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制备猪瘟荧光抗体并用于猪只活体扁桃体组织抹/涂片检测。将制备的猪瘟高免血清采用硫酸铵分级沉淀法纯化免疫球蛋白,反向透析法标记荧光素,过葡聚糖凝胶柱(Sephadex G-50)去除游离荧光素,通过扁桃体组织匀浆吸附去除异嗜性或交叉反应抗体。以5?S做稀释液,以RT-PCR检测阳性样本的冰冻切片确定其最大稀释度为1/60,最佳工作浓度为1/30,阻抑试验证明荧光为特异荧光。用标记的荧光抗体检测活体扁桃体样本39份,检出阳性11份,与IDEXX猪瘟抗原检测试剂盒复核结果完全吻合。结果表明,本实验所制备的猪瘟荧光抗体具有很高的灵敏性和特异性,可用于猪瘟净化中扁桃体组织抹/涂片检测。 相似文献
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In this paper we describe a study of the use of the white blood cell count (wbcc) as a parameter for detecting outbreaks of Classical Swine Fever (CSF). Meta-analysis of the results of challenge experiments revealed that oronasal infection of SPF-pigs with the virulent CSF virus (CSFV) strains Brescia or NL9201 resulted in a significant decrease in the average white blood cell count during the first week after inoculation of the virus. Challenge of conventional finishing pigs and sows with the moderately virulent strain Paderborn also resulted in a significant decrease in the average wbcc. However, this decrease was not observed after inoculation of SPF pigs with the mildly virulent CSFV strains Henken, Zoelen, or Bergen. The usefulness of clinical inspection in combination with wbcc to detect CSF outbreaks in the field was examined using the results of 214 EDTA blood specimens collected from 22 infected herds and 7250 EDTA blood specimens collected from 1450 non-infected herds. Half of the infected herds had been infected with the moderately virulent CSFV strain Venhorst (closely related to strain Paderborn) during the 1997-98 epidemic in the Netherlands. The other half had been infected with the moderately virulent CSFV strain Loraine. Using these data as a starting point, 1000 samples of one to ten specimens were generated by Monte Carlo simulation. These simulated samples and the samples of the non-infected herds were analysed by use of Receiver Operating Characteristic curves. On the basis of that analysis, the optimal number of animals whose wbcc needed to be determined to detect a CSF outbreak was five. With this number of animals, in conjunction with the threshold of 8000 white blood cells per mm3 (meaning that a herd is designated as CSF suspect if one or more of the five specimens has a white blood cell count of 8000 leukocytes/mm3 or less), the test procedure had a herd sensitivity (HSE) of 94.5% and a herd specificity (HSP) of 97.2%). The HSE is defined as the percentage of samples of infected herds with a positive result of the test procedure; HSP is defined as the percentage of uninfected herds with a negative result of the test procedure. We conclude that the wbcc can help the veterinary practitioner to detect outbreaks of CSF caused by (moderately) virulent CSFV strains. However, for the detection of outbreaks caused by mildly virulent CSFV strains, the contribution of the wbcc is doubtful. Development of additional tools that can improve the clinical diagnosis of the veterinary practitioner remains desirable. 相似文献
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为了探讨母猪的猪瘟疫苗免疫情况和猪瘟病毒之间的关系,本研究采用阻断ELISA技术和RT-PCR技术,对某规模化猪场随机采取57头母猪的血清样品和扁桃体样品进行了猪瘟抗体水平和猪瘟病毒检测。结果表明：猪瘟抗体阳性率为74.07%,抗体离散度为41.79%,表明猪瘟抗体阳性率刚达到国家规定的标准,抗体离散度偏高。猪瘟抗体和猪瘟病毒之间存在一定关系,无论猪瘟疫苗免疫是否合格,均有可能感染猪瘟野毒,猪瘟抗体阴性组,以及抗体阻断率大于90%组中的猪瘟病毒阳性率较高。 相似文献
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Ji-Hyun Sung Won-Jung Lee Sung-In Lim Jae-Young Song 《Research in veterinary science》2011,90(2):329-335
Classical swine fever (CSF) is a highly contagious disease of pigs that causes fever, diarrhea and paralysis, often resulting in death. E2 is the major structural protein of the CSF virus (CSFV) and mediates the entrance of the virus, subsequently inducing a neutralizing immune response. In this study, the E2 gene of a recent Korean isolate of CSF, SW03, was cloned and the DNA sequence was compared to other strains via phylogenetic analysis. With the purified E2 protein, an enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of CSFV infection. The sensitivity and specificity of the E2-ELISA were 96.1% and 94.8%, respectively. A total of 17 out of 485 field-collected pig sera tested demonstrated conflicting results between two ELISA methods, a commercial kit and the E2-ELISA. Of these sera, 60% were determined to be CSFV positive by a virus neutralization test (VNT), suggesting involvement of different immune responses in the cases of CSFV infection. As the E2-ELISA was developed using a recent Korean isolate, SW03, this assay is capable of rapidly identifying newly emerging CSFV strains. 相似文献
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Gonzalez C Pijoan C Ciprian A Correa P Mendoza S 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2001,63(9):991-996
The airborne transmission of Classical Swine Fever (CSF) virus to susceptible pigs, as well as the effect of vaccination with the CSF virus PAV-250 strain was investigated on this mode of transmission. Experiment I: four pigs were inoculated with the ALD CSFV strain (10(4.3) 50% TCID) by the intramuscular route, and at the onset of fever, they were introduced into an enclosed chamber. At the end of the experiment surviving pigs were sedated, anesthetized and euthanatized. Experiment II: four pigs were previously vaccinated with the CSF virus PAV-250 strain, and at 14 days post-vaccination they were challenged with the CSF virus ALD strain. In both experiments, four susceptible pigs were exposed to infectious aerosols by placing them in a chamber connected by a duct to the adjacent pen containing the infected animals and were kept there for 86 hs. In Experiment I, pigs exposed to contaminated air died as a result of infection with CSF virus on days 14, 21 and 28 post-inhalation. These four pigs seroconverted from day 12 post-inhalation. CSF virus was isolated from these animals, and the fluorescent antibody test on tonsils was positive. In Experiment II, a vaccinated pig exposed to contaminated air did not seroconvert, nor was CSF virus isolated from lymphoid tissues. However, mild fluorescence in tonsil sections from these pigs was observed. In conclusion, CSF virus was shown to be transmitted by air at a distance of 1 m to susceptible pigs. Vaccination with the PAV-250 CSF virus strain protected the pigs from clinical disease under the same conditions. 相似文献
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Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens 总被引：8，自引：0，他引：8
An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n=47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n=96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection. 相似文献
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猪圆环病毒2型感染对猪瘟疫苗体液免疫应答的影响 总被引：3，自引：0，他引：3
采用ELISA方法对单独接种猪瘟疫苗组(CSFV组,n=3)、PCV2感染且出现病毒血症后接种猪瘟疫苗组(PCV2/CSFV组,n=3)及PCV2感染同时接种猪瘟疫苗组(CSFV/PCV2组,n=3)不同时相血清中的猪瘟抗体进行检测;并对PCV2感染对照组(PCV2组)及PCV2/CSFV和CSFV/PCV2组血清中PCV2特异的抗体和核酸分别进行ELISA和PCR检测.结果表明,在接种后52 d CSFV组血清中抗体的阻断值显著高于CSFV/PCV2组(P<0.05);接种后42 d和52 d CSFV组平均抗体效价明显高于PCV2/CSFV和CSFV/PCV2组,其中在52 d CSFV组抗体阳性率这100%(3/3)而PCV2/CSFV和CSFV/PCV2在相应时相抗体阳性率仅为67%(2/3).结果提示PCV2感染可在一定程度上抑制猪瘟疫苗特异性的抗体反应. 相似文献
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In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods. 总被引：8，自引：0，他引：8
Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available. 相似文献
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河南平顶山某猪场母猪出现较严重的流产和产死胎现象,且50日龄~70日龄仔猪出现神经症状,根据临床表现初步诊断为伪狂犬病。为排除猪繁殖与呼吸综合征和猪瘟,进行了实验室诊断。应用ELISA方法检测发病保育猪及母猪血清的伪狂犬病病毒野毒株gE抗体,并对发病仔猪病料进行了伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的实时荧光定量PCR检测。结果显示,伪狂犬病病毒野毒抗体阳性,实时荧光定量PCR检测确定仔猪病料中PRV核酸阳性,PRRSV和CSFV核酸阴性。结合临床症状及实验室检测,确诊该猪场发生的是猪伪狂犬病。 相似文献
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猪瘟单克隆抗体的制备及ACI-ELISA检测猪瘟病毒的研究 总被引：1，自引：0，他引：1
本研究用猪瘟石门毒(CSFV-Shimen)免疫BALB/C小鼠,按常规单克隆抗体(McAb)技术方法制作,最终获得4株McAb,分别命名为AC9、CF8、DG5和EC9,4株McAb与基因工程CSFV E2蛋白反应结果表明:AC9、CF8和EC9是抗CS-FV E2蛋白的McAb.用AC9和CF8McAb对CSFV进行抗原捕获间接ELJSA试验(ACI-ELISA),通过一元McAb和二元McAb CAI-ELISA试验的比较,结果表明AC9与CF8两种McAb有协同作用,其捕获CSFV的能力比一元McAb显著提高.方阵试验结果表明:McAb和血清多抗(PcAb)的最佳工作稀释度分别为1:400和1:200.特异性试验和敏感性试验结果显示本法特异性强,敏感性高.最后用ACI-ELISA与PCR对30份病料的检测结果比较,表明ACI-ELISA与PCR检测结果相符.上述结果说明本研究所获得AC9和CF8可用作猪瘟诊断试剂盒的研制,是检测CSFV的有效方法. 相似文献
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R. Jayakumar K. Nachimuthu V. D. Padmanaban 《Comparative immunology, microbiology and infectious diseases》1995,18(4):269-273
A modified enzyme linked immunosorbent assay (Dot ELISA) is described for visual detection of rabies antigen in animals. The test materials were dotted onto the nitrocellulose paper and allowed to react with rabies antiserum. The bound antigen—anti-body were reacted with a peroxidase conjugated antirabbit immunoglobulin. Positive reactions were easily visualized as brown dots after enzyme degradation of the substrate. A total of 400 specimens from various geographical locations were tested with the dot ELISA technique, and also with the fluorescent antibody test (FAT), which was used as a reference method. The concordance between the two tests was 95.25%. The dot ELISA may have potential applications as a rapid, simple and economical field test in the diagnosis of rabies. 相似文献
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为了解新疆北疆地区某规模化猪场几种主要传染病的抗体水平,便于及时发现猪场潜在的疾病风险。本试验采用间接酶联免疫吸附试验（ELISA）方法对某规模化猪场各阶段猪群进行猪瘟病毒（CSFV）抗体、猪繁殖与呼吸障碍综合征病毒（PRRSV）抗体、猪圆环病毒2型（PCV2）抗体、猪O型口蹄疫病毒（FMDV-O）抗体,及猪伪狂犬病病毒（PRV）gB与gE蛋白抗体进行检测。试验结果表明,该场的CSFV、PRRSV、PCV-2、FMDV-O、PRV-gB蛋白、PRV-gE蛋白的平均抗体阳性率分别为83.57%、90.56%、90.29%、71.86%、82.84%、18.29%;不同类别猪群间抗体水平参差不齐,种猪群的PRRSV抗体阳性率仅为52.63%,保育猪群的CSFV抗体阳性率仅为44.12%,育肥猪群的PRV-gB抗体阳性率为30.00%,种公猪的PRV-gE抗体阳性率为30.00%,育肥猪群的FMDV-O抗体阳性率为6.25%,基于以上试验结果,为该场免疫程序的制定和优化提供参考依据,以期达到有效控制及逐渐净化疫病的目的。 相似文献
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为建立一种快速、特异、灵敏的检测猪瘟病毒（classical swine fever virus,CSFV）巢式RT-PCR（nested RT-PCR）方法,本研究参照GenBank中公布的CSFV E2基因保守区域序列设计2对特异性引物,以CSFV总RNA为模板,优化反应条件,建立CSFV nested RT-PCR方法,对其进行特异性、敏感性和重复性试验;利用所建立的方法对35份临床疑似CSFV感染样品进行了应用检测,并对阳性PCR产物进行克隆测序鉴定。结果表明,本研究成功建立了CSFV nested RT-PCR检测方法,能够特异性地扩增CSFV,但对ST正常细胞对照和其他8种病原对照未扩增出任何条带;稳定性和重复性好;敏感性高,最低检测病毒含量为1 TCID50;自35份疑似CSFV感染样品中检出19份阳性样品,PCR阳性产物克隆测序结果表明均为CSFV E2基因片段。本研究成功建立了CSFV nested RT-PCR检测方法,可用于CSFV的快速检测,为CSF的早期检测诊断提供了特异、灵敏的方法。 相似文献
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Abstract AIM: To investigate the cause of classical swine fever (CSF) virus-seropositive animals in a nucleus pig-breeding herd in New Zealand, where porcine circovirus-associated disease had been diagnosed. CASE HISTORY AND CLINICAL FINDINGS: An exotic disease investigation was undertaken to exclude CSF and porcine reproductive and respiratory syndrome (PRRS) on a nucleus pig-breeding herd comprising approximately 300 breeding sows, 1,000 weaners, and 650 grower pigs. The herd was experiencing poor reproductive performance in sows, and breeding records showed a declining farrowing rate attributable to a single manager. The growing pigs (10–15 weeks old) were experiencing respiratory disease and wasting, and the mortality rate by pen varied between 9 and 20%. Post-mortem changes in affected grower pigs were consistent with circovirus-associated diseases. DIAGNOSTIC TESTING: Serological screening using an IDEXX-ELISA gave negative results for PRRS virus antibodies, but two grower pigs and one sow tested positive for CSF virus antibodies. These three seropositive animals remained positive to CSF virus, using three commercial ELISA test kits, over 27 weeks. A newly developed virus neutralisation test (VNT), using a New Zealand isolate of border disease (BD) virus, demonstrated that the seropositive pig sera had higher antibody titres to BD virus than to bovine viral diarrhoea (BVD) virus and CSF virus. PCR performed on tonsil, kidney, ileum and spleen gave negative results for CSF virus, and histopathology on lymph nodes, intestine, lung, kidney, liver and brain showed no evidence of the disease. Virus isolation performed on a number of samples was negative. CLINICAL RELEVANCE: The seropositive samples for CSF virus found in this investigation were likely to be a cross reaction to a pestivirus other than CSF virus. The finding of a possible endemic pestivirus capable of being transmitted between sheep and pigs on this farm may explain findings from previous serological survey work in New Zealand, and supports experience elsewhere, where BD virus was found to be the predominant ruminant pestivirus infecting pigs. The results show that pestivirus cross reactivity can result in unexpectedly high titres, and that testing with a full set of (local) pestiviruses is necessary to reach the correct conclusion. The investigation has direct relevance where pig herds with a low seroprevalence are encountered during surveillance for CSF. 相似文献
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Uttenthal A Le Potier MF Romero L De Mia GM Floegel-Niesmann G 《Veterinary microbiology》2001,83(2):85-106
Two commercial marker vaccines against classical swine fever virus (CSFV) and companion diagnostic tests were examined in 160 conventional pigs. To test the vaccines in a "worst case scenario", group of 10 weaners were vaccinated using a single dose of an E2 (gp55) based vaccine at days -21, -14, -10 or -7, and subsequently challenged at day 0. The challenge virus was CSFV 277, originating from a recent outbreak of classical swine fever (CSF) in Germany. In all groups, only 5 out of 10 pigs were challenged; the remaining 5 pigs served as vaccinated contact controls. Also, three control groups, each consisting of 10 non-vaccinated pigs, were challenged in parallel to the vaccinated animals. CSFV could be isolated from all non-vaccinated pigs. Among these pigs 40% displayed a chronic course of the infection (virus positive for more than 10 days). Pigs vaccinated 21 or 14 days before challenge displayed no clinical signs of CSFV after challenge. However, they were still able to replicate CSFV when challenged, as measured by reisolation of CSFV from leukocytes of the directly challenged pigs. CSFV could be isolated from the leucocytes of 25% of the pigs vaccinated 21 days before challenge and 50% of the pigs vaccinated 14 days before challenge. Chronic infection was not observed, but transmission to one vaccinated contact pig occurred. From all pigs vaccinated 10 or 7 days before challenge, CSFV could be reisolated. We observed a chronic course of infection in 5% of pigs vaccinated 10 days before challenge and in 30% of pigs vaccinated 7 days before challenge. The mortality rate was 20% in the pigs vaccinated 10 days before challenge, and varied between 20 and 80% in pigs vaccinated 7 days prior to challenge. The contact animals had lower mortality (0-20%) than directly challenged pigs, probably mirroring the delayed time point of infection. There was thus some protection against clinical illness by both marker vaccines, but not a solid protection against infection and virus shedding. The efficacy of the vaccine was best if used 3 weeks before challenge and a clear correlation between time interval from vaccination to challenge and the level of virus shedding was observed. Each vaccine had its own accompanying discriminatory ELISA, but 18% of the virus positive pigs never seroconverted in these tests. 相似文献
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Host-virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population. 相似文献