Enrichment for isolating Salmonella Choleraesuis and other Salmonella spp. from pigs

The growth of Salmonella Choleraesuis was examined in Rappaport Vassiliadis broth (RV) and Hajna-tetrathionate broth (HTT) at 37 and 42 degrees C. As the enrichment in RV at 37 degrees C was satisfactory for isolating S. Choleraesuis, we used this enrichment for isolation from the samples collected from 15 asymptomatic pigs reared on a S. Choleraesuis contaminated farm. S. Choleraesuis was frequently isolated from six pigs (40.0%) under field conditions. The isolation of other Salmonella serovars than S. Choleraesuis was attempted by using both RV enrichment at 37 degrees C and HTT enrichment at 42 degrees C. Salmonella organisms were isolated from 156 (44.8%) of 348 fecal samples and more frequently with HTT at 42 degrees C (43.4%) than with RV at 37 degrees C (20.9%). If other serovars in addition to S. Choleraesuis are to be surveyed, HTT enrichment should be used in combination with RV enrichment.     

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

Comparison of a commercial enzyme-linked immunosorbent assay with electron microscopy, fluorescent antibody, and virus isolation for the detection of bovine and porcine rotavirus

The purpose in this study was to compare the sensitivity of a commercial enzyme-linked immunosorbent assay (ELISA) with electron microscopy (EM), fluorescent antibody (FA), and virus isolation (VI) for the detection of bovine and porcine rotavirus (RV). Seventy-three bovine and 116 porcine accessions were evaluated by 1 or all 4 diagnostic tests, where suitable specimens were available. For the bovine samples, agreement was 33% between FA and EM, 33% between FA and ELISA, and 92% between EM and ELISA. For the porcine samples, agreement was 79% between EM and FA, 72% between EM and ELISA, and 82% between ELISA and FA. Virus was isolated from 68% and 41% of the bovine and porcine fecal samples, respectively. Commercial ELISA was as sensitive as EM, but was more sensitive than FA or VI for the detection of RV in bovine feces. Electron microscopy was more sensitive than FA, ELISA, or VI for detection of RV in porcine feces. The ELISA was an advantageous alternative to the conventional methods of EM, FA, and VI for the diagnosis of RV in calf feces, but not for porcine feces.     

Comparison of an antigen-capture enzyme-linked immunosorbent assay with bacterial culture for detection of Salmonella in poultry-hatchery environmental samples

An antigen-capture, monoclonal-antibody-based enzyme-linked immunoassay (ELISA) that detects a broad range of Salmonella serovars in various serogroups was developed and compared with standard culture procedures for detection of Salmonella in 1055 field samples collected from poultry-hatchery environments. The diagnostic sensitivity of the ELISA relative to culture was 99.9% and the diagnostic specificity 99.6%. The extensive culture procedure included nonselective enrichment (NSE) as well as primary selective enrichment (PSE) and delayed secondary enrichment (DSE) with Hajna tetrathionate (TT) and Rappaport–Vassiliadis (RV) selective-enrichment broths. Significantly more Salmonella-positive samples were detected by ELISA and culture at the DSE stage than at the NSE and PSE stages (P < 0.05). Significantly more RV than TT broths were positive for Salmonella by culture and ELISA by the DSE stage (P < 0.05). This ELISA procedure could be a reliable screening test for the detection of Salmonella in hatchery samples.     

Comparison of sampling and isolation procedures for recovery of Yersinia enterocolitica serotype O:3 from the oral cavity of slaughter pigs

Yersinia enterocolitica serotype 0:3/biotype 4 was isolated from the oral cavity of altogether 32 (68.1 %) of 47 freshly eviscerated slaughter pigs. Most efficient recovery was achieved by cultivation of tissue samples from both tongue and tonsils of the same individual. The isolation rate so obtained was significantly higher than that obtained by separate examination of either tonsil swabs or tongue swabs. However, the isolation frequency achieved by combined swabbing of the 2 sites was not significantly different. In general, tonsils were more productive for the recovery of 0:3 strains than were tongues, and tissue samples yielded higher isolation rates than did swabs. Three-week cold enrichment in a low selective medium proved essential for optimal recovery. However, the highest number of isolates was obtained using a combination of methods, including direct plating and selective enrichment in a modified Rappaport broth in addition to cold enrichment.     

Effects of sample storage and delayed secondary enrichment on detection of Salmonella spp in swine feces

OBJECTIVE: To determine effects of fecal sample storage and delayed secondary enrichment (DSE) on detection of Salmonella spp in swine feces. Sample Population-Fecal samples obtained from 84 pigs in a commercial herd. PROCEDURE: Each fecal sample underwent 3 storage treatments: no storage (ie, processed on the day of collection), storage at 4 C for 6 days, and storage at -15 C for 14 days. After assigned storage treatments, all samples were enriched in Rappaport-Vassiladias (RV) broth (single enrichment) and plated on XLT4 agar. Delayed secondary enrichment was performed, using single enrichment broths that were stored for 4 days at room temperature. RESULTS: Of 504 cultures, 186 (36.9%) were Salmonella positive. A difference in proportions of samples with positive results was not found between same-day processing and storage at 4 C for 6 days. Compared with use of single enrichment for 24 hours (34% positive), use of DSE resulted in a greater proportion (40%; P < 0.001) of samples with positive results. Estimated relative sensitivities for the storage methods were 0.90, 0.85, and 0.71 for same-day processing, storage at 4 C for 6 days, and storage at -15 C for 14 days, respectively. CONCLUSIONS: Where practical, processing of fecal samples on the day of collection is recommended, although storage at 4 C for several days does not result in marked loss of sensitivity. Improved detection associated with DSE warrants further investigation and optimization.     

Development of monoclonal antibody ELISA for simultaneous detection of bovine coronavirus, rotavirus serogroup A, and Escherichia coli K99 antigen in feces of calves.

A rapid ELISA was developed for simultaneous detection of bovine coronavirus (BCV), rotavirus (RV) serogroup A, and Escherichia coli K99 antigen in feces of calves. A mixture of 3 monoclonal antibodies specific for BCV, RV, or K99 was used successfully to capture the antigens; the same antibodies labeled with peroxidase were used to detect BCV, RV, or K99. The triple ELISA was compared with standard reference diagnostic methods by examining feces from experimentally and naturally infected and healthy calves. All the components of the test were highly specific (greater than 90%) and sensitive (BCV, 77%; K99, 93%; RV, 100%) when used in a format requiring short incubation steps at 20 C and visual recording of results.     

Improved culture methods for isolation of Salmonella organisms from swine feces

OBJECTIVE: To compare 3 alternative culture techniques for the detection of Salmonella organisms in swine feces with a modification of the International Standard Organization (ISO) 6579 standard protocol. SAMPLE POPULATION: Fecal samples from swine herds suspected of having Salmonella infections. PROCEDURE: 4 experiments were performed to evaluate the following: 1) diagnostic sensitivity of the selective preenrichment and rapid isolation novel technology (SPRINT) protocol, compared with that of the modified ISO protocol; 2) detection limit of the SPRINT protocol for Salmonella organisms; 3) use of tetrathionate-novobiocin (TTN) broth, compared with selenite cysteine (SC) broth for selective enrichment; and 4) use of universal preenrichment (UPE) broth, compared with buffered peptone water (BPW) for preenrichment of samples prior to the use of modified semisolid Rappaport-Vassiliadis (MSRV) plates. RESULTS: Comparing the Salmonella culture results of 183 swine fecal samples, the diagnostic sensitivity of the SPRINT protocol (0.86) was not significantly different than the diagnostic sensitivity of the modified ISO protocol (0.80), although it was 24 hours faster. The SPRINT protocol could detect 5 of the 6 investigated Salmonella serotypes at inoculation concentrations of < 10 colony-forming units (CFU)/25 g of uncontaminated feces. The TTN broth performed significantly better than the SC broth for selective enrichment of Salmonella organisms. There was no significant difference in results of preenrichment of samples between the use of UPE broth or BPW. CONCLUSIONS AND CLINICAL RELEVANCE: The SPRINT protocol may provide a faster alternative for isolation of Salmonella organisms from swine fecal samples. Furthermore, the use of TTN broth instead of SC broth may increase the sensitivity of the modified ISO 6579 protocol.     

Optimization of a culture technique for the isolation of Listeria monocytogenes from faecal samples

A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.     

Detection of Vibrio parahaemolyticus in Mediterranean mussels (Mytilus galloprovincialis) in Slovenia

The aim of this study was to determine the prevalence of Vibrio parahaemolyticus in shellfish samples harvested along the Slovenian coast. Shellfish samples of Mediterranean mussels (Mytilus galloprovincialis) were collected along the Slovenian coast at four locations (Se?a, Piran, Strunjan and Debeli Rti?) between 2006 and 2008. Samples were examined and analysed for the presence of V. parahaemolyticus by conventional and molecular methods. The presence of Vibrio in the samples was examined by conventional methods on plate grown bacterial cells before and after enrichment in alkaline saline peptone water (ASPW). PCR methods were used for the detection of V. parahaemolyticus-specific toxR and tlh genes and of the virulence-associated tdh and trh genes. Out of 168 samples examined, 24 were positive for toxR and tlh genes by PCR from enrichment broth. Five out of 62 (8.1%), 4 out of 32 (12.5%) and 15 out of 74 (20.2%) samples were positive in 2006, 2007 and 2008, respectively. Colonies of V. parahaemolyticus were isolated from only one sample positive for V. parahaemolyticus by PCR.     

Effect of preculture freezing and incubation on bacteriological isolation from subclinical mastitis samples

In this study the sensitivity of three methods of isolation of udder pathogens from milk samples from subclinical mastitis cases was compared. For analysis 1827 quarter milk samples were selected. Milk was cultured using a standard culture technique (0.01 ml of fresh milk streaked on a sheep blood agar plate and on Edward's medium). In addition, an inoculum of 0.01 ml of the original milk sample was incubated for 24h at 37 degrees C in broth, followed by culture using the standard culture technique. In the third method, the whole milk sample was frozen for 24h, and then incubated for 24h at 37 degrees C, followed by culture using standard culture technique. The isolation percentage of Staphylococcus aureus was 4.7% for standard culture technique, 14.2% for incubation in broth, and 21.5% for the combination of freezing plus incubation. Isolation percentage of Streptococcus dysgalactiae and Streptococcus agalactiae was highest using the standard culture technique, while isolation rate of Streptococcus uberis was not different among the three methods used. With increasing somatic cell count, the likelihood of S. aureus, S. dysgalactiae and S. uberis isolation increased.Based on the relative sensitivity, defined as the isolation rate using a single technique compared to the isolation rate of the three techniques together, a combination of standard culture technique and freezing plus incubation was most attractive for achieving a high isolation rate of S. agalactiae and S. dysgalactiae. Relative sensitivity of S. uberis isolation was highest using the standard culture technique and incubation in broth, while S. aureus was most often isolated using a combination of incubation in broth and freezing plus incubation. A combination of the three methods increased the isolation rate for S. dysgalactiae, S. uberis and S. aureus. The standard culture technique, together with the combination of freezing plus incubation, can be recommended for isolating major udder pathogens. If S. aureus is the pathogen of main interest, using incubation in broth together with the combination of freezing plus incubation performed best.     

Comparison of Different Sampling Strategies and Laboratory Methods for the Detection of C. jejuni and C. coli from Broiler Flocks at Primary Production

Summary The objective of the study was to evaluate the performance of different combinations of sample type, transport medium and culture methods for the recovery of Campylobacter jejuni and C. coli from broiler flocks at primary production. Boot swabs moistened with one of four different transport media [maximum recovery diluent (n = 120), Exeter broth (EX) (n = 120), buffered peptone water (n = 120) and modified semi‐solid Cary‐Blair (n = 120)], caecal samples (n = 40) and faecal samples (n = 120) from 40 broiler flocks were compared and sensitivity estimates obtained using a Bayesian model. Samples were cultured onto mCCDA before and after enrichment in EX and incubated microaerobically at 41.5°C. Campylobacter suspect colonies were identified to the species level by multiplex PCR. Results from the Bayesian model indicated that boot swabs after enrichment had higher sensitivity (90–94%) than caecal contents before or after enrichment (84% and 89%, respectively) and faecal samples after enrichment (82%) for the detection of Campylobacter spp., although these differences were not statistically significant. Enrichment significantly increased the sensitivity of boot swab and caecal samples for detection of Campylobacter spp. and C. jejuni, respectively. However, the enrichment of caecal samples resulted in a significant decrease in the sensitivity of these samples for detection of C. coli. There was much greater variation in the sensitivity estimates of the methods for detecting C. coli than for C. jejuni, and the ranking of methods was different between the two species. Boot swabs gave the best sensitivity values for detection of C. jejuni, and enrichment culture of faecal samples was the most sensitive method for detection of C. coli.     

Temporal patterns of diagnostic results in serial samples from cattle with advanced paratuberculosis infections.

As part of investigating diagnostic strategies for Mycobacterium avium subsp. paratuberculosis (Map), serial results from polymerase chain reaction (PCR) on extraintestinal tissues (blood, milk, and liver) were compared with those from more conventional detection methods including serum enzyme-linked immunosorbent assay (ELISA), fecal culture, and fecal PCR. Three cows previously identified as being subclinically infected with Map were selected for the study. Blood, milk, and feces were collected daily and liver biopsies were obtained weekly for a 30-day period. Unexpectedly, a substantial daily variation in serum ELISA sample to positive (S/P) ratios was observed in all 3 cows. In contrast, fecal culture results were consistently positive. However, whereas fecal culture colony counts were consistently high for 2 cows throughout the study, colony counts from the third cow varied from day to day. Diagnostic sensitivity of PCR for fecal, blood, milk, and liver samples in these advanced subclinically infected cows was 87%, 40%, 96%, and 93%, respectively.     

Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.     

Comparison of bacterial enriched-broth culture, enzyme linked immunosorbent assay, and broth culture-polymerase chain reaction techniques for identifying asymptomatic infections with Salmonella in swine

A polymerase chain reaction (PCR) assay was combined with a broth-culture enrichment system to detect Salmonella shed in feces from subclinically infected swine. The effectiveness of the broth culture-polymerase chain reaction (BC-PCR) assay to identify pigs shedding Salmonella in feces was compared with a microbiological culture and a commercial enzyme linked immunosorbent assay (ELISA) kit to detect Salmonella-specific serum antibody. A total of 67 pigs were tested by each of the 3 methodologies. Forty-one pigs tested positive for Salmonella by BC-PCR and ELISA identified 6 positives and 23 suspicious samples. It was shown that the BC-PCR assay is a rapid diagnostic tool for detecting of Salmonella shed by asymptomatic swine compared with current diagnostic technologies.     

Evaluation of an immunochromatography strip assay for the detection of Salmonella sp. from poultry.

The present study was conducted to evaluate the sensitivity and specificity of an immunochromatography-based diagnostic kit for Salmonella. The analytical sensitivity of the test when using pure colonies of different Salmonella species was in the range of 1 X 10(4) to 1 x 10(5) colony-forming units per milliliter. The strip detected 19 of 22 strains of Salmonella spp. but failed to detect S. worthington, S. choleraesuis var. kunzendorf and S. johannesburg. The strip did not detect 27 different enteric bacteria, including Escherichia coli O157:H7, Campylobacter jejuni, Shigella sonnei, and Vibrio parahaemolyticus. In direct testing of feces (n = 66) from chickens infected with Salmonella typhimurium, the strip had a sensitivity of 12.3% and a specificity of 100%. Evaluation of the strip assay (n = 510) after sample pre-enrichment in 2% buffered peptone water (BPW) yielded a sensitivity of 93.8% and specificity of 89% when compared to isolation and identification with xylose-lysine-tergitol 4 (XLT4) selective plating media. Subsequent enrichment in Hajna tetrathionate (TT) broth yielded a higher sensitivity (94.7%) and specificity (96.8%). The agreement (kappa) between the strip test and isolation was 0.004 in direct fecal testing, 0.82 in BPW, and 0.89 in TT broth. The assay could detect Salmonella sp. as early as 18-48 hours during pre-enrichment and enrichment compared to isolation on XLT4, which required an overnight incubation step for the presumptive isolation and identification of Salmonella.     

Sensitivity of methods for the isolation of Escherichia coli O157 from naturally infected bovine faeces

At present, no standard protocol has been described to detect the presence of Escherichia coli O157 in cattle faeces. Therefore, the sensitivity of 26 different isolation methods was determined in order to recommend a method of choice. Faeces samples from 17 different beef cattle at a farm previously found positive for E. coli O157 were subdivided into a total of 40 samples. It was not known whether the 17 cattle shed E. coli O157 at the time of sampling. At another farm where cattle have been found negative for E. coli O157 on different occasions, five faeces samples were collected. Two methods yielded the highest sensitivity (74%): 6h enrichment in modified tryptone soya broth supplemented with novobiocin (mTSBn) followed by an immunomagnetic separation (IMS) with (i) Dynal beads or (ii) Captivate beads and selective plating on Rainbow agar (RA) plates. Enrichment for 6h was significantly better than 24h enrichment. Only after 24h, buffered peptone water (BPw) was significantly better than mTSBn. A sensitivity of 82% was obtained only when the two most sensitive tests were done simultaneously. Because none of the tests gave 100% sensitivity, it can be concluded that isolation rates of E. coli O157 from bovine faeces using only one of the tested procedures results in an underestimation of the incidence of E. coli O157 in cattle. Performing more than one test on the samples must be considered.     

Evaluation of a recombinant enzyme-linked immunosorbent assay for detecting Chlamydophila psittaci antibodies in turkey sera

Chlamydophila psittaci (formerly Chlamydia psittaci) is one of the major pathogens associated with turkey respiratory disease. Devastating outbreaks with high mortality rates, similar to those of 1950 to 1970 in the USA occasionally occur, but respiratory signs without or with low mortality mostly characterize outbreaks now a day. Accurate diagnostic methods should be made available. The present study examined the sensitivity and specificity of a recombinant ELISA (rMOMP ELISA) for detecting Cp. psittaci major outer membrane specific antibodies in turkey sera. Test results were compared to those of immunoblotting and of a competitive ELISA (Chlamydia-psittaci-AK-EIA, R?hm Pharma, Germany) and an indirect ELISA (LPS/LGP) detecting antibodies to the lipopolysaccharide/lipoglycoprotein complex. The rMOMP ELISA was most sensitive as determined on serial dilutions of positive control sera originating from experimentally infected SPF turkeys. The competitive ELISA gave false positives since three negative controls reacted positive. For conventional sera, the sensitivities of the competitive ELISA, immunoblotting and the indirect ELISA were found to be 99.4, 93.1 and 82.2%, respectively, as compared to the rMOMP ELISA (100%). The specificities of the rMOMP ELISA, immunoblotting and the indirect ELISA were found to be 100% while the specificity of the competitive ELISA was only 2.7%. The rMOMP ELISA was chosen to compare the prevalence of chlamydiosis in 2002 with the one from 1992. In 2002, 188 on 200 (94%) turkey sera reacted positive compared to 175 on 200 (87.5%) in 1992 and like 10 years ago all examined farms were seropositive at slaughter. Interestingly, Belgian as well as French farms were seropositive.     

Evaluation of the enzyme-linked immunosorbent assay for detecting Brucella abortus antibodies.

The specificity of enzyme-linked immunosorbent assays (ELISA) corresponds to conventional methods for detecting brucella antibodies in bovine serum. The ELISA test detected brucella antibodies early in only 12.5% of the cattle sera tested. Also, the sensitivity of ELISA was comparable to complement-fixation and Rivanol methods, but less sensitive than the standard tube agglutination method.     

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.