IN VITRO CULTIVATION OF FASCIOLA HE’PATICA

METACERCARIAE AND OF PARTIALLY DEVELOPED

FLUKES RECOVERED FROM MICE

CAROLINE DAVIES and J. D. SMYTH

Department of Zoology and Applied Entomology, Imperial College of Science and Technology, Prince Consort Road,
London SW7 2BB, England

(Received 22 March 1977 ; Received J’brpublication 3 1 August 1977)

Abstract-DAVIES C. and SMYTH J. D. 1978. In vitro cultivation of Fasciolu hepaticu metacercariae

and of partially developed flukes recovered from mice. ~~~er~~~j~~~~ Journalfor Purusitoiogy 8: 12S-
131. Attempts were made to culture the metacercariae of Fusciula heputicu under a wide variety of
conditions. Of the media tested, the most successful was NCTC 135 plus 50% heat inactivated chick
serum and sheep red blood cells at 37”-38°C. In this medium, somatic development of newly excysted
juveniles was similar to that of flukes recovered from the liver of a mouse 11 days post-infection.
There was, however, no corresponding development of the genital rudiment. Various supplements,
such as liver extract, bile, yeast extract, embryo extract, egg products, monolayer cells and diphasic
media were tested, but none enhanced development. The effects of various physical parameters on
growth and development in vitro were examined. Cultured metacercariae appeared to be in a state of
‘suspended animation’; when injected intraperitoneally into mice they developed into egg-producing
adults. Flukes recovered from the abdomen and liver of mice continued their somatic growth in vitro
but their genitalia failed to develop further.

INDEX KEY WORDS: Fusciulu bepu&kz; in vitro culture; development in vitro; development in
viva; somatic development; genital rudiment development.

INTRODUCTION which other workers have so far failed to do. The

THE in vitro cultivation of Fasciclla hepatica has
been reviewed recently by Smyth (1976). Previous
work has concentrated mainly on the maintenan~
of adults (Stephenson, 1947; Dawes, 1954; Clegg,
1957; Rohrbacher, 1957; BCnex, 1966; Martinetto &
Capucinelli, 1968 ; Ratcliffe, Guevara-Pozo
Lopez-Roman, 1969; Foster, 1970; Locatelli &
Paoletti, 1970) although some attempts have been
made to culture the metacercariae to maturity in
vitro (Wikerhauser & Cvetnic, 1967; Wikerhauser,
Cvetnic & Brudnjak, 1968; Dawes & Hughes,
1970; Carillo de Albornoz & Guevara-Pozo, 1974).
These latter workers have used various complex
media, sera and cells (monolayer cells or red blood
cells) but with only limited success, The maximum
growth achieved was similar to that of flukes
recovered from the livers of mice eight days post
infection, i.e. soon after the flukes had reached
the liver (Dawes, 1962). Once the flukes had reached
this stage of development in vitro they were able to
continue in a state of ‘sus~nded animation’ for up
to 108 days but were unable to develop further
(Dawes & Hughes, 1970).
The aim of this study was to culture the metacer-
cariae of F. hepatica to maturity it! vitro by initiating
and maintaining the liver phase of development
criteria used to assess the development of the cultured
metacercariae were based on the developmental
pattern in mice (Dawes, 1962). Three factors were
chosen-
1. The increase in size of the fluke.
& 2. The branching of the digestive caeca.
3. The development of the genital rudiment.
In addition, flukes which had been grown to
various stages of development in the mouse were
transferred to culture in an attempt to answer certain
basic problems-i.e.
1. Is the penetration of the intestinal wall neces-
sary in order to trigger further development in the
way that penetration of the skin is necessary for the
subsequent development of the cercariae of Schisto-
som sPP.?
2. Since very little development occurs until the
fluke reaches the liver (Dawes. 1962), is the subse-
quent development within the liver triggered by an
initial ‘all or none’ stimulus?
3. Once the fluke has reached a certain develop-
mental stage itt viva will it continue to develop
further in vitro?
In order to test the suggestion of Dawes & Hughes
(1970) that cultured flukes can remain in a state of
‘suspended animation’, metacercariae grown in vitro
125
I26 CAROLINF DAVIES and J. D. SMYTH I.J.P. voL. x. 197x

for various lengths of time were injected intraperi- the criteria described in the Introduction and compared to
toneally into mice to determine whether they were the optimum development in Medium RC.
still capable of developing to adults. Flukes removedfbom mice. Flukes were recovered from
the abdomens of mice 2 days post-infection and from the
livers of mice 7, IO and I4 days post-infection. They were
MATERIALS AND METHODS washed in Hanks BSS and antibiotics and transferred to
small Falcon flasks containing Medium RC (approx. IO
Culture tnerhods. Many different cultivation systems flukes per vessel).
were tested; they are described briefly below. The prep- C~4lturedjfukes i:ljected into mice. Flukes were injected
aration of media and principles of in vitro culture tech- intraperitoneally into mice after 4, 8, 12 and 30 days
niques have been described in detail by Davies (1976). culture in Medium RC. Five flukes were injected into
1. Defined media. Hanks BSS (DIFCO), NCTC I35 each of three mice on each occasion. The liver and bile
(GIBCO). ducts of these mice were examined after a further 40 days.
2. Sera. Chick, rabbit, lamb, calf, horse, foetal calf
(Flow or BioCult). (These were heated at 56°C for 30 RESULTS
min to inactivate complement.)
Cl4ltiva:ion oj’metacevcasiac
3. Diphasic media. Solid phase: coagulated calf serum,
macerated coagulated calf serum, coagulated blood/ The growth and development of F. hepaticcz meta-
serum, blood/agar, coagulated egg macerate (Kannan- cercariae in the routine culture medium (Medium
gara & Smyth, 1974), or crude liver macerate. Liquid RC) is summarised in Table 1 and the various test
phase: NCTC 135 plus 20% inactivated foetal calf serum. media are compared to it.
4. Supplements. Horse or sheep defibrinated blood cells Although a wide variety of cultivation systems
(Tissue Culture Services); chick, mouse or bovine embryo were tested it proved impossible to grow the excysted
extracts (EE,,) (Paul, 1970 or Flow); liver extract metacercariae much beyond the stage achieved by
(Davi-s. 1976 or Sigma): yeast extract (Oxoid) in NCTC
previous workers. The most successful media proved
I35 ; sheep or mo& biic; sodium taurocholate (Sigma);
to te a relatively simple one; it consisted of NCTC
egg yolk medium (Berntzen & Macy, 1969).
135 with 50 “/, inactivated chick serum and sheep red
5. Monolayer cell cultures. BHK. Chang iivcr, chick
embryo, mouse embryo cells (all from Flow); newborn
blood cells. The other conditions were as described
mouse liver explant grown on coagulated chick szrum. for the routine culture medium. In this medium
6. Physical conditions. Media change-never, once or (Medium CS) the rate of growth and the degree of
twice weekly. Tempzraturc-37”C-38°C. Agitation- development was surprisingly uniform and the
stationary, shaking or rolling. Gas phase-air; aerobic majority of metacercariae grew from about 0.2 mm
(5% CO,, 15% O,, 80% N,); anaerobic (10% COP, to a maximum of I .2 mm in 13 days (Fig. I ). How-
90% N,). pH-approx. 7.4. Culture vessels-small ever, growth tailed off after this and the culture
Falcon flasks (Flow) or Leighton tubes (Belico). became contaminated with a fungal infection. When
A routine cultivation system was devised and used as a this experiment was repeated under identical con-
standard against which other cultivation systems could
ditions the maximum length achieved in 50% chick
be compared and assessed.
serum was 0.8 mm. The repeat culture was continued
Routine Culture Medium (Medium RC): Temperature
for 51 days after which time stocks of that particular
-37”-38°C. Gas phase-air. Culture vessel-Leighton
tubes. Agitation-continuously shaking water bath. batch of chick serum (Batch A) were no longer
Replacement of media-once weekly. Volume of media- available. Subsequent batches of chick serum (e.g.
IO ml. Culture media-NCTC 135 (with 100 pg/ml Batch B) proved no better than the routine culture
streptomycin and 100 I.U./ml penicillin) plus 20% medium.
inactivated foetal calf strum and 2 drops (i.e. approx. The growth in 507; chick serum (Batch A) was
150 pl) of defibrinated horse or sheep blood. Number of superior to that in 20% serum (Fig. I). In loo:‘,
metacercariae per culture vessel-50-100. chick serum the flukes only grew to 0.5 mm and
Cultivation of metacercariae. The cysts of F. hepatica appeared dark and sluggish. In 20 “/, and 50 ‘:/; serum
were excysted according to the methods of Wikerhauser
there was significant development of the digestive
(1960) or Sewell & Purvis (1969). The former involves the
caeca (Fig. 2B). After 7 days in vitro the simple
use of digestive enzymes and bile and the latter employs
the physiological criteria for excystation determined by bilobed caeca of the metacercaria (Fig. 2C) had
Dixon (1966). The newly excysted juveniles were washed grown considerably and lateral outgrowths had
repeatedly in sterile Hanks BSS plus streptomycin and developed. By Day 13 these had divided at their tips
penicillin before being transferred to culture. The cultures to produce secondary branches. This degree 01
were examined regularly and a record was kept of the somatic growth and caecal development is similar
flukes’ mobility, gut development, length and general to that of flukes recovered from the livers of mice I I
condition. Flukes were removed from culture, during the days post infection (Dawes, 1962) (Fig. 2A). How-
course of the experiments, in order to examine the ever, the genital rudiment of the cultured flukes did
development of the genital rudiment by staining in
not show a corresponding degree of development.
aceto-orcein or Gower’s carmine. The cultures were
maintained until the flukes died, or until they became After I I days in rGo the rudimentary bilobed genital
moribund and showed no further signs of growth or rudiment of the metacercaria showed considerable
development. The maximum degree of growth or develop- development (Dawes, 1962); the posterior portion
ment in each particular system was assessed according to (which gives rise to the ovary, Mehlis’ gland and
I.J.P. VOL. 8. 1978 //I vitro culture of Fasciolu hepalica 127

TABLE I-GROWTH AND DEVELOPMENT OF Fusciolahepalica METACERCARIAE in vitro

Max.
length Gut Genital Days
Culture conditions (in mm.) development rudiment in vitro
- ____~._
Medium KC 0.55 SB o-3t 43
Test media
Hanks BSS NG NG NG 3
NCTC 135 NC NC NG 5
NCTC 135 +- 2076 foetal calf serum 0.35 LB+ o-3
NCTC 135 + 20% chick serum (A) + r.b.c. 0.7 SB 3-5 &*
NCTC 135 + 50% chick serum (A) f r.b.c. I.2 SB 3-5 (18)
100% chick serum (A) + r.b.c. 0.55 LB o-3 (61)
NCTC 135 + 20% chick serum (B) + r.b.c. 0.44 LB o-3 51
NCTC + 50% chick serum (B) + r.b.c. 044 LB o-3
NCTC + 20% rabbit serum $ r.b.c. 0.55 LB+ o-3 r:;,
NCTC + 20% lamb serum + r.b.c. 06 LBi- o-3 24
NCTC + 20% calf serum f r.b.c. 0.45 LB NG 24
NCTC 7 20% horse serum f r.b.c. NG NG NG 6
RC + coagulated calf serum (w/o r.b.c.) 0.3 NG NG
RC -+ macerated coagulated calf serum (w/o r.b.c.) 0.4 LB o-3 (f!)
RC + serum/blood coagulate (w/o r.b.c.) NC NG NG 8
RC + agar/blood coagulate (w/o r.b.c.) NC NG NG IO
RC ;- 20% egg macerate (w/o r.b.c.) NG NG NG
RC + 10% chick EE,, (Flow) 0.6 LB+ O-3 (25s)
RC -I 10% chick EE5,, (prepared) 0.5 LB; o-3 (29)
RC + 2 % mouse EEjo (prepared) 0.66 LB.- o-3 55
RC + 10% bovine EE,, (Flow) NG NG NG 6
RC + 0.25 % liver extract (Oxoid) 0.6 SB o-3
RC $ 10% sheep liver extract (prepared) 0.6 SB o-3 (ii)
RC + 1% sheep bile NG NG NG 8
RC $- 0.05% mouse bile NG NG NG 23
RC + 0.04% sodium taurocholate 0.33 LB NG 53
RC -t 10% yeast extract 0.33 LB NG 14
RC + egg yolk medium (w/o r.b.c.) NG NG NG 12
RC + BHK cells (w/o r.b.c.) 0.4 LB o-3 I7
RC t Chang liver cells (w/o r.b.c) NG NG NG 28
RC + chick embryo cells (w/o r.b.c.) 0.4 LB O-3 51
RC + mouse embryo cells (w/o r.b.c.) 0.55 LB o-3 51
RC f new born mouse liver explant (w/o r.b.c.) 0.5 LB o-3 20
RC:-stationary 0.35 LB+ NG 30
RC:-rolling NG NG NG
RC:-anaerobic gas 0.55 LB; o-3 (::)
RC :-aerobic gas 0.55 LBi o-3 (22)
RC:-media not changed 0.33 LB NG 24
NG, no growth; LB, lateral branches; LB+, bifurcations of lateral branches; SB, secondary branches: *( ) con-
taminated-discarded, t, corresponding development in days p.i. in the mouse (Dawes, 1962).
two testes) had divided into three separate parts tested was there any further growth of the genital
and the anterior portion (which produces the cirrus rudiment.
and cirrus sac) was beginning to take on a recognis- Those flukes which were cultured in a cell-free
able shape (Fig. 2A). In contrast to this, the genital medium were generally smaller and less well develop-
rudiment of the cultured flukes either failed to ed than those which had been grown in a medium
develop at all or was similar to that of flukes re- containing blood cells, monolayer cells or explant
covered from the livers of mice 3 days post infection hepatocytes. In Medium RC, the flukes ingested the
(Dawes, 1962). In the latter case, the posterior rudi- red blood cells but, after about a week, black solids
ment had a bilobed appearance and the strand which began to accumulate in the caecal lumen. These solids
connects the two parts of the mctacercarial rudiment were presumably partially digested or degenerating
had elongated slightly. red blood cells and they often built up to such an
The degree of somatic growth in 50% chick serum extent that the caeca became swollen and deformed.
was not obtained in any other media. The digestive Once the flukes reached this condition there was
caeca showed the same degree of development in the usually no further growth or development. However,
routine culture medium and in some of the test in Medium CS, there was rarely any build up of
media. In none of the other culture media solids within the gut.
128 CAROLINEDAVIESand J. D. SMYTH l.J.1’. VOL. 8. 1978

rA

The size, caecal development and genital rudiment

development achieved by flukes recovered from
niice 2, 7, 10 and 14 days post-infection is described
in detail by Dawes (1962). Table 2 shows the flukes’
maximum length (measured live) and the degree of
caecal development both before and after in vitro
culture.
Although there was considerable somatic growth
and development ir7 vitro in flukes removed from the
livers of mice on Days 7, 10 and 14, there was no
further development of the genital rudiment. On the
contrary, the partially formed genitalia-especially
the developing testes-appeared to degenerate and
were difficult to recognise when stained in aceto-
orcein or Cower’s carmine.

Cultured.flukes /@wed Mo mice

In all the mice, exceptfor one, that were injected
with cultured flukes, one or more egg-producing
adults were recovered after 40 days.

I , I
’ 2 4 6 8 IO 12 14 16 18
DISCUSSION
DAYS
Although a wide variety of culture conditions were
FIG. I. Emio/u hrpafica: incr-nse in length nwxsured i,z
tested during the course of this study in an attempt
viva and itr vitro.
Growth in mice (after
to initiate the liver phase of development of Fasciola
A. Dawes, 1962)-average
length. lzepatica in vitro, they proved generally unsuccessful.
B-D. Growth in NCTC 135 + inactivated chick serum + Many of the ‘cultivation’ media were virtually only
red blood cells-maximum length, measured live. maintenance media. The inability of the various liver
B-50% serum; C-20% Serum: D-1000/, serum. cell cultures, extracts and products (e.g. bile) to

Frc. 2. Fasciola hepah-u: A. II day fluke from mouse liver (redrawn after Dawes, 1962). B. Fluke
cultured in NCTC 135 -i_ 50% chick serum + red blood cells for 13 days. C. Newly excysted juvenile.
CR, cirrus rudiment: DC, digestive caecae; CR, genital rudiment; MG, Mehlis’ gland rudiment:
OR, ovary rudiment: TR. testes rudiment.
1.1.~. VOL. 8. 1978 In vitro culture of Fasciola hepatica 129

TABLE 2---CULTIVATION OF FLUKES RECOVERED FROM MICE

-
Original Length after Caecal
Days post infection Days let&h in- vitro Original caecal development
in the mouse in vitro (mm.) culture (mm.) development in vitro
2 (abdomen) 27 0.2 0.35 unbranched lateral branches
7 (liver) 32 0.6 1.6 lateral branches tertiary branches
10 (liver) 78 1.3 4.0 secondary branches multiple branches
14 (liver) 31 2.1 3.2 secondary branches multiple branches
--
stimulate development was particularly disappoint- patent infection after intraperitoneal injection of
ing. cysts. Flukes from the liver failed to continue their
Various diphasic media, which have proved so normal development in vitro suggesting that the
successful in the cultivation of other helminths (e.g. liver phase of development requires the continued
Erlzitzococcus guarzulosus, Smyth, 1967; Diplostomzmz presence of various factors found in the liver rather
spp., Kannangara & Smyth, 1974), were unsuitable than an initial ‘trigger’.
in this case. The egg yolk medium of Bertzen & Adults were recovered from mice following intra-
Macy (1969) is particularly rich in cholesterol (Dr. B. peritoneal injection of cultured metacercariae thus
Fried, personal communication) and has been supporting the suggestion of Dawes & Hughes
successfully used to culture Sphaeridiotrema globu- (1970) that cultured flukes can remain in a state of
los (Berntzen & Macy, 1969) and Leucochloridio- ‘suspended animation’ in vitro whilst retaining their
mnrpha constantiae (Fried & Contos, 1973). Choles- ability to develop if transferred to a suitable host.
terol is found in mammalian liver and bile (Smyth & Similar results were obtained by Hughes & Cristo-
Haslewood, 1963) and in extracts of adult liver fluke finis (unpublished results quoted by Hughes &
(van Brand, 3928), however, its addition in the Harness, 1973) with F. hepatica and by Hanna &
form of egg yolk medium failed to enhance growth or Jura (1976) with F. gigantica.
development. Similar results were obtained by Voge During this study, one of the major problems has
& Jeong (I 97 1) with Cotylurus lutzi. Yeast extract is a been the inconsistency of the results-a problem well
rich source of B vitamins and has been widely used recognised in in vitro culture (Smyth & Z. Davies,
in many culture systems (Smyth, 1967; Wyllie, 1974). The micro-environment which a fluke inhabits
Williams & Hopkins, 1960), but it had a marked within the cultivation system may be of the utmost
inhibitory effect on the development of F. hepatica importance in determining whether that particular
i/z vitro. Embryo extracts are considered to promote fluke will develop or not. This depends on the inter-
the growth of cells in culture (Paul, 1970) but for action of various physico-chemical factors, e.g. the
F. hepatica metacercariae they were generally shape of the vessel, agitation, frequency of media
inhibitory and the results varied from one batch of change, number of flukes per vessel as well as the
the extract to another. Embryo extracts have also p02, pCO*, pH and Eh. The difficulty of exactly
been shown to inhibit the development of Schisto- reproducing these conditions from one culture to
soma mansotri (Clegg, 1965) and Cotylarrrs latzi another may help to account for apparently inex-
(Voge & Jeong, 1971). plicable variations.
The best medium for the cultivation of F. hepatica The use of non-defined components, e.g. sera and
metacercariae was, in fact, a relatively simple one embryo extracts in the culture media, presents con-
consisting of a complex defined media (NCTC 135), siderable problems since different batches often
serum (50% inactivated chick) and red blood cells possess widely different growth promoting proper-
(horse or sheep). In this medium there was a 6-fold ties. Many attempts have been made to replace serum
increase in length and considerable development of with defined substitutes (e.g. Lasfargues, Coutinho,
the digestive caeca but the lack of development of the Lasfargues & Moore, 1973 ; Hayashi & Sato, 1976)
genital rudiment indicated that the liver phase of but the substitutes were often only capable of
development had not been properly initiated. In this supporting the growth of one or two particular cell
experiment, a relatively high level of serum produced lines. However, the search for a completely defined
the optimum growth. The use of cells in the culture culture medium must continue as, only then, can
medium confirmed the observations of Wikerhauser the source of these variations be eliminated.
& Cvetnic (1967) and Wikerhauser rt a/. (1968) that It appears from these results that somatic and
F. lzepatica metec?rcariae survive longer in cultures germinal development, which normally proceed side
containing cells than those without. by side in a co-ordinated manner in vivo, can become
Flukes from the abdomens of mice developed no unsynchronised in vitro. A similar situation has been
further in culture than did excysted metacercariae recorded during the cultivation of E. maltiIocularis
indicating that the penetration of the host’s intestinal (Smyth & Z. Davies, 1975) with the production of
wall is not a pre-requisite for subsequent develop- sexually mature monozoic or unsegmented forms.
ment. This reinforces the results of Wikerhauser, The techniques traditionally employed in the
Magud, Dzakula & Cvetnic (1973) who obtained a cultivation of helminths are largely empirical and
130 CAROLINE DAVIESand J. D.
rely heavily on the addition of non-defined com-
ponents whose growth promoting properties are
poorly understood. The disappointing nature of the
results presented here indicates that a new approach
is now necessary based on an understanding of the
parasite’s normal environment in vivo and the way
in which it interacts with its host.
Acknowledgements-The metacercarial cysts of Fasciola
hepafica were kindly supplied by Dr. C. P. Ollerenshaw of
the Central Veterinary Laboratories, Weybridge, Surrey.
The work was undertaken when one of us (C.D.) was in
receipt of a Postgraduate Studentship from the Medical
Research Council.
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