Improvements in the modified direct complement fixation test and its application in the detection of bluetongue antibodies in cattle and sheep sera.

In this study, improvements were made in the technique and the preparation of the antigen. It was possible to perform three extractions and elutions resulting in a soluble reactive preparation from each batch of infected mouse brain. This led to an appreciable increase in the yield of highly reactive antigen. The presence of bluetongue antibodies was not detected in 13,210 sheep sera. Of the 13,486 bovine sera tested, only three questionable reactions were obtained. It was possible to determine that two of these animals were imported. Various isolation methods, including transmission trials to susceptible sheep followed by serological tests on the sheep sera, failed to confirm the infection in the three reactors.

Equine infectious anemia: sensitivity of the agar-gel immunodiffusion test, and the direct and the indirect complement-fixation tests for the detection of antibodies in equine serum.

The comparative values of the direct, the indirect complement-fixation and the agar-gel immunodiffusion tests were assessed for the diagnosis of equine infectious anemia. Antibodies were detected on the agar-gel immunodiffusion test as early as 18 days post-inoculation in the serums of experimentally infected horses and were readily detectable in all the subsequent bleedings. Complement-fixing antibodies, demonstrable by the direct method, were detected commencing about the same time. However, these were not long-lasting and were replaced by the non-complement-fixing antibodies demonstrable by the indirect method; although both types of antibodies could be detected in some sera at the same time. In a herd of 55 horses, 28 were positive on the agar-gel immunodiffusion test, and among these 28 horses, 24 of them reacted on either the direct or indirect complement-fixation test or both. Thirteen horses that were negative on the three tests at the first sampling, reacted on the agar-gel immunodiffusion test 43 days later. Ten of these positive animals had direct type of complement-fixing antibodies; only one had the indirect; and two of them were negative on both tests. It appeared that the AGI test was a more reliable technique than either the direct or indirect complement-fixation tests, particularly when dealing with serums which contained small amounts of antibody. The sequential appearance of the two different types of complement-fixing activity might be used to determine the evolution of the disease on a herd basis.

Equine infectious anemia: activity of liquid antigen extracts in the agar-gel immunodiffusion and complement-fixation tests.

Twenty-nine lots of acetone-ether extracted liquid antigen were prepared from the pulp of 11 spleens collected from horses at the acute phase of experimental infection. The lots prepared from the highly reactive pulp resulted in general in a liquid antigen of greater activity than those extracted from weakly reactive pulps. Some variations in activity between lots of antigen prepared from the same spleen were also observed. No matter what the results, given a wide enough variation, all results were reproducible. The procedure permitted production of a greater number of antigen test doses from reactive spleens and rendered usable the spleens which failed to give sufficient reactivity when used as pulp antigen in the agar-gel immunodiffusion test. The activity of each lot of liquid antigen was standardized, first by the complement-fixation test and finally by matching with a reference antiserum in the agar-gel immunodiffusion test.

Equine infectious anemia: preparation of a liquid antigen extract for the agar-gel immunodiffusion and complement-fixation tests.

An agar-gel immunodiffusion test recommended for the diagnosis of equine infectious anemia was evaluated. Our preliminary observations confirmed those of Coggins concerning the mechanism of the test and the results obtained. Furthermore, emphasis was put on the difficulties encountered in the production of spleen antigens with an optimum amount of reactivity. Acetone-ether extraction procedures for the preparation of a liquid antigen extract are described. This type of antigen was reactive in the complement-fixation test in 1:8 or greater dilution and it is proposed to use the complement-fixation test in assessing and standardizing the liquid antigen extract activity to be used in the immunodiffusion test. This antigen can also be concentrated or diluted, if required, to meet the reactivity of a standard antigen used in the test.

Rate of Clearance of Vibrio fetus var. venerealis in the Rabbit.

The rate of clearance of Vibrio fetus var. venerealis was measured by using in vivo and in vitro methods. The rate of clearance from the circulating blood increased in immune rabbits and reached a plateau 4 days after immunization. Thereafter in vivo clearance did not increase further despite rising agglutinin titers. Vibriocidal activity of freshly drawn blood paralleled the rate of in vivo clearance although it was less effective at 4 days. Vibriocidal activity of immune serum was not demonstrated at the same intervals of time as the freshly drawn blood. Vibriocidal activity of immune sera plus complement was demonstrated after 1 hr of incubation at 37 C in a candle jar. An effective systemic immune response to V. fetus var. venerealis can be elicited in the rabbit. These results suggest that humoral antibodies play an important role in effecting acquired immunity against V. fetus var. venerealis.

Bactericidal effect and serological response of blood and secretions in bovine vibriosis.

Udder infection of vaccinated heifers with Vibrio fetus var. venerealis led to an early and severe reaction consisting of local swelling, hyperthermia, and increased blood leukocyte counts. This reaction was absent or less pronounced in heifers not previously vaccinated. This was interpreted as an immediate hypersensitivity reaction elicited in the vaccinated heifers. Specific antibodies were found in udder exudate from vaccinated heifers. Immune serum and udder exudate were moderately bactericidal and had a strong immunosuppressive and opsonophagocytic effect in rabbits. Immune cervicovaginal mucus was neither bactericidal nor opsonophagocytic or immunosuppressive. This would suggest that antibodies found in cervicovaginal mucus are not protective. It appeared that the immediate hypersensitivity observed and the subsequent transfer of antibodies from serum to udder exudate would provide by analogy a possible explanation of the mechanisms of immunity to bovine vibriosis.