Genome-wide association studies to identify quantitative trait loci affecting milk production traits in water buffalo.

Water buffalo is the second largest resource of milk supply around the world, and it is well known for its distinctive milk quality in terms of fat, protein, lactose, vitamin, and mineral contents. Understanding the genetic architecture of milk production traits is important for future improvement by the buffalo breeding industry. The advance of genome-wide association studies (GWAS) provides an opportunity to identify potential genetic variants affecting important economical traits. In the present study, GWAS was performed for 489 buffaloes with 1,424 lactation records using the 90K Affymetrix Buffalo SNP Array (Affymetrix/Thermo Fisher Scientific, Santa Clara, CA). Collectively, 4 candidate single nucleotide polymorphisms (SNP) in 2 genomic regions were found to associate with buffalo milk production traits. One region affecting milk fat and protein percentage was located on the equivalent of Bos taurus autosome (BTA)3, spanning 43.3 to 43.8 Mb, which harbored the most likely candidate genes MFSD14A, SLC35A3, and PALMD. The other region on the equivalent of BTA14 at 66.5 to 67.0 Mb contained candidate genes RGS22 and VPS13B and influenced buffalo total milk yield, fat yield, and protein yield. Interestingly, both of the regions were reported to have quantitative trait loci affecting milk performance in dairy cattle. Furthermore, we suggest that buffaloes with the C allele at AX-85148558 and AX-85073877 loci and the G allele at AX-85106096 locus can be selected to improve milk fat yield in this buffalo-breeding program. Meanwhile, the G allele at AX-85063131 locus can be used as the favorable allele for improving milk protein percentage. Genomic prediction showed that the reliability of genomic estimated breeding values (GEBV) of 6 milk production traits ranged from 0.06 to 0.22, and the correlation between estimated breeding values and GEBV ranged from 0.23 to 0.35. These findings provide useful information to understand the genetic basis of buffalo milk properties and may play a role in accelerating buffalo breeding programs using genomic approaches.

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.

Effects of FUT1 gene mutation on resistance to infectious disease.

Alpha-(1,2)-fucosyltransferase (FUT1) gene has been identified as a candidate gene for regulating the expression of Escherichia coli F18 receptor gene (ECF18R) which promotes adherence of Enterotoxigenic (ETEC) and Verotoxigenic (VTEC) Escherichia coli (E. coli) via F18 fimbriae. In order to illustrate the polymorphisms of FUT1 and their effects on resistance to natural infection by Porcine Respiratory and Reproductive Symdrome Virus (PRRSV) and Haemophilus parasuis, the distributions of different genotypes and the relative risks of disease incidence in pigs were investigated. A total of 1,041 pigs representing three European breeds (Duroc, Landrace and LargeWhite), five Chinese local breeds (Wild pig, Small MeiShan, QinPing, JinHua, and JianLi) and three commercial populations (LargeWhite × JianLi, Duroc × Landrace × LargeWhite and Duroc × wild pig) were selected to analyze the genotype of the FUT1 gene by PCR-RFLP. Only the GG genotype associated with susceptibility to ECF18 bacteria was detected in Chinese local pig breeds and a population of LargeWhite × JianLi, while the AA genotype which confers resistance to ECF18 was detected in two European breeds (Duroc and LargeWhite), two populations of Duroc × wild pig and Duroc × Landrace × LargeWhite. Regarding relative risk of incidence, Duroc × Landrace × LargeWhite with genotypes GG or AG showed greater relative risk (OR = 2.040, P = 0.025; OR = 1.750, P = 0.081, respectively) than those with genotype AA during natural infection by both PRRSV and Haemophilus parasuis. It can be concluded that the mutation of FUT1 gene might play a role in pig infection by multi-pathogens, and that AA may be a favourable genotype for increasing the resistance to disease.

An immunological method to screen sex-specific proteins of bovine sperm.

This study was designed to identify sex-specific antibodies (SSAb) in rabbit antisera against bovine sex-sorted sperm, and capture sex-specific proteins of bovine X- or Y- proteins by SSAb. The rabbit antisera against bovine X- or Y-sperm were first produced by a series of immunological approaches, and further purified through immuno-neutralization with excess sex-sorted Y- or X-sperm, respectively, to remove non-sex specific antibodies and enrich sex-specific antibodies. After removal of non-sex specific antibodies, the purified rabbit sera with enriched sex-specific antibodies were screened for sex-specific antibodies by immunofluorescence staining and flow cytometry. The results showed that 3.0, 2.2, and 4.2% of unsorted sperm, sex-sorted X-sperm, and sex-sorted Y-sperm were recognized by the purified rabbit antisera against Y-sperm, respectively, whereas 29.2, 19.7, and 3.9% of unsorted sperm, sex-sorted X-sperm, and sex-sorted Y-sperm were recognized by the purified rabbit antisera against X-sperm. These results suggested that the purified rabbit antisera against X-sperm contained SSAb that preferentially bound to sex-sorted X-sperm. Subsequently, the purified rabbit antisera against X- or Y-sperm were used to immunoprecipitate sex-specific proteins in bovine sperm proteins, and a 30-kDa protein was specifically captured by the rabbit antisera against X-sperm. In conclusion, our results implied that this 30-kDa protein might be a sex-specific protein in bovine X-sperm, which has the potential to be used in immunological procedures for sexing sperm.

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.

Polymorphism of the growth hormone gene and its association with growth traits in Boer goat bucks.

In the present study, the polymorphism of growth hormone (GH) gene was analyzed as a genetic marker candidate for growth traits in Boer goat bucks. Two single nucleotide polymorphisms (SNPs) - A781G (Ser/Gly35) and A1575G (Leu147), were identified by GH gene sequencing and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis. AA genotype resulted in a significant decrease in birth chest girth (P=0.03) and weaning weight (P=0.014) comparing to AB genotype, while CC genotype contributed to weaning height (P=0.04) greater than CD genotype. When in combination, AACD genotype was undesired for lower scores in a series of growth traits including body weight, length, height, and chest girth at birth and weaning, as well as the pre-weaning daily gain and body weight at age of 11 months. These results indicate that new molecular markers associated with caprine growth traits can be used in MAS (marker-assisted selection) in Boer goat bucks.

Development and evaluation of a novel DNA vaccine expressing inhibin alpha (1-32) fragment for improving the fertility in rats and sheep.

Inhibin is an important protein hormone in regulating folliculogenesis. Immunization against inhibin can improve follicle developments. The objective of present study is to investigate inhibin DNA immunization as a potential tool for improving follicle development and litter sizes of female animals. In our study, the inhibin DNA vaccine was constructed with inhibin alpha (1-32) fragment inserted into the C termination of HBsAg-S. Ninety rats and forty sheep were immunized with inhibin DNA vaccine. In rats, immunization against inhibin resulted in increase of positive sera ratio (P<0.01). The treatment was accompanied by a significant increase in the total number of mature follicles of >0.8mm in diameter on the onset of estrous cycle after twice immunization (31.0+/-3.9 in the test groups versus 27.4+/-5 in control groups) and after third immunization (35.2+/-6.7 in the test groups versus 30.3+/-5.2 in control groups). Litter sizes were significantly (P<0.05) bigger in rats treated with inhibin DNA vaccine (12.7+/-4.5 n=6) than in control (9.8+/-4.5). In sheep, twinning rate in test groups (39.2%) was significantly higher than that in control groups (10%) after immunization (P<0.05). These results indicated that inhibin was an important factor in improvement of fertility in rats and sheep, and demonstrated that DNA immunization against inhibin could induce more mature follicles resulting in increased litter sizes. Our results revealed that inhibin DNA vaccine may be an alternative to the use of exogenous gonadotrophins for increasing ovarian follicular development and improving animal fertility.

Evaluation of the VP22 gene adjuvant for enhancement of DNA vaccine against somatostatin in mice.

Somatostatin (SS) is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. A novel SS-VP22 fused vaccine, pEGS2SS-V, was constructed from pEGS2SS plasmid with a VP22 gene fragment. Two times of immunization with pEGS2SS-V-induced anti-SS antibodies in mice. Compared with mice immunized with pEGS2SS and 0.85% saline, the growth performance of mice immunized with pEGS2SS-V was increased by 14.1% (P < 0.05) and 48.4% (P < 0.01) on the 2nd week after the first vaccination, respectively. The results indicated that the effects of the somatostatin DNA vaccine could be improved effectively by VP22 gene adjuvant.

[Epidemiologic survey of dental fluorosis and caries in school students in Wensu county in Xinjiang].

OBJECTIVE: To assess the relationship between the dental fluorosis and caries,and their prevalence features in school students in Wensu country in Xinjiang. METHODS: The study groups consisted of 1527 Weuer and Han students at the age of 6 to 16. Dental fluorosis and caries disease were assessed strictly by Dean's Classification Standard and WHO "Oral Health Surveys Basic Methods" (the 3rd ed). The concentration of fluorine in water and urine was measured by using selective electrode. RESULTS: The prevalence and index of dental fluorosis in Weuer and Han students were 73.70%, 64.67%, 1.647, 1.303,respectively. The prevalence of dental caries and DMFT were 61.19%, 42.66%, 1.648, 1.023 respectively. The corresponding values were 51.94%, 52.99%, 1.305, 1.449 for students of fluorosis group and non-fluorosis group, respectively. The fluorine degree of water was 2-5mg/L,the average value of fluorine in urine was 3.64 mg/L in Han students, and 5.28 mg/L in Weuer students. CONCLUSION: The prevalence of dental caries didn't decrease, even though the grevalence of fluorosis was high in Weuer country. The prevalence of fluorosis dental caries in Weuer students were significantly higher than those in Han students. It showed no significant different between the group of fluorosis and the group of non-fluorosis in the prevalence of dental caries, perhaps due to the high fluorine intake, poor oral hygiene, and unqualified medical service.