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Successful cryopreservation of Brugia pahangi third-stage larvae in liquid nitrogen
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPKA~
Successful
cryopreservation
815
MEDICINE AND HYGIENE, VOL. 77, No. 6, 8154319 (1983)
of Brugia pahangi liquid nitrogen
third-stage
larvae in
P. J. HAM AND E. R. JAMES London School of Hygiene and Tropical Medicine, Winches Farm Field Station, 39.5 Hatfield Road, St. Albans,
Herts AL4
OXQ, UK
Summary Experiments are described which lead to the retention of infectivity of Brugia pahangi third-stage larvae after cooling to -196°C. Methanol, a well documented cryoprotectant was used at a concentration of 20% (v/v). The schedule consisted of a 5°C min-’ cool to an intermediate temperature of -21°C and a subsequent rapid cool into liquid nitrogen. A rapid thaw of the parasites led to approximately 34% of motile cryopreserved larvae developing in multimammate rats (Mastomys natalensis) compared to unfrozen control larvae. Cooling rate and intermediate temperature were both found to be crucial variables affecting survival levels of the larvae.
Introduction Over the last few years there have been several reports concerning the cryopreservation of Brugia bahati microfilariae (OGUNBA.1969: OBIAMIWE& -MACDONALD,1971; MINJAS & TO&ON, 1980). Cryopreservation of the infective stage larvae of this parasite and, indeed, other species of filarial nematodes appears, however, to have received little attention. MCCALL et al. (1975) reported the survival of Dipetalonema viteae third-stage larvae after exposure of infected ticks to -196”C, a small proportion retaining their infectivity for the mammalian host and developing into adults which produced patent microfikuaemias. They used 5% (v/v) dimethvlsulnhoxide (Me2SO) as a &yoprotectant, .cooling the iicks at aooroximatelv 1°C rnin- ’ to -70°C and transferring t&m to liquid nitrogen (LN2). Later studie: (MCCALL & WEATHERSBY,1978) resulted in retention of infectivity of D. viteae third-stage larvae frozen free of ticks. SCHILLERet al. (1979) stored blackflies infected with Onchocerca volvulus in LN2 and reported that the larvae were motile on dissection of the flies after thawing. Work performed on the third-stage larvae of B. pahangi and D. viteae (HAM, 1977, 1980)showed that a two-stage cooling schedule might be useful. This paper describes experiments on he cryopreservation of B. pahangi third-stage larvae leading to the development of a technique with which a proportion of larvae retain their infectivity after thawing. Materials and Methods 1. Parasites B. pahangi larvae were produced in a susceptible strain of Aedes aegvpti (see MACDONALD, 1962) that had been previously fed on an infected microfilaraemic cat with three microfilariae per mm3 of peripheral blood). 2. Cryoprorectanttoxicity tests The larvae were incubated at 37°C or in an ice bath in Hanks balanced salt solution containing various concentrations of methanol for 30 min. This period was considered to
be the maximum combined pre-freeze and post-thaw handling time. The cryoprotectant was removed by lo-fold dilution in 199 medium at 37°C. 3. Freezing and thawing procedures After incubation in the cryoprotectant, the larvae were pipetted in 40~1drops on to an ahuninium block set at O”C, forming part of the duc&g of a Planer R101/201 programmable freeze/thaw machine (Planer Products Ltd.). Cooling was performed at various specified rates and drops were thawed at various subzero temperatures by pushing them into 2 ml of warm (37°C) medium and shaking. Other drops were pushed into LN2 before thawing in the same way. 4. Survival assessment This was performed by counting the number of normally motile parasites on a microscope warm stage(Microtec) set at 37°C. This number was expressed asa percentage of the total number of larvae counted. The mean (*SE) of triplicate samples was calculated for each experimental treatment. Infectivity was assessedby injecting larvae subcutaneously into male multimammate rats (Mustomys nata/ensis). This rodent has been described as a laboratory host for 6Iariids by AHMED(1967) and bv BENIAMIN& SOULSBY(1976). Males were u&d, a$ fernal& are- less susceptible tb B. ‘pahangi infections than males (ASH, 1971). Animals were dissected at intervals after inoculation; the tissues were examined for developing worms and then soaked overnight in Dulbecco’s PBS to allow any remaking larvae to migrate out. The medium was then examined. Worms were fixed in hot (60°C) 70% (v/v) alcohol and two days later transferred to glycerol alcohol (95% v/v of 70% alcohol : 5% v/v glycerol); they were measured after four weeks.
Experiments and Results Experiment I. Toxicity of methanol
Larvae were incubated in 1 ml volumes of Hanks balanced salt solution (HBSS) containing up to 40% (v/v) methanol either at 0°C or at 37”C, as described earlier. The results of the toxicity tests in Fig. 1 indicate that relatively low concentrations of methanol are toxic to the larvae at 37°C with 5% (v/v) reducing survival to 75% after 30 mins’ exposure and all dying with concentrations greater than 20%. However, concentrations of up to 20% are non-toxic if incubation is performed at 0°C on crushed ice. Because of this, all subsequent incubations of parasites were performed on crushed ice.
816
CRYOPRESERVATION
OF
B. pahangi
~3's
NORMAL MOTILITY
ANY MOTILITY
’
0
IO
20
PERCENT
30
METHANOL
40 (v/v)
Fig. 1. Toxicity of methanol at different concentrations temperatures to E. pahungithird-stage larvae.
and
0
Experiment 2. The effect of cooling rate on survival levels
Larvae were incubated in HBSS with 15% (v/v) methanol and 20% (v/v) new-born calf serum at 0°C for 10 min. They were cooled at rates of 1, 2, 5 and 10°C min-’ +before either thawing from - 15°C or plunging into LN2 before thawing. This intermediate temperature was chosen as preliminary studies (unpublished observations) suggested that it resulted in somedegreeof survival from LNz. The percentageof larvae showing any motility was assessedas well as those showing normal motility. Fig. 2 shows the motility levels of the larvae after cryopreservation using the four cooling rates. That of 5°C min-’
NP P
1
II I: NPP
NPP
2 5 COOL1NG RATE
NP P
10 OCmin-1
Fig. 2. The effect of the 1st step cooling rate during an interrupted cool on survival levels of B. pahangithird-stage larvae. NP = no plunge, i.e., thawed directly from -15°C; P = plunge.
resulted in the highest levels of survival with 95% of larvae showing somemotility. Survival levels at faster and slower cooling rates were decreased both for larvae frozen to - 196°Cand for those thawed directly from - 15°C.
0-e 5°C min-l non plunge M II II plunge a- a 3°C tin-1 ran plunge 0-4 II II plunge
intermediate Fig. 3. The effect of intermediate
temperature
temperature and cooling rate during an interrupted
( OC >
slow cool on survival levels of E.
pahangithird-stage
larvae.
P.
J.
HAM
Experiment 3. The effect of intermediate temperature using cooling rates of 3 and S”C mine1 in the first step of an interrupted cool After incubation at. 0°C in HBSS with 20% (v/v) methanol and 20% (v/v) new-born calf serum, larvae were cooled at 3 or 5°C min-‘. At various intermediate temperatures they were plunged into LNz and thawed and their motility assessed. The results in Fig. 3 show that the maximum level of survival from LNz using a 5°C rnin-’ cool was obtained with an intermediate temperature of -20°C. This level dropped with higher and lower intermediate temperatures so that by -45°C no survival could be obtained after plunging the drops into LNz. Survival from samples thawed directly declined with temperature. There was a large increase in damage using the slightly slower cooling rate of 3°C min- ‘. A statistical comparison between the two cooling rate survival levels was performed using the ANOVA test. This showed that levels using 5°C min-’ were significantly higher than those for 3°C min-’ (RO.05 thawed directly, RO.01 plunged to -196°C before thawing). Ex;ay’ments 4A and 4B. Infectivity
of cyopreserved
A. 150 third-stage larvae were injected subcutaneously into each of six male multimammate rats. Three received frozen larvae which had been cryopreserved using trhe technique described in Experiment 2, with a cooling rate of 5°C mint and an intermediate temperature of -21°C. Only normally motile larvae were injected into the rats. The rats were necropsied 40 days after infection and the larvae recovered were counted and measured as described earlier. A small proportion (1.6%) of the larvae did develop using this cooling and thawing technique as shown in Table IA. This represents 6.4% of the unfrozen controls. The sizes are shown in Table IB and male worms at 40 days seemed to show no stunting. As only one cryopreserved female was recovered, a comparison with unfrozen larvae was not possible. B. In this experiment five groups of male M. natalensis were each injected subcutaneously with either unfrozen or cryopreserved larvae which were frozen and thawed as above. Details of the groups and larvae injected are shown in Table II. In groups B, C and D larvae were thawed, counted and injected directly into the rats. Larvae injected into group E were only those showing normal motility and were ‘separated’ from the damaged larvae before injection. The results in Table III indicate that no larvae could be recovered from rats injected with 50 or 100 unseparated cryopreserved larvae+nly with a dose of 500 larvae per rat were developing worms recovered. However, if only normally motile larvae were used then a dose of 100 larvae was sufficient to produce an adult worm burden of approximately 34% of the unfrozen controls. Fig. 4 shows the lengths of the worms recovered and indicates that some retardation of growth of female worms occurred during the early stages of development. Discussion
& WEATHERSBY (1978) reported that using dimethylsulphoxide and polyvinylpyrrolidone, which are respectively intra- and extra-cellular cryoMCCALL
AND
E.
R.
JAMES
817
Table IA-Recoveries of B. pahangi larvae from multimammate rats 40 days after injection of third-rate larvae No. wmns from
unfrozen larvae Male Female Total
Animal 1
15
2 3
44 16
6 22 7
Mean (SD) % of inoculum
21 66 23
No. wcwms from cryopreserved larvae Male Female Total 4
1 1
1 0 0
5 1 1
36.7 (25.4)
2.3 (2.3)
24.5
1.6
recovered
Table IB-Lengths of B. pahangi larvae recovered from multimammate rats 40 days after injection of third-stage larvae
Unfrozen (mm) Male Female Mean (SD) Il.
12.31 (2.12)
19.28 (1.85)
11
8
Cryopreserved (mm) Male
Female
11.09 (3.48)
13.43 -
5
1
Males: 0.25>P>O.20
(Student’s t-test indicates no significant difference in length)
Table II-The groups of Masromys narulensis used in Experiment 4B showing numbers and type of B. puhungi third-stage larvae per animal No. M. nutulensis
Group
per group
A B C D E
2 4 2
5
5
No. of larvae per animal 100 unfrozen 50 unseparated frozen 100 unseparated frozen 500 unseparated frozen 100 separated frozen
protectants, viable third-stage larvae of Dipetakmema viteae could be recovered from LNr. The study described here shows that methanol also protects the larvae against freezing and thawing damage and it is clear that by using a two-step cooling system some of the damage is averted. The size measurements of the developing worms show that a considerable difference occurred between the growth rate of female cryopreserved and unfrozen larvae. Unfortunately, no animals were hilled earlier than 33 days post-infection, as the proportion of larvae developing to the fifth stage (i.e. those that moulted twice to become adults) was considered a more valuable result. It appears that the growth of cryopreserved female worms was retarded during these critical stages of infection. SCHACHER(1962) observed that female and male Brugia pahangi in cats grew at similar rates during the first 20 or so days but after this the female growth rate accelerated so that at 33
818
CRYOPRESERVATION
Table III-The effect of inoculum size and activity development in multimammate rats Animal No.
Days post inoculation
knfrozen
:
40
controls) 100 L3 per rat
:
:; 75 156
Group
5
OF
B. pahangi
of cryopreserved
~3’s
B. pahangi third-stage
larvae on their
No. worms recovered Female
Male
Total
; 4
fi 9
26 13
z
1:
2; Mean, 10 f 7.3
CRYOPRESERVED
GROUPS Both negative
: 40
1 to 4
C Unseparated 100 L3 per rat D
All negative
3 156 ::
“2 1
:3
:.
::
2
2
3 4 5
75 75 75
: Negative Negative
z
:3
~$se~rated per rat E Separated 100 L3 per rat
: Mean, 4 f 1 4 z
b 0 Mean, 3.4 + 3.4
In all positive groups larvae were found in heart, subcutaneous tissue and testes. FEMALE
$
25-
y
20-
MALE
15lo50’1
’
0
a
20
’
f
8
LO DAYS
I
60
.
f
80
F?I.
Fig. 4. The lengths (mm) of B. pahangi larvae during development in Mastomys mm&m-is after cryopreservation at -196°C.
days post-infection males had a mean length of 15.5 mm and females 18.3 mm. At 55 days, males were 17.0 mm and females 38.4 mm. Male growth plateaued after about 50 days and female growth after about 110 days. In normal infections in this study with M. natalensis the growth rate was much slower than that observed by Schacher with means of 12.71 and 15.95 mm for males and females at 59 days post-infection and 12.16 and 21.13 at 75 days post-infection, i.e., the male growth rate appears to have stabilized out by 40 days (at around 12 mm) whereas in cats they grow to 17.0 mm (SCHACHER, 1962). However, in jirds (ASH & RILEY, 1970) by 157 days males reach means of 13.1 mm (10.9-14.4 range) and females reach 36.3 mm (336-43.9 range). This compares favourably with the worms in M. nataknsis infected for six months in this study. The apparent retardation of the growth of female worms after cryopreservation may have resulted in the low numbers observed in Experiment 4A with B. pahangi. Only one female was found in three animals whereas six males were found. However, in Experiment 4B 41.7% and 52.9% of the worms recovered in the two cryopreserved groups were female, whereas 37.9% were females in the unfrozen control group. This suggests that females survived cryopreservation equally as well as males. JAMES (1980) working with schistosomula of Schistosoma mansoni demonstrated that methanol probably
I’.
J.
HAM
AND
permeatescells very rapidly (within approximately 10 sets). If some shrinkage of the B. pahangi larvae occurred during the first few minutes in methanol, then this would be increased with a cooling rate of Yrnin- ‘. As ice formed outside the cells of the larvae further shrinkage of the organisms would occur. This, in addition to a oreliminarv shrinkage at 0°C mav be beyond a minimum critical level (MERYMA~, 1970). However, a critical level of shrinkage may not in itself be the injurious factor, but someconsequencesuch as dilution shock on thawing as found with Onchocerca microfilariae (HAM & JAMES, 1982). Recovery levels of third-stage larvae appear to be more erratic and lower than those of microfilariae, perhaps because of their more advanced state of development. The increase in cell numbers and types and the over-all dimensions of the larvae may make these third-stage larvae more susceptible to injury as a result of freezing and thawing. These studies demonstrate that viability (assessed as infectivity) can be retained from - 196°C which is low enough to be a stable storage temperature. Acknowledgements We thank the Wellcome Trust and the Edna McConnell Clark Foundation for financial support and Mr. Tony Dobinson for technical assistance. Thanks also ao to Dr. D. A. Denham for supplying the parasitic material, Dr. A. E. Bianco for helpful advice and Professor G. S. Nelson in whose department the work was conducted. References Ahmed, S. S. (1967). Studies on the laboratory transmission of sub-periodic Brugia malayi and B. pahangi. II. Transmission to intact and splenectomized rats and cotton rats. Annals of Tropical Medicine and Hygiene, 61,
._- .__.
4?2-436.
Ash, L. R. (1971). Preferential susceptibility of male jirds (Meriones unguiculatus) to infection with Brugia pahangi. Journal of Parasitology, 57, 777-780. Ash, L. R. & Riley, J. M. (1970). Development of Brugia pahangi in the jird, (Meriaes unguiculatus), with notes on infections in other rodents. 3ournal of Parasitology, 56, 962-968. Benjamin, D. B. & Soulsbv, E. J. L. (1976). The homocytotropic and hemagglutinating antibody response to Brugia pahangi infection in the multimammate rat (Mastomys natalensis). AmericanJournal of Tropical Medicine and Hygiene, 25, 266-272.
E.
R.
819
JAMES
Ham, P. J.(1977). Studies on the cryopreservation if twofilarial nematodes, Brugia pahangi and Dipetalonema viteae. M.Sc. Thesis. University of London, 66 pp. Ham, P. J. (1980). The recovery of viable third-stage larvae of Brugia pahangi from liquid nitrogen. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 677. Ham, P. J. & James, E. R. (1982). Protection of cryopreserved Onchocerca microfilariae (Nematoda) from dilution shock by the use of serum. Cryobiology, 19, 448-457. James, E. R. (1980). Cryopreservation of Schistosoma mansoni schistosomula using 40% v/v (1OM) methanol and rapid cooling. Ctyo-Letters, 1, 535-544. Macdonald, W. W. (1962). The selection of a strain of Aedes aegvpti susceptible to infection with semi-periodic Brugia malayi. Annals of Tropical Medicine and Parasitology, 56, 368-372. McCall, J. W., Jun, J. & Thompson, P. E. (1975). Cryopreservation of infective larvae of Dipetalonema viteae. 3oumal of Parasitology, 61, 340-342. McCall, J. W. & Weathersby, A. B. (1978). Survival and infectivitv of infective larvae of Dipetulonema viteae free of the tick. 4th International Congress of Parasitology, 19-26 August, Warsaw. Short Communications, Section c, p. 155. Meryman, H. T. (1970). The exceeding of a minimal tolerable cell volume in hypertonic suspension as a cause of freezing injury. In: The Frozen Cell, G. E. W. Wolstenholme & M. O’Connor (Editors). London: Churchill, pp. 5 l-64 Minjas, J. N. & Townson, H. (1980). The successful cryopreservation of microfilariae with hydroxyethyl starch as cryoprotectant. Annals of Tropical Medicine and Parasitology, 74, 571-573. Obiamiwe, B. A. & Macdonald, W. W. (1971). The preservation of Brugia pahangi microlilariae at sub-zero temperatures and their subsequent development to the adult stage. Annals of Tropical Medicine and Parasitology, -. 65, 547-354. Ogunba, E. 0. (1969). Preservation of frozen Brugia pahangt using dimethyl sulphoxide. Journal of Parasitology, 55, 1101-1102. Schacher, J. F. (1962). Developmental stages of Brugia pahangi in the final host. Journal of Parasitology, 48, 693-706. Schiller, E. L., Turner, V. M., Marroqt’iin, H., F. & D’Antonio, R. (1979). The cryopreservation ani;ltz cultivation of larval Onchocerca volvulus. 3oumal of Tropical Medicine and Hygiene, 28, 997-1009.
Accepted for publication
30th March,
1983.
Successful
cryopreservation
815
MEDICINE AND HYGIENE, VOL. 77, No. 6, 8154319 (1983)
of Brugia pahangi liquid nitrogen
third-stage
larvae in
P. J. HAM AND E. R. JAMES London School of Hygiene and Tropical Medicine, Winches Farm Field Station, 39.5 Hatfield Road, St. Albans,
Herts AL4
OXQ, UK
Summary Experiments are described which lead to the retention of infectivity of Brugia pahangi third-stage larvae after cooling to -196°C. Methanol, a well documented cryoprotectant was used at a concentration of 20% (v/v). The schedule consisted of a 5°C min-’ cool to an intermediate temperature of -21°C and a subsequent rapid cool into liquid nitrogen. A rapid thaw of the parasites led to approximately 34% of motile cryopreserved larvae developing in multimammate rats (Mastomys natalensis) compared to unfrozen control larvae. Cooling rate and intermediate temperature were both found to be crucial variables affecting survival levels of the larvae.
Introduction Over the last few years there have been several reports concerning the cryopreservation of Brugia bahati microfilariae (OGUNBA.1969: OBIAMIWE& -MACDONALD,1971; MINJAS & TO&ON, 1980). Cryopreservation of the infective stage larvae of this parasite and, indeed, other species of filarial nematodes appears, however, to have received little attention. MCCALL et al. (1975) reported the survival of Dipetalonema viteae third-stage larvae after exposure of infected ticks to -196”C, a small proportion retaining their infectivity for the mammalian host and developing into adults which produced patent microfikuaemias. They used 5% (v/v) dimethvlsulnhoxide (Me2SO) as a &yoprotectant, .cooling the iicks at aooroximatelv 1°C rnin- ’ to -70°C and transferring t&m to liquid nitrogen (LN2). Later studie: (MCCALL & WEATHERSBY,1978) resulted in retention of infectivity of D. viteae third-stage larvae frozen free of ticks. SCHILLERet al. (1979) stored blackflies infected with Onchocerca volvulus in LN2 and reported that the larvae were motile on dissection of the flies after thawing. Work performed on the third-stage larvae of B. pahangi and D. viteae (HAM, 1977, 1980)showed that a two-stage cooling schedule might be useful. This paper describes experiments on he cryopreservation of B. pahangi third-stage larvae leading to the development of a technique with which a proportion of larvae retain their infectivity after thawing. Materials and Methods 1. Parasites B. pahangi larvae were produced in a susceptible strain of Aedes aegvpti (see MACDONALD, 1962) that had been previously fed on an infected microfilaraemic cat with three microfilariae per mm3 of peripheral blood). 2. Cryoprorectanttoxicity tests The larvae were incubated at 37°C or in an ice bath in Hanks balanced salt solution containing various concentrations of methanol for 30 min. This period was considered to
be the maximum combined pre-freeze and post-thaw handling time. The cryoprotectant was removed by lo-fold dilution in 199 medium at 37°C. 3. Freezing and thawing procedures After incubation in the cryoprotectant, the larvae were pipetted in 40~1drops on to an ahuninium block set at O”C, forming part of the duc&g of a Planer R101/201 programmable freeze/thaw machine (Planer Products Ltd.). Cooling was performed at various specified rates and drops were thawed at various subzero temperatures by pushing them into 2 ml of warm (37°C) medium and shaking. Other drops were pushed into LN2 before thawing in the same way. 4. Survival assessment This was performed by counting the number of normally motile parasites on a microscope warm stage(Microtec) set at 37°C. This number was expressed asa percentage of the total number of larvae counted. The mean (*SE) of triplicate samples was calculated for each experimental treatment. Infectivity was assessedby injecting larvae subcutaneously into male multimammate rats (Mustomys nata/ensis). This rodent has been described as a laboratory host for 6Iariids by AHMED(1967) and bv BENIAMIN& SOULSBY(1976). Males were u&d, a$ fernal& are- less susceptible tb B. ‘pahangi infections than males (ASH, 1971). Animals were dissected at intervals after inoculation; the tissues were examined for developing worms and then soaked overnight in Dulbecco’s PBS to allow any remaking larvae to migrate out. The medium was then examined. Worms were fixed in hot (60°C) 70% (v/v) alcohol and two days later transferred to glycerol alcohol (95% v/v of 70% alcohol : 5% v/v glycerol); they were measured after four weeks.
Experiments and Results Experiment I. Toxicity of methanol
Larvae were incubated in 1 ml volumes of Hanks balanced salt solution (HBSS) containing up to 40% (v/v) methanol either at 0°C or at 37”C, as described earlier. The results of the toxicity tests in Fig. 1 indicate that relatively low concentrations of methanol are toxic to the larvae at 37°C with 5% (v/v) reducing survival to 75% after 30 mins’ exposure and all dying with concentrations greater than 20%. However, concentrations of up to 20% are non-toxic if incubation is performed at 0°C on crushed ice. Because of this, all subsequent incubations of parasites were performed on crushed ice.
816
CRYOPRESERVATION
OF
B. pahangi
~3's
NORMAL MOTILITY
ANY MOTILITY
’
0
IO
20
PERCENT
30
METHANOL
40 (v/v)
Fig. 1. Toxicity of methanol at different concentrations temperatures to E. pahungithird-stage larvae.
and
0
Experiment 2. The effect of cooling rate on survival levels
Larvae were incubated in HBSS with 15% (v/v) methanol and 20% (v/v) new-born calf serum at 0°C for 10 min. They were cooled at rates of 1, 2, 5 and 10°C min-’ +before either thawing from - 15°C or plunging into LN2 before thawing. This intermediate temperature was chosen as preliminary studies (unpublished observations) suggested that it resulted in somedegreeof survival from LNz. The percentageof larvae showing any motility was assessedas well as those showing normal motility. Fig. 2 shows the motility levels of the larvae after cryopreservation using the four cooling rates. That of 5°C min-’
NP P
1
II I: NPP
NPP
2 5 COOL1NG RATE
NP P
10 OCmin-1
Fig. 2. The effect of the 1st step cooling rate during an interrupted cool on survival levels of B. pahangithird-stage larvae. NP = no plunge, i.e., thawed directly from -15°C; P = plunge.
resulted in the highest levels of survival with 95% of larvae showing somemotility. Survival levels at faster and slower cooling rates were decreased both for larvae frozen to - 196°Cand for those thawed directly from - 15°C.
0-e 5°C min-l non plunge M II II plunge a- a 3°C tin-1 ran plunge 0-4 II II plunge
intermediate Fig. 3. The effect of intermediate
temperature
temperature and cooling rate during an interrupted
( OC >
slow cool on survival levels of E.
pahangithird-stage
larvae.
P.
J.
HAM
Experiment 3. The effect of intermediate temperature using cooling rates of 3 and S”C mine1 in the first step of an interrupted cool After incubation at. 0°C in HBSS with 20% (v/v) methanol and 20% (v/v) new-born calf serum, larvae were cooled at 3 or 5°C min-‘. At various intermediate temperatures they were plunged into LNz and thawed and their motility assessed. The results in Fig. 3 show that the maximum level of survival from LNz using a 5°C rnin-’ cool was obtained with an intermediate temperature of -20°C. This level dropped with higher and lower intermediate temperatures so that by -45°C no survival could be obtained after plunging the drops into LNz. Survival from samples thawed directly declined with temperature. There was a large increase in damage using the slightly slower cooling rate of 3°C min- ‘. A statistical comparison between the two cooling rate survival levels was performed using the ANOVA test. This showed that levels using 5°C min-’ were significantly higher than those for 3°C min-’ (RO.05 thawed directly, RO.01 plunged to -196°C before thawing). Ex;ay’ments 4A and 4B. Infectivity
of cyopreserved
A. 150 third-stage larvae were injected subcutaneously into each of six male multimammate rats. Three received frozen larvae which had been cryopreserved using trhe technique described in Experiment 2, with a cooling rate of 5°C mint and an intermediate temperature of -21°C. Only normally motile larvae were injected into the rats. The rats were necropsied 40 days after infection and the larvae recovered were counted and measured as described earlier. A small proportion (1.6%) of the larvae did develop using this cooling and thawing technique as shown in Table IA. This represents 6.4% of the unfrozen controls. The sizes are shown in Table IB and male worms at 40 days seemed to show no stunting. As only one cryopreserved female was recovered, a comparison with unfrozen larvae was not possible. B. In this experiment five groups of male M. natalensis were each injected subcutaneously with either unfrozen or cryopreserved larvae which were frozen and thawed as above. Details of the groups and larvae injected are shown in Table II. In groups B, C and D larvae were thawed, counted and injected directly into the rats. Larvae injected into group E were only those showing normal motility and were ‘separated’ from the damaged larvae before injection. The results in Table III indicate that no larvae could be recovered from rats injected with 50 or 100 unseparated cryopreserved larvae+nly with a dose of 500 larvae per rat were developing worms recovered. However, if only normally motile larvae were used then a dose of 100 larvae was sufficient to produce an adult worm burden of approximately 34% of the unfrozen controls. Fig. 4 shows the lengths of the worms recovered and indicates that some retardation of growth of female worms occurred during the early stages of development. Discussion
& WEATHERSBY (1978) reported that using dimethylsulphoxide and polyvinylpyrrolidone, which are respectively intra- and extra-cellular cryoMCCALL
AND
E.
R.
JAMES
817
Table IA-Recoveries of B. pahangi larvae from multimammate rats 40 days after injection of third-rate larvae No. wmns from
unfrozen larvae Male Female Total
Animal 1
15
2 3
44 16
6 22 7
Mean (SD) % of inoculum
21 66 23
No. wcwms from cryopreserved larvae Male Female Total 4
1 1
1 0 0
5 1 1
36.7 (25.4)
2.3 (2.3)
24.5
1.6
recovered
Table IB-Lengths of B. pahangi larvae recovered from multimammate rats 40 days after injection of third-stage larvae
Unfrozen (mm) Male Female Mean (SD) Il.
12.31 (2.12)
19.28 (1.85)
11
8
Cryopreserved (mm) Male
Female
11.09 (3.48)
13.43 -
5
1
Males: 0.25>P>O.20
(Student’s t-test indicates no significant difference in length)
Table II-The groups of Masromys narulensis used in Experiment 4B showing numbers and type of B. puhungi third-stage larvae per animal No. M. nutulensis
Group
per group
A B C D E
2 4 2
5
5
No. of larvae per animal 100 unfrozen 50 unseparated frozen 100 unseparated frozen 500 unseparated frozen 100 separated frozen
protectants, viable third-stage larvae of Dipetakmema viteae could be recovered from LNr. The study described here shows that methanol also protects the larvae against freezing and thawing damage and it is clear that by using a two-step cooling system some of the damage is averted. The size measurements of the developing worms show that a considerable difference occurred between the growth rate of female cryopreserved and unfrozen larvae. Unfortunately, no animals were hilled earlier than 33 days post-infection, as the proportion of larvae developing to the fifth stage (i.e. those that moulted twice to become adults) was considered a more valuable result. It appears that the growth of cryopreserved female worms was retarded during these critical stages of infection. SCHACHER(1962) observed that female and male Brugia pahangi in cats grew at similar rates during the first 20 or so days but after this the female growth rate accelerated so that at 33
818
CRYOPRESERVATION
Table III-The effect of inoculum size and activity development in multimammate rats Animal No.
Days post inoculation
knfrozen
:
40
controls) 100 L3 per rat
:
:; 75 156
Group
5
OF
B. pahangi
of cryopreserved
~3’s
B. pahangi third-stage
larvae on their
No. worms recovered Female
Male
Total
; 4
fi 9
26 13
z
1:
2; Mean, 10 f 7.3
CRYOPRESERVED
GROUPS Both negative
: 40
1 to 4
C Unseparated 100 L3 per rat D
All negative
3 156 ::
“2 1
:3
:.
::
2
2
3 4 5
75 75 75
: Negative Negative
z
:3
~$se~rated per rat E Separated 100 L3 per rat
: Mean, 4 f 1 4 z
b 0 Mean, 3.4 + 3.4
In all positive groups larvae were found in heart, subcutaneous tissue and testes. FEMALE
$
25-
y
20-
MALE
15lo50’1
’
0
a
20
’
f
8
LO DAYS
I
60
.
f
80
F?I.
Fig. 4. The lengths (mm) of B. pahangi larvae during development in Mastomys mm&m-is after cryopreservation at -196°C.
days post-infection males had a mean length of 15.5 mm and females 18.3 mm. At 55 days, males were 17.0 mm and females 38.4 mm. Male growth plateaued after about 50 days and female growth after about 110 days. In normal infections in this study with M. natalensis the growth rate was much slower than that observed by Schacher with means of 12.71 and 15.95 mm for males and females at 59 days post-infection and 12.16 and 21.13 at 75 days post-infection, i.e., the male growth rate appears to have stabilized out by 40 days (at around 12 mm) whereas in cats they grow to 17.0 mm (SCHACHER, 1962). However, in jirds (ASH & RILEY, 1970) by 157 days males reach means of 13.1 mm (10.9-14.4 range) and females reach 36.3 mm (336-43.9 range). This compares favourably with the worms in M. nataknsis infected for six months in this study. The apparent retardation of the growth of female worms after cryopreservation may have resulted in the low numbers observed in Experiment 4A with B. pahangi. Only one female was found in three animals whereas six males were found. However, in Experiment 4B 41.7% and 52.9% of the worms recovered in the two cryopreserved groups were female, whereas 37.9% were females in the unfrozen control group. This suggests that females survived cryopreservation equally as well as males. JAMES (1980) working with schistosomula of Schistosoma mansoni demonstrated that methanol probably
I’.
J.
HAM
AND
permeatescells very rapidly (within approximately 10 sets). If some shrinkage of the B. pahangi larvae occurred during the first few minutes in methanol, then this would be increased with a cooling rate of Yrnin- ‘. As ice formed outside the cells of the larvae further shrinkage of the organisms would occur. This, in addition to a oreliminarv shrinkage at 0°C mav be beyond a minimum critical level (MERYMA~, 1970). However, a critical level of shrinkage may not in itself be the injurious factor, but someconsequencesuch as dilution shock on thawing as found with Onchocerca microfilariae (HAM & JAMES, 1982). Recovery levels of third-stage larvae appear to be more erratic and lower than those of microfilariae, perhaps because of their more advanced state of development. The increase in cell numbers and types and the over-all dimensions of the larvae may make these third-stage larvae more susceptible to injury as a result of freezing and thawing. These studies demonstrate that viability (assessed as infectivity) can be retained from - 196°C which is low enough to be a stable storage temperature. Acknowledgements We thank the Wellcome Trust and the Edna McConnell Clark Foundation for financial support and Mr. Tony Dobinson for technical assistance. Thanks also ao to Dr. D. A. Denham for supplying the parasitic material, Dr. A. E. Bianco for helpful advice and Professor G. S. Nelson in whose department the work was conducted. References Ahmed, S. S. (1967). Studies on the laboratory transmission of sub-periodic Brugia malayi and B. pahangi. II. Transmission to intact and splenectomized rats and cotton rats. Annals of Tropical Medicine and Hygiene, 61,
._- .__.
4?2-436.
Ash, L. R. (1971). Preferential susceptibility of male jirds (Meriones unguiculatus) to infection with Brugia pahangi. Journal of Parasitology, 57, 777-780. Ash, L. R. & Riley, J. M. (1970). Development of Brugia pahangi in the jird, (Meriaes unguiculatus), with notes on infections in other rodents. 3ournal of Parasitology, 56, 962-968. Benjamin, D. B. & Soulsbv, E. J. L. (1976). The homocytotropic and hemagglutinating antibody response to Brugia pahangi infection in the multimammate rat (Mastomys natalensis). AmericanJournal of Tropical Medicine and Hygiene, 25, 266-272.
E.
R.
819
JAMES
Ham, P. J.(1977). Studies on the cryopreservation if twofilarial nematodes, Brugia pahangi and Dipetalonema viteae. M.Sc. Thesis. University of London, 66 pp. Ham, P. J. (1980). The recovery of viable third-stage larvae of Brugia pahangi from liquid nitrogen. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 677. Ham, P. J. & James, E. R. (1982). Protection of cryopreserved Onchocerca microfilariae (Nematoda) from dilution shock by the use of serum. Cryobiology, 19, 448-457. James, E. R. (1980). Cryopreservation of Schistosoma mansoni schistosomula using 40% v/v (1OM) methanol and rapid cooling. Ctyo-Letters, 1, 535-544. Macdonald, W. W. (1962). The selection of a strain of Aedes aegvpti susceptible to infection with semi-periodic Brugia malayi. Annals of Tropical Medicine and Parasitology, 56, 368-372. McCall, J. W., Jun, J. & Thompson, P. E. (1975). Cryopreservation of infective larvae of Dipetalonema viteae. 3oumal of Parasitology, 61, 340-342. McCall, J. W. & Weathersby, A. B. (1978). Survival and infectivitv of infective larvae of Dipetulonema viteae free of the tick. 4th International Congress of Parasitology, 19-26 August, Warsaw. Short Communications, Section c, p. 155. Meryman, H. T. (1970). The exceeding of a minimal tolerable cell volume in hypertonic suspension as a cause of freezing injury. In: The Frozen Cell, G. E. W. Wolstenholme & M. O’Connor (Editors). London: Churchill, pp. 5 l-64 Minjas, J. N. & Townson, H. (1980). The successful cryopreservation of microfilariae with hydroxyethyl starch as cryoprotectant. Annals of Tropical Medicine and Parasitology, 74, 571-573. Obiamiwe, B. A. & Macdonald, W. W. (1971). The preservation of Brugia pahangi microlilariae at sub-zero temperatures and their subsequent development to the adult stage. Annals of Tropical Medicine and Parasitology, -. 65, 547-354. Ogunba, E. 0. (1969). Preservation of frozen Brugia pahangt using dimethyl sulphoxide. Journal of Parasitology, 55, 1101-1102. Schacher, J. F. (1962). Developmental stages of Brugia pahangi in the final host. Journal of Parasitology, 48, 693-706. Schiller, E. L., Turner, V. M., Marroqt’iin, H., F. & D’Antonio, R. (1979). The cryopreservation ani;ltz cultivation of larval Onchocerca volvulus. 3oumal of Tropical Medicine and Hygiene, 28, 997-1009.
Accepted for publication
30th March,
1983.