An agar gel immunodiffusion (AGID) test was used over a 3-year period to examine 1,871 serum samples from sheep representing 5 Mycobacterium paratuberculosis infected flocks and 4 flocks presumed to be uninfected. Of 1,032 sheep, 31 had positive AGID test results (scoring 1 to 5), and 23 of these 31 were ecropsied. Infection with M paratuberculosis was confirmed by 1 or more of the following findings: observation of typical lesions on histologic examination of sections of ileum or ileocecal lymph nodes, observation of clumps of acid-fast bacteria in mucosal smears of ileum, and isolation of the organism from feces or tissue. False-positive results on AGID testing were not found in sheep from flocks known to have exposure to Cotynebacterium pseudotuberculosis. Diarrhea in infected sheep was observed infrequency; chronic, severe weight loss was the most common sign observed. On histologic examination of tissues from 20 infected sheep, 16 (80%) had diffuse lesions of the ileum and 13 (65%) had acid-fast bacteria in areas of ileal inflammation; 4 had discrete granulomas and peripheral lymphocytic infiltrates in the ileum. Sheep with diffuse lesions tended to have higher mean scores on AGID testing and examination for acid-fast bacteria, compared with those from sheep with more discrete lesions. Bacteriologic culture yielded M paratuberculosis from only 3 sheep with paratuberculosis. On the basis of results of this study, we suggest that the nature of the response to infection with M paratuberculosis may influence the results of diagnostic tests for paratuberculosis, and that AGID testing may be useful to identify M paratuberculosis infection in sheep with chronic weight loss and in flock-screening programs.

Two agar gel immunodiffusion (AGID) kits for the serodiagnosis of bovine leukemia virus (BLV) were imported from Europe and were compared with North American kits. The BLV AGID kits from North America and from Europe differed significantly. The punches were different, as were the pattern distribution in the agar of the reference and the test sera, resulting in differences in the reading of the immunoprecipitation lines. Based on the testing of 1200 serum samples from cattle, the European kits gave a good correlation with the American kits, as indicated by their respective kappa values. However, the European kits were found to be less sensitive when evaluated against weakly positive samples from field specimens or following a dilution trial. Only 65% and 50% of the weakly positive samples detected by the American kit #1 were detected by the European kits #2 and #3, respectively. The American kit was also capable of detecting BLV antibodies in 45% of strongly positive samples diluted 1/50 in negative sera, while antibodies were detected in only 15% of the samples with the European kit #2 and in none of the samples with the European kit #3. False negatives were also detected with the European kits. Among the false negatives, the degree of expected reactions was weak (European kit #2) or of varying degrees of positivity (European kit #3). Besides the differences in format and performance, the BLV-AGID kits in Europe are evaluated with the National Standard Serum E4 while a proficiency panel composed of a quadruplicate set of 10 reference sera is used in Canada to monitor the kits. Based on the overall observations, we noted a lack of standardization between the BLV-AGID kits used in North America and in Europe.

A study was performed to determine whether experimentally induced bovine leukemia virus (BLV) infection in cattle could be detected earlier by use of polymerase chain reaction (PCR) amplification of genomic DNA extracted from leukocytes than by use of conventional agar-gel immunodiffusion (AGID). The PCR primers were designed to amplify a 375-basepair region of the proviral gag gene. Five cows were identified that were BLV-negative on the basis of AGID and PCR results. At day 0, these cows were inoculated IM with blood pooled from 3 naturally infected cows. Blood samples were taken on days 0, 1, and 7, and every 2 weeks thereafter until 3 months after inoculation. Three of the cows were BLV-positive by AGID test results 3 weeks after inoculation, and the remaining 2 seroconverted at 5 weeks. In contrast, all 5 cows were BLV-positive by PCR results 7 days after inoculation and remained positive for the duration of the study. Five cows that were BLV-positive by AGID test and PCR results on day 0 and from which samples were obtained at the same times as those from the other 5 cows, remained BLV-positive by results of both tests during the course of the study. Results indicate that under experimental conditions, BLV infection in cattle can be detected as much as 2 to 4 weeks earlier by use of PCR than by use of the AGID test.

The performance of the serum complement fixation (CF) test was compared with that of a serum agar gel immunodiffusion (AGID) test on 74 subclinically infected and 154 uninfected cattle in 6 commercial midwestern dairy herds with Mycobacterium paratuberculosis infection and on 30 cattle in a herd that was free of infection. Infection status of cattle within herds was established by performance of a series of 3 or more fecal cultures and of ileocecal lymph node cultures of culled cattle. In cattle with subclinical infection detected by culturing, the sensitivity estimates of the CF and AGID tests were 10.8% (3.6% SE) and 18.9% (4.5% SE), respectively. In the cattle classified as disease free, the specificity estimates of the CF and AGID tests were 97.4% (1.3% SE) and 99.4% (0.6% SE), respectively. Neither set of estimates was significantly different. Negative test results obtained with the use of either test in apparently normal cattle from suspect herds should be interpreted with caution because both tests suffer from low sensitivities in subclinically infected animals. However, the AGID test may be more useful in regulatory situations in which the CF test is currently used because the AGID test is easier to perform and to interpret.

Ontogeny of the third component of complement (C3) was monitored in 10 neonatal swine, using a radial immunodiffusion technique. Significant differences in mean serum C3 concentrations, expressed as percentage of C3 concentration in a pooled standard drawn from 15 adult swine, were not observed between serum samples collected before pigs suckled and at 2 days of age (P = 0.2583). Serum C3 concentrations increased significantly between 2 and 7 days of age (P less than 0.0001) and 7 and 14 days of age (P less than 0.0001). Concentrations comparable with those in adults were not observed at 14 days of age and significant changes were not observed thereafter. Acquisition of adult concentrations of C3 appeared to be a function of endogenous production by the neonate, rather than by passive colostral transfer.

Results of a double agar gel immunodiffusion (Ouchterlony) test that contained a polysaccharide (poly-B) antigen of Brucella melitensis strain B115 were compared with those of 5 other serotests. To determine the sensitivity and specificity of the immunodiffusion, standard tube, 2-mercaptoethanol, Rivanol, card, and complement fixation tests, sera obtained from 1,328 vaccinated, infected and seronegative cattle, 56 of which had been examined bacteriologically, were used to evaluate the humoral response to Brucella sp. The poly-B antigen confirmed infection in 87.5% of the 56 cattle from which Brucella abortus biotype 1 had been isolated, and in 96.6% (205/212) of a group of cattle suspected to be infected on the basis of results of conventional serotests. Likewise, sera from 4 groups of vaccinated cattle did not react with poly-B antigen, whereas they did not react in conventional tests. The poly-B antigen was more specific in detecting infected cattle even in a group of vaccinated adults. A useful strategy to identify infected cattle might be screening, using a combination of the Rivanol and card tests together with the agar-gel immunodiffusion test containing poly-B antigen.

An agar gel immunodiffusion (AGID) test was used over a 3-year period to examine 1,871 serum samples from sheep representing 5 Mycobacterium paratuberculosis infected flocks and 4 flocks presumed to be uninfected. Of 1,032 sheep, 31 had positive AGID test results (scoring 1 to 5), and 23 of these 31 were ecropsied. Infection with M paratuberculosis was confirmed by 1 or more of the following findings: observation of typical lesions on histologic examination of sections of ileum or ileocecal lymph nodes, observation of clumps of acid-fast bacteria in mucosal smears of ileum, and isolation of the organism from feces or tissue. False-positive results on AGID testing were not found in sheep from flocks known to have exposure to Cotynebacterium pseudotuberculosis. Diarrhea in infected sheep was observed infrequency; chronic, severe weight loss was the most common sign observed. On histologic examination of tissues from 20 infected sheep, 16 (80%) had diffuse lesions of the ileum and 13 (65%) had acid-fast bacteria in areas of ileal inflammation; 4 had discrete granulomas and peripheral lymphocytic infiltrates in the ileum. Sheep with diffuse lesions tended to have higher mean scores on AGID testing and examination for acid-fast bacteria, compared with those from sheep with more discrete lesions. Bacteriologic culture yielded M paratuberculosis from only 3 sheep with paratuberculosis. On the basis of results of this study, we suggest that the nature of the response to infection with M paratuberculosis may influence the results of diagnostic tests for paratuberculosis, and that AGID testing may be useful to identify M paratuberculosis infection in sheep with chronic weight loss and in flock-screening programs.

Quantitative immunodiffusion in one dimension was performed in 6-mm Duran tubes containing a 1% Nobel agar solution and various dilutions of antisera. A series of dilutions of pure myoglobin in equine sera as well as plasma from horses with rhabdomyolysis were tested. Standard curves were prepared of the migration distance of the formed precipitate from the meniscus of the gel after 3, 6, 12, and 24 hours. The clearest line of precipitate was formed with a 1:20 dilution of antisera in agar. Standard curves were nonlinear and plasma myoglobin could be detected at 2 micrograms of myoglobin/ml or greater. The test was optimal, with an error of 5.6%, when read at 24 hours at approximately 25 C. Tubes with agar could be stored for 6 months at 4 C without affecting the accuracy of the test. The specificity of myoglobin for skeletal or cardiac muscle, and its rapid clearance from serum after muscle necrosis, make it ideally suited for evaluating acute muscle damage and for testing the susceptibility of horses for rhabdomyolysis following an exercise test.

Concurrent bacteriologic culture of feces and agar gel immunodiffusion (AGID) testing was performed on all cows and bred heifers over 14 months old in 10 dairy herds during a 32-month period to determine the effectiveness of the AGID test for the detection of subclinical paratuberculosis. Herds were sampled 5 times and, when possible, culled animals were tested again at slaughter. During 5 herd-wide samplings, Mycobacterium paratuberculosis was isolated from 139 fecal specimens obtained from 109 cattle. Results of the AGID test were simultaneously positive 40 of 139 times (28.8%). Thirty-six of the 109 cattle (33.0%) determined to be infected had a positive AGID test result at some point during the 5 herd-wide samplings. When results of tests performed at time of slaughter were included, 117 cattle were identified as infected by culture methods; 55 of these (47.0%) were AGID test-positive at some point during the study. The upper limit of the maximal false-positive rate for the AGID test was 2.1%. On the basis of colony counts from cultures, subclinically infected cows shedding higher numbers of M paratuberculosis in the feces were more likely to have positive AGID test results (P less than 0.0001). In known infected cattle, neither the culture nor AGID test results were consistently positive on repeated testing. Of 48 official calfhood paratuberculosis vaccinates tested as adults, 3 had positive AGID test results and in 1 of these, M paratuberculosis was also isolated from the feces, indicating that the rate of false-positive AGID test results in calfhood vaccinates is low.

Blood samples were obtained from sentinel beef cattle at monthly intervals, and the sera were tested for antibodies, using a bluetongue virus (BTV) immuodiffusion test (IDT) and virus-neutralization test (VNT), for 5 BTV serotypes (2, 10, 11, 13, and 17) and 2 epizootic hemorrhagic disease virus (EHDV) serotypes (1 and 2). The cattle tested were transported from Tennessee to Texas in 1984 and 1985. All cattle were seronegative by the BTV IDT at the initial bleeding in Texas in 1984 and 1985. In 1984, 16 of 40 (40%) cattle seroconvertedas assessed by results of the BTV IDT. In 16 seropositive cattle in 1984, neutralizing antibodies were detected to BTV serotypes 10 (n = 7), 11 (n = 3), and 17 (n = 11), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1984, no cattle seroconverted to BTV-2 or BTV-13. In 1985, 10 of 36 (27.8%) cattle seroconverted as assessed by results of the IDT. Of the 10 seropositive cattle in 1985, neutralizing antibodies were detected to BTV serotypes 10 (n = 10), 11 (n = 10), 13 (n = 7), and 17 (n = 5), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1985, no catttle seroconverted to BTV-2. Clinical diseases attributable to BTV or EHDV was not detected in these cattle in 1984 or 1985.