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Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro
- Hong-Bo Liu, Pei-Ru Lv, Xiao-Gan Yang, Xiao-E Qin, Dao-Yuan Pi, Yang-Qing Lu, Ke-Huan Lu, Sheng-Sheng Lu, Dong-sheng Li
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Miniature pigs are valuable for research in xenotransplantation and as models for investigating human diseases. Although many mammalian species have been cloned, the success rates have been very low, especially in the pig. In the present study, an attempt was made to optimize somatic cell nuclear transfer (SCNT) protocols for use in the production of the Guangxi Bama mini-pig. Firstly, mini-pig fibroblast cells from a new-born Guangxi Bama piglet were isolated and cultured. Cell type was identified by fluorescence immunocytochemistry (ICC); the cells expressed cimentin, but not cytoceratin and follicular stimulation hormone receptor (FSHR). Secondly, the optimal cell cycle synchronization protocol for treating fibroblast cells from the newborn piglet's testicle was investigated by contact inhibition and serum starvation. When fibroblast cells were treated by contact inhibition, a higher fusion (66.0% vs. 58.3%, p > 0.05) and blastocyst production (20.8% vs. 15.1, p > 0.05) rates were obtained than with serum starvation. Thirdly, to examine the ability of old cells to be morphologically remodelled after activation, testicular fibroblasts (passage 10–14) were introduced into enucleated oocytes; enlarged nuclei were formed in most of the reconstructed embryos at 6 h and enlarged nuclei or distinct pseudopronuclei were formed in nearly all the reconstructed embryos at 12 h. The old donor cell could be morphologically remodelled correctly and was competent to support embryo development to the blastocyst in vitro. Fourthly, the in vitro development potential of the cloned embryos was investigated using two types of donor cell: ear fibroblasts and low or high passage testicular fibroblasts. The rate of fusion was highest using low passage testicle fibroblasts (84.5% vs. 69.8% and 80.0%, p < 0.05), as was development to the blastocyst stage (14.6% vs. 7.7% and 6.3%, p < 0.05). Finally, the effect of phytohaemagglutinin (PHA) on parthenogenetic and cloned embryo development was examined. The PHA had no significant effect on the parthenogenetic embryos, but cloned embryo development to the blastocyst stage was significantly increased by PHA (10μg/ml), (13.4% vs. 5.6% and 5.6%, p < 0.05).
Comparison of four serological tests for the diagnosis of Chagas disease in a Colombian endemic area
- R. GUTIERREZ, V. M. ANGULO, Z. TARAZONA, C. BRITTO, O. FERNANDES
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- Parasitology / Volume 129 / Issue 4 / October 2004
- Published online by Cambridge University Press:
- 14 September 2004, pp. 439-444
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The performance of 4 serological tests for the diagnosis of Chagas disease was evaluated in Santander, Colombia, a region still presenting active transmission. Serum samples from 638 individuals were submitted to an enzyme immunoassay test (EIA), using total lysate of a local Trypanosoma cruzi strain and 52·5% were positive (335/638). A subset of this group (94 positive individuals and 90 seronegatives) was randomly selected for further serological confirmation. Three additional tests were used – indirect immunofluorescence (IIF) and 2 distinct enzyme-linked immunosorbent assays using total lysate of the Y strain (EIA BM) and a mixture of 2 recombinant antigens (EIA RA). Seventy-nine patients were seropositive in all tests (84·0% – 79/94). The number of positive sera with the IIF, EIA RA and EIA BM was 84/94 (89·4%), 80/94 (85·1%) and 79/94 (84·0%), respectively. In 15 out of the 94 EIA seropositive patients (16·0%), 10 individuals were negative in all 3 tests (10·6% – 10/94). One was negative in the EIA BM and positive in the other two tests (1·1% – 1/94) and 4 patients were positive, solely, in the IIF assay (4·3% – 4/94). Relative to the 90 EIA negative individuals, 89 were confirmed in all other tests (98·9% – 89/90). One individual, although seronegative in the IIF, was positive in both confirmatory EIA tests (1·1% – 1/90). In addition, 120 blood specimens were submitted to PCR amplification. This group consisted of 79 confirmed seropositive cases, 16 individuals with discordant serological results and 25 validated seronegative individuals. The PCR was able to detect the presence of parasite DNA in 67 out of the 79 seropositive patients (84·8%), in 8 individuals with discordant serology (50·0%) and in only one seronegative individual (4·0%). The results pointed to the necessity for performing more than one serological test, preferentially with antigens from autochthonous strains, to achieve a reliable diagnosis of Chagas disease in Colombia.