Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination

A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus-neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do.     

An indirect fluorescent antibody test for antibodies to transmissible gastroenteritis of swine.

The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transmissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20 degrees C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advantages over the virus neutralization test.     

猪传染性胃肠炎病毒单克隆抗体的制备与鉴定

以纯化的猪传染性胃肠炎病毒(TGEV)作为抗原免疫BALB/c小鼠,经3次免疫后,通过聚乙二醇方法进行融合,利用有限稀释法在HAT培养基上筛选杂交瘤细胞,共获得12株既能稳定生长并可以分泌特异性抗TGEV的单克隆抗体的杂交瘤细胞系,分别命名为8D2、4H4、5D6、7H10、4C3、9G1、3A2、8G1、5G6、2D7、4D6、6B10。间接ELISA、Western blot、间接免疫荧光结果表明,获得的12株单抗能特异性识别TGEV,通过间接ELISA做病原检测,显示12株单抗与猪流行性腹泻病毒(PEDV)和猪轮状病毒(PrV)不发生交叉反应。经抗体亚类鉴定,该12株单克隆抗体均为IgG2b。12株抗TGEV的单抗制备成功,为猪传染性胃肠炎病原特性研究和病原快速检测提供了物质基础。     

Infection of pigs with transmissible gastroenteritis virus from contaminated carcases

Sixteen 6-month-old pigs were exposed to transmissible gastroenteritis (TGE) virus by placing them in close contact with piglets infected at 1 week of age. Fourteen of the older pigs were slaughtered between 1 and 5 d after exposure to infection and their carcases dressed in simulated abattoir conditions. Samples of muscle, bone marrow and carcase lymph nodes were stored at -25 degrees C for at least 30 d and then homogenised and fed to groups of 1-week-old and 3-week-old pigs. Four of 12 one-week-old pigs died and TGE virus was isolated from intestinal contents of one of these. All pigs of both age groups developed neutralising antibody to TGE virus over the ensuing 4 w. The results indicate that carcases from pigs infected with TGE virus can represent a source of infection for susceptible pigs given access to them.     

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.     

猪乳汁中抗猪传染性胃肠炎病毒IgA抗体间接ELISA检测方法的建立

为建立检测猪乳汁中抗猪染性胃肠炎病毒(TGEV)Ig A抗体的间接ELISA方法,本研究以TGEV重组N蛋白为包被抗原,以辣根过氧化物酶标记羊抗猪Ig A为检测抗体,并采用方阵法确定包被抗原和待检乳汁的最佳工作浓度,对各种反应条件进行优化,建立了猪乳汁中TGEV Ig A抗体的间接ELISA检测方法。该方法检测稀释160倍的阳性乳清仍呈阳性;与猪流行腹泻和猪轮状病毒感染阳性乳清无交叉反应;批内和批间变异系数均小于10%。利用建立的ELISA方法与间接免疫荧光方法分别对134份临床样品进行检测,阳性检出率分别为58.2%(78/134)和59.7%(80/134),总符合率为85.6%。本研究建立的检测方法能够有效评估乳汁中TGEV Ig A抗体的水平。     

猪传染性胃肠炎病毒卵黄抗体的研制

针对猪传染性胃肠炎病尚无有效的治疗药物,本文研究开发抗猪传染性胃肠炎病毒卵黄抗体,以期为该病的防治提供技术支持.用猪传染性胃肠炎灭活疫苗按程序免疫90～140日龄健康商品海兰褐蛋鸡,三免14天后收集高免蛋进行卵黄抗体提取、ELISA抗体效价检测及攻毒保护试验.高免蛋三免后14天效价可达到1:256,并维持3个月,三免后...     

Recovery of transmissible gastroenteritis virus from chronically infected experimental pigs.

Transmissible gastroenteritis (TGE) virus was reisolated from pulmonary and intestinal tissues from 6 of 9 chronically infected experimental pigs (principals) necropsied 30 to 104 days after inoculation. Tissue homogenates (lung and small intestine) from the principals were prepared and inoculated into 3- to 5-day-old gnotobiotic pigs. The virus reisolated from the tissue homogenates produced a milder disease on 1st passage and a more severe disease on 2nd passage. The chronically infected experimental pigs (principals) developed serum-neutralization titers to TGE of 1:30 to 1:525. There appeared to be no relationship between serum titers and reisolation of TGE virus from the 9 principals. The persistence of virus in lung or intestine to 104 days indicates the recovered (or carrier) pig may be considered the primary source of TGE virus infection.     

Inhibition of virus attachment to CPK cells by monoclonal antibody to transmissible gastroenteritis virus.

Five MAbs, which recognized E2 glycoprotein of TGE virus TO-163 and showed neutralizing activity, were examined to see if they inhibit virus attachment to the susceptible cells (CPK cells). Only one (160/4) MAb blocked the virus attachment to the cells, indicating that the inhibition of virus attachment is one of important mechanisms of neutralizing by antibody.     

猪传染性胃肠炎病毒重组N蛋白间接ELISA抗体检测方法的建立

为建立一种检测猪传染性胃肠炎病毒(TGEV)血清抗体的间接ELISA方法,将TGEV的N基因片段克隆到p ET-28a载体中,转化至大肠杆菌BL21感受态细胞中进行诱导表达,并对表达的蛋白进行Western-blot鉴定。以纯化的重组N蛋白作为包被抗原,最终建立了检测TGEV抗体的间接ELISA方法。该ELISA方法与猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)、猪繁殖与呼吸系统综合征病毒(PRRSV)、口蹄疫病毒(FMDV)、猪圆环病毒2型(PCV2)等5种病毒阳性血清不发生交叉反应,表明建立的ELISA方法具有良好的特异性。本研究可为猪传染性胃肠炎的流行病学调查、诊断与防控奠定基础。     

Intestinal phospholipase B activity in pigs inoculated with transmissible gastroenteritis virus

Intestinal phospholipase B activity in pigs inoculated with transmissible gastroenteritis (TGE) virus was studied. Phospholipase activity was quantified by measuring the hydrolytic release of free fatty acids in homogenized intestinal tissue incubated with lysophosphatidylcholine. An increase in enzyme activity was observed in the cranial and caudal portions of the ileum and jejunum in pigs killed 3, 6, and 8 days after inoculation with TGE virus. Seemingly, phospholipase B may be part of the host immune response against TGE viral infection.     

Oral transmission of transmissible gastroenteritis virus by muscle and lymph node from slaughtered pigs

A study was conducted in the USA to determine whether transmissible gastroenteritis (TGE) virus could be transmitted from carcases of slaughtered pigs. Transmissible gastroenteritis virus was transmitted to 6-day-old piglets by dosing with homogenates of muscle and lymph node collected from 500 clinically normal pigs at the time of slaughter. All piglets in 2 separately housed litters showed clinical signs of TGE with 5 piglets dying within 10 d of oral dosing with homogenates. Transmissible gastroenteritis virus was isolated from 2 of these piglets and all piglets developed TGE antibody. Transmissible gastroenteritis virus was not isolated in tissue culture from muscle and lymph node homogenates, but was isolated from 4 (0.8%) of 500 tonsil samples collected from the same pigs. A survey of 250 serum samples provided an estimate of the prevalence of slaughtered pigs with TGE antibody of 34.8% in the sample population. The results indicate that carcases of some pigs from TGE endemic areas contain viable TGE virus, and that there would be a substantial risk of introducing TGE virus into Australia by the importation of uncooked pig meat from these areas.     

Financial impact of transmissible gastroenteritis in pigs

The financial impact of an epizootic of transmissible gastroenteritis in pigs was evaluated in a California sow herd through estimating growth, feed, and profit functions. Two groups of pigs were studied: pigs born before and surviving the epizootic (epizootic [E] pigs), and pigs born after the epizootic (postepizootic [PE] pigs). Short-term profits were maximized at 165 days for both groups of pigs, ranging from $47.14 for female E pigs to $60.32 for male PE pigs. Accordingly, it was concluded that pigs surviving or born shortly after a transmissible gastroenteritis epizootic are profitable to raise, if raised under management conditions similar to those in the study herd.     

Serum antibody responses of neonatal and young adult pigs to transmissible gastroenteritis coronavirus

Serum titers of virus-neutralizing (VN) antibody were 10 to 16 times higher in neonatal pigs than in young adult pigs, after single oral doses of virulent transmissible gastroenteritis virus (TGEV). To determine the reason for this higher response, sera from neonatal and young adult pigs, 18 to 21 days after exposure to TGEV, were collected and assayed for VN antibody by plaque reduction. In addition, sera of VN-positive and VN-negative neonatal pigs were analyzed for immunoglobulin classes by radial immunodiffusion technique.The competence of neonatal pigs to produce VN antibody with increased IgG levels was demonstrated. The higher antibody response seen in neonatal pigs, when compared to sera of young adult pigs, may be attributed to the increased replication of TGEV in the intestinal tracts of neonatal pigs or to the lack of other immunogens that may interfere or compete with the production of specific antibody.