An ELISA with Brucella lipopolysaccharide antigen for the diagnosis of B. melitensis infection in sheep and for the evaluation of serological responses following subcutaneous or conjunctival B. melitensis strain Rev 1 vaccination.

An indirect enzyme-linked immunosorbent assay (ELISA) with unpurified Brucella melitensis smooth lipopolysaccharide (S-LPS) as antigen was evaluated for the serological diagnosis of B. melitensis infection in sheep in comparison with the Rose Bengal (RB), complement fixation (CF), radial immunodiffusion (RID), microplate agglutination (MA) and rivanol agglutination (RIV) tests. Tests RB and CF detected as positive each of the 77 sera from B. melitensis-infected animals tested, the RID (74), MA (76) and the RIV (72) were less sensitive. However, all tests compared were negative when 77 sera from Brucella-free rams were tested. While subcutaneous Rev 1 vaccination induced high response levels in any of the tests, low level responses were obtained upon conjunctival vaccination, particularly in ELISA and RID tests.     

Comparison of response to immunotherapy by intradermal skin test and antigen-specific IgE in canine atopy

The intradermal skin test (IDST) and serologic allergy test (SAT) has been developed for confirming a diagnosis of canine atopy and determining allergens for immunotherapy. To determine the prevalence of causative allergens for canine atopic dermatitis in Japan, IDST and SAT were performed with the CMG Immunodot strips on 95 atopic dogs using 9 allergens. In addition, we compared agreement rate, sensitivity and specificity between them (using IDST as the standard). The allergen most commonly positive in both tests was house dust mites (IDST: 69.5%, SAT: 48.4%). Moreover, Japanese cedar, mugwort and grass mix were detected as attendant causative allergens. Agreement rates between the two tests ranged from 67.4% to 96.8%; the overall mean agreement rate were 81%. SAT was shown to have sensitivity to IDST ranging from 16.7 to 68.2%. The specificities were very high for all allergens, on the order of 94.9-100% (median=98.7%). Finally, the efficacy of immunotherapy was evaluated on 27 atopic dogs based on IDST (15 dogs) and SAT (12 dogs) results. Overall, 60% (9/15) of the IDST group and 66.8% (8/12) of the SAT group experienced a 50% to 100% reduction in their symptomatology. No significant differences were found in response to immunotherapy during the follow-up period between allergen selection methods. These results indicate the value of serologic tests as an aid to identifying an allergen solution for immunotherapy.     

检测布氏杆菌病的四种血清学方法比较及国际标准单位的应用

本研究通过布氏流产杆菌（Brucella abortus)国际标准参考血清（ISABS）的使用，对目的应用的补体结合试验（CFT）、酶联免疫吸附测定法（ELISA）、虎红平板凝集试验（RBPT）及试管凝集试验（SAT）等四种血清学检测方法的测定结果进行了校正和临界点的设定。并对42份不同抗体效价血清进行了测定，同时通过计算和应用标准参考血清校正系数，对不同方法间的测定结果如何换算成国际标准单位进行了研究和探讨。结果表明，该校准方法可对不同实验室间的布氏杆菌病检测结果校准到统一的国际标准上来，可作为血清学测定国际标准化一条途径。     

细胞壁抗原用于布氏杆菌病诊断试验的研究

用培养的布氏杆菌菌体，通过超声波裂解、反复离心制备出布氏杆菌细胞壁抗原。将细胞壁抗原作1：128稀释，用作牛种布氏杆菌酶联免疫吸附试验（ELISA)的抗原；将布氏杆菌细胞壁抗原作1：32稀释，用作平板凝集试验抗原；把细胞壁抗原作1：16稀释用作试管凝集试验抗原，分别建立了牛布氏杆菌ELISA试验、平板凝集试验和试管凝集试验。用这3种方法，检测已知200份平板凝集试验阴性血清，5份平板凝集试验阳性血清。结果5份阳性血清在平板凝集试验、试管凝集试验和ELISA试验中均为阳性；200份阴性血清在平板凝集试验和试管凝集试验中均为阴性，在ELISA试验中有1份为阳性?试验证明，细胞壁抗原，既能用于传统的平板凝集试验和试管凝集试验，又能用于ELISA试验。     

Brucellosis of camels in Kuwait

This study investigated the presence of Brucella antibodies in serum and milk obtained from camels in Kuwait. Brucella strains were also isolated from the foetus using standard technique (Webridge Lab Techniques). Three serological tests for serum were adopted. These tests were Rose Bengal Plate Test (RBPT), the Serum Tube Agglutination Test (SAT) and the Complement Fixation Test (CFT). The RBPT was used for all sera samples, and both SAT and CET were used for the positive RBPT. Camels that showed a titer of 1:40 in SAT or 1:5 in CFT or greater were considered positive. Thirteen of the samples examined were found positive by CFT (at 1:5); by SAT, they showed a titer of 1:20. One serological test, the Milk Ring Test (MRT), was used for milk. Here 3 and 2 were considered positive reactors but 1+ was considered suspicious. We were unable to isolate the Brucella organism from Sedemine and Cream of milk, but we isolated them from Foetus Brucella abortus and it is confirmed by Webridge Laboratory, U.K. It is Brucella abortus (Biovar 1). The prevalence rate was 14.8% from serum by the CFT and RBPT methods and 10.8% by the SAT method. For milk, the prevalence rate was 8.0%. Two Brucella abortus were isolated from 5 foetuses.     

Evaluation of the indirect hemagglutination assay as a practical serodiagnostic test for mycoplasmal pneumonia of swine

Sera from swine experimentally or naturally infected with Mycoplasma hyopneumoniae (the etiological agent of mycoplasmal pneumonia of swine, MPS) were tested by the indirect hemagglutination assay (IHA), the enzyme-linked immunosorbent assay (ELISA) and the complement fixation (CF) test. The IHA detected antibody at comparable times and levels to the other 2 serological tests following experimentally-induced infection. In the late antibody response (greater than or equal to 86 days post-infection), the ELISA titres were higher than either the IHA or the CF test. The IHA appeared least satisfactory when it was used to test sera from commercial swine herds. When 1000 sera were tested, the IHA was positive for only 30 (22%) of 135 sera which were positive by the ELISA and the CF test. The IHA titres were low; 20 of the 30 sera had a titre of only 10. The end-points for the IHA were difficult to read for sera of this low titre. The relationship between positive IHA results for the herd sera obtained at necropsy, and the occurrence of gross or microscopic lesions typical of MPS was poor (41 and 50% agreement, respectively). An agreement of 39% was noted between positive IHA results and the localization of mycoplasmal antigens by an indirect immunofluorescence (IIF) test. However, IHA results correlated significantly (P less than 0.05) with gross and microscopic lesions, but not with the IIF test. No significant correlation was noted between the IHA (or the other 2 serologic tests) and the cultural isolation of M. hyopneumoniae or M. flocculare. On the basis of these results, the IHA appears to have limited promise as a practical test for the diagnosis of MPS in commercial swine herds because of the low titres observed, poor correlation of the IHA and other indicators of MPS, the necessarily subjective determination of end-points, and other inherent technical limitations of the test.     

Laboratory diagnosis of African horse sickness: comparison of serological techniques and evaluation of storage methods of samples for virus isolation

Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA described in this paper was group specific. It did not require calibration with a standard positive serum but did yield elevated values with negative sera that were repeatedly frozen and thawed or heat inactivated. The IFA test was sensitive but could not be used on some field sera as the control cells exhibited fluorescence, possibly due to the animal being recently vaccinated with cell culture material. Sixty-two experimental sera were compared by VN, CF, AGID, and ELISA. Forty sera, 10 positive and 30 negative, were correctly classified by the 5 serologic assays. The 22 remaining sera gave mixed reactions. The AGID had no false positive results but had false negative results for up to 20% of the samples, depending upon the comparison. The VN, CF, and ELISA were similar in their variability. The length of time that virus could be recovered from a viremic blood sample was compared in an evaluation of storage methods for virus isolation samples. Washed erythrocytes were held at 4 C, washed erythrocytes plus stabilizer were held at -70 C, and blood that was drawn into a preservative (oxalate/phenol/glycerol) was held at 4 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of the Fluorescence Polarisation Assay (FPA) for the serological diagnosis of brucellosis

In order to evaluate suitability of Fluorescence Polarisation Assay (FPA) for serological Brucella diagnostic, 1739 samples of sera from cattle, pigs, sheep and goats (65 Brucella-positive, 960-negative and 714 false-positive sera) were investigated at a dilution of 1:10. The cut-off was adjusted by means of ROC analysis. Furthermore, the serum samples were examined for Brucella antibodies using SAT, CFT and ELISA and the results were evaluated regarding sensitivity and specificity. FPA, SAT, CFT and ELISA attained a sensitivity of 92.3, 98.5, 84.6 and 86.2%. In comparison, specificity varied with 87.8, 72.6, 92.5 and 85.8%, respectively. Accordingly, FPA is a suitable test for serodiagnosis of brucellosis.     

Evaluation of four serological techniques to determine the seroprevalence of Neospora caninum in foxes (Vulpes vulpes) and coyotes (Canis latrans) on Prince Edward Island, Canada

The objectives of this study were (1) to evaluate the performance and agreement of serological assays (ELISA, IFAT, Neospora caninum agglutination test and immunoblot) using reference sera and field sera from foxes and coyotes and (2) to estimate the N. caninum seroprevalence in foxes and coyotes on Prince Edward Island, Canada. With fox and coyote reference sera the test performance of the ELISA, IFAT and IB was excellent (100% sensitivity and specificity). NAT showed a low sensitivity (50%). Serum was collected from 201 coyotes and 271 foxes. The seroprevalence observed in the different assays ranged from 0.5 to 14.0% in coyotes and 1.1 to 34.8% in foxes. The seroprevalence, when taking more than one test positive as cut-off value was 3.3 and 1.1% for coyotes and foxes, respectively. From the N. caninum-positive group, all coyotes were older than 3 years. Agreement among assays (measured as prevalence-adjusted bias-adjusted kappa) using the field sera ranged from 0.17 to 0.97. Best agreement was observed between ELISA and IFAT, poor agreement was observed between NAT and the other assays. Positive agreement was moderate to poor among all assays utilized in this study. Although the seroprevalence observed was low, N. caninum antibodies are present in foxes and coyotes on Prince Edward Island (PEI) and their role in the N. caninum epidemiology needs further study.     

Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México.

A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipoplysaccharide (20-30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test (n = 490) and some with the rivanol-agglutination (RIV) test (n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions.     

Comparison of an enzyme-linked immunospecific assay (ELISA) with the cold complement fixation test for the serodiagnosis of Brucella ovis infection

An enzyme-linked immunospecific assay (ELISA) for the serodiagnosis of Brucella ovis infection in sheep is described and compared with the cold complement fixation (CF) test. ELISA was performed in microtiter plates, using horse-radish peroxidase conjugated to anti-normal sheep serum globulins, and hydrogen peroxide plus o-phenylenediamine as substrate. A heated, cell-free B. ovis extract was used as antigen in both tests. ELISA was easier to perform, distinguished better between positive and negative sera, and did not need heat-inactivated sera.     

A comparative study of ELISA and other methods for the detection of Brucella antibodies in bovine sera

Enzyme-linked immunosorbent assay (ELISA), using β-galactosidase and a fluorigenic substrate, was used for the detection of antibodies to Brucella abortus in bovine sera.Among 677 animals from 9 brucellosis-free herds, none reacted in the ELISA. Among 785 animals from 23 brucellosis-infected herds, 336 were positive in ELISA, 229 in the slow agglutination test (SAT), 185 in the complement fixation test (CFT), and 165 in the Rose-Bengal test (RBT).Experimental infections were conducted with two B. abortus strains. At slaughter on day 101, after intraconjunctival infection of heifers with B. abortus strain 19 organisms, 3 animals were positive in the SAT, 3 in the CFT, 4 in the RBT and 11 in the ELISA, and Brucella organisms could be cultivated from 10 animals; among these, 2 scored positive in the SAT, 3 in the CFT, 3 in the RBT and 8 in the ELISA test. Seventeen heifers were infected with organisms of B. abortus strain 2308. On day 101, 11 heifers were found to be carriers, all of which yielded positive results in the CFT, RBT and ELISA tests, but not in the SAT.     

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.     

Dot enzyme-linked immunosorbent assay (Dot-ELISA): comparison with standard ELISA and complement fixation assays for the diagnosis of human visceral leishmaniasis

The dot enzyme-linked immunosorbent assay (Dot-ELISA), standard ELISA and the complement fixation (CF) tests were compared in the serodiagnosis of African visceral leishmaniasis (kala-azar). Assay sensitivity was determined using sera from 44 patients with parasitologically confirmed kala-azar. Using the Dot-ELISA, 42 of 44 patients (95%) were positive at a reciprocal titer of greater than or equal to 32 (titer range 512-524 288). In the standard ELISA technique, 43 of 44 patients (98%) were positive (titer range 32-32 768). At a reciprocal titer of greater than or equal to 8 in the CF test, 35 patients (80%) were positive, 1 (2%) was negative and 8 patients (18%) showed anticomplementary (AC) activity (titer range 8-2048). Specificity, determined using 33 sera from healthy individuals not living in endemic areas, was 97% in both the Dot-ELISA and the standard ELISA (32 of 33 sera); in he CF test, all sera were negative except 1 (3%) which showed AC activity. Sera from patients with Chagas' disease cross-reacted in the dot-ELISA up to a titer of 512. In the standard ELISA, cross-reactions occurred mainly using sera from patients with Chagas' disease, malaria and syphilis, and to a lesser extent with sera from amebiasis, schistosomiasis and trichinosis patients. Overall titer agreement in replicate experiments was highest in the Dot-ELISA (89%), followed by the standard ELISA (80%) and the CF test (72%).     

NO/ADMA在布鲁菌侵染小鼠机体过程中的响应特征

本试验旨在研究布鲁菌侵染小鼠过程中一氧化氮(NO)的作用,以及NO和非对称性二甲基精氨酸(ADMA)在此过程的相互作用关系。以布鲁菌标准疫苗株M5侵染小鼠,首先用小鼠的血清进行虎红平板凝集(RBPT)和试管凝集试验(SAT),再用Griess试剂法和ELISA法测定对照组和侵染组不同组织和血清中的NO和ADMA含量,对小鼠肝脏和脾脏的各个侵染时间段进行CFU计数观察。结果显示,用布鲁菌M5侵染小鼠后,RBPT和SAT在14d时检测到有阳性反应。侵染组和空白对照组NO总含量上变化不大,但侵染组血清中的NO含量随时间出现下降,而肝脾中NO含量随时间出现上升,其他各组织NO含量波动不明显。而ADMA的总趋势与NO相反。CFU计数结果显示,小鼠肝脾内布鲁菌的数量在14d时一直处于增长状态,28d时布鲁菌的数量下降,表明布鲁菌的分裂繁殖和机体NO的含量存在一定的关系。     

The ELISA for the examination of hare sera for anti-Brucella antibodies

Hare brucellosis is caused primarily by Brucella suis biovar 2. Hares along with wild boars are the natural reservoir of this microorganism. In view of restriction of applicability of traditional serological methods the work aimed to develop the ELISA to examine hare sera for the presence of anti-Brucella antibodies. Lipopolysaccharide (LPS) antigen obtained from the strain S19 of Brucella abortus and the conjugate of antibodies against rabbit immunoglobulin with horseradish peroxidase were used in the test. Hares' sera positive and negative in the CFT were used as controls of the ELISA. The sera collected from 9 hares suspected to be infected with Brucella organisms, positive in CFT (in this number 7 hares revealed clinical symptoms or anathomopathological lesions characteristic of brucellosis), 6 sera from hares showing no symptoms of the disease, negative in CFT and 520 sera from hares monitored for brucellosis were tested. All serum samples from hares suspected for Brucella infection were positive in ELISA and 2 of them were negative in RBPT. Additionally among the samples from hares monitored 12 sera were positive in ELISA and CFT, whereas 9 sera from 12 ones were also positive in the RBPT. The obtained results indicated that the ELISA developed in our laboratory proved to be equivalent in specificity to CFT. In addition, ELISA proved to be more sensitive than RBPT for the diagnosis of Brucella infection in hares.     

Comparison of dot enzyme-linked immunosorbent assay (dot-ELISA) with other serological tests for the detection of Brucella antibodies in bovine sera

A dot Enzyme-linked Immunosorbent Assay (dot-ELISA), using whole cell Brucella abortus antigen dotted on the nitrocellulose membrane bound to a plastic strip (dipstick) was employed for the detection of Brucella antibodies in bovine sera. The results were compared with that of serum agglutination (SAT), Rose Bengal plate agglutination (RBPT) and Complement Fixation test (CFT). All the four tests gave negative reaction in 127 sera obtained from a brucellosis free herd. Testing of 549 sera from a chronically infected herd revealed 57 positive and 447 negative animals in all the four assays. Of the remaining 45 sera, 34 were positive in dot-ELISA. Six of these cases were independently detected by dot-ELISA while 28 showed positive reactions in combination with other tests. When serum samples from 158 aborted cases were subjected to dot-ELISA, 79 were found positive. Of these dot-ELISA positive cases, 71 gave positive reaction in SAT, 72 in RBPT and 78 in CFT. B. abortus biotype 3 was isolated from 34 of the 98 aborted fetuses examined.     

New Zealand national mastitis survey: 1965–6 1. Preliminary studies

An enzyme-linked immunospecific assay (ELISA) for the serodiagnosis of Brucella ovis infection in sheep is described and compared with the cold complement fixation (CF) test. ELISA was performed in microtiter plates, using horse-radish peroxidase conjugated to anti-normal sheep serum globulins, and hydrogen peroxide plus o-phenylenediamine as substrate. A heated, cell-free B. ovis extract was used as antigen in both tests.

ELISA was easier to perform, distinguished better between positive and negative sera, and did not need heat-inactivated sera.     

牛副结核ELISA诊断方法研究

本文建立了牛副结核病ELISA诊断方法。采用亲和层析抗原。被检血清以高压粉碎草分枝杆菌吸收原进行吸收。该方法的敏感性76％,特异性97％,通过对2483头奶牛进行检测,检出率为6.1％,并与常规的补反和变态反应进行了比较。作者认为:牛副结核ELISA可以替代补反,做为检测牛副结核的一种有力手段。     

Comparison of the dot-immunobinding assay with the serum agglutination test, the rose bengal plate test and the milk ring test for the detection of Brucella antibodies in bovine sera and milk.

In this study, Brucella antibodies in bovine sera and milk were detected using the dot-immunobinding assay (DIA), the serum agglutination test (SAT), the Rose Bengal plate test (RBPT) and the milk ring test (MRT). For this purpose, a total of 116 paired blood and milk samples collected at the same time from 56 aborted and from 60 healthy dairy cows was examined. In DIA, a nitrocellulose membrane (NCM) was used as the solid phase. Antigen adsorbed on the NCM was extracted from Brucella abortus S99 by heat treatment. The results obtained by DIA were compared with those of SAT, RBPT and MRT. Of the 116 paired blood and milk samples, 24 were positive and 72 were negative by all tests used. Serum samples of six aborted cows were positive by DIA, SAT and RBPT but the milk samples were negative by DIA and MRT. Serum and milk samples of four aborted cows gave positive reaction only by DIA tests. The remaining six aborted cows were negative only by MRT and two of them were negative by both RBPT and MRT. Four sera of healthy cows were found to be positive only by SAT.