内源和外源性禽白血病病毒鉴别PCR检测方法的建立

为建立简单快捷的内、外源性禽白血病病毒(ALV)的鉴别检测方法,本研究根据外源性ALVA亚群标准株RAV-1的p27、env基因以及内源性ALVE亚群标准株ev1的env基因的保守序列,设计3对特异性引物,可分别对ALV、外源性ALV(A、B亚群)和内源性ALV(E、J亚群)进行扩增,扩增产物分别为793bp、387bp和234bp,通过对各反应条件的优化建立了同时检测并鉴别内、外源性ALV的多重PCR方法。该方法特异性良好、灵敏度可达到2×103copies,利用该方法和ELISA对5份现地病鸡组织样品和10枚疑似感染内源性ALV的鸡胚进行检测,结果表明:4份病鸡样品均扩增出3条特异性片段;而9枚鸡胚仅扩增出793bp和234bp的特异性片段,2种检测方法的符合率为100%。该方法为内、外源性ALV的临床鉴别诊断奠定了基础。     

某蛋用型父母代鸡群种蛋中禽白血病病毒的检测

为了检测某蛋用型父母代鸡群是否存在外源性禽白血病病毒（ALV）的感染,收集该鸡群的92枚鸡胚,每枚鸡胚无菌取蛋清2份,一份用禽白血病病毒p27抗原检测试剂盒直接检测群特异性p27抗原,同时分别将92枚鸡胚蛋清接种DF1细胞,培养维持8d后取细胞上清检测p27抗原。将p27抗原阳性样品,分别用针对ALV-J和ALV-AB的单因子血清做间接免疫荧光检测（IFA）。比较p27阳性检出率。结果表明,直接检测蛋清的检出率高于蛋清接种DF1后的细胞上清,接种DF1细胞后p27抗原阳性样品的IFA检测结果显示ALV-J的阳性率为100%,ALV-AB全部为阴性。说明该蛋用型父母代鸡群存在的外源性ALV感染主要是ALV-J,该研究为针对ALV的净化提供了可行性检测方法。     

禽白血病抗原快速检测试纸条的研制及初步应用

禽白血病给养鸡业带来极大损失,目前检测该病病原的方法主要有ELISA、PCR及RT-PCR、原位杂交(ISH)、间接免疫荧光(IFA)等。本研究应用2株抗禽白血病病毒(ALV)p27蛋白特异性单克隆抗体5D3和4F12研制了ALV快速检测试纸条。结果证明该试纸条具有良好的特异性,与H9亚型禽流感病毒、新城疫病毒、马立克氏病病毒、小鹅瘟病毒、鸡贫血病病毒没有交叉反应;采用p27表达蛋白检测试纸条灵敏性,检测下限达到70ng/mL;应用该试纸条检测71份临床样品,与商业ELISA试剂盒检测结果比较,二者符合率达到93%。该试纸条的研制,为基层快速检测ALV提供了条件。     

不同ELISA检测方法在检测出壳雏鸡禽白血病病毒感染中的应用

通过雏鸡胎粪中群特异性衣壳蛋白27(p27)抗原检测以判定禽白血病病毒(ALV)垂直传播是实施禽白血病净化的关键步骤,为对比不同ALV-p27抗原ELISA试剂盒对我国四类广泛流行的亚群ALV垂直传播的检测效果,本试验设置ALV-A、ALV-B、ALV-J和ALV-K感染组,以无菌生理盐水作为对照组,在8胚龄时以卵黄囊接种的方式感染SPF鸡胚,建立鸡胚携带ALV-A、ALV-B、ALV-J和ALV-K的垂直感染模型。采集各组雏鸡胎粪样品和血浆样品,死亡鸡胚肝脏样品,死亡雏鸡胎粪样品、血浆样品和肝脏样品,其中血浆和肝脏样品需接种鸡胚成纤维细胞系(DF-1细胞)进行病毒分离,各样品以4家公司提供的ALV-p27抗原ELISA试剂盒进行检测,并以实时荧光定量PCR(RT-qPCR)进行平行检测,比较阳性检出率和灵敏度。结果显示,4家公司的ALV-p27抗原ELISA试剂盒对于同一样品的阴阳性判定结果基本一致,但灵敏度存在差异;胎粪样品直接ELISA检测不能将ALV阳性鸡只全部检出,相对于胎粪样品,血浆和肝脏病毒分离样品ELISA检测更具有灵敏度优势。本试验所建立的对比方法不仅有助于评估4家公...     

土鸡源ALV-K HB2018003株的分离鉴定

为确认湖北某土鸡养殖场鸡只消瘦并伴有个别死亡的现象是否由禽白血病所引起,试验采用禽白血病群特异性抗原P27 ELISA和特异性针对禽白血病病毒(ALV)-A/J/K的多重PCR(Multi-PCR)的方法对送检的20只病鸡进行检测,对阳性样品进行病毒分离。结果表明:送检的20只病鸡中有11只为P27抗原阳性,阳性率为55%,将群特异性抗原P27和PCR检测均为阳性的病鸡血液接种DF-1细胞,成功分离到一株ALV-K,命名为HB2018003。说明该湖北土鸡养殖场鸡只感染ALV-K。     

PCR检测血液样品方法在禽白血病净化中的应用研究

本研究选择山东某地方品种祖代种公鸡的177份血液样品和177份泄殖腔棉拭子样品为试验材料,应用ELISA、病毒分离、PCR等方法对禽白血病病毒(Avian leukosis virus,ALV)p27抗原进行检测。结果显示,血清、泄殖腔棉拭子ALV-p27抗原阳性率分别为20.34%(36/177)、26.55%(47/177);病毒分离率为1.70%(3/177),血液样品PCR方法检出J亚群ALV(ALV-J)阳性率为0.56%(1/177),序列分析结果显示为ALV-J,但该血液样品的病毒分离结果为阴性。研究表明,PCR检测血液样品方法的应用有助于减少禽白血病净化所需的病毒分离结果的可能缺漏。在此基础上,优化并进一步拓展对其他亚群ALV的PCR方法检测,可作为禽白血病经典净化方法的有力补充,并加快我国地方品种鸡禽白血病的净化进程。     

禽白血病病毒衣壳蛋白p27基因绝对定量PCR方法的建立及应用

研究旨在建立一种检测禽白血病病毒(ALV)衣壳蛋白p27(ALV-p27)基因实时荧光定量PCR的方法。通过设计ALV-p27特异性的荧光定量PCR引物扩增基因并制备质粒标准品,同时建立检测病毒衣壳蛋白p27基因的实时荧光定量PCR方法,并利用该方法检测6日龄胚胎感染,1日龄出壳雏鸡不同组织中p27基因的分布和表达情况。结果表明,ALV-p27基因在1日龄雏鸡脑组织中表达水平最高,而在胸腺组织中表达水平最低。研究建立的检测ALV-p27基因绝对定量PCR方法为进一步研究其生物学功能提供了一种技术手段。     

乌骨鸡禽白血病病毒感染病例的诊断

从湖南某乌骨鸡种鸡场采集病鸡病料,通过临床症状、病理变化观察,以及ELISA及RT-PCR方法进行禽白血病病毒的检测,结果表明该样品中ALV p27抗原阳性,经RT-PCR检测为ALV感染,病理变化可见肝脏出现明显灰白结节或弥散病灶,组织学可见成淋巴细胞浸润,诊断为ALV感染所致的淋巴细胞性白血病。     

核酸斑点杂交法与ELISA在SPF鸡检测弱毒疫苗中ALV污染的应用与比较

本研究对SPF鸡检查法在检测弱毒疫苗中低剂量禽白血病病毒(ALV)污染时的核酸斑点杂交法和ELISA进行了比较。分别以10 TCID_(50)/羽份和5 TCID50/羽份ALV-A人为污染一批商品化新城疫(ND)活疫苗,将污染ND疫苗各接种10只SPF鸡,以ELISA定期检测ALV抗体并利用PCR结合核酸斑点杂交法检测血液中ALV核酸。结果显示,在免疫污染疫苗后连续3周内ALV抗体全部为阴性,而核酸斑点杂交法检测表明自免疫后两周开始就出现ALV阳性。结果提示在利用经典的SPF鸡检查法检测弱毒疫苗中ALV污染时,结合对病原核酸的检测有助于节省检测时间和降低漏检率。     

广西龙胜县某凤鸡养殖场禽白血病检测

为了解广西纯地方品种龙胜凤鸡中禽白血病（AL）的流行情况,采集龙胜凤鸡的肛拭子217份、蛋清样品59份和血清样品276份,采用禽白血病病毒（ALV）群特异性抗原p27和A/B、J亚群抗体检测试剂盒分别对肛拭子、蛋清样品和血清样品进行检测.结果显示,p27抗原的总阳性率为26.45％（68/276）;A/B亚群抗体的总阳性率为24.64％（10/276）;J亚群抗体的阳性率为3.62％（73/276）.结果表明,龙胜凤鸡的种群已存在ALV的感染,以A/B亚群感染为主.     

Diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in individual milk and bulk milk samples of dairy herds

The objective of the study was to determine the diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Map) in individual milk samples and in bulk milk samples. For individual milk samples the specificity of the Pourquier ELISA was estimated by testing a panel of individual milk samples from certified Map-free herds. The relative sensitivity of the assay in individual milk samples and agreement of the results with those of serum samples was estimated by testing panels of paired serum-milk samples from seropositive cattle, whole-herd investigations, and moderate or heavy shedders. The specificity of the ELISA for individual milk samples was still 99.8% at a cut-off of 20% sample to positive (S/P) value, clearly lower than the cut-off defined by the manufacturer (30% S/P). The relative sensitivity for individual milk samples as compared with positive serum samples was 87% for a cut-off of 20% S/P, and 80% for a cut-off of 30% S/P. The sensitivity of this ELISA for detection of high shedders was >90% both for individual milk and serum samples, also agreement was very good (kappa=0.91 for all paired samples). The specificity of the Pourquier ELISA in bulk milk samples was investigated by testing bulk milk samples from certified Map-free herds. Feasibility of bulk milk testing was investigated by titrating ELISA positive individual milk samples in negative milk. In addition, 383 bulk milk samples from herds with a known within-herd seroprevalence were tested. The specificity of the ELISA for bulk milk samples was 100% at a cut-off of 12.5% S/P. At the cut-off recommended by the manufacturer (30% S/P) performance of the bulk milk ELISA related to herd status (> or =2 seropositive cows) was rather poor, corresponding with a sensitivity of 24% and a specificity of 99% relative to serology. However, at the revised cut-off for bulk milk of 12.5% S/P and a within-herd seroprevalence of > or =3%, sensitivity and specificity relative to serology were 85% and 96%, respectively. Given the current herd-level seroprevalence in The Netherlands, these test characteristics corresponded with positive and negative predictive values for bulk milk of 67% and 94%, respectively. In conclusion, the diagnostic performance of the Pourquier ELISA for individual milk samples creates opportunities for a cheaper and more feasible testing scheme, while the diagnostic performance for bulk milk samples warrants further consideration.     

Evaluation of radiolabeled and colorimetric DNA probes in comparison with an antigen screening assay for the detection of Salmonella from poultry farms

A total of 48 environmental drag-swab samples from various poultry farms were tested for the presence of Salmonella spp. by culture, an enzyme-linked immunosorbent assay-based Salmonella antigen screening (SAS) assay, and two DNA probes (radiolabeled and colorimetric). The radiolabeled DNA probe was allowed to hybridize with culture-positive samples (n = 8) and was found to detect Salmonella spp. in all cases (100%). Both of the probes, subsequently hybridized with culture-negative samples (n = 8), were observed to yield good agreement (91%) with the culture findings. The remaining samples (n = 32) were tested by the SAS assay, and where there was no agreement between the culture and SAS, samples were further examined by the DNA probes. Results using both probes agreed with those obtained by culturing the samples but did not agree with the SAS assay result when the ratio of samples tested to samples positive (S/P) cutoff value used was 0.5.     

Verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking ELISA methods

Monoclonal antibodies to group A rotavirus Vp6 protein were prepared and used for verification of three blocking enzyme-linked immunosorbent assay (ELISA) modifications to detect rotavirus A. Selected competitive blocking ELISA (CB-ELISA) and electron microscopy (EM) were used for examination of 194 field faecal samples of piglets affected with diarrhoea. Rotavirus was detected in 43 samples (22.2%) by CB-ELISA method, whereas in 26 (13.4%) samples by EM examination. However, of 26 samples positive by EM, rotavirus A was detected by CB-ELISA in 19 (73.1%) samples; indicating the share of group A rotavirus in all cases of gastroenteritis caused by rotavirus. The sensitivity and specificity of the CB-ELISA was verified both by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV) in each analysis and by comparative examination of samples with the commercial ELISA kit. The CB-ELISA sensitivity was positively affected by examination of samples in the presence of chelating agent.     

Evidence of mixed infection of peste des petits ruminants virus and bluetongue virus in a flock of goats as confirmed by detection of antigen, antibody and nucleic acid of both the viruses

A mixed infection with peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) occurred in goats which exhibited symptoms characteristic of PPR. A number of samples were collected from ailing or dead goats for labrotory diagnosis. Antibody to BTV and PPRV was detected in sera samples by competitive ELISA. No PPRV antigen was detected in tissue samples like lung and spleen, however, presence of PPRV antigen in some sera samples was confirmed by sandwich ELISA. All the blood samples collected from the ailing animals were found positive for BTV antigen by a sandwich ELISA. BTV- and PPRV nucleic acids were amplified from the pooled blood and tissue samples respectively by RT-PCR assays. The identity of the amplicons was confirmed by cloning and sequencing. All these tests confirm that the goats were infected with PPRV and BTV simultaneously. Isolation of viruses from the clinical samples is underway.     

广东省东莞市猪乙型脑炎流行病学调查

为了解东莞市猪乙型脑炎的流行情况,于2009年-2011年对东莞市内猪养殖场采集猪血清4 282份,进行血清学与病原学检测。其中,应用ELISA检测方法对3 918份血清进行血清学检测,检测抗体阳性数2217份,抗体阳性率56.58%;应用实时定量RT-PCR检测方法对364份血清进行病原学检测,检测病毒阳性10份,病毒阳性检出率2.75%。结果表明,东莞市内饲养的猪存在乙型脑炎带毒的情况,免疫接种猪群的免疫效果比较理想,未免猪群存在乙型脑炎感染情况。     

胶乳凝集抑制试验快速鉴定肉种类研究

以羧化聚苯乙烯胶乳作载体,将动物血清分别共价交联到载体微球表面,制成免疫微球。将此微球同相应抗血清配对,进行胶乳凝集抑制试验以鉴定牛肉、马肉、羊肉、狗肉和猪肉。通过对162份牛肉、43份马肉、100份猪肉、34份羊肉、30份狗肉、10份骡肉、4份驴肉和7份山羊肉的鉴定,制备的5种胶乳试剂敏感性均为100％。除马肉胶乳试剂和羊肉胶乳试剂有种属内交叉反应外,其它胶乳试剂特异性为100％。实验具有良好的重复性,试剂稳定性强,易于保存。5分钟内出结果,操作简便,适于基层单位应用。     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.     

The effect of Linola and W92/72 transgenic flax seeds on the rabbit caecal fermentation--in vitro study

The effect of W92/72 transgenic flax seeds taken from a variety of Linola on the production of SCFA, ammonia and methane by bacteria inhabiting rabbit caecum was studied. The in vitro method was used where caecal contents from rabbits was incubated with W92/72 transgenic or Linola flax seeds, or without any additives (control samples). The total concentration of SCFA was higher in samples with the addition of flax seeds than in the control samples. The increase in concentrations of acetic, propionic and butyric acids was the highest in samples with Linola seeds added. A higher percentage of propionic and butyric acids was observed in the contents incubated with addition of flax seeds as compared to the control samples. This increase was the result of a percentage decrease in acetic acid. No differences were observed in the concentration of ammonia between fermented samples. Moreover, the addition of flax seeds resulted in slight decrease of pH in incubated samples. In gas samples, the methane level was higher in samples with flax seeds added, although the highest level was found in samples with transgenic seeds. In addition, gas pressure was significantly higher in samples with flax seeds added as compared to control samples, and this may indicate a higher intensity of microbiological fermentation processes. These studies suggest that neither Linola nor W92/72 flax seeds have any unfavorable effect on the caecal microflora activity of rabbits. A beneficial influence of flax seeds on the microbiological fermentation process in rabbit caecum was observed, based on an increase in percentage ratio of propionic acid in samples with flax seeds added.     

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.