immunodiffusion studies of purified equine infectious ... of nacl in agar, incubation temperature,...

Immunodiffusion Studies of Purified Equine Infectious ... of NaCl in agar, incubation temperature, and
Immunodiffusion Studies of Purified Equine Infectious ... of NaCl in agar, incubation temperature, and
Immunodiffusion Studies of Purified Equine Infectious ... of NaCl in agar, incubation temperature, and
Immunodiffusion Studies of Purified Equine Infectious ... of NaCl in agar, incubation temperature, and
Immunodiffusion Studies of Purified Equine Infectious ... of NaCl in agar, incubation temperature, and
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  • INFECnON AND IMMUNITY, March 1971, p. 373-377 Copyright ( 1971 American Society for Microbiology

    Vol. 3, No. 3 Printed in U.S.A.

    Immunodiffusion Studies of Purified Equine Infectious Anemia Virus HIDEO NAKAJIMA AND CHUZO USHIMI

    Equine Inifectious Anemia Research Division, National Institute of Animal Health, Kodaira, Tokyo, Japan

    Received for publication 20 October 1970

    Antigenicity of purified equine infectious anemia (EIA) virus was examined by immunodiffusion against sera obtained from horses experimentally infected with EIA virus. The purified virus reacted with the infected horse serum, and virus- specific precipitating antibody was demonstrated. Furthermore, it was found that purified EIA virus reacted against the serum of horses infected with all st;ains of EIA virus which were antigenically different from one another. From the result, group-specific components of the virus rather than strain-specific ones were con- sidered to be involved in the reaction. Serological reactivity was lost by adding anti- serum from the infected horse to the antigen. The precipitating antibody usually appeared in the serum 1 to 2 weeks after the first febrile attack of EIA and re- mained for a longer period. Some characteristics of the purified antigen and speci- ficity of the reaction for EIA are described.

    Equine infectious anemia (EIA) is worldwide in distribution and has been extensively investigated for many years. Many aspects of the disease, however, still require further investigation. Studies on the causative agent and its propaga- tion, immunology, pathogenesis, and diagnosis and on prevention of the disease are particularly needed. Many attempts have been made to elucidate the

    physicochemical and morphological properties of ETA virus, and some characteristics of the virus have been previously reported (9, 10, 12-15). During these investigations a procedure for virus purification was developed and highly purified and concentrated virus was successfully obtained (10). In the present investigation, the serological

    reactivity of the purified virus, as measured against the serum obtained from horses experi- mentally infected with ETA virus, was examined by immunodiffusion. As a result, the purified virus reacted effectively, and viral specific antibody was demonstrated in the infected equine serum. The present report describes a standardization

    of the immunodiffusion reaction and some char- acteristics and antigenicity of the purified antigen. Specificity of the test in EIA is also discussed.


    Cell culture and preparation of virus material. Hepa- rinized blood was obtained from horses, and leukocyte culture was carried out by a procedure previously de-

    scribed (10) to obtain the starting virus material for purification of the antigen. The horse leukocyte- adapted cloned strain (the P337 strain) of EIA virus was used as the inoculum. Infectivity of virus materials was titrated by using the horse leukocyte culture tech- nique as previously reported (3, 11).

    Purification of viral antigen. The purification proce- dure for EIA virus has been previously described (10). Briefly, the virus was precipitated by ultracentrifuga- tion from approximately 1,000 ml of starting material and suspended in 0.01 M phosphate buffer (pH 7.4; PB). This material was loaded onto a diethylamino- ethyl cellulose column, and the virus was eluted with 1.0 M NaCl solution after contaminating protein was removed with 0.15 M NaCl solution. The eluate was concentrated and centrifuged in cesium chloride (CsCl) solution at 40,000 rev/min for 22 hr in an SW 65 rotor of a Beckman model L2 ultracentrifuge. After centrifugation, 1.5 ml of purified fraction which showed the highest infectivity was obtained and dia- lyzed against 0.01 M PB. This fraction was used as the purified antigen.

    Analytical ultracentrifugation. The purified and dialyzed virus fraction was analyzed by a Spinco model E ultracentrifuge at an average speed of 25,000 and 27,000 rev/min, and sedimentation coefficients of the virus were measured by using ultraviolet adsorption and schlieren optical systems.

    Sera. Sera used in the present investigation were obtained at various stages during the disease from nine horses experimentally infected with EIA virus. Four strains of EIA virus, namely, the P337 strain, the Wyoming strain, the German strain, and the Goshun strain (2), were used as inoculum.

    Agar gel diffusion. Gel diffusion plates were pre- pared on microscope slides (26 by 76 mm) by using


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    4.5 ml of 0.8% purified agar (Eiken Comp., Japan), 0.85% NaCi, and 0.01% thimerosal in 0.01 M PB. The wells were 3 mm in diameter, and six circumferen- tial wells were placed at a distance of 4 mm from the central well. The central well was usually filled with the purified antigen, and the peripheral wells were filled with sera. The plate was incubated in a moist chamber at 22 C for 4 days. When a precipitin line was formed, the plate was photographed unstained. It was next washed for 3 days in normal saline and rinsed for a few hours in distilled water. Finally, the slide was dried, stained with amido black, destained, and photo- graphed again.

    Neutralization test. Two strains of EIA virus which were antigenically different from each other were used as indicator virus for the neutralization test. One was the P337 strain which had adapted to horse leukocyte culture and was used as the seed virus for preparation of the virus material. The other was the Wyoming strain which had been supplied by the courtesy of C. D. Stein in 1955 and had been serially passaged only through horses. To facilitate viral propagation in horse leukocyte cultures, the latter was passaged through horse leukocytes four times by Y. Kono and once by the senior author. The serological reactivity was not altered during passages. A 0.1-ml amount of the mixture of indicator virus, which had approxi- mately 1050 median tissue culture infectious dose (TCID50)/0.l ml, and serially diluted serum was inocu- lated into 1-day cultures of horse leukocytes. The titer of neutralizing antibody was expressed in a manner previously described (5).

    Other measurements. Electron microscopy was carried out as previously reported (10). The density of viral fractions was determined by a gravimetric method by using 10 microliter capillaries (Drummond Scientific Corp., Philadelphia, Pa.). The complement fixation test was performed by a method previously described (4). Complement-fixing (CF) antigenicity was expressed as the reciprocal of dilution.

    RESULTS Standardization of immunodiffusion procedure.

    Purified EIA virus was tentatively examined by immunodiffusion to determine whether it would have serological reactivity with the serum of experimentally infected horses (no. 520 and 569) which had clinically shown typical symptoms of EIA. As a result, a fairly clear and distinct pre- cipitin line was formed between the antigen and the serum after 24 hr. With the purified virus and above-mentioned

    sera, the concentration of agar gel, concentration of NaCl in agar, incubation temperature, and incubation period were examined to determine the best conditions for immunodiffusion. Conse- quently, it was found that 0.8% agar gel, 0.85% NaCl and incubation at 22 C were comparatively better among the conditions examined. A pre- cipitin line was developed within 24 hr in most cases. It seemed, however, to become clearer when the slide was allowed to incubate for 3 more

    days. Therefore, an incubation period of 4 days was used throughout the experiment.

    Three kinds of antigen were prepared as fol- lows. One was purified and dialyzed virus with no further treatment (see above). The second was ob- tained by freezing, thawing, and shaking the purified virus more than 10 times. The third was prepared by treating the purified virus with an equal volume of ether at room temperature for 5 min. Ether was immediately removed. Viral infectivity was completely lost by this treatment. All of the antigen prepared above reacted with serum, and precipitin lines developed approxi- mate strength. The antigen and sera were diluted by a serial

    twofold dilution and a reaction was carried out to determine the serological reactivity and the anti- body titer. The strongest reaction was observed when undiluted antigen and sera were used. Pre- cipitin lines became almost invisible when the antigen and sera were diluted twofold. From the results obtained above, the virus

    which had been purified and dialyzed to remove CsCl was used as antigen, and the reaction was performed without dilution of both the antigen and sera. Some characteristics of the purified antigen. The

    viral antigen used in experiments was purified from 1,000 ml of infected horse leukocyte cul- tures, having a titer of approximately 106.5 TCID0/0.5 ml, and concentrated to 1.5 ml. The virus ranging between 1.12 and 1.18 g/ml

    in CsCl gradients was collected since the virus showed broad distribution in the gradient. Infec- tivity and CF reactivity of the final material increased to approximately 108.0 TCID5o/0.5 ml and 100, respectively. Its optical density at 280 nm was mostly between 1.5 and 3.0, and the material was slightly white and turbid. The sedi- mentation coefficients measured by analytical ultracentrifugation were between 110 and 120S. Viral particles were observed by use of the elec- tron microscope, and most ofthem were disrupted (Fig. 1).

    Reaction with sera obtained from horses infected with antigenically different strains of ELT virus. Recent evidence suggests that there are