河北省鸡柔嫩艾美耳球虫地方株的耐药性检测
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摘要
河北省鸡柔嫩艾美耳球虫地方株的生物学特性：运用单卵囊分离技术,从河北省主要养鸡地区秦皇岛、唐山、石家庄分离并纯化得到鸡柔嫩艾美耳球虫秦皇岛株、唐山株、石家庄株,观察和测定了柔嫩艾美耳球虫地方株的生物学特性,并与柔嫩艾美耳球虫敏感株和耐盐霉素株进行对比,结果表明：(1)各虫株卵囊均寄生于盲肠,卵囊呈卵圆形,壁光滑,分两层。(2)唐山株的卵囊潜隐期最长,为142h,,而秦皇岛株、石家庄株的潜隐期与敏感株、耐药株相似,分别为137h、133h、132h、135h；(3)秦皇岛株、唐山株、石家庄株、敏感株和耐药株的最短孢子化时间分别为19h、17h、20h、20h和18h。(4)唐山株、石家庄株、秦皇岛株和耐药株的卵囊大小平均为24.11μm×19.07μm、23.41μm×19.34μm、24.89μm×20.14μm和24.06μm×19.50μm,卵形指数基本一致,分别为1.26、1.21、1.24和1.23；而敏感株的卵囊大小平均为24.13μm×20.37μm,卵形指数最小,为1.18。(5)不同地方株OPG最高值出现在感染后的第7～8d,各虫株在感染后7～9d的排卵囊量占总量值相近,达75.62%～86.06%。本试验结果为进一步开展药物的疗效试验和鸡球虫耐药机理等研究奠定了基础。
河北省鸡柔嫩艾美耳球虫地方株耐药性的测定及耐药谱变迁分析：通过药敏试验,以最适抗球虫活性百分率(POAA)、病变记分减少率(RLS)、相对卵囊产量(ROP)、抗球虫指数(ACI)为指标,测定了秦皇岛株、唐山株、石家庄株柔嫩艾美耳球虫对克球粉、氨丙啉、马杜霉素、瑞冠球、尼卡巴嗪、金三特、球敌等常用抗球虫药的耐药性,结果表明,秦皇岛株柔嫩艾美耳球虫对球敌呈现完全耐药性,对克球粉、瑞冠球和金三特呈现中度耐药,对马杜霉素、尼卡巴嗪和氨丙啉则呈现轻度耐药。唐山株柔嫩艾美耳球虫对尼卡巴嗪和氨丙啉均表现无耐药性,而对马杜霉素、金三特和球敌均表现完全耐药性；对克球粉和瑞冠球则呈中度耐药性。石家庄株柔嫩艾美耳球虫对金三特和氨丙啉均表现无耐药性,对瑞冠球呈轻度耐药性,而对马杜霉素、克球粉和球敌均表现完全耐药性。同时,采用回顾性调查法,分析了2004～2008年分离的秦皇岛株柔嫩艾美耳球虫的耐药谱,结果提示该虫株对常用抗球虫药物的耐药谱增宽,耐药率呈递增趋势,对已经产生完全耐药的抗球虫药物如磺胺喹噁啉钠,在停用一段时间后,对该药物的敏感性得到了一定程度的恢复。该试验结果为当地鸡球虫病的合理用药和高效防治提供了可靠的依据。
河北省鸡嫩艾美耳球虫田间多重耐药虫株的耐药相关基因的筛选：以敏感株作对照,采用Trizol试剂法提取秦皇岛、唐山和石家庄柔嫩艾美耳球虫多重耐药虫株的总RNA。用3种锚定引物和20种随机引物进行RT-PCR,产物经变性聚丙烯酰胺凝胶电泳后银染,共回收26条差异条带,进行2次PCR扩增,产物回收纯化,回收12条差异条带,其浓度大约20-50ng。对12条质粒克隆鉴定的差异带进行反向Northern Dot-blotting点杂交鉴定3个片段为阳性,对阳性克隆测序、同源性比较。其中来自于石家庄田间多重耐药性虫株mRNA的S116序列与Genebank和Sanger上已经发表的柔嫩艾美耳球虫第一段染色体上882bp长的序列同源性高达99%,为未知蛋白；分别来自唐山和秦皇岛田间多重耐药性虫株mRNA的T311和Q19序列为未知新序列。这些耐药相关基因片段可能参与了球虫抗球虫药耐药性产生的过程。
Study on the Biological Characheristics of Field Strains of E.tenella in Hebei Province Using single-oocyst separation technology, three Eimeria tenella (E.tenella) field strains in Hebei province were isolated and identified as E.tenella Qinhuangdao strain, Tangshan strain, and Shijiazhuang strain, then their biological characheristics were detected and compared with E.tenella sensitive strain and resistat salinomycin strain. The results showed that (1) Oocysts of all strains parasitized in mecums,and were broadly ovoid with a smoth wall.(2)The prepatent period of Tangshan strain was the longest, being 142h, but the prepatent periods of Qinhuangdao strain and Shijiazhuang strain were similar to those of sensitive strain and resistant salinomycin strain,being 137h,133h,132h and 135h respectively.(3)The minimum sporulation time of Qinhuangdao strain,Tangshan strain,Shijiazhuang strain,sensitive strain and resistant salinomycin strain was 19h,17h,20h,20h and 18h, respectively.(4)The oocysts sizes of Tangshan strain,Shijiazhuang strain, Qinhuangdao strain and resistant salinomycin strain were 24.11μm×19.07μm,23.41μm×19.34μm, 24.89μm×20.14μm and 24.06μm×19.50μm respectively, their shape indexes were 1.26,1.21,1.24 and 1.23, but the oocyst size of sensitive strain was 24.13μm×20.37μm, its shape index was the smallest,being 1.18. (5) The perk production of oocysts of different strains was 7 to 8 days after infection, and its ration of yield of oocysts to total yield from 7 to 9 day after infection was 75.62% to 86.06%.The results provided a basis for drug control and study on drug-resistant mechanism.
Detection of Drug Resistance to the Field Strains of E.tenella and Analysis on Change of Drug-Resistant Spectrum in Hebei Province Based on the criteria of the percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS),relative oocyst production (ROP) and anticoccidial index (ACI), the drug resistances of E.tenella Qinhuangdao strain, Tanshan strain and Shijiazhuang strain in Hebei provoince to common used anticoccidial drugs such as keqiufen, amprolium, maduramicin, ruiguanqiu, nicarbazin, jinsante and qiudi were detected by drug sensitive test. The results revealed that Qinhuangdao strain was complete resistant to qiudi, middle resistant to keqiufen, ruiguanqiu and jinsante, but light resistant to maduramicin, nicarbazin and amprolium. Tanshan strain was no resistant to nicarbazin and amprolium, middle resistant to keqiufen and ruiguanqiu, but complete resistant to maduramicin, jinsante and qiudi. Shijiazhuang strain was no resistant to jinsante and amprolium, light resistant to ruiguanqiu, but complete resistant to maduramicin, keqiufen and qiudi. At the same time the change on the drug-resistant spectrums of E. tenella Qinhuangdao strain isolated from 2004 to 2008 year was analysed by the method of review investigation. The results revealed that resistant spectrum of E. tenella to common anticoccidial drugs was broad, resistant rate was increasing in recent years, and the sensitive was recoved in certain degree after anticoccidial drugs were stoped using for some times. The results provided a reliable basis on effectivelly controlling chicken coccidosis.
Isolation on the Related Genes of Drug Resistance of Field Strains of E.tenella in Hebei Province The total RNA of E.tenella sensitive strain and drug-resistant strains from Qinhuangdao,Tangshan and Shijiazhuang were extracted with Trizol. DDRT-PCR of three strains was established by using 3 anchored primers and 20 arbitrary primers. Their products of PCR were analysed on the denaturing polyacrylamide gels by silver-staining.Twenty-six differential fragments were excised from the gels and reamplified with the same sets of primers. Then the PCR products were purified and extracted 12 differential expression fragments with about 20 to 50ng in concentration. Twelve differential expression fragments were cloned with PMD-18T vector and identifed by reverse Northen dot hybridization, so three positive gene fragments related to drug resistance of E.tenella field resistant strains were isolated. After sequence analysis with sequence in Genebank and Sanger, the sequence S116 from mRNA of Shijiazhuang drug-resistant strain was similar to the sequence of first chromosome with 882bp in length.The similarity was 99% and it was an unknown protein,but the sequences T311 and Q19 from mRNA of Tangshan and Qinhuang drug-resistant strains were respectively unknown. These drug-resistant relative genes maybe be involved in the development of drug resistance.
河北省鸡柔嫩艾美耳球虫地方株耐药性的测定及耐药谱变迁分析：通过药敏试验,以最适抗球虫活性百分率(POAA)、病变记分减少率(RLS)、相对卵囊产量(ROP)、抗球虫指数(ACI)为指标,测定了秦皇岛株、唐山株、石家庄株柔嫩艾美耳球虫对克球粉、氨丙啉、马杜霉素、瑞冠球、尼卡巴嗪、金三特、球敌等常用抗球虫药的耐药性,结果表明,秦皇岛株柔嫩艾美耳球虫对球敌呈现完全耐药性,对克球粉、瑞冠球和金三特呈现中度耐药,对马杜霉素、尼卡巴嗪和氨丙啉则呈现轻度耐药。唐山株柔嫩艾美耳球虫对尼卡巴嗪和氨丙啉均表现无耐药性,而对马杜霉素、金三特和球敌均表现完全耐药性；对克球粉和瑞冠球则呈中度耐药性。石家庄株柔嫩艾美耳球虫对金三特和氨丙啉均表现无耐药性,对瑞冠球呈轻度耐药性,而对马杜霉素、克球粉和球敌均表现完全耐药性。同时,采用回顾性调查法,分析了2004～2008年分离的秦皇岛株柔嫩艾美耳球虫的耐药谱,结果提示该虫株对常用抗球虫药物的耐药谱增宽,耐药率呈递增趋势,对已经产生完全耐药的抗球虫药物如磺胺喹噁啉钠,在停用一段时间后,对该药物的敏感性得到了一定程度的恢复。该试验结果为当地鸡球虫病的合理用药和高效防治提供了可靠的依据。
河北省鸡嫩艾美耳球虫田间多重耐药虫株的耐药相关基因的筛选：以敏感株作对照,采用Trizol试剂法提取秦皇岛、唐山和石家庄柔嫩艾美耳球虫多重耐药虫株的总RNA。用3种锚定引物和20种随机引物进行RT-PCR,产物经变性聚丙烯酰胺凝胶电泳后银染,共回收26条差异条带,进行2次PCR扩增,产物回收纯化,回收12条差异条带,其浓度大约20-50ng。对12条质粒克隆鉴定的差异带进行反向Northern Dot-blotting点杂交鉴定3个片段为阳性,对阳性克隆测序、同源性比较。其中来自于石家庄田间多重耐药性虫株mRNA的S116序列与Genebank和Sanger上已经发表的柔嫩艾美耳球虫第一段染色体上882bp长的序列同源性高达99%,为未知蛋白；分别来自唐山和秦皇岛田间多重耐药性虫株mRNA的T311和Q19序列为未知新序列。这些耐药相关基因片段可能参与了球虫抗球虫药耐药性产生的过程。
Study on the Biological Characheristics of Field Strains of E.tenella in Hebei Province Using single-oocyst separation technology, three Eimeria tenella (E.tenella) field strains in Hebei province were isolated and identified as E.tenella Qinhuangdao strain, Tangshan strain, and Shijiazhuang strain, then their biological characheristics were detected and compared with E.tenella sensitive strain and resistat salinomycin strain. The results showed that (1) Oocysts of all strains parasitized in mecums,and were broadly ovoid with a smoth wall.(2)The prepatent period of Tangshan strain was the longest, being 142h, but the prepatent periods of Qinhuangdao strain and Shijiazhuang strain were similar to those of sensitive strain and resistant salinomycin strain,being 137h,133h,132h and 135h respectively.(3)The minimum sporulation time of Qinhuangdao strain,Tangshan strain,Shijiazhuang strain,sensitive strain and resistant salinomycin strain was 19h,17h,20h,20h and 18h, respectively.(4)The oocysts sizes of Tangshan strain,Shijiazhuang strain, Qinhuangdao strain and resistant salinomycin strain were 24.11μm×19.07μm,23.41μm×19.34μm, 24.89μm×20.14μm and 24.06μm×19.50μm respectively, their shape indexes were 1.26,1.21,1.24 and 1.23, but the oocyst size of sensitive strain was 24.13μm×20.37μm, its shape index was the smallest,being 1.18. (5) The perk production of oocysts of different strains was 7 to 8 days after infection, and its ration of yield of oocysts to total yield from 7 to 9 day after infection was 75.62% to 86.06%.The results provided a basis for drug control and study on drug-resistant mechanism.
Detection of Drug Resistance to the Field Strains of E.tenella and Analysis on Change of Drug-Resistant Spectrum in Hebei Province Based on the criteria of the percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS),relative oocyst production (ROP) and anticoccidial index (ACI), the drug resistances of E.tenella Qinhuangdao strain, Tanshan strain and Shijiazhuang strain in Hebei provoince to common used anticoccidial drugs such as keqiufen, amprolium, maduramicin, ruiguanqiu, nicarbazin, jinsante and qiudi were detected by drug sensitive test. The results revealed that Qinhuangdao strain was complete resistant to qiudi, middle resistant to keqiufen, ruiguanqiu and jinsante, but light resistant to maduramicin, nicarbazin and amprolium. Tanshan strain was no resistant to nicarbazin and amprolium, middle resistant to keqiufen and ruiguanqiu, but complete resistant to maduramicin, jinsante and qiudi. Shijiazhuang strain was no resistant to jinsante and amprolium, light resistant to ruiguanqiu, but complete resistant to maduramicin, keqiufen and qiudi. At the same time the change on the drug-resistant spectrums of E. tenella Qinhuangdao strain isolated from 2004 to 2008 year was analysed by the method of review investigation. The results revealed that resistant spectrum of E. tenella to common anticoccidial drugs was broad, resistant rate was increasing in recent years, and the sensitive was recoved in certain degree after anticoccidial drugs were stoped using for some times. The results provided a reliable basis on effectivelly controlling chicken coccidosis.
Isolation on the Related Genes of Drug Resistance of Field Strains of E.tenella in Hebei Province The total RNA of E.tenella sensitive strain and drug-resistant strains from Qinhuangdao,Tangshan and Shijiazhuang were extracted with Trizol. DDRT-PCR of three strains was established by using 3 anchored primers and 20 arbitrary primers. Their products of PCR were analysed on the denaturing polyacrylamide gels by silver-staining.Twenty-six differential fragments were excised from the gels and reamplified with the same sets of primers. Then the PCR products were purified and extracted 12 differential expression fragments with about 20 to 50ng in concentration. Twelve differential expression fragments were cloned with PMD-18T vector and identifed by reverse Northen dot hybridization, so three positive gene fragments related to drug resistance of E.tenella field resistant strains were isolated. After sequence analysis with sequence in Genebank and Sanger, the sequence S116 from mRNA of Shijiazhuang drug-resistant strain was similar to the sequence of first chromosome with 882bp in length.The similarity was 99% and it was an unknown protein,but the sequences T311 and Q19 from mRNA of Tangshan and Qinhuang drug-resistant strains were respectively unknown. These drug-resistant relative genes maybe be involved in the development of drug resistance.
引文
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