高致病性猪繁殖与呼吸综合征、猪圆环病毒病和副猪嗜血杆菌病混合感染的诊断与防制

目前在集约化养殖场的猪群中经常出现病毒病和细菌病的混合感染,严重影响中国养猪业的发展。2011年9月,广东某个千头母猪场的猪群大量发病,表现为高热、咳嗽、食欲下降、皮肤发绀、关节肿大等临床症状。通过流行病学调查、剖检、细菌分离鉴定、药敏试验、PCR和RT-PCR等实验室检测最终确诊为高致病性猪繁殖与呼吸综合征、猪圆环病毒病和副猪嗜血杆菌病的混合感染,在此基础上提出和实施了该病的防控方案。     

广东部分猪场副猪嗜血杆菌病的流行病学调查

为了解副猪嗜血杆菌病(Haemophilus parasuis,HPS)在广东省部分地区的流行情况,通过针对副猪嗜血杆菌外膜蛋白P2(OMPP2)的间接ELISA法对广东省部分猪场的376份猪血清样本进行了抽样检测。结果显示,副猪嗜血杆菌的阳性率为23.8%,其中母猪阳性率高达31%;保育仔猪为29%,育肥猪为33.6%,育肥猪阳性率显著高于母猪和保育仔猪;冬季(11～1月)副猪嗜血杆菌抗体阳性检出率最高为31.4%、秋季(8～10月份)的阳性检出率最高为18.7%,冬季比秋季高出12.7%,差异极显著(P<0.01)。     

福建省部分地区副猪嗜血杆菌病的流行病学调查

为了解副猪嗜血杆菌病在福建省的流行情况,通过间接血凝检测方法对福建省部分猪场猪副猪嗜血杆菌病的流行情况进行了调查,结果表明,福建部分地区猪副猪嗜血杆菌病的阳性率为6.5%,其中母猪阳性率偏高,为8.75%。     

副猪嗜血杆菌对猪气管上皮细胞的黏附作用及其黏附模型的建立

为了探讨副猪嗜血杆菌(Haemophilus parasuis,Hps)与原代培养的猪气管黏膜上皮细胞的相互作用,应用气管支气管结扎灌注酶冷消化法分离气管黏膜上皮细胞,于胶原覆盖的盖玻片上培养,使上皮细胞分化成假复层黏膜纤毛上皮细胞,然后以对数生长期不同Hps含量的菌液感染上皮细胞和猪脐静脉血管内皮细胞,分别作用1、2、34、h后,通过革兰氏染色法观察Hps对细胞的黏附情况。结果表明,Hps可黏附在上皮细胞和内皮细胞表面;Hps对气管上皮细胞有毒性作用,致细胞变性坏死和凋亡;Hps对细胞的黏附力与其在菌液中含量和作用时间有关,以菌落形成单位计,当含量为1.0×108mL-1且感染4 h后,黏附效果最好。高倍显微镜下可见大量Hps黏附于猪气管上皮细胞,只有个别Hps黏附于猪血管内皮细胞表面。说明Hps对猪气管黏膜上皮细胞的黏附能力远强于猪血管内皮细胞,可以利用猪气管黏膜上皮细胞建立副猪嗜血杆菌的黏附细胞模型。     

副猪嗜血杆菌的分离与鉴定

从福建省龙岩市l例临床症状、病理剖检变化疑似副猪嗜血杆菌病的患病猪的关节液中分离到1株革兰氏染色阴性可疑菌株,经卫星现象、生化鉴定等实验室诊断,进一步以副猪嗜血杆菌的16S rRNA基因设计特异性引物进行PCR鉴定,确定该分离菌为副猪嗜血杆菌。药敏试验结果表明,分离株对环丙沙星、卡那霉素、强力霉素、氨苄西林、阿米卡星、庆大霉素药物敏感;对泰妙菌素、诺氟沙星有抵抗力。     

浙江地区副猪嗜血杆菌分离株的生物学特性

为明确浙江地区副猪嗜血杆菌(HPS)病的流行特点,提高对该病的免疫防控能力,本研究对HPS分离株进行生物学鉴定。结果表明,HPS在含有2%小牛血清和16μg/mL NAD的TSB培养基中生长良好,在TSB液体培养基中培养16 h～18 h后,活菌数达到峰值;6个分离株对小鼠的LD50分别为2.03×109、5.3×109、1.34×1010、7.0×109、1.17×1010和1.62×109;可在感染HPS豚鼠的心血中分离到该菌;ERIC-PCR指纹图谱分析结果表明,4个分离株与4型、5型和9型标准菌株图谱一致,但有2个分离株与15个血清型标准菌株图谱不吻合。研究提示浙江地区内HPS的散发、流行的原因可能是由于该细菌血清型复杂性造成的。     

Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens

From December 2003 to July 2006, a total of 131 (28.4%) Haemophilus parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. These strains were isolated from clinically diseased pigs, and 50 of them along with 15 reference strains of all known serovars were subjected to PCR–FRLP (restriction fragment length polymorphism) analysis by tbpA gene. The analysis of the 1.9-kb tbpA amplicon using TaqI, AvaI and RsaI endonucleases produced 9 RFLP patterns for the15 reference strains and 13 patterns for the 50 field isolates. And the first three prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). Meanwhile, co-infection of H. parasuis, PRRSV and PCV2 was examined in the 462 pig herds. It is indicated that 11.5% cases (53), 27.9% cases (129) and 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens. It is confirmed that H. parasuis existed widely in southeast China with numerous genotypes.     

一起猪繁殖与呼吸综合征与副猪嗜血杆菌混合感染的诊治

为查明猪场发病原因,对发病猪进行剖检及实验室检测,剖检发现淋巴结肿大,胸腔、腹腔有纤维素性渗出物;通过细菌分离鉴定发现所分离的菌株培养特性与HPS基本一致,通过病原学检测发现发病猪只存在PRRSV和HPS混合感染。     

副猪嗜血杆菌分离鉴定及药敏试验

副猪嗜血杆菌能够引起猪的多发性浆膜炎、关节炎和脑膜炎等,是影响猪的最重要细菌之一,目前在所有的主要养猪国家均有存在。为了弄清河南省副猪嗜血杆菌病流行情况,2012年-2015年,从河南不同地区猪场送检的疑似病料,进行副猪嗜血杆菌分离和鉴定,共分离到5株细菌,通过细菌形态观察、培养特征鉴定、生化试验、PCR检测,鉴定为副猪嗜血杆菌,分别命名为A6-fei、C3-xin、C12-xin、D2-fei和E1-fei。采用纸片扩散法,对分离5株副猪嗜血杆菌进行药敏试验,其结果表明所分离的5株副猪嗜血杆菌的药物敏感性不尽相同,各分离菌株对头孢噻肟、氟苯尼考星最敏感,对复方新诺明敏感性最差,其中菌株C3-xin对复方新诺明、庆大霉素、卡那霉素、青霉素完全耐药。表明副猪嗜血杆菌病在河南省依然存在,并且不同地区菌株对常用药物的敏感性各不相同,应当引起养猪场重视。     

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.