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In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica
International Journal for Parasitology.
1977.Vol.
7. pp. 367-371. Pergarnon Press. Printed in Great Britain.
IN VIP’0 AND IN VITRO STUDIES OF THE EFFECTS OF IMMUNE RAT SERUM ON FASCZOLA HEZ’ATZCA M. J. HOWELL, R. M. SANDEMANand G. R. RAJASEKARIAH Department
of Zoology, Australian National University, Box PO 4, Canberra, ACT 2600, Australia (Received
21 December
1976)
Abstract-HowELL M. J., SANDEMANR. M. and RAJASEKARIAH G. R. 1977. In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica. International Journal for Parasitology 7: 367-371. Significant protection against infection with 10 or 30 metacercariae of Fusciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components,presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established. INDEX KEY WORDS: Fasciola hepatica; metacercariae; immunity; passive transfer; intraperitoneal infection.
INTRODUCTION IT
shown that a primary infection of in rats confers appreciable resistance to a challenge infection (Hayes, Bailer & Mitrovic, 1972, 1973, 1974~; Goose & McGregor, 1973; Armour & Dargie, 1974). Immunological factors appear to be involved in this phenomenon since resistance can be passively transferred to naive recipients by serum and lymphoid cells from infected rats (Corba, Armour, Roberts & Urquhart, 1971; Hayes, Bailer & Mitrovic, 19746, c; Armour & Dargie, 1974). The present report describes infection schedules for the production of serum in DA and Wistar rats which passively protects naive recipients against infection with Fasciola hepatica. In addition, preliminary findings concerning the in vitro effects of this serum on metacercariae are outlined. HAS
been
Fkciola
hepatica
MATERIALS
AND METHODS
Rats. Two strains of rats were used: male, outbred Wistar rats and male, inbred DA rats. These were obtained from the John Curtin School of Medical Research, ANU. All animals were given food and water adlib. Parasites.
Fasciola hepatica metacercariae were obtained from a laboratory colony of Lymnuea tomentosa which had been infected with miracidia derived from
367
rats; immune serum;
in vitro culture;
eggs recovered from the gall bladders of infected sheep. They were kept at 4°C until required. Rats were infected with viable metacercariae and later autopsied as previously described (Rajasekariah & Howell, 1977). Production of serum. Three batches of serum were prepared for passive transfer experiments. Batch A was derived from DA rats which had been given a primary infection of 5 metacercariae at 5 weeks of age, challenged 7 weeks later with 30 metacercariae, and exsanguinated 8 weeks later. Batch B was derived from Wistar rats which were the same age and which had been exposed to the same levels of infection as the DA rats; they were exsanguinated 1 week after challenge. Batch C was also derived from Wistar rats as for Batch B, but the levels of primary and challenge infection were 10 and 20 metacercariae respectively. Normal serum was obtained from uninfected rats of the same strain and age as infected rats. Blood was allowed to clot and centrifuged at 4000 rev./min for 30 min; the serum was drawn off, sterilized by Seitz filtration, and stored at -20°C until required. Passive transfer experiments. Three experiments were performed, each with test and control groups of 6, 5 weeks old, male rats as recipients. In experiment 1, DA rats and Batch A serum (see above) were used. Each recipient received 10 ml of normal or immune serum intraperitoneally immediately before and after infection with 30 metacercariae. In experiment 2, Wistar rats and Batch B serum were used, following the same protocol as experiment 1. In experiment 3, Batch C serum was used and Wistar rats were given 5 ml intraperitoneally immediately following infection with 10 metacercariae.
M. J. HOWELL, R. M. SANDEMAN and G. R. RAJASEKARIAH
368
Experiment 1 also contained a group of 6 rats, each of which was infected with 30 metacercariae but not given serum. All rats were examined for adult worms 7 weeks after infection. In vitro cultures. Metacercariae were excysted by pepsin (3 x crystalline; Calbiochem) and trypsin (2 ’ crystalline, salt free; Nutritional Biochemicals) plus bile treatment as described by Wikerhauser (1961). The bile was obtained from uninfected sheep at Canberra abbatoir. Excysted metacercariae were rinsed several times in Medium 199 (Commonwealth Serum Laboratories) containing 100 units/ml penicillin, 100 ug/ml streptomycin and 2.5 pg/ml amphotericin B (E. R. Squibb & Sons), and then transferred in groups of about 50 to sterile Leighton tubes containing 5 ml of culture medium. The culture medium contained 20”/, normal or immune serum (Batch C, see Results) in Medium 199 plus antibiotics. The pH was maintained at 7.2 - 7.4 by a gas phase of 5% CO, in air (Commonwealth Industrial Gases). Cultures were incubated at 37’C in a water bath and agitated by means of a shaker operating at 120 agitationsimin. The medium was not changed during the period of culture. Fluorescent antibody (FAB) test. A direct FAB test was carried out on metacercariae cultured in immune and normal sera using fluorescein isothiocyanate labelled rabbit anti-rat immunoglobulin (Burroughs Wellcome). Metacercariae were gently washed 3 times with phosphate buffered saline (PBS), exposed for 30 min to the labelled anti-rat immunoglobulin diluted I :16, then washed 3 times with PBS. Examination was carried out under a Leitz U.V. microscope equipped with a BG12 exciter filter and blue absorbent barrier filters. Metacercariae which had not been exposed to labelled conjugate were also examined. Viability tests. Live mctacercariae which had been cultured for 36 h in immune or normal serum were washed 3 times in PBS. Ten mctacercariae were then injected intraperitoneally into each member of groups of 6. 5 weeks old, male Wistar rats using an ISG needle connected to a syringe by a I5 cm length of polythene tubing. A control, in which freshly excysted metacercariae were injected into a group of 6 rats, was also carried out. The rats were examined for adult worms 7 weeks later.
Immunogenicity
of itrxune
serum precipitate to rats.
When metacercariae are cultured in immune serum a precipitate forms on their teguments and in the culture medium (see Results). The immunogenicity of this precipitate was examined by injecting it into rats and testing the effects of sera from these animals on metacercariae in vitro. It was postulated that this procedure would give rise to an antiserum which would react with metacercariae in a similar manner to immune serum. The amount of precipitate used for injection was not quantified in terms of protein concentration, but for both the initial and booster injections it was derived from one culture containing 50 metacercariae which had been incubated in immune serum for 10 days. The metacercariae were rinsed thoroughly and discarded: the precipitate was then separated from the culture medium and rinsings by centrifugation. The pellet was washed 3 times with PBS, resuspended in I.0 ml PBS and then emulsified with 1.0 ml of Freund’s complete adjuvant (FCA, Difco). Two, 5 weeks old, male Wistar rats each received I ml of this emulsion: 0.2 ml intramuscularly
I.J.P. VC!I
7. 1977
and 0.8 ml intrapcritoneally. A control rat received an emulsion of 0.5 ml PBS plus 0.5 ml FCA. Five weeks later all rats received booster injections following the same procedures as described. One week later the rats wcrc exsanguinated and serum prepared.
RESULTS
The
results
of
passive
transfer
Fasciola hepatica infection I. The mean
serum
donors
(+s.D.)
experiment
used
worm
for
burden
_ I .3S,
2, 3.0
experiments
experiment
of 2.7
with
in Table
in rats are shown
1 had
i 0.2,
a
for
those
and those for experiment
3,
3.4 + 0.7.
TABLE I-Fasciola hepatica IN RATS: WORM RECOVERIES FOLLOWING INTRAPERITONEAL ADMINISTRATION OF SERUM IMMEDIATELY PRIOR TO AND/OR FOLLOWING INFECTION WITH 30 METACERCARIAE (EXPERIMENTS I & 2) OR IO METACERCARIAE (EXPERIMENT 3) Mean worm
Experiment
Immune serum recipients
recoveries Normal serum recipients
4.2 ! 29 3.6 t 1.7” I.6 I 0.8“
9.2 ! 3.5” 8.5 f 2.1” 3.4 _ 1.1”
1 2 3
In each experiment a b. P 0.05 In experiment I, b not significantly different from c. P 0.1. The
serum
doses
of
experienced tion;
donorc
primary
different
(see
in
experiments
significant with
were
Materials
the
degree
of
and
control
(experiment in worm Jn vitro
donor
groups
serum) was
no
of
conferred
a
challenge
1 and
(experiments
there
volumes
against
(no
infec-
Nevertheless.
serum
3). When
culturing
immune
from
rats
for
serum
used
a precipitate
about
2) or
normal
10
serum
were
compared
significant
difference
oral
sucker
to develop
and
excretory
were
appeared
also
to be derived
tate by dislodgment, result
of
of
parasite
which
medium.
An
which
on
free
from
although
a separate
immune were attempt
serum
Small
reaction
have
precipi-
between into
the to
of This
developed
secretions
made
of
of the
amounts
the tegument
not
3 199.
the tegument
it may
discharged was
experiment
in the medium.
and
of
derived
in Medium
in the vicinity
pore.
found
was
for
donors
particularly
precipate
ponents
C,
to be protective)
began
metacercaria,
I2 h in the presence
(Batch
as serum
and shown
each
a
different
Methods).
and
to
recovery.
20”/,
as
exposure
experiments
After
above
of
(experiment I),
varying
metacercariae
protection
30 metacercariae
metacercariae
I I.0 \ 3.3‘
administered
given
and
Control (no serum)
Mann-Whitney U-test.
challenge periods
the recipients
serum all
were
and
(is.0.)
comof
the
culture
distinguish
I J.P.
VOL.
7. 1977
Immune
rat strum
and F. /wpctica
FIG. 1. Precipitate on the tegument of a metacercaria cultured in immune rat serum for 36 h. Note that some precipitate is only loosely attached and the tegument appears to be intact. FIG. 2. Metacercariae cultured in normal rat serum for 36 h. Note the absence of precipitate on the tegument. FIG. 3. Metacercariae cultured in immune rat serum for 36 h and then exposed to FITC labelled rabbit anti-rat immunoglobulin and examined under U.V. The precipitate on the tegument and in the medium is strongly fluorescent, indicative of the presence of rat antibody. (Scale lines for Figs. l-3 - @I mm,)
3 70
M. J.
HOWELL, R.
M. SANDEMAN and G. R.
between these possibilities; for the remainder of this report, these precipitates will be regarded as having the same origin. After 36 h, the precipitate on each metacercaria was more extensive, covering most of the tegument (Fig. 1); larger amounts were also present in the culture medium. The tegument appeared to remain intact. Subsequently, no marked changes were evident although inactive metacercariae had more precipitate adhering to them than active metacercariae. Similar effects to those described were observed when immune serum was heated to 56’C for 30 min prior to being added to the culture medium. No precipitate formed on metacercariae or in culture media in the presence of intact normal serum or normal serum which had been heated to 56 ‘C for 30 min (Fig. 2). Mortality
qf metaccrcariae
This was assessed by their inactivity and opaque appearance. Pooled results from 3 cultures show an apparently higher mortality rate over the first 2 days for metacercariae in immune serum (Fig. 4). After about 60 h, mortality rates in both normal and immune serum are similar. Most metacercariae died within IO days in Iaitro, irrespective of the type of serum present.
cercariae paired. TABLE
RAJASEKARIAH
incubated
2-Fasciola
FOLLOWING
I.J P. Vol..
in normal
hepatica
INTRAPERITONEAL
IN
serum
RATS:
was also im-
WORM
INJECTION
7. 1917
OF
RECOV~KY 10
META-
CERCARIAE Mean
Source
Freshly excysted Cultured in normal serum for 36 h Cultured in immune serum for 36 h a h a
worm
recovery ( i
of metacercariae
s.D.)
4.5 1 0.8” 2.8 + 2.7” 0’
c, P -: 0.005 (‘. P G’ 0.05 h, P ~ 0.05
Composition
1
Mann-Whitney
U-test.
J
qf’ the immune
serum precipitate
It was considered
that the precipitate which formed in immune serum consisted of one or more rat antibody-parasite antigen complexes. The presence of antibody was confirmed by treating metacercariae with FITC labelled rabbit anti-rat immunoglobulin. The precipitate on the tegument fluoresced strongly (Fig. 3), as did that free in the medium. No fluorescence was observed on metacercariae cultured in normal serum or on specimens which had not been treated with the labelled conjugate. Confirmation that the immune serum precipitate was immunogenic to rats was obtained as follows: serum from rats which had been injected with an emulsion of precipitate and FCA reacted with metacercariae in vitro in a similar manner to immune serum. However, the precipitate which formed both on metacercariae and in the culture medium was relatively more copious. The direct FAB test showed that rat antibody was associated with this precipitate. No precipitate formed in cultures containing serum from rats which received an emulsion of FCA and PBS. DISCUSSION
FIG. 4. Mortality rates for metacercariae cultured in vitro in the presence of normal (A) and immune ( 0) rat serum. The curves for normal (-_) and immune (-) serum have been fitted by inspection. Viability of cultured
metacercariae
Table 2 shows adult worm recoveries from the bile ducts of rats 7 weeks after intraperitoneal injection of freshly excysted metacercariae and metacercariae cultured for 36 h in either normal or immune serum. Exposure of metacercariae to immune serum prior to injection into rats resulted in a loss of viability. No liver lesions were detected in rats injected with these metacercariae. By comparison with freshly excysted metacercariae, the viability of some meta-
The passive transfer experiments showed that a significant degree of protection against F. hepatica infection in naive rats was conferred by serum from infected animals. These results are in agreement with previous work (Hayes et al,, 19746, c; Armour & Dargie, 1974). Such experiments can be taken as evidence for the view that resistance to challenge is mediated, at least in part, by serum antibody (Sinclair, 1970; Kelly, 1973). Lang (1974) demonstrated that a 4 h incubation of partly matured F. hepatica in immune mouse serum significantly reduced the ability of the worms to re-establish in uninfected mice. This points to the involvement of antibody in resistance in these hosts as well. Serum was taken from infected donors when they were 13-20 weeks old. At this age, the rats would have developed a degree of age resistance (Rajasekariah & Howell, 1977). Since there was no difference in worm recovery between rats which did
Immune rat serum and F. hepaf~ea
I.J.P. VOL. 7. 1977
not receive serum and those which received normal serum (experiment I, Table I) it is concluded that the factors involved in age resistance arc not apparently transmitted in serum. The results of in V&U studies of the effects of immune rat serum on metacercariae were simiIar to those of Wikerhauser (1961) who used serum from cattle, rabbits, horses and humans. Thus, precipitate formation on metacercariae occurred in the presence of serum from infected animals, and this effect was still observed if the serum had been heated to 56°C for 30 min. The present study showed that the ability of metacercariae to undergo development in vivo was completely impaired by prior exposure to immune rat serum. The viability of some meta~er~ariae exposed to normal serum was also impaired; this points to inadequacies in the culture technique. The results of similar tests carried out by Wikerhauser (1961 f, using metacercariae incubated in infected cattle serum and then injected into guinea pigs, were apparently less striking. However, since no details of worm recovery were given and the cultures were contaminated with microorganisms, the significance of these results cannot be gauged. Moreover, the protective capacity of the infected cattle serum was not established with any degree of certainty. This is essential if in vitro effects are to bear any relationship to protection in vim. Metacercariae of F. ~e~~~~ca survived longer in immune rat serum than schistosumula of Schistocoma mansoni in immune monkey serum (Clegg & Smithers, 1972). This suggests that either protective activity in the serum was low or inactivated within a short period of culture, or that the co-operation of cells is required for the full effects of antibody to be realised. Frequent changes of the culture medium, raising the concentration of immune serum in the medium and the addition of cell populations to cultures are ways in which these possibilities could be tested. The precipitate which forms on metacercariae (and in the culture medium) in the presence of immune rat serum was shown to contain both antibody and immunogenic (presumably of parasite origin) components. However, whether it represents one or moreantibody/antigen complexesand whether non-specific serum factors are also involved in its formation has yet to be clarified. Neither complement nor reaginic antibodies are apparently responsible since these factors are heat labile (Davis, Dulbecco, Eisen, Ginsberg & Wood, 1967) and precipit&e formation takes place in their absence. An impo~ant question yet to be resolved is whether the precipitate is a manifestation of protective antibody-functional
antigen interaction,
and thus respon-
371
sible for impairing
the viability of metacercariae. It may well be that the deleterious effects of immure rat serum on metacercariae are less obvious. This, and other questions raised above, are currently under investigation. REFERENCES ARMOURJ. & DARGIEJ. R. 1974. Imn~unity to Fusci~la hepafica in the rat. Successful transfer of immunity by lymphoid cells and by serum. Experimental farasitology 35: 381-388.
CLEGG J. A. & SMITHERSS. R. 1972. The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni international Jorrrnalfbr
during
cultivation
in vitro.
Parasitology 2: 79-98.
C~RBAJ., ARMOURJ., ROBERTSR. J. & URQUHART G. M. 1971. Transfer of immunity to Fasriola heparica infection by lymphoid cells. Research in Veterinary Science 12: 292-295.
DAVIS B. D., DULBECCOR.. EISEN H. N.. GINSBERG H. S. & WOOU W. B. 1967. Microbioloiy. Hoeber Medical Division. Haruer & Row. New York. GOOSE J. & MACGREGOR M. 1973: Naturally acquired immunity to Fcisciofa hepariea in the rat. Brit!.sh Veterinary Journal 129: xlix-Iii. HAYEST. J., BAILERJ. & MI~R~VI~ M. 1972. immunity in rats to superinfection with Fasciola hepafica. Journal of Parasitology 58
: 1103.-1105 _
HAYEST. J., BAILERJ. & MITROVICM. 1973. Immunity to Fasciola heputica in rats: the effect of two different Levels of primary exposure on superinfectjon. ~~f~~~ff~ of Parasilo/ogy 59: 8 i O-8 I 2. HAYES T. J., BAILERJ. & MITROV~CM. 1974a. Acquired immunity and age resistance in rats with chronic fascioliasis. Journu~ of Purasi~oZo~y 60: 247-250 HAYES T. J., BAILERJ. & M~TROVICM. 1974b. Serum transfer of immunity to Fasciola hepaticu in rats. Journal of Parasitology 60: 722-723.
HAYEST. J., BAILERJ. & MITROVICM. 1974~. Studies on the serum transfer of immunity to Fasciola hepatica in the rat. Journal of Parasitology 60: 930--934. KELLYJ. D. 1973. Mechanisms of imniunity to intestinal helminths. Australian Veterinary Journal 49: 91-97. LANG B. Z. 1974. Host parasite relationships of Fasciala hepatiea in the white mouse. VI. Studies on the effects of immune and normal sera on the viability of young worms transferred to normal recipients. Journal of Parasitology 60: 925-929.
RAJASEKARIAHC. R. & HOWELL M. J. 1977. Fascioia hepatica in rats: effects of host age and infective dose. International Journal for Parasitology I: 119--l2 I. SINCLAIR1. J. 1970. The relationship between circulating antibodies and immunity to helminthic infections. Advances in Parasitology 8: 97-138. WIKERHAUSERT. 1961. lmmunobiologic diagnosis of fascioliasis. II. The in vitro action of immune serum on the young parasitic stage of F. &par&a-a new precipitin test for fascioliasis. Veterinarski arch&, Zagreb 31: 71-80.
1977.Vol.
7. pp. 367-371. Pergarnon Press. Printed in Great Britain.
IN VIP’0 AND IN VITRO STUDIES OF THE EFFECTS OF IMMUNE RAT SERUM ON FASCZOLA HEZ’ATZCA M. J. HOWELL, R. M. SANDEMANand G. R. RAJASEKARIAH Department
of Zoology, Australian National University, Box PO 4, Canberra, ACT 2600, Australia (Received
21 December
1976)
Abstract-HowELL M. J., SANDEMANR. M. and RAJASEKARIAH G. R. 1977. In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica. International Journal for Parasitology 7: 367-371. Significant protection against infection with 10 or 30 metacercariae of Fusciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components,presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established. INDEX KEY WORDS: Fasciola hepatica; metacercariae; immunity; passive transfer; intraperitoneal infection.
INTRODUCTION IT
shown that a primary infection of in rats confers appreciable resistance to a challenge infection (Hayes, Bailer & Mitrovic, 1972, 1973, 1974~; Goose & McGregor, 1973; Armour & Dargie, 1974). Immunological factors appear to be involved in this phenomenon since resistance can be passively transferred to naive recipients by serum and lymphoid cells from infected rats (Corba, Armour, Roberts & Urquhart, 1971; Hayes, Bailer & Mitrovic, 19746, c; Armour & Dargie, 1974). The present report describes infection schedules for the production of serum in DA and Wistar rats which passively protects naive recipients against infection with Fasciola hepatica. In addition, preliminary findings concerning the in vitro effects of this serum on metacercariae are outlined. HAS
been
Fkciola
hepatica
MATERIALS
AND METHODS
Rats. Two strains of rats were used: male, outbred Wistar rats and male, inbred DA rats. These were obtained from the John Curtin School of Medical Research, ANU. All animals were given food and water adlib. Parasites.
Fasciola hepatica metacercariae were obtained from a laboratory colony of Lymnuea tomentosa which had been infected with miracidia derived from
367
rats; immune serum;
in vitro culture;
eggs recovered from the gall bladders of infected sheep. They were kept at 4°C until required. Rats were infected with viable metacercariae and later autopsied as previously described (Rajasekariah & Howell, 1977). Production of serum. Three batches of serum were prepared for passive transfer experiments. Batch A was derived from DA rats which had been given a primary infection of 5 metacercariae at 5 weeks of age, challenged 7 weeks later with 30 metacercariae, and exsanguinated 8 weeks later. Batch B was derived from Wistar rats which were the same age and which had been exposed to the same levels of infection as the DA rats; they were exsanguinated 1 week after challenge. Batch C was also derived from Wistar rats as for Batch B, but the levels of primary and challenge infection were 10 and 20 metacercariae respectively. Normal serum was obtained from uninfected rats of the same strain and age as infected rats. Blood was allowed to clot and centrifuged at 4000 rev./min for 30 min; the serum was drawn off, sterilized by Seitz filtration, and stored at -20°C until required. Passive transfer experiments. Three experiments were performed, each with test and control groups of 6, 5 weeks old, male rats as recipients. In experiment 1, DA rats and Batch A serum (see above) were used. Each recipient received 10 ml of normal or immune serum intraperitoneally immediately before and after infection with 30 metacercariae. In experiment 2, Wistar rats and Batch B serum were used, following the same protocol as experiment 1. In experiment 3, Batch C serum was used and Wistar rats were given 5 ml intraperitoneally immediately following infection with 10 metacercariae.
M. J. HOWELL, R. M. SANDEMAN and G. R. RAJASEKARIAH
368
Experiment 1 also contained a group of 6 rats, each of which was infected with 30 metacercariae but not given serum. All rats were examined for adult worms 7 weeks after infection. In vitro cultures. Metacercariae were excysted by pepsin (3 x crystalline; Calbiochem) and trypsin (2 ’ crystalline, salt free; Nutritional Biochemicals) plus bile treatment as described by Wikerhauser (1961). The bile was obtained from uninfected sheep at Canberra abbatoir. Excysted metacercariae were rinsed several times in Medium 199 (Commonwealth Serum Laboratories) containing 100 units/ml penicillin, 100 ug/ml streptomycin and 2.5 pg/ml amphotericin B (E. R. Squibb & Sons), and then transferred in groups of about 50 to sterile Leighton tubes containing 5 ml of culture medium. The culture medium contained 20”/, normal or immune serum (Batch C, see Results) in Medium 199 plus antibiotics. The pH was maintained at 7.2 - 7.4 by a gas phase of 5% CO, in air (Commonwealth Industrial Gases). Cultures were incubated at 37’C in a water bath and agitated by means of a shaker operating at 120 agitationsimin. The medium was not changed during the period of culture. Fluorescent antibody (FAB) test. A direct FAB test was carried out on metacercariae cultured in immune and normal sera using fluorescein isothiocyanate labelled rabbit anti-rat immunoglobulin (Burroughs Wellcome). Metacercariae were gently washed 3 times with phosphate buffered saline (PBS), exposed for 30 min to the labelled anti-rat immunoglobulin diluted I :16, then washed 3 times with PBS. Examination was carried out under a Leitz U.V. microscope equipped with a BG12 exciter filter and blue absorbent barrier filters. Metacercariae which had not been exposed to labelled conjugate were also examined. Viability tests. Live mctacercariae which had been cultured for 36 h in immune or normal serum were washed 3 times in PBS. Ten mctacercariae were then injected intraperitoneally into each member of groups of 6. 5 weeks old, male Wistar rats using an ISG needle connected to a syringe by a I5 cm length of polythene tubing. A control, in which freshly excysted metacercariae were injected into a group of 6 rats, was also carried out. The rats were examined for adult worms 7 weeks later.
Immunogenicity
of itrxune
serum precipitate to rats.
When metacercariae are cultured in immune serum a precipitate forms on their teguments and in the culture medium (see Results). The immunogenicity of this precipitate was examined by injecting it into rats and testing the effects of sera from these animals on metacercariae in vitro. It was postulated that this procedure would give rise to an antiserum which would react with metacercariae in a similar manner to immune serum. The amount of precipitate used for injection was not quantified in terms of protein concentration, but for both the initial and booster injections it was derived from one culture containing 50 metacercariae which had been incubated in immune serum for 10 days. The metacercariae were rinsed thoroughly and discarded: the precipitate was then separated from the culture medium and rinsings by centrifugation. The pellet was washed 3 times with PBS, resuspended in I.0 ml PBS and then emulsified with 1.0 ml of Freund’s complete adjuvant (FCA, Difco). Two, 5 weeks old, male Wistar rats each received I ml of this emulsion: 0.2 ml intramuscularly
I.J.P. VC!I
7. 1977
and 0.8 ml intrapcritoneally. A control rat received an emulsion of 0.5 ml PBS plus 0.5 ml FCA. Five weeks later all rats received booster injections following the same procedures as described. One week later the rats wcrc exsanguinated and serum prepared.
RESULTS
The
results
of
passive
transfer
Fasciola hepatica infection I. The mean
serum
donors
(+s.D.)
experiment
used
worm
for
burden
_ I .3S,
2, 3.0
experiments
experiment
of 2.7
with
in Table
in rats are shown
1 had
i 0.2,
a
for
those
and those for experiment
3,
3.4 + 0.7.
TABLE I-Fasciola hepatica IN RATS: WORM RECOVERIES FOLLOWING INTRAPERITONEAL ADMINISTRATION OF SERUM IMMEDIATELY PRIOR TO AND/OR FOLLOWING INFECTION WITH 30 METACERCARIAE (EXPERIMENTS I & 2) OR IO METACERCARIAE (EXPERIMENT 3) Mean worm
Experiment
Immune serum recipients
recoveries Normal serum recipients
4.2 ! 29 3.6 t 1.7” I.6 I 0.8“
9.2 ! 3.5” 8.5 f 2.1” 3.4 _ 1.1”
1 2 3
In each experiment a b. P 0.05 In experiment I, b not significantly different from c. P 0.1. The
serum
doses
of
experienced tion;
donorc
primary
different
(see
in
experiments
significant with
were
Materials
the
degree
of
and
control
(experiment in worm Jn vitro
donor
groups
serum) was
no
of
conferred
a
challenge
1 and
(experiments
there
volumes
against
(no
infec-
Nevertheless.
serum
3). When
culturing
immune
from
rats
for
serum
used
a precipitate
about
2) or
normal
10
serum
were
compared
significant
difference
oral
sucker
to develop
and
excretory
were
appeared
also
to be derived
tate by dislodgment, result
of
of
parasite
which
medium.
An
which
on
free
from
although
a separate
immune were attempt
serum
Small
reaction
have
precipi-
between into
the to
of This
developed
secretions
made
of
of the
amounts
the tegument
not
3 199.
the tegument
it may
discharged was
experiment
in the medium.
and
of
derived
in Medium
in the vicinity
pore.
found
was
for
donors
particularly
precipate
ponents
C,
to be protective)
began
metacercaria,
I2 h in the presence
(Batch
as serum
and shown
each
a
different
Methods).
and
to
recovery.
20”/,
as
exposure
experiments
After
above
of
(experiment I),
varying
metacercariae
protection
30 metacercariae
metacercariae
I I.0 \ 3.3‘
administered
given
and
Control (no serum)
Mann-Whitney U-test.
challenge periods
the recipients
serum all
were
and
(is.0.)
comof
the
culture
distinguish
I J.P.
VOL.
7. 1977
Immune
rat strum
and F. /wpctica
FIG. 1. Precipitate on the tegument of a metacercaria cultured in immune rat serum for 36 h. Note that some precipitate is only loosely attached and the tegument appears to be intact. FIG. 2. Metacercariae cultured in normal rat serum for 36 h. Note the absence of precipitate on the tegument. FIG. 3. Metacercariae cultured in immune rat serum for 36 h and then exposed to FITC labelled rabbit anti-rat immunoglobulin and examined under U.V. The precipitate on the tegument and in the medium is strongly fluorescent, indicative of the presence of rat antibody. (Scale lines for Figs. l-3 - @I mm,)
3 70
M. J.
HOWELL, R.
M. SANDEMAN and G. R.
between these possibilities; for the remainder of this report, these precipitates will be regarded as having the same origin. After 36 h, the precipitate on each metacercaria was more extensive, covering most of the tegument (Fig. 1); larger amounts were also present in the culture medium. The tegument appeared to remain intact. Subsequently, no marked changes were evident although inactive metacercariae had more precipitate adhering to them than active metacercariae. Similar effects to those described were observed when immune serum was heated to 56’C for 30 min prior to being added to the culture medium. No precipitate formed on metacercariae or in culture media in the presence of intact normal serum or normal serum which had been heated to 56 ‘C for 30 min (Fig. 2). Mortality
qf metaccrcariae
This was assessed by their inactivity and opaque appearance. Pooled results from 3 cultures show an apparently higher mortality rate over the first 2 days for metacercariae in immune serum (Fig. 4). After about 60 h, mortality rates in both normal and immune serum are similar. Most metacercariae died within IO days in Iaitro, irrespective of the type of serum present.
cercariae paired. TABLE
RAJASEKARIAH
incubated
2-Fasciola
FOLLOWING
I.J P. Vol..
in normal
hepatica
INTRAPERITONEAL
IN
serum
RATS:
was also im-
WORM
INJECTION
7. 1917
OF
RECOV~KY 10
META-
CERCARIAE Mean
Source
Freshly excysted Cultured in normal serum for 36 h Cultured in immune serum for 36 h a h a
worm
recovery ( i
of metacercariae
s.D.)
4.5 1 0.8” 2.8 + 2.7” 0’
c, P -: 0.005 (‘. P G’ 0.05 h, P ~ 0.05
Composition
1
Mann-Whitney
U-test.
J
qf’ the immune
serum precipitate
It was considered
that the precipitate which formed in immune serum consisted of one or more rat antibody-parasite antigen complexes. The presence of antibody was confirmed by treating metacercariae with FITC labelled rabbit anti-rat immunoglobulin. The precipitate on the tegument fluoresced strongly (Fig. 3), as did that free in the medium. No fluorescence was observed on metacercariae cultured in normal serum or on specimens which had not been treated with the labelled conjugate. Confirmation that the immune serum precipitate was immunogenic to rats was obtained as follows: serum from rats which had been injected with an emulsion of precipitate and FCA reacted with metacercariae in vitro in a similar manner to immune serum. However, the precipitate which formed both on metacercariae and in the culture medium was relatively more copious. The direct FAB test showed that rat antibody was associated with this precipitate. No precipitate formed in cultures containing serum from rats which received an emulsion of FCA and PBS. DISCUSSION
FIG. 4. Mortality rates for metacercariae cultured in vitro in the presence of normal (A) and immune ( 0) rat serum. The curves for normal (-_) and immune (-) serum have been fitted by inspection. Viability of cultured
metacercariae
Table 2 shows adult worm recoveries from the bile ducts of rats 7 weeks after intraperitoneal injection of freshly excysted metacercariae and metacercariae cultured for 36 h in either normal or immune serum. Exposure of metacercariae to immune serum prior to injection into rats resulted in a loss of viability. No liver lesions were detected in rats injected with these metacercariae. By comparison with freshly excysted metacercariae, the viability of some meta-
The passive transfer experiments showed that a significant degree of protection against F. hepatica infection in naive rats was conferred by serum from infected animals. These results are in agreement with previous work (Hayes et al,, 19746, c; Armour & Dargie, 1974). Such experiments can be taken as evidence for the view that resistance to challenge is mediated, at least in part, by serum antibody (Sinclair, 1970; Kelly, 1973). Lang (1974) demonstrated that a 4 h incubation of partly matured F. hepatica in immune mouse serum significantly reduced the ability of the worms to re-establish in uninfected mice. This points to the involvement of antibody in resistance in these hosts as well. Serum was taken from infected donors when they were 13-20 weeks old. At this age, the rats would have developed a degree of age resistance (Rajasekariah & Howell, 1977). Since there was no difference in worm recovery between rats which did
Immune rat serum and F. hepaf~ea
I.J.P. VOL. 7. 1977
not receive serum and those which received normal serum (experiment I, Table I) it is concluded that the factors involved in age resistance arc not apparently transmitted in serum. The results of in V&U studies of the effects of immune rat serum on metacercariae were simiIar to those of Wikerhauser (1961) who used serum from cattle, rabbits, horses and humans. Thus, precipitate formation on metacercariae occurred in the presence of serum from infected animals, and this effect was still observed if the serum had been heated to 56°C for 30 min. The present study showed that the ability of metacercariae to undergo development in vivo was completely impaired by prior exposure to immune rat serum. The viability of some meta~er~ariae exposed to normal serum was also impaired; this points to inadequacies in the culture technique. The results of similar tests carried out by Wikerhauser (1961 f, using metacercariae incubated in infected cattle serum and then injected into guinea pigs, were apparently less striking. However, since no details of worm recovery were given and the cultures were contaminated with microorganisms, the significance of these results cannot be gauged. Moreover, the protective capacity of the infected cattle serum was not established with any degree of certainty. This is essential if in vitro effects are to bear any relationship to protection in vim. Metacercariae of F. ~e~~~~ca survived longer in immune rat serum than schistosumula of Schistocoma mansoni in immune monkey serum (Clegg & Smithers, 1972). This suggests that either protective activity in the serum was low or inactivated within a short period of culture, or that the co-operation of cells is required for the full effects of antibody to be realised. Frequent changes of the culture medium, raising the concentration of immune serum in the medium and the addition of cell populations to cultures are ways in which these possibilities could be tested. The precipitate which forms on metacercariae (and in the culture medium) in the presence of immune rat serum was shown to contain both antibody and immunogenic (presumably of parasite origin) components. However, whether it represents one or moreantibody/antigen complexesand whether non-specific serum factors are also involved in its formation has yet to be clarified. Neither complement nor reaginic antibodies are apparently responsible since these factors are heat labile (Davis, Dulbecco, Eisen, Ginsberg & Wood, 1967) and precipit&e formation takes place in their absence. An impo~ant question yet to be resolved is whether the precipitate is a manifestation of protective antibody-functional
antigen interaction,
and thus respon-
371
sible for impairing
the viability of metacercariae. It may well be that the deleterious effects of immure rat serum on metacercariae are less obvious. This, and other questions raised above, are currently under investigation. REFERENCES ARMOURJ. & DARGIEJ. R. 1974. Imn~unity to Fusci~la hepafica in the rat. Successful transfer of immunity by lymphoid cells and by serum. Experimental farasitology 35: 381-388.
CLEGG J. A. & SMITHERSS. R. 1972. The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni international Jorrrnalfbr
during
cultivation
in vitro.
Parasitology 2: 79-98.
C~RBAJ., ARMOURJ., ROBERTSR. J. & URQUHART G. M. 1971. Transfer of immunity to Fasriola heparica infection by lymphoid cells. Research in Veterinary Science 12: 292-295.
DAVIS B. D., DULBECCOR.. EISEN H. N.. GINSBERG H. S. & WOOU W. B. 1967. Microbioloiy. Hoeber Medical Division. Haruer & Row. New York. GOOSE J. & MACGREGOR M. 1973: Naturally acquired immunity to Fcisciofa hepariea in the rat. Brit!.sh Veterinary Journal 129: xlix-Iii. HAYEST. J., BAILERJ. & MI~R~VI~ M. 1972. immunity in rats to superinfection with Fasciola hepafica. Journal of Parasitology 58
: 1103.-1105 _
HAYEST. J., BAILERJ. & MITROVICM. 1973. Immunity to Fasciola heputica in rats: the effect of two different Levels of primary exposure on superinfectjon. ~~f~~~ff~ of Parasilo/ogy 59: 8 i O-8 I 2. HAYES T. J., BAILERJ. & MITROV~CM. 1974a. Acquired immunity and age resistance in rats with chronic fascioliasis. Journu~ of Purasi~oZo~y 60: 247-250 HAYES T. J., BAILERJ. & M~TROVICM. 1974b. Serum transfer of immunity to Fasciola hepaticu in rats. Journal of Parasitology 60: 722-723.
HAYEST. J., BAILERJ. & MITROVICM. 1974~. Studies on the serum transfer of immunity to Fasciola hepatica in the rat. Journal of Parasitology 60: 930--934. KELLYJ. D. 1973. Mechanisms of imniunity to intestinal helminths. Australian Veterinary Journal 49: 91-97. LANG B. Z. 1974. Host parasite relationships of Fasciala hepatiea in the white mouse. VI. Studies on the effects of immune and normal sera on the viability of young worms transferred to normal recipients. Journal of Parasitology 60: 925-929.
RAJASEKARIAHC. R. & HOWELL M. J. 1977. Fascioia hepatica in rats: effects of host age and infective dose. International Journal for Parasitology I: 119--l2 I. SINCLAIR1. J. 1970. The relationship between circulating antibodies and immunity to helminthic infections. Advances in Parasitology 8: 97-138. WIKERHAUSERT. 1961. lmmunobiologic diagnosis of fascioliasis. II. The in vitro action of immune serum on the young parasitic stage of F. &par&a-a new precipitin test for fascioliasis. Veterinarski arch&, Zagreb 31: 71-80.