应用ELISA间接法检测猪流行性腹泻抗体的研究

应用猪流行性腹泻病毒(PEDV)吉(J)毒株猪胎肠单层细胞培养物,以冻融法制备抗原,建立了酶联免疫吸附试验(ELISA)间接法检测PED抗体的方法。对30头份经直接免疫荧光技术证实的PED病猪群血清测定,97％为阳性;检测32头份无PED猪场猪血清,93％为阴性。对1份PED“华毒株”,1份“川毒株”以及3头份“吉毒株”PED免疫血清测定,均为阳性。对猪传染性胃肠炎血清、猪轮状病毒病血清、疑似猪血球凝集性脑脊髓炎血清、鸡传染性支气管炎血清及猪梭菌性肠炎血清共16头份检测均为阴性,证明PEDV不与TGEV等其它3种冠状病毒血清发生交叉反应。89头份疫区猪血清的区域性试验阳性率为75％(67/89);对82头份屠宰猪血清测定,53％阳性。抗原包被板保存期试验表明,包被板在-20℃可保存2个月。     

间接ELISA对鸡猪空肠弯曲菌快速检验的实验研究

以方阵滴定法确定了空肠弯曲菌抗血清、酶结合物的最佳工作浓度和孵育时间,从而立了快速检测空肠弯曲菌的间接ELISA。该试验可检出含8～10个空肠弯曲菌经24h培养的标本(含30万个菌/ml的肉汤培养物);不与沙门氏菌等15种对照菌及其混合液发生交叉反应。以鸡源和猪源空肠弯曲菌抗血清分别作为ELISA的第一抗体,对144份鸡泄殖腔粪便标本和116份猪直肠粪便标本的选择性增菌肉汤培养物进行了检测。其阳性率分别为83.3％和79.3％,可于28h内报告结果;常规法对上述标本的阳性检出率分别为85.4％和81％,但程序繁琐,需5～7d方能获得最终检查结果。两种方法对鸡、猪粪便标本检测结果的阳性符合率,分别为97.5％与97.9％。     

采用不同血清学检测方法对猪蓝耳病免疫效果调查

本文采用猪蓝耳病通用型抗体ELISA检测法和猪蓝耳病通用型抗体快速检测卡检测法(胶体金法),对180份已免高致病性猪繁殖与呼吸障碍综合征苗的猪血样进行免疫抗体检测。结果显示,采用ELISA检测法检测的猪蓝耳病免疫抗体阳性率为82.2%;用胶体金法检测的猪蓝耳病免疫抗体阳性率为60.6%。二者相比,阳性符合率高(100%)即胶体金法抗体滴度大于1∶40以上者在ELISA法检测中为阳性;阴性符合率低即胶体金法抗体滴度大于1∶20小于1∶40之间者在ELISA法检测中也为阳性,只有小于1∶20者2种方法检测结果都为阴性。胶体金法简单,快速,易操作,可用于乡镇畜牧兽医站或养殖场的自行检测。     

上海地区猪和奶牛血清中TTV（Torque Teno virns）感染的检测

于2009年3～5月采集上海地区的200份牛血清和400份猪血清,采用ELISA和荧光PCR方法检测其TTV（Torque Teno virus）感染状况。经ELISA检测,牛血清样品中检出阳性数2份,阳性率1．0％;猪血清样品中共检出阳性数15份,阳性率3．75％,其中生产母猪和育肥肉猪的阳性率分别为5．5％和2．0％,而荧光PCR检测结果均为阴性。结果表明上海地区猪和牛TTV感染现状处于较低水平。     

应用斑点酶联免疫吸附试验快速检测猪弓形虫抗体

将斑点酶联免疫吸附试验(Dot—ELISA)用于检测猪弓形虫抗体,并与常规ELISA和IHA法进行了比较。结果,对102份滴度下降的猪阳性血清检测,弓形虫抗体阳性检出率,Dot—ELISA为66.67％(68/102),常规ELISA为48.04％(49/102),IHA为27.45％(28/102);对675头商品猪血清检测,弓形虫抗体阳性检出率,Dot—ELISA为48.15％(325/675),常规ELISA为41.93％(283/675),IHA为33.80％(228/675);与3种寄生虫(猪囊虫、猪旋毛虫、住肉孢子虫)阳性血清无交叉反应;对123份弓形虫抗体阳性和158份阴性猪血清进行3次重复性试验,结果完全一致。结果证明,该法敏感性高,特异性强,操作简便快速(于接到病料后2h报告结果),便于在基层推广使用。     

某猪场“僵猪”致病原因分析

为分析某猪场出现"僵猪"的可能原因,对该猪场的11头"僵猪"采集血液样品,用RT-PCR方法检测猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV),用PCR方法检测猪伪狂犬病毒(PRV)和猪圆环病毒2型(PCV-2)。结果11份样品的CSFV、PRRSV及PRV检测均为阴性,PCV-2经套式PCR检测均为阳性。对11份样品分离血清,应用酶联免疫吸附试验(ELISA)检测PCV-2血清抗体,结果均为阳性。随后又对与11头"僵猪"同舍的部分健康猪进行了上述4种病毒的RT-PCR及PCR检测,结果均为阴性。由此推测PCV-2极有可能是该猪场形成"僵猪"的主要因素之一。     

用BA-ELISA法诊断猪囊虫病研究

用方阵滴定确定的BA－ELISA法对宰后检验确认后为猪囊虫病的阳笥血清26份，阴性血清808份进行检测，其阳性符合率为100％，阴性附合率为99．9％，进行重复性试验，重复率为100％，经与旋毛虫，弓形虫、细颈囊尾蚴和猪蛔虫等病的阳性血清试验均无交叉反应，BA－ELISA法的敏感性高于常规ELISA法。     

两种奶牛结核病检测方法与PPD检测符合率比较

对新疆某规模化奶牛场的2 106头奶牛用PPD进行普检,共检出结核病阳性牛26头;随机采集20头阴性牛和26头阳性牛抗凝血和全血,进行牛结核γ-干扰素ELISA试验和胶体金检测,然后将检测结果与PPD检测结果进行比对。结果显示:3种方法检测均为阳性的16份,均为阴性的10份,总符合率为57%。PPD和γ-干扰素ELISA相比,均为阳性的23份,阳性符合率为88%(23/26),均为阴性的11份,阴性符合率为55%(11/20);PPD和胶体金检测相比,均为阳性的19份,阳性符合率为73%(19/26),均为阴性的19份,阴性符合率为95%(19/20)。结果表明:3种检测方法的符合率较低;每种检测方法各有优缺点,γ-干扰素ELISA敏感性较高,而胶体金特异性较高。因此,在实际操作中应根据需要,合理选择相应检测方法。     

应用斑点酶联免疫吸附试验诊断猪旋毛虫病的研究

利用差速离心,TritonX-100处理,超速离心,在国内首次制得旋毛虫S_3抗原,其抗原性优于ES抗原。用该抗原建立诊断猪旋毛虫病的Dot-ELISA比常规ELISA敏感,与猪囊虫血清、猪弓形虫血清无交叉反应。对70份感染猪旋毛虫的阳性血清进行Dot-ELISA和常规ELISA检测,分别检出68份和66份,阳性符合率分别为97.1％和94.3％。对182份采自非疫区的被检仔猪血清进行检测,均末出现阳性,阴性符合率为100％。结果表明,Dot-ELISA除和常规ELISA一样具有敏感性高、特异性强、稳定性好等优点外,还具有快速简便的优点。     

猪瘟抗体阴性猪筛选方法的比较

评价ELISA法用于筛选猪瘟抗体阴性猪的可行性,分别采用两种商品化猪瘟抗体ELISA检测试剂盒检测了61份猪血清样品,并与兔体中和试验方法进行了比较.兔体中和试验法检测出4份阳性、2份可疑、55份阴性,而两种ELISA试剂盒均检测出6份阳性、55份阴性;两种ELISA方法与兔体中和试验检测结果阴性符合率均为100%(55/55).结果表明,ELISA法更加敏感,可以替代兔体中和试验方法用于筛选猪瘟抗体阴性猪.     

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffin embedded sections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues.     

囊虫病猪循环抗原的研究(初报)

用琼脂凝胶双扩散试验发现了囊虫病猪的循环抗原,并用反向间接血凝技术作了检测。103份囊虫病猪血清琼扩试验对循环抗原的检出率为22.33％(23/103);反向间接血凝试验检出率为32.03％(33/103),其余70份呈阴性反应的血清经免疫复合物解离后,又有25份呈阳性反应,总检出率为56.31％(58/103)。     

山西某鸡棚改造育肥猪场腹泻病因诊断

为诊断山西省某鸡棚改猪场80~100日龄猪群发生腹泻病的病因。采集40份猪的肛拭子样品,通过实时荧光扩增和细菌分离鉴定的方法,进行实验室诊断。检测结果显示,腹泻样品中轮状病毒（Porcine rotavirus, PoRV）核酸100%为阳性,猪圆环病毒2型（Porcine circovirus type 2, PCV-2）核酸30%为阳性。猪流行性腹泻病毒(Porcine epidemic diarrhea, PED)、猪伪狂犬野毒(Pseudorabies virus, PrV)、猪瘟病毒(Swine fever virus, SFV)核酸检测均为阴性,大肠杆菌（Escherichia coli, E.coli）和沙门氏菌(Salmonella enteriditis, SE)100%呈阳性,仅对万古霉素和头孢曲松同时敏感。     

Herd-level risk factors for Salmonella enterica subsp. enterica in U.S. market pigs

Midwest U.S. herds (n = 63) were studied to identify risk factors for harboring Salmonella enterica among slaughter-weight pigs. Samples collected on farms (feces) and at slaughter (distal colonic content, cecal content and ileocolic lymph nodes) were cultured using conventional means. Approximately 15 pigs were studied per herd, for a total of 3754 samples. The proportion of pigs positive in one or more samples was calculated for each herd. Herd characteristics were described by a combination of interview and written survey. Logistic regression was used to detect relationships between the detection of Salmonella and potential herd-level risk factors. The mean individual pig prevalence was 5% for feces, 4% for distal colonic content, 15% for ileocolic lymph nodes, and 17% for cecal contents. One or more Salmonella isolates were detected in at least one sample type in every herd. The five most common serovars were S. Agona, S. Derby, S. Schwarzengrund, S. Typhimurium and S. Senftenberg, with 25 additional serovars detected. Salmonella prevalence estimates were positively correlated among all samples except distal colonic content and ileocolic lymph nodes. Pigs with culture positive fecal samples were at increased odds of being detected positive for each of the slaughter-collected samples examined, namely distal colonic content (OR = 30.5), ileocolic lymph nodes (OR = 12.9) and cecal content (OR = 23.2). Herds with positive fecal sample(s) had increased odds of having positive cecal content (OR > 1.5), distal colonic content (OR = 15.3) and ileocolic lymph nodes (OR = 12.7). Pigs from herds with at least some bowl drinkers had eight-fold higher odds of testing Salmonella positive than did pigs from herds with only nipple drinkers. Pigs from herds with only dry feeders had five-fold higher odds of testing Salmonella positive when compared with pigs from herds with combinations of wet/dry style feeders. Interventions at these two points should be considered when designing growing pig facilities to reduce Salmonella shedding.     

空肠弯曲菌共同抗原单克隆抗体的研制及其在细菌检验中应用——Ⅲ.在细菌快速检验中的应用

应用抗空肠弯曲菌共同抗原的单克隆抗体(4A7),建立了适用于空肠弯曲菌快速检验的间接-ELISA和BA-ELISA。两种方法的特异性检查表明,对受试的32种其它细菌,除与幽门弯曲菌发生交叉反应外,其余均为阴性反应。对空弯菌纯培养物检测的敏感性,BA-ELISA为3.2×104个菌/mL,间接-ELISA为1.6×105个菌/mL;对细菌动态培养物检测,BA-ELISA可检出接种约5个菌/mL、间接-ELISA可检出接种约10个菌/mL的24 h增菌培养物。应用BA-ELISA对人(140份)、猪(140份)、禽类(364份)及特种经济动物(265份)标本进行检测,阳性率依次为121%、52%、58.2%和32.8%;应用间接-ELISA对人(140份)、禽类(222份)及特种经济动物(265份)标本进行检测,阳性率依次为11.4%、61.7%、31.3%。两种方法的结果均与常规培养法相差不显著,可在27h内报告结果。     

Control of Isospora suis-induced coccidiosis on a swine farm

Results of a program designed to control neonatal porcine coccidiosis on a total confinement, farrow-to-finish swine farm are reported. The control program consisted of washing, phenol disinfection, and steam cleaning of farrowing houses and treatment of sows with amprolium HCl before and after farrowing. Before initiation of the control program, 88.9% of the sows examined in the farrowing house were negative for coccidian oocysts, 9.9% were positive for Eimeria spp, and 1.2% were positive for Isospora suis. Most pigs nursing on sows before initiation of the control program had diarrhea at 5 to 10 days of age, which led to dehydration and weight loss. Morbidity was high, and mortality was moderate. Composite fecal samples from these litters were all positive (100%) for I suis. After initiation of the control program, 99.6% of the sows examined in the farrowing house were negative for coccidian oocysts and 0.4% were positive for Eimeria spp. Clinical signs of coccidiosis were rarely present in nursing pigs examined after the control program was initiated; however, I suis was still present in 19.8% of the composite fecal samples from pigs examined. An association between oocyst production in sows and I suis infections in pigs was not found in the present study. Oocysts of Eimeria spp were not found in the feces from the pigs.     

猪病毒性腹泻分子流行病学调查

为了解近年来中国猪病毒性腹泻的发生现状,于2011年4月至2012年4月利用多重RT-PCR方法对采集于13个省市的猪腹泻样品和临床健康的样品进行了检测,结果显示,腹泻猪群样品中TGEV阳性率为2.65%,PEDV阳性率为24.49%,ARV阳性率为3.20%;在肠道组织样品中,TGEV的阳性率为3.11%,PEDV为14.83%,ARV为1.67%;粪便样品中,TGEV的阳性率为2.80%,PEDV为28.42%,ARV为4.86%;母猪(所产仔猪腹泻)乳汁中,TGEV阳性率为1.08%,PEDV为31.89%,ARV为0.54%。健康猪群样品中,保育与育肥阶段猪中PEDV阳性率为2.13%,ARV阳性率为1.42%;哺乳仔猪中ARV阳性率为12.86%。由此可知,目前中国猪病毒性腹泻以PED为主要病因,PEDV和TGEV在幼龄猪群中存在隐性感染现象,且3种病毒性腹泻均可通过母乳传播病毒。     

广西猪流行性腹泻流行病学调查

对广西6个市的6个规模化猪场进行了猪流行性腹泻流行病学调查。对其中4个猪场统计,在11090头猪中,患流行性腹泻的猪4658头,发病率为42%;死亡265头,病死率为5.69%。对6个猪场的170头份血清检测,阳性血清14份,阳性率为8.24%。结果表明广西的一些规模化猪场有猪流行性腹泻存在。     

Enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the coronavirus-like agent in feces of pigs naturally affected with porcine epidemic diarrhea (PED) or experimentally infected with the CV777 isolate. The assay was specific and more sensitive than electron microscopy. An ELISA blocking assay is described for the detection and titration of antibodies. Specific antibody formation was demonstrated in pigs experimentally infected with CV777 and in swine naturally affected with PED.     

猪流行性腹泻病毒间接ELISA检测方法的建立

猪流行性腹泻（porcine epidemic diarrhea,PED）是由猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）引起的猪的一种急性、高度传染性肠道疾病,给养猪业造成了严重的经济损失。本研究以纯化的PEDV 为包被抗原,通过优化 ELISA 反应条件,建立了间接 ELISA 抗体检测方法,其反应条件为：抗原最佳包被浓度为 20 μg/mL,血清标准品最佳稀释度为 1：500,包被时间为4℃过夜,5%小牛血清37℃封闭1 h,二抗 1：10 000稀释,37℃作用1 h,抗体临界值为 D_{450 nm}≥ 0.289判为阳性,D_{450 nm}≤ 0.236判为阴性,介于二者之间为可疑。检测猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、伪狂犬病毒、细小病毒和口蹄疫病毒标准血清抗体均为阴性,重复试验变异系数均小于10%,对江苏、江西、福建、广东地区74份猪血清样品进行检测,抗体阳性率达84%,表明该方法具有较好敏感性、特异性和重复性,可用于 PEDV 抗体检测和流行病学调查。