猪繁殖与呼吸综合征病毒与副猪嗜血杆菌混合感染的诊断

为确诊广东省阳江市某规模化猪场（存栏800头母猪）保育猪发病死亡的原因,本试验对从该发病猪场采集的3份肺脏、肝脏、脾脏临床样品进行细菌学检测及药敏试验,采用PCR/RT-PCR检测临床样品中猪伪狂犬病病毒（PRV）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、猪圆环病毒3型（PCV3）和猪肺炎支原体等病原。对特异性扩增的3株PRRSV的ORF5基因产物进行序列测定,与VR2332、HuN4、JXA1、CH-1a等代表毒株进行核苷酸序列同源性分析,并构建系统进化树。结果表明,试验分离鉴定出1株副猪嗜血杆菌（Hps）,对7种临床常用药如阿莫西林、头孢拉定等均有较强的敏感性。同源性比对结果表明,3株PRRSV （LJW1、LJW2和LJW3）ORF5基因核苷酸同源性为99.3%~99.8%,与欧洲型代表毒株Lelystad核苷酸同源性为64.0%~64.2%,与HP-PRRSV毒株JXA1、HuN4、CH-1a和TJ核苷酸同源性较高,分别为99.2%~99.5%、99.0%~99.3%、94.5%~94.9%和98.8%~99.2%;与中国河南和广西分离的HP-PRRSV毒株HeNzm1-16和GXLZ05-2015核苷酸同源性较高,分别为99.3%~99.7%和99.2%~99.7%,与美洲型经典疫苗株MLV、美洲型标准株NC、美洲型经典株VR2332核苷酸同源性较低,分别为88.5%~88.8%、85.2%~85.5%和82.3%~82.6%。PRRSV ORF5基因系统进化树分析表明,3株PRRSV均属于美洲型毒株,与国内HP-PRRSV代表毒株JXA1、HuN4和TJ等处于同一分支,亲缘关系较近。本研究揭示了该场保育猪发病病原,并从分子水平上明确了分离的3株PRRSV与不同代表毒株的亲缘关系,为弱毒疫苗的合理选择使用和综合防控PRRSV提供了参考依据。     

三种鉴别诊断PRRSV美洲型经典毒株和变异株的方法比较

我国流行的猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）包括美洲型经典毒株和变异株。本研究应用RT—PCR、直接免疫荧光（ direct immunofluorescence asay, DFA ）及病毒分离3种检测方法对99份不同类型的猪繁殖与呼吸综合征临床疑似病料和人工感染病料进行检测。结果显示,RT—PCR检测试剂盒敏感性最高,DFA鉴别诊断试剂盒次之,病毒分离最差。DFA与病毒分离、RT—PCR的总符合率分别为92％和85．5％。RT—PCR灵敏性高,可作为PRRSV鉴别诊断的首选方法;DFA鉴别诊断法所需时间较短、特异性好,但需要一定的操作经验;病毒分离敏感性低、操作繁琐,阳性分离需要其它方法验证,不适合于PRRSV的鉴别诊断。     

猪瘟、猪圆环病毒病和猪繁殖与呼吸综合征寡核苷酸芯片诊断方法的建立与初步应用

猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）和猪圆环病毒Ⅱ型（PCV-2）是引发猪繁殖障碍的主要病原,建立其基因芯片快速检测体系,对开发猪繁殖障碍快速检测试剂盒具有重要意义。根据GenBank中已发表的PRRSV、CSFV和PCV-2的病毒基因组序列,设计合成特异性引物和特异性较强的60mer的寡核苷酸探针,并将探针按所设计阵列固定于表面经氨基化修饰的玻片上,制备出寡核苷酸芯片。通过引物标记及特异性验证,建立了带标记引物的多重PCR检测体系,从而产生大量可与寡核苷酸探针特异性互补的带标记的DNA片段。将标有荧光染料的扩增产物与芯片上寡核苷酸探针杂交,扫描、分析芯片上荧光信号。结果表明,芯片上各样本对应探针位点呈现阳性荧光信号,而阴性对照和空白对照则基本不能检测到荧光信号。分别用基因芯片检测方法和PCR/RT-PCR检测60份临床病料,两者符合率高达92%,表明该检测技术能够用于临床病料的检测及快速诊断PRRSV、CSFV和PCV-2。     

猪繁殖与呼吸综合征重组核衣壳蛋白(N)-ELISA诊断方法的建立与应用

将猪繁殖与呼吸综合征病毒国内分离株ＣＨ－ｌａ的核衣壳蛋白基因定向克隆到ｐＢｌｕｅＢａｃＨｉｓＢ转移载体ＰＨ启动子下游,与苜蓿银蚊夜蛾多核型多角体病毒线性化ＤＮＡ共转染Ｓｆ９细胞。获得能隐定表达核衣壳蛋白（Ｎ 蛋白）的重组杆状病毒。利用昆虫杆状病毒表达系统表达的猪繁殖与呼吸综合征病毒重组Ｎ蛋白作为抗原,包被聚苯乙烯微量反应板,建立了猪繁殖与呼吸综合征重组Ｎ蛋白－ＥＬＩＳＡ诊断方法。试验证明,该方法对其他８种猪疫病阳性血清均无交叉反应,对标准阳性样品的检出率为１００％,具有敏感性高、特异性强的特点。     

Mucopolysaccharidosis IIIA (Sanfilippo syndrome) in a New Zealand Huntaway dog with ataxia

Aim: To investigate the nature of a progressive ataxia in a New Zealand Huntaway dog.

Methods: The affected dog was examined clinically before being humanely killed and necropsied. Selected tissues were submitted to light and electron microscopy and to biochemical analyses.

Results: The histological lesions were interpreted as indicative of one of the forms of mucopolysaccharidosis type-III (dMPS-III), a lysosomal storage disease. Biochemically there was a deficiency of heparan sulphamidase. All the heparan sulphate chains had non-reducing-end glucosamine-N-sulphate residues.

Conclusion: The disease is dMPS-IIIA (Sanfilippo syndrome). An autosomal recessive mode of inheritance can be provisionally assumed from the nature of this disease in other species.     

Construction and Identification of PRRSV ORF5 Gene Prokaryotic Expression Vector

The present study was designed to construct recombinant plasmids,which could express porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene.RNA was extracted from spleen and lung samples of the suspected pigs which were infected with PRRSV.According to PRRSV ORF5 gene,a pair of primers was designed for RT-PCR amplification.The ORF5 target gene was cloned into pMD19-T vector and then the recombinant pMD19-ORF5 was achieved.According to the sequencing results and the characteristics of expression vectors,a pair of primers with NcoⅠand XbaⅠenzyme cleavage sites was designed.Target fragment dORF5 was amplified and then connected to pProEXHTb and pNZ8149 vectors,respectively.And recombinant HTb-dORF5/DE3 and pNZ8149-dORF5/NZ3900 was induced by IPTG and Nisin,respectively,and analyzed by SDS-PAGE and Western blotting.Recombinant HTb-dORF5/DE3 induced by 1.5 mmol/L IPTG was expressed in the highest quantity.There were specific band at about 22 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.Recombinant pNZ8149-dORF5/NZ3900 induced by 20 ng/mL Nisin was expressed in the highest quantity.There were specific band at about 19 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.The IFA result showed specific green fluorescence.This study successfully constructed recombinant plasmids HTb-dORF5 and pNZ8149-dORF5 and expressed,the result laid a solid foundation for further development of PRRS vaccines.     

Canine renal failure syndrome in three dogs

Three dead dogs were brought to the College of Veterinary Medicine, Kyungpook National University for study. Clinically, all the dogs showed emaciation, anorexia, depression, hemorrhagic vomiting and diarrhea for 7~10 days before death. All the clinical signs were first noted for about one month after feeding the dogs with commercial diets. At necropsy, all 3 dogs had severe renal damage with the same green-yellowish colored nephroliths in the renal pelvis. They also showed systemic hemorrhage and calcification of several organs, which might have been induced by uremia. Microscopically, necrosis, calcification and calculi were detected in the renal tubules, and especially in the proximal convoluted tubules and collecting ducts of the kidney. These findings were supportive of a mycotoxic effect, and especially on their kidneys. However, the precise cause of the toxic effect in these cases of canine renal failure could not be determined.     

Double in situ hybridization for simultaneous detection and differentiation of porcine circovirus 1 and 2 in pigs with postweaning multisystemic wasting syndrome

Double in situ hybridization using a digoxigenin-labelled porcine circovirus 1 (PCV1) and biotinylated PCV2 probe, was developed for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The combination of an alkaline phosphatase conjugated antidigoxigenin system with alkaline phosphatase conjugated streptavidin-biotin system allowed identification of PCV1 and/or PCV2. No evidence of cross-reaction was observed. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. Co-localization of hybridization signal for both PCV1 and PCV2 was present in macrophages and multinucleated giant cells of the lymph node and spleen. This double-labelling technique for the differentiation between PCV1 and PCV2 is suitable for pathogenesis studies and diagnostic applications.     

肉鸡发育障碍与矮小综合征

肉鸡发育障碍与矮小综合征是一种包括肠道损害等多症状的传染性疾病,目前已遍布世界各地,在蛋鸡、珍珠鸡和火鸡中均有发生,是一个严重威胁现代养禽业的重大问题。论文从肉鸡发育障碍与矮小综合征的临床症状、病理变化和病原学对其进行了综述,以期为本病的诊断和预防提供参考。