Comparison of a newly developed enzyme-linked immunosorbent assay with complement fixation and neutralisation tests for serology of bovine respiratory syncytial virus infections

An indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and titration of serum antibodies to bovine respiratory syncytial virus (BRSV). The ELISA was compared with a complement fixation (CF) test and a test for virus neutralising antibody in serum (virus neutralisation [VN] test). Testing sera collected in dairy herds revealed the closest correlation between the results of the ELISA and the CF test with respect to BRSV antibody titres. The VN test detected BRSV antibodies in a higher percentage of acute phase sera compared to the other two tests in field samples and in early bleedings of experimentally infected calves. However, the VN test was less effective in making a diagnosis of BRSV infections on the basis of a significant titre increase in paired sera. For this purpose the ELISA was found to be the most sensitive test.     

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses

OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.     

Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.     

Functional analysis of antibody responses of feedlot cattle to bovine respiratory syncytial virus following vaccination with mixed vaccines.

The antibody response of cattle to bovine respiratory syncytial virus (BRSV) immunization was investigated using 4 different commercially available mixed vaccines. Forty, 5-6 month old, beef calves, randomly assigned to groups of 10, were vaccinated on day 0 and 21 with 1 of 3 inactivated vaccines, (3 groups), or a modified live virus (MLV) vaccine. BRSV-specific antibody responses were measured prior to vaccination and on day 35 by using an enzyme linked immunosorbent assay (ELISA), virus neutralization assay (VN), a fusion inhibition assay (FI); and responses were also measured for their ability to facilitate antibody dependent, complement mediated cytotoxicity (ADCMC) of BRSV infected cells. Sera from day 35 were, in addition, analyzed by use of an IgG1, IgG2 isotype specific ELISA. All vaccines induced significant increases in BRSV specific IgG antibody as measured by ELISA, but only one inactivated and the MLV vaccine induced significant increases in VN titers. Fusion inhibiting antibody titers were low or undetected in calves vaccinated with the inactivated vaccines. Vaccination with modified live virus induced significantly higher titers of fusion inhibiting antibodies, which are considered to be most highly correlated with protection. The VN to ELISA and FI to ELISA ratio of the calves that received MLV vaccine were significantly greater than the calves receiving the 3 inactivated vaccines. Vaccination with MLV induced the highest IgG2/IgG1 ratio. This difference was small, and only significant relative to 2 of the inactivated vaccine groups, which were not significantly different from each other. The higher proportion of IgG2 isotype in the MLV sera was not associated with lower ADCMC, a function not attributed to this isotype. The VN and FI titers, but not the ELISA value of the sera, were most predictive of ADCMC. The inactivation processes apparently alter epitopes and affect the induction of functional antibodies.     

Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox

Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus. The VN test was conducted in 96-well tissue-culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr. The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age. However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable. On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge. Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period. This study showed that the ELISA was considerably more sensitive and practical than the VN test.     

Single dilution ELISA for detection of serum antibody to foot-and-mouth disease virus in cattle

A single dilution blocking ELISA was developed and evaluated for measuring serum antibody to foot-and-mouth disease virus (FMDV). Basic parameters of the assay were established and a positive-negative threshold determined from testing 176 specific antibody negative sera from Australian cattle. Sera collected from immunised animals in Thailand were tested by ELISA and virus-neutralisation (VN) tests and the results compared. A positive correlation between ELISA and VN titres was recorded for each of the 3 FMDV serotypes endemic in Thailand, with the overall correlation coefficient being r = 0.8990. A positive correlation for each of the serotypes was also found between ELISA titre and the degree of blocking (percentage inhibition) of each test serum at a dilution of 1:16, with the overall correlation being r = 0.8704. This simplified ELISA was sensitive, specific and gave reproducible results, and had the potential to test quickly and efficiently a considerable number of sera.     

Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus

Four tests for detection of antibodies to bovine leukemia virus (BLV) were compared. The sera that were tested came from cattle in naturally infected commercial dairy herds, cattle that were infected under experimental conditions, and cattle in an isolated BLV-free herd. The tests that were compared included a radioimmunoprecipitation assay (RIA) with p24 antigen, a RIA with glycoprotein (gp) antigen, an agar-gel immunodiffusion (AGID) test with gp antigen, and a virus-neutralization (VN) test that was based on inhibition of BLV-induced syncytia in cell culture. Results of the 4 serologic tests agreed for 96.8% of the sera from cattle in commercial herds. The gp RIA detected the greatest number of positive sera (188); it was followed in turn by the p24 RIA (187), the VN test (183), and the AGID test (176). The gpd RIA titers of the 12 sera that gave negative AGID results were 175 or less. In RIA, the percentage of precipitation of labeled antigen by positive sera was almost always higher with gp antigen than with p24 antigen. Satisfactory sensitivity in the p24 RIA required the acceptance of a low level of antigen precipitation, 15%, as a positive test. In the gp RIA, however, almost all positive sera precipitated at least 50% of the labeled antigen. Nonspecific precipitation of antigen in the RIA by sera from BLV-free cattle ranged from 4% to 10%. Examination of sequential serum samples from 17 experimentally infected cattle showed that BLV antibody was first detected 2 to 8 weeks after inoculation. In 9 cattle, seroconversion was detected simultaneously by all of the tests. Results from the other 8 cattle indicated that seroconversion could be detected first by p24 RIA, followed by the gp RIA and the VN test. The longest interval between RIA seroconversion and AGID seroconversion was 10 days. Monthly tests of sera from 10 laboratory cattle that were infected by contact exposure showed that 7 animals seroconverted in all tests at the same time. Two cattle were positive first in RIA, but the next month they were also positive in the VN and AGID tests. One animal was positive in the RIA and the VN test for 2 months before antibody was detected by AGID.     

Comparison of a tissue-culture virus-neutralization test and the enzyme-linked immunosorbent assay for measurement of antibodies t infectious bronchitis

Two serological tests, the virus-neutralization (VN) test in tissue culture using a tissue-cell-adapted virus and the enzyme-linked immunosorbent assay (ELISA), were compared to detect antibodies against Massachusetts 41 and Connecticut 46 strains of Infectious Bronchitis Virus (IBV). The VN test was conducted in wells of microplates by the usual procedure. The two strains of IBV were adapted after 20 serial passages to induce CPE in 24 hours in chickens embryos kidney cells (CEKC). The ELISA test was carried out using partially virus following ultracentrifugation of each stain of IBV as antigen. The ELISA test detected higher geometric mean antibody titers (GMT) against both strains of IBV than did the VN test. One hundred four serum samples taken at 1, 3, 5, 9, 22, 24, and 26 weeks of age from a flock of chickens vaccinated with the Mass strain three times and the Conn strain of IBV two times during the growing period showed higher antibody titer responses to the Conn 46 than to the Mass 41 strain. Maternal antibodies in chicks one week of age were readily detected by the ELISA test, whereas low or insignificant titers were found by the VN test. Sera of vaccinated chickens collected following challenge with Mass 41 or Conn 46 strain of IBV showed that the ELISA was more sensitive and showed higher titers than did the VN test. Although the VN test showed no rise in GMT in the same sera tested with the heterologous virus, the ELISA showed a slight increase or cross-reaction. The serum samples from the unchallenged control group showed no change in GMT with either test or IBV strain.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

Application of a modified indirect fluorescent antibody test to the detection of antibodies to bovine respiratory syncytial virus in Ontario cattle.

A modified indirect fluorescent antibody test for the detection of serum antibodies to bovine respiratory syncytial virus was developed. The test made use of Terasaki plastic microtiter plates in which bovine respiratory syncytial virus (Saskatchewan strain) infected Georgia bovine kidney cells were grown and fixed in situ by a modified acetone fixation procedure. Evans blue dye was used as a counterstain to reduce nonspecific fluorescence. In a study of 986 field sera from a geographically broad cross-section of mature Ontario cattle, 95% of the samples were found to be positive at or above a 1:2 dilution. No seronegative regions, counties or herds were identified. When representative samples covering a range of indirect fluorescent antibody titers were further examined by a microtiter virus neutralization assay, a significant agreement was found between the two tests. Up to a fourfold decrease in titer was observed when antigen coated plates were stored at -70 degrees C for four months. The modified indirect fluorescent antibody test for bovine respiratory syncytial virus antibody detection proved to be a rapid, practical procedure for use in the diagnostic laboratory. This study confirms that bovine respiratory syncytial virus is widespread in the Ontario cattle population and that most mature cattle can be assumed to have been exposed to this virus.     

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)     

Studies on bovine leukosis. VII. Further experience with an ELISA for the detection of antibodies to bovine leukosis virus

An enzyme linked immunosorbent assay (ELISA) and the agar gel immunodiffusion test with bovine leukosis virus glycoprotein as antigen (AGIDT-BLV gp) were further used to test 633 bovine sera for antibodies to BLV. Both tests detected the same number of sera positive (149) or negative (464) for antibodies. Nine sera were negative in the ELISA but found to be weakly positive (2 sera) or bending the control line (7) in the AGDT-BLV gp. On the other hand 11 sera were scored negative in the AGIDT-BLV gp but were weakly positive (9 sera), positive (1), and strongly positive (1) in the ELISA. Both tests are used routinely in this Institute as they complement each other, specially if sera with low antibody titers are under investigation. It is concluded that ELISA can fully replace radioimmunoassays in the serodiagnosis of enzootic bovine leukosis.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of enzyme-linked immunosorbent assays for the detection of Fusobacterium necrophorum antibody in animal sera

Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.     

Evaluation of the critical parameters of a sensitive ELISA test using purified infectious bovine rhinotracheitis virus antigens

An enzyme-linked immunosorbent assay (ELISA) for the survey or titration of bovine sera for the presence of IgG antibodies against infectious bovine rhinotracheitis (IBR) virus was developed. The optimal conditions of serum dilution, antigen concentration, conjugate dilution, substrate concentrations, and reaction time were established using the signal/ noise (S/N) ratio as the determining criterion. Equilibrium density gradient purified IBR virus was used as antigen at an optimal concentration of 0.60 μg/cuvette. The use of purified antigen allowed the testing of sera at a 1 : 10 dilution without nonspecific reaction.The conditions of conjugate dilution, substrate concentration and reaction time were shown to have significant effects on the ELISA test. Results from 35 sera showed this optimized ELISA procedure to be as much as 1000-fold more sensitive than the serum neutralization plaque reduction assay. Numerous sera showing no neutralizing titer to IBR virus were found to be positive when examined by this ELISA method.     

Syncytium-induction inhibition test with complement for detection of antibodies against bovine leukemia virus.

A modified syncytium-induction inhibition test which is more sensitive than the immunodiffusion test, was developed using rabbit complement. In this test, fetal lamb kidney cells continuously infected with bovine leukemia virus were used as effector cells, and the CC81 cat cells transformed with murine sarcoma virus, were used as indicator cells. The syncytium-induction inhibition effect of anti-bovine leukemia virus serum was enhanced significantly by the addition of rabbit complement. The syncytium-induction inhibition titers had a statistically significant correlation with the immunodiffusion titers and were four to 64 times higher than immunodiffusion titers. In 12 experimentally infected cattle, the syncytium-induction inhibition test detected the antibodies earlier than the immunodiffusion test and continuously detected them when immunodiffusion antibody changed to negative. In the 81 sera from naturally infected herds, 35 (43.2%) were positive by the immunodiffusion test and 55 (67.9%) by the syncytium-induction inhibition test.     

A comparative examination of swine sera for antibody to Aujeszky virus with the conventional and a modified virus-serum neutralization test and a modified direct complement fixation test.

Sera of pigs from élite breeding herds, of boars and sows collected at slaughter-houses, and of pigs from herds known to be infected, were examined for antibody to Aujeszky virus. The conventional and a modified virus-neutralizing antibody (VNA) test and a modified direct complement fixation (CF) test were employed. In simultaneous titrations of positive sera the modified VNA test gave titers approx. 4 log2 units above the titers obtained by the conventional test. The conventional VNA test was found insufficiently sensitive. Unspecific neutralization in the modified VNA test was infrequent in serum dilution 1/2 and rare in dilution 1/4. The GF tests on sera of slaughter sows and animals from known infected herds showed a remarkable consistency with the VNA tests. Inconsistent results were obtained with but few sera. Abt. 5 % of the sera could not be examined because of complement fixation with control antigen.