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.

Studies on bovine virus diarrhea: serum neutralization, complement-fixation and immunofluorescence.

The complement-fixation, the serum neutralization tests and the fluorescent-antibody technique were the serological methods applied in this laboratory for the detection of antigens for bovine virus diarrhea (BVD). As observed previously, the modified direct complement-fixation (MDCF) test was required to demonstrate antibodies against virus infections of cattle. At a certain stage of infection, the MDCF test was found to be as accurate and less time-consuming than the serum neutralization test for the detection of antibodies in bovine sera. The modified direct complement-fixing antibodies were detectable in the serum from approximately three weeks up to a few months after infection as compared to several years for the serum neutralization test. Thus, as in most other viral diseases, the MDCF test was of value for detecting recent infections while the serum neutralization test detects both recent and long-standing infections. The fluorescent antibody technique was of value to detect viral antigens of both cytopathogenic and noncytopathogenic strains of BVD in primary fetal kidney cell cultures inoculated with field specimens. In addition, the virus was detected in six of 220 fetuses collected at a local slaughter house for the preparation of primary cell cultures. The length of time required for the detection and identification of specific viral antigens by immunofluorescence was considerably reduced over that of the serum neutralization and virus interference tests.

Anaplasmosis: control of the first outbreak in Canada by serological identification and slaughter.

On August 13, 1968 Canada experienced its first outbreak of anaplasmosis. The initial diagnosis based on hematological and clinical evidence was made by the Provincial Veterinary Laboratory, Winnipeg, Manitoba, and later confirmed in our laboratory by use of the complement-fixation test, hematology, and animal transmission studies. Sixteen herds (1,717 cattle) were examined but the outbreak was found to be localized mainly in one herd of 830 cattle. A low degree of infection was also found in four other herds. None of the remaining 11 herds in the area were infected.The infection was controlled by serological testing, and a slaughter policy. In the four herds with low grade infection, no clinical signs were evident, and serological tests made five and six months after the discovery of the outbreak were negative. In the main herd, the tests were negative at six and nine months. Even though no clinical manifestations of anaplasmosis were detected, surveillance of the animals in the area was continued. Sera from all the cattle were tested 16 months after the initial test. Four reactors were detected in the herd in which the main infection had previously been located. In addition, single borderline reactions were observed in a herd which previously had only one questionable reactor, and in another herd which had heretofore been negative. All of these reactive animals were slaughtered including the two with low grade reactions of doubtful significance. Following the removal of the reactive animals, tests were performed until negative results were obtained twice at six week intervals. The last test was conducted at the end of January 1970, 18 months after the original test.

Equine infectious anemia: preliminary investigation of the complement-fixation test for the demonstration of antibodies and antigen.

Clinical field cases of equine infectious anemia were studied and the disease was reproduced experimentally in horses. Attempts were made to adapt the complement-fixation test to the detection of antibodies in the serum of infected animals and to the demonstration of antigens in tissue extracts.A moderate complement-fixing antibody response was demonstrated in the serum of horses shortly after primary exposure to the infectious agent. However, this reactivity was of short duration and occurred with normal as well as with infected saline tissue extracts. It was therefore concluded that this reaction was not specific for equine infectious anemia. Possibly it is due to the appearance of auto-tissue antibodies. The value of this reaction in the diagnosis of the infection was limited because of its short duration and absence in chronic infection and following re-exposure to the infectious agent.

A histological study of the effects of enzootic abortion of ewes virus in the lactating bovine mammary gland.

Inoculation of the udders of cows with the virus of enzootic abortion of ewes resulted in a severe acute mastitis. The mastitis was characterized clinically by pyrexia, anorexia, decreased milk production, swelling and marked alteration in the physical quality of the milk. The basic lesion was necrosis of alveolar and duct epithelial cells. Virus particles were demonstrated in all levels of the mammary gland three days after inoculation illustrating the rapid spread of the agent. Cells resembling Reed-Sternberg cells and similar to those seen in experimental mastitis produced by the virus of infectious bovine rhinotracheitis were seen in tissues collected six and nine days after inoculation.

Studies on bluetongue. 3. Comparison of two complement-fixation methods.

The complement-fixation test used at Onderstepoort was compared with the method used at A.D.R.I. on infected calf and sheep sera. In the first method, the tests are incubated at 37 degrees C for 90 minutes and the test sera are inactivated at 53 degrees C; whereas in the A.D.R.I. method, the test sera are inactivated at 60 degrees C for 30 minutes, incubation is at 9 degrees C for 18 hours, and guinea-pig complement is supplemented with 5 per cent fresh, non-inactivated, normal calf serum. Serial serum samples from one of six experimentally infected calves were negative in the Onderstepoort test, three calves gave only trace reactions and two showed maximum titres of 1:10 whereas all six had maximum serum titres of 1:10 to 1:80 in the A.D.R.I. test. A good correlation was obtained, however, between the results of the two methods with the sera of experimentally inoculated sheep although titres 3 to 8 times higher were obtained with the A.D.R.I.'s test. Post inoculation bleedings from each sheep reacted in both tests.

Studies on bluetongue. IV. Studies of three strains in primary bovine foetal kidney cell cultures.

Three different strains of bluetongue virus were adapted to grow in primary bovine foetal kidney cell cultures. The cytopathic effects observed from the three strains were similar, and were characterized by shrinkage of cells and increased granularity. The specificity of the changes was confirmed by the fluorescent antibody technique. No significant immunological cross-reaction was detected by serum-virus neutralization tests from the strains studied.

Studies on bluetongue. V. Detection of the virus in infected materials by immunofluorescence.

The fluorescent-antibody technique was employed for the detection of bluetongue virus in bovine foetal kidney cell cultures inoculated with tissues and blood from experimentally infected animals. In the first series, a total number of 79 inoculated suckling-mouse brains were examined, 36 as frozen sections alone and 43 as impression slides in conjunction with tissue culture inoculation of the same material. With the combined tissue culture immunofluorescent methods, 36 suspicious were detected by impression smears and 37 positives by the tissue culture out of 43 brains examined. Twenty-two were suspicious out of the 36 examined as frozen sections. Results obtained with the second series, using sheep tissues, showed that the combined tissue culture-fluorescent antibody method was satisfactory for demonstrating the virus in blood of infected animals 1 to 9 days postinfection, and in some organs after death. No false positive reactions were obtained.

Studies on bluetongue. VI. Animal transmission studies.

The Cyprus strain of bluetongue virus was successfully transmitted through six passages and the Station strain through one passage in calves. Although the animals developed no visible evidence of infection, viremia as shown by both passage and fluorescent antibody examination of infected foetal bovine kidney culture, and by serological conversion was nevertheless demonstrated. No enhancement of virulence for calves or sheep was shown following bovine passage. A ewe inoculated in late pregnancy with blood drawn from a calf 59 days after its infection, gave birth to a lamb from whose blood the virus was isolated. Significant complement-fixation titres persisted for at least 200 days.

African swine fever. 3. The use of the agar double-diffusion precipitation test for the detection of the virus in swine tissue.

The agar double-diffusion precipitation test was applied successfully in the demonstration of ASF viral antigen in spleen and liver from swine experimentally infected by the oral route. Positive reactions were obtained with tissues collected as early as 24 hours after the onset of pyrexia and before other clinical manifestation of the disease. Cross-reactions were observed between the various ASF strains used in the study, making the test practical for routine diagnosis in which different strains may be encountered.

African swine fever. IV. Demonstration of the viral antigen by means of immunofluorescence.

African swine fever immunofluorescent conjugates were prepared in swine and used successfully in the demonstration of viral antigen in frozen tissue sections and in inoculated tissue culture cells. Cross reactivity was observed with the six strains used in the inoculation of swine. The high antibody content of the serum of immune swine did not interfere with demonstration of the antigen in frozen tissue sections of certain of their organs. The localisation and extent of antigen varied with the stage of infection. The virus was demonstrated in spleen and other organs as early as after one day of pyrexia and until after death of the animal. A pool of hog cholera and African swine fever conjugates stained with dyes of different colours was used in the localisation of respective antigens in experimental mixed infection.

African swine fever. V. Cultivation of the virus in primary pig kidney cells.

Six strains of African swine fever (ASF) virus were propagated in culture of primary pig kidney (PK) cells. The course of virus growth was followed by means of the fluorescent antibody staining technique. All 6 strains multiplied in the cultures, and 5 of these eventually showed cytopathic effects leading to cell death. Three of the strains were tested for pathogenicity in pigs at various passage levels. Each showed evidence of modification in virulence after a relatively few passages in PK cells. In one case modified virus produced resistance to challenge with homologous virulent virus. All strains rendered the PK cultures capable of hemadsorption of pig erythrocytes.

African swine fever. II. Detection of the virus in swine tissues by means of the modified direct complement-fixation test.

The modified direct complement-fixation test, supplemented with unheated normal calf serum, was used to demonstrate antibodies in sera of swine immunized to African swine fever virus. These antibodies did not react in the ordinary direct non-supplemented complement-fixation test.African swine fever complement-fixing antigen in infected swine tissue is not denatured by extraction with fat solvents. Consequently, good antigens devoid of non-specific reactivity were obtained by extraction with a mixture of acetone and ether. The virus was detected in infected swine tissue harvested one day after beginning of pyrexia. The modified direct complement-fixation test demonstrated cross-reactions between the six strains of virus studied.

Anaplasmosis: comparison of complement fixation methods and study of the cattle population of southern Alberta.

The standard procedure for the complement-fixation test adopted in 1958 by the Animal Disease Eradication Division of the U.S. Department of Agriculture for testing of anaplasmosis was compared with the routine method used in our laboratory. In general a good agreement was observed between the two methods, although some standard control sera having a low titre in the U.S.D.A. test gave a slightly higher reaction in the A.D.R.I. test, whereas the reverse was observed with certain high titre control sera. None of the differences in titre were sufficient to change the interpretation of the tests.A survey of 3090 field samples collected from southern Alberta close to United States border detected 3 serological reactions in 3 different herds. In one of these, the animal was negative when retested 3 months later. In a second animal the serum titre was still present 11 weeks later but the blood from this animal failed to transmit infection to a susceptible splenectomized calf. In the case of the third herd the animal had been disposed of at the time of retest but all other animals in this herd at the time the reacting animal was examined were still serologically negative. This survey failed to reveal the presence of anaplasmosis in the Canadian animals investigated.