A simple, rapid syncytial-inhibition test for antibodies to bovine leukemia virus.

Cocultivation of equal numbers of cells from a fetal lamb kidney line infected with bovine leukemia virus and African green monkey (Vero) cells results in the rapid production of syncytia. The effect was blocked or inhibited by serum containing antibodies to bovine leukemia virus. A serological test based on syncytial inhibition was compared to the agar gel immunodiffusion test and the modified direct complement fixation test for the detection of bovine leukemia virus antibodies in sera from leukosis-free cattle, cases of adult enzootic bovine lymphosarcoma and cattle from herds in contact with enzootic lymphosarcoma. The results showed the syncytial inhibition test to react positively with sera from all cases of adult enzootic lymphosarcoma, but to be much less sensitive than the other tests in detecting bovine leukemia virus antibodies in sera of exposed animals.     

The role of virus dose in experimental bovine leukemia virus infection in sheep.

Twenty-four, six month old lambs were assembled into four groups of five animals each and one group of four animals. All groups were inoculated with lymphocytes from a single donor lamb infected with bovine leukemia virus. The inoculum varied from 250 to 250,000 lymphocytes, in tenfold increments. Animals were exposed by intradermal injection in the neck region immediately anterior to the left shoulder joint. All groups were monitored at 0, 3, 7 and 12 weeks after inoculation using the following procedures: a. Syncytia induction assay for detection of bovine leukemia virus in peripheral blood lymphocytes. b. Agar gel immunodiffusion against the gp51 antigen of bovine leukemia virus for the detection of antibovine leukemia virus gp51 antibody. c. Lymphocyte stimulation test for the assessment of cell-mediated immunity using mitogen, nonfractionated bovine leukemia virus antigen, and partially purified bovine lymphoma tumor-associated antigen for the in vitro activation of lymphocytes from bovine leukemia virus-inoculated and sham-inoculated, control animals. d. Routine hematological techniques for the assessment of total leukocyte and lymphocyte counts. The median infectious dose for lymphocytes from the single bovine leukemia virus-infected donor used in this study was determined to be 2000 cells. The syncytia induction assay detected more infected individuals (13/23) at an earlier time than did the agar gel immunodiffusion assay (10/23). Using either serological or virus isolation techniques, infected animals were first detected at three weeks postinoculation in the group receiving the high-dose inoculum and at seven weeks postinoculation in groups receiving low- or medium-dose inocula.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Precipitating antibodies in experimental visna and natural progressive pneumonia of sheep

Serological responses of Icelandic sheep experimentally infected with visna virus (vv) were contrasted with responses in American Targhee sheep naturally infected with progressive pneumonia virus (PPV). Precipitating antibodies assayed by immunodiffusion were compared with the neutralising and complementing fixing antibody response. In experimental infections with vv, complement fixing and neutralising antibodies appeared early after infection and rose to high levels in all sheep, while precipitating antibodies were detected only at minimal titre. In natural infections with PPV, immune responses were less consistent and precipitating antibodies were detected more frequently than complement fixing or neutralising antibodies against PPV. These results may suggest important biological differences between the lytic fibroblast-tropic virus strains used for experimental infection of Icelandic sheep and the nonlytic macrophage-tropic strains of PPV circulating in nature. Lytic strains evoke a brisk response against the viral glycoprotein with high titre neutralising antibody while nonlytic strains induce a less consistent response to the glycoprotein.     

Evaluation of serologic methods for the detection of antibodies to encephalomyocarditis virus in swine fetal thoracic fluids.

Hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) tests were compared to the serum neutralization (SN) test to evaluate their ability to detect antibodies to encephalomyocarditis virus (EMCV). Swine fetal thoracic fluids of known EMCV SN antibody titers (200 samples greater than or equal to 1:2, 100 samples less than 1:2) were selected from a collection of field cases. The thoracic fluids were tested for EMCV antibodies by HI and AGID, and the results were compared to those of the SN test. Of 200 SN antibody-positive samples, 183 (91.5%) and 173 (86.5%) were positive in HI and AGID tests, respectively. Of 100 SN-negative samples, 81 (81%) and 94 (94%) were negative in HI and AGID tests, respectively. Agreement between the tests was analyzed by calculating Kappa values. The values were 0.73 between SN and HI tests and 0.77 between SN and AGID tests, indicating very good to excellent agreement for HI and AGID tests with the SN test. Of 200 SN-positive samples, 19 samples with low SN titers (1:2-1:16) were further tested by Western immunoblotting, and all were confirmed as positive. Interpretation of the present results suggests that both HI and AGID tests can be used as alternatives to the SN test.     

Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.     

Eradication of bovine leukemia virus infection in commercial dairy herds using the agar gel immunodiffusion test.

Demands for bovine leukemia virus test negative breeding cattle and for semen from bovine leukemia virus test negative bulls by several countries have encouraged the eradication of bovine leukemia virus infection from selected herds in Canada. This project was undertaken to evaluate the suitability of the agar gel immunodiffusion test, standardized to detect anti-bovine leukemia virus glycoprotein antibodies, for eradication of bovine leukemia virus from commercial dairy herds. Of nine participating herds, the prevalence rate of bovine leukemia virus infection was low (less than 10%) in three, medium (11-30%) in four and high (greater than 30%) in two. The herds were tested by the agar gel immunodiffusion test, reactors were removed and the herds were then retested at regular intervals. The results indicate that it is possible to eliminate bovine leukemia virus infection from the herds after two to three cycles of agar gel immunodiffusion tests and prompt removal of the reactors.     

Antigens and antibodies of malignant catarrhal fever herpesvirus detected by immunodiffusion and counter-immunoelectrophoresis

Using hyperimmune rabbit and cattle sera, immunodiffusion (ID) and counter-immunoelectrophoresis (CIEP) tests detected three or four and two or three malignant catarrhal fever (MCF) virus antigens, respectively, in infected cells. The ID test detected precipitating antibodies to MCF virus in $\text{3}\text{9}$ experimentally infected rabbits, $\text{0}\text{14}$ experimentally infected cattle, $\text{1}\text{13}$ naturally infected cattle, $\text{62}\text{176}$ wildebeest and $\text{3}\text{20}$ hartebeest. The CIEP test detected specific antibodies in $\text{3}\text{9}$ rabbit sera, but non-specific reactions prevented its use with bovine sera.The CIEP test was 2 to 4 times more sensitive than ID for detecting antibodies to MCF virus, but both tests were less sensitive than indirect immunofluorescence.The ID test demonstrated an antigenic relationship between wildebeest and hartebeest strains of MCF virus. Neither ID nor CIEP detected MCFV antigens in tissues infected with MCF virus.     

Estimation of the sensitivity and specificity of the agar gel immunodiffusion test for bovine leukemia virus: 1,296 cases (1982-1989).

A retrospective study of the results of 12,549 agar gel immunodiffusion tests for bovine leukemia virus, conducted on 1,296 dairy bulls over an 8-year period, was performed to estimate the sensitivity and specificity of the test. The number of tests performed on each bull ranged from 5 to 35, with a mean of 9.7 tests per bull. Bulls were categorized by their agar gel immunodiffusion test responses; 1,069 (82.5%) were noninfected and 227 (17.5%) were infected. Eighteen false-positive results were reported from the noninfected bulls. Test specificity was estimated to be 99.8%. Thirty-one false-negative results were reported from the infected bulls. Test sensitivity was estimated to be 98.5%. Fifty-six bulls had 1 or more positive responses when less than 6 months old. In 26 (46%), these results were thought to be attributable to colostral immunity.     

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the efficacy of different adjuvants in live stock specific bacterial vaccines for turkeys against Bordetella infection and onset of antibody titers in respect to the age of the turkey poults

For evaluating the influence of the age of the vaccinated birds on the development of antibodies, five groups of turkey poults were inoculated subcutaneously at day 1, 7, 10, 14 and 21 of life with vaccine containing inactivated Bordetella avium and Freund's incomplete adjuvant. No matter which vaccine schedule was used, serum antibodies with the ELISA were first detected at the 28th day of life and increased continuously until the 49th day, when it exhibited either a peak or a plateau. Aluminium hydroxide, Freund's complete and incomplete adjuvant and a mineral oil-arlacel-tween-mixture being permitted adjuvants (appendix II EWG 2377/90) and the adjuvant Gerbu 100 were evaluated for their suitability. Turkeys were vaccinated at the age of three weeks and examined clinically as well as serologically up to the 11th week. Humoral antibodies were detected quantitatively using an ELISA for IgG and a microagglutination test for IgM and qualitatively using immunodiffusion. The damage at the application site was rated by measurement of the swelling of the tissue. In the 10th week, the animals were infected with the agent for challenge. The serological examination for IgG antibodies in the ELISA both treatments with Freund's adjuvants resulted in high titers, which differed significantly from the unvaccinated control after 21 days. IgM could be detected from day 7 onwards in all vaccinated groups and showed the highest titers when aluminium hydroxide was used as adjuvant. In the immunodiffusion assay, precipitating antibodies could be found from the first week after vaccination onwards. There was no correlation between the occurrence of precipitating antibodies and ELISA titers. The measurements of the swelling of the tissue in the beginning showed the largest swellings in the animals injected with Freund's incomplete adjuvant and differed significantly from the unvaccinated control. In the 10th week, the animals were infected with Bordetella avium for challenge. In comparison to the unvaccinated animals, all vaccinated turkeys, no matter which adjuvant was used, showed a distinct and significant reduction of the reisolation rate.     

Antigen and Antibody in Aleutian Disease in Mink II. The Reaction of Antibody with the Aleutian Disease Agent Using Immunodiffusion and Immunoelectroosmophoresis

Aleutian disease viral (ADV) antigen was prepared by fluorocarbon extraction of spleen, liver, and lymph nodes from mink experimentally infected ten days previously. Using a potent ADV antigen, antibody was detected by immunodiffusion (ID) and immunoelectroosmophoresis (IEOP). Utilizing these precipitin tests, antibody was detected in all the mink sera tested as early as seven days after experimental infection. Titer of antibody increased throughout the infection period. Titers of more than 100 were reached by 15 days post infection, titers of 1,000 at one month, and titers of more than 5,000 to 10,000 were achieved at two months post infection and thereafter. The immunodiffusion test gave similar or slightly lower titers than those detected by the IEOP.

The IEOP test promises to be a most useful technique for the diagnosis of aleutian disease because it is simple, rapid and specific and is capable of detecting infection early in the course of the disease. It is suggested that this test should be utilized especially for the screening of animals purchased or imported as breeding stock onto ranches.

     

An epidemiological study of natural in utero infection with bovine leukemia virus.

The purpose of this study was to examine rates of natural in utero infection with bovine leukemia virus for association with breed, sex, dam age, dam parity and time of maternal seroconversion. Analyses conducted for breed and sex, dam age and parity and time of maternal seroconversion were the FUNCAT procedure for categorical data, Wilcoxon Rank Sums test and Fisher's exact test, respectively. A total of 223 calves born between July 1979, and September 1980, to cows infected with bovine leukemia virus in the University of Florida Dairy Research Unit herd were tested for detectable bovine leukemia virus antibodies prior to the consumption of colostrum. Sera were tested for antibodies by agar-gel immunodiffusion and radioimmunoprecipitation using the glycoprotein-51 antigen. In a group of 125 calves in which in utero infection could be confirmed through serological follow-up (group A), eight calves (6.4%) had precolostral bovine leukemia virus antibodies. For all 223 calves (group B), 18 (8.1%) had detectable bovine leukemia virus antibodies. For calves in group A, no associations were detected between precolostral bovine leukemia virus antibodies and breed (p = 0.66), dam age (p = 0.86), dam parity (p = 0.83), or time of maternal seroconversion to bovine leukemia virus (p = 0.50). However, precolostral bovine leukemia virus antibodies were found in 17.4% of the males and 3.6% of the females in group A (p = 0.11) and in 12.4% of the males and 3.6% of the females in group B (p = 0.04).     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

Enhancement of Leptospira hardjo agglutination titers in sheep and goat serum by heat inactivation.

Heat inactivation of sheep serum samples resulted in the detection of an additional 9% reactors to Leptospira hardjo that were negative on the initial test of fresh samples. Treatment with EDTA gave results generally similar to heat inactivation suggesting that complement was responsible for the inhibition of agglutination. Tests on heat inactivated serum from experimentally infected sheep and goats revealed enhanced titers or reactions which were not detected in fresh serum.     

Detection of bovine leukemia virus antibodies using the cytotoxicity test in comparison with other serologic methods

In ninety-five serum samples taken in a herd of five-year to seven-year cattle that was heavily infected by bovine leukosis virus, the four serological assays were used for demonstration of the antibodies to bovine leukosis virus; cytotoxic test, immunodiffusion test in agar-agar, immunoenzymatic test and serum neutralizing test. The serum neutralizing test was found to be the most sensitive: further seven positive reagents were diagnosed in comparison with immunoenzymatic test; cytotoxic and immunodiffusion tests in agar-agar have the lowest sensitivity and the results of these tests are almost identical. It was found out in forty titrated samples that serum neutralizing test was by as much as 20 times more sensitive than immunoenzymatic test, the latter being about 50 times more sensitive than cytotoxic and immunodiffusion tests.     

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.     

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.     

Indirect hemagglutination test in equine infectious anemia.

An indirect hemagglutination was developed for the diagnosis of equine infectious anemia using sheep red blood cells coated with group specific virus antigen which had been highly purified by affinity chromatography. The presence of indirect hemagglutination antibodies was demonstrated in horses with equine infectious anemia since the cells were specifically agglutinated by all the serum samples obtained from experimentally infected horses. Antibodies appeared within 35 days after inoculation, and development of which coincided well with that of precipitating and complement fixing antibodies. Titer of indirect hemagglutination antibodies were ten to 320 times greater than those of precipitating antibodies. Test results could be read more clearly by the indirect hemagglutination test especially in weakly positive cases. Ninety-six samples from suspected field cases collected from every region of Japan which were positive on the immunodiffusion test were also positive on indirect hemagglutination test. Serum samples from 420 horses in one race track were examined by both the indirect hemagglutination and immunodiffusion tests to determine the reliability of the indirect hemagglutination test for diagnosis of equine infectious anemia. The same result was obtained on both tests. Based on this evidence, the indirect hemagglutination test can be employed as a very sensitive serological test for the diagnosis of equine infectious anemia.