Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.     

A reappraisal of the complement fixation test using soluble Mycobacterium avium antigen for the detection of M. paratuberculosis infection in cattle

Serums from 263 cattle suspected of having paratuberculosis on the basis of clinical signs, were tested for antibodies to Mycobacterium paratuberculosis with a complement fixation test (CFT) employing a heat extracted, soluble M. avium antigen. Microscopic examination confirmed that 172 (65.4%) clinically affected animals had paratuberculosis, the remainder being disease-free. The specificity and sensitivity of the CFT was 92.3% and 74.4% respectively. Phenol treatment of serums before testing was compared with no treatment and was found to have no significant effect on the CFT titres. Results obtained are discussed in relation to the cause of false negative and false positive reactions.     

Evaluation of two commercial assays for the detection of Chlamydophila abortus antibodies

Two commercial enzyme-linked immunosorbent assays (ELISA), the CHEKIT-CHLAMYDIA which uses inactivated Chlamydophila psittaci antigen, and the Chlamydophila abortus ELISA produced by the Institut Pourquier which uses a recombinant fragment of the 80-90 kDa protein, were evaluated with the objective to determine whether the new ELISAs would perform as improved alternatives to the complement fixation test (CFT) for the serological diagnosis of ovine enzootic abortion (OEA). The results were compared to those obtained by the CFT and the competitive ELISA (cELISA). The tests were assessed with a panel of 17 serum samples from specific pathogen-free (SPF) lambs experimentally infected with various subtypes of Chlamydophila pecorum, with sera from 45 C. abortus-infected pregnant sheep and from 54 sheep free of OEA. The C. abortus ELISA was identified as being more specific and sensitive than the other tests. The 4 assays were evaluated further with 254 sera from flocks with documented OEA, from flocks with no history of abortion and from animals after abortion of unknown cause. The C. abortus ELISA by the Institut Pourquier identified less OEA-positive sera than the other assays though it identified correctly 9 of 10 OEA-positive flocks. The basis of the discordant results is discussed.     

Evaluation of two absorbed enzyme-linked immunosorbent assays and a complement fixation test as replacements for fecal culture in the detection of cows shedding Mycobacterium avium subspecies paratuberculosis.

Control of paratuberculosis in dairy herds is based on preventing the transmission of Mycobacterium avium subsp. paratuberculosis (Mptb) from cows to calves by management measures, supported by removal of cows excreting these bacteria by the fecal route (Mptb shedders). Fecal culture is the most accurate test for identifying Mptb shedders, but this technique is expensive and takes up to 16 weeks for results to be available. Serologic tests are inexpensive, rapid, and easy to perform. Of serologic tests, the complement fixation test (CFT) and absorbed enzyme-linked immunosorbent assay (ELISA) are the serologic tests used most frequently; the CFT is considered less accurate than the ELISA with respect to sensitivity and specificity. The commonly accepted absorbed ELISA is from the Australian Central Serum Laboratory. However, a European supplier has marketed a second ELISA that is supposed to be more sensitive in detecting Mptb shedders. These 2 absorbed ELISAs, designated ELISA-A and ELISA-B, and an in-house CFT were compared with data from 2 serum panels. The Mptb shedding panel consisted of sera from 198 culture-positive cows from 53 infected herds. The method used for culture of fecal samples was a modified J?rgensen method on individual samples. The Mptb shedder detection rate by the 3 serologic tests ranged from 29.8% to 39.4%. Detection rate for ELISA-A was lower than that for ELISA-B and CFT. For all 3 tests, detection rate was dependent on the level of Mptb shedding and the age of the animals. Detection rates increased as cattle age increased to 4 years. The specificity panel was initially composed of sera from 811 cows randomly selected from 41 herds without clinical paratuberculosis that were negative for Mptb based on whole-herd fecal culture. The modified J?rgensen method for culture was used on pooled fecal samples. Serologic test specificity ranged from 93.4% to 99.8%. The specificity of ELISA-A was higher than that of ELISA-B and CFT. Specificity of ELISA-B between herds was 75-100%. Specificity of CFT between herds was 62-100%. The low specificity of ELISA-B and CFT could not be explained by a higher sensitivity for Mptb-infected cows before onset of shedding, because in the 19 herds with 8 more subsequent negative whole-herd fecal cultures in the 4 years after sampling, specificity was not improved. The insufficient specificity of ELISA-B was not corrected sufficiently by heightening the cutoff value because Mptb shedder detection rate was lowered to 28.9%, equal to that of ELISA-A, and specificity only rose to 97%, much lower than that of ELISA-A. Taking into account the different test characteristics, serologic tests are a cost-effective alternative to fecal culture in high-prevalence herds. For certification programs, only ELISA-A is recommended because in a large number of nonsuspect herds specificity remained almost 100%.     

Experience with simple ELISA test systems for Brucella serology in cattle, sheep and goats

The objective of this work was to use the ELISA technique for the serological surveillance for freedom of brucellosis of cattle, sheep and goats. By comparing 28 cattle sera taken after a brucellosis outbreak, 15 bovine sera supplied by the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) and 497 serum slow agglutination test (SSAT) and complement fixation test (CFT) negative bovine sera from herds officially declared free of brucellosis, the ELISA technique not only shows higher sensitivity as compared to SSAT and CFT but also distinguishes clearly between positive and negative reactions. The serological comparison by SSAT, CFT and ELISA of 615 cattle, 624 sheep and 630 goat sera from herds acknowledged as brucellosis free showed equivalent specificities for both CFT and ELISA. The specificity of the SSAT was much lower, 81.1% in cattle and 96.2% in goat sera. The examination of 5796 cattle, 1337 calf, 5031 sheep and 1796 goat sera demonstrates the advantage of the ELISA technique as routine method. The possible application of the ELISA technique as a screening method for serological brucellosis tests in sheep, goats and possibly also in pigs is discussed.     

Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera

OBJECTIVE: To compare the sensitivity and specificity of Chlamydophila abortus antibody assays, to find a suitable serological assay for testing sheep for export. DESIGN: Comparison of results from known positive and negative sheep populations. PROCEDURE: Fifty-five positive and fifty negative sera were analysed by four enzyme linked immunosorbent assays (ELISA), three using recombinant antigens based on the chlamydial polymorphic outer membrane proteins (POMP90-3, POMP90-4, POMP80-90) and one using a synthetic peptide based on chlamydial major outer membrane proteins (MOMP-P). They were also analysed by complement fixation tests (CFT) using crude antigens from chlamydia isolated from an Australian sheep, a Californian parakeet and a Texan turkey. Assay sensitivity and specificity were expressed as point estimates and 95% confidence intervals. Results were compared using McNemar's test for paired samples. RESULTS: ELISA sensitivity ranged from 70 to 98% and complement fixation test sensitivity from 60 to 96%; with POMP90-3 > POMP90-4 > CFT (parakeet) > CFT (turkey) > POMP80-90 > MOMP-P > CFT (sheep). There was no significant difference from POMP90-3 to POMP80-90 (P > 0.05). ELISA specificity ranged from 88 to 100% and CFT specificity was 100% for all three antigens; with CFT and POMP90-4 > MOMP-P > POMP80-90 > POMP90-3. There was no significant difference from CFT to POMP80-90 (P > 0.05). Changing the CFT cut-off from 1:32 to 1:4 substantially reduced the specificity with little improvement in sensitivity. CONCLUSION: Assays using POMP90-4, POMP80-90, CFT (parakeet) and CFT (turkey) had equivalent sensitivity and specificity; none of the ELISAs were more specific than any CFT. The POMP80-90 ELISA is recommended as an alternative to CFT (parakeet) but as its specificity is not ideal the search for a more specific assay should continue.     

Comparison of seroreactivity of rams with brucellosis in a complement fixation test, whole cell ELISA and by immunoblotting

In rams with ovine brucellosis, a high degree of serological correlation exists between the complement fixation (CF) test which utilises antigen extracted from bacteria with hot saline, and the ELISA reactivity using methanol-fixed Brucella ovis as the assay reagent. Since the whole cell ELISA (CELISA) detects mainly antibodies against surface antigens of B. ovis, it was concluded that the similar findings of the two serological tests is due in part to the presence of membrane antigens in the CF test antigen following hot saline extraction of intact bacteria. Immunoblots with pooled sera representing different CF titres confirmed that the major immunoreactive antigens of B. ovis were located in four zones: alpha, beta, gamma 1 and 2 with corresponding apparent molecular masses of 55 and 60 kDa; 27 and 29 kDa; 18.5-20 kDa and 17-18 kDa, respectively. These zones of reactivity were consistently present in immunoblots when assayed against different B. ovis isolates even though Coomassie brilliant blue staining of SDS-PAGE gels revealed some differences in polypeptide banding patterns. However, these intensely-stained CBB bands located at 38 and 40 kDa which distinguished three of the seven B. ovis isolates were considerably less reactive in immunoblots compared to polypeptides that were located at positions equivalent to alpha, beta or gamma reactivities. Intensity of immunoblot reactivity against polypeptides located in the alpha, beta and gamma zones intensified with increasing CF titre. Sera with CF titres greater than 32 also tended to react against bands of higher apparent molecular masses located at 65, 70, 73, 78, 80 and 86 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum antibodies against particular antigens of Borrelia burgdorferi sensu stricto and their potential in the diagnosis of canine Lyme borreliosis

Dog sera (n = 118) were tested for antibodies recognizing Borrelia (B.) burgdorferi sensu stricto strain B31 (ATCC 35210) antigens. In total, 18 of the dog sera gave positive results in a whole cell sonicate ELISA (WCS ELISA). These positive sera were further evaluated by immunoblot assay, utilizing a whole bacterial lysate as antigens. 94.4% (17 of 18) of the dog sera reacted with immunodominant antigens at 20-22 kDa (protein C, pC), 31 kDa (outer surface protein A, OspA), 34 kDa (outer surface protein B, OspB), 41 kDa (flagellin), 60 kDa ("common antigen"), and/or 100 kDa (presumably p100). Sera recognizing pC (20-22 kDa) and antigens > 94 kDa always detected the highest number of antigen bands, indicating the specificity of those antigens in serological diagnosis. The results clearly demonstrate that the WCS ELISA is a useful tool for testing sera of dogs for antibodies against B. burgdorferi. However, positive results should be confirmed by immunoblot, using WCS as antigen. According to the presented data, we recommend criteria for B. burgdorferi immunoblots using dog sera as follows: sera have to be considered as positive if they detect the 41 kDa flagellin, and two of the 5 immunodominant antigens, namely > 94 kDa (presumably p100), 60 kDa ("common antigen"), 34 kDa and 29-31 kDa (OspB and OspA, respectively) and 20-22 kDa (pC). If sera only recognize the 41 kDa flagellin, this result is equivocal, requiring testing a second serum sample 4 to 8 weeks later.     

Agreement between ELISA and complement fixation test used for diagnosing of paratuberculosis in goats

An ELISA with a lipoarabinomannan as an antigen, developed for diagnosis of bovine paratuberculosis, has been adapted for use in goats, and compared with complement fixation test. Kappa value of 0.62 indicated good agreement between CFT and the adapted ELISA and proved that the investigated ELISA may be helpful in diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats. The ELISA has been used to screen a randomly selected representative sample of Polish breeding goat population (21.78% of herds, 21.33% of goats). It has been demonstrated that only 2.42% of animals coming from 15.79% of herds were seropositive. Within-herd seroprevalence varied from 1.69% to 38.10%. Most of the infected animals (67.07%) were 3- 4-years-old. No seropositive cases were found in group up to 1-year-old animals.     

The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.     

Comparison of the Johne''s absorbed EIA and the complement-fixation test for the diagnosis of Johne''s disease in cattle

A commercially available absorbed ELISA for the diagnosis of Johne's disease (JD) (paratuberculosis) in cattle, the Johne's Absorbed EIA, was compared with the conventional complement-fixation test (CFT) used in Australia. Stored plasma from 3 Victorian dairy herds with a history of JD, sera from specimens submitted from animals showing clinical signs of JD and sera from the US National Repository for Paratuberculosis Specimens were used to determine the sensitivity of each test. The EIA detected 48.8% of 43 Australian animals with subclinical JD, while the CFT detected only 12 (21.4%) of 56 subclinically affected cattle. Of 150 subclinically infected US cattle, the EIA detected 47.3% and the CFT detected 52.0%. The EIA detected 59.7% of animals which at the time of sampling were shedding Mycobacterium paratuberculosis in their faeces, but showed no clinical signs of JD, while the CFT detected 57.3%. The EIA correctly identified 88.2% of 136 histologically confirmed clinical cases, and the CFT detected 83.4%. The specificity of each test was determined by testing sera collected at slaughter from animals residing in a known JD-free area of Australia, and from samples from the US National Repository of Paratuberculosis Specimens collected from certified-free herds in Wisconsin. The EIA was found to have a specificity of 99.8% when 998 Australian animals were used as the test population, and 99.0% when 196 US animals were used. The specificity of the CFT using Australian samples was 96.9% and 95.2% using American samples.     

Comparison of an enzyme-linked immunosorbent assay using monoclonal antibodies and a complement fixation test for cattle vaccinated and infected with Brucella abortus

A competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody conjugated to horseradish peroxidase MA(A) and a complement fixation test (CFT) were applied to sera collected over a two-year period from 60 cattle challenged with Brucella abortus strain 544. Forty-eight of the cattle were previously vaccinated with B. abortus strain 19 (S19) or B. abortus strain 45/20 (45/20). After challenge 33 of the cattle remained uninfected and nine of the 27 infected cattle showed aberrant reactions by the CFT. The performance of the MA(A) ELISA was as follows: after vaccination, the MA(A) ELISA, like the CFT, was unable to differentiate infected cattle from those recently vaccinated with S19. After challenge the MA(A) ELISA gave results comparable with the CFT for those cattle with aberrant reactions. For the non-infected cattle there was a similar number of weeks after challenge when both tests were negative. It is suggested that the main advantage of the MA(A) ELISA when compared with the CFT lies in its relatively simple test procedure.     

Comparative evaluation of eight serological assays for diagnosing Chlamydophila abortus infection in sheep

Chlamydophila abortus is one of the principal causes of late-term abortion (enzootic abortion of ewes or EAE) in sheep across Europe. Serological diagnosis of EAE is routinely carried out by the complement fixation test, although the interpretation of results can often be difficult because of cross reaction with Chlamydophila pecorum, which also commonly infects sheep. The purpose of this study was to evaluate and compare four ELISAs developed at Moredun Research Institute and based on whole C. abortus elementary bodies (EBs), an outer membrane preparation of the whole organism (SolPr) and two recombinant polymorphic outer membrane protein fragments (rOMP90-3 and rOMP90-4), with 3 commercial tests, the CHEKIT Chlamydophila Abortus, Pourquier ELISA Chlamydophila abortus and ImmunoComb Ovine Chlamydophila Antibody tests. The tests were evaluated using a panel of 202 sera from experimentally and naturally infected animals, as well as from EAE-free flocks. The EB, SolPr and CHEKIT ELISAs performed similarly to the CFT, all lacking in specificity by cross reacting with sera from C. pecorum infected animals. The ImmunoComb also lacked specificity with C. pecorum sera, but also badly cross reacted with sera from EAE-free flocks. The rOMP90-3, rOMP90-4 and Pourquier ELISAs were the most specific, although the Pourquier test appeared less sensitive with sera from naturally infected animals. Overall, the rOMP90-3 ELISA performed the best, with high sensitivity (96.8%) and no cross reaction with sera from C. pecorum infected animals or from EAE-free flocks (100% specificity) and so would be a suitable alternative to the CFT for the serological diagnosis of EAE.     

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle

OBJECTIVE: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. ANIMALS: 1043 cattle from 10 herds in Michigan. PROCEDURE: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. RESULTS: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.     

Performance of complement fixation test and confirmatory immunoblot as two-cascade testing approach for serodiagnosis of glanders in an endemic region of South East Asia

Various serological tests were used for the diagnosis of glanders in the past but still complement fixation test (CFT) is the internationally prescribed test for trading equines. A new immunoblot (IB) technique has recently been introduced to overcome the well known shortcomings of CFT i. e. a considerable number of false positive and negative results and anticomplementary effects of sera. The objective of this study was the comparative evaluation of two glanders CFT antigens commercially available at Central Veterinary Institute ofWageningen UR, Lelystad, NL (CIDC) and at c.c.pro GmbH, Oberdorla, DE (c.c.pro) in a glanders endemic area regarding specificity and sensitivity. A total of 1678 serum samples from the endemic region (Province Punjab, Pakistan) and a non-endemic area (Germany) were analysed. All sera tested positive or suspicious with CFT were analysed by the confirmatory IB to exclude CFT false positive results. Both CFT antigens showed 100% sensitivity. The use of CIDC or c.c.pro antigen resulted in specificities of 77.45% or 75.71% for sera from endemic area and 93.75% or 94.79% for sera from non-endemic areas, respectively. The results demonstrate the different performances of identical tests in different epidemiologically settings. Based on these results, the combined use of CFT and IB is highly suggestive for the serodiagnosis of glanders. Good agreement was calculated between CFT (using either c.c.pro or CIDC antigen) and immunoblot.     

Evaluation of four commercial enzyme-linked immunosorbent assays for the diagnosis of bovine paratuberculosis in Chilean dairy herds.

The accuracy of 4 commercial enzyme-linked immunosorbent assays (ELISAs) for diagnosis of bovine paratuberculosis was compared using sera from 53 Mycobacterium avium subsp. paratuberculosis (MAP) fecal culture-positive dairy cows (cases) and sera from 345 dairy cattle resident in 11 fecal culture-negative herds on 2 consecutive occasions 1 year apart (controls). The specificity of all 4 ELISA kits was >99%, and their diagnostic sensitivity ranged from 30.2% to 41.5%. Pairwise comparison of ELISAs found no significant differences (McNemar's chi-square test > 0.05), and assay agreement for categorical assay interpretation (positive or negative) was high (>98%) with kappa values ranging from 0.84 to 0.95. Receiver operating characteristic (ROC) curve analysis and the corresponding area under the ROC curves indicate that kit B had the highest overall accuracy. Thus, all 4 ELISA kits for bovine paratuberculosis had comparable accuracy when tested on Chilean dairy cattle, with kit B having a slight statistical advantage based on ROC area under the curve analysis. This suggests that any of the 4 kits could be appropriate for herd certification and for paratuberculosis control programs on Chilean dairy cattle.     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

Carboxyl terminus of the 34 kDa protein of Mycobacterium paratuberculosis shares homologous B-cell epitopes with Mycobacterium avium and Mycobacterium intracellulare

Monoclonal antibodies (mAbs) against a recombinant carboxyl terminus of the 34 kDa protein of Mycobacterium paratuberculosis were produced in mice. Two of the mAbs cross-reacted with Mycobacterium avium and Mycobacterium intracellulare in both an elisa and immunoblot. The recombinant protein also reacted with polyclonal sera produced in rabbits against all three mycobacteria, indicating the presence of cross-reactive epitopes in the protein. To determine the reactivity of cattle sera against epitopes recognised by the mAbs, competition assays between bovine sera and the mAbs were carried out. Two mAbs were significantly inhibited by sera from cattle that were naturally infected with M paratuberculosis. The results indicate that epitopes on the carboxyl terminus of the 34 kDa protein common to M paratuberculosis, M avium and M intracellulare readily induce antibody production in naturally infected cattle. These epitopes reduce the diagnostic specificity of the carboxyl terminus of the 34 kDa protein, which was originally thought to contain only M paratuberculosis-specific epitopes.     

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.     

A Serological Investigation into Contagious Caprine Pleuropneumonia (CCPP) in Ethiopia

For a comparison of serological tests for CCPP, sera from 767 goats were examined. They were subjected to three tests: complement fixation test (CFT) with Mycoplasma capricolum subspecies capripneumoniae antigen; blocking ELISA (B-ELISA) with Mycoplasma capricolum subspecies capripneumoniae antigen; and CFT with Mycoplasma mycoides subspecies mycoides small colony type antigen. Antibodies were detected by these three tests in 23%, 2% and 12%, respectively, of sera from districts in which CCPP had not been reported, and in 60%, 83% and 87%, respectively, in sera from areas in which CCPP had been reported. The specificity of the tests is discussed. The use of the B-ELISA test for the diagnosis and for epidemiological studies of CCPP is strongly recommended.