动物布鲁氏菌病实验室诊断与检测技术进展

实验室诊断与检测对防治动物布鲁氏菌病（brucellosis）至关重要,其不仅可早期发现传染源和隐性感染动物,而且可检测动物通过疫苗免疫后产生的抗体水平,为合理制定免疫程序提供科学依据。实验室诊断与检测动物布鲁氏菌病常规方法包括：一般实验室检查、免疫血清学试验和病原学检查;高新生物技术包括：应用分子生物学技术检测及荧光偏振试验（FPA）。目前,我国对该病诊断的国家标准主要是依据细菌学和血清学检测结果来确定。其中免疫血清学试验是我国最常用的诊断与检测方法,该方法不仅可用于动物布鲁氏菌病的诊断与检测,而且还用于人感染布鲁氏菌病的诊断与检测。但是,这种诊断与检测方法在我国已经使用半个多世纪,并且存在着许多弊端,诸如：非特异性反应、假阳性、前带现象或动物自身免疫抑制等,因此还远不能满足对动物布鲁氏菌病的防控需求。探索动物布鲁氏菌病的实验室诊断与检测新技术以及高新分子生物学技术的研究进展,对于早期开展动物布鲁氏菌病的分子流行病学调查、早期确诊和防控均具有重要意义。     

布鲁氏菌病研究进展

布鲁氏菌病的早期诊断及鉴定是该病防控的重要环节,其诊断技术主要包括细菌学诊断技术、血清学诊断技术以及分子生物学诊断技术。布鲁氏菌病防控应坚持预防为主的策略,其免疫主要以弱毒活疫苗为主,随着分子生物学及重组DNA技术的发展,基因工程疫苗成为近年研究热点,但目前尚未有鉴别疫苗免疫和自然感染的鉴别诊断技术及相应的疫苗制品。本文对布鲁氏菌病的病原学、流行病学、诊断技术和疫苗研究进展等方面进行概述,以期为建立灵敏性高、特异性强、高效便捷且易推广的布鲁氏菌病诊断技术和研发更加安全有效的布鲁氏菌病疫苗提供基础。     

我国家畜布鲁氏菌病流行现状与诊断技术研究进展

布鲁氏菌病是由细胞内寄生的布鲁氏菌引起的一种危害极其严重的人畜共患传染病。本文详细分析了布鲁氏菌病在我国人畜间最新流行现状,重点介绍了布鲁氏菌分型及诊断技术,特别是免疫与自然感染鉴别诊断技术的最新研究进展,同时对今后我国布鲁氏菌病的防控提出了建议。     

动物布鲁氏菌病实验室诊断与检测技术进展

实验室诊断与检测对防治动物布鲁氏菌病(brucello-sis)至关重要,其不仅可早期发现传染源和隐性感染动物,而且可检测动物通过疫苗免疫后产生的抗体水平,为合理制定免疫程序提供科学依据.实验室诊断与检测动物布鲁氏菌病常规方法包括:一般实验室检查、免疫血清学试验和病原学检查;高新生物技术包括:应用分子生物学技术检测及荧光偏振试验(FPA).目前,我国对该病诊断的国家标准主要是依据细菌学和血清学检测结果来确定.其中免疫血清学试验是我国最常用的诊断与检测方法,该方法不仅可用于动物布鲁氏菌病的诊断与检测,而且还用于人感染布鲁氏菌病的诊断与检测.但是,这种诊断与检测方法在我国已经使用半个多世纪,并且存在着许多弊端,诸如:非特异性反应、假阳性、前带现象或动物自身免疫抑制等,因此还远不能满足对动物布鲁氏菌病的防控需求.探索动物布鲁氏菌病的实验室诊断与检测新技术以及高新分子生物学技术的研究进展,对于早期开展动物布鲁氏菌病的分子流行病学调查、早期确诊和防控均具有重要意义.     

庆阳市2009～2013年间布鲁氏菌病流行病学调查

基于分析庆阳市畜间布鲁氏菌病（Brucellosis）流行特征、疫情变化趋势及原因,为制定布鲁氏菌病防治对策提供科学评价依据.按照《动物布鲁氏菌病诊断技术》（GB/T 18646～2002）虎红平板凝集试验（RBPT）和试管凝集试验（SAT）操作方法进行采样和检测.共检测羊、牛、猪、犬血清共计209 864份,检出布病阳性血清1415份,阳性率为0.67％.     

对免疫布鲁氏菌A19株疫苗奶牛的检测

通过虎红平板凝集试验（RBPT）和标准试管凝集试验（SAT）,对免疫布鲁氏菌A19株疫苗后的奶牛进行抽检,阳性头数分别为3头和2头。同时,在SAT检测阳性奶牛的奶样中,培养分离到疑似布鲁氏菌,经染色,在显微镜下观察到布鲁氏菌。     

布鲁氏菌S19疫苗应用及研究进展

布鲁氏菌病（布病）是由布鲁氏菌引起的一种重要的人兽共患病,该病对畜牧业和人类健康均构成严重威胁,使用疫苗免疫是防控布病的重要措施之一。光滑型牛种布鲁氏菌19（S19）活疫苗是世界上第一个被广泛应用且效果良好的布病疫苗,至今为止仍是使用最广泛的疫苗之一。本文主要从应用情况及目前研究进展两大方面对S19疫苗进行概述,以期为日后使用该疫苗预防布鲁氏菌病提供借鉴及为疫苗研究提供思路。     

牛布鲁氏菌A19号疫苗免疫血清几种检测方法的比较

为选择科学适用的牛布鲁氏菌病净化检测方法,对1 721份免疫布鲁氏菌A19号疫苗18个月后的牛血清,分别用虎红平板凝集试验（RBT）、试管凝集试验（SAT）、竞争酶联免疫吸附试验（cELISA）、间接酶联免疫吸附试验（iELISA）和荧光偏振试验（FPA）5种血清学检测方法,以及环介导等温扩增技术（LAMP）和荧光定量 PCR病原学检测方法进行检测,分析比对不同检测方法的特异性、敏感性、误诊率、漏诊率、符合率、Kappa值等。结果显示：与SAT相比,其他4种血清学检测方法的敏感性、特异性和符合率从高到低依次为iELISA、FPA、cELISA、RBT,其中敏感性均在85.00%以上,特异性均在97.60%以上,符合率均在97.50%以上,Kappa值均≥1.00;与PCR相比,LAMP方法敏感性低（9.09%）,但特异性强（99.65%）,与PCR符合率高（98.49%）。结果表明：血清学检测方法较为敏感特异,符合率和一致性均较高,而分子生物学检测方法特异性强,不易误诊和漏诊。因此,对于牛群的布鲁氏菌病净化,要结合牛群污染和免疫情况以及净化的不同阶段选择适用的检测方法,仅用一种方法可能会存在偏差。建议先用iELISA或FPA或RBT进行初筛,再用cELISA或SAT进行确诊,进而对确诊为阳性牛的阴道拭子或奶样进行分子生物学检测,以确定是否存在布鲁氏菌感染。本研究为免疫牛群的布鲁氏菌病净化检测方法选择提供了参考。     

动物布鲁氏菌病实验室检测技术研究进展

布鲁氏菌病是由布鲁氏菌引起的人畜共患传染病,对我国畜牧业生产和公共卫生安全造成严重的威胁。科学准确诊断是布鲁氏菌病防控的重要技术依据,然而布鲁氏菌病的检测和确诊一直以来都并非易事,常常需要通过多种检测结果的综合分析,因此发展敏感性高、特异性好的布鲁氏菌的检测技术十分重要。总结并分析了近年来布鲁氏菌检测技术基础研究和临床应用研究方面的最新进展,结合国家动物布鲁氏菌病参考实验室工作经验,对各类检测方法的优缺点和适用范围进行了分析,旨在为动物布病诊断技术的选择和推广应用提供理论和实践依据,为动物布鲁氏菌诊断技术的研究提供方向性思考。     

牛布鲁氏菌抗体荧光快速检测试纸卡的研制

为研制一种快速检测牛布鲁氏菌抗体的便捷试纸卡,采用间接荧光免疫层析技术,以荧光微球为标记物,与兔抗牛IgG偶联,以布鲁氏菌脂多糖（LPS）抗原包被硝酸纤维素膜,通过反应条件优化,研制牛布鲁氏菌抗体荧光微球快速检测试纸卡。结果显示：该试纸卡灵敏度可达0.8 IU/mL,与牛口蹄疫病毒O型、牛口蹄疫病毒A型、牛病毒性腹泻病毒、牛传染性鼻气管炎病毒的抗体阳性血清均无交叉反应;对469份临床牛血清进行检测,同时与荧光偏振方法进行比较,发现两种方法的符合率为100%。结果表明,本次研制的牛布鲁氏菌抗体荧光微球检测试纸卡灵敏度高,特异性好,适用于临床样品的快速检测。本试纸卡的成功研制为牛布鲁氏菌病免疫效果评估和准确诊断提供了一种简便的血清学诊断方法。     

猪圆环病毒检测技术研究进展

猪圆环病毒病是由猪圆环病毒(PCV)感染引起的,以免疫抑制为特征的一类病毒性传染病,临床表现主要是由PCV-2引起的仔猪断奶后多系统衰竭综合征。文章就近几年国内外对该病毒检测技术,包括病毒分离、电镜观察、聚合酶链式反应、间接免疫荧光试验、免疫组织化学技术、酶联免疫吸附试验、原位核酸杂交试验等的研究进展做了阐述。     

A real-time polymerase chain reaction assay to detect single nucleotide polymorphisms at codon 171 in the prion gene for the genotyping of scrapie susceptibility in sheep.

The objective of this study was to report a reliable real-time polymerase chain reaction assay compatible with the Roche LightCycler 2.0 capable of genotyping sheep for scrapie susceptibility at codon 171. The single nucleotide polymorphisms (SNPs) in the prion protein gene in sheep that may govern resistance to scrapie at codon 171 encode for lysine (K), histidine (H), glutamine (Q), and arginine (R). A modified proteinase K method for leukocytes or whole blood was used to isolate genomic DNA from sheep blood. Fluoresentric developed and optimized primers and probes for the codon 171 SNP assay. The assay was initially validated using 218 determinations from whole blood of known genotypes with 100% correct identity. The assay was further validated through a whole-blood check test provided annually by the National Veterinary Services Laboratory with a correct identification rate of 100%. From January 2005 to December 2006, 3,672 samples from blood were genotyped at codon 171. The genotypes were QR(171) (n = 1,838, 50.05%), RR(171) (n = 1,423, 38.75%), QQ(171) (n = 407, 11.08%), HR(171) (n = 2, 0.05%), and HQ(171) (n = 2, 0.05%). The combination of this simple extraction method and the novel Fluoresentric assay is very accurate, is capable of identifying all 4 SNPs at codon 171 in one reaction, and has proven to be a useful tool for producers in their selective breeding programs.     

An avidin-biotin enhanced dot-immunobinding assay for the detection of Mycoplasma gallisepticum and M. synoviae serum antibodies in chickens

A dot-immunobinding assay was enhanced by the incorporation of avidin and biotin reagents into the test system (DAB assay). This assay was used to detect serum antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS) from chickens. Serum samples were tested by rapid serum plate (RSP), hemagglutination-inhibition (HI), and DAB assay methods. These results were compared. The DAB assay was at least 20 times more sensitive in detecting antibodies for MS and at least 75 times more sensitive in detecting antibodies for MG than the HI test. The DAB assay was as specific as the HI test. The DAB assay was also more sensitive and specific than the RSP test. Some cross-reactions occurred when low dilutions of high-titer sera were used in the DAB assay. Parameters for determining negative, suspicious, and positive samples were established. The DAB assay for MG and MS may have several applications, including use as a screening test and a confirmatory test.     

Use of an avidin-biotin enhanced dot-immunobinding assay to detect antibodies for avian mycoplasma in sera from Iowa market turkeys

A dot-immunobinding assay, amplified with avidin and biotin (DAB assay), was used to detect serum antibodies to Mycoplasma iowae in immunized turkeys. The DAB assay was used to test serum samples from 122 commercial market turkey flocks obtained from four Iowa processing plants. The samples were pooled and tested for the presence of antibodies to four species of Mycoplasma spp. considered to be important pathogens for turkeys: M. gallisepticum (MG), M. iowae (MI), M. meleagridis (MM), and M. synoviae (MS). The occurrence of antibodies against these mycoplasmas, as determined by the DAB assay, were 5.7% for MG, 18.0% for MI, 77.9% for MM, and 9.8% for MS.     

Evaluation of canine lymphocyte proliferation: comparison of three different colorimetric methods with the 3H-thymidine incorporation assay.

To evaluate canine lymphocyte stimulation the radioactive thymidine incorporation assay is still the method of choice. In order to find a suitable non-radioactive alternative to the standard 3H-thymidine incorporation assay, proliferation of canine peripheral blood lymphocytes (PBL) was measured with three different colorimetric assays, using the two tetrazolium salts MTT and XTT and 5-bromo-deoxyuridine (BrdU). Isolated canine PBL were stimulated with two different mitogens, Concanavalin A (Con A) and Phytohemagglutinin (PHA), using different culture conditions. Applying statistical analysis we found that BrdU and MTT showed a high correlation to the 3H-thymidine incorporation assay, although the BrdU assay proved to be more sensitive than the MTT assay. No significant correlation between the XTT assay and the radioactive method was demonstrated. Consequently, the BrdU assay is the most suitable alternative to the radioactive method.     

营养酸模对产奶母牛饲喂效果的试验报告

试验用营养酸模鲜茎叶替代奶牛部分精料观测其对泌乳牛的饲喂效果。试验共设三个试验组和一个对照组，其试验组营养政模的替代率分别为１２．５％、２５．０％及 ３７．５％，经三期 ３００天的饲喂，其结果为试验组比对照组头日产奶量分别高 ０．９６ｂｄ、２．２５ａｂ、２．６７ｋｇ（Ｐ＜０．０１或 Ｐ＜０．０５），比对照组（ＣＫ）提高 ７．７％、１８．０％、２１．３％；平均乳脂率分别提高 ０．６３Ａ，０．３５Ｂ，０．３１Ｂ个百分点（Ｐ＜０ ．０１或 Ｐ＜０．０５），比ＣＫ组提高１７．３％、９．６％、８．５％。经济效益显著，适应性良好。试验证实，用营养酸模替代部分精料对提高奶牛泌乳量和改善牛奶品质及口感是有利的。     

Polyclonal activation of canine B lymphocytes evaluated by a protein A reverse hemolytic plaque assay

This paper describes the optimal culture and assay conditions for the polyclonal activation of canine lymphocytes with pokeweed mitogen and the quantitation of immunoglobulin secreting plaque-forming cells (PFC) using a staphylococcal protein A-reverse hemolytic plaque assay. The assay permits the quantitation of total immunoglobulin secreting PFC as well as class-specific immunoglobulin secreting PFC. On the optimal day of culture, a mean of 176 IgA PFC/10(6), 575 IgM PFC/10(6), 1276 IgG PFC/10(6), and 2158 total PFC/10(6) cells were generated following polyclonal activation. This study provides a simple and reproducible assay for the delineation of the immunoregulatory mechanisms involved in the differentiation of canine B lymphocytes.     

Characterization of antibody response to porcine reproductive and respiratory syndrome virus ORF5 product following infection and evaluation of its diagnostic use in pigs.

The sensitivity and specificity of recombinant open reading frame 5 products used in the Western blotting assay for confirmation of porcine reproductive and respiratory syndrome virus (PRRSV) serologic status were evaluated. The recombinant antigen-based assays were specifically compared with a commercial enzyme-linked immunosorbent assay (ELISA) for PRRSV antibodies using 1) PRRSV antibody-negative reference sera (n = 30), 2) naturally infected pig sera (n = 40), 3) sequential sera obtained from 24 experimentally infected pigs, and 4) sera submitted to 3 state diagnostic laboratories (n = 200). The recombinant antigen assay yielded an average increased sensitivity of 10% over the commercial PRRSV ELISA. The negative controls (group 1 sera) showed no difference between the 2 assays. This comparison confirmed that the recombinant antigen-specific assay was more sensitive than the commercial ELISA and is well suited for routine confirmation of the presence of PRRSV antibodies.     

一种鉴别尼帕病毒和高致病性猪繁殖与呼吸综合征病毒二重荧光RT-PCR检测方法的建立

为建立一种快速、敏感和特异地鉴别尼帕病毒(NiV)和高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的检测方法,本试验以NiV M基因和HP-PRRSV nsp2基因为靶序列,通过优化反应条件建立了一种二重荧光RT-PCR检测方法,并对该方法的特异性、定量线性范围、敏感性和重复性进行了评价及初步应用.结果显示,用该方法检测NiV M基因和HP-PRRSV nsp2基因的RNA标准对照(NiV-M-RNA和HP-PRRSV-nsp2-RNA),线性范围分别为4.6×10¹~4.6×10⁷和4.1×10¹~4.1×10⁸拷贝/μL;最低检出限分别为46和4.1拷贝;该方法组内试验和组间试验的变异系数均小于2.0%,显示出良好的可重复性;该方法仅对NiV和HP-PRRSV呈现特异性扩增曲线,不与猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、猪流感病毒(SIV)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV2)发生交叉反应.用该方法对236份猪实际样品进行NiV和HP-PRRSV核酸检测,所有样本的NiV检测结果均为阴性,8份样本的HP-PRRSV检测结果为阳性.本研究建立的方法为猪实际样本中NiV和HP-PRRSV的鉴别检测提供了一种快速、敏感和特异的技术手段.