液相色谱-串联质谱法测定蜂产品中20种喹诺酮类药物的残留量

建立了同时测定蜂蜜和蜂王浆中20种喹诺酮类药物的超高效液相色谱-串联质谱方法。以磷酸盐缓冲液(p H 3.0)溶液提取样品中的待测物,HLB固相萃取柱净化,液相色谱-串联质谱法测定,外标法定量。药物在1~50 ng/m L浓度范围内线性关系良好,相关系数均大于0.99。方法的检出限为0.5μg/kg,定量限为1.0μg/kg,在蜂蜜和蜂王浆中添加浓度范围为1.0~5.0μg/kg,其平均回收率为69.0%~105.1%,相对标准偏差在1.4%~14.7%之间。方法抗干扰能力强、分析速度快、灵敏度高,适用于蜂产品中喹诺酮类药物残留量的测定。     

采用全自动在线固相萃取-高效液相色谱法检测畜禽产品中氟喹诺酮类药物残留量

为建立畜产品中6种氟喹诺酮全自动在线固相萃取-高效液相色谱的检测方法,实验采用全自动在线固相萃取-高效液相色谱替代了前处理使用固相萃取小柱净化过程,净化效果较好。实现了恩诺沙星、环丙沙星、洛美沙星、培氟沙星、诺氟沙星和丹诺沙星6种氟喹诺酮类药物同时检测。本方法采用1%乙酸乙腈溶液提取,稀释后使用全自动在线固相萃取-高效液相色谱仪检测。6种氟喹诺酮平均回收率为81.9%~106.8%;相对标准偏差小于3.52%;定量限均为2.5μg/kg。该方法操作简单、经济、灵敏度高,结果准确可靠。     

超高效液相色谱法检测鸡肉中氟喹诺酮类药物残留

本研究建立了检测鸡肉中氟喹诺酮类药物的超高效液相色谱分析方法,样品采用磷酸盐缓冲溶液提取,提取液经Bond Elut C18固相萃取柱富集净化,在荧光检测器下进行定性与定量分析。结果表明,该方法对4种氟喹诺酮类药物在0.000 2~0.5μg/mL范围内呈良好的线性关系,相关系数(r~2)均大于0.999 8,在3个添加水平范围内的回收率为67.6%~97.1%,相对标准偏差为3.1%~5.4%,方法检出限为3μg/kg,定量限为10μg/kg。     

液相色谱-串联质谱法测定鸡蛋中7种氟喹诺酮类药物的残留量

建立了一种液相色谱-串联质谱法测定鸡蛋中7种氟喹诺酮类药物残留的方法。样品经EDTA-Mcllvaine缓冲液提取,冷冻离心,然后用正己烷脱脂,有效地去除杂质,HLB固相萃取柱净化,氮气吹干浓缩,流动相溶解,进行LC-MS/MS定性定量分析。其中5种氟喹诺酮类药物含量在2.5μg/kg~100μg/kg范围内,线性关系良好,环丙沙星和恶喹酸含量分别在6μg/kg~240μg/kg和0.25μg/kg~10μg/kg范围内,线性关系良好,相关系数均大于0.995 9;添加量在100、200、500μg/kg时,该方法的加标回收率为36.9%~81.2%,相对标准偏差均小于15.8%。该方法对环丙沙星和恶喹酸的检测限分别为1.2μg/kg和0.05μg/kg,定量限为24μg/kg和1μg/kg,其他5种氟喹诺酮类药物的检测限为0.5μg/kg,定量限为10μg/kg,表明所建立的方法适用于鸡蛋中7种氟喹诺酮类药物残留的定性和定量检测。     

超高效液相色谱法同时测定鸡肝中五种氟喹诺酮类药物残留

建立了一种同时测定鸡肝中五种氟喹诺酮类药物残留的超高效液相色谱法(UPLC)。样品经磷酸盐缓冲溶液提取、C18柱净化,采用ACQUITY UPLCTMBEH C18色谱柱(50 mm×2.1mm,1.7μm)分离,以乙腈溶液和0.1%甲酸的水溶液作为流动相进行等度洗脱,荧光检测器检测。五种药物在0.005~0.5μg/mL范围内线性关系良好,相关系数r均大于0.999;以0.05、0.15、0.30μg/g三个浓度水平进行添加回收试验,平均回收率为70.2%~98.2%,相对标准偏差为4.6%~12.1%,方法的检出限(LOD)为0.02μg/g,定量限(LOQ)为0.05μg/g。本方法重现性好、灵敏度高、简单、快捷,适用于鸡肝中五种氟喹诺酮类药物多残留的检测。     

超高效液相色谱-串联质谱法检测牛奶中7种氨基糖苷类药物残留

应用超高效液相色谱-串联质谱技术,建立了牛奶中7种氨基糖苷类药物(链霉素、双氢链霉素、庆大霉素、卡那霉素、丁胺卡那霉素、安普霉素、大观霉素)的检测方法。样品经乙酸铵缓冲液提取,固相萃取柱净化,CORTECS HILIC为分离色谱柱,采用甲酸铵溶液作为流动相进行梯度洗脱,通过多反应监测模式进行测定。在0.1~5μg/m L的范围内线性关系良好,相关系数均大于0.996。在20、50、200μg/kg添加水平下,平均回收率为80%~117.5%,相对标准偏差均小于15%。检测限为10μg/kg,定量限为20μg/kg,该方法准确、简单、快速。     

超高效液相色谱串联质谱法测定饲料中7种氟喹诺酮类药物

本试验建立了超高效液相色谱串联质谱同时测定饲料中7种喹诺酮类药物残留的方法。饲料样品中喹诺酮药物经酸化乙腈提取,电喷雾离子源三重四级杆串联质谱对喹诺酮类药物进行测定,外标法定量。试验结果表明,7种喹诺酮药物在0~500 ng/m L的浓度范围内,其回归标准曲线方程的相关系数为0.997 4~0.999 7。对空白饲料添加5、50、100μg/kg 3种浓度的喹诺酮类药物,回收率在74.7%~92.3%范围内,相对标准偏差为3.99%~8.29%,方法检出限为2μg/kg以下。说明该方法操作简单快速,定性定量准确。     

HPLC-MS/MS检测双黄连注射液中非法添加13种喹诺酮类药物的研究

建立了高效液相色谱-串联质谱法检测兽用中药制剂双黄连注射液中非法添加13种喹诺酮类药物的方法。样品经0.2%甲酸溶液溶解后稀释,以0.2%甲酸溶液与甲醇作为流动相进行梯度洗脱,采用多反应监测模式进行测定。13种喹诺酮类药物检测限(LOD)为0.1~0.5 mg/m L,定量限(LOQ)为1~5 mg/m L,在5.0~200 ng/m L范围内线性关系良好,相关系数均大于0.99,在5、10、20 mg/m L三个添加浓度下,平均回收率为90.9%~101.9%,相对标准偏差为0.6%~3.8%。     

HPLC-MS/MS检测双黄连注射液中非法添加13种喹诺酮类药物的研究

建立了高效液相色谱-串联质谱法检测兽用中药制剂双黄连注射液中非法添加13种喹诺酮类药物的方法。样品经0.2%甲酸溶液溶解后稀释,以0.2%甲酸溶液与甲醇作为流动相进行梯度洗脱,采用多反应监测模式进行测定。13种喹诺酮类药物检测限(LOD)为0.1~0.5 mg/m L,定量限(LOQ)为1~5 mg/m L,在5.0~200 ng/m L范围内线性关系良好,相关系数均大于0.99,在5、10、20 mg/m L三个添加浓度下,平均回收率为90.9%~101.9%,相对标准偏差为0.6%~3.8%。     

液相色谱-串联质谱法测定猪血浆和尿液中8种同化激素的残留

为建立猪血浆和尿液中8种同化激素的液相色谱-串联质谱(LC-MS/MS)检测方法,样品酶解后,用乙酸乙酯超声提取,经C18固相萃取小柱净化,进行LC-MS/MS检测。结果表明,采用基质添加标准曲线消除基质干扰,8种同化激素在1.5μg/L~100μg/L内,相关系数均大于0.99,线性关系良好。除炔诺酮定量限为1.5μg/L,其他7种药物定量限均为1μg/L。在2、10、50μg/L添加水平下,药物的回收率为71.5%~92.3%,日内相对标准偏差(RSD)为2.2%~14.6%,日间RSD为3.2%~12.7%。本方法灵敏度高,适用于同化激素类药物残留的检测分析。     

The effects of adjuvants on immune responses in cattle injected with a Brucella abortus soluble antigen.

Five different adjuvants were examined for potentiation of humoral and cell-mediated immune (CMI) responses in cattle to a Brucella abortus soluble antigen (BASA). Two separate experiments were performed involving a total of 64 steers, divided among six groups (Experiment 1) and 9 groups (Experiment 2). The adjuvants used were: muramyl dipeptide, Freund's incomplete adjuvant, dimethyl-dioctadecyl ammonium bromide (DDA), Bordetella pertussis and Propionibacterium acnes. In each experiment, three groups received BASA (2 mg protein) subcutaneously with adjuvant, one group received a reduced dose of B. abortus Strain 19 (S19), one group served as unvaccinated controls, and another group received BASA alone. Primary responses were studied following a single immunization in comparison to the single inoculation with S19. For each experiment serum antibody responses and CMI responses were sequentially determined over a period of 56 days. Antibody responses to B. abortus were measured using the brucellosis card, rivanol precipitation-plate agglutination, complement fixation, and fluorometric immunoassay tests, and as well as with an enzyme-linked immunosorbent assay. The CMI response was measured using antigen-specific lymphoproliferation (LP) and skin testing for delayed-type hypersensitivity (DTH) to BASA (Experiment 2). Specific aspects of induced CMI responses investigated were macrophage activation (IL-1 production), helper T cell activation (IL-2 production), and release of soluble suppressor factor(s). In general, mean antibody responses were significantly higher (P less than 0.05) in immunized steers than in control steers and those receiving BASA alone. The LP responses to heat-killed B. abortus were generally higher in immunized groups than in the controls. The LP and DTH responses were greatest in the groups receiving S19 and BASA + DDA. Increased induction of IL-1 was largest in the group receiving BASA + DDA whereas IL-2 release was greatest in S19 vaccinated steers. Suppressor T cell responses were most obvious in the groups receiving S19, BASA + B. pertussis, and P. acnes. These studies demonstrated that DDA potentiates CMI responses to a soluble B. abortus antigen and may be useful as an adjuvant for future vaccines, particularly subunit vaccines.     

Effect of dose of Brucella abortus strain 19 in yearling heifers on the relative risk of developing brucellosis from challenge exposure with strain 2308

Yearling heifers were given SC injections of 10(8) (n = 40), 10(9) (n = 44), or 10(10) (n = 44) colony-forming units of Brucella abortus strain 19 (S19). The proportion of heifers with positive serologic test results at 1 month following vaccination increased as the dose of S19 increased. These proportions decreased with time, and all heifers had negative card, rivanol, and complement fixation test results within 4 months. Positive ELISA results persisted beyond 4 months in all three S19 dose groups; however, all heifers were ELISA-negative within 9 months after vaccination. Comparable lymphocyte transformation activity was stimulated by S19 dose of 10(9) or 10(10) and approximately half of the heifers in both groups had a positive stimulation index at 9 months. Immunity of the pregnant heifers was challenged 9 months after vaccination with 10(7) B abortus strain 2308 as follows: diluent controls (n = 69); 10(8) B abortus S19 (n = 40); 10(9) B abortus S19 (n = 39); and 10(10) B abortus S19 (n = 39). Tissue specimens from heifers were obtained at parturition and necropsy for culturing of B abortus. The proportion of heifers that developed brucellosis, ie, had positive culture results, increased as gestation days at challenge exposure increased. The effect of gestational age was controlled in the analysis using logistic regression. The relative risk of brucellosis was reduced to 0.38, 0.15, and 0.06 for B abortus S19 doses of 10(8), 10(9), and 10(10), respectively, compared with diluent controls at 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells

Real-time quantitative PCR (RT-qPCR) is a critical tool used to evaluate changes in gene expression. The precision of this tool is reliant upon the selection of reference genes whose expression remains unaltered in culture conditions and following stimulation. Stably expressed reference genes are used to normalize data so observed changes in expression are not due to artifacts but rather reflect physiological changes. In this study, we examined the expression stability of the porcine genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), eukaryotic elongation factor 1 gamma-like protein (eEF1), ribosomal protein L19 (RPL19), beta-actin (ACTB) and ATP synthase mitochondrial F0 complex (ATP5G1) in peripheral blood mononuclear cells (PBMCs), monocytes, monocyte-derived dendritic cells (MoDCs), blood isolated dendritic cells (BDCs) and T cells with or without stimulation with lipolysaccharide (LPS). An M value was used as a measure of gene stability as determined using geNORM software. Recommendations for the use of reference genes include using GAPDH and B-actin in PBMCs: RPL19 and SDHA in T cells; RPL19 and B-actin in monocytes; RPL-19 and SDHA in BDCs: and RPL-19 and ATP5GA in MoDCs.     

Validation of an Anaplasma marginale cELISA for use in the diagnosis of A. ovis infections in domestic sheep and Anaplasma spp. in wild ungulates

A commercially available (cELISA) kit for diagnosing Anaplasma marginale infection in cattle was validated for diagnosing A ovis infection in sheep using the bovine serum controls as supplied by the manufacturer (BcELISA) and sheep serum controls from pathogen-free sheep (OcELISA). True positives were identified using two previously established assays, a nested PCR (nPCR) test and an indirect immunofluorescent assay (IFA). The BcELISA was also applied to sera from various species of wild ruminants, comparing the results with the IFA. Receiver operating characteristic (ROC) analysis indicated that the predicted threshold inhibition for the BcELISA was 19.2. The sensitivity for the BcELISA was 98.2% and the specificity was 96.3%. The predicted threshold inhibition decreased to 14.3 for the OcELISA; the sensitivity was 96.5% and the specificity was 98.1%. There was >/=90% concordance between IFA and nPCR, as well as between the BcELISA at 19% inhibition cutoff and either IFA or PCR. Concordance between the cELISA and IFA using sera from elk, mule deer, bighorn sheep, pronghorn antelope, and black-tailed deer ranged from 64% to 100%. This commercially available cELISA test kit can be used very effectively to test domestic sheep for infection with A. ovis using the kit-supplied controls (i.e. the BcELISA) and a 19% inhibition cutoff; the kit may also be useful for detecting intra-erythrocytic Anaplasma infections in wild ruminants.     

Prevalence of thyroglobulin autoantibodies detected by enzyme-linked immunosorbent assay of canine serum in hypothyroid, obese and healthy dogs in Japan

Thyroglobulin autoantibodies (TgAA) were detected in sera of hypothyroid (n=19), obese (n=28) and clinically healthy dogs (n=52) using a commercially available immunoassay kit. TgAA-positive results occurred in 10 of 19 hypothyroid, 1 of 28 obese and 1 of 52 clinically healthy dogs. The clinically healthy TgAA-positive dog had additional evidence of hypothyroidism supported by low total T(4), low free T(4) and high canine TSH. Among the breeds, Golden Retriever had the highest frequency of hypothyroid (9/19) and TgAA-positive hypothyroid dogs (6/10). This study was the first survey about the prevalence of canine TgAA in Japan and could be a useful reference for clinicians.     

野生马蹄金种质遗传多样性的SRAP研究

为评价野生马蹄金种质的遗传多样性,从80对SRAP引物中筛选出19对多态性高、稳定性好的引物,用于研究21份马蹄金材料,获得如下结果:1)19对引物共扩增出212条带,平均每对引物扩增出11.1条,其中多态性条带156条,平均每对引物8.2条,多态率为73.58%。材料间遗传相似系数变化范围为0.444~0.980,平均值为0.711 2。结果表明,供试马蹄金材料具有较为丰富的遗传多样性。2)对所有材料的聚类分析发现,在GS值为0.78处所有供试材料可聚为3个类群,大部分来源地相同或生态环境相似的材料聚为一类,表明供试材料的聚类和生态地理环境具有一定的相关性。     

DNA homology of Brucella abortus strains 19 and 2308

The restriction endonuclease digestion DNA patterns from Brucella abortus strains 19 and 2308 were examined with 11 restriction enzymes (AvaI, BamHI, BglII, BstEII, DdeI, EcoRI, HindIII, KpnI, PstI, XbaI, and SalI). The DNA electrophoretic banding patterns between the 2 strains were highly similar, using this restriction enzyme analysis. Differences were not discernable between B abortus strains 19 and 2308 in any of the restriction banding patterns examined. Methylation at CCGG or GATC sites was not detectable on the basis of digestion with isoschizomers (HpaII and MspI, and DpnI, Sau3AI and MboI). Homology between B abortus strains 19 and 2308 was assessed, using solution-hybridization techniques followed by S1 nuclease assays. Results of these reassociation experiments indicated 98.6 to 99.3% homology between B abortus strains 19 and 2308 with 13.5 to 18.6% homology between B abortus (strains 19 and 2308) and the E coli HB101 control. We concluded that any DNA differences between the 2 B abortus strains are small and will require analysis at the DNA sequence level.     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor associated with tumor cell proliferation in canine cutaneous squamous cell carcinomas and trichoepitheliomas

The expression of 5 markers associated with angiogenesis was studied in canine squamous cell carcinomas (SCCs) (n = 19) and canine trichoepitheliomas (TCPs) (n = 24). SCCs were assigned histologic grades, and tissue sections from both tumor types were immunohistochemially stained for the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2), as well as intratumoral microvessel density (iMVD), tumor proliferation index (PI), and tumor apoptotic index (AI), using antibodies against VEGF, VEGFR-2, von Willebrand's factor, Ki-67 antigen, and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate end-labeling method (TUNEL), respectively. VEGF and VEGFR-2 were detected in 17/19 (89.4%) and 19/19 (100%) SCCs and in 17/24 (70.8%) and 20/24 (83.3%) TCPs, respectively. In SCCs, there was substantial correlation between histologic grade and PI (r = 0.51); and moderate correlation between VEGF and histologic grade (r = 0.43), VEGFR-2 and histologic grade (r = 0.47), VEGF and PI (r = 0.47), and VEGFR-2 and PI (r = 0.47) (Spearman rank correlation coefficient). In TCPs, there was substantial correlation between VEGF and PI (r = 0.51) and a moderate correlation between VEGFR-2 and iMVD (r = 0.36). The median iMVD of SCCs (15.5) was significantly higher than the median iMVD of TCPs (9.05) (P value < .05). It was concluded that VEGF and VEGFR-2 may promote tumor cell proliferation in TCPs and SCCs. An autocrine pathway for VEGF probably operates in canine SCCs and TCPs, as VEGF and VEGFR-2 expression was found in most tumors and was associated with evidence for tumor cell proliferation.     

中国牛种布鲁菌弱毒疫苗株A19 SNP位点的研究

为鉴别我国牛种布鲁菌弱毒疫苗株A19与野生菌株,运用生物信息学方法结合基因测序,对疫苗株A19基因组SNP位点分析筛选,选取其中部分SNP位点,通过与布鲁菌常见种、生物型标准参考菌株和弱毒疫苗株基因组SNP位置核苷酸测序比较,验证SNP位点的A19特异性。结果表明,共筛选获得A19基因组29个SNP位点,验证ClpX G825-C825、LysR A605-C605、Omp2b G503-A503这3个SNP位点为A19（或S19）特异,揭示了A19基因组SNP位点分布情况,为疫苗株 A19与野生菌株鉴别提供了分子依据。