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.     

Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis.

A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the areaunder the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpreting weak positive WB results as positive test results, the sensitivities and 95% confidence intervals (CI) of all 3 tests were identical and equal to 88.9% (51.8-99.7%). The specificities and 95% CIs of the IFAT, WB, and mWB test were 100% (91-100%), 87.2% (72.6-95.7%), and 69.2% (52.4-83%), respectively. The overall accuracy of the IFAT was shown to be better than that of the WBs and, therefore, the test has potential for use in the diagnosis of EPM caused by Sarcocystis neurona.     

Serologic response of Babesia equi-infected horses as measured by complement-fixation and indirect fluorescent antibody tests

Both the complement-fixation test (CFT) and the indirect fluorescent antibody test (IFAT) were conducted on weekly serum samples from nine Arab geldings for 28 days before and 256 days after their exposure to Babesia equi of European origin. On an average the IFAT became positive 8 days before the CFT and showed higher relative serum titer increases. Both test procedures successfully detected infection and neither showed an appreciable drop in titer during this time frame, with the exception of the CFT, which showed a transient drop immediately following treatment with imidocarb. A test conducted 540 days after infection showed four of the eight surviving, and presumably infected, horses to be negative on CFT, where as all eight were still positive on IFAT. Comparisons made with the IFAT, on horse sera from B. equi infection of both European and North American origin, utilizing homologous and heterologous antigens, showed significantly higher titers with homologous antigens.     

Comparison of serological tests based on outer membrane or internal antigens for detecting antibodies to Brucella ovis in infected flocks.

The aim of this work was to compare the performance of 6 serological tests using outer or internal antigens from Brucella for the diagnosis of Brucella ovis infection in sheep in an endemic area. Outer membrane antigens included a hot saline extract (HS) and the rough lipopolysaccharide (R-LPS) from B. ovis. Internal antigens were LPS-free total cytosolic proteins (CP) and an 18-kDa cytosolic protein (p18) from Brucella spp. Sera from 200 sheep from naturally infected flocks were assayed by agar gel immunodiffusion test (AGID) and by complement fixation test (CFT), both using HS, and by 4 ELISA using HS, R-LPS, CP, and p18, respectively. The percentage of positive results was 45.5% for ELISA with HS, 42.0% for ELISA with p18, 39.5% for CFT, 33.5% for ELISA with R-LPS, 29.0% for ELISA with CP, and 18.0% for AGID. Taking CFT as the reference test for calculating relative test parameters, the ELISA with HS had the best sensitivity (96.2%), while AGID and the ELISA with R-LPS had the best specificity (96.6%). The ELISA with CP was not more sensitive than the ELISA with p18 (67.1% vs. 79.7%) in spite of the higher number of antigens in CP. The lower relative sensitivity of tests using internal antigens might reflect a lack of antibodies to cytosolic proteins in some infected animals or a shorter persistence of these antibodies relative to antibodies to outer membrane components after recovery from infection.     

The use of the fluorescent antibody test to detect Escherichia coli with K88 and 987P pilus antigens

Direct fluorescent antibody tests were used to detect Escherichia coli possessing K88 and 987P antigens. Identification of bacteria was accomplished on suspensions of organisms from clinical isolates, on frozen sections and impression smears from small intestine and on faecal smears. This assay makes possible the rapid identification of E. coli possessing K88 and 987P pilus antigens.     

Validation of the indirect fluorescent antibody and the complement fixation tests for the diagnosis of Theileria equi

The indirect fluorescent antibody (IFA) test for Theileria equi was evaluated to assess test's suitability for the serological diagnosis of equine piroplasmosis, to provide performance parameters for the purpose of test validation, and to compare it with the complement fixation (CF) test. Using a protocol that included Evan's blue, the specificity of the IFA test was estimated at 99.0% for T. equi by the classical method of analysis, and 96.6% by the Bayesian method. The use of Evan's blue in the test protocol increased test specificity and contributed to an excellent test agreement between two collaborating laboratories (kappa = 0.96). Using Bayesian analysis, the sensitivity estimate for the IFA test was 89.2%. The CF test sensitivity and specificity estimates for T. equi were 63.1 and 96.4%, respectively, as determined by Bayesian analysis. The IFA test was more sensitive than the CF test but the specificity estimates were similar.     

Preparation and evaluation of antigens used in serological tests for caprine syncytial retrovirus antibody in sheep and goat sera

Methods used to prepare antigens from caprine syncytial retrovirus (CSR) for use in the agarose gel immunodiffusion test (AGID) or an indirect enzyme-linked immunosorbent assay (ELISA) are described. Caprine and ovine sera were tested for antibody to CSR using the AGID test and ELISA incorporating a caprine system (CSR antigen and rabbit anti-goat IgG) or an ovine system (maedi-visna virus antigen and rabbit anti-sheep IgG). Good correlation was achieved in the results of the 3 tests when sera were devoid of antibody or were strongly positive. Variations in the results on weakly positive sera were considered to be more a matter of interpretation than due to basic differences in the reagents employed.     

Comparison of IS900 tissue PCR, bacterial culture, johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.     

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.     

Specificity of absorbed ELISA and agar gel immuno-diffusion tests for paratuberculosis in goats with observations about use of these tests in infected goats

OBJECTIVE: To determine the specificity of serological tests that are currently used in veterinary diagnostic laboratories in Australia for detection of Mycobacterium avium subsp paratuberculosis infection in goats. DESIGN: A laboratory study. PROCEDURE: Four tests were studied, comprising AGID with M. a. paratuberculosis antigen derived from cattle isolates of caprine or bovine origin, the EMAI caprine Johne's disease absorbed ELISA and the CSL PARACHEK Johne's absorbed EIA. The specificities of AGID and ELISA for paratuberculosis (Johne's disease) were estimated after examining a panel of 1000 serum samples collected from goats in Western Australia, a region free of paratuberculosis. In addition a comparison was made of test performance in a small number of paratuberculous goats from New South Wales using sera from two archival collections. RESULTS: The specificity of the AGID tests was 100% while the specificities of the two absorbed ELISA were 99.7 to 99.8% at appropriate positive-negative cut-offs. Based on testing the small sample of sera from infected goats, the absorbed ELISA tests detected about twice as many goats with Johne's disease as the AGID. Each test detected paratuberculous animals regardless of whether infection was caused by cattle or sheep strains of M. a. paratuberculosis. CONCLUSIONS: Both ELISA and AGID tests for paratuberculosis have high specificity and can be used in a market assurance program without risk of generating large proportions of false positive test results. However, the results suggested the ELISA is more sensitive for detection of infected goats and should be used in preference to the AGID. The two formats of ELISA evaluated in this study have similar characteristics and could be used in paratuberculosis control programs for the goat industries, but further data on sensitivity would increase confidence in their application.     

The specificity of antibody response in experimental and natural bovine paratuberculosis studied by crossed immunoelectrophoresis with intermediate gel

The sonicate antigen (MPS) of a local strain (IVRI) of Mycobacterium paratuberculosis and a commercial lysate of Strain 18 were analysed using hyperimmune rabbit and calf antisera to MPS in crossed immunoelectrophoresis with intermediate gel (CIE-ig) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The rabbit antiserum was more potent than the calf antiserum and it precipitated 35 and 15 antigens, respectively, among MPS and lysate antigens. SDS-PAGE resolved 50 and 32 peptides among these antigens respectively, of which, 35 and 15 were precipitated by rabbit antiserum. A CIE-ig reference system, with 30 MPS antigens, was standardized and used to analyse antibody specificities among sera derived from animals experimentally and naturally infected with bovine paratuberculosis. Fourteen antigens of MPS were found to be reactive with these sera and among these, Antigens 2 and 5 were found to be serodominant; sonicate antigens of M. bovis BCG and M. avium did not contain these antigens. Both were high molecular weight (greater than 60 kDa) antigens which may be of serodiagnostic value.     

Comparison of IgG antibody levels to Clostridium botulinum antigens between euthanased and surviving cases of chronic grass sickness

Serum from 12 horses suffering from chronic grass sickness (CGS) were assayed for IgG antibodies against botulinum neurotoxins C and D (BoNT/C and BoNT/D) and to a surface antigen extract of a neurotoxin negative strain of Clostridium botulinum type C. Collectively, the six surviving CGS cases demonstrated significantly higher initial IgG levels (P=0.05) against surface antigens than the six that were subsequently euthanased. The surviving animals also demonstrated higher initial IgG levels against the BoNT/C but not reaching significance (P=0.06). The two groups demonstrated no difference between IgG levels against BoNT/D. This study supports existing evidence of the involvement of C. botulinum type C in the aetiology of grass sickness.     

An enzyme-linked immunosorbent assay method for the simultaneous measurement of antibody titer to multiple viral, bacterial or protein antigens

A model enzyme-linked immunosorbent assay (ELISA) system was developed which permits the user to evaluate replicate single sera dilutions of 46 test and control serum samples for the presence of specific antibody against up to eight different antigens in one assay. The system makes use of a transplanting device for the simultaneous transfer of aliquots of independently diluted replicate test samples along with conjugate and substrate from a serum reservoir plate or from a reagent reservoir onto different antigen coated target plates. Kinetically read, raw absorbance data from tests are transmitted to a microcomputer, where absorbance values from all tests are quickly reduced to predicted titer levels by computer analysis. All titer computations involve the use of a single prescribed standard curve for predicting antibody titer against the different antigens. Between assay repeatability of this procedure is high and its potential for replacing a number of different conventional assays for epidemiological studies has been evaluated.     

Analysis by ELISA and Western blotting of antibody reactivities in cattle infected with Mycobacterium paratuberculosis after absorption of serum with M phlei

Preabsorption of cattle serum with Mycobacterium phlei was of value in eliminating falsely positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against M paratuberculosis. Specific antibody titres from 16 animals naturally infected with M paratuberculosis were unaffected by absorption. Analysis by Western blotting indicated that a different set of antigens of M paratuberculosis were recognised by serum from falsely positive reactors compared with that from animals with established infection. After experimental infection the time required for seroconversion in the ELISA in nine calves lay between 10 and 28 months, although one animal had not seroconverted after 30 months when the experiment ended. All animals shed M paratuberculosis in their faeces before seroconversion.     

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.     

Comparison of results using electron microscope, immunodiffusion and fluorescent antibody analyses to detect rotavirus in diarrheic fecal samples of calves.

Seventy-nine diarrheic calf fecal samples were examined by electron microscopy, immunodiffusion and the fluorescent antibody technique for the presence of rotavirus (reovirus-like agent). Thirty-eight (48%) of the samples were positive by electron microscopy, 59% by immunodiffusion and 20% positive by fluorescent antibody technique analyses. Another 9% were suspect-positive by fluorescent antibody technique. Chymotrypsin treatment of the fecal samples increased the ease of observing the viral particles by electron microscopy and also intensified the immunodiffusion arcs obtained. Immunodiffusion analyses using specific antisera to the virus would appear to be a practical method of detecting rotavirus in diarrheic fecal samples.