氯霉素间接竞争ELISA(ciELISA)检测方法的建立

以人工合成的氯霉素－牛血清白蛋白（ＣＡＰ－ＢＳＡ）为包被抗原,氯霉素（Ｃｈｌｏｒａｍｐｈｅｎｉｃｏｌ,ＣＡＰ）为竞争的半抗原,两者与一定量的抗ＣＡＰ单抗（ＣＡＰ－ＭｃＡｂ）反应。实验结果表明,理想的包被抗原浓度为１．２５μｇ／ｍｌ,抗ＣＡＰ－ＭｃＡｂ工作浓度为１：１２０００,酶标二抗工作浓度为 １： ５０００,可测最适范围为 １ｎｇ／ｍｌ－１００ｎｇ／ｍｌ,最小检测量为０．１ｎｇ／ｍｌ,批内和批间变异系数分别为３． ６２％和 ５． １９％。得到回归方程 ｙ ＝１．２７３０－ ０．６７４５ｘ（ｒ２＝ ０． ９７７９）和标准曲线,从而建立了快速定量测定 ＣＡＰ含量的间接竞争酶联免疫吸附试验（ＥＬＩＳＡ）。整个测定时间为６小时。     

多杀巴氏杆菌的一种选择性分离培养基

以马丁培养基为基础，加入促生长因子牛全血和抑菌剂硫酸新霉素和盐酸洁霉素制成多杀巴氏杆菌分离培养基．当培养基中硫酸新霉素的浓度低于２．５μｇ／ｍｌ时，对多杀巴氏杆菌生长无影响；浓度为２．５μｇ／ｍｌ时．对其个别菌株的生长稍有抑制作用；当浓度增大到到５μｇ／ｍｌ时，荚膜Ａ型菌株的生长明显受到抑制，Ｂ型菌株部分受到抑制，而Ｄ型菌株基本不受影响；当浓度达到１０μｇ／ｍｌ时，各型菌株均不能生长．当培养基中含硫酸新霉素２．５μｇ／ｍｌ和盐酸洁霉素１．０μｇ／ｍｌ时，仅个别菌株的生长稍受抑制，而绝大多数菌株生长不受影响．当培养基中含硫酸新霉素２．０μｇ／ｍｌ和盐酸洁霉素１．０μｇ／ｍｌ时．对各型多杀巴氏杆菌的生长均无抑制作用，并可抑制环境中绝大多数杂菌的生长。因此，可以作为选择性培养基分离多杀巴氏杆菌。     

多杀巴氏杆菌的一种选择性分离培养基

以马丁培养基为基础，加入促生长因子牛全血和抑菌剂硫酸新霉素和盐酸洁霉素制成多杀巴氏杆菌分离培养基。当培养基硫酸新霉素的浓度低于２．５μｇ／ｍｌ时，对多杀巴氏杆菌生长无影响；浓度为２．５μｇ／ｍｌ时，对其个别菌株的生长销有抑制作用；当浓度增大到５μｇ／ｍｌ时，英膜Ａ型菌株的生长明显受到抑制，Ｂ型菌株部分受到抑制，而Ｄ型菌株基本不受影响；当浓度达到１０μｇ／ｍｌ时，各型菌株均不能生长。当培养基中含硫酸     

猪肌注硫酸安普霉素的血清浓度测定

给６头健康猪单剂量肌肉注射国产硫酸安普霉素（２０ｍｇ／ｋｇ）,采取血样,用微生物法测定硫酸安普霉素的血清药物浓度。结果平均回收率为９９．０３％,血清是低检测浓度为０．１μｇ／ｍｌ,日内、日间变异系数为２．２％～５．１％,血清药物浓度在０．１～３．０μｇ／ｍｌ范围呈良好线性关系（ｒ＝０．９９６５）,用此法对６头猪肌注硫酸安普霉素的血清药物浓度测定,得出药时,曲线数据。     

全血法鸡淋巴细胞转化试验最佳试验条件的研究

通过丝裂原浓度、血液稀释度、灭活犊牛血清浓度和培养时间四个参数的测定，确定了鸡全血淋巴细胞转化试验的最佳培养条件。最佳试验方法如下：采用不含灭活犊牛血清的ＲＰＭＩ１６４０培养液，刺激管中ＣｏｎＡ浓度为４５μｇ／ｍｌ或ＬＰＳ浓度为２５μｇ／ｍｌ，血液稀释度为１：２０，４０℃培养５６ｈ，在培养结束前１６ｈ加＾３Ｈ－胸腺嘧啶核苷（＾３Ｈ－ＴｄＲ）。本法简单方便，可检测Ｔ、Ｂ细胞的功能状况及用于鸡病病理发     

动物类人乙型肝炎的调查

作者采用人的乙型肝炎ＥＬＩＳＡ诊断试剂盒检测动物的乙型肝炎。结果ＨＢｓＡｇ检测黄牛血清４３份，阳性率为２．３％（１／４３）；肝浸液４３份，阳性率为２７．９％（１２／４３）；肌浸液４３份，阳性率为９．３％（３／４３）；鸡血清７２份，阳性率为１２．５％（９／７２）；牛奶３７份，阳性率为１０．８％．ＨＢｅＡｇ检测黄牛血清４３份，阳性率为２．８％（２／７２）。其结果表明黄牛，鸡，奶牛均感染类人乙型肝炎病毒     

反相HPLC测定猪血浆中隐丹参酮及其活性代谢物的血药浓度

本试验建立了用反相ＨＰＬＣ同时进行猪血浆中隐丹参酮（ＣＴ）及其活性代谢物丹参酮ⅡＡ（Ｔｓ－ⅡＡ）的快速定量检测方法。采用ＹＷＧＣ１８键合相柱作为分析柱，甲醇－水（８５１５）为流动相，检测波长２５４ｎｍ，二苄基为内标。平均回收率分别为ＣＴ：８８．０９％，Ｔｓ－ⅡＡ：８８．５１％；原药及代谢物的检测限为１０ｎｇ，血浆中最低检出浓度为１６ｎｇ／ｍｌ。日内、日间的变异系数为１．５４％～６．２８％，血浆浓度０．０４～２．５μｇ／ｍｌ范围内呈线性关系，ｒ＝０．９９１４（ＣＴ）及０．９９８８（Ｔｓ－ⅡＡ）。用此法对５头猪进行了ｉｖＣＴ后的ＣＴ及Ｔｓ－ⅡＡ的血浆浓度测定，得出了药时曲线数据。     

鸡血清中磺胺二甲嘧啶残留的酶免疫法测定

用直接竞争酶免疫测定法测定磺胺二甲嘧啶（ＳＭ２）在鸡血清中的残留,血清中的ＳＭ２与酶标ＳＭ２竞争酶联板上的抗ＳＭ２抗体的结合位点,最低检出限为５０μｇ／Ｌ。在１００μｇ／Ｌ处,回收率为９１％。对１１种磺胺类和非磺胺类药物进行测定,最大交叉反应只有０．５％。     

单抗夹心ELISA检测产蛋下降综合征病毒的研究

利用３株特异性单抗,建立了检测ＥＤＳＶ的夹心ＥＬＩＳＡ方法。经测定,ＭｃＡｂ最适包被浓度为２μｇ／ｍｌ,ＭｃＡｂ－ＨＲＰ最佳工作浓度为１∶４００,该方法最小检出浓度为０．０３μｇ／ｍｌ,与常见的几种鸡的传染病的病原无交叉反应。对某发病鸡场的５０份泄殖腔拭子样本进行了检测,阳性率为８４％（４２／５０）。     

鸡T淋巴细胞IL－2的体外诱生及活性检测

经Ｌ１６（４５）正交试验选择，并经实验验证，鸡脾脏Ｔ淋巴细胞白细胞介素２（ＩＬ－２）体外诱生和活性检测的最优水平组合及适宜条件为２．５μｇ／ｍＬ伴刀豆蛋白Ａ（ＣｏｎＡ）、ＩＬ－２体外诱生时间２０ｈ、１×１０７／ｍＬ淋巴细胞、１０％小牛血清、靶细胞培养时间４８ｈ、４×１０６／ｍＬ靶细胞、靶细胞接触时间３６ｈ、５ｍｇ／ｍＬＭＴＴ、ＭＴＴ加入时间３ｈ和甲溶解时间２ｈ；胸腺Ｔ淋巴细胞ＩＬ－２体外诱生和活性检测的最优水平组合及适宜条件为５μｇ／ｍＬＣｏｎＡ、ＩＬ－２体外诱生时间４８ｈ，余同脾脏Ｔ淋巴细胞ＩＬ－２体外诱生及活性检测。比较试验表明，ＭＴＴ比色分析法和３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入法有显著的直线回归关系。     

Mitogen-induced transformation of sheep and goat peripheral blood lymphocytes in vitro: The effects of varying culture conditions and the choice of an optimum technique

Peripheral blood lymphocytes (PBL) prepared by centrifugation of heparinized sheep or goat jugular venous blood on Ficoll-Triosil were shown to incorporate methyl-[H³]-thymidine ([H³]-Tdr) in vitro in response to lymphocyte mitogens.Optimal conditions for transformation included the culture of 2.5 × 10⁵ viable cells per round bottomed culture well in 250μl medium RPMI-1640 supplemented with fetal calf serum (FCS) at 10% for goat or 15% for sheep lymphocytes. Optimum incorporation of [H³]-Tdr by sheep PBL was recorded after 3–5 days and was achieved in response to 100μg/ml phytohaemagglutinin (PHA), 20μl/ml pokeweed mitogen (PWM), 10μg/ml Concanavalin-A (Con-A) and 50μg/ml bacterial lipopolysaccharide (LPS). For goat PBL the optimum mitogen concentrations were 50μg/ml PHA, 20μl/ml PWM, 5μg/ml Con-A and 50μg/ml LPS. Optimum PHA concentrations were influenced by the level of FCS supplementation, higher concentrations of PHA being required for optimum response when the concentration of FCS was increased.While variability within preparations was small there was considerable variation in the magnitude of the response between preparations, which was sufficient to confound comparisons between different experiments and between animals. The variability between preparations could not be attributed to changes in sensitivity of PBL to mitogens or to the influence of erythrocyte contamination of the PBL preparations. While these results are in general agreement with previous reports of optimal conditions for the measurement of ruminant PBL to mitogens, there are some important differences which are discussed in the context of the available literature.     

Development of optimal conditions for lymphokine production by chicken lymphocytes

Chicken thymus, spleen, and bursa lymphocytes were isolated by different methods and incubated under differing conditions in order to obtain and characterize avian lymphokines. The biological activity of lymphokine-containing cell culture supernatants was measured by their antiviral activity (interferon(IFN)-units) and by their capacity to induce cytostatic effects in bone-marrow-derived macrophages (50% cytostasis-inducing dose, CID). Lymphokine production by thymus lymphocytes required concanavalin A (ConA)-stimulation, while spleen cells, when cultured at high density, released CID and IFN activities into the culture medium even without mitogen-stimulation. By way of comparison, the highest lymphokine content was found in the supernatant of lymphocyte cultures, which were incubated for 72 hours at 41 degrees C after stimulation with an optimal ConA dose. For stimulation of thymus lymphocytes 30 micrograms ConA/ml were found to be optimal, independent of serum content and cell density in the cultures. In contrast, the optimal ConA dose for spleen lymphocytes not only depended on the serum content but also on the cell density in the cultures and varied within a range of 2.5 micrograms and 45 micrograms ConA/ml.     

A zoospore inhibition technique to evaluate the activity of antifungal compounds against Batrachochytrium dendrobatidis and unsuccessful treatment of experimentally infected green tree frogs (Litoria caerulea) by fluconazole and benzalkonium chloride

Effective and safe treatments of chytridiomycosis in amphibians, caused by Batrachochytrium dendrobatidis, are needed to help prevent mortality in captive programs for threatened species, to reduce the risk of spread, and to better manage the disease in threatened populations. We describe a simple method to determine minimum inhibitory concentrations (MICs) of antifungal agents that involves adding zoospores to various drug concentrations in 96 well plates and microscopic observation after four days. We report results from testing 10 commercially available antifungal compounds: benzalkonium chloride (<0.78 μg/ml), povidone iodine (312.5 μg/ml), amphotericin B (3.125 μg/ml), fluconazole (<1.56 μg/ml), itraconazole (<1.56 μg/ml), enilconazole (<1.56 μg/ml), mercurochrome (6.25 μg/ml), sodium chloride (12.5 mg/ml), methylene blue (<1.56 μg/ml) and Virkon (3.125 μg/ml). For treatment trials of juvenile Litoria caerulea, baths of benzalkonium chloride at 1 mg/L and fluconazole at 25 mg/L were used on 18 experimentally infected frogs per treatment. Although these treatments resulted in longer survival times (mean 43.7 ± 11.3 days) than in the untreated controls (37.9 ± 9.3 days), the mortality rate was still 100%. Higher doses of fluconazole are suggested for further animal trials.     

肌注硫酸庆大霉素在大肠杆菌感染鸡体内的药动学试验

为分析硫酸庆大霉素在健康和鸡大肠杆菌感染鸡体内的药物动力学特征,试验通过给健康鸡腹腔注射大肠杆菌O157,以临床症状、病理剖检和微生物检查为指标,成功建立鸡大肠杆菌感染模型。选取健康鸡和患病鸡各8只,分别以20 mg/kg体重单剂量肌内注射硫酸庆大霉素,分别于0.167、0.25、0.5、0.75、1、2、3、4、6、8和12 h时间点采血,采用管碟法测定血浆中庆大霉素的浓度。结果显示:试验所建立的标准曲线相关性好,相关系数均达0.990以上,日内、日间变异系数均小于10%。肌注给药后,硫酸庆大霉素在鸡体内吸收迅速,房室模型分析表明,健康鸡与患病鸡药时数据均符合有二室开放模型,硫酸庆大霉素在健康鸡体内峰浓度(Cmax)为(15.01±3.51)μg/mL,药时曲线下面积(AUC)为(100.79±5.14)μg/mL·h,消除半衰期(t1/2β)为(4.41±1.32)h,达峰时间(Tp)为(1.27±0.50)h。硫酸庆大霉素在患病鸡体内峰浓度(Cmax)为(12.50±2.19)μg/mL,药时曲线下面积(AUC)为(83.38±4.19)μg/mL·h,消除半衰期(t1/2β)为(4.18±1.17)h,达峰时间(Tp)为(0.97±0.05)h。结果表明:硫酸庆大霉素在患病鸡体内的峰浓度和药时曲线下面积低于健康鸡(P<0.05),因此对于已感染大肠杆菌的病鸡可以考虑适当增加给药剂量。     

Effects of a standardized purified dry extract from Echinacea angustifolia on proliferation and interferon gamma secretion of peripheral blood mononuclear cells in dairy heifers

This study was performed to ascertain whether a standardized extract from Echinacea angustifolia (Polinacea™) affects proliferation and interferon gamma (IFN-γ) secretion in bovine peripheral blood mononuclear cells (PBMC).PBMC from six Holstein heifers were incubated with 0, 6.3, 20, 60, or 180 μg/ml of the tested compound. Proliferation was stimulated by concanavalin A (ConA) or pokeweed-mitogen (PWM). Secretion of IFN-γ was stimulated by ConA.All concentrations of Polinacea™ exerted a mitogenic effect. With respect to control PBMC (0 μg/ml), the lowest and highest increase of proliferation were observed with Polinacea™ at 6.3 (2-fold increase) or 180 (10-fold increase) μg/ml, respectively. Polinacea™ at 180 μg/ml reduced ConA-driven proliferation, whereas at 20 and 60 μg/ml improved proliferation of PWM-stimulated PBMC. IFN-γ secretion was not affected. In conclusion, Polinacea™ modulates bovine PBMC proliferation, and deserves to be tested in vivo to define conditions that may benefit from its utilization.     

Optimum conditions for the chicken lymphocyte transformation test.

Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.     

Preliminary clinical pharmacological investigations of tylosin and tiamulin in chickens

Summary The minimal inhibitory concentrations (M1C) of tiamulin and tylosin for mycoplasma. Gram-positive, and Gram-negative micro-organisms isolated from chickens were determinated by the agar dilution method. Median M1C values for tiamulin against Mycoplasma gallisepticum (0.05 μg/ml) and Mycoplasma synoviae (0.10 μg/ml) were 2 to 4 times lower than the corresponding values for tylosin. Tiamulin was also slightly more effective in vitro in inhibiting Escherichia coli, Pasteurella multocida, and beta-haemolytic streptococci than was tylosin. Groups of chicken were offered tiamulin medicated drinking water at rates of 125 and 250 mg/litre for 48 hours. Average serum tiamulin concentrations were 0.38 and 0.78 μg/ml, respectively. When tylosin tartrate was added to the drinking water at 500 and 700 mg/litre, average serum drug levels were 0.12 and 0.17 μg/ml, respectively. Tiamulin was 45% bound in chicken serum, as against 30% serum protein binding or tylosin. Correlations were made between free (non protein bound) serum drug levels and the MIC values of the two drugs. Such comparisons suggest that when tiamulin is given in the drinking water at rates of 125 to 250 mg/litre, better antimycoplasmal activity is to be expected in vivo than by giving tylosin tartrate in the drinking water at 500 to 700 mg/litre. Based on these data, no clinical efficacy of these dose rates can be expected in flocks infected by gram-negative microorganisms such as E. coli or P. multocida. The tylosin tartrate rate of 500 to 700 mg/litre, may be clinical ineffective the treatment of Staphylococcus aureus infections.     

Feline-specific serum total IgE quantitation in normal, asthmatic and parasitized cats

Immunoglobulin E (IgE) plays an important role in defense against parasitic infections as well as allergy. Knowledge of serum total IgE concentrations may have value in diagnosis and prognostication of various disorders; however, to date, no studies have reported feline serum total IgE concentrations. We hypothesize that serum total IgE concentrations will be greater in spontaneously parasitized and asthmatic cats compared to healthy pet cats. Healthy (n=10), parasitized (10) and asthmatic cats (eight) had measurement of serum total IgE by ELISA. Data were analyzed using a t-test with P<0.05 considered significant. Serum total IgE was higher in parasitized (mean±SEM, 328.4±123.8μg/ml; P<0.028) and asthmatic cats (85.5±19.5μg/ml; P<0.047) compared to healthy cats (45.9±19.6μg/ml). However, serum total IgE had poor discriminatory capability between diseased and healthy cats. In conclusion, this assay can detect small quantities of feline serum total IgE, which may be beneficial in future studies of parasitism or allergic disease.     

高效液相色谱荧光检测法同时检测鸡肉中氟苯尼考及氟苯尼考胺残留

经丙酮、二氯甲烷提取,饱和正己烷脱脂,氮吹仪吹干浓缩后,以乙腈-磷酸二氢钠溶液(0.01 mol/L,含0.005mol/L十二烷基硫酸钠和0.1%三乙胺)(35 :65)为流动相,流速为1.0 mL/min,荧光检测激发波长为225 nm,发射波长285 nm.氟苯尼考在0.01～10.0 mg/L、氟苯尼考胺在0.002 5～2.5 mg/L浓度范围内,本方法线性关系良好,相关系数分别为0.999 7和0.999 8.当添加水平氟苯尼考为15～500 μg/kg、氟苯尼考胺为5～500μg/kg时.该方法平均回收率分别为79.5%～84.6%和80.7 0A～88.2%,相对标准偏差分别为2.8%～6.4%和2.4%～5.3%;检测限分别为5μg/kg和μg/kg.该方法样品处理简单,可同时检测氟苯尼考和氟苯尼考胺的残留,且准确度和精密度均符合残留分析的要求.     

Purification of cattle oviduct specific proteins and their effect on in vitro embryo development

The purpose of this study was to determine the effect of oviduct specific proteins as a media supplement for in vitro embryo development in cattle. The proteins were extracted from oviducts of cows and precipitated by ammonium sulfate (30%, 40%, 50% and 60%) followed by dialysis in 50 mM Tris–HCl (pH 7.0) buffer. The dialyzed proteins were fractionated into acidic, basic and neutral fractions using SP sephadex cation exchange and DEAE sephadex anion exchange column chromatography respectively. Cow oviduct specific proteins (cOSPs) constituting all the extracted proteins were used as media supplement in three different concentrations (10, 50 and 100 μg/ml) for in vitro maturation, fertilization and culture (IVMFC) of cow oocytes. Acidic, basic and neutral (unbound) fractions were also used as media supplement in three different concentrations (10, 30 and 50 μg/ml) for IVMFC. Cumulus oocytes complexes were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24 h in 5% CO₂ at 38.5 °C with maximum humidity. In vitro matured oocytes were co-incubated with in vitro capacitated sperm in Fert-BO media at 38.5 °C for 18 h in 5% CO₂. The fertilized oocytes were washed and cultured in embryo development media for cleavage. After 40–42 h cleavage was observed and embryos were put in the replacement media for further development. The cleavage rates (%) for cOSPs were observed as 68.24±2.46, 69.28±2.05, 61.77±0.93 and 42.62±1.31 at concentrations of 0, 10, 50 and 100 μg/ml respectively. Rates of blastocyst stage development were 14.49±3.61, 21.17±2.77, 14.66±1.06 and 11.98±1.84. These results indicate that addition of cOSP at10 μg/ml increased blastocyst formation as compared to other concentrations (0, 50 and 100 μg/ml). Although acidic, basic and neutral fractions seemed to have no major effect on cleavage rate, but both acidic and neutral fraction of oviduct specific proteins improved the cleavage rate at 30 μg/ml concentration and basic fraction improved the blastocyst formation at 10 μg/ml concentration.