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Dictyocaulus viviparus: Migration in agar of larvae subjected to a variety of physicochemical exposures
EXPERIMENTAL
PARASITOLOGY
Dictyocaulus
49, 106- 115 (1980)
viviparus: Migration in Agar of Larvae Subjected Variety of Physicochemical Exposures
to a
ROLF JEW JORGENSEN Institute
of Veterinary
Microbiology and Hygiene, Royal Veterinary and Agricultural Biilowsvej 13. DK-I870 Copenhagen V. Denmark
university,
(Accepted for publication 19 July 1979) JORGENSEN, R. J. 1980.Dictyocaulus viviparus: Migration in agar of larvae subjected to a variety of physiochemical exposures. Experimental Parasitology 49, 106- 115. Dictyocaulus vivipurus larvae were exposed to ox bile and CO, at intervals during their cultivation to the infective stage. Preinfective and young infective larvae were stimulated by COP, Bile slightly inhibited preinfective larvae, but stimulated the infective stage. Old coiled, resting infective larvae were stimulated by bile down to a concentration of 10 ppm of bile dry matter. by vertebrate biles of pig, sheep, newborn calf, cow, guinea pig, dog, and chicken, as well as by defatted bile dry matter and by glyco-, tauro-, glycodeoxy-, and taurodeoxycholates. Continuous bile exposure appeared necessary to maintain high larval activity. A high pC0, as well as a low redox potential potentiated the effect of bile, but had no effect alone. Exposure to pepsin-HCl and to trypsin had only a minor stimulatory effect. INDEX DESCRIPTORS: Dictyocaulus viviparus; Nematode, parasitic; Larval triggers; Host response; Migration, agar gel; Bile; Bile salts; Pepsin; Trypsin; Triggers.
cultured in water they show lower mortality and a higher degree of infectivity (Jarrett et al. 1954). Resting third-stage larvae of Dictyocaulus viviparus become activated when exposed to ox bile at body temperature (Jorgensen 1973). Such activated larvae were capable of migration in agar gel (Jorgensen 1975a) so that larval motility could be expressed quantitatively and far more accurately than by conventional observation of movements. The purpose of the reported experiments was to follow the response of developing larvae to simulated host stimuli and to relate any such changes in response to the stages in development which occur during cultivation to the infective stage. Furthermore, an attempt was made to study the activation process of coiled, resting infective larvae in more detail.
Host stimuli which initiate the further development of parasitic helminth larvae have been studied by several workers, mostly as studies in vitro. In species where the infective stage is inactive, the reassumption of activity may be used as a criterion for host stimulus, e.g. the evagination of the proboscis of cystacanths (Lackie 1974), the evagination of cysticerci (Campbell 1963), and the excystation of metacercariae (Wikerhauser 1960; Dixon 1966). In the trichostrongyles, exsheathment rather than increased locomotion is the criterion most commonly used (Rogers 1960; Taylor and Whitlock 1960; Mapes 1972). The infective stage of Dictyocaufus viviparus, parasitic in cattle, shows a low level of motility (Soliman 1953), and when larvae are cultured to the infective stage on feces, MATERIALS AND METHODS their sluggish movements gradually cease so that such cultures eventually consist of Bile. Ox bile was collected at the resting coiled larvae. Compared to larvae slaughterhouse, quickly cooled, and lyoph106 0014-4894/80/010106-10$02.00/O Copyright @ 1980 by Academic Press, Inc. AU rights of reproduction in any form reserved.
Dictyocaulus
viviparus:
STIMULATION
ilized. Only clear, bright green bile was collected. A rehydrated 10% of ox bile dry matter was referred to as “standard bile.” Bile from other vertebrate species listed in Table III was stored in the frozen state and thawed on the day of testing. Bile fractions and bile salts. Highly purified glyco-, tauro-, glycodeoxy-, and taurodeoxycholate as well as defatted bile dry matter were provided by G. A. D. Haslewood, Biochemistry and Chemistry Department, Guys Hospital Medical School, London. Test solutions. Six commercially available detergents were used. They are listed in Table V. A 2% solution of pepsin in water was made and adjusted to pH 2.5 with dilute HCl. A 2% solution of trypsin was adjusted to pH 8.9 with bicarbonate. A 1% solution of dithionite (Merck) was prepared. Larvae. With the exception of Experiments 1 and 2, Dictyocaulus viviparus larvae were cultured to the infective stage on feces at 16 C and subsequently stored at 4 C in shallow water for 2 to 6 months. The larvae were all coiled and showed no movements when examined under the microscope. General procedure. Each experiment consisted of one or more tests in which defined volumes of a suspension of larvae in water were exposed to solutions of various chemicals or to gases. As a parallel to this, identical volumes were processed as controls. The principle and the basic procedures used were as previously described (Jorgensen 1975a). Freshly prepared liquid agar, kept at 50 C in a water bath, was mixed with the test substance, cooled to 40 C, mixed with the larval suspension, and poured into petri dishes. The petri dishes were allowed to set at room temperature before being incubated at 38 C. Thus exposure took place from the time of mixing and was terminated when larvae, present on the surface of the agar gel after 90 min of incubation, were washed off and counted. Dif-
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107
ferences between larval counts of test and control reflected differences in the speed of migration representing the extent of the response. The details of the procedures in each experiment were as follows. Experiment 1. Feces were collected over a 24-hr period from the rectum of an artificially infected calf and stored at 4 C. At the end of the period, larvae were extracted in water in pointed bottom sedimentation glasses (Nevenic et al. 1962) at 10 C and on a later occasion at 22 C. During the experiment the larvae were cultured in water at the respective temperatures in Ehrlenmeyer flasks under constant aeration with atmospheric air saturated with water. At intervals during cultivation the larvae were tested against 10% bile. Six agar plates in petri dishes were produced for each test by mixing agar containing larvae with bile (triplicates) as well as with water. The morphological appearance as well as the motility of the larvae were noted before each test was carried out. Experiment 2. A proportion of feces collected per rectum was baermannized at 16 C. The isolated larvae were cultured at 16 C as described in Experiment 1. Hot liquid agar was divided into two Ehrlenmeyer flasks and CO, and air, respectively, were bubbled through it for 1 hr. The portions were subdivided and bile and water, respectively, were added. The four mixtures were kept at 50 C. After cooling to 40 C equal volumes of the larval suspension were added and six plates were produced from each mixture. Plates produced from CO,-treated agar were incubated in a 50% mixture of CO, in air. Experiment 3. Two-milliliter serial dilutions of lyophilized bile were kept in test tubes at 38 C in a water bath. Eight milliliters of a larval suspension in liquid agar was added to each tube by means of a prewarmed syringe. The contents of each tube was mixed and immediately poured into petri dishes.
108
ROLFJESSJORGENSEN
Experiment 4. Eight-milliliter aliquots of a suspension of larvae in liquid agar were added to test tubes containing 2 ml of 50% of bile from the following species: pig, sheep, cattle (cow and newborn calf), guinea pig, dog, and chicken. Experiment 5. Three test tubes, A, B, and C, each containing 0.5 ml of larval suspension, were kept at 38 C in a water bath. Standard bile, 0.05 ml, was added to C. After 30 min exposure 20 ml of 10% bile was added to A, and 20 ml of water was added to B and C. The contents were immediately mixed with 20 ml of 3% agar each, in beakers, and aliquots of 4 ml were distributed into small (50 mm diameter) petri dishes. Experiment 6. One hundred twenty milliliters of 1.5% agar, kept at 50 C, was cooled to 38 C; 1 ml of the larval suspension was added. After thorough mixing 36 ml was transferred into each of three beakers kept at 38 C. These contained the test substance in 4 ml of water (A), an equal amount of standard bile in 4 ml of water (B), and 4 ml of water (C), respectively. The contents ,of each beaker were immediately mixed and aliquots of 4 ml distributed into ten 50mm-diameter petri dishes. The stimulating activity of a test compound was calculated as a percentage of that obtained by using standard bile, after subtraction of the spontaneous larval count obtained in water. Thus the activity was calculated using the following formula where L, stands for larval count:
to each tube, mixed, and poured into a petri dish. Experiment 8. Four rows of test tubes containing larval suspensions were kept in a 38 C water bath. Before the larvae were embedded in agar and incubated, acid-pepsin solution was added to two of the rows. Following 30 min exposure, the larvae were spun down and neutralized by sodium bicarbonate -trypsin solution. Bile was added to one of these rows and to one which received no pretreatment. Experiment 9. Aliquots of a mixture of larvae in agar were added to test tubes containing 0.5% dithionite, 10% bile, dithionite, and bile or water, respectively. Experiment IO. Six-month-old infective larvae were embedded in agar treated with CO, and atmospheric air with and without the addition of bile. For details, see Experiment 2. RESULTS
Experiment I: The response of the Free-Living Stages,to Bile Exposure
The purpose of this experiment was to see if the stimulatory effect of bile was a characteristic of the Dictyocaulus viviparus infective stage only. Furthermore it was attempted to follow and compare the rate of development morphologically as well as physiologically at the two temperature levels. It appears from Table I that preinfective larvae respond to bile in that their motility becomes slightly depressed. However, L, compound - L, Hz0 x looo/ during their development to the infective 0. L, standard - L, H,O stage an increasing proportion of larvae was recovered after bile treatment. A decrease The final mixtures of larvae in agar con- in observed motility was observed simultatained 0.23% of test substance or standard neously with the liberation of the second bile, respectively. sheath from the larval cuticle, indicating Experiment 7. Half a milliliter of the de- that the majority of the larvae had reached tergents in water in a concentration IO-fold the infective stage. At 10 C this took place the concentration to be tested was kept in after 6 days and at 22 C after 3 days. Simultest tubes in a 38 C water bath. Liquid 1.5% taneously with this development the ratio agar, 4.5 ml, containing larvae, was added between the number of larvae recovered
Dictyocaulus
viviparus:
STIMULATION
OF LARVAE
109
TABLE I Response of Preinfective and Infective Dictyocaulus viviparus Larvae Toward Bile Exposure, Expressed as the Ratio between No. of Larvae Recovered with and without Bile in the Agar Plate@ Age of larvae 0 hr
4 hr
6 hr
24 hr
2 days
3 days
4 days
6 days
8 days
10 c Motility No. of sheaths Ratio +I- bile
+++ 1’ 0.364
+++ 1’ 0.368
ne -
+++ 1 0.384
+++ 1 0.54
ne -
+++ 1 0.1
++ 2 1.6
++ 2 4.1
22 c Motility No. of sheaths Ratio +I- bile
ne -
ne -
+++ 1’ 0.23
+++ 1 0.54
+++ I 0.39
++ 2 2.4
++ 2 6.6
+ 2 16.4
+ 2 43.7
o Motility and morphological appearance of the larvae were observed before each test was carried out. * +++ = full motility. + = larvae mostly inactive. ne = not examined. c Only visible at the tip of the tail at high power magnification.
with and without bile exposure passed 1.0. In tests carried out on later occasions the larval cultures showed a more and more markedly positive bile response. Naturally this change occurred more rapidly at 22 C than at 10 C, so that at the end of the observation period (Day 8) 4.7-fold more larvae were recovered from the 10 C culture after bile exposure than without, whereas 43.7fold more larvae were recovered after cultivation at 22 C. From this experiment it may be concluded that bile has a slightly depressive effect on the motility of the spontaneously moving preinfective D. viviparus larvae, but that a stimulatory effect occurred simultaneously with the morphological development to the infective third stage. The observed separation of tail end and sheath of newly extracted larvae is discussed later.
in the rumen, a comparative study was set up where newly harvested larvae were exposed to COz, to bile, and to a combination of CO, and bile simultaneously, at intervals during their cultivation to the infective stage. Microscopical examination. During the first few days of cultivation, the tip of the larval tail was observed to be separated from the cuticle on most of the larvae. On the fourth day, the sheath was clearly visible at the posterior end as well as the anterior end of practically all larvae. At Days 5 and 6 the larvae appeared less active. At Day 7 the larvae were active again and the second sheath became visible on some. At this point, most took up iodine (staining) at a much slower rate than on previous occasions. The slow uptake of iodine is a characteristic of the infective stages of trichostrongyles and may indicate the presence of Experiment 2: Effect of CO, on the a protective cuticula. From Day 10 and onFree-Living Stages during Cultivation ward, the second sheath was visible on at 16 C most larvae, but during further cultivation The infective stage as well as the newly some larvae lost their first sheath. From hatched larvae of D. viviparus enter the di- Day 33 onward, most larvae were seen gestive system of the host during the life coiled but still slightly granulated. Most of cycle of the parasite. In order to determine the remaining larvae also appeared inactive whether preinfective larvae differ, not only but were stretched or slightly bent. Probamorphologically, but also physiologically in bly these larvae were dead or dying. Larval counts. A much more accurate astheir response to a high pCO,, as it is found
110
ROLF JESS JORGENSEN
o- - - -.- -.-.---.- -u -33 Age of larvae
FIG. 1. The response of Dicfyocauhs
tdays)
38
larvae toward CO, and bile during their cultivation in water at 16 C. (0) Nonexposed larvae, (A) bile exposure, (0) CO, exposure, (A) bile plus CO, exposure. Each determination is the mean larval count of six plates in petri dishes. viviparus
sessment of the spontaneous motility appears from Fig. 1, where the speed of migration is reflected in the larval counts obtained with nonexposed larvae. A low number of nonexposed larvae were recovered at Days 5 and 6. It is also interesting to note that at 16 C the change in bile response took place between Days 4 and 5. From the figure it appears that preinfective larvae are stimulated by a high pC0,. Apparently young infective larvae are also stimulated to a certain degree, before they coil up. One-week-old infective larvae showed a marked response to simultaneous bile plus CO, exposure. Although this exposure provided the best stimulus for older larvae, the larval count decreased considerably toward the end of the experiment. It may be the result of an increasing mortality observed in the culture, but it may also indicate that the in viva stimulus resulting from the physicochemical properties in the alimentary tract is of a more complex nature than the in vitro stimulus provided in this experiment.
Experiment 3: Sensitivity of Resting Infective Larvae to Different Concentrations of Bile The result of this experiment is shown in Table II. It appears that no D. viviparus larvae were recovered in the absence of bile. Although a decrease in the number of larvae recovered was experienced with decreasing concentrations of bile, it is evident that the larvae were highly sensitive to bile exposure since they responded to concentrations down to 10 ppm of bile dry matter. Experiment 4: The Response of Resting Infective Larvae to Bile from Various Vertebrate Species The composition of vertebrate bile may vary with species as well as with changes in feeding (Smyth and Haslewood 1963). The results in Table III show that these 56-dayold infective larvae still showed some spontaneous activity since a mean of 12 larvae was recovered in the absence of bile. When compared with this spontaneous ac-
Dictyocaulus viviparus: STIMULATION
OF LARVAE
111
TABLE II Number of 4-month-old Infective Dictyocaulus viviparus Larvae Recovered after Exposure to Different Concentrations of Bile in Water Dilution of bile dry matter
Larval counts mean of four plates (range)
Larval count in percentage of maximum count
1:50 1:lOO 1:200 l:l,OOO 1:10,000 1:100,008 1:500,000 H,O
267 (244-290) 250 (197-304) 231 (216-257) 124 (118-131) 13 (10-21) 2 (l-3) 0 0
100 94 87 47 5 0.8 0 0
As can be seen from the table, the 3month-old larval suspension showed practically no spontaneous activity. A 30-min pretreatment was able to activate the larvae to a certain degree, but this activation only resulted in 25% of the activation observed after continuous exposure. The experiment indicates that exposure to bile for a period Experiment 5: Short-Term versus greater than 30 min is necessary in order to Continuous Exposure of Resting ensure high activity. Thus it may be conInfective Larvae to Bile cluded, within the testing period used, that Larvae were exposed for 30 min to 10% the larval response to bile is not working bile and subsequently the bile concentra- through a so-called trigger mechanism tion was diluted 80 times during the 90 min (Bullock 1957). of incubation (Table IV, A). As a parallel to this, equal amounts of larvae were exposed Experiment 6: The Effect of Purified Bile Salts on Resting Infective Larvae continuously to 10%bile (Table IV, B) or not In order to determine which active comexposed at all (Table IV, C). ponents of bile produced stimulation, defatted bile dry matter was tested. In conTABLE III secutive tests an effect of 90 and 104% of Response of 2-month-old Infective Dictyocau/us tivity it can be seen that whole bile from all species examined was able to stimulate the larvae. Determination of dry matter content was not carried out, and therefore it is not possible to make a quantitative comparison between counts obtained from the various species.
Larvae to Exposure to 10% Solutions of Bile from Different Vertebrate Species
viviparus
Vertebrate species
Larval count Mean of three plates (range)
Pig Sheep Newborn calf cow Guinea pig Dog Chicken H,O
154 (138- 175) 130 (129-131) 151 (145-159) 156 (151-164) 133 (115-143) 131 (118-142) 116 (100-125) I2 (9-14)
TABLE IV Comparison of the Effect of 30 min Exposure of 3-month-old Infective Dicrjxxahs viviparus Larvae to Bile (A) with that of Continuous Exposure (120 min) (B), as well as no Exposure (C)
A B C
Larval count Mean of 10 plates (range)
Response in percentage of B
14 (12-17) 58 (48-64) 0.4 (O-l)
25 100 1
ROLF JESS JORGENSEN
112
that of standard bile was obtained, indicating that the active components were to be found among the nonlipids. Triplicate testings of highly purified bile salts gave the following results: Na-glycocholate: 60, 79, and 79%; Na-taurocholate: 17, 35, and 46%; Na-glycodeoxycholate: 104, 95, and 129%; Na-taurodeoxycholate: 99, 74, and 90%. These results show that at least four common bile salts were able to stimulate Dictyocaulus larvae. Experiment 7: Exposure of Resting Infective Larvae to Detergents
was used as a pretreatment and when used alone, i.e., without subsequent bile exposure. Experiment 9: Effect of a Reducing agent on Resting Infective Larvae Table VII shows that dithionite alone had no effect, but that the near-anaerobic condition resulting from the addition of dithionite was able to increase the effect exhibited by bile. Experiment 10: Effect of CO, on Resting Infective Larvae
This experiment is similar to Experiment The results presented in Table V show that none of the tested common detergents 2, but in this case, a much more uniform had any significant stimulatory effect on suspension of resting coiled larvae cultured 3-month-old infective Dictyocaulus larvae. on feces was used. The effect of CO, is indicated in Table Experiment 8: Effect of Pepsin and VIII. Apparently, CO, in itself is not able to Trypsin on Resting Infective Larvae activate the coiled, resting D. viviparus larSilverman and Podger (1964) reported vae. However, it has a stimulatory effect that exsheathment of D. viviparus larvae when such larvae are activated by bile exwas dependent on exposure to pepsin. posure. Its effect therefore seems to be Parker and Croll (1976) on the other hand, similar to that experienced after exposure showed that exsheathment took place in a to reducing agents. range of proteolytic enzymes. DISCUSSION From Table VI it appears that bile was by The lack of effect of detergents indicates far the most important factor in stimulating the larvae. However, exposure to enzymes that stimulation is due to the chemical comincreased the activity slightly, both when it position of bile salts rather than to their
Response of 3-month-old Infective
Dicfyocaulus
TABLE V rivpunrs Larvae to Commercially Available Detergents Concentrations
Test substance
l:l,Ooo
1:10,000
1:50,000
1:100,000
1:1,ooo,oOo
Haemo-sol
1
0
0
0
0
Dioctyl-sulfosuccinate
0
0
I
0
0
Laurylsulfate
0
0
0
0
0
Hyamin-104
0
0
0
0
0
Teepol
0
0
0
1
0
Lissapol
0
0
2
0
0
90
7
0
0
0
Bile H,O mean of ten determinations
_.___.____.___________________________ 0.4 (range O- 1)
_.____.________.______________________
Dictyocaulus
viviparus:
STIMULATION
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OF LARVAE
TABLE VI Stimulatory Effect of Enzymatic Pretreatment on 6-month-old Infective Dictyocaulus Subsequent exposure to bile
Row No.
Enzymatic pretreatment
I
Yes
Yes
No Yes No
Yes No No
II III Iv
surface active property. This is in contrast to Cysticercus bovis, which responds to detergents (Campbell 1963) as well as to bile. The fact that infective Dictyocaulus viviparus larvae are sensitive to concentrations down to the parts per million level suggests that a specific chemoreceptor is involved. This assumption is supported by the findings that the potentiated stimulation shown in Tables VII and VIII required the presence of bile. In the present experiments the only exception was the slight effect of enzymatic treatment without subsequent bile exposure. A possible explanation to this may be that stimulation resulted from exsheathment, since infective D. viviparus larvae were shown by Parker and Croll (1976) to exsheath in the presence of pepsin and other proteolytic enzymes. The more pronounced effect of the enzymatic exposure when given as a pretreatment to bile exposure may partly be explained by the presence of free COZ or a low pH rather than the result of enzymatic exposure. A comparison with similar work carried out with the infective stages of other parasite species suggests that infective D. viviTABLE VII Effect of Reducing Agent (Dithionite) on the Number of 6-month-old Infective Dictyocndus viviparus Larvae Recovered
Exposure 0.5% Dithionite 10% Bile Dithionite + bile Hz0
Larval count Mean of five plates (range) 1 (O-2) 31 (27-34) 87 (70- 108) 0 (O-l)
viviparus
Larvae
Larval counts Mean (k SE) of six plates
Corrected larval counts in percentage of row No. I
220 2 7 187 + 8 15 + 1 2-tl
100 85 6 0
parus larvae respond to basically the same exposures. A positive response to bile is common to a variety of infective stages of zooparasitic species such as Trichostrongylus colubriformis (Mapes 1972), Polymorphus minutus (Lackie 1974), Parorchis acanthus (Fried and Roth 1974), Eimeria tenella (Pratt 1937), Fusciola hepatica (Dixon 1966), oncospheres of Taenia pisiformis (Leonard and Leonard 1941) and of Echinococcus granulosus (Berberian 1957), and cysticerci (Campbell and Richardson 1960). The detailed work of Dixon (1966) showed that activation of metacercariae was initiated by high concentrations of carbon dioxide, reducing conditions, and a temperature about 39 C; that reducing conditions increased the rate of action and that bile was essential for the emergence of such activated metacercariae. Taylor (1951) stated that the larvae passed in feces are in their first stage, and Soliman (1953) defined larvae passed in feces as first-stage larvae. This point of view is generally accepted (Soulsby 1960). However, original observations containing detailed descriptions on preinfective and infective stages have been made by DaubTABLE VIII Effect of CO, on Number of 6-month-old Infective Dictyocaulus viviparus Larvae Recovered Larval count Mean of five plates Exposure Air co, Bile + air Bile + CO,
(range) 0 0 41 (35-50) 7.5 (59-84)
ROLF JESS JORGENSEN
114
ney (1920) who worked with D. viviparus larvae cultures from eggs. He observed that larvae lie almost motionless during both first and second lethargus. The finding of only one period of inactivity in both Experiments 1 and 2 before the larvae reached the infective stage indicates that only one lethargus took place. This is consistent with the microscopical finding that larvae examined immediately after they were isolated from fresh feces were in the process of liberating themselves from the first-stage cuticle, i.e., they have initiated their first molt indicating that such larvae have entered their second stage. The reported experiments are based on migration in agar gel. Since the introduction of the technique (Jorgensen 1975a) this principle has been developed for particular purposes, such as the isolation of infective D. vivipavus larvae (Jorgensen 1975b), of infective Ostertagia sp. and Haemonchus sp. larvae (Mwegoha and Jorgensen 1977), as well as of a variety of adults and immatures of gastrointestinal species parasitizing sheep (van Wyk and Gerber 1978). Technically the principle worked satisfactorily in the present experiments and it is suggested that it would be equally useful in similar work with species other than Dictyocaulus viviparus, with different species for comparison, and in other exposure studies such as anthelminthic screening tests. ACKNOWLEDGMENTS I wish to express my thanks to Professor G. A. D. Haslewood, Biochemistry and Chemistry Department, Guy’s Hospital Medical School, London, for preparing the bile fractions and the purified salts used in Experiment 6. I am also grateful to Dr. D. Poynter of Allen and Hanburys Research Ltd., England, for providing the infective Dictyocaulus taiviparus larvae used in Experiment 4. The technical assistance of Mrs. Karen Madsen and Mrs. Bente Hein is greatly acknowledged. This work was supported by the Danish Agricultural and Veterinary Research Council. REFERENCES BERBERIAN, D. A. 1957. Host specificity and the effect of digestive juices on ova ofE. granulosus. 10th Report Orient Hospital.
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PARASITOLOGY
Dictyocaulus
49, 106- 115 (1980)
viviparus: Migration in Agar of Larvae Subjected Variety of Physicochemical Exposures
to a
ROLF JEW JORGENSEN Institute
of Veterinary
Microbiology and Hygiene, Royal Veterinary and Agricultural Biilowsvej 13. DK-I870 Copenhagen V. Denmark
university,
(Accepted for publication 19 July 1979) JORGENSEN, R. J. 1980.Dictyocaulus viviparus: Migration in agar of larvae subjected to a variety of physiochemical exposures. Experimental Parasitology 49, 106- 115. Dictyocaulus vivipurus larvae were exposed to ox bile and CO, at intervals during their cultivation to the infective stage. Preinfective and young infective larvae were stimulated by COP, Bile slightly inhibited preinfective larvae, but stimulated the infective stage. Old coiled, resting infective larvae were stimulated by bile down to a concentration of 10 ppm of bile dry matter. by vertebrate biles of pig, sheep, newborn calf, cow, guinea pig, dog, and chicken, as well as by defatted bile dry matter and by glyco-, tauro-, glycodeoxy-, and taurodeoxycholates. Continuous bile exposure appeared necessary to maintain high larval activity. A high pC0, as well as a low redox potential potentiated the effect of bile, but had no effect alone. Exposure to pepsin-HCl and to trypsin had only a minor stimulatory effect. INDEX DESCRIPTORS: Dictyocaulus viviparus; Nematode, parasitic; Larval triggers; Host response; Migration, agar gel; Bile; Bile salts; Pepsin; Trypsin; Triggers.
cultured in water they show lower mortality and a higher degree of infectivity (Jarrett et al. 1954). Resting third-stage larvae of Dictyocaulus viviparus become activated when exposed to ox bile at body temperature (Jorgensen 1973). Such activated larvae were capable of migration in agar gel (Jorgensen 1975a) so that larval motility could be expressed quantitatively and far more accurately than by conventional observation of movements. The purpose of the reported experiments was to follow the response of developing larvae to simulated host stimuli and to relate any such changes in response to the stages in development which occur during cultivation to the infective stage. Furthermore, an attempt was made to study the activation process of coiled, resting infective larvae in more detail.
Host stimuli which initiate the further development of parasitic helminth larvae have been studied by several workers, mostly as studies in vitro. In species where the infective stage is inactive, the reassumption of activity may be used as a criterion for host stimulus, e.g. the evagination of the proboscis of cystacanths (Lackie 1974), the evagination of cysticerci (Campbell 1963), and the excystation of metacercariae (Wikerhauser 1960; Dixon 1966). In the trichostrongyles, exsheathment rather than increased locomotion is the criterion most commonly used (Rogers 1960; Taylor and Whitlock 1960; Mapes 1972). The infective stage of Dictyocaufus viviparus, parasitic in cattle, shows a low level of motility (Soliman 1953), and when larvae are cultured to the infective stage on feces, MATERIALS AND METHODS their sluggish movements gradually cease so that such cultures eventually consist of Bile. Ox bile was collected at the resting coiled larvae. Compared to larvae slaughterhouse, quickly cooled, and lyoph106 0014-4894/80/010106-10$02.00/O Copyright @ 1980 by Academic Press, Inc. AU rights of reproduction in any form reserved.
Dictyocaulus
viviparus:
STIMULATION
ilized. Only clear, bright green bile was collected. A rehydrated 10% of ox bile dry matter was referred to as “standard bile.” Bile from other vertebrate species listed in Table III was stored in the frozen state and thawed on the day of testing. Bile fractions and bile salts. Highly purified glyco-, tauro-, glycodeoxy-, and taurodeoxycholate as well as defatted bile dry matter were provided by G. A. D. Haslewood, Biochemistry and Chemistry Department, Guys Hospital Medical School, London. Test solutions. Six commercially available detergents were used. They are listed in Table V. A 2% solution of pepsin in water was made and adjusted to pH 2.5 with dilute HCl. A 2% solution of trypsin was adjusted to pH 8.9 with bicarbonate. A 1% solution of dithionite (Merck) was prepared. Larvae. With the exception of Experiments 1 and 2, Dictyocaulus viviparus larvae were cultured to the infective stage on feces at 16 C and subsequently stored at 4 C in shallow water for 2 to 6 months. The larvae were all coiled and showed no movements when examined under the microscope. General procedure. Each experiment consisted of one or more tests in which defined volumes of a suspension of larvae in water were exposed to solutions of various chemicals or to gases. As a parallel to this, identical volumes were processed as controls. The principle and the basic procedures used were as previously described (Jorgensen 1975a). Freshly prepared liquid agar, kept at 50 C in a water bath, was mixed with the test substance, cooled to 40 C, mixed with the larval suspension, and poured into petri dishes. The petri dishes were allowed to set at room temperature before being incubated at 38 C. Thus exposure took place from the time of mixing and was terminated when larvae, present on the surface of the agar gel after 90 min of incubation, were washed off and counted. Dif-
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ferences between larval counts of test and control reflected differences in the speed of migration representing the extent of the response. The details of the procedures in each experiment were as follows. Experiment 1. Feces were collected over a 24-hr period from the rectum of an artificially infected calf and stored at 4 C. At the end of the period, larvae were extracted in water in pointed bottom sedimentation glasses (Nevenic et al. 1962) at 10 C and on a later occasion at 22 C. During the experiment the larvae were cultured in water at the respective temperatures in Ehrlenmeyer flasks under constant aeration with atmospheric air saturated with water. At intervals during cultivation the larvae were tested against 10% bile. Six agar plates in petri dishes were produced for each test by mixing agar containing larvae with bile (triplicates) as well as with water. The morphological appearance as well as the motility of the larvae were noted before each test was carried out. Experiment 2. A proportion of feces collected per rectum was baermannized at 16 C. The isolated larvae were cultured at 16 C as described in Experiment 1. Hot liquid agar was divided into two Ehrlenmeyer flasks and CO, and air, respectively, were bubbled through it for 1 hr. The portions were subdivided and bile and water, respectively, were added. The four mixtures were kept at 50 C. After cooling to 40 C equal volumes of the larval suspension were added and six plates were produced from each mixture. Plates produced from CO,-treated agar were incubated in a 50% mixture of CO, in air. Experiment 3. Two-milliliter serial dilutions of lyophilized bile were kept in test tubes at 38 C in a water bath. Eight milliliters of a larval suspension in liquid agar was added to each tube by means of a prewarmed syringe. The contents of each tube was mixed and immediately poured into petri dishes.
108
ROLFJESSJORGENSEN
Experiment 4. Eight-milliliter aliquots of a suspension of larvae in liquid agar were added to test tubes containing 2 ml of 50% of bile from the following species: pig, sheep, cattle (cow and newborn calf), guinea pig, dog, and chicken. Experiment 5. Three test tubes, A, B, and C, each containing 0.5 ml of larval suspension, were kept at 38 C in a water bath. Standard bile, 0.05 ml, was added to C. After 30 min exposure 20 ml of 10% bile was added to A, and 20 ml of water was added to B and C. The contents were immediately mixed with 20 ml of 3% agar each, in beakers, and aliquots of 4 ml were distributed into small (50 mm diameter) petri dishes. Experiment 6. One hundred twenty milliliters of 1.5% agar, kept at 50 C, was cooled to 38 C; 1 ml of the larval suspension was added. After thorough mixing 36 ml was transferred into each of three beakers kept at 38 C. These contained the test substance in 4 ml of water (A), an equal amount of standard bile in 4 ml of water (B), and 4 ml of water (C), respectively. The contents ,of each beaker were immediately mixed and aliquots of 4 ml distributed into ten 50mm-diameter petri dishes. The stimulating activity of a test compound was calculated as a percentage of that obtained by using standard bile, after subtraction of the spontaneous larval count obtained in water. Thus the activity was calculated using the following formula where L, stands for larval count:
to each tube, mixed, and poured into a petri dish. Experiment 8. Four rows of test tubes containing larval suspensions were kept in a 38 C water bath. Before the larvae were embedded in agar and incubated, acid-pepsin solution was added to two of the rows. Following 30 min exposure, the larvae were spun down and neutralized by sodium bicarbonate -trypsin solution. Bile was added to one of these rows and to one which received no pretreatment. Experiment 9. Aliquots of a mixture of larvae in agar were added to test tubes containing 0.5% dithionite, 10% bile, dithionite, and bile or water, respectively. Experiment IO. Six-month-old infective larvae were embedded in agar treated with CO, and atmospheric air with and without the addition of bile. For details, see Experiment 2. RESULTS
Experiment I: The response of the Free-Living Stages,to Bile Exposure
The purpose of this experiment was to see if the stimulatory effect of bile was a characteristic of the Dictyocaulus viviparus infective stage only. Furthermore it was attempted to follow and compare the rate of development morphologically as well as physiologically at the two temperature levels. It appears from Table I that preinfective larvae respond to bile in that their motility becomes slightly depressed. However, L, compound - L, Hz0 x looo/ during their development to the infective 0. L, standard - L, H,O stage an increasing proportion of larvae was recovered after bile treatment. A decrease The final mixtures of larvae in agar con- in observed motility was observed simultatained 0.23% of test substance or standard neously with the liberation of the second bile, respectively. sheath from the larval cuticle, indicating Experiment 7. Half a milliliter of the de- that the majority of the larvae had reached tergents in water in a concentration IO-fold the infective stage. At 10 C this took place the concentration to be tested was kept in after 6 days and at 22 C after 3 days. Simultest tubes in a 38 C water bath. Liquid 1.5% taneously with this development the ratio agar, 4.5 ml, containing larvae, was added between the number of larvae recovered
Dictyocaulus
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STIMULATION
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109
TABLE I Response of Preinfective and Infective Dictyocaulus viviparus Larvae Toward Bile Exposure, Expressed as the Ratio between No. of Larvae Recovered with and without Bile in the Agar Plate@ Age of larvae 0 hr
4 hr
6 hr
24 hr
2 days
3 days
4 days
6 days
8 days
10 c Motility No. of sheaths Ratio +I- bile
+++ 1’ 0.364
+++ 1’ 0.368
ne -
+++ 1 0.384
+++ 1 0.54
ne -
+++ 1 0.1
++ 2 1.6
++ 2 4.1
22 c Motility No. of sheaths Ratio +I- bile
ne -
ne -
+++ 1’ 0.23
+++ 1 0.54
+++ I 0.39
++ 2 2.4
++ 2 6.6
+ 2 16.4
+ 2 43.7
o Motility and morphological appearance of the larvae were observed before each test was carried out. * +++ = full motility. + = larvae mostly inactive. ne = not examined. c Only visible at the tip of the tail at high power magnification.
with and without bile exposure passed 1.0. In tests carried out on later occasions the larval cultures showed a more and more markedly positive bile response. Naturally this change occurred more rapidly at 22 C than at 10 C, so that at the end of the observation period (Day 8) 4.7-fold more larvae were recovered from the 10 C culture after bile exposure than without, whereas 43.7fold more larvae were recovered after cultivation at 22 C. From this experiment it may be concluded that bile has a slightly depressive effect on the motility of the spontaneously moving preinfective D. viviparus larvae, but that a stimulatory effect occurred simultaneously with the morphological development to the infective third stage. The observed separation of tail end and sheath of newly extracted larvae is discussed later.
in the rumen, a comparative study was set up where newly harvested larvae were exposed to COz, to bile, and to a combination of CO, and bile simultaneously, at intervals during their cultivation to the infective stage. Microscopical examination. During the first few days of cultivation, the tip of the larval tail was observed to be separated from the cuticle on most of the larvae. On the fourth day, the sheath was clearly visible at the posterior end as well as the anterior end of practically all larvae. At Days 5 and 6 the larvae appeared less active. At Day 7 the larvae were active again and the second sheath became visible on some. At this point, most took up iodine (staining) at a much slower rate than on previous occasions. The slow uptake of iodine is a characteristic of the infective stages of trichostrongyles and may indicate the presence of Experiment 2: Effect of CO, on the a protective cuticula. From Day 10 and onFree-Living Stages during Cultivation ward, the second sheath was visible on at 16 C most larvae, but during further cultivation The infective stage as well as the newly some larvae lost their first sheath. From hatched larvae of D. viviparus enter the di- Day 33 onward, most larvae were seen gestive system of the host during the life coiled but still slightly granulated. Most of cycle of the parasite. In order to determine the remaining larvae also appeared inactive whether preinfective larvae differ, not only but were stretched or slightly bent. Probamorphologically, but also physiologically in bly these larvae were dead or dying. Larval counts. A much more accurate astheir response to a high pCO,, as it is found
110
ROLF JESS JORGENSEN
o- - - -.- -.-.---.- -u -33 Age of larvae
FIG. 1. The response of Dicfyocauhs
tdays)
38
larvae toward CO, and bile during their cultivation in water at 16 C. (0) Nonexposed larvae, (A) bile exposure, (0) CO, exposure, (A) bile plus CO, exposure. Each determination is the mean larval count of six plates in petri dishes. viviparus
sessment of the spontaneous motility appears from Fig. 1, where the speed of migration is reflected in the larval counts obtained with nonexposed larvae. A low number of nonexposed larvae were recovered at Days 5 and 6. It is also interesting to note that at 16 C the change in bile response took place between Days 4 and 5. From the figure it appears that preinfective larvae are stimulated by a high pC0,. Apparently young infective larvae are also stimulated to a certain degree, before they coil up. One-week-old infective larvae showed a marked response to simultaneous bile plus CO, exposure. Although this exposure provided the best stimulus for older larvae, the larval count decreased considerably toward the end of the experiment. It may be the result of an increasing mortality observed in the culture, but it may also indicate that the in viva stimulus resulting from the physicochemical properties in the alimentary tract is of a more complex nature than the in vitro stimulus provided in this experiment.
Experiment 3: Sensitivity of Resting Infective Larvae to Different Concentrations of Bile The result of this experiment is shown in Table II. It appears that no D. viviparus larvae were recovered in the absence of bile. Although a decrease in the number of larvae recovered was experienced with decreasing concentrations of bile, it is evident that the larvae were highly sensitive to bile exposure since they responded to concentrations down to 10 ppm of bile dry matter. Experiment 4: The Response of Resting Infective Larvae to Bile from Various Vertebrate Species The composition of vertebrate bile may vary with species as well as with changes in feeding (Smyth and Haslewood 1963). The results in Table III show that these 56-dayold infective larvae still showed some spontaneous activity since a mean of 12 larvae was recovered in the absence of bile. When compared with this spontaneous ac-
Dictyocaulus viviparus: STIMULATION
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TABLE II Number of 4-month-old Infective Dictyocaulus viviparus Larvae Recovered after Exposure to Different Concentrations of Bile in Water Dilution of bile dry matter
Larval counts mean of four plates (range)
Larval count in percentage of maximum count
1:50 1:lOO 1:200 l:l,OOO 1:10,000 1:100,008 1:500,000 H,O
267 (244-290) 250 (197-304) 231 (216-257) 124 (118-131) 13 (10-21) 2 (l-3) 0 0
100 94 87 47 5 0.8 0 0
As can be seen from the table, the 3month-old larval suspension showed practically no spontaneous activity. A 30-min pretreatment was able to activate the larvae to a certain degree, but this activation only resulted in 25% of the activation observed after continuous exposure. The experiment indicates that exposure to bile for a period Experiment 5: Short-Term versus greater than 30 min is necessary in order to Continuous Exposure of Resting ensure high activity. Thus it may be conInfective Larvae to Bile cluded, within the testing period used, that Larvae were exposed for 30 min to 10% the larval response to bile is not working bile and subsequently the bile concentra- through a so-called trigger mechanism tion was diluted 80 times during the 90 min (Bullock 1957). of incubation (Table IV, A). As a parallel to this, equal amounts of larvae were exposed Experiment 6: The Effect of Purified Bile Salts on Resting Infective Larvae continuously to 10%bile (Table IV, B) or not In order to determine which active comexposed at all (Table IV, C). ponents of bile produced stimulation, defatted bile dry matter was tested. In conTABLE III secutive tests an effect of 90 and 104% of Response of 2-month-old Infective Dictyocau/us tivity it can be seen that whole bile from all species examined was able to stimulate the larvae. Determination of dry matter content was not carried out, and therefore it is not possible to make a quantitative comparison between counts obtained from the various species.
Larvae to Exposure to 10% Solutions of Bile from Different Vertebrate Species
viviparus
Vertebrate species
Larval count Mean of three plates (range)
Pig Sheep Newborn calf cow Guinea pig Dog Chicken H,O
154 (138- 175) 130 (129-131) 151 (145-159) 156 (151-164) 133 (115-143) 131 (118-142) 116 (100-125) I2 (9-14)
TABLE IV Comparison of the Effect of 30 min Exposure of 3-month-old Infective Dicrjxxahs viviparus Larvae to Bile (A) with that of Continuous Exposure (120 min) (B), as well as no Exposure (C)
A B C
Larval count Mean of 10 plates (range)
Response in percentage of B
14 (12-17) 58 (48-64) 0.4 (O-l)
25 100 1
ROLF JESS JORGENSEN
112
that of standard bile was obtained, indicating that the active components were to be found among the nonlipids. Triplicate testings of highly purified bile salts gave the following results: Na-glycocholate: 60, 79, and 79%; Na-taurocholate: 17, 35, and 46%; Na-glycodeoxycholate: 104, 95, and 129%; Na-taurodeoxycholate: 99, 74, and 90%. These results show that at least four common bile salts were able to stimulate Dictyocaulus larvae. Experiment 7: Exposure of Resting Infective Larvae to Detergents
was used as a pretreatment and when used alone, i.e., without subsequent bile exposure. Experiment 9: Effect of a Reducing agent on Resting Infective Larvae Table VII shows that dithionite alone had no effect, but that the near-anaerobic condition resulting from the addition of dithionite was able to increase the effect exhibited by bile. Experiment 10: Effect of CO, on Resting Infective Larvae
This experiment is similar to Experiment The results presented in Table V show that none of the tested common detergents 2, but in this case, a much more uniform had any significant stimulatory effect on suspension of resting coiled larvae cultured 3-month-old infective Dictyocaulus larvae. on feces was used. The effect of CO, is indicated in Table Experiment 8: Effect of Pepsin and VIII. Apparently, CO, in itself is not able to Trypsin on Resting Infective Larvae activate the coiled, resting D. viviparus larSilverman and Podger (1964) reported vae. However, it has a stimulatory effect that exsheathment of D. viviparus larvae when such larvae are activated by bile exwas dependent on exposure to pepsin. posure. Its effect therefore seems to be Parker and Croll (1976) on the other hand, similar to that experienced after exposure showed that exsheathment took place in a to reducing agents. range of proteolytic enzymes. DISCUSSION From Table VI it appears that bile was by The lack of effect of detergents indicates far the most important factor in stimulating the larvae. However, exposure to enzymes that stimulation is due to the chemical comincreased the activity slightly, both when it position of bile salts rather than to their
Response of 3-month-old Infective
Dicfyocaulus
TABLE V rivpunrs Larvae to Commercially Available Detergents Concentrations
Test substance
l:l,Ooo
1:10,000
1:50,000
1:100,000
1:1,ooo,oOo
Haemo-sol
1
0
0
0
0
Dioctyl-sulfosuccinate
0
0
I
0
0
Laurylsulfate
0
0
0
0
0
Hyamin-104
0
0
0
0
0
Teepol
0
0
0
1
0
Lissapol
0
0
2
0
0
90
7
0
0
0
Bile H,O mean of ten determinations
_.___.____.___________________________ 0.4 (range O- 1)
_.____.________.______________________
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STIMULATION
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TABLE VI Stimulatory Effect of Enzymatic Pretreatment on 6-month-old Infective Dictyocaulus Subsequent exposure to bile
Row No.
Enzymatic pretreatment
I
Yes
Yes
No Yes No
Yes No No
II III Iv
surface active property. This is in contrast to Cysticercus bovis, which responds to detergents (Campbell 1963) as well as to bile. The fact that infective Dictyocaulus viviparus larvae are sensitive to concentrations down to the parts per million level suggests that a specific chemoreceptor is involved. This assumption is supported by the findings that the potentiated stimulation shown in Tables VII and VIII required the presence of bile. In the present experiments the only exception was the slight effect of enzymatic treatment without subsequent bile exposure. A possible explanation to this may be that stimulation resulted from exsheathment, since infective D. viviparus larvae were shown by Parker and Croll (1976) to exsheath in the presence of pepsin and other proteolytic enzymes. The more pronounced effect of the enzymatic exposure when given as a pretreatment to bile exposure may partly be explained by the presence of free COZ or a low pH rather than the result of enzymatic exposure. A comparison with similar work carried out with the infective stages of other parasite species suggests that infective D. viviTABLE VII Effect of Reducing Agent (Dithionite) on the Number of 6-month-old Infective Dictyocndus viviparus Larvae Recovered
Exposure 0.5% Dithionite 10% Bile Dithionite + bile Hz0
Larval count Mean of five plates (range) 1 (O-2) 31 (27-34) 87 (70- 108) 0 (O-l)
viviparus
Larvae
Larval counts Mean (k SE) of six plates
Corrected larval counts in percentage of row No. I
220 2 7 187 + 8 15 + 1 2-tl
100 85 6 0
parus larvae respond to basically the same exposures. A positive response to bile is common to a variety of infective stages of zooparasitic species such as Trichostrongylus colubriformis (Mapes 1972), Polymorphus minutus (Lackie 1974), Parorchis acanthus (Fried and Roth 1974), Eimeria tenella (Pratt 1937), Fusciola hepatica (Dixon 1966), oncospheres of Taenia pisiformis (Leonard and Leonard 1941) and of Echinococcus granulosus (Berberian 1957), and cysticerci (Campbell and Richardson 1960). The detailed work of Dixon (1966) showed that activation of metacercariae was initiated by high concentrations of carbon dioxide, reducing conditions, and a temperature about 39 C; that reducing conditions increased the rate of action and that bile was essential for the emergence of such activated metacercariae. Taylor (1951) stated that the larvae passed in feces are in their first stage, and Soliman (1953) defined larvae passed in feces as first-stage larvae. This point of view is generally accepted (Soulsby 1960). However, original observations containing detailed descriptions on preinfective and infective stages have been made by DaubTABLE VIII Effect of CO, on Number of 6-month-old Infective Dictyocaulus viviparus Larvae Recovered Larval count Mean of five plates Exposure Air co, Bile + air Bile + CO,
(range) 0 0 41 (35-50) 7.5 (59-84)
ROLF JESS JORGENSEN
114
ney (1920) who worked with D. viviparus larvae cultures from eggs. He observed that larvae lie almost motionless during both first and second lethargus. The finding of only one period of inactivity in both Experiments 1 and 2 before the larvae reached the infective stage indicates that only one lethargus took place. This is consistent with the microscopical finding that larvae examined immediately after they were isolated from fresh feces were in the process of liberating themselves from the first-stage cuticle, i.e., they have initiated their first molt indicating that such larvae have entered their second stage. The reported experiments are based on migration in agar gel. Since the introduction of the technique (Jorgensen 1975a) this principle has been developed for particular purposes, such as the isolation of infective D. vivipavus larvae (Jorgensen 1975b), of infective Ostertagia sp. and Haemonchus sp. larvae (Mwegoha and Jorgensen 1977), as well as of a variety of adults and immatures of gastrointestinal species parasitizing sheep (van Wyk and Gerber 1978). Technically the principle worked satisfactorily in the present experiments and it is suggested that it would be equally useful in similar work with species other than Dictyocaulus viviparus, with different species for comparison, and in other exposure studies such as anthelminthic screening tests. ACKNOWLEDGMENTS I wish to express my thanks to Professor G. A. D. Haslewood, Biochemistry and Chemistry Department, Guy’s Hospital Medical School, London, for preparing the bile fractions and the purified salts used in Experiment 6. I am also grateful to Dr. D. Poynter of Allen and Hanburys Research Ltd., England, for providing the infective Dictyocaulus taiviparus larvae used in Experiment 4. The technical assistance of Mrs. Karen Madsen and Mrs. Bente Hein is greatly acknowledged. This work was supported by the Danish Agricultural and Veterinary Research Council. REFERENCES BERBERIAN, D. A. 1957. Host specificity and the effect of digestive juices on ova ofE. granulosus. 10th Report Orient Hospital.
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