232

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL. MEDICINE AND HYGIENE, VOL. 76, No. 2, 1982

Successful cryopreservation of Wuchereria bancrofii microfilariae

DAWN G. OWEN’ AND M. ANANTARAMAN' ‘Medical Research Council Laboratoy Animals Centre, Woodmansterne Road, Carshalton,

Surrey SM.5 4EF, UK; ‘Principal Investigator, Zoological Survey of India, 100 Santhome High Road, Madras 600 034, India

A fast freezing technique -developed by JAMES (1981) significantly improved the yield of schistosome larvae that could be stored in liquid nitrogen. This was adapted by HAM et al. (1981) to the freezing of microfilariae of Onchocerca spp. with excellent results. They found 70 to 80% of the cryopreserved larvae remained viable using this method and since then Ham (personal communication) has successfully ap- plied the same technique to Brugia malayi microfil- ariae. The further application to Wuchereria bancrofti is described.

Introduction The present study arose from a need for a steady

supply of Wuchereria bancrofti L3 larvae. Bearing in mind the successful cryopreservation of Brugia sp. there seemed a reasonably good chance that W. bancrofti microfilariae might also withstand cryo- preservation and that, & this way, quantities- of microfilariae collected m the endemic areas could be stock-piled and retained for use when required.

A practice attempt was made to complete the whole cycle of development in the laboratory, commencing with frozen microfilariae of B. pahangi. The micro- filariae were collected from worms cultured in the peritoneal cavity of the jird, frozen in the manner described by HAM et al. (1981), and thawed and fed artificially to mosquitoes using the method described by PONNUDURAI et al. (1971). Active third-stage larvae (Ls) were recovered at the end of two weeks from the mosquitoes and, when inoculated into jirds, adult worms develoFd to maturity.

Materials, methods and results The W. banuoftz microfilariae were collected from

patients in endemic areas in the Indian states of Kerala and Tamil Nadu. The blood was collected between 10.30 and 11.30 at night into heparinized tubes. It was filtered through a nuclepore membrane in a Seitz filter using the method described by DENNIS & KEAN (1971) and the microfilariae were washed off the filter into 199 tissue culture medium containing foetal calf serum. Thereafter the microfilariae were processed according to the Ham technique; centri- fuged at 650 P for 10 min. the suoernatant removed a&i the worn& resuspendid in 10% v/v ethanediol in 199 tissue culture medium to a volume of either O-5 ml or 1 ml, depending on the worm count of the sample involved, and the suspension incubated at 37°C for 15 min. The second incubation, at O’C, was

carried out by placing 10 ~1 drops of 70% v/v ethanediol in 199 tissue culture medium on thin slips of glass resting on a strip of metal cooled by crushed ice. To each of these drops was added 10 ~1 of microfilarial suspension (containing 10% v/v ethanediol) resulting in a final cryoprotectant concen- tration of 40% v/v. This incubation period was extended from the 40 to 45 set recommended by HAM et al. (1981) for Brugia sp. to 60 set in an attempt to accommodate the slightly larger larva. Rapid test thawings were made shortly after collection and freezing by plunging one of the 20 ~1 droplets from each batch into 199 tissue culture medium with 10% foetal calf serum at a temperature of 37°C (HAM et al., 1981). Approximately 50% of the larvae resuscitated in this way exhibited a characteristically lively activity.

On return to the UK, the viability of the stored microfilariae was assessed by their ability to infect mosquitoes. Six of the 20 ~1 frozen samples were thawed and fed to a small batch of Glen sp. at a concentration of 17/10 ~1. After infection the mos- quitoes were maintained in the insectary at the London School of Hvgiene and Tropical Medicine for 14 days before transport to the Laboratory Animals Centre. Dissection of anoroximatelv 150 mosauitoes produced 60 normal i; W. bankofti on day 15 together with five immature or damaged larvae.

Because of possible losses during a period when liquid nitrogen ran low, no accurate assessment of the freezing technique using the viability of the micro- fdariae brought back from this field collection can be made. However, the Ham technique has proved suitable for the collection of this species, even under adverse conditions, and the surviving larvae have been proved viable.

Acknowlednements Thanks are extended to &. R. Suswillo, London

School of Hygiene and Tropical Medicine, London and Dr. P. Ham, London School of Hygiene and Tropical Medicine, Winches Farm, St. Albans, Herts, UK.

References Dennis, D. T. & Kean, B. H. (1971). Isolation of

microfilariae: report of a new method. Journal of Parasitology, 57, 1146.

Ham, P. J., Townson, S., James, E. R. & Bianco, A. E. (1981). An improved technique for the cryo- preservation of Onchocerca microfilariae. Parasit- ology (in press).

James, E. R. (1981). Schistosoma mansoni: cryo-

D. G. OWEN AND M. ANANTARAMAN 233

preservation of schistosomula by 2-step addition of ported between laboratories. Journal of Helmin- ethanediol and rapid cooling. Experimental Para- thology, 45, 415-418. sitology, (in press).

Ponnudurai, T., Denham, D. A. & Nelson, G. S. (1971). The use of a membrane feeding technique for infecting mosquitoes with filarial worms trans- Accepted for publication 23rd July, 1981.

New Fellows

Elected 10 December 1981

Ahmed, A. H., MB, ChB, Britain Ahmed, M., MB, BS, Nigeria Ahmed, S. I., MB, BS, India Akande, G. I., BSc, Nigeria Ali, K. N., MB, ChB, Britain Al-Shafi, K. M., MB, ChB, Britain Amajoh, Chioma N., BSc., Nigeria Bahety, S., MB, BS, India Beidas, M. F., BSc, United Arab Emirates Brown, M., MB, BS, DTM&H, BSc, FRCP, Britain Buck, R. L., MD, MPH, U.S.A. Burger, P. J., MB, ChB, M.Med.Path, South Africa Burhanuddin, M. D., MB, BS, DTCD, Bangladesh Carr, W. E., MD, BSc, U.S.A. Chakraborty, A., MB, BS, India Chatterjee, A., MB, BS, India Chew, M. W. K., BSc, DIC, PhD, Britain Chiotha, S., B.Ed., Malawi Choudhury, S. K., MB, BS, India Dala, G. R. M., MB, BS, Britain Das, D. P., MB, BS, India Das, S. K., MB, BS, Dip.Card, India Das, S. P., MB, BS, India Daud, K. H., B.Tech., Brunei De, K. K., MB, BS, India De Bonilla, L. C., MD, Venezuela Dey, K. C., MB, BS, India Dirie, M. F., MVSc, MSC, Somali Dutta, R. S., MB, BS, India Duttachoudhury, J., MB, BS, India Edem, C. B., BM, BCh DTCD, Britain Franke, Eileen D., PhD, U.S.A. Ganguly, B., MB, BS, India Gwayer, A. A. H., MB, ChB, Kuwait Haruna. A. S.. MB. BS. Nigeria Henshaw, E. I?., BSc Nigerii Ingole, D. L., MB, BS, MD, MSc, India Iimenez, V. E., MD Peru Jose-John, K., BSc, MB, BS, India

Ketchurn, D. ti., MD, MPHTM, U.S.A. Khader, M. A., MB, BS, India Kitolo, H. K., BSc, Kenya Kumar, P. V., MB, BS, ECFMG, MRCP, India Kundu, D., MSc, India Kundu, S., MB, BS, India Lewis, Jane E. C., BSc, MSc, Britain Londner, M. V., PhD, Israel Lovelace, Cheryl E. A., BSc, PhD, Zambia McManus, D. I’., BSc, PhD, Britain Macpherson, C. N. L., BSc, PhD, DIC, Britain Mandal, K. S., MB, BS, India Mechiel, C. K. C., MB, BS, DTPH, Malaysia Miri, F. S., MB, BS, Nigeria Mondal, D. K., BSc, MB, BS, Dip.Card., DCP,

India Mondal, N. C., MB, BS, India Mukhtar, A., BSc&Ed, Britain Nash, T.:, MD, U.S.A. Nwokedi, Josephine N., BSc, Nigeria Palmer, K., MS, MPH, PhD, U.S.A. Pardhy, V., MB, BS, MRCS, LRCP, MRCP,

DTM&H. U.S.A. Pien, F. D., MD, MPH, MS, U.S.A. Pilakasiri, C., BSc, Thailand Rahman, N. A., BSc, Britain Rakshit, S. K., MB, BS, India Raseroka, B. H., MSc, South Africa Ray, S. K., MB, BS, India Rossi, B. C., BSc, Brazil Saha, S. K., MB, BS, MIPHA, India Sanyal, R., MB, BS, DPH, India Schwartzman, J. D., MD, U.S.A. Sen Gupta, U. K., MB, BS, DDV, India Seroney, I. K., BSc, Kenya Sharma, R. A., MB, BS, India Tjokrosonto, S., MD, M.Comm.H, Indonesia Tun-Lin, W., MB, BS, DP&TM, Burma Udezue, E. O., MB, BS, DHMSA, Britain Unuane, M. B., MD, German Federal Republic Wittner, M., MD, PhD, U.S.A.