Differential expression of luteinizing hormone receptor, androgen receptor and heat-shock protein 70 in the testis of long-distance transported mice.

Spermatogenesis, is a complex process of precisely regulated intracellular events, where it is affected by many factors. Long-distance transport of animals is one of the stressors that may influence spermatogenesis and sperm quality. The present study chose luteinizing hormone receptor (LHR), androgen receptor (AR), and heat-shock protein 70 (HSP70) as our target genes to investigate their mRNA and protein expression in the testes of long-distance transported (about 1000 km) mice. Histological analysis showed that there was a reduction in the thickness of the seminiferous epithelium in the transported mice, and a significant decrease in body weight and sperm count in the epididymis was also observed. mRNA expression was determined by QPCR in the testis of transported and control mice. The levels for AR decreased significantly in transported mice. LHR and HSP70 expression in the testes of the transported mice was slightly higher than that of control mice but did not reach a significant level. A similar tendency of protein expression was also observed by Western blot analysis. The levels of LHR and HSP70 increased slightly after transportation. However, none of the changes were statistically significant compared with the control mice. In conclusion, long-distance transport has an adverse effect on reproductive organs and spermatozoa in adult mice.

Efficacy and safety of an oral somatostatin DNA vaccine without antibiotic resistance gene in promoting growth of piglets.

This study aimed to evaluate the efficacy and safety of an oral DNA vaccine against somatostatin (SS) (pGS/2SS-asd, encoding two copies of somatostatin genes) mediated by attenuated Salmonella choleraesuis C500 without antibiotic resistance gene on piglets growth. A total of 50 piglets were uniformly divided into five groups. The animals in the first three groups were orally given vaccine in dose of either 5 9 1010, 5 9 109 or 5 9 108 colony-forming units (CFU).The remaining two groups were orally administered with either bacteria C500(containing pVAX-asd plasmid without somatostatin gene) or phosphate buffered saline (PBS) as controls. The results indicated that the vaccine induced SS-specific antibodies in a dose-dependent pattern. Compared with the PBS control, animals in the high-dose group showed lower SS levels and higher growth hormone (GH) levels in sera. Average daily gain of animals in the high dose group was increased by 32.88% and 26.46% during 4 and 8 weeks,respectively. Anti-SS antibodies were positively correlated with either GH levels or average daily gain at week 8 after primary immunization (P < 0.05). Faecal,soil and water samples originating from immunized piglets and surrounding environment were collected. The target gene (the fusion gene GS/2SS) of C500(pGS/2SS-asd) was not detected by PCR amplification in these samples,indicating that the surrounding environment was not contaminated by residual recombinant bacteria. In conclusion, the vaccine without antibiotic resistance gene is attributable to improve growth performance of piglets through an influence on GH secretion. Moreover, the immunization did not contaminate the surrounding environment of animals.

Oral and intranasal administration of somatostatin DNA vaccine mediated by attenuated Salmonella enterica serovar Typhimurium to promote growth of piglets.

An attenuated strain of Salmonella typhimurium has been used as a carrier for oral and intranasal genetic immunization. Here, we evaluate the efficacy of a vaccine strain of S. typhimurium. CSO22 (pGM-CSF/SS, plasmid granulocyte-macrophage colony-stimulating factor/somatostatin) expressing two copies of SS genes. A total of 115 piglets, aged 2 months old, were either orally or intranasally immunized against the vaccine strain CSO22 (pGM-CSF/SS) with three dosages (5 × 10(10) colony forming units (CFU), 5 × 10(9) CFU and 5 × 10(8) CFU). For oral immunization, the specific anti-SS antibodies were detected in the immunized piglets. The levels of SS antibodies in the high-dose immunized group (5 × 10(10) CFU) were significantly higher than that in the phosphate buffered saline immunized group (P < 0.01) and 40% of animals were positive in SS antibodies in the high-dose immunized group. Moreover, the weight gain of the high-dose group was increased by 20.86%, 10.26% and 15.30% during 4, 8 and 12 weeks, respectively, after immunization in comparison to the control. For intranasal immunization, the growth of the low-dose group was increased by 10.23% in the whole test period (12 weeks). In conclusion, our results suggest that the recombinant strain could elicit anti-SS antibodies and improve the growth performance of immunized piglets, and that the oral immunization program is better than the intranasal program.

Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines.

A novel plasmid pGS/2SS-M4GFP was constructed in the present study by recombination of GS/2SS gene and enhanced green fluorescent protein (M4GFP) sequence. The GS/2SS fusion gene encoding two copies of somatostatin genes was firstly introduced into pVAX-asd vector in which the kanamycin resistance cassette was replaced by the asd cassette. The M4GFP gene was then fused into 3' end of GS/2SS gene in the proper reading frame. After purified, plasmid pGS/2SS-M4GFP was transfected into different cell lines derived from pig kidney and human cancer cells. The transcription process of GS/2SS gene was confirmed by RT-PCR, and the localization as well as expression of GS/2SS-M4GFP fusion protein was observed by confocal microscopy and ELISA. Transfection results revealed that sole M4GFP was localized within the cytosol and the nucleus, while fusion protein GS/2SS-M4GFP was localized only in the cytoplasm. Furthermore, it should be noted that subcellular localization of GS/2SS-M4GFP was not specific to one cell line, but appeared to be common across a variety of cell lines. These results provide for the first time valuable evidence that M4GFP is a versatile tool to trace GS/2SS protein and pave the way for further study on its tissue distribution and immunological mechanism in vivo.

Serological survey of bovine herpesvirus type 1 infection in China.

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).

Infection of HeLa cells by avian infectious bronchitis virus is dependent on cell status.

To investigate the adaptation of avian infectious bronchitis virus (IBV) in a human cell line may be beneficial to understanding the potential mechanisms of coronavirus interspecies infection. The current study addressed the poor replication of IBV in the HeLa human cell line demonstrated in previous reports. We showed that IBV strains M41, H52, H120 and Gray could be propagated in HeLa cells with distinct cytopathic effect. The virus titre in freshly dispersed HeLa cells was 1000-fold higher than in cell monolayers. Trypsin was not the determinant for the viral replication, suggesting that the restriction of IBV replication in HeLa cells is the result of intracellular events rather than the binding to or fusion with host cells. These IBV strains replicated to an average titre of 10(3.4+/-0.2)/0.1 ml median tissue culture infectious doses in freshly dispersed HeLa cells and maintained this titre for the first 12 passages. Then an approximately 10-fold increase (10(4.20+/-0.19)/0.1 ml) occurred in passage 13, which was maintained to passage 16, after which there was another, bigger rise to 10(6.6+/-0.3)/0.1 ml in passage 17. This titre was maintained until passage 24 when the experiment was terminated. The IBV M41 S1 gene was amplified and sequenced for passages 0, 5 and 21. There was only one amino acid replacement in the S1 protein, in passage 21. The presence of sialic acid on HeLa cells contributed to efficient virus replication, while human aminopeptidase N was not involved in the infection. Haemagglutinin activity gradually reduced with increased passages. These results indicated that the virus adaptation would probably be determined by host cell modification such as receptor glycosylation and different receptor utilization instead of viral gene mutation.