This study aimed to establish a cost-effective duplex TaqMan real-time quantitative PCR （qPCR） assay for the rapid detection of Brucella infection in animals and animal products， and to differentiate B.abortus from other Brucella species. This method provides a scientifically valid diagnostic tool for distinguishing currently used vaccine strains and novel vaccines under development. Specific primers and TaqMan probes targeting the galU and Omp31 genes were designed based on GenBank sequences. Recombinant plasmids were constructed. The assay's specificity， sensitivity， and repeatability were rigorously evaluated. Clinical serum and tissue samples from animals with brucellosis were tested. A duplex TaqMan qPCR assay for Brucella detection was successfully developed. The limit of detection （LOD） for recombinant plasmid standards was 1.93×10⁰copies·μL^-1. The assay exhibited no cross-reactivity with other Gram-negative bacteria. Both intra-assay and inter-assay reproducibility were excellent， with coefficients of variation （CV） consistently below 2%. Testing of 156 samples （including laboratory-prepared and clinical specimens） revealed a positive agreement rate of 69.75% compared to BCSP31-PCR. Furthermore， the duplex TaqMan qPCR demonstrated significantly higher sensitivity （100%） than conventional PCR （69.75%） and the RBPT （58.65%）. The established duplex TaqMan qPCR assay provides a robust technical foundation for the clinical differential diagnosis， epidemiological surveillance， and eradication programs of brucellosis.

In recent years， diarrhea in cattle has been spreading in cattle farms with a high mortality rate， causing economic losses. This experiment aims to isolate and identify Shigella flexneri， which causes diarrhea in a Beijing cattle farm， and to clarify the biological characteristics and genomic features of the pathogen’s serotype， drug resistance， and pathogenicity. In this study， fecal samples from diarrheal cattle were collected for bacterial isolation and purification， 16S rRNA amplicon sequencing analysis， virulence gene detection， and whole-genome sequencing of isolated strains. The results showed that one strain of Shigella flexneri Fc was identified through bacterial isolation and purification， and PCR identification. Whole-genome sequencing revealed that the Shigella flexneri Fc strain has a genome size of 4.98 Mb and a GC content of 50.76%. It contains 91 tRNA genes， 7 23S rRNA， 7 16S rRNA， 8 5S rRNA， 283 misc_RNA， and 97 carbohydrate-active enzyme genes. TVBD annotation analysis identified 1 363 transporter genes； CARD annotation analysis identified 80 resistance genes； VFDB and PHI-base annotation analysis identified 1 410 potential virulence factor-related genes and 2 052 phenotypic mutation genes. Phylogenetic tree analysis revealed that Shigella flexneri is closely related to Shigella flexneri 29903， belonging to the same branch. This study completed the isolation and identification of a bovine-origin Shigella flexneri strain and whole-genome sequencing analysis， providing a theoretical basis for the prevention and control research of shigellosis.

The aim of this study was to understand the epidemic characteristics and drug resistance of Clostridium perfringens in Xizang’s Tibetan pig population， and to provide theoretical basis for disease prevention and control. In this study， 273 Tibetan pig fecal samples collected from 9 districts and counties in Xizang from 2021-2024 were isolated and identified by bacterial isolation and purification， PCR identification， drug sensitivity test， and drug resistance gene detection and other methods， and the toxin typing and drug resistance phenotype were determined. The results showed that a total of 180 strains of C. perfringens from Tibetan pigs were obtained， with an isolation rate of 65.93% （180/273）； According to toxin gene testing， all strains were identified as type A C. perfringens， with 142 strains containing the cpb2 gene. The results of antibiotic susceptibility tests showed that the isolated strains had high resistance to 6 drugs， including gentamicin， streptomycin， erythromycin， polymyxin B， sulfamethoxazole， trimethoprim. 97.22% （175/180） of the isolated bacteria were multidrug-resistant strains. The proportion of strains resistant to 4-6 types of antibiotics is 71.67% （129/180）. In addition， the isolated strains carried 12 resistance genes， with a detection rate of 23.08%（12/52）. The detection rate of the aminoglycoside resistance gene aadA was the highest， with a detection rate of 85.00%（153/180）. This study clarified the prevalence of C. perfringens from Tibetan pigs in Xizang， providing data support for taking effective prevention and control measures.

The purpose of this study was to prepare feline PD-L1 antigen and screen monoclonal antibody， and preliminary application was carried out in detection and diagnosis of canine and feline oncology diseases. In addition， feline derived chimeric single chain variable Fragment-Feline Fragment crystallizable （scFv-fFc） was prepared， which provided a tool for the treatment of cat tumor in future. The PD-L1 gene of feline was amplified by PCR and prokaryotic expression recombinant plasmid PET-MPD-L1 was constructed， and the antigen of PD-L1 was prepared by expression with host BL21（DE3）. After immunization with mice， cell was fused， and an ELISA was established to screen antibody produced by hybridoma cells. After subcloning three times， the cell line of MPD-L1 hybridoma were established， subtypes were identified， and the ascites were prepared， and titer of the ascites was determined. Characteristics were determined by Western blot and immunohistochemistry （IHC）. Total RNA was extracted from the hybridoma cells， and the heavy chain variable region （VH） and light chain variable region （VL） genes were amplified by RT-PCR， which were compared with the antibody light and heavy chain genes in the IMGT database for confirmation. RNA from peripheral blood of a cat in clinic was extracted， and the Fc gene of IgG1 was amplified by RT-PCR. The recombinant plasmids of pFast-MPD-L1-scFv and PFAST-MPD-L1-scFv-fFc were constructed by splicing VH and VL into scFv and scFv-Fc into scFv-fFc by landing PCR， then expressed in the baculovirus expression system. The results showed that feline PD-L1 was highly expressed in the supernatant of cultures and the purified protein could be used antigen for immunization. The monoclonal antibody of 2A5A2F4D7 belonged to IgG 2a subtype， and the light chain is subtype κ. The titer of the supernatant， ascites was 1：3200， 1：102400 respectively. Western-Blot results showed that monoclonal antibody 2A5A2F4D7 reacted with expressed feline PD-L1， and IHC results showed that the monoclonal antibody secreted by 2A5A2F4D7 hybridoma cells can recognize PD-L1 of tumor cells both canine and feline. The VH and VL genes amplified were consistent with genes in the IMGT database， with 4 framework regions and 3 hypervariable regions. The recombinant plasmids of pFast-MPD-L1-scFv and PFAST-MPD-L1-scFv-fFc were constructed successfully and expressed in the supernatants of insect baculovirus expression system. The prepared feline PD-L1 monoclonal antibody have certain value in the detection and prognosis of canine and feline tumors， while scFv-fFc might hold great potential for application in the treatment of tumors in canine and feline.

To establish a diagnostic ELISA method for distinguishing RHDV infection and vaccination， the RdRp proteins of RHDV1 and RHDV2 were expressed by prokaryotic expression system. And these two soluble RdRp proteins were successfully purified. RdRp protein was used as coated antigen， an indirect ELISA method was established to detect serum antibody against RdRp. After purification and restriction enzyme digestion， the RdRp protein without tag was obtained with a size of 57 ku. Western blot analysis confirmed that both RdRp proteins reacted specifically with RHDV1 or RHDV2 infection sera. And there was no difference between the types of RdRp proteins. So RHDV2-RdRp was selected as a coating antigen at a coating concentration of 2.5 μg·mL^-1. The optimal serum dilution was 1∶100 and the incubation was 60 min at 37 ℃. The optimal dilution of enzyme-labeled antibody was 1∶10 000. The cut-off value was 0.225. It was proved that the ELISA method has good specificity and sensitivity. The coefficient of variation in both intra-batch and inter-batch repeatability test was less than 10%， which indicating a good stability. The samples after RHDV challenge and the serum samples after genetic engineering vaccine immunization were tested， and the results showed that RHDV infection can induce the production of serum antibodies against RdRp protein， but the serum tests after genetic engineering vaccine immunization were negative. In this study， the ELISA method based on RHDV-RdRp protein can distinguish between genetic engineering vaccine immunity and RHDV infection， which provides an effective detection method for clinical screening of RHDV infection.

This study aimed to understand the histopathological changes of goats infected with Mycoplasma ovipneumoniae. An infection group and a control group （5 goats each） were established. Serum， nasal swabs， lung tissue， trachea， and bronchi were collected from each group. Mycoplasma ovipneumoniae-specific antibodies in serum were detected by ELISA， Mycoplasma ovipneumoniae-specific nucleic acids in nasal swabs were identified by PCR， and histopathological examination was performed on lung， tracheal， and bronchial tissues. The results showed that there were varying degrees of cellulosic pneumonia， liver hepatization and carnification in the lung tissue during the autopsy； There were Mycoplasma ovipneumoniae antibodies in the sample serum； The PCR amplification produced specific bands； The lung tissue， trachea and bronchus showed different degrees of pathological characteristics. Goats infected with Mycoplasma ovipneumoniae pneumonia showed obvious histopathological changes， including lung inflammation， the production of serologicalantibodies and positive detection of Mycoplasma ovipneumoniae nucleic acids in lung tissues. These results provide a strong scientific basis for the diagnosis and research of Mycoplasma ovipneumoniae in goats.

The purpose of this study was to investigate the therapeutic effects of deer blood polypeptide （ARPBP） in the treatment of androgenic alopecia and to reveal underlying. By using ARPBP to treat testosterone （TES）-induced C57BL mouse alopecia model， HE staining， immunohistochemical staining qPCR and Western blot experiments were performed on the skin tissue of its back. Results showed that it significantly promoted hair regeneration， and the efficacy exhibited a concentration-dependent manner. Histological analysis showed that the skin thickness， follicle density and diameter in TES group were significantly reduced， which was consistent with the pathological characteristics of androgenic alopecia. After the treatment with ARPBP， the skin thickness and parameters of hair follicle shape in the low-dose ARPBP treatment group （15 mg·mL^-1） recovered nearly to the physiological level， and the hair density and area of hair coverage were better than those of the high-dose treatment group （75 mg·mL^-1）， indicating that the low-dose ARPBP group could reverse the testosterone induced hair follicle miniaturization more effectively. Immunohistochemical results showed that low-dose （15 mg·mL^-1） ARPBP significantly up-regulated the expression of follicle proliferation markers KI67 and PCNA， and enhanced the nuclear localization signal of upstream factor LEF in Wnt/β-catenin pathway. Western blot analysis further confirmed that low-dose ARPBP significantly reversed the inhibitory effect of androgen on LEF1 protein expression， and inhibited the activation of androgen on BMP4 signaling pathway. This study revealed the effective dose-dependent effect of ARPBP in the treatment of androgen alopecia. The mechanism underlying these achieved results could be via promoting the transition of androgen-induced hair follicles from resting period to growth period by activating LEF1-Wnt/β-catenin signaling pathway and inhibiting BMP signaling pathway， thus promoting hair regeneration. We believe our study has laid the foundation for the development of precision medicine strategies based on natural peptides.

The aim of this study was to explore the effect and molecular mechanism of peroxisome proliferator-activated receptor α （PPARα） on skeletal muscle development. We utilized young （6-8 weeks）， adult （3-4 months）， and old （18-20 months） mice （n=8/group） to examine the expression of PPARα in skeletal muscle during development. The role of PPARα in skeletal muscle development was further investigated using a whole-body knockout mouse model. HE staining was used to observe the morphological differences of gastrocnemius muscle of mice of different ages， and immunofluorescence staining was used to detect the expression changes of P21 and P53 in gastrocnemius muscle of mice of different ages to determine the age difference of mice. The expression of PPARα in skeletal muscle was detected by common PCR， and the expression of PPARα in gastrocnemius muscle of mice of different ages was detected by Western blot， real-time quantitative PCR （qRT-PCR） and immunofluorescence staining （IF）. qRT-PCR， Western blot and immunofluorescence staining were used to detect the expression of MyoD， MyoG and Pax7 in gastrocnemius muscles of WT and PPARα knockout mice （PPARα^-/-）. Western blot was used to detect the expression of PPARα downstream signaling pathways AMPK and P38 MAPK. The results showed that PPARα was expressed in the skeletal muscle of mice at all developmental stages， and its expression level gradually decreased with age. Immunofluorescence staining showed that PPARα protein was widely distributed in the nucleus and cytoplasm of mouse gastrocnemius muscle. Further studies found that PPARα gene knockout led to a significant decrease in the expression of MyoD， MyoG and Pax7 in gastrocnemius muscle， while the phosphorylation levels of AMPK and P38 increased significantly.

The purpose of this study is to establish an efficient method for purifying plasminogen from pig blood. In this study， sPLG was purified from pig blood using lysine affinity chromatography combined with size exclusion chromatography. Meanwhile， recombinant swine tissue plasminogen activator （st-PA） and staphylokinase （SAK） were expressed in Escherichia coli. Then， st-PA was purified by refolding of inclusion bodies， and SAK was purified by nickel column affinity chromatography combined with size exclusion chromatography. The activities of the prepared sPLG， SAK and st-PA were evaluated by a plasmin-specific substrate hydrolysis assay， a fibronectin degradation test， and an in vitro pig blood clot dissolution assay. The results showed that sPLG was highly purified by the method established by this study. Approximately 50 mg of pure sPLG was obtained from 250 mL of pig blood plasma. The purified sPLG could be activated by st-PA or SAK， and the resulting plasmin could then cleave the plasmin-specific substrate， degrade fibronectin， and lyse pig blood clots in vitro. In conclusion， this study established an efficient method for the preparation of sPLG， st-PA and SAK. In addition， the complete plasminogen activity of sPLG was verified using st-PA and SAK as well. This study provide essential materials for research on the mechanisms by which pathogens invade pigs via sPLG.

The purpose of this study was to understand the prevalence and drug resistance characteristics of Klebsiella pneumoniae in dairy cow mastitis in Xinjiang. Two thousand and eighty-three milk samples of dairy cows with clinical mastitis or subclinical mastitis were collected from 15 large-scale dairy farms in 9 areas of intensive dairy farming in Xinjiang. Klebsiella pneumoniae in the samples was isolated and cultured， Gram stained. PCR and 16S rRNA sequencing were conducted to identified the isolated strains， and the drug resistance of the isolated strains was detected by micro-broth dilution method. The results showed that 266 strains of Klebsiella pneumoniae were isolated from the milk samples， and the isolation rate was 12.8% （266/2 083）.There was no significant difference in the isolation rate of Klebsiella pneumoniae between clinical mastitis and subclinical mastitis （P>0.05）. The sensitivity of 266 isolates to 16 antimicrobial agents and the production of extended-spectrum β-lactamases （ESBLs） were determined by broth microdilution method. The results showed that the resistance rates of the isolates to tetracycline， ceftiofur， cefepime， cefotaxime and cefazolin were high （23.7%-41.0%）. And 16.9% （45/266） of the isolates were multidrug-resistant bacteria， and 6.4% （17/266） of the isolates produced ESBLs. The results suggest that the average separation rate of mastitis Klebsiella pneumoniae in 15 large-scale dairy farms in Xinjiang is at a relatively low level in the whole country. The isolated strains had strong resistance to tetracycline and cephalosporin antibiotics. This study provided a basis for mastering the epidemiological characteristics of Klebsiella pneumoniae in dairy cow mastitis and guiding clinical medication.