Cryopreservation of an attenuated vaccine strain of the protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that infects birds and mammals, including humans. T. gondii T-263 is an attenuated mutant strain that is being developed as a live vaccine to protect cats from shedding oocysts. A cryopreservation procedure for T. gondii T-263 bradyzoites has been developed to meet the requirement for product stability. A Me2SO-based procedure for the cryopreservation of tachyzoites was used as a basis for process optimization. A modified cell culture plaque assay was used to determine the effects of selected cryobiological parameters on bradyzoite viability. The major parameters evaluated were: (i) cooling rates; (ii) intermediate plunge temperature; and (iii) thawing and dilution rates and temperatures. The optimized cryopreservation protocol comprised incubation in 12.5% Me2SO and 4% BSA for 30 min at room temperature, cooling at 1 degree C min-1 to -40 degrees C, followed by direct transfer into liquid nitrogen. Rapid thawing (approximately 120 degrees C min-1) followed by slow dilution of cryoprotectant over 15 min resulted in the highest survival. The optimized procedure increased survival 10,000-fold over that obtained using an established tachyzoite protocol. This procedure is to be adapted for the large-scale cryopreservation of T. gondii T-263 bradyzoites in individual vaccine doses.

Quantification of Toxoplasma gondii bradyzoites.

A simple 2-step method to selectively quantify functional bradyzoites of Toxoplasma gondii is described. The method was developed to quantify bradyzoites produced in cell cultures that also contain tachyzoites. The selection step comprises incubation of bradyzoites and tachyzoites in 0.026% pepsin at 37 C for 30 min. This treatment kills all tachyzoites, whereas all bradyzoites survive. A modified plaque assay is then used to quantify the surviving bradyzoites. Plaque assay cultures are scored according to levels of infection that correlate with numbers of bradyzoites added to each well. The assay can detect log differences in the range of 2 x 10(0)-2 x 10(4) bradyzoites per sample. This procedure is simple to perform and provides an efficient way of comparing numbers of functional bradyzoites in multiple samples that also contain tachyzoites.

On-host viability and fecundity of Ctenocephalides felis (Siphonaptera: Pulicidae), using a novel chambered flea technique.

The on-host viability and fecundity of cat fleas, Ctenocephalides felis (Bouché), confined within a novel chambering system are described. Using this system, all fleas and flea eggs are recovered from chambers after fleas have fed on cats. Thus, accurate calculations of both adult flea survival and female flea fecundity can be made. The technique provides a microenvironment in which adult fleas exhibit > 90% survival over 14 d. Female fleas lay an average of 9.5 eggs per day on the 2nd d of feeding, 22.1 eggs per day between days 3 and 7, and 19.6 eggs per day between days 3 and 14. These numbers are similar to values previously reported for studies in which fleas were not confined. The technique permits accurate, multiple sampling of experimental flea populations during a study, and does not require the use of pesticides or extensive combing to collect surviving fleas at the end of a study. Moreover, the technique does not require that cats be caged or prevented from grooming. Collecting data from fleas confined in chambers is much less time consuming and labor intensive than studies with free-roaming fleas.

Immunization of cats with tissue cysts, bradyzoites, and tachyzoites of the T-263 strain of Toxoplasma gondii.

Previous studies have demonstrated that oral administration to cats of tissue cysts of the oocyst-negative mutant strain of Toxoplasma gondii, T-263, induces immunity to oocyst shedding following challenge. Experiments were designed to compare the levels of protection induced by T. gondii T-263 when tissue cysts, bradyzoites released from tissue cysts, and tachyzoites were administered to cats. In 1 experiment, groups of cats received 2 oral doses of intact tissue cysts or released bradyzoites of T. gondii T-263 and were challenged 47 days later with the oocyst-producing strain of T. gondii T-265. All cats seroconverted following immunization and none of them shed oocysts following challenge. In a second experiment, groups of cats received tachyzoites of T. gondii T-263 as a single oral dose and either 1 or 2 intraduodenal doses; they were challenged 60 days after the last vaccination. All cats seroconverted following immunization. Following challenge, all cats shed oocysts except for 2 of 7 cats that received 2 intraduodenal doses of tachyzoites. Thus, orally administered bradyzoites of T. gondii T-263, either contained in intact tissue cysts or liberated from cysts, induced immunity to oocyst shedding. In contrast, tachyzoites did not completely protect against oocyst shedding, even when delivered directly to the duodenum and despite the development of high antibody titers.

Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora.

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 mul volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.

Liquid Culture of the Entomogenous Nematode Steinernema feltiae with Its Bacterial Symbiont.

The insect-parasitic nematode, Steinernema feltiae Filipjev strain 42, was reared in liquid culture along with its bacterial symbiont, Xenorhabdus nematophilus Thomas &Poinar. First-stage juveniles developed into reproducing adults in a maintenance salts medium containing resuspended Xenorhabdus cells and the yeast Kluyveromyces marxianus (Hansen) van der Walt or cholesterol. Cultures with media depths greater than 4 mm required aeration. Nematode populations increased as bacterial density increased. An optimal culture system was obtained when the bacteria and nematodes developed in a semidefined medium containing tryptic soy, yeast extract, and cholesterol and were incubated on a rotary shaker at 25 +/- 1 C. Under these conditions, up to 86% of the final population were infective juveniles.

Schistosoma mansoni: cholesterol uptake by paired and unpaired worms.

Mature males and females of Schistosoma mansoni were incubated for 24 hr in medium containing [3H]cholesterol. Worms thus labeled were paired for 24 hr with unlabeled partners, in vitro or in vivo by surgical implantation into hamsters. Controls consisted of additional unlabeled worms that did not pair. Scintillation counting of thin layer chromatographic separations of lipid extracts of schistosome tissues and of the culture medium indicated that [3H]cholesterol underwent no major metabolic changes during the course of the experiment. During the period of time allowed for pairing, labeled worms lost up to 65% of their [3H]cholesterol, which was detected in the pairing medium. In both unlabeled males and females which had paired with labeled partners, levels of [3H]cholesterol were higher than in unpaired controls. This suggests that normal cholesterol transfer in worm pairs is bidirectional and that it is facilitated by physical contact between juxtaposed membranes. Cholesterol exchange in schistosome worm pairs may be partly or wholly a consequence of normal tegumental turnover of the molecule.

Male-stimulated female maturation inSchistosoma: A review.

InSchistosoma mansoni and other schistosome species, pairing of the female with a male partner is necessary for the completion of reproductive morphogenesis and growth. Permanent contact with a male is also necessary for the maintenance of reproductivity in the sexually mature female. This phenomenon appears to be unique within the animal kingdom. The mechanism of male-stimulated female reproductive development in schistosomes remains unknown however. In this paper, the theories for the nature of the process are reviewed briefly, recent findings are added, and the biological and technical problems associated with its study are highlighted.

Passage of Salmonella enteritidis and Salmonella thompson through chick ileocecal mucosa.

The passage of Salmonella enteritidis and S. thompson across the cecal mucosa has been visualized in an electron microscope study with the freshly hatched chick as a model. The uptake of salmonellae by macrophages took place in the cecal lumen; the macrophages became abnormal and often ruptured to release organisms back into the lumen. The entry of bacteria into the epithelial cells was associated with a series of pathological changes, beginning with the appearance of active Golgi apparatus and the production of a variety of lysosomal vesicles. Salmonellae became sequestered within lysosomes but were unaffected by the presence of hydrolytic enzyme. Epithelial cell death was related to particularly large numbers of bacteria. Fragments of invaded epithelial cells, especially those undergoing cell death, contributed to the cytoplasmic debris and released further salmonellae into the lumen. Bacteria were never observed in large numbers below the basement membrane, and there was no significant pathology in the lamina propria tissue. Wandering cells, identified as macrophages and containing the bacteria, were observed spanning the epithelial and lamina propria regions through breaks in the basement membrane. It is suggested that the passage of bacteria from the epithelium to the lamina propria is primarily the result of capture and transport within host macrophages.

Wound healing in the trematode Schistosoma.

The ability of adult Schistosoma mansoni to effect wound healing over an exposed surface has been demonstrated. In transected worm segments a new external plasma membrane formed over the exposed tegumental cytoplasm. An elevated leading edge of tegument developed around the margin of the wound; the surface of this region was highly convoluted and there was a proliferation of membranous bodies within its cytoplasm. Inward migration of the leading edge over the exposed internal tissues took place. The resulting new tegument lacked spines and sensory endings. There was no regeneration of basal lamina or tegumentary cytons. In vitro maintenance of worm segments for 3 weeks did not give rise to any major ultrastructural changes in the tissues away from the wound.

Schistosoma mansoni: ultrastructure of adults from mice treated with oxamniquine.

The in vivo effects of a single oral dose (50 mg/kg) of oxamniquine on the ultrastructure of Schistosoma mansoni were investigated. In male worms, severe disruption of the tegument and gastrodermis took place, and extensive extracellular spaces developed between the cells of the internal tissues. Elimination of the damaged worms was associated with complete tegumental breakdown and encapsulation by host cells. A small proportion of females showed similar drug-induced changes and were also eliminated. In the residual females, no drug-induced morphological damage was observed even after a second dose of oxamniquine. However, these females became much reduced in size, and regression of the organs of the reproductive system took place. It is suggested that such regressive changes resulted from discontinued male stimulation rather than the direct effect of oxamniquine.

Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments.

Maturation and maintenance of normal reproductive function in female Schistosoma mansoni require a permanent association with the male, but the nature of this relationship is not well understood. The regional localization of a stimulatory factor in the male and its target in the female were investigated. Unisexual female and mature male worms were transected into segments of various lengths. Various combinations of transected male and female segments and intact worms were transferred to the mesenteric veins of recipient hamsters and were also maintained in vitro. In hamsters and in vitro, pairing took place between intact worms of each sex and segments of the other, and between segments of both sexes. The majority of female worms and segments so paired showed some reproductive development, as assessed by vitelline gland differentiation. In intact unisexual females paired with small male segments, vitelline gland development was limited to that portion of the worm that had been held by the male. Worm segments continued to display normal body contractions throughout 24 days of in vitro maintenance and morphological integrity was retained. It is concluded that 1) in the absence of a functioning gut, worm segments can survive for prolonged periods on nutrients absorbed through the tegument; 2) worm pairing, male stimulation, and the female developmental response are independent of central nervous control by the cerebral ganglia; 3) males have no centralized localization for the female-stimulating factor; 4) vitelline gland differentiation in the female requires local stimulation through male contact, and this is not propagated throughout the worm.

Putative polypeptide transfer from male to female Schistosoma mansoni.

Mature males and females, and unisexual females of Schistosoma mansoni were incubated in medium containing [14C]leucine. By use of polyacrylamide gel electrophoresis and fluorography no differences were detected in their ability to synthesize polypeptides. Male worms thus labelled were paired for various periods with unlabelled mature or unisexual females both in vitro and by surgical implantation into hamsters. Female worms paired in vitro had two controls: an accompanying female that did not pair and an additional female placed in the conditioned medium after removal of the other worms. After separation and washing, only small amounts of label were found in females by liquid scintillation counting despite the release by males of substantial amounts of label into the medium. No label was detected in fluorograms of polyacrylamide electrophoresis gels of re-paired females after 7 days' exposure to X-ray film, but a faint pattern of normal polypeptides above 50 kDa did develop after 6 weeks. In light microscope autoradiographs a low grain count was found over sections of unlabelled females paired with labelled males but this was no greater than that over females incubated in medium that had formerly held males. We have been unable to detect any polypeptide reported to be synthesized exclusively by male worms and transferred in large amounts to females. Although female worms did take up small amounts of male-derived metabolic products, the transfer of a specific polypeptide of 66 kDa in significant quantities did not take place under the conditions investigated.

Schistosoma mansoni: the survival and reproductive status of mature infections in mice treated with oxamniquine.

A single oral dose of 50 mg/kg of oxamniquine administered to mice with mature infections of Schistosoma mansoni gave rise to a hepatic shift and almost total elimination of the male worms. Regression of the reproductive system and a conspicuous reduction in size took place in the residual females which eventually resembled sexually immature adults. A second dose of oxamniquine had no apparent effect on these females within a period of 28 days following this treatment and it is suggested that this change in reproductive status is the result of discontinued male stimulation.