柔嫩艾美耳球虫对四种抗球虫药的交叉抗药性试验

为了测试柔嫩艾美耳球虫的４种抗药虫株（抗地克珠利、抗拉沙里菌素、抗盐霉素和抗克球粉虫株）对４种抗球虫药（地克珠利、拉沙里菌素、盐霉素和克球粉）的敏感性进行了本试验。结果表明：柔嫩艾美耳球虫地克珠利抗药虫株对拉沙里菌素、盐霉素和克球粉均表现敏感,拉沙里菌素抗药虫株、盐霉素抗药虫株和克球粉抗药虫株对地克珠利也表现为高度敏感,因此,柔嫩艾美耳球虫对地克珠利与拉沙里菌素、地克珠利与盐霉素、地克珠利与克球粉３组药物不存在交叉抗药性。     

广东省鸡球虫的抗药性研究进展

文章分析了广东省不同地区分离的柔嫩艾美耳球虫（Eimeria tenella) 对球净，地克珠利，氯羟吡啶，球痢灵，氯苯胍和氨丙啉等6种化学合成抗球虫药和马杜拉霉素，盐霉素，拉沙霉素和森杜拉霉素等4种抗生素类抗球虫药的抗药性的现状，80％以上虫株仅对1种或2种药物敏感；各虫株至少对2种药物抗药，且80％以上虫株同时对4种或4种以上药物抗药。E.tenella野外分离株对抗生素类抗球虫药的抗药性要比对化学合成类药物的抗药性相对严重得多，部分虫株对早期上市的一些化学药物如氯苯胍，球痢灵等敏感，说明如果基于药物敏感性试验，有限制地使用这些药物会取得较好效果，在此基础上，重点对我省球虫病的药物防制对策进行了讨论并提出了合理用药原则和方案。     

四种鸡艾美耳球虫实验室保存株的PCR鉴定

以4种鸡球虫实验室保存株(毒害、巨型、柔嫩、堆型艾美耳球虫)的基因组DNA为模板,分别用针对4种球虫的ITS-1序列设计的种特异性引物进行PCR扩增,扩增产物进行琼脂糖凝胶电泳分析,观察特异性条带。结果显示,只有在模板和扩增引物为同一种球虫时,方能扩增出特异性条带。表明本实验室保存的4种鸡球虫均为纯种株,建立的PCR检测方法可以用于鸡球虫纯种的鉴定。     

球虫抗药性分子生物学检测技术的建立

选择在敏感虫株中沉默、抗药虫株中表达的特异序列AGD5设计检测引物，采用RT—PCR方法。以总RNA反转录的cDNA为模板，RT—PCR方法能够鉴别马杜霉素抗药虫株和3株马杜霉素敏感虫株（2株对所有药物敏感的不同地理株，1株地克珠利抗药性虫株对马杜霉素敏感），此鉴定结果与鸡体试验的结果一致，说明敏感虫株和抗药虫株在该序列上的差异发生在转录水平。而以卵囊总DNA为模板的PCR方法不能鉴别敏感和马杜霉素抗药性虫株。     

柔嫩艾美耳球虫交叉抗药性试验

以雏鸡为试验对象,以抗球虫指数(ACI)为判断指标,检测5 mg/kg马杜霉素,70 mg/kg盐霉素,1 mg/kg 地克珠利对抗盐霉素株、抗地克珠利株和实验室保存的敏感株的控制效果。结果表明,马杜霉素对3 个虫株都有很好的抑杀效果,地克珠利也可有效地控制抗盐霉素株,同时,盐霉素也可有效地控制抗地克珠利株,说明盐霉素和地克珠利2种药物之间没有交叉抗药性。     

柔嫩艾美耳球虫拉沙里菌素抗性虫株的实验室诱导

采用实验室建立抗性虫株的方法,用４０、６０、７５、１０５、１２５、１３５μｇ／ｇ梯度的拉沙里菌素作用于柔嫩艾美耳球虫盐霉素敏感株和盐霉素抗性株,经过１５次继代,诱导产生了对１３５μｇ／ｇ拉沙里菌素的抗药性虫株。在同一诱导条件下,盐霉素敏感株和盐霉素抗性株对拉沙里菌素产生抗药性的时值和强度没有差异。     

银染mRNA差异显示法克隆柔嫩艾美耳球虫(Eimeria tenella)抗药性相关基因

以柔嫩艾美耳球虫（Eimeria tenella）敏感株、地克珠利抗药株和马杜拉霉素抗药株孢子化卵囊为材料，用银染mRNA差异显示方法筛选和克隆与球虫抗药性相关的基因。首先提取这3个虫株孢子化卵囊的总RNA为模板．用Oligo（dT）12GG为锚定引物和2个10碱基随机引物组合进行RTPCR，产物经变性聚丙烯酰胺凝胶电泳后银染。分别切取5务差异条带，进行2次PCR扩增，产物回收后与PGEM—T—easy Vector连接转化。经PCR鉴定正确后，再进行斑点杂交试验、序列分析和同源性比较。结果发现，地克珠利抗药株和马杜拉霉素抗药株分离的差异片段中都有2个cDNA片段（可能为新的基因片段），这为克隆全长cDNA和探索球虫抗药性产生的分子机理奠定了一定的基础。     

柔嫩艾美耳球虫2个抗药株的抗药性评估

为检测实验室长期保存的柔嫩艾美耳球虫马杜拉霉素抗药株和地克珠利抗药株的遗传稳定性,采用鸡体试验法,以抗球虫指数、最适抗球虫活性百分率、病变记分减少率和相对卵囊产量4项指标,评价2个抗药株对地克珠利、癸氧喹酯、马杜拉霉素等3种抗球虫药物的抗药性,并以柔嫩艾美耳球虫敏感株为对照。结果显示,柔嫩艾美耳球虫敏感株对3种抗球虫药物均无抗药性,4项指标全部阴性。柔嫩艾美耳球虫马杜拉霉素抗药株对马杜拉霉素为完全耐药,4项指标全部阳性;对地克珠利、癸氧喹酯则表现为无抗药性,4项指标全部阴性。柔嫩艾美耳球虫地克珠利抗药株对地克珠利表现为完全耐药,4项指标全部阳性;对马杜拉霉素表现为中度耐药,2项指标阳性;对癸氧喹酯则表现为无抗药性,4项指标全部阴性。结果表明,在实验室已繁殖保存10余年的柔嫩艾美耳球虫2个抗药虫株对其诱导药物具有明显抗药性,实验室保种繁殖方法可行,虫株遗传稳定性良好,为利用该虫株进行球虫抗药性机制等相关研究奠定了基础。     

柔嫩艾美耳球虫扬州分离株对8种抗球虫药的抗药性

将柔嫩艾美耳球虫(Eimeria tenella)扬州分离株感染14日龄苏禽黄羽肉鸡,设立8个用药组、1个感染不用药对照组和1个不感染不用药对照组,以相对卵囊产量、病变记分减少率、最适抗球虫百分数和抗球虫指数为指标对该虫株对盐霉素、马杜拉霉素、球痢灵、氯羟吡啶、氨丙啉、尼卡巴嗪、甲基三嗪酮、地克珠利等8种常用抗球虫药的抗药性进行了综合评定,分析了该地区球虫的抗药性及对策。结果表明,该虫株对甲基三嗪酮敏感,对氨丙啉具有中度抗药性,对盐霉素、马杜拉霉素、球痢灵、氯羟吡啶、尼卡巴嗪、地克珠利等其他6种药物具有完全抗药性。提示目前在扬州地区甲基三嗪酮抗球虫疗效最好,氨丙琳效果稍差,应合理使用,盐霉素等其他6种药物应慎用、减少或暂停使用。     

柔嫩艾美耳球虫野外抗药性虫株的RAPD分析

用RAPD方法对柔嫩艾美耳球虫（Eimeria tenella）的8个分别来源于黑龙江、北京、四川、甘肃和广东的中山、新会、东莞以及广州近郊的江村镇的野外分离虫株和2个药物敏感性虫株进行了遗传多态性分析。同时用9种抗球虫药物即马杜霉素（5mg/kg）、盐霉素（60mg/kg）、莫能霉素（120mg/kg）、拉沙菌素（90mg/kg）、克球多（150mg/kg）、常山酮（3mg/kg）、氯氢苯乙氰（1mg/kg）、尼卡巴嗪（125mg/kg）和球痢灵（125mg/kg）分别对8个野不进行抗药性试验，8个虫株对上述药物均有不同程度的抗药性。RAPD分析表明：10个样品都有相似而清晰的主带，每个泳道有5－13条带不等，大小为0.18-2.10kb。它们之间的相似率（SI）大于40.58%，最大的为90.72%，这种相似率属种内变异水平。根据SI值可把10个样品划分为3个类群：广东虫株类群，群内比较，平均SI值为76.60%；广东省外虫株类群，群内比较的平均SI值72.14%；敏感株类群，其间的SI值为89.27%。在另一方面，广东株与广东省外株、敏感株之间比较，其SI的平均值分别仅为63.03%和47.25%，说明柔嫩艾美耳球虫抗药性虫株之间有遗传差异法。     

柔嫩艾美耳球虫耐药株与鸡3种球虫的同工酶的研究

对不同离子载体抗生素具有抗药性的柔嫩艾美耳球虫和药物敏感的柔嫩区美耳球虫、布氏匀美耳球虫和堆型艾耳球虫的孢子化卵囊,采用聚安凝胶垂直板电泳,进行了乳酸脱氢酶（ＬＤＨ）、葡萄糖磷酸异构酶（ＧＰＩ）和葡萄糖－６－磷酸脱氢酶（Ｇ６ＰＤ）等同工酶华不同离子载体抗生素具有抗药性的柔嫩艾美耳球虫和药物同工酶酶谱可以反应出球虫种间差异;而柔嫩艾耳球虫抗性虫株的ＬＤＨ酶谱是２条带,敏感虫株是３条带,抗性株比敏感株     

Complementary randomly amplified polymorphic DNA (RAPD) analysis patterns and primer sets to differentiate Mycoplasma gallisepticum strains.

Randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate 7 strains of Mycoplasma gallisepticum. Six commercially available primers or primer combinations were screened for their ability to differentiate vaccine and type strains. Although major and minor bands were produced with each primer, many of the primers were unsuitable for strain differentiation. The use of primer 6 and combined primers 3 and 4 resulted in complementary RAPD banding patterns for each M. gallisepticum strain. Eleven different isolates representing 7 different strains were segregated into 7 different patterns, corresponding to the 7 strains.     

Comparison of Mycobacterium avium complex (MAC) strains from pigs and humans in Sweden by random amplified polymorphic DNA (RAPD) using standardized reagents

Infections with atypical mycobacteria belonging to the Mycobacterium avium/intracellulare complex (MAC) can cause infection in both animals and humans. Using a standardized reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis, 49 MAC strains isolated from 32 slaughter pigs and 17 humans in Sweden were identified and sorted out, yielding 6 RAPD types. By combining the results of RAPD primers 4 and 5 and the primer IS1245A, we found that pigs and humans may be infected with the same types of MAC strains, since 14 strains from humans and 8 strains from pigs were essentially identical and together, comprised RAPD type 2, the largest group of strains (44.8% of strains). With respect to grouping of strains, serotype and RAPD type were uncorrelated, except for serotype 20 and RAPD type 6. Using standardized beads, RAPD analysis is a reproducible technique for typing MAC strains, as the indistinguishable banding patterns obtained with repeated analyses of two isolates from each strain in this study demonstrate. However, primer selection and DNA purity were crucial for differentiating closely related strains.     

柔嫩艾美耳球虫盐霉素耐药株的实验室诱导

为了研究耐药性柔嫩艾美耳球虫产生耐药性的机理,本研究在实验室条件下,采用药物浓度递增的方法,以15 mg/kg为起始诱导浓度,对柔嫩艾美耳球虫盐霉素耐药性进行诱导。经过10次传代,获得了对60 mg/kg盐霉素耐药的柔嫩艾美耳球虫虫株。以抗球虫指数、最适抗球虫活性百分率、病变记分减少率和相对卵囊产量四项指标综合判定柔嫩艾美耳球虫对盐霉素的耐药性。其中抗球虫指数为156.92,最适抗球虫活性百分率为32.38,病变记分减少率为53.85,相对卵囊产量为28.17。结果显示,成功获得了柔嫩艾美耳球虫对盐霉素完全耐药的虫株,为进一步从分子水平研究球虫抗药性奠定基础。     

Genomic fingerprinting of pigeon Streptococcus gallolyticus strains of different virulence by randomly amplified polymorphic DNA (RAPD) analysis

Randomly amplified polymorphic DNA (RAPD) analysis was performed on 95 pigeon S. gallolyticus strains of different virulence and belonging to different biotypes and different culture supernatant phenotypes as determined by SDS-PAGE. Four distinct RAPD patterns, designated A, B, C and D, were distinguished using primer OPM6 (5'CTGGGCAACT). All 76 strains generating RAPD pattern A or B were designated highly virulent on the basis of their SDS-PAGE pattern. Five of seven strains generating RAPD pattern C and 11 of 12 strains generating RAPD pattern D belonged to the moderately virulent and low virulent culture supernatant phenotype groups, respectively. Only one RAPD group C strain belonged to a highly virulent culture supernatant phenotype group. There was a correlation between biotype and RAPD patterns. These findings indicate that there is a high correlation between RAPD pattern and virulence for pigeons. Therefore, RAPD typing seems a rapid, reliable method to distinguish pigeon S. gallolyticus strains of high, moderate and low virulence.     

中、西蜂纯种及其不同品系蜂的基因组DNA多态性分析

使用P8(5‘-CCCACCCTTG-3‘)和W2（5‘-CGGCCCCGGT-3‘)随机引物，通过PRAPD（Random Amplified Polymorphic DNA)-PCR(polymerase chain reaction)遗传学分析，分别对中、西纯种蜂的不同品系的基因组DNA进行多态性分析。结果：中、西纯种蜂的不同品系的DNA多态4性图谱均具有明显的差异，从而为中、西纯种蜂的品系之间的鉴别提供了科学依据。     

Randomly amplified polymorphic DNA (RAPD) analysis of Mycoplasma gallisepticum isolates from turkeys from the central valley of California.

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the molecular epidemiology of 26 Mycoplasma gallisepticum (MG) isolates obtained from turkeys located in the central valley of California. The MG isolates were recovered from 5 different companies and 13 ranches. Each company had unique MG strains. No evidence of spread of MG between companies was detected. RAPD analysis of MG isolates within a ranch during an outbreak revealed only a single strain involved in each outbreak. RAPD analysis identified an isolate from 1 ranch with a banding pattern identical to that of the 6/85 vaccine strain, which had been used on that particular ranch. Similar RAPD banding patterns of isolates from different ranches within the same company suggested horizontal spread of MG between ranches. The use of 2 primer sets in RAPD analysis was critical to prevent misinterpretation of relationships between different isolates.     

利用单孢子囊接种技术建立鸡柔嫩艾美耳球虫克隆

利用显微操作从柔嫩艾美耳球虫长春株、河北株、山东株(由单卵囊接种建立)挑选出单孢子囊接种于雏鸡,接种成功率为17.8%。传代后提取DNA,用7条随机引物PCR扩增,鉴别RAPD图谱及特异条带,结果获得柔嫩艾美耳球虫长春株2个克隆、河北株2个克隆、山东株1个克隆。