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Axenic culture of the rodent tapeworms Hymenolepis diminuta and H. nana
EXPERIMENTAL
PARASITOLOGY
11,
Axenic
176-187 (1961)
Culture
of
Hymenolepis Angela Zoology
Department, (Submitted
the
diminuta
Rodent and
Tapeworms
H. nana
E. R. Taylor1
University
of California,
for publication,
Los Angeles, California
9 December
1960)
1. Cysticercoid larvae of Hymenolepis diminuta have been maintained in vitro for 6 days at 25” C in simple media containing proteose peptone, Ringer’s solution, and glucose. The larvae were still infective to rats at the end of this time. When rat serum was added to the medium the larvae remained infective for 6 days at 30” C but not at 25” C. 2. Cysticercoid larvae of H. nana survived for 5 days in vitro at 25” C in a simple salt solution with glucose and they were still infective to mice at the end of this period. 3. The early beetle stages of H. diminuta and H. nana were maintained in a healthy condition in vitro for 7 days and some development was obtained with H. diminuta larvae (30” C). 4. The juveniles of H. diminuta have been kept alive for 7 days in vitro at 38” C in a mixture of horse serum and medium 199; they retained their infectivity to rats only for the first 4 days. 5. The juveniles of H. nana have survived for 9 days in vitro at 38” C in a mixture of horse serum and medium 199 supplemented with amino acids; they retained their infectivity to mice for the first 8 days when the horse serum was replaced by calf serum. Some growth in length occurred when an extract of mouse intestine was added to the medium.
and was included in all the media (unless otherwise indicated). Phenol red was included to indicate pH. In general the media used for the cultivation of the “insect-stages” were of lower osmotic pressure than those used for the “mammalian-stages.” The parasites were cultured in small petri dishes (3/4 inch in diameter) or in 15 ml roller culture tubes. The dishes or tubes containing the “insectstages” were incubated at 25” or 30” C, whereas those tubes containing excysted juveniles were incubated at 37”, 38’, or 38.5” C. The latter parasites had their media changed on alternate days. Parasites were removed at frequent intervals for microscopic examination, or to test their infectivity to the rodent host. Several extracts were used in the various culture media.
Apart from the preliminary experiments on the cultivation of the scolex and neck from adult Hymenolepis diminuta reported by Schiller, Read, and Rothman (1959)) and the maintenance of adult H. nana in vitro (Wardle and Green, 1941) for 20 days, no attempts have been made to culture H. diminuta or H. nana axenically in vitro. This paper is concerned with attempts to obtain growth of the beetle stages of H. diminuta and H. nana in axenic culture and to cultivate excysted cysticercoid larvae (juveniles) of both species. METHODS
Sterile techniques were employed throughout these experiments. Dicrysticin (penicillin 400,000 units and streptomycin 0.5 g supplied by E.R. Squibb & Sons, New York) was found to be well tolerated by the parasites
Preparation
of Extracts
(a) Ckick embryo extract. Ten-day-old chick embryos were homogenized in a brass
1 Present address: Biology Department, Sir John Atkins Laboratories, Queen Elizabeth College, Campden Hill, London, W.8, England.
tissue 176
grinder
(with
a chrome
plunger)
and
the homogenate was diluted with an equal volume of Tyrode’s solution. After standing for 3 hours, the cellular debris was removed by centrifugation and the resulting extract was kept at -20” C until required. (b) Tenebrio or Tribolium extracts [based on the technique described by Wyatt (1956)) for the preparation of tyrosinase-free insect hemolymph] . Young beetle larvae were homogenized at 60” C in an all-glass tissue grinder [three Tenebrio larvae or six Tribolium larvae per milliliter of dilute Ringer’s solution (50: 50 with distilled water) 1. The extract was kept at 60” C for a further 5 minutes in order to inactivate the enzyme tyrosinase which is present in the hemolymph of these larvae. (Tyrosinase in insect hemolymph produces melanin via intermediary quinones which are believed to be toxic in insect tissue culture: Wyatt, 1956). The extract was then centrifuged to remove the cellular debris. Glucose (O.S$) and trehalose (0.5%) were added to the supernatant which was bacterially filtered (using a fritted glass bacterial filter) and stored at -20” C until required. (c) Intestinal and tapeworm extracts. The distal third of rat or mouse small intestine, or whole adult tapeworms, were washed thoroughly in Ringer’s solution before being finely chopped with scissors. The intestinal fragments, with twice their own volume of Tyrode’s solution, or the worm fragments, in their own volume of Tyrode’s solution, were homogenized in an all-glass tissue grinder. After the cellular debris had been removed by centrifugation the extracts were bacterially filtered through a fritted glass bacterial filter and stored at -20” C until required. (d) Bacterial metabolites. Bacteria, from the small intestine of rats or mice, were cultured for one week in a liquid culture medium designed for Escherichia co2i (medium F, Adams, 1959). The bacteria were removed by filtration and the filtrate used as the source of bacterial metabolites. It was stored at 4’ C until required. Parasitic Strains and Their Maintenance The parasites were maintained in the laboratory by the method of Voge and Heyneman (1957). In all experiments the Rice In-
stitute strains of H. diminuta and H. nana were used. Hymenolepis diminuta was carried in Sprague-Dawley albino rats bred in the vivarium of the Zoology Department and H. nana was carried in white Swiss parasitefree mice obtained from the Cancer Genetics Laboratory at the University of California at Berkeley or from Jackson Laboratories, Bar Harbor, Maine. The tapeworms were transmitted by the confused flour beetle (Tribolium confusum), originally obtained from the Carolina Supply Co., Elon College, North Carolina, and subsequently maintained in this laboratory. The infected beetles were kept at 30” C in l-inch stendor dishes during the development of the parasites (the beetles were starved for one week prior to their infective feed and then allowed to feed for 4 hours on previously separated tapeworm eggs) and were maintained on sterile enriched flour. Culture of the Beetle Stages Infected beetles were dissected into dilute Ringer’s solution at intervals after their infective feed. Some of these larvae were set aside at this point to serve as controls. The remaining larvae were washed four times in dilute Ringer’s solution, the final washing containing antibiotics (Dicrysticin-0.005 ml/ ml Ringer. Dicrysticin diluted with 5.0 ml of distilled water). Initially, cysticercoid larvae were cultured in small petri dishes (3/d inch in diameter) at 25” or 30” C. Each dish contained 0.5 ml of culture medium and five larvae were placed in each dish. Control cysticercoids were fed to rats or mice at the beginning of each experiment and the experimental cysticercoids were treated likewise when each experiment was concluded. The majority of the experiments on the earlier beetle stages were conducted in 1.5 ml roller tube cultures containing 1 ml of culture medium and five larvae per tube. These were maintained at 30’ C. Controls were fixed in Bouin’s fluid and stained with Meyer’s Haem Alum (3 drops/100 ml distilled water, overnight) at the beginning of each experiment. Five to 7 days later the experimental larvae were similarly stained for comparison with the controls (Table III). The media used
178
TAYLOR
Media Used for the Cultivation Tapeworm Medium A B C D G H J
JI
Beetle Hd. Hn. Hd. Hn. Hd. Hd. Hn. Hn. Hd. Hd. Hn.
J2 K Kl
L M N 0 P
Hd. Hd. Hn. Hd. Hd. Hd. Hd.
Q R S T U
Hd. Hd. Hd. Hn. Hd.
Ul u2
V W X
TABLE I of Hymenolepis
stage Juvenile
Ingredients Serum
Hd. Hd.
RS 50 HS 50
Hd. Hd. Hd. Hd. Hn. Hd.
RS HS HS HS HS HS HS
Hd. Hd. Hd. Hd. Hd. Hn. Hd. Hn. Hd.
Hd. Hd. Hn.
Hn. Hn. Hn. Hn.
Hn. Hn. Hn. Hn. Hn. Hn. Hn.
diminuta and H. nana
50 50 25 10 17 25 30
HS 25 HS 20 HSUF 25 CS 25 HuS 25 HS 30 HS 25 HS 20 HS 15 HS 25 HS 25 cs 25
199a
Salt solution DR 50 DR 50 DR 35 B 50 R 50 DR 50
(percentage) Extract
TE 50
TE 50
50 50 75 90 58 65
AA 25 AA 10 EYD EYD
50 50 55 65 65 65
in the above experiments are listed in Table I. Culture of the Juvenile Worms Cysticercoid larvae were dissected from beetles and washed twice in dilute Ringer’s solution. They were then induced to excyst by the method described by Rothman (1959)) which was briefly as follows. The cysticercoids were placed in a stender dish (1.5 inch in diameter) containing a freshly prepared 1% solution of pepsin in Ringer’s solution (pH 1.5 using 1 NHCl). They were then gently shaken while being incubated in a water bath at 37” C (30 minutes for H. diminuta; 15 minutes for H. nana). At the end of this time the larvae were washed in dilute Ringer’s solution and similarly incubated in a trypsin and bile salt solution
20 20
TE 50 TE 80 TE 50
Gey’s 25 TE 20
EYD 65 60 45 25 25 65
Other ingredients PB 50, G 1 PB So, G 1, PP 2 PB 35, G 1, PP 2 G 0.9 Gl Gl
EYD EYD
20
15 15
TriE 50 CEE 10 CEE 20 CEE 40 TE 25 TriE 25 CEE 10
AA AA AA AA AA
25 5 lo 10 10
AA AA AA AA AA AA
10 10 10 10 10 10
(H. diminuta: 10 minutes in 0.5% trypsin plus 0.6% sodium taurocholate solution in buffered Ringer’s solution pH 6.7; H. nana: 30 minutes in 0.5% trypsin plus 0.3 % sodium taurocholate solution in similarly buffered Ringer). After this treatment, the excysted juveniles were washed three times in dilute Ringer’s solution, the final washing containing antibiotics (Dicrysticin, 0.005 ml/ml). They were then transferred with a fine pipet to 15 ml roller culture tubes (2 to 3 juveniles per tube), each containing 1 ml of culture medium and incubated at 37, 38, or 38.5” C. Controls were fixed in Bouin’s fluid and stained with Giemsa for comparison with the similarly treated juveniles at the end of each experiment. The infectivity of the
AXENIC
CULTURE
TABLE Tapeworm Medium
Beetle
Xl
x2
OF RODENT
179
TAPEWORMS
I (ContintLed) Ingredients
stage Juvenile
Serum
199a
Extract
Other ingredients
Hn. Hn.
cs 20 cs 15
60 45
CEE 20 CEE 40
AA lo AA 10
HS
25
HS HS HS HS HS HS HS
25 25 25 25 25 25 20
60 45 55 55 55 55 55 SO
CEE 15 CEE 30 MIE 20 HNE 20 HDE 20 RIE 20 BM 20 HDE 15, CEE 15 BM 15, MIE 15 BM 15, MIE 15 HDE 20
AA A.4 AA AA AA AA AA
Y
Hn.
Yl Z AA BB cc DD EE
Hd. Hn. Hn. Hd. Hd. Hd. Hd.
FF
Hn.
HS 20
50
GG
Hn.
HS 20
50
HH
Hn.
HuSN
Hn.
Hn. Hn. Hn. Hn.
25
55
Salt solution
(percentage)
10 10 10 10 10 10 10
AA 10 AA A.4 T AA
10 10, P 0.0001, 0.0001, BS 0.0001 10
Abbreviations: = Hymenolepis diminuta; Hn. = Hymenolepis nana; RS = Rat serum; = Horse serum; HSUF = Horse serum ultra filtrate; CS = Calf serum; HuS = Human serum (pregnant woman) ; DR = Dilute Ringer’s solution (50:50 with distilled H,G) ; = Ball and Chao (1960) + 400 mg/liter trehalose; R = Ringer’s solution; B EYD = Egg yolk dialyzate; TE = Tenebrio extract; TriE = Tribolium extract; CEE = Chick embryo extract; MIE = Mouse intestine extract; RIE = Rat intestine extract; HNE = H. nana extract; HDE = H. diminuta extract; BM = Bacterial metabolites; = Phosphate buffer ph 7.0; G = Glucose; PP = Proteose peptone; PB = Amino acid mixture (Eagle et al., 1957) ; P = Pepsin; T = Trypsin; AA = Sodium taurocholate; HuSN = Human serum (Normal). BS a Morgan, Morton, and Parker (1950). Hd.
HS
juveniles before and after culture was tested by injecting them directly into the small intestine of the rodent host while it was anesthetized with ether. Two or 3 weeks later the animals were autopsied and their small intestines examined for the presence of adult hymenolepids. The culture media used in these experiments are shown in Table I. RESULTS
The parasites their infectivity but the majority for the further stages.
remained alive and retained in vitro for limited periods, of media were not adequate development of the different Beetle Stages
Preliminary experiments were carried out to determine how long the cysticercoid larvae
could be maintained in vitro and still remain infective to the rodent host. Table II shows that the cysticercoid larvae of H. diminuta survived for a maximum of 6 days, and remained infective to rats, in simple media (A and B) when kept at 25’ C. The cysticercoids did not survive quite as long at 30’ C. When Tenebrio extract was added (medium C) the larvae remained infective both at 25” and 30” C for 5 days. However, when serum was added to the medium (D) the cysticercoids remained infective for 6 days at 30” C but not at 25” C as was the case in the simpler media. On the other hand, the cysticercoid larvae of H. nana survived for a maximum of 5 days in a simple salt solution (medium A) at 25’ C and were still infective to mice, but they survived for only 3 days in media D and G.
180
TAYLOR
TABLE Maintenance Time Medium A
of Hymenolepis
II Cysticercoids
in vitro
Species
at 25” C
H. diminuta
30 Hours
in Vitro Time
A/C
a/9 6/6 2/6
in vitro
at 30” C 30 Hours
A/C
6/9
A
2 Days
A B B B
6 Days
5 Days
2/6
B
6 5 5 6
Days Days Days Days
3/6 3/3 3/6 O/6
6 Days 5 Days 5 Days
3/5
6 Days
4/6
20 Hours 2 Days
214 3/4
12 Hours 1 Day
3/5 3/5
A B B
5 Days 2 Days 2 Days
3/4 l/12 ll/ll
2 Days 12 Hours
O/5 O/4
B
2 Days
3/3
D G
3 Days 3 Days
4/4 2/3
3 Days 3 Days
2/4 414
C D D H. nana
A A
A,C
=
Number of adult worms obtained from Number of cysticercoids fed to rat
2 6 2 2 5
Days Days Days Days Days
5/7 O/6 3/5 5/5 2/6 O/6 fv-5
rat or mouse or mouse
Subsequent experiments were designed to determine whether the earlier beetle stages could be induced to develop in vitro. The results of these experiments are shown in Table III. One of the difficulties with these experiments was that although the beetles were given the same short infective feed (4 hours), when the larvae were dissected from them at intervals later, even those from the same beetle were not all at the same stage of development. Thus some difficulty was experienced in assessing the results. This was overcome to some extent by the use of large numbers of controls in each experiment; also beetles which were dissected up to 3 days after their infective feed were found to contain only spherical morulae, which meant that any development in vitro could easily be seen as the next stage involved elongation of the larvae. It can be seen from Table III that, with the exception of media K1 and N which appeared to be toxic, all the larvae seemed normal and healthy after being kept in vitro for periods of up to 7 days. The use of rat, horse, calf, or pregnant human sera, medium 199, Tenebrio extract, Tribolium extract, and
chick embryo extract in the media were equally well tolerated by the larvae. The morula stages of these worms showed only slight development in vitro in media L and M and the use of horse serum ultra filtrate, medium 199, Gey’s fluid, Tribolium extract or egg yolk dialyzate (media N, 0, P, T and W) did not improve these results. Somewhat better results were obtained in the fourth stage larvae (6 days after the beetle’s infective feed). Here there was some change in vitro from stage 4 of H. diwzinuta to early stage 5. The scolex became well differentiated with the rostellum showing well and the suckers becoming well defined with alignment of the muscles. The spaces in the peripheral layers of the larvae also disappeared but there was no sign of growth in this region. When larvae, which had an everted scolex, were kept in vitro they did not develop further; thus the scolex did not become withdrawn. Juveniles
of H. diminuta
The juveniles of H. diminuta survived for a maximum of 7 days in vitro in media J,
AXENIC
CULTURE
OF
RODENT
181
TAPEWORMS
TABLE III Beetle Stages of Hymenolepis in Vitro
at 30” C
Time in vitro Medium
Species
Morula Stage 2 Stage 2 Morula Stage 2 Stage 2 Morula Stage 2 Stage 4 Stage 2 Morula Morula Morula
A A A H
J Kl
L L L L M N N 0 0 0 0 0 P P P P R S T T T T T U V V V w W w W W L T
Stage of controlsa
H. diminuta
H. nana
Stage 2 Stages 3 Morula Stage 2 Stages 3 Stage 2 Morula Stage 2 Stage 2 Stage 2 Stage 2 Stage 4 Stage 2 Stage 2 Morula Stage 2 Stage 3 Stage 3 Stages 3 Stages 3 Stages 3 Morula Stage 2 Stage 2 Stages 3
(days)
7 2 9 6 3
& 4
7 7
Stage in r&rob
Appearance
Morula 318 Stage 3 Stage 2 Early stage 2 Stage 2 Early stage 5 Stage 2 Early stage 2 Morula Morula
Normal Normal Degenerate Normal Normal Normal Normal Normal Degenerate Degenerate
S/S Stage 3 lo/10 Stages 3 & 4
Normal Normal
3/9 Stage 5 Stage 2
Normal Normal
l/7 Stage 3 4/7 Stage 3 Early Stage 5 l/3 Stage 3 2/S Stage 3
Normal Normal Normal Normal Normal
3/14 Stage 4 3/4 Stage 4 12/12 Stage 3 2/10 Stage 5 II/II Stage 3
Normal Normal Normal Normal Normal
2/13 Stage 5
Normal
Stale 4 Sage 3
Normal Normal
7 & 4 6 7
7 5 6 7
& 4 & 4 & 4
7
7 & 4
Stage 4 Stage 3
6
a These describe the stages reached by the larvae 01 dissection from the infected beetles and before being cultured in vitro. b These describe the stages of the larvae after being cultured in vitro.
J 1, and J2 (Table IV). However, they were
no longer infective at the end of this time and remained so for only 4 days in medium J (50% horse serum: 50% medium 199). Supplementing medium 199 with a mixture
of amino acids (medium K,) or using calf serum (medium R) produced no better results although some of the juveniles retained their infectivity for 2 or 3 days in both these media and survived for a maximum of 3 days.
TAYLOR
182
TABLE IV diminuta Juveniles
Survival of Hymenolepis
Medium
:1 JZ
Kl KI Kl KI KI Kl Kl 0
i R R R R R R s u Yl Yl Yl
BB BB BB cc DD EE
Temperature (“0 30 38.5 38 38 38 37 38 38.5 38 38 38 38.5 38.5 38.5 38 38 38.5 38 38 38 38.5 38 38.5 38 38 38 38 38 38 38 38 38
Maximum survival time (days) and E/S in parentheses
7 7 7 2
Controls
Experimental
A/J
(days)
24/44
1 (O/12) 4 (10/20)
213 2/4 2/4
(4/4) (4/4) (4/4) (U/22)
1 (9/9)
214 3/5 213 17/17
2 1 1 3 1 1 0
2 (13/H) (6/9) (6/6) (l/6) (l/4) (7/9) (l/4) (O/15)
alI degenerate
1 3 3 0
(4/6) (6/6) (6/10) (O/15)
2 2 3 2 2 2 2
(3/9) (5/9) (5/6) (l/16) (6/g) (3/6) (3/6)
1 (213) 1 (4/6)
degenerate
O/11 (1) 3/3 (3); O/2 (7) O/2 O/2 11/15
(7) (7) (2)
l/3
(2) (2)
17/17 616 2/3
O/l O/g O/2 O/l5
(3) (2) (I)
213 17117
3/5 O/6
(2) (3);
6/6 all degenerate
2/3 17/17
A/J and time
in parentheses
3/7
fv6
2 (3/15) 2 (6/9) 2 (g/9)
in vitro
6/6 (4)
O/3 (2)
2/7 (3) O/15 (1) O/9
(3)
O/3
(1)
2 days
3 (2/6)
E/S =
Number of juveniles still active Number of juveniles at start of experiment
A/J =
Number of adult worms obtained from rat Number of juveniles injected into small intestine of rat
The addition of chick embryo extract, pregnant human serum, Tenebrio extract, horse serum ultra filtrate, H. diminuta extract, rat intestine extract, bacteria1 metabolites or a mixture of H. diminuta extract, and chick embryo extract (media U, Y, S, P, Q, BB, CC, DD and EE, respectively) did not support the activty of these parasites for more than 2 days and in fact media Q, S, and DD appeared to be toxic.
Juveniles of H. nana Table V shows that the maximum period of survival (9 days) was obtained in one experiment with medium K but that the organisms were not infective to mice at the end of this time. On the other hand, the juveniles survived for 8 days and still retained their infectivty to mice in medium R, the difference between these media being that R contains calf serum whereas K con-
AXENIC
Survival Temperature (” C)
Medium
37 38 37 37 37 38 38 38.5 38.5 38 38 38.5 38 38 38.5 38 38 38 38 38 38.5 38.5 38.5 38.5 38 38.5 38.5 38.5 38 38 38 38 38 38 38 38 38 38 38 38
:
JI J2 K Kl Kl Kl Kl Kl
0 0 P R R R S T U U U U
Ul u2 u2
x
Xl x2 Y Yl 2
AA BB BB cc DD EE FF GG HH E/S = A,J
=
CULTURE
OF RODENT
TABLE V of Hymenolepis nana Juveniles
Maximum survival time (days) and E/S in parentheses 4 4 4 4 4 9 5 3 3 4 9 2 5 8 3 4 3 5 3 6 3
(2/S) (S/15) (2/10) (2/6) (4/S) (3/15)
(2/15)
degenerate
(J/6) (8/15) (3/15) (3/9)
2 (S/12) 1 (6/12) 2 (2/12) 3 (S/6) 0; 1 day degenerate
2 (l/12) 0; 2 4 3 2 4 3 2 0 0 0 0 3
A/J 112 112 112 112
(4/9) (l/12) (4/6) (l/15) (6/9) (7/15) (4/9) (l/6) (2/15)
Controls
4/S
(6/W
1 day degenerate (4/6) (2/6) ; 3 (4/6) (6/6) (3/6) (l/6); 2 (3/6) (3/3) (3/6) (O/6) toxic (O/6) toxic (O/6) toxic (O/6) toxic (3/6)
183
TAPEWORMS
4/S 6110 4/S l/10 o/17 4/S 4/S 6110 6/10 l/10 o/17 2/3 4/S 21f3 6110 3/S l/10 l/10 l/10 o/17 l/10 l/10 l/10 o/17 o/17 o/17 O/5 o/17
W3 O/5
in vitro Experimental A/J and time (days) in parentheses O/l o/3 O/2 O/l O/l o/2 o/3 l/S o/3 3/3 O/2
(4) (4) (4) (4) (4); (9) (5); (3) (2) (3); (9)
l/l
(2)
l/3
(3)
O/l
(4)
l/l
(7)
O/l
(4)
016 (2) O/3 l/3 O/3 2/2 O/2 O/3 O/4 O/3 l/4
(5) (8); (2) (3); (3) (5) (3) (6) (3)
l/f5 (2) O/3 (2) O/3 (2) 3/3 (3) O/7 (2)
O/2 (4) Growth in length O/3 O/2 (4); o/2 (3) 3/3 O/3
5/6 5/6 5/6 5/6 8/S
2/3
Number of juveniles still active Number of juveniles at start of experiment Number Number
of adult worms obtained from rat at biopsy of juveniles injected into small intestine of rat
tains horse serum (Table I). In one experiment, rat intestine extract was included in the medium (Z). The juveniles survived for 3 days in this and doubled their length during
this time (Figs. 1 and 2). This was an encouraging result but unfortunately time did not permit any further experiments. Apart from the above, survival was only
184
TAYLOR
FIG. 1.
I).
Juvenile
of Hymenolepis
FIG. 2. Juvenile of Hymnolepis Stained Giemsa, X 700.
nana;
Control.
Stained
Giemsa,
nana; 4 days in vitro in culture
X 700.
medium
Z (Table
AXENIC
CULTURE
OF RODENT
TAPEWORMS
185
caution was that large numbers of controls were used for each experiment. There is some controversy in the literature over the normal mode of development of H. nana in T. con&sum. Volge and Heyneman (1957) considered that the scolex of the cysticercoid larva first develops externally and is then withdrawn into the cavity of the mid-body of the larva, whereas Schiller (1959) has presented evidence that “the scolex and internal membrane develop within the capsule of the cysticercoid.” He has observed that those forms with extruded scoDISCUSSION lices lack uniformity in their morphological The approach used in these experiments appearance except for a poorly formed scolex has been to dissect the larval tapeworms from external to the capsule. For this reason and the intermediate host and to try to induce that no particular morphological type could these larvae to develop to sexual maturity. be associated with a definite period of time One great advantage of such a starting point during development, he considers these orwas that the larvae were dissected from an ganisms to be abnormal. If that is so, then already sterile environment and hence asepsis it is not surprising to find that in the present presented no special problem. No complicated experiment the stages with an everted scolex sterilization procedure, such as that used by did not show signs of development in vitro. Wardle and Green (1941) when they at- However, one would have expected to have tempted to culture adult tapeworms, was found some development of the morula stage had the culture conditions been suitable. The necessary. lack of growth at this stage was therefore Beetle stages an indication of inadequate culture condiA thorough knowledge of the normal tions. morphology of the parasite is essential for Wyatt and Kalf (1957) reported that the assessment of the effects of culture. In trehalose was a major blood sugar of insects. the case of H. diminuta and H. nana exten- Consequently trehalose was added to several sive studies of the morphology of the beetle of the media. Unfortunately it did not apstages had already been carried out by Voge pear to prolong survival or stimulate growth (1960) and Voge and Heyneman (1957, at the concentrations used here. The majority 1960), and their work provided a basis for of the media used appeared to be well the present study. tolerated by the morulae for they showed Although the beetles were given a rela- no signs of degeneration in vitro and in a few tively short infective feed (4 hours), when embryos early indications of growth were observed. With the more advanced embryos the larvae were dissected out at intervals later, they were found to be at various de- some further development was observed in velopmental stages, even those dissected media V, T, and L. However, in view of the from the same beetle. Thus it was difficult to small numbers of embryos recovered from obtain eperimental embryos of the same age the staining procedure, these experiments can only be regarded as of a preliminary nature. and the same stage prior to culture. This difficulty was overcome to some extent by the Much more work is required to elucidate the use of 2.5day or 6-day embryos, since 2.5 in vitro requirements of the early beetle stages of these organisms. days after the infective feed beetles contained The cysticercoid larvae of H. diminuta and embryos which had not developed beyond the morula stage and 6 days after the in- H. nana differed somewhat in their ability fective feed the embryos had not quite de- to survive in vitro; those of H. diminuta surveloped to the infective stage. Another pre- vived and remained infective for 7 days,
maintained for about 3 to 5 days in the other media used. Thus the addition of chick embryo extract, pregnant human serum, normal human serum, Tenebrio extract, horse serum ultra filtrate, H. nana extract, H. diminuta extract, rat intestine extract, bacterial metabolites, or a mixture of H. dimifluta extract and chick embryo extract (media U, Y, yl, s, HH, P, Q, AA, BB, CC, DD, and EE, respectively) had no effect on the survival of the juveniles, at the concentrations used here.
186
TAYLOR
which was slightly longer than cysticercoid larvae of H. nana at 25”. Rausch and Jentoft (1957) cultured the scolices of Echinococcus multilocularis in vitro obtaining proliferation and survival up to 30 days. These authors also used infectivity as a criterion for assessing their results for, when the cultured scolices were introduced into the peritoneal cavity of a natural intermediate host (the vole), they proliferated and produced new scolices. Unfortunately Rausch and Jentoft had too few cultured scolices to attempt to infect the canine definitive host. Juveniles The experiments using excysted juveniles have shown that there are differences in ability to survive in vitro between the two species used. The juveniles of H. diminuta survived for a maximum of 7 days in media J, J1, and Jz, but were only infective for the first 4 days in medium J at 38’ C. On the other hand, the juveniles of H. nana survived for 9 days in media K and 0, and 8 days in medium R; they remained infective for the full culture period in medium R. Further experiments of this nature may be helpful in indicating some of the physiological differences between the two species. However, it was not altogether surprising that H. nana proved to be more amenable to culture than H. diminuta since it can complete the whole of its life-cycle within the mammalian host alone. A surprising result was that the addition of serum to medium 199 prolonged the survival of these intestinal parasites. An interesting result in passing was that possibly the gonadotropic hormones, as supplied by pregnant human serum, were toxic whereas normal human to the juveniles, serum was tolerated for 3 days with the retention of their infectivity. The fact that some growth was obtained in one experiment with H. nana in medium Z was most encouraging. Medium Z contained an extract of mouse intestine (see Methods) which probably provided them with some essential growth factor. Schiller, Read, and Rothman (1959) have also reported growth in H. diminuta. They cultured the scolex and neck from adult worms in a medium containing 50% horse serum and an extract of
the adult worm, which resulted in a 30-fold increase in size by the 11th day. This type of growth really constitutes regeneration of the strobila from the scolex and may differ somewhat from the type of growth that might be obtained from the culture of a juvenile worm. It would appear that juvenile worms may require some “trigger” to be present in the medium before growth will occur. Berntzen (1960) has reported an effective method for the in vitro culture of H. diminuta. In this he stated that larvae of H. diminuta were isolated from infected beetles and grown in vitro (using several types of media) to adults which contained onchospheres. He used an apparatus designed to provide a continuous flow of medium over the worms and this culture experiment was terminated on the 15th day. It is to be hoped that this experiment will soon be reported in greater detail since it may represent a considerable advance in the study of parasites by their culture in vitro. ACKNOWLEDGMENTS
It Ball
is a pleasure and
his staff
they extended
to for
thank the
Professor facilities
and
to me. I am very grateful
Gordon
H.
hospitality to Professor
Ball, Dr. Marietta Voge, Dr. Donald Heyneman, and Dr. Jowett Chao for several stimulating discussions during this work, and I would also like to thank Mr. Glen Harrington for his willing and skilful technical assistance. made to Mrs. Nora
Grateful acknowledgment is Liu for histological preparation
of the beetle stages. This work was supported by a Post Doctoral Trainee Fellowship (ZE-70) from the U. S. Public Health Service, and the travel to California was made possible by a Fulbright Travel Award. REFERENCES ADAMS, M. H. 1959. Bacteriophages. Publishers Inc., New York, p. 446.
Interscience
BALL, G. H., AND CHAO, J. 1960. In vitro development of the mosquito phase of Plasmodium relicturn. Experimental Parasitology 9, 47-55. BERNTZCEN, A. K. 1960. An effective method for the in vitro cultivation of Hymenolepis diminuta. Journal of Parasitology 46 (Sec. 2), 47. EAGLE, H., OYAMA, V. I., LEVY, M., AND FREEMAN, A. E. 1957. Myo-inositol as an essential growth factor for normal and malignant cells in tissue culture. Journal of Biological Chemistry 226, 191.
AXENIC
CULTURE
OF RODENT
MORGAN, J. F., MORTON, H. J., AND PARKER, R. C. 1950. Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proceeding of the Society for Experimental Biology and Medicine 73, 1. RUJSCH, R. L., AND JENTOFT, V. L. 1957. Studies on the helminth fauna of Alaska. XxX1. Observations on the propagation of the larval Echinococcus multilocularis Leuckart, 1863, in vitro. Journal of Parasitology 43, 1-8. ROTHMAN, A. H. 1959. Studies on the excystment of tapeworms. Experimental Parasitology 8, 336364. SCHILLER, E. L. 1959. Experimental studies on morphological variations in the cestode genus Hymenolepis. I. Morphology and development of the cysticercoid of H. nana in Tribolium conjusum. Experimental Parasitology 8, 91-118. SCHILLER, E. L., REaD, C. P., AND ROTHMAN, A. H. 1959. Preliminary experiments on the growth of a cyclophyllidean cestode in vitro. Journal of Parasitology 46 (4 sect. 2), 45. VOGE, M. 1960. Studies in cysticercoid histology. I. Observations on the fully developed cysticercoid
TAPEWORMS
187
of Hymenolepis diminuta (Cestoda, Cyclophyllidea). Proceedings of the Helminihological Society of Washington 27 (1) 32-36. VOGE, M., AND HEYNEMAN, D. 1957. Development of Hymenolepis nana and Hymenolepis diminuta (Cestoda Hymenolepididae) in the intermediate host Tribolium confusum. University of California Publications in Zoology 59, 549-580. VOCE, M., AND HEYNEMAN, D. 1960. Studies in cysticercoid histology. II. Observations on the fully developed cysticercoid of Hymenolepsis nana (Cestoda, Cyclophyllidea) . Proceedings of the Helminthological Society of Washington 27, 185. WARDLE, R. A., AND GREEN, N. K. 1941. The cultivation of tapeworms in artificial media. Canadian Journal of Research D19, 240. WYATT, G. R., AND KALF, G. F. 1957. The chemistry of insect haemolymph. II. Trehalose and other carbohydrates. Journal of General Physiology 40, 833-847. WYATT, S. S. 1956. Culture in vitro of tissue from the silkworm Bombyx mori L. Journal of General Physiology 39, 841.
PARASITOLOGY
11,
Axenic
176-187 (1961)
Culture
of
Hymenolepis Angela Zoology
Department, (Submitted
the
diminuta
Rodent and
Tapeworms
H. nana
E. R. Taylor1
University
of California,
for publication,
Los Angeles, California
9 December
1960)
1. Cysticercoid larvae of Hymenolepis diminuta have been maintained in vitro for 6 days at 25” C in simple media containing proteose peptone, Ringer’s solution, and glucose. The larvae were still infective to rats at the end of this time. When rat serum was added to the medium the larvae remained infective for 6 days at 30” C but not at 25” C. 2. Cysticercoid larvae of H. nana survived for 5 days in vitro at 25” C in a simple salt solution with glucose and they were still infective to mice at the end of this period. 3. The early beetle stages of H. diminuta and H. nana were maintained in a healthy condition in vitro for 7 days and some development was obtained with H. diminuta larvae (30” C). 4. The juveniles of H. diminuta have been kept alive for 7 days in vitro at 38” C in a mixture of horse serum and medium 199; they retained their infectivity to rats only for the first 4 days. 5. The juveniles of H. nana have survived for 9 days in vitro at 38” C in a mixture of horse serum and medium 199 supplemented with amino acids; they retained their infectivity to mice for the first 8 days when the horse serum was replaced by calf serum. Some growth in length occurred when an extract of mouse intestine was added to the medium.
and was included in all the media (unless otherwise indicated). Phenol red was included to indicate pH. In general the media used for the cultivation of the “insect-stages” were of lower osmotic pressure than those used for the “mammalian-stages.” The parasites were cultured in small petri dishes (3/4 inch in diameter) or in 15 ml roller culture tubes. The dishes or tubes containing the “insectstages” were incubated at 25” or 30” C, whereas those tubes containing excysted juveniles were incubated at 37”, 38’, or 38.5” C. The latter parasites had their media changed on alternate days. Parasites were removed at frequent intervals for microscopic examination, or to test their infectivity to the rodent host. Several extracts were used in the various culture media.
Apart from the preliminary experiments on the cultivation of the scolex and neck from adult Hymenolepis diminuta reported by Schiller, Read, and Rothman (1959)) and the maintenance of adult H. nana in vitro (Wardle and Green, 1941) for 20 days, no attempts have been made to culture H. diminuta or H. nana axenically in vitro. This paper is concerned with attempts to obtain growth of the beetle stages of H. diminuta and H. nana in axenic culture and to cultivate excysted cysticercoid larvae (juveniles) of both species. METHODS
Sterile techniques were employed throughout these experiments. Dicrysticin (penicillin 400,000 units and streptomycin 0.5 g supplied by E.R. Squibb & Sons, New York) was found to be well tolerated by the parasites
Preparation
of Extracts
(a) Ckick embryo extract. Ten-day-old chick embryos were homogenized in a brass
1 Present address: Biology Department, Sir John Atkins Laboratories, Queen Elizabeth College, Campden Hill, London, W.8, England.
tissue 176
grinder
(with
a chrome
plunger)
and
the homogenate was diluted with an equal volume of Tyrode’s solution. After standing for 3 hours, the cellular debris was removed by centrifugation and the resulting extract was kept at -20” C until required. (b) Tenebrio or Tribolium extracts [based on the technique described by Wyatt (1956)) for the preparation of tyrosinase-free insect hemolymph] . Young beetle larvae were homogenized at 60” C in an all-glass tissue grinder [three Tenebrio larvae or six Tribolium larvae per milliliter of dilute Ringer’s solution (50: 50 with distilled water) 1. The extract was kept at 60” C for a further 5 minutes in order to inactivate the enzyme tyrosinase which is present in the hemolymph of these larvae. (Tyrosinase in insect hemolymph produces melanin via intermediary quinones which are believed to be toxic in insect tissue culture: Wyatt, 1956). The extract was then centrifuged to remove the cellular debris. Glucose (O.S$) and trehalose (0.5%) were added to the supernatant which was bacterially filtered (using a fritted glass bacterial filter) and stored at -20” C until required. (c) Intestinal and tapeworm extracts. The distal third of rat or mouse small intestine, or whole adult tapeworms, were washed thoroughly in Ringer’s solution before being finely chopped with scissors. The intestinal fragments, with twice their own volume of Tyrode’s solution, or the worm fragments, in their own volume of Tyrode’s solution, were homogenized in an all-glass tissue grinder. After the cellular debris had been removed by centrifugation the extracts were bacterially filtered through a fritted glass bacterial filter and stored at -20” C until required. (d) Bacterial metabolites. Bacteria, from the small intestine of rats or mice, were cultured for one week in a liquid culture medium designed for Escherichia co2i (medium F, Adams, 1959). The bacteria were removed by filtration and the filtrate used as the source of bacterial metabolites. It was stored at 4’ C until required. Parasitic Strains and Their Maintenance The parasites were maintained in the laboratory by the method of Voge and Heyneman (1957). In all experiments the Rice In-
stitute strains of H. diminuta and H. nana were used. Hymenolepis diminuta was carried in Sprague-Dawley albino rats bred in the vivarium of the Zoology Department and H. nana was carried in white Swiss parasitefree mice obtained from the Cancer Genetics Laboratory at the University of California at Berkeley or from Jackson Laboratories, Bar Harbor, Maine. The tapeworms were transmitted by the confused flour beetle (Tribolium confusum), originally obtained from the Carolina Supply Co., Elon College, North Carolina, and subsequently maintained in this laboratory. The infected beetles were kept at 30” C in l-inch stendor dishes during the development of the parasites (the beetles were starved for one week prior to their infective feed and then allowed to feed for 4 hours on previously separated tapeworm eggs) and were maintained on sterile enriched flour. Culture of the Beetle Stages Infected beetles were dissected into dilute Ringer’s solution at intervals after their infective feed. Some of these larvae were set aside at this point to serve as controls. The remaining larvae were washed four times in dilute Ringer’s solution, the final washing containing antibiotics (Dicrysticin-0.005 ml/ ml Ringer. Dicrysticin diluted with 5.0 ml of distilled water). Initially, cysticercoid larvae were cultured in small petri dishes (3/d inch in diameter) at 25” or 30” C. Each dish contained 0.5 ml of culture medium and five larvae were placed in each dish. Control cysticercoids were fed to rats or mice at the beginning of each experiment and the experimental cysticercoids were treated likewise when each experiment was concluded. The majority of the experiments on the earlier beetle stages were conducted in 1.5 ml roller tube cultures containing 1 ml of culture medium and five larvae per tube. These were maintained at 30’ C. Controls were fixed in Bouin’s fluid and stained with Meyer’s Haem Alum (3 drops/100 ml distilled water, overnight) at the beginning of each experiment. Five to 7 days later the experimental larvae were similarly stained for comparison with the controls (Table III). The media used
178
TAYLOR
Media Used for the Cultivation Tapeworm Medium A B C D G H J
JI
Beetle Hd. Hn. Hd. Hn. Hd. Hd. Hn. Hn. Hd. Hd. Hn.
J2 K Kl
L M N 0 P
Hd. Hd. Hn. Hd. Hd. Hd. Hd.
Q R S T U
Hd. Hd. Hd. Hn. Hd.
Ul u2
V W X
TABLE I of Hymenolepis
stage Juvenile
Ingredients Serum
Hd. Hd.
RS 50 HS 50
Hd. Hd. Hd. Hd. Hn. Hd.
RS HS HS HS HS HS HS
Hd. Hd. Hd. Hd. Hd. Hn. Hd. Hn. Hd.
Hd. Hd. Hn.
Hn. Hn. Hn. Hn.
Hn. Hn. Hn. Hn. Hn. Hn. Hn.
diminuta and H. nana
50 50 25 10 17 25 30
HS 25 HS 20 HSUF 25 CS 25 HuS 25 HS 30 HS 25 HS 20 HS 15 HS 25 HS 25 cs 25
199a
Salt solution DR 50 DR 50 DR 35 B 50 R 50 DR 50
(percentage) Extract
TE 50
TE 50
50 50 75 90 58 65
AA 25 AA 10 EYD EYD
50 50 55 65 65 65
in the above experiments are listed in Table I. Culture of the Juvenile Worms Cysticercoid larvae were dissected from beetles and washed twice in dilute Ringer’s solution. They were then induced to excyst by the method described by Rothman (1959)) which was briefly as follows. The cysticercoids were placed in a stender dish (1.5 inch in diameter) containing a freshly prepared 1% solution of pepsin in Ringer’s solution (pH 1.5 using 1 NHCl). They were then gently shaken while being incubated in a water bath at 37” C (30 minutes for H. diminuta; 15 minutes for H. nana). At the end of this time the larvae were washed in dilute Ringer’s solution and similarly incubated in a trypsin and bile salt solution
20 20
TE 50 TE 80 TE 50
Gey’s 25 TE 20
EYD 65 60 45 25 25 65
Other ingredients PB 50, G 1 PB So, G 1, PP 2 PB 35, G 1, PP 2 G 0.9 Gl Gl
EYD EYD
20
15 15
TriE 50 CEE 10 CEE 20 CEE 40 TE 25 TriE 25 CEE 10
AA AA AA AA AA
25 5 lo 10 10
AA AA AA AA AA AA
10 10 10 10 10 10
(H. diminuta: 10 minutes in 0.5% trypsin plus 0.6% sodium taurocholate solution in buffered Ringer’s solution pH 6.7; H. nana: 30 minutes in 0.5% trypsin plus 0.3 % sodium taurocholate solution in similarly buffered Ringer). After this treatment, the excysted juveniles were washed three times in dilute Ringer’s solution, the final washing containing antibiotics (Dicrysticin, 0.005 ml/ml). They were then transferred with a fine pipet to 15 ml roller culture tubes (2 to 3 juveniles per tube), each containing 1 ml of culture medium and incubated at 37, 38, or 38.5” C. Controls were fixed in Bouin’s fluid and stained with Giemsa for comparison with the similarly treated juveniles at the end of each experiment. The infectivity of the
AXENIC
CULTURE
TABLE Tapeworm Medium
Beetle
Xl
x2
OF RODENT
179
TAPEWORMS
I (ContintLed) Ingredients
stage Juvenile
Serum
199a
Extract
Other ingredients
Hn. Hn.
cs 20 cs 15
60 45
CEE 20 CEE 40
AA lo AA 10
HS
25
HS HS HS HS HS HS HS
25 25 25 25 25 25 20
60 45 55 55 55 55 55 SO
CEE 15 CEE 30 MIE 20 HNE 20 HDE 20 RIE 20 BM 20 HDE 15, CEE 15 BM 15, MIE 15 BM 15, MIE 15 HDE 20
AA A.4 AA AA AA AA AA
Y
Hn.
Yl Z AA BB cc DD EE
Hd. Hn. Hn. Hd. Hd. Hd. Hd.
FF
Hn.
HS 20
50
GG
Hn.
HS 20
50
HH
Hn.
HuSN
Hn.
Hn. Hn. Hn. Hn.
25
55
Salt solution
(percentage)
10 10 10 10 10 10 10
AA 10 AA A.4 T AA
10 10, P 0.0001, 0.0001, BS 0.0001 10
Abbreviations: = Hymenolepis diminuta; Hn. = Hymenolepis nana; RS = Rat serum; = Horse serum; HSUF = Horse serum ultra filtrate; CS = Calf serum; HuS = Human serum (pregnant woman) ; DR = Dilute Ringer’s solution (50:50 with distilled H,G) ; = Ball and Chao (1960) + 400 mg/liter trehalose; R = Ringer’s solution; B EYD = Egg yolk dialyzate; TE = Tenebrio extract; TriE = Tribolium extract; CEE = Chick embryo extract; MIE = Mouse intestine extract; RIE = Rat intestine extract; HNE = H. nana extract; HDE = H. diminuta extract; BM = Bacterial metabolites; = Phosphate buffer ph 7.0; G = Glucose; PP = Proteose peptone; PB = Amino acid mixture (Eagle et al., 1957) ; P = Pepsin; T = Trypsin; AA = Sodium taurocholate; HuSN = Human serum (Normal). BS a Morgan, Morton, and Parker (1950). Hd.
HS
juveniles before and after culture was tested by injecting them directly into the small intestine of the rodent host while it was anesthetized with ether. Two or 3 weeks later the animals were autopsied and their small intestines examined for the presence of adult hymenolepids. The culture media used in these experiments are shown in Table I. RESULTS
The parasites their infectivity but the majority for the further stages.
remained alive and retained in vitro for limited periods, of media were not adequate development of the different Beetle Stages
Preliminary experiments were carried out to determine how long the cysticercoid larvae
could be maintained in vitro and still remain infective to the rodent host. Table II shows that the cysticercoid larvae of H. diminuta survived for a maximum of 6 days, and remained infective to rats, in simple media (A and B) when kept at 25’ C. The cysticercoids did not survive quite as long at 30’ C. When Tenebrio extract was added (medium C) the larvae remained infective both at 25” and 30” C for 5 days. However, when serum was added to the medium (D) the cysticercoids remained infective for 6 days at 30” C but not at 25” C as was the case in the simpler media. On the other hand, the cysticercoid larvae of H. nana survived for a maximum of 5 days in a simple salt solution (medium A) at 25’ C and were still infective to mice, but they survived for only 3 days in media D and G.
180
TAYLOR
TABLE Maintenance Time Medium A
of Hymenolepis
II Cysticercoids
in vitro
Species
at 25” C
H. diminuta
30 Hours
in Vitro Time
A/C
a/9 6/6 2/6
in vitro
at 30” C 30 Hours
A/C
6/9
A
2 Days
A B B B
6 Days
5 Days
2/6
B
6 5 5 6
Days Days Days Days
3/6 3/3 3/6 O/6
6 Days 5 Days 5 Days
3/5
6 Days
4/6
20 Hours 2 Days
214 3/4
12 Hours 1 Day
3/5 3/5
A B B
5 Days 2 Days 2 Days
3/4 l/12 ll/ll
2 Days 12 Hours
O/5 O/4
B
2 Days
3/3
D G
3 Days 3 Days
4/4 2/3
3 Days 3 Days
2/4 414
C D D H. nana
A A
A,C
=
Number of adult worms obtained from Number of cysticercoids fed to rat
2 6 2 2 5
Days Days Days Days Days
5/7 O/6 3/5 5/5 2/6 O/6 fv-5
rat or mouse or mouse
Subsequent experiments were designed to determine whether the earlier beetle stages could be induced to develop in vitro. The results of these experiments are shown in Table III. One of the difficulties with these experiments was that although the beetles were given the same short infective feed (4 hours), when the larvae were dissected from them at intervals later, even those from the same beetle were not all at the same stage of development. Thus some difficulty was experienced in assessing the results. This was overcome to some extent by the use of large numbers of controls in each experiment; also beetles which were dissected up to 3 days after their infective feed were found to contain only spherical morulae, which meant that any development in vitro could easily be seen as the next stage involved elongation of the larvae. It can be seen from Table III that, with the exception of media K1 and N which appeared to be toxic, all the larvae seemed normal and healthy after being kept in vitro for periods of up to 7 days. The use of rat, horse, calf, or pregnant human sera, medium 199, Tenebrio extract, Tribolium extract, and
chick embryo extract in the media were equally well tolerated by the larvae. The morula stages of these worms showed only slight development in vitro in media L and M and the use of horse serum ultra filtrate, medium 199, Gey’s fluid, Tribolium extract or egg yolk dialyzate (media N, 0, P, T and W) did not improve these results. Somewhat better results were obtained in the fourth stage larvae (6 days after the beetle’s infective feed). Here there was some change in vitro from stage 4 of H. diwzinuta to early stage 5. The scolex became well differentiated with the rostellum showing well and the suckers becoming well defined with alignment of the muscles. The spaces in the peripheral layers of the larvae also disappeared but there was no sign of growth in this region. When larvae, which had an everted scolex, were kept in vitro they did not develop further; thus the scolex did not become withdrawn. Juveniles
of H. diminuta
The juveniles of H. diminuta survived for a maximum of 7 days in vitro in media J,
AXENIC
CULTURE
OF
RODENT
181
TAPEWORMS
TABLE III Beetle Stages of Hymenolepis in Vitro
at 30” C
Time in vitro Medium
Species
Morula Stage 2 Stage 2 Morula Stage 2 Stage 2 Morula Stage 2 Stage 4 Stage 2 Morula Morula Morula
A A A H
J Kl
L L L L M N N 0 0 0 0 0 P P P P R S T T T T T U V V V w W w W W L T
Stage of controlsa
H. diminuta
H. nana
Stage 2 Stages 3 Morula Stage 2 Stages 3 Stage 2 Morula Stage 2 Stage 2 Stage 2 Stage 2 Stage 4 Stage 2 Stage 2 Morula Stage 2 Stage 3 Stage 3 Stages 3 Stages 3 Stages 3 Morula Stage 2 Stage 2 Stages 3
(days)
7 2 9 6 3
& 4
7 7
Stage in r&rob
Appearance
Morula 318 Stage 3 Stage 2 Early stage 2 Stage 2 Early stage 5 Stage 2 Early stage 2 Morula Morula
Normal Normal Degenerate Normal Normal Normal Normal Normal Degenerate Degenerate
S/S Stage 3 lo/10 Stages 3 & 4
Normal Normal
3/9 Stage 5 Stage 2
Normal Normal
l/7 Stage 3 4/7 Stage 3 Early Stage 5 l/3 Stage 3 2/S Stage 3
Normal Normal Normal Normal Normal
3/14 Stage 4 3/4 Stage 4 12/12 Stage 3 2/10 Stage 5 II/II Stage 3
Normal Normal Normal Normal Normal
2/13 Stage 5
Normal
Stale 4 Sage 3
Normal Normal
7 & 4 6 7
7 5 6 7
& 4 & 4 & 4
7
7 & 4
Stage 4 Stage 3
6
a These describe the stages reached by the larvae 01 dissection from the infected beetles and before being cultured in vitro. b These describe the stages of the larvae after being cultured in vitro.
J 1, and J2 (Table IV). However, they were
no longer infective at the end of this time and remained so for only 4 days in medium J (50% horse serum: 50% medium 199). Supplementing medium 199 with a mixture
of amino acids (medium K,) or using calf serum (medium R) produced no better results although some of the juveniles retained their infectivity for 2 or 3 days in both these media and survived for a maximum of 3 days.
TAYLOR
182
TABLE IV diminuta Juveniles
Survival of Hymenolepis
Medium
:1 JZ
Kl KI Kl KI KI Kl Kl 0
i R R R R R R s u Yl Yl Yl
BB BB BB cc DD EE
Temperature (“0 30 38.5 38 38 38 37 38 38.5 38 38 38 38.5 38.5 38.5 38 38 38.5 38 38 38 38.5 38 38.5 38 38 38 38 38 38 38 38 38
Maximum survival time (days) and E/S in parentheses
7 7 7 2
Controls
Experimental
A/J
(days)
24/44
1 (O/12) 4 (10/20)
213 2/4 2/4
(4/4) (4/4) (4/4) (U/22)
1 (9/9)
214 3/5 213 17/17
2 1 1 3 1 1 0
2 (13/H) (6/9) (6/6) (l/6) (l/4) (7/9) (l/4) (O/15)
alI degenerate
1 3 3 0
(4/6) (6/6) (6/10) (O/15)
2 2 3 2 2 2 2
(3/9) (5/9) (5/6) (l/16) (6/g) (3/6) (3/6)
1 (213) 1 (4/6)
degenerate
O/11 (1) 3/3 (3); O/2 (7) O/2 O/2 11/15
(7) (7) (2)
l/3
(2) (2)
17/17 616 2/3
O/l O/g O/2 O/l5
(3) (2) (I)
213 17117
3/5 O/6
(2) (3);
6/6 all degenerate
2/3 17/17
A/J and time
in parentheses
3/7
fv6
2 (3/15) 2 (6/9) 2 (g/9)
in vitro
6/6 (4)
O/3 (2)
2/7 (3) O/15 (1) O/9
(3)
O/3
(1)
2 days
3 (2/6)
E/S =
Number of juveniles still active Number of juveniles at start of experiment
A/J =
Number of adult worms obtained from rat Number of juveniles injected into small intestine of rat
The addition of chick embryo extract, pregnant human serum, Tenebrio extract, horse serum ultra filtrate, H. diminuta extract, rat intestine extract, bacteria1 metabolites or a mixture of H. diminuta extract, and chick embryo extract (media U, Y, S, P, Q, BB, CC, DD and EE, respectively) did not support the activty of these parasites for more than 2 days and in fact media Q, S, and DD appeared to be toxic.
Juveniles of H. nana Table V shows that the maximum period of survival (9 days) was obtained in one experiment with medium K but that the organisms were not infective to mice at the end of this time. On the other hand, the juveniles survived for 8 days and still retained their infectivty to mice in medium R, the difference between these media being that R contains calf serum whereas K con-
AXENIC
Survival Temperature (” C)
Medium
37 38 37 37 37 38 38 38.5 38.5 38 38 38.5 38 38 38.5 38 38 38 38 38 38.5 38.5 38.5 38.5 38 38.5 38.5 38.5 38 38 38 38 38 38 38 38 38 38 38 38
:
JI J2 K Kl Kl Kl Kl Kl
0 0 P R R R S T U U U U
Ul u2 u2
x
Xl x2 Y Yl 2
AA BB BB cc DD EE FF GG HH E/S = A,J
=
CULTURE
OF RODENT
TABLE V of Hymenolepis nana Juveniles
Maximum survival time (days) and E/S in parentheses 4 4 4 4 4 9 5 3 3 4 9 2 5 8 3 4 3 5 3 6 3
(2/S) (S/15) (2/10) (2/6) (4/S) (3/15)
(2/15)
degenerate
(J/6) (8/15) (3/15) (3/9)
2 (S/12) 1 (6/12) 2 (2/12) 3 (S/6) 0; 1 day degenerate
2 (l/12) 0; 2 4 3 2 4 3 2 0 0 0 0 3
A/J 112 112 112 112
(4/9) (l/12) (4/6) (l/15) (6/9) (7/15) (4/9) (l/6) (2/15)
Controls
4/S
(6/W
1 day degenerate (4/6) (2/6) ; 3 (4/6) (6/6) (3/6) (l/6); 2 (3/6) (3/3) (3/6) (O/6) toxic (O/6) toxic (O/6) toxic (O/6) toxic (3/6)
183
TAPEWORMS
4/S 6110 4/S l/10 o/17 4/S 4/S 6110 6/10 l/10 o/17 2/3 4/S 21f3 6110 3/S l/10 l/10 l/10 o/17 l/10 l/10 l/10 o/17 o/17 o/17 O/5 o/17
W3 O/5
in vitro Experimental A/J and time (days) in parentheses O/l o/3 O/2 O/l O/l o/2 o/3 l/S o/3 3/3 O/2
(4) (4) (4) (4) (4); (9) (5); (3) (2) (3); (9)
l/l
(2)
l/3
(3)
O/l
(4)
l/l
(7)
O/l
(4)
016 (2) O/3 l/3 O/3 2/2 O/2 O/3 O/4 O/3 l/4
(5) (8); (2) (3); (3) (5) (3) (6) (3)
l/f5 (2) O/3 (2) O/3 (2) 3/3 (3) O/7 (2)
O/2 (4) Growth in length O/3 O/2 (4); o/2 (3) 3/3 O/3
5/6 5/6 5/6 5/6 8/S
2/3
Number of juveniles still active Number of juveniles at start of experiment Number Number
of adult worms obtained from rat at biopsy of juveniles injected into small intestine of rat
tains horse serum (Table I). In one experiment, rat intestine extract was included in the medium (Z). The juveniles survived for 3 days in this and doubled their length during
this time (Figs. 1 and 2). This was an encouraging result but unfortunately time did not permit any further experiments. Apart from the above, survival was only
184
TAYLOR
FIG. 1.
I).
Juvenile
of Hymenolepis
FIG. 2. Juvenile of Hymnolepis Stained Giemsa, X 700.
nana;
Control.
Stained
Giemsa,
nana; 4 days in vitro in culture
X 700.
medium
Z (Table
AXENIC
CULTURE
OF RODENT
TAPEWORMS
185
caution was that large numbers of controls were used for each experiment. There is some controversy in the literature over the normal mode of development of H. nana in T. con&sum. Volge and Heyneman (1957) considered that the scolex of the cysticercoid larva first develops externally and is then withdrawn into the cavity of the mid-body of the larva, whereas Schiller (1959) has presented evidence that “the scolex and internal membrane develop within the capsule of the cysticercoid.” He has observed that those forms with extruded scoDISCUSSION lices lack uniformity in their morphological The approach used in these experiments appearance except for a poorly formed scolex has been to dissect the larval tapeworms from external to the capsule. For this reason and the intermediate host and to try to induce that no particular morphological type could these larvae to develop to sexual maturity. be associated with a definite period of time One great advantage of such a starting point during development, he considers these orwas that the larvae were dissected from an ganisms to be abnormal. If that is so, then already sterile environment and hence asepsis it is not surprising to find that in the present presented no special problem. No complicated experiment the stages with an everted scolex sterilization procedure, such as that used by did not show signs of development in vitro. Wardle and Green (1941) when they at- However, one would have expected to have tempted to culture adult tapeworms, was found some development of the morula stage had the culture conditions been suitable. The necessary. lack of growth at this stage was therefore Beetle stages an indication of inadequate culture condiA thorough knowledge of the normal tions. morphology of the parasite is essential for Wyatt and Kalf (1957) reported that the assessment of the effects of culture. In trehalose was a major blood sugar of insects. the case of H. diminuta and H. nana exten- Consequently trehalose was added to several sive studies of the morphology of the beetle of the media. Unfortunately it did not apstages had already been carried out by Voge pear to prolong survival or stimulate growth (1960) and Voge and Heyneman (1957, at the concentrations used here. The majority 1960), and their work provided a basis for of the media used appeared to be well the present study. tolerated by the morulae for they showed Although the beetles were given a rela- no signs of degeneration in vitro and in a few tively short infective feed (4 hours), when embryos early indications of growth were observed. With the more advanced embryos the larvae were dissected out at intervals later, they were found to be at various de- some further development was observed in velopmental stages, even those dissected media V, T, and L. However, in view of the from the same beetle. Thus it was difficult to small numbers of embryos recovered from obtain eperimental embryos of the same age the staining procedure, these experiments can only be regarded as of a preliminary nature. and the same stage prior to culture. This difficulty was overcome to some extent by the Much more work is required to elucidate the use of 2.5day or 6-day embryos, since 2.5 in vitro requirements of the early beetle stages of these organisms. days after the infective feed beetles contained The cysticercoid larvae of H. diminuta and embryos which had not developed beyond the morula stage and 6 days after the in- H. nana differed somewhat in their ability fective feed the embryos had not quite de- to survive in vitro; those of H. diminuta surveloped to the infective stage. Another pre- vived and remained infective for 7 days,
maintained for about 3 to 5 days in the other media used. Thus the addition of chick embryo extract, pregnant human serum, normal human serum, Tenebrio extract, horse serum ultra filtrate, H. nana extract, H. diminuta extract, rat intestine extract, bacterial metabolites, or a mixture of H. dimifluta extract and chick embryo extract (media U, Y, yl, s, HH, P, Q, AA, BB, CC, DD, and EE, respectively) had no effect on the survival of the juveniles, at the concentrations used here.
186
TAYLOR
which was slightly longer than cysticercoid larvae of H. nana at 25”. Rausch and Jentoft (1957) cultured the scolices of Echinococcus multilocularis in vitro obtaining proliferation and survival up to 30 days. These authors also used infectivity as a criterion for assessing their results for, when the cultured scolices were introduced into the peritoneal cavity of a natural intermediate host (the vole), they proliferated and produced new scolices. Unfortunately Rausch and Jentoft had too few cultured scolices to attempt to infect the canine definitive host. Juveniles The experiments using excysted juveniles have shown that there are differences in ability to survive in vitro between the two species used. The juveniles of H. diminuta survived for a maximum of 7 days in media J, J1, and Jz, but were only infective for the first 4 days in medium J at 38’ C. On the other hand, the juveniles of H. nana survived for 9 days in media K and 0, and 8 days in medium R; they remained infective for the full culture period in medium R. Further experiments of this nature may be helpful in indicating some of the physiological differences between the two species. However, it was not altogether surprising that H. nana proved to be more amenable to culture than H. diminuta since it can complete the whole of its life-cycle within the mammalian host alone. A surprising result was that the addition of serum to medium 199 prolonged the survival of these intestinal parasites. An interesting result in passing was that possibly the gonadotropic hormones, as supplied by pregnant human serum, were toxic whereas normal human to the juveniles, serum was tolerated for 3 days with the retention of their infectivity. The fact that some growth was obtained in one experiment with H. nana in medium Z was most encouraging. Medium Z contained an extract of mouse intestine (see Methods) which probably provided them with some essential growth factor. Schiller, Read, and Rothman (1959) have also reported growth in H. diminuta. They cultured the scolex and neck from adult worms in a medium containing 50% horse serum and an extract of
the adult worm, which resulted in a 30-fold increase in size by the 11th day. This type of growth really constitutes regeneration of the strobila from the scolex and may differ somewhat from the type of growth that might be obtained from the culture of a juvenile worm. It would appear that juvenile worms may require some “trigger” to be present in the medium before growth will occur. Berntzen (1960) has reported an effective method for the in vitro culture of H. diminuta. In this he stated that larvae of H. diminuta were isolated from infected beetles and grown in vitro (using several types of media) to adults which contained onchospheres. He used an apparatus designed to provide a continuous flow of medium over the worms and this culture experiment was terminated on the 15th day. It is to be hoped that this experiment will soon be reported in greater detail since it may represent a considerable advance in the study of parasites by their culture in vitro. ACKNOWLEDGMENTS
It Ball
is a pleasure and
his staff
they extended
to for
thank the
Professor facilities
and
to me. I am very grateful
Gordon
H.
hospitality to Professor
Ball, Dr. Marietta Voge, Dr. Donald Heyneman, and Dr. Jowett Chao for several stimulating discussions during this work, and I would also like to thank Mr. Glen Harrington for his willing and skilful technical assistance. made to Mrs. Nora
Grateful acknowledgment is Liu for histological preparation
of the beetle stages. This work was supported by a Post Doctoral Trainee Fellowship (ZE-70) from the U. S. Public Health Service, and the travel to California was made possible by a Fulbright Travel Award. REFERENCES ADAMS, M. H. 1959. Bacteriophages. Publishers Inc., New York, p. 446.
Interscience
BALL, G. H., AND CHAO, J. 1960. In vitro development of the mosquito phase of Plasmodium relicturn. Experimental Parasitology 9, 47-55. BERNTZCEN, A. K. 1960. An effective method for the in vitro cultivation of Hymenolepis diminuta. Journal of Parasitology 46 (Sec. 2), 47. EAGLE, H., OYAMA, V. I., LEVY, M., AND FREEMAN, A. E. 1957. Myo-inositol as an essential growth factor for normal and malignant cells in tissue culture. Journal of Biological Chemistry 226, 191.
AXENIC
CULTURE
OF RODENT
MORGAN, J. F., MORTON, H. J., AND PARKER, R. C. 1950. Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proceeding of the Society for Experimental Biology and Medicine 73, 1. RUJSCH, R. L., AND JENTOFT, V. L. 1957. Studies on the helminth fauna of Alaska. XxX1. Observations on the propagation of the larval Echinococcus multilocularis Leuckart, 1863, in vitro. Journal of Parasitology 43, 1-8. ROTHMAN, A. H. 1959. Studies on the excystment of tapeworms. Experimental Parasitology 8, 336364. SCHILLER, E. L. 1959. Experimental studies on morphological variations in the cestode genus Hymenolepis. I. Morphology and development of the cysticercoid of H. nana in Tribolium conjusum. Experimental Parasitology 8, 91-118. SCHILLER, E. L., REaD, C. P., AND ROTHMAN, A. H. 1959. Preliminary experiments on the growth of a cyclophyllidean cestode in vitro. Journal of Parasitology 46 (4 sect. 2), 45. VOGE, M. 1960. Studies in cysticercoid histology. I. Observations on the fully developed cysticercoid
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of Hymenolepis diminuta (Cestoda, Cyclophyllidea). Proceedings of the Helminihological Society of Washington 27 (1) 32-36. VOGE, M., AND HEYNEMAN, D. 1957. Development of Hymenolepis nana and Hymenolepis diminuta (Cestoda Hymenolepididae) in the intermediate host Tribolium confusum. University of California Publications in Zoology 59, 549-580. VOCE, M., AND HEYNEMAN, D. 1960. Studies in cysticercoid histology. II. Observations on the fully developed cysticercoid of Hymenolepsis nana (Cestoda, Cyclophyllidea) . Proceedings of the Helminthological Society of Washington 27, 185. WARDLE, R. A., AND GREEN, N. K. 1941. The cultivation of tapeworms in artificial media. Canadian Journal of Research D19, 240. WYATT, G. R., AND KALF, G. F. 1957. The chemistry of insect haemolymph. II. Trehalose and other carbohydrates. Journal of General Physiology 40, 833-847. WYATT, S. S. 1956. Culture in vitro of tissue from the silkworm Bombyx mori L. Journal of General Physiology 39, 841.