Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera.

Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.

     

Survey of anaplasmosis in elk of the Clearwater National Forest (Idaho).

Samples of blood from 27 free-roaming elk (Cervus canadensis canadensis) from the Clearwater National Forest in north central Idaho were tested by the rapid card agglutination test and complement-fixation test for the presence of antibodies against Anaplasma marginale. The serum card test and complement-fixation test gave incomplete and false-positive reactions; the plasma card test did not give any reactions. Anaplasma bodies or other blood parasites were not observed in stained smears of elk blood. Blood from 11 elk, including 2 that were serum card test-positive, did not produce clinical, hematologic, or serologic evidence of infection in 3 anaplasmosis-susceptible bovine calves.     

Toxoplasmosis I. Studies by the Fluorescein-labelled Antibody Technique

The fluorescein-labelled antibody technique was investigated for the diagnosis of toxoplasmosis. The direct method, the inhibition and indirect modifications are suitable for the demonstration of Toxoplasma gondii in fluid and tissue-impression slides from animals in the acute phase of infection. The method was not applicable with the frozen tissue sections.

The fluorescein-labelled antibody inhibition technique detected antibodies in immune sera from various species of animal. However the titres obtained were lower than with the complement-fixation test.

     

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.

     

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.     

Paratuberculosis in cattle: a comparison of three serologic tests with results of fecal culture

Feces and blood were collected from cattle in 13 herds known to be infected with Mycobacterium paratuberculosis to evaluate a complement-fixation (CF) test, an agar gel immunodiffusion (AGID) test and an enzyme-linked immunosorbent assay (ELISA) for the serologic diagnosis of paratuberculosis. M. paratuberculosis was isolated from the feces of 36 of 192 cattle examined. Twenty-three culture-positive animals had CF test titers regarded as suspect or positive, 10 were positive by the AGID test and 34 were suspect or positive by the ELISA. Of the 156 culture-negative animals, the CF test agreed on 136, the ELISA on 129 and the AGID on 151.     

Comparison of three serological tests in the diagnosis of Brucella infection in unvaccinated cattle in Eritrea

Three serological methods, the Rose-Bengal test (RBT), the complement-fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (I-ELISA) were compared for the detection of Brucella-infected animals in unvaccinated cattle herds in Eritrea. In this study, 71 herds first were classified as positive or negative for Brucella infection on the basis of at least one animal being seropositive by RBT and CFT. All the 159 RBT-positive samples from the 26 seropositive herds and 214 RBT-negative samples randomly selected from the seropositive herds and from the 45 negative herds were tested further by CFT and I-ELISA. Using the ELISA titer as main predictor, and incorporating the RBT results, a logistic model was built to predict the CFT-negative or -positive status of individual sera and to estimate sensitivity and specificity. Whilst the ELISA titers (< or =20) accurately predicted all the negative sera in herds that were also negative by the CFT, the number of seropositive animals was higher by ELISA in herds that had positive animals. Serum samples which give higher degrees of agglutination with the RBT need not be re-tested with CFT; consideration of the seropositive status of a herd should be taken into consideration on defining the cut-off optical density readings for ELISA.     

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.

     

Experimental maedi virus infection in sheep: early cellular and humoral immune response following parenteral inoculation

The early immune response (19 weeks) in sheep inoculated parenterally with maedi virus was studied, using the lymphocyte transformation test, immunodiffusion test, complement-fixation test, and neutralization test. Cellular immune responses were detected between 3 and 7 weeks after the sheep were inoculated. Antibodies were detected by immunodiffusion test in 3 of 5 animals in weeks 7 to 11 and by complement-fixation test in weeks 15 to 19. Neutralizing antibodies were not detected. There was no sign of virus-induced immunosuppression as determined by lymphocyte responsiveness to mitogens or by in vivo and in vitro immune responses following BCG vaccination.     

Isolation of specific peptides from Mycobacterium paratuberculosis protoplasm and their use in an enzyme-linked immunosorbent assay for the detection of paratuberculosis (Johne's disease) in cattle

An antigen was isolated from the protoplasm of Mycobacterium paratuberculosis by a combination of gel filtration, ion exchange, and affinity chromatography. The purified antigen constituted 7.8% of the total protein in the protoplasm. The specificity and sensitivity of the enzyme-linked immunosorbent assay (ELISA) for paratuberculosis, using the purified antigen, were evaluated with sera from 104 cattle which were examined (surveyed) for M paratuberculosis infection by fecal cultural technique. The ELISA was positive in 50 of 60 infected animals. Five of 44 noninfected animals were also test-positive. When a crude protoplasmic extract was used as antigen in the ELISA, sera from 37 infected and from 18 noninfected animals were test-positive. Cross-reactions were encountered in both complement-fixation test and the ELISA between crude or partially purified M paratuberculosis antigens and antisera to Nocardia asteroides, M avium, M phlei, and M fortuitum. The purified antigen gave no complement-fixation reaction with any of these antisera. In the ELISA, cross-reaction was not found when purified antigen was used and the sera were screened at 1:40 dilution.     

Evaluation of different antigens in the complement-fixation test for diagnosis of Haemophilus pleuropneumoniae (parahaemolyticus) infections in swine

The identification of new serotypes of Haemophilus pleuropneumoniae (parahaemolyticus) and the frequency of pleural adhesions due to contagious pleuropneumonia in many fattening swine herds have prompted the study of the complement-fixation (CF) test as a diagnostic tool for use in swine. Whole cell antigens, mixed antigens, autoclaved antigens, and phenol-water-extracted antigens derived from different serotypes were prepared and tested with immunized-swine sera by the CF test. Mixed antigen consisting of whole cells from all known serotypes was the best screening antigen for routine use. This antigen gave positive titers with all sera in which a positive reaction against the separate serotype antigen was registered. The most highly serotype-specific reactions were obtained with antigens prepared by phenol-water extractions of whole cells. When whole-cell antigens were used in the CF test, antibodies to superficial serotype-specific and common species-specific antigens could be detected.     

Incidence of antibodies to Toxoplasma gondii in cats from Brno and the surrounding area

In the course of three years, 620 cats of all age categories were investigated for the occurrence of antibodies to Toxoplasma gondii. The serological examinations included the Sabin-Feldman reaction (SFR), complement-fixation reaction (CFR) and microprecipitation in agar gel (MPA), according to methods prescribed for use in Czechoslovakia. When the Sabin-Feldman reaction was used, 250 (40.3%) of the examined cats were found to be positive at the titre of 4-128. By the complement-fixation reaction, antibodies were demonstrated in 144 (23.2%) of the cats at the titre of 5-80. Microprecipitation in agar gel was positive in 106 animals, i.e. 17.1%. Antibodies to Toxoplasma gondii were demonstrated by one serological reaction in 150 (24.2%) cats; coincidence of two serological reactions occurred in 104 (16.8%) of the animals, and coincidence of all the three serological reactions was demonstrated in 48 (7.7%) cats. Specific antibodies were detected by minimally one serological reaction (which means one to three positive findings) in 302 (48.7%) of the examined cats. The diagnostic value of all serological reactions is discussed. It is recommended to repeat the examination after two to three weeks.     

LEPTOSPIRA INTERROGANS SEROVAR HARDJO INFECTION OF CATTLE

A survey of normal cattle in the Southern Victorian statistical divisions revealed that microscopic agglutination titres to L. hardjo occur at high frequency and are distributed throughout the cattle population. These titres are difficult to interpret as they may represent recent or old exposure, with or without disease. L. hardjo infection of dairy cattle was studied in 4 herds using the microscopic agglutination and complement-fixation tests. Statistical comparisons of individual titres obtained indicated that the sensitivity of the complement-fixation test was satisfactory for diagnostic purposes, but the test was unable to differentiate between current or past infections.     

A latex agglutination test for field diagnosis of contagious caprine pleuropneumonia

Latex beads were sensitised with a polysaccharide isolated from a F38 culture supernatant and used in a slide agglutination test to detect serum antibodies in goats with contagious caprine pleuropneumonia. The latex agglutination test detected antibodies in the sera of goats by 22 +/- 2 (mean +/- 1 sd) days after contact exposure to contagious caprine pleuropneumonia, whereas the complement-fixation test detected antibodies by 24 +/- 4 days after contact exposure. Both tests were negative with 181 sera from a farm which was free of the disease. When the same tests were done on 763 sera from two different farms with outbreaks of classical contagious caprine pleuropneumonia, 63 per cent were positive by the latex agglutination test and 23 per cent were positive by the complement-fixation test. Besides being more sensitive than complement fixation, the latex agglutination test can be performed in the field using undiluted serum or whole blood and a result obtained within two minutes.     

Comparisons of the complement-fixation, indirect fluorescent antibody, and card agglutination tests for the diagnosis of bovine anaplasmosis.

Results of complement-fixation (CF), indirect fluorescent antibody (IFA), and card agglutination (CT) tests were statistically compared, using 380 serum samples obtained from 140 cattle which were disease-free or naturally or experimentally infected with Anaplasma marginale of Colombian origin. The IFA test was significantly the most sensitive for detection of amimals infected with anaplasmosis (97%); the CT test and the CF test were less so (84% and 79%, respectively). However, the most efficient test for identifying noninfected animals was the CF test (100%), and the CT and the IFA tests were less efficient (98% and 90%). A linear regression analysis performed on the average IFA and CF titers of 10 calves artificially infected with A marginale during a 20-week period showed significant regression coefficients for both tests. The regression line for the CF titers decreased below the sensitivity threshold at 14 weeks after calves were inoculated, whereas the regression line for the IFA titers continued above the sensitivity threshold 20 weeks after inoculation. The CT test also detected antibodies until the end of the observation period.     

Antibodies in calves on feed supplemented with chlortetracycline after vaccination with Brucella abortus strain 19

Twenty dairy heifers each consumed 350 mg of chlortetracycline/day in their feed. Four tests were performed on serum specimens from these and 20 control calves after vaccination with Brucella abortus strain 19. The numbers of positive test results on the card test and mean titers on the tube and rivanol agglutination and complement-fixation tests were compared in the 2 groups. Using the rivanol and complement-fixation tests, there were differences in the mean titers at weeks 5 and 6 after vaccination, but by week 10, differences were not found. The results suggest that addition of low concentration of chlortetracycline in feeds have minimal effects on postvaccinal serologic reactions determined after strain-19 inoculation.     

Etiologic significance of bacterial isolates from rams with palpable epididymitis

Identified and partly identified bacterial isolates were obtained from 48 rams of various breeds that had unilateral or bilateral epididymitis. Most of the animals were approximately 1 year of age; a few were older. Brucella ovis, Actinobacillus spp, Corynebacterium spp, Haemophilus spp, Acinetobacter spp, Escherichia coli, Moraxella spp, Staphylococcus spp, Pasteurella spp, Streptococcus spp, and Chlamydia psittaci were isolated. A vaccine strain of B ovis, isolated species of bacteria, and mixtures of isolates of tissue homogenates containing all isolates except B ovis and C psittaci were inoculated via the mucous membranes of the eyes, nares, and prepuce. Palpable epididymitis was not produced by the inoculations. The vaccine strain of B ovis induced complement-fixation reaction in 11 of 20 rams.     

Comparison of complement fixation test and enzyme-linked immunosorbent assay for detection of early infection with Mycoplasma hyopneumoniae

The relative merits of the complement-fixation test (CF) and enzyme-linked immunosorbent assay (ELISA) for the detection of the early antibody response to Mycoplasma hyopneumoniae were evaluated. Discriminant analysis, a statistical procedure, was used to avoid difficulties associated with variation in background color and nonspecific reactions obtained with ELISA with different sera. Specific-pathogen-free pigs were exposed by contact to other specific-pathogen-free pigs which had been inoculated with M hyopneumoniae intratracheally (experiment A) or intranasally (experiment B) 18 to 21 days previously. Sera were collected from each pig before contact exposure and once a week until necropsy. Antibodies were detected by CF at postexposure (PE) week 3 in animals in experiment A (6 of 18) and at PE week 5 in experiment B (3 of 12). The ELISA antibodies were detected at 2 weeks after beginning of contact exposure in experiments A (4 of 18) and B (1 of 12). Examination of pooled data for experiments A and B indicated that ELISA was substantially (P less than 0.05) more sensitive for detection of antibodies than was the CF test at 3 to 5 weeks after contact exposure began. At PE weeks 6 and 7, both tests were similarly effective in detecting M hyopneumoniae antibodies.     

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.

     

Evaluation of the fluorescent antibody test for diagnosis of paratuberculosis

Results of indirect fluorescent-antibody microscopy did not differ significantly from complement-fixation test results in diagnosing paratuberculosis (Johne's disease) in cattle. Neither test had acceptable sensitivity or specificity for detecting subclinical cases.