E.R. James

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

Abstract A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ( v v ) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ( v v ) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min −1 . Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.

Schistosoma haematobium in the baboon (Papio anubis): effect of vaccination with irradiated larvae on the subsequent infection with percutaneously applied cercariae

Groups of five baboons were vaccinated three times at approximately six-weekly intervals at a rate of 1,000 organisms per kg of gamma-irradiated Schistosoma haematobium larvae. Five vaccines were tested: 3 and 20 Krad cercariae applied percutaneously; fresh 3 and 20 Krad mechanically transformed schistosomula injected intramuscularly; and cryopreserved 20 Krad schistosomula injected intramuscularly. These five groups and an unvaccinated control group were challenged percutaneously with 7,500 S. haematobium cercariae three months after the last vaccination. The efficacy of the vaccines was judged by faecal egg excretion, and by adult worm and tissue egg recoveries at necropsy 4.5 months after challenge. Significant protection, with 64 to 89% reductions in worm burden and parallel reductions in egg production, was achieved by all but the cryopreserved vaccine, although egg production was not significantly reduced in those female worms which did mature. Cercariae tended to give more protection than schistosomula and 20 Krad more protection than 3 Krad. No significant pathology could be detected in an additional baboon vaccinated with 20 Krad schistosomula but not challenged with cercariae. This is an encouraging result for the development of a live vaccine against S. haematobium.

Recovery of infective Schistosoma mansoni schistosomula from liquid nitrogen: A step towards storage of a live schistosomiasis vaccine

Successful recovery of infective schistosomula of Schistosoma mansoni following storage at -196 degrees C is reported. The technique involves a two-step cooling procedure--slow cooling (0.65 degrees C min-1) to an intermediate temperature of -28 degrees C, followed by rapid cooling into liquid nitrogen (10,000 degrees C min-1). Rewarming (10,000 degrees C min-1) and rapid dilution to remove the cryoprotectant (17.5% methanol) yielded motile organisms some of which developed to adult worms in mice after intramuscular injection. The percentage of schistosomula developing to adult worms was small (0.44%), but is a significant step towards storage of trematode larvae and of a live attenuated vaccine for schistosomiasis.

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

Abstract Microfilariae of Onchocerca gutturosa , O. cervicalis and O. 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 min −1 to an intermediate temperature of between −14 and −17 C, followed by a rapid cool into liquid nitrogen (taking about 1 sec). Upon rapid warming to 37 C, a high percentage of microfilariae showed normal motility. Following subcutaneous injection into T.O. mice, the microfilariae of O. 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 . Microfilariae of O. volvulus also developed into third-stage larvae in this insect, while those of O. cervicalis developed similarly in their natural vector, Culicoides nubeculosus . This technique of preservation provides a good and reliable method for storage of viable microfilariae of these bovine, equine, and human Onchocerca spp.

Schistosoma mansoni: Influence of the mouse host's sex, age, and strain on resistance to reinfection

Abstract Male and female CBA strain mice varying in age from 2 to 7 months were observed to acquire the same degree of resistance to reinfection with Schistosoma mansoni . Random bred Tylers Original (TO) strain mice obtained from different commercial suppliers showed significant variation in mortality rate and the levels of resistance they respectively acquired to homologous reinfection with S. mansoni . Measurement of the mandibles of representative mice from the three TO mouse colonies confirmed the presence of genetic differences. A further comparison of seven strains of mice showed there to be significant differences, with respect to resistance to reinfection, between the inbred strains C57BL and C3H. However, in relation to the intermediate responses of other strains, this difference could not be attributed to H-2 specificity. In all of the strains tested there was a significant correlation between the number of worm pairs derived from the primary infection and the degree of resistance to challenge.

Cryopreservation of Plasmodium chabaudi. II: Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial protection of baboons against Schistosoma mansoni using radiation-attenuated cryopreserved schistosomula

Three groups of five baboons were vaccinated in Kenya using three doses of 10,000 viable cryopreserved schistosomula attenuated with either 10, 20 or 60 krad 60Co-irradiation prepared in England. Animals were vaccinated at four-week intervals, challenged after a further six weeks with 2,000 cercariae and perfused at 10 weeks after challenge. High antibody titres to schistosomula mediating in vitro cytoadherence with P 388D1 macrophage-like cells were demonstrated in all vaccinated animals but not in controls. Significant titres to soluble egg antigen (SEA) were also demonstrated by ELISA in the 10 and 20 krad vaccinated groups following the first vaccination. The subsequent vaccinations and the challenge boosted this response considerably. Mean anti-SEA titres were only elevated above background in the 60-krad group six weeks after the third vaccination and in the challenge controls six weeks after challenge. Peripheral eosinophil counts were slightly reduced and neutrophil counts slightly elevated before challenge while eosinophil and erythrocyte counts were elevated and neutrophil counts depressed after challenge. PCV values were erratic in all groups. Eggs appeared in the faeces from six weeks after challenge and excretion rates were higher in all three vaccinated groups than in the challenge controls by necropsy 10 weeks after challenge. Body-weights were depressed in all groups after challenge but subsequently rose in the 10 and 20 krad groups. The 60 krad and challenge control groups lost 12.4% and 7.9% of body-weight respectively after challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me2SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me2SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min−1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min−1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% vv) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 106 parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% vv and Me2SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% vv glycerol or 15% vv Me2SO and with 15% wv glucose in the diluent medium.

Protection of cryopreserved Onchocerca microfilariae (nematoda) from dilution shock by the use of serum

This paper describes the use of newborn calf serum during the cooling and warming/dilution phases of the cryopreservation of Onchocerca gutturosa microfilariae using an interrupted slow cool to −196 °C in the presence of 5% (v/v) methanol. Serum proved detrimental at concentrations above 20% (v/v) in the cooling medium unless it was also present in high concentrations, 60% (v/v) in the warming/ dilution medium. Damage to the organisms occurred predominantly during the thawing/dilution phase of cryopreservation and not the cooling phase and could be reduced greatly by the presence of high serum concentrations when thawing. This indicates that the major protective action of serum is that of reducing dilution shock—shock produced by a rapid influx of water and/or the effects of high solute concentrations established during cooling.

Studies on preservation of schistosomula of Schistosoma mansoni and S. mattheei

Abstract Schistosomula were not damaged by exposure for 1 hr at room temperature to the cryoprotectant dimethylsulphoxide (DMSO) providing that concentrations greater than 10% were not used. Rapid dilution to remove the DMSO was less harmful to the organisms than was slow dilution. Schistosomula were not damaged by thermal shock (cooling in the absence of freezing) but were damaged by conditions produced by freezing. Although the freezing damage rendered schistosomula noninfective they retained flame cell activity and certain contractile properties in the oral sucker, gut, and musculature. The least damage was produced by slow cooling (at approximately 0.3 °C/min) and fast warming (approximately 300 °C/min). Schistosomula remained infective following freezing and slow cooling to −20 °C in DMSO (10%) and storage for 2 hr at this temperature but were damaged at temperatures below −26 °C and at −20 °C for longer time periods.