评价基于致密颗粒蛋白GRA1为抗原检测猫弓形虫感染的血清学诊断方法

为了研究以弓形虫致密颗粒蛋白GRA1为抗原检测猫弓形虫感染的血清学诊断及评价,试验采用标准弓形虫抗体阳性、阴性猫血清建立ELISA方法,最终确定重组GRA1最佳蛋白包被浓度为5μg/m L、血清最佳稀释度为1∶64,用此方法对已通过MAT/IFA法共同确定的185份猫弓形虫抗体血清进行检测并评价。结果表明:应用MAT/IFA法检出阳性血清39份、阴性血清146份,而通过重组GRA1-ELISA法检出阳性血清37份、阴性血清148份,二者共同检出阳性血清33份、阴性血清142份,GRA1假阳性率为10.8%,假阴性率为4.1%。比较重组GRA1-ELISA与MAT/IFA的结果,二者不一致部分无显著性差异(P0.05);一致性部分经Kappa检验(K=0.83),一致率为94.6%。说明以重组GRA1作为包被抗原建立的ELISA方法检测效果没有弓形虫体裂解蛋白TLA好。因此,重组GRA1不是用于检测猫弓形虫感染的流行病学调查的最佳诊断抗原。     

1种检测弓形虫急性感染的夹心ELISA方法

弓形虫（Toxoplasma gondii）是一种人畜共患机会性致病原虫，其急性感染可导致宿主产生明显的临床症状和严重的病理损伤。弓形虫致密颗粒蛋白1（dense granuleprotein 1，GRA1）是一种良好的诊断抗原，也是弓形虫急性感染的标志物循环抗原（circulating antigen，CAg）的重要组分。本研究利用TgGRA1单克隆抗体建立双抗体夹心ELISA方法，为急性弓形虫感染的检测提供依据。将免疫GRA1-His的小鼠脾细胞与SP2/0进行融合，筛选出能稳定分泌抗体的杂交瘤细胞。选择其中一种单抗与HRP标记后的鼠源GRA1多抗配对，建立1种双抗体夹心ELISA方法，检测人工感染弓形虫的猪和小鼠血清样品，并将检测效果与巢式PCR（nest PCR，nPCR）和商品化试剂盒进行比较。结果筛选到4株杂交瘤细胞，腹水效价为10⁶~10⁷，亚型均为IgG1；IFA和Western blot结果显示，4株单抗均具有良好的反应性和特异性。选择1G2单抗和HRP标记多抗配对，建立了循环抗原双抗体夹心ELISA方法，最低能够检测到血清中1.563 ng·mL^-1 GRA1抗原，或者100 ng·mL^-1 ESA。该方法与nPCR相比具有较高的一致性，较市售商品化试剂盒更为准确可靠。本研究第1次将GRA1抗原作为急性弓形虫感染的诊断指标，建立相应的检测方法，成功地在人工感染样品中检测到弓形虫急性感染，可为弓形虫急性感染的诊断提供参考，对临床上急性弓形虫病的治疗有指导意义。     

青海省牛弓形虫病的ELISA诊断及试剂盒建立

用弓形虫的重组蛋白SAG1作为ELISA诊断抗原,建立了ELISA诊断试剂盒,并对青海省牛群进行弓形虫病的血清学诊断。经对所收集的1856份牛血清进行弓形虫抗体检测,共检出阳性血清71份,阳性率约为3.83%。结果表明青海省牛群中存在弓形虫病的感染。     

青海地区喜马拉雅旱獭弓形虫病的ELISA检测

用弓形虫的重组蛋白GST-SAG1作为ELISA诊断抗原,进行了ELISA诊断方法的建立,并对青海省喜马拉雅旱獭进行了弓形虫病的血清学调查。经对所收集的68份喜马拉雅旱獭血清抗体进行检测,共检出阳性血清15份,阳性率约为22.06%,表明青海省喜马拉雅旱獭中存在弓形虫病的感染。     

小反刍兽疫病毒N蛋白抗体与H蛋白抗体在羊体内代谢消长规律的研究

为研究小反刍兽疫病毒N蛋白抗体与H蛋白抗体在羊体内的代谢消长规律,试验通过建立小反刍兽疫N蛋白双抗原夹心ELISA抗体检测方法、H蛋白阻断ELISA抗体检测方法与血清中和抗体试验方法,分别检测了羊免疫疫苗后体内N蛋白抗体与H蛋白抗体在每个免疫阶段的代谢消长变化。结果表明:羊免疫小反刍兽疫疫苗后,血清内的N蛋白抗体与H蛋白抗体在6~8周时血清抗体效价较高,对应的羊体内中和抗体效价为1∶512,且效价至少可以持续到10周以上,2种抗体具有一定消长代谢规律的相关性。该研究结果也为以N抗原与H抗原为基础建立相应的抗原捕获ELISA方法、竞争ELISA检测方法、间接ELISA检测方法以及双抗原夹心ELISA检测方法奠定了一定的理论依据。     

猪弓形虫病的ELISA诊断及试剂盒建立

用弓形虫的重组蛋白P30作为ELISA诊断抗原,进行了ELISA诊断试剂盒的建立,并对互助县猪弓形虫病的血清学诊断。经对所收集的139份猪血清抗体进行检测,共检出阳性血清8份,阳性率为5.76%。结果说明互助县猪群中存在弓形虫病的感染。     

弓形虫TgERP基因的原核表达及间接ELISA方法的建立

为原核表达弓形虫的TgERP基因，参照GenBank中弓形虫GTl株的TgERP基因序列设计引物，通过PCR扩增分离的一株猫源弓形虫（TC株）的TgERP基因，将其克隆至原核表达载体pET-28a中，通过IPTG诱导表达蛋白，应用带His标签的重力柱纯化蛋白，采用BCA方法测定蛋白浓度，利用重组蛋白免疫Balb/c小鼠制备多克隆抗体，并对重组蛋白进行SDS-PAGE和Western blotting分析。结果成功表达了TgERP蛋白，且蛋白以上清形式表达，相对分子质量约16 ku。将纯化的重组蛋白pET-28-TgERP作为诊断抗原，初步建立了间接ELISA方法；利用间接ELISA、改良凝集试验（MAT）和间接血凝试验（IHA）3种方法分别检测472份羊、犬、猫血清样品，发现阳性率分别为1.0%、3.8%、3.4%，验证了建立的间接ELISA方法可区分动物感染弓形虫的途径。本研究为弓形虫病快速诊断方法的建立和疫苗的研发奠定了基础。     

青海省乌兰地区马焦虫病的血清学调查

由驽巴贝虫和马巴贝虫引起中国北方地区的马焦虫病的流行情况仍然不清楚,在本研究中我们调查了青海省乌兰地区马焦虫病的流行情况。从乌兰县的铜普地区的牧户中收集了97份马血清样品,并分别用重组P48蛋白作为ELISA诊断抗原,进行马巴贝斯原虫的诊断检查,调查青海省乌兰地区马焦虫病的流行情况。1材料和方法1.1通过载体pGEX大肠杆菌表达的P48作为ELISA诊断抗原来诊断驽巴贝虫(Babesia caballi),方法简述如下:重组P48蛋白用谷胱苷肽琼脂糖4B球珠纯化,作为ELISA诊断抗原,检测巴贝虫抗体。巴贝虫阴、阳性标准血清,均为日本国家原虫中心…     

大豆抗原蛋白血清抗体间接ELISA检测方法的建立

旨在建立检测血清大豆抗原蛋白抗体的间接ELISA方法。经琼脂糖凝胶层析纯化大豆抗原蛋白,以不同剂量皮下注射免疫小鼠,采用方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测血清大豆抗原蛋白抗体的间接ELISA方法,利用该方法检测小鼠免疫后血清抗体水平。通过方阵滴定法确定11S蛋白最佳包被浓度为5.0μg/mL,血清稀释倍数为1∶800;7S蛋白抗原最佳包被浓度为2.5μg/mL,血清稀释倍数为1∶1 600;两者的批内、批间系数均小于10%,重复性较好,通过ELISA法确定11S和7S蛋白的最佳免疫次数为2次,免疫剂量为1 000μg/kg。结果表明本试验初步建立大豆抗原蛋白抗体检测间接ELISA方法,具有很强的特异性、敏感性和重复性,可用于大豆抗原蛋白过敏反应的临床检测。     

陇西县牛巴贝斯虫病的流行病学调查

将重组的牛巴贝斯虫棒状体相关蛋白(Bo-RAP-1)作为诊断抗原建立ELISA检测方法,对2016—2017年采自甘肃省陇西县的188份黄牛血清样品进行抗Bo-RAP-1特异性抗体检测。结果检出47份阳性,阳性率为25%,说明该地区牛群存在牛巴贝斯虫病的感染。     

Determination of the More Adequate Modified Agglutination Test Cut‐off for Serodiagnosis of Toxoplasma gondii Infection in Sheep

Toxoplasmosis is one of the most common food borne disease world‐wide. Among food animals, sheep seems to having higher prevalence of Toxoplasma gondii infection. However, there is no consensus about the best cut‐off for serodiagnosis in sheep. To estimate the more adequate cut‐off value of Modified Agglutination Test (MAT) for serodiagnosis in sheep, a commercial ELISA kit was used as a golden standard. Evaluation of the optimal sensitivity and specificity was calculated using Youden’s J‐statistics. Values obtained were used to estimate the prevalence of sheep toxoplasmosis. One thousand four hundred and sixty seven blood samples were collected randomly from 160 farms from northern Portugal, representing approximately 10% of the ovine population from the region. All sera were tested for anti‐T. gondii antibodies using the MAT. One hundred nine sheep (7.4%) presented a MAT titer ≥1 : 80; 45 (3.0%) a MAT titer of 1 : 40; 97 (6.6%) a MAT titer of 1 : 20 and 1216 (83.0%) a MAT titer ≤1 : 20. The best Youden’s J‐statistic was obtained at 1 : 20 titer (0.752), with 86.15% of sensitivity and 89.09% of specificity with negative and positive predictive values of 90.32% and 84.48% respectively, suggesting that the 1 : 20 was the most appropriate cut‐off for serodiagnosis of toxoplasmosis in sheep. Assuming this cut‐off, the prevalence of toxoplasmosis in the studied population was 17.1% and 92 (57.5%) of the 160 studied flocks having one or more positive sheep. Those results indicate that toxoplasmosis in Portugal should be considered in the differential diagnosis of abortions in sheep and neurological signs in lambs. Furthermore, while Portugal produces ovine meat for internal consumption and for exportation, isolation of T. gondii from ovine meat and further characterization of the isolates will be needed to understand the risk that ovine toxoplasmosis may represent for human health.     

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis.     

Diagnostic value of recombinant nanoluciferase fused Toxoplasma gondii antigens in Luciferase-linked Antibody Capture Assay (LACA) for Toxoplasma infection in pigs

Toxoplasmosis is a widespread protozoan zoonosis. Since ingesting undercooked meat harboring Toxoplasma gondii cyst is considered one of the major transmission routes to humans, the screening of T. gondii in meat-producing animals can reduce the risk of food-borne toxoplasmosis in humans. Among serological diagnostic methods, Luciferase-linked Antibody Capture Assay (LACA) has been found to be a promising platform with high sensitivity and specificity. In this study, we aimed to evaluate recombinant nanoluciferase fused-T. gondii antigens (rNluc-GRA6, rNluc-GRA7, rNluc-GRA8 and rNluc-BAG1) for their potential use in LACA for pigs. As a result, the sensitivity of GRA6-, GRA7-, GRA8- and BAG1-LACA were 70.0%, 80.0%, 80.0% and 30.0% with specificity 87.0%, 81.5%, 74.1% and 50.0%, respectively. The cocktail LACA using a mixture of rNluc-GRA6, rNluc-GRA7 and rNluc-GRA8 indicated higher sensitivity (90.0%) and a similar specificity (96.3%) in comparison with the commercial ELISA kit. Compared to the Dye-Test as a reference test, cocktail LACA showed strong agreement (kappa value=0.811) when we assessed pig sera collected at the slaughterhouse. In addition, we also successfully established the rapid LACA format for the detection of Toxoplasma infection in pigs (called Rapid-LACA) in which the test could be performed within 30 min. In Rapid-LACA, the protein A pre-coated/blocked plates could be preserved at −30°C, 4°C or room temperature conditions for at least two months without compromising on the quality of assay.     

High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis

Toxoplasmosis, caused by Toxoplasma gondii, is a disease of economic importance in livestock, especially in sheep and goats, where it causes abortion. Although several serological tests are in use for diagnosis of infection, production of reliable reagents is a constraint. An 814 bp sequence coding for a truncated surface antigen surface antigen 1 (SAG1), a tachyzoite stage-specific protein, as well as a 657 bp sequence coding for granule protein 7 (GRA7), a dense granule protein were PCR amplified from the genomic DNA of T. gondii. The amplified products were ligated in pET-32b(+) and pET-32c(+) expression vectors, respectively and subsequently transformed into BL21(DE3)pLysS cells. A high-level expression of the histidine-tagged SAG1 and GRA7 fusion proteins were obtained after 7h of incubation. The recombinant proteins were purified using Ni-NTA column and were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis using reference positive sera from goat, rabbit and humans at 1:100 dilution. Subsequently, the diagnostic efficiency of the recombinant proteins, either individually or as a cocktail of the recombinant proteins, was assessed with 56 reference goat sera by enzyme-linked immunosorbent assay (ELISA). The immunoreactivity of the refolded SAG1 and GRA7 was evidenced by high OD values. The reactivity of the recombinant proteins as a cocktail preparation was more than that of individual proteins in ELISA and could detect accurately the infection in goats. This is the first report of serological detection of caprine toxoplasmosis by ELISA using a cocktail of recombinant Toxoplasma proteins.     

Sensitivity and specificity of various serological tests for the detection of Toxoplasma gondii infection in naturally infected sheep

Comparative serological examination of 300 serum samples from sheep slaughtered in the main abattoir in Cairo, Egypt revealed a higher prevalence of toxoplasmosis (43.7%) with the modified agglutination test (MAT), followed by the enzyme linked immune-sorbant assay (ELISA) (41.7%) and the indirect fluorescent antibody test (IFAT) (37%), while the lowest prevalence was detected with the dye test (DT) (34%). When the data from the first three serological tests were compared with that of the DT test, which was used as a reference test for toxoplasmosis, MAT had the highest sensitivity (96%), followed by ELISA (90.1%) and IFAT, which demonstrated the lowest sensitivity (80.4%). Conversely, IFAT had the highest specificity (91.4%), followed by MAT (88.9%) and ELISA (85.9%).     

绵羊痒螨兔亚种MMP2基因的转录水平及其重组蛋白间接ELISA的建立

本研究旨在分析绵羊痒螨兔亚种MMP2基因(PocMMP2)的虫体转录水平,及建立针对重组PocMMP2(rPocMMP2)蛋白抗体的间接ELISA检测方法(iELISA).通过实时荧光定量PCR(qRT-PCR)测定PocMMP2在不同发育阶段虫体、饱食与饥饿虫体的转录水平,采用原核表达系统获得rPocMMP2,经方阵...     

Comparison of enzyme-linked immunosorbent assay, indirect fluorescent antibody test, and direct agglutination test for detecting Toxoplasma gondii antibodies in naturally aborted ovine fetuses

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to greater than 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.     

Biologic and genetic characteristics of Toxoplasma gondii isolates in free-range chickens from Costa Rica, Central America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 144 free-range chickens (Gallus domesticus) from Costa Rica was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 60 (40.1%) of 144 chickens with titers of 1:5 in 16, 1:10 in 5, 1:20 in 2, 1:40 in 3, 1:80 in 5, and 1:160 or higher in 29. Tissues of all chickens were bioassayed for T. gondii in mice or cats. Hearts and brains of 52 chickens with titers of 1:5 or higher and 16 chickens with doubtful titers were pooled and bioassayed in mice. Tissues from 76 chickens with MAT titers of 1:10 or less were pooled and fed to three T. gondii-free cats. Fecal floats of cats were bioassayed orally in mice but were negative for T. gondii oocysts. T. gondii was isolated by bioassay in mice from 32 chickens with MAT titers of 1:10 or higher. All infected mice from 4 of the 32 isolates died of toxoplasmosis. Genotyping of these 32 isolates using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed five genotypes. Five isolates had type I alleles and one isolate had type III alleles at all loci. The rest 26 isolates contained the combination of type I and II or I and III alleles and were divided into three genotypes. None was found to have genotype II alleles at all five loci. This is the first report of genetic characterization of T. gondii isolates from Costa Rica, Central America.     

西农萨能羊乙酰/丙二酸单酰基转移酶基因重组腺病毒载体的构建

本研究旨在通过构建西农萨能羊脂肪酸合酶(FASN)基因乙酰/丙二酸单酰基转移酶(MAT)区域的重组腺病毒载体,为研究其在奶山羊乳腺上皮细胞中过表达以及功能和作用机制做准备。根据GenBank收录的西农萨能羊MAT序列设计引物,PCR扩增并克隆测序。将目的基因连接到穿梭载体pAdTrack/CMV上并线性化后,转化含有腺病毒骨架载体pAdEasyⅠ的E.coli Bj5183感受态细胞进行同源重组,得到重组腺病毒质粒pAd-MAT-HIS,并用PacⅠ酶切鉴定,将经过PacⅠ线性化的pAd-MAT-HIS转染HEK 293细胞进行病毒包装和扩增,用LaSRT法测定病毒滴度。将本次克隆的MAT序列与GenBank收录的山羊序列对比发现,在601bp处碱基由G转变为A,导致氨基酸序列由丙氨酸(Ala)201转变为苏氨酸(Thr)201。酶切鉴定、绿色荧光蛋白观察、PCR及Western blot检测均证明,重组腺病毒质粒构建成功,病毒滴度为2×109 PFU.mL-1。本研究成功构建了重组腺病毒pAdEasy-MAT-HIS。     

Activities of adenine nucleotide and nucleoside degradation enzymes in platelets of rats infected by Trypanosoma evansi

Pigeons (Columba livia) cohabit with humans in urban and rural areas, representing a public health problem since microorganisms are transmitted through the inhalation of dust from their dry feces (chlamydiosis) and through ingestion of their undercooked or poorly refrigerated meat (toxoplasmosis). This study aimed to evaluate the presence of Chlamydophila psittaci and Toxoplasma gondii in pigeons from four cities in São Paulo State, Brazil. C. psittaci was evaluated through hemi-nested polymerase chain reaction (hnPCR) using cloacal and tracheal swabs, whereas T. gondii specific antibodies were assessed by means of modified agglutination test (MAT), mouse brain and muscle bioassay, and polymerase chain reaction (PCR). To confirm the infection in mice, T. gondii antibodies were assayed by using indirect fluorescent antibody test (IFAT). Considering C. psittaci, 40/238 (16.8%; 95%CI 12.6–22.1%) samples were positive according to hnPCR, especially for the cities of São Paulo (42.5%) and Bauru (35%). As regards T. gondii, 12/238 (5%; 95%CI 2.9–8.6%) serum samples were positive according to MAT. Of these, five samples had titer equal to 1:8; six samples, 1:16; and one sample, 1:32. Bioassay, IFAT and PCR were negative for mouse toxoplasmosis. The absence of T. gondii antibodies suggests that pigeons may be infected with a low concentration of the agent, not detected by the antigen test. Thus, C. psittaci represents an actual problem concerning bird health.