Use of schizont and piroplasm antigens of Babesia equi in the indirect fluorescent antibody and complement fixation tests

Eight ponies infected with Babesia equi were investigated for their serological response to B. equi schizont and piroplasm antigen with the indirect fluorescent antibody test (IFAT) and complement fixation test (CFT). Piroplasm antigen was prepared from an infected splenectomized pony, while schizont antigen was produced from cultured lymphoid cells which contained B. equi macroschizonts. The IFAT detected a rise in antibody titres to schizont antigen as well as to piroplasm antigen, but differences were obtained in the duration of antibody detection. Significant antibody titres to piroplasm antigen were detectable for a longer period post infection than to schizont antigen. The complement fixation test was not effective in detecting specific antibodies to schizont antigen in contrast to piroplasm antigen. The schizont antibody titres were in general extremely low and not detectable in 3 horses. Neither test showed any serological cross-reaction with B. caballi and B. bigemina antiserum using schizont antigen.     

Contagious caprine pleuropneumonia: Some observations in a field vaccination trial using inactivatedMycoplasma strain F38

The efficacy of an inactivated Mycoplasma strain F38-saponin vaccine in natural infection with contagious caprine pleuropneumonia was investigated. A total of 10,000 goats were vaccinated, out of which 400 were regularly monitored for a period of six months post-vaccination. Immunised animals remained free from infection throughout the period of observation. The antibody response was followed using complement fixation and slide agglutination tests. Both tests could detect F38 antibody in the majority of vaccinated goats but the slide agglutination test was found to be more sensitive than complement fixation. The significance of the results is discussed.     

Comparison of serological tests for the detection of antibody to natural and experimental murine cytomegalovirus.

Three serological tests, i.e. complement fixation test, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA) were compared for sensitivity in the detection and titration of murine cytomegalovirus antibody. The three tests were compared using sera from experimentally inoculated and naturally infected mice bled at intervals from 3 to 140 days postinfection. In the acute infection, complement fixation and indirect immunofluorescent assay tests were of comparable sensitivity for early detection of antibody, whereas the ELISA was less sensitive. In persistent infection, higher titers were recorded with ELISA. Since murine cytomegalovirus has been shown to exert significant effects on the immune response of infected mice, this antigen should be included routinely in viral antibody screening programs.     

Complement fixation titers in cattle following intranasal inoculation of Hemophilus somnus.

Five bulls were inoculated intranasally with a live culture of Hemophilus somnus originally isolated from a clinical case of Hemophilus septicemia. Preinoculation and postinoculation blood samples were taken at weekly intervals for nine weeks for measuring complement fixation titers and daily postinoculation temperatures were taken for one week. Three animals had transient fever and slight lethargy was observed in two animals had a transitory rise in complement fixation titers in the second to fifth weeks postexposure while one animal which had been seronegative on preinoculation testing produced little serological response to the organism. The experiment demonstrated that the nasal instillation of young cattle using an originally pathogenic H. somnus isolate is capable of stimulating only transitory complement fixation antibody titer.     

Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae.

Caesarean-derived, colostrum-deprived swine were exposed to a broth culture of a low passage field isolate of Mycoplasma hyopneumoniae by intranasal inoculation. The intranasal-inoculated swine subsequently were commingled with their litter-mates to effect transmission via contact-exposure. Sera were collected from the swine at two to four week intervals for approximately one year postexposure and evaluated by the enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination and complement fixation tests. The intranasal-exposed swine seroconverted earlier, developed higher titers and remained indirect hemagglutination and complement fixation positive longer than the contact-exposed swine. It was concluded that the antibody response of intranasal-exposed swine was artificially high and that sera from such swine were not suitable for evaluating the sensitivity of mycoplasmal pneumonia of swine serodiagnostic tests. The indirect hemagglutination test was relatively insensitive and technically cumbersome and the least promising as a practical field test. The complement fixation test appeared to be slightly more sensitive in detecting early antibody production (especially in contact-exposed swine) but it was the least sensitive in detecting late antibodies. The ELISA was generally the most sensitive procedure. Individual high ELISA titers were from ten to 32 times greater than maximum complement fixation and indirect hemagglutination titers. The most striking difference among the three tests was the persistence of high ELISA titers late in the study. All swine were ELISA positive at necropsy approximately one year postexposure despite the fact that lungs were devoid of lesions and culturally and immunofluorescent negative for M. hyopneumoniae.     

Serological responses of specific pathogen-free foals to equine herpesvirus-1: primary and secondary infection, and reactivation.

Serum antibody (virus neutralisation, complement fixation, IgM and IgG) responses to equine herpesvirus-1 (EHV-1) infection were measured in six foals which were initially free from EHV-1 and EHV-4 infection and maternally-derived antibodies. Following primary infection, high titres of virus neutralisation and complement fixation antibodies were detectable against EHV-1, however, corresponding antibody levels against EHV-4 were low or inapparent, although the two viruses share a number of cross-reactive epitopes. In addition, following the primary infection with EHV-1, IgM levels increased before those of IgG, virus neutralisation and complement fixation antibodies, peaked sooner and thereafter declined. Stimulation of IgM levels was observed on secondary infection with EHV-1 given 61 days later. In contrast, IgG, virus neutralisation and complement fixation antibodies following primary infection were more sustained and no increase in their levels was observed on secondary infection. No consistent changes in IgM or IgG levels were seen after administration of dexamethasone to reactivate latent virus.     

The bovine immune response to Brucella abortus. II. Elimination of some sporadic serological reactions by chelation of divalent cations.

The standard agglutination tests for detecting antibody to Brucella abortus were modified by addition of chelating agents (EDTA and EGTA) to the antigens. Approximately 80% of "singleton" agglutination test reactions, negative on the diagnostic complement fixation test, obtained with cattle sera were eliminated while no decrease in titer was apparent when sera from B. abortus infected or vaccinated cattle were tested.     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. V. Residual Complement Activity

Complement-fixation tests of three different antigen-bovine antibody systems, two antibacterial and one antiviral, were set up with or without normal bovine serum supplement. At the end of the fixation period all mixtures were tested for whole complement activity and for first, second, third and fourth complement components using the conventional, crude reagents R1, R2, R3 and R4. The increased fixation in mixtures containing the bovine serum supplement mainly reflected a greater decrease in second and fourth component activity than in the antigen-bovine antibody mixtures with non-supplemented complement. The decline in first component activity was relatively less. The results of tests for residual third component activity were not consistent.     

The effects of Leptospira serotype pomona in sheep of different haemoglobin types

A warm complement fixation test that will detect antibody in sheepserum in the virtual absence of anti-complementary activity is described. Sheep antibody-antigen complexes were detected by fixation of sheep complement. Sheep serum, heparinized or Mg⁺⁺—EGTA plasma was used as the source of sheep complement. Sheep-antibody-sensitized human erythrocytes were used as the haemolytic indicator cells for sheep complement. As the modified complement-fixation test was performed in the presence of Mg⁺⁺—EGTA, sheep C probably reacts with sheep Ab-Ag complexes by a different mechanism than does guinea-pig complement.     

The Effect of Unheated Normal Bovine Serum on The Complement Fixing Activity of Heat Inactivated Bovine Antiserum With Homologous Antigen: II. Aggregative Properties

To determine whether the greater fixation of complement observed in “modified” complement-fixation tests is related to an increased aggregation of the antigen-antibody complexes, parallel tests by the two methods have been made with two different particulate bacterial antigens and corresponding bovine antisera. At the end of the fixation period the mixtures were centrifuged, the supernatant fluids removed carefully, the sediments washed twice and re-suspended in a small volume of buffer. Smears of each sediment were examined by immunofluorescence microscopy using a fluorescein-labelled rabbit antibody for a globulin fraction of fresh guinea-pig serum containing first component of complement.

A greater degree of aggregation of the antigen-antibody complexes was observed in the sediments from tests with modified complement, that is complement supplemented with a diluted bovine serum globulin fraction prepared by dialysis. Aggregates from mixtures showing increased fixation of complement, as determined by titration of the residual hemolytic activity of the supernatant, appeared somewhat more brightly fluorescent.

Very faint or no fluorescence was evident in the stained washed sediments from mixtures of antigen and antibody without complement or from mixtures of antigen, heat-inactivated normal bovine serum and complement.

     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.     

A comparison of five serological tests for bovine brucellosis.

Five serological assays: the buffered plate antigen test, the standard tube agglutination test, the complement fixation test, the hemolysis-in-gel test and the indirect enzyme immunoassay were diagnostically evaluated. Test data consisted of results from 1208 cattle in brucellosis-free herds, 1578 cattle in reactor herds of unknown infection status and 174 cattle from which Brucella abortus had been cultured. The complement fixation test had the highest specificity in both nonvaccinated and vaccinated cattle. The indirect enzyme immunoassay, if interpreted at a high threshold, also exhibited a high specificity in both groups of cattle. The hemolysis-in-gel test had a very high specificity when used in nonvaccinated cattle but quite a low specificity among vaccinates. With the exception of the complement fixation test, all tests had high sensitivities if interpreted at the minimum threshold. However, the sensitivities of the standard tube agglutination test and indirect enzyme immunoassay, when interpreted at high thresholds were comparable to that of the complement fixation test. A kappa statistic was used to measure the agreement between the various tests. In general the kappa statistics were quite low, suggesting that the various tests may detect different antibody isotypes. There was however, good agreement between the buffered plate antigen test and standard tube agglutination test (the two agglutination tests evaluated) and between the complement fixation test and the indirect enzyme immunoassay when interpreted at a high threshold. With the exception of the buffered plate antigen test, all tests were evaluated as confirmatory tests by estimating their specificity and sensitivity on screening-test positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Foot-and-mouth disease typing and serology in Zimbabwe

A small foot-and-mouth disease diagnostic unit was established in Zimbabwe. Twenty-six out of 27 field specimens were successfully typed using the complement fixation technique. The antibody response in cattle to the SAT II vaccine was determined using a microtitre neutralisation method. Techniques are described which should be within the capacity of laboratories in developing countries.     

Reciprocal Antibody and Complement Responses of Two Chicken Breeds to Vaccine Strains of Newcastle Disease Virus, Infectious Bursal Disease Virus and Infectious Bronchitis Virus

Serum antibody responses and haemolytic complement activity were evaluated in White Leghorn (WLH) and Rhode Island Red (RIR) chickens that were vaccinated with live-attenuated vaccines of Newcastle disease virus, or infectious bronchitis virus, or infectious bursal disease virus by means of ocular challenge at 10 times the normal vaccination dose. Complement titres in non-vaccinated birds were significantly higher in WLH birds compared to RIR birds. The lentogenic viral infection resulted in an immediate stimulation of complement activity, followed by a decrease to initial complement levels within 2 weeks post vaccination, when the antibody response took over immune defence. As compared to WLH chickens, RIR birds mounted a faster and significantly higher antibody response to the vaccine viruses used. In WLH hens, significantly higher haemolytic complement activity post vaccination was found as compared to RIR hens. Possible consequences of the observed differences in immune responsiveness of the two breeds to viral vaccines are discussed.     

Diagnostic sensitivity and specificity of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams.

An enzyme-linked immunosorbent assay (ELISA) for antibody to Brucella ovis was compared with a standard complement fixation test. Sera of 176 rams from uninfected flocks gave 175 negative and one suspect ELISA reaction (diagnostic specificity 99.4%) whereas the complement fixation test yielded 167 negative, seven suspect and two anticomplementary reactions (diagnostic specificity of 96.0%). Diagnostic sensitivity was evaluated on sera of 79 rams from which B. ovis had been isolated. The ELISA showed 75 positive and four suspect reactions, while complement fixation test revealed 64 positive, 13 suspect and two negative results. Considering both positive and suspect reactions, the diagnostic sensitivity was 100% for ELISA and 97.5% for complement fixation test. The ELISA method was considered more specific, more sensitive and technically more advantageous than complement fixation test as a serodiagnostic test for B. ovis infection in rams.     

Myopathy of dogs resembling white muscle disease of sheep

Automation of the complement fixation test for diagnosing Brucella abortus infection in cattle has allowed this difficult, time-consuming and labour-intensive hut most specific test to he used for large scale serodiagnosis in New Zealand's Brucellosis Eradication Scheme. The automated test has the advantages of eliminating much human error, of improving accuracy and of reducing the costs of labour and reagents.

The Auto-Analyzer performs a warm complement fixation test at a single serum dilution. Each batch of 39 serum samples is compared with a standard positive control serum with a complement fixing antibody activity equivalent to 1/30 of the second International Standard Ani-Brucella abortus Serum. All sera with an antibody level equal to, or greater than, the standard serum are regarded as positive to the test. A titre for serum tested by the automated method is not obtained, and positive sera that prozone strongly may be recorded as negative. Where necessary, the semi-automated microtitre complement fixation test is used to delineate titres and the status of doubtful automated CFT results. The automated and microtitre complement fixation tests use the same reagents; only relative concentrations differ.     

Comparison of serological techniques to measure antibody to Pasteurella haemolytica A1.

Analysis of 45 sera was performed employing five techniques which are currently in use in three laboratories to measure anti-Pasteurella haemolytica antibodies. The enzyme linked immunosorbent assay, passive hemagglutination, complement fixation and direct and indirect bacterial agglutination assays were employed and a relationship between tests in the measurement of anti-P. haemolytica antibodies was demonstrated. Regression analysis together with prediction and confidence intervals were tabulated also. The conclusion drawn from statistical analysis was that all five tests are similar in their ability to detect immune responses (antibody and antigen(s) interactions) to Pasteurella haemolytica.     

Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci

A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.     

Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests.

The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.     

A COMPLEMENT-FIXATION TEST FOR ENZOOTIC PNEUMONIA OF PIGS USING A COMPLEMENT DILUTION METHOD

Complement-fixing antibody to Mycoplasma hyopneumoniae in the serums of pigs experimentally infected with enzootic pneumonia was demonstrated by comparing the haemolytic titre of guinea-pig complement titrated in the presence of heated test serum, M. hyopneumoniae antigen and unheated normal pig serum with the titre obtained when the antigen was omitted. The haemolytic titres against sensitised sheep erythrocytes were determined after a fixation period of 16 to 18 hours at 5°C. When serums, collected at intervals of 3 to 7 days, from 43 pigs exposed to pigs experimentally infected with enzootic pneumonia were tested, 4.6 or more complement units were first fixed 14 to 44 (mean 23.4) days after contact began. Serums collected subsequently fixed from 4.6 to more than 31 complement units. This positive reaction usually persisted until the pigs were killed 4 to 35 weeks after contact began. Thirty-three had gross enzootic pneumonia lesions and 9 had lung lesions detected microscopically. Serum antibody was not detected in 73 weaned pigs aged 7 weeks in a pneumonia-free herd but serums from 9 of 15 unweaned piglets aged 9 to 14 days in the same herd, fixed between 3 and 7 complement units.