Changfa Wang

Analysis of biofilm formation and associated gene detection in Staphylococcus isolates from bovine mastitis

The objective of this study was to investigate the biofilm-forming ability and distribution of biofilm associated genes in clinically isolated Staphylococcus in bovine mastitis. Silver staining, scanning electron microscopy (SEM) and crystal violet staining were conducted for the detection of biofilmforming ability in 24-well plates. The bap, icaAD, icaBC, Staphylococcal accessory regulator (sar), accessory gene regulator (agr), sigB, clumping factor A (clfA), clfB, fibronectin-binding proteins (fnbpA) and fnbpB genes were amplified by polymerase chain reaction (PCR). Formation of biofilms was found macroscopically in 120 of the 137 strains after being stained with silver (biofilm-forming rate 87.6%). Five strains did not adhere to the surface of the silica gel after being stained with crystal violet, while the remaining 132 strains did adhere. Bap was amplified in 57 isolates, and icaAD and icaBC were isolated in 43 and 54 strains, respectively. SigB, sar and agr were amplified in 73, 49 and 38 isolates, respectively, and clfA and clfB were isolated in 76 and 50 strains, respectively. FnbpA was present in 52 strains and fnbpB in 26 isolates. Our study reveals that bap, sigB, sar, icaAD and icaBC may be crucial biofilm associated genes since these genes were present more often in biofilm-positive strains than in biofilm-negative strains. There was no obvious difference between the frequencies of agr in the biofilmpositive strains and biofilm-negative strains, which indicates that the role of agr in biofilm development is still controversial. The distribution of clfA, clfB, fnbpA and fnbpB in biofilm-positive strains were not greatly different from that in biofilm-negative strains. Key words: Bovine mastitis Staphylococcus , biofilm, silver staining, crystal violet staining.

Reconstruction of metabolic network in the bovine mammary gland tissue

Understanding bovine metabolism and its relationship with milk products is important in cow breeding. In the present work, the metabolic network in the mammary gland tissue of cattle was reconstructed with the available bovine genome information using several public datasets from NCBI, Uniprot, and KEGG. The network consisted of 1,743 metabolites named by KEGG compound numbers as nodes and 657 enzymes that catalyzed the corresponding reactions as edges. The characteristics of the network were analyzed. The top 20 hub metabolites were determined, and the mean path length was identified to be 6.52. Moreover, 11 key enzymes with significant changes in expression under the condition of mastitis were identified and analyzed by integrating the microarray expression data of normal and clinical mastitis. Aside from the GATM gene, 10 downregulated enzymes were detected in bovine with mastitis. In addition, many of the identified enzymes were involved in amino acid metabolisms or had a direct connection to amino acid metabolisms. These results indicate that mastitis could affect the expression of enzymes, which is vital in some amino acid metabolisms, resulting in the reduction of milk proteins. The present work provides information that may improve the understanding on bovine milk production and mastitis.

Alternative splicing and mRNA expression analysis of bovine SLAMF7 gene in healthy and mastitis mammary tissues

The signaling lymphocyte-activating molecule family 7 (SLAMF7) proteins serve as adhesion molecules on the surface of a variety of mature hematopoietic cells, and also partially control certain innate and adaptive immune responses. We characterized three novel bovine SLAMF7 splice variants, designated as SLAMF7-AS1, AS2, and AS3. All three novel SLAMF7 isoforms are derived from the complete transcripts (SLAMF7-complete) via alternative splicing (AS). The patterns of the three splice variants are exon skipping and alternative 5′ splice sites. Bovine SLAMF7 transcripts are expressed in mammary tissue, as demonstrated by real-time PCR. The levels of the complete transcript expression in the normal mammary tissues were higher than that in Staphylococcus aureus (Staph. aureus)-induced mastitis mammary tissues. However, it was not significant for the mRNA expression level comparison between these two kinds of mammary. The SLAMF-AS2 isoforms are expressed the lowest levels among the three transcripts in both normal and infected mammary tissues. This study provides clues for a better understanding of bovine SLAMF7 gene function.

Study on Red Coat Color Gene and Prediction of the Secondary Structure in Chinese Holstein

Abstract The nucleotide sequence of the melanocortin-1-receptor (MC1R) gene was studied with the help of the polymerase chain reaction (PCR), in which the protein structure in Chinese Holstein was predicted, and the molecular mechanism of the red coat color was investigated. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to genotype the individuals. The bioinformatics and biotechnology softwares were used to predict the secondary structure of MC1R. The results showed that the EE genotype was the dominant genotype in Chinese Holstein Black and White herd, whereas, it was ee in Chinese Holstein Red and White herd. The secondary structure of the mutational MC1R protein was changed and the deletion mutation caused an earlier termination in translation, which led to the formation of the red coat color. The allele E was mainly associated with the black coat color, whereas, e was associated with red.

The polymorphisms of κ-casein gene and their associations with milk production traits and expression analysis in Chinese Holstein cattle

The polymorphisms of exon 4 and 5 of κ-casein (CSN3 ) gene and their associations with milk production traits and expression pattern in Chinese Holstein cattle were investigated. Nine mutational sites, of which seven were novel mutational sites, were identified and genotyped by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-RFLP) and sequencing methods in 398 cows. Linkage disequilibrium analysis showed that SNP-1 (g.10891 T > C rs 43703015, g.10927 C > A rs 43703016, g.10988 G > A ss 256302464 and g.10966 A > T ss 256302465) and SNP-2 (g.12907 A > G ss 256302466, g.12950 G > A ss 256302468, g.12989 C > T ss 256302469 and g.13028 A > G ss 256302470) were completely linked, respectively. Correlation analysis showed that SNP-1, SNP-2 and SNP-3 (g.12980 T > C ss 256302467) markers were closely correlated to the fat content. The SNP-3 marker had a remarkable effect on the protein content (P < 0.05). 16 combined genotypes of the three SNPs were found. Fat and protein content in combinations of genotypes were varied significantly (P < 0.05). Genotypes BBCCEE and ABTCDD individuals had the highest fat and protein content, respectively, which may be useful for marker assisted selection program in dairy cattle. The expression of CSN3 mRNA in the mammary tissue was higher than that of in the liver tissue (P < 0.05) and the expression in the spleen of BB genotype was higher than that of AA genotype in the SNP1 (P < 0.05) by fluorescent quantitation real-time PCR (Q-PCR) assay.