Onchocerca spp: Cryopreservation of microfilariae and subsequent development in the insect host

EXPERIMENTAL

47, 384-391 ( 1979)

PARASITOLOGY

Onchocerca spp: Cryopreservation of Microfilariae Subsequent Development in the Insect Host

and

P. J. HAM, E. R. JAMES, AND A. E. BIANCO London

School of Hygiene and Tropical Medicine, Winches Farm, Hertforckhire, England, United Kingdom

St. Albans,

(Accepted for publication 26 January 1979) HAM, P. J., JAMES, E. R., AND BIANCO, A, E. 1979. Onchocerca spp: Cryopreservation of microfilariae and subsequent development in the insect host. Experimental Parasitology 47, 384-391. Microfilariae of Onchocerca gutturosa, 0. cervicalis and 0. volvulus were successfully recovered after freezing, storage at -196 C, and thawing. The technique that produced maximum viability involved a two-step cooling schedule consisting of an initial slow cool of 1 C mine1 to an intermediate temperature of between -14 and -17 C, followed by a rapid cool into liquid nitrogen (taking about 1 set). Upon rapid warming to 37 C, a high percentage of microfilariae showed normal motility. Following subcutaneous injection into T.O. mice, the microfilariae of 0. gutturosa migrated to the skin of the ears and nose, and a proportion of them developed into third-stage larvae in the insect vector, Simulium ornatum. Microflariae of 0. VOZVUZUS also developed into third-stage larvae in this insect, while those of 0. cervicalis developed similarly in their natural vector, Culicoides nubecuZosus. This technique of preservation provides a good and reliable method for storage of viable microfilariae of these bovine, equine, and human Onchocerca spp. gutturosa, 0. volvulus, 0. certiicalis; Nematodes, INDEX DESCRIPTORS : Onchocerca parasitic; Filaria; Microfilariae; Cryopreservation; Cooling rates; Migration in proxy host; Development in vectors; Insect vectors; Simulium ornatum, Culicoides nubeculosus.

There have been several reports, for exon Litomosoides carinii and Wuchereria bancrofti ( Weinmann and McAllister 1947), Dirofilaria immitis (Bemrick et al. 1965) and D. repens (Restani 1968)) of the successful cryopreservation of microfilariae without the use of cryoprotectant additives. The use of cryoprotectants was found to be essential for freezing the microfilariae of Brugia pahangi, since no microfilariae survived in the absence of additives (Ogunba 1969). Both Ogunba ( 1969) and Obiamiwe and Macdonald (1971) reported that, with this species, dimethylsulphoxide (DMSO) was more effective than glycerol. ample

Motile microfilariae of Onchocerca have been shown to survive following storage at -25 C (Mazzoti 1953) and at -196 C, using methanol as a cryoprotectant (Ham et al. 1978). Although motility can be used as an indication of survival, it does not necessarily follow that motile organisms are infective and will develop normally. Demonstration of the infectivity of cryopreserved Onchocerca microfilariae has not previously been reported. This paper describes experiments identifying the best cryoprotectant and conditions of cooling which produced the optimum survival of these microfilariae. Their motility following cryopreservation, migra384

0014-4894/79/0303,&G08$02.00/O Copyright All rights

0 of

1979 by Academic Press, in any form

reproduction

Inc. reserved.

Onchocerca

spp: CXYOPRJZSERVEDMICROFILARIAE INTO INSERTS

tion in a temporary (or proxy) host model, and development in their natural insect vectors is also described. MATERIALS AND METHODS

(1) Origin

of Parasites

Microfilariae were obtained from the skin of infected animals; those of Onchocerca gutturosa from the umbilical region of the cow, and those of 0. cervicalis from the mid ventral region of the horse belly. These skin microfilariae were identified specifically by comparing them with those from the uteri of adult female worms. Skin, taken from freshly killed animals was shaved, cleaned, pinned to a cork board, and swabbed with absolute alcohol. Dry slivers of epidermis and dermis were cut and placed in sterile Tyrode’s solution supplemented with 20% deactivated cow serum and 200 units of penicillin and streptomycin per milliliter (TCS). These operations were performed in a sterile hood under aseptic conditions. Microfilariae were collected at intervals between 2-18 hr later. 0. volvulus microfilariae were obtained by the same procedure using small sterile skin snips from an experimentally infected chimpanzee. (2) Cryoprotectant

Toxicity

Tests

Microfilariae were incubated for 30 min at 37 C in 1 ml of Tyrode’s solution containing one of four cryoprotectants (methanol, DMSO, glycerol, or ethanediol) at varying concentrations. Thirty minutes was considered to be the maximum handling time before and after freezing. Rapid removal of the cryoprotectant was achieved by the addition of 9 ml of Tyrode’s solution at 37 C. (3) Freezing and Thawing

Schedule

(i) Prefreeze. Microfilariae were incubated in TCS for 5 min at 37 C. When cryoprotectants were used they were added

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at the beginning of this step to allow them to permeate into the organism. Samples, each containing 50 to 100 microfilariae per 20-p drop, were then pipetted onto an aluminium block. The temperature was measured on a potentiometric flat-bed recorder connected to a fine thermocouple probe inserted into one of the 20-p drops on the block. (ii) Cooling rate. The freezing experiments were initially performed using a cooling rate of 1.0 C min-I. This rate was attained by placing the aluminium block into a Let CLlOB refrigerator at -80 C. The initial temperature of the block was about 21 C. Other cooling rates used in this work were obtained by placing the same block in the chamber of a PLANER R201/101 freezing machine. This machine can be programmed to cool at rates between 0.1 C and 50 C min-I. (iii) Intermediate temperature. Drops containing parasites were plunged into liquid nitrogen from various intermediate temperatures during the slow cooling step of 1 C min-I, and then thawed to 37 C. Control drops were thawed directly to 37 C at the same time as the experimental drops were plunged. (iv) Thawing and dilution. Drops were thawed in glass tubes containing 1 ml of TCS at 37 C, the tubes shaken rapidly, and the drops thawed in less than 1 sec. The volume of TCS, large in relation to the size of the drop, also ensured that the cryoprotectant additives were diluted quickly, in order to reduce any toxic effects on the parasites. (4) Viability

Assay

Viability was assessed (i) by motility, (ii) migration in a mammalian proxy host model, and (iii) by development in the insect vector. (i) Observations to assess the motility of the worms were performed using a microscope warm stage at 37 C. A scale from

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3+ (normally active) to 0 (dead or nonmotile) was used to score motility and the percentage of worms with each score was recorded. (ii) This work is based on the use of a model by Nelson et al. (1966). Six- to seven-week-old T.O. mice, inoculated subcutaneously with microfilariae, were killed on Day 5 and the ears snipped into fine fragments in TCS. These were incubated for 24 hr at 24 C and the emergent microfilariae were counted. This count was a measure of the ability of the injected worms to migrate from the subcutaneous tissue to the dermis. Day 5 was chosen as the day of assay, as normal microfilariae had been found to reach a peak in the skin at this time. Subcutaneous inoculation gave higher recoveries of worms than intramuscular, intravenous, and intraperitoneal routes, and more migrated to the ears than other regions of the body (manuscript in preparation). (iii) A further assessment of viability is the ability to develop into third-stage larvae within the insect vector. Steward (1937) reported that 0. gutturosa is transmitted, in England, by Simulium ornutum and in our laboratory this fly has been used extensively to produce third-stage larvae of 0. gut&rosa. Infectivity studies with this parasite, and with 0. volvu~u.s, were carried out by injecting normal or cryopreserved microfilariae into the thoracic of adult flies. Injection was performed using an extruded glass pipet and flies were kept at 22 C in the dark and killed on Day 12 to demonstrate infective stage larvae of 0. gutturosa, and on Day 10 for 0. volvulus. Quantitative assessment of the developing larvae was made on Day 5 when the worms had reached the late first stage of development. To produce infections with 0. cervicalis, the natural vector Culicoides nubeculosus was fed blood containing microfilariae through a membrane at 37 C. Cryopreserved microfilariae were concentrated by

AND

BIANCO U 4

w

methanol DMSO ethanediol glycerol

a0 &

60

0 0

5

10

percent

15

20

additive

25

30

(v/v)

FIG. 1. Toxicity of cryoprotectant Onchocerca gutiurosa microfilariae.

additives

for

centrifugation and resuspended in heparinixed whole cow blood to give a concentration of 120,000 microfilariae/ml. Infected midges were kept at 25 C and the head, thorax, and abdomen of each fly were examined for infective stage larvae on Day 10. RESULTS

Cryoprotectant gutturosa

Toxicity

to

Onchocerca

Experiment 1. Four chemicals (methanol, DMSO, ethanediol, and glycerol) with well-documented cryoprotective properties, were tested for their toxicity to microfilariae at concentrations between 5 and 3070 (v/v). From the results in Fig. 1, 5% was chosen as the maximum nontoxic concentration for each additive for use in later experiments. Preliminary experiments (not reported here) with cryopreservatives, using higher concentrations of glycerol, indicated that survival was lower than with 570,. Effect of Slow Cooling (1 C min-I) to Various Intermediate Temperatures on the Recovery of hlicrofilariae of Onchocerca gutturosa Experiment 2. The experiment was done either without a cryoprotectant or with 5% of each of the additives. Samples at tem-

Onchocerca

0

-10 -20

-30 -40 45 intermediate

spp: CRYOPFUZSERVED MICROF&iRIAE

0 -10

-20

teIll!Xr~tW~

-30

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INSECTS

-40 45 intermediate

(‘C 1

FIG. 2. Plunge plunge filariae

INTO

into liquid nitrogen and nosurvival of Onchocerca gutturosa microin the presence of cryoprotectants.

peratures from 0 to -45 C were either plunged into liquid nitrogen and thawed, or thawed directly. Motile organisms were not recovered from any samples without a cryoprotectant cooled below -5 C and thawed directly, nor from any of those which were plunged into liquid nitrogen before thawing. Results using cryoprotectants are shown in Fig. 2 and indicate that survival was better with methanol than with the other additives. Experiment 3. Five drops of medium containing methanol at 5% (v/v) were maintained at each of the intermediate temperatures. Two drops were thawed directly to 37 C in separate vials, while three drops were plunged to -196 C and then thawed separately. The results in Fig. 3 show the means and standard deviations for each of the groups of samples, and indicate that the optimum intermediate for a cool of 1 C min-l and storage at -196 C, is -17 C. Experiment 4. In this experiment, using 5% methanol (v/v), the intermediate temperature of -17 C was used in conjunction with different cooling rates. The results shown in Fig. 4 indicate that optimum survival was obtained with a cooling rate of 1 C min-l for the initial slow cooling step to -17 c.

temperature

(‘C

1

FIG. 3. Plunge into liquid nitrogen and noplunge survival of Onchocerca gutturosu microfilariae in the presence of 5% methanol (Means and SE).

Viability Studies Using the Mouse Proxy Host Model Experiment 5. Two groups of four mice were each injected subcutaneously with 5000 normal or cryopreserved microfilariae. The cryopreservation schedule was basically as outlined earlier but with slight modification as follows: five minutes incubation at 21 C with 5% methanol in TCS followed by 1 C min-l cool to -17 C, before plunge into liquid nitrogen. The drops were thawed rapidly in TCS at 37 C

O-l

1 cooling

rate

10 (Wmins-1)

35

FIG. 4. Plunge into liquid nitrogen and noplunge survival of Onchocercu gutturosu microfilariae after cooling at different rates in the presence of 5% methanol.

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HAM,

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AND

BIANCO

-

no plunge plunge

50

bp

0

g

100

E”

0 cervicalis

50

FIG. 5. Survival of Onchocerca gutturosa microfilariae with serum (S) and without serum (No S) during freezing in the presence of 5% methanol (Means and SE).

lIsI2kl 0

and the worms stabilized at that temperature for 5 min before injection. The results indicated that a mean of 90 (SD * 14.4) microfilariae migrated to the ears of mice receiving normal parasites and 27 (SD * 5.6) microfilariae migrated to the ears in the cryopreserved group. Experiment 6. Preliminary experiments had suggested that the presence of serum is important in the cryopreservation of Onchocerca microfilariae. Serum-supplemented medium had been used in Experiments 1-5, and the aim of this experiment was to compare the cryopreservation of Onchocerca microfilariae in medium with or without serum. Control groups consisted TABLE Recoveries

Experiment 7B Normal microfilariae Cryopreserved microfilariae

-30

-20

-36

temperature PC)

FIG. 6. Plunge into liquid nitrogen and noplunge survival of Onchocerca ceroicalis and Onchocerca VOZUUZUSmicrofilariae in the presence of 5% methanol.

of worms maintained in medium with methanol at 0 C, and microfilariae cooled to -17 C were either thawed directly or plunged to -196 C before thawing. The motility of the microfilariae in each of the six groups was assessed, from aliquots, just prior to injection into the mice. Motility and migration results are shown in Fig. 5 and indicate that the addition I

of First-Stage Larvae of Onchoccrca gutturosa, from Simulium ornatum Flies Number of flies injected

Experiment 7A Normal microfilariae Cryopreserved microfilariae

-10 intermediate

No. positive

NO. negative

at Day 5,

Mean No. larvae

SD

20

1G

4

3.4

3.95

20

15

5

1.55

1.47

12

11

1

3.08

1.44

29

18

11

0.9

0.86

Onchocerca of serum survival.

produces

spp: CRYOPRESERVED MICROFILARIAE INTO INSECTS

significantly

Viability studies using development insect vector, Simulium ornatum

higher

in the

Experiment 7. Two groups of flies were injected intrathoracically with 10 microfilariae per fly. The first group received normal microfilariae and the second group received microfilariae which had been frozen, stored, and thawed under the same conditions as described earlier for the proxy host model. Assessment of the numbers of late firststage larvae developing in the flies was performed 5 days postinjection since fly mortality was high subsequently. Infective stage larvae were demonstrated in the heads of flies 12 days after injection. This experiment was repeated and both sets of results are shown in Table I. They indicate that between 29 and 46% of the cryopreserved microfilariae developed to late first-stage larvae. Studies with cervicalis

Onchocerca

volvulus

and 0.

Experiment 8. Studies on cryopreservation using motility to assess viability. Microfilariae of 0. volvulus and 0. cervicalis were frozen in TCS with 5% methanol to various intermediate temperatures in a similar manner to that described earlier and then thawed directly or plunged into liquid nitrogen and then thawed. This was repeated twice, and the optimum intermediate temperature for 0. volvulus and 0. cervicalis appears to be broadly similar to that for 0. gutturosa. The results are presented in Fig. 6. Experiment 9. Development of cryopreserved 0. volvulus and 0. cervicalis microf&zria to third stage larvae. Microfilariae were frozen to -196 C using the optimum intermediate temperatures found from Experiment 8. After thawing, they were injected into their respective hosts (Culi-

389

coi&s nubeculosus for 0. cervicalis and S. ornatum for 0. volvulus). Third-stage larvae were recovered from the head region of the flies for both species of parasite and second stage larvae from the thoraci. However, mortality in uninfected Culicoides was high. Twenty microfilariae were injected into each fly and of the 6 that survived a range of 1 to 5 third-stage larvae and 0 to 6 second-stage larvae were recovered. Means of 2 and 2.5 per fly, respectively, occurred. DISCUSSION

The results from Experiment 2 (Fig. 3) showed that there was an optimum intermediate temperature of -17 C from which microfilariae of Onchocerca gutturosa could be cooled rapidly by plunging into liquid nitrogen. Both above and below this temperature, survival, assessed by motility and by the mouse temporary host model, was lower. Similar observations have been reported for two-step cooling schedules in other systems by Walter et al. (1975) for lymphocytes, by McGann and Farrant (1976) for Chinese hamster cells, and by James and Farrant (19177)for schistosomula of Schistosoma munsoni. It is thought that damage using higher intermediate temperatures is due to insufficient shrinkage of cells which leads to the formation of large amounts of intracellular ice during the second rapid cooling step in liquid nitrogen. At intermediate temperatures, lower than the optimum, it is thought that damage is due to excessive shrinkage of the cells when the high concentrations of intracellular salts become toxic. The results of Walter et al. (1975) also suggest that, at low intermediate temperatures, the cells may be shrunken beyond the limit from which they can return to their normal size. The same theories are thought to account for the effect, on survival, of cooling at different rates down to the intermediate temperature. At the higher rates, intra-

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cellular ice is produced, whereas at rates lower than the optimum for the initial cooling step, the cells are held for a long time in a shrunken state and therefore in high salt concentrations. The optimum cooling rate therefore, is that rate which matches the rate of shrinkage of the organism, to produce the least amounts both of intracellular ice and of irreversible salt damage. This view is expressed by Morris and Farrant (1972,1973) and by Pribor ( 1975). In this study, the optimum cooling rate for microfilariae was found to be 1 C min-I. Either side of this the recovery of motile larvae dropped off markedly and at 0.5 C min-l there was no recovery even in the no-plunged control group. At faster cooling rates, a drop in motility of plunged organisms also occurred up to 35 C min-I, although the no-plunged organisms were still motile. These observations appear to fit the theories of the interaction of cooling rate and damage outlined above. Damage to microfilariae can also result from ice recrystallisation within the cells during the thawing process. This occurs as ice melts and reaggregates within the cells into larger crystals. Throughout this work a rapid thawing rate was used during warming, so that the opportunity for this kind of damage to occur was minimised. A significant finding of the present study was that the demonstration of motility in organisms recovered after freezing is not alone a sufficient criterion to assess their viability. Thus, in experiments on Onchocera gutturosa microfilariae, SS% of the organisms showed normal motility after freezing while only 30% migrated in proxy hosts and 29 to 46% developed further in the insect vector. ACKNOWLEDGMENTS Mr. P. J. Ham would like to thank the Wellcome Trust for financial support and this investigation also received financial support from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

AND BL4NCO Thanks are also due to “Alf Reading for their invaluable help supplying fresh cow necropsy Professor G. S. Nelson in whose work was carried out.

Meade Ltd” of and patience in material and to department this

REFERENCES BEMRICK, W. J., BUCHLI, B. L., AND GRIFFITHS, H. J. 1965. Development of DirofZaria immitis in Anopheles quadrimaculatus after exposure of the microfilariae to a freezing temperature. Journal of Parasitology 51, 954-957. HAM, P. J., JAMES, E. R., AND BIANCO, A. E. 1978. Preliminary studies on the cryopreservation of Onchocerca microfilariae using a proxy host model to assess viability. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 434. JAMES, E. R., AND FARRANT, J. 1977. Recovery of infective Schistosoma mansoni schistosomula from liquid nitrogen: A step towards storage of a live schistosomiasis vaccine. Transactions of the Royal Society of Tropical Medicine and Hygiene 71, 498-500. MAZZOTTI, L. 1953. Supervivencia de las microfilarias de Dirofilaria immitis y de Onchocerca reticulata a la temperature de 25°C bajo cero. Revista de1 Institute de Salubridad y Enfermedades Tropicales Mexico 13, 289-291. MCGANN, L. E., AND FARRANT, J. 1976. Survival of tissue culture cells frozen by a two-step procedure to -196°C. I. Holding temperature and time. Cryobiology 13, 261-268. MORRIS, G. J., AND FARRANT, J. 1972. Interactions of cooling rate and protective additive on the survival of washed human erythrocytes frozen to -196°C. Cryobiology 9, 173-181. MORRIS, G. J., AND FARRANT, J. 1973. Effects of cooling rate on thermal shock hemolysis. Cryobiology 10, 119-125. NELSON, G. S., AMIN, M. A., BLACKIE, E. J., AND ROBSON, N. 1966. The maintenance of Onchocerca gutturosa microfilariae in vitro and in vivo. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 17. OBIAMIWE, B. A., AND MACDONALD, W. W. 1971. The preservation of Brugia pahangi microfilariae at sub-zero temperatures and their subsequent development to the adult stage. Annals of Tropical Medicine and Parasitology 65, 547554. OGUNBA, E. 0. 1969. Preservation of frozen Brugia pahangi using dimethylsulphoxide. Journal of Parasitology 55, 1101-1102. PRIBOR, D. B. 1975. Biological interactions between cell membranes and glycerol or DMSO. Clyobiology 12, 309-320.

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CRYOPRESERVED

RESTANI, R. 1968. Ricerche sulla resisteuza di microfilarie di Dir&&a repens a diverse temperature. Parassitologia 10, 75-83. STEWARD, J. S. 1937. The occurrence of Onchocerca gutturosa Neumann in cattle in England with an account of its life-history and development in Simulium ornutum (Meigen, 1818). Parasitology 29, 212-219. WALTER, C. A., KNIGHT, S. C., AND FARRANT, J.

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1975. Ultrastructural appearance of freeze-substituted lymphocytes frozen by rapid cooling with a period at -26°C. Cryobiology 12, 103109. WEINMANN, D., AND MCALLISTER, J. 1947. Prolonged storage of human pathogenic protozoa with conservation of virulence: Observations on the storage of hehninths and leptospiras. American Journal of Hygiene 45, 102-121.