Identification, molecular characterisation and differential expression of caveolin-1 in Trichinella spiralis maturing oocytes and embryos.

Caveolins are integral membrane proteins implicated in cholesterol homeostasis and transport, endocytosis mechanisms and regulation of signal transduction in differentiated cells. In this work a caveolin-1 gene from the nematode Trichinella spiralis (Ts-cav-1) was cloned and identified as an adult-specific antigen. For this, a cDNA library of T. spiralis 3-day-old adult worms was screened using a stage-specific cDNA-labelled probe. One positive clone contained a cDNA insert of 1427-bp and a full-length open reading frame (ORF) of 687-bp, which encodes for a 229 amino acid polypeptide with a theoretical molecular weight of 26kDa. BLAST and FASTA searches revealed a 36% and 57% identity with Caenorhabditis elegans caveolin-1, respectively. Confocal laser microscopy analysis using antibodies generated against Ts-CAV-1 protein and cross-sections of adult parasites showed that Ts-CAV-1 gradually accumulates on the surface of Trichinella oocytes and embryos, reaching a maximum at 3days p.i., and decreasing during new-born larvae (NBL) development. RT-PCR assays of parasites from 1 to 4days p.i. showed a similar gene expression profile to that observed for Ts-CAV-1 which suggests a specific developmental regulation. Free cholesterol was mainly distributed in the female germ line and it displayed increasing membrane accumulation, similar to the pattern obtained for Ts-CAV-1 protein, which suggests a temporal membrane association with Ts-CAV-1 that in turn will perform the functions mentioned above. Our results strongly indicate that Ts-cav-1 from T. spiralis plays a role in oocyte maturation and embryogenesis during development, demonstrating gender-specific expression.

[Genetic variability of Trichinella spiralis Oven, 1835, and Trichinella pseudospiralis Garkavi, 1972, detected by polymerase chain reaction with random primers]. / Geneticheskaia izmenchivost' Trichinella spiralis Oven, 1835, i Trichenella pseudospiralis Garkavi, 1972, vyiavliaemaia metodom polimeraznoi tsepnoi reaktsii so sluchainymi praimerami.

DNA polymorphisms in two parasitic nematode species, Trichinella spiralis Oven, 1835, and Trichinella pseudospiralis Garkavi, 1972, were revealed via random amplification of polymorphic DNA by the polymerase chain reaction (RAPD PCR). The diagnostic value of seven 10-bp oligonucleotide primers was evaluated, and the extent of the homology between the genomes of the two species was estimated. The intraspecific variation of RAPD markers was revealed in larvae of both species isolated from experimentally infected white rats. The variation was higher in larvae from nonlinear rats than in larvae from linear rats. When animals were infected with both Trichinella species simultaneously, "hybrid" progeny were obtained that had capsule that somewhat differed in shape from one characteristic of the parental species, T. spiralis. In RAPD spectra, the hybrids showed higher similarity of T. spiralis than to T. pseudospiralis. Intra- and interspecific differentiation, genome divergence, and factors inducing the intraspecific variation in Trichinella species are discussed.

Characterization of kinetics of anti-Trichinella spiralis newborn larvae immunity in rats.

The kinetics of anti-T. spiralis newborn larvae (NBL) immunity and its dose effects were studied in vivo. Rats were either immunized with newborn larvae i.v. or muscle larvae per os and challenged with newborn larvae either i.v. or i.p. on day 7 up to day 27 after immunization. Immunity was assessed by examining the muscle larvae burden or the larval recovery from the peritoneal cavity. Recovered newborn larvae were further examined for cell adherence and viability. Results indicate that as early as 9 days after infection and only 3 days after newborn larvae production in vivo, specific anti-newborn larvae immunity was developed. Peritoneal cells as well as blood cells adhered to the cuticles of the larvae and killed them. When different doses of immunization were examined, it was found that 2,000 muscle larvae per os induced the strongest immunity as compared to 500, 5,000 or 6,000. Such immunity maintained its strength when challenge infection with newborn larvae reached 50,000 dosage and it declined significantly when the dose reached 100,000. This indicates that the immune cells and antibodies are not re-deployed.

Ultrastructural study of Trichinella spiralis with emphasis on adult female reproductive organs.

The ultrastructure of the reproductive organs of adult female T. spiralis was described on the basis of extensive observation under an electron microscope. The organisms were recovered from the host intestine 7 days after oral infection. The organs consisted of a single ovary, seminal receptacle, uterus and vagina, confirming the reports described by previous authors at light microscopical level. All the organs were surrounded by a basal lamina and epithelial cells, and were bathed in haemolymph. Ova were formed in a germinal zone on the ovary wall exhibiting a half-moon shape, and spread to other sides of the ovary. Mature ova exhibited a smooth cell surface, cuboidal shape, prominent cytoplasmic polysomes, a clear nucleus and a well-developed nucleolus. Embryos in early stage of development consisted of numerous small cells and were surrounded by a sheath. As they matured they shed the sheath and left a pool of sheaths in the uterus. During oogenesis and embryogenesis of adult worms in normal development, three occurred lipid droplets and the degradation of embryos; reportedly signs of worm damage.