Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera

OBJECTIVE: To compare the sensitivity and specificity of Chlamydophila abortus antibody assays, to find a suitable serological assay for testing sheep for export. DESIGN: Comparison of results from known positive and negative sheep populations. PROCEDURE: Fifty-five positive and fifty negative sera were analysed by four enzyme linked immunosorbent assays (ELISA), three using recombinant antigens based on the chlamydial polymorphic outer membrane proteins (POMP90-3, POMP90-4, POMP80-90) and one using a synthetic peptide based on chlamydial major outer membrane proteins (MOMP-P). They were also analysed by complement fixation tests (CFT) using crude antigens from chlamydia isolated from an Australian sheep, a Californian parakeet and a Texan turkey. Assay sensitivity and specificity were expressed as point estimates and 95% confidence intervals. Results were compared using McNemar's test for paired samples. RESULTS: ELISA sensitivity ranged from 70 to 98% and complement fixation test sensitivity from 60 to 96%; with POMP90-3 > POMP90-4 > CFT (parakeet) > CFT (turkey) > POMP80-90 > MOMP-P > CFT (sheep). There was no significant difference from POMP90-3 to POMP80-90 (P > 0.05). ELISA specificity ranged from 88 to 100% and CFT specificity was 100% for all three antigens; with CFT and POMP90-4 > MOMP-P > POMP80-90 > POMP90-3. There was no significant difference from CFT to POMP80-90 (P > 0.05). Changing the CFT cut-off from 1:32 to 1:4 substantially reduced the specificity with little improvement in sensitivity. CONCLUSION: Assays using POMP90-4, POMP80-90, CFT (parakeet) and CFT (turkey) had equivalent sensitivity and specificity; none of the ELISAs were more specific than any CFT. The POMP80-90 ELISA is recommended as an alternative to CFT (parakeet) but as its specificity is not ideal the search for a more specific assay should continue.     

羊流产嗜衣原体病和弓形虫病IHA与ELISA诊断方法的比较

通过比较研究羊流产嗜衣原体与弓形虫抗体的间接血凝试验(IHA)与酶联免疫吸附试验(ELISA)检测结果,选择适宜贵州省山羊流产血清学调查的检测方法。通过IHA和ELISA两种方法对贵州省195份山羊血清进行羊流产嗜衣原体与弓形虫抗体的检测,统计并分析两种疫病的阳性符合率、阴性符合率和总符合率。结果表明,羊流产嗜衣原体与弓形虫IHA与ELISA的总符合率分别为77.95%和78.97%,阳性符合率均仅为50%,阴性符合率为80.45%和79.27%。说明在检测两种疫病血清抗体方面,IHA比ELISA更适合在贵州省基层推广。     

山羊流产嗜衣原体OmpA基因重组质粒构建及对小鼠免疫效果研究

为探讨山羊流产嗜衣原体重组真核质粒进入临床试验的可行性,本试验用PCR方法扩增出山羊流产嗜衣原体OmpA基因,克隆至真核表达载体pcDNA3.1（+）中,经PCR和双酶切鉴定后,将重组质粒pcDNA3.1-OmpA转染至PK-15细胞,观察目的基因表达情况,并将制备的大肠杆菌基因组单链DNA作为分子佐剂与重组质粒共同免疫小鼠,检测重组质粒在小鼠体内分布情况及血清抗体水平。结果表明,经PCR、双酶切和测序鉴定表明成功构建重组质粒pcDNA3.1-OmpA;转染PK-15细胞后,荧光抗体试验结果证实重组质粒pcDNA3.1-OmpA得到有效表达。首免后14 d,重组质粒加分子佐剂组的抗体效价显著高于重组质粒组和灭活疫苗组（P<0.05）;随着免疫次数增加和时间推移,免疫小鼠抗体水平均呈现上升趋势,至35 d时达到最高峰,此后抗体滴度逐渐下降,但仍维持较高水平。首免后21 d,在小鼠心脏、肝脏、脾脏、肾脏、肺脏、脑、空肠和腿肌中均可检测到质粒的分布,此后逐渐消失,49 d在所检组织中均未检测到重组质粒的存在。表明试验成功构建了基于OmpA基因的山羊流产嗜衣原体重组真核质粒,且加入分子佐剂后可诱导小鼠产生较高水平的血清抗体。     

Seroprevalence of Antibodies to Chlamydophila psittaci in Zoo Workers in Brazil

To evaluate the prevalence of antibodies to Chlamydophila psittaci 364 serum samples were collected from veterinarians, biologists, animal scientists, veterinary students, animal keepers and others employees in 20 zoos, and from veterinary practitioners in 10 Brazilian states. Subjects ranged from 15 to 64 years of age, with 268 (74%) males and 96 (26%) females. Chlamydial antibodies were determined by the complement fixation test (CFT) and specific anti‐C. psittaci IgG antibodies were determined by the microimmunoflurescence (MIF) test. Complement fixation test showed 23.9% (87/364) and MIF test showed 4.7% (17/364) positive serum samples. Titres ranged from 16 to 256 in both assays, demonstrating evidence of recent or current infection. Although chlamydial antibodies were detected in workers of seventeen zoos, MIF test only detected specific C. psittaci antibodies in seven of them. Previous psittacosis infection was suspected in eight workers of two zoos, five of whom reported having pneumonia, while employed at the zoos. However, diagnosis was not established in any of these cases in the past. Results indicated the occurrence of infection and previous contact of Brazilian zoo workers with C. psittaci, as well as the zoonotic potential of psittacosis in this risk population. Other studies are necessary to evaluate the risk factors of infection in this population. This seroepidemiological survey confirmed the need to adopt preventive measures to control avian chlamydiosis and protect the health of zoo workers in the country.     

Comparison of the Q-fever complement fixation test and two commercial enzyme-linked immunosorbent assays for the detection of serum antibodies against Coxiella burnetii (Q-fever) in ruminants: Recommendations for use of serological tests on imported animals in New Zealand

Abstract

AIM: To make valid recommendations on the use of serological test methods for the detection of serum antibodies in ruminants against Coxiella burnetii (Q-fever), by comparing the performance of the complement fixation test (CFT) and two ELISA, and by identifying reasons for discrepancies between the test methods.

METHODS: A total of 73 serum samples from infected cattle, 69 from infected goats, and 100 samples from non-infected cattle and 57 samples from non-infected sheep, as well as 95 samples from infected cattle herds (mix of seropositive and seronegative samples), were tested using the CFT, the IDEXX ELISA (I-ELISA) and the Pourquier ELISA (P-ELISA). A mixed panel of 12 serum samples from sheep from inter-laboratory proficiency testing (proficiency panel) was also tested using the CFT and both ELISA, and further investigated using IgG- and IgM-specific ELISA.

RESULTS: Generally, the two commercial ELISA were more sensitive than the CFT for the detection of infected ruminants. Good agreement between ELISA for positive and negative results was found for samples from the infected herd, while results for the positive panels varied between the two ELISA. For the total of the positive serum panels, the I-ELISA detected 95% of samples as positive or suspicious, while the P-ELISA detected only 81%. In the P-ELISA, more samples were considered suspicious (18%) than in the I-ELISA (14%). All sera from noninfected sheep and cattle tested negative in the serological test methods employed, except for one positive sample from a sheep in the P-ELISA. Further investigation revealed that a CFT-positive but ELISA-negative result was due to high IgM and low IgG reactivity.

CONCLUSIONS: The two commercial ELISA were more sensitive than the CFT in all panels from infected ruminants. However, they could only detect IgG. The I-ELISA should be the serological test method of choice for cattle, sheep and goats for import testing of animals into New Zealand because it was more sensitive than the P-ELISA and was equally specific to the PELISA and the CFT. For other animal species, such as deer and camelids, the CFT should still be used since none of the ELISA has been evaluated for these species. This study has shown that the two commercial ELISA will detect the majority of infected ruminants but may miss animals that have not developed an IgG response.     

Construction of Recombined Plasmid with OmpA Gene of Goat Chlamydophila abortus and Immunogenicity Effect in Mice

To explore the feasibility of entering clinical trials of the goat Chlamydophila abortus eukaryotic plasmids,Chlamydophila abortus OmpA gene was amplified by PCR and cloned into the eukaryotic expressing plasmid pcDNA3.1(+）to construction the recombinant vetor.After identification by PCR and restriction enzyme digestion,this vector was transfected into PK-15 cells and its expression were observed by fluorescent antibody test,the distribution of serum antibodies and plasmid were detected in mice after the immunization of the recombinant vector and molecular adjuvant which was single-stranded DNA of E.coli bacterial genome.The results showed that the recombinant plasmid pcDNA3.1-OmpA was successfully constructed after detecting by PCR,enzyme digestion and sequencing.The OmpA gene was effectively expressed in PK-15 cells.The anti-OmpA antibody levels of the pcDNA3.1-OmpA with molecular adjuvant group was significantly higher than that in pcDNA3.1-OmpA group and the inactivated vaccine group at 14 d after immunization（P<0.05).This levels showed an upward trend following numbers of immunization and times.The highest levels was at 35 d after immunization.Then the antibody titers were gradually decreased which still maintain a higher antibody levels than before.The pcDNA3.1-OmpA could be detected in the heart,liver,spleen,kidney,lung,brain,jejunum and leg muscle of mice on 21 d after immunization,and couldn't be detected in any organs at 49 d after immunization.The results above indicated that the the recombinant vetor based on OmpA gene of Chlamydophila abortus was successfully constructed in this experiment,after joining the molecular adjuvant could induce to a higher level of serum antibodies in mice.     

山羊流产衣原体ompA和CPAF基因的序列分析及原核表达

为了解山羊流产衣原体陕西分离株ompA和CPAF基因的遗传变异情况及制备检测抗原,根据GenBank收录的ompA和CPAF基因序列设计合成特异性引物,分别进行ompA基因和CPAF基因的基因克隆、序列分析及原核表达.结果表明,成功克隆出大小为1 053 bp的ompA基因和大小为1 056 bp的CPAF基因;核酸序...     

Immunopathology of Chlamydophila abortus infection in sheep and mice

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.     

禽流感病毒夹心ELISA诊断试剂盒的研制及应用

将成熟的禽流感病毒（AIV）夹心ELISA快速检测方法组装成诊断试剂盒。试剂盒由1～11号试剂、一块酶标板和一份说明书组成。对试剂盒内各组份进行了特异性、敏感性、重复性及保存期的测定。结果表明．在-10℃下能保存6个月．而且特异性强、敏感性高、重复性好，操作简单．方便．能快速、准确地检测出样品中是否带有AIV抗原。先后制备了8个批次的诊断试剂盒．于湖北、安徽及河南等地的养禽场、兽医站、农科院、农业院校等化验室应用．取得了良好的效果。     

Establishment of ELISA Detection Method for Neomycin in Import and Export Animal

This experiment used carbodiimide get egg albumin synthesis of artificial antigens coupling neomycin,through square test to determine the best antigen packaged concentration and antibody dilution ratio,mass concentration logarithm of neomycin as abscissa,inhibition rate of antibody as the ordinate drawing standard curve,established ELISA fast detection techniques of neomycin in edible animal for import and export.Methods of recovery and specific test had been done in further research.Results showed that the test dilution ratio of optimum synthetic antigen was 1:200,the best dilution multiple of antibodies was 1:2 000, half inhibitory concentration(IC₅₀) was 6.99 ng/mL;The minimum detection limit of the method was 40 ng/mL,and cross reaction rate of gentamicin, kanamycin, streptomycin,tobramycin,amikacin and dihydrostreptomycin were less than 0.1%,the average of recovery rate was 83.77%.The range of standarding curve in 1.5~40 ng/mL was linear,the correlation coefficient was 0.987,the equation of standarding curve was y=-37.66x+94.592;Compared with HPLC method,the operation was simple and fast.The test results showed that the enzyme-linked immunosorbent method of detecting neomycin was rapid,efficient and specific.     

进出口动物中新霉素ELISA检测方法的建立

应用碳二亚胺法合成人工抗原得到卵清蛋白偶联新霉素,通过方阵试验确定最佳抗原包被浓度与抗体稀释倍数,以新霉素质量浓度对数为横坐标、抗体抑制率为纵坐标绘制标准曲线,建立了进出口食用动物中新霉素ELISA快速检测技术,并对建立的方法进行了回收率和特异性试验。结果表明,建立的检测方法包被人工合成抗原的最适稀释度为1：200,抗体的最佳稀释度为1：2 000,半数抑制浓度（IC₅₀）为6.99 ng/mL;该方法的最低检测限达40 ng/mL,与庆大霉素、卡那霉素、链霉素、托普霉素、阿米卡星、双氢链霉素的交叉反应率均小于0.1%,平均加标回收率83.77%。标准曲线在1.5~40 ng/mL范围内是线性的,相关系数为0.987,标准曲线方程为y=-37.66x+94.592;与常规液相检测方法相比操作简单、快速。本研究建立了一种快速、高效、特异的新霉素ELISA检测方法。     

酶联免疫检测试剂盒应用于牛奶中四环素残留的测定

为了加强牛奶中四环素残留量的监控，以四环素-卵清白蛋白为包被抗原，四环素-牛血清白蛋白免疫的产生的抗血清为抗体，辣根过氧化物酶-羊抗兔IgG为酶标记物，研制了四环素快速酶联免疫分析试剂盒，在3．9-2000ng/mL四环素标准浓度范围内，呈线性相关，相关系数为0．9882，批内误差小于批间误差，但其变异系数均在10％以下。与土霉素、金霉素的交叉反应分别为24.31％、6．22％。该试剂盒具有快速、灵敏、准确的特点，适用于牛奶中四环素残留量的快速筛选测定。     

快速凝集试验与补体结合试验检测牛边缘无浆体感染的效果对比

从138份血清样品的比较试验结果显示，快速凝集试验（RCA）比补体结合试验（CF）检测边缘无浆体感染的敏感性高（88．9％：81．5％），假阴性率低（11．1％：18．5％），两者都具有良好的特异性和预测性，检测阳性符合率高，快速凝集试验对一次感染牛的持续检出阳性时间更长久（303天：92天）     

阻断ELISA与中和试验检测猪瘟疫苗免疫猪血清抗体的比较

应用阻断ELISA和中和试验2种不同的方法对猪瘟疫苗免疫前后的猪血清抗体进行了检测,在所检测的6头仔猪免疫前和免疫后的12份血清中,2种方法的检测结果相符.应用Western blot对部分血清进行了验证,结果与2种方法检测结果一致,表明阻断ELISA和中和试验结果确实、特异,能真实反映免疫猪群的抗体水平.     

Role of polymorphonuclear neutrophils (PMNs) and NK cells in the protection conferred by different vaccines against Chlamydophila abortus infection

Ovine enzootic abortion (OEA) is caused by Chlamydophila abortus, an intracellular bacterium which acts by infecting the placenta, causing abortion in the last term of gestation. The main prevention strategy against OEA is the vaccination of flocks. An effective vaccine against C. abortus must induce a Th1-like specific immune response, which is characterized by the early production of IFN-gamma and the activation of CD8(+)T cells. Moreover, vaccine effectiveness could be modulated by the functioning of the innate immunity. The purpose of this study was to ascertain how polymorphonuclear neutrophils (PMNs) and NK cells might influence vaccine-induced protection. The live attenuated 1B vaccine and two inactivated experimental vaccines, adjuvated with aluminium hydroxide (AH) or QS-21 (QS), were used in PMN-depleted or NK cell-depleted mice. For PMN depletion, RB6-8C5 monoclonal antibody, which recognizes GR1(+) receptors (Robben, P.M., LaRegina, M., Kuziel, W.A., Sibley, L.D. 2005. Recruitment of Gr-1(+) monocytes is essential for control of acute toxoplasmosis. The Journal of Experimental Medicine 201, 1761-1769.) was used, while for NK cell-depletion the anti-asialo GM1 polyclonal antibody was used. The depletion of PMNs caused 100% mortality in non-vaccinated mice (NV) and 60% mortality in the AH-vaccinated mice by day 10 p.i., while both groups showed a significant increase in their bacterial burden in the liver by day 4 p.i. The depletion of NK cells caused mortality only in the NV group (50% by day 10 p.i.), although this group and the 1B vaccinated mice showed an increased bacterial burden in the liver at day 4 p.i. Our results suggest that the importance of PMNs in inactivated vaccines depends on the adjuvant chosen. The results also demonstrated that the importance of NK cells is greater in live vaccines than in inactivated vaccines.     

非繁殖季节中国美利奴羊血清E2、P4、FSH、LH激素变化规律研究

为了在非繁殖季节判断绵羊的发情状况,试验以新疆高海拔地区中国美利奴羊为研究对象,采用酶联免疫测定(ELISA)法测定绵羊血清中雌二醇(E2)、孕酮(P4)、促卵泡素(FSH)、促黄体生成素(LH)的含量,研究激素诱导发情羊、发情症状不明显羊、不发情羊体内激素分泌规律的差异。结果表明:激素诱导发情羊的E2含量比不发情羊高,差异显著(P<0.05);但是在P4、FSH、LH水平上,激素诱导发情羊要低于不发情羊,差异不显著(P>0.05)。发情羊与发情不明显羊在4种激素含量上差异不显著(P>0.05),说明公羊试情存在一定缺陷,而通过测定E2含量判定绵羊发情状态将成为未来研究的重点。     

Slide Enzyme-Linked Immunosorbent Assay for the Diagnosis of Babesia bigemina Infection in Bovines

A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of Babesia bigemina infection in cattle was standardized. Acetone-fixed whole Babesia bigemina-infected erythrocytes on micro-slides were immunoreacted with bovine serum samples followed by antibovine horseradish peroxidase conjugate and developed using diaminobenzidine tetrahydrochloride as a substrate. The positive immunoreactivity (staining pattern) was visualized in the form of dark brown piroplasms. Using the laboratory-standardized SELISA with a sensitivity of 94.4%, the seroprevalence of babesiosis was studied in cattle from two endemic areas of the disease. In comparison to IFAT, SELISA detected higher number of serum samples positive for bovine babesiosis.