Quorum sensing (QS) system is a microbial cell-to-cell communication process that can influence the transcriptional expression of multiple genes by one or more signaling molecules to coordinate the population response of multiple bacterial pathogens. It relies on the production and detection of chemical signals called autoinducers to monitor cell density and species complexity in the population. The system includes a variety of types, which LuxS/AI-2 type is one of the hottest types, luxS as a highly conserved gene, involved in the regulation of a variety of gene expression, thereby regulating the growth and virulence of bacteria and other life activities, to adapt to different environments. LuxS protein cleaves S-ribosylhomocysteine to form homocysteine and 4, 5-dihydroxy-2, 3-pentanedione (DPD) which is then spontaneously cyclized to form AI-2. Streptococcus suis (SS) is a major swine pathogen and an emerging zoonotic agent of human meningitis and streptococcal toxic shock-like syndrome. It is of great significance to study the SS LuxS/AI-2 system and uses LuxS/AI-2 system to prevent and treat SS disease. This review focuses on the general mechanism of QS system in SS, the discovery, structure, metabolism, expression and regulation of LuxS/AI-2.

With the extensive and unreasonable use of fluoroquinolones in the treatment of Mycoplasma gallisepticum disease, the drug resistance of Mycoplasma on fluoroquinolone becoming more and more serious. In order to regulate the application of fluoroquinolones in the treatment of Mycoplasma disease and to reduce the occurrence of drug resistance to a certain extent, it is necessary to develop the standard of resistance to Mycoplasma gallisepticum on fluoroquinolones. The author introduces the definition and formulation process centered on the criteria for drug resistance, and summarized the related studies on the determination of resistance to fluoroquinolones in Mycoplasma at home and abroad, including wild type Cutoff, PK-PD Cutoff and clinical Cutoff. Finally, the shortcomings of the current researches were reviewed and put forward our own ideas, in order to provide a reference to slow down the resistance of Mycoplasma gallisepticum to fluoroquinolones.

This experiment was conducted to investigate the interaction between porcine heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein and non-receptor tyrosine protein kinase (c-Src) containing SRC Homology (SH3) domain. The yeast two-hybrid expressing vectors pGBKT7-hnRNPK, pGADT7-c-Src, pGADT7-ΔSH3, pGADT7-ΔSH2, pGADT7-ΔPTKc and pGADT7-SH3 were constructed using PCR, enzyme digestion and ligation methods, and then transformed into the yeast strain AH109 by PEG-LiAc method, and self-activation activity of bait and prey proteins in yeast cells were tested. Furthermore, the interaction between porcine hnRNPK and different mutants of c-Src protein were analyzed using yeast two-hybrid assay in vivo. With the DNA recombinant technology, the plasmids pET28a-hnRNPK, pGEX-6p1-c-Src, pGEX-6p1-ΔSH3, pGEX-6p1-ΔSH2, pGEX-6p1-ΔPTKc and pGEX-6p1-SH3 were constructed. Then the plasmids were transformed into E. coli BL21 and induced to express the target proteins by IPTG. GST and His tagged fusion proteins were purified by affinity chromatography. The binding of porcine hnRNPK and different mutants of c-Src protein were verified via GST pull-down assay in vitro. The results showed that the yeast two-hybrid expressing vectors of porcine hnRNPK and different mutants of c-Src were successfully constructed. The bait and prey plasmids were transformed into yeast cells and proved to have no self-activation activity in yeast cells. In yeast, the yeast colonies co-transformed with pGBKT7-hnRNPK and pGADT7-c-Src or pGADT7-ΔPTKc or pGADT7-SH3 grew well in SD-Trp -Leu -His-Ade medium compared with negative controls, indicating that porcine hnRNPK directly interacted with SH3 domain at c-Src protein N-terminal in yeast. The expression vectors of porcine hnRNPK and different mutants of c-Src were successfully constructed, and the soluble His-hnRNPK, GST-c-Src, GST-ΔSH3, GST-ΔSH2, GST-ΔPTKc and GST-SH3 fusion proteins were obtained via induction expression by IPTG and purification. The further GST pull-down assay showed strong interaction between porcine hnRNPK protein and N-terminal of c-Src protein, but weak interaction between hnRNPK protein and SH2, PTKc domains of c-Src protein. The results indicate that porcine hnRNPK protein can interact with the N-terminal domain (SH3 domain) of c-Src protein, which is convenient for studying the regulation sites of porcine c-Src protein, and find a technical platform for researching the mechanism of hnRNPK protein regulated kinase activity of c-Src protein.

The aim of this study was to reveal the regulatory roles of TAC1 and PRLR genes in seasonal estrus and transition between reproductive stages of sheep. In this study, the expression patterns of TAC1 and PRLR genes in 10 tissues (hypothalamus, pituitary, pineal body, brain, cerebellum, ovary, uterus, oviduct, kidney, adrenal gland) of adult Sunite (seasonal estrus breed) ewes under different photoperiods and adult Small Tail Han (STH, year-round estrus breed) ewes at different reproductive stages were analyzed by q-PCR method. The results showed that:1) The expression of TAC1 and PRLR genes were detected in all the 10 tissues of the two sheep breeds, and the expression characteristics were basically similar. TAC1 gene was mainly expressed in gonadal axis tissues, and PRLR gene was mainly expressed in pituitary and adrenal gland. 2) The expression of TAC1 and PRLR genes were higher in Sunite tissues under long photoperiod than those under short photoperiod, and for most tissues of STH sheep, the expression levels of the 2 genes were higher in luteal phase than that in follicular phase. 3) The expression level of TAC1 gene in the pituitary was increased slowly from short photoperiod to long photoperiod, and reached the highest level at LP49D, which was significantly higher than short and other long photoperiod points(P<0.01). The PRLR gene expression level in the pituitary of Sunite sheep was significantly increased from LP3D (P<0.05), reaching peak at LP21D; in hypothalamus, the expression level was significantly higher from LP15D (P<0.05) than that short photoperiod, and there was a tendency to continue to rise. These results indicate that TAC1 and PRLR genes may be involved in the regulation of sheep seasonal reproduction and the transition of reproductive stages, and identify the main tissues where the 2 genes may play important roles. Meanwhile this study reveal the expression trend of the 2 genes from short photoperiod to long photoperiod and different response patterns of PRLR gene in pituitary and the hypothalamus.

The aim of this study was to reveal EYA 3 and TSH β regulatory roles in seasonal estrus and reproduction stages transition.we investigated the expression patterns of eyes absent 3 (EYA 3) and thyrotropin beta subunit (TSH β) genes in different tissues of seasonal estrous sheep(Sunite sheep, SNT)at different photoperiods (long photoperiod (LP) and short photoperiod(SP)) and year-round estrous sheep (Small Tail Han sheep, STH) at different reproduction stages (luteal phase and follicular phase) through q-PCR technology. The results showed that EYA 3 was widely expressed in different tissues of 2 breeds, while TSH β was expressed highly in pituitary. In most tissues, the expression levels of those 2 genes under long photoperiod (LP) were higher than those under short photoperiod (SP) in SNT, and the expression levels were higher at luteal phase than those at follicular phase in STH. When SP turned into LP in SNT, the expression levels of those 2 genes rose in pituitary, and EYA 3 showed a transient peak at LP 3, and the peak of TSH β was at LP 21. The expression of EYA 3 declined prior to TSH β. The results elucidated the expression characteristics of EYA 3 and TSH β in year-round estrous and seasonal estrous sheep, the expression characteristics of those 2 genes at different photoperiods and different reproduction stages in pineal and pituitary of sheep, which suggested that EYA 3 played an important role in pineal and might regulate seasonal estrous of sheep. In addition, EYA 3 and TSH β might be involved in the transition of estrous stages. When SP turned to LP, EYA 3 played a regulatory role earlier than TSH β.

In order to deeply understand the efficiency of crossbreeding system using Simmental sire×Holstein cows, fixed and mixed models (GLM and Mixed procedures, SAS9.2) were used to analyze 8 economically important traits of 4 dairy cattle groups, namely Montbeliarde×Holstein (MO×HO), Fleckvieh×Holstein (FL×HO), Chinese Holstein (CHO) and Holstein imported from Australia (AHO), based on information collected from a large scale farm with 4 393 milking cows. The results showed that, in terms of milk performance, daily milk yield and herd test adjusted corrected milk (HTACM) of MO×HO cows were significantly higher than those of AHO (P<0.05), and MO×HO had the highest milk fat percentage and milk protein percentage among the 4 groups. No significant difference among 4 groups were observed (P>0.05) for somatic cell score (SCS) and milk urea nitrogen (MUN);Referring to reproductive traits, the age at first service (AFS) of MO×HO heifers was significantly lower than those of CHO and AHO (P<0.05), and crossbred cows had shorter calving to first service interval (CTFS) than that of the pure breed groups. Number of services (NS) of AHO heifers was significantly lower than that of CHO cows (P<0.05). Furthermore, days open (DO) of MO×HO was 38.43 and 20.93 d fewer than that of CHO and AHO cows, respectively; For growth performance, all traits of crossbred cattle were greater than those of CHO cows except for body depth, and there was a significant difference in chest circumference between MO×HO and CHO(P<0.05); Indexes related to the heat stress were measured on smaller number of animals, results showed that, the morning rectal temperature (RT) of crossbred cows was lower than that of both CHO and AHO cows, the difference between FL×HO and AHO was significant (P<0.05), and no significant difference was found in afternoon RT among groups (P>0.05); In addition, crossbred cows had significantly higher body condition score (BCS) than that of CHO cows (P<0.05), while no significant difference in temperament score and locomotion score were found among breeds groups (P>0.05). Skinfold thickness measured on the skin over the mid-point of the last rib of FL×HO was significantly greater than that measured on the same position of the other groups(P<0.05). In conclusion, preliminary results indicate that crossbred F1 cows of Simmental sire×Holstein dam have better overall performance than pure Holstein cows in the experimental farm.

The objective of this study was to investigate the genetic diversity and paternal origin of breeder sika deer by Y chromosome genes, and provide data support for the protection and breeding of sika deer. AMELY, DBY, USP9Y, SRY gene fragments on Y chromosome of 171 breeder sika deer (Shuangyang, Aodong, Siping, Dongfeng, Xingkaihu, Changbaishan, Dongda breeder sika deer) were amplified by PCR, and the PCR products were sequenced directly and analyzed by bioinformatics. The results showed that 8 SNPs were screened from 5 gene fragments, and AMELY1, AMELY2, DBY, USP9Y, SRY gene fragments had 3, 2, 1, 1, 1 mutation sites, respectively. Ten haplotypes (H1, H2, H3, H4, H5, H6, H7, H8, H9 and H10) were defined by DNASP5.1. H1 was the dominant haplotype, accounting for 49.1% of all breeder sika deer. H5 and H10 were unique to Dongfeng breeder sika deer. H9 was the ancestral haplotype. Haplotype diversity and nucleotide diversity of 171 breeder sika deer were 0.696 80 and 0.000 36, respectively. The results of NJ phylogenetic tree and structure population analysis showed that breeder sika deer had 3 paternal types (Y1, Y2 and Y3). Shuangyang and Siping breeder sika deer had a single paternal type(Y3), the other 5 breeder sika deer populations had multiple paternal types. The results indicate that Y chromosome of Chinese breeder sika deer have high haplotype diversity and very low nucleotide diversity.

The aim of this study was to clarify the expression characteristics of yak KDM4A gene and its role in yak oocytes and granulosa cells. The primer was designed according to the published bovine (Bos taurus) KDM4A gene sequence on the GenBank, and the expression pattern of KDM4A was detected by RT-PCR in yak various tissues which included heart, liver, spleen, lung, kidney, small intestine, ovary, uterus, stomach, testicle and cerebrum.While the coding sequence of KDM4A gene was cloned by RT-PCR.The structure and function of KDM4A were analyzed by ExPASY software on line. The mRNA expression level of KDM4A in different periods of yak oocytes and granulosa cells were detected by qRT-PCR. Results showed that KDM4A gene of yak contained a 3 289 bp cDNA fragment, and had high homology with other mammals in nucleotide sequence through sequence alignment and phylogenetic tree, indicating that KDM4A was relatively conservative in the evolutionary process. The complete CDs of yak KDM4A gene was 3 201 bp, encoding 1 066 amino acids, and the molecular weight was 122.48 ku. The protein encoded by KDM4A gene in yak was an unstable, soluble and acidic protein without transmembrane regions and signal peptide. Random coil and α-helix were mainly in the secondary structure of KDM4A, consistent with the results of the tertiary structure analysis. KDM4A gene was expressed in various tissues of yak, and high abundance in ovary, spleen and testicle.The qRT-PCR results showed that the mRNA expression level in MⅡ phase granulosa cell was significantly higher than that in GV and MI phases (P<0.05). Meanwhile, the mRNA expression level of KDM4A was significantly higher in GV phase oocyte than that in MI and MⅡ phases (P<0.05). The complete CDs of KDM4A gene was successfully cloned and the KDM4A had a significant difference expression among the 3 periods of yak oocytes and granulosa cells, which indicated that KDM4A played role in the maturing process of yak oocytes and granulosa cells.

This study aimed to screen genes associated with bovine follicular development and investigate their expression patterns.Nine young cows were collected.The small follicle at onset of predeviation (PDF2) and the largest follicle at onset of deviation (ODF1) follicles were collected after estrus synchronization. The total RNA was extracted from separated granulosa cells for the transcriptome sequencing. The Gene Ontology analysis was carried out after searching the bovine Refseq database and screening differentially expressed genes. MAPK13,CYP19A1,GREB1,SERPINE2,GSTA5 genes were selected randomly for verification using qRT-PCR technology. Results suggested that 15 413 genes were obtained from PDF2 and ODF1 transcriptomes (RPKM value ≥ 0.5), of which 651 differentially expressed genes were selected (Fold change ≥ 2). GO assignments were used to classify the functions of the 651 genes, and which could be categorized into 3 main catrgories, biological process (41.66%), molecular function (17.24%), cellular component (41.10%). Combining with Genecards, 13 down-regulated and 17 up-regulated genes screened were associated with follicle development. qRT-PCR results indicated that the expression of CYP19A1, GREB1 and SERPINE2 in dominant follicles were very significantly greater than that in subordinate follicles (P<0.01). MAPK13 mRNA amounts in subordinate follicles were very significantly greater than that in dominant follicles (P<0.01). GSTA5 mRNA amounts in subordinate follicles were significantly greater than that in dominant follicles (P<0.05). qRT-PCR results were entirely consisted with transcriptome sequencing results, and further proved the reliability of the transcriptome sequencing, which provided a foundation for future investigation of the regulatory mechanisms involved in follicular development in cattle.

The aim of this study was to clone the CDS of yak (Bos grunniens) Cyclin D1 gene, to analyse its biological characteristics, and to research the effects of insulin-like growth factor-I (IGF-I) on Cyclin D1 gene and protein expression in Sertoli cell (SC). The testicular samples were collected from 5-to 8-month-old healthy yaks for isolation and culture of SC. The Cyclin D1 gene was cloned by RT-PCR and its biological characteristics were analyzed by ORF Finder, MEGA7.0 and DNAMAN. The expression of Cyclin D1 protein in SC was localized by immunofluorescence staining. IGF-I was added into the culture medium of SC in the concentration of 0(control), 25, 50, 100, 150 ng·mL^-1, and then the relative expressions of Cyclin D1 mRNA and protein were detected by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The results were as follows:1) Yak Cyclin D1 gene (GenBank accession number:KY 420723) had the high homology with other species. 2) Cyclin D1 protein belonged to nuclear protein because of high fluorescence intensity on cell nucleus. 3) The level of Cyclin D1 mRNA in 25 and 150 ng·mL^-1 groups were not significantly different with the control group (P>0.05), while 50 and 100 ng·mL^-1 groups were significantly higher than other groups (P<0.05); the difference of Cyclin D1 protein expression between 100 ng·mL^-1 group and the control group was not significantly different (P>0.05), however, a significant difference existed between 25, 50, 150 ng·mL^-1 groups and control group (P<0.05); the relative expression of Cyclin D1 mRNA and protein were all reached the highest levels when IGF-I was 50 ng·mL^-1, which were 1.65 and 1.20 times higher than that of the control group, respectively. The results suggested that Cyclin D1 gene was highly conserved during evolutionary process; IGF-I could regulate the expression of Cyclin D1 in SC and its optimal concentration was 50 ng·mL^-1.

This study aimed to investigate the protective effect of methionine hydroxy analog chelated zinc (MHZn) against cadmium inducing organ damage in weaned piglets. 24 barrows (Landrace×Large white, 45 day of age, (13.22±1.36)kg BW) were randomly allotted to 4 dietary groups (6 replicates per group, one piglet per replicate per pen):basal diet (control group, Con group), basal diet+30 mg·kg^-1 Cd (Cd group), basal diet + 30 mg·kg^-1 Cd+100 mg·kg^-1 MHZn (LMHZn group), basal diet+30 mg·kg^-1 Cd + 200 mg·kg^-1 MHZn (HMHZn group). The trial lasted for 30 days. On the morning of day 31, all of the piglets were weighted and blood samples were collected, then, all of the piglets were slaughtered and sampled. Liver, spleen and kidney were weighted to calculate organ coefficients. Average daily gain (ADG), average daily feed intake (ADFI) and the ratio of feed/gain (F/G) were calculated. We also conducted the histopathology examination of liver, kidney, duodenum, jejunum and ileum, the contents of Cd and metal trace elements in liver and kidney were detected, and the biochemical parameters in plasma were analyzed. The results showed that:1) Compared with Con group, piglets in Cd group had lower ADG and higher F/G ratio (P<0.05); dietary Cd also significantly decreased the villus length of duodenum and jejunum (P<0.05), increased crypt depth of duodenum and jejunum(P<0.05), decreased the ratio of V/C (P<0.05), increased Cd concentrations in liver and kidney (P<0.05). Hepatocellular degeneration and renal tubular degeneration were observed in piglets of Cd group. The concentrations of albumin in plasma significantly decreased after Cd treatment (P<0.05). 2) When piglets were fed diet with Cd and MHZn together, the ADG increased and F/G ratio decreased compared with piglets fed diet with Cd alone (P<0.05), and the value of ADG and F/D in LMHZn and HMHZn groups were not significantly different to Con group (P>0.05). Compared with Cd treatment, MHZn increased the villus length of duodenum and jejunum(P<0.05), suggesting that it could prevent the damage of intestinal villus. The liver coefficient in HMHZn group significantly decreased compared with Cd group (P<0.05), and the value of liver coefficient in HMHZn group was not significantly different to Con group (P>0.05), suggesting that MHZn relived hepatomegaly effectively. Besides, MHZn relived the granular degeneration and steatosis in liver, reduced renal tubular degeneration and lymphocyte proliferation in kidney. Moreover, MHZn (200 mg·kg^-1) decreased Cd accumulation in liver and kidney compared with Cd group(P<0.05), reduced the impact of Cd on albumin and total protein in plasma, suggesting that MHZn was helpful in reliving the Cd inducing damage on liver, kidney and gut. In summary, MHZn has beneficial effect on reliving and repairing the damage of small intestine, liver and kidney caused by Cd.

This study aimed to investigate the effects of dietary fat levels on growth, body size, preweaning diarrhea and serum parameters of early-weaned male twin Hu lambs. Thirty pairs of heathy male twin Hu lambs weaned at 7 days of age were selected and randomly allotted to two groups according to the paired design. One group was fed normal fat level (NF) diet with milk replacer (EE:15%) and starter (EE:2.8%), and another group was fed high fat level (HF) diet with milk replacer (EE:27%) and starter (EE:5%). Milk replacer and starter were fed to lambs from weaning to 60 days of age. Then lambs in NF and HF groups were fed unified starter (US) from 60 to 120 days of age. Body weight of all lambs was measured before morning feeding, daily feed intake and diarrhea status were recorded; At 60, 90 and 120 days old, blood samples were collected from jugular vein before morning feeding, and the blood routine indices were determined.The results showed that:1) Body weight of lambs in HF group was significantly higher than that in NF group on 30,50, 90 and 120 days of age (P<0.05). The weaning BW of the lambs in the HF group tended to be higher than that in the NF group (0.05 < P < 0.10). From 7 to 60 days of age, average intake of milk replacer in HF group was significantly higher than that in NF group (P<0.05), but intake of starter, average daily gain and feed conversion ratio were not significant (P>0.05) between the two groups. From 60 to 90 days of age, the ADG and intake of starter of lambs in HF group were significantly higher than that in NF group (P<0.05). Feed conversion ratio of lambs in HF group was significantly lower than that in NF group(P<0.05). From 90 to 120 days of age, intake of starter of lambs in HF group was significantly higher than that in NF group (P<0.05), the feed conversion ratio of the lambs in the HF group tended to be higher than that in the NF group (0.05 < P < 0.10). During the whole experiment period, milk replacer and starter intake of lambs in HF group were significantly higher than that in NF group (P<0.05), but the ADG and feed conversation ratio were not significantly different (P>0.05) between the two groups. 2) The diarrhea rate before weaning in HF group tended to be lower than that in NF group (0.05 < P < 0.10). There was no significant difference in body size indices between the two groups(P>0.05). High fat diet can improve the growth performance of male twin Hu lambs before and after weaning. The high fat diet can reduce the diarrhea rate before weaning, and in the present experimental condition, high fat diet have less effect on serum parameters of male twin Hu lambs, which are all within permissible values by their autoregulation.

The aim of this study was to investigate the effect of stubble height on the response to the low temperature and cold-resistance overwintering. Three alfalfa cultivars of "Gold empress", "Zhungeer" and "WL323" were selected as experimental materials. The alfalfa with 4 stubble heights(0, 0-5, 5-10, 10-15 cm)were measured for the water soluble sugar(WSS), proline(Pro), MDA, soluble protein(SP) contents, SOD and CAT activities in the roots under natural environment, the cold-resistance of different cultivars with different stubble heights were evaluated comprehensively. The results showed that:1) The WSS, Pro, SP contents and CAT activity showed opposite trend to temperature; the trend of SOD activity and MDA content of different cultivars and stubble heights showed different variation with the temperature variation. 2) The cold-resistance of different alfalfa cultivars were "Zhungeer" > "Gold empress" > "WL323", and the cold-resistance of the same alfalfa cultivar with different stubble heights were A3 > A2 > A1 > A0. The results indicate that the cold-resistance of different alfalfa cultivars are different, and the recommended stubble height is 10-15 cm.

Marek's disease virus (MDV) is one of the oncogenic herpes viruses that can induce tumors in their natural hosts, which is considered to be an excellent model for investigating the biology, genetics, and immunology of tumorigenesis. Recently, a lot of microRNAs (miRNAs) have been identified in MDV genomes and the miR-M4-5p encoded by MDV-1 has been identified as a viral analog of cellular miR-155. Since miR-155 is a host miRNA associated with several cancers, miR-M4-5p may play a critical role in MDV oncogenesis. The present work was performed to construct a cDNA library and to primarily screen the putative host mRNA targets for miR-M4-5p. All hybrid-PCR products amplified from CEF RNA were harvested and cloned into pMD19-T vectors, then transformed into E. coli JM109 to produce a pool. The clones were selected and sequenced, using the online basic local alignment search tool (BLAST) to analyze the target sequences. A total of 88 candidate mRNA genes were obtained, 29 of which contained the predicted binding miRNA sites in the 3'-UTRs, complementary to the seed sequence of miR-M4-5p. After three rounds of dual fluorescence reporter assays (DLRA), five host genes including PRICKLE1, COLA, BCAT1, ANTXR1 and TECPR1 were primarily identified as the biological targets for miR-M4-5p. Our work provides an important basis for further studies on the molecular regulatory mechanism mediated by miR-M4-5p.

In order to inhibit replication of foot-and-mouth disease virus (FMDV) by RNAi technology, two pairs of short hairpin RNA (shRNA) targeting 3D, 3C and VP1 gene of foot-and-mouth disease virus ON strain were designed separately and named as VP1-1,VP1-2, 3C-1, 3C-2, 3D-1 and 3D-2. According to the principle of RNAi, the six shRNA expression plasmid were constructed and transfected into BHK21 cell that was infected with the FMDV ON strain. TCID₅₀ and real-time fluorescent quantitative PCR were used to analyze inhibition efficiency of shRNA recombinant plasmids on FMDV. ShRNAs with highest inhibition efficiency were chose to produce lentivirus particles and infect BHK21 cell lines, TCID₅₀ and real-time fluorescent quantitative PCR were used to analyze lentivirus inference vectors inhibition efficiency on foot-and-mouth disease virus. The results showed that the inhibition rate of shRNAs was between 71.5%-93.2%, and the most obvious inhibitory effect were PLKO.1-VP1-2 as 93.2% and PLKO.1-3D as 90.8%, respectively; lentivirus inference vectors PLKO.1-VP1-2 and PLKO.1-3D inhibition rate of FMDV were between 88.3%-95.49%. Our result screened out shRNAs that can inhibit foot-and-mouth disease virus replication at the cellular level. The study laid the foundation for transgenic sheep against FMDV production and cultivation.

In order to detect S. suis and S. suis serotype 2 rapidly, two sets of specific primers and TaqMan probes were designed according to the gdh and cps2J gene of S. suis serotype 2 to establish a duplex real-time PCR assay for detection of S. suis and S. suis serotype 2. Then the duplex real-time PCR was used to detect clinical samples and compare with conventional PCR. Results showed that S. suis and S. suis serotype 2 could be identified in 1.5 hours by the duplex real-time PCR assay and without any cross reaction with other bacteria. Besides, the detection limit was 5 copies/reaction. The correlation coefficient of standard curves was over 0.999, and intra-and inter-assay CV was less than 1.25%. Positive rate for sixty-seven clinical samples was 79.1% and 35.8%, respectively. Coincidence rates of duplex real-time PCR with conventional PCR for S. suis and S. suis serotype 2 were 92.5% and 89.6%, kappa values were 0.800 and 0.757, respectively. A sensitive, specific, reproducible assay is established successfully to detect S. suis and S. suis serotype 2 simultaneously and rapidly, and this assay is very suitable for detection of mass clinical samples.

To study the pathogenic mechanism of Mycoplasma hyopneumoniae (Mhp), an air-liquid interface (ALI) cultivation model of continuous STEC cell line, closer to the status in vivo, was developed for Mhp infection. Mhp strains with different virulence (virulence strains NJ, intermediate virulence strain AH, and attenuated strain 168L) were selected for respective infection with the same dose. The integrity of cell monolayer and the vitality of the infected cells were respectively evaluated by TEER value, Alamar Blue assay and lactate dehydrogenase (LDH) release assay. The ability of the differentiated cells to secrete MUC5B mucin against Mhp infection was measured by laser confocal and fluorescence spectrophotometer. To further study the pathogenic mechanism of Mhp infection, the cellular oxidation system of the infected STEC cells, including the secretion of NO and endogenous ROS were detected, respectively. The effect of NAC antioxidant on cell damage of Mycoplasma infection group was observed and was further demonstrated. The results revealed that the infection of virulence strains NJ and intermediate virulence strains AH could significantly roughen, disorder or damage the microvillus and cilia on the surface of STEC cells, but not attenuated strains 168L. The resistance of cell monolayer was decreased after infection, which was positively correlated with the virulence of Mhp strains. Mhp strains NJ and AH could significantly reduce the vitality of the infected cells using Alamar Blue and LDH release assays, especially at 48 h post-infection. Mhp strains could also promote the differentiated cells to secrete MUC5B mucus against Mhp infection, and the stronger the virulence of the strain was, the more mucous was secreted. It was found that except the 168L strain, AH strain and NJ strain could both significantly stimulate STEC cells to secrete NO and endogenous ROS, which could cause the oxidative stress reaction of cells significantly. After the anti-oxidation treatment of NAC, the ROS and mucus secretion of virulence strains NJ, intermediate virulence strains AH and the attenuated strains 168L groups decreased significantly, while the cell activity and resistance value increased significantly, while control group were not significantly changed. This study has been proved that Mhp infection can comprehensively disorder the growth characteristics, vitality and secretory function of the host cells, which are positively correlated with the virulence of Mhp strains. Moreover, the pathogenic mechanism of Mhp infection is closely related to the ability to induce the oxidative stress to host cells.

This experiment was conducted to investigate the prevalence of Salmonella, the distribution of serotypes and the antibiotic resistance of Salmonella enteritidis, and resistant genes they carried, in chicken of Guangzhou city. According to the national standard GB4789.4-2016, the chicken samples were collected. Salmonella serotypes were determined using diagnostic sera from S&A, Thailand. The antibiotic resistance of Salmonella enteritidis was determined by using the Kirby-Bauer method. The resistance gene of Salmonella enteritidis was detected by PCR. In 316 samples of chicken, the positive rate was 76.9%. A total of 23 serotypes were identified. The main serotypes include Salmonella Agona (19.8%), Salmonella Kottbus(14.0%), Salmonella Mbandaka (11.9%), Salmonella Kentucky(10.3%), Salmonella enteritidis (7.8%) and Salmonella Branederup (7.4%). The resistance rates of Salmonella enteritidis to nalidixic acid, peptidylsulfanilamide, ampicillin, streptomycin, tetracycline, gentamicin, cefotaxime and ceftazidime were 100.0%, 79.0%, 57.9%, 36.8%, 21.0%, 21.0%, 15.8% and 5.3%, respectively. The multiple antibiotic resistance rate was 68.4%. The mutation rates of gyrA, gyrB and parC were 94.7%, 73.7% and 15.8%, respectively. The most main mutations were Phe-414→Ser (73.7%), followed by Asp-87→Gly (42.1%),Asp-87→Trp(31.6%) and Ser-83→Trp(21.1%). The detection rate of quinolone-resistant plasmids was low. The detection rates of bla_TEM and bla_CTX-M were 42.1% and 10.5%, respectively. The pollution rate of Salmonella in chicken of Guangzhou city is high and the serotype is complex. Salmonella enteritidis are resistant to Nalidixic acid, peptidylsulfanilamide, ampicillin. Multiple antibiotic resistance is high. The mutation rate of the gene in the quinolone resistance-determining region is high, which is highly consistent with the resistance of Salmonella to quinolones.

E. coli K12 was the host of phage Bp7, and its mutant strain K12-R was resistant to phage Bp7. To understand the resistance reason and the mutation effect on biological properties of K12-R, the genome of K12-R was sequenced and compared with that of E. coli K12 to find the mutation sites, the growth ability and the autoaggregation ability of K12-R were measured by turbidity test and the biofilm formation ability was detected by gram staining method, the biological properties of K12-R were compared with E. coli K12. The results showed that a mutant gene hldE, which was associated with Lipopolysaccharide synthesis, in K12-R caused the resistance to phage Bp7. Compared with E. coli K12, the growth ability of K12-R was reduced, but the cell membrane permeability, the autoaggregation ability and the biofilm formation ability were enhanced. The hldE gene of E. coli K12 was associated with LPS synthesis, the mutation of hldE gene changed the LPS character of K12-R, which caused the resistance to phage Bp7. The results provides the basis to understand the tolerance mechanism of K12-R to phage Bp7.

This study was designed to investigate effects of acid on survival of Salmonella enteritidis. In this study, Box-Behnken design with response surface methodology was used to create the optimal conditions to evaluate the growth of Salmonella under acetic acid pressure environment. The changes of S. enteritidis survivability and the relative expression of acid tolerance-related genes were compared before and after optimization. This model was also used to compare the different relative expression of acid tolerance-related genes in S. enteritidis which induced by other common acids. The results showed that the optimum favorable conditions were as follows:incubation temperature 22.5℃, shaking time of 6 h, amount of bacteria added 15%.Validation experiments showed that bacterial colonies count were 3.966×10⁸ CFU·mL^-1, which basically met with the standard value. At the same time, the survival of S. enteritidis was highest in this environmental condition, and the relative expression of acid tolerance genes were significantly higher (P<0.01). The expressions of acid tolerance genes were also significantly higher (P<0.01) under acidic stress conditions created by other commonly used acids. Our study indicates that the model established by response surface methodology has a reliable tool which could be reasonably used in bacterial acid tolerance induction conditions during optimization.

Foot-and-mouth disease virus (FMDV) is the causative agent of contagious and economically devastating diseases that affects cloven-hoofed livestock worldwide. An effective defense barrier against FMD would be established if the respiratory tract transmission for virus was being cut off due to FMDV is mainly transmitted by the respiratory tract. In the current study, health cattle were immunized by inactivated FMDV along with Bacillus subtilis administered through intranasal spray to explore the impact on mucosal immune response (respiratory tract) and systemic immunity in cattle. The results showed that the IgA secreting cells significantly increased in cattle nasal mucosae and pulmonary bronchial (P<0.01) after intranasal immunization with inactivated FMDV along with Bacillus subtilis, but the conventional inactivated FMDV vaccine had no significant effect on the number of IgA secreting cells (P>0.05). At the same time, the level of IL-12 and TNF-α significantly increased in nose, trachea, pulmonary and pulmonary bronchus (P<0.01), especially in the trachea and pulmonary bronchus, but IL-6 level have no significantly difference (P>0.05). O type FMDV specific SIgA from nasal, saliva and O type FMDV specific antibodies in serum significantly increased at 3 days after intranasal immunization, and maintained for 3 months (P<0.01 or P<0.05). These results indicated that both cellular immunity and local humoral immunity of the bovine respiratory tract were enhanced by intranasal immunization with inactivated FMDV along with Bacillus subtilis, as well as the systemic immune response. Our study provided an easier method and prospect for application of whole inactivated virus.

P-glycoprotein (P-gp), encoded by ABCB1/Mdr1, is a most important member of ABC family efflux transporter dictating the bioavailability of the various xenobiotics. The expression and function of P-gp can be influenced by other xenobiotics. The aim of the study is to explore the effect of quercetin, a promising herbal additive, on the expression and function of P-gp as well as the drug-drug interaction. Randomly selected rats were grouped and treated by quercetin (15 and 60 mg·kg^-1) for 5 and 15 days, respectively. mdr1a, mdr1b, PXR and CAR gene mRNA expression level and P-gp protein expression level and location were detected by real-time RT-PCR, Western bolt and immunohistochemistry, respectively. Single-pass intestinal perfusion was used to explore the effect of quercetin on ivermectin permeability in jejunum for another 12 rats wite quercetin for 15 days. Finally, ATPase activity was detected in quercetin-treated Caco-2 cells by using ATP-kit assay. Results were as follows:Quercetin can upregulate the mRNA expression level of mdr1a, mdr1b, PXR and CAR in rats liver and jejunum (P<0.05, P<0.01) as well as P-gp expression level (P<0.01). There is a correlation between P-gp and CAR expression level (P<0.05). Single pass intestinal perfusion experiment proved that quercetin affected the permeability of ivermectin in jejunum with declining K_a and P_app values (P<0.05). Compared with the control group, quercetin significantly induced the ATPase activity in Caco-2 cells (P<0.01). Quercetin upregulated the P-gp expression in liver and jejunum which was transcriptionally regulated through the activation of nuclear receptors (CAR), accordingly, affected the efflux transport of ivermectin from jejunum in the rats. This may have significant implications for long term use of quercetin causing drug-drug interaction during pharmacotherapy.

In order to investigate the antimicrobial activity and anti-infective activity of Total Magnolol on mastitis-related pathogens (mainly Staphylococcus aureus), a broth microdilution method was employed to determine the minimal inhibitory concentrations (MIC) of the Total Magnolol against S. aureus strains. Hemolysis assay, Western blot analysis, real-time RT-PCR assay and cytotoxic assay were further performed to evaluate the influence of this compound on S. aureus virulence. The results showed that the minimal inhibitory concentration of Total Magnolol against S. aureus were 8-16 μg·mL^-1. Furthermore, treatment with Subinhibitory concentration of Total Magnolol could inhibit the expression of α-hemolysin, reduce the pathogenicity of Staphylococcus aureus, and significantly alleviate the injury induced by α-hemolysin of Staphylococcus aureus.

This experiment was conducted to investigate the effects of dietary energy concentrations on production performance, egg quality and hatching performance in primiparous laying Sichuan White geese, and explore the optimal dietary energy concentration of primiparous laying Sichuan White geese. Two hundred fifty 26 weeks old female Sichuan White geese were randomly allotted into 5 dietary treatments with 5 dietary metabolizable energy (ME) concentrations(9.14, 10.01, 10.88, 11.75 and 12.62 MJ·kg^-1) containing 10 replicate pens with 5 birds per pen. And 1 male goose was put into each repeating group, respectively. The experiment lasted for 12 weeks. According to replicate, the egg weight and egg number for everyday was recorded to examine the egg production performance. Two goose eggs from each replicate group were selected for determination of egg quality.All qualified eggs were selected to hatch for determination of hatching performance. The results showed that:1) The final body weight and average daily gain of birds fed diets containing 10.88,11.75 and 12.62 MJ·kg^-1 ME were significantly higher than those birds fed diets containing 9.14 and 10.01 MJ·kg^-1 ME (P<0.05). 2) The average egg weight, daily laying rate of birds fed diets containing 10.88, 11.75 and 12.62 MJ·kg^-1 ME were significantly higher than those birds fed diets with 9.14 and 10.01 MJ·kg^-1 ME (P<0.05). The feed egg ratio of birds fed diets containing 10.88, 11.75 and 12.62 MJ·kg^-1 ME were significantly lower than those birds fed diets containing 9.14 and 10.01 MJ·kg^-1 ME (P<0.05). The dietary energy concentration did not significantly affect the age at the first egg and the weight of the first egg in Sichuan White geese (P>0.05).3) The albumen height and Haugh unit of geese egg in 10.01, 10.88 and 11.75 MJ·kg^-1 ME groups were significantly higher than those in 9.14 and 12.62 MJ·kg^-1 ME groups(P<0.05). The yolk color of birds fed diets with 10.88, 11.75 and 12.62 MJ·kg^-1 ME were significantly higher than those birds fed diets with 9.14 and 10.01 MJ·kg^-1 ME (P<0.05). The birds fed diets with 10.01, 10.88 and 11.75 MJ·kg^-1 ME had higher egg specific gravity than those birds fed diets with 9.14 MJ·kg^-1 ME (P<0.05). 4) Dietary energy concentration did not significantly affect fertility of birds (P>0.05). The hatchability of fertile eggs in birds fed diet containing 11.75 MJ·kg^-1 ME was higher than those birds fed diets with 9.14, 10.01 and 12.62 MJ·kg^-1 ME (P<0.05). 5) Based on cubic curve model analysis, the optimal energy concentration in primiparous laying Sichuan White geese were 11.93, 11.91 and 11.83 MJ·kg^-1 ME, the average egg weight, daily laying rate and hatchability of fertile eggs were as the indicators, respectively. Collectively, the 11.83 MJ·kg^-1 ME can meet the energy requirement of primiparous laying Sichuan White geese.

This experiment was conducted to investigate the effects of wheat straw (WS) or ammonium bicarbonate treated wheat straw (ABWS) basal diets supplemented with different levels of alfalfa on associative effects(AE) in vitro. There were 6 concentrate-roughage ratio(70:30, 60:40, 50:50, 40:60, 30:70, 20:80) and 4 alfalfa supplementation levels (0%, 10%, 20%, 30%) in this study. Roughage contained WS/ABWS and alfalfa. Gas production (GP) of 48 feed combination groups (24 groups of WS and 24 groups of ABWS)and 4 diets was recorded at 2, 4, 6, 9, 12, 24, 36, 48, 72, 96 h for culture. The AE values were calculated by in vitro GP during 24 h and weighted values of different combinations. The results showed as follows:1) In WS basal diet, the AE of 20% group was significantly higher than that of 30%, 10% and 0 groups (P<0.01), the AE of 30% group was significantly higher than that of 10% and 0 groups (P<0.01), the AE of 10% group was significantly higher than that of 0 group (P<0.01) when C:R was 70:30. The AE of 30% group was significantly higher than that of 10% group (P<0.01) and 0 group (P<0.05) when C:R was 50:50. The AE of 30% group was significantly higher than that of 0 group (P<0.01), and the AE of 10% and 20% groups were significantly higher than that of 0 group (P<0.05) when C:R was 40:60. 2) In ABWS basal diet, the AE of 30% and 10% groups were significantly higher than that of 0 group (P<0.05) when C:R was 70:30, the AE of 10% group was significantly higher than that of 30% group (P<0.05) when C:R was 60:40, the AE of 10%, 20% and 0 groups were significantly higher than that of 30% group (P<0.01) when C:R was 50:50, the AE of 0 group was significantly higher than that of 20% group (P<0.05) when C:R was 40:60. It is concluded that ABWS diet need less alfalfa (10%) than WS (30%, 20%) diet to develop positive AE. ABWS is better than WS in reduing the supplement of alfalfa.