Evaluation of a Macroscopic Plate Test and an Indirect Immunofluorescence Test to Detect Leptospiral Antibodies in Bovine Serum

A macroscopic plate test was found to be reliable and convenient for detection of Leptospira serotype pomona antibodies in bovine sera. However, the procedure was unreliable for detecting L. serotype canicola antibodies because of false positive reactions. An indirect immunofluorescence test and the microscopic agglutination test provided comparable results and they effectively detected serotype pomona antibodies in bovine sera.     

A Micro-passive Hemagglutination Test for the Rapid Detection of Antibodies to Infectious Bovine Rhinotracheitis Virus

Serum antibodies to infectious bovine rhinotracheitis virus are most commonly detected using the serum-virus-neutralization test, a time-consuming and expensive procedure. A passive hemagglutination test was developed for the rapid detection of antibodies using the Microtiter System. Two cloned strains of the virus were used in the investigation, namely the BF strain and the Los Angeles strain. Erythrocytes from several species were used. The best results were obtained using the Los Angeles strain of virus adsorbed to sheep cells treated with formalin and tannic acid. Sera from field cases of the disease as well as hyperimmune bovine and rabbit sera were tested using both the micro-passive hemagglutination test and the standard plaque-neutralization procedures. Comparable titers were obtained in the two tests. The micro-passive hemagglutination test is rapid, accurate and economical.     

Complement-Fixing And Neutralizing Antibody Response To Bovine Viral Diarrhea And Hog Cholera Antigens

In calves inoculated with bovine viral diarrhea (BVD) viruses and soluble antigen, the complement-fixing (CF) antibodies appeared before serum-neutralizing (SN) antibodies and remained at high levels throughout the test period. A rapid rise in SN antibodies occurred after challenge with homologous virus with no apparent effect on CF antibody levels.

The CF antibody responses in calves infected with cytopathogenic NADL-MD and noncytopathogenic CG-1220 viruses were similar whereas SN antibody responses indicated strain specificity by reciprocal cross-neutralization tests.

The CF antibody levels in 5 hog cholera (HC) antisera were assayed using the soluble antigen of NADL-MD BVD virus. No demonstrable SN antibodies were present in four HC antisera tested against NADL-MD virus, but a significant titer was present in the commercially prepared antiserum.

Virus was reisolated from animals infected with BVD viruses by buffy coat culture technique during 3 weeks postinoculation, even when significant levels of CF and SN antibodies were present.

     

Detection of Infectious Bovine Rhinotracheitis (IBR) by Immunofluorescence

Infectious bovine rhinotracheitis can be readily and accurately diagnosed by direct fluorescent antibody techniques on infected bovine embryo kidney monolayers. Non-specific fluorescence was encountered in direct observation of bovine nasal secretions and cotyledon impressions.     

Noncytopathogenic Bovine Viral Diarrhea Viruses Detected and Titrated by Immunofluorescence

Noncytopathogenic (NCP) bovine viral diarrhea (BVD) disease agents can be detected and titrated in tissue culture systems by a method employing immunofluorescence. Cytopathogenic (CP) and NCP viruses cross react with fluorescein-conjugated serum globulins produced against either CP and NCP viruses, but the fluorescence is more intense in the homologous system. Serum neutralization titers of sera against both CP and NCP groups were compared for both groups of viruses, and results of cross reactions were in agreement with results from immunofluorescence tests. Results of these two tests were discussed as to possible antigenic groupings of CP and NCP viruses. Use of immunofluorescence as a diagnostic test for BVD and as an alternate method of titrating NCP viruses in tissue culture systems is proposed.     

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。     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. V. Residual Complement Activity

Complement-fixation tests of three different antigen-bovine antibody systems, two antibacterial and one antiviral, were set up with or without normal bovine serum supplement. At the end of the fixation period all mixtures were tested for whole complement activity and for first, second, third and fourth complement components using the conventional, crude reagents R1, R2, R3 and R4. The increased fixation in mixtures containing the bovine serum supplement mainly reflected a greater decrease in second and fourth component activity than in the antigen-bovine antibody mixtures with non-supplemented complement. The decline in first component activity was relatively less. The results of tests for residual third component activity were not consistent.     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for “supplementing” properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56°C. for 30 min. destroyed the supplementing activity of each of these fractions.

Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C¹1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C¹2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C¹1 by this method.

     

Studies on Bovine Virus Diarrhea: Serum Neutralization, Complement-fixation and Immunofluorescence

The complement-fixation, the serum neutralization tests and the fluorescent-antibody technique were the serological methods applied in this laboratory for the detection of antigens for bovine virus diarrhea (BVD). As observed previously, the modified direct complement-fixation (MDCF) test was required to demonstrate antibodies against virus infections of cattle.

At a certain stage of infection, the MDCF test was found to be as accurate and less time-consuming than the serum neutralization test for the detection of antibodies in bovine sera. The modified direct complement-fixing antibodies were detectable in the serum from approximately three weeks up to a few months after infection as compared to several years for the serum neutralization test. Thus, as in most other viral diseases, the MDCF test was of value for detecting recent infections while the serum neutralization test detects both recent and long-standing infections.

The fluorescent antibody technique was of value to detect viral antigens of both cytopathogenic and noncytopathogenic strains of BVD in primary fetal kidney cell cultures inoculated with field specimens. In addition, the virus was detected in six of 220 fetuses collected at a local slaughter house for the preparation of primary cell cultures. The length of time required for the detection and identification of specific viral antigens by immunofluorescence was considerably reduced over that of the serum neutralization and virus interference tests.

     

The Effect of Unheated Normal Bovine Serum on the Complement-Fixing Activity of Heat Inactivated Bovine Antiserum with Homologous Antigen: III. Immunodiffusion Studies

Examination by immuno-diffusion methods in agar gel plates demonstrated that the supplementing fraction precipitated from normal bovine serum by dialysis against dilute buffer, pH 5.0 to 6.6, contains at least three main antigens, and a weaker fourth. Two of these antigens or antigen determinants appeared to be present in globulin fractions prepared by dilution with distilled water or differential precipitation with ammonium or sodium sulphate.     

The Detection of Specific Complement-Fixing Antibodies in Serum of White-Tailed Deer (Odocqileus Virginianus) with Theileria Infection

A complement-fixation (CF) antigen which has been prepared from Theileria infected erythrocytes is capable of reacting to specific serum antibodies of deer acutely infected with Theileria.

No sera from 17 deer known to be free of Theileria infection reacted positively to the CF test. Of 35 tests on sera from 12 infected deer having a parasitemia of 2% or less and no accompanying anemia, only 10 (29%) were positive, 2 (6%) were suspicious, and 23 (66%) were negative. Of 65 tests on 8 acutely infected deer, 49 (75%) were positive, 4 (6%) were suspicious and 12 (18%) were negative. Of the 8 deer in which acute theileriasis occurred all reacted to Theileria antigen at one time or another.

A significant correlation was found between CF titers and the degree of parasitemia in acute infections.

Rabbits were hyperimmunized using erythrocytes from either normal or Theileria infected deer. Reciprocal absorption of the hyperimmune sera with Theileria and normal erythrocytic antigens demonstrated the presence of antibodies specific for Theileria.

     

Use of Monoclonal Antibodies for Immunohistochemical Study of Bovine Spleen on Frozen Sections

Many monoclonal antibodies reactive with bovine leukocyte differentiation antigens are now available. Immunohistochemical staining on frozen sections using these monoclonal antibodies permits study of the functional morphology of bovine spleen. This study confirms accepted notions (B and T dependent-zones) and supplies complementary data about the repartition of CD4 and CD8 cells. γδ cells. MHC (Major Histocompatibility Complex) II expression, and macrophages.     

Detection of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea Viruses in the Nasal Epithelial Cells by the Direct Immunofluorescence Technique

Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 µL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell cultures for virus isolation. In the experimental studies for infectious bovine rhinotracheitis, 29/35 specimens were positive by fluorescent antibody technique and 32/35 by cell culture and in the field cases, 22/119 were positive by fluorescent antibody technique and 19/119 by cell culture. In the field cases of bovine viral diarrhea, 28/69 samples were positive by fluorescent antibody technique and 14/69 by cell culture. When fluorescent antibody technique was performed on inoculated cell cultures a total of 24/69 specimens were positive for bovine viral diarrhea. The sensitivity of fluorescent antibody technique was thus comparable to that of cell culture method for infectious bovine rhinotracheitis and bovine viral diarrhea.     

用大肠杆菌K99和F41菌毛抗原检测牛血清抗体的琼脂扩散试验的研究

用含２ｍｏｌ／Ｌ尿素的磷酸盐缓冲液以热处理方法提取的Ｋ９９和Ｆ４１菌毛作为抗原,对琼脂扩散试验方法进行研究。结果表明本试验方法简便,特异性强,敏感性高,可用于血清抗体检测;所制备的抗原稳定、重复性好。