泰地罗新注射液体外抑菌试验及对感染副猪嗜血杆菌仔猪的保护作用

【目的】开展了泰地罗新注射液的体外抗菌活性及对仔猪感染副猪嗜血杆菌的保护作用研究。【方法】体外抑菌活性中,采用试管二倍稀释法,以泰拉霉素为对照,测定泰地罗新对副猪嗜血杆菌、胸膜肺炎放线杆菌、多杀性巴氏杆菌、支气管败血性波氏杆菌的最小抑菌浓度（MIC）;在对仔猪感染副猪嗜血杆菌的保护作用中,选择菌量为1×10 ¹⁰ CFU·mL ^-1的副猪嗜血杆菌细菌悬液0.5 mL·kg ^-1bw作为攻毒剂量,进行腹腔注射,复制病理模型。将泰地罗新以2、4、8 mg·kg ^-1bw 3个剂量,泰拉霉2.5 mg·kg ^-1bw单次肌肉注射,治疗人工感染副猪嗜血杆菌的仔猪;在病死猪病料病原菌分离与鉴定中,挑取分离纯化后的副猪嗜血杆菌菌落进行基因组DNA提取,针对副猪嗜血杆菌16sRNA序列设计1对特异性引物,PCR扩增后检验目的DNA片段碱基序列长度。 【结果】体外抑菌结果显示,泰地罗新注射液对副猪嗜血杆菌、支气管败血性波氏杆菌、胸膜肺炎放线杆菌及多杀性巴氏杆菌的MIC分别为0.06—8.00、0.06—8.00、0.25—1.00、2.00—32.00 μg·mL ^-1,MIC₅₀分别为0.5、2.0、4.0、0.5 μg·mL ^-1,MIC₉₀为2、8、16、1 μg·mL ^-1,结果表明,泰地罗新注射液对副猪嗜血杆菌、支气管败血性波氏杆菌抑菌效果较强,对胸膜肺炎放线杆菌、多杀性巴氏杆菌抑菌效果较弱;DNA目标片段PCR扩增结果显示,PCR扩增目标片段长度均与文献报道中预测条带一致,特异性片段长度为820bp左右,结果表明,病死猪只均死于副猪嗜血杆菌感染;体内治疗试验结果显示,泰地罗新注射液低、中、高剂量组增重分别为（2.5±0.2）、（2.9±0.2）、（2.9±0.3）kg,死亡率分别为40%、0、0,有效率分别为40%、100%、100%,治愈率分别为40%、100%、100%;泰拉霉素注射液组增重为（3.0±0.2）kg,死亡率为10%,有效率为90%,治愈率为80%;感染不给药组增重为（2.1±0.1）kg,死亡率为70%。结果表明,泰地罗新注射液高、中剂量组与泰拉霉素对照药物组无显著性差异（P>0.05）,均能迅速的减轻临床症状,具有显著的治疗效果。泰地罗新低剂量组治疗效果与感染不给药组相当,无显著性差异（P>0.05）。 【结论】泰地罗新注射液按4 mg·kg ^-1bw临床推荐给药剂量,可有效治疗由副猪嗜血杆菌感染引起的猪呼吸道疾病。     

应用抑制性差减杂交技术构建副猪嗜血杆菌基因组差减文库

为构建副猪嗜血杆菌(Haemophilus parasuis,HPS)有毒力菌株SW124(血清4型)和无毒力菌株H465(血清11型)间的差异表达基因文库,采用抑制性差减杂交技术(Suppression subtractive hybridization,SSH)分析SW124和H465菌株细菌基因组的表达差异,并进行两轮差减杂交和两次PCR扩增,将第2次PCR产物与pMD19-T载体相连,转化大肠杆菌E.coli DH5α感受态细胞并进行文库扩增和蓝白斑筛选,RT-PCR鉴定差异表达文库。结果共获得327个阳性克隆,筛选100个克隆制备质粒进行PCR检测,均扩增出100～2 000 bp大小的片段。差异DNA差减文库的构建为进一步筛选、克隆HPS特异的未知新基因、建立分子鉴别新体系奠定了基础。     

Drug Resistance and Evolution of Haemophilus parasuis Isolates from Guizhou Province During 2013 to 2019

This study was aimed to reveal the trend of drug resistance about Haemophilus parasuis (Hps) in Guizhou province and guide clinical use of Glasser's disease then slow the emergence of drug-resistant Hps strains.K-B slip-agar diffusion method was used to test the 15 common antibiotics resistance of 517 Hps strains isolated from 9 districts in Guizhou province from 2013 to 2019.The drug resistance differences of strains isolated from different time and districts were analyzed.The results showed that 14.36% to 70.58% isolates indicated drug resistance to 15 antibiotics,the proportion of EFT resistant strains was the lowest and SXT resistant strains was the highest.Compared with 2013,the resistance rate of Hps strains isolated in 2019 to 13 antibiotics increased by 4.52% to 324.68%.Among them,the proportion of drug-resistant strains of 13 drugs increased which EFT resistant strains increased at the fastest rate to 324.68% while STR resistant strains slowest rate to only 4.52%.The rate of TET resistant strains showed negative growth which decreased by 2.21% and 8.63%,respectively.Compared with Kaili,Bijie and Xingyi of Guizhou,the strains isolated from Anshun,Zunyi and Guiyang districts showed higher drug resistance rate of EFT,MC and DO which with low resistance in Guizhou province,but lower of SXT,AML and STR which with high resistance in Guizhou province.The results indicated that the resistance of Hps strains to 15 commonly antibiotics in Guizhou province was serious with rising tendency and the resistance rate of certain strains showed obvious differences between different districts.     

Preparation and use of an autogenous bacterin against infectious coryza in chickens

An experiment was conducted to determine the efficacy of an autogenous bacterin against infectious coryza from a local strain of Haemophilus paragallinarum in Morocco compared with a commercial vaccine. Hens were vaccinated with a single dose or two doses of the bacterin at 15 and 18 weeks of age. Both the autogenous and the commercial vaccine conferred significant protection against experimental challenge (94% and 88%, respectively). A single dose was less protective with both vaccines.     

Identification and Analysis of Serotype and Genotype of 89 Clinical Isolates of Haemophilus parasuis

In this study, we aimed to determine the tissue distribution of clinical isolates of Haemophilus parasuis (HPS) to explore the epidemic distribution and correlation of its serotypes and genotypes, and to provide scientific basis for the effective prevention and control of Glässer's disease. According to the 89 strains of HPS isolated from the clinic, the serotypes of HPS were identified by PCR, and the number of HPS strains in different parts of the isolates was counted. Sequence type (ST) identification analysis, site polymorphism analysis, BURST cluster analysis and UPGMA phylogenetic tree cluster analysis were performed by using the multilocus sequence typing method. Nine serotypes (1, 2, 4, 5, 7, 11, 12, 13, and 14) and undetermined serotypes (NT) were identified from 89 HPS clinical isolates. Serotypes 4,13,7 and 5 were the predominant serotypes, accounting for 28.09%, 22.47%, 13.48% and 10.11%, respectively, and sixty-four strains of HPS were isolated from the lung tissues. ST267, ST268, ST387 and ST365 were dominant genotypes, accounting for 26.97%, 21.35%, 8.99% and 5.62%, respectively, there were 3-13 alleles at each locus, and the polymorphisms ranged from 3(g3pd) to 71(6pgd). The BURST analysis showed that the 89 HPS were divided into 2 single ST types and 11 clonal groups (CC). The phylogenetic tree of UPGMA had 4 branches, the dominant ST type was found in 2, 3 and 4 branches, which was corresponded to the HPS isolates with virulent serotype. The epidemic serotypes and genotypes of HPS are diversified and have obvious genetic heterogeneity. At the same time, ST types and serotypes have some crossover and are related to HPS clinical pathogenicity.     

Establishment, validation and use of the Kielstein-Rapp-Gabrielson serotyping scheme for Haemophilus parasuis

OBJECTIVES: To produce antisera to the 15 recognised reference strains of the Kielstein-Rapp-Gabrielson (KRG) serotyping scheme for Haemophilus parasuis, validate those sera and use them to serotype 46 Australian field isolates of H parasuis. DESIGN: Antisera were produced in rabbits and validated by cross-testing with the reference strains and re-testing 15 Australian field isolates of H parasuis that had been previously serotyped in the United States of America. The validated antisera were then used to determine the serovar of 46 Australian isolates. RESULTS: Monospecific antisera were produced for 14 of the 15 KRG serovars of H parasuis. Two Australian field isolates, confirmed previously as serovars 1 and 7, were used to produce monospecific antisera for serovars 1 and 7 respectively. The antiserum for serovar 4 gave a one-way cross reaction with the antigen of serovar 14. The typing antisera correctly typed all 15 H parasuis that had been previously typed by antisera produced overseas. The 46 field isolates were shown to belong to serovars 2 (two isolates), 4 (one isolate), 5 (18 isolates), 12 (two isolates) and 13 (four isolates). The remaining 19 isolates were non-typable. CONCLUSION: Serotyping of H parasuis isolates is now available in Australia. H parasuis serovars 5 and 13 remain the predominant serovars present in Australian pigs.     

2013－2019年贵州省副猪嗜血杆菌分离株耐药性及其演变

试验旨在揭示贵州省副猪嗜血杆菌（Haemophilus parasuis，Hps）的耐药性及其变化趋势，指导Glasser's病临床防控用药，减缓Hps耐药菌株出现。通过K-B纸片琼脂法，检测2013－2019年分离自贵州省9个地区517株Hps对15种常用抗菌药的耐药性，分析不同分离时期和分离地区的菌株的耐药性差异。结果显示，针对15种抗生素分别有14.36%~70.58%的分离菌株表现耐药，其中耐头孢噻呋菌株的比例最低，耐复方新诺明菌株的比例最高；与2013年相比，2019年分离的Hps菌株对13种抗生素的耐药率分别升高了4.52%~324.68%，其中耐头孢噻呋菌株比例上升速度最快，达324.68%，耐链霉素菌株比例上升最慢，仅4.52%，耐土霉素和四环素菌株的比例分别下降2.21%和8.63%，出现负增长。相对于凯里、毕节和兴义地区，安顺、遵义、贵阳地区流行的菌株对头孢噻呋、替米考星、多西环素等在全省范围内耐药菌株比例较低的药物耐药率相对较高，而对复方新诺明、阿莫西林、链霉素等在全省范围内耐药菌株比例较高的药物耐药率相对较低。这一结果说明，贵州省Hps菌株对15种常用药物的耐药性较为严重，且总体呈现上升趋势，不同地区分离株对部分抗菌药的耐药率呈现明显的地区差异性。     

副猪嗜血杆菌的分离鉴定与药敏试验研究

副猪嗜血杆菌病是一种猪的传染病,主要引起断奶仔猪和保育猪发病。试验对天津某规模化猪场疑似副猪嗜血杆菌病猪采集病变明显的组织进行细菌分离培养、显微镜镜检、生化试验等实验室诊断,结合病猪的临床症状和病理变化特点,判定该细菌分离株为副猪嗜血杆菌。药敏试验结果显示,该株副猪嗜血杆菌对头孢噻呋、阿米卡星等药物高度敏感。     

副猪嗜血杆菌Apd-ELISA抗体检测试剂盒的制备和初步应用

旨在对副猪嗜血杆菌（Haemophilus parasuis,HPS）的黏附素蛋白（autotransporter passenger domain,Apd）ELISA抗体检测方法进行参数优化、临界值确定和应用评价,获得性能稳定的副猪嗜血杆菌病抗体检测试剂盒。首先通过猪的免疫攻毒试验获得HPS阴、阳性对照血清,优化Apd-ELISA的反应参数;然后用背景确认的阴、阳性血清确定其临界值,并对封闭液及酶标板的包装方式进行选择;最后用Apd-ELISA试剂盒对临床样品进行检测并与荷兰Biocheck的OppA-ELISA试剂盒进行比较。结果表明,抗原包被质量浓度为0.5 μg·mL^-1、被检血清以1：200倍稀释后反应45 min以及二抗稀释度为1：15 000时,Apd-ELISA的反应背景下降,区分度提高。根据背景确认的27份阳性和40份阴性血清的OD_{630 nm}值以及临界值计算公式（S-N）/（P-N）,得到Apd-ELISA的阴阳性临界值是0.33。证实用封闭液K封闭和真空包装的酶标板可以在50℃保存4 d（相当于在4℃保存22个月）。用Apd-ELISA试剂盒检测1 179份临床血清,表明母猪、后备和育肥猪以及仔猪的HPS血清阳性率分别为83.76%、61.09%和27.48%。与荷兰的Biocheck试剂盒进行比较,发现Apd-ELISA与Biocheck试剂盒（OppA-ELISA）对HPS临床阴性血清和疫苗免疫血清检测的符合率为100%,对HPS临床阳性血清的符合率仅为4.76%（6/126）。然而,Apd-ELISA与OppA Western blot的阳性符合率可达到73.02%（92/126）。本研究获得了能有效区分HPS阴、阳性血清和稳定保存的Apd-ELISA抗体检测试剂盒,可用于HPS灭活疫苗免疫后的抗体检测和副猪嗜血杆菌病的血清抗体检测。     

副猪嗜血杆菌疫苗研究进展

近年来,副猪嗜血杆菌所引起的全身性炎性疾病使全球的养猪业造成巨大的经济损失,如何预防和控制副猪嗜血杆菌病发生成为关注的焦点,疫苗的研究开发也受到了重视。近年使用的疫苗有灭活苗、亚单位疫苗、DNA疫苗和基因工程疫苗等,论文对目前副猪嗜血杆菌应用的疫苗种类、研究现状以及发展方向进行了综述。