2 weeks p.i., whereas in the remaining two sheep, F. hepatica ES antigens were first detected at 6 or 8 weeks p.i. Maximum detection occurred in four ani- mals at 8 weeks p.i.

In the group infected with S. mansoni, none of the sera had positive OD values for FILES circulating antigens through the 10 weeks following infection (Fig. 2 ). When these animals were challenged with F. hepatica, FhES antigens were de- tected in one lamb 2 weeks later ( 12-20 weeks after the primary S. mansoni in- fection), 4 weeks after challenge in three animals, and at 8 weeks after F. hepatica challenge in the fifth animal. The mean values of all sheep per week of infection

62 J. Rodrlguez-P~rez, G. V. Hillyer / Veterinary Parasitology 56 (1995) 57-66

are shown in Fig. 2. The mean OD of each post challenge (p.ch.) dates were significantly different from normal sera mean (Week 0) and from pre-challenge dates (Week 10) (P< 0.01 and P< 0.05, respectively). No statistical differences were found when comparing the different p.i. or p.ch. dates between them. More- over, comparing the group infected with F. hepatica (2-10 weeks) with the dou- bly infected group ( 12-20 weeks), no statistical differences were found between the two. Thus, sheep infected with S. mansoni did not develop detectable F. he- patica ES circulating antigens during the l0 weeks of Schistosoma infection tested.

3.3. Antibody detection

Positive values were the mean OD of all preinfection sera plus three standard deviations. Mean preinfection, normal serum OD values were x=0.340 with a standard deviation of 0.092. Thus, positive anti-F, hepatica antibody values were 0.616 or higher.

Using the FAST-ELISA, all animals infected with F. hepatica showed evidence ofanti-FhES antibodies in all p.i. dates examined (Fig. 3 ). The mean OD at each p.i. date was significantly different from normal sera mean (P< 0.01 ). No statis- tical differences were found in mean ODs at all p.i. dates examined.

In the group infected first with S. mansoni, four of five sheep had low but dearly detectable cross-reactive antibodies to F. hepatica above the decided positive level by 4-6 weeks p.i. Only one lamb with Schistosoma infection did not have positive values until after F. hepatica challenge. All sheep with double infections showed high antibody levels to F. hepatica by Week 2 after homologous infection. (Fig. 4). The mean OD at each time point was compared with the mean of normal sera. The mean OD at Weeks 4, 6, 8 and l 0 p.i. were significantly different from

E

I,o ¢,D

(n

,<

1.50

1.25

1.00

0.75 -

0 .50"

i

0.25-

0.00

Weeks

Fig. 3. Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) of sera from sheep infected with Fasciola hepatica. OD value for each date is the mean of the five individual sera. Standard error bars are shown.

J. Rodriguez-Pbrez, G. V. Hillyer / Veterinary Parasitology 56 (1995) 57-66 63

2.5'

2.0'

E ~" 1.5

1.0

0.5

0.0 0 2 4 6 8 10 12 14 16 18 20

t Weeks F- henat lca I n fec t i on

S. manson i In fect ion

Fig. 4. Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) of sera from sheep infected with Fasciola hepatica and from sheep infected with Schistosoma mansoni and chal- lenged with F. hepatica. OD value for each date is the mean of the five individual sera. Standard error bars are shown.

normal sera (P< 0.05 ). All weeks after F. hepatica challenge (after 10 weeks of S. mansoni infection) were also statistically different from normal sera (P< 0.01 ). Comparison of the anti-FhES antibody levels in the two groups showed that at the tenth week (before F. hepatica challenge), sheep infected with S. rnansoni had significantly higher mean values than normal serum values. OD values at Weeks 16, 18 and 20 p.i. (6, 8 and 10 weeks p.ch., respectively) were statistically greater (P< 0.01 ) than those measured in the F. hepatica infected group at the same time points. Mean OD values obtained at Weeks 12 and 14 p.i. (2 and 4 weeks p.ch. ) did not differ statistically from those found in the F. hepatica group.

4. Discussion

Immunological diagnosis of parasitic infections has focused on the direct de- tection of parasite circulating antigens and/or immune complexes (reviewed by Langley and Hillyer (1989a) ). In fascioliosis, efforts have been directed towards the detection of these substances in naturally infected sheep and goats and in experimentally infected rabbits (Peinado-Pelaez et al., 1988 ), mice (Langley and Hillyer, 1989a), cattle (Langley and Hillyer, 1989b), and humans (Espino et al., 1990). Antigen detection has the advantage over antibody-based tests in that an- tigenemia implies current infection (Hillyer, 1993). Also, measurement of cir- culating antigens as a parameter of active infection may be of potential use in the management of clinical disease. One of the main characteristics that a diagnostic

64 J. Rodrlguez-P~rez, G. V. Hillyer / Veterinary Parasitology 56 (1995) 57-66

test should possess, especially if it is to be used for human infection, is early de- tection of infection. This is one of the main advantages of antigen immunodi- agnostic detection tests over antibody tests. In parasitic infections, particularly those involving tissue parasites, early therapeutic treatments are usually most successful, as once parasites become established in the target organs or viscera, it is much harder to eradicate them.

In the present study, detection of circulating FhES antigens in the sera of F. hepatica-infected sheep showed that the highest absorbances values were reached at 8 weeks p.i. The group with S. mansoni-F, hepatica double infection exhibited exactly the same pattern of circulating antigen, with the highest OD values at 8 weeks after F. hepatica challenge ( 18 weeks after primary S. mansoni infection).

It is noteworthy that OD values for circulating FhES antigens in the group in- fected with F. hepatica alone were slightly higher than those found in the doubly infected group, although the difference was not statistically significant. A de- crease in FhES antigen levels could be explained because in those double infected animals there were antibodies cross-reactive to FhES from previous S. mansoni exposure. The phenomenon of antigen forming immune complexes probably oc- curred as soon as first antigens began circulating. Decreasing FhES circulating antigen levels 10 weeks after F. hepatica infection or challenge were observed although the decrease was not significant. Langley and Hillyer (1989b) obtained a similar pattern when they studied the presence of immune complexes in F. he- patica-infected cattle. In the single infection group, FhES antigens decreased but antibodies reached maximum levels, after a period coincident with the end of fascioliosis prepatent periods in Puerto Rico (De Leon et al., 1981 ), and once parasite establishment in the bile ducts occurs, relatively less antigen may then be in circulation.

Sheep are susceptible to natural infection with F. hepatica and have been pro- posed as susceptible hosts to reinfection with F. hepatica. This is because the evidence suggests that primary sensitization of sheep with a primary F. hepatica infection fails to stimulate any resistance to a second challenge infection in terms of worm burden reduction after challenge (Haroun and Hillyer, 1986 ). In con- trast, there is ample evidence for cross-resistance between Schistosorna and Fas- ciola using several hosts, including both experimental animals (Hillyer, 1976, 1981; Christensen et al., 1978, 1980) and farm animals (Monrad et al., 1981; Haroun and Hillyer, 1988). In these studies a primary infection with one of these trematodes resulted in significant worm burden reduction with the other chal- lenge trematode infection (reviewed by Hillyer, 1984; Haroun and Hillyer, 1986).

Santiago and Hillyer ( 1988 ) studied the antibody reactivity patterns of sheep with primary fascioliosis vs. sheep infected with S. mansoni and then challenged with F. hepatica by enzyme-linked immunoelectrotransfer blot (EITB). They found that polypeptide patterns were almost identical in both groups, which im- plies no qualitative difference between the two groups.

In the present study, a quantitative technique such as ELISA clearly showed that antibody levels to FhES in the sheep infected first with S. mansoni and then challenged with F. hepatica were higher than those infected singly with F. hepa- tica. Thus, although the single S. mansoni infection induced slight, but signifi-

J. Rodriguez-P~rez, G. V. Hillyer / Veterinary Parasitology 56 (1995) 57-66 65

cant, levels of antibodies which cross-reacted with FhES antigens, the group dou- ble infected (S. mansoni followed by F. hepatica) had steadily increasing antibody levels to FhES through the 10 weeks of F. hepatica infection tested. This suggests that the S. mansoni infection induced an anamnestic response to common epi- topes shared between the two genera of trematodes. This is reinforced by the fact that the group with the highest antibody levels to FhES, that infected with S. mansoni and challenged with F. hepatica, had half the F. hepatica worm burden of the group with a single trematode F. hepatica infection. In single species rein- fections in fascioliosis in sheep, suppression of the humoral (Sandeman and Howell, 1981 ) and lymphocytic (Zimmerman et al., 1983) immune response have been reported. This is not seen in the double infections of the present study.

This cross-reactivity makes the obtaining of a specific immunodiagnosis more difficult when using antibody detection tests. However, F. hepatica antigen detec- tion appeared to be specific because in the doubly infected animals, the FhES antigen detection results were not affected by primary Schistosoma infection. This was supported by the finding of similar FhES antigen profiles in both groups of sheep infected either singly with F. hepatica or doubly with S. mansoni and F. hepatica.

Acknowledgments

J.R.-P. received support from Ministerio de Educaci6n y Ciencia, Madrid, Spain. This work was supported by the National Science Foundation Advanced Development Program, Grant EHR-9108775.

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