猪血凝性脑脊髓炎病毒抗原捕获ELISA诊断方法的建立

采用实验室保存的杂交瘤细胞,通过扩大培养、接种小鼠、收集腹水、腹水纯化等程序得到了猪血凝性脑脊髓炎病毒(HEV)单克隆抗体,并对其效价进行了测定.用HEV单克隆抗体作为检测抗体,猪多抗作为捕获抗体,建立了检测HEV的抗原捕获ELISA方法,通过对各步反应条件的优化,最终获得最佳工作条件为:猪多抗1:2 000稀释,单抗1:4 000稀释,酶标抗体为1:4 000稀释.特异性和敏感性试验结果表明:该方法对TGEV、HCV、PEDV和PRV等病毒无特异性交叉反应,其最低检测下线为3.75 mg/L.与RT-PCR方法的对比试验证明,抗原捕获ELISA阳性检出结果与PCR方法相一致.上述结果说明,已成功建立特异、敏感的用于HEV检测的抗原捕获ELISA方法,为临床快速诊断HEV试剂盒的研制奠定了基础.     

Porcine encephalomyelitis caused by hemagglutinating encephalomyelitis virus.

Six epizootics of encephalomyeltis in suckling pigs in Minnesota were attributed to infection with hemagglutinating encephalomyelitis virus. The disease occurred in 74 litters of pigs and was characterized by sudden onset of tremors, inappetence, weakness, atazia, and hyperesthesia, with high morbidity and case fatality rate. Pathologic changes consisted of marked nonsuppurative, nondemyelinating encephalomyelitis characterized by perivascular mononuclear cuffing, gliosis, neuronal death, and satellitosis. Clinical disease was limited principally to suckling pigs during a single farrowing period and did not recur in the herds involved during the ensuing 18 months.     

猪血凝性脑脊髓炎病毒抗体的调查

应用血凝和血凝抑制试验检测了从吉林省部分地区采集的猪血清中血凝性脑脊髓炎病毒（HEV）抗体。结果，212份样品中有94份呈现HEV抗体阳性反应，阳性率高达44．3％。被采集血清的猪未表现临床症状，说明该地区的猪群中存在HEV隐性感染。     

猪血凝性脑脊髓炎实验室诊断方法综述

猪血凝性脑脊髓炎是引起仔猪神经系统障碍的重要传染病之一,严重危害养猪业健康发展。正确诊断猪血凝性脑脊髓炎对防治该病极为关键。文章围绕该病病原分离鉴定、血清学诊断、分子生物学诊断这三方面研究进展分别展开阐述,以期了解该病诊断方面的研究进展。     

Virus isolated and immunofluorescence in different organs of pigs infected with hemagglutinating encephalomyelitis virus

Hemagglutinating encephalomyelitis virus (HEV; also designated vomiting and wasting disease virus) was inoculated oronasally in 26 colostrum-deprived pigs. Anorexia and vomition were seen after an incubation period of 4 to 6 days. In pigs killed during the incubation period or within 2 days after the onset of the clinical signs, HEV could be isolated regularly from the tonsils and the respiratory tract, irregularly from the digestive tract, rarely from the blood, and never from lymph nodes and spleen. The brainstem almost always contained virus after clinical signs appeared, but was only one positive during the incubation period. Olfactory bulb, cerebrum, cerebellum, and vagal nerve were also frequently virus positive in pigs which were ill when killed. The results of the examination by immunofluorescent antibody technique indicated that HEV multiplies in the epithelium lining the respiratory tract and the tonsillar crypts, in neuroepithelium of the nasal mucosa, and in neurons of the digestive tract. The neuronotropism of HEV was also shown by the presence of fluorescence in the perikaryon of neurons in the brainstem and in the trigeminal ganglion without the involvement of other cell types. The presence of viral antigens in the perikaryon of trigeminal sensory ganglion cells in pigs killed during the incubation period was considered as positive evidence for viral spread via nerves.     

Immunofluorescence studies on the pathogenesis of hemagglutinating encephalomyelitis virus infection in pigs after oronasal inoculation

Hemagglutinating encephalomyelitis virus (HEV; also designated vomiting and wasting disease virus) was inoculated oronasally in 14 colostrum-deprived pigs at the day of birth. Anorexia and vomition were seen after 4 days. Pigs were killed at different times after inoculation, and the results of the examination by immunofluorescent antibody technique revealed that the epithelial cells of nasal mucosa, tonsils, lungs, and small intestine served as sites of primary viral replication. After the local replication near the sites of entry, the virus spread via peripheral nervous system to the CNS. During the incubation period, viral antigens were detected in the trigeminal ganglion,the inferior vagal ganglion, the superior cervical ganglion, the intestinal nervous plexuses, the solar ganglion, and the dorsal root ganglia of the lower thoracic region. In the brain stem, the infection started in the trigeminal and vagal sensory nuclei and spread to other nuclei and to the rostral part of the brain stem. In later stages of the infection, viral spread into the cerebrum, cerebellum, and spinal cord was sometimes also observed. Viral replication in nervous plexuses of the stomach was not present during the incubation period, but was detected in all except 1 of the pigs that were ill when killed. The question whether the vomition is induced centrally by viral replication in the brain stem or is due to viral replication in peripheral nervous tissues remains unanswered.     

Persistence of passively acquired antibodies to hemagglutinating encephalomyelitis virus in swine

The persistence of passively acquired antibodies to hemagglutinating encephalomyelitis virus (HEV) was determined in 4 pigs in each of the litters of 10 sows. At time of delivery by the sows, the colostrum and serum samples (from the 10 sows) had hemagglutination-inhibiting antibodies to HEV. All of the pigs also had hemagglutination-inhibiting antibodies to HEV at 2 days of age. The level of circulating antibodies to HEV decreased at a nonlinear rate and persisted for about 4 to 18 (mean 10.5) weeks in the circulation of pigs. All 40 pigs were seronegative at 20 weeks of age.     

猪血凝性脑脊髓炎病毒抗体间接ELISA检测方法的建立及山东省猪血清抗体检测分析

首先应用血凝抑制试验(HI)对从山东省部分地区采集的178份猪血清样品进行了猪血凝性脑脊髓炎病毒(HEV)抗体检测。结果显示,这些猪群中HEV的HI抗体总体阳性率高达61.24%(109/178),说明HEV的感染较为普遍。同时,以纯化的病毒作为包被抗原,通过优化反应条件,成功建立了HEV抗体间接酶联免疫吸附试验(ELISA)检测方法。应用该ELISA方法对从山东地区猪血清中随机抽取的44份样品进行检测,结果显示,HEV抗体阳性率为72.73%(32/44);而HI抗体阳性率为56.82%(25/44)。两种检测方法的阳性符合率为92.00%。结果表明,建立的ELISA方法较HI方法敏感性高。     

Pathogenicity of an attenuated strain of transmissible gastroenteritis virus for newborn pigs.

The pathogenicity of a cell culture-attenuated strain of transmissible gastroenteritis virus for newborn pigs was investigated. Newborn (1- to 2-day-old) pigs were orally given 2 x 10(6) plaque-forming units of attenuated virus. All pigs developed mild diarrhea, but deaths did not occur. As determined by immunofluorescence and villous atropy, infection of the small intestine was limited to the caudal 50 to 66%. Fluorescing cells and atrophic villi were seen from 2 to 3 days until 6 to 7 days after exposure. Attenuated virus-exposed pigs produced circulating virus-neutralizing antibodies detectable as early as 5 days after exposure. By contrast, all pigs orally given 1 x 10(2) pig infective doses of virulent transmissible gastroenteritis virus developed severe diarrhea, and almost all of those not killed died within 2 to 5 days after exposure. In the latter pigs, the entire length of the small intestine, except for the first 4 to 5 cm, was infected with virus by 24 to 36 hours after exposure.