抗双氯青霉素单克隆抗体的制备与初步鉴定

采用碳化二亚胺法,将双氯青霉素(dicloxacillin)与牛血清白蛋白偶联制备免疫原,免疫BALB/c小鼠,取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,间接ELISA筛选,阳性杂交瘤细胞用有限稀释法克隆,获得1株可稳定分泌McAb的杂交瘤细胞5D4-C9-C8;间接ELISA方法测定,其细胞上清抗体效价为1∶300,腹水效价为1∶3.5×105,为IgG1,与其结构类似物邻氯青霉素、苯唑青霉素的交叉反应率分别为17.6%和26.1%,与苄青霉素、羟氨苄青霉素和氨苄青霉素均无交叉反应性。体外传代培养和冻存复苏后抗体分泌稳定。为进一步研制检测双氯青霉素的ELISA试剂盒奠定了基础。     

磺胺氯吡嗪钠二甲氧苄啶溶液质量标准研究

研究磺胺氯吡嗪钠二甲氧苄啶溶液的质量标准。以高效液相色谱法测定溶液中磺胺氯吡嗪钠和二甲氧苄啶含量,采用Sino Chrom ODS-BP色谱柱,以磷酸二氢钾溶液∶甲醇为流动相,磺胺氯吡嗪钠和二甲氧苄啶在0.5-100.0μg/m L和0.3-45.0μg/m L范围内线性关系良好,平均回收率分别为99.9%和99.1%。该研究建立的色谱条件可同时检测2种成分,简便快捷。     

药物代谢动力学的研究(第二报)——青霉素G钾、普鲁卡因青霉素 苄星青霉素、复方苄星青霉素在马体内代谢动力学的比较研究

本研究应用青霉素G钾、普鲁卡因青霉素、苄星青霉素和复方苄星青霉素四种制剂,分别给各4匹蒙古马单剂量(10,000u/kg)肌肉注射,用生物管碟法分别测出其不同时间的血药浓度。前三种制剂按有吸收因素一室模型回归方程C=M(e~(-ket)-e~(-kat))估算出血药浓度理论值;复方苄星青霉素按有吸收因素二室模型回归方程C=Ae~(-αt)+Be~(-βt)-(A+B)e~(-kat)估算出血药浓度理论值。然后按模型公式算出每匹马的药代动力学参数,再求出平均值与标准差。通过参数对四种制剂的药代动力学特点进行了比较,为兽医临床合理用药提供了依据。     

高效液相色谱法测定饲料中的氯苄青霉素

本实验对饲料中氨苄青霉素的高效液相色谱(HPLC)检测方法进行了研究.采用磷酸盐缓冲液提取,高效液相色谱-二极管矩阵检测器(HPLC-DAD)检测,在检洲波长为220 nm的条件下,对饲料进行添加5.0～500mg/kg氨苄青霉素的回收实验.结果表明:氨苄青霉素的平均回收率为76.1%～94.8%(n=6),变异系数为1.7%～7.4%(n=6),检出限为1.0 mg/kg,定量限为5.0 mg/kg.     

鸡血浆中磺胺氯吡嗪钠和甲氧苄啶含量HPLC检测方法的建立

建立了鸡血浆中磺胺氯吡嗪钠和甲氧苄啶含量测定的HPLC方法。对鸡血浆中磺胺氯吡嗪和甲氧苄啶采用乙腈提取和净化,梯度洗脱,高效液相色谱紫外检测法测定,外标法定量。流动相为甲醇-0.02 mol/L磷酸二氢钾水溶液。紫外测定时采用双波长检测:磺胺氯吡嗪检测波长为268 nm,甲氧苄啶检测波长为240 nm。空白血浆添加磺胺氯吡嗪和甲氧苄啶分别在0.1～70.0μg/m L和0.05～5.00μg/m L范围内线性关系良好(R~2均大于0.9999),磺胺氯吡嗪和甲氧苄啶的检测限分别为0.05μg/m L和0.025μg/m L,定量限分别为0.1μg/m L和0.05μg/m L。空白血浆添加磺胺氯吡嗪和甲氧苄啶在各自线性范围内,批内变异系数分别小于6.84%和7.55%,批间系数分别小于5.23%和6.81%。本研究建立的鸡血浆中磺胺氯吡嗪和甲氧苄啶的提取和净化方法,适用于鸡血浆中磺胺氯吡嗪和甲氧苄啶含量的单个或同时测定。     

苄星氯唑西林国家对照品的研制

为了研制首批苄星氯唑西林国家对照品。对质量标准中的关键项目进行考察。采用红外分光光度法、高效液相色谱法对原料进行鉴别,采用高效液相色谱法进行含量测定。结果显示,红外光吸收图谱与USP溯源对照品图谱一致,高效液相色谱法测定氯唑西林与二苄基乙二胺的含量和为92.1%。研究建立的苄星氯唑西林国家对照品可用于苄星氯唑西林及其制剂的鉴别。     

120万u/瓶注射用苄星青霉素的无菌检验方法学验证

采用直接接种法对120万u/瓶注射用苄星青霉素进行无菌检查。实验结果表明,每瓶供试品用6ml青霉素酶稀释,向每支装量为120ml的培养基接入1.0ml供试液后培养,可消除苄星青霉素的抑菌活性,且培养基能够澄清,便于观察是否有微生物生长。     

猪血浆中青霉素高效液相色谱检测法

本文建立了猪血浆中青霉素浓度的高效液相色谱(HPLC)快速检测方法。血浆经等体积乙腈沉淀蛋白及提取药物后,经高速离心、衍生化、过膜后直接进样。色谱柱为Hypersil BDS-C18柱,流动相为0.05 mol.L-1磷酸盐缓冲液-乙腈(体积比为62∶38),流速为1 mL.min-1,检测波长为325 nm。血浆中青霉素质量分数为0.1～10μg.mL-1时,青霉素的质量分数与其峰面积之间存在良好的线性关系(R2>0.999,n=5)。本方法最低检测限(LOD)为0.05μg.mL-1,最低定量限(LOQ)为0.1μg.mL-1,萃取回收率>97%,批内和批间变异系数均≤4.2%。该方法重现性好,灵敏度高,简便,可用于猪及其他动物血浆中青霉素及其部分同类药物的快速检测。     

高效液相色谱法测定甲氧苄啶含量

建立甲氧苄啶含量高效液相色谱测定方法。采用Diamonsil C18色谱柱(200mm×4.6mm,5μm),以甲醇∶0.02mol/L磷酸二氢钾溶液(30∶70)为流动相,流速为1.0mL/min,柱温为25℃,检测波长为271nm。甲氧苄啶对照品浓度在10μg/mL～160μg/mL范围内与峰面积呈线性关系,线性回归方程A=0.389 2C-0.369 6,r=0.999 9(n=5);平均回收率为99.61%,RSD=0.41%(n=5)。高效液相色谱法快速、简便,专属性强,重现性好,准确度高,可用于甲氧苄啶原料含量的测定。     

高效液相色谱法测定磺胺氯达嗪钠含量

建立高效液相色谱(HPLC)法测定磺胺氯达嗪钠的含量。色谱柱为Agilent ZORBAX Eclipse Plus C18柱(150mm×1.6mm,5μm),流动相为0.1%磷酸溶液-甲醇(85∶15),流速为1.0mL/min,柱温为25℃,检测波长为270nm。磺胺氯达嗪钠对照品质量浓度在40～200μg/mL范围内与峰面积呈良好的线性关系,线性回归方程A=23.34C-10.58,r=0.9999(n=5);平均回收率为99.7%,RSD=0.32%(n=5)。HPLC法快速、简便,专属性强,重现性好,准确度高,可用于磺胺氯达嗪钠原粉的含量测定。     

苄星氯唑西林乳房注入剂无菌检查方法研究

本试验旨在建立苄星氯唑西林乳房注入剂无菌检查法并进行验证.采用离心—薄膜过滤—酶中和相结合的方法,按照《中国兽药典》2010年版一部附录进行试验.结果显示采用离心—薄膜过滤—酶中和相结合的方法,以0.5%聚山梨酯-80 pH 7.0氯化钠—蛋白胨缓冲液冲洗,冲洗量400 mL/膜,在最后的培养基中加入不少于300万单位的青霉素酶可有效消除样品对各试验菌株的抗菌活性;验证试验结果显示各验证菌生长良好,各供试品管均未见菌生长.结果表明本试验成功建立了可靠、准确、可用于苄星氯唑西林乳房注入剂的无菌检查法.     

Ocular and serum disposition kinetics of cloxacillin after topical administration of benzathine cloxacillin and intravenous administration of sodium cloxacillin to calves

Disposition kinetics of cloxacillin were examined in calves after topical administration of benzathine cloxacillin and single IV administration of sodium cloxacillin, and the susceptibility of 17 field isolates of Moraxella bovis was measured. For the IV pharmacokinetic phase, sodium cloxacillin was administered at dosage of 10 mg/kg of body weight to male Holstein calves (n = 6, weighing 146 to 170 kg), and serum concentration of cloxacillin was measured thereafter for 10 hours. For the ocular pharmacokinetic phase, 6 calves were given either of 4 benzathine cloxacillin topical formulations consisting of 50-, 125-, 250-, or 375-mg doses. Treatment was repeated every 10 days until all 4 benzathine cloxacillin dosages were tested in the same 6 calves. Blood and tears were collected for 72 hours after each benzathine cloxacillin formulation was administered, and the concentration of cloxacillin in each specimen was measured, using a bioassay. The minimal inhibitory concentration of cloxacillin for 17 field isolates of M bovis was determined by use of an agar pour-plate dilution assay. After single IV administration of sodium cloxacillin, its half-life, body clearance, and volume of distribution were 19.5 +/- 12.8 minutes, 18.3 +/- 2.2 ml/min.kg, and 496 +/- 290 ml/kg, respectively. After topical administration of benzathine cloxacillin, cloxacillin concentration in lacrimal fluid peaked between 30 and 45 minutes and ranged between 963 micrograms/ml and 3,256 micrograms/ml for the 125- and 375-mg doses, respectively. There was no detectable cloxacillin activity in the lacrimal fluid of any calf by 36 hours after topical administration of benzathine cloxacillin, and cloxacillin was not detected in the serum at any time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skin sensitivity to tuberculins in cattle vaccinated against Johne's disease

Aim: To evaluate the efficacy of a dry-cow antibiotic preparation containing cloxacillin plus ampicillin in a formulation that gives a 10-week duration of action, in comparison to products containing cephalonium (10-week action) or cloxacillin alone (7-week action).

Methods: A total of 493 cows were selected from 6 spring-calving dairy herds in the Manawatu region of New Zealand, according to the criteria of the SAMM plan, to receive intramammary antibiotic therapy at the end of lactation (drying off). Cows were randomly allocated to receive 1 of the 3 dry-cow antibiotic products under investigation. Cows were examined twice during the dry period and twice daily during the first 10 days of their subsequent lactation for the presence of mastitis. Milk samples were collected from individual quarters at the time of drying off and at 7 and 28-35 days after calving, for determination of milk somatic cell counts (SCC). Bacteriology was carried out on milk samples taken from cows that developed mastitis during the first 10 days after calving.

Results: No cows developed mastitis during the dry period. Sixteen cows developed clinical mastitis within 10 days of calving; there was no difference in incidence between treatments. Streptococcus uberis was the most commonly isolated organism. Mean SCC on Day 7 were lower (p = 0.019) in cephalonium-treated quarters (189.9 ± 28.4 × 10³ cells/ml) than in cloxacillin-treated quarters (388.7 ± 71.2 x 10³ cells/ml); values in quarters receiving cloxacillin plus ampicillin were intermediate (252.0 ± 47.0 × 10³ cells/ml). SCC were similar between treatment groups on Day 28–35.

Conclusions: The use of a combination of cloxacillin plus ampicillin was effective for the prevention of mastitis during the dry- and peri-calving-periods in pastured dairy cattle.     

Persistence of antibiotics in bovine mammary secretions following intramammary infusion at cessation of milking

A study was conducted to determine the persistence of antibiotic preparations for use in nonlactating cows in bovine mammary secretions following intramammary infusion at cessation of milking. Five commercially available antibiotic formulations were evaluated using 311 cows. All quarters of each cow were sampled once only during the nonlactating period and most cows were sampled at or near parturition. Antibiotic residues were detected qualitatively by the Bacillus stearothermophilus disc assay. Great variation between different antibiotics in persistence in mammary secretion was observed. In general, mammary secretions from most mammary glands infused with cloxacillin or penicillin-dihydrostreptomycin were positive at 28–35 days after infusion and some were positive at 42–49 days after infusion. On the other hand, <13% of mammary secretions at 7 days after infusion of novobiocin and 50% of mammary secretions at 14 days after infusion of penicillin-novobiocin were positive for antibiotics. Cephapirin benzathine persisted for about 21 days after infusion. Some samples that were positive for antibiotics after initial testing were negative following heating of samples, suggesting that component(s) of dry secretion can inhibit growth of B. stearothermophilus and influence the interpretation of results. Colostrum samples from all quarters except one were negative for antibiotics. These data suggest that nonlactating-cow antibiotic formulations persist primarily during the early to mid-nonlactating period. Based upon present methods of formulation, it would appear that antibiotic preparations for use in nonlactating cows most likely provide little protection during the periparturient period, at a time when mammary glands are highly susceptible to new intramammary infections.     

Assessment of antimicrobial transfer from treated to untreated mammary gland quarters by use of high-pressure liquid chromatography for detection of cloxacillin in milk samples from nonlactating dairy cows

OBJECTIVE: To determine whether half-udder intramammary infusion of cloxacillin results in transfer of cloxacillin from treated to untreated mammary gland quarters within nonlactating cows, and, if so, at what concentrations, and to determine whether selection of ipsilateral versus diagonal-contralateral quarters for treatment affects cloxacillin transfer among quarters. ANIMALS: 20 Holstein-Friesian cows from a dairy herd. PROCEDURES: A within-cow half-udder comparison trial was used in which 2 of 4 mammary gland quarters (ipsilaterally or diagonally) received an intramammary infusion of cloxacillin on day 1 of the nonlactating period. Three days later, milk samples were taken from all untreated quarters and high-pressure liquid chromatography was used to detect and quantify milk cloxacillin concentrations. RESULTS: Cloxacillin was detected in 25% of all untreated mammary gland quarters. Mean cloxacillin concentration in untreated quarters was below minimum inhibitory concentrations for targeted mastitis pathogens. No significant difference in cloxacillin concentrations was found in the ipsilateral or diagonal treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Within-cow half-udder comparison trials are valid for antimicrobial trials in nonlactating cows, although transfer of antimicrobials does occur in trace concentrations. Ipsilateral or diagonal-contralateral treatment designs perform similarly. This type of design is economical for researchers, although care must be taken to account for within-cow clustering of mammary gland quarter data.     

Influence of the genotype of Staphylococcus aureus, determined by pulsed-field gel electrophoresis, on dry-period elimination of subclinical mastitis in Canadian dairy herds

By combining information from 2 databases, we investigated the possibility of an association between the genotype of Staphylococcus aureus causing bovine intramammary infection and dry-period cure of subclinical infection. The 1st database contained bacteriologic and cow data from a field study evaluating the efficacy in such infections of a new intramammary dry-cow therapy (DCT) containing tilmicosin phosphate, in comparison with a commercially available DCT containing benzathine cloxacillin. Isolates of S. aureus from that study were frozen and later independently analyzed by pulsed-field gel electrophoresis (PFGE) and macrorestriction DNA fingerprinting. The molecular information, summarized and published elsewhere, constituted the 2nd database. Data from 121 subclinically infected quarters of 92 cows from 40 herds were studied by univariate and multivariable regression analysis. Infection by an isolate of PFGE lineage group D was more likely than infection by an isolate of group A or F to be cured (P < 0.05). Cows infected by lineage group D had a higher linear somatic cell count score (LS) from the last Dairy Herd Improvement test before the dry period than did cows infected by the other lineage groups (P = 0.04). Although the probability of cure was significantly lower for cows with an LS at or above the mean of 5.7 for the study population (P = 0.05), when such a cow was infected with lineage group D, cure was significantly more likely (P < 0.001) than when it was infected by another lineage group. Significantly more (P = 0.02) of the infections treated with tilmicosin (74%) than of those treated with benzathine cloxacillin (53%) were cured, and significantly more (P = 0.05) of the infections by group D (81%) than of those by group A (57%) or group F (54%) were cured. However, there was no difference in cure rate for any PFGE genotype when tilmicosin phosphate was administered; when benzathine cloxacillin was administered, 87% of lineage group D isolates were eliminated, as compared with 46% of group A and 33% of group F isolates (P < 0.05). This research demonstrates that certain genotypes of S. aureus may naturally elicit a greater inflammatory response, yet be more susceptible to elimination by antibiotics in the dry period, than other genotypes.     

Enhanced killing of penicillin-treated S. aureus by host defences: Effects of amoxycillin, cloxacillin and nafcillin in vitro and in experimental mastitis

The effects of pretreatment of Staphylococcus aureus with subMIC or 10 × MIC amoxycillin, cloxacillin and nafcillin on subsequent killing by bovine neutrophils or sensitivity to lysostaphin were examined in vitro, using penicillinase positive and negative strains. All penicillins increased the susceptibility of S. aureus to bovine neutrophils; with amoxycillin this was most marked with the penicillinase negative strain. Cloxacillin pretreatment was significantly more effective than nafcillin or amoxycillin in some tests and equally effective in others. The sensitivity of S. aureus to lysostaphin was increased following cloxacillin or nafcillin exposure. In a mouse model of mastitis, more cloxacillin and nafcillin-treated S. aureus (at 1/4 MIC) were killed than untreated bacteria 1 hr after infection but by 4 hr no differences were found. No differences were seen after intramammary infection with 10 × MIC penicillin-treated S. aureus. In glands with established chronic mastitis both cloxacillin and nafcillin at high doses failed to kill staphylococci. These findings indicate that prior exposure to penicillins increases staphylococcal susceptibility to killing by neutrophils but following phagocytosis, intracellular bacteria are protected from the lethal action of penicillins.     

利福昔明乳房注入剂对小鼠的急性毒性试验

为评估利福昔明乳房注入剂的急性毒性,采用标准的急性毒性试验方法进行了利福昔明乳房注入剂对小鼠的急性毒性试验。结果表明,最高组剂量达到5 000 mg/（kg·BW）时,小鼠未见不良反应,饮食活动正常,观察期内均未出现死亡,即利福昔明乳房注入剂对小鼠经口灌胃的LD50大于5 000 mg/（kg·BW）,属于实际无毒级物质。     

A note on effectiveness of dry cow therapy in New Zealand dairy herds

Two dry-cow therapy products were evaluated in seven factory-supply dairy herds in the Waikato area. A product containing neomycin sulphate and the benzathine salt of penicillin (Neopen D.C. White; Smith-Biolab) was used in five herds, and one containing benzathine cloxacillin (Orbenin, Beecham) was used in two herds. Non-treated control cows were included in each herd. Both products were effective in eliminating intramammary infections with Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. Efficacy of dry-cow therapy against S. aureus was 83.8% and 85.2% respectively. Spontaneous cure rate among controls was 30.8% for S. aureus during the dry period. Spontaneous cure rate for Str. uberis was 50%, while dry-cow therapy eliminated 100% and 77.8%, respectively, for the two products. Dry-cow therapy with either product eliminated more than 90% of Str. agalactiae infections while spontaneous cure rate was only 28.6%. These results further support the effectiveness of dry-cow therapy in reducing the level of subclinical mastitis in dairy herds by shortening the duration of intramammary infections.