Therapeutic potential of amniotic fluid stem cells to treat bilateral ovarian dystrophy in dairy cows in a subtropical region.

Amniotic fluid is a rich source of multipotent mesenchymal stem cells (MSCs). Amniotic fluid stem cells (AFSCs) have become a new source of stem cells; they have low immunogenicity and are easily harvested. For this reason, they may be useful in clinical tissue engineering. Moreover, AFSCs have anti-inflammatory properties and can repair tissues. This study evaluated the utility of AFSC injection to treat bilateral ovarian dystrophy in Holstein-Friesian cows. Bovine AFSCs (BAFSCs) were collected at slaughter from Holstein-Friesian cows during the third or fourth month of pregnancy and cultured in vitro. The BAFSCs began to show a fibroblast-like morphology. They were positive for ß-integrin, CD44, CD73, CD106 and Oct4 and negative for CD34 and CD45. After induction, the cells differentiated into mesodermal lineages. Bilateral ovarian dystrophy was confirmed by ultrasonography in 16 lactating cows. The subsequent experiment lasted 15 weeks. Serum was collected weekly to analyse progesterone concentrations, and weekly ultrasonography recorded ovarian changes. Each cow was equipped with an automatic heat detection system to facilitate oestrus observation and breeding records. The progesterone concentration of two cows in the treatment group (25%) significantly increased during weeks 10-15. On ultrasonography, the treatment group demonstrated mature follicles after BAFSCs injection, and foetuses were visualized approximately 40 days after artificial insemination (AI). Oestrus rates in the control and treatment groups were 0% (0/8) and 50% (4/8), respectively; pregnancy rates were 0% (0/8) and 25% (2/8), respectively. Calves were successfully delivered in both cases of pregnancy. These results show that BAFSCs can alleviate bovine ovarian dystrophy and restore fertility.

Molecular cloning and nucleotide sequence determination of three envelope genes of classical swine fever virus Taiwan isolate p97.

A strain of classical swine fever virus (CSFV) has been isolated in Taiwan. The cDNA coding for three envelope glycoproteins E1, E2 and E3 were molecularly cloned from purified viral particles using the reverse transcription-polymerase chain reaction (RT-PCR) method and sequence-specific primers. The resulting PCR products (1113 bp for E1. 699 bp for E2 and 567 bp for E3) were cloned into the SmaI site of pUC19 and then subjected to DNA sequence analysis. Data showed that nucleotide sequence of the three envelope genes shared a 82-83% homology with the corresponding genes of three other strains (Alfort, Brescia and Weybridge). However, the homology of the deduced amino acid sequence was greater than 90% among the four strains. The potential asparagine-linked glycosylation sites for E1 (5 sites), E2 (7 sites) and E3 (2 sites) were conserved. This suggests that the Taiwan p97 strain is distinct from other three strains described. The variations may have implications for future vaccine development. The sequence has been submitted to GenBank. The accession numbers are U43924 and U03290.

The presence of RNA splicing signals in the cDNA construct of the E2 gene of classical swine fever virus affected its expression.

E2 is the major neutralizing antigen for classical swine fever virus (CSFV) infection. Previously, we have cloned and sequenced the E2 cDNA of Taiwan strain p97 by the reverse transcription-polymerase chain reaction (RT-PCR) method from CSFV-infected tissue. The presence of RNA splicing donor and acceptor sites were found in the cDNA sequence. In this study, transfection of E2 cDNA into mammalian cells resulted in the production of a spliced RNA. Site-directed mutagenesis of the donor and acceptor sites prevented the RNA splicing event and generated a full length transcript in COS7 cells. Although the spliced E2 transcript has not been reported in natural infection of CSFV, this study suggested that the potential splicing sites affected the E2 gene expression when the plasmid-based E2 gene was introduced into mammalian cells.