Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum

An enzyme-linked immunosorbent assay (ELISA) was evaluated for detection of antibodies (Ab) against Mycoplasma hyopneumoniae and M. flocculare in sera from swine experimentally infected with these agents. In addition, the ELISA was compared with the complement fixation test (CFT), and radial immunodiffusion enzyme assay (RIDEA) for the demonstration of Ab against M. hyopneumoniae. Twenty two 6-week-old swine from a respiratory disease-free herd were divided into five groups. Two or three pigs from each of the four groups were inoculated, respectively, with M. hyopneumoniae or with M. flocculare while two pigs in each group were contact exposed to the inoculated penmates. A fifth group, consisting of three pigs, served as inoculated controls. Pigs inoculated with M. hyopneumoniae began coughing 13 days post inoculation (PI). Antibodies were first detected 2 weeks PI with the CFT, 3 weeks PI with the ELISA, and 5 weeks PI with the RIDEA. With the ELISA and RIDEA, Ab were still detectable one year PI at a very low level. With the CFT, Ab were not detectable in sera from any swine beyond 5 months PI. At necropsy 1 year PI, no lesions were detected in lungs of any of the animals nor were mycoplasmas detected. M. flocculare inoculated or contact-exposed pigs never evidenced clinical signs. Antibodies against M. flocculare were first detected 5 to 12 weeks PI with CFT, and 6 to 12 weeks PI with the ELISA. Peak optical density (OD) values obtained in the ELISA with M. flocculare Ab were as high as the values obtained with peak M. hyopneumoniae Ab titers. Levels of Ab against M. flocculare were at relatively higher OD at 1 year PI than Ab against M. hyopneumoniae. Sera with high levels of Ab against M. flocculare cross-reacted slightly with M. hyopneumoniae antigen in immunoblotting and ELISA.     

A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae

A blocking ELISA was developed by using a monoclonal antibody (4082-05-344-18) which specifically detected an epitope on the Mycoplasma hyppneumoniae 40 kDa membrane protein without cross-reacting with M flocculare or M hyorhinis. The results obtained with sera from specific pathogen-free pigs inoculated with M flocculare or M hyorhinis confirmed the specificity of the assay. An immunoblotting procedure was used to characterise the antibody response of pigs experimentally infected with M hyopneumoniae. Antibodies to the 40 kDa antigen were detected two weeks after infection and remained as major markers for at least 20 weeks. Cross-reacting antibodies to this antigen were not detected in convalescent sera from piglets infected with M flocculare or M hyorhinis. Sera from experimentally infected pigs were compared by means of the blocking ELISA and an indirect ELISA. The kinetics of ELISA antibodies after experimental inoculation were also studied. The detection of antibody was rather more stable for a longer time with the blocking ELISA than with the indirect ELISA. In an evaluation of more than 1000 sera from the field there was excellent agreement between the two methods.     

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.     

Evaluation of the indirect hemagglutination assay as a practical serodiagnostic test for mycoplasmal pneumonia of swine

Sera from swine experimentally or naturally infected with Mycoplasma hyopneumoniae (the etiological agent of mycoplasmal pneumonia of swine, MPS) were tested by the indirect hemagglutination assay (IHA), the enzyme-linked immunosorbent assay (ELISA) and the complement fixation (CF) test. The IHA detected antibody at comparable times and levels to the other 2 serological tests following experimentally-induced infection. In the late antibody response (greater than or equal to 86 days post-infection), the ELISA titres were higher than either the IHA or the CF test. The IHA appeared least satisfactory when it was used to test sera from commercial swine herds. When 1000 sera were tested, the IHA was positive for only 30 (22%) of 135 sera which were positive by the ELISA and the CF test. The IHA titres were low; 20 of the 30 sera had a titre of only 10. The end-points for the IHA were difficult to read for sera of this low titre. The relationship between positive IHA results for the herd sera obtained at necropsy, and the occurrence of gross or microscopic lesions typical of MPS was poor (41 and 50% agreement, respectively). An agreement of 39% was noted between positive IHA results and the localization of mycoplasmal antigens by an indirect immunofluorescence (IIF) test. However, IHA results correlated significantly (P less than 0.05) with gross and microscopic lesions, but not with the IIF test. No significant correlation was noted between the IHA (or the other 2 serologic tests) and the cultural isolation of M. hyopneumoniae or M. flocculare. On the basis of these results, the IHA appears to have limited promise as a practical test for the diagnosis of MPS in commercial swine herds because of the low titres observed, poor correlation of the IHA and other indicators of MPS, the necessarily subjective determination of end-points, and other inherent technical limitations of the test.     

Serological, pathological and cultural evaluations of swine infected experimentally with Mycoplasma flocculare.

Fourteen caesarean-derived, colostrum-deprived pigs and seven conventional swine were exposed to low passage, cloned, field isolates of Mycoplasma flocculare. Sera were collected at varying intervals postexposure (PE) and tested against M. flocculare and M. hyopneumoniae antigens in a semi-automated ELISA. Swine were killed six to 17 weeks PE and their lungs examined grossly for lesions and culturally for mycoplasmas. Pure cultures of M. flocculare were recovered from the lungs of 11 of 14 swine killed six to 12 weeks PE. Mycoplasmas were not isolated from the swine killed 15 to 17 weeks PE. Only one pig had gross lesions of pneumonia. Immunoassays revealed that swine were slow to seroconvert and titers (expressed in terms of optical density) were low. Three of 21 swine had antibodies to M. flocculare five weeks PE, five of 17 had seroconverted at seven to eight weeks and all surviving swine had antibodies to M. flocculare 76 days PE and beyond. Net optical density of positive sera was in the range of 0.201 to 0.412 (an optical density of 0.2 regarded as the breakpoint between negative and positive reactions in our ELISA). All of the sera were ELISA-negative when tested against M. hyopneumoniae antigen. This is regarded as a very significant finding. There has been concern that field sera might contain antibodies to M. flocculare and that such antibodies could render serodiagnostic tests for mycoplasmal pneumonia of swine nonspecific. Results of the present study suggest that swine infected with M. flocculare do not develop sufficient levels of antibodies to interfere with enzyme immunoassays for M. hyopneumoniae.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of Mycoplasma hyopneumoniae antibody in porcine serum

An enzyme-linked immunosorbent assay (ELISA) for detecting antibody to Mycoplasma hyopneumoniae in porcine serum is described. The results are presented as an ELISA ratio, calculated by dividing the absorbance of the test sample by the mean absorbance of control negative sera. In known infected pigs, the ELISA ratio was highest when the serum concentration applied to the ELISA plate was diluted 1 in 20 in PBS - Tween. Mean ELISA ratios ranged from 1.2 +/- 0.3 for pigs without porcine enzootic pneumonia (PEP) lesions to 5.5 +/- 1.5 for pigs observed with a PEP lesion reacting positively with immunofluorescent histopathology. Pigs observed with typical PEP lesions at slaughter, but not confirmed by immunofluorescent histopathology had a mean ELISA ratio of 4.9 +/- 1.7. The ELISA was highly sensitive (95.6%) and specific (98.8%) when pig sera from commercial piggeries of known M hyopneumoniae infection status were assessed. No cross-reactivity with serum from a pig hyperimmunised with killed M flocculare was detected, and reactivity with serum from another pig hyperimmunised with killed M hyorhinis showed only weak cross-reactivity, which failed to reach the ELISA positive threshold (ELISA ratio 3) for M hyopneumoniae.     

An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. III. Serological findings and their relationship to pathomorphological and microbiological findings

Blood samples from 777 pigs, originating from 9 different herds, were collected at slaughter and examined for antibodies to Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae by the indirect hemagglutination assay (IHA) and the complement fixation (CF) test, respectively. Results were compared to pathological and microbiological findings. Antibodies to M. hyopneumoniae in positive titers of 1/80 or higher were found in 62% of the samples. The relationship between positive IHA titers to M. hyopneumoniae and gross findings indicative of enzootic pneumonia of pigs (EPP), histological findings indicative of EPP, the isolation of M. hyopneumoniae and the demonstration of M. hyopneumoniae by indirect immunofluorescent testing ranged from 64% to 68%. No correlation was noted between positive IHA titers and the isolation of Mycoplasma flocculare. Positive antibody titers to A. pleuropneumoniae of 1/10 or higher were detected in 5% to 85% of the samples from individual herds. Positive titers to A. pleuropneumoniae serotype 2 were found in 71% to 79% of the sampled animals from herds with high frequencies of pneumonic lesions indicative of pleuropneumonia. In herds with low frequencies of pleuropneumonia, positive titers were recorded in from 0 to 4% of the tested pigs. However, no statistical association was found between pleuropneumonia and positive titers to A. pleuropneumoniae serotype 2 in individual animals. Twenty-one per cent of samples with positive CF titers to A. pleuropneumoniae showed antibodies to more than one serotype.     

Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies

The 36kDa L-lactate dehydrogenase (LDH) and a 29kDa partial fragment of an ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potential as diagnostic antigens. Recombinant LDH was genetically engineered to contain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni(2+)-chelate affinity chromatography. A partial 262 amino acid segment representing the C-terminal end of the PR2 protein was cloned as a glutathione S-transferase (GST) fusion protein, expressed in E. coli and purified by urea extraction. Purified recombinant LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assays. Our immunoblots showed that both proteins detected anti-M. hyopneumoniae antibodies in field and experimentally infected pig sera but not in any of the SPF control sera. The two proteins were specific for M. hyopneumoniae as they did not react with sera of pigs infected with the closely related Mycoplasma flocculare and Mycoplasma hyorhinis which are frequently isolated in pigs but are not of particular concern.     

Establishment of swine interleukin-6 sandwich ELISA

We established a sandwich enzyme-linked immunosorbent assay (ELISA) for swine interleukin-6 (SwIL-6), which was applied for detection of SwIL-6 in vitro and in vivo. Anti-SwIL-6 rabbit- and goat-polyclonal antibodies, and monoclonal antibody (mAb) were prepared, conforming that all of the antibodies were reactive with recombinant SwIL-6 by Western blotting and indirect ELISA. A sandwich ELISA was developed using the mAb as a capture antibody and biotinylated goat-polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA for rSwIL-6 was 49pg/ml and did not show cross-reactivity with swine IL-1b, IL-4, IL-8, IL-18, IL-12, and IFN-g. Using the ELISA, SwIL-6 was detected in culture medium of the monocytes stimulated with PHA-P and PMA, and the plasma or the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae or Mycoplasma hyopneumoniae. This ELISA for SwIL-6 may be useful for understanding the role of this cytokine in various swine diseases.     

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.     

Enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the coronavirus-like agent in feces of pigs naturally affected with porcine epidemic diarrhea (PED) or experimentally infected with the CV777 isolate. The assay was specific and more sensitive than electron microscopy. An ELISA blocking assay is described for the detection and titration of antibodies. Specific antibody formation was demonstrated in pigs experimentally infected with CV777 and in swine naturally affected with PED.     

Sensitive glycoprotein gIII blocking ELISA to distinguish between pseudorabies (Aujeszky's disease)-infected and vaccinated pigs.

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV) (Aujeszky's disease virus) -infected pigs from those immunized with a glycoprotein g92 (gIII) deletion mutant, PRV (dlg92dltk) [OMNIMARK-PRV]. This blocking ELISA test utilizes an anti-PRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate, TMB for color development and a cloned PRVg92 (gIII) antigen to coat wells of microtiter test plates. Undiluted sera are used to block the binding of the mAbgIII-HRPO conjugate to the antigen. The gIII blocking ELISA is specific and has a sensitivity comparable to screening ELISA and latex agglutination tests. PRV-negative sera and sera from pigs vaccinated once, twice, or four times with the gIII-negative vaccine all showed negative S/N values of greater than 0.70 (S/N defined as the optical density at 630 nm of test sera/optical density at 630 nm of negative control sera). Sera from PRV-infected herds, sera from pigs experimentally infected with virulent PRV, and sera from pigs vaccinated with modified-live or inactivated gIII+ vaccines were positive for gIII antibodies (S/N less than 0.7). Sera from pigs experimentally infected with 200 PFU virulent PRV seroconverted to gIII+ antibodies 7-10 days postinfection. Sera from pigs vaccinated with gpX- and gI- vaccines seroconverted to gIII+ antibodies 7-8 days after vaccination. The gIII antibodies persisted after gIII+ vaccinated for at least 376 days postvaccination. Sera from pigs protected by vaccination with PRV (dlg92dltk) and then challenge exposed to virulent PRV at 21 days postvaccination showed gIII+ antibodies by 14 days postchallenge. The specificity and sensitivity of the gIII blocking ELISA assay was further demonstrated on the United States Department of Agriculture-National Veterinary Services Laboratory (USDA-NVSL) sera from the 1988 PRV check set and the 1989 gIII PRV check set by comparing the gIII blocking ELISA assay with virus neutralization, screening/verification ELISA and latex agglutination assays.     

Field validation of a commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds.

A commercially available blocking ELISA was analyzed for its ability to identify antibodies to porcine coronaviruses (transmissible gastroenteritis virus [TGEV] or porcine respiratory coronavirus [PRCV]), to differentiate antibodies to TGEV and PRCV, and to identify TGEV-infected herds. Nine sera from uninfected pigs, 34 sera from 16 pigs experimentally infected with TGEV, and sera from 10 pigs experimentally infected with PRCV were evaluated using both the TGEV/PRCV blocking ELISA and a virus neutralization (VN) assay. The ELISA was not consistently effective in identifying pigs experimentally infected with TGEV until 21 days postinfection. Sera from 100 commercial swine herds (1,783 sera; median 15 per herd) were similarly evaluated using both tests. Thirty of these commercial herds had a clinical history of TGEV infection and a positive TGEV fluorescent antibody test recorded at necropsy within the last 35 months, while 70 herds had no history of clinical TGEV infection. The blocking ELISA and the VN showed good agreement (kappa 0.84) for the detection of porcine coronavirus antibody (TGEV or PRCV). The sensitivity (0.933) of the ELISA to identify TGEV-infected herds was good when considered on a herd basis. The ELISA was also highly specific (0.943) for the detection of TGEV-infected herds when the test results were evaluated on a herd basis. When sera from specific age groups were compared, the ELISA identified a greater proportion (0.83) of pigs in herds with TGEV antibody when suckling piglets were used. In repeatability experiments, the ELISA gave consistent results when the same sera were evaluated on different days (kappa 0.889) and when sera were evaluated before and after heating (kappa 0.888). The blocking ELISA was determined to be useful for herd monitoring programs and could be used alone without parallel use of the VN assay for the assessment of large swine populations for the detection of TGEV-infected herds.     

Evaluation of a monoclonal blocking ELISA and IHA for antibodies to Mycoplasma hyopneumoniae in SPF-pig herds.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) and an indirect haemagglutination assay (IHA) were applied to serum samples from 124 specific pathogen-free (SPF) breeding and multiplying herds, which participate in the routine serological surveillance of the Danish SPF programme. Clinical and pathological observations of the herds and microbiological culturing of Mycoplasma hyopneumoniae were used to calculate herd sensitivity, herd specificity and herd predictive values for the two serological assays. The ELISA was superior to the IHA in herd sensitivity and herd specificity, with values of 93 per cent and 96 per cent, respectively, for the ELISA, and 61 per cent and 92 per cent for the IHA. During the six month period of evaluation 2.5 per cent of the herds were infected with M hyopneumoniae each month. At this level the IHA was found to have a positive herd predictive value of 16 per cent, compared with 39 per cent for the ELISA. The negative herd-predictive value on the same level was 99.8 per cent for the ELISA and 98.9 per cent for the IHA. If the assays were applied to a group of herds with a herd prevalence of M hyopneumoniae infection of 30 per cent (as is the case with the production herds in the Danish SPF programme) the predictive value of a positive herd diagnosis would be 91 per cent for the ELISA and 76 per cent for the IHA, and the predictive value of a negative herd diagnosis would be 97 per cent with the ELISA and 85 per cent with the IHA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).     

Japanese encephalitis virus immunoglobulin M antibodies in porcine sera

A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of porcine immunoglobulin (Ig)M antibodies to Japanese encephalitis virus (JEV). Antibodies in sera were captured onto the solid phase of Microtiter plates sensitized with mouse monoclonal antibodies to porcine mu heavy chain. Virus antigen binding to the lawn of IgM was quantitated by subsequent binding of peroxidase-labeled human hyperimmune anti-JEV IgG, which in the final step, catalyzed a substrate color change. In sucrose density-gradient fractionated sera from recently infected pigs, the peak of ELISA JEV IgM activity corresponded to the peak of 18-S, 2-mercaptoethanol-sensitive hemagglutination-inhibiting (HAI) antibody activity. Within 2 to 3 days, JEV-infected sentinel pigs developed high JEV IgM activity; this activity decreased within 2 weeks. Among specimens collected from 99 random swine at abattoirs in Thailand during a period of low JEV transmission, none of 25 JEV HAI-negative sera had JEV IgM activity, 7 of 74 JEV HAI-positive sera did have JEV IgM activity, and the remaining 67 sera had readily detectable JEV HAI antibodies, but lacked JEV IgM. The JEV IgM solid-phase ELISA was useful for rapidly diagnosing active or recent JEV infections in swine.     

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

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

Cell-mediated and humoral immune response to Mycoplasma hyopneumoniae in pigs enhanced by dextran sulfate

The effect of dextran sulfate (DS), known to be cytotoxic to macrophages, on the cell-mediated and humoral immune response to nonviable Mycoplasma hyopneumoniae in pigs was investigated. The cell-mediated immune response was determined by means of lymphocyte transformation a test, using uptake of [3H]thymidine in a microculture system and the humoral immune response by means of a microplate complement-fixation test. Peripheral blood lymphocytes from pigs vaccinated with nonviable M hyopneumoniae and DS incorporated substantially more [3H]thymidine than did those from pigs given Mycoplasma or DS alone. The transformation of lymphocytes from M hyopneumoniae-DS vaccinated pigs was enhanced when M hyopneumoniae cells used in the assay system were heated at 60 C for 30 minutes. Similarly prepared M flocculare and M hyorhinis cells also stimulated lymphocytes from M hyopneumoniae-DS vaccinated pigs, but not nearly as great as when M hyopneumoniae cells were used. The humoral antibody response and the cell-mediated immune response to nonviable M hyopneumoniae was markedly enhanced by DS. Pigs were vaccinated with nonviable M hyopneumoniae and/or DS 4 times and challenge exposed intratracheally with viable M hyopneumoniae. Pigs vaccinated with M hyopneumoniae and DS had less severe pneumonia than did nonvaccinated pigs.     

Use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus.

A blocking ELISA was developed to detect antibodies directed against porcine epidemic diarrhea virus (PEDV). The PEDV antigen was first incubated with dilutions of test sera. Any antigen that was not blocked by antibodies in the serum was assayed in a double-antibody sandwich ELISA, using 2 monoclonal antibodies directed against different antigenic sites on PEDV as capture and detecting antibodies, respectively. The blocking ELISA was compared with a fixed-cell ELISA that used monolayers of Vero cells infected with PEDV prototype strain CV777 as a solid phase and a conjugate of an IgG-specific monoclonal antibody for antibody detection. Pigs were inoculated with PEDV strain CV777 or 1 of 2 field isolates, and antibody responses were measured by use of the 2 tests. Antibodies were detected by the blocking ELISA as early as postinoculation day 7 and, by the fixed-cell ELISA, as early as postinoculation day 14. From day 14 on, antibody titers for both tests correlated highly. Titers for the fixed-cell ELISA were 5.4 times higher than those for the blocking ELISA. The latter technique is easier to perform and discriminates well between infected and noninfected pigs, which makes this test useful for routine diagnosis and serologic surveys of porcine epidemic diarrhea.     

Assessment of antibody response of swine infected with Mycoplasma hyopneumoniae by immunoblotting

An immunoblot procedure was used to evaluate porcine antibody response to inoculation with Mycoplasma hyopneumoniae. Mycoplasmas solubilized with sodium dodecyl sulfate were used as antigens. Antibodies to 5 antigens, estimated to be of molecular weight (mol wt) 110,000, 64,000, 50,000, 41,000, and 36,000, were detected in sera collected during the course of induced mycoplasmal pneumonia. Mycoplasma hyopneumoniae antigens, mol wt 110,000, 50,000, 41,000, and 36,000, cross-reacted with M flocculare when antigen prepared from M flocculare or hyperimmune serum against it were used in the immunoblot procedure. The 36,000-dalton (D) antigen reacted with M hyopneumoniae and M hyorhinis convalescent sera. The 64,000-D M hyopneumoniae antigen was the only antigen that did not cross-react with M flocculare or M hyorhinis. Exposure of immunoblot strips with antigens to trypsin before reacting them with the convalescent sera abolished binding ability of the 110,000-D and 36,000-D antigens, but had no effect on binding by 64,000-D, 50,000-D, or 41,000-D antigens. None of the 5 antigens bound to 11 lectins.